Wojtasz 1 

 

Supplemental Material

Contents:

Supplemental Materials and Methods

Supplemental Figure Legends

Supplemental References

Supplemental Figures

Supplemental Fig. S1, S2, S3, and S4 are related to Fig. 1, Supplemental Fig. S5 is

related to Fig. 2, Supplemental Fig. S6 is related to Fig. 3, Supplemental Fig. S7 is

related to Fig. 4, Supplemental Fig. S8 is related to Fig. 5, and Supplemental Fig. S9 is

related to Fig. 6.

Supplemental Materials and Methods:

Targeting Hormad2: The Hormad2 targeting vector was designed according to a multi-

purpose allele strategy (Testa et al. 2004), and the vector was constructed using

recombineering methods available on request (Zhang et al. 1998). In brief, the Splice-

Acceptor (SA)-IRES-LacZneo-pA and PGK-Blasticidin-pA cassettes flanked by FRT

sites were inserted in intron 3 (Supplemental Fig. S1). The frame-shifting exon No. 4

was flanked by loxP sites.

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Cell culture and transfection: To modify the Hormad2 locus, mouse R1 embryonic

stem cells (mES) were cultured and electroporated with the linearised targeting

construct. Mouse R1 embryonic stem cells (mES) were cultured on Mitomycin-C

inactivated mouse embryonic fibroblasts (MEFs) using the following ES-medium

(DMEM, 15% fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 1%

penicillin/streptomycin, 100 M non-essential amino acids (all Invitrogen), 100 M ß-

mercaptoethanol (Sigma) containing leukemia inhibitory factor (LIF)). ES cells (107)

were electroporated using a BIORAD electroporator (250 V, 500 µF) with 40 µg of

linearised targeting constructs and selected with 200 µg/ml G418 (Invitrogen). Colonies

were picked in 96-well plates and screened by Southern hybridization (data not shown).

Southern hybridization: To screen for targeted ES cell clones, genomic DNA was

extracted from cells by proteinase K digestion and isopropanol precipitation. DNA was

digested overnight with EcoRV and PstI, EcoRV or the combination of MunI and BamHI

for Southern blots with a 5’-probe (upstream of the 5’ homology arm of the targeting

construct), an internal probe or a 3’-probe (downstream of the 3’ homology arm of the

targeting construct), respectively. DNA fragments were separated on 0.8% agarose gels

and blotted onto nylon membranes (PALL). Probes were made with 32P by random

priming (Roche Diagnostics) and membranes were hybridized using the Church and

Gilbert protocols.

Generation of knockouts, genotyping and animal experiments: Two independent

ES cell clones with a single integration of the targeting construct into the Hormad2 locus

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were used to generate chimeric animals (ES12 and ES25). Progeny of the chimeric

animals crossed to C57BL/6JOlaHsd females were genotyped by Southern blot and

PCR (Supplemental Fig. S1). In subsequent crosses, only PCR was used for

genotyping the various alleles of Hormad2 from tail tip DNA. Genotyping primers:

H2lox3 5’-CACTTTAGCCCATATGAACAGCC, H2lox5 5’-

AATACTTTATTAGCCCTCTTTCC, H2FRT 5’-GTCTACAGAGTGAGTTTAAAATGC.

The product sizes for H2lox3/H2lox5 PCR were 361 bp on the wt template, 469 bp on

Hormad2insertion and Hormad2restored alleles, and no specific product was amplified from

Hormad2deletion template. The product sizes for H2FRT/H2lox3 PCR were 456 bp on

Hormad2deletion allele, 897 bp on Hormad2restored allele, and no specific product was

amplified from wt template and from Hormad2insertion allele (theoretical product size is

8768 bp in the latter case).

To generate Hormad2+/restored mice, we crossed Hormad2+/insertion animals derived from

the ES12 line with hACTB:FLPe transgenic mice (Rodriguez et al. 2000). To generate

Hormad2+/deletion mice, we crossed Hormad2+/restored with PGK-Cre transgenic mice

(Lallemand et al. 1998).

Lack of HORMAD2 in Hormad2insertion/insertion and in Hormad2deletion/deletion mice was

reconfirmed by IF on nuclear spreads of spermatocytes and immunoblot analysis of

testes extracts (data not shown and Supplemental Fig. S1C). The initial analysis of

HORMAD2 functions was carried out in both ES12 Hormad2insertion and ES25

Hormad2insertion lineages. Since the phenotypes of these lines were very similar in

respect of sex body formation and the SC formation, we decided to further study only

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one of them (ES12). We generated ES12 Hormad2deletion/deletion mice, and all the

described experiments were performed at least twice in these mice. Whenever possible,

experimental animals were compared to controls from the same litter or from age-

matched mice from other litters of the same colony. For staging embryonic

development, the day of detection of a vaginal plug was marked as 0.5 days post

coitum (dpc). All animals were used and maintained according to regulations provided

by the animal ethics committee of the Technische Universität Dresden.

Immunofluorescence (IF): For quantification of γH2AFX signal in spread

spermatocytes of Spo11-/- and Hormad2-/- Spo11-/- mutants, matched exposure

images were taken as described earlier (Wojtasz et al. 2009). With the help of the

ImageJ program package, we measured total IF signal intensity of γH2AFX in squares

that covered spread nuclei identified by DAPI staining. Signal intensities were also

measured in four regions around the examined nuclei in order to estimate the

background. The signal intensity values shown in Fig. 5D are background corrected. To

compare oocyte numbers in adults, we sectioned both ovaries of each mouse (8 μm

thick sections), identified oocytes by anti-NOBOX (oocyte marker (Suzumori et al.

2002)) immunostaining and counted oocytes in every eighth section. In addition to

antibodies that were used in two previous studies (Wojtasz et al. 2009; Daniel et al.

