Lewin Benjamin (ed.) Genes IX

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inactive

region may

then be extended by the

ability of

further HPI molecules

to

interact

with

one another.

The

existence

of a common

basis

for silenc-

ing in

yeast

is suggested by its reliance on a com-

mon set of

genetic

loci. Mutations in any one

of a

number of

genes

cause HML and HMR to

become activated,

and also relieve the inacti-

vation of

genes

that

have

been

integrated near

telomeric

heterochromatin. The

products

of

these

loci therefore

function

to

maintain the

inactive state of

both types of heterochromatin.

il{;il+ii: ,'; li

.ii: proposes

a

model for

actions

of

these

proteins.

Only one of them is a sequence-

specific

DNA-binding

protein.

This is RAPl,

which binds

to the C1-3A repeats at the telo-

meres, and also binds

to

the

crs-acting silencer

elements

that are needed for repression of

HML

and

HMR. The

proteins

Sir3 and Sir4

interact

with RapI

and also with one another

(they

may function as

a heteromultimer). Sir3/Sir4

interact with the N-terminal

tails of the

his-

tones

H3

and

H4.

(In

fact, the first evidence

that

histones might be

involved directly in

for-

mation of

heterochromatin was

provided

by

the discovery that

mutations abolishing silenc-

ing at

HML/HMR

map

to

genes

coding for

H3

and

H4.)

Rapl

has the crucial

role

of

identifying the

DNA sequences

at which heterochromatin

forms.

It recruits Sir3/Sir4,

and they interact

directly with

the histones

H3lH4.

Once

SirS/Sir4

have bound to

histones HllH4, the complex

may

polymerize further and spread along the

chromatin

fiber.

This may inactivate the

region,

either because

coating with SirS/Sir4

itself

has

an inhibitory

effect, or because binding

to his-

tones

Hf /H4

induces some further change

in

structure.

We do

not know what limits the

spreading

of the

complex. The C-terminus

of

Sirl has a

similarity to

nuclear lamin

proteins

(constituents

of

the nuclear matrix)

and may

be responsible

for tethering

heterochromatin

to

the nuclear

periphery.

A similar series

of events forms the

silenced

regions

atHMRandHML

(see

also Section

19.22,

Silent Cassettes

aL

HML and HMR

Are

Repressed).

Three sequence-specific

factors are

involved

in triggering

formation of the

com-

plex:

Rapl,

Abf I

(a

transcription

factor),

and

ORC

(the

origin

replication complex).

In this

case, Sirl

binds to

a sequence-specific

factor

and recruits

Sir2,

-3,

and

-4

to form the

repres-

sive structure.

Sir2

is

a

histone deacetylase.

The

deacetylation

reaction

is necessary to

maintain

binding

of the Sir complex

to chromatin.

i::i:ll!ii. :l i" I Binding

of

HP1 to

methytated

histone

H3

forms

a trigger

for

sitencing

because

further

molecutes

of

HP1 aggregate

on the

nucteosome

chain.

i':,fiq.l$i :1.1

.r Formation

of

heterochromatin

is initiated

when

Rapl binds

to

DNA.5ir3/4

bind

to

Rapl

and atso

to

histones

H3/H4.Ihe

comptex

potymerizes a[ong

chromatin

and may connect

telomeres

to

the

nuctear

matrix.

31.3

Heterochromatin

Depends

on

Interactions

with

Histones

823

Formation

of heterochromatin

in

the

yeast

S.

pombe

depends

on a complex

that

contains

several

RNAi

molecules

(see

Section ll.I0,

RNA

Interference

Is Related

to Gene

Silenc-

ing).

These

RNAi molecules

are

produced

by

transcription

of

centromeric

repeats

to

give

RNAs

that are

cleaved into

smaller

units. The

complex

also contains proteins

that

are

homologs

of those involved

in

heterochromatin

formation

in

other organisms,

including

Arg-

onaute, which

is involved

in

targeting RNA-

induced

silencing

complex

(RISC)

remodeling

complexes

to chromatin.

The

RNAi

compo-

nents

are

responsible

for localizing

the com-

plex

at centromeres.

The

complex

then

promotes

dimethylation

of histone

H3 by

a his-

tone

methyltransferase.

