Lewin Benjamin (ed.) Genes IX

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Fully methylated

sites

Replication

Hemimethylated sites

Hemimethylated

sites

Fi{;l-i€i --t i.tt

The

state of methylated sites cou[d be

perpetuated

by an enzyme that recognizes onty hemimethy-

lated sites as substrates.

Such a site is described

fully methyl-

ated. Consider, though, the consequences of

replicating this site. F:ii*.ql

t"'i:: shows that each

daughter duplex has one methylated strand and

one unmethylated

sgand.

Such a site

is called

hemimethylated.

The

perpetuation

of the methylated site

now depends on

what happens

hemimeth-

ylated

DNA.

If methylation

of the unmethylated

strand occurs, the site is restored to the fully

methylated condition.

If replication

occurs

first,

though,

the hemimethylated condition will be

perpetuated

on one

daughter

duplex, but

the site

will become

unmethylated on the other daugh-

Perpetuation

methylase

Demethylase

t-tc'>X1"

\rve

F5{.i1.1fiil

"":

li .Ii=:

The state of

methylation

is controlted

three types ofenzyme.

De novo and

perpetuation

methyl

ases are

known, but demethytases

have not been

identified.

ter duplex. fi:*ilFiil

.i .i"t*

shows

that the

state of

methylation of

DNA is controlled

by meth-

ylases,

which

add

methyl

groups

the 5

posi-

tion of cytosine,

and

demethylases,

which

remove the methyl

groups.

(The

formal

name

for the enzymes

uses

methyltransferase

as the description.)

There are two

types of

DNA

methylase,

whose actions are

distinguished

by the

state of

the

methylated

DNA. To

modify

DNA at a

new

position

requires

the action

the de novo

methylase,

which

recognizes

DNA

by virtue

of a specific

sequence.

Ir acts only

on nonmeth-

ylated

DNA, to

add a

methyl

group

one strand.

There are two de

novo

methylases

(Dnmt3A

and

Dnmt3B)

in mouse;

they

have different

target

sites, and

both are

essential

for development.

A maintenance

methylase

acts

constitu-

tively

lnly on hemimethylated

sites

to convert

them

to fully

methylated

sites.

Its existence

means

that any methylated

site

perpetuated

after

replication.

There is one

maintenance

meth-

ylase (Dnmtt

)

in mouse,

and

it is essential:

mouse

embryos

which

its

gene

has been

disrupted

not

survive

past

early

embryogenesis.

Maintenance

methylation

is virtually

100%

efficient,

ensuring

that

the situation

shown

31.7

DNA

Methytation

Is Perpetuated

by a

Maintenance

Methytase 831

the left of Figure

31.19 usually

prevails

in vivo.

The result is

that, if. a de novo methylation

occurs

one allele but not

on the other, this

differ-

ence will be

perpetuated

through

ensuing cell

divisions, maintaining

a difference

between the

alleles that

does not depend

on their sequences.

Methylation

has various

types of rargets.

Gene

promoters

are the most

common target.

The

promoters

are methylated

when

the

gene

is inactive,

but unmethylated

when it is active.

The absence

of DnmtI in

mouse causes

wide-

spread

demethylation

promoters,

and we

assume

this is lethal

because of the

uncontrolled

gene

expression.

Satellite DNA

is another

tar-

get.

Mutations

in Dnmt38

prevent

methylation

of satellite DNA,

which causes

centromere insta-

bility at

the cellular level.

Mutations in

the cor-

responding

human

gene

cause a

disease called

ICF

(immunodeficiency/centromere

instabil-

ity,

facial

anomalies). The importance

methy-

Iation

is emphasized

by another

human disease,

Rhett

syndrome,

which is

caused

mutation

of the

gene

for the

protein

MeCP2

that binds

methylated

CpG

sequences.

The

methylases

are conventional

enzymes

that act

on a DNA target.

There may, however,

also

be a methylation

system

that uses a

short

RNA

sequence

to target

a corresponding

DNA

sequence

for methylation

(see

Section 13.6,

Antisense

RNA Can Be

Used to Inactivate

Gene

Expression)

Nothing is known

about the mech-

anism

operation of

this system.

How

are demethylated

regions

established

and maintained?

If a DNA

site has not

been

methylated,

protein

that recognizes

the

unmethylated

sequence

could

protect

against

methylation.

Once

a site has

been methylated,

there

are

two

possible

ways to

generate

demethylated

sites.

One is to

block the main-

tenance methylase

from

acting

on the site when

it is replicated.

After a

second replication

cycle,

one of the

daughter

duplexes

will be unmeth-

ylated (as

shown

on the right

side

of Fig-

ure

31. l9). The

orher is

acrively

to demethylate

the site,

as shown

in i:li.i,:*I

"ii".;t

either by

removing

the

methyl

group

directly from

cyto-

sine,

or by

excising the methylated

cytosine

cytidine

from

DNA for replacement

by a repair

system.