2011), we used human anti-Human-centromere antibodies (15-235) from Antibodies

Incorporated at 1:700 dilution and rabbit anti BRCA1 antibodies (1:1000, gift from

Satoshi Namekawa (Ichijima et al. 2011)).

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Staging meiotic prophase: nuclear spreads were staged based on axis development:

dotty-very short stretches of axes corresponds to leptotene stage, short stretches of

axes corresponds to early zygotene, relatively long stretches of axes that are

incomplete corresponds to mid-zygotene, fully formed axes corresponds to late-

zygotene and pachytene stages. Spermatocytes are eliminated in Hormad2-/- mice at a

stage that corresponds to the beginning of mid-pachytene in wt mice. Therefore, it was

important to identify sub-stages in wt pachytene spermatocytes in order to compare the

same stages in wt and Hormad2-/- pachytene spermatocytes. Depending on the

experiments, we relied on three alternative approaches: 1. Anti-histone H1t staining was

used to identify mid- and late-pachytene wt spermatocytes, which are characterized by

high histone-H1t expression. 2. Low intensity γH2AFX staining can be detected along

synapsed autosomal axes in early pachytene cells. In late-pachytene spermatocytes

this type of staining is virtually absent. 3. Axis-configuration and the morphology of sex

chromosomes were also used for staging: relatively long stretches of SC can form

between XY chromosomes in early-pachytene cells, while X and Y chromosomes are

synapsed only at their very end in late-pachytene.

Statistics: Statistical analysis was performed with GraphPad Prism5. For the

comparison of independent samples, the two-tailed non-parametric Wilcoxon–Mann–

Whitney two-sample rank-sum test was used. In the RNA FISH experiments, one-way

ANOVA and Tukey’s multiple comparison test was used.

RNA FISH: RNA FISH in combination with IF staining was carried out as described

before (Mahadevaiah et al. 2009). BACs were used to prepare FISH probes: Utx: from

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CITB Mouse BAC library, Research Genetics/Invitrogen, #96022_612O14, Zfx: from

Mouse bMQ BAC library, bMQ-372M23, Scml2: from CHORI BACPAC library, RP24-

204O18.

Extracts, IPs and pull down experiments:

Preparation of soluble nuclear testis extracts: Testis was chopped up by a scalpel in

a petri dish and cell suspension was prepared by dounce cell/tissue grinder (15 strokes)

in ice cold PBS pH 7.4 supplied with protease inhibitor cocktail, phosphatase inhibitor

cocktail 1+3, NaVO3 and PMSF. Single cell suspensions were span at 1020 rcf, 10 min,

4° C and cells were resuspended in ice cold hypotonic buffer (40 mM Tris pH=7.5, 5

mM KCl, 2 mM EDTA, 0.5 mM spermidine, protease and phosphatase inhibitors) for

nuclear preparations. The testis cells were lysed with the help of a Tenbroek cell/tissue

grinder (37strokes). Following cell-lysis, nuclei were pelleted (1020 rcf, 10 min) and

resuspended in nuclear lysis buffer (50 mM Tris, pH=7.5, 150 mM NaCl, protease and

phosphatase inhibitors) supplemented with 0.5% Brij®-58. Nuclei were lysed for 30 min

at 4° C in lysis buffer, then sonicated, after which Brij-58 soluble and insoluble (crude

chromatin) fractions were prepared by ultra-centrifugation at 100000 rcf for 30 min.

IPs: anti-HORMAD1 (10 µg) and HORMAD2 (3 µg) and anti-ATR (10 µg) antibodies

were cross-linked to 30 µl ProteinA- or ProteinG-sepharose resin (20333 or 20398,

Pierce) with 20 mM Dimethyl Suberimidate according to standard protocols (Harlow and

Lane 1999). Aliquots of soluble nuclear extracts were diluted with nuclear lysis buffer

without Brij®-58, and the concentration of proteins and Brij®-58 were adjusted to 500

mg/ml and 0.1%, respectively. In each IP, one ml aliquot of this extract was incubated

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with affinity resin overnight at 4° C. Following three washes with nuclear lysis buffer

supplemented with 0.1% Brij®-58, IPed material was eluted from the resin by incubating

the resin in 50 µl 100 mM glycine pH 2.5 at room temperature for 10 min. IPed material

was analyzed by SDS-PAGE and western blot.

Detailed pull down protocol: GST, GST-HORMAD1, GST-HORMAD2, MBP-

HORMAD1 and MBP-HORMAD2 proteins were expressed in E. Coli Rosetta (DE3)

pLysS (Novagen) cells using standard techniques. cDNA of the mouse proteins were

sub-cloned into modified pET expression vectors (maps are available upon request) by

PCR (pET-GST for baits or pET-MBP for preys) and sequences were verified by DNA

sequencing. These vectors allow HORMAD1 and HORMAD2 proteins to be expressed

with an N-terminal GST- or MBP- and a C-terminal His6-tag. Recombinant proteins

were then first purified on Ni-sepharose (GE-Healthcare) beads. After elution with

imidazol all final protein samples were dialyzed into storage buffer containing 20 mM

Tris pH 8.0, 100 mM NaCl and 2 mM DTT. GST fusion proteins were loaded to

glutathione-sepharose beads (GE-Healthcare) and used as baits in pull-down assays

while MBP fusion proteins – in order to increase solubility of HORMAD1 and HORMAD2

proteins - were used as preys. Glutathione beads were equilibrated with binding buffer

(20 mM Tris, 100 mM NaCl, 0.1% IGEPAL, 2 mM DTT). 20 μg immobilized GST-fusion

proteins (and GST protein alone as negative control) were incubated in the presence of

10 μM MBP-proteins in 200 μl binding buffer for 20 min at room temperature. