Genetic

Dissection

of

Transcriptional

Regulation

in

Budding

Yeast

Rachel

B.

Brem

et

al.

Science

-LRB-

-RRB-

DOI

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served

similarity

in

fitness

effects

of

interacting

proteins

was

sufficient

to

explain

the

similarity

in

their

rates

of

evolution

Path

analysis

based

on

the

causal

model

shown

in

Fig.

indicated

that

the

correlation

between

the

fitness

effects

of

interacting

proteins

contributes

only

slightly

to

the

correlation

between

their

evolutionary

rates

Thus

similarity

in

fitness

effects

is

not

sufficient

to

explain

the

observed

similarity

in

the

evolutionary

rates

of

interacting

proteins

We

also

considered

two

other

alternatives

to

the

coevolutionary

hypothesis

First

interacting

proteins

might

evolve

at

similar

rates

simply

because

they

have

similar

numbers

of

interactors

and

as

shown

in

Fig.

the

number

of

interactors

influences

the

rate

of

evolution

However

we

found

that

proteins

that

interact

do

not

have

similar

numbers

of

P

-RRB-

A

second

actors

-LRB-

possibility

is

that

interacting

proteins

evolve

at

similar

rates

because

they

exhibit

structural

homology

and

therefore

have

similar

distributions

of

constrained

sites

The

most

likely

origin

of

structural

homology

between

interacting

proteins

is

duplication

of

the

gene

that

encodes

a

homodimeric

protein

followed

by

evolution

of

one

copy

of

the

gene

This

process

would

result

in

homology

not

only

between

the

structures

but

also

between

the

sequences

of

interacting

proteins

Hence

we

have

ensured

that

of

the

interactions

in

our

data

set

occur

between

proteins

that

exhibit

detectable

sequence

similarity

Thus

to

account

for

the

similarity

in

evolutionary

rates

that

we

observe

structural

similarity

would

have

to

be

independent

of

sequence

which

would

be

difficult

to

explain

evolutionarily

In

sum

having

considered

a

number

of

alternative

hypotheses

we

conclude

that

the

coevolution

of

interacting

proteins

may

be

largely

responsible

for

the

observed

similarity

in

their

rates

of

evolution

Beyond

describing

the

relation

between

a

protein

's

interactions

and

its

rate

of

evolution

the

correlations

presented

here

could

find

application

in

the

rapid

assessment

of

functional

genomic

data

Much

as

gene

expression

levels

have

recently

been

used

to

assess

protein-protein

interaction

data

sets

-LRB-

-RRB-

the

correlation

between

protein

interaction

and

evolutionary

rate

may

allow

one

to

use

simple

genomic

sequence

comparisons

to

statistically

assess

the

quality

of

large

interaction

data

sets

More

generally

correlations

between

protein

interaction

fitness

effect

and

evolutionary

rate

may

provide

a

means

by

which

multiple

bioinformatic

data

sets

can

be

quickly

cross-referenced

to

assess

the

reliability

of

any

single

method

or

data

set

A.

C.

Wilson

S.

S.

Carlson

T.

J.

White

Annu

Rev.

Biochem

-LRB-

-RRB-

E.

A.

Winzeler

et

al.

Science

-LRB-

-RRB-

S.

A.

Chervitz

et

al.

Science

-LRB-

-RRB-

A.

E.

Hirsh

H.

B.

Fraser

Nature

-LRB-

-RRB-

E.

Zuckerkandl

J.

Mol

Evol

-LRB-

-RRB-

R.

E.

Dickerson

J.

Mol

Evol

-LRB-

-RRB-

B.

Schwikowski

P.

Uetz

S.

Fields

Nature

Biotechnol

-LRB-

-RRB-

P.

Uetz

et

al.

Nature

-LRB-

-RRB-

T.

Ito

et

al.