We know

that

active

demethylation

can occur

to the

paternal

genome

soon after

fertilization,

but we do not

know

what mech-

anism is

used.

One interesting possibility

is that

the cytidine

deaminase

AID

may

involved;

it can

deaminate

methylated

residues,

cre-

ating

a mismatched

base

pair

that a repair

system

might

then

correct

to a standard

(unmethylated)

C-G

pair.

Epigenetic

Effects

Are Inherited

.- fl

| /

\ /'-

-\,1

kH-

l/-.;

-1l\

(

,n/

rw-

1-l

---v

}#U

3',

/\\

Removal

of Removal

methyl

group

S-Me-C base

nucleotide

::i**itf,

-::;i.i:li DNA

coutd be demethylated

by removing

the methyl

group.

the

base. or the nucleotide.

Removal

of the

base or

nucteotide

woutd reouire

its replacement

by a repair

system.

DNA Methylation

Responsible for

Imprinting

Paternal

and maternal

altetes may have

different

patterns

methytation

at fertilization.

Methylation is

usua[[y associated

with inactivation

of the

gene.

When

genes

are differentiatty

imprinted,

survival

the embryo may require

that

the

functional

altele

provided

by the

parent

with

the unmethytated

attete.

Survival of heterozygotes

for imprinted

genes

different, depending

on the direction

the cross.

Imprinted

genes

occur in clusters

and may

depend

on a [oca[

control site where

norzo

methylation

occurs unless

specifica[[y

prevented.

The

pattern

of methylation

germ

cells is estab-

Iished

each sex during

gametogenesis

by a

two-stage

process:

First

the existing

pattern

erased

by a

genome-wide

demethylation,

and

then the

pattem

specific

for each

sex is imposed.

832

CHAPTER

All allelic

differences

are losr

when

pri-

mordial

germ

cells develop

the embryo;

irre-

spective of

sex, the

patterns

methylation

are

erased, and

a typical

gene

then unmethylated.

males,

the

pattern

develops

in two

stages. The

methylation

pat-

tern

that

characteristic

of mature

sperm is

established in

the spermatocyte,

but further

changes are made

in this

pattern

after fertiliza-

tion. In females,

the maternal pattern

imposed

during

oogenesis,

when

oocytes

mature

through meiosis

after

birth.

As may

be expected from

the inactivity

genes

gametes,

the typical

state is

to be meth-

ylated.

There are

cases of differences

between

the two sexes,

though, for

which

locus

unmethylated

one sex. A

major

question

how the

specificity of methylation

determined

the male and female gametes.

Systematic

changes

occur in

early embryo-

genesis.

Some sites will

continue

to be meth-

ylated,

whereas others

will

be specifically

unmethylated in

cells in

which a

gene

expressed. From

the

pattern

changes, we may

infer

that individual

sequence-specific

demeth-

ylation

events

occur during

somatic

development

of the

organism as

particular

genes

are activated.

The specific

pattern

of methyl groups

germ

cells

responsible

for the

phenomenon

of imprinting,

which describes

a difference in

behavior

between the alleles

inherited

from each

parent.

The

expression

of certain

genes

in mouse

embryos depends

upon the

sex of the

parent

from which they

were inherited.

For example,

the allele coding for IGF-II

(insulin-like

growth

factor II) that

is inherited

from the father

expressed, but the

allele that is inherited

from

the mother is not

expressed. The

IGF-II

gene

oocytes is methylated,

but the IGF-II

gene

sperm is not methylated,

so that the

two alleies

behave

differently in the zygote.

This is the most

common

pattern,

but

the dependence

on sex is

reversed for

some

genes.

In fact,

the opposite

pattern (expression

of maternal

copy) is

shown

for

IGF-IIR, the receptor

for IGF-II.

This sex-specific mode

of inheritance

requires

that

the

pattern

of methylation

is established

specifically during

each

gametogenesis.

The

fate

of a hypothetical locus

in a mouse is

illustrated

liiji.iiii:

i..:1.j.

the early embryo,

the

pater-

nal

allele

nonmethylated

and expressed, and

the matemal allele is methylated

and

silent. What

happens

when this mouse itself forms

gametes?

If it is

male,

the allele contributed

to the sperm

must

nonmethylated,

irrespective

of whether

was originally methylated

not.

Thus when

the maternal allele finds itself in

a sDerm. it must

be demethylated. If the mouse

female, the

allele contributed to the egg must be

methylated;

if it was

originally the

paternal

allele, methyl

groups

must be added.

The

consequence

of imprinting is that an

embryo requires a

paternal

allele

for

this

gene.

