Food-Caching

Western

Scrub-Jays

Keep

Track

of

Who

Was

Watching

When

Joanna

M.

Dally

et

al.

Science

-LRB-

-RRB-

DOI

science

The

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The

title

Science

is

a

registered

trademark

of

AAAS

REPORTS

postsynaptic

neuron

This

then

leads

to

activation

of

calcium-dependent

protein

kinases

and

NOS

which

causes

amplification

of

painrelated

information

at

the

first

synapse

in

pain

pathways

Blockade

of

unmasked

LTD

of

synaptic

strength

in

C-fibers

induced

by

LFS

This

suggests

that

LFS

may

simultaneously

induce

synaptic

plasticity

of

opposite

polarity

involving

divergent

signal

transduction

pathways

Hyperalgesia

in

human

-LRB-

-RRB-

and

in

animal

studies

-LRB-

-RRB-

and

the

synaptic

pain

amplifier

described

in

this

work

share

induction

mechanisms

relevant

neuron

populations

in

spinal

cord

pharmacological

profile

and

signal

transduction

pathways

This

strongly

suggests

that

LTP

at

the

first

synapse

in

pain

pathways

between

nociceptive

C-fibers

and

spinal

lamina

I

projection

neurons

is

a

cellular

key

mechanism

of

inflammatory

hyperalgesia

and

perhaps

other

forms

of

low-level

afferent-induced

hyperalgesia

-LRB-

-RRB-

References

and

Notes

C.

J.

Woolf

Nature

-LRB-

-RRB-

J.

L.

K.

Hylden

R.

L.

Nahin

R.

J.

Traub

R.

Dubner

Pain

-LRB-

-RRB-

D.

A.

Simone

et

al.

J.

Neurophysiol

-LRB-

-RRB-

S.

G.

Khasabov

et

al.

J.

Neurosci

-LRB-

-RRB-

A.

J.

Todd

et

al.

J.

Neurosci

-LRB-

-RRB-

A.

D.

Craig

J.

Comp

Neurol

-LRB-

-RRB-

W.

D.

Willis

Ann

N.Y.

Acad

Sci

-LRB-

-RRB-

S.

P.

Hunt

P.

W.

Mantyh

Nat

Rev.

Neurosci

-LRB-

-RRB-

M.

Cavazzini

T.

Bliss

N.

Emptage

Cell

Calcium

-LRB-

-RRB-

Materials

and

methods

are

available

as

supporting

material

on

Science

Online

A.

J.

Todd

M.

M.

McGill

S.

A.

S.

Shehab

Eur

J.

Neurosci

-LRB-

-RRB-

H.

Ikeda

B.

Heinke

R.

Ruscheweyh

J.

Sandkuhler

Science

-LRB-

-RRB-

S.

Puig

L.

S.

Sorkin

Pain

-LRB-

-RRB-

W.

D.

Willis

Jr.

R.

E.

Coggeshall

Sensory

Mechanisms

of

the

Spinal

Cord

Primary

Afferent

Neurons

and

the

Spinal

Dorsal

Horn

-LRB-

Kluwer

Academic/Plenum

New

York

ed

-RRB-

pp.

T.

L.

Yaksh

X.

Y.

Hua

I.

Kalcheva

N.

Nozaki-Taguchi

M.

Marsala

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

S.

T.

Meller

G.

F.

Gebhart

Pain

-LRB-

-RRB-

R.

Ruscheweyh

A.

Goralczyk

G.

Wunderbaldinger

A.

Schober

J.

Sandkuhler

Neuroscience

in

press

I.

Ismailov

D.

Kalikulov

T.

Inoue

M.

J.

Friedlander

J.

Neurosci

-LRB-

-RRB-

R.

C.

Malenka

B.

Lancaster

R.

S.

Zucker

Neuron

-LRB-

-RRB-

S.

N.

Yang

Y.

G.

Tang

R.

S.

Zucker

J.

Neurophysiol

-LRB-

-RRB-

J.

Lisman

Philos

Trans

R.

Soc

London

B

Biol

Sci

-LRB-

-RRB-

E.

R.

Perl

in

Handbook

of

Physiology

Section

The

Nervous

System

vol

I.

Darian-Smith

Ed

-LRB-

American

Physiological

Society

Bethesda

MD

-RRB-

pp.

H.

E.

Torebjork

L.

E.

Lundberg

R.

H.

LaMotte

J.

Physiol

-LRB-

-RRB-

R.

C.

Spike

Z.

Puska

D.

Andrew

A.

J.

Todd

Eur

J.

Neurosci

r

-LRB-

-RRB-

R.

Ruscheweyh

H.

Ikeda

B.

Heinke

J.

Sandku

hler

J.

Physiol

-LRB-

-RRB-

Because

most

lamina

I

spino-PAG

neurons

have

collaterals

to

the

parabrachial

area

they

constitute

a

subgroup

of

about

one-third

of

the

spino-PB

population

Nevertheless

they

have

been

shown

to

differ

from

other

spino-PB

neurons

e.g.

with

respect

to

expression

and

active

and

passive

membrane

properties

See

-LRB-

-RRB-

for

further

discussion

T.

Klein

W.

Magerl

H.

C.

Hopf

J.

Sandku

hler

R.-D

Treede

J.

Neurosci

-LRB-

-RRB-

P.

W.

Mantyh

et

al.

Science

-LRB-

-RRB-

M.

L.

Nichols

et

al.

Science

-LRB-

-RRB-

F.

Cervero

R.

Gilbert

R.

G.

Hammond

J.

Tanner

Pain

-LRB-

-RRB-

W.

Koppert

et

al.

Anesthesiology

-LRB-

-RRB-

Supported

by

grant

from

the

Austrian

Science

Fund

-LRB-

FWF

-RRB-

to

J.S.

The

Japan

Society

for

the

Promotion

of

Science

partially

supported

H.I.

We

thank

R.-D

Treede

and

A.

H.

Dickenson

for

reading

an

earlier

version

of

the

manuscript

Supporting

Online

Material

Materials

and

Methods

Figs

to

References

March

accepted

May

science

Food-Caching

Western

Scrub-Jays

Keep

Track

of

Who

Was

Watching

When

Joanna

M.

Dally

Nathan

J.

Emery

Nicola

S.

Western

scrub-jays

-LRB-

Aphelocoma

californica

-RRB-

hide

food

caches

for

future

consumption

steal

others

caches

and

engage

in

tactics

to

minimize

the

chance

that

their

own

caches

will

be

stolen

We

show

that

scrub-jays

remember

which

individual

watched

them

during

particular

caching

events

and

alter

their

recaching

behavior

accordingly

We

found

no

evidence

to

suggest

that

a

storer

's

use

of

cache

protection

tactics

is

cued

by

the

observer

's

behavior

ocial

living

is

suggested

to

have

selected

for

increased

sociocognitive

skills

in

animals

-LRB-

-RRB-

Several

species

use

information

about

the

dominance

rank

-LRB-

-RRB-

and

social

relationships

-LRB-

-RRB-

of

conspecifics

when

forming

alliances

or

competing

for

resources

-LRB-

-RRB-

Corvids

rely

on

observational

spatial

memory

to

steal

others

caches

-LRB-

-RRB-

and

they

engage

in

a

variety

of

behaviors

to

reduce

cache

theft

-LRB-

-RRB-

It

may

be

advantageous

to

remember

who

was

watching

during

specific

caching

events

because

scrub-jays

can

only

defend

caches

against

subordinates

-LRB-

-RRB-

Scrub-jays

S

Sub-department

of

Animal

Behavior

University

of

Cambridge

Cambridge

UK

of

Experimental

Psychology

University

of

Cambridge

Cambridge

UK

To

whom

correspondence

should

be

addressed

E-mail

should

therefore

engage

in

cache

protection

when

they

are

caching

in

view

of

dominant

birds

but

might

refrain

from

doing

so

when

watched

by

their

partner

because

they

often

defend

their

caches

and

tolerate

cache

theft

by

their

mate

-LRB-

-RRB-

Nine

birds

-LRB-

storers

-RRB-

cached

in

two

trays

one

near

and

one

far

from

an

observer

Storers

cached

in

the

presence

of

a

dominant

bird

-LRB-

Bdominant

-LSB-

-RRB-

a

subordinate

bird

-LRB-

Bsubordinate

-LSB-

-RRB-

their

partner

-LRB-

Bpartner

-LSB-

-RRB-

or

when

an

view

was

obscured

-LRB-

Bin

private

-LSB-

-RRB-

After

hours

storers

recovered

their

caches

Bin

private

-LSB-

-LRB-

-RRB-

Partnerships

and

dominance

indices

were

determined

before

this

experiment

when

the

birds

were

aviary

housed

-LRB-

-RRB-

The

total

number

of

items

cached

did

not

differ

significantly

between

conditions

analysis

of

variance

-LRB-

ANOVA

-RRB-

VOL

SCIENCE

P

Birds

cached

predominantly

in

the

far

tray

in

the

Bdominant

-LSB-

-LRB-

sign

test

S

0 7/7

P

-RRB-

and

Bsubordinate

-LSB-

condition

-LRB-

S

0 7/7

P

-RRB-

but

not

in

the

Bpartner

-LSB-

condition

-LRB-

S

0 5/7

P

-RRB-

or

Bin

private

-LSB-

-LRB-

S

0 3/7

P

-RRB-

-LRB-

table

-RRB-

The

proportion

of

items

that

were

recached

at

recovery

was

greatest

in

the

Bdominant

-LSB-

condition

-LRB-

Fig.