How

does a

silencing

complex repress

chro-

matin

activity?

It could

condense

chromatin

so

that

regulator proteins

cannot

find

their tar-

gets.

The

simplest

case would

be

to suppose

that

the

presence

of a silencing

complex

is mutually

incompatible

with

the

presence

of transcription

factors

and RNA

polymerase.

The

cause

could

be that

silencing

complexes

block remodeling

(and

thus

indirectly

prevent

factors

from

bind-

ing)

or that

they directly

obscure

the

binding

sites

on DNA

for the

transcription

factors.

The

situation

may not

be this

simple,

though,

because

transcription

factors

and

RNA

pory-

merase

can

be found

at

promoters

in

silenced

chromatin.

This could

mean

that

the silencing

complex prevents

the factors

from

working

rather

than

from

binding

as such. In

fact,

there

may

be

competition

between

gene

activators

and the

repressing

effects

of chromatin,

so

that

activation

of a

promoter

inhibits

spread

of the

silencing

complex.

Another

specialized

chromatin

structure

forms

at the

centromere.

Its

nature is

suggested

by the properties

oI an Saccharlmyces

cerevisiae

mutation,

cse4,

that disrupts

the

structure

of the

centromere.

Cse4 is

a

protein

that

is related

to

histone

H3.

A mammalian

centromeric protein,

centromere protein

A

(CENP-A),

has a related

sequence.

Genetic interactions

between

cse4

and

CDE-II,

and

between

cse4

anda

mutation

in the

H4

histone gene,

suggest

that

a histone

octamer

may

form

around

a

core of

Cse4-H4,

and

then

the

centromeric

complexes (core

binding

fac-

tors)

CBFI

and CBF3

may

attach

to form

the

centromere.

The

centromere

may then

be

associated

with

the formation

of

heterochromatin

in

the region.

In

human

cells,

the centromere-specific

protein

CENP-B

is

required

to initiate

modifications

of

Effects

Are

Inherited

histone

H3

(deacetylation

of

elys

and

lalys,

fol-

lowed

by methylation

of

elys)

thar trigger

an

association

with the

protein

Swi6 that

leads to

the formation

of heterochromatin

in

the resion.

Polycomb

and Trithorax

Are Antagonistic

Repressors

and

Activators

o

Polycomb

group proteins

(Pc-G) perpetuate

a state

of repression

through ce[[

divisions.

r

The PRE is

a DNA sequence

that is required

for

the

action

of Pc-G.

r

The PRE

provides

a nucteation

center from

which

Pc-G

proteins

propagate

an inactive

structure.

.

No individual

Pc-G

protein

has

yet

been found

that

can bind the PRE.

o

Trithorax

group proteins

antagonize

the

actions

of

the Pc-G.

Heterochromatin

provides

one

example

of

the

specific repression

of chromatin.

Another

is

pro-

vided by

the

genetics

of homeotic genes

in

Drosophila,

which have led

to the

identification

of a

protein

complex

that may

maintain

certain

genes

in

a repressed

stale. Pc

mutants

show

transformations

of cell type

that

are equivalent

to

gain-of-function

mutations

in

the

genes

Antennap

e dia

(Antp

)

or U ltrab

ithorax,

becatse

these

genes

are

expressed

in tissues

in

which

usually

they are repressed.

This

implicates

pc

in

regulating

transcription.

Furthermore,

Pc is

the

prototype

for

a class

of

-I5

loci

called

the

pc-

group

(Pc-G);

mutations

in

these

genes

gener-

ally have

the

same result

of

derepressing

homeotic genes,

which

suggests

the

possibility

that

the

group

of

proteins

has

some

common

regulatory

role.

The

Pc

proteins

function

in

large

complexes.

The

PRCI

(Polycomb-repressive

complex)

con-

tains

Pc itself,

several

other Pc-G proteins,

and

five

general

transcription

factors.

The

Esc-E(z)

complex

contains

Esc,

E(z),

other Pc-G proteins,

a histone-binding

protein,

and

a

histone

deacetylase.

Pc itself

has

a chromodomain

that

binds

to methylated

H3,

and E(z)

is

a merhyl-

transferase

that acts

on H3.