Thus

in the case of a

heterozygous cross where

the allele of one

parent

has an

inactivating muta-

tion, the

embryo

will survive

the wild-type

allele

comes

from

the

father, but will die

the

wild-type

allele is from the

mother. This type of

dependence

on the

directionality of

the

cross

(in

contrast with Mendelian

genetics)

is an

example of epigenetic inheritance,

where some

factor

other than

the sequences of

the

genes

themselves influences their

effects

(see

Section

ll.l0. Epigenetic Effects Can

Be Inherited).

Although the

paternal

and

maternal alleles have

identical

sequences,

they display different

prop-

erties, depending on

which

parent provided

them. These

properties

are inherited through

meiosis and the subsequent

somatic mitoses.

Imprinted

genes

are sometimes

clustered.

More than half of the

l7 known imprinted

genes

in mouse are contained

in two

particular

regions,

each containing

both

maternally and

paternally

expressed

genes.

This suggests

the

possibility

that imprinting

mechanisms

may

function

over

long

distances.

Some

insights into

llt:{l!'ll :i

",:

The

typicaI

pattern

for imprinting

is that

a methylated [ocus

is inactive.

this

is the maternaI

attete, on[y the

paternal

altele

active,

and wit[ be essen-

tia[

for

viabitity.

The methytation

pattern

is reset when

gametes

are formed, so that

a[[ sperm

have the

paternal

type and a[[ oocytes

have the

maternal type.

31.8

DNA Methytation

Is Responsibte

for imprinting 833

this

possibility

come from deletions in the human

population

that cause

the Prader-Willi

and

Angel-

man

diseases. Most cases are caused by the same

4 Mb deletion, but the syndromes are different,

depending

which

parent

contributed the dele-

tion. The reason is that the

deleted

region

con-

tains at

least

one

gene

that

paternally

imprinted

and at least one that is maternally imprinted.

There

are some

rare

cases.

however.

with

much

smaller deletions. Prader-Willi syndrome can be

caused by a 20 kb deletion that silences

genes

that are distant on either side

it. The

basic effect

of the deletion

prevent

a father from reset-

ting the

paternal

mode

to a chromosome

inher-

ited from his

mother. The result is that these

genes

remain in matemal mode, so that the

pater-

nal

as well as maternal alleles are silent in the

offspring. The inverse

effect

is found in

some

small deletions that cause Angelman's syndrome.

The implication

is that this region comprises

some

sort of

"imprint

center" that acts at a distance to

switch one

parental

tlpe to the

other.

Methylation is also responsible for

epige-

netic effects that control the

expression of

rRNA

genes.

The

phenomenon

of nucleolar domi-

nance describes the transcription

of only one

set

parental

rRNA

genes.

It results from methy-

lation

at cytosines in the

promoters

for

the

genes

inherited

from one

Darent and

not

the other-

0ppositeLy

Imprinted

Genes

Can

Controlled

by a SingLe

Center

Imprinted

genes

are controlted

methylation

crs-acting

sites.

Methylation may

responsibte

for either

inactivating

or activating a

gene.

Imprinting

determined by the state of meth-

ylation

of a cli-acting

site

near

a target

gene

genes.

These regulatory

sites are known as dif-

ferentially

methylated

domains

(DMDs)

imprinting

control regions

(ICRs).

Deletion of

these sites removes

imprinting,

and the target

loci

then behave the same in

both maternal and

paternal

genomes.

The

behavior

of a

region

containing

two

genes,

Igp and I119, illustrates

the ways in

which methylation

can control gene

activity.

::ii,i.:::i

:i..i*i

shows that these

two

genes

react

oppositely to the

state of methylation

at the

ICR

located

between them. The ICR is methylated

on the

paternal

allele. I1.19 shows

the typical

Epigenetic

Effects Are

Inherited

lgt2 ICR H79

Enhancer

Paternal allele

ACTIVE

INACTIVE

Maternal allele

INACTIVE ACTIVE

f,i{**F

.i:.":ii ICR is methytated

on the

paternaI

atlete,

where

lgf2 is

active and

H19 is inactjve. ICR is

unmethy-

lated on the

maternal

atlele,

where IgF2 is inactive

and

H19 is

active.

+:3*LiRf

i.t*

The ICR is

insulator

that

prevents

enhancer from actjvating lgf2. Ihe insulator functions

onty when it binds CTCF to unmethytated DNA.

response of inactivation.

Note.

however,

that

Igf2 is expressed. The reverse situation

is found

on a maternal allele, where

the ICR is not

methylated. H19 now

becomes expressed,

but

Igf2 is inactivated.

The

control of

lgp is

exercised by an insu-

lator function

of the

ICR.

fl**it$.

-q1.il4

shows

that when

the

ICR is

unmethylated, it

binds the

protein

CTCF. This creates an insulator

func-

tion that

blocks an enhancer from

activating

Ihre lgp

promoter.

