Divergence

of

Transcription

Factor

Binding

Sites

Across

Related

Yeast

Species

Anthony

R.

Borneman

et

al.

Science

-LRB-

-RRB-

DOI

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AAAS

Downloaded

from

on

May

REPORTS

cause

these

are

the

only

mutations

that

will

fix

This

might

help

explain

why

similar

beneficial

mutation

rates

are

estimated

in

very

diverse

organisms

under

very

diverse

environments

These

estimates

are

obtained

in

populations

with

very

large

effective

sizes

-LRB-

-RRB-

which

are

likely

to

produce

strong

underestimations

of

Ua

It

is

plausible

that

in

natural

habitats

population

sizes

will

be

large

If

the

effective

size

of

a

bacterial

species

is

much

higher

than

-LRB-

-RRB-

then

our

results

imply

that

clonal

interference

plays

a

major

role

in

limiting

the

adaptation

of

these

asexual

organisms

As

such

if

there

is

a

chance

for

recombination

clonal

interference

will

be

much

lower

and

organisms

will

adapt

faster

This

has

been

predicted

theoretically

-LRB-

-RRB-

although

the

empirical

evidence

is

still

very

preliminary

-LRB-

-RRB-

Given

our

results

we

anticipate

that

clonal

interference

is

important

in

maintaining

sexual

reproduction

in

eukaryotes

Notably

mutation

accumulation

experiments

in

Saccharomyces

cerevisiae

and

Arabidopsis

thaliana

have

detected

a

significantly

large

number

of

mutants

with

increased

fitness

-LRB-

-RRB-

Given

the

estimates

for

the

overall

mutation

rate

in

E.

coli

-LRB-

-RRB-

and

its

genomic

deleterious

mutation

rate

-LRB-

-RRB-

our

estimate

of

Ua

implies

that

in

newly

arising

mutations

is

beneficial

and

that

in

fitness-affecting

mutations

increases

the

fitness

of

the

individual

carrying

it

Hence

an

enterobacterium

has

an

enormous

potential

for

adaptation

and

may

help

explain

how

antibiotic

resistance

and

virulence

evolve

so

quickly

References

and

Notes

T.

Bataillon

Heredity

-LRB-

-RRB-

R.

A.

Fisher

The

Genetical

Theory

of

Natural

Selection

-LRB-

Clarendon

Press

Oxford

UK

-RRB-

H.

A.

Orr

Nat

Rev.

Genet

-LRB-

-RRB-

M.

Imhof

C.

Schlotterer

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

R.

Miralles

P.

J.

Gerrish

A.

Moya

S.

F.

Elena

Science

-LRB-

-RRB-

C.

L.

Burch

L.

Chao

Genetics

-LRB-

-RRB-

C.

Zeyl

Genetics

-LRB-

-RRB-

S.

Estes

M.

Lynch

Evol

Int

J.

Org

Evol

-LRB-

-RRB-

R.

D.

Barrett

R.

C.

MacLean

G.

Bell

Biol

Lett

-LRB-

-RRB-

D.

R.

Rokyta

P.

Joyce

S.

Caudle

H.

Wichman

Nat

Genet

-LRB-

-RRB-

R.

Kassen

T.

Bataillon

Nat

Genet

-LRB-

-RRB-

M.

Kimura

The

Neutral

Theory

of

Molecular

Evolution

-LRB-

Cambridge

Univ.

Press

Cambridge

-RRB-

A.

J.

Betancourt

J.

P.

Bollback

Curr

Opin

Genet

Dev

-LRB-

-RRB-

W.

G.

Hill

A.

Robertson

Genet

Res

-LRB-

-RRB-

P.

Gerrish

R.

Lenski

Genetica

-LRB-

-RRB-

D.

E.

Rozen

J.

A.

de

Visser

P.

Gerrish

Curr

Biol

-LRB-

-RRB-

M.

Hegreness

N.

Shoresh

D.

Hartl

R.

Kishony

Science

-LRB-

-RRB-

C.

Schlotterer

M.

Imhof

H.

Wang

V.

Nolte

B.

Harr

J.

Evol

Biol

-LRB-

-RRB-

Materials

and

methods

are

available

as

supporting

material

on

Science

Online

J.

F.

Crow

M.

Kimura

An

Introduction

to

Population

Genetics

Theory

-LRB-

Harper

Row

New

York

-RRB-

Ua

k

-LRB-

Ne

-LRB-

sa

-RRB-

T

-RRB-

where

k

is

the

number

of

observed

mutations

T

is

the

number

of

generations

and

E

-LRB-

sa

-RRB-

is

the

mean

selection

coefficient

This

assumes

that

there

is

no

clonal

interference

If

its

effect

is

major

the

value

of

Ua

will

be

greatly

underestimated

Also

small

effect

mutations

are

likely

to

be

missed

because

the

time

it

takes

for

a

mutation

to

increase

in

frequency

is

The

expected

fitness

increase

over

generations

without

clonal

interference

is

Ne

Ua

E

-LRB-

sa

-RRB-

E

-LRB-

sa

-RRB-

in

the

small

size

populations

H.

A.

Orr

Genetics

-LRB-

-RRB-

R.

D.

H.

Barrett

S.

P.

Otto

L.

K.

M'gonigle

Genetics

-LRB-

-RRB-

J.

Charlesworth

A.

Eyre-Walker

Mol

Biol

Evol

-LRB-

-RRB-

B.

Grimberg

C.

Zeyl

Evol

Int

J.

Org

Evol

-LRB-

-RRB-

N.

Colegrave

Nature

-LRB-

-RRB-

S.

B.

Joseph

D.

W.

Hall

Genetics

-LRB-

-RRB-

R.

G.

Shaw

D.

Byers

E.

Darmo

Genetics

-LRB-

-RRB-

J.

W.

Drake

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

This

work

was

supported

by

and

through

Funda

o

para

a

Ci

ncia

e

Tecnologia

We

thank

M.

Imhof

for

help

in

the

initial

setup

of

this

work

and

R.

Azevedo

D.

Bachtrog

A.

Coutinho

F.

Dionisio

K.

Xavier

and

anonymous

reviewers

for

comments

on

the

manuscript

Supporting

Online

Material

Materials

and

Methods

SOM

Text

Figs

to

Table

References

March

accepted

July

science

Divergence

of

Transcription

Factor

Binding

Sites

Across

Related

Yeast

Species

Anthony

R.

Borneman

Tara

A.

Gianoulis

Zhengdong

D.

Zhang

Haiyuan

Yu

Joel

Rozowsky

Michael

R.

