Lewin Benjamin (ed.) Genes IX

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N ucLeosome

0rganization

May Be

Changed

at the

Promoter

Remodeling

complexes are recruited

promoters

by sequence-specifi c activators.

The factor may

reteased

once

the

remodeling

comptex has

bound.

The MMTV

promoter

requires

a change in rotational

positioning

of a nucleosome

to attow an activator

to bind to DNA on the nucleosome.

How are remodeling complexes

targeted to

spe-

cific

sites

chromatin? They

do not them-

selves contain subunits

that bind specific DNA

sequences.

This

suggests the model

shown in

!.1.-",r-ri,:i:

:;;:.1,,

in which

they are recruited

by acti-

vators

(sometimes)

by repressors.

The interaction

between

transcription fac-

tors and remodeling complexes

gives

a key

insight into their modus

operandi. The

tran-

scription

factor

Swi5 activates

the HO locus in

yeast. (Note

that Swi5 is not

member

of the

SWI/SNF complex.)

Swi5 enters nuclei toward

the

end of

mitosis

and binds to the IIO

promoter.

then

recruits

SWI/SNF to the

promoter.

Swi5

is then released, Ieaving

SWI/SNF at the

pro-

moter. This

means that a transcription factor

can activate a

promoter

by a

"hit

and run" mech-

anism,

which its function is fulfilled

once the

remodeling complex has

bound.

The involvement

remodeling

complexes

gene

activation was discovered

because the

complexes are necessary

for the ability of cer-

tain transcription factors

to activate their tar-

get genes.

One of the

first

examples was the

GAGA

factor,

which activates the hsp70

Drosophila

promoter

invitro. Binding

of GAGA

to four

(CT),-rich

sites on the

promoter

disrupts

the nucleosomes, creates

hypersensitive

region, and causes the adjacent nucleosomes

to be rearranged so that they

occupy

preferen-

tial instead of random

positions.

Disruption

an energy-dependent

process

that requires the

NURF

remodeling

complex. The organization

of nucleosomes is altered

so as to create a bound-

ary that determines the

positions

of the adjacent

nucleosomes. During this

process,

GAGA binds

to its target sites and DNA, and its

presence

fixes

the remodeled state.

The

PHO

system was one of the first in

which it

was

shown that

a change

in nucleo-

some organization

is involved

gene

activation.

At the PHO5

promoter,

the bHLH regulator

A remodeting

complex binds

to chromatin

via

an activator

(or

repressor).

PHO4 responds

phosphate

starvation

inducing the disruption

four

precisely

posi-

tioned nucleosomes.

This event

independent

of transcription

(it

occurs

in a TATA-

mutant)

and independent of

replication.

There

are two

binding sites

for PHO4 at

the

promoter.

One

located between

nucleosomes,

which

can

bound by the isolated

DNA-binding

domain of

PHO4, and the other

lies within

nucleosome,

which cannot

recognized.

Disruption

of the

nucleosome to allow

DNA binding

at the

sec-

ond site

is necessary

for

gene

activation.

This

action requires

the

presence

the transcrip-

tion-activating

domain.

The activator

sequence

of VPl6 can substitute

for the

PHO4 activator

sequence

in nucleosome

disruption.

This sug-

gests

that disruption

occurs

protein-protein

interactions that

involve

the same

region

that

makes

protein-protein contacts

to activate

30.4

Nucleosome

Organization

May Be Changed

at the

Promoter

transcription. In

this case, it is not known which

remodeling

complex is involved in

executing

the effects.

survey of nucleosome

positions

large

region

of the

yeast

genome

showed that

most

sites that bind transcription factors

are

free

nucleosomes.

Promoters for

RNA

polymerase

II typically

have a nucleosome-free region

-200

bp upstream

of the startpoint, which is flanked

positioned

nucleosomes

on either side.

It is not always

the case, however, that

nucleosomes

must

be excluded in

order

per-

mit initiation

of transcription.

Some activators

can

bind to DNA

on a

nucleosomal

surface.

Nucleosomes

appear to be

precisely positioned

some steroid hormone response

elements in

such a way that receptors

can bind. Receptor

binding may

alter the interaction

DNA

with

histones,

and may even lead

to exposure of new

binding sites. The

exact

positioning

nucleo-

somes

could be required

either because the

nucleosome

"presents"

DNA in a

particular

rota-

tional

phase

or because there

are

protein-

protein

interactions

between

the activators and

histones

or other

components of chromatin.

Thus

we have

now moved some

way from

view-

ing

chromatin exclusively

as a repressive

struc-

ture

to considering

which interactions

between

activators

and

chromatin can

recuired

for

activation.

