Lewin Benjamin (ed.) Genes IX

Enhancers

Work

by

Increasing

the Concentration

of

Activators Near

the

Promoter

.

Enhancers usuatly work onty in crs configuration

with a target

promoter.

.

Enhancers can be made

to

work in

trans

configuration by tinking the DNA that contains

the target

promoter

to the DNA that contains the

enhancer

via a

protein

bridge or by catenating the

two molecutes.

.

The

princip[e

is that an enhancer works in

any

situation in which it is constrained to be in the

same

proximity

as the

promoter.

How can an enhancer stimulate initiation at a

promoter

that can be located

any distance away

on either side of

it?

When enhancers were first

discovered,

several

possibilities

were

consid-

ered for their action as elements distinctly dif

-

ferent from

promoters:

.

An enhancer could change the overall

structure of the template-for exam-

ple,

by

influencing

the density of

supercoiling.

.

It could be responsible for locating the

template

at

a

particular place

within

the cell-for example, attaching

it

to

the

nuclear

matrix.

.

An enhancer could

provide

an

"entry

site"-a

point

at which RNA

polymerase

(or

some other essential

protein)

ini-

tially associates with chromatin.

Now we take the view that enhancer

func-

tion involves the same sort of interaction with

the basal apparatus as

the interactions spon-

sored by

upstream

promoter

elements. En-

hancers are modular, like

promoters.

Some

elements

are found

in

both enhancers and

pro-

moters. Some individual elements found in

pro-

moters share

with enhancers the ability to

function at variable distance and

in

either

ori-

entation.

Thus the distinction between en-

hancers and

promoters

is

blurred:

Enhancers

might be

viewed

as containing

promoter

ele-

ments, which

are

grouped

closely together, and

as

having the ability to function at

increased

distances

from the

startDoint.

coactivators

in more

detail in Section 25 .5, Acti-

vators

Interact with the Basal Apparatus.

The essential

role of

the enhancer

may be

to

increase the concentration

of activator

in the

vicinity of the

promoter

(vicinity

in this sense

being a

relative term).

TWo types

of experiment

illustrated in

1:l:,ri.ii;li:

:l'i.;;i.i suggest

that this

is the

case.

A fragment of

DNA

that

contains an

enhancer at one

end and

a

promoter

at the

other

is not

effectively

transcribed,

but the

enhancer

can stimulate transcription

from the

promoter

when they

are connected

by a

protein

bridge.

Structural effects,

such

as changes

in supercoil-

ing, could

not be transmitted

across

such a

bridge;

this suggests

that

the critical

feature

is

bringing the enhancer

and

promoter into close

proximity.

A bacterial

enhancer

provides

a binding

site

for the

regulator NtrC,

which

acts upon

RNA

polymerase

using

promoters recognized

by o5a.

When the enhancer

is

placed upon a circle

of

DNA that

is catenated

(interlocked)

with a

cir-

cle

that contains

the

promoter, initiation

is

almost as effective

as when

the enhancer

and

promoter

are

on the

same

circular

molecule.

There is, however,

no initiation

when

the

enhancer

and

promoter are on

separated

cir-

cles. Again,

this suggests

that

the

critical

fea-

ture

is localization

of the

protein

bound

at the

enhancer,

which

increases

the enhancer's

chance

of contacting

a

protein bound

at the

Dromoter.

i:tr*t-iirii:

l{r,-l,r'r An enhancer

may function

by bringing

proteins

into the

vicin-

ity

of the

promoter.

An enhancer

does

not act on

a

promoter

at the

opposite

end ofa [ong

[inear

DNA, but

becomes

effective

when the

DNA isjoined

into

a circle

by a

protein

bridge.

An enhancer

and

promoter

on separate

circutar

DNAs do not

interact, but

can

interact

when the

two

molecutes are

catenated.

Promoter

Enhancer

+

lnterlocked

circles

24.i,7

Enhancers Work by

Increasing

the

Concentration

of

Activators

Near the

Promoter

631

If

proteins

bound at an

enhancer several

kb

distant from

a

promoter

interact

directly with

proteins

bound in

the vicinity

of the startpoint,

the

organization

of DNA

must be flexible

enough

to allow the

enhancer and

promoter

to

be closely located.

This requires

the interven-

ing DNA

to be extruded

as a large

"loop."

Such

loops

have been

directly observed

in the case

of

the

bacterial

enhancer.

There is

an interesting

exception

to the rule

that

enhancers

are cli-acting in natural

situa-

tions.

This is

seen in the

phenomenon

of trans-

vection. Pairing

of somatic

chromosomes

allows

an enhancer

on one chromosome

to

activate a

promoter

on the

partner

chromosome.