Seringhaus

Lu

Yong

Wang

Mark

Gerstein

Michael

Characterization

of

interspecies

differences

in

gene

regulation

is

crucial

for

understanding

the

molecular

basis

of

both

phenotypic

diversity

and

evolution

By

means

of

chromatin

immunoprecipitation

and

DNA

microarray

analysis

the

divergence

in

the

binding

sites

of

the

pseudohyphal

regulators

and

was

determined

in

the

yeasts

Saccharomyces

cerevisiae

S.

mikatae

and

S.

bayanus

under

pseudohyphal

conditions

We

have

shown

that

most

of

these

sites

have

diverged

across

these

species

far

exceeding

the

interspecies

variation

in

orthologous

genes

A

group

of

targets

was

shown

to

be

bound

only

in

S.

mikatae

and

S.

bayanus

under

pseudohyphal

conditions

Many

of

these

genes

are

targets

of

during

mating

in

S.

cerevisiae

indicating

that

specialization

between

the

two

pathways

has

occurred

in

this

species

Transcription

factor

binding

sites

have

therefore

diverged

substantially

faster

than

ortholog

content

Thus

gene

regulation

resulting

from

transcription

factor

binding

is

likely

to

be

a

major

cause

of

divergence

between

related

species

The

detection

of

binding

sites

with

chromatin

immunoprecipitation

and

microarray

-LRB-

ChIP-chip

-RRB-

analysis

-LRB-

-RRB-

offers

the

ability

to

globally

map

TF

binding

locations

experimentally

rather

than

computationally

For

species

such

as

yeasts

where

genome

sequences

of

numerous

related

species

are

available

-LRB-

-RRB-

this

approach

can

allow

for

the

evolutionary

comparison

of

binding

sites

of

conserved

TFs

across

species

We

have

used

this

approach

to

investigate

evolutionary

divergence

in

the

targets

of

two

developmental

regulators

in

the

Saccharomyces

sensu

stricto

yeasts

S.

cerevisae

S.

mikatae

and

S.

bayanus

In

S.

cerevisiae

diploids

and

act

cooperatively

to

regulate

genes

during

pseudohyphal

development

-LRB-

-RRB-

whereas

in

haploid

cells

regulates

mating

genes

-LRB-

-RRB-

The

binding

sites

of

and

were

mapped

in

all

three

species

under

low-nitrogen

Department

of

Molecular

Cellular

and

Developmental

Biology

Yale

University

New

Haven

CT

USA

Program

in

Computational

Biology

Yale

University

New

Haven

CT

USA

of

Molecular

Biophysics

and

Biochemistry

Yale

University

New

Haven

CT

USA

Data

Systems

Department

Siemens

Corporate

Research

Princeton

NJ

USA

Department

of

Computer

Science

Yale

University

New

Haven

CT

USA

D

ifferences

in

related

individuals

are

generally

attributed

to

changes

in

gene

composition

and/or

alterations

in

their

regulation

Previous

efforts

to

examine

divergence

of

regulatory

information

have

relied

on

the

analysis

of

conserved

sequences

in

putative

promoter

regions

-LRB-

-RRB-

However

these

approaches

are

limited

because

transcription

factor

-LRB-

TF

-RRB-

binding

sites

are

often

short

and

degenerate

making

their

computational

detection

difficult

-LRB-

-RRB-

In

addition

requiring

the

conservation

of

motifs

across

species

precludes

the

detection

of

sequences

that

are

evolutionarily

divergent

SCIENCE

VOL

Present

address

Australian

Wine

Research

Institute

Glen

Osmond

Adelaide

SA

Australia

To

whom

correspondence

should

be

addressed

E-mail

michael.snyder@yale.edu

AUGUST

Downloaded

from

on

May

REPORTS

-LRB-

pseudohyphal

-RRB-

conditions

with

the

use

of

triplicate

ChIP-chip

experiments

and

species-specific

high-density

oligonucleotide

tiling

microarrays

-LRB-

fig

-RRB-

-LRB-

-RRB-

bound

to

and

discrete

sites

whereas

bound

to

and

sites

in

S.

cerevisiae

S.

mikatae

and

S.

bayanus

respectively

-LRB-

tables

to

-RRB-

For

each

species

the

two

factors

bound

to

a

high

proportion

of

common

regions

-LRB-

and

for

S.

cerevisiae

S.

mikatae

and

S.

bayanus

respectively

-RRB-

suggesting

that

the

cooperative

interaction

observed

between

and

in

S.

cerevisiae

is

conserved

across

the

three

Saccharomyces

species

Analysis

of

the

signal

tracks

allowed

for

global

comparisons

in

TF

binding

to

be

made

among

the

species

revealing

qualitative

and

quantitative

differences

in

ChIP

binding

regions

-LRB-

Fig.

-RRB-

To

systematically

perform

interspecies

comparisons

we

removed

regions

that

were

not

represented

across

all

three

yeast

genomes

-LRB-

-RRB-

Comparison

of

the

overlap

in

binding

across

species

as

a

function

of

rank

order

revealed

significant

binding

differences

throughout

the

rank

order

indicating

that

even

strong

targets

from

one

species

may

not

be

bound

in

the

others

-LRB-

Fig.

-RRB-

As

a

control

replicate

experiments

from

S.

cerevisiae

-LRB-

-RRB-

displayed

concordance

in

binding

Overall

three

classes

of

TF

binding

events

were

observed

those

conserved

across

all

three

species

those

present

in

two

of

the

three

species

and

species-specific

binding

events

-LRB-

Fig.

-RRB-

Of

the

and

targets

bound

in

total

by

and

respectively

only

-LRB-

-RRB-

and

-LRB-

-RRB-

targets

were

conserved

across

all

three

species

-LRB-

Figs.

and

-RRB-

The

conserved

binding

events

were

present

throughout

the

rank

order

indicating

that

both

highly

occupied

and

less-occupied

regions

are

conserved

-LRB-

tables

and

-RRB-

To

ensure

that

these

binding

differences

were

not

due

to

the

scoring

threshold

used

we

calculated

signal

distributions

for

unbound

orthologs

of

target

regions

-LRB-

-RRB-

Of

the

unbound

orthologous

regions

had

signals

similar

to

background

indicating

that

most

will

be

unaffected

by

threshold

changes

-LRB-

fig

-RRB-

Even

when

identical

binding

regions

were

used

differed

in

their

intensity

by

at

least

fold

between

species

-LRB-

between

S.

cerevisiae

replicates

-RRB-

suggesting

that

quantitative

differences

exist

in

site

occupation

or

binding

strength

between

species

-LRB-

Fig.

and

tables

and

-RRB-

Thus

most

target

genes

were

bound

in

only

one

or

two

of

the

three

species

indicating

considerable

divergence

in

binding

sites

across

A

S.

cerevisiae

bp

ORF

bp

bp

bp

bp

ORF

ORF

B

Cumulative

overlap

with

S.

cerevisiae

Replicate

Cumulative

overlap

with

S.

cerevisiae

Replicate

S.

cerevisiae

replicate

S.

mikatae

S.

bayanus

C

S.

cerevisiae

replicate

S.

mikatae

S.

bayanus

S.

cerevisiae

S.

bayanus

S.

mikatae

S.

cerevisiae

28 50 100 150

RANK

-LRB-

descending

binding

strength

-RRB-

S.

mikatae

S.

bayanus

RANK

-LRB-

descending

binding

strength

-RRB-

Fig.

