Molecular

Systems

Biology

Article

number

doi

Citation

Molecular

Systems

Biology

EMBO

and

Nature

Publishing

Group

All

rights

reserved

On

the

relation

between

promoter

divergence

and

gene

expression

evolution

Itay

Adina

Dana

Mark

and

Naama

Department

of

Molecular

Genetics

Weizmann

Institute

of

Science

Rehovot

Israel

and

Department

of

Physics

of

Complex

Systems

Weizmann

Institute

of

Science

Rehovot

Israel

Corresponding

author

Department

of

Molecular

Genetics

Weizmann

Institute

of

Science

Rehovot

Israel

Tel.

Fax

E-mail

naama.barkai@weizmann.ac.il

Received

accepted

Recent

studies

have

characterized

significant

differences

in

the

cis-regulatory

sequences

of

related

organisms

but

the

impact

of

these

differences

on

gene

expression

remains

largely

unexplored

Here

we

show

that

most

previously

identified

differences

in

transcription

factor

-LRB-

TF

-RRB-

binding

sequences

of

yeasts

and

mammals

have

no

detectable

effect

on

gene

expression

suggesting

that

compensatory

mechanisms

allow

promoters

to

rapidly

evolve

while

maintaining

a

stabilized

expression

pattern

To

examine

the

impact

of

changes

in

cis-regulatory

elements

in

a

more

controlled

setting

we

compared

the

genes

induced

during

mating

of

three

yeast

species

This

response

is

governed

by

a

single

TF

-LRB-

-RRB-

and

variations

in

its

predicted

binding

sites

can

indeed

account

for

about

half

of

the

observed

expression

differences

The

remaining

unexplained

differences

are

correlated

with

the

increased

divergence

of

the

sequences

that

flank

the

binding

sites

and

an

apparent

modulation

of

chromatin

structure

Our

analysis

emphasizes

the

flexibility

of

promoter

structure

and

highlights

the

interplay

between

specific

binding

sites

and

general

chromatin

structure

in

the

control

of

gene

expression

Molecular

Systems

Biology

January

doi

Subject

Categories

functional

genomics

chromatin

and

transcription

Keywords

chromatin

structure

evolution

gene

expression

This

is

an

open-access

article

distributed

under

the

terms

of

the

Creative

Commons

Attribution

Licence

which

permits

distribution

and

reproduction

in

any

medium

provided

the

original

author

and

source

are

credited

Creation

of

derivative

works

is

permitted

but

the

resulting

work

may

be

distributed

only

under

the

same

or

similar

licence

to

this

one

This

licence

does

not

permit

commercial

exploitation

without

specific

permission

Introduction

The

unique

phenotype

of

each

organism

is

defined

by

a

combination

of

its

gene

content

and

the

regulation

of

these

genes

Evolution

of

protein

sequence

and

its

contribution

to

phenotypic

adaptation

has

been

studied

extensively

-LRB-

Pal

et

al

-RRB-

while

most

of

what

we

know

about

regulatory

evolution

comes

from

the

study

of

individual

genes

-LRB-

Wray

-RRB-

Regulatory

evolution

reflects

changes

in

genomic

sequences

that

influence

-LRB-

either

directly

or

indirectly

-RRB-

gene

expression

Among

the

multiple

mechanisms

that

control

gene

expression

the

binding

of

transcription

factors

-LRB-

TFs

-RRB-

to

sequence-specific

binding

sites

within

the

upstream

promoters

is

arguably

the

best-characterized

regulatory

scheme

The

short

lengths

of

TF-binding

sites

and

their

sensitivity

to

even

a

small

number

of

mutations

make

them

ideal

candidates

for

driving

gene

expression

divergence

-LRB-

ED

-RRB-

in

cis

Recent

studies

have

begun

to

compare

the

promoters

of

related

organisms

in

search

for

such

regulatory

differences

These

analyses

are

hindered

by

the

difficulty

to

distinguish

differences

in

functional

-LRB-

e.g.

TF-binding

sites

-RRB-

from

EMBO

and

Nature

Publishing

Group

non-functional

promoter

elements

Nonetheless

leveraging

on

prior

knowledge

of

TF-binding

motifs

several

studies

predicted

the

gain

and

loss

of

thousands

of

TF-binding

sites

both

in

yeast

and

mammalian

species

-LRB-

Donaldson

and

Gottgens

Doniger

and

Fay

-RRB-

Furthermore

two

very

recent

studies

have

used

chromatin

immunoprecipitation

to

directly

identify

differences

in

TF

binding

among

related

species

-LRB-

Borneman

et

al

Odom

et

al

-RRB-

While

these

studies

rely

on

the

premise

that

observed

differences

in

TF-binding

sequences

represent

gene

ED

and

ultimately

phenotypic

evolution

this

was

not

directly

examined

In

fact

various

evidences

have

indicated

that

extensive

promoter

divergence

including

differences

in

TF-binding

sequences

may

evolve

neutrally

with

no

influence

on

gene

expression

First

several

studies

found

that

promoters

from

different

species

which

have

extensively

diverged

still

drive

the

same

reporter

expression

patterns

-LRB-

Ludwig

et

al

Takahashi

et

al

Romano

and

Wray

Ruvinsky

and

Ruvkun

Oda-Ishii

et

al

Fisher

et

al

Wang

et

al

-RRB-

Second

it

was

shown

that

changes

in

TF-binding

sequences

are

poorly

correlated

with

divergence

of

gene

Molecular

Systems

Biology

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

expression

among

yeast

paralogs

-LRB-

Zhang

et

al

-RRB-

Third

Doniger

and

Fay

-LRB-

-RRB-

introduced

mutations

in

binding

sequences

that

were

found

in

Saccharomyces

paradoxus

and

S.

mikatae

to

the

orthologous

promoters

in

S.