This

reinforces

the

view that enhancers

work

by

proximity.

What limits

the activity

of an

enhancer?

\pically

it works

upon the nearest

promoter.

There

are situations in

which

an enhancer is

located

between

two

promoters,

but activates

only

one of them

on the

basis of specific

pro-

tein-protein

contacts

between

the complexes

bound

at the

two elements.

The action

of an

enhancer

may be limited

by an insulator-an

element in

DNA that

prevents

the enhancer

from

acting

on

promoters

beyond the insula-

tor

(see

Section

29.I4, Insulators

Block the

Actions

of Enhancers

and Heterochromatin).

The

generality

of

enhancement

is not

yet

clear.

We

do not know

what

proportion

of

cel-

lular

promoters

require

an enhancer

to achieve

their usual

level

of expression, nor

do

we

know

how

often an

enhancer

provides

a

target for

regulation.

Some enhancers

are

activated

only

in

the

tissues in

which their

genes

function,

but

others

could

be active in

all cells.

l@

Gene Expression

Is Associated

with

Demethylation

e

Demethylation

at the

5'end of

the

gene

is

necessary

for

transcription.

Methylation

of

DNA

is

one of several

regula-

tory

events

that influence

the activity

of a

pro-

moter.

Methylation

at the

promoter

prevents

transcription,

and the methyl groups

must

be

removed

in

order

to activate

a

promoter.

This

effect is

well

characterized

at

promoters

for

both

RNA

polymerase

I and RNA polymerase

II.

In

effect, methylation is

a

reversible

regulatory

event. It is triggered

by

modifications

to his-

tones that include deacetylation

and

protein

methylation

(see

Section 30.9, Methylation

of

Histones

and DNA Is Connected).

Methylation

also occurs as

an epigenetic

event.

In

this case, modification

may

occur

specifically in sperm

or oocyte, with

the result

that there may

be a difference

between

two

alleles in

the next

generation.

This can result

in

differences in the

expression of

the

paternal

and maternal

alleles

(see

Section

31.8, DNA

Methylation

Is Responsible for

Imprinting).

In

this chapter we are

concerned

with the

means

by which methylation

influences

tran-

scription, which is

the same whether

the methyl

groups

were added

or

removed

as

a local regu-

latory

event or as an

epigenetic event.

Methylation

at

promoters

for

RNA

poly-

merase II

occurs at CG doublets.

The

distribu-

tion of methyl

groups

can be

examined

by taking

advantage

of restriction

enzymes

that cleave

target sites containing

the CG

doublet. TVvo

tlpes

of restriction

activity

are compared

in

ilt*-

iiRil li4.F*s.

These isoschizorners

are enzymes

that cleave the

same target sequence

in DNA,

but have

a different response

to its

state of

methylation.

The

enzyme HpaII

cleaves the

sequence

CCGG

(writing

the sequence of

only one

strand

of DNA). If

the second C is methylated,

though,

the enzyme

can no longer

recognize

the site.

The

enzyme MspI, however,

cleaves

the

same

fi{;ii*t-

Jri

.;:$

The

restriction

enzyme

MspI

cteaves

at[

CCGG sequences

whether or not

they are

methytated

at

the second

C. but HpaII

cteaves only nonmethytated

CCGG

tetramers.

Sites are

cleaved

irrespective

of methvlation

N/ethyiated

MsPl

Nonmethylated

CCGG

UUUU

lvle

Methylated

site

Nonmethylated

site

is not

cleaved

is

cleaved

632

CHAPTER

24 Promoters

and Enhancers

target site irrespective of the

state of methylation

at this C. Thus MspI

can be used

to

identify

all

the CCGG sequences, and HpaII

can be used to

determine whether they are methylated.

With a substrate

of

nonmethylated

DNA,

the two enzymes would

generate

the same

restriction bands. In methylated

DNA, however,

the

modified

positions

are not cleaved

by

HpaIL

For every such

position,

one

larger

HpaII frag-

ment replaces two MspI fragments.

fI*iift{ :il+"i{';

gives

an example.

Many

genes

show a

pattern

in which the

state of

methylation is

constant at most sites

but

varies at others. Some of the sites are methylated

in all tissues examined;

some sites are unmeth-

ylated

in all tissues . A minority

of sites are meth-

ylated

in tissues in which

the

gene

is not expressed, but

are nlt methylated in tissues in which the

gene

is

active. Thus an active

gene

may

be described as

undermethylated.

