IN VITRO ACETYLATION OF HISTONES
IN RAT LIVER CHROMATIN

By
LOUISE ADELE RACEY

A DISSERTATION PRESENTED TO THE GRADUATE COUNaL OF

THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1970

imJn,^,'?.!,':^ °^ FLORIDA

3 1262 08552 3099

■lilllll

ACKN0V7LEDGMENTS

I would ] ike to thank the members of my comiaittee for
their help during the progress of this research and the
preparation of this dissertation. I would especially like
to thank Dr. Paul Byvoet for his guidance and critical
evaluation of my research and preparation of this manu-
script. His continued interest in my progress and stim.\.ilat-
ing discussions have been an invaluable contribution to this
study. I thank Dr. John VJ. Brookbank, who has been a source
of encc)urag6nent and guidance in this work. Dr. E. Marsliall
Johnson and Dr. Thom?i.3 W. O'Brien contributed greatly in
discussion throughout my graduate career and by critically
reading this manuscript. 1 would like to thank Dr. Steven
Zam and Dr. Ann Larkin for their suggestions and criticisms
in the preparation of this manuscript. To my friend, Use
Ortabasi, I. extend many thanks for her help in introducing
me to rhe techniques of electrophoresis and photography,
and her critical reading of this manuscript. To alj the
faculty or Cellular and Molecular Biology at the University
of Florida I extend man^' thanks.

XX

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ii

LIST OF CHARTS vi

LIST OF TABLES vii

LIST OF FIGURES ix

ABSTRACT X

INTRODUCTION 1

Composition of Lhe Interphase Nucleus 3

Restriction of the Genome 5

Possible Mechanisms for Gene Control 6

Repetition of DNA sequences 6

RNA polymerase and associated factors. ... 6

Histones as repressors 7

Nonhistone chromosomal proteins as

antagonists to DNA-histone inLoraction . . 9
Nonhistone chromosomal proteins as
antagonists to RNA i^olymerase-

histone interaction 11

Arguments against histones as repressors . . 11

Modification of liistone structure 13

Mothylation of Histones 13

Phosphorylation of Histones 14

Acebylation of Histones 16

Rationale of Proposed Study 24

MATERIAIiS TVND METHODS 28

Isolation of Nuclei 28

Isolation of Dooxyribonucleoprotein (DNP) .... 29

Conditions of Incubation 30

Extraction of Histones and Determination of

Si^ecific Radioactivity 31

Determination of Acidic and Res.i.dual Proteins . . 33

111

TABLE OF CONTENTS (CONTINUED)

Methods for Extraction of Acetylating Enzymes . . 36
Extraction of acetylating enzyme from

acetone powder of rat liver nuclei .... 36
Extraction of acetylating enzyme from

rat liver nuclei with saline 3 7

Extraction of acetylating enzyme from

rat liver DNP 39

Method for Determining the Extent of 0-
Acetylation of Free Histones and that

Occurring in DNP 41

Determination of Incorporation of Radioactive

Acetate into the Various Histone Fractions. . . 43

Fractionation according to Johns 43

Electrophoresis of histone fractions .... 47

RESULTS 49

Conditions Influencing the Acetylation of

Histones in DNP 52

Divalent cations 52

Medium components 52

Temperature 54

Concentration of DNP 58

Concentration of acetyl-CoA and Kin

determination 58

Effect of pfl in diffex-ent buffer systems . . 59
Enzymatic Nature of the DNP Catalyzed

Acetylation of Histones 64

Inhibition studies 64

Comparison of DNP catalyzed acetylation with

spontaneous acetylation of free histones . 68

Buffer Effects 68

Temperature Effects 68

Determination of the Extent of 0-

Acetylation by Hydroxylamine Test . . 70

Enzyme isolation 74

Acetylating Enzymes from Rat

Liver Nuclei 74

A.cetone powder extracts 74

Saline extracts 75

Acetylating Enzymes from Rat

Liver DNP 78

Relative acetylation of the various

histone fractions 81

iv

TABLE OF CONTENTS (CONTINUED)

Page
DISCUSSION 84

ADDENDUM 92

Comp^irison of Acetylating Activity in

Novikoff Hepatoma and Livor DNP 92

Deacetylating Activity in Nuclear Extracts. ... 95
Effects of Treatments Known to Increase RNA
Synthesis on the Acetylation of Histones

in DNP 98

Purity of hydrocortisone preparations. ... 99

Use of more samples 100

Use of adrenalectoraized rat 100

Preincubation of DNP with hydrocortisone . . 102
Media changes designed to increase the

solubility of DNP during the incubation. . 102
Effect of in vivo administration of
hydrocortisone on the In vitro

acetylation of histones 105

Effect of phcnobarbital and liver
regeneration on the in vitro
acetylation of histones in DNP 105

LIST OF REFERENCES 108

BIOGRAPIilCAL SKETCH 118

V

LIST OF CHARTS

Page
CHART

1 OUTLINE OF GENERAL PROCEDURE 35

2 OUTLINE OF SEPARATION OF HISTONE FRACTIONS . 46

VI

LIST OF TABLES

Page

Table

I Effect of Various Medium Components on the
Transfer of Acetate from Acetyl-CoA to
Histones in DNP 55

II Effect of Increasing Concentrations of
Acetyl-CoA on the Transfer of; Acetate from
Acetyl-CoA to Histones in DNP 51

III Effect of Various Conditions on In Vitro
Transfer of Acetate from Acctyl-CoA

to Histones 67

IV Comparison of the Extent of 0-Acetylation
in DNP with that Occurring Spontaneously

as Measured by Lability to Hydroxy lamine . . 73

V Restoration of Acotylating Activity in

Heated DNP by 0.3 5 M Extracts from Nuclei. . 76

VI Acetylation of Isolated Histones with a

0.35 M NaCl Enzyme Extract from Nuclei. . . 77

VII Extraction of Histone Acetyltransf erase
Activity from DNP 80

VIII Acetylation of Various Histone Fractions

in DNP 82

IX In. Vitro Uptake of Acetate into Histones:

Comparison of Liver and Novikoff Hepatoma. . 94

X Deacetylation Activity in Nuclear Sap

Extracts 97

XI Effect of Increasing Concentrations of
Hydrocortisone on In Vitro Acetylation
of Histones in DNP 101

vii

LIST OF TABLES (CONTINUED)

Page

XII Effect of Hydrocortisone on ^n Vitro
Acetylation of Histones in Liver DNP
from an Adrenalectomized Rat 101

iin Effect of Preincubation of DNP with

Hydrocortisone on Iii Vitro Acetylation of
Histones 103

XIV Effect of Increasing the Solubility of
DNP on In Vitro Acetylation of Histones

in the Presence of Hydrocortisone 104

XV Effect of In. Vivo Administration of Hydro-
cortisone on In, Vitro Acetylation of His-
tones in DNP 105

XVI Effect of Phenobarbital and Liver Re-
generation on Iri Vi tro Acetylation of
Histones in DNP 106

V. 1.1.1

LIST OF FIGURES

Page

Effect of Temperature on the Transfer

of Acetate from Acetyl-CoA to Histones

in DNP 57

Effect of the Concentration of DNP in the

Incubation Mixture on the In Vitro Transfer

of Acetate from Acetyl-CoA to Histones ... 60

Effect of Increasing Concentrations of

Acetyl-CoA on the ^ Vitro Transfer of

Acetate from Acetyl-CoA to Histones

in DNP Plotted According to Lineweaver

and Burk 62

Effect of pH in Different Buffer Systems
on the Transfer of Acetate from Acetyl-
CoA to flistones in DNP 63

Comparison of the Effect of Increasing pH
in Different Buffer Systems on the Spon-
taneous and Enzymatic Acetylation
Reactions 71

Effect of Temperature on the Transfer of
Acetate from Acetyl-CoA to Histones in
Rat Liver DNP and to Free Plis tones in
Different Buffer Systems . . 72

Acrylamide Gel Pattci^ns Obtained with

Various Histone Fractions 83

IX

Abstract of DissorLation Presented to the Graduate Council

of the University of Florida in Partial Fulf iJ Imont
of tlie Requirements for the Degree of Doctor of Philosophy

IN VITRO ACETYL/\TION OF HISTONES IN RAT LIVER CHROMATIN

By

Louise Adele Racey

December, 1970

Chairman: Jolin W. Brookbank
Co-chairman: Paul Byvoet
Major Department: Zoology

Rat liver chromatin or deoxyribonucleoprotein (DNP) was
found to exhibit acetyltransf erase activity in vitro, which
seems to be closely associated with it. Evidence is pro-
vided v/hich indicates that this transfer of acetate from
acetyl-CoA to lysine in "arginine-rich" liistones represents
an enzymatic reaction. A number of comparisons have re-
vealed that the in. vitro reaction occurring within DNP is
much less random than the spontaneous acetylation of free
histones in solution in the presence of acetyl-CoA. Reports
fj:om other laboratories have indicated that a similar enzyme
could be extracted from an acetone powder of either v/hole
liver or nuclei. The study reported seams to involve a
different enzyrae since no acetyltransferase activity can be
obtained from an acetone powder of chromatin using these
m.ethods. A procedure similar to that used for the extraction
of mammalian RNA polymerase has been successfully applied to
the extraction of acetyltransferase activity from rat liver

X

chromatin, although even in this case, about lial.f of the
activity remains bound to the DNP complex. The extraction
of acetyltransf erase activity from chromatin provides
strong support for the assumption that the described in
vitro reaction is enzymatic.

XI

INTRODUCTION

The mechanism of gene control remains today as one of
the major unex^^lained areas of biology. It is obvious from
an analysis of living systems that some form of control

must exist. One example is the circadian rhythm of liver

1

enzyme activity exhibited by rats and other mammals.

Although oscillations in the activity of some enzymes may
be explained by enzyme stimulation or inhibition, many
enzymes are subject to induction and repression at the
level of the genome. In this case the production of a
new enzyme is dependent on deoxyribonucleic acid (DNA)
directed ribonucleic acid (Rl^TA) synthesis as an initial
step. The mechanism by which the control of the expression
of certain regions of the genome occurs in eukaryotic cells
is largely unknown. In prokaryotic cells a model system has
been proposed involving repressor proteins which interact
with DNA, thereby influencing its transcription by RNA
polymerase.'^ ° Small effector molecules interact with the
repressor protein, changing its configuration in such a way
as to influence its interaction with DNA and thereby the

transcriptive process. These effector molecules can be
metabolites or other substances capable of allowing the cell
to become responsive and adaptable to changes in its environ-
ment. It is not known whether a similar mechanism is opera-
tive in eukaryotic cells.

The differentiation process undergone by eukaryotic
systems is another area where gene control is operative.
Here a totipotent cell produces offspring of a particular
type, the characteristics of whicli are maintained from one
generation to another. Since within an organism each cell
type contains the same amount of DNA and hybridization

studies have shovv^n the DNA of each cell type to be similar

7-10
to that of every other type, the question arises as to

how the cells acquire and maintain the selective expression
of specific genetic information from one generation to
another. The eukaryotic system is therefore more compli-
cated than the prokaryotic coll in that it is not only
capable of reversible changes in gene expression, such as
occurs in enzyme induction, but also undergoes more per-
manent chc-mges which are relatively constant from generation
to generation. That these changes are not completely ir-
reversible "nas been shown by nuclear transplantation studies
wherein nuclei from the intestinal cells of Xenopus appeared
to be cajjable of supporting the development of a complete

11

embryo from an enucleated egg. However, Br3.ggs and King,
liave demonstrated that nuclei taken from frog embryo cells

at successive stages of development are progressively less

12
able to support normal development. Although the total

complement of genetic information is present, the expression
of this information is restricted by its passage through
developing cytox^lasm.

Composition of the InLerphase Nucleus
Attempts to ox^^lain the mechanism of genetic control
must take into consideration the structure of the nucleus.
In the eukaryotic cell the nucleus is a defined area sur-
rounded by a semipermeable membrane. This membrane in-
fluences a dynamic interchange of materials between the
nucleus and the cytoplasm, vrithin the nucleus is contained
DNA and its associated proteins. Nuclear sap proteins can
be removed by isotonic saline v/ashes whereas other proteins

along with the DNA form part of the insoluble chromatin

13

complex. In the interphase nucleus part of the chromatin

has a diffuse appearance and consists of m.any fibers. In
some regions the chromatin appears in a more condensed con-
dition. Condensed regions of a single chromosome are called
heterochromo tin, as opposed to the less condensed or euchro-
matic regions. The term "heterochromatin" is also used to

refer to a condensed state of chromatin, and is applied to

14
such regions of the interphase nucleus as well.

The componexits of the chromatin include DNA and histone

15, 16
proteins, found m approximately equivalent amounts.

Nonhistone proteins compi'ise about 27 percent of the DNA

17
associated protein in calf thymus, and 43 percent in

18
liver. These proteins are subdivided into acidic pro-
teins, which are alkali soluble, and residual proteins,

which remain behind after acidic extraction of histones and

19
basic extraction of acidic proteins. RNA polymerase and

other enzymes are among those which make up the nonhistone

20, 21

chromosomal proteins. Also some species of RNA have

been found associated with the chromatin. '

The arrangement of tlie chromosome has been a subject
of considerable controversy among cytologists. Interphase
chromatin fibers of 200-300 A in diameter have been de-
scribed and Ris has proposed that these consist of two 100
A° units, and that each of these further consist of two
40 A° units representing single DNA-protein molecules.
This has been questioned and more recent data utilizing
the techniques of trypsin digestion of chromatin fibers in
conjunction with electron microscopy shows that each 230 A
fiber consists of a single DNA molecule packed in a pro-
teinaceous sheath. ' According to the model this unit

is further induced to supercoil by the addition of more

28
protein. Different chromosomal proteins may play specific

roles in the maintenance of this structure.

Restriction of the Genome

The need for selective gene expression implies that
certain regions of the chromsome will be active at a given
time whereas others will remain quiescent. Evidence that
this is the case comes from hybridization studies. Georgiev
observed that RJ>IA synthesized on free DNA hybridized to a
much greater extent with DNA than that synthesized on chroma-
tin. He explaxned thxs by the observation that there are
many repeated DNA sequences in the chromatin which are re-
stricted in transcription. However, RNA synthesized on
chromatin competes with iH. vivo synthesized messenger RNA
(mRNA) for sites on DNA more efficiently than does RNA
synthesized on free DNA. This implies that RNA synthesized
on a chromatin template more closely approximates the in
vivo situation.

Heterochromatic or tightly condensed regions of chroma-
tin are considered to be in the repressed condition, whereas
the more diffuse euchromatic regions are believed to be
metabolically active. Electron microscope siutoradiography

has revealed that the diffuse, presumable e^ichromatic,

3

regions actively incorporate H-uridine in contrast to the

30
heterochromatic regions. Frenster has isolated bulk

fractions of condensed and diffuse chromatin from interphase

31

calf thymus lymphocytes. He has shown that the condensed

6
regions contain up to 80 percent of the nuclear DNA, but
only 14 percent of tlie nuclear RNA. This observation would
seem to support the concept that the condensed state of the
chromatin is associated with repression of genetic expression,
Possible Mechanisms for Gene Control
Repetition of DNA sequences
One finding which has recently attracted attention is

that more than one third of the DNA of higher organisms is

3
made up of sequences which recur anywhere from a 1 x 10

6 32

1 X 10 Limes per cell. The nucleolus of amphibian

oocytes contains many replicas of the genes which code for

33, 34
ribosomal RNA. This repetition of specific genes has

been viewed as a unique means of controlling the expression

of genetic information by allowing for the production of

certain products in greater quantity at a given time in

the life of the cell.

RNA polymerase and associabed factors

In considering the ways in which the regulation of gene

expression may occur, RNA polym.erase and its associated

sigma factor must be mentioned. In bacterial systems the

sigma factor has been found to be responsible for the initi-

35
at ion of TiNA chains by DNA-depondcnt RNA polymerase. RNA

synthesis in isolated nuclei of avian erthryocytes was

stimulated by a heat stable factor present in extracts of

36

HcLa colls. MorG recently, a protein-like factor fx-ora

calf thymus with a sedimentation constant of 3 S has been

37
described. This factor stimulated DNA-dependent RNA

synthesis from Lhe same tissue after the enzyme had already
become bound to DNA. These findings have a bearing on ex-
planations of gene control in that specificity of initiation
site and rate of RNA synthesis are influenced by these
factors .

Histones as repressors
In 1951 the Stedmans suggested that histones may play

a role in the regulation of gene activity by acting as

38
repressors of RNA synthesis. The observation by Huang

and Bonner that histones suppress RNA polymerase activity

when added to DNA in vitro prompted an increased investiga-

39
tion into this area. The relevancy of these findings has

been questioned and it has been suggested that the inhibi-
tion of RNA polymerase activity is due simply to the pre-

40
cipitation and removal of DNA from solution. Bonner and

Huang have answered this objection by describing conditions

41
which prevent DNA precipitation, and Butler and Chipper-
field shov/ed that the amount of inhibition continues to

increase with increasing histone concentrations even after

42
the DNA has been fully precipitated. Clark and Byvoet

atterapited to resolve the question of whether or not a corre-

8
lation exists between the activity and the solubility of
the DNA template by plotting the logarithm of the percent
inhibition of template activity against the amount of un-

aggregated DNA present in the reaction mixtures at various

43
histone/DNA ratios. This type of plot revealed a close

correlation between the solubility of the DNA template and
the inhibition of polymerase activity by histone. In any
case, it seems likely that reconstitution of the DNA-
histone complex by simple titration of DNA with histones
preserves very little of the specificity of the original
relationship and there is a question as to whether or not
these Jji^ vitro findings have any relation to the _in vivo
situation.

Further support for the involvement of histones in
repression of RNA synthesis came from studies v/hich
selectively removed different histone fractions from chro-
matin. Successive extractions of pea-bud chromatin with

44
NaCl solutions resulted in increased template activity.

Extraction of histones from the heterochromatin of the male

mealy bug was shown to derepress RNA synthesis and to in-

crease actinomycin D binding. ^Vhen all the histone was

removed from the heterochromatin it became equivalent to the

euchromatin in these x'espccts.

Nonhistone chromosomal proteins as
antagonists to DMA-histone interaction

It has been suggested that nonhistone proteins act as

antagonists to the DNA--histone interaction which serves to

. 46
repress RN/\ synthesis. In support of this, it has been

found that basic proteins, such as histones, are closely

associated witli DNA, occurring in highest concentrations

47

where DNA appears to be tightly coiled and condensed.

During puff formation in dipteran giant salivary chromo-
somes, i.e., at regions of intense RNA synthesis, there is
no measurable difference in the amount of stainable basic
protein, implying a continuity of structure during the
transcriptive process. Interestingly, however, there seems

to be an increase in the acidic proteins in these regions

48
during puff formation. In isolated chromatin fractions the

ratio of total histones to DNA did not significantly differ
within either the repressed or active fractions, and the
relative proportions of each of the distinct types of his-
tones were similar in both forms of chromatin. However,
when nuclear polyanion contents of active and repressed
chromatin fractions were determined relative to the DNA
contents, the active fractions were found to contain more

total nonhistone proteins, RNA and phospholipids, and phos-

49-53
phoprotein phosphorous. ■ About 15 percent of the non-

. ■ . . . ' 54-55

histcne proteins consist of such nuclear phosj^hoproteins .

10

In vitro experiments hcivo shown that acidic proteins

complex with free his tones and prevojit liistone inhibition

56
of RNA synthesis by bacterial Rl\iA polymerase. However,

these proteins cannot dissociate the histone-DNA con\plex

to reverse histono inhibition of RNA synthesis. Therefore,

Gpelsberg and Hnilica have suggested that their action may

reside in the prevention of DNA-histone interaction rather

than. tliG dissociation of DNA-histono complexes.

It has been shown by Paul and GiJmour that a] 1 the

protein components of the chromosome are necessary for tlie

57
production of FiNA which resembles that found in^ vivo .

Using hybridization teclmiques they found that his tone re-
pression of DKA directed RNA synthesis in. vitro is non-
specific. However, if they added the residual and acidic
proteins back to DNA and histone, the new mRNA synthesized
does compete effectively with mRNA produced _in vivo. Similar
experiments by Bekhor, Kung, and Bonner showed that DNA,

histones and chromosomal RNA produced a primer capeible of

58
yielding mRNA equivalent to the natural product. They

therefore suggested that nonhistone protein is not re-
pressive. Marusliige, B.rutlag, and Bonner, in contrast to

Paul and GiJinour, found that DNA saturated with nonliistonc

59

chromosoma.l proteins was as good a pr.imer as naked DNA.

These differences may result from contaminated preparations

11

and have yet to be resolved. It is difficult to imagine
a mechanism for gene control in which any one of the ele-
ments of the chromosome does not play a role, either directly
or indirectly. The observations of these interactions is,
therefore, liniited probably by experimental technique.

Nonhistone chromosomal proteins as
antagonists to RNA polymerase-histone interaction

Association of chromosomal proteins with RNA polymerase

is another means by which repression of transcription could

take place. Histones form complexes with bacterial and

mammalian RNA polymerase leading to the inhibition of in

60-62
vitro RNA synthesis. Since it has been observed that

salt and polyanions (e.g., acidic nuclear proteins) acti-

63, 64
vate RNA polymerase in chromatin, it has been suggested

that this may be due to dissociation of enzyme-histone com-

65
plexes, thereby stimulating RNA synthesis. This sugges-
tion is supported by the observation that the ionic strength
required for maximum dissociation of the enzyme-histone
complex is within the range required for the activation of
endogenous RNA polymerase. In contrast, no detectable dis-
sociation of DNA-histone complexes was seen at these ionic
strengths.

Arguments against histones as repressors
One of the strongest criticisms against the idea that
histones function as genetic repressors is the fact that

12

histones lack specificity. Thoro are five main histone

fractions: very lysine-rich (fj^), moderately lysino-rich

if
(f ), and the arginine-rich (f , f , and f ) histones.
^o 2al<ia^ 3

Although there are major differences between the five his-

66-69
tone fractions, there is very little difference in the

composition of similar fractions from different species.
Sequence studies of an arginine-fraction (f., ,) from organ-
isms as far removed from each other as calf (thymus) and

pea seeds revealed essentially identical primary struc-

70-75
tures. Recent data on very lysine-rich (f-,) histones

showed that they are more numerous and have some phenotypic

76-79
specif J.cihy, but not enough to account for the observed

phenotypic differences. It has been suggested, therefore,

that chromosomal RNA plays a role in gene repression and

46
derepression. This is an attractive idea since it offers

a means for specific recognition of DNA sites for selected

repression of transcription. In support of this, Huang and

*

Older terminology referred to just the "arginme-rich"

histones, v/hich included the moderately lysine-rich (f2-b)
fraction, and the "lysine-rich" histones, v/hich only
referred to the very lysine-rich (fi) fraction. Hereafter,
"arginine-rich" enclosed in quotation marks refers to the
arginine-rich {f„^,, f-,. ^, and f ) and moderately lysine-

rich ( f o-u) fractions conibined .

13

Bonner have found a chromosomal i^A bound to a nonhistone

22, 23
protein, which they have postulated is associated in

80

complexes with histones.

Another major" objection to the simple repression of
transcription by histono binding to DNA comes from the ob-
servations that histones turn over at the same rate as

81-83
DNA, are synthesized at about the same time of the

84-88
cell cycle as DNA, , and are present at tlie same levels

3]
within the chromosome duriny RNA synthesis. These obser-
vations mediate against a simple explanation of histones
acting alone as gene repressors simply by combination with
and dissociation from DNA. It is for these reasons that
attention has focused on structural modifications of his-
tones, taking place after their synthesis, which may influence

their binding to DNA, and thereby perhaps the transcriptive

89
process .

Modification of histone structure

Three methods of histone modification are known. These

90-92
are methylation of lysine and histidine residues,

^ -> . • ^ 1 • ■. 71, 74, 93, 94
acetylacion of lysine residues, and phos-

95
phorylation of serine residues.

Methylation of Histones

Isolated nuclei from calf thymus and chromatin from

96-98 90

P<?as, rat liver, and Ehrlich ascites cells can

14

91

methylate histones iri vitro. Methionine is the methyl

97
donor via S-adenosyl-methionine. Gershey et al have found

that the methylation of histones occurs much more rapidly

in immature erythroid cells, but as the nucleus matures and

loses its biosynthetic capacities, it also loses its caj^acity

92
to methylate histxdine xn its histones. Tidwell et al.

have compared the time courses of histone synthesis and

99

methylation during regeneration of the liver m the rat.

They found that maximal histone methylation occurs at a time
when the rates of histone and DNA synthesis have already
begun to decline and does not correlate v/ith an increase in
RNA or nonhistone protein synthesis. rhey suggest, there-
fore, that methylation is not involved in gene activation
for RNA synthesis.
Phosphorylation of Histones

The phosphorylation of histones occurs both in vivo
and in vitro. Ord and Stocken have found a 2-fold increase
in the rate of phosphorylation and phosphate content of

very lysine-rich (fi) histone of regenerating rat liver near

. , 100

the first peak of DNA synthesis after partial hepatectomy.

Langan and Smith have isolated an enzyme from liver which

specifically transfers pliosphate from adenosine triphosphate

95

(ATP) to .'?orine residues of protamines and histones.

The most rapidly phosphorylated histones are the lysine-rich

15

fractions, f, and f . It appears that there is more than
1 2b

one histone kinase and that they differ in their rate of

101
phosphorylation of f, and f histones. A phosphatase

1 2b

has also been partially purified which is specific for

102

phosphorylated histones and protamine.

Since phosphorylation by histone kinase is stimulated
by adenosine-3 ' , 5 ' -monophosphate (cyclic AMP) Langan has
suggested a mechanism for the induction of RNA synthesis

by those hormones that cause increases in the concentration

103

of cyclic AjMP. The administration of glucagon or insulin

to rats causes an in_ vivo increase in phosphorylation of

lysine-rich histones in the liver involving the same specific

104

serine residue that becomes phosphorylated in vitro.

The phosphorylation of chromosomal proteins is not

restricted to histones. Kleinsmith et al.have observed an

32

increase m P-phosphate incorporation into nuclear phos-

phoproteins of lymphocytes stimulated to grow and divide by

105
phytohemagglutinin. This increase was particularly evi-
dent in nonhistone protein and occurs prior to an increase
in the rate of RNA syntliesis occuring under these con-
ditions. Gershey and Kleinsmith have followed changes in

nuclear phosphox^roteins during Lhe course of development of

106
the avian erythrocyte. As maturation proceeds, the

nuclear levels of both phosphoprotein kinase and protein-

16
bound phosphorous correlate with a decrease in tlie bio-
synthetic activities of the nucleus. The bulk of the
incorporation of phosphate occurs as in the case of the
lymphocyte in the nonhistone protein fraction. Since
Langan has reportod that phosphoproteins can interact with
histones in vitro, and that complex formation with phos-
phoprotein decreased the inhibitory effects of added histone
on DNA dependent RNA synthesis, it has been suggested that
this interaction may play a role in the regulation of gene
expression." Further support for this idea comes from
studies on isolated chromatin fractions, which revealed that

phosphoprotein concentrations are highest in fractions which

107
are active in RNA synthesis. Dipteran salivary gland

chromosomes also show active phosphorylation of nonhistone

] 08
proteins, particularly in the puff regions.'

Acetylation of Histones

The acctyli^tion of histones has been studied in intact

109, 110 111-113 .

animals, in cells in tissue cultui-e, m iso-

94, 96, 114-116
lated nuclei, and in isolated enzyme prep-

.117-119 , . ,. ^ ^^ .

arations. Early studies measured the incorporation

14
of C-acetate, but it v/as later found that acetyl Coenzyme

] 14

A (acetyl-CoA) is the direct acetyl donor. In every case

examined the incorporation of labeled acetate occurred pri-
marily in the argininc-rich (f.,, f-.,,, and f-..^) fractions,

17

v/hile very lysine-rich (f]^) ^nd moderately lysine-rich (f^-, )
fractions incorporated minor amounts. This is in contrast

to the predominant phosphorylabion of the lysine-rich frac-

101
tions. The low level of acetate incorporation into thymus

f-j histone also contrasts with the finding that this fraction

is almost completely acetylated at its amino terminal

, 120, 121 , , , , , ^ -, 1

end. These alpha-N-acetyl groups appear to be rela-

tively stable, whereas the internal epsilon-N-acetyl groups in
the fp-,-1 and f^ fractions are involved in a dynamic process
of acetylation and deacetylation . Evidence for the fact that
acetyl groups are attached to histones after synthesis of the
polypeptide chain is provided by the finding that puromycin
in concentrations sufficient to block protein synthesis in

calf thymus nuclei did not inhibit the acetylation of his-

96
tones. In agreement with this, Byvoet has observed that

the turnover of acetyl groups in histones is much more rapid

than the turnover of histones themselves and the ratio of the

81

tv7o turnover rates vriry with the tissues. Gershey et al .

have prepared histone fractions from calf thymus nuclei which

14 93

had been incubated in the presence of sodium- C-acetate.

These were digested with trypsin and pronase and the result-
ing peptides and amino acids were separated by chromatog-
raphy. Only the arginine-rich fractions f„ . and f_, were

2al 3

appreciably labeled and the radioactivity was associated with

18

epsilon-N-acetyl-lysino. Studies on t]ie sequence of f,, , his-

tones have shovn that 50 percent of the lysine residues at

71
position 16 are acetylated in calf thymus, whereas in pea

74
bud the epsilon-N-acetyl content is only 6 percent.