This is

an unusual

effect in

which methylation

indirectly activates

gene

by blocking an insulator.

The regulation

I1l9

shows the more

usual

direction

of control

which methylation

cre-

ates an inactive imprinted

state. This

could

reflect a direct

effect of methvlation

on Dro-

moter

activity.

Maternal

allele

CTCF binds to unmethylated ICR

tgf2

Enhancer is blocked

834 CHAPTER

Epigenetic

Effects

Can Be Inherited

Epigenetic

effects can resutt

from modification

a nucleic

acid after

has

been

synthesized or

the

perpetuation

protein

structures.

Epigenetic inheritance

describes

the ability

different states,

which may

have

different

phe-

notypic consequences,

to be inherited

without

any

change in the

sequence

DNA.

How

can

this

occur? We can

divide epigenetic

mecha-

nisms

into two

general

classes:

DNA

may be modified

by the covalent

attachment

of a moiety

that is then

per-

petuated.

TWo

alleles with

the same

sequence may

have

different states

of methylation

that

confer

different

properties.

self

perpetuating

protein

state may be

established. This

might involve

assem-

bly of a

protein

complex,

modification

specific

protein(s),

or establishment

of an alternative

protein

conformation.

Methylation

establishes

epigenetic inheri-

tance so long as the maintenance

methylase

acts constitutively

to restore the

methylated

state after each

cycle of replication,

as shown in

Figure

31.19. A state

of methylation

can be

per-

petuated

through

an indefinite

series of somatic

mitoses.

This is

probably

the

"default"

siruarion.

Methylation

can also be

perpetuated

through

meiosis:

for example, in

the fungus Ascobolus

there are epigenetic

effects that

can be trans-

mitted through

both mitosis

and meiosis

maintaining the

state of methylation.

In mam-

malian

cells, epigenetic

effects are created

resetting

the state of methylation

differently in

male and female meioses.

Situations in

which epigenetic

effects appear

to be maintained by means

protein

states are

less well understood

in molecular

terms. Posi-

tion effect variegation

shows that

constitutive

heterochromatin

may

extend for a variable

dis-

tance, and the structure is

then

perpetuated

through somatic divisions.

There is no methy-

Iation of DNA in

Saccharomyces

and a vanish-

ingly

small amount inDrosophila,

and as a result

the inheritance of epigenetic

states of

position

effect

variegation

or telomeric silencing in

these

organisms is likely

to be due to the

perpetuation

protein

structures.

i:iiiliF.l I

i.;t:; considers

two extreme

possi-

bilities

for

the fate of a

protein

complex at

replication.

A complex could

perpetuate

itself if it

splits

symmetrically,

so that

half

com-

plexes

associate

with each daughter

duplex. If the

half

complexes

have the

capacity to nucleate

formation of full

complexes, the original state will be

restored.

This is

basically

analogous to

the

maintenance of methylation.

The

problem

with this model

is that there is

no evident

reason

why

protein

com-

plexes

should behave

in this way.

A complex could be

maintained as a unit

and

segregate

to one of the

two daugh-

ter duplexes.

The

problem

with this

model is that

it requires a new complex

assembled de

novl

orr

the other

daughter duplex,

and

it is not evident

why this should

happen.

Consider

now the need to

perpetuate

a het-

erochromatic structure

consisting of

protein

complexes. Suppose that a

protein

is distrib-

uted

less

continuously

along

a stretch

of heterochromatin, as

implied in

Figure 31.4.

If individual subunits are distributed

at random

to each

daughter

duplex at

replication, the two

daughters will continue

to be marked by

the

protein,

although

its density

will be reduced to

half

of the level before

replication.

If the

pro-

tein has a self-assembling

property

that causes

ll+Ljitl- .i{'1

,il:

What happens

protein

comptexes on

chromatin during

replication?

31.10

Epigenetic

Effects Can Be

Inherited 83s

iii-]..:i:i

=:: I.iri-_:

Acetytated

cores are conserved and djstributed at random

to the

daughter chromatin fibers

reptication.

Each daughter fiber has a

mixture

ofotd

(acetytated)

cores and new

(unacetylated)

cores.

new

subunits to associate

with

it,

the original

situation may

restored. Basically,

the existence

of epigenetic

effects

flrces

us t0 the view that a

protein

responsible

for

such a situatiln must h6ve slme slrt

s e

lf-

temp lating or se lf- as

emb ling

cap acity.

In some

cases,

it may

be the state of

pro-

tein modification, rather

than the

presence

the

protein per

se, rhar is responsible for an epi-

genetic

effect.

There

is a

general

correlation

between the activity

of chromatin and the state

of acetylation

of the histones, in

particular

the

acetylation of histones H3

and H4, which occurs

on their

N-terminal tails. Activation of tran-

scription

is associated with

acetylation in the

vicinity

of the

promoter;

and

repression

of tran-

scription is

associated with deacetylation

(see

Section 30.7, Acetylases

Are Associated with

Activators).