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

Our

data

set

of

yeast

protein-protein

interactions

was

compiled

from

-LRB-

-RRB-

the

core

data

of

-LRB-

-RRB-

and

interactions

identified

by

the

two-hybrid

array

method

-LSB-

described

in

-LRB-

-RRB-

-RSB-

listed

at

Putative

orthologs

were

defined

as

reciprocal

best

hits

-LRB-

-RRB-

with

P

and

protein

sequence

alignment

in

reciprocal

searches

using

Washington

University

Basic

Local

Alignment

Search

Tool

for

Proteins

-LRB-

WU-BLASTP

-RRB-

-LRB-

-RRB-

Thus

if

a

query

of

all

nematode

open

reading

frames

-LRB-

ORFs

-RRB-

with

yeast

ORF

i

yielded

the

set

of

hits

-LCB-

W

-RCB-

with

P

and

alignment

and

a

query

of

all

yeast

ORFs

with

nematode

ORF

j

yielded

the

set

of

hits

-LCB-

Y

-RCB-

with

P

and

alignment

then

the

pair

-LRB-

ORF

i

ORF

j

-RRB-

was

considered

a

putative

ortholog

only

if

i

was

the

member

of

-LCB-

Y

-RCB-

with

the

lowest

P

value

and

j

was

the

member

of

-LCB-

W

-RCB-

with

the

lowest

P

value

To

estimate

the

number

of

substitutions

per

amino

acid

site

we

numerically

solved

the

equation

q

-LSB-

ln

-LRB-

-RRB-

-RSB-

where

is

the

proportion

of

identical

sites

between

aligned

sequences

This

method

is

described

in

-LRB-

-RRB-

and

tested

further

in

-LRB-

-RRB-

Unless

otherwise

specified

the

correlation

coefficient

rX

Y

represents

Spearman

's

rank

correlation

between

variables

X

and

Y.

Y.

Ho

et

al.

Nature

-LRB-

-RRB-

A.

C.

Gavin

et

al.

Nature

-LRB-

-RRB-

Supplementary

fig.

is

available

on

Science

Online

at

V.

Smith

K.

N.

Chou

D.

Lashkari

D.

Botstein

P.

O.

Brown

Science

-LRB-

-RRB-

H.

Jeong

S.

P.

Mason

A.

L.

Barabasi

Z.

N.

Oltvai

Nature

-LRB-

-RRB-

The

rIF

and

rFK

correlations

were

also

significant

when

genetic

footprint

data

-LRB-

-RRB-

were

used

instead

of

parallel

analysis

data

When

essential

proteins

are

excluded

from

the

analysis

which

is

expected

to

strengthen

the

correlation

between

fitness

P

fect

and

evolutionary

rate

-LRB-

-RRB-

rFK

for

nonessential

proteins

in

parallel

analysis

D.

Kaplan

in

Advanced

Quantitative

Techniques

in

the

Social

Sciences

J.

de

Leeuw

R.

Berk

Eds

-LRB-

Sage

Publications

Thousand

Oaks

CA

-RRB-

pp.

J.

D.

Gibbons

in

Sage

University

Papers

M.

S.

LewisBeck

Ed

-LRB-

Sage

Publications

Newbury

Park

CA

-RRB-

pp.

Examples

of

interprotein

coevolution

are

described

in

-LRB-

-RRB-

A.

Grigoriev

Nucleic

Acids

Res

-LRB-

-RRB-

M.

C.

Rivera

R.

Jain

J.

E.

Moore

J.

A.

Lake

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

N.

V.

Grishin

J.

Mol

Evol

-LRB-

-RRB-

D.

Feng

R.

Doolittle

J.

Mol

Evol

-LRB-

-RRB-

V.

Smith

D.

Botstein

P.

O.

Brown

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

P.

D.

Rawson

D.

A.

Brazeau

R.

S.

Burton

Gene

-LRB-

-RRB-

K.

K.

Koretke

A.

N.

Lupas

P.

V.

Warren

M.

Rosenberg

J.

R.

Brown

Mol

Biol

Evol

-LRB-

-RRB-

B.

Dunn

provided

genetic

footprinting

data

P.

Uetz

assisted

with

interaction

data

J.

Davis

M.

Eisen

B.

Kerr

D.

Petrov

and

C.

Winter

provided

helpful

discussion

and

comments

on

the

manuscript

December

accepted

March

Genetic

Dissection

of

Transcriptional

Regulation

in

Budding

Yeast

Rachel

B.