Enzyme fractions which acetylate histones liave been iso-

117
lated from an acetone powder of who.Te pigeon liver. Two

fractions were found, of which one activates acetate by con-
verting it in the presence of ATP, Mg , and coenzyme A to
acetyl-CoA, while the other transfers the acetate from
acetyl-CoA to histones. Using similar procedures Gallwitz

reported the isolation of acetokinase from an acetone powder

118
of I'at liver nuclei. This enzyme was capable of trans-
ferring acetate from acotyl-CoA to isolated histones and was

especially active for arginine-rich (f_ and f ) fractions.

3 2al

More recently Gallv/itz reported the isolation of two

histone-specif ic transacetylases from rat liver nuclei

119
using a different approach. Rat liver nuclei were

sonicated in the presence of 1 M ammonium sulfate and 20

percent glycerol, followed by precipitation with 3.5 M

ammonium sulfate. A 50-fold purification was accomplished

by Ciiroma L"ography . No preference of the two enzymes has

been found for one specific histoae fraction.

The isolation of a dea::etylating enzyme v/as reported by

122
Inoue ana I'ujimotf) from a salt extract of calf ttiymus.

19

This enzyme showed a preference for histones which were

14
acetylated by incubating calf thymus nuclei with C-acetic

anhydride. Libby has also reported deacetylating activity

123

in nuclei of rat liver and Novikoff hepatoma.

Studies by Allfrey, Mirsky, and their colleagues have
attempted to correlate histone acetylation and chromosomal
activity. Arginine-rich histones from calf thymus inhibited

RNA polymerase in an "aggregate enzyme" from calf tliymus as

124 125
well as purified RNA polymerase from bacterial sources. '

In the case of the manmialian RNA polymerase studies, the

problem v/as complicated by difficulties in the purification

of the enzyme because it remained bound to the chromatin

124
complex. Mien external histones are added to this com-
plex, inhibition of RNA synthesis is observed, but the
significance of this observation is questionable and it may
represent a nonspecific effect. When RNA polymerase from
Escherichia coli was used, this problem was circumvented as

it can be obtained in purified form. In. vitro RNA synthesis

14

by E_^ Coll RJSIA polymerase was followed using C-ATP (ade-

14
nosine triphosphate) or C-UTP (uridine triphosphate) as

precursors. It was found that the incorporation into RNA

was decreased by the addition of arginine-rich histone.

This degree of inhibition of RNA synthesis was progressively

decreased experimentally by the chemical addition of acetyl

20

96

yroups bo the isolated his tones. The technique used to

acetylate the hi.stones for the inhibition of IINA snythesis
was selected to give maximum acetylation of alpha amino
groups and minimum acetylation of epsilon amino groups of
lysine. It has subsequently been found that ijl vivo ace-
tylation involves jprimarily the epsilon amino group of ly-
sine. Therefore, it v/ould be interesting to study tliis
effect using histones which have been acetylated in a more
physiological manner.

Histone acetylation has been compared in chromatin
fractions v/hich are active or inactive in RNA synthesis.
Fractionation of thymus chromatin after labeling nuclei in
vitro with isotopic RNA precursors has shown that RNA syn-
thesis proceeds faster in the diffuse chromatin than in the

30, 31
condensed chromatin. Comparisons of the rates of

acetate incorijoration into the histones of each of these

fractions show that histone acetylation, like that of RNA

synthesis, proceeded 2-3 times more rapidly in the diffuse

chromatin fraction. Autoradiography of calf thymus nuclei

3

after H-acetate incorporation indicates that acetylation

occurs in both diffuse and condensed regions but is par-
ticularly evident at the boundaries between the two re-'

126 ...

gions. Thj.s would be expected if acetylation is associ-
ated with the transitj.on of chromatin from a condensed to a
more diffuse, active state.

21
Since puffing in dipteran polytene chromosomes has been

associated with RNA synthesis., attempts to correlate this

127
with acetylation of histones have been made. Autoradio-

gr^iphic studies indicated uptake of radioactivity in chromo-
somes when salivary glands of Chironomus thummi v/ere incu-

3

bated in the presence of H-acetate, The graj.n densities

over t]\e puff regions however were not particularly intense
and A13 f rey has suggested that the methods used in the fix-
ation process extract most of the acetylated arginine-rich
histones from t>ie chromatin. However, using new methods of
fixation, which retain incorporated acetate. Clever and
Ellga^rd also failed to observe any accumulation of label

over puff regions and it is th.eir opinion that puff forma-

128
tion does not include acetylation of histones.

The mature nucleated red cells of birds synthesize
little or no RNA, whereas lymphocytes do. Comparisons have
been made of the rates of histone acetylation and RNA syn-
thesis in nucleated erythrocytes and in isolated lymphocyte
nuclei. These reveal that the lymphocyte nuclei were far

more active in acetylating their histones than were the

1 9 r

erythrocytes. Although th:is may be coincidental, it is

further evidence for the hypothesis that acetylation of
histones may be associated with gene activation.

Comparison of the rates of histone acetylation and RNA

22

synthesis in lymphocytes at different times ;•< fter the addi-
tion of phytohemagglutinin (PIIA) to the culture medium shows
that RNA synthesis is stimulated and that there is an increase
in acetylation of aryinine-rich histones preceding the in-
crease in RNA synthetic activity. In contrast to the be-
havior of lymphocytes upon exposure to PHA, polymorphonu-
clear leucocytes curtail the synthesis of Rhil^i and under
these conditions histone acetylation is also depressed. '
Monjardino and MacGillivray have suggested, T-iowever^ that
these effects may be nonspecific since some preparations of

PHA were found to increase RNA synthesis in lymi^hocytes

129
while decreasing histone acetylation. Kil lander and

Rigler have shewn that the amount of acridine orange dye
bound to the chromatin of PFIA stimulated lynipihocytes in-
creased rapidly over a time course which is similar to that

130
for histone acetylation. This reflects a change in

chromatin molecular structure as a result of PFIA treatment,

which as suggested by Allfrey may be initiated by the acety-

126
lation reaction.

It has been shovm that in liver cells g^one activation

occurs \vith consequent appearance of new species of RNA as

131

a result of partial hepatectomy. Increa.Vrfi'S m DNA tem-

132

plate activity of isolated liver nuclei, and increases

in template activity and RNA x^olymerase activity in chro-

23

mat .in and in "aggregate" enzyme preparations have been re-

44, 13 3 - 13 5 _
ported. With this j.n mind Allfrey and others

have studied the acetylation of histones during the course

110, 126
ot li\'er regeneration. The specific activity of

different histone fractions from control and regenerating

rat liver were measured at various time intervals after an

3

in vivo injection of h-acetate. The results m sham-
operated controls indicated a high rate of acetate uptake,
the maximum specific acti\/ity being reached in .15 minutes.
Turnover at 60 minutes v;es such that only one third of the
acetyl-groups o.riginally incorporated into the argi.nine-rich
fraction vi^ieleft. This pattern was altered for regenerating
liver. In th.is crise histone acetylation was increased by
300 percent at 3 hours after partial hepatectomy and in the
period between 1-2 hours after the operation the histone^s
lost 13.7 percent of their original acetyl content while
the cont.rols had lost 70 percent. Allf.rey has suggested
that this is due to an increase in the rate of acetylation
and lower rates of deacetylation for regenerating liver.
The inc?.ea3e in the percent retention of the acetyl groups
in histones from regenerating liver just precedes a rise in
PJSTA polymerase activity in .rogenerat.i.ng rat liver nuclei
and reaches its peak 2 hou.rs before the riuclei reach the
first i:>lateau in RNA polymerase activity. These findings

24

are consistent with the view that acetylation of histones
modifies DNA-histone interactions, and the subsequent changes
j.nfluence the template activity of the chromatin for RNA
synthesis .

A correlation has also boon observed between the pat-
terns of RNA synthesis and histone acetylation in liver re-
sponding to stimulation by steroid hormones. ' Ad-
ministration of Cortisol to adrenalectomized rats leads to
increases in the amounts and changes in the types of RNA
synthesized. As in the case of liver regeneration, Cortisol
treatment of adrenalectomized rats leads to early increases
in the rate of acetylation of arginine-rich histones and a
suppression of turnover of previously incorporated acetyl
groups. Cortisol stimulation of the liver is only one
example of hormone-induced gene activation. Takaku et al.
observed increases in the acetylation of histones in spleen
cells of polycythemic mice just preceding an increase in

RNA synthesis at 4 and 8 hours after erythropoietin injec-

136
tion. Another example is provided by estradiol which m-

137-139
creases RNA synthesis in the uterus. Estradiol has

also been reported to stimulate the acetylation of histones

140
by cell free extracts of the rat uterus.

Rationale of Proposed Study

Although none of these observations show a direct cause

25

and effect relationship, they suggest that the acetylation
of histones may be related to genetic expression. The fact
that only two of the five known histone fractions are acety-
lated ujion incubation of nuclei in the presence of radio-
active acetate suggests that t:his is an extremely specific
reaction. The complete elucidation of the amino iicid se-

auence of the f , histone has even established that in the

2al

entire molecule only one specific lysine residue is acety-

71, 74
lated at the epsilon amino position. It does not seem

likely that the acetylation of only one lysine residue would
change the structure of the histone sufficiently to influ-
ence gene activity. Kov/ever, the location of this lysine
residue within an unusual cluster of five basic residues
would seem rather suggestive of some role which this acety-
lation may play in histone--DNA interaction.

It appeared that the biological implications of the
postulated changes in histone-DNA interactions, resulting
from histone acetylation, are sufficiently important to
warrartt a thorough study of this process at the molecular
level. In order to carry out such a study, the development
of an iri vitro system is essential in view of the many ad-
vantages of such a system over the myriad of possible in-
direct effects which m.ay influence the outcome of _in vivo
studies. Some of these may be hormonal influences, pool

26

sizes, and degradation of labeled precursor. Even in Lhe
case of isolated nuclei there is a possibility of cytoplas-
mic contamination. In addition, nuclei contain an enzyme
wliich dcacetylatos histones (see Addendum). The development
of the _in. vitro system described in this study which utilizes
deoxyribonucleoi^rotein (DNP) has eliminated many of these
disadvantages and possesses all the advantages of ini vitro
systems in general. The most important of those is the
isolation of Lhe enzyme to be studied from the many vari-
ables which cannot be controlled j^ vivo. Other advantages
are greater reproducibility, control of medium composition,
temperature, and pH. In the first stages of the development
of t.his system, it appeared that DNP could be employed in-
stead of isolated nuclei, especially if the most immediate
precursor, acetyl-CoA was used. It was found that isolated
rat liver DNP was capable of transferring acetate from
acetyl-CoA to histones. A problem which presented itself
at thi.s point was the possibility that this transfer might
not be enzymatically catalyzed since it was found that free
histones are spontaneously acetylated upon incubation v/ith
acetyl-CoA. It, therefore, became essential to establish
that this transfer is an enzymatically catalyzed reaction
and attention has been directed toward this problem. This
dissertation describes the conditions influencing the trans-

27

fer of acetate from acetyl-CoA to histones in rat liver
chromaLin and provides evidence that this is an enzymatic
reaction .

MATERIALS AND METHODS
Isolation of Nuclei

Although in the beginning these studies v/ere done v/ith
calf thymus, this was replaced by rat liver since fresh
preparations were found to exhibit higher acetylating
activities in contrast to frozen material, O'cher factors
in favor of rat liver include availability of previously
described methods for the isolation and purification of
nuclei and deoxyribonucl eoprotein (DNP) . It v/as also
desirable to compare experiments on Novikoff hepatoma and
the tissue from which the tumor presumably originated.

Male Holtzman rats weighing approximately 300-500 gm
were decapitated and the liver excised. All preparations
v.'ore carried out at 4°C. The liver was homogenized in 10
volumes (weight/volume) 0.25 M sucrose containing 1.5 rcM
CaCl2 in a Potter type homogenizer, filtered througli cheese-
cloth to remove connective tissue, and centrifuged at 600 x

141
g for 10 minutes. The pellet v/as resuspended in 5

volumes of 0.25 M sucrose, 1.5 niM CaCl„ and recentifuged

as a.bove. The nuclear fraction thus obtained v.'as purified

28

29

by rosuspension and centrifugation at 40,000 x g for 30

142
minutes in 2.1 M sucrose, 0.5 mM CaCl„.

It has been found that this procedure removes surface

cytoplasm from rat liver nuclei even when loose homogenizers

21

were employed. Examination of the 2.1 M sucrose, 0.5 mM

CaClp pellet by phase microscopy showed clean nuclei with
little visible debris in the preparation. Nuclei have also
been prepared according to more recent procedures using
detergent, Triton N-101, which has been shown by electron

microscopy to remove the outer nuclear membrane and perinu-

143
clear ribosomes.

Where calf thymus was used, the 2.1 M sucrose purifi-
cJition of nviclei was omitted because there is less of a
problem of cyLoplasmic contamination with this tissue. If
Novikoff hepatoma was used, fibrous connective tissue was
removed by filtration through a wire screen rather than
cheesecloth.

Isolation of Deoxyribonucleoprotein (DNP)

DNP v/as prepared from nuclei by homogenization in 10-
15 volumes of 0.14 M NaCl, 0.01 M sodium citrate in a micro
Waring Blendor (Virtis) , for 1 minute followed by centri-
fugation at 2000 X g for 10 minutes. The sediment was re-
suspended by light homogenization in a loose fitting Potter
type homogenizer and recentrifuged. This procedure, which

30

13
removes solub.l e nuclear proteins, was repeated once.

Chart 1 illustrates the outline of the general procedure

used.

Conditions of Incubation

Unless otherwise stated, samples of isolated DNP or

purified nucle.i containing about 0.3-0.5 mg of histones were

o
incubated at 37' C m a medium originally designed for studies

144
of protein synthesis by isolated calf thymus nuclei.

This medium contr'ined 0.19 M sucrose, 20 mM glucose, 25 ^M

phosphate buffer, pJI 6.75, 12 mM NaC]. , 0,75 mM Ca"^"^, 5 mM

++ 14

Mg and 0.01 ^ic acetyl- C-CcT^ (spec. act. 56 mc/mM, New Eng-
land Nuclear), in a final volume of 2 ml (step 3 in Chart

3

1) . In prolindnary experiments H-acetate was used as a

14
precursor, but replaced by acetyl- C-CoA since it was

14
found that this improved incorporation of C-acetate. Low

3

incorporation of acetate into histones when H-acetate was

used as a precursor may be due to lack of activating enzymes

converting acetate to acetyl-CoA (see Introduction) , es-

3

pecially since there was no stimulation of Il-acetate in-
corporation into liistones upon the addition of ATP. Studies
reported below revealed that divalent cations, glucose, and
sucrose v/ere not necessary for the transfer of acetate from
acetyl-CoA to histones. They were therefore eliminated from
the medium. Latr.r, experiments were also run at a pH of 8,
rather tlian 6.75.

' 31

After a 15 minute incubation, the reaction was stopped
by cooling the flasks and adding trichloroacetic acid (TCA)
to 5%. The resultJ.ng mixture was centrifuged at 2000 x g
for 10 minutes (step 4 in Chart 1).