The most

dramatic

correlation

that the inactive X

chromosome in mammalian

female

cells is

underacetylated on histone H4.

The inactivity

of constitutive heterochro-

matin

may require that

the histones are not

acetylated. If

a histone acetyltransferase

is teth-

ered to a region

of telomeric heterochromatin

yeast,

silenced

genes

become active.

When

yeast

exposed to trichostatin

(an

inhibitor of

deacetylation),

centromeric heterochromatin

CHAPTER

Epigenetic

Effects

Are Inherited

becomes

acetylated, and silenced

genes

in cen-

tromeric

regions may become active. The effect

may

persist

even after trichostatin has been removed.

In fact, it may be

perpetuated

through mitosis

and meiosis. This suggests that an epigenetic

effect

has

been

created by changing the state of

histone acetylation.

How might the state of acetylation be

per-

petuated?

Suppose that the H)2-H42 tetramer

is distributed at

random

to the two daughter

duplexes.

This creates the situation shown in

tiGi-iftil

]1.1$, in

which

each daughter

duplex

contains some

histone

octamers that are fully

acetylated on the H3 and H4 tails, whereas oth-

ers are completely unacetylated.

account lor

the epigenetic

effect, we could suppose

that the

presence

of some

fully

acetylated histone

octamers

provides

a signal that causes

the

unacetylated octamers

to be acetylated.

(The

actual situation

probably

more com-

plicated

than the one shown in the figure,

because transient acetylations occur during

replication. If they are simply reversed follow-

ing

deposition

of histones into nucleosomes,

they

may

irrelevant. An

alternative

possibil-

ity is that the usual deacetylation is

prevented,

instead

of, or as well

as, inducing

acetylation.)

Yeast Prions

Show

UnusuaL

Inheritance

The

Sup35

protein

in its wild-type

soluble form is

a termination

factor for

transtation.

can also exist

an atternative form of

otigomeric aggregates.

in which it

is not actjve in

protein

synthesis.

The

presence

of the otigomeric form causes newty

synthesized

protein

to acquire the inactive

structure.

Conversion between the two

forms'is

influenced

cnaDer0nes.

The witd-type form has

the

recessive

genetic

state

psr

and the mutant form has the dominant

genetic

state P5.[+.

One of the clearest cases

of the dependence

epigenetic inheritance

the

condition

of a

protein

provided

the

behavior of

prions.

They have

been characterized in

two circum-

stances: by

genetic

effects in

yeast,

and as

the

causative agents of neurological

diseases in

mammals, including human

beings. A striking

epigenetic

effect

is found

yeast,

where two

different states can be inherited

that map to a

single

genetic

locus, although the

sequence of the

836

Termination

[PSl+]

state: no termination

---*

Sup35

[psl-]

Sup35

[PS/.]

-.>

:ilii-jt:::

i,.i:r

The

state

of the Sup35

protein

determines

whether termination

of transtation

occurs.

gene

is the

same in both

states. The two

different

states are

lpsi-l

and

[PSI+].

switch in condi-

tion occurs at

low

frequency

as the result

a spontaneous

transition

between the

states.

The

[ps{

genotype

maps to the locus

SUP35,

which

codes for a translation

termination fac-

tor.

l:l,i.i!iir

-i: .':l'

summarizes the

effects of the

Sup35

protein

yeast.

In wild-type

cells, which

are characterized

lpsi),

the

gene

is active,

and Sup35

protein

terminates

protein

synthe-

sis. In cells

the mutant

[PSI+]

type, the factor

does not function,

which causes a failure

to ter-

minate

protein

synthesis

properly.

(This

was

originally detected

by the lethal

effects of the

enhanced efficiency

of suppressors

of ochre

codons in

[PSI+]

strains.)

[PS1+]

strains have unusual

genetic

prop-

erties. When a

lpsi-l

strain is crossed

with a

[PSI+]

strain, all of the

prlgeny

are

IPSF).

This is

pattern

inheritance

that

would be expected

of an extrachromosomal

agent, but the

[PSI+]

trait cannot be mapped

to any such nucleic

acid.

The

[PSI+]

trait is metastable,

which means that,

although it is inherited

by most

progeny,

it is

Iost

at a higher rate than is

consistent with muta-

tion. Similar behavior is

shown also by the locus

URE2, which codes for

protein

required for

fi{.;tiiii -i

r.,:r::

Newly synthesized

Sup35

protein

con-

verted into

the

IPSI+]

state by the

presence

preexist-

ing

IPSI+]

protein.

nitrogen-mediated

repression of certain

cata-

bolic enzymes. When a

yeast

strain is converted

into

an alternative state,

called

lURE3l,

the Ure2

protein

is no longer

functional.