The MMTV

promoter presents

an example

of the need for specific nucleosomal

organiza-

tion. It contains

an array of six

partly palin-

dromic sites, each bound by one dimer

hormone

receptor

(HR),

which constitute

the

HRE. It also has a single binding

site

for

the fac-

tor

NFl,

and two adjacent

sites for the factor

OTF. HR and NFI cannot bind simultaneously

their sites

in free

DNA. i:I{li,iS*

;iii.!" shows

how the nucleosomal structure

controls bind-

ing

the factors.

The HR

protects

its

binding sires

at the

pro-

moter when hormone is added,

but does not

affect the micrococcal nuclease-sensitive

sites

that mark either side of the nucleosome.

This

suggests

that

HR is

binding to the DNA

on the

nucleosomal

surface; however,

the rotational

positioning

of DNA on the nucleosome prior

hormone addition

allows access to

only two of

the four sites. Binding to

the other two

sites

requires

a change in rotational

positioning

the nucleosome. This can

be detected by

the

appearance

of a sensitive site at the axis

of dyad

symmetry

(which

is in

the center

of the bind-

ing

sites that constitute the HRE).

NFI can

footprinted

on the nucleosome after

hormone

induction,

so these

structural changes may

necessary to

allow NFI to bind,

perhaps

because

they

expose

DNA

and abolish

the steric hin-

drance

by which HR blocks

NFI binding to free

DNA.

Histone

Modification

Is a Key Event

Histones

are

modified

by methytation,

acety[ation,

and

phosphorylation.

Whether a

gene

expressed

depends

the

structure of chromatin

both locally

(at

the

promoter)

and

the surrounding

domain.

Chromatin structure

correspondingly

can

regulated

by individual

activation

events

or by

changes

that affect a

wide chromosomal

region.

The

most localized

events concern

an individ-

ual target

gene,

where changes

in nucleosomal

structure and

organization

occur in the

imme-

diate vicinity

of the

promoter.

general

changes may

affect regions

as large

as a whole

chromosome.

Changes

that affect large

regions

control

the

potential

of a

gene

to be expressed.

The term

silencing is

used to refer

repression

gene

i:ir'ili;;i.

;ir

Hormone receptor

and NF1

cannot bind

simuttaneously

to the MMTV

promoter

the form

of [in-

ear DNA,

but can

bind when the DNA

oresented on a

nuc[eosomaI

surface,

CHAPTER

30 Controtting

Chromatin

Structure

activity in a local

chromosomal

region.

The term

heterochromatin is

used to

describe chromo-

somal regions

that are large

enough

to be seen

to have a

physically

compact structure in

the microscope. The

basis for

both types of

change is the same: Additional

proteins

bind to

chromatin and either

directly or indirectly

pre-

vent transcription factors

and RNA

polymerase

from

activating

promoters

the region.

Changes at an individual promoter

control

whether transcription is initiated

for

particu-

lar

gene.

These

changes may

be either activat-

rng

represslng.

All of these

events depend

on interactions

with histones. Changes in

chromatin

struc-

ture are initiated

modifying

the N-termi-

nal

tails of the histones,

especially H3 and H4.

The histone tails

consist of 15-30

amino acids

at the

N-termini of all four

core histones and

the C-terminus of H2A. The

tails of H2B and

pass

between the turns

of DNA

(see

Fig-

tre 29.21 in

Section 29.7

Organization

of the

Histone Octamer).

: 1i,,i.if

:',

shows that they

can be modified at

several sites

methyla-

tion.

acetylation, or

phosphorylation.

Other

modifications, such as mono-ubiquitylation

or sumoylation, also occur

but are

less

well

characterized.

Acetylation

and methylation

occur on the

free

(e)

amino

group

of lysine. As seen in

acetylation

removes the

positive

charge that

resides

on the

NHI

form

of the

group.

Methylation also occurs

arginine.

Phosphory-

lation

occurs on the

hydroxyl

group

of serine and

also on threonine. This introduces

a negative

charge

the

form

of the

phosphate

group.

Lysine

can be mono-, di-, or trimethylated

(all

still

pos-

itively charged), and arginine

can be mono- or

dimethylated

(symmetrically

or asymmetrically).

These modifications are transient.

They can

change

the

charge of the

protein

molecule, and

as a result they are

potentially

able

to change

the functional

properties

of the octamers. Mod-

ification

histones is

associated with structural

changes that occur in chromatin

replication

and transcription. Phosphorylations

on specific

positions

and on different histones may be

required for

particular

processes,

for

example,

the Serr0

position

of Hl is

phosphorylated

when

chromosomes condense

at mitosis.