-RRB-

In

the

Bdominant

-LSB-

and

Bsubordinate

-LSB-

conditions

items

were

predominantly

moved

from

the

near

tray

Surprisingly

items

were

also

moved

but

from

both

trays

after

the

Bin

private

-LSB-

and

Bpartner

-LSB-

conditions

-LRB-

Fig.

-RRB-

Irrespective

of

condition

items

were

always

moved

to

out-of-tray

sites

around

the

home

cage

The

pattern

of

caching

and

recaching

suggests

that

scrub-jays

engage

in

cache

protection

to

combat

the

specific

risk

that

nonpartners

pose

to

their

caches

even

though

observers

did

not

have

the

opportunity

to

pilfer

Specifically

birds

cached

mainly

in

the

far

tray

when

observed

by

a

nonpartner

and

recached

the

few

items

hidden

in

the

near

tray

at

recovery

It

may

be

difficult

for

observers

to

see

the

location

of

caches

in

the

far

tray

thereby

decreasing

their

ability

to

use

observational

spatial

memory

to

facilitate

cache

pilferage

This

strategy

was

not

implemented

when

the

partner

was

present

perhaps

because

storers

prefer

to

spend

time

near

to

their

partner

and

do

not

perceive

them

as

a

risk

to

cache

safety

There

are

three

alternate

explanations

for

the

preference

to

cache

in

the

far

tray

in

the

Bdominant

-LSB-

and

Bsubordinate

-LSB-

conditions

JUNE

Downloaded

from

on

May

REPORTS

First

being

near

nonpartners

might

be

aversive

However

in

a

previous

experiment

scrub-jays

spent

similar

amounts

of

time

near

and

in-view

and

far

and

out-of-view

of

nonpartners

during

caching

-LRB-

-RRB-

Second

jays

might

be

predisposed

to

cache

at

a

distance

from

nonpartners

Yet

our

youngest

cohort

of

birds

do

not

discriminate

between

near

Emedian

interquartile

range

-LRB-

IQR

-RRB-

and

far

trays

-LRB-

median

IQR

Z

P

-RRB-

-LRB-

table

-RRB-

Unlike

the

birds

in

this

experiment

-LRB-

age

-RRB-

these

young

birds

-LRB-

age

-RRB-

have

not

experienced

stealing

others

caches

an

experience

that

seems

essential

for

the

expression

of

recaching

-LRB-

-RRB-

and

other

protective

behaviors

Finally

storers

might

have

learned

that

caching

in

far

sites

reduces

cache

theft

However

storers

received

a

single

trial

in

each

caching

condition

and

because

caches

were

never

stolen

they

received

no

feedback

as

to

the

success

of

their

caching

strategies

Critically

these

explanations

can

not

explain

the

differences

in

the

birds

recaching

behavior

because

cache

recovery

always

occurred

Bin

private

-LSB-

Differences

in

recaching

must

therefore

be

based

on

a

memory

for

the

social

status

of

who

watched

them

cache

To

test

whether

scrub-jays

remember

which

particular

individual

was

present

when

they

made

specific

caches

eight

birds

were

given

eight

trials

in

which

they

cached

successively

in

two

trays

each

in

view

of

a

different

observer

Consequently

individual

A

observed

the

storer

caching

in

one

tray

and

individual

B

observed

the

storer

caching

in

a

second

tray

immediately

afterwards

-LRB-

AB

trial

-RRB-

or

vice

versa

-LRB-

BA

trial

-RRB-

During

each

caching

period

both

trays

were

placed

in

the

cage

but

Perspex

strips

covered

one

tray

to

prevent

caching

in

that

tray

Observers

therefore

saw

the

storer

caching

in

one

-LRB-

observed

tray

-RRB-

but

not

the

other

tray

After

hours

storers

recovered

their

caches

either

Bin

private

-LSB-

in-view

of

individual

A

or

individual

B

-LRB-

Bobserved

-LSB-

condition

-RRB-

or

watched

by

a

Bcontrol

-LSB-

bird

that

had

not

witnessed

the

storer

caching

-LRB-

-RRB-

To

control

for

any

affect

of

observer

social

status

on

caching

behavior

birds

of

similar

rank

were

used

and

storer-observer

dominance

relationships

were

counter

balanced

across

trials

No

differences

were

detected

in

the

number

of

items

cached

in

the

presence

of

individual

A

and

individual

B

during

AB

and

BA

trials

-LRB-

P

-RRB-

The

proportion

of

caches

that

were

recached

at

recovery

differed

significantly

between

conditions

-LRB-

P

G

-RRB-

-LRB-

table

-RRB-

and

significantly

more

caches

were

recached

during

the

Bobserved

-LSB-

condition

relative

to

the

Bcontrol

-LSB-

condition

-LRB-

Fig.

-RRB-

A

similar

proportion

of

caches

were

recached

from

either

tray

Bin

private

-LSB-

-LRB-

S

0 8/12

P

-RRB-

whereas

in

the

Bobserved

-LSB-

condition

storers

recached

specifically

from

the

Bobserved

-LSB-

tray

-LRB-

Fig.

-RRB-

Few

items

were

recached

from

the

Bother

-LSB-

tray

in

the

Bobserved

-LSB-

condition

or

from

either

tray

in

the

Bcontrol

-LSB-

condition

Recaching

in

the

Bobserved

-LSB-

and

Bin

private

-LSB-

conditions

differed

in

two

respects

First

Bin

private

-LSB-

items

were

recached

in

Bout-of

Fig.

Median

proportion

-LRB-

percentile

and

percentile

-RRB-

of

items

recached

after

each

caching

condition

-LRB-

A

-RRB-

Recaching

was

greatest

after

the

dominant

condition

-LRB-

P

-RRB-

When

the

dominant

condition

was

removed

from

the

analysis

no

significant

difference

was

identified

between

the

remaining

conditions

-LRB-

P

-RRB-

The

asterisk

indicates

P

G

ns

not

significant

-LRB-

B

-RRB-

The

median

proportion

-LRB-

percentile

and

percentile

-RRB-

and

median

number

-LRB-

in

parentheses

-RRB-

of

items

recached

from

near

-LRB-

open

bars

-RRB-

and

far

-LRB-

shaded

bars

-RRB-

trays

is

shown

for

birds

that

cached

in

both

trays

The

data

could

not

be

analyzed

statistically

because

several

birds

did

not

cache

in

both

trays

-LRB-

two

in

the

dominant

condition

four

in

the

partner

condition

and

two

in

private

-RRB-

and

their

recovery

behavior

would

therefore

be

confined

to

one

location

Fig.

-LRB-

A

-RRB-

Median

-LRB-

percentile

and

percentile

-RRB-

proportion

and

median

number

-LRB-

in

parentheses

-RRB-

of

items

recached

from

the

observed

and

other

trays

in

the

observed

condition

and

the

mean

of

trays

in

the

control

and

in

private

conditions

-LRB-

P

-RRB-

The

proportion

that

was

recached

from

the

observed

and

other

trays

differed

significantly

-LRB-

S

0 25/25

P

G

-RRB-

The

triple

asterisk

indicates

P

G

-LRB-

B

-RRB-

Total

number

of

times

items

were

moved

from

-LRB-

i

-RRB-

the

observed

tray

-LRB-

ii

-RRB-

the

other

tray

-LRB-

iii

-RRB-

the

in

private

trays

and

-LRB-

iv

-RRB-

the

trays

in

the

control

condition

Caches

in

the

observed

tray

were

moved

more

often

than

caches

in

the

in

private

trays

-LRB-

P

-RRB-

Recaching

from

the

other

tray

and

the

trays

in

the

control

condition

was

too

low

to

allow

further

analysis

SCIENCE

VOL

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REPORTS

tray

-LSB-

locations

By

contrast

items

from

the

Bobserved

-LSB-

tray

were

predominantly

moved

to

cache

sites

within

that

tray

-LRB-

P

G

-RRB-

-LRB-

table

-RRB-

Second

caches

in

the

Bobserved

-LSB-

condition

were

moved

twice

as

often

as

those

recached

Bin

private

-LSB-

-LRB-

Fig.