-LRB-

A

-RRB-

and

bind

to

discrete

regions

of

chromosome

IX

of

S.

cerevisiae

and

to

orthologous

regions

of

S.

mikatae

and

S.

bayanus

ChIP-chip

enrichment

by

and

-LRB-

log

ratios

-RRB-

is

shown

relative

to

ORFs

of

S.

cerevisiae

-LRB-

red

-RRB-

S.

mikatae

-LRB-

blue

-RRB-

and

S.

bayanus

-LRB-

green

-RRB-

bp

base

pairs

-LRB-

B

-RRB-

Rank-order

analysis

of

and

ChIP-chip

targets

in

S.

cerevisiae

-LRB-

red

-RRB-

S.

mikatae

-LRB-

blue

-RRB-

and

S.

bayanus

-LRB-

green

-RRB-

-LRB-

-RRB-

-LRB-

C

-RRB-

Gene

target

overlap

across

the

three

Saccharomyces

species

SCIENCE

AUGUST

VOL

Downloaded

from

on

May

S.

bayanus

S.

mikatae

REPORTS

these

yeasts

-LRB-

Fig.

-RRB-

Because

the

fraction

of

nonconserved

genes

among

S.

cerevisiae

S.

mikatae

and

S.

bayanus

is

less

than

-LRB-

-RRB-

the

amount

of

variation

in

TF

binding

is

substantially

larger

than

that

of

gene

variation

One

possible

cause

for

the

interspecies

differences

in

the

ChIP

binding

locations

is

divergence

in

binding

site

sequences

To

examine

this

possibility

we

investigated

sequence

motifs

in

both

bound

and

orthologous

unbound

regions

across

the

three

Saccharomyces

species

Position

weight

matrices

-LRB-

PWM

-RRB-

representing

the

putative

binding

motifs

for

and

were

generated

from

the

ChIP-chip

data

-LRB-

-RRB-

Analysis

of

the

targets

of

the

three

species

revealed

an

overrepresented

sequence

motif

that

matched

the

known

consensus

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

whereas

the

targets

of

in

S.

cerevisiae

and

S.

mikatae

revealed

a

motif

that

was

similar

to

the

known

binding

sequence

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

This

sequence

was

not

overrepresented

in

S.

bayanus

With

the

use

of

the

PWM

sequences

ChIP

bound

regions

and

orthologous

unbound

regions

from

each

species

were

then

scored

for

the

presence

of

each

motif

-LRB-

-RRB-

There

were

several

significant

classes

of

TF

binding

events

with

those

genes

bound

by

all

three

factors

present

near

the

top

of

both

the

-LRB-

all

bound

motif

in

all

-RRB-

and

-LRB-

all

bound

with

and

without

motif

-RRB-

lists

-LRB-

Fig.

C

and

D

-RRB-

For

promoter

regions

that

displayed

evolutionarily

conserved

ChIP

binding

in

all

three

Saccharomyces

species

-LRB-

-RRB-

and

-LRB-

-RRB-

of

the

regions

contained

at

least

one

significant

occurrence

of

the

PWM

motif

for

that

factor

in

each

species

-LRB-

Fig.

E

and

F

-RRB-

In

contrast

and

of

the

promoters

that

displayed

conserved

ChIP

binding

did

not

contain

a

match

to

the

PWM

in

at

least

two

of

the

three

species

Thus

the

motif

is

not

present

in

a

high

proportion

of

pseudohyphalresponsive

genes

implying

that

may

target

to

these

regulatory

regions

-LRB-

-RRB-

In

contrast

to

the

previous

results

in

which

experimentally

determined

binding

correlated

with

the

presence

of

predicted

motifs

there

were

many

examples

where

a

species-specific

loss

of

binding

and/or

a

loss

of

sequence

have

occurred

There

were

-LRB-

of

total

binding

events

-RRB-

and

-LRB-

of

total

binding

events

-RRB-

experimentally

bound

regions

that

contained

a

PWM

match

where

the

orthologous

region

in

at

least

one

other

species

neither

was

bound

nor

contained

a

motif

match

For

these

loci

loss

of

ChIP

binding

is

concordant

with

the

loss

of

the

motif

for

this

factor

providing

clear

examples

of

regions

where

network

evolution

occurred

through

the

gain

or

loss

of

regulatory

sequences

Furthermore

there

were

-LRB-

-RRB-

and

-LRB-

-RRB-

instances

where

a

PWM

match

occurred

in

all

three

species

but

where

that

region

was

experimentally

bound

in

only

two

species

-LRB-

Fig.

-RRB-

Either

these

loci

are

occupied

at

other

times

in

the

life

cycle

or

they

are

not

functional

Conversely

in

-LRB-

-RRB-

and

-LRB-

-RRB-

instances

genomic

regions

displayed

conserved

ChIP

binding

but

at

least

one

species

was

missing

a

PWM

motif

match

-LRB-

Fig.

-RRB-

Thus

sequence

conservation

does

not

readily

predict

binding

To

further

examine

the

role

of

conserved

versus

nonconserved

ChIP

binding

events

and

motifs

we

compared

these

results

with

expression

microarray

studies

of

pseudohyphal

formation

in

S.

cerevisiae

-LRB-

-RRB-

Of

the

ChIP

binding

gene

targets

that

had

significantly

altered

expression

A

Sc

B

Sc

Sc

Sc

Sm

Sm

Sm

Sm

Sb

Sb

Sb

Sb

C

Sc

D

E

Sc

Sc

Sb

Sb

Sb

Sm

Sm

Sm

Sc

Sm

Sb

Fig.

Comparison

of

binding

by

and

across

S.

cerevisiae

-LRB-

red

-RRB-

S.

mikatae

-LRB-

blue

-RRB-

and

S.

bayanus

-LRB-

green

-RRB-

-LRB-

A

-RRB-

Conserved

binding

-LRB-

B

-RRB-

Conserved

binding

with

quantitative

signal

differences

-LRB-

C

-RRB-

Conserved

binding

with

loss

of

consensus

sequences

in

one

species

-LRB-

D

-RRB-

Species-specific

binding

despite

conserved

SCIENCE

consensus

sequences

-LRB-

E

-RRB-

Binding

only

in

S.

mikatae

and

S.

bayanus

ChIp-chip

enrichment

signals

are

shown

-LRB-

log

ratios

-RRB-

Circles

and

squares

represent

matches

to

PWM

and

PWM

respectively

Triangles

nonconserved

peaks

difference

in

peak

signal

intensity

difference

in

peak

signal

intensity

VOL

AUGUST

Downloaded

from

on

May

REPORTS

A

B

C

S.c

S.m

S.b

freq

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

D

p-value

S.c

S.m

S.b

freq

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

p-value

-LRB-

-RRB-

-LRB-

-RRB-

PWM

match

NO

PWM

match

-LRB-

-RRB-

-LRB-

-RRB-

Key

region

bound

in

chIP

E

Bound

PWM

F

Bound

PWM

Fig.