In synchronized cells in culture, both the

preexisting

and newly

synthesized core histones

appear to be acetylated and methylated

during

phase

(when

DNA is replicated and the his-

tones also are synthesized). During the cell

rycle,

the modifying

groups

are later removed.

The

coincidence

of modification

and repli-

cation suggests that acetylation

(and

methyla-

tion)

could

be connected with

nucleosome

assembly. One speculation

has been

that the

reduction

positive

charges on

histones might

lower their affinity for

DNA, thus allowing

the

reaction to

better controlled.

The

idea has lost

some

ground

in view of the observation

that

nucleosomes can be

reconstituted,

at least invitro,

with

unmodified

histones.

Histone acetylation

is essential for nucleosome

assembly

yeast,

and is

probably

required

for some

of the

protein-protein

interactions

that occur during

later

stages

of the reaction

(see

Section

10.6, His-

tone Acetylation

Occurs

in TWo Circumstances).

The N-terminaL tai[s of

histones

H3 and H4

can be acetytated,

methylated, or

phosphorylated

severaI

positions.

Acetylation of

Lysine or

phosphorylation

Sites of modification

in H3

1 2 3

5 6

7 I I

1011

121314

Sites of modiiication

in H4

7 8 I 1011

121314

Lysine Serine

CH,

serine reduces the

overatl

positive

charge

of a

protein.

30.5

Histone

Modification

Is a Kev

Event

803

cycle of

phosphorylation

and dephos-

phorylation

occurs with HI,

but

its

timing

different from the modification

cycle of the other

histones.

With

cultured

mammalian

cells, one

or two

phosphate groups

are introduced at

phase.

The major

phosphorylation

event

the later addition

groups

at mitosis,

though, which

brings the total number up to

as many

as six. AII the

phosphate groups

are

removed

at the end of the

process

of division.

The

phosphorylation

of HI is catalyzed

the

M-phase

kinase that

provides

an essential trig-

ger

for mitosis. In fact,

this enzyme is now

often

assayed

in terms of its Hl kinase

activity. Nor

much is

known about

phosphatase(s)

that

remove

the

groups

later.

The timing

of the major Hl

phosphoryla-

tion has

prompted

speculation

that it is involved

mitotic condensation.

In Tetrahymena

pro-

f,:Slifif

-:*.1* Acetylation

of H3 and H4 is

associated

with

active chromatin,

whereas methvlation is

associated

with

inactive

chromatin.

f?fi ijR$

i*".! 1

Most modified

s'ites in histones have

a single.

specific type of

modification,

but some

sites can have more

than one

type of modification.

IndividuaI

functions

can be

associated with some

of the modifications.

tozoarrl, however, it is

possible

to delete all the

genes

for Hl without significantly affecting the

overall

properties

of chromatin. There

is a rel-

atively small effect on the ability

of chromatin

to condense

mitosis.

Some

genes

are acti-

vated and others are repressed by this

change,

which suggests that

there are alterations in local

structure.

Mutations

that eliminate sites

phos-

phorylation

Hl have no effect, but mutations

that mimic the effects of

phosphorylation

pro-

duce a

phenotype

that resembles the

deletion.

This suggests that

the effect of

phosphorylating

HI is

to eliminate its effects on local

chromatin

structure.

Do histone modifications

affect nucleosome

structure directly, or is their effect

upon chro-

matin indirect?

There is not in fact

much evi-

dence for any

difference

the

properties

nucleosomes depending on the

state of the mod-

ification of the histones.

Several cases have

now

been

characterized, though, in which

histone

modification

creates binding

sites for the attach-

ment of nonhistone

proteins

that change

the

properties

oI chroma tin.

The range of nucleosomes

that is

targeted

for modification

can vary. Modification

can be

local

event, for example, restricted

to nucle-

osomes at the

promoter.

also can

be a

gen-

eral event,

extending, for example,

to an entire

chromosome.

FSli#R*

3*,:!*

shows that

there is

general

correlation

which

acetylation is

associated

with active chromatin,

whereas

methylation is

associated with inactive

chro-

matin. This

is not, however,

a simple rule,

and

the

particular

sites that are modified

(as

well as

combinations of specific modifications)

may

important, so

there are certainly

exceptions in

which (for

example) histones methylated

certain

position

are found in active

chromatin.

Mutations in

one of the histone

acetylase

com-

plexes

yeast

have

the opposite

effect from

usual

(they prevent

silencing

of some

genes);

this emphasizes the lack

of a uniform

effect

acetylation.

The

specificity

the modifications

is indi-

cated by the fact

that many

of the modifying

enzymes

have individual

target sites

in specific

histones. fg$ilftil

3*"?'i summarizes

the

effects of

some of the modifications.