Brem

Gael

Yvert

Rebecca

Clinton

Leonid

Kruglyak

To

begin

to

understand

the

genetic

architecture

of

natural

variation

in

gene

expression

we

carried

out

genetic

linkage

analysis

of

genomewide

expression

patterns

in

a

cross

between

a

laboratory

strain

and

a

wild

strain

of

Saccharomyces

cerevisiae

Over

genes

were

differentially

expressed

between

the

parent

strains

Expression

levels

of

genes

were

linked

to

one

or

more

different

loci

with

most

expression

levels

showing

complex

inheritance

patterns

The

loci

detected

by

linkage

fell

largely

into

two

categories

cis-acting

modulators

of

single

genes

and

trans-acting

modulators

of

many

genes

We

found

eight

such

trans-acting

loci

each

affecting

the

expression

of

a

group

of

to

genes

of

related

function

Genetic

linkage

analysis

has

traditionally

focused

on

mapping

loci

that

affect

one

or

a

small

number

of

organism-level

phenotypes

DNA

microarray

technology

-LRB-

-RRB-

makes

it

possible

to

apply

such

analysis

to

global

patterns

of

gene

expression

with

the

transcript

abundance

of

each

of

thousands

of

genes

Fred

Hutchinson

Cancer

Research

Center

-LRB-

FHCRC

-RRB-

Fairview

Avenue

North

Seattle

WA

USA

and

Howard

Hughes

Medical

Institute

These

authors

contributed

equally

to

this

work

To

whom

correspondence

should

be

addressed

Email

leonid@fhcrc.org

References

and

Notes

treated

as

a

quantitative

phenotype

-LRB-

-RRB-

Although

it

has

recently

become

clear

that

genetic

variation

has

a

strong

effect

on

gene

expression

-LRB-

-RRB-

little

is

known

about

the

genetic

basis

of

natural

variation

in

expression

levels

-LRB-

the

number

and

type

of

loci

involved

the

effect

of

each

locus

and

the

interaction

between

loci

-RRB-

We

carried

out

linkage

analysis

of

global

expression

levels

in

a

cross

between

two

strains

of

the

budding

yeast

Saccharomyces

cerevisiae

The

parents

were

haploid

derivatives

of

a

standard

laboratory

strain

-LRB-

BY

-RRB-

and

a

wild

isolate

from

a

California

vineyard

APRIL

VOL

SCIENCE

Downloaded

from

on

May

REPORTS

-LRB-

RM

-RRB-

-LRB-

-RRB-

We

first

measured

expression

of

genes

in

six

independent

cultures

of

each

parent

undergoing

log-phase

growth

in

a

defined

medium

and

found

profound

differences

in

expression

-LRB-

-RRB-

A

total

of

genes

showed

differential

expression

at

P

whereas

only

are

expected

by

chance

-LRB-

-RRB-

At

P

we

observed

differences

compared

to

expected

by

chance

suggesting

that

nearly

half

-LRB-

out

of

-RRB-

of

all

the

genes

in

the

genome

are

differentially

expressed

Of

the

messages

that

were

different

at

P

differed

by

twofold

by

twofold

by

fourfold

and

by

eightfold

Expression

measurements

in

haploid

segregants

from

a

cross

between

the

two

parents

showed

that

parental

differences

in

expression

were

highly

heritable

the

median

proportion

of

the

observed

variation

that

is

genetic

was

estimated

to

be

-LRB-

-RRB-

Genetic

markers

were

identified

with

oligonucleotide

microarrays

using

a

method

previously

described

by

Winzeler

et

al.

-LRB-

-RRB-

The

resulting

genetic

map

of

markers

covered

of

the

genome

Analysis

of

four

segregants

from

one

tetrad

showed

marker

segregation

-LRB-

Fig.

-RRB-

with

crossovers

observed

across

the

genome

which

is

close

to

the

expected

number

of

-LRB-

-RRB-

In

the

analysis

of

the

cross

we

found

that

mating

type

kanamycin

resistance

and

auxotrophies

to

lysine

uracil

and

leucine

were

correctly

linked

with

LOD

scores

to

regions

containing

the

genes

MAT

HO

and

respectively

We

also

carried

out

linkage

analysis

of

flocculation

-LRB-

agglutination

of

cells

in

liquid

culture

-RRB-

Although

neither

parent

is

flocculent

tetrad

analysis

showed

flocculent

nonflocculent

segregation

in

the

cross

We

found

linkage

to

a

pair

of

loci

one

containing

the

gene

-LRB-

chr

V

-RRB-

and

the

other

containing

the

gene

-LRB-

chr

I

-RRB-

-LRB-

-RRB-

encodes

a

cell

wall

protein

responsible

for

agglutination

of

cells

and

encodes

a

transcription

factor

that

regulates

expression

-LRB-

-RRB-

Sequencing

of

the

corresponding

BY

and

RM

alleles

showed

that

BY

but

not

RM

carried

the

mutation

-LRB-

-RRB-

and

that

RM

but

not

BY

carried

a

short

deletion

in

We

next

tested

for

linkage

between

markers

and

the

abundance

of

each

message

-LRB-

-RRB-

Expression

levels

of

messages

showed

linkage

to

at

least

one

locus

at

P

-LRB-

are

expected

by

chance

-RRB-

Two

examples

of

segregation

of

gene

expression

with

the

genotype

of

a

linked

marker

are

shown

in

Fig.