These properties

directly

support

the connection

between

chro-

matin

remodeling

and repression

that

was ini-

tially

suggested

by the

properties

of

brahma,

a

fly

counterpartlo

SW2.

brahma

codes

for a

com-

ponent

of

the SWI/SNF

remodeling

complex,

and

loss

of brahma

function

suppresses

muta-

tions

in Polycomb.

824

CHAPTER

31 Epigenetic

Consistent with

the

pleiotropy

of

Pc muta-

tions, Pc is a nuclear

protein

that can be visu-

alized at

-80

sites on

polytene

chromosomes.

These sites include rhre Antp

gene.

Another

member of

the Pc-G,

polyhomeotic,

is

visualized

at a set of

polytene

chromosome bands that are

identical with those bound by Pc. The two

pro-

teins coimmunoprecipitate

in

a complex of

-2.5

x

106 D thatcontains l0 to 15

polypeptides.

The

relationship between these

proteins

and the

products

of the 40

Pc-G

genes

remains to be

established.

One

possibility

is that some of these

gene products

form a

general

repressive com-

plex,

and then some of the other

proteins

asso-

ciate with

it

to determine

its

specificity.

The Pc-G

proteins

are not conventional

repressors. They are not responsible for deter-

mining the

initial

pattern

of expression of the

genes

on which they act.

In

the absence

of Pc-

G

proteins,

these

genes

are initially

repressed

as usual, but

later in development the repres-

sion

is lost without

Pc-G

group

functions.

This

suggests that

the Pc-G

proteins

in some way rec1g-

nize the state of repressionwhen

itis

established,

and

they

then act t0

perpetuate

it thrlugh cell

division of

the daughter

cells.

;:::t,:':,:t- ,,

I

r

shows a model

in

which Pc-G

proteins

bind

in conjunction with

a repressor,

but the Pc-G

proteins

remain bound

after

the repressor

is no longer

available.

This

is

necessary to maintain repression, so that

if

Pc-G

proteins

are absent, the

gene

becomes

activated.

A

region of DNA that is sufficient to

enable

the

response to the

Pc-G

genes

is called a PRE

(Polycomb

response element). It can be defined

operationally

by

the

property

that it maintains

repression in

its

vicinity

throughout develop-

ment.

The assay for a PRE

is

to

insert it close to

a

reporter

gene

that is controlled by an enhancer

that is

repressed in early development, and

then

to determine

whether

the reporter becomes

expressed

subsequently

in

the descendants.

An

effective

PRE will

prevent

such

reexpression.

The

PRE is a complex structure that

mea-

sures

-

I 0 kb. TWo

proteins,

Pho and Pho I

,

with

DNA-binding

activity for sites within

the PRE

have been

identified, but there could be oth-

ers. When

a locus is

repressed

by

Pc-G, how-

ever, the

Pc-G

proteins

occupy a

much larger

Iength of

DNA than the PRE itself.

Pc is found

locally

over a few

kilobases of DNA surround-

ing a PRE.

This suggests

that the

PRE may

provide

a

nucleation center,

from which a structural

state

depending

on

Pc-G

proteins

may

propagate.

This model

is supported by the observation

of

tirrri,t',,

I:,r

Pc-Gproteinsdonotinitiaterepression.butareresponsib[efor

maintaining

it.

effects

related to

position effect

variegation

(see

Figure 3I.4), that

is, a

gene near to

a locus

whose

repression

is maintained

by

Pc-G

may become

heritably

inactivated

in some

cells but

not

oth-

ers.

In one typical

situation,

crosslinking

exper-

iments

in vivo showed

that

Pc

protein is found

over

large

regions

of the

bithorax

complex

that

are

inactive, but

the

protein is excluded

from

regions that

contain

active

genes. The idea

that

this could be

due

to cooperative

interactions

within

a multimeric

complex

is supported

by

the existence

of

mutations

in Pc that

change

its

nuclear

distribution

and

abolish

the

ability

of

other Pc-G

members

to

localize

in the

nucleus.

The role of

Pc-G

proteins

in maintaining,

as

opposed

to establishing,

repression

must

mean

that

the formation

of the

complex

at the

PRE

also depends

on

the

local state

of

gene

expressron.