cerevisiae

and

examined

their

effect

on

gene

expression

using

reporter

assays

The

expected

effects

on

gene

expression

were

found

only

in

out

of

the

cases

that

were

examined

Finally

a

comprehensive

analysis

of

of

the

human

genome

-LRB-

The

ENCODE

Project

Consortium

-RRB-

has

shown

that

a

large

percentage

of

the

functional

regulatory

elements

are

not

conserved

among

mammals

suggesting

the

neutral

evolution

of

these

sequences

Thus

it

appears

that

promoters

are

highly

flexible

and

are

capable

of

maintaining

stabilized

gene

expression

pattern

through

many

realizations

of

sequences

even

when

binding

motifs

are

concerned

Here

we

examine

the

impact

of

changes

in

TF-binding

sequences

on

the

associated

gene

expression

on

a

genomewide

scale

using

comparative

expression

data

sets

of

related

organisms

-LRB-

Ranz

et

al

Rifkin

et

al

Su

et

al

Khaitovich

et

al

Gilad

et

al

Tirosh

et

al

-RRB-

combined

with

comparative

data

sets

of

predicted

TF-binding

sites

We

find

that

most

predicted

changes

in

TF-binding

sequences

in

both

yeast

and

mammals

have

only

little

effect

on

gene

expression

To

examine

the

connection

between

changes

in

TF-binding

sequences

and

ED

in

a

more

controlled

setting

we

measured

the

transcription

response

of

three

closely

related

yeast

species

to

mating

pheromone

Analysis

of

this

response

allows

us

to

assess

the

relative

contribution

of

specific

cis-regulatory

elements

and

of

general

promoter

structure

to

the

divergence

of

gene

expression

Results

Reported

changes

in

TF-binding

sequences

have

only

little

detectable

impact

on

gene

expression

A

recent

study

has

characterized

thousands

of

matches

to

binding

site

motifs

that

were

conserved

in

the

promoters

of

both

chimpanzee

and

mouse

-LRB-

and

are

thus

likely

to

be

functional

in

both

organisms

-RRB-

but

are

mutated

and

do

not

match

the

binding

site

motif

in

human

-LRB-

Donaldson

and

Gottgens

-RRB-

To

examine

the

impact

of

these

mutations

on

gene

expression

we

assembled

two

data

sets

comparing

gene

expression

between

human

and

either

chimpanzee

or

mouse

The

first

data

set

compares

the

expression

levels

of

human

and

chimpanzee

genes

across

five

tissues

-LRB-

Khaitovich

et

al

-RRB-

and

the

second

data

set

compares

the

expression

patterns

of

human

and

mouse

genes

across

orthologous

tissues

-LRB-

Su

et

al

-RRB-

In

both

cases

we

found

that

the

ED

of

genes

with

diverged

sequence

motifs

in

their

proximal

promoters

-LRB-

kb

-RRB-

was

indistinguishable

from

the

ED

of

genes

with

conserved

motifs

-LRB-

Supplementary

Figure

-RRB-

Analysis

of

mammalian

regulatory

divergence

is

hindered

by

several

limitations

including

the

inherent

complexity

of

mammalian

promoters

the

presence

of

multiple

-LRB-

often

far

away

-RRB-

enhancers

and

the

poor

knowledge

of

TFs

and

their

binding

specificities

To

try

and

circumvent

these

problems

we

turned

to

yeast

whose

promoters

are

significantly

shorter

-LRB-

bp

-RRB-

and

well

defined

Doniger

and

Fay

-LRB-

-RRB-

have

recently

analyzed

the

conservation

of

sequence

motifs

in

Molecular

Systems

Biology

promoters

of

closely

related

yeast

species

and

predicted

the

loss

of

TF-binding

sites

We

examined

the

impact

of

predicted

changes

in

TF-binding

sites

on

ED

using

a

comparative

data

set

that

we

have

recently

reported

where

we

examined

the

genome-wide

expression

programs

of

the

same

species

to

several

environmental

stresses

-LRB-

Tirosh

et

al

-RRB-

Also

here

genes

predicted

to

loose

TF-binding

sites

-LRB-

i.e.

whose

promoters

contained

diverged

sequence

motifs

-RRB-

had

the

same

level

of

ED

as

genes

with

conserved

sequence

motifs

-LRB-

Figure

-RRB-

To

further

verify

these

results

we

have

also

analyzed

the

promoters

of

these

species

and

generated

another

set

of

predicted

changes

in

TF-binding

sites

-LRB-

see

Materials

and

methods

-RRB-

Notably

both

our

analysis

and

that

of

Doniger

and

Fay

-LRB-

-RRB-

predicted

the

loss

of

a

TF-binding

site

only

if

there

were

no

other

matches

to

that

sequence

motif

at

the

same

promoter

thus

effectively

removing

cases

of

binding

site

turnover

-LRB-

Dermitzakis

and

Clark

-RRB-

However

also

for

these

predictions

we

observed

similar

levels

of

ED

-LRB-

Figure

see

Supplementary

Figure

for

results

with

different

parameters

-RRB-

Since

the

expression

data

are

taken

from

stress

conditions

it

could

be

expected

that

only

changes

in

sequence

motifs

for

TFs

that

participate

in

the

stress

response

will

have

an

impact

in

this

data

set

We

thus

separately

analyzed

the

impact

of

changes

in

sequence

motifs

for

each

TF

-LRB-

Figure

-RRB-

Although

changes

in

sequence

motifs

for

stressrelated

TFs

were

in

general

associated

with

higher

ED

than

other

TFs

of

these

TFs

were

significantly

associated

with

high

ED

One

possible

explanation

for

the

lack

of

correlation

between

the

divergence

of

sequence

motifs

and

that

of

gene

expression

is

that

the

reported

sequence

variations

do

not

affect

the

binding

of

the

respective

TFs

to

promoters

To

explore

this

possibility

we

examined

the

binding

of

multiple

TFs

with

conserved

or

diverged

motifs

to

S.

cerevisiae

promoters

-LRB-

Harbison

et

al

-RRB-

-LRB-

Figure

-RRB-

We

found

that

S.

cerevisiae

promoters

with

diverged

motifs

are

bound

by

the

respective

TF

less

often

than

promoters

with

conserved

motifs

but

more

often

than

promoters

without

sequence

motifs

for

that

TF

in

any

of

the

species

-LRB-

Figure

-RRB-

Thus

in

certain

cases

TFs

retain

their

binding

to

promoters

despite

divergence

of

the

respective

sequence

motifs

although

this

may

also

represent

the

differences

between

the

promoter

regions

examined

by

sequence

analysis

and

those

experimentally

tested

However

in

other

cases

promoters

with

diverged

motifs

are

not

bound

by

the

respective

TF

suggesting

that

TF

binding

has

also

diverged

Notably

also

for

these

promoters

the

percentage

of

genes

with

diverged

expression

is

not

higher

than

average

-LRB-

Supplementary

Figure

-RRB-

Thus

despite

the

apparent

loss

of

TF

binding

gene

expression

remained

conserved

perhaps

through

compensation

by

other

regulatory

elements

Since

divergence

of

sequence

motifs

corresponds

only

partially

to

divergence

of

TF

binding

interspecies

differences

in

TF

binding

should

be

experimentally

determined

The

binding

of

four

TFs

-LRB-

and

-RRB-

to

orthologous

gene

pairs

in

human

and

mouse

liver

cells

was

recently

analyzed

by

chromatin

immunoprecipitation

-LRB-

Odom

et

al

-RRB-

and

extensive

differences

were

identified

between

the

binding

of

these

TFs

to

orthologous

genes

To

examine

the

impact

of

these

differences

we

compared

the

expression

levels

of

human

and

mouse

liver

cells

-LRB-

Xing

et

al

EMBO

and

Nature

Publishing

Group

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

d

d

P

P

d

Figure

Expression

divergence

of

yeast

genes

with

diverged

TF

sequence

motifs

-LRB-

A

-RRB-

The

percentage

of

genes

with

conserved

intermediate

or

diverged

expression

among

those

with

conserved

or

diverged

motifs

as

predicted

by

Doniger

and

Fay

-LRB-

-RRB-

and

by

a

similar

analysis

-LRB-

see

Materials

and

methods

and

Supplementary

Figure

-RRB-

The

difference

between

any

pair

of

the

three

sets

is

not

statistically

significant

-LRB-

-RRB-

-LRB-

B

-RRB-

Average

expression

divergence

for

genes

with

conserved

or

diverged

motifs

for

various

TFs

Some

stress-related

TFs

-LRB-

e.g.