Two

hundred

and

five

of

the

linkages

remained

significant

at

P

-LRB-

is

expected

by

chance

-RRB-

Message

levels

for

all

engineered

auxotrophies

linked

to

regions

containing

the

respective

genes

Of

the

messages

with

parental

differences

at

P

showed

linkage

to

at

least

one

locus

at

P

An

additional

messages

were

not

called

different

between

the

parents

at

P

but

showed

linkage

in

the

cross

This

observation

can

be

explained

in

three

ways

First

a

linkage

may

be

a

false

positive

as

noted

above

we

expect

false-positive

linkages

at

P

Second

a

true

difference

in

expression

levels

may

exist

between

the

parents

and

be

statistically

significant

in

a

comparison

of

segregants

separated

by

parental

genotype

but

not

be

statistically

significant

in

a

comparison

of

six

replicates

from

each

parent

Third

each

parent

may

harbor

several

loci

with

alleles

of

opposite

effect

on

message

levels

reducing

the

parental

difference

relative

to

the

range

of

the

segregants

This

phenomenon

called

transgressive

segregation

is

common

-LRB-

-RRB-

and

is

observed

for

a

number

of

messages

an

example

is

shown

in

Fig.

Conversely

levels

of

messages

differed

between

the

parents

at

P

but

did

not

show

linkage

to

any

locus

as

noted

above

we

expect

only

false-positive

differences

at

P

Simulations

showed

that

if

each

of

these

differences

in

expression

were

caused

by

a

single

locus

we

would

expect

to

detect

linkage

for

of

them

-LRB-

-RRB-

Simulations

also

showed

that

if

multiple

loci

were

involved

and

for

each

differentially

expressed

message

the

locus

with

the

strongest

effect

accounted

for

more

than

a

third

of

the

difference

we

would

expect

to

detect

linkage

for

of

these

messages

-LRB-

-RRB-

Thus

detection

of

linkage

for

just

of

the

Fig.

Chromosome

XII

genotypes

of

four

segregants

-LRB-

a

b

c

and

d

-RRB-

isolated

from

a

single

tetrad

Each

vertical

bar

represents

one

genetic

marker

colored

red

green

or

blue

when

the

genotype

of

the

segregant

at

the

marker

is

RM

BY

or

ambiguous

respectively

The

crosses

indicate

inferred

positions

of

crossovers

The

centromere

position

is

shown

by

a

black

circle

The

scale

bar

represents

kb

Fig.

Expression

levels

of

parents

and

segregants

for

two

genes

that

show

linkage

In

each

panel

the

first

column

shows

expression

levels

for

all

segregants

and

the

second

and

third

columns

show

expression

levels

for

six

replicates

of

each

parent

The

fourth

and

fifth

columns

show

expression

levels

for

segregants

that

inherited

the

linked

marker

from

BY

and

RM

respectively

-LRB-

A

-RRB-

The

gene

is

and

the

marker

lies

in

-LRB-

B

-RRB-

The

gene

is

-LRB-

-RRB-

and

the

marker

lies

in

Note

that

in

this

example

the

effect

of

the

locus

is

in

the

opposite

direction

from

the

difference

between

the

parents

illustrating

transgressive

segregation

SCIENCE

VOL

APRIL

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from

on

May

REPORTS

differences

in

the

real

data

-LRB-

out

of

-RRB-

indicates

that

most

messages

are

affected

by

multiple

loci

and

that

most

loci

account

for

less

than

a

third

of

the

total

parental

expression

differences

We

also

used

simulations

to

estimate

the

number

of

loci

involved

under

a

model

in

which

each

difference

is

caused

by

n

loci

of

equal

effect

-LRB-

-RRB-

The

results

showed

that

we

would

expect

to

detect

linkage

for

of

the

differences

if

n

if

n

if

n

and

if

n

The

fact

that

in

the

real

data

set

we

detected

linkage

for

only

of

the

differentially

expressed

messages

implies

that

under

this

model

loci

affect

each

message

In

reality

both

the

number

of

loci

contributing

to

expression

differences

and

the

distribution

of

their

effects

undoubtedly

vary

among

genes

but

our

data

are

inconsistent

with

one

or

two

major

loci

explaining

the

observed

differences

in

expression

for

more

than

a

small

fraction

of

genes

Because

the

estimates

above

assume

that

all

loci

from

one

parent

act

in

the

same

direction

existence

of

transgressive

segregation

implies

even

greater

complexity

To

determine

whether

the

loci

found

by

linkage

act

in

cis

or

in

trans

we

looked

for

messages

whose

levels

were

linked

to

markers

within

kb

of

their

own

gene

Such

self-linkages

suggest

that

a

polymorphism

affecting

a

gene

's

expression

lies

within

the

Fig.