A

working

model

for

Pc-G

binding

at a

PRE

is suggested

by the

properties of the

individual

proteins. First

Pho and

PhoI

bind

to specific

sequences

within

the PRE.

Esc-E(z)

is recruited

to Pho/Phol;

it then

uses

its methyltransferase

activity to

methylate

27Lys

of histone

Hl.

This

creates

the binding

site

for the

PRC,

because

the chromo

domain

of

Pc

binds

to the

methy-

Iated lysine.

The

Polycomb

complex

induces

a

more compact

structure

in

chromatin;

each

31.4

Potycomb

and

Trithorax

Are

Antagonistic

Repressors

and

Activators

825

PRC

I complex

causes

about three

nucleosomes

to become

less accessible.

In fact,

the

chromo domain

was first

iden-

tified

as a region

of homology

between

Pc and

the

protein

HPI found

in heterochromatin.

Binding

of the

chromo domain

of Pc

to

27lys

on H3

is analogous

to HPI's

use

of its chromo

domain

to

bind to

elys.

Variegation

is

caused

by the

spreading

of

inactivity

from constitutive

heterochromatin,

and as

a result it is Iikely

that

the

chromo

domain is

used by Pc

and HPI in

a

similar

way to induce

the formation

of het-

erochromatic

or inactive

structures

(see

Sec-

tion

10.13,

Some

Common Motifs

Are Found

in

Proteins

That Modify

Chromatin).

This

model

implies

that

similar mechanisms

are used

to

repress

individual

loci

or to

create heterochro-

matin.

The

trithorax group (trxG)

of

proteins

have

the

opposite

effect

to the Pc-G

proteins:

They act

to

maintain genes

in

an active

state.

There may

be some

similarities

in

the actions

of the

two

groups:

Mutations

in

some loci prevent

both

Pc-G

and

trx from

functioning,

suggesting

that

they

could rely

on

common

components.

The

GAGA factor,

which

is coded

by rhe

trithorax-like

gene,

has

binding

sites in

the PRE.

In fact,

the

sites where

Pc

binds

to DNA

coincide

with the

sites

where

GAGA factor

binds.

What

does

this mean?

GAGA

is

probably

needed

for

activating

factors,

including

trxG

members,

to bind

to DNA. Is

it also

needed for

Pc-G

proteins

to

bind and

exercise

repression?

This

is not

yet

clear, but

such

a model

would

demand

that

something

other

than GAGA

deter-

mines

which

of

the alternative

types of

com-

plex

subsequently

assemble

at

the site.

X

Chromosomes

Undergo

Gl.obaI

Changes

o

One

of the

two X

chromosomes

is inactivated

at

random

in

each celt during

embryogenesis

of

eutherian

mammats.

o

In

exceptional

cases where

there

are

>2

X

chromosomes,

at[ but

one are inactivated.

o

The Xrc (X

inactivation

center)

is

a crs-acting

region

on

the X chromosome

that is necessary

and

sufficient

to ensure

that

onLy

one

X

chromosome

remains

active.

r

Xrc

inctudes

the Xrsf

gene.

which

codes for

an RNA

that is found

onty on inactive

X

chromosomes.

o

The

mechanjsm

that is responsible

for

preventing

Xtsf

RNA from

accumutating

on the

active

chromosome

is

unknown.

+.1*iJ**

.it t.1{i Different

means

of dosage

compensation

are used to

equatize X chromosome

expression in

mare

and female.

Sex

presents

an

interesting problem

for

gene

regulation,

because of

the

variation

in the num-

ber

of

X

chromosomes.

If X-linked genes

were

expressed

equally well in

each

sex, females

would have

twice

as much

of each

product

as

males. The

importance

of avoiding

this situa-

tion

is shown

by the existence

of

dosage

com-

pensation,

which

equalizes

the level

of

expression

of X-linked

genes

in

the two

sexes.

Mechanisms

used in

different

species

are sum-

marized

in

!:lil*qil

"!.i.:*:

.

In mammals,

one of the

two female

X

chromosomes

is

inactivated

completely.

The result

is that females

have

only

one

active

X chromosome,

which is

the same

situation found

in males.