Motif

analysis

of

ChIP

binding

targets

Logo

representations

of

the

PWM

for

-LRB-

A

-RRB-

and

-LRB-

B

-RRB-

-LRB-

-RRB-

-LRB-

C

and

D

-RRB-

Classes

of

binding

targets

after

classification

by

both

the

conservation

of

ChIP

binding

and

the

presence

or

absence

of

consensus

motifs

-LRB-

E

and

F

-RRB-

Compiled

proportions

of

binding

targets

and

PWM

matches

for

and

Fig.

-LRB-

A

-RRB-

and

bind

to

common

and

distinct

sets

of

genes

across

the

Saccharomyces

sensu

stricto

lineage

Overrepresented

GO

terms

are

listed

for

each

combinatorial

category

-LRB-

B

-RRB-

Mating

genes

bound

specifically

by

in

S.

mikatae

and

S.

bayanus

-LRB-

C

-RRB-

TF

network

conservation

in

S.

cerevisiae

-LRB-

red

-RRB-

S.

mikatae

-LRB-

blue

-RRB-

and

S.

bayanus

-LRB-

green

-RRB-

A

S.

cerevisiae

growth

-LRB-

p

x

-RRB-

B

S.

mikatae

S.

bayanus

S.

mikatae

S.

bayanus

S.

mikatae

S.

bayanus

S.

mikatae

S.

bayanus

S.

mikatae

S.

bayanus

S.

mikatae

S.

bayanus

S.

mikatae

S.

bayanus

Gene

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

ORFN

S.

cerevisiae

hom

ol

ogue

-LRB-

-RRB-

-LRB-

YN

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

YG

-RRB-

-LRB-

YI

-RRB-

-LRB-

YO

-RRB-

R

ati

os

carbohydrate

transport

-LRB-

p

x

-RRB-

S.

mikatae

S.

bayanus

filamentous

growth

-LRB-

p

x

-RRB-

regulation

of

transcription

from

RNA

pol

II

promoter

-LRB-

p

x

-RRB-

reproduction

of

a

single-celled

organism

-LRB-

p

x

-RRB-

S.

cerevisiae

carboxylic

acid

transport

-LRB-

p

x

-RRB-

pseudohyphal

growth

-LRB-

p

x

-RRB-

S.

mikatae

S.

bayanus

filamentous

growth

-LRB-

p

x

-RRB-

signal

transduction

-LRB-

p

x

-RRB-

C

S.

cerevisiae

S.

mikatae

S.

bayanus

AUGUST

VOL

SCIENCE

Downloaded

from

on

May

-LRB-

-RRB-

-LRB-

of

the

ChIP

targets

a

several-fold

enrichment

-RRB-

there

was

no

enrichment

for

genes

with

conserved

binding

-LRB-

bound

versus

unbound

-RRB-

or

PWM

matches

-LRB-

with

motif

versus

without

motif

-RRB-

-LRB-

table

-RRB-

Thus

sequence-based

motif

analyses

in

the

absence

of

experimentally

determined

binding

data

are

not

sufficient

for

the

accurate

prediction

of

TF

binding

profiles

and

gene

function

In

addition

the

presence

of

nonconserved

ChIP

targets

upstream

of

pseudohyphal-regulated

genes

at

the

same

frequency

as

conserved

targets

indicates

that

nonconserved

sites

are

likely

to

be

functional

To

elucidate

the

biological

importance

of

both

the

conserved

and

species-specific

gene

targets

we

mapped

each

bound

region

to

its

downstream

target

genes

by

identifying

open

reading

frames

-LRB-

ORFs

-RRB-

that

were

of

and

directly

flanking

each

ChIP

binding

event

-LRB-

tables

and

-RRB-

Conserved

and

gene

targets

displayed

enrichment

for

two

gene

ontology

-LRB-

GO

-RRB-

-LRB-

-RRB-

categories

filamentous

growth

and

regulation

of

transcription

from

RNA

polymerase

II

promoters

-LRB-

Fig.

-RRB-

Because

most

of

the

genes

from

within

the

second

category

encode

TFs

the

predicted

downstream

TF

networks

of

S.

bayanus

and

S.

mikatae

were

compared

to

those

of

S.

cerevisiae

-LRB-

-RRB-

to

determine

which

connections

had

been

maintained

during

the

evolution

of

the

Saccharomyces

sensu

stricto

group

-LRB-

Fig.

-RRB-

The

binding

of

and

to

downstream

TFs

was

shown

to

be

highly

conserved

-LRB-

across

the

three

species

-RRB-

The

network

of

S.

mikatae

was

most

diverged

and

had

several

key

REPORTS

regulatory

omissions

including

-LRB-

not

bound

by

either

or

-RRB-

and

-LRB-

not

bound

by

-RRB-

Thus

although

important

differences

can

be

found

TF

binding

to

the

promoters

of

other

TFs

was

highly

conserved

between

species

relative

to

the

level

of

conservation

observed

for

other

genes

From

those

groups

of

genes

that

did

not

display

conserved

binding

across

the

three

species

one

notable

class

was

bound

by

specifically

in

S.

mikatae

and

S.

bayanus

and

was

enriched

in

genes

involved

in

mating

-LRB-

GO

category

reproduction

in

single-celled

organisms

-RRB-

in

S.

cerevisiae

-LRB-

Fig.

A

and

B

-RRB-

Unlike

the

gene

targets

in

the

diploid

cells

used

in

this

study

these

genes

are

targets

of

in

haploid

S.

cerevisiae

cells

-LRB-

-RRB-

and

this

differential

binding

occurs

despite

the

presence

of

conserved

binding

motifs

-LRB-

fig

-RRB-

Thus

binding

targets

may

be

occupied

under

different

conditions

across

related

species

In

S.

cerevisiae

binds

to

these

sites

only

during

mating

whereas

in

S.

mikatae

and

S.

bayanus

binds

to

these

same

regions

in

diploid

cells

To

extend

this

study

outside

of

Saccharomyces

yeasts

we

also

mapped

the

binding

of

the

Candida

albicans

ortholog

-LRB-

-RRB-

functions

in

the

dimorphic

switch

of

this

yeast

a

process

that

shares

many

genetic

components

with

pseudohyphal

growth

-LRB-

-RRB-

A

total

of

significant

ChIP

binding

events

-LRB-

table

-RRB-

was

detected

under

dimorphic

growth

conditions

with

many

residing

upstream

of

known

pathogenicity

determinants

-LRB-

-RRB-

From

these

gene

targets

have

recognizable

orthologs

in

S.

cerevisiae

and

of

these

orthologs

and

displayed

conserved

binding

with

S.

cerevisiae

S.

mikatae

and

S.

bayanus

respectively

Although

most

gene

targets

of

in

C.

albicans

are

not

conserved

with

the

Saccharomyces

species

the

C.

albicans

orthologs

bound

by

like

those

from

S.

mikatae

and

S.

bayanus

included

a

significant

number

of

genes

that

function

during

reproduction

and

mating

in

S.