-RRB-

Repeatedly

moving

items

may

decrease

the

accuracy

with

which

potential

thieves

can

relocate

caches

because

memories

for

successive

locations

should

interfere

with

one

another

Storing

jays

have

been

shown

to

recover

caches

they

did

or

did

not

move

multiple

times

with

a

comparable

degree

of

accuracy

-LRB-

-RRB-

It

is

possible

that

in

experiment

storers

were

simply

reacting

to

cues

provided

by

the

observer

-LRB-

-RRB-

For

example

observers

may

direct

more

-LRB-

or

less

-RRB-

attention

to

trays

they

have

seen

a

bird

caching

in

In

experiment

we

repeated

the

Bobserved

-LSB-

condition

of

experiment

and

contrasted

it

with

an

Bobserver-control

-LSB-

condition

-LRB-

Fig.

-RRB-

The

Bobserver-control

-LSB-

condition

was

identical

to

the

Bobserved

-LSB-

condition

except

that

in

the

Bobserved

-LSB-

condition

the

observer

saw

the

same

bird

at

caching

and

recovery

whereas

in

the

Bobserver-control

-LSB-

condition

the

control

bird

watched

a

different

individual

recover

its

caches

from

the

one

it

saw

caching

-LRB-

-RRB-

If

cache

recovery

is

mediated

by

a

memory

for

who

was

present

during

caching

the

birds

recovery

behavior

should

differ

between

the

Bobserved

-LSB-

and

Bobserver-control

-LSB-

conditions

Based

on

experiment

storers

in

the

Bobserved

-LSB-

condition

should

recache

predominantly

from

the

tray

in

which

the

observer

at

recovery

had

seen

them

cache

-LRB-

Bobserved

-LSB-

tray

-RRB-

However

because

the

control

bird

was

not

present

when

the

storer

cached

few

items

should

be

recached

from

either

tray

in

the

Bobserver-control

-LSB-

condition

A

behavior-cueing

account

leads

to

a

different

prediction

If

observers

primarily

attend

to

trays

in

which

they

have

seen

caching

and

if

storers

use

these

cues

to

guide

recovery

then

storers

should

recache

specifically

from

the

Bobserved

-LSB-

tray

in

both

conditions

The

number

of

items

cached

in

the

Bobserved

-LSB-

and

Bobserver-control

-LSB-

conditions

did

not

differ

statistically

-LRB-

P

-RRB-

nor

was

there

any

preference

for

caching

in

either

tray

-LRB-

Bobserved

-LSB-

S

0 7/14

P

Bobservercontrol

-LSB-

S

0 7/12

P

-RRB-

-LRB-

table

-RRB-

The

proportion

of

caches

that

were

recached

at

recovery

did

not

differ

statistically

between

conditions

-LRB-

P

-RRB-

In

the

Bobserved

-LSB-

condition

items

were

predominantly

recached

from

the

Bobserved

-LSB-

and

not

from

the

Bother

-LSB-

tray

By

contrast

in

the

Bobservercontrol

-LSB-

condition

items

were

recached

from

both

trays

without

selectivity

-LRB-

Fig.

-RRB-

In

both

conditions

few

items

were

recached

in

Bout-oftray

-LSB-

sites

-LRB-

P

-RRB-

-LRB-

table

-RRB-

Furthermore

in

terms

of

the

behavior

of

the

birds

that

were

observing

at

recovery

there

was

no

evidence

that

observers

and

control

birds

-LRB-

that

saw

a

different

bird

at

caching

and

recovery

-RRB-

differed

in

the

amount

of

time

they

spent

nearest

to

or

oriented

toward

either

tray

-LRB-

-RRB-

These

results

suggest

that

scrub-jays

remember

who

observed

them

make

specific

caches

and

are

not

consistent

with

a

behaviorcueing

account

In

our

experiments

observers

were

never

given

the

opportunity

to

pilfer

the

caches

However

in

previous

studies

the

birds

not

only

had

their

caches

pilfered

-LRB-

-RRB-

but

witnessed

observers

stealing

their

caches

-LRB-

-RRB-

The

unpredictability

of

cache

safety

might

have

motivated

storers

to

consistently

engage

in

Fig.

-LRB-

A

-RRB-

The

observed

and

observer-control

conditions

of

experiment

The

clear

bird

indicates

the

storer

in

the

observed

and

observer-control

conditions

the

horizontal

striped

bird

indicates

an

additional

storer

in

the

observer-control

condition

the

lightly

shaded

bird

indicates

individual

A

the

vertical

striped

bird

indicates

individual

B

and

the

black

bird

indicates

the

control

bird

The

black

X

indicates

Perspex

strips

the

rectangle

indicates

the

observed

tray

-LRB-

B

-RRB-

Median

-LRB-

percentile

and

percentile

-RRB-

proportion

and

median

number

-LRB-

in

parentheses

-RRB-

of

items

recached

from

the

observed

and

other

trays

in

the

observed

-LRB-

S

0 6/6

P

-RRB-

and

observer-control

conditions

-LRB-

S

0 4/6

P

-RRB-

The

asterisk

indicates

P

G

ns

not

significant

VOL

SCIENCE

JUNE

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May

REPORTS

cache

protection

Indeed

it

was

necessary

to

use

different

birds

in

experiments

and

because

the

birds

in

experiment

ceased

to

cache

in

the

presence

of

an

observer

Cache

cessation

appeared

to

be

a

direct

consequence

of

observer

presence

as

the

birds

continued

to

cache

Bin

private

-LSB-

a

finding

perhaps

indicative

of

a

mechanism

that

acts

to

reduce

caching

levels

with

increased

observer

presence

It

is

possible

that

scrub-jay

caching

and

recovery

behavior

might

be

based

on

a

series

of

conditional

rules

rather

than

an

assessment

of

risk

That

is

jays

might

form

specific

associations

between

particular

observers

and

cache

locations

To

generate

the

flexibility

demonstrated

in

these

experiments

however

these

rules

would

have

to

be

highly

conditional

and

integrated

with

the

associations

between

a

specific

tray

and

a

particular

individual

Scrub-jays

encode

the

Bwhat-where-when

-LSB-

of

specific

caching

episodes

-LRB-

-RRB-

Our

findings

suggest

that

scrub-jays

also

remember

who

was

present

during

earlier

caching

events

This

ability

need

not

be

based

on

a

humanlike

episodic

memory

because

these

what-where-when

memories

may

exist

without

the

jay

mentally

reconstructing

the

past

-LRB-

-RRB-

Similarly

keeping

track

of

who

was

watching

when

need

not

require

a

humanlike

Btheory-of-mind

-LSB-

in

terms

of

attributing

unobservable

metal

states

-LRB-

-RRB-

but

it

may

result

from

behavioral

predispositions

in

combination

with

specific

learning

algorithms

or

from

reasoning

about

future

risk

The

propensity

for

subordinates

to

adjust

their

behavior

in

response

to

the

presence

or

absence

of

a

dominant

conspecific

has

been

suggested

as

evidence

for

knowledge

attribution

in

chimpanzees

-LRB-

-RRB-

and

ravens

-LRB-

-RRB-

Although

the

ability

of

nonhuman

animals

to

reason

about

mind

continues

to

elude

definitive

study

our

study

provides

evidence

to

suggest

that

a

nonhuman

animal

might

discriminate

between

individuals

with

different

knowledge

states

References

and

Notes

N.

Humphrey

in

Machiavellian

Intelligence

Social

Expertise

and

the

Evolution

of

Intellect

in

Monkeys

Apes

and

Humans

R.

Byrne

A.

Whiten

Eds

-LRB-

Clarendon

Press

Oxford

-RRB-

pp.

R.

Byrne

A.

Whiten

Eds.

Machiavellian

Intelligence

Social

Expertise

and

the

Evolution

of

Intellect

in

Monkeys

Apes

and

Humans

-LRB-

Clarendon

Press

Oxford

-RRB-

J.

Silk

Anim

Behav

-LRB-

-RRB-

D.

Cheney

R.

Seyfarth

Anim

Behav

-LRB-

-RRB-

A.

Engh

E.

Siebert

D.

Greenberg

K.

Holekamp

Anim

Behav

-LRB-

-RRB-

R.

Connor

M.

Heithaus

L.

Barre

Proc

R.

Soc

London

Ser

B

-LRB-

-RRB-

P.

Bednekoff

R.

Balda

Behaviour

-LRB-

-RRB-

P.

Bednekoff

R.

Balda

Anim

Behav

-LRB-

-RRB-

N.