The

number

of

linkages

plotted

against

genome

location

The

genome

is

divided

into

bins

of

kb

each

shown

in

chromosomal

order

from

the

start

of

chromosome

I

to

the

end

of

chromosome

XVI

The

dashed

line

is

drawn

at

linkages

no

bin

is

expected

to

contain

linkages

by

chance

-LRB-

-RRB-

The

regions

with

an

unusually

large

number

of

linkages

are

marked

through

and

correspond

to

the

groups

in

Table

Table

Groups

of

messages

linking

to

loci

with

widespread

transcriptional

effects

The

location

of

the

center

of

the

linked

bin

is

shown

as

chromosome

base

pair

Lists

of

genes

in

each

group

are

available

as

supplementary

information

-LRB-

-RRB-

Group

Number

of

messages

Common

function

Budding

daughter

cell

separation

Leucine

biosynthesis

Mating

Uracil

biosynthesis

Heme

fatty

acid

metabolism

Subtelomerically

encoded

helicases

Mitochondrial

induction

in

acid

Linkage

bin

II

III

III

V

XII

XII

XIV

XV

Putative

regulator

MAT

Unknown

Unknown

APRIL

VOL

SCIENCE

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from

on

May

gene

itself

or

its

regulatory

region

rather

than

elsewhere

in

the

genome

We

found

that

-LRB-

-RRB-

of

the

messages

that

show

linkage

at

P

fell

into

this

category

Because

no

message

is

expected

to

link

to

its

own

gene

by

chance

-LRB-

-RRB-

a

more

accurate

estimate

of

for

the

fraction

of

cis-acting

loci

is

obtained

by

dividing

the

number

of

self-linkages

by

the

expected

number

of

true

positives

-LRB-

-RRB-

We

next

considered

whether

there

are

many

trans-acting

regulatory

polymorphisms

each

affecting

one

or

a

few

messages

or

a

small

number

of

such

polymorphisms

with

effects

on

many

messages

We

divided

the

genome

into

bins

and

counted

the

number

of

linkages

to

markers

within

each

bin

-LRB-

Fig.