Most

modified

sites

are subject to

only a single type

of modification.

some cases, modification

one site may

acti-

vate or inhibit modification

of another

site. The

idea that

combinations

of signals

may be

used

to define chromatin

types has

sometimes

been

called the histone

code.

Histone

Site Modilication

Function

Lys-4 Methylation

Activation

Lys-9 l,4ethylation

Chromatin

condensation

Lys-9 Methylation Required

for DNA methylation

Lys-9

Acetylation Activation

Ser-10

Phosphorylation

Activation

Lys-14

Acetylation Prevents

methylation

at Lys-g

Lys-79 Methylation

Telomeric

silencing

Arg-3

Methylation

Lys-S

Acetylation

Assembly

Lys-12

Acetylation

Assembly

Lys-16

Acetylation

Nucleosome

assembly

Lys-16

Acetylation Fly

X activation

804

CHAPTER

30 Controllinq

Chromatin

Structure

Histone Acetylation

0ccurs in Two

Circumstances

Histone acetytation

occurs transiently

rentication.

Histone

acetylation is associated

with activation

gene

expression.

All

the core

histones

can be acetylated. The

major targets for acetylation

are lysines in the

N-terminal tails of histones H3

and H4. Acety-

lation

occurs

two different circumstances:

during DNA replication,

and

when

genes

are activated.

When chromosomes are replicated.

which

occurs during the S

phase

of the cell cycle, his-

tones are transiently acetylated.

i: :

i,i iji I

;,{

:.r

* -i ;i

shows that this acetylation

occurs before the

histones are incorporated into

nucleosomes.

We know that histones Hl and H4 are acety-

lated at the stage

when

they are associated with

one another in the H)2-H42

tetramer.

The

tetramer is then incorporated into nucleosomes.

Quite

soon after, the acetyl

groups

are removed.

The importance of the acetylation is indi-

cated by the

fact that

preventing

acetylation of

both histones H3 and H4 during replication

causes loss of viability in

yeast.

The two his-

tones are redundant as substrates.

because

yeast

can manage

perfectly

well

long as

they

can

acetylate either one of these histones during

phase.

There are two

possible

roles for the

acetylation: It could

needed for

the

histones

to be recognized by factors that incorporate

them

into nucleosomes,

or it could be

required

for the assembly and/or structure of the new

nucleosome.

The factors that are known to be involved

in chromatin assembly do not

distinguish

between acetylated and nonacetylated histones,

which suggests that the modification is more

likely to be required for

subsequent

interac-

tions.

It has been thought for a long time that

acetylation

might

needed

to help control

pro-

tein-protein interactions that occur as histones

are

incorporated into nucleosomes.

Some evi-

dence for such a

role

is that the

yeast

SAS his-

tone acetylase complex binds to chromatin

assembly complexes at the

replication fork,

where

it acetylates

r6lys

of histone H4.

This

may

part

of the system that establishes the

histone acetylation

patterns

after replication.

Outside of S

phase,

acetylation

of histones

chromatin

generally correlated

with

the

state of

gene

expression.

The

correlation was

first noticed because

histone acetylation

increased in a domain

containing

active

genes,

and acetylated chromatin

more sensitive to

DNAase I and

(possibly)

micrococcal

nucle-

ase.

i.i*:ritiS:

ii.r.:lri

shows

that this

involves

the

acetylation of

histone

tails in

nucleosomes. We

now know that

this occurs

largely because

acetylation of the

nucleosomes

in the vicinity

the

promoter

when

gene

is activated.

irI{.il,lHf

i+-i.i.i

Acetytation at

rep[ication

occurs on

his-

tones before they are

incorporated

into nucleosomes.

lI{,ilfiii

;ii,t.1.:i

Acetytation

associated

with

gene

activa-

tjon occurs by

directty

modifoing

histones

in nucleosomes.

30.6

Histone

Acetytation

Occurs

in Two Circumstances

addition to events

individual

promot-

ers,

widescale changes in

acetylation occur

sex chromosomes. This

part

of the mecha-

nism by which

the activities

genes

on the X

chromosome

are altered to

compensate for the

presence

of two X chromosomes

one species

but only

one

chromosome

(in

addition to the

Y chromosome)

other species

(see

Sec-

tion 31.5, X

Chromosomes

Undergo Global

Changes). The

inactive X chromosome

in female

mammals

has underacetylated

H4. The

super-

active

X chromosome

in Drosophila males

has

increased

acetylation of H4. This

suggests that

the

presence

of acetyl

groups

may

be a

prereq-

uisite for a less

condensed,

active structure. In

male Drosophila,

the X

chromosome is

acety-

Iated

specifically at

r6lys

of histone H4. The

his-

tone

acetyltranoferase

(HAI)

that is responsible

an enzyme

calied MOF that is

recruited to

the

chromosome

part

of a

large protein

complex.