-RRB-

have

relatively

high

ED

of

genes

with

diverged

motifs

but

in

of

these

cases

it

is

significantly

higher

than

the

respective

ED

of

genes

with

conserved

motifs

-LRB-

C

-RRB-

Percentage

of

S.

cerevisiae-bound

promoters

at

two

different

binding

P-values

-LRB-

Harbison

et

al

-RRB-

among

promoters

with

different

patterns

of

motif

conservation

and

divergence

The

difference

between

each

pair

of

different

patterns

is

significant

-LRB-

-RRB-

-LRB-

D

-RRB-

Expression

divergence

between

human

and

mouse

liver

cells

of

genes

with

conserved

diverged

or

no

binding

by

four

liver-related

TFs

-RRB-

However

we

found

no

connection

between

differences

in

TF

binding

and

ED

-LRB-

Figure

-RRB-

Genome-wide

analysis

of

the

mating

response

in

three

yeast

species

The

difficulty

to

predict

expression

changes

from

analysis

of

sequence

motifs

could

stem

from

the

coordinated

activity

of

multiple

TFs

that

affect

gene

expression

through

combinatorial

regulation

The

binding

of

multiple

TFs

to

multiple

promoter

elements

may

conceal

the

influence

of

specific

differences

but

at

the

same

time

provide

more

raw

material

for

regulatory

changes

and

is

in

general

correlated

with

an

increased

ED

-LRB-

Tirosh

et

al

Landry

et

al

-RRB-

We

thus

sought

to

analyze

a

simpler

situation

where

gene

expression

is

defined

primarily

by

a

single

TF

The

yeast

mating

response

appears

suitable

This

response

is

relatively

isolated

and

is

induced

by

the

activation

of

the

TF

whose

consensus

binding

motif

as

well

as

target

promoters

is

well

defined

-LRB-

Roberts

et

al

Zeitlinger

et

al

Bardwell

-RRB-

Mating

in

yeast

occurs

when

two

haploid

cells

of

the

opposite

mating

types

-LRB-

a

and

a

-RRB-

fuse

and

form

a

single

diploid

cell

Each

cell

secrets

a

unique

pheromone

-LRB-

a-factor

or

a-factor

respectively

-RRB-

that

is

sensed

by

the

other

cell

triggering

it

to

initiate

the

mating

response

Mating

involves

extensive

changes

in

the

gene

expression

program

-LRB-

Roberts

et

al

-RRB-

with

more

than

upregulated

genes

and

additional

EMBO

and

Nature

Publishing

Group

genes

downregulated

Many

of

the

upregulated

genes

have

specific

roles

in

mating

and

are

regulated

by

while

the

downregulated

ones

are

associated

with

cell-cycle

arrest

The

a-factor

peptide

secreted

by

S.

cerevisiae

has

been

isolated

and

synthesized

in

vitro

Currently

most

studies

of

the

mating

response

are

performed

by

subjecting

haploid

yeast

cells

of

the

a-type

to

this

synthetic

a-factor

-LRB-

e.g.

Roberts

et

al

-RRB-

Since

the

gene

coding

for

the

a-factor

peptide

is

highly

conserved

within

the

sensu

stricto

complex

we

used

the

synthetic

a-factor

from

S.

cerevisiae

to

elicit

the

mating

response

in

three

closely

related

species

S.

cerevisiae

S.

paradoxus

and

S.

mikatae

As

expected

all

species

responded

to

this

a-factor

by

arresting

their

cell

cycle

and

growing

a

visible

shmoo

-LRB-

Supplementary

Figure

-RRB-

We

measured

the

gene

expression

response

to

a-factor

using

microarrays

containing

complete-ORF

probes

for

the

S.

cerevisiae

genes

To

control

for

technical

variations

we

performed

biological

repeats

-LRB-

three

in

S.

cerevisiae

and

S.

paradoxus

and

four

in

S.

mikatae

-RRB-

with

all

experiments

-LRB-

for

all

species

and

repeats

-RRB-

executed

in

parallel

The

sequence

of

S.

paradoxus

and

S.

mikatae

genes

is

highly

similar

to

those

of

S.

cerevisiae

-LRB-

and

on

average

respectively

-RRB-

and

accordingly

produced

significant

and

reproducible

hybridization

Notably

while

absolute

hybridization

intensities

are

affected

by

sequence

mismatches

our

analysis

is

based

solely

on

the

ratios

of

hybridization

intensities

in

samples

taken

with

and

without

pheromone

Indeed

this

cross-species

hybridization

platform

was

validated

in

both

yeast

and

other

organisms

Molecular

Systems

Biology

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

by

us

and

others

-LRB-

Sartor

et

al

Tirosh

et

al

Oshlack

et

al

-RRB-

In

each

of

the

three

species

a-factor

induced

significant

-LRB-

-RRB-

expression

changes

of

more

than

genes

As

expected

about

genes

were

upregulated

by

at

least

twofold

and

these

genes

were

enriched

with

previously

known

mating-related

genes

-LRB-

in

each

species

-RRB-

The

response

of

each

species

was

highly

reproducible

with

a

genome-wide

correlation

of

among

biological

repeats

-LRB-

Figure

-RRB-

The

correlations

between

the

responses

of

the

different

species

were

significantly

lower

-LRB-

-RRB-

although

clearly

far

from

being

random

Thus

the

overall

transcriptional

response

is

conserved

but

also

includes

substantial

species-specific

differences

As

additional

controls

we

compared

our

results

with

those

of

a

previous

study

of

the

mating

response

in

S.

cerevisiae

-LRB-

Roberts

et

al

-RRB-

and

with

expression

measurements

of

S.

cerevisiae

cells

undergoing

natural

mating

As

expected

both

data

sets

had

high

correlations

with

the

response

of

the

three

species

to

a-factor

and

especially

with

the

response

of

S.

cerevisiae

-LRB-

Supplementary

Figure

-RRB-

We

identified

genes

that

are

differentially

expressed

between

at

least

one

pair

of

yeast

species

-LRB-

see

Materials

and

methods

and

Supplementary

Table

-RRB-

Interestingly

these

diverged

genes

had

high

ED

also

in

the

stress-related

comparative

data

-LRB-

Tirosh

et

al

-RRB-

-LRB-

P

-RRB-

and

were

enriched

with

TATA-containing

genes

-LRB-

compared

with

P

-RRB-

This

suggests

that

genes

vary

in

their

tendency

for

ED

such

that

the

same

genes

diverge

in

expression

at

different

processes

Furthermore

this

tendency

may

be

related

to

the

presence

of

TATA

boxes

as

we

and

others

have

previously

suggested

-LRB-

Tirosh

et

al

Landry

et

al

-RRB-

In

contrast

these

diverged

genes

do

not

have

higher

then

average

protein

sequence

divergence

-LRB-

Wall

et

al

-RRB-

-LRB-

P

-RRB-

suggesting

a

decoupling

between

evolution

of

protein

sequence

and

expression

To

better

understand

the

pattern

of

differential

expression

we

classified

the

differentially

expressed

genes

into

classes

and

separately

analyzed

the

genes

within

each

class

-LRB-

Figure

-RRB-

Each

class

corresponds

to

genes

that

are

up

or

downregulated

only

in

a

specific

subset

of

the

three

species

-LRB-

Materials

and

methods

-RRB-

Interestingly

the

largest

class

corresponds

to

genes

with

an

S.

paradoxus-specific

upregulation

-LRB-

Figure

-RRB-

and

the

smallest

class

corresponds

to

genes

with

the

opposite

pattern

-LRB-

Figure

no

upregulation

only

in

S.

paradoxus

-RRB-

We

analyzed

the

enrichment

of

gene

ontology

-LRB-

GO

-RRB-

annotations

and

the

presence

of

mating-related

genes

within

each

class

-LRB-

Figure

-RRB-

The

most

notable

class

was

composed

of

genes

that

were

upregulated

in

S.

cerevisiae

and

S.

paradoxus

but

not

in

S.

mikatae

-LRB-

Figure

-RRB-

This

class

is

comprised

of

genes

and

includes

six

mating-related

genes

-LRB-

and

and

-RRB-

This

class

as

well

as

the

entire

set

of

differentially

expressed

genes

is

also

enriched

with

cell

wall

genes

-LRB-

for

both

gene

sets

-RRB-

which

are

important

for

various

aspects

of

the

mating

response

such

as

shmoo

formation

cell

adhesion

and

fusion

Additional

studies

are

needed

to

elucidate

the

possible

impact

of

these

expression

differences

on

the

mating

behavior

of

these

yeast

species

TF-binding

sequences

versus

ED

in

the

mating

response

The

mating

response

is

orchestrated

by

a

single

TF

whose

binding

site

motif

-LRB-

TGAAACA

-RRB-

is

well

characterized

This

binding

consensus

as

well

as

the

coding

sequence

of

and

specifically

its

binding

domain

are

well

conserved

between

the

species

analyzed

-LRB-

Supplementary

Figure

-RRB-

As

mentioned

above

these

properties

enable

a

more

focused

analysis

of

evolution

of

TF-binding

sequences

that

minimizes

trans-acting

and

combinatorial

effects

We

thus

searched

for

genes

with

a

conserved

sequence

motif

that

has

diverged

in

at

least

one

of

the

three

yeast

species

for

which

expression

was

measured

-LRB-

Materials

and

methods

-RRB-

This

analysis

identified

diverged

sequence

motifs

In

each

species

we

classified

diverged

sequence

motifs

as

a

conserved

motif

if

the

sequence

motif

was

conserved

in

that

species

but

lost

in

another

species

and

lost

motif

if

the

sequence

motif

was

conserved

in

two

other

species

but

lost

in

that

species

Divergence

of

sequence

motifs

was

highly

correlated

with

ED

in

the

mating

response

In

each

of

the

three

species

genes

with

conserved

motif

were

significantly

more

likely

to

be

upregulated

in

response

to

a-factor

than

genes

with

lost

motif

-LRB-

Figure

-RRB-

For

example

more

than

half

of

the

conserved

motifs

in

S.

paradoxus

were

associated

with

upregulated

S.

paradoxus

genes

-LRB-

out

of

-RRB-

while

of

the

lost

motifs

in

S.

paradoxus

were

associated

with

upregulated

S.

paradoxus

genes

Thus

genes

with

an

site

that

is

conserved

in

two

species

but

lost

in

the

third

species

tend

to

respond

to

a-factor

only

in

the

species

in

which

the

site

is

conserved

-LRB-

Figure

-RRB-

For

example

the

promoter

of

-LRB-

flavin

adenine

dinucleotide

synthetase

-RRB-

has

EMBO

and

Nature

Publishing

Group

I

type

Figure

Correlations

between

the

mating

expression

program

in

different

species

We

isolated

a-type

cells

from

S.

cerevisiae

S.

paradoxus

and

S.

mikatae

subjected

them

to

S.

cerevisiae

a-factor

and

measured

their

genome-wide

expression

profiles

using

S.

cerevisiae

arrays

Each

species

was

measured

with

three

or

four

biological

repeats

The

correlations

among

these

genomic

responses

were

calculated

over

genes

with

a

significant

response

in

at

least

one

experiment

Molecular

Systems

Biology

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

A

A

B

S

C

D

E

C

F

S

Figure

Differential

expression

pattern

in

the

mating

response

Differentially

expressed

genes

were

classified

into

patterns

of

up

or

downregulation

in

a

subset

of

the

species

-LRB-

see

Materials

and

methods

-RRB-

Each

subfigure

shows

the

expression

ratios

of

genes

from

two

classes

corresponding

to

up

and

downregulation

in

a

specific

subset

of

species

-LRB-

A

-RRB-

S.

cerevisiae

-LRB-

B

-RRB-

S.

paradoxus

-LRB-

C

-RRB-

S.

mikatae

-LRB-

D

-RRB-

S.

paradoxus

S.

mikatae

-LRB-

E

-RRB-

S.

cerevisiae

S.

mikatae

-LRB-

F

-RRB-

S.

cerevisiae

S.

paradoxus

Cer

par

and

mik

indicate

the

columns

corresponding

to

expression

of

S.

cerevisiae

S.

paradoxus

and

S.

mikatae

respectively

The

corresponding

subset

of

species

number

of

genes

within

each

class

enriched

GO

annotations

and

selected

genes

are

indicated

at

the

top

of

each

subfigure

Red

and

green

correspond

to

up

and

downregulation

respectively

a

perfect

match

to

the

sequence

motif

in

S.

paradoxus

and

S.

mikatae

but

not

in

S.

cerevisiae

Accordingly

was

upregulated

only

in

S.

paradoxus

and

S.

mikatae

A

significant

number

of

exceptions

were

still

observed

however

-LRB-

Figure

-RRB-

For

example

-LRB-

b

subunit

of

the

farnesyltransferase

complex

which

prenylates

the

a-factor

-RRB-

was

upregulated

in

S.

mikatae

despite

a

mutation

in

the

sequence

motif

and

was

not

upregulated

in

S.

cerevisiae

despite

the

conservation

of

its

sequence

motif

In

yet

other

cases

differential

expression

was

found

despite

the

presence

of

conserved

sequence

motifs

-LRB-

e.g.

-RRB-

We

next

asked

how

much

of

the

observed

interspecies

differential

expression

can

be

accounted

for

by

differences

in

sequence

motifs

-LRB-

Figure

-RRB-

Since

controls

the

upregulation

but

not

downregulation

in

response

to

a-factor

we

examined

the

presence

and

divergence

of

sequence

motifs

in

promoters

of

genes

that

are

upregulated

only

in

a

subset

of

the

three

yeast

species

We

found

that

only

of

this

differential

expression

can

be

accounted

for

by

divergence

of

sequences

If

we

restrict

this

analysis

to

genes

with

sequence

motifs

in

at

least

one

species

which

are

thus

more

likely

to

be

directly

regulated

by

divergence

of

sequences

is

consistent

with

approximately

one-third

of

the

expression

differences

While

the

mating

response

is

predominately

activated

by

other

TFs

may

also

impact

the

observed

gene

EMBO

and

Nature

Publishing

Group

expression

We

thus

examined

whether

divergence

of

sequence

motifs

for

other

TFs

is

correlated

with

expression

differences

in

the

mating

response

Divergence

of

sequence

motifs

for

seven

additional

TFs

were

found

to

be

significantly

-LRB-

-RRB-

associated

with

differential

upregulation

-LRB-

and

-RRB-

For

example

out

of

pairs

of

orthologs

which

are

differentially

upregulated

and

also

have

a

diverged

sequence

motif

divergence

of

the

motif

is

correlated

with

the

loss

of

upregulation

in

gene

pairs

-LRB-

-RRB-

consistent

with

the

predicted

cooperative

binding

of

and

-LRB-

Banerjee

and

Zhang

Das

et

al

-RRB-

Conversely

loss

of

sequence

motifs

is

correlated

with

the

gain

of

upregulation

consistent

with

the

role

of

in

shifting

the

binding

of

from

matingrelated

genes

to

pseudohyphal-related

genes

-LRB-

Zeitlinger

et

al

-RRB-

Moreover

in

four

of

these

genes

the

differential

appearance

of

the

motif

in

S.

cerevisiae

and

S.