-RRB-

With

a

random

distribution

of

linkages

across

the

genome

no

bin

is

expected

to

contain

linkages

-LRB-

-RRB-

In

our

data

bins

contained

linkages

ranging

from

to

In

two

cases

two

nearby

bins

contained

linkages

and

were

combined

into

one

group

for

future

analyses

Over

of

all

linkages

-LRB-

-RRB-

fell

into

one

of

the

eight

groups

-LRB-

Table

-RRB-

Groups

through

contain

known

members

of

the

leucine

biosynthesis

mating

and

uracil

biosynthesis

pathways

and

link

to

MAT

and

respectively

Because

and

are

biosynthetic

en

zymes

rather

than

transcription

factors

these

linkages

illustrate

indirect

transcriptional

effects

on

other

genes

in

the

perturbed

pathways

The

other

five

loci

represent

natural

polymorphisms

between

the

parent

strains

with

large

transcriptional

effects

The

genes

within

each

group

appear

to

be

functionally

related

based

on

annotated

group

members

Group

contains

genes

of

whose

products

contain

heme

regulate

heme

synthesis

or

are

involved

in

fatty

acid

or

sterol

metabolism

Five

of

them

are

known

to

be

regulated

by

the

heme-dependent

transcriptional

activator

-LRB-

essential

for

anaerobic

growth

which

requires

ergosterol

metabolism

-RRB-

and

the

gene

encoding

is

in

the

linkage

region

for

this

group

The

allele

was

previously

shown

to

carry

a

insertion

that

reduces

transcriptional

activation

of

cytochrome

c

-LRB-

-RRB-

by

-LRB-

-RRB-

We

amplified

and

sequenced

the

BY

and

RM

alleles

and

found

that

BY

but

not

RM

carries

this

insertion

consistent

with

our

observation

that

and

are

underexpressed

in

all

segregants

inheriting

the

BY

allele

These

results

strongly

suggest

that

the

other

genes

in

group

are

also

regulated

by

We

searched

for

the

known

binding

site

consensus

sequence

in

the

upstream

regions

of

these

genes

and

found

sites

containing

at

most

one

mismatch

for

genes

Group

contains

genes

of

which

were

found

to

be

co-regulated

in

previous

array

experiments

with

six

expressed

specifically

in

daughter

cells

during

budding

-LRB-

-RRB-

The

gene

controlling

this

group

may

be

which

is

in

the

region

of

linkage

shares

the

group

's

expression

pattern

and

is

also

expressed

specifically

in

daughter

cells

during

budding

-LRB-

-RRB-

Group

consists

of

genes

all

putative

subtelomerically

encoded

helicases

these

closely

related

genes

may

cross-hybridize

on

arrays

a

known

transcriptional

silencer

active

at

telomeres

is

within

the

linked

region

Group

contains

genes

with

known

to

function

in

mitochondria

of

which

function

as

mitochondrial

ribosomal

proteins

This

group

shares

genes

with

a

previously

defined

mitochondrial

expression

cluster

-LRB-

-RRB-

Several

genes

in

the

region

of

linkage

function

in

mitochondria

Group

consists

of

genes

of

which

were

previously

shown

to

be

expressed

under

acidic

conditions

in

the

presence

of

the

transcription

factors

and

-LRB-

-RRB-

and

have

at

least

one

and

binding

site

in

their

upstream

region

Unlike

experiments

that

measure

the

correlations

among

transcript

levels

under

different

conditions

our

approach

allows

causal

connections

to

be

made

between

modulator

loci

and

the

genes

whose

expression

they

directly

or

indirectly

affect

In

addition

studying

naturally

occurring

alleles

in

the

REPORTS

context

of

segregating

variation

allows

the

discovery

of

subtle

effects

obscured

in

strains

with

engineered

knockouts

We

have

found

that

regulatory

genetic

variation

is

characterized

by

a

high

rate

of

cis-acting

alleles

and

a

small

number

of

transacting

alleles

with

widespread

transcriptional

effects

Finally

genetic

variation

in

physiological

and

behavioral

quantitative

phenotypes

is

known

to

be

highly

complex

Our

results

indicate

that

even

in

a

single-cell

organism

grown

in

a

controlled

environment

variation

in

gene

expression

typically

also

has

a

polygenic

basis

J.

L.

DeRisi

V.

R.

Iyer

P.

O.

Brown

Science

-LRB-

-RRB-

L.

Wodicka

H.

Dong

M.

Mittmann

M.

H.

Ho

D.

J.

Lockhart

Nature

Biotechnol

-LRB-

-RRB-

R.

C.

Jansen

J.

P.

Nap

Trends

Genet

-LRB-

-RRB-

R.

Sandberg

et

al.

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

M.

Primig

et

al.

Nature

Genet

-LRB-

-RRB-

W.

Jin

et

al.

Nature

Genet

-LRB-

-RRB-

D.

Cavalieri

J.

P.

Townsend

D.

L.