Experiments with the drug 5-azacytidine

produce

indirect evidence

that demethylation

can result

in

gene

expression. The

drug

is incor-

porated

into DNA in

place

of cytidine

and can-

not be methylated, because

the 5'position is

blocked.

This leads

to the appearance of

demethylated sites in DNA as the

consequence

of

replication

(following

the scheme on the right

of

Figure I5.7).

The

phenotypic

effects of 5-azacytidine

include the induction of changes in

the state of

cellular differentiation. For

example, muscle

cells are

induced

to develop from nonmuscle

cell

precursors.

The drug also

activates

genes

on a silent

X

chromosome, which raises the

pos-

sibility that the state of methylation could

be

connected with chromosomal inactivity.

As well as examining the state of methyla-

tion

of resident

genes,

we can compare the

Mspl digest Hpall

digest

Band

unique to Hpall

replaces Mspl bands

Bands unique to Mspl

=

methvlated

sites

Band at same

oosition

=

nonmethylated site

fIfii-lF.g

ii4.t? The

resutts

of MspI and HpaII cteavage

are compared by

gel

etectrophoresis of the

fragments.

results

of

introducing

methylated or

nonmeth-

ylated

DNA into

new host cells.

Such experi-

ments

show

a clear correlation;

The methylated

gene

is inactive, but the

nonmethylated

gene

is

active.

What

is the extent of

the undermethylated

region? In the chicken

a-globin

gene

cluster

in

adult erythroid

cells, the

undermethylation

is

confined to sites

that extend

from

-500

bp

upstream of the

first of the two

adult cr

genes

to

-500

bp

downstream

of the second.

Sites of

undermethylation

are

present

in the

entire

region, including the spacer

between

the

genes.

The region

of

undermethylation

coincides with

the region of

maximum sensitivity

to DNAase

I.

This argues that undermethylation

is a

feature

of a domain that

contains

a transcribed

gene

or

genes.

As

with

other

changes

in chromatin,

it seems likely

that the

absence

of methyl

groups

is associated

with the

ability

to be tran-

scribed

rather than

with the

act of transcrip-

tion

itself.

Our

problem

in interpreting

the

general

association

between

undermethylation

and

gene

activation

is that

only a

minority

(some-

times a small

minority) of

the

methylated sites

are involved.

It is likely

that the

state of

methylation

is critical

at specific

sites or

in a

restricted region.

It is also

possible

that a

reduction in the

level of

methylation

(or

even

the complete

removal

of

methyl

groups

from

some stretch

of DNA)

is

part

of some structural

change

needed to

permit

transcription

to

proceed.

In

particular,

demethylation

at the

promoter

may be necessary

to make

it available

for the

ini-

tiation

of transcription.

In the

y-globin gene,

for

example,

the

presence

of

methyl

groups

in the

region around the

startpoint.

between

-200

and

+90,

suppresses

transcription.

Removal

of the

three methyl

groups located upstream

of the

startpoint,

or of the

three

methyl

groups

located

downstream,

does

not relieve

the suppression.

Removal of all

methyl

groups, though,

allows

the

promoter

to

function.

Transcription

may

therefore

require a

methyl-free

region at the

promoter

(see

Section

24.I9,

CpG Islands

Are

Regulatory Targets).

There are

exceptions

to

th

is

general

relationship.

Some

genes

can

be expressed

even

when

they are extensively

methylated.

Any connec-

tion

between

methylation

and

expression

thus

is not universal

in an

organism,

but

the

general

rule is that

methylation

prevents

gene

expres-

sion and

demethylation

is

required

for ex-

pression.

24.1,8 Gene

Expression

Is Associated

with Demethytation

633

l@

CpG Islands Are

Regulatory

Targets

.

CpG islands

surround the

promoters

of

constitutivety

expressed

genes

where

they are

un methytated.

.

CpG

istands

also

are

found

at the

promoters

of

some tissue-regulated

genes,

r

There

are

-29,000

CpG

is[ands in

the human

gen0me.

o

Methytatjon

of a CpG

istand

prevents

activation of

a

promoter

within it.

o

Repression is

caused by

proteins

that

bind to

methylated

CpG doubtets.

The presence

of CpG islands in

the 5'regions

of some

genes

is connected

with the effect

of

methylation

on

gene

expression. These

islands

are detected

by the

presence

of an increased

density

of the dinucleotide

sequence, CpG.

(CpG

=

5'-CG-3'l

The

CpG doublet occurs in

vertebrate DNA

at

only

-20oh

of the

frequency

that would be

expected from

the

proportion

of G-C base

pairs.