The

active

X

chromosome

of females

and the

single

X chromosome

of males

are expressed

at

the same level.

.

In

Drosophila,

tll'e

expression

of

the sin-

gle

male

X chromosome

is doubled

rel-

ative to

the expression

of

each female

X

chromosome.

.

In

Caenorhabditis

elegans,

the

expression

of each female

X

chromosome

is

halved

relative

to

the expression

of the

single

male

X chromosome.

The

common feature

in

all

these

mecha-

nisms

of dosage

compensation

is

that the

entire

chromosome

is the

target

for

regulation.

A

global

change

occurs

that

quantitatively

affects

all

of

the

promoters

on the

chromosome.

We know

most

about the inactivation

of the X

chromo-

some in

mammalian

females,

where

the

entire

chromosome

becomes

heterochromatic.

The

twin

properties

of heterochromatin

are

its

condensed

state

and associated

inactivity.

It

can

be divided

into

two types:

.

Constitutiveheterochromatin

con-

tains

specific

sequences

that

have no

coding

function.

In

general

these

include

Inactivate

Double

Halve

one9X

expression

ix

two I X

X

r-j

r:

x

i._.,-_.ij...)

|

Xo

826

CHAPTER

31 Epigenetic

Effects

Are

Inherited

satellite DNAs,

and they are

often

found

at the centromeres. These

regions are

invariably heterochromatic

because of

their intrinsic nature.

.

Facultative heterochromatin

takes

the

form

of entire chromosomes that

are

inactive in

one cell lineage, although

they can be expressed in

other

lineages.

The example

par

excellence is

the

mam-

malian X

chromosome. The inactive X

chromosome is

perpetuated

in a het-

erochromatic state, whereas

the

active X

chromosome

is

part

of the euchromatin.

Thtrs identical DNA sequences

are involved

in both states.

Once the inactive state has

been established, it is inherited by

descendant cells. This is

an example of

epigenetic

inheritance,

because it does

not depend on the DNA

sequence.

Our basic

view

of the situation of the

female

mammalian

X

chromosomes was formedby the

single X hypothesis in 1961 . Female mice that

are

heterozygous for X-linked

coat color

muta-

tions have a variegated

phenotype

in which

some areas

of the coat are

wild-type, but oth-

ers are mutant. shows that this can

be explaine

d if one of the two X

chromosomes

ts

inactivated at

random in

each cell of a small

precur-

sor

population

Cells in which the X chromosome

carrying the wild-type

gene

is inactivated

give

rise to

progeny

that express only the mutant

allele on

the active chromosome.

Cells derived

from a

precursor

where the

other chromosome

was inactivated have an active wild-type

gene.

In the case of coat

color,

cells descended

from

a

particular

precursor

stay together and thus

form a

patch

of the same color, creating the

pat-

tern of visible variegation. In other cases, indi-

vidual cells

in a

population

will

express

one or

the other of

X-linked

alleles;

for

example,

in

heterozygotes for the X-linked locus G6PD, any

particular

red

blood cell will express

only one

of the

two allelic forms.

(Random

inactivation

of one

X

chromosome

occurs in eutherian marrr-

mals. In

marsupials, the

choice

is

directed:

It is

always the

X

chromosome

inherited from the

father

that

is inactivated.)

Inactivation of the

X

chromosome

in

females is

governed

by the n-l rule: However

many

X

chromosomes

are

present,

all but one

will be

inactivated. In normal females there are

of

course two

X

chromosomes, but

in rare cases

where

nondisjunction has

generated

a 3X or

greater

genotype,

only one

X

chromosome

remains

active. This suggests a

general

model in

which

a specific event is limited to one

X

chro-

,

'

X-tinked variegation

is

caused

by the

ran-

dom

inactivation of one

X chromosome

in each

precursor

ce[t. Cetts

in which the

+ attete

is on the

active chromo-

some

have wil"d

phenotype;

cetls

in which

the

-

altete

js

on the active

chromosome

have

mutant

phenotype.

mosome and

protects

it

from an

inactivation

mechanism that

applies

to all

the others.

A single

locus on

the

X chromosome

is

suf-

ficient for

inactivation.