The

[PSI+]

state is determined

by the

con-

formation

of the

Sup35

protein.

In a wild-type

[psi-]

cell, the

protein

displays

its normal

func-

tion. In a

[PSI+]

cell, though,

the

protein

pres-

ent in an alternative

conformation

in which

its

normal function has been

lost. To explain

the

unilateral dominance

[PSI+]

over

[psi-]

genetic

crosses, we

must suppose

that the

pres-

ence of

protein

in the

lPSfl

rtatu causes

all the

pro-

tein in the cell to enter

this state.

This requires an

interaction

between

the

[PSI+]

protein

and

newly synthesized

protein,

which

probably

reflects the

generation

an oligomeric

state

which the

[PSI+]

protein

has a

nucleating role,

as illustrated

iii':i.iFlf; -i i.iil-i.

A feature common

to both the

Sup35 and

Ure2

proteins

is that

each consists

of two

domains that function

independently.

The

C-terminal domain

is sufficient

for the activity

of the

protein. The

N-terminal

domain

3L.11

Yeast Prions Show

Unusual

Inheritance 837

sufficient for formation of the structures that

make the

protein

inactive. Thus

yeast

in which

the N-terminal domain of Sup35 has been

deleted cannot acquire the

[PS/+]

state, and the

presence

of a

[PS1+]

N-terminal domain is suf-

ficient to maintain Sup35

protein

in the

[PS1+]

condition. The critical feature of the N-termi-

nal domain is that it is rich in

slutamine

and

asparagine residues.

Loss

o{ function in the

[PSI+]

state is due to

the sequestration of the

protein

in an oligomeric

complex. Sup35

protein

[PSI+]

cells

clus-

tered in

discrete foci, whereas the

protein

[psr]

cells is

diffused

in the

cytosol. Sup3 5

pro-

tein from

[PS1+]

cells forms amyloid fibers ir

vitro-these

have a characteristic hish content

B-sheet

structures.

The involvement

protein

conformation

(rather

than covalent modification) is

suggested

by the

effects of conditions that affect

protein

structure.

Denaturing treatments cause loss of

the

[PS1+]

state. In

particular,

the chaperone

Hsp

104 is involved in inheritance

[PSI+].

Its

effects are

paradoxical.

Deletion

of. HSPl04pre-

vents maintenance of the

[PSI+]

state, and over-

expression of

Hsp104 also

causes

loss

the

[PSI+]

state. This suggests that Hspl04 is required

for some change in the structure of Sup3 5 that

is necessary for acquisition of the

[PSI+]

state,

but that

must

transitory.

Using the ability of Sup35 to form the inac-

tive structure

in vitro, it is

possible

provide

biochemical

proof

for the role of the

protein.

ji;i.iii::

.i..i:;r

illustrates a striking experiment

which

the

protein

was converted to

the

inac-

tive form in vitro,

put

into

liposomes

(where

effect the

protein

is surrounded

by an arti-

ficial membrane), and then introduced directly

into

cells by

fusing the liposomes

with

[psr]

yeast.

The

yeast

cells were converted to

[PS1+]

This experiment refutes all of the objections

that

were

raised to the conclusion

that the

pro-

tein has the ability to confer the epigenetic

state.

Experiments in which

cells are mated,

which extracts are taken

from

one cell to

treat another cell, always are susceptible to the

possibility

that a nucleic acid has

been

trans-

ferred.

When the

protein

itself

does not con-

vert target cells, though

(even

though

protein

converted to the inactive state can do so), the

only difference is the treatment

of the

protein-

which must therefore be responsible

for the

converslon.

The

ability of

yeast

form

the

[PSI+]

prion

state depends on the

genetic

background. The

yeast

must

[PIN+]

order for the

[PS1+]

state

form. The

[PIN+]

condition itself is

an epige-

netic state. It can be created by the formation

prions

from any one of several

different

pro-

teins.

These

proteins

share the characteristic

Sup35 that they have Gln/Asn-rich

domains.

Overexpression of these domains in

yeast

stim-

ulates formation of the

[PSI+]

state.

This

sug-

gests

that there

a common model for

the

formation of the

prion

state

that

involves

aggre-

gation

the

Gln/Asn domains into

self-

propagating

amyloid structure.

How

does the

presence

of one Gln/Asn

pro-

tein influence

the

formation

prions

another? We know

that the formation

of Sup35

prions

specific to Sup35

protein.

that is, it

does not

occur by cross-aggregation

with other

proteins.

This suggests

that the

yeast

cell

may

contain

soluble

proteins

that antagonize

prion

formation. These

proteins

are not

specific for

any

one

prion.

a result, the introduction

any

Gln/Asn domain

protein

that

interacts

with

these

proteins

will reduce

the concentration.

