Here is your pdf: Ensuring meiotic DNA break formation in the mouse pseudoautosomal region

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Acquaviva

et al.,

30 Jan

, 2019

Ð bioRxiv

preprint v

1 1 !”#$%&”‘(

)*&+,&-(./0

(1%*23(4+%)2,&+”(&”(,5*()+$#*(6#*$7+2$,+#+)28(%*’&+”

Laurent Acquaviva

1*, Michiel Boekhout

1€, Mehmet

E. Karasu

1,2

, Kevin Brick

3, Florencia Pratto

3, Megan van Overbeek

1à, Liisa Kauppi

1¤, R. Daniel Camerini

-Otero

3, Maria Jasin

2,4

* and Scott Keeney

1,2,5

* 1 Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY

, USA

. 2 Louis V. Gerstner, Jr., Graduate School of Biom

edical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY

, USA

. 3 Genetics & Biochemistry Branch, NIDDK, NIH, Bethesda, MD, USA

. 4 Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY

, USA

. 5 Howard Hughes Medical

Institute, Memorial Sloan Kettering Cancer Center, New York, NY

, USA

. *!Correspondence to

acquavil@mskcc.org

, m-jasin@ski.mskcc.org

or

s-keeney@ski.mskcc.org

Current

address

es: €UMC Utrecht, Oncode Insitute, Utrecht University, Utrecht, Netherlands

; àCaribou Biosciences, Inc., Berkeley, CA, USA

; ¤Faculty

of Medicine, University of Helsinki

, Helsinki, Finland.

Sex chromosomes in males share only a diminutive homologous

segment, the pseudoautosomal region (PAR), wherein meiotic

double

-strand breaks (DSBs), pairing, and crossing over must occur

for correct segregation. How cells

ensure PAR recombination is

unknown. Here we delineate cis

– and trans

-acting factors that control

PAR ultrastructure and make the PAR the hottest area of DSB

formation in the male mouse genome. Prior to DSB formation, PAR

chromosome axes elongate, sister c

hromatids separate, and DSB

-promoting factors hyperaccumulate. These phenomena are linked to

mo-2 minisatellite arrays and require ANKRD31 protein. We

propose that the repetitive PAR sequence confers unique chromatin

and higher order structures crucial for

DSB formation, X

ÐY pairing,

and recombination. Our findings establish a mechanistic paradigm

of mammalian sex chromosome segregation during spermatogenesis.

9″,%+7$-,&+”

(Meiotic recombination forms connections between homologous

chromosomes that ensure accurate segregation

(1). In many speci

es,

every chromosome must recombine, so a crucial challenge is to ensure

that every chromosome pair acquires at least one SPO11

-generated DSB

to initiate recombination

(2). This challenge is especially acute in most male placental mammals

for sex chromosomes

(X and Y), on which a DSB can only support

recombination if it occurs in the tiny PAR

(3-7). The PAR in laboratory

mice is the shortest thus far mapped in mammals, at ~700 kb

(5, 6

). Since

only one DSB is f

ormed per ten megabases on average in the mouse, the

PAR would risk frequent recombination failure if it behaved like a typical

autosomal segment

(7, 8

). However, the PAR is not typical, having

disproportionately frequent DSB formation and recombination

(4, 7, 9,

10). The mechanisms

promoting

such frequent DSBs are not known in

any species.

Higher order chromosome structure plays an important role in

meiotic recombination. DSBs arise concomitantly w

ith development of

linear axial structures that anchor arrays of chromatin loops within which

DSBs occur

(1, 11

-13). The axis begins to form between sister

chromatids during pre

-meiotic repl

ication (pre

-leptonema) and includes

SYCP2 and SYCP3

(14, 15

), cohesin complexes

(16), and HORMA

domain proteins (HORMAD1 and

HORMAD

2)

(17-20). Axes

are

also

assembl

y sites for

IHO1, MEI4, and REC114 complexes

(13, 21

-24), whose functions as essential promoters of SPO11 activity are

incompletely understood

(25, 26

). We previously showed that PAR chromatin is organized into short

loops on a long axis,

(7). However, only a low

-resolution view of PAR

struct

ure was available and the cis

– and trans

-acting factors controlling

PAR structure and DSB formation have remained largely unknown.

:*#$8,#

(A distinctive PAR ultrastructure rich in pro

-DSB factors

We applied a cytogenetic approach to

investigate

the

mouse

PAR

str

ucture

in the C57BL/6J strain (B6)

. In most of the genome, axes

elongate

and DSBs begin to form during lepto

nema

, then h

omologous

chromosomes pair and axes

are juxtaposed by the transverse filament

protein SYCP1 (forming the synaptonemal complex, or SC) du

ring

zygonema

. S

ynapsis and recombination are completed during

pachynema

, then the SC disassembles during diplonema with homologs

remaining attached at sites of crossovers (chiasmata) until anaphase I

(1,

27). X and Y usually pair late, with PARs paired in less than 2

0% of

spermatocytes at late zygonema when nearly all autosomes are paired

(7,

8). At this stage,

we found

by conventional immunofluorescence

microscopy

that

unsynapsed PA

R axial elements

(SYCP2/3 staining)

appeared thickened relative to

other unsynapsed

axes and had bright

HORMAD1/2 staining (

Fig. 1A and fig. S1

A,B) (28). We found that the PAR was highly enriched for

DSB

-promoting

factors REC114, MEI4, MEI1, and IHO1 [proteins essential for genome

-wide DSB formation

(22-24, 29

)] as well as ANKRD31

, a REC114

partner essential for PAR DSB formation

(30, 31

). All five proteins

(hereafter RMMAI for simplicity

) colocalized in several bright, irregular

ÒblobsÓ

for most of prophase I (

Fig

. 1A

ÐC and fig. S1C).

Two

blobs

were

on

the X and Y PARs

as judged by chromosome morphology at late

zygonema (

Fig

. 1A) and

particularly bright

fluorescence in situ

hybridization

(FISH

) with a probe for the PAR boundary (PARb) (

Fig

. 1C). Other blobs highlighted

the distal ends of

specific

autosomes

(Fig

. 1C), which

we r

evisit below.

Undefined

blobs

are

also

apparent in

published micrographs but

were not

explored further

(21-24). Consistent

with

and extending

other

studies

(21-24, 30, 31

), all

five proteins

also

colocalized in

numerous small foci along unsynapsed chromosome axes

(Fig

. 1B and fig.

S1C), but PAR staining was much brighter.

ANKRD31

, MEI1 and REC114

enrichment on

the

PAR was already detectable in pre

-leptotene cells

during premeiotic S phase

(fig. S1D

), as shown for MEI4

and IHO1

(21, 23

). Structured illumination microsc

opy (SIM)

resolved the

thickened

PAR axe

s as two strands of axial core

(Fig

. 1D and fig.

S2A,B

) that were

heavily decorated along

thei

r length

s with RMMAI proteins (

Fig

. 1E). At the

zygotene

Ðpachytene

transition, X and Y

pair and initiate synapsis,

then SC spreads bidirectionally

Ñhomologously

in the

PAR and non

-homologously between the non

-PAR axes

, which

definitively marks

early pachyne

ma

in mice

(28, 32, 33

) (Fig

. 1D). Separation of PAR axes

was readily observed by SIM in late zygonema before X

and Y pairing

and synapsis, remained apparent after synapsis, then disappeared during

early pachynema (

Fig

. 1D).

A multi

-core

structure

was also seen in

earlier

electron microscopy

studies, but

was staged incorrectly as

occurring at mid

-to-late pachynema

(34) (fig.

S2C,D

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