mikatae

is

also

consistent

with

their

differential

binding

by

in

pseudohyphal

conditions

-LRB-

Borneman

et

al

-RRB-

while

show

the

opposite

pattern

Altogether

divergence

of

sequence

motifs

for

and

these

seven

additional

TFs

could

account

for

of

the

observed

differential

upregulation

and

up

to

of

the

differential

upregulation

of

genes

with

an

sequence

motif

-LRB-

Figure

-RRB-

In

contrast

divergence

of

these

motifs

is

inconsistent

with

differential

upregulation

-LRB-

e.g.

loss

of

motifs

coincides

with

gain

of

upregulation

-RRB-

in

of

all

genes

and

of

genes

Molecular

Systems

Biology

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

upregulated

genes

Conserved

motifs

Lost

motifs

A

Divergence

of

other

motifs

-LRB-

-RRB-

Divergence

of

motifs

-LRB-

-RRB-

S.

cerevisiae

S.

cerevisiae

S.

paradoxus

S.

paradoxus

S.

mikatae

S.

mikatae

B

Expression

ra

tio

-LRB-

log

-RRB-

Gene

Without

motifs

-LRB-

-RRB-

With

motifs

-LRB-

-RRB-

C

Figure

Differential

upregulation

classified

by

the

presence

and

divergence

of

TF-binding

sites

Venn

diagram

showing

all

the

pairwise

species

differences

in

upregulation

classified

by

the

presence

of

sequence

motifs

-LRB-

yellow

no

motif

green

motif

in

at

least

one

of

the

species

-RRB-

the

divergence

of

sequence

motifs

-LRB-

red

expression

differences

are

consistent

with

divergence

of

sequence

motifs

-RRB-

and

the

divergence

of

additional

TFs

which

are

significantly

associated

with

differences

in

the

mating

response

-LRB-

blue

expression

differences

are

consistent

with

divergence

of

TF

sequence

motifs

-RRB-

Numbers

in

parentheses

indicate

the

number

of

genes

in

each

class

Gene

Figure

Mating

response

of

genes

with

conserved

or

diverged

sequence

motifs

-LRB-

A

-RRB-

The

percentage

of

upregulated

genes

in

response

to

a-factor

is

significantly

higher

among

genes

whose

sequence

motif

is

conserved

-LRB-

blue

-RRB-

than

those

whose

sequence

motifs

has

diverged

-LRB-

red

-RRB-

in

each

species

-LRB-

P

for

S.

cerevisiae

S.

paradoxus

and

S.

mikatae

respectively

-RRB-

Only

sequence

motifs

which

are

conserved

in

two

species

and

diverged

in

another

were

considered

and

these

were

defined

as

conserved

and

lost

in

the

respective

species

-LRB-

including

and

genes

lost

in

S.

cerevisiae

S.

paradoxus

and

S.

mikatae

respectively

-RRB-

Upregulation

was

defined

by

ratio

yet

other

thresholds

gave

similar

results

-LRB-

B

-RRB-

Four

examples

of

genes

in

which

divergence

of

sequence

motifs

was

associated

with

reduced

response

to

a-factor

Conserved

and

mutated

sequence

motifs

are

shown

beneath

each

gene

mutated

positions

are

indicated

by

black

and

lowercase

-LRB-

C

-RRB-

Four

examples

of

genes

in

which

the

presence

of

sequence

motifs

is

not

correlated

with

the

response

to

a-factor

Flanking

promoter

sequences

and

their

impact

on

chromatin

structure

Taken

together

changes

in

TF

sequence

motifs

can

account

for

approximately

half

of

the

ED

of

genes

To

understand

the

genetic

basis

of

the

remaining

expression

differences

we

examined

the

divergence

of

promoter

sequences

that

flank

the

sequence

motifs

-LRB-

Figure

-RRB-

Interestingly

we

found

that

these

flanking

sequences

are

less

conserved

in

genes

with

unexplained

differential

upregulation

than

either

in

genes

with

conserved

upregulation

or

in

those

with

differential

upregulation

that

could

be

accounted

by

diverged

binding

sequences

This

difference

did

not

extend

to

the

entire

promoter

and

was

only

significant

at

the

region

surrounding

the

sequences

-LRB-

to

-RRB-

Moreover

this

region

was

less

conserved

than

the

remaining

promoter

among

genes

with

unexplained

differential

Molecular

Systems

Biology

upregulation

but

not

among

other

genes

-LRB-

Figure

-RRB-

These

results

suggest

that

sequences

flanking

sites

are

important

for

binding

and

that

divergence

of

these

sequences

is

responsible

for

some

of

the

observed

differential

upregulation

Flanking

sequences

could

influence

gene

expression

if

they

contain

unrecognized

sites

that

are

bound

by

either

or

by

related

TFs

However

we

find

no

enrichment

of

matches

-LRB-

or

weak

matches

-RRB-

to

TF

sequence

motifs

at

these

regions

Alternatively

these

sequences

could

influence

gene

expression

through

other

mechanisms

most

notably

by

modulating

chromatin

structure

To

examine

this

possibility

we

applied

a

computational

model

to

predict

the

nucleosome

occupancy

in

promoters

of

the

different

yeast

species

-LRB-

Segal

et

al

-RRB-

We

found

that

divergence

of

the

sequences

flanking

sequence

motifs

indeed

affects

the

predicted

nucleosome

occupancy

at

the

location

of

the

sequence

motifs

and

that

this

effect

is

larger

among

genes

with

unexplained

differential

upregulation

compared

with

other

genes

-LRB-

Figure

-RRB-

For

most

genes

the

predicted

differences

in

nucleosome

occupancy

are

quite

small

and

do

not

appear

to

influence

gene

expression

However

among

the

three

genes

with

the

largest

predicted

changes

in

nucleosome

occupancy

-LRB-

differences

larger

than

at

the

conserved

sequence

motifs

-RRB-

these

changes

were

exactly

consistent

with

the

observed

differential

upregulation

-LRB-

Figure

E

-RRB-

For

example

the

mating-related

gene

is

upregulated

in

S.

cerevisiae

and

S.

paradoxus

but

not

in

S.

mikatae

and

the

predicted

nucleosome

occupancy

of

the

conserved

site

at

its

promoter

is

significantly

higher

in

S.