cerevisiae

-LRB-

P

-RRB-

-LRB-

-RRB-

Thus

in

C.

albicans

like

in

S.

mikatae

and

S.

bayanus

the

ortholog

also

binds

to

genes

required

for

mating

in

S.

cerevisiae

under

filamentous

growth

conditions

raising

the

possibility

that

these

genes

have

become

more

specialized

in

S.

cerevisiae

We

find

that

extensive

regulatory

changes

can

exist

in

closely

related

species

which

is

consistent

with

a

recent

study

that

showed

that

distinct

regulatory

circuits

can

produce

similar

regulatory

outcomes

in

S.

cerevisiae

and

C.

albicans

-LRB-

-RRB-

Furthermore

although

S.

cerevisiae

and

S.

mikatae

are

quite

similar

to

one

another

at

the

nucleotide

sequence

level

they

are

equally

different

to

each

other

and

to

S.

bayanus

in

their

TF

profiles

We

expect

that

the

extensive

binding

site

differences

observed

in

this

study

reflect

the

rapid

specialization

of

these

organisms

for

their

distinct

ecological

environments

and

that

differences

in

transcription

regulation

between

related

species

may

be

responsible

for

rapid

evolutionary

adaptation

to

varied

ecological

niches

References

and

Notes

P.

Cliften

et

al.

Science

-LRB-

-RRB-

M.

Kellis

N.

Patterson

M.

Endrizzi

B.

Birren

E.

S.

Lander

Nature

-LRB-

-RRB-

M.

Tompa

et

al.

Nat

Biotechnol

-LRB-

-RRB-

V.

R.

Iyer

et

al.

Nature

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-RRB-

B.

Ren

et

al.

Science

-LRB-

-RRB-

J.

Piskur

R.

B.

Langkjaer

Mol

Microbiol

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-RRB-

H.

D.

Madhani

G.

R.

Fink

Science

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-RRB-

V.

Gavrias

A.

Andrianopoulos

C.

J.

Gimeno

W.

E.

Timberlake

Mol

Microbiol

-LRB-

-RRB-

H.

Liu

C.

A.

Styles

G.

R.

Fink

Science

-LRB-

-RRB-

S.

Fields

I.

Herskowitz

Cell

-LRB-

-RRB-

A.

R.

Borneman

et

al.

Funct

Integr

Genomics

in

press

preprint

available

at

See

supporting

material

on

Science

Online

X.

S.

Liu

D.

L.

Brutlag

J.

S.

Liu

Nat

Biotechnol

-LRB-

-RRB-

J.

W.

Dolan

C.

Kirkman

S.

Fields

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

T.

L.

Bailey

M.

Gribskov

Bioinformatics

-LRB-

-RRB-

S.

Chou

S.

Lane

H.

Liu

Mol

Cell

Biol

-LRB-

-RRB-

S.

Prinz

et

al.

Genome

Res

-LRB-

-RRB-

E.

I.

Boyle

et

al.

Bioinformatics

-LRB-

-RRB-

A.

R.

Borneman

et

al.

Genes

Dev

-LRB-

-RRB-

C.

T.

Harbison

et

al.

Nature

-LRB-

-RRB-

J.

Zeitlinger

et

al.

Cell

-LRB-

-RRB-

T.

Jones

et

al.

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

C.

Sanchez-Martinez

J.

Perez-Martin

Curr

Opin

Microbiol

-LRB-

-RRB-

C.

E.

Birse

M.

Y.

Irwin

W.

A.

Fonzi

P.

S.

Sypherd

Infect

Immun

-LRB-

-RRB-

B.

R.

Braun

W.

S.

Head

M.

X.

Wang

A.

D.

Johnson

Genetics

-LRB-

-RRB-

B.

R.

Braun

A.

D.

Johnson

Genetics

-LRB-

-RRB-

T.

C.

White

S.

H.

Miyasaki

N.

Agabian

J.

Bacteriol

-LRB-

-RRB-

A.

E.

Tsong

B.

B.

Tuch

H.

Li

A.

D.

Johnson

Nature

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-RRB-

The

authors

would

like

to

thank

P.

Chambers

and

D.

Gelperin

for

comments

on

the

manuscript

Funding

was

provided

by

NIH

grants

-LRB-

to

M.S.

and

M.G.

-RRB-

and

by

the

Burroughs

Wellcome

Foundation

Detailed

lists

of

binding

regions

conservation

information

and

motif

scores

are

available

from

Supporting

Online

Material

Materials

and

Methods

Fig

to

Tables

to

References

February

accepted

June

science

High-Speed

Imaging

Reveals

Neurophysiological

Links

to

Behavior

in

an

Animal

Model

of

Depression

Raag

D.

Airan

Leslie

A.

Meltzer

Madhuri

Roy

Yuqing

Gong

Han

Chen

Karl

The

hippocampus

is

one

of

several

brain

areas

thought

to

play

a

central

role

in

affective

behaviors

but

the

underlying

local

network

dynamics

are

not

understood

We

used

quantitative

voltagesensitive

dye

imaging

to

probe

hippocampal

dynamics

with

millisecond

resolution

in

brain

slices

after

bidirectional

modulation

of

affective

state

in

rat

models

of

depression

We

found

that

a

simple

measure

of

real-time

activity

stimulus-evoked

percolation

of

activity

through

the

dentate

gyrus

relative

to

the

hippocampal

output

subfield

accounted

for

induced

changes

in

animal

behavior

independent

of

the

underlying

mechanism

of

action

of

the

treatments

Our

results

define

a

circuit-level

neurophysiological

endophenotype

for

affective

behavior

and

suggest

an

approach

to

understanding

circuit-level

substrates

underlying

psychiatric

disease

symptoms

he

hippocampus

as

an

integral

component

of

the

limbic

system

is

a

focus

of

depression

research

-LRB-

-RRB-

drives

other

brain

regions

implicated

in

depression

and

appears

to

hippocampal

activity

Antidepressant-induced

hippocampal

neurogenesis

is

linked

to

behavioral

responses

-LRB-

-RRB-

moreover

excitatory

hippocampal

neurons

are

injured

by

chronic

stress

-LRB-

-RRB-

Animal

models

have

proven

useful

in

identifying

molecular

and

cellular

markers

relevant

to

depression

-LRB-

-RRB-

but

have

not

identified

neurophysiological

final

common

pathways

relevant

to

behavior

Voltage-sensitive

dye

imaging

-LRB-

VSDI

-RRB-

could

allow

analysis

of

disease-related

neural

activity

on

millisecond

time

scales

with

micrometer

spatial

resolution

and

a

scope

spanning

entire

brain

networks

-LRB-

-RRB-

We

applied

VSDI

to

hippocampal

physiology

in

the

chronic

mild

stress

-LRB-

CMS

-RRB-

model

a

well-validated

rodent

Department

of

Bioengineering

Stanford

University

Stanford

CA

USA

Program

Stanford

University

Stanford

CA

USA

of

Electrical

Engineering

Stanford

University

Stanford

CA

USA

of

Statistics

Stanford

University

Stanford

CA

USA

of

Psychiatry

and

Behavioral

Sciences

Stanford

University

Stanford

CA

USA

T

serve

as

a

primary

site

of

action

for

antidepressants

that

inhibit

pathological

hyperactivity

-LRB-

-RRB-

Complicating

this

picture

however

is

evidence

suggesting

that

antidepressants

can

stimulate

SCIENCE

VOL

These

authors

contributed

equally

to

this

work

To

whom

correspondence

should

be

addressed

E-mail

deissero@stanford.edu

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

divergence evolution yeast transcription


Top-Down

Versus

Bottom-Up

Control

of

Attention

in

the

Prefrontal

and

Posterior

Parietal

Cortices

Timothy

J.