This

"dosage

compensation"

complex is respon-

sible for introducing general

changes in

the X

chromosome

that enable it to

be more highly

expressed.

The increased

acetylation

is only

one

of its activities.

Acetylases

Are

Associated

with Activators

Deacetytated

chromatin may

have a more

condensed

structure.

Transcription

activators are

associated with

histone

acetytase

activities in

[arge comptexes.

Histone

acetytases vary in

their target specificity.

Acetytation

could affect

transcription in

quantitative

quatitative

way.

Acetylation

is reversible.

Each

direction of the

reaction

catalyzed

by a specific type

of enzyme.

Enzymes

that can acetylate

histones

are called

histone

acetyltransferases

or HATs;

the

acetyi

groups

are removed

by histone

deacety-

lases

or HDACs.

There

are two

groups

of HAT

enzymes:

those in

group

act on histones in

chromatin

and are involved

with

the control

transcription;

those in

group

act on newly

synthesized

histones in

the cytosol,

and

are

involved

with

nucleosome

assembly.

lWo inhibitors

have

been

useful in analyz-

ing

acetylation.

Trichosratin

and butyric

acid

inhibit

histone

deacetylases,

and

cause acety-

lated

nucleosomes

accumulate.

The

use of

these

inhibitors

has supported

the

general

view

that

acetylation

associated

with

gene

expres-

CHAPTER

30 Controtling

Chromatin

Structure

sion;

in fact,

the ability of butyric

acid to cause

changes in chromatin resembling

those found

upon

gene

activation

was one of the first

indi-

cations of

the connection between

acetylation

and

gene

activity.

The breakthrough in

analyzing the role

histone acetylation

was

provided

by the char-

acterization of the acetylating

and deacetylat-

ing

enzymes, and their association

with other

proteins

that are involved in

specific events

activation and repression. A

basic change in

our

view

histone

acetylation was

caused by the

discovery that HATs are not

necessarily

dedi-

cated enzymes

associated with

chromatin:

rather,

it turns out that known

activators

transcription have

HAT activity.

The connection

was established

when

the

catalytic

subunit of a

group

A HAI

was identified

as a homolog of the

yeast

regulator protein

Gcn5.

It then

was

shonm that Gcn5 itself

has HAI

activ-

ity

(with

histones H3

and H4 as

substrates).

Gcn5

part

of an adaptor complex

that is necessary

for

the interaction between

certain

enhancers

and their target

promoters.

Its HAT

activity is

required for

activation

of the target

gene.

This enables

us to

redraw

our

picture

for

the

action

of coactivators as

shown in

f],{-iiit,Ji:

il{"}.11

.:.,

where

RNA

polymerase

bound

at a hypersen-

sitive site and coactivators

are acetylating

histones

on the nucleosomes in

the

vicinity.

Many

exam-

ples

are now

known of interactions

of this t1pe.

Gcn5 leads us into

one of the

most impor-

tant acetylase

complexes. In

yeast,

Gcn5 is

part

Coactivators

Basal

apparatus

Activators

I 5r;iili!: .;tl,,.i

Coactivators may have

HAT activities

that

acety-

late

the tai[s of nucleosomaI

histones.

806

of the 1.8 MDa Spt-Ada-Gcn5-acetyltransferase

(

SAGA) complex, which contains

several

proteins

that are involved in transcription.

Among these

proteins

are several TAF1s. In

addition, the

TAFtr145 subunit of TFnD is

an acetylase.

(Yeast

TAF1l4S is

the

homolog

of mammalian TAF1250;

both are known as TAFI.) There

are some func-

tional overlaps between TFnD

and SAGA,

most

notably that

yeast

can manage with either

TAFrrl45 or Gcn5, but is damaged

by the deletion

of both.

This

suggests that an acetylase activity is

essential

for

gene

expression, but

can be

provided

by either

TFnD

or SAGA. As might be expected

from the size of the SAGA complex,

acetylation

only

one of its functions,

although its other

func-

tions

gene

activation

are

less

well characterized.

One of the first

general

activators to be char-

acterized as HAT was

p300/CREB-binding pro-

tein

(CBP). (Actually, p300

and CBP are different

proteins,

but they are

so closely

that

they are often

referred

to as a single type of

activity.)

p300/CBP

is a coactivator

that

links

activator to the basal

apparatus

(see

Fig-

ure

25.7).

p300/CBP

interacts

with various acti-

vators,

including horrnone receptors, AP- I

(c-Jun

and c-Fos), and

MyoD.