When

a translocation

occurs between

the

X

chromosome

and

an auto-

some, this

iocus

is

present on only

one

of the

reciprocal

products,

and

only

that

product

can

be

inactivated.

By comparing

different

translo-

cations, it

is

possible to map

this

locus, which

is

called

the Xic

(X-inactivation

center).

A cloned

region of

450 kb contains

all the

properties of

tlne Xic. When

this sequence

is

inserted

as a

transgene

on to

an autosome,

the autosome

becomes subject

to

inactivation

(in

a cell

cul-

ture system).

Xic is a cis-acting

locus that

contains

the

information

necessary

to

count

X chromosomes

and inactivate

all copies

but one.

Inactivation

spreads

from

Xic along

the

entire

X chromo-

some. When

Xlc

is

present on an

X chromosome-

autosome

translocation,

inactivation

spreads

into the autosomal

regions

(although the effect

is not

always

complete).

Xic conrains

a

gene,

called

Xist, that

is

expressed

only

on the

inactive

X

chromosome.

The behavior

of this

gene

is effectively

the oppo-

site from all

other

loci

on the

chromosome,

which are

turned

off .

Deletion

of Xlsl

prevents

Both

X chromosomes

are

active

in

precursor

cell

Wild-type

coat

color

.

Mutant

coat

color

gene

/

n:2,:,:z:"n:'""Jl:"\

active

allele

.,--..,:l

active

allele

.-

-i?--J

31.5

X Chromosomes

Undergo

GlobaI

Changes

827

X-inactivation

invo[ves

stabitization

of

Xrst

RNA, which

coats the inactive

chromosome.

an X

chromosome

from being

inactivated.

It

does

not, however,

interfere

with

the counting

mechanism

(because

other X chromosomes

can

be inactivated).

Thus

we can

distinguish

two

features

of. Xic:

an unidentified

element(s)

required

for counting,

and

the Xist

gene

required

for inactivation.

i

ir,

,

::

i:

,

.

. illustrates

the role

of

Xlsf

RNA

in X-inactivalion.

Xist

codes for

an RNA that

Iacks

open reading

frames.

The XzslRNA

"coaIs"

the

X chromosome

from

which it is

synthesized,

which

suggests that it

has a

structural role.

Prior

to X-inactivation,

it is

synthesized

by both female

X

chromosomes.

Following

inactivation,

the

RNA

is found

only

on the inactive

X

chromo-

some. The

transcription

rate remains

the

same

before

and after inactivation,

so the

transition

depends

on

posttranscriptional

events.

Prior

to X-inactivation,

Xist

RNA

decays

with

a

half

life

of

-2

hours.

X-inacrivation

is

mediated

by

stabilizing

the XrilRNA

on the inac-

tive X

chromosome.

The XlslRNA

shows a

punc-

tate

distribution

along

the X

chromosome,

which

suggests

that

association

with

proteins

to form

particulate

structures

may

be the means

of

stabilization.

We

do not know

yet

what other

factors

may

be involved

in

this reaction

and

how

the Xlsl RNA

is limited

to spreading in

cli

along

the

chromosome.

The

characteristic

fea-

tures

of the inactive

X

chromosome,

which

include

a lack

of acetylation

of histone H4,

and

methylation

of

CpG sequences (see

Section

Epigenetic

Effects

Are

Inherited

24.19,

CpG

Islands

Are Regulatory

Targets).

pre-

sumably occur later as

part

of the mechanism

of inactivation.

The n-l

rule suggests

that stabilization

of

Xrsl RNA

is the

"default,"

and that some

block-

ing

mechanism

prevents

stabilization

at one X

chromosome

(which

will be the

active X). This

means

that, although Xic is

necessary

and suf-

ficient for

a chromosome to

be inactivated,

the

products

of other loci may

be necessary

for

the

establishment

of an active X chromosome.

Silencing

of Xlsl expression

is necessary

for

the active X. Deletion

of the

gene

for DNA

methyltransferase

prevents

silencing

of. Xist,

probably

because methylation

at

Ihe Xist

pro-

moter is necessary for

cessation

of transcription.

Chromosome

Condensation

Is

Caused

by Condensins

.