If nuclei were incubated in the above described manner,
the reaction was stopped after 10 minutes by rapid cooling
and centrifugation of the samples at 2000 x g for 10 min-
utes. The nuclei were then washed in saline-citrate to re-
move soluble nucleoproteins as described for the preparation
of DNP.

In experiments where proteinaceous extracts were added
to DNP preparations, the reaction was not stopped by TCA,
but only by centrifugation after cooling, to prevent co-
precipitation of the added proteins with the DNP upon ad-
dition of TCA. These proteins coiald contaminate subse-
quently extracted histones.

Extraction of Histones and Determination
of Specific Radioactivity

After incubation, the total histones were extracted

from the DNP sediment with 0.25 N PICl (step 5 of Chart 1).

Chromatographic analysis and subsequent amino acid analysis

of histones extracted in this manner have shown that less

than 0.5% of the total nitrogen is due to contamination

^ ,^ , . 145, 146

from ocher proteins. After centrifugation the

supernatant was made 5% with TCA which precipitates the

32

-arginine-rich" (includes f^^ fraction) histones leaving
the very lysine-rich hi stones behind in solution. After
standing overnight the TCA precipitate was washed with ace-
tone-1% HCl and ether and allowed to dry (stop 6 in Chart 1).
This procedure, therefore, selectively isolates the "ar-
ginine-rich" histones, which have been found to be by far
the most actively acetylated fraction (see Introduction) .

Determinations of total counts contained in histones,
DNA, acidic and residual proteins (described in next sec-
tion) show that 76% of the radioactive label is contained in
the "arginine-rich" histone fraction and therefore cannot be
ascribed to contamination with a hypothetical nonhistone
fraction which is very highly labeled. AlmO'St all of the
remaining label was contained in the acidic protein frac-
tion. Purity of histone fractions was tested by amino acid
analysis and acrylamide gel electrophoresis of "arginine-
rich" histones isolated according to this procedure.

In experiments where the spontaneous acetylation of
free histones was studied, "arginine-rich" calf thymus
histones were used. Essentially, these histones are com-
parable to those obtainable from rat liver, but were
used in place of rat liver histones because larger quan-
tities could be obtained more easily from calf thymus.
Tliese histones were extracted from 0.14 M NaCl washed calf

thymus with 0.2 5 N HCl and the "arginino-rich" fractions
were x^i'<^cipitated by 5% TCA and dried in acetone-1% HCl
and ether.

Protein concentrations were determined by the method of

147
Lowry et al . using bovine serum albumin as a standard.

Radioactivity was determined by liquid scintillation count-
ing after dissolving the histones in a small aliquot of
water (step 7 in Chart 1 lists this procedure) . One liter
of liquid scintillation fluid contained 72 ml of spectra-
fluor Butyl-PBD (Nuclear Chicago), 300 ml of ethanol, and
628 ml of toluene.

Determination of Acidic and Residual Proteins
Amounts of DNA in DNP subjected to various treatments
were determined prior to incubation to ensure that histone/
DNA ratios were unchanged by these treatments. Therefore,
any differences in acetylating activity observed between
pretreated DNP and control preparations cannot have been
due to changes in the essential composition of the complex,
such as might occur by the selective removal of histones.
The precipitate remaining after the 0.25 N HCl extraction
of histones was washed with cold 5% TCA, to remove any small
organic molecules, such as sucrose, which might interfere
with the DNA determinations. After centrifugation, the
precij)itate was washed with 95% ethanol, absolute ethanol.

34
chloroformimenfbanol 2:1, and twice with ether to remove
lipids. ^Vlien DNA was determined by tlie TJ SO. reaction,
the mixture was dissolved in 1 N NaOII and incubated at 3 7°C
for 16-20 hours. Upon acidification with IICl at 0 C the
DNA precipitates leaving hydrolyzed RNA in solution. After

hydrolysis in perchloric acid at 90 C for 20 minutes it

149
was reacted with JI^SO^ to give a colorimetric reaction.

The Burton test which is a modified diphenylamine test for

150
DNA was also used. In this case it was not necessary to

remove RNA as this reaction is specific for deoxyribose.
Specific activity of acidic proteins was determined by
dissolving the proteins remaining in the precipitate after
hot acid exti^action in 1 N NaOH. Any residual protein un-
dissolved by this procedure was dissolved in NCS solubilizer
(Nuclear Chicago) and counted.

35

CHART 1: OUTLINE OF GENERAL PROCEDURE

1. Isolated nuclei washed 3x in saline-citrate-:?'

2. Deoxyi ibonucleoprotcin (DNP) .

3. Nuclei or DNP incubated in 2 ml final volume, containing;

0.19 M sucrose, 20 mM glucose, 25 mM phosphate buffer,
pH 6.75, 12 ml-I NaCl, 0.75 mM Ca''""'', 5 ml^l Mg++, 0.01 uc
acetyl-^^C-CoA (0.6 muM) .

4. After 15 minutes brought to 0 C and trichloroacetic
acid (TCA) added to 5% and centrifuged.

5. Sediment extracted with 0.2 5 N HCl "Arginine-rich
histones"

6. Precipitated with 5% TCA, v/ashed with acetcne-1% HCl,
ether, dry.

7. Dissolved in 0 ,. 6 ml HoO/ protein concentration (Low.ry) ,
radioactivity (liquid scintillation), DNA (H^SO^) .

36

Mebhods for Extraction of Acetylatinq Enzymes

Extraction of acetylating enzyme from
acetone powder of rat liver nuclei

Nohara et al. have reported the isolation of acetylating

117
enzymes from an acetone powder of whole pigeon liver. In

addition Gallwitz has extracted an acetokinase from an ace-

118
tone powder of rat liver nuclei. An attempt v^as there-
fore made to use a similar procedure to extract the acetyl-
transferase from nuclei and DNP . A modification of the
original procedure was used, which was claimed by Bondy and

Roberts to be successful in the isolation of a histone

151
acetokinase from rat brain and liver nuclei.

Nuclei or DNP obtained from 2 5 gm of rat liver were

suspended in 7 ml of water. This preparation was then added

dropwise with continuovis stirring to 70 ml of acetone kept

o
at -30 C. The resulting suspension was passed through

Whatman No. 1 filter paper on a Buchner funnel. The pre-

o

cipitate was washed 3 times with 50 ml acetone at -30 C and

o
dried in a dessicator at 0 C. The dry powder obtained was

llien liomogenized in 13 ml of 0.1 M tris-IICl buffer at pH 8.2

and centrifuged at 2000 x g for 10 minutes. The resulting

supernatant was treated with neutral saturated ammonium

sulfate, Tlie fraction v/hich precipitated between 3 5-60%

saturation witli ommoin.um sulfate was dissolved in 6.5 ml of

tris buffer and dialyzed against two successive 500 ml

37

portions of a solution wliicli contained 0.0G8 M KCl, 0.001
M 2--niorcaptoethanol and 0,02 M NaHCO at pll 8.0 for 1 hour
each. This dialysed preparation contained the soluble aco-
tylating enzyme and 1 ml containing 0.1-0,5 nvy of protein
v/as incubated at 37^0 with 0.5 mg of isolated "arginine-

rich" calf thymus histoncs in 25 \\M phosphate buffer, pH 8,

.14

12 mM NaCl in the presence of acetyl--"' C-CoA. The reaction

was stopped by prc;cipitating the protein onto filter paper
v/ith cold 15% TCA,

In a preliminary experiment Bio Gel filters v/ere used,
which were washed twice v/ith 2 ml of incubation medium
containing 10 times the ojriginal concentration of unlabeled
acetyl- CoA, twice with 2 ml of acctone-1% KCl, and twice
with 1 ml of ether. Since this filter paper did not dis-
solve in scintillation fluid, corrections were made for loss
of counts by counting a sample before and after absorption
onto filter paper. In subsequent experiments millipore

(AAWPO2500) filters were used v/hich could be dissolved in

152 o

Bray's scintillation fluid. These were dried at 90 C for

15 minutes after the TCA precipitation step and subsequently

counted.

Extraction of acetylating enzyme from
rat liver nuclei with saline

Johnfj aiid Forrester have found that acidic x->i-Oteins

38

w}i.i.ch remain bound to the chromatin complex during the 0.14
M NaCl procedures used to prepare DNP from nuclei can be
removed by extraction witli 0.35 M NaCl. It seemed
plaiisibJo that the acetylating enzyme was among this group
of proteins and could be extracted from the DNP complex
v/ith 0.3 5 M NaCl. Therefore, attempts were made to extract
the cicctyltransferase witli 0.35 M NaCl from nuclei and DNP.

Pui-ified rat liver nuclei or DNP fi"om 6 gm of liver
were therefore blended for 1 minute in the Virtis homogeni-
zer in 4 ml of 0.35 M NaCl. After a preliminary centrifuga-
tion at 2000 x g for 10 minutes to remove the m.ajor portion
of the DNP, the supernatant was diluted to 0.14 M NaCl
and centxifuged at 40,000 x g for 30 ma nates. To check
whether ariv DNP remained in the supernatant, the DNA con-
centration of the supernatants was determined whicli showed
less than 0.15 mg of total DNA present. This supernatant
was then used as an enzyme source.

DNP was inactivated by lieating ?.t 65^C for 10 minutes
in 0.14 M NaCl- 0.01 M citrste. The salt extract from
nuclei or DNP derived from 1 gm of liver was incubated with
inactivated DNP from 1 gm of liver at 37'^C for 15 minutes
in 0.1 M NaC] , 25 mM phosphate buffer, pH 8. To pi-event
precipitation of the extract v/ith th.e DNP, the reaction v/as
not stopped with TC/A, but the DNP f;pun down and Tii stones

39

extracted find dried as usual. vVliere isolated "arginine-
rich" calf thymus histories v/ere used as a substrate, the in-
cubation reaction was stopped by precipitation w.i th 15% TCA
(since isolated histones are soluble in the medium) and the
resulting precipitate was dissolved in 1 M hyamine and
counted. Extracts alone and inactivated extracts plus sub-
strate were used as controls.

Extraction of acetylating enzyme
from rat liver DNP

Since previous extraction procedures were effective for

the isolation of acetylating enzymes from rat liver nuclei,

but not fi~om. DNP, the possibility was considered that these

enzymes represented cytoplasmic contaminants and were not

the acetyltransf erase in tlie chromatin complex. Therefore,

an attempt was made to extract the acetylating activity from

the DNP complex using a method which had been proven to be

154
successful .IB the isolation of PI\!A polymerase. Earlier

studies on the isolation of Rl^A polymerfise had revealed

that this enzyme is tightly bound to the chromatin complex,

similar to the chromatin acetyltransf erase . In studies on

endogenous RNA polymerase activity the crude chromatin was

155
consequently often referred to as the "aggregate enzyme."

Nuclei and DNP from 12 gm of rat liver were gently

homogenized in 12 ml of 15 mM phosphate buffer, pll 8, 0.5

40
mM ethelenediamine totraacctic acid (EDTA) , and 1.0 mM
2-mGr.captoethanol. This mixturo was inculcated at 30 C for
50 minutes with gentle shaking. In the original polymerase

extraction procedure a tris-phosphate buffer, pH 8.8, was

154
used, but since phosphate buffer had been shown to be

much more favorable to the DNP catalyzed transfer of acetate

from acetyl-CoA to histones than tris buffer, the phosphate

buffer system v;as substituted. After the extraction, the

mixture was ceutrifuged at 115,000 x g for 40 minutes and

the supernatant served as the enzyme source. One ml of

this supernatant contained the enzyme extracted from DNP

isolated from 1 gra of rat liver. One ml of enzyme solution

14
v/as incubated in the presence of acetyl- C-CoA either with

inactivated (by heating at 65°C, 10 minutes) DNP from 1 gra

of rat liver or 0.5 mg of isolated "arginine-rich" calf

thymus histones. The enzyme was also added back to DNP

from 1 gm of liver which had been extracted by the above

procedure, i.e., to the 115,000 x g precipitate, in an

attempt to restore the preparatioii to full activity. The

final volume was 2 ml and contained 0.25 mM EDTA, 0.5 mM

2-mercaptoethanol, 33 rnM phosphate buffer, pH 8, 12 mM

14 / 1

NaCl, and 0.01 uc of acetyl- C-CoA (spec. act. 56 mc/mM) .

After 15 minutes at 37°C, the fractions containing isolated

histones were cooled and made 15% with respect to TCA, and

41

were filtered on raillipore filters. The filters were

dried in a hot air oven, dissolved in Bray's scintillation

fluidy and counted. The samples containing DNP were cooled

rapidly and centrifuged to prevent co-precipitation of the

enzyme in 15% TCA. The histones were extracted from the

sediment as described above.

Method for Determining the Extent of 0-Acetylation of
Free Histones and that Occurring in DNP

It is possible to estimate the degree of 0--acetylation

156

occurring in proteins by a method devised by Narita. In

this procedure one takes advantage of the lability of the

0-acetyl bond in the presence of hydroxylamine . Histones

or DNP prelabeled with radioactive acetyl-CoA can therefore

be incubated in the presence of hydroxylamine. The degree

of 0-acetylation can subsequently be estimated by measuring

the amount of radioactivity released as a result of this

treatment.

Pogo et al . have shown that the acetate incorporated

in the f his tone fraction of regenerating rat liver was
2al

110
stable to treatment with 2 M hydroxylamine, "^ indicating

that this fraction does not contain 0-acetyl groups. Other

studies on the site of acetylation of histones in calf

thymus nuclei f fraction showed that acetylation only

2a J.

93
takes place at the epsilon-amino group in lysine. The

42

f^ fraction of regenerating rat liver, however, showed a

release of 55% of the acetyl groups as a resulL of hydro-

110
xylaraine treatment, which agrees with the work of Nohara

et al. who reported considerable 0-acetylation of the f^

fraction by pigeon liver enzymes xn vitro. In tlie case

of calf thymus, all of the acetate incorx^orated in the f^

fraction upon incubation of nuclei in the presence of C-

acetate was recovered as epsilon-N-acetyllysine after en-

94
zymatic digestion and ion exchange chromatography. Thus,

tliere is, at least in calf t'nymus, no evidence for the forma-
tion of 0-acetyl linkages under those conditions.

Since it may be expected that an enzymatically cata-
lyzed reaction will show greater specificity of acetylation
and therefore perhaps a lower level of 0-acetylation than
that occurring spontaneously, DNP catalyzed acetylation in
the presence of acotyl-CoA was compared with that occurring
spontaneously by free histones with respect to 0-acetyla-
tion. Conditions were selected to yield a similar amount
of radioactivity by eitlier process. Therefore, for DNP

catalyzed acetylation, DNP equivalent to 1 gm of liver was

14

incubated m the presence of 0.01 uc of acetyl- C-CoA,

12 mM NaCl and 2 5 mM. phosphate buffer, pll 8, for 15 minutes
at 3 7°C. The reaction v;as stopped with 5% TCA, and the
histones were dried with acctone-1% IICl and ether.