mikatae

These

results

suggest

that

divergence

of

the

sequences

flanking

conserved

TF-binding

sites

have

influenced

nucleosome

positioning

and

therefore

also

the

accessibility

of

promoters

to

TFs

To

further

examine

this

possibility

we

analyzed

the

predicted

nucleosome

occupancy

among

genes

with

interspecies

differences

in

the

binding

of

and

at

EMBO

and

Nature

Publishing

Group

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

C

D

E

Figure

Divergence

of

flanking

promoter

sequences

and

chromatin

structure

-LRB-

A

-RRB-

The

average

conservation

of

promoter

sequences

flanking

conserved

sequence

motifs

is

shown

as

a

function

of

the

distance

from

the

sequence

motif

-LRB-

x-axis

-RRB-

and

the

conservation

of

the

expression

response

-LRB-

color

-RRB-

The

difference

in

conservation

between

genes

with

unexplained

differential

expression

and

the

other

two

gene

sets

is

significant

only

at

the

to

interval

-LRB-

-RRB-

-LRB-

B

-RRB-

Maximal

interspecies

differences

in

the

predicted

nucleosome

occupancy

at

the

location

of

conserved

sites

-LRB-

C

-RRB-

Predicted

nucleosome

occupancy

at

the

regions

of

conserved

sites

for

the

three

genes

with

the

largest

differences

in

nucleosome

occupancy

among

those

with

unexplained

differential

upregulation

-LRB-

interspecies

differences

larger

than

-RRB-

The

three

species

are

indicated

by

different

colors

-LRB-

blue

purple

and

orange

correspond

to

cerevisiae

paradoxus

and

mikatae

respectively

-RRB-

-LRB-

D

-RRB-

Expression

response

-LRB-

log

ratio

-RRB-

of

the

three

genes

shown

in

-LRB-

C

-RRB-

-LRB-

E

-RRB-

Predicted

configuration

of

binding

-LRB-

circles

-RRB-

and

nucleosomes

-LRB-

ovals

-RRB-

at

the

sites

of

the

three

genes

pseudohyphal

conditions

-LRB-

Borneman

et

al

-RRB-

Similarly

we

found

that

the

largest

predicted

changes

in

nucleosome

occupancy

are

consistent

with

differences

in

TF

binding

-LRB-

Supplementary

Figure

-RRB-

predicted

changes

in

nucleosome

occupancy

could

account

for

six

out

of

seven

differences

in

TF

binding

among

the

genes

with

the

largest

-LRB-

-RRB-

variations

-LRB-

P

-RRB-

Discussion

Our

goal

in

this

study

was

to

examine

systematically

the

impact

of

changes

in

cis-regulatory

elements

on

the

divergence

of

gene

expression

To

this

end

we

first

analyzed

existing

data

comparing

the

gene

expression

profiles

of

related

species

for

which

promoter

analyses

predicted

the

gain

and

loss

of

TF-binding

sites

Both

in

mammalian

and

in

yeast

data

sets

we

observed

only

a

poor

correlation

between

the

divergence

of

sequence

motifs

and

gene

expression

Furthermore

analysis

of

experimentally

mapped

differences

in

human

and

mouse

TF

binding

led

to

similar

conclusions

Although

surprising

these

results

are

consistent

with

various

previous

studies

as

described

above

Several

reasons

may

explain

the

stabilized

expression

we

and

others

have

observed

despite

the

divergence

of

TF

sequence

motifs

First

the

identification

of

diverged

motifs

may

not

be

optimal

and

may

not

correspond

to

divergence

in

TF

binding

We

tried

to

overcome

this

difficulty

by

using

different

data

sets

and

restricting

our

analysis

to

the

most

significant

cases

of

EMBO

and

Nature

Publishing

Group

sequence

divergence

Second

it

might

be

that

divergence

of

sequence

motifs

does

impact

gene

expression

but

only

under

conditions

which

do

not

appear

in

our

gene

expression

data

sets

However

also

for

the

stress-related

TFs

in

yeast

divergence

of

sequence

motifs

could

not

predict

divergence

of

gene

expression

during

environmental

stress

Third

although

we

tried

to

control

for

binding

sites

turnover

a

compensatory

motif

may

have

appeared

in

more

distant

regions

not

considered

in

our

analysis

Again

this

is

an

important

limitation

concerning

the

mammalian

system

where

transcription

is

controlled

by

multiple

enhancers

but

is

less

of

a

problem

in

the

yeast

system

where

the

relevant

controlling

region

is

believed

to

be

rather

limited

Finally

gene

expression

might

be

compensated

through

other

mechanisms

such

as

chromatin

structure

or

interactions

among

TFs

For

example

loss

of

binding

sites

may

be

buffered

by

the

presence

of

other

binding

sites

for

additional

factors

that

are

involved

in

the

same

process

or

that

bind

cooperatively

with

the

factor

whose

binding

site

has

diverged

Indeed

both

the

mammalian

tissue-specific

expression

and

the

response

of

yeast

to

stressful

conditions

involve

a

coordinated

function

of

multiple

TFs

Thus

the

observed

variability

in

TF-binding

sequences

among

related

species

may

reflect

the

complexity

of

the

underlying

regulatory

network

and

the

possibility

to

encode

the

same

expression

pattern

with

many

different

promoter

designs

perhaps

even

more

than

it

reflects

the

actual

divergence

of

gene

expression

To

overcome

the

above

limitations

we

extended

the

analysis

to

a

more

controlled

setting

and

generated

a

new

comparative

Molecular

Systems

Biology

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

data

set

describing

the

transcriptional

response

of

three

closely

related

yeast

species

to

mating

pheromone

This

response

is

predominantly

orchestrated

by

a

single

TF

whose

binding

specificity

is

well

characterized

While

the

response

was

largely

conserved

the

expression

of

genes

significantly

differed

between

the

species

In

this

case

divergence

of

sequence

motifs

was

correlated

with

the

changes

in

gene

expression

and

together

with

other

TF

sequence

motifs

could

account

for

approximately

one-third

of

the

observed

differential

upregulation

and

up

to

half

of

that

of

genes

Importantly

predictions

based

on

sequence

analysis

are

only

partially

consistent

with

the

actual

binding

of

TFs

In

fact

a

number

of

studies

over

the

past

few

years

indicate

that

binding

does

occur

at

highly

diverged

and

non-consensus

sites

reflecting

additional

regulatory

aspects

which

are

still

poorly

understood

For

example

approximately

half

of

the

genes

that

are

bound

by

in

S.

cerevisiae

contain

an

exact

match

to

the

motif

and

only

one-third

of

the

genes

with

conserved

matches

to

the

motif

are

bound

by

in

S.

cerevisiae

-LRB-

Harbison

et

al

-RRB-

Accordingly

the

impact

of

binding

site

divergence

could

be

actually

larger

than

that

inferred

from

our

analysis

of

binding

site

sequences

alone

Chromatin

structure

has

a

central

role

in

controlling

the

accessibility

of

promoters

to

TFs

and

the

mechanisms

by

which

it

is

regulated

are

widely

studied

-LRB-

Li

et

al

-RRB-

The

influence

of

DNA

sequence

on

nucleosome

positions

has

been

extensively

analyzed

and

several

models

have

been

developed

to

predict

nucleosome

positioning

based

on

DNA

sequences

alone

-LRB-

Ioshikhes

et

al

Segal

et

al

-RRB-

We

applied

the

model

developed

by

Segal

et

al

to

examine

the

effect

of

sequence

divergence

in

regions

surrounding

sites

and

found

that

sequence

divergence

may

indeed

affect

nucleosome

positions

and

that

at

least

in

certain

cases

this

could

account

for

the

observed

differential

expression

Similarly

we

found

that

some

of

the

interspecies

differences

in

the

binding

of

and

at

pseudohyphal

conditions

are

correlated

with

changes

in

nucleosome

occupancy

Thus

the

location

of

nucleosomes

can

evolve

among

closely

related

species

and

influence

gene

ED

Notably

our

analysis

may

capture

only

a

small

proportion

of

the

actual

influence

of

chromatin

structure

as

it

is

based

on

computational

predictions

of

the

intrinsic

preference

of

nucleosomes

for

certain

DNA

sequences

but

ignores

additional

regulatory

mechanisms

and

particularly

the

dynamic

nature

of

chromatin

structure

It

would

be

interesting

although

significantly

more

complicated

to

directly

measure

the

location

of

nucleosomes

among

multiple

species

at

similar

conditions

such

as

during

mating

and

examine

their

influence

on

expression

differences

Our

results

provide

a

detailed

account

of

the

impact

of

promoter

sequence

variations

on

gene

expression

within

the

mating

system

Such

a

detailed

analysis

is

enabled

by

the

relative

simplicity

of

this

system

and

would

be

much

more

difficult

in

typical

regulatory

systems

such

as

the

yeast

stress

response

or

the

mammalian

liver

Similar

analysis

of

additional

regulatory

systems

may

indicate

the

generality

of

the

results

shown

here

and

begin

to

uncover

the

relative

importance

of

different

mechanisms

in

generating

interspecies

differences

in

gene

expression

Molecular

Systems

Biology

Finally

we

note

that

while

our

study

focused

on

the

genetic

basis

of

gene

expression

evolution

a

complementary

challenge

is

to

describe

the

contribution

of

such

changes

to

phenotypic

diversity

In

this

aspect

the

data

set

generated

here

might

be

beneficial

as

it

provides

insights

into

the

modulation

of

the

mating

response

First

we

have

found

that

several

mating-related

genes

are

not

upregulated

in

S.