Clayton

D.

Griffiths

N.

Emery

A.

Dickinson

Philos

Trans

R.

Soc

London

Ser

B

-LRB-

-RRB-

N.

Emery

N.

Clayton

Nature

-LRB-

-RRB-

T.

Bugnyar

K.

Kotrschal

Anim

Behav

-LRB-

-RRB-

K.

Clarkson

S.

Eden

W.

Sutherland

A.

Houston

J.

Anim

Ecol

-LRB-

-RRB-

J.

Dally

N.

Emery

N.

Clayton

Proc

R.

Soc

London

Ser

B

-LRB-

-RRB-

J.

Dally

N.

Emery

N.

Clayton

Anim

Behav

-LRB-

-RRB-

B.

Heinrich

J.

Pepper

Anim

Behav

-LRB-

-RRB-

J.

Dally

N.

Emery

N.

Clayton

Behaviour

-LRB-

-RRB-

Materials

and

methods

are

available

as

supporting

material

on

Science

Online

D.

Povinelli

J.

Vonk

Trends

Cogn

Sci

-LRB-

-RRB-

N.

Emery

J.

Dally

N.

Clayton

Anim

Cogn

-LRB-

-RRB-

N.

Clayton

A.

Dickinson

Nature

-LRB-

-RRB-

T.

Suddendorf

J.

Busby

Trends

Cogn

Sci

-LRB-

-RRB-

T.

Suddendorf

J.

Busby

Learn

Motiv

-LRB-

-RRB-

B.

Hare

J.

Call

M.

Tomasello

Anim

Behav

-LRB-

-RRB-

T.

Bugnyar

B.

Heinrich

Proc

R.

Soc

London

Ser

B

-LRB-

-RRB-

N.S.C.

received

grants

from

the

Biotechnology

and

Biological

Sciences

Research

Council

Clare

College

and

the

University

of

Cambridge

N.J.E.

was

funded

by

a

Royal

Society

University

Research

Fellowship

J.M.D.

received

financial

support

from

Clare

College

the

University

of

Cambridge

the

Sir

Richard

Stapeley

Educational

Trust

and

the

Sefton

Educational

Trust

We

thank

T.

Dickinson

N.

Davies

D.

Byrne

S.

de

Kort

C.

Rutte

N.

Humphrey

C.

Teufel

A.

Seed

D.

Alexis

A.

Miller

and

H.

Prior

for

comments

and

discussion

S.

Stevens

and

S.

de

Kort

for

allowing

reference

to

their

data

and

D.

Alexis

C.

Teufel

and

A.

Seed

for

acting

as

observers

We

are

indebted

to

B.

Brown

for

his

statistical

expertise

Supporting

Online

Material

Materials

and

Methods

Tables

to

References

February

accepted

May

Published

online

May

science

Include

this

information

when

citing

this

paper

Regulation

of

B

Cell

Tolerance

by

the

Lupus

Susceptibility

Gene

Kirthi

Raman

Kumar

Liunan

Li

Mei

Yan

Madhavi

Bhaskarabhatla

Angela

B.

Mobley

Charles

Nguyen

Jill

M.

Mooney

John

D.

Schatzle

Edward

K.