The interaction is inhib-

ited by the viral regulator

proteins

adenovirus

EIA and SV40 T antigen, which

bind

p300/CBP

prevent

the

interaction

with tran-

scription factors; this explains how these viral

proteins

inhibit cellular transcription.

(This

inhi-

bition is important for the ability of the

viral

proteins

to contribute to the tumorigenic state.)

p300/CBP

acetylates the N-terminal tails of

in nucleosomes. Another coactivator, PCAF,

preferentially

acetylates

H3 in

nucleosomes.

p300/CBP

and PCAF form a complex that func-

tions

transcriptional activation.

some

cases

yet

another

HAT is involved:

the coactivator

ACTR, which functions with hormone receptors,

is itself an HAT that acts on H3 and H4, and aiso

recruits both

p300/CBP

and

PCAF

form a

coactivating

complex. One explanation for the

presence

of multiple HAT activities in a coacti-

vating complex

is that each HAT has a differ-

ent specificity,

and that multiple different

acetylation events

are required for activation.

general

feature

oI acetylation

is that

group

A HAT is

part

of a large complex.

,''!r.ri:r,:

1r..

shows a simplified

model for their

behavior. HAT complexes can be targeted

DNA

interactions with DNA-binding

factors.

This determines the target

for

the

HAT. The com-

plex

also contains effector subunits that

affect

chromatin structure or act directly on

transcrip-

tion.

It is likely that at least some of the

effec-

tors

require the acetylation

event

in order

act. Deacetylation,

catalyzed

by an

HDAC, may

work in a

similar

way.

Acetylation occurs

at both

replication

(when

it is transient) and

at transcription

(when it is

maintained while the

gene is active).

Is it

play-

ing the same

role in each

case? One

possibility

is that the

important effect

nucleosome

structure.

Acetylation

may

be necessary

"loosen"

the

nucleosome

core.

At replication,

acetylation of

histones could

necessary

allow them

to be

incorporated

into

new cores

more easily.

At transcription,

similar effect

could be

necessary

to allow

a related

change

structure,

possibly

even

to allow

the

histone core

to be displaced

from

DNA. Alternatively,

acety-

Iation could

generate

binding

sites

for other

pro-

teins that

are required

for

transcription.

In either

case, deacetylation

would

reverse

the effect.

the

effect of

acetylation

quantitative

qualitative?

One

possibility is that

a certain

number of acetyl

groups are

required to

have

effect, and the

exact

positions at which

they

occur

are largely

irrelevant.

An alternative

that individual

acetylation

events

have specific

effects. We

might

interpret

the existence

of com-

plexes

containing

multiple

HAT activities

either way-if

individual

enzymes

have differ-

ent specificities,

we may

need

multiple

activi-

ties either

to acetylate

sufficient

number

different

positions

because

the

individual

events are

necessary

for

different

effects

upon

transcription.

At replication,

it appears

(at

least

with

respect to

histone

H4) that

acetylation

any two of

three available

positions is adequate,

favoring a

quantitative model

in this

case.

Where

chromatin

structure

changed

to affect

tran-

scription, acetylation

at specific

positions is

important

(see

Section

31.3,

Heterochromatin

Depends on

Interactions

with

Histones).

ii-ii

jrri

ii.,. r

Comptexes

that

modify chromatin

structure

activity

have targeting

subunits

that

determine

their

sites of

action,

HAT or HDAC

enzymes

that acetytate

or deacetytate

his-

tones, and effector

subunits

that

have other

actions on

chro-

matin or

DNA.

30.7

Acetytases

Are

Associated

with

Activators

Deacetylases

Are

Associated

with

Repressors

Deacetytation

associated with repression

gene

activity.

Deacetytases

are

present

complexes with

repressor

activity.

yeast,

mutations

in SlNf

and RPD3 behave

though

these

loci repress

a variety

genes.

The

proteins

form

a complex

with the DNA-binding

protein

Ume6, which

binds

to the URS/

ele-

ment. The

complex represses

transcription

the

promoters

containing

URSl, as illustrated

in :.:i='"titi.:

';i.i.ii.

Rpd3 has

histone

deacetylase

activity;

do not know

whether

the function

of SinS is

just

to bring

Rpd3 to

the

promoter,

whether

it has

an additional

role in repression.

A similar

system for

repression

is found in

mammalian

cells. The

bHLH family

of transcrip-

tion regulators

includes

activators

that function

as heterodimers,

including

MyoD

(see

Sec-

tion

25. I 5,

Helix-Loop-Helix

Proteins

Interact

Combinatorial

Association).