Buschman

et

al.

Science

-LRB-

-RRB-

DOI

science

The

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in

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at

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Online

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at

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on

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Web

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Downloaded

from

on

May

REPORTS

Top-Down

Versus

Bottom-Up

Control

of

Attention

in

the

Prefrontal

and

Posterior

Parietal

Cortices

Timothy

J.

Buschman

and

Earl

K.

Miller

Attention

can

be

focused

volitionally

by

top-down

signals

derived

from

task

demands

and

automatically

by

bottom-up

signals

from

salient

stimuli

The

frontal

and

parietal

cortices

are

involved

but

their

neural

activity

has

not

been

directly

compared

Therefore

we

recorded

from

them

simultaneously

in

monkeys

Prefrontal

neurons

reflected

the

target

location

first

during

top-down

attention

whereas

parietal

neurons

signaled

it

earlier

during

bottom-up

attention

Synchrony

between

frontal

and

parietal

areas

was

stronger

in

lower

frequencies

during

top-down

attention

and

in

higher

frequencies

during

bottom-up

attention

This

result

indicates

that

top-down

and

bottom-up

signals

arise

from

the

frontal

and

sensory

cortex

respectively

and

different

modes

of

attention

may

emphasize

synchrony

at

different

frequencies

V

olitional

shifts

of

attention

are

thought

to

depend

on

top-down

signals

derived

from

knowledge

about

the

current

task

-LRB-

e.g.

finding

your

lost

keys

-RRB-

whereas

the

automatic

bottom-up

capture

of

attention

is

driven

by

properties

inherent

in

stimuli

that

is

by

salience

-LRB-

e.g.

a

flashing

fire

alarm

-RRB-

-LRB-

-RRB-

Imaging

and

neurophysiological

studies

have

found

neural

correlates

of

both

types

in

the

frontal

and

posterior

parietal

cortices

-LRB-

-RRB-

but

their

respective

contributions

are

not

clear

they

have

largely

been

studied

in

separate

experiments

rendering

comparisons

difficult

and

obscuring

timing

differences

that

could

give

clues

to

information

flow

-LRB-

-RRB-

We

therefore

recorded

from

multiple

electrodes

simultaneously

implanted

in

the

frontal

and

parietal

cortices

as

monkeys

-LRB-

Macaca

mulatta

-RRB-

found

a

visual

target

under

two

conditions

-LRB-

Fig.

-RRB-

The

target

was

randomly

located

in

an

array

of

four

stimuli

with

conditions

differing

in

how

the

distractors

related

to

the

target

In

pop-out

the

distractors

were

identical

and

differed

from

the

target

along

two

dimensions

-LRB-

color

and

orientation

-RRB-

so

the

target

's

salience

automatically

drew

attention

to

it

-LRB-

-RRB-

During

search

each

distractor

independently

differed

from

the

target

Because

the

target

matched

some

of

the

distractors

in

each

dimension

it

was

not

salient

and

had

to

be

sought

using

only

its

remembered

appearance

-LRB-

-RRB-

The

monkeys

showed

the

behavioral

hallmarks

of

bottom-up

versus

top-down

attention

Psychophysical

testing

showed

a

shallower

increase

in

reaction

time

with

more

distractors

during

pop-out

than

during

search

-LRB-

ms

per

item

for

pop-out

ms

per

item

for

search

P

t

test

of

least-squares

linear

regression

-RRB-

During

recording

when

three

distractors

were

always

presented

the

monkeys

reaction

time

was

significantly

longer

and

more

variable

for

search

than

for

pop-out

-LRB-

Fig.

-RRB-

We

focused

on

the

lateral

intraparietal

area

-LRB-

LIP

-RRB-

in

the

parietal

cortex

and

the

lateral

prefrontal

cortex

-LRB-

LPFC

-RRB-

and

frontal

eye

fields

-LRB-

FEF

-RRB-

in

the

frontal

cortex

-LRB-

-RRB-

For

each

recording

session

we

implanted

up

to

electrodes

-LRB-

in

frontal

and

in

parietal

cortex

-RRB-

We

recorded

the

activity

of

neurons

over

sessions

-LRB-

-RRB-

Picower

Institute

for

Learning

and

Memory

RIKEN-MIT

Neuroscience

Research

Center

and

Department

of

Brain

and

Cognitive

Science

Massachusetts

Institute

of

Technology

Cambridge

MA

USA

To

whom

correspondence

should

be

addressed

E-mail

ekmiller@mit.edu

Fig.

-LRB-

A

-RRB-

Behavioral

task

comparing

visual

search

and

pop-out

Red

circle

indicates

eye

position

-LRB-

B

-RRB-

Histogram

of

reaction

times

-LRB-

RTs

-RRB-

during

search

and

pop-out

tasks

across

all

recording

sessions

for

one

target

and

three

distractors

Average

RTs

for

search

-LRB-

ms

-RRB-

and

pop-out

-LRB-

ms

-RRB-

differed

significantly

-LRB-

P

t

test

-RRB-

The

variance

in

RT

also

differed

significantly

-LRB-

SDs

of

ms

for

search

and

ms

for

pop-out

P

test

-RRB-

VOL

SCIENCE

MARCH

Downloaded

from

on

May

We

determined

when

each

neuron

first

found

-LRB-

reflected

-RRB-

the

target

location

by

computing

when

the

amount

of

information

in

its

firing

rate

about

target

location

first

reached

significance

-LRB-

-RRB-

The

data

for

each

trial

were

grouped

by

condition

-LRB-

pop-out

or

search

-RRB-

and

by

target

location

thus

factoring

out

information

about

target

features

The

top

row

of

Fig.

shows

the

distribution

of

these

times

relative

to

the

start

of

the

saccade

During

popout

there

was

a

bimodal

distribution

-LRB-

Fig.

-RRB-

For

each

area

there

was

a

population

of

neurons

that

first

found

the

target

well

before

the

saccade

-LRB-

i.e.

shortly

after

visual

array

onset

-RRB-

and

a

separate

population

that

found

the

target

after

the

saccade

The

early

population

consisted

of

of

all

target

location

selective

LIP

neurons

-LRB-

-RRB-

of

selective

LPFC

neurons

-LRB-

-RRB-

and

of

selective

FEF

neurons

-LRB-

-RRB-

There

were

clear

differences

in

timing

LIP

neurons

found

the

target

first

followed

by

LPFC

neurons

and

then

FEF

neurons

Fits

of

bimodal

Gaussians

-LRB-

Fig.