Hartl

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

Parent

strains

were

an

derivative

-LRB-

MATa

-RRB-

-LRB-

-RRB-

and

-LRB-

MATa

HO

kan

-RRB-

a

haploid

derived

from

-LRB-

-RRB-

a

natural

isolate

collected

by

Robert

Mortimer

as

in

-LRB-

-RRB-

Auxotrophic

deletions

were

used

to

select

diploids

during

mating

and

as

positive

controls

for

linkage

Both

strains

were

gifts

from

Lee

Hartwell

FHCRC

Seattle

WA

Cultures

were

grown

to

cells/mL

in

synthetic

C

medium

at

C

and

shaken

at

rpm

in

a

shaker

bath

RNA

was

isolated

with

hot

acid

phenol

and

chloroform

-LRB-

-RRB-

followed

by

the

RNA

cleanup

protocol

for

RNeasy

columns

-LRB-

Qiagen

Valencia

CA

-RRB-

Samples

were

labeled

and

hybridized

to

cDNA

microarrays

containing

open

reading

frames

as

described

-LRB-

-RRB-

Images

were

analyzed

with

GenePix

software

Each

two-color

experiment

involved

one

sample

and

one

reference

with

the

same

RNA

reference

being

used

for

all

experiments

Two

hybridizations

were

carried

out

for

each

sample

one

with

the

sample

labeled

with

and

the

reference

with

and

one

with

the

fluors

reversed

for

each

gene

the

two

log

ratios

were

averaged

Differences

in

the

expression

between

parent

strains

were

assessed

by

comparing

the

six

RM

values

to

the

six

BY

values

with

the

Wilcoxon-Mann-Whitney

test

-LRB-

-RRB-

and

nominal

P

values

are

reported

To

estimate

the

number

of

differences

expected

by

chance

at

a

given

P-value

threshold

we

randomly

assigned

of

the

measurements

to

one

group

and

to

the

other

without

regard

for

strain

identity

We

then

compared

the

two

groups

using

the

same

statistical

test

we

applied

in

comparing

the

two

strains

and

recorded

how

many

genes

had

P

values

below

the

threshold

The

randomization

procedure

was

repeated

times

and

the

results

were

averaged

Heritability

was

estimated

as

-LRB-

segregant

variance

parent

variance

-RRB-

segregant

variance

E.

A.

Winzeler

et

al.

Science

-LRB-

-RRB-

GeneChip

Yeast

Genome

microarrays

were

purchased

from

Affymetrix

-LRB-

Santa

Clara

CA

-RRB-

Genomic

DNA

was

isolated

using

Qiagen

columns

digested

with

DnaseI

and

end-labeled

as

described

-LRB-

-RRB-

and

hybridized

following

Affymetrix

instructions

We

developed

algorithms

to

select

oligos

as

reliable

genetic

markers

and

to

genotype

them

in

the

segregants

These

are

available

as

supplementary

information

-LRB-

-RRB-

J.

M.

Cherry

et

al.

Nature

-LRB-

-RRB-

For

each

pair

of

markers

with

all

flocculent

segregants

sharing

one

parental

genotype

at

one

marker

and

the

opposite

parental

genotype

at

the

other

a

Chi-square

test

of

independence

was

performed

on

a

contingency

table

of

all

segregants

according

to

their

flocculation

phenotype

-LRB-

or

-RRB-

and

genotype

-LRB-

BY/BY

BY/RM

RM/BY

or

RM/RM

-RRB-

at

the

two

markers

A

single

pair

showed

linkage

with

P

O.

Kobayashi

H.

Yoshimoto

H.

Sone

Curr

Genet

-LRB-

-RRB-

S.

J.

Kron

Trends

Microbiol

-LRB-

-RRB-

We

tested

for

linkage

between

a

marker

and

a

message

by

partitioning

the

segregants

into

two

groups

according

to

marker

genotype

and

comparing

the

message

levels

between

the

groups

with

the

Wilcoxon-Mann-Whitney

test

-LRB-

-RRB-

The

number

of

linkages

expected

by

chance

was

estimated

by

empirical

permutation

tests

-LRB-

-RRB-

M.

C.

deVicente

S.

D.

Tanksley

Genetics

-LRB-

-RRB-

L.

H.

Rieseberg

M.

A.

Archer

R.

K.

Wayne

Heredity

-LRB-

-RRB-

For

each

message

differentially

expressed

between

the

parents

at

P

we

computed

the

expression

difference

x

in

units

of

standard

deviation

We

simulated

a

locus

explaining

a

fraction

f

of

this

difference

by

randomly

assigning

parent

genotypes

at

the

locus

for

segregants

and

drawing

the

expression

level

of

each

segregant

from

one

of

two

Gaussian

distributions

of

unit

variance

one

with

mean

and

the

other

with

mean

fx

conditional

on

the

genotype

We

recorded

the

proportion

q

of

messages

for

which

linkage

was

detected

at

P

in

the

simulated

data

averaged

over

simulations

In

the

text

q

for

f

and

q

for

f

For

n

loci

each

explaining

a

fraction

f

of

the

difference

x

we

computed

the

rate

of

detecting

at

least

one

locus

as

-LRB-

q

-RRB-

n

The

genome

was

divided

into

bins

of

kb

each

-LRB-

the

bins

at

ends

of

chromosomes

were

smaller

-RRB-

Excluding

self-linkages

we

found

linkages

at

P

If

these

were

randomly

distributed

across

the

genome

the

number

of

linkages

in

any

one

bin

would

follow

a

Poisson

distribution

with

a

mean

of

The

probability

that

the

bin

with

the

most

linkages

would

contain

is

M.

Gaisne

A.

M.

Becam

J.