(This

may

be because

CpG doublets

are meth-

ylated

on C,

and spontaneous

deamination of

methyl-C

converts it to T,

which introduces a

mutation

that removes

the doublet.) In

certain

regions,

however,

the density

of CpG doublets

reaches

the

predicted

value;

in fact,

it is increased

by l0x relative

to the rest

of the

genome.

The

y-globin

lritiltlil]il

5'**&d

ffi

Exon Exon

12

500 1000

bp

APRT

5'r*-y

Exon Exon

12

i:i--*i

:i+..-:iJ

The

typicaI density

of CpG doubtets in

mammatian

DNA

is

-1/100

bp, as seen for

a

y-gtobin

gene.

In a

CpG-rich island,

the density is

increased

to

>10

dou-

btets/100

bp. The istand

jn

the APRT

gene

starts

-100

bp

upstream

of the

promoter

and extends

-400

bp into

the

gene.

Each

vertical

Line represents

a CpG doubtet.

CpG doublets in these regions

are

generally

unmethylated.

These

CpG-rich islands have

an average

G-C content of

-60"/",

compared

with the 40%

average in bulk DNA. They

take the form

of

stretches

of

DNA

typically I to 2 kb long.

There

are

-45,000

such islands in the human genome.

Some of the islands are

present

in repeated

Alu

elements, and may

just

be the consequence

of

their high

G-C-content. The human

genome

sequence confirms that,

excluding these,

there

are

-29,000

islands.

There are

fewer in

the

mouse

genome,

-15,500.

About 10,000

of the

predicted

islands in both

species appear

to

reside

in a context of

sequences that are

conserved

between the

species, suggesting that

these may

be the

islands

with regulatory

significance. The

structure of chromatin in

these regions

has

changes

associated with

gene

expression

(see

Section 30.1 I, Promoter Activation

Involves

an

Ordered Series of Events);

there is

a reduced

content

of

histone

Hi

(which

probably

means

that the structure is less

compact),

the other

histones

are extensively

acetylated

(a

feature

that tends to

be associated with

gene

expres-

sion), and there are hypersensitive

sites

(as

would be expected of active

promoters).

In several

cases. CpG-rich islands

begin

just

upstream

of a

promoter

and

extend

down-

stream into the transcribed region

before

peter-

ing out. I?iltitiil ;lri.#*i

compares

the density

of

CpG

doublets in a

"general"

region

of the

genome

with a CpG island identified

from

the

DNA

sequence. The

CpG island

surrounds

the 5'region

of the APRT

gene.

which is

con-

stitutively expressed.

All of the

"housekeeping"

genes

that

are

constitutively

expressed have

CpG islands;

this

accounts for

about half of

the

islands.

The

other

half

of the islands occur

at the

promoters

of

tis-

sue-regulated

genes;

only a minority (<40%)

of these

genes

have islands.

In these

cases,

the

islands are

unmethylated irrespective

of the

state of expression

of the

gene.

The

presence

of

unmethylated

CpG-rich islands

may

be neces-

sary, but

therefore is not

sufficient,

for tran-

scription. Thus

the

presence

of unmethylated

CpG islands

may

be taken as an indication

that

a

gene

is

potentially

active rather

than inevitably

transcribed. Many

islands

that are

nonmeth-

ylated

in the

animal become

methylated

in

cell

lines

in tissue

culture, and

this could

be

con-

nected

with the inability

of these lines

to

express

all of the functions

typical of the

tissue from

which they

were derived.

CHAPTER

24 Promoters

and Enhancers

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stim-

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the CTD.

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Goodrich,

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direct

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C., and Timmers,

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Opening

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promoter

occurs in

two distinct

steps and

requires

the basal transcription

factors IIE

and

tIH.

EMBO J t5, 1666-1677.

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melt-

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by transcription

factor IIH

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W., Conaway,

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action in

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and

promoter

escape

requires

distinct regions

of downstream pro-

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Connection between Transcriotion

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Lehmann, A. R.

(2001).

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T. H., ICm,

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638

CHAPTER 24 Promoters

and Enhancers

Enhancers

Work

by

Increasing

the

CpG

Islands Are Regulatory

Targets

Concentration

of Activators Near

the

Promoter

Review

Blackwood, E. M.

and I(adonaga,

J.

T.

(1998).

Going the distance: a

current view of

enhancer action. Science 281,

6O-6j.

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Mueller-Storm,

H. P., Sogo,

J.

M.,

and Schaffner,

W.

(

1989). An enhancer

stimulates

transcription

in trans when

attached to the

promoter

via a

protein

bridge. CeIl 58, 7

67

-777

.

Zenke, M. et al.