mikatae

as

in

S.

cerevisiae

and

S.

paradoxus

-LRB-

Figure

-RRB-

Second

we

identified

a

large

class

of

genes

that

were

only

upregulated

in

S.

paradoxus

-LRB-

Figure

-RRB-

Third

studies

of

protein

sequence

evolution

have

shown

that

proteins

involved

in

mating

and

fertility

are

among

the

most

rapidly

evolving

genes

-LRB-

Swanson

and

Vacquier

-RRB-

In

the

future

as

more

data

accumulate

it

would

be

interesting

to

examine

our

data

set

from

this

respect

and

to

see

whether

the

same

is

also

true

for

the

evolution

of

gene

expression

and

to

examine

the

possible

contribution

to

phenotypic

adaptation

and

speciation

Materials

and

methods

Prediction

of

changes

in

human

TF-binding

sequences

We

downloaded

the

mammalian

promoters

multiple

alignments

from

the

UCSC

genome

browser

We

focused

on

proximal

promoters

-LRB-

kb

-RRB-

of

human

chimpanzee

mouse

and

rat

and

searched

them

for

the

presence

of

exact

matches

to

the

mammalian

promoter

motifs

defined

by

Xie

et

al

-LRB-

-RRB-

This

initial

search

was

performed

after

excluding

the

alignment

gaps

We

restricted

this

analysis

to

the

promoter

motifs

with

the

highest

confidence

those

which

matched

the

binding

sites

of

known

TFs

and

the

promoter

motifs

with

the

highest

conservation

significance

We

searched

for

promoters

that

contain

an

exact

match

to

a

specific

motif

in

several

species

but

not

in

another

Because

the

human

sequences

are

more

accurate

than

those

of

the

other

species

and

because

our

expression

data

sets

compare

human

with

either

chimpanzee

or

mouse

we

focused

only

on

matches

that

are

conserved

in

chimpanzee

and

mouse

-LRB-

or

rat

-RRB-

but

mutated

in

human

Thus

we

demanded

that

the

chimpanzee

and

mouse

promoters

contain

exact

matches

to

the

motif

and

that

these

matches

are

aligned

-LRB-

at

the

same

position

in

the

multiple

alignments

-RRB-

The

entire

human

proximal

promoter

was

required

to

have

no

exact

matches

The

number

of

functional

mutations

was

estimated

by

the

number

of

differences

from

the

motif

in

the

sequence

which

was

aligned

to

the

matches

in

the

other

species

This

analysis

resulted

in

prediction

of

human

promoters

with

at

least

two

mutations

in

a

TF-binding

site

which

is

conserved

in

chimpanzee

and

mouse

-LRB-

or

rat

-RRB-

Prediction

of

changes

in

yeast

TF-binding

sequences

Promoters

-LRB-

bp

-RRB-

of

S.

cerevisiae

S.

paradoxus

S.

mikatae

S.

kudriavzevii

and

S.

bayanus

were

downloaded

from

SGD

and

aligned

by

clustelw

We

searched

these

promoters

for

the

presence

of

TF

sequence

motifs

as

defined

by

MacIsaac

et

al

-LRB-

-RRB-

For

each

TF

we

calculated

the

position

weight

matrix

scores

across

all

promoters

and

defined

binding

sites

as

matches

with

scores

greater

than

a

predefined

threshold

-LRB-

-RRB-

Diverged

promoters

were

defined

as

those

with

a

conserved

and

aligned

match

exceeding

in

at

least

three

of

the

five

species

but

no

match

in

another

species

that

exceeds

a

second

threshold

-LRB-

-RRB-

Thus

promoters

with

multiple

binding

sequences

for

the

same

TF

would

be

recognized

as

diverged

only

if

all

these

binding

sequences

have

diverged

in

one

of

the

species

The

thresholds

-LRB-

and

-RRB-

were

chosen

based

on

their

specificity

in

predicting

the

binding

of

TFs

-LRB-

Harbison

et

al

-RRB-

the

specificity

of

a

given

threshold

is

the

fraction

of

bound

promoters

among

those

with

a

conserved

match

exceeding

that

threshold

We

chose

as

the

minimal

threshold

with

EMBO

and

Nature

Publishing

Group

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

specificity

greater

than

and

as

the

maximal

threshold

with

specificity

lower

than

Other

thresholds

and

parameters

were

also

examined

and

led

to

similar

conclusions

-LRB-

see

Supplementary

Figure

-RRB-

Raw

and

normalized

expression

data

are

available

at

the

GEO

-LRB-

Gene

Expression

Omnibus

-RRB-

database

with

the

series

accession

Expression

divergence

Yeast

ED

was

calculated

from

the

response

of

four

yeast

species

to

various

environmental

stresses

-LRB-

Tirosh

et

al

-RRB-

Tissue-specific

ED

between

human

and

chimpanzee

was

taken

from

Khaitovich

et

al

-LRB-

-RRB-

centered

and

normalized

by

its

s.d.

Expression

patterns

of

human

and

mouse

across

orthologous

tissues

were

taken

from

Su

et

al

-LRB-

-RRB-

ED

was

calculated

as

in

Liao

and

Zhang

-LRB-

-RRB-

centered

and

normalized

by

its

s.d.

ED

between

human

and

mouse

liver

cells

was

calculated

as

the

of

the

absolute

difference

in

log-transformed

expression

levels

as

measured

with

exon

arrays

-LRB-

Xing

et

al

-RRB-

Diverged

genes

were

defined

in

each

data

set

as

those

with

ED

higher

than

mean

s.d.

Differential

expression

in

the

mating

response

To

identify

genes

that

are

differentially

expressed

among

the

three

species

we

performed

a

two-sampled

t-test

for

each

gene

and

for

each

pairwise

species

comparison

A

false

discovery

rate

of

was

used

to

generate

a

list

of

differentially

expressed

genes

We

noted

that

many

of

the

genes

identified

by

the

t-test

as

differentially

expressed

are

highly

regulated

and

display

the

same

general

trend

in

all

species

-LRB-

up

or

downregulated

-RRB-

These

differences

may

arise

from

technical

artifacts

such

as

inflated

log

ratios

due

to

low

signals

and

different

sensitivity

of

the

different

arrays

Furthermore

these

differences

are

less

biologically

meaningful

than

differences

between

up/downregulation

in

one

species

and

no

regulation

-LRB-

or

opposite

regulation

-RRB-

in

another

species

We

thus

employed

another

test

for

differential

expression

which

takes

into

account

not

only

the

difference

in

log

ratios

but

also

the

magnitude

of

overall

up

or

downregulation

We

used

the

formula

from

Tirosh

et

al

-LRB-

-RRB-

to

estimate

the

ED

of

each

gene

which

can

be

rewritten

as

EDi

j

g

jxi

g

xj

g

j

jxi

g

xj

g

j

Analysis

of

mating

and

response

to

a-factor

in

three

yeast

species

Haploids

isolation

S.