43
For the liyd.roxylamine test, 0.45 ml of liydroxylamine
solution (pH 6.4) containing 3 volumes of 40% hydx-oxylamine
hydrochloride and 2 voJ.umes of 3.5 N NaOH were added to 0.3

ml of 0.1 N acetate buffer, pH 5.4, and 0.3 ml of H^O con-

14
tamxng 0.3-0.5 mg of C-acetylated histones obtained as

described above. After standing at room temperature for 5
hours the histones were precipitated with 15% TCA, centri-
fuged after 4 hours, and dried in acetone- 1% HCl and ether.
The percent 0 acetylation was determined by counting the
radioactivity released in an aliquot of the 15% TCA super-
natant, whereas the extent of N-acetylation was deduced from
the radioactivity remaining in the histones after the ■
hydroxy lamine treatment. . _ _

Determination of Incorporation of Radioactive
Acetate into the Various Histone Fractions

157
Fractionation according to Johns

Since it has been observed by others iihat acetylation

of histones occurs primarily in the arginine-rich (f and

^ ^ 2al

f ^) fractions (see Introduction) , it was deemed important to
compare this labeling pattern with that of histones ace-
tylated iii vitro in DNP. Such a comparison would determine
how closely the _in vitro reaction approximated the in vivo
situation. Therefore, histones v/hich had been acetylated
were separated into the five major fractions according to

44

li37
Johns and their specific eictivity determined. These

fractions were examined by acrylamidc gel electrophoresis to
determine the purity of the separation.

For this experiment, DNP was obtained as usual from
15 gm of rat liver and incubated at 3 7°C for 15 minutes in '
5 gm samples containing 10 ml total volume per sample of 12

mM NaCl, 2 5 roM phosphate buffer, pH 8, and 0.1 ^uc acetyl-

14

C-CoA. After the xncubation, the reaction was stopped by

centrifugation and the unbound label was rinsed out by

wasliing once with a large volume of saline-citrate. Then,

the very lysino-rich (fi) fraction was extracted from the

DNP with 8 ml of 5% perchloric acid (PCA) , and centrifugcd

at 1100 X g for 20 minutes. This procedure was repeated

once with 4 ml of 5% PCA. This fraction was precipitated

from the combined supernatants with 25% TCA, dried with

acetone-- 1% HCl, and ether. The residual DNP was extracted

for 18 hours with 20 ml of ethanol-1.25 N HCl (4:1), with

stirring, and centrifugcd at 1100 x g for 15 minutes. This

extraction was rcTseated once with 10 ml of ethanol-HCl

(4:1), for 2 hours. After centrifugation the combined

supernatants contained the arginlne-rich (f and f ) frac--

^a J

tions, whereas the moderately lysine -rich {^^h^ fraction
remained still bound to the residual DNP. The supernatants
containing the f^ and f_ fvp.ctions were dialyzed against

Z3l 3

45

ethauol for 18 hours, which causes the f fraction to pre-

3

cipitate, leaving the f fraction in solution. After cen-

trifugation, Lhe f^ histones were further separated by the

addition of an equal volume of acetone. This caused the

precipitation of f„ „. After centrifugat ion f„ , was pre-

z a ^ 2 a -L

cipitated from the supernatant with 3 volumes of acetone.
The precipitates were dried in ether. The moderately lysine-
rich (fpv,) fr iction v/as then extracted from the residue with
0.2 5 N HCl, and precipitated with 5 volumes of acetone, and
dried in ether.

46

CilART 2: OUTLINE OF SEPARATION OF HISTONE FRACTIONS

DNP from
15 grams of rat liver

extract with 8 ml 5% PCA
centrifuge 1100 x g, 20 min

repeat with 4 ml 5%PCA

ocipitate

nkl

supernatant

extract 18 'lours with 20 ml
ethanol-1.2'3 N HCl (4:1)

centrifuge I l.OO x g, 20 min,

repeat with 10 ml for 2 hrs

add TCA to
25%

centrifuge
1100 X g 20 min.

Precipitate=f ^
wash with acetone-1% HCl, ether

ecipitate

extract with 0.25 N HCl

centrifuge 1100 x g, 20 min.

superhatant

diiilyze against ethanol
18 hrs. I

ecipitate supernatant

precipitate=f

add 5 vol.
acetone

centrifuge
1100 X g
20 min.

precipitate=f o
wash with ether

precipita a=f.

supernatant

add 1 volunie
acetone

1 su

pernatant

2a2

wash with ether

add 3 vol . acetone

2b

precipitate=f „ ,

wash with etlier

47
Electrophoresib of histone fractions
The histono fractions obtained according to Johns were
checked for purity by polyacrylaniide gel electrophoresis
in a vertical electrophoresis apparatus from tlie E. C.

Apparatus Co., Philadelphia, Pennsylvania. Acid gel con-

158
d-itions, designed for the resolution of basic proteins,

were used as I'ollows:

Gel and SJimple Buffer: Tris 0.12 H adjusted to pH 2.9

with citric acid.

Electrode Buffer: Glycine 0.3 7 M, adjusted to pH 4.0

with citric acid.

Sample Solvent: Tris-citric acid buffer pK 2.9 con-
taining 6 M urea. The sample solvent
was saturated with sucrose to facili-
tate the settling of the sample in
the slots.

Polymer Solution: 12% Cyanogum-41 in tris-citric

acid buffer, pH 2.9, containing
3 M urea. Total volume was 150
ml for each gel.

Catalyst: 0.1% ascorbic acid, 0.002 5% ferrous sulfate,
and 0.02% HO.

Besides the addition of sucrose to the sample solvent,
the only modification of the procedure as originally de-
signed was that the HO concentration was dropped from
0.03% to 0.02% to increase the polymerization time long
enough to pour the gel.

After mixing the polymer solation, the ascorbic acid
and ferrous sulfate v.ere added with stirring. Immediately

48

after mixing, the H^O^ was added and the gel was poured.
The electrophoresis apparatus was precoolod Ijefore pouring
the gel by running water at 8 C through Lhc circulation
system. This was done to prevent contraction of tlio gel
upon poliTnerization. Since the gel hardened within a few
minutes, it was necessary to remove any bubbles that formed
immediately. After about 10 minutes the gel had suf f icientJ.y
hardened to fill the apparatus with 2 liters of the electrode
buffer. It was only after this step that Lhe teflon slot
former could be easily removed.

Samples of 15 ul containing 30 rig of protein were
placed in each slot and ran at 250 v., 8^C, for 4 hours.
It was found in preliminary experiments that no pre-run
was necessary using these gel conditions. Staining of the
gel v^/as done in 0.2% Amido Schwartz, 1% acetic acid, and 40%
ethanol, for 20 minutes, and dcstaining accomplished elec-
trophoretically by a destainer from E. C. Apparatus Company.

RESULTS

In early experiments the in vitro acetylation of his-
tones by isolated nuclei was studied. These nuclei were
purified by centrifugation through 2.1 M sucrose as described
in Materials and Methods , In the course of these investi-
gations it was discovered that if these purified nuclei
were washed with isotonic saline, the resulting DNP prepara-
tion possessed an acetylating activity approximately 3 times
that of the nuclei. This was an indication that perhiips an
acetylating enzyme responsible for the transfer of acetate
from acetyl-CoA to histones was present in or at least
closely associated with the chromatin complex. Since other

laboratories had only reported the isolation of acetylating

117-119

enzymes from whole tissue or nuclei, this finding

stimulated interest to characterize this reaction more
carefully. An enzyme obtained from the DNP complex had less
chance of being a cytoplasmic contaminant tlian those iso-
lated from nuclei, and, in view of its localization in the
chromatin itself, took on more meaning in viev/ of the pos-
sibility that histone acetylation plays a role in chromo-
somal function., e.g., the control of gene expression.

49

50

To verify that the acetylation reaction was not due to
cytoplasmic contamination, DNP was also prepared from
nuclei isolated in detergent. This procedure removes the
outer nuclear memljrane . DNP prepared from these nuclei v/as
found to possess an acetylating activity approximately
equivalent to that of DNP obtained from nuclei isolated by
tlie usual procedure. This v/ould support the contention
that the acetylating activity observed in DNP prepared from
nuclei by the usual procedure is not of cytoplasmic origin.

Further support for this was obtained from experiments
in which DNP isolated according to the usual procedure was
further purified by centrifugation at 22,000 rpm for 3 hours
in the Spinco S. W. 25 head in 1.7 M sucrose. Marushige

and Bonner have shown that rat liver chromatin purified by

44
this procedure is characterized by a low RNA content, and

others have reported that the nonhistone protein contained

in this preparation is not a cytoplasmic contaminant, but a

159
real constituent of chromatin. DNP treated in this

manner showed acetylating activity tv/o or three times

greater than that of the usual preparation. These results

confirmed that the acetylation reaction was not due to a

cytoplasmic contaminant and was closely associated with the

chromatin. Having established the localization of the

transferase activity in DNP, the following experiments were

51

designed to characterize Lhe conditions influencing this

reaction, using rat liver DNP as the source for enzyme
activity as well as the acetate acceptor.

52

Conditions Influencing the
Acetylabion of Histones in DNP

Among the conditions influencing the in vitro reaction
that were studied were divalent cations, medi.um components ,
temperature, concentration of substrates, and pH. These
are discussed below in that order.

Divalent cations

Nohara et al. has found a Mg requirement for two
pigeon liver fractions which act respectively as acetate

activating and transferring enzymes in the acetylation of

117 ++ .

i.Golated histones. Gallwitz has suggested that Mg is

only necessary for the activating enzyme since a trans-
ferring enzyme which he has isolated from rat liver nuclei

118
does not require it. In view of these findings it was

decided to study the effect of several divalent cations

on the DNP catalyzed transfer of acetate from acetyl-CoA

to histones.

Rat liver DMP washed in 0.075 M NaCl and 0.024 M EDTA

160
(chelates divalent cations) , pH 8, was incubated in the

presence of 5 mM EDTA or different concentrations of divalent

cations. Tlie results indicate a depression in activil-.y at

10 mM Mn and 8 mM Ca . The presence of Mg from 0-10

mM did not seem to affect the activity signif icanLly .

Medium components (Table I)

Since the original incubation iriedium was designed for

53

the study of protein synthesis by isolated calf thymus

nuclei, it seemed desirable to analyze the effects of the
various components of this medium on the acetylation re-
action. The results of 1 experiment using 4 samples for
each condition is reported in Table I. The term "complete
medium" refers to the one described in .f4atc;rials and Methods,
except that phosphate buffer, i:)H 8, rather than pll 6.75, was
used and the divalent cations were omitted. These changes
were made on the basis of other experiments to be reported
here. Components of the complete medium were omitted and
the molarity of the buffer was gradually lov/ered. The re;-
sults indicate that the presence of sucrose, and glucose is
not essential for the DNP catalyzed acetylation of h5 stones,
and the opcimum concentration of the phosphate buffer was
25 mM. The effect of ionic strength on the reaction mix-
ture was studied by changing the concentration of NaCl in
the incubation medium. Between 0.0]. K to 0.2 M NaCl there
was a slight rise in activity up to 0.1 M NaCl, followed
by a decrease at 0.2 M NaCl. This is in agreement with a

report by Gallwitz and Sekeris on the acetylation of histones

115
by rat liver nuclei. The optim.um conditions for this

reaction v/ere therefore assumed to be 12 mM NaCl, and 25 mJ^i

i:)hosx:-'hnte buffer, pK 8, according to the pa.rameters studied.

54

Temper aturo (Figure 1)
Figure 1 shows the course of the reaction with time
at two different temperatures. The medium for this experi-
ment was as described for the original procedure (Materials

14
and Methods) except that the concentration of acetyl- C-CoA

was 4 times higher. From these data a Q,-^ of about 1.8

could be calculated. Allfrcy has reported a Q, ^ of 2.09

J26
for t-his reaction in nuclei. In gonei-al the velocity

of enzymatic reactions is doubled for a 10 rise in

temperature.

55

Tabic I

Effect of Various Medium Components on the Transfer of
Acetate from Acetyl-CoA to Histones in DNP

Concentration
Incubation Media Specific Activity of Phosphate
( cpm/mg ) Buffer

Complete medium 8243 + 611 25 mM

-sucrose
-glucose 9075 + 910 25 mM

-sucrose
-glucose
-NaCl 7037 + 444 25 mM

-sucx"Ose
-glucose
-NaCl 1729 + 224 2 . 5 mM

-sucrose
-glucose
-NaCl 998 + 85 0.25 mM

-sucrose
~g].ucose
-NaCl 769 + 76 0.025 mM

The results are expressed as specific activity (counts
per minute per mg) of histones plus or minus the standard
error.

Figure 1: Effect of Tcmperatvire on the Transfer of
Acetate Prom Acetyl-CoA to Histones in DNP.

The results are expres_^.cd as specific activity (counts
per minute per mg x 10 ) of histones as a function
of t i ine .

57

Minutes

58

Concentration of DNP (Figure 2)
Since in this particular experimental set-up the acc-
Late-accepting substrate and enzyme are both present in
the DNP preparation, the hi.stone concentration varies with
that of the enzyme when the DNP concentration in the re-
action mixture changes. Therefore, it seemed interesting
to determine the optimum concentration of DNP for the trans-
fer reaction. l^at liver DNP was incubated at different
concentrations in the presence of acetyl- " C-CoA in the
usual m.anner. The results are represented graphically in
Figure 2, either as total counts per minute or as specific
activity (counts per minute per mg histone) . As can be
seen, there is a rise in both curves with an optimum con-
centration of DNP equivalent to 0.3-0.5 mg of histones per
2 ml incubation miixture. Past this point increasing con-
centrations of DNP affected the total incorporation to a
minor degree, but caused a lapid drop in the specific
activity of histones.

Concentration of acetyl-CoA (Table II)
and Km determination (Figure 3)

Tlie I<m of the transfer reaction for acetyl-CoA was
determined by incubating the optimal amount of DNP with in-
creasing concentrations of acetyl-CoA. Results were ob-
tained from 2 separate experiments using isotope solutions
containing 3 different specific activities of acetyl- " C~

59

CoA. The data arc given in Table II below. The results

161

were plotted (Figure 3) according to Linev/eaver and Burk,

giving a I-Cin value of 2 . 5 x 10 M.

Effect of pH in different buffer systems (Figure 4)
In these experiments 25 mM phosphate buffer was sub-
stituted by 2 5 mM tris-HCl buffer or 2 5 niM glycine-NaOH
buffer. The optimum pH for the DNP catalyzed acetylation of
histones in glycine-NaOH buffer was found to be 8.5-9.6.
Tlie phosphate buffer system appeared the most favorable for
the DNP catalyzed reaction, and since pH 8 was the highest
pH that can be obtained v^ith this buffer, it represents the
optimum buffer conditions for DNP catalyzed acetylation of
histones .

60

5000 h
4000

^^2000

E

Q.

E

Q.
u

1000

500

0

1.0 mg. Hist.