It also includes

repressors,

particular

the

heterodimer

Mad:Max,

where

Mad can

be any one

of a

group

of closely

proteins.

The Mad:Max

het-

erodimer

(which

binds to

specific DNA

sites)

interacts

with

a homolog

of Sinl

(called

mSin3

mouse

and hSinS in

human

beings). mSin3

part

of a repressive

complex

that includes

his-

iriiltliii;

"i;;.

-i+ A repressor

comptex contains

three

com-

ponents:

a DNA

binding subunit,

corepressor,

and a his-

tone deacety[ase.

CHAPTER

Controlting

Chromatin

Structure

tone binding

proteins

and the histone

deacety-

Iases HDAC I

and

HDAC2.

Deacetylase

activity

is required

for repression.

The modular nature

of this system is

emphasized

by other means

employment: A corepressor

(SMRT),

which

enables retinoid hormone

receptors

to repress

certain target

genes,

functions

binding mSin3,

which in turns

brings the HDAC

activities

the

site. Another means

of bringing HDAC

activ-

ities to the

site may be a

connection

with

MeCP2,

protein

that

binds to methylated

cytosines

(see

Section

24.19,

CpG Islands Are

Regulatory Targets).

Absence

of histone acetylation

also a fea-

ture

of heterochromatin.

This is

true of

both

constitutive

heterochromatin

(

typically

involv-

ing

regions of centromeres

telomeres)

and

facultative heterochromatin (regions

that are

inactivated

in one cell

although they

may

active in another). T\upically

the

N-terminal

tails

of histones H3

and H4 are not

acetylated

in het-

erochromatic regions.

Methylation

of Histones

and DNA Is

Connected

Methytation

of both DNA and histones

feature

inactive

chromatin.

The

two types

methytation

event

may be

con nected.

Methylation

of both histones

and

DNA is

asso-

ciated

with inactivity.

Sites that

are methylated

in histones

include

two lysines

in the

tail of H3

and an

arginine in the

tail of H4.

Methylation

of H3

elys

a feature

of con-

densed regions

chromatin,

including

het-

erochromatin

seen in bulk

and also

smaller

regions

that are known

not to

be expressed.

The

histone

methyltransferase

enzyme

that

targets

this lysine

called SUV39Hl.

(We

see rhe

ori-

gin

of this

peculiar

name

in Section

30.13,

Some

Common Motifs

Are Found

in Proteins

That

Modify Chromatin).Its

catalytic

site has a region

called the

SET domain.

Other histone

methyl-

transferases

act on arginine.

addition,

meth-

ylation

may occur

Telys

the

globular

core

region

of H3; this may

be necessary

for

the for-

mation

of heterochromatin

at telomeres.

Until recently,

was thought

that

histone

methylation

was irreversible.

Histone

demeth-

ylases

have now

been identified,

though, includ-

ing

a lysine-specific

demethylase (LSDI)

that

acts on

I(4

of histone H3,

and an enzyme

that

demethylates arginine on histones

Hl and H4.

We do not

yet

know

how

demethylation is

regulated.

Most of the methylation

sites in DNA

are

CpG islands

(see

Section 24.19,

CpG Islands

Are Regulatory Targets).

CpG

sequences in het-

erochromatin are

usually methylated.

Con-

versely, it is necessary

for the

CpG

islands

located in

promoter

regions

to be unmeth-

ylated

in order for

gene

to be expressed

(see

Section

24.18,

Gene Expression

Is Associated

with

Demethylation).

Methylation of DNA

and methylation of

histones is

connected in a mutually reinforcing

circuit. Methylation

H3 is

the signal that

recruits the DNA methylase

to chromatin. The

order of events is that H3

el,ys

deacetylated

create

the substrate

for methylation. H3 is

then converted to the Meelys

or the

Me2elys

condition, which

provides

a binding site

for

the

DNA methylase. Some histone

methyltrans-

ferase enzymes contain

potential

binding sites

for the methylated CpG doublet,

so the

DNA

methylation reinforces

the circuit by

providing

a large| for the histone methyltransferase

bind.

The important

point

is that

one

type

modification can be the

trigger for another.

These

systems are widespread, as seen by evi-

dence for these connections in fungi,

plants,

and animal cells, and for regulating transcrip-

tion at

promoters

used by both RNA

poly-

merases I and II, as well

maintaining

heterochromatin in an inert

state.

Chromatin States

Are Interconverted

Modification

Acetytation of histones is associated with

gene

activation.

Methytation of DNA and

histones is

associated

with heterochromatin.

irii:i:ltir

::ij. i

summarizes three types of differ-

ences

that are found between

active chromatin

and inactive chromatin:

Active chromatin is acetylated on the

tails of histones H3 and H4.