(

1986). Multiple

sequence motifs

are

involved

in SV40

enhancer function.

EMBO J. 5. 387-397.

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Bird, A.

(20021.

DNA methylation

patterns

and

epigenetic

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Dev. 16, 6-21.

Research

Antequera, F. and Bird,

A.

(I99)l

. Number

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islands and

genes

in human and

mouse. Proc.

Natl. Acad. Sci. USA

90, 11995-11999.

Bird, A.

et al.

(1985).

A fraction

of the

mouse

genome

that

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derived

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methylated, Cp-G-rich

DNA. Cel/

40, 9l-99.

Boyes,

J. and

Bird, A.

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DNA

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inhibits

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indirectly

via

a

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CpG binding

protein.

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64,

ll23-lli4.

References

639

Activati

n

g

Tran

scri

pti

on

CHAPTER

OUTLINE

Introduction

r

Eukaryotic

aene

expression

'is

usually controtted at the [eve[

of initiation

of

transcription.

There Are

SeveraI Types

of Transcription

Factors

r

The

basaI apparatus

determines

the startpoint for

transcription.

o

Activators

determine the frequency

of

transcription.

o

Activators

work by making

protein-protein

contacts with

the

basaI factors.

r

Activators

may

work via

coactivators.

r

Some components

of

the transcriptionaI

apparatus work

by

changing

chromatin

structure.

Independent

Domains

Bind DNA

and Activate

Tra nscri

otio n

o

DNA-binding

activity

and transcription-activation

are car-

ried

by independent

domains of an

activator.

.

The ro[e

of the DNA-binding

domain is

to bring the

transcription-activation

domain into

the vicinity of the

promoter.

The

Two Hybrid

Assay

Detects Protein-Protein

Interactio

ns

o

The

two hybrid

assay

works by requiring

an interaction

between

two

proteins,

where one has

a

DNA-binding

domain

and

the other has

a transcription-activation

domain.

Activators

Interact

with

the BasaI Apparatus

r

The

principle

that

governs

the

function

of atl activators is

that

a

DNA-binding

domain

determines

specificity for

the

target

promoter

or enhancer.

o

The DNA-binding

domain

is responsible

for localizing

a

transcription-activating

domain in

the

proximity

of

the

L^--l ---^--r..-

uq)dL dPPdrdLu5.

r

An

actjvator

that works

directly has

a DNA-binding

domain

and

an activating

domain.

r

An

activator

that does not

have an

activating domain may

work

by binding

a coactivator

that has

an activating

domain.

o

Several factors

jn

the basaI

apparatus are

targets with which

activators

or

coactivators interact.

r

RNA

polymerase

may

be associated

with various

atternative

sets

of transcription

factors

in

the

form

of a holoenzyme

complex.

Some

Promoter-Binding Proteins Are

Repressors

r

Repression is usuatly

achieved by affecting chromatin

struc-

ture, but there

are

repressors

that act by binding

to specific

promorers.

Response E[ements Are

Recognized by Activators

.

Response

etements

may

be [ocated in

promoters

or

en

nancers.

o

Each response element is recognized

by a specific activator.

o

A

promoter

may have

many response elements,

which in

turn may

activate transcription independently

or

in

certajn

combi nations.

There Are

Many Types of DNA-Binding Domains

r

Activators

are classified according to the type

of DNA-binding

domain.

r

Members

of the same

group

have

sequence variations

of a

specific motif that confer specificity for individuaI

target

sites.

A Zinc Finger Motif Is

a

DNA-Binding

Domain

r

A zinc finger is

a loop of

-23

amino acids

that

protrudes

from a zinc-binding

site formed by His and

Cys amino

acids.

r

A zinc finger

protein

usua[[y

has

multipte zinc fingers.

o

The C-terminal

part

of each finger forms

an cr,-heUx that

binds one turn of the major

groove

of DNA.

.

Some zinc finger

proteins

bind RNA instead

of. or as wetl

as,

DNA.

Steroid Receptors Are Activators

.

Steroid receptors

are examptes of ligand-responsive

activa-

tors that are actjvated

by binding a steroid

(or

other related

molecules).

o

There

are separate DNA-binding

and ligand-binding

domains.

Steroid Receptors Have Zinc Fingers

r

The DNA

binding domain of a

steroid

receptor

is

a type of

zinc finger

that has Cys

but

not His

residues.

.

Gtucocorticoid

and estrogen receptors

each have

two zinc

fingers,

the

first

of which determines

the DNA

target

sequence.

r

Steroid receDtors

bind to DNA as dimers.

640

Lewin Benjamin (ed.) Genes IX

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