Figure 2: Effect of the Concentration of DNP in

the Incubation Mixture on the In Vitro Transfer of

Acetate from Acetyl-CoA to Histones

DNP was incubated at increasing concentrations in the
presence of acetyl-14c-coA as described in Materials
and Methods. Results arc expressed as total counts

per minute recovered in isolated histones ri Fl

or as specific activity (counts per minute per mcj)~'^

ISJ 0 of histones. DNP concentrations

are expressed in terms of histone content of DNP
sample in 2 ml medium.

61

Table II

Effect of Increasing Concentrations of Acetyl-CoA
on the Transfer of Acetate
from Acetyl-CoA to Histones in DNP

Concentration

Spec. Act.

Spec. Act.

pmcles

Acetate/

of Acetyl-CoA

Histones

Acetyl-CoA

mq Histones

(cpm/mq)

(cpm/jarnole)

EXP.

1 1.6 X lO'^M

798

27.7

X 10 ^'

1.6 X 10~ M

6139

213.2

X 10-^^

1.6 X lo" M

23892

6
28.8 X 10

829,5

X 10' ^'

1.6 X 10 M

33892

1176.8

X 10"^

1 X lO" M

3 751

1631.0

X 10"^'

1 X 10 M
2 1 X lO'^M

3168

2.3 X 10^'

13 77.0

-6
X 10

EXP .

2123 9

21 2. .4

X 10-^

0.5 X 10"*^M

29338

293.4

X 10"^

0.33 X 10"^M

18954

8

1.0 X 10

189.7

X lO"*^

0.25 X 10~^M

16072

160.7

X 10""^

0.20 X 10~^M

14338

143.4

X 10~^

-6

0.166 X 10 M

12202

122.0

X 10-^

62

1.0

0.8

o 0.6 -

H> 0.4 -

0.2 -

Figure 3: Effect of Increasing Concentrations of Acetyl-CoA
on the In^ Vitro Transfer of Acetate from Acetyl-
CoA to Histones in DNP Plotted According
to Lineweaver and Burk

Rat liver DNP equivalent to 0.3-0,5 mg histones was incubated in
2 5 mM phosphate buffer, pll 8, 12 niM NaCl, 0.19 M sucrose
and 20 mil glucose in the presence of increasing concentra-
tions of acetyl-CoA as described in Materials and Methods.
Calcium and magnesium were omitted. Ordinate: V-pmoles
of acetate incorporated per mg histone. Abscissa: S-molar
concentration of acetyl-CoA.

-4000

6000 /

/
/

/
/

_/ (phosphate)

-2000

y^

^

y* (trii-HCI)

-2000

.A

® \

-4000

Iglycine-NaOH]

-2000

63

70

8.0 8.0

9.0

8.5 9.0

10.0

pH

Figure 4: Effect of pH in Different Buffer Systems on
the Transfer of Acetate from Acetyl-CoA to
Histones in DNP

The results are expressed as specific activity (counts per
minute per mg) of histones as a function of pH.

64

Enzymatic Nature of the DNP Catalyzed
Acetylation of Histones

Since it had been reported, and svibsequently recon-
firmed in til is laboratory, that isolated histones will be-
come acetylated spontaneously when incubated in the presence
of acetyl-CoA, considerable attention has been given to the
possibility that the acetylation of histones occurring v/ith-
in the DNP complex may not be enzymatic.

Inhibition studies (Table III)

The first approach taken to rule out nonenzymatic
transfer of acetate from acetyl-CoA to histones was an in-
direct one. In these studies the DNP complex was treated
IDrior to incubation in various ways to either wash oiat or
extract the acetylating enzyme, or to inhibit its action by
methods which are in general considered to be deleterious to
enzymes. The effects of these various treatments on the
transfer of labeled acetate during subsequent incubation
are shown in Table III.

Treatment of DNP in 1 M NaCl causes it to dissociate

17, 162, 163
into histones and DNA. Subsequent dilution of this

solution to 0.14 iM will result in recombination of histone
and DNA. Nonhistone proteins, however, remain in solution.
It was found that if liver DNP v/as subjected to such a
treatment the activity decreased to 66% of untreated con-
trols. Although this treatment is expected to remove non-

65
histone proteins bound within the DNP complex, it appears
as though most of the activity remains tightly bound to
the DNP.

Recently Johns and Forrester reported that extraction
of calf thymus DNP with 0.35 M NaCl removes acidic x^roteins

v/hich have become bound to it during the isolation proce-

153
dure. An attempt was therefore made to extract the

factor responsible for acetylating activity from rat liver
DNP with 0.3 5 M NaCl (see Materials and Methods). The re-
sults of these experiments indicate that DNP subjected to
such treatment retains 80% of its acetylating activity.

Heating briefly at 65 C, or washing with ethanol,
acetone, and ether, practically abolished activity. The
histone acetylation in these preparations appeared to be
negligible, both under conditions (phosphate buffer) which
favor an enzymatic reaction and those (tris buffer) favoring
a chemical process (see below). Prolonged incubation of DNP
in the regular medium at 0 C and 3 7 C for different time
periods resulted in a decreased activity. Neither the salt
extractions, treatment with organic solvents, or heat,
changed the histone/DNA ratio of the DNP. It could be
argued that the decrease in acetylating activity observed
upon incubatJon of DNP for prolonged periods at different
temperatures could be due to an alteration in the histones

66
raLher than a change in an enzyme within the DNP. To check
this possibility, "arginino-rich" histoncs from DKP which
had been incubated for 20 hours at 0 C and for 4 hours at
37 C were isolated and their ability to become acctylated
spontaneously or enzymatically in the presence of the ace-
tyltraiisferase (sec below) was compared with that of his-
tones isolated from control preparations of DNP. This ex-
periment showed that histones from these pretreated prepara-
tions vyere still capable of becoming acetylated to the same
degree as the controls or even higher. These findings argue
against the possibility that the histones were degraded or
extracted from, the DNP complex during these treatments.
Therefore the observed decrease in acetylation cannot be
explained by a defective substrate, but presumably results
from enzymatic denaturation.

Free histones subjected to treatment with organic
solvents or heated at 65°C were still capable of becoming
spontaneously acetyJ.ated. In contrast the acetylation re-
action within the DNP is completely abolished by these
treatments. These findings suggest that acetylation is
somehow prevented in histones which are bound within the
DNP coraplox.

67

Table III

Effect of Viirious Conditions on jCn Vitro Transfer of
Acetate from Acetyl-CoA to Histones

Percent
Pretreatment of Deoxyribonucleoprotoin of

Control

Extraction with 1 M NaCl 66.0

Extraction with 0.3 5 M NaCl 80.0

Organic solvents 4.0

65°G for 5 minutes 1.3

Organic solvents (Incub. at pH 9, 12 mM tris) 1.0
65 C for 5 minutes (Incub. at pH 9, 12 mM tris) 0

Incubated at 0°C for 20 hours in regular medium 70

Incubated at 3 7°C for 4 hours in regular medium 17

The results are expressed as percent of untreated con-
trols on the basis of specific activity (counts per minute
per mg of histones) .

68

Comparison of DNP catalyzed acetylabion with
spontaneous acetylation of free histones

Duffer Effects (Figure 5)

Comparisons between the pH and buffer conditions in-
fluencing the spontaneous acetylation of free histones and
that occurring within the DNP were made in an attempt to
show that they are, indeed, two different reactions. Figure
5 represents a composite graph which summarizes data from a
number of separate experiments in which the spontaneous
acetylation o I: free histones was compared with that occurring
within DNP in different buffer systems, at increasing pH.
As can be seen the DNP catalyzed reaction is greatly favored
by the x^hosphate buffer system, whereas the spontaneous
reaction is more active in tris-HCl buffer. The spon-
taneous reaction shows an increase with pH in tris buffer,
and at pH 9 actually exceeded that occur ing in the DNP.
Due to the range of the phosphate buffer system, it was im-
possible to observe the DNP catalyzed reaction under optimum
phosphate buffer conditions at a pll higher than 8. Both
the spontaneous and the DNP mediated reactions show a pH
optimum at pH 8.6-9.6 in glycine-NaOH buffer.
Temperature Effects ( F i g vi r e 6 )

Since most mamma].ian enzyme reactions show temperature
optima close to 37'~'c, whereas noncnzymatic reactj.ons in-
crease with temperature, it was decided to compare the

69
spontaneous acetylatiun of histoncs with that: occurring
in DNP at increasing temperatures.

Previous experiments indicated th.at a phosphate buffer
system, pH 8, rejjresented tlie most favorable conditions ob-
tainable for the DNP catalyzed acetylation of histoncs and
that a tris-HCl, pH 9, buffer represented the optimum con-
dition for the spontaneous acetylation of free histones.
Therefore, the transfer of acetate from acetyl-CoA to his-
tones in DNP as well as free calf thymus histones were com-
pared under these conditions at increasing temperature.

The data shown in Figure 6A were obtained at pH 8 in
25 mM phosphate buffer (most favorable conditions for DNP
acetylation) ; those in 6B at pH 9 in 25 mM tris-HCl buffer
kaost favorable for spontaneous acetylation of free his-
tones). As can be seen, there is a pronounced differential
effect of the ionic environment on the two reactions. In
both m.edia, however, the qualitative effect of increasing
temperature on each of the two processes was the sam.e: the
enzymatic reaction in the DNP complex indicating an optimum
at 37 C, followed by a sharp decline; while the spontaneous
acetylation of isolated histones showed a progressive in-
crease v/ith increasing temperature up to 7 7°C. This ex-
periment offers further evidence that the acetate transfer
occurring in the DNP coniplex is catalyzed by an enzyme.

70

Determination of bhe Extent of 0-Acetylation
by Hydroxylamine Test (Table IV)

Since most evidence available at present indicates that
the acetylation of histones occurs only at the epsilon-
amino group of internal lysine residues and at alpha-
araino gi'oups of terminal amino acids, it may be expected
that 0-acetylation often found in Jji vitro systems is an
artifact occurring only as a result of spontaneous acetyla-
tion. The DNP catalyzed acetylation of histones was there-
fore compared with that occurring spontaneously by comparing
the extent of 0-acotylation occurring in either case. The
results in Table IV show that 3% of the acetyl-gx'oups of
"arginine-rich" histones acetylated by DNP can be released
by hydroxylaraine treatment, whereas about 28% can be re-
leased from "arginine-rich" histones acetylated spon-
taneously. This suggests a greater degree of specificity
occurring in the DNP catalyzed reaction and im.plies an
enzymatic rather than a random spontaneous process.

71

8000-

6000

E

4000

C/}

2000

I I Spontaneous reaction with free histones

H Enzyme reaction within DNP

T

7.0

I

m

I

8.0
Tris Buffer

9.0

I

_I*L

if

I

i

I

i

A

PH 7.0 8.0
Phosphate Buffer

Figure 5: Comparison . of the Effect of
Increasing pH in Different Buffer Systems
on the Spontaneous and Enzymatic Acetylation Reactions

Results are expressed as specific activity (counts per
minute per mg) of Jiistones. Range of data is given in
brackets .

72

8000 r

O)

E
E

Q.
u

.i 4000

<

(J
o>

Q.
CO

Figure 6: Effect of Temperature on the Ti-ansfer of
Acetate from Acetyl-CoA to Histoncs in Rat Liver

DNP 0 0 , and to Free

nistones pi r-i in Different Buffer Systems "

In Figure 6A, the incubations were carried out in 25 mM
phosphate buffer, pH 8; in Figure 6B, in 25 inM tris buffer,
pll 9. Calcium and magnesium were omitted in botli. Results
are expressed as specific activity (counts per minute per
mg) of histones.

73

Table IV

Comparison of the Extent of 0-Acetylation in DNP with that

Occurring Spontaneously as
Measured by Lability to Hydroxylaraine

Type of Labile Acetyl Stable Acetyl Percent

Reaction Group (cpm) Group (cpm) Q--Acetylation

Acehyl-

Transf erase Exp. 1 67 2267 2.8

in

DNP Exp. 2 47 1149^ 3.9

Spontaneous

Acetylation Exp, 1 1340 3994 25.1

of Free

liistones Exp. 2 579 1287 31.0

Results are expressed as total counts per minute. Per-
cent O-acetylation was derived from the amount of label re-
leased by the hydroxy lamine and the total counts recovered.

74

Enzyme isolation

Although the evidence reported above suggests the
presence of an enzyme within the DNP complex which is re-
sponsible for the transfer of acetate from acetyl-CoA to
histonos, conclusive proof of this hypothesis would be the
isolation of the enzyme (acctyltransferase) . The results
of attempts to this effect are reported below.
Acotylatinq Enzymes from Rat Liver Nuclei
Acetone powder extracts

The first attemi:)ts at isolation followed procedures re-
ported to have been successful for the isolation of histone
acetokinases from rat brain and liver nuclei." ' ■
Following the procedure given in Materials and Methods, it
was found that a low activity acetylating enzyme could be
isolated from nuclei using this procedure, but not from DNP.
In fact, protein determinations of the tris extract of the
acetone powder preparation from DNP showed that no protein
whatsoever could be extracted by this procedure. VHien the
acetone powder of DNP or nuclei remaining after tris ex-
traction was tested for acetylating activity, it showed
about 50-70% of the activity usually observed in normal DNP
preparations. This suggests that the enzyme responsible for
the reaction in the DNP complex is still tightly bound to tlie
DNP and must therefore be decidedly different from that
which is extractable from nuclei.

75

Saline extracts (Tables V, VI)

Since it has been reported that washing DNP in 0.35 M

153
NaCl removes acidic proteins, saline extracts were made

of nuclei and DNP which were examined for acetylating ac-
tivity. Table V shows the results of 3 different ex-
periments in which these extracts, in quantities comparable
to the native preparation, were added back to inactivated
DNP. As can be seen, no activity was restored to inacti-
vated preparations by the addition of extracts from DNP,
although the addition of extracts from nuclei did restore
tlie acetylating activity to 10% of that of the controls.
Table VI shov/s the activity of salt extracts of nuclei
using isolated "arginine-rich" calf thymus histones and
poly lysine as acetate acceptors. It can be seen that under
these conditions this enzyme lacks specificity, as poly-
lysine was acetylated to the same degree as isolated his-
tones.

76

Table V

Restoration of Acetylating Activity
in Heated DNP by 0.3 5 M Extracts from Nuclei

Experiment

Control DNP 8505 + 1340 10997 + 758 6718 + 474

65°C inactivated DNP 306 + 64 292 + 156 54 -f- 46

6 5°C inactivated DNP and

0,35 M extract of DNP 284 + 101 171 + 6

65°C inactivated DNP and

65°C inactivated 0.35 M

extract of DNP 196 + 36 0

65°C inactivated DNP and

0.35 M extract of nuclei 1306 + 27 792 + 50

Results are expressed as specific activity (counts per
minute pei- mg) of histones plus or minus the standard
error.