Inactive chromatin is methylated on

el-ys

histone H3.

Inactive

chromatin

is methylated

cytosines of CpG doublets.

The reverse types of

events occur

com-

pare

the activation

of a

promoter

with the

gen-

eration of

heterochromatin.

The actions

of the

enzymes that

modify chromatin

ensure that acti-

vating events are

mutually exclusive

with

inac-

tivating events.

Methylation

el-ys

and

acetylation of H3

lalys

are mutually

antagonistic.

Acetylases and deacetylases

may trigger

the

initiating events.

Deacetylation

allows

methy-

lation to occur, which

causes

formation of a

het-

erochromatic

complex

(see

Section

31.3,

Heterochromatin

Depends on

Interactions with

Histones).

Acetylation

marks a region

as active

(see

Section

10. I l,

Promoter

Activation

Involves

an Ordered Series

Events).

Promoter

Activation

Involves an

0rdered

Series

Events

The remode[ing comptex

may recruit the

acetylating comp[ex.

Acetytation of

histones

may be the

event that

maintajns the complex

in the activated

state.

How are acetylases

(or

deacetylases)

recruited

to their specific targets?

As we have

seen with

remodeling

complexes,

the

process is likely to

l.tiiiii:t:

..rii.

Acetylation

histones activates

chro-

matin, and methylation

DNA and

histones

inactivates

ch ro mati

30.11

Promoter

Activation

Involves an

0rdered

Series of

Events

be indirect. A

sequence-specific activator

(or

repressor) may

interact

with

a component of

the

acetylase

(or

deacetylase)

complex to recruit

to a

promoter.

There may

also be direct interactions

between remodeling

complexes

and

histone-

modifying

complexes. Binding

by the SWI/SNF

remodeling

complex may

lead in turn to

bind-

ing

by the SAGA acetylase

complex. Acetyla-

tion of histones may

then in fact

stabilize the

association

with the SWI/SNF

complex, mak-

ing

mutual

reinforcement

of the changes in

the

components

at the

promoter.

Ii*ijRl

-:tii.:+: Promoter

activation

invotves

binding

of a

sequence-specific

actjvator, recruitment

and actjon

of a

remoo-

eling

comptex,

and recruitment

and

action of

an acetytating

comptex. The

order

ofevents can

differ or

can even be sjmut-

taneous

depending

on the

gene.

We can connect all of the

events at the

pro-

moter into the

series summarized in ftf:#gtil

3{,i.ti.*.

The initiating

event is binding

of a sequence-

specific component

(which

able to find its

tar-

get

DNA sequence in the

context of chromatin).

This recruits

remodeling

complex.

Changes

occur in nucleosome

structure. An

acetylating

complex binds, and the acetylation

of target his-

tones

provides

a covalent mark

that the locus

has been activated.

Modification

of DNA also

occurs at the

pro-

moter. Methylation of cytosine

at CpG

doublets

is associated

with

gene

inactivity

(see

Sec-

Iion24.I8, Gene Expression Is

Associated

with

Demethylation). The

basis for recognition

DNA as

atargel for methylation is

not very

well

established

(see

Section 31.8,

DNA Methyla-

tion Is Responsible for Imprinting).

It is clear

that chromatin remodeling

at the

promoter

requires a

variety of changes

that

affect nucleosomes,

including

acetylation,

but

what changes are required

within

the

gene

allow an RNA

polymerase

to traverse

it?

know

that RNApolymerase

can transcribe DNA

in vitro

at rates comparable

to the in

vivo rare

(-25

nucleotides

per

second)

only with

a tem-

plate

of free DNA.

Several

proteins

have been

charactedzed for

their abilities

improve

the

speed with

which RNA

polymerase

transcribes

chromatin in vivo. The

common

feature is

that

they

act on chromatin. A

current model

for their

action is

that they associate

with RNA

poly-

merase

and travel

with

along

the template,

modifying nucleosome

structure

by acting

histones. Among

these factors

are histone

acety-

Iases.

One

possibility

is that

the first RNA

poly-

merase

to transcribe

gene

is a

pioneer

polymerase

carrying factors

that

change

the

structure of the

transcription unit

so as to make

easier

for

subsequent

polymerases.

Histone

Phosphorylation

Aftects

Chromatin

Structure

least two histones

are targets for

phosphorylation,

possibty

with

opposing

effects.

Histones

are

phosphorylated

two circumstances:

cyclically

during the

cell cycle,

and

in association

with chromatin

re-

modeling.

810

CHAPTER

30 Controtting

Chromatin

Structure