Wakeland

Chandra

The

susceptibility

locus

for

the

autoimmune

disease

lupus

on

murine

chromosome

and

the

orthologous

human

locus

are

associated

with

production

of

autoantibody

to

chromatin

We

report

that

the

presence

of

impairs

B

cell

anergy

receptor

revision

and

deletion

Members

of

the

SLAM

costimulatory

molecule

family

constitute

prime

candidates

for

among

which

the

isoform

of

the

gene

was

most

highly

expressed

in

immature

B

cells

from

lupus-prone

mice

The

normal

allele

but

not

the

lupus-associated

allele

was

found

to

sensitize

immature

B

cells

to

deletion

and

RAG

reexpression

As

a

potential

regulator

of

tolerance

checkpoints

may

censor

self-reactive

B

cells

hence

safeguarding

against

autoimmunity

oss

of

B

cell

tolerance

is

the

hallmark

of

systemic

lupus

erythematosus

-LRB-

SLE

-RRB-

or

lupus

an

antibody-mediated

chronic

autoimmune

disease

affecting

multiple

organs

However

the

precise

means

by

which

tolerance

is

breached

in

lupus

and

the

underlying

genes

and

molecules

responsible

remain

obscure

The

origin

of

lupus

in

both

mice

and

humans

appears

to

be

polygenic

involving

more

than

a

dozen

potent

loci

-LRB-

-RRB-

although

of

these

at

least

one

L

locus

on

chromosome

appears

to

be

shared

by

both

species

The

z

allele

of

and

its

component

sublocus

derived

from

the

lupus-prone

strain

of

mice

are

linked

to

a

variety

of

lupus-related

disease

phenotypes

including

antinuclear

antibodies

-LRB-

ANAs

-RRB-

splenomegaly

and

glomerulonephritis

-LRB-

-RRB-

A

locus

on

human

chromosome

orthologous

to

murine

has

also

been

implicated

in

lupus

susceptibility

-LRB-

-RRB-

Located

within

SCIENCE

VOL

the

sublocus

the

SLAM

gene

family

encoded

costimulatory

molecules

are

among

the

first

candidate

genes

to

be

identified

as

being

linked

to

spontaneous

lupus

in

mice

-LRB-

-RRB-

Extensive

polymorphisms

and

expression

differences

were

documented

in

several

members

of

this

gene

cluster

between

the

normal

-LRB-

-RRB-

strain

and

the

lupus-prone

z

congenic

mice

bearing

the

z

haplotype

of

SLAM

-LRB-

-RRB-

To

determine

the

mechanisms

by

which

B

cell

tolerance

might

be

infringed

in

lupus

the

B

cell

repertoire

of

z

congenic

mice

-LRB-

-RRB-

was

modified

to

an

essentially

monoclonal

specificity

by

breeding

onto

them

a

B

cell

receptor

-LRB-

BCR

-RRB-

transgene

specific

for

lysozyme

-LRB-

HEL

-RRB-

-LRB-

-RRB-

In

these

mice

the

HELIg

BCR

transgene

leads

to

a

near-uniform

pool

of

HEL-specific

B

cells

bearing

a

BCR

heavy

chain

of

immunoglobulin

Ma

-LRB-

IgMa

-RRB-

allotype

and

high

titers

of

serum

antibodies

to

HEL

of

IgMa

allotype

-LRB-

-RRB-

Developmental

exposure

of

these

B

cells

to

a

strong

cross-linking

surrogate

Department

of

Internal

Medicine

-LRB-

Rheumatology

-RRB-

for

Immunology

of

Pathology

University

of

Texas

Southwestern

Medical

Center

Dallas

TX

USA

To

whom

correspondence

should

be

addressed

E-mail

chandra.mohan@utsouthwestern.edu

JUNE

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May

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


letters to nature breaks induced at ectopic transcription units 21 activated by demethylation . The data presented here show that Dnmt3L is required for heritable silencing of retrotransposons in male germ cells . Temporary deprivation of Dnmt3L through drug-induced conditional alleles of the Dnmt3L gene might allow the controlled mobilization of endogenous retrotransposons without meiotic catastrophe and could form the basis of a system of retrotransposon insertional mutagenesis that would be useful in forward genetic screens in the mouse . A Received 22 May ; accepted 26 July 2004 ; doi : 10.1038 / nature02886 . Published online 18 August 2004 . 1 . Aapola , U. et al. . Isolation and initial characterization of a novel zinc finger gene , DNMT3L , on 21q22 .3 , related to the cytosine-5-methyltransferase 3 gene family . Genomics 65 , 293 298 -LRB- 2000 -RRB- . 2 . Hata , K. , Okano , M. , Lei , H. & Li , E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice . Development 129 , 1983 1993 -LRB- 2002 -RRB- . 3 . Bourc ` his , D. , Xu , G. L. , Lin , C. S. , Bollman , B. & Bestor , T. H. Dnmt3L and the establishment of maternal genomic imprints . Science 294 , 2536 2539 -LRB- 2001 -RRB- . 4 . La Salle , S. et al. . Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells . Dev . Biol . 268 , 403 415 -LRB- 2004 -RRB- . 5 . Dobson , M. J. , Pearlman , R. E. , Karaiskakis , A. , Spyropoulos , B. & Moens , P. B. Synaptonemal complex proteins : occurrence , epitope mapping and chromosome disjunction . J. Cell Sci . 107 , 2749 2760 -LRB- 1994 -RRB- . 6 . Odorisio , T. , Rodriguez , T. A. , Evans , E. P. , Clarke , A. R. & Burgoyne , P. S. Meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-independent apoptosis . Nature Genet . 18 , 257 261 -LRB- 1998 -RRB- . 7 . Enders , G. C. & May , J. J. Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female mice . Dev . Biol . 163 , 331 340 -LRB- 1994 -RRB- . 8 . Yoder , J. A. , Walsh , C. P. & Bestor , T. H. Cytosine methylation and the ecology of intragenomic parasites . Trends Genet . 13 , 335 340 -LRB- 1997 -RRB- . 9 . Lin , S. P. et al. . Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1 Gtl2 imprinted cluster on mouse chromosome 12 . Nature Genet . 35 , 97 102 -LRB- 2003 -RRB- . 10 . Davis , T. L. , Trasler , J. M. , Moss , S. B. , Yang , G. J. & Bartolomei , M. S. Acquisition of the H19 methylation imprint occurs differentially on the parental alleles during spermatogenesis . Genomics 58 , 18 28 -LRB- 1999 -RRB- . 11 . Ueda , T. et al. . The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development . Genes Cells 5 , 649 659 -LRB- 2000 -RRB- . 12 . Bestor , T. H. Cytosine methylation mediates sexual conflict . Trends Genet . 19 , 185 190 -LRB- 2003 -RRB- . 13 . Walsh , C. P. , Chaillet , J. R. & Bestor , T. H. Transcription of IAP endogenous retroviruses is constrained by cytosine methylation . Nature Genet . 20 , 116 117 -LRB- 1998 -RRB- . 14 . Kuff , E. L. & Lueders , K. K . The intracisternal A-particle gene family : structure and functional aspects . Adv. Cancer Res . 51 , 183 276 -LRB- 1988 -RRB- . 15 . Ostertag , E. M. & Kazazian , H. H. Jr Biology of mammalian L1 retrotransposons . Annu . Rev. Genet . 35 , 501 538 -LRB- 2001 -RRB- . 16 . Kaneda , M. et al. . Essential role for de novo methyltransferase 3a in paternal and maternal imprinting . Nature 429 , 900 903 -LRB- 2004 -RRB- . 17 . Bestor , T. H. & Tycko , B. Creation of genomic methylation patterns . Nature Genet . 12 , 363 367 -LRB- 1996 -RRB- . 18 . Chedin , F. , Lieber , M. R. & Hsieh , C. L . The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a . Proc . Natl Acad . Sci . USA 99 , 16916 16921 -LRB- 2002 -RRB- . 19 . Cambareri , E. B. , Jensen , B. C. , Schabtach , E. & Selker , E. U. Repeat-induced G-C to A-T mutations in Neurospora . Science 244 , 1571 1575 -LRB- 1989 -RRB- . 20 . Maloisel , L. & Rossignol , J.-L . Suppression of crossing-over by DNA methylation in Ascobolus . Genes Dev . 12 , 1381 1389 -LRB- 1998 -RRB- . 21 . Petes , T. D. Meiotic recombination hot spots and cold spots . Nature Rev. Genet . 2 , 360 369 -LRB- 2001 -RRB- . 22 . Peters , A. H. , Plug , A. W. , van Vugt , M. J. & de Boer , P . A drying-down technique for the spreading of mammalian meiocytes from the male and female germline . Chromosome Res . 5 , 66 68 -LRB- 1997 -RRB- . 23 . Ponzetto-Zimmerman , C. & Wolgemuth , D. J. Methylation of satellite sequences in mouse spermatogenic and somatic DNAs . Nucleic Acids Res . 12 , 2807 2822 -LRB- 1984 -RRB- . 24 . Sanford , J. , Forrester , L. & Chapman , V. Methylation patterns of repetitive DNA sequences in germ cells of Mus musculus . Nucleic Acids Res . 12 , 2822 2835 -LRB- 1984 -RRB- . 25 . Pietras , D. F. et al. . Construction of a small Mus musculus repetitive DNA library : identification of a new satellite sequence in Mus musculus . Nucleic Acids Res . 11 , 6965 6983 -LRB- 1983 -RRB- . ... . Transcriptional regulatory code of a eukaryotic genome Christopher T. Harbison1 ,2 * , D. Benjamin Gordon1 * , Tong Ihn Lee1 , Nicola J. Rinaldi1 ,2 , Kenzie D. Macisaac3 , Timothy W. Danford3 , Nancy M. Hannett1 , Jean-Bosco Tagne1 , David B. Reynolds1 , Jane Yoo1 , Ezra G. Jennings1 , Julia Zeitlinger1 , Dmitry K. Pokholok1 , Manolis Kellis1 ,3,4 , P. Alex Rolfe3 , Ken T. Takusagawa3 , Eric S. Lander1 ,2,4 , David K. Gifford3 ,4 , Ernest Fraenkel1 ,3 & Richard A. Young1 ,2,4 1 Whitehead Institute for Biomedical Research , Nine Cambridge Center , Cambridge , Massachusetts 02142 , USA 2 Department of Biology , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , USA 3 MIT Computer Science and Artificial Intelligence Laboratory , 32 Vassar Street , Cambridge , Massachusetts 02139 , USA 4 Broad Institute , One Kendall Square , Building 300 , Cambridge , Massachusetts 02139 , USA * These authors contributed equally to this work ... . Acknowledgements We thank B. Tycko for suggestions , P. Moens for antibodies against synaptonemal complex proteins and H1t and for discussions , G. Enders for antibodies against GCNA1 , J. Walonoski for advice on in situ hybridization , and K. Anderson , M. Damelin and M. Goll for criticism of the manuscript . This work was supported by grants from the National Institutes of Health to T.H.B. and by a fellowship from the Rett Syndrome Research Foundation to D.B. Competing interests statement The authors declare that they have no competing financial interests . Correspondence and requests for materials should be addressed to T.H.B -LRB- thb12@columbia.edu -RRB- . Dnmt3L tm1Bes mutant mice are available from the Jackson Labs -LRB- http://jaxmice.jax.org/ -RRB- . NATURE | VOL 431 | 2 SEPTEMBER 2004 | www.nature.com/nature DNA-binding transcriptional regulators interpret the genome 's regulatory code by binding to specific sequences to induce or repress gene expression1 . Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation2 6 , but this information alone does not reveal if or when transcriptional regulators occupy these binding sites . We have constructed an initial map of yeast 's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species . The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed . We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast 's transcriptional regulators . We used genome-wide location analysis7 10 to determine the genomic occupancy of 203 DNA-binding transcriptional regulators in rich media conditions and , for 84 of these regulators , in at least 1 of 12 other environmental conditions -LRB- Supplementary Table 1 , Supplementary Fig. 1 ; http://web.wi.mit.edu/young/regulatory_ code -RRB- . These 203 proteins are likely to include nearly all of the DNA-binding transcriptional regulators encoded in the yeast genome . Regulators were selected for profiling in an additional environment if they were essential for growth in that environment or if there was other evidence implicating them in the regulation of gene expression in that environment . The genome-wide location data identified 11,000 unique interactions between regulators and promoter regions at high confidence -LRB- P # 0.001 -RRB- . To identify the cis-regulatory sequences that are likely to serve as recognition sites for transcriptional regulators , we merged information from genome-wide location data , phylogenetically conserved sequences , and prior knowledge -LRB- Fig. 1a -RRB- . We used six motif discovery methods11 13 to discover 68,279 DNA sequence motifs for the 147 regulators that bound more than ten probes -LRB- Supplementary Methods , Supplementary Fig. 2 -RRB- . From these motifs we derived the most likely specificity for each regulator through clustering and stringent statistical tests . This motif discovery process identified highly significant -LRB- P # 0.001 -RRB- motifs for each of 116 regulators . We determined a single high-confidence motif for 65 of these regulators by using additional criteria including the requirement for conservation across three of four related yeast species . Examples of discovered and rediscovered motifs are depicted in Fig. 1b , and comparisons of the discovered motifs with those described previously are shown in Supplementary Table 2 . 99 2004 Nature Publishing Group letters to nature The discovered motifs provide significantly more information than was previously available ; for 21 of the regulators there was no prior specificity information in the literature , and detailed probability matrices had previously been determined for only 17 regulators for which we report motifs14 . For Cin5 , which showed the largest difference between the computationally derived motif -LRB- TTACRTAA -RRB- and the previously reported site -LRB- TTACTAA ; Supplementary Table 2 -RRB- , we found that the motif we report is also the preferred target in vitro -LRB- Supplementary Fig. 3 -RRB- . We supplemented the discovered motifs with additional motifs from the literature that also passed conservation tests , and we used this compendium of sequence motifs for 102 regulators -LRB- Supplementary Table 3 -RRB- in all subsequent analysis . We constructed an initial version of the transcriptional regulatory code by mapping on the yeast genome sequence the motifs that are bound by regulators at high confidence -LRB- P # 0.001 -RRB- and that are conserved among sensu stricto Saccharomyces species -LRB- Fig. 2 ; http : / / web.wi.mit.edu/fraenkel/regulatory_map -RRB- . This map includes 3,353 interactions within 1,296 promoter regions . Maps of regulatory sites encompassing larger numbers of promoters , constructed with lower-confidence information , can also be viewed on the authors ' website . Because the information used to construct the map includes binding data from multiple growth environments , the map describes transcriptional regulatory potential within the genome . During growth in any one environment , only subsets of the binding sites identified in the map are occupied by transcriptional regulators , as we describe in more detail below . Where the functions of specific transcriptional regulators were established previously , the functions of the genes they bind in the regulatory map are highly consistent with this prior information . For example , the amino-acid biosynthetic regulators Gcn4 and Leu3 bind to sites in the promoter of BAP2 -LRB- chromosome II -RRB- , which encodes an amino-acid transporter -LRB- Fig. 2a -RRB- . Six well-studied cell cycle transcriptional regulators bind to the promoter for YHP1 -LRB- chromosome IV -RRB- , which has been implicated in the regulation of the G1 phase of the cell cycle . The regulator of respiration Hap5 binds upstream of COX4 -LRB- chromosome VII -RRB- , which encodes a component of the respiratory electron transport chain . Where regulators with established functions bind to genes of unknown function , these target genes are newly implicated in such functional processes . The utility of combining regulator binding data and sequence conservation data is illustrated in Fig. 2b . All sequences matching the regulator DNA binding specificities described in this study -LRB- Supplementary Table 3 -RRB- that occur within the 884-base-pair intergenic region upstream of the gene BAP2 are shown in the upper panel . The subset of these sequences that have been conserved in multiple yeast species , and are therefore likely candidates for Figure 1 Discovering binding-site specificities for yeast transcriptional regulators . a , Cis-regulatory sequences likely to serve as recognition sites for transcriptional regulators were identified by combining information from genome-wide location data , phylogenetically conserved sequences and previously published evidence , as described in Supplementary Methods . The compendium of regulatory sequence motifs can be found in 100 Supplementary Table 3 . b , Selected sequence specificities that were rediscovered and were newly discovered are shown . The total height of the column is proportional to the information content of the position , and the individual letters have a height proportional to the product of their frequency and the information content30 . 2004 Nature Publishing Group NATURE | VOL 431 | 2 SEPTEMBER 2004 | www.nature.com/nature letters to nature regulator interactions , is shown in the middle panel of Fig. 2b . The presence of these conserved regulatory sites indicates the potential for regulation through this sequence but does not indicate whether the site is actually bound by a regulator under some growth condition . The incorporation of binding information -LRB- Fig. 2b , bottom panel -RRB- identifies those conserved sequences that are used by regulators in cells grown under the conditions examined . The distribution of binding sites for transcriptional regulators reveals constraints on the organization of these sites in yeast promoters -LRB- Fig. 2c -RRB- . Binding sites are not uniformly distributed over the promoter regions but instead show a sharply peaked distribution . Very few sites are located in the region 100 base pairs -LRB- bp -RRB- upstream of protein-coding sequences . This region typically includes the transcription start site and is bound by the transcription initiation apparatus . The vast majority -LRB- 74 % -RRB- of the transcriptional regulator binding sites lie between 100 and 500 bp upstream of the protein-coding sequence , far more than would be expected at random -LRB- 53 % -RRB- . Regions further than 500 bp upstream contain fewer binding sites than would be expected at random . It seems that yeast transcriptional regulators function at short distances along the linear DNA , a property that reduces the potential for inappropriate activation of nearby genes . We note that specific arrangements of DNA binding sites occur within promoters , and we suggest that these promoter architectures provide clues to regulatory mechanisms -LRB- Fig. 3 -RRB- . For example , the presence of a DNA binding site for a single regulator is the simplest promoter architecture and , as might be expected , we found that sets of genes with this feature are often involved in a common biological Figure 2 Drafting the yeast transcriptional regulatory map . a , Portions of chromosomes illustrating locations of genes -LRB- grey rectangles -RRB- and conserved DNA sequences -LRB- coloured boxes -RRB- bound in vivo by transcriptional regulators . b , Combining binding data and sequence conservation data . The diagram depicts all sequences matching a motif from our compendium -LRB- top -RRB- , all such conserved sequences -LRB- middle -RRB- and all such conserved NATURE | VOL 431 | 2 SEPTEMBER 2004 | www.nature.com/nature sequences bound by a regulator -LRB- bottom -RRB- . c , Regulator binding site distribution . The red line shows the distribution of distances from the start codon of open reading frames to binding sites in the adjacent upstream region . The green line represents a randomized distribution . 2004 Nature Publishing Group 101 letters to nature function -LRB- Supplementary Table 4 -RRB- . A second type of promoter architecture consists of repeats of a particular binding site sequence . Repeated binding sites have been shown to be necessary for stable binding by the regulator Dal80 -LRB- ref . 15 -RRB- . This repetitive promoter architecture can also permit a graded transcriptional response , as has been observed for the HIS4 gene16 . Several regulators , including Dig1 , Mbp1 and Swi6 , show a statistically significant preference for repetitive motifs -LRB- Supplementary Table 5 -RRB- . A third class of promoter contains binding sites for multiple different regulators . This promoter arrangement implies that the gene might be subject to combinatorial regulation , and we expect that in many cases the various regulators can be used to execute differential responses to varied growth conditions . Indeed , we note that many of the genes in this category encode products that are required for multiple metabolic pathways and are regulated in an environment-specific fashion . In the fourth type of promoter architecture we discuss here , binding sites for specific pairs of regulators occur more frequently within the same promoter regions than would be expected by chance -LRB- Supplementary Table 6 -RRB- . This ` co-occurring ' motif architecture implies that the two regulators interact physically or have related functions at multiple genes . By conducting genome-wide binding experiments for some regulators under multiple cell-growth conditions , we learned that regulator binding to a subset of the regulatory sequences is highly dependent on the environmental conditions of the cell -LRB- Supplementary Fig. 4 -RRB- . We observed four common patterns of regulator binding behaviour -LRB- Fig. 4 , Supplementary Table 7 -RRB- . Prior information about the regulatory mechanisms employed by