-RRB-

indicated

that

the

early

population

of

LIP

neurons

was

centered

at

ms

before

the

saccade

-LSB-

confidence

interval

-LRB-

CI

-RRB-

to

ms

-RSB-

followed

by

the

early

populations

in

LPFC

and

FEF

ms

-LRB-

CI

to

ms

-RRB-

and

ms

-LRB-

CI

to

ms

-RRB-

before

the

saccade

respectively

-LRB-

LIP

PFC

P

LIP

FEF

P

PFC

FEF

REPORTS

P

t

test

-RRB-

-LRB-

-RRB-

The

distribution

of

the

neurons

that

found

the

target

after

the

saccade

was

overlapping

in

all

three

areas

and

centered

about

ms

after

saccade

-LRB-

Fig.

-RRB-

The

same

ordering

was

seen

in

the

cumulative

distributions

of

these

data

-LRB-

Fig.

-RRB-

-LRB-

-RRB-

LIP

LPFC

and

FEF

neurons

began

finding

the

target

ms

ms

and

ms

before

saccade

respectively

-LRB-

LIP

PFC

P

LIP

FEF

P

PFC

FEF

P

randomization

test

-RRB-

-LRB-

-RRB-

One-quarter

of

the

selective

LIP

neurons

-LRB-

-RRB-

began

encoding

the

target

location

before

the

LPFC

population

first

carried

significant

information

This

is

more

than

expected

by

chance

-LRB-

P

tested

against

binomial

distribution

-RRB-

When

aligning

trials

on

visual

array

onset

instead

of

saccade

we

found

similar

results

-LRB-

-RRB-

In

the

search

task

neurons

began

finding

the

target

later

than

in

pop-out

just

before

the

saccade

and

in

the

reverse

order

The

frontal

areas

-LRB-

LPFC

and

FEF

-RRB-

showed

selectivity

first

followed

by

LIP

-LRB-

Fig.

B

and

D

-RRB-

About

onethird

of

all

selective

neurons

in

FEF

and

LPFC

began

to

reflect

the

target

location

before

the

saccade

-LRB-

and

respectively

-RRB-

whereas

only

-LRB-

-RRB-

of

selective

LIP

cells

did

so

This

is

greater

than

expected

by

chance

for

FEF

and

LPFC

but

not

for

LIP

-LRB-

P

P

and

P

respectively

tested

against

binomial

distribution

-RRB-

The

cumulative

distributions

in

Fig.

show

that

target

location

information

reached

significance

in

the

FEF

and

LPFC

at

and

ms

before

the

saccade

respectively

followed

by

LIP

-LRB-

LPFC

and

FEF

were

earlier

than

LIP

P

and

P

respectively

randomization

test

-RRB-

-LRB-

-RRB-

Whereas

during

pop-out

the

LIP

neurons

found

the

target

first

-LRB-

and

well

before

the

saccade

-RRB-

during

search

target

location

information

in

LIP

did

not

reach

significance

until

ms

after

the

saccade

As

with

pop-out

similar

results

were

observed

when

trials

were

aligned

on

visual

array

onset

-LRB-

-RRB-

Synchrony

of

neural

activity

may

increase

the

effectiveness

of

connections

between

brain

areas

and

enhance

the

representation

of

attended

stimuli

-LRB-

-RRB-

To

investigate

this

we

quantified

the

degree

of

synchrony

between

local

field

potentials

-LRB-

LFPs

-RRB-

in

the

parietal

and

frontal

cortices

Synchrony

was

measured

between

all

pairs

of

simultaneously

recorded

electrodes

that

had

at

least

one

neuron

selective

for

target

location

-LRB-

pairs

-RRB-

The

degree

of

synchrony

was

captured

in

the

coherence

statistic

a

measure

of

the

co-spectrum

between

two

signals

normalized

for

the

power

-LRB-

-RRB-

Significance

was

determined

by

randomization

tests

-LRB-

-RRB-

Because

similar

results

were

found

between

LIP

and

LPFC

and

between

LIP

and

FEF

we

combined

data

from

the

LPFC

and

FEF

During

both

search

and

pop-out

there

was

an

increase

in

coherence

between

LIP

and

frontal

cortex

in

a

middle

-LRB-

to

Hz

-RRB-

and

upper

-LRB-

to

Hz

-RRB-

frequency

band

-LRB-

Fig.

-RRB-

that

peaked

during

the

perisaccadic

period

-LRB-

i.e.

around

the

time

of

the

attention

shift

-RRB-

-LRB-

Fig.

-RRB-

The

increase

in

coherence

for

each

frequency

band

differed

between

bottom-up

and

top-down

-LRB-

Fig.

-RRB-

This

was

highlighted

by

subtracting

coherence

during

pop-out

from

coherence

during

search

Figure

shows

a

greater

increase

in

middle-frequency

-LRB-

to

Hz

-RRB-

coherence

between

LIP

and

frontal

cortex

during

top-down

search

than

during

bottom-up

pop-out

By

contrast

the

increase

in

upper

Fig.

Timing

of

target

location

selectivity

during

pop-out

-LRB-

left

column

-RRB-

and

search

-LRB-

right

column

-RRB-

Significance

was

determined

through

randomization

tests

-LRB-

-RRB-

-LRB-

A

and

B

-RRB-

Distribution

of

times

at

which

neurons

first

began

to

carry

significant

information

about

the

target

location

relative

to

the

saccade

Vertical

black

line

indicates

saccade

gray

shaded

regions

indicate

mean

-LRB-

SD

-RRB-

of

distribution

of

visual

array

onset

-LRB-

C

and

D

-RRB-

Normalized

cumulative

sums

of

the

histograms

shown

in

-LRB-

A

-RRB-

and

-LRB-

B

-RRB-

respectively

A

z-score

for

the

observed

distribution

was

calculated

through

randomization

tests

and

was

corrected

for

multiple

comparisons

-LRB-

-RRB-

SCIENCE

VOL

Fig.