Verdiere

C.

J.

Herbert

Curr

Genet

-LRB-

-RRB-

A.

Colman-Lerner

T.

E.

Chin

R.

Brent

Cell

-LRB-

-RRB-

T.

R.

Hughes

et

al.

Cell

-LRB-

-RRB-

H.

C.

Causton

et

al.

Mol

Biol

Cell

-LRB-

-RRB-

C.

B.

Brachmann

et

al.

Yeast

-LRB-

-RRB-

R.

K.

Mortimer

P.

Romano

G.

Suzzi

M.

Polsinelli

Yeast

-LRB-

-RRB-

The

protocol

for

RNA

isolation

from

yeast

developed

by

P.

O.

Brown

's

lab

at

Stanford

University

can

be

found

online

at

jsp?ProtocolId

VersionNo

T.

G.

Fazzio

et

al.

Mol

Cell

Biol

-LRB-

-RRB-

B.

W.

Lindgren

Statistical

Theory

-LRB-

Macmillan

New

York

ed

-RRB-

Supplemental

material

is

available

at

www

G.

A.

Churchill

R.

W.

Doerge

Genetics

-LRB-

-RRB-

We

thank

J.

Delrow

M.

Eberle

I.

Furman

B.

Garvik

J.

Hartman

L.

Iwasaki

C.

Neal

J.

Pincus

E.

Ramos

C.

Saunders

and

P.

Tata

for

technical

assistance

and

H.

Coller

A.

Colman-Lerner

E.

Giniger

D.

Gottschling

L.

Hartwell

K.

Markianos

J.

Olson

and

G.

Smith

for

helpful

discussions

and

comments

on

the

manuscript

Supported

by

the

Howard

Hughes

Medical

Institute

-LRB-

of

which

L.K.

is

an

associate

investigator

-RRB-

and

NIH

postdoctoral

fellowship

-LRB-

R.B.

-RRB-

L.K.

is

a

James

S.

McDonnell

Centennial

Fellow

January

accepted

March

Published

online

March

science

Include

this

information

when

citing

this

paper

References

and

Notes

Transcription

Control

by

and

MAP

Kinase

Pathway

via

Mediator

Subunit

Jennitte

L.

Stevens

Greg

T.

Cantin

Gang

Wang

Andrej

Shevchenko

Anna

Shevchenko

Arnold

J.

is

a

metazoan

Mediator

subunit

that

interacts

with

the

adenovirus

protein

and

functions

in

a

mitogen-activated

protein

kinase

pathway

required

for

vulva

development

in

Caenorhabditis

elegans

We

generated

embryonic

stem

cells

to

analyze

its

function

as

a

mammalian

Mediator

component

Our

results

show

that

forms

a

subcomplex

of

the

Mediator

with

two

other

subunits

and

Knock-out

of

prevents

activation

by

and

the

mitogen-activated

protein

kinase

regulated

ETS

transcription

factor

but

not

by

multiple

other

transcription

factors

These

results

imply

that

specific

activation

domains

stimulate

transcription

by

binding

to

distinct

Mediator

subunits

Activation

by

and

requires

recruitment

of

Mediator

to

a

promoter

by

binding

to

its

subunit

Regulation

of

transcription

by

RNA

polymerase

II

-LRB-

Pol

II

-RRB-

is

controlled

by

specific

combinations

of

sequence-specific

DNA

binding

regulatory

transcription

factors

-LRB-

TFs

-RRB-

bound

to

a

gene

's

promoter

and

enhancer

regions

These

TFs

interact

with

several

multiprotein

complexes

that

remodel

the

chromatin

context

of

the

promoter

integrate

signals

from

multiple

TFs

to

control

the

frequency

of

transcription

initiation

and

regulate

the

efficiency

of

transcription

elongation

-LRB-

-RRB-

One

such

complex

is

the

Mediator

an

complex

of

to

subunits

that

is

believed

to

function

as

a

molecular

bridge

by

simultaneously

interacting

with

both

DNA-bound

TFs

and

Pol

II

-LRB-

-RRB-

The

human

Mediator

subunit

was

identified

through

its

interaction

with

adenovirus

conserved

region

-LRB-

-RRB-

a

potent

activation

domain

that

regulates

early

adenovirus

genes

-LRB-

-RRB-

In

C.

elegans

mutations

in

suppress

an

activated-ras

multivulva

phenotype

and

produce

SCIENCE

VOL

APRIL

Downloaded

from

on

May

O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O