well-studied regulators in each of the four groups suggests hypotheses to account for the environment-dependent binding behaviour of the other regulators . ` Condition-invariant ' regulators bind essentially the same set of promoters -LRB- within the limitations of noise -RRB- in two different growth environments -LRB- Fig. 4 -RRB- . Leu3 , which is known to regulate genes involved in amino-acid biosynthesis , is among the best studied of the regulators in this group . Binding of Leu3 in vivo has been shown to be necessary but not sufficient for the activation of Leu3-regulated genes17 . Rather , regulatory control of these genes requires the association of a leucine metabolic precursor with Leu3 to convert it from a negative to a positive regulator . We note that other zinc cluster type regulators that show ` condition-invariant ' behaviour are known to be regulated in a similar manner18 ,19 . It is therefore reasonable to propose that the activation or repression functions of some of the other regulators in this class have requirements in addition to DNA binding . ` Condition-enabled ' regulators do not bind the genome detectably under one condition , but bind a substantial number of promoters with a change in environment . Msn2 is among the best-studied regulators in this class , and the mechanisms involved in Msn2-dependent transcription provide clues to how the other regulators in that class might operate . Msn2 is excluded from the nucleus when cells grow in the absence of stresses but accumulates rapidly in the nucleus when cells are subjected to stress20 ,21 . This condition-enabled behaviour was also observed for the thiamine biosynthetic regulator Thi2 , the nitrogen regulator Gat1 and the developmental regulator Rim101 . We suggest that many of these transcriptional regulators are regulated by nuclear exclusion or by another mechanism that would cause this extreme version of condition-specific binding . ` Condition-expanded ' regulators bind to a core set of target promoters under one condition but bind an expanded set of promoters under another condition . Gcn4 is the best-studied of the regulators that fall into this ` expanded ' class . The levels of Gcn4 are reported to increase sixfold when yeast cells are introduced into media with limiting nutrients22 , owing largely to increased nuclear protein stability21 ,23 , and under this condition we find that Gcn4 binds to an expanded set of genes . The probes bound when Gcn4 102 Figure 3 Yeast promoter architectures : single regulator architecture , promoter regions that contain one or more copies of the binding site sequence for a single regulator ; repetitive motif architecture , promoter regions that contain multiple copies of a binding site sequence of a regulator ; multiple regulator architecture , promoter regions that contain one or more copies of the binding site sequences for more than one regulator ; co-occurring regulator architecture , promoters that contain binding site sequences for recurrent pairs of regulators . For the purposes of illustration , not all sites are shown and the scale is approximate . Additional information can be found in Supplementary Tables 4 6 . levels are low contain better matches to the known Gcn4-binding site than probes that are bound exclusively at higher protein concentrations , which is consistent with a simple model for specificity based on intrinsic protein affinity and protein concentration -LRB- Supplementary Fig. 5 -RRB- . The expansion of binding sites by many of the regulators in this class might reflect increased levels of the regulator available for DNA binding . ` Condition-altered ' regulators exhibit an altered preference for the set of promoters bound in two different conditions . Ste12 is the best-studied of the regulators whose binding behaviour falls into this ` altered ' class . Depending on the interactions with other regulators , the specificity of Ste12 can change and alter its cellular function24 . For example , under filamentous growth conditions , Ste12 interacts with Tec1 , which has its own DNA-binding specificity25 . This condition-altered behaviour was also observed for the transcriptional regulators Aft2 , Skn7 and Ume6 . We propose that the binding specificity of many of the transcriptional regulators might be altered through interactions with other regulators or through modifications -LRB- such as chemical -RRB- that are dependent on environment . Substantial portions of eukaryotic genome sequence are believed to be regulatory2 ,3,26 , but the DNA sequences that actually contribute to regulation of genome expression have been ill-defined . By mapping the DNA sequences bound by specific regulators in various environments , we identify the regulatory potential embedded in the genome and provide a framework for modelling the mechanisms NATURE | VOL 431 | 2 SEPTEMBER 2004 | www.nature.com/nature 2004 Nature Publishing Group letters to nature Figure 4 Environment-specific use of the transcriptional regulatory code . Four patterns of genome-wide binding behaviour are depicted on the left , where transcriptional regulators are represented by coloured circles and are placed above and below a set of target genes / promoters . The lines between the regulators and the target genes/promoters represent binding events . Specific examples of the environment-dependent behaviours are depicted on the right . Coloured circles represent regulators and coloured boxes represent their DNA binding sequences within specific promoter regions . We note that regulators might exhibit different behaviours when different pairs of conditions are compared . that contribute to global gene expression . We expect that the approaches used here to map regulatory sequences in yeast can also be used to map the sequences that control genome expression in higher eukaryotes . A Methods Strain information For each of the 203 regulators , strains were generated in which a repeated Myc-epitopecoding sequence was integrated into the endogenous gene encoding the regulator . Polymerase chain reaction -LRB- PCR -RRB- constructs containing the Myc-epitope-coding sequence and a selectable marker flanked by regions of homology to either the 5 0 or 3 0 end of the targeted gene were transformed into the W303 yeast strain Z1256 -LRB- refs 8 , 9 -RRB- . Genomic integration and expression of the epitope-tagged protein were confirmed by PCR and western blotting , respectively . Genome-wide location analysis Genome-wide location analysis was performed as described previously8 ,9 . Bound proteins were crosslinked by formaldehyde to DNA in vivo , followed by cell lysis and sonication to shear DNA . Crosslinked material was immunoprecipitated with an anti-Myc antibody , followed by reversal of the crosslinks to separate DNA from protein . Immunoprecipitated DNA and DNA from an unenriched sample were amplified and differentially fluorescently labelled by ligation-mediated PCR . These samples were hybridized to a microarrray consisting of spotted PCR products representing the intergenic regions of the Saccharomyces cerevisiae genome . Relative intensities of spots were used as the basis for an error model that assigns a probability score -LRB- P -RRB- to binding interactions . All microarray data are available from ArrayExpress and from the authors ' website -LRB- http : / / web.wi.mit.edu/young/regulatory_code -RRB- . discovery programs to the intergenic sequences identified by the binding data . The resulting specificity predictions were filtered for significance with uniform metrics and then clustered to yield representative motifs -LRB- Supplementary Fig. 2 -RRB- . We used six methods to identify the specific sequences bound by regulators : AlignACE11 , MEME13 , MDscan12 , the method in ref . 2 and two additional new methods that incorporate conservation data : MEME_c and CONVERGE . MEME_c uses the existing MEME program without change but applies it to a modified set of sequences in which bases that are not conserved in the sensu stricto Saccharomyces species were replaced with the letter ` N ' . CONVERGE is a novel expectation-maximization -LRB- EM -RRB- - based algorithm for discovering specificities by using sequence information from multiple genomes . Rather than searching for sites that are identical across the sensu stricto species , as occurs with MEME_c , CONVERGE searches for loci at which all aligned sequences are consistent with the same specificity model . See Supplementary Methods for runtime parameters and additional details for all of these methods . Each of the programs we used attempts to measure the significance of its results with one or more statistical scores . However , we observed that these programs report results with high scores even when applied to random selections of intergenic regions . To distinguish the true motifs we chose a set of statistical measures that are described in Supplementary Methods and converted these scores into the empirical probability that a motif with a similar score could be found by the same program in randomly selected sequences . To estimate these P values we ran each program 50 times on randomly selected sets of sequences of various sizes . We accepted only those motifs that were judged to be significant by these scores -LRB- P # 0.001 -RRB- . Significant motifs from all programs were pooled and clustered with the use of a k-medoids algorithm . Aligned motifs within each cluster were averaged to produce consensus motifs and filtered according to their conservation . This procedure typically produced several distinct consensus motifs for each regulator . To choose a single specificity for each regulator we compared the results with information in the TRANSFAC27 , YPD28 and SCPD29 databases . When no prior information was available we chose the specificity with the most significant statistical score . Growth environments We profiled all 203 regulators in rich medium . In addition , we profiled 84 regulators in at least one other environmental condition . The list of regulators is given in Supplementary Table 1 . Regulatory code Potential binding sites were included in the map of the regulatory code if they satisfied two criteria . First , a locus had to match the specificity model for a regulator in the S. cerevisiae genome and at least two other sensu stricto Saccharomyces genomes with a score of at least 60 % of the maximum possible . Second , the locus had to lie in an intergenic region that also contained a probe bound by the corresponding regulator in any condition -LRB- P # 0.001 -RRB- . All Regulator binding specificity The putative specificities of regulators were identified by applying a suite of motif NATURE | VOL 431 | 2 SEPTEMBER 2004 | www.nature.com/nature 2004 Nature Publishing Group 103 letters to nature analyses of promoter architecture and environment-specific binding were based on this map and can be found in Supplementary Information . Received 11 March ; accepted 1 July 2004 ; doi : 10.1038 / nature02800 . 