SMC

proteins

are

ATPases

that inctude

the

condensins

and the cohesins.

r

A heterodimer

of SMC

proteins

associates

with

other subunits.

r

The

condensins

cause chromatin

to be more

tightty

coited by

introducing

positive

supercoits

into DNA.

o

Condensins are responsibte

for condensing

chromosomes at mitosis.

.

Chromosome-specific

condensins

are respons'ibte

for

condensing inactive X

chromosomes

in

C.

elegons.

The

structures of entire

chromosomes

are influ-

enced by interactions

with

proteins

of the SMC

(structural

maintenance

of

chromosome)

family.

They are ATPases

that fall

into

two func-

tional

groups.

Condensins

are involved

with

the control of

overall structure,

and

are respon-

sible for the

condensation

into compact

chro-

mosomes

at mitosis.

Cohesins

are

concerned

with connections

between

sister

chromatids

that must

be released

at mitosis.

Both

consist

of

dimers formed

by SMC

proteins.

Condensins

form

complexes

that have

a core

of the het-

erodimer

SMC2-SMC4

associated

with

other

(non-SMC)

proteins.

Cohesins have

a similar

organization

based

on the heterodimeric

core of

SMCI-SMC3.

i li,i.;l;

I

:.

i ,: shows

that an

SMC

protein

has

a coiled-coil

structure in

its center

that

is inter-

rupted

by a flexible

hinge region.

Borh

the

amino and

carboxyl

termini have

ATP-

and

DNA-binding

motifs.

Different

models

have

been

proposed

for

the actions

of these

proteins

CHAPTER

31

ATP- and DNA-

binding site

Coiled-coil

Hinge

Coiled-coil

ATP-

and

DNA-

binding site

C-terminus

il5iitlftE: ;i

-i.i:i An

SMP

protein

has

a

"Walker

modute"

with an ATP-binding motif

and

DNA-binding

site at each

end, which are connected bv

coiled coi[s that are [inked

by a

hinge region.

F..I$*i{{ i:.::i

SMC

proteins

dimerjze

by antiparatteI

interactions between

the centraI coited

coits.

Both terminaI

regions of

each subunit

have

ATP- and

DNA-binding motifs.

Cohesins

may

form an extended

structure

that atlows two

different DNA

motecutes to be

[inked.

Condensin

ATP

-

86"

ATP

Cohesin

Flt]t"!l-l{ -:;1.:r-J

The two halves

of a condensin are

fotded

back at an angte

of

6o. Cohesins

have

a

more

open con-

formation with

an angle of 86o between the two

halves.

depending on whether they dimerize by intra-

or

intermolecular interactions.

Experiments

with the bacterial

homologs

of

the SMC

proteins

suggest that a dimer is formed

by an antiparallel

interaction

between the coiled

coils, so that the N-terminus of one subunit

bonds

to the C-terminus of the other subunit.

The existence of

a flexible hinge region could

allow cohesins and condensins to depend on a

different

mode

of action by the

dimer.

f:*it-i*i i.1.1.! shows that cohesins have a V-

shaped structure,

with the arms separated by an

86'angle, whereas

condensins are more sharply

bent back, with

only 6o

between the

arms. This

enables cohesins to hold sister chromatids

together,

whereas condensins instead condense

an individual chromosome. *:.i*li*L: :'*1":.* shows

that a cohesin

could take the form of an extended

dimer

that crosslinks two DNA molecules.

Ft*L{**

3:: . .r

+

shows that a condensin could take

the form of a

V-shaped

dimer-essentially

bent at the

hinge-that

pulls

together distant

ilItiLigfi

.1 1""'itl

Condensins

may

form a compact

structure

by bending at

the hinge, causing

DNA to become

compacted'

sites on the

same

DNA

molecule,

causing

it to

condense.

An

alternative

model

is suggested

by

exper-

iments to suggest

that

the

yeast

proteins dimer-

ize by intramolecular

interactions,

that

is, a

homodimer

is formed

solely

by

interaction

between two

identical

subunits.

Dimers

of two

different

proteins

(in

this

case,

SMC

I and SMCS)

may then

interact

at both

their

head

and hinge

regions to

form a circular

structure

as

illustrated

in ru*i.:El-: iri.,l

,r.