LFP

coherence

between

LIP

and

frontal

cortex

-LRB-

LPFC

and

FEF

-RRB-

across

frequencies

for

-LRB-

A

-RRB-

pop-out

and

-LRB-

B

-RRB-

search

tasks

Coherence

was

calculated

around

the

time

of

the

attention

shift

-LRB-

in

a

perisaccadic

period

beginning

ms

before

saccade

to

ms

afterward

-RRB-

and

compared

to

a

baseline

an

intertrial

interval

-LRB-

ITI

-RRB-

epoch

-LRB-

a

window

starting

ms

before

trial

onset

-RRB-

Shaded

regions

are

confidence

intervals

around

average

coherence

Frequencies

below

Hz

are

not

meaningful

-LRB-

and

are

not

shown

-RRB-

because

of

the

relatively

short

time

epochs

used

MARCH

Downloaded

from

on

May

REPORTS

frequency

-LRB-

to

Hz

-RRB-

coherence

was

greater

during

pop-out

than

during

search

Thus

bottomup

and

top-down

attention

may

rely

on

different

frequency

bands

of

coherence

between

the

frontal

and

parietal

cortex

The

source

of

top-down

signals

has

largely

been

inferred

from

indirect

evidence

such

as

patterns

of

anatomical

connections

-LRB-

-RRB-

In

the

case

of

visual

attention

previous

research

has

shown

its

neural

correlates

throughout

the

cortex

with

control

attributed

to

parietofrontal

networks

-LRB-

-RRB-

Our

results

suggest

that

within

this

network

fast

bottom-up

target

selection

occurs

first

in

LIP

whereas

longerlatency

top-down

selection

occurs

first

in

the

frontal

cortex

This

supports

the

hypothesis

that

parietal

neurons

form

saliency

maps

for

bottom-up

selection

-LRB-

-RRB-

as

well

as

studies

showing

that

stimulation

of

the

frontal

cortex

causes

attention-like

effects

in

the

extrastriate

cortex

-LRB-

-RRB-

It

also

fits

with

attenuation

of

topdown

effects

in

the

posterior

cortex

after

PFC

damage

-LRB-

-RRB-

Although

both

the

frontal

and

parietal

cortex

are

involved

in

attention

our

results

illustrate

that

bottom-up

signals

appear

first

in

LIP

and

top-down

signals

appear

first

in

the

frontal

cortex

Localized

synchrony

of

activity

within

a

brain

area

may

help

resolve

competition

for

attentional

selection

-LRB-

-RRB-

and

interareal

synchrony

may

aid

in

long-range

communication

between

areas

-LRB-

-RRB-

Our

results

suggest

that

the

flow

of

top-down

and

bottom-up

information

is

aided

by

coherence

emphasizing

different

frequency

bands

Lowerfrequency

bands

are

more

robust

to

spike

timing

delays

and

thus

may

be

better

suited

for

longer-range

coupling

between

multiple

distant

areas

-LRB-

-RRB-

The

increase

in

lowfrequency

synchrony

during

search

could

reflect

a

broadcast

of

top-down

signals

on

a

larger

anatomical

scale

Synchrony

at

higherfrequency

bands

might

support

the

local

interactions

needed

to

enhance

stimulus

representations

-LRB-

-RRB-

The

emphasis

of

higherfrequency

synchrony

during

pop-out

could

reflect

local

enhancement

of

stimulus

representations

that

are

passed

forward

from

parietal

to

frontal

cortex

This

suggests

that

the

brain

may

emphasize

coherence

at

different

frequency

bands

for

the

dynamic

modulation

of

interareal

connections

which

in

turn

engages

the

network

best

suited

for

the

current

task

References

and

Notes

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J.

Duncan

Annu

Rev.

Neurosci

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S.

Kastner

L.

Ungerleider

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A.

M.

Treisman

G.

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Corbetta

G.

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Shulman

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Reynolds

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R.

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Pare

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H.

Wurtz

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-RRB-

See

supporting

material

on

Science

Online

A

sliding

window

of

ms

was

stepped

every

ms.

Significance

was

determined

by

constructing

a

distribution

through

randomization

tests

-LRB-

-RRB-

A

neuron

was

said

to

first

indicate

the

target

location

when

significant

at

P

for

two

bins

in

a

row

The

cumulative

distribution

is

the

cumulative

sum

of

the

histogram

of

when

each

cell

first

carried

significant

information

about

the

target

location

-LRB-

Fig.

top

row

-RRB-

The

cumulative

sum

was

then

normalized

for

the

number

of

significant

cells

expected

at

each

point

in

time

by

chance

This

procedure

corrected

for

multiple

comparisons

and

determined

when

an

area

carried

information

about

the

target

location

allowing

us

to

infer

the

order

of

selectivity

See

-LRB-

-RRB-

for

details

A.

M.

Aertsen

G.

L.

Gerstein

M.

K.

Habib

G.

Palm

J.

Neurophysiol

-LRB-

-RRB-

E.

Salinas

T.

J.

Sejnowski

Nat

Rev.

Neurosci

-LRB-

-RRB-

P.

Fries

Trends

Cognit

Sci

-LRB-

-RRB-

P.

Fries

J.

H.

Reynolds

A.

E.

Rorie

R.

Desimone

Science

-LRB-

-RRB-

N.

P.

Bichot

A.

F.

Rossi

R.

Desimone

Science

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M.

R.

Jarvis

P.

P.

Mitra

Neural

Comput

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J.

W.

Bisley

M.

E.

Goldberg

Science

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M.

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J.

Neurosci

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Itti

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Koch

Nat

Rev.

Neurosci

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Moore

M.

Fallah

Proc

Natl.

Acad

Sci

U.S.A.

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H.

Tomita

M.

Ohbayashi

K.

Nakahara

I.

Hasegawa

Y.

Miyashita

Nature

-LRB-

-RRB-

M.

Eglin

L.

C.

Robertson

R.

T.

Knight

Cereb

Cortex

-LRB-

-RRB-

L.

L.

Chao

R.

T.

Knight

Cereb

Cortex

-LRB-

-RRB-

A.

K.

Engel

P.

Fries

W.

Singer

Nat

Rev.

Neurosci

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A.

von

Stein

J.

Sarnthein

Int

J.

Psychophysiol

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N.

Kopell

G.

B.

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M.

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Whittington

R.

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Proc

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Supported

by

NSF

grant

and

National

Institute

of

Neurological

Disorders

and

Stroke

grant

We

thank

S.

Henrickson

J.

Roy

and

M.

Wicherski

for

comments

on

the

manuscript

W.

Asaad

and

K.

Maccully

for

technical

and

other

support

and

E.

N.

Brown

for

help

with

the

statistical

analyses

Fig.

-LRB-

A

-RRB-

Level

of

coherence

for

pop-out

and

search

during

the

middle

-LRB-

left

to

Hz

-RRB-

and

upper

-LRB-

right

to

Hz

-RRB-

frequency

bands

in

different

trial

epochs

P

P

-LRB-

t

test

-RRB-

-LRB-

B

-RRB-

Differences

in

LFP

coherence

between

LIP

and

frontal

cortex

during

pop-out

and

search

for

the

perisaccadic

period

-LRB-

green

-RRB-

and

ITI

-LRB-

black

-RRB-

Pop-out

coherence

was

subtracted

from

search

coherence

-LRB-

Fig.

-RRB-

Dashed

lines

indicate

significance

levels

-LRB-

P

corrected

for

multiple

comparisons

-RRB-

Differences

above

the

upper

dashed

line

indicate

significantly

more

coherence

during

search

than

during

popout

differences

below

the

lower

dashed

line

denote

significantly

more

coherence

during

pop-out

than

during

search

Supporting

Online

Material

Materials

and

Methods

Figs

to

Table

November

accepted

March

science

MARCH

VOL

SCIENCE

Downloaded

from

on

May

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