1 . Jacob , F. & Monod , J. Genetic regulatory mechanisms in the synthesis of proteins . J. Mol . Biol . 3 , 318 356 -LRB- 1961 -RRB- . 2 . Kellis , M. , Patterson , N. , Endrizzi , M. , Birren , B. & Lander , E. S. Sequencing and comparison of yeast species to identify genes and regulatory elements . Nature 423 , 241 254 -LRB- 2003 -RRB- . 3 . Cliften , P. et al. . Finding functional features in Saccharomyces genomes by phylogenetic footprinting . Science 301 , 71 76 -LRB- 2003 -RRB- . 4 . Pritsker , M. , Liu , Y. C. , Beer , M. A. & Tavazoie , S. Whole-genome discovery of transcription factor binding sites by network-level conservation . Genome Res . 14 , 99 108 -LRB- 2004 -RRB- . 5 . Wang , T. & Stormo , G. D. Combining phylogenetic data with co-regulated genes to identify regulatory motifs . Bioinformatics 19 , 2369 2380 -LRB- 2003 -RRB- . 6 . Blanchette , M. & Tompa , M. FootPrinter : A program designed for phylogenetic footprinting . Nucleic Acids Res . 31 , 3840 3842 -LRB- 2003 -RRB- . 7 . Iyer , V. R. et al. . Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF . Nature 409 , 533 538 -LRB- 2001 -RRB- . 8 . Ren , B. et al. . Genome-wide location and function of DNA binding proteins . Science 290 , 2306 2309 -LRB- 2000 -RRB- . 9 . Lee , T. I. et al. . Transcriptional regulatory networks in Saccharomyces cerevisiae . Science 298 , 799 804 -LRB- 2002 -RRB- . 10 . Lieb , J. D. , Liu , X. , Botstein , D. & Brown , P. O. Promoter-specific binding of Rap1 revealed by genome wide maps of protein-DNA association . Nature Genet . 28 , 327 334 -LRB- 2001 -RRB- . 11 . Roth , F. P. , Hughes , J. D. , Estep , P. W. & Church , G. M. Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation . Nature Biotechnol . 16 , 939 945 -LRB- 1998 -RRB- . 12 . Liu , X. S. , Brutlag , D. L. & Liu , J. S . An algorithm for finding protein-DNA binding sites with applications to chromatin-immunoprecipitation microarray experiments . Nature Biotechnol . 20 , 835 839 -LRB- 2002 -RRB- . 13 . Bailey , T. L. & Elkan , C. Proc . Int . Conf . Intell . Syst . Mol . Biol . Vol . 3 21 29 -LRB- AAAI Press , Menlo Park , California , 1995 -RRB- . 14 . Knuppel , R. , Dietze , P. , Lehnberg , W. , Frech , K. & Wingender , E. TRANSFAC retrieval program : a network model database of eukaryotic transcription regulating sequences and proteins . J. Comput . Biol . 1 , 191 198 -LRB- 1994 -RRB- . 15 . Cunningham , T. S. & Cooper , T. G . The Saccharomyces cerevisiae DAL80 repressor protein binds to multiple copies of GATAA-containing sequences -LRB- URSGATA -RRB- . J. Bacteriol . 175 , 5851 5861 -LRB- 1993 -RRB- . 16 . Donahue , T. F. , Daves , R. S. , Lucchini , G. & Fink , G. R . A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast . Cell 32 , 89 98 -LRB- 1983 -RRB- . 17 . Kirkpatrick , C. R. & Schimmel , P. Detection of leucine-independent DNA site occupancy of the yeast Leu3p transcriptional activator in vivo . Mol . Cell . Biol . 15 , 4021 4030 -LRB- 1995 -RRB- . 18 . Axelrod , J. D. , Majors , J. & Brandriss , M. C. Proline-independent binding of PUT3 transcriptional activator protein detected by footprinting in vivo . Mol . Cell . Biol . 11 , 564 567 -LRB- 1991 -RRB- . 19 . Ma , J. & Ptashne , M . The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80 . Cell 50 , 137 142 -LRB- 1987 -RRB- . 20 . Beck , T. & Hall , M. N . The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors . Nature 402 , 689 692 -LRB- 1999 -RRB- . 21 . Chi , Y. et al. . Negative regulation of Gcn4 and Msn2 transcription factors by Srb10 cyclin-dependent kinase . Genes Dev . 15 , 1078 1092 -LRB- 2001 -RRB- . 22 . Albrecht , G. , Mosch , H. U. , Hoffmann , B. , Reusser , U. & Braus , G. H. Monitoring the Gcn4 proteinmediated response in the yeast Saccharomyces cerevisiae . J. Biol . Chem . 273 , 12696 12702 -LRB- 1998 -RRB- . 23 . Kornitzer , D. , Raboy , B. , Kulka , R. G. & Fink , G. R. Regulated degradation of the transcription factor Gcn4 . EMBO J. 13 , 6021 6030 -LRB- 1994 -RRB- . 24 . Zeitlinger , J. et al. . Program-specific distribution of a transcription factor dependent on partner transcription factor and MAPK signaling . Cell 113 , 395 404 -LRB- 2003 -RRB- . 25 . Baur , M. , Esch , R. K. & Errede , B. Cooperative binding interactions required for function of the Ty1 sterile responsive element . Mol . Cell . Biol . 17 , 4330 4337 -LRB- 1997 -RRB- . 26 . Waterston , R. H. et al. . Initial sequencing and comparative analysis of the mouse genome . Nature 420 , 520 562 -LRB- 2002 -RRB- . 27 . Matys , V. et al. . TRANSFAC : transcriptional regulation , from patterns to profiles . Nucleic Acids Res . 31 , 374 378 -LRB- 2003 -RRB- . 28 . Hodges , P. E. , McKee , A. H. , Davis , B. P. , Payne , W. E. & Garrels , J. I . The Yeast Proteome Database -LRB- YPD -RRB- : a model for the organization and presentation of genome-wide functional data . Nucleic Acids Res . 27 , 69 73 -LRB- 1999 -RRB- . 29 . Zhu , J. & Zhang , M. Q. SCPD : a promoter database of the yeast Saccharomyces cerevisiae . Bioinformatics 15 , 607 611 -LRB- 1999 -RRB- . 30 . Schneider , T. D. & Stephens , R. M. Sequence logos : a new way to display consensus sequences . Nucleic Acids Res . 18 , 6097 6100 -LRB- 1990 -RRB- . ... . Structure of the acrosomal bundle Michael F. Schmid1 , Michael B. Sherman1 * , Paul Matsudaira2 & Wah Chiu1 1 National Center for Macromolecular Imaging , Verna and Marrs McLean Department of Biochemistry and Molecular Biology , Baylor College of Medicine , Houston , Texas 77030 , USA 2 Whitehead Institute , Department of Biology and Division of Biological Engineering MIT , Cambridge , Massachusetts 02142 , USA * Present address : Purdue University , Department of Biological Sciences , West Lafayette , Indiana 47907-139 , USA ... . Supplementary Information accompanies the paper on www.nature.com/nature . Acknowledgements We thank T. Ideker and S. McCuine for help in selecting regulators to study in environmental conditions ; E. Herbolsheimer , G. Bell , R. Latek and F. Lewitter for computational assistance ; and E. McReynolds for technical assistance . E.F. is a Whitehead Fellow and was funded in part by Pfizer . D.B.G. was supported by a NIH/NIGMS NRSA award . This work was supported by an NIH grant . Competing interests statement The authors declare competing financial interests : details accompany the paper on www.nature.com/nature . Correspondence and requests for materials should be addressed to R.A.Y. -LRB- young@wi.mit.edu -RRB- or E.F. -LRB- efraenkel@wi.mit.edu -RRB- . ArrayExpress number E-WMIT-10 has been given for microarray data . In the unactivated Limulus sperm , a 60-mm-long bundle of actin filaments crosslinked by the protein scruin is bent and twisted into a coil around the base of the nucleus . At fertilization , the bundle uncoils and fully extends in five seconds to support a finger of membrane known as the acrosomal process . This biological spring is powered by stored elastic energy and does not require the action of motor proteins or actin polymeriza tion1 . In a 9.5-A electron cryomicroscopic structure of the extended bundle , we show that twist , tilt and rotation of actin scruin subunits deviate widely from a ` standard ' F-actin filament . This variability in structural organization allows filaments to pack into a highly ordered and rigid bundle in the extended state and suggests a mechanism for storing and releasing energy between coiled and extended states without disassembly . The actin bundle of the Limulus acrosomal process is an unusual actin-based engine that is highly ordered in its structure2 . The coiled state has a difference in average twist of 0.238 per subunit relative to the extended form . Change in the twist accompanies uncoiling of the bundle into the final extended state3 . Mechanical measurements of the extended bundle yield a Young 's modulus -LRB- a measure of stiffness -RRB- of 2 GPa -LRB- refs 4 6 -RRB- . This stiffness enables the bundle to penetrate the jelly layer surrounding the egg membrane . A 40-A tomographic reconstruction7 revealed periodic and dense packing of the scruin-crosslinked actin filaments . However , to understand all these functions , a higher resolution structure is necessary . An electron micrograph of the native extended bundle shows its crystallinity -LRB- Fig. 1a -RRB- . We merged data from 153 such bundles at various orientations8 to produce a three-dimensional map at a resolution of 9.5 A -LRB- Fig. 1b -RRB- . As far as we know , this is the highest resolution of any electron cryomicroscopic reconstruction containing an F-actin filament . We can identify each actin subunit and both domains of scruin , referred to as elongated -LRB- E -RRB- and spheroidal -LRB- S -RRB- on the basis of their shape9 , and secondary structure elements of both molecules . We used correlation analysis and non-crystallographic symmetry -LRB- NCS -RRB- averaging within the asymmetric unit to determine the azimuthal and axial positions of the 14 actin scruin subunits . The azimuthal rotation between adjacent subunits ranges from 2176.58 to 2142.58 -LRB- Fig. 1c , d -RRB- , and thus deviates from the canonical actin helix by 210.48 under-twist to 23.68 over-twist . Nevertheless , the rise per subunit in the axial direction had less than one pixel -LRB- 1.33 A -RRB- deviation . This amount of individual azimuthal distortion is larger than the distortions found in averaged reconstructions from F-actin filaments10 15 , but such studies can not reveal individual subunit variations . On the basis of this two-dimensional cross-correlation , we created an average actin monomer from the 14 subunits in the asymmetric unit . In this map -LRB- Fig. 2a -RRB- , three a-helices in subdomain 1 of actin were detected by an a-helix search -LRB- using the program ` helixhunter ' -RRB- 16 . Their locations match a-helices in the actin crystal structure -LRB- amino acids 79 91 , 113 125 and 137 144 -RRB- 17 . Because we obtained this map without referring to the actin crystal structure during either the reconstruction process8 or the NCS NATURE | VOL 431 | 2 SEPTEMBER 2004 | www.nature.com/nature 104 2004 Nature Publishing Group

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