77

Table VI

Acetylation of Isolated Histones with a
0.3 5 M NaCl Enzyme Extract from Nuclei

Incubated Material

Total Counts per Minute

Isolated Histones

131 + 23

Histones and extract

1677 + 275 (enzyme extracted from
1 gm liver)

Histones and extract

1199 +_ 1075 (enzyme extracted from
0.5 gm liver)

Histones and 65 C
inactivated extract

113 + 26 (enzyme extracted from
0.5 gm liver)

Extract alone

268 + 4 (enzyme extracted from 0.5
gm liver)

Poly lysine

267 H- 48

Polylysino and extract 1288 + 368 (enzyme extracted from

0.6 gm liver)

Results are expressed as total counts per minute plus
or minus the standard error.

78

Acetylatinq En?;ymos from Rat Livor DNP (Table VII)

A method similar to one used for the isolablon of RNA
polymerase from the "aggregate enzyme complex" proved to be

successful for the extraction of the acetyltransferase ac~

154
tivity from DMP. Table VII shows the results of 2

experiments in -which the extract containing acetyltrans-
ferase activity from DNP from 1 gm of liver v/as added back
to inactivated DNP, free histones, or to DNP which had been
extracted by this procedure. In each instance, the sub-
strate contained histones in quantities equivalent to that
found in 1 gm of liver. In the case of samples in which
isolated histones served as the substrate, the specific
activity v.'as calculated by di.viding the total counts ob-
tained by the canount of histones added to the reaction mix-
ture. Since there were no histones present in the case of
the incubation of the extract alone, this hypothetical
figure represents the specific activity calculated by as-
suming that the standard amount of histones was present.
This figure was deri.vcd by dividing the total counts per
minute in tliese samples by 0.5 mg of histones.

As can be .seen, the extract can only restore activity
to heated DNP to 10% of the control level, although about
50% of tlie acetylating activity appears to be extiacted by
this prooeduirc. Wlion the extract is added bock to DNP

79

which has been extracted by this procedure, again, only
approximately a 10% increase is observed. However, when
the extract is added to free histones, there is a high rate
of acotylation.

80

Tiib-le VII
Extraction of Ilistone Accty Itransferease Activity from DhV

Incubated Material

Exp. 1 Exp. 2
Specific Specific
Activity Activity
( cpm/ing ) (cpm/mg) _

Normal DNP

11204 H- 1608 5857 + 1046

DNP he-iatcd at 65'-^C, 10 min

o,-.

172 + 131 134 -I 82

Heated DNP and extract

1140 + 75 537 + 35

"Arginine-rich" histones (0.5 mg) 1142 ± 108 528 + 47

Extract

474 + 18* 440 + 8*

"Argininc- rich " histones (0.5 mg )
and extract

9668 + 335 4445 + 287

Extracted DNP

6107 + 228 3523 + 79

Extracted DNP and extract
added back

70] 3 + 198 4898 + 708

The amount of DNP or extract used for each incubation
was obtained from 1 gm of rat liver. The result.s are ex-
pressed as specific activity (counts per minute per mg) of
histones plus or minus the standard error.

Specific activity was calculated on the assumj-ition that
0.5 mg of histon.es was present in the reaction mixtui-e.

81

Relative acetylation of the various
histone fractions (Table VIII ) (Figure 7)

After incubation of DNP in the presence of acetyl-CoA

as described, tlie histones were extracted and fractionated

15 7
according to Johns into the five major histone groups.

Table VIIl shows the specific activity of each of these

fractions. As can be seen, the arginine-ricli histones,

f and f , are the most actively acetylated fractions,
2al 3

v/itli some activity also in tlic f fraction. The radio-

2a2

activity of the lysine-rich fractions, f and f , was low

v/hich correlated with iji vivo findings.

The purity of these fractions was checked by acrylamide

gel electrophoresis and Fi.gure 7 illustrates the pcitterns

observed . As can be seen the f ^ sample shows a band

2a2 ^

corresponding to the f band and therefore this fraction

2al

is probably slightly contaminated v/ith f,, -, histones. This

^a X

could account for some of tlie activi ty observed in the f

sample, as the f fraction is very highly labeled. Frac--

2al

tions f^, f_.„, and f are not completely separated by
J zcxA 2b

this procedure, when all the fractions are combined, but
when run separately f^ and f each show only one distinct
major band and therefore v;ere considered to be relatively
homogeneous .

82

Table VIII

Acetylation of Various Ilistone Fractions in DNP

Fraction

Specific
Activity
(cpm/mq)

Lysine-rich histones

557

■2b

2,909

■2al

16,041

Arginine-rich histones

2a2

8,713

12,904

The results are exi:)ressed as spGci.fic activity (counts
per minute per ing) of histones.

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DISCUSSION

Since previous work has revealed h.hat free histones are

118
spontaneously acetylated upon incubation with acetyl-CoA,

Lhe possibility arose that the acetylation of histones oc-
curring in DNP might not be enzymatically catalyzed. There-
fore, attention has been given to this problem and evidence
is provided for the presence of an acetyltransf erase bound
to DN? which is responsible for this reaction .

Indirect evidence suggesting that the jji vitro acetyla-
tion is catalyzed by an enzyme was provided by experiments
in which DNP was pretreated in a way which would be expected
to denature a complexed enzyme. Heat or organic solvents,
which are generally considered to be inhibitory to enzyme
reactions, practically destroyed the acetylating activity
of DNP. The fact that after these procedures the histone/
DNA ratios v;ere unchanged implies that the physical compo-
sitJ.on of the DNP complex was the same even after treatment.
Therefore, the inhibitory action observed can be attributed
to denaturation of an enzyme responsible for the acetylation
reaction, especially since similar pretreaLraent of histones
did not affect thoir spontaneous acetylation. DNP subjected

84

85

to heat or organic solvents does not catalyze the acetyla--
tion roachion, even under conditions most f^ivorable for a
spontaneous transfer of acetate to h.i.stones , Apparently,
spontaneous acetylation is somehow prevented in h. istones
bound to DNZ\ in the DNP complex. This may have biological
implications for the mechanism of gene control in that only
an enzymatically catalyzed reaction, capable of a gretit deal
of specificity, rather than a random ^xrocess, is permitted
v/ithin the DNP complex.

In the case where DNP showed a loss in acetylating
activity as a result of preincubation at 37 C for 4 hours,
it could be argued that the histones themselves were de-
graded by this treatment and thereby were incapable of be-
coming acetyJ.ated. However, if histones are isolated from
thusly treated DNP, they are still capable of becoming spon-
taneously acetylatcd or acetylated in the presence of a
subsequently prepared extract containing acetyltransf erase.
These histones as well as the acetyltransf erase are there-
fore relatively unche-inged with respect to their ability to
becom.e acetylated.

Although the above mentioned pretreatments of DNP,
especially heating at 65*^0, did not change the composition
of the DNP with respect to hisloiie/DNA ratios or the ability
of the subsequently isolated histones to become spontane-

86
ously acctylatcd, it could bo arguod that the structure of

the complex was altered in sucli a way as to prevent a non-
enzymatic transfer of acetate causing the observed inhibi-
tion. This seems unlikely, however, as it has been demon-
strated that the DNA of liver chromatin is stabilized against

heat denaturation as compared v;ith deproteinized liver

44

DNA. The temperature of half- melting (T^^^) is 68°C in the

case of naked DNA, but is increased to 81 C for chi'omatin.

o
Preparations of DNP heated at 65 C according to the methods

described herein, therefore, probal^ly represent a native

structure.

Comparisons between acetyiation of coraplexed histones
in DMP and that of free histone oc-:urring spojitaneously re-
vealed that, although the pH optin.a of the two reactions
wais the same, the DNP reaction was much more efficient in a
phospViate buffer system, whereas the spontaneous reaction
was greatly favored by a tris-HCl buffer. Furthermore, the
DNP catalyzed reaction showed a marked temperature optimum
of 37 C, v.'hich is common for mammalian enzyme reactions,
while tlie spontaneous reaction rate increased with increas-
ing temperature. These data v/ould seem to indicate that
one is dealing with two comxjletely different processes.

Another difference between the acetylation of histones
in DNP and that of free histones was the degree of 0-acetyla-

8 7
tiori. Acetyl groups of "argininc-rich" histones acctylated
by DNP showed a much greater degree of stability to hydroxy la-
inine treatment than those acetyj.ated spontaneously. This
indicates that most of the acetate is N-linked, which corx*e-

lates with findings by others on the acetylation of histones

93, 94

by calf thymus nuclei. Although findings by others

on regenerating rat liver showed 55% 0-acetylation for the

f3 fraction, acetate incorporation in the f^-i fraction was

110

stable to hydroxylamine treatment. Using the same pro-
cedure Gallwitz and Sekeris also found 35% 0--acetylation

among the acetate groups of the f^ fraction acetylated in

115
'/itro in rat liver nuclei, but none in the other fractions.

These findings are probably an exaggerated estimate of the

degree of 0-acetylation since Perlraann has reported that the

epsilon-N-acetyl groups of the lysine residues of pepsinogen

. . 154

are a], so splxt by hydroxy lamn.ne treatment. Furthermore,

direct chromatographic analysis of tryptic and pronase

digests of labeled histones according to Gershey et al .

indicates that 80% of the radioactivity was present, as

93
epsilon-N-acetyllysine .

Using similar methods, Videili et al. have also reported

that the radioactive acetate of isolated histones acetylated

in calf thymvis nuclei in_ vitro can be recoverf^d as a single

chromatogrciphic peak which was identified a?, epsilon-N-

88

94
acetyl lysine. Since the latter represent more careful

and reliable studies, it is probably safe to assume that N-

acetylation is the i^hysiological mode of acetylation which

occurs J^ vivo, whereas 0-acetylation is an artifact of the

spontaneous reaction. The results reported in this study

show only a 3% release of acetate from "arginine-rich"

histones acetylated by DNP ui:>on exposure to hydroxylamine .

The conclusion that the DNP catalyzed acetylation of

histones approximates the natural process and does not

represent an artifact is supported by the finding that the

in vitro pattern of labeling, in which the most actively

acetylated fractions are f„ , and f , is similar to that
^ 2al 3'

14 110

obtained in. vivo after administration of C-acetate.

Therefore, in studies using whole animals, nuclei or DNP,

only the arginine-rich fractions are capable of becoming

acetylated whereas the lysine-rich (f, and f ) are not.
^ 1 2b

A further similarity between the acetyltransferase

acting in DNP with acetyltransf erring enzymes described by

++
others is the lack of a Mg requirement. Previovis investi-

++
gat ions have shown that Mg is required for the transfer

of acetate to acotyl-CoA by a fraction from an acetone powder

117
extract from pigeon liver, but not for the transfer of

16 5
acetate from acetyl-CoA to is-ammobenzoic acid.

++
Similarly Gallv/itz showed that there is no Mg required for

89

tliG transfer of acetate from acetyl-CoA to liistones by an

118
acetokinase isolated from rat liver nuclei. C]-ioline

acetyltransf erase activity from pigeon and sheep liver is

++ 166

also not affected by addition of Mg up to 30 mM.

Another similarity between the histone acetyltrans-
ferase reported here and other acetyltransf erases con-
cerns the pH requirements. Arylaraine acetyltransferase,
showing a wide variety of specificities, has a broad pH
optimum from pH 6-9.5 when p-nitroaniline is the sub-
strate.' When histamine is the substrate, the reaction
falls off sharply below pH 8.5, which is similar to the DNP
acetyltransferase. Gallwitis has claimed that an acetokinase
isolated from rat liver nuclei is inactive at pH 9 although

he has found an increase in acetylating activity on raising

113, 118
the pH 7.5 to 9.0 in whole nuclei. He attributes

the incorporation of acetate into histones at pH 9 in

nuclei to a spontaneous process, but his data show that

heat inactivates 90% of this reaction, suggesting that it

is primarily enzymatic.

The most conclusive evidence for the demonstration that

this reaction is enzymatically catalyzed was the extraction

of acetyltransferase activity from the DNP complex. This

v/as achieved using a m.ethod found successful for the ex-

154
traction of RNA polymerase from chromatin. ' After the

90

extraction, 50% of the acetylating activity still remained
bound to the DNP complex. However, when the extract from
an equivalent amount of DNP was added back to heat in-
activated or extracted DNP, only a 10% increase was ob-
served. This difference represents a loss which is equal
to inactivation of 40% of the total enzyme activity. Another
possibility is that the enzyme in the natural state is
structurally cornxDloxed witli the DNP in a certain way.
After extraction of the enzyme, it cannot regain this native
state by readdition to the DNP. The same amount of extract
was able to acetylate free histones actively. It is pos-
sible that ace tyltransf erase as it occurs in the DNP com-
plex, in contrast to the free state, is restricted in its
action. Perhaps this restriction may play a role in the
regulation of genetic activity by allowing only specifically
selected sites of the histones to become available for
acetylation. This would in turn affect the DNA-histone
interaction and could reduce repression of transcription in
certain areas of the genome.

In conclusion, this study demonstrates the presence of
an enzyme, histone acetyltransferase, present in and closely
associated with rat liver chromatin which is responsible
for the transfer of acetate from acotyl-CoA to the lysine
residue of "arginine-rich" histones. This finding is con-

91

sistent with the hypothesis that hi stone acetylation may
be biologically significant as a mechanism involved in
the control of gene activity.

ADDENDUM

Preliminary attempts have been made to correlate the

acetyltransf erase activity with some biological phenomena.

Comparison of Acetylating Activity
in Novikoff Hepatoma and Liver DNP (Table IX)

One of these investigations involved a comparison be-

tv/een the acetyltransferase activity of DNP from Novikoff

hepatoma with that of liver. In vivo experiments from this

laboratory had shown that the turnover of acetyl-groups in

Novikoff hepatoma histones was very slow relative to that

163
of normal liver or other tissues. It was therefore de-
cided to compare the activity of acetyltransferase in tumor
DNP with that in normal liver DNP. Talkie IX shows the
results of 3 separate experiments in which the iji vitro
rate of acetate incorporation into histones from liver and
Novikoff hepatoma DNP were compared, and one in which nuclei
were used rather than DNP. Althouc^i the specific activity of
the tumor histones was about half of that of liver histones,
the magnitude of the difference did not soom adequate to
account for the almost complete lack of turnover of acetyl
groups by Novikoff hepatoma observed in vivo.

92

93

A corrollary to thj s finding was the observation that
DNP possessed an acetyl^iting activity about 3 times that
of nuclei. Therefore, it was hypothesized that perhaps a
deacetylating enzyme was present in the nucleopj.asm and
differences in the activity of this enzyme in tamor as
compared with liver could account for the in vivo observed
differences .

94
Table IX

In Vitro Uptake of Acetate into Histones:
Comparison of Liver and Novikoff Ilepatoma

14
Uptake of C-Acetate as cpm/mg Histone

Experiment Nuclei DNP

Tumor Liver Tumor Liver

1 1052 + 47 2213 ± 77 3031 + 531 7132 + 259

2 3107 + 141 6307 + 231

3 3062 + 63 4334 + 152

Incubations were conducted as described in Materials
and Methods. The resvilts are expressed as specific activi-
ties (counts per minute per mg) of histones plus or minus
standard error.