This will allow

other Gln/Asn

proteins

aggre-

gate

more easily.

state'of

u.rr,

affid

protein

can convert the

CHAPTER 31 Epigenetic

Effects

Are inherited

[psi-]

protein

[PSl']

Converl rn vitro

Incornoraltr inlo lrnnsome

Fuse

liposome with

[psi

]

yeast

east remains

Lpsi-]

i'i)i.iil

l-:ttt":::1,:l

iF$1']

838

lpsrl

'tn

Prions

Cause Diseases

Mam

ma[s

The

protein

responsibte

for

scrapie

exists in two

forms: the

witd-type noninfectious

form

PrPc,

which is

susceptible

proteases.

and

the disease-

causing form

PrPsc, which

is resistant

proteases.

The neurotogical

disease can

be transmitted

mice by injecting

the

purified

PrPsc

protein

into

mrce.

The recipient mouse

must

have

a copy of the PrP

gene

coding

for

the mouse

protein.

The PrPsc

protein

can

perpetuate

itsetf

by causing

the newly

synthesized PrP

protein

take up the

PrPsc

form instead

of the PrPc form.

Multiple

strains of PrPs.

may have

different

conformations of the

orotein.

Prion

diseases have

been found

in sheep, in

human

beings, and, more

recently,

in cows. The

basic

phenotype

an ataxia-a

neurodegener-

ative disorder

that is manifested

inability

to remain

upright. The

name

of the disease in

sheep,

scrapie, reflects

the

phenotype:

The

sheep rub against

walls

in order

to stay upright.

Scrapie can

perpetuated

by inoculating

sheep

with

tissue extracts from

infected

animals. The

disease kuru

was

found in

New Guinea, where

it appeared

to be

perpetuated

cannibalism,

particular

the eating of brains.

diseases

in Western

populations

with a

pattern

genetic

transmission include

Gerstmann-Straussler

syn-

drome and the related

Creutzfeldt-Jakob

dis-

ease

(CJD),

which occurs

sporadically. Most

recently, a disease resembling

CJD appears to

have

been transmitted

by consumption

of meat

from cows suffering

from

"mad

cow" disease.

When

tissue from

scrapie-infected

sheep

inoculated

into mice,

the disease

occurs in a

period

ranging from

75 to I50

days. The active

component is a

protease-resistant

protein.

The

protein

is coded

by a

gene

that is normally

expressed

the brain. The

form of the

protein

in normal

brain, called PrPc,

is sensitive to

pro-

teases. Its conversion

to the resistant

form, called

Prps', is

associated with

occurrence of the dis-

ease. The inf ectious

preparation

has

detectable nucleic acid, is

sensitive to UV irra-

diation at wave lengths

that damage

protein,

and has a low infectivity

infectious

unit / 105

PrPs'proteins). This

corresponds

to an epige-

netic inheritance in

which

there is no change

genetic

information

(because

normal and dis-

eased cells have the same PrP

gene

sequence),

but the PrPs'form of the

protein

is the infectious

agent

(whereas

PrPc is harmless).

The PrPs'

form

has a high content of

sheets, which

form

an amyloid fibrillous structure that

absent

from the PrPc form.

The basis for the difference between

the

PrPs'and Prpc forms appears to

lie

with a change

conformation rather than with

any covalent

alteration. Both

proteins

are

glycosylated

and

Iinked

to the membrane by

a GPl-linkage.

The

assay

for infectivity

in mice

allows

the

dependence

protein

sequence to be tested.

i.1l:,t;l{t

r; i.

ii;

illustrates the

results of some crit-

ical experiments. In the normal situation,

PrPs'

protein

extracted

from an infected

mouse

will

induce disease

(and

ultimately

kill) when it is

injected into

recipient mouse.

If the PrP

gene

"knocked

out,

a mouse becomes

resistant to

infection.

This experiment

demonstrates

two

things. First, the endogenous

protein

is neces-

sary for

infection,

presumably

because

pro-

vides the raw material that

converted

into

the infectious

agent.

Second. the cause

of dis-

ease is not the removal

of the PrPc

form

the

protein,

because

a mouse with

no PrPc survives

normally:

The disease

is caused by a

gain-of-

function in PrPs'. If the PrP

gene

is altered to

pre-

vent the GPl-linkage

from occurring,

mice

infected

with PrPs'do

not develop disease,

which

suggests

that the

gain

function involves

an altered signalling

function for which

the GPI-

linkage is required.

The

existence of

species barriers

allows

hybrid

proteins

to be

constructed

to delineate

irii;lJliri

;i i,:,i.i A

Prps'protein can onty

infect an animal

that

has

the

same

type of endogenous

PrPc

protein.

31.12

Prions Cause

Diseases

in Mammats 839

the features required for infectivity. The origi-

nal

preparations

of scrapie

were

perpetuated

several types of animal, but these cannot always

be transferred readily.