Instead

of binding

directly

to

DNA, a structure

of

this type

could

hold

DNA

molecules

together

by

encircling

them.

Visualization

of mitotic

chromosomes

shows

that condensins

are

located

all along

the

length

of

the chromosome,

as

can

be seen

in

If.{;Ltit{:1 i.t*i.

(By

contrast,

cohesins

are

found

at discrete

locations.)

The condensin

complex

was

named

for its ability

to

cause

chromatin

to

31.6

Chromosome

Condensation

Is Caused

by Condensins

829

i'ii:.iRi

--:.i.:...r

Cohesins may dimerize

by intramotecutar

connections,

and

then

form

muttimers

that are

connected

at

the

heads

and at the hinge.

Such a structure

coutd ho[d

two motecutes

of DNA together

by surrounding

them.

I.iiL:liI

i:i.]S

Condensins

are located

atonq the

entire

Length

of a mitotic

chromosome. DNA

is red;

condensins

are

yetlow.

Photo

courtesy

ofAna Losada

and Tatsuya Hirano.

condense

in vitro.It

has

an ability

to introduce

positive

supercoils

into

DNA in

an action

that

uses

hydrolysis

of AIP

and depends

on the

pres-

ence

of topoisomerase

I. This

ability is

controlled

by

the

phosphorylation

of the non-SMC

sub-

units.

which

occurs at mitosis.

We do not know

yet

how

this connects

with

other modifications

of chromatin-for

example,

the

phosphoryla-

rion

of histones.

The

acrivation

of the

condensin

ilkff

ffi *1iL1t*"J",#iisH*T;

Global

changes

occur in

other types

of

dosage

compensation.In

Drosophila,

a complex

of

proteins

is

found in

males,

where it localizes

CHAPTER

31 Epigenetic

Effects

Are Inherited

on

the

X

chromosome. In C. elegans,

a

protein

complex associates with both X

chromosomes in

XX

embryos, but the

protein

components remain

diffusely distributed in

the

nuclei

of XO

embryos.

The

protein

complex contains an

SMC core, and

is similar to the

condensin complexes

that are

associated

with mitotic chromosomes

in other

species. This suggests that it has

a structural role

in

causing the chromosome

to take

up a more

condensed, inactive

state. Multiple

sites on the

X

chromosome may

be

needed

for the

complex

to be fully

distributed along it. The

complex

binds

to these sites, and then

spreads along

the chro-

mosome

to cover it more

thoroughly.

Changes

affecting all the

genes

on a chro-

mosome/

either negatively

(mammals

and

C. elegans) or

positively

(Drosophila),

are there-

fore

a common feature

of dosage

compen-

sation. The

components

of the dosage

compensation

apparatus may vary,

however,

as well

as the means by

which it is localized

to

the chromosome,

and of course its

mechanism

of action is

different in each

case.

DNA Methylation

Is Perpetuated

by

a

Maintenance

Methylase

o

Most methyl

groups

in DNA

are

found

on cytosine

on both

strands of the CpG

doublet.

r

RepUcation

converts a fuLty methytated

site to a

hemimethylated

site.

r

Hemimethytated

sites are

converted

to

futty

methytated

s'ites

by a maintenance

methylase.

Methylation

of DNA occurs

at specific

sites. In

bacteria, it is

associated

with identifying

the

bac-

terial

restriction-methylation

system

used for

phage

defense, and

also with

distinguishing

replicated

and nonreplicated

DNA

(see

Section

20.7,

Controlling

the Direction

of Mismatch

Repair). In

eukaryotes, its

principal

knor,rm

func-

tion is

connected with

the control

of transcrip-

tion;

methylation is

associated

with

gene

inactivation

(see

Section

24.18,

Gene Expres-

sion Is Associated with

Demethylation).

From

2o/o

to

7

o/o

of the

cytosines

of animal

cell DNA

are methylated

(the

value

varies

with

the

species). Most

of the

methyl

groups

are

found in

CG

"doublets,"

and, in

fact,

the major-

ity

of the

CG sequences

are methylated.

Usu-

ally the

C

residues

on both strands

of this

short

palindromic

sequence

are methylated, giving

the

structure.

830

Lewin Benjamin (ed.) Genes IX

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