95

Doacetylatinq Activity in Nviclear Extracts (Table X)

Inoue and Fujimoto had found deacetylating activity in

122

a 0.14 M NaCl extract of calf thymus. Therefore, a

similar extract was made of rat liver nuclei and tested for

deacetylating activity.

14
To measure deacetylation, C-acetate labeled DNP was

prepared as follows: DNP from 5 grams of rat liver was

isolated as described and incubated for 15 minutes at 37 C

in a final volume of 10 ml, containing 12 mM NaCl, 2 5 mM

-6 14

phosphate buffer, pH 8, and 0.5 uc (1 x 10 M) acetyl- C-

CoA. The reaction was stopped by rapid cooling and centri-

fugation (10 min . , 2000 x g) . The precipitates were

washed an additional 2 times with saline-citrate contain.ing

-5 -6

10 M unlabeled acetyl-CoA, and 1.7 x 10 M acetic acid

designed as carriers to remove any label not covalently

^ ^ 14

bound. C-acetyl labeled "arginine-rich" histones were

prepared from the thusly isolated DNP according to the pro-
cedure described above. A crude nuclear deacetylating
enzyme extract was prepared by homogenizing nuclei from 16
grains of rat liver in 3 m.l of 0.14 M NaCl and centra fuging
at 8,000 X g for 15 minutes. 0.3 ml of this supernatant
containing 1.5-3.0 mg of protein was added to 0.3 mJ of
50 mM tris-IICl buffer pH 7.3 containing 0.1-0.2 ma of the
labeled liistones and rhe mixture was incubated at 3 7*1: for

96
20 minutes. The reaction v/as stopped by Lhe addition of
0.1 ml of 0.1 N IICl containing 5 nrr.oles of carrier acetic
acid. The acidified incubation media were extracted with
ethyl acetate and their radioactivity determined by liquid
scintillation counting.

Table X sh.ov/s the results of such an experiment, v/Viich
clearly demonstrates tlie presence of a deacotylating ewzyme
in the nuclear sap. The results indicate that exposure to
the crude deacetylating enzyme released 72% of the labeled
acetate from free hi stones and 13% from DNP containing an
equivalent amount of histones. It is interesting to note
that less acetate is removed from. DNP than fr.om free his-
tones. This is a parallel to the finding that the isolated
acetyltransferase is also more active in the transfer of
acetate to free histones than v/hen they are bound to DNA
within the DNP complex. This suggests a restrictive action
by DNA and/or the nonhistone proteins in the DNP influencing
the availability of the sites for acetylation and deacetyla-
tion. Perhaps this may be related to transcriptional con-
trol m.echanisms and the activity of RNA polymerase.

WTien the activity of nuclear extracts containing de-
acetylating enzymes (same amount of protean in each) Erom
liver and tumor v;ere compared preliminary results suggested
that the deacetylating activity is mucli less in the tumor

97

Table X
Deacetylation Activity in Nuclear Sap Extracts

14 14

Acetyl- C-DNP Acetyl- C-Histones

Radioactivity of start-
ing material (cpm) 12668 10798

cpm extract able with
ethyl acetate from the
incubation medium after
incubation :

1. in absence of nu-
clear sap extract 884 9

2. in presence of
heat inactivated

(10 rain. , 70°C)
nuclear sap ex-
tract 970 17

3. in presence of
nuclear sap ex-
tract 2599 7859

Results are expressed as total counts per minute

98

than in liver. As these studies were being conducted, Libby

published a roi^ort showing that whole nuclei from liver
incubated with acetylated histones prepar^ed from calf thymus

could release acetate about 7 times faster than nuclei from

169
Novikoff hepatoma, Libby ' s experiments seem to support

the findings reported here and perhaps offer an explanar.ion

for the ]ack of acetate turnover observed _in vivo in Novikoff

hepatoma .

Effects of Treatments Known to Increase Bl^A
Synthesis on the Acetylation of Histones in DNP

Acetylation of histones has been implicated as a
mechanism for influencing gene expression. Moreover, others
have shown that an increase of ijT. vivo acetylation precedes
an increase in RNA synthesis (see Introduction) . Therefore,
the effect of treatments known to stimulate RNA synthesis
in vivo was studied on iji vitro acetylation of histones in
DNP.

The most extensive group of experiments involved a
study of the jjl v^itro transfer of acetate to histones by
rat liver DNP in the presence of Vc^rious concentrations of
hydrocortisone. Preliminary experiments gave indications
that a stimulation of histone acetylation may occur under
these conditions, however, upon closer investigation, it
was concluded that this is not the case.

Unless otherwise stated tlie incubation mediiun for these

99

experiments contained 0.19 M sucrose, 20 mM glucose, 25 mM

14
iohosphatc buffer, pH 8, 12 mM NaCJ^ and 0.01 uc acetyl- C-

CoA. Other conditions, including the isolation of histones
and determination of specific activities, were as described
in Materials and Methods.

Purity of hydrocortisone preparations
In preliminary experiments hydrocortisone preparations
were used which wei-e obtained from the hospital pharmacy.
Since this material would not dissolve completely in water
at the required dilution it was suspected that this prepara-
tion contained some contaminants. An interesting report
related to this finding was the observation that 21-dehydro-
corticosteroids bind irreversibly to arginine-rich histones
and that these compounds frequently contaminate steriod

preparations, loading to the suggestion that corticosteroid

170

alcohols react with histones. Other findings have shown

that 21-dehydrocortisol was much more effective than hydro-
cortisone in the Jji vitro stimulation of RNA synthesis in

171
rat liver nuclei. This may explain the preliminary

findings in which there appeared to be a positive effect on
the acetylation of histones iri vitro. Subsequent experi-
ments reported here were carried out with hydrocortisone
from Sigma Chemical Company.

UsG of more samples (Table XI)
In some of the preliminary experiments the difference
between samples was not .statistically significant although

there was a suggestion of an increase with increasing con-

-10 -7
centration of hydrocortisone from 10 -]0 M. When 5 sam-
ples were run for each condition the data summarized in
Table XI were obtained. As can be seen no significant dif-
ferences could be observed between controls and experimen-
tals.

Use of adrenalectomized rat (Table XII)
Because the adrenal gland is responsible for hydro-
cortisone production, there is a possibility that removcil
of the adrenal gland might lower the levels of circulating
hydrocortisone and that under these conditions perhaps an
in vitro effect of the hormone on acetyltransf erase activity
may be observed. A male rat kept on a salt water diet for
72 hours after adrenalectomy was sacrificed along with a
control. The DNP was isolated and incubated as described
in Materials and Methods. The results in Table XII were
obtained. Prom this experiment it was concluded that there
is little or no affect of hydi'ocortisone on acetyltrans-
f erase activity under these conditions.

101

Table XI

Effect of Increasing Concentrations of Hydrocortisone on
In Vitro Acetylation of Ills tones in DNP

Cone. Hydrocortisone
ill Incubation Media

Specific Activity
(cpm/mg)

lo-i^M

10 ^M

10 ^M

6040 + 388
6461 + 407
6530 + 361
5969 t 644

'Ilie data presented are the results of 1 experiment and are
expressed as specific activity (counts per minute per mg)
of histones plus or minus the standard error.

Table XII

Effect of Hydrocortisone on jUn Vitro
Acetylation of Histones in Liver
DNP from an Adrenalectomized Rat

Cone. Hydrocortisone
in Incubation Media

Specific Activity
( cpm/mg )

Adrenalectomized Rat Control Rat

10 ^M

5323 + 1050
6371 + 1050

6821 + 423
7247 -1- 279

The data presented are the results of 1 experiment using 5
samples for each condition. They are expressed as specific
activity (counts per minute per mg) of histones plus or
minus the standard error.

102
Preincubation of DNP with hydrocortisone (Table XIII)
Because Lukas and Sekeris have demonstrated an increase

in mammalian RNA polymerase activity in vitro ui^on x^rcincu-

172
babion of rat liver nuclei with hydrocortisone, it seemed

worthwhile to try if an increase in acetyl transferase activ-
ity might be observed upon preincubation of DNP with hydro-
cortisone. In this experiment DNP was preincubated with
10" M hydrocortisone in the usual incubation medium at 4 C
and 3 7°C for 10 minutes prior to addition of acetyl- C-CoA.
The results in Table XIII were obtained. No significant dif-
ferences could be observed by the differ"ent treatments.

Media changes designed to increase the solubility of
DNP during the incubation (Table XIV)

Because DNP is in a suspended particulate state in the
salt conditions of the usual incubation medium, it seemed
possible that this could be Lhe reason for its unresponsive-
ness to hydrocortisone, which might otherwise have an effect
on its acetylating ability. Therefore, DNP was washed in
water, which causes it to swell and become gelatinous, and

incubated either in 12 mT-I NaCl, 2 5 mM phosphate buffer, pH 8,

14
or water in the presence of acetyl- C-CoA. From the re-
sults shown in Table XIV it was concluded that changing the
colloidal state of DNP in this manner does not make it any
more responsive to the presence of hydrocortisone, and incu-
bating DNP in the presence of water only decreases acetyl-
transferase activity.

103

Table XIII

Effect of Preincubation of DNP with
Hydrocortisone on j[n Vitro Acetylation of Histones

Cone, of Hydrocortisone Specific Activity Conditions of

(cpm/mg) Preincubation

0 4848 +354 0 time

10 "^M 4280 + 816 0 time

4713 + 178 10 min. at 4 C

-7 o

10 M 4254 + 499 10 mm. at 4 C

0 4011 + 319 10 min. at 37'^C

10 "^M 3163 + 785 10 min. at 3 7°C

After preincubation, the acetyl-l^c-CoA was added to the
samples and the same medium was used for the incubation.
The data are the results of 1 experiment in which 4 samples
were used for each condition. They are expressed as spe-
cific activity of histones (counts per minute per mg) plus
or minus standard error.

104

Table XIV

Effect of Increasing the Solul^i.liLy of DNP on
Tn Vitro Acotylation of Histones
in the Presence of Hydrocortisone

Pre- treatment of DNP Incubation Media Specific Activity

(cpm/mg)

Usual

Usual

9891 + 117

Usual

H^O

2931 + 128

H^O

Usual

12006 + 365

"2°

"2°

346 + 138

Usual

H2O + 10 ^M
hydrocortisone

2467 + 23

HjO

Usual +10 ^M
hydrocortisone

9259 + 521

H2O

H2O + 10 'm
hydrocortisone

201 h 87

Tlie results are expressed as specific activity (counts per
minute per rag) of histones p].u3 or minus the standard error,

105

Effect of in vivo admini stration of hydrocortisone on
the in vitro acetylation of histones (Table XV)

Another approach to this problem was a study of the

effect of the J^ vivo injection of hydrocortisone on the in

vitro acetylation of histones. By measuring the in vivo

3

uptake of H-acetate into histones others (see Introduction)

have concluded that the in vivo injection of hydrocortisone
increased the acetylation of histones. Following similar
conditions (time course, dose, etc.) we found no stimulation
of the in vitro acetylation of histones by DNP following
the injection of hydrocortisone.

In this experiment a 450 gm male rat was injected with
10 mg of hydrocortisone subcutaneously between the shoulder
and sacrificed l^ hours later. DNP was isolated and incu-
bated as usual. The results are summarized in Table XV.

Effect of phenobarbital and liver regeneration on
the in vitro acetylation of histones in DNP (Table XVI)

Injection of phenobarbital or removal of part of the
liver are knov/n means of stimulating RNA synthesis in liver
cells. In the case of liver regeneration an increase in the
in vivo acetylation of histones can be observed. The effect
of these treatments on the in vitro acetylation of histones
in DNP was therefore studied. The median and left lateral
lobe of the liver of one 450 gm rat was surgically extir-
pated 3h hours before sacrifice. DNP was isolated from the

106

Table XV

Effect of In_ Vivo Administration of Hydrocortisone on
In Vitro Acotylation of Histones in DNP

Treatment Sx^ecific Activity

(cpm/mg)

Control 7858 + 688

Hydrocortisone 8220 ± 565

These data represent the results of 1 experiment utilizing
7 samples from each rat. They are expressed as specific
activity (counts per minute per mg) of histones plus or
minus the standard error.

Table XVI

Effect of Phonobarbital and Liver Regeneration on
In Vitro Acetylation of Histones in DNP

Treatment Specific Activity

( cpm/mg )

Control 7858 + 688

Phenobarbital 8559 + 518

Regenerating Liver 6899 + 605

These data represent the results of 1 expex-iment utilizing
7 samples from each rat. Tliey are expressed as specific
activity (counts per minute per mg) of histones plus or
minus the standard error.

107
remaining regenerating portion and incubated as usual. One
390 gm rat was injected v/ith phenobarbital (75 rag/kg) 21
hours before sacrifice. DNP was isolated from the liver
and incubated as usual. Results are shown in Table XVI.
As can be seen, no significant differences were observed
between the control and the treated rats.

These experiments demonstrate the need for caution when
drawing conclusions concerning cause and effect relation-
ships from J^ vivo observations. Under these conditions no
stimulation of acetylating activity of DNP by hydrocortisone
can be demonstrated. Therefore, it is likely that increased
acetate incorporation into histones observed in_ vivo is an
indirect effect of the hormone, and is not due to a modifi-
cation in the amount or activity of acetyltransf erase. The
same conclusion appears to be true for the experiment in
which the effects of phenobarbital and liver regeneration
were studied.

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BIOGRAPHICAL SKETCH

Louise Adeie Racey v/as born March b, 194]., in New York
CJ.Ly. In Juno, 1959, she was graduated from V.hite Plains
High School. She received the degree of Bachelor of Artu
with a major in Biology in June, 1963, from Trinity College,
Washi'igtoji, D, C. lii September, 1963, she entered graduate
school at The CathoJ.ic University of Aiiierica. She hold a
teaoViir.g ass istantship from SeptenilDer, 1963, until June,
196'"/, v/aon ohn received Iho degree of Master of Sciemce
with 'i :.':ajor in Col] Physiology. In 1965-56 she taught
high school Biology at Trinity Preparatory School, and in
1966-67 she was an instructor at Marymount Manhattan College,
She has spent summers at The Mount Desert Island Marine
Biological Lah'oratory (1962) and Woods Hole Marine Bio-
logical Laborai ory (1965). From September, 1967, to the
precept: she hc-s p-arsuod th.e requirements for the degree of
Doctor of Philosophy. She presented a paper entitled "In
Vitro Ai-etylation of Histone^' at the American Cancer
Society Mec.tir.gs in San Francisco in March, 1969. 3he is
a memboi- of Sinma Xi.

118

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

'. Brookbank, Chairma

l-tU^iyL-

Professor of Zoology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

Paul/Byvoet, Ub-Ltiairman

Associate Professor, Division of Biological Sciences

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

fw^'^->^A

Thomas W. O'Brien

Assistant Professor of Biochemistrv

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

Zam
Assistant Professor

Zoology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

Ann R. Larkin

Assistant Professor of Anatomical Sciences

rhis dissertation was submitted to the Dean of the College of Arts and
Sciences and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

December, 1970

Dean, College of Arts and ScieMes

Dean, Graduate School

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