paradoxus

-LRB-

hoDHKAN

-RRB-

and

S.

mikatae

-LRB-

hoDHKAN

-RRB-

cells

were

grown

in

liquid

YPD

to

saturation

then

transferred

to

sporulation

plates

-LRB-

K-acetate

yeast

extract

glucose

and

agar

-RRB-

and

incubated

at

for

days

Haploid

cells

were

isolated

by

random

spore

analysis

Asci

were

collected

from

the

sporulation

plates

resuspended

in

buffer

A

-LRB-

mM

DTT

mM

pH

-RRB-

and

incubated

for

h

at

with

gentle

agitation

Next

the

asci

were

resuspended

in

buffer

B

-LRB-

M

Sorbito

mM

K-phosphate

buffer

pH

-RRB-

treated

with

yeast

lytic

enzyme

-LRB-

ICN

-RRB-

-LRB-

cells

-RRB-

for

h

at

and

transferred

to

Triton

Asci

were

disrupted

by

sonication

-LRB-

three

rounds

of

s

-RRB-

and

then

spread

on

YPD

plates

Haploids

mating

type

was

determined

using

PCR

-LRB-

Huxley

et

al

-RRB-

r

r

a-Factor

treatment

S.

cerevisiae

S.

paradoxus

and

S.

mikatea

a-type

cells

were

grown

to

log

phase

in

YPD

at

Cells

-LRB-

-RRB-

were

treated

with

mM

a-factor

for

min

Samples

were

collected

at

the

indicated

time

points

Cell

counting

was

performed

in

a

standard

hemocytometer

Microarray

analysis

a-Factor

experiments

S.

cerevisiae

S.

paradoxus

and

S.

mikatae

a-type

cells

-LRB-

of

each

-RRB-

were

grown

on

YPD

at

to

early

log

phase

Cells

were

treated

with

mM

a-factor

for

min

As

reference

we

used

the

same

cells

before

addition

of

a-factor

Three

to

four

biological

repeats

-LRB-

different

colonies

-RRB-

were

processed

in

parallel

for

each

species

Natural

mating

experiments

S.

cerevisiae

a

and

a-type

cells

-LRB-

of

each

-RRB-

were

mixed

and

applied

on

to

filter

The

filters

were

placed

on

YPD

agar

plates

at

for

min

and

then

snap-frozen

with

liquid

nitrogen

For

reference

S.

cerevisiae

a

and

a-type

cells

were

placed

on

separate

filters

and

incubated

on

YPD

agar

plates

at

for

min

The

cells

were

mixed

only

prior

to

mRNA

extraction

Sample

labeling

hybridization

and

scanning

cDNA

of

experiment

and

reference

samples

was

synthesized

from

total

RNA

using

M-MLV

Reverse

Transcriptase

RNase

H

Minus

-LRB-

Promega

-RRB-

and

labeled

with

and

respectively

by

the

indirect

amino-allyl

method

Hybridization

and

scanning

were

carried

out

as

described

in

Tirosh

et

al

-LRB-

-RRB-

Expression

data

were

and

normalized

by

intensity-dependent

correction

-LRB-

subtracting

a

lowess

regression

-RRB-

and

by

position-dependent

correction

-LRB-

subtracting

the

median

of

each

subarray

from

all

the

genes

in

it

-RRB-

To

account

for

differences

in

the

overall

signal

in

different

arrays

we

calculated

the

linear

regression

between

the

first

S.

cerevisiae

array

and

each

subsequent

array

and

divided

the

expression

ratios

of

that

array

by

the

regression

coefficient

where

EDi

j

-LRB-

g

-RRB-

is

the

expression

divergence

of

gene

g

between

species

i

and

j

and

xi

-LRB-

g

-RRB-

is

the

normalized

expression

ratio

of

gene

g

in

species

i

The

genes

with

the

highest

ED

were

considered

to

be

differentially

expressed

These

two

tests

agreed

on

the

differential

expression

of

genes

which

we

further

analyze

We

classified

the

differentially

expressed

genes

into

patterns

of

differential

expression

Each

pattern

corresponds

to

higher

expression

in

a

subset

of

the

species

compared

with

the

remaining

species

We

first

defined

characteristic

expression

vectors

for

six

patterns

with

values

of

in

a

subset

of

the

species

and

in

the

remaining

species

and

classified

genes

according

to

the

vector

with

which

their

expression

pattern

had

the

highest

correlation

Next

we

divided

each

class

into

two

subclasses

The

first

subclass

corresponds

to

genes

that

can

be

estimated

as

upregulated

in

one

subset

of

the

species

and

not

regulated

in

the

other

species

and

the

second

corresponds

to

genes

that

are

better

estimated

as

downregulated

in

one

subset

of

the

species

and

not

regulated

in

the

other

species

This

was

done

by

comparing

the

absolute

values

of

the

highest

and

lowest

response

of

those

genes

if

the

maximum

magnitude

of

upregulation

in

any

of

the

species

was

higher

than

the

maximum

magnitude

of

downregulation

in

any

of

the

species

then

we

classified

the

gene

as

upregulated

and

in

the

opposite

case

as

downregulated

Each

class

-LRB-

and

subclass

-RRB-

was

analyzed

by

searching

for

enrichment

of

GO

annotations

and

presence

of

matingannotated

genes

Impact

of

changes

in

TF

sequence

motifs

on

mating

ED

For

each

pair

of

orthologs

that

are

differentially

upregulated

we

examined

whether

there

are

non-conserved

matches

to

TF

sequence

motifs

-LRB-

MacIsaac

et

al

-RRB-

Divergence

in

sequence

motifs

for

and

five

other

TFs

were

significantly

-LRB-

-RRB-

associated

with

loss

of

upregulation

and

divergence

in

sequence

motifs

for

two

other

TFs

were

significantly

associated

with

gain

of

upregulation

Thus

the

differential

upregulation

of

each

pair

of

orthologs

with

diverged

matches

to

any

of

these

eight

motifs

in

which

the

difference

in

expression

was

consistent

with

the

difference

in

the

sequence

motif

-LRB-

e.g.

the

gene

with

the

site

is

upregulated

and

the

gene

without

this

binding

site

is

not

upregulated

-RRB-

was

considered

to

be

accounted

by

changes

in

TF

sequence

motifs

Nucleosome

occupancy

Nucleosome

occupancy

was

estimated

across

the

promoters

of

the

three

yeast

species

using

the

working

version

of

the

yeast

model

from

Molecular

Systems

Biology

EMBO

and

Nature

Publishing

Group

Promoter

divergence

and

gene

expression

evolution

I

Tirosh

et

al

the

Segal

lab

-LRB-

index.html

-RRB-

We

examined

the

differences

in

predicted

nucleosome

occupancy

at

the

positions

of

sites

Few

promoters

had

large

differences

-LRB-

or

higher

-RRB-

and

the

predicted

changes

in

nucleosome

occupancy

among

these

promoters

were

correlated

with

differential

upregulation

Supplementary

information

Supplementary

information

is

available

at

the

Molecular

Systems

Biology

website

-LRB-

-RRB-

Acknowledgements

This

study

was

supported

by

the

Helen

and

Martin

Kimmel

Award

for

Innovative

Investigation

and

grants

from

the

Pasteur

Weizmann

joint

research

program

the

Kahn

fund

for

Systems

Biology

at

the

Weizmann

Institute

of

Science

the

Tauber

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