For

example,

mice

are

resistant

to infection from

prions

of hamsters.

This means that hamster-PrPst cannot convert

mouse-PrPc

PrPsc. The

situation changes,

though, if the mouse PrP

gene

is replaced by a

hamster PrP

gene. (This

can be done by

intro-

ducing the hamster PrP

gene

into the PrP knock-

out mouse.) A mouse with a hamster PrP

gene

is sensitive

infection

hamster PrPsc. This

suggests that the conversion of cellular PrPc

protein

into the Sc state requires

that the

PrPsc

and PrPc

proteins

have

matched sequences.

There

are different

"strains"

of PrPs', which

are distinguished by characteristic incubation

periods

upon inoculation into mice. This implies

that

the

protein

is not restricted solely to alter-

native states

PrPc

and

PrPsc,

but

rather

that

there may

be multiple Sc states. These differ-

ences must depend on some

self-propagating

property

of the

protein

other than

its

sequence.

If conformation is

the feature that distinguishes

PrPs'from

PrPc, then there must

multiple

conformations,

each of which has a self-

templating

property

when it converts PrPc.

The

probability

of conversion from PrPc to

PrPsc

is affected by the

sequence of

PrP.

Gerstmann-Straussler

syndrome in human

beings

caused by a single amino

acid change

in PrP. This is inherited

as a dominant trait. If

the

same change is made in the mouse PrP

gene,

mice

develop the disease. This

suggests that the

mutant

protein

has an increased

probability

spontaneous conversion into

the Sc state. Sim-

ilarly, the

sequence of the PrP

gene

determines

the susceptibility of

sheep to develop the dis-

ease spontaneously;

the combination of amino

acids at three

positions (codons

I)6, I54, and

l7l

)

determines

susceptibility.

The

prion

offers an extreme case

of epige-

netic inheritance,

in which

the

infectious

agent

is a

protein

that can adopt multiple

conforma-

tions.

each of

which

has

a self-templating

prop-

erty. This

propefiy

is likely

to involve rhe

srare

of aggregation of the

protein.

Summary

Ihe formation

of heterochromatin

occurs by

proteins

that bind

to specific chromosomal

regions

(such

telomeres) and that interact

xt:l,llT

Jffi:T:,H

:i J :,":il:T;

CHAPTER

31 Epigenetic

Effects Are Inherited

thread from an initiation center. Similar events

occur

silencing

of the inactive

yeast

mating

type loci. Repressive structures that are

required

to maintain the inactive states of

particular genes

are formed by the Pc-G

protein

complex in

Drosophila. They share with heterochromatin

the

property

propagating

from

initiation

center.

Formation of

heterochromatin

may be ini-

tiated

at certain sites and then

propagated

for

a distance that

is not

precisely

determined.

When

a heterochromatic state has been estab-

Iished.

it is inherited through

subsequent cell

divisions. This

gives

dse

to a

pattern

of epige-

netic inheritance, in which two identical

sequences of

DNA may

be associated with dif-

ferent

protein

structures, and therefore have

different abilities to be expressed. This

explains

the occurrence of

position

effect variegation in

Drosophila.

Modification of

histone

tails is a trigger for

chromatin reorganization. Acetylation is

gen-

erally

associated with

gene

activation. Histone

acetylases are found

activating complexes,

whereas histone deacetylases are found in

inac-

tivating complexes. Histone methylation is asso-

ciated with

gene

inactivation.

Some

histone

modifications may be exclusive

or synergistic

with others.

Inactive chromatin at

yeast

telomeres

and

silent

mating

type

loci

appears to have a com-

mon cause, and involves the interaction

of cer-

tain

proteins

with

the

N-terminal tails of histones

and

H4. Formation

of the

inactive

complex

may

initiated

by binding of one

protein

to a

specific sequence of DNA; the

other compo-

nents may

then

polymerize

in a cooperative

manner along the chromosome.

Inactivation

of one

chromosome in female

(eutherian)

mammals

occurs at random. The

Xrclocus is necessary and

sufficient to count

the

number

chromosomes. The n-I rule

ensures that allbut one X

chromosome are inac-

tivated. Xic contains the

gene

Xist,

which codes

for an RNA

that

expressed only on the inac-

tive X

chromosome. Stabilization

Xist

RNA

is the mechanism

by which the inactive X

chro-

mosome

is distinguished.

Methylation

DNA is

inherited

epigenet-

ically. Replication

of DNA creates hemimethy-

lated

products,

and a maintenance

methylase

restores

the fully methylated state.

Some methy-

lation

events depend on

parental

origin.

Sperm

and eggs contain specific

and different

patterns

of methylation,

with the result that

paternal

and maternal

alleles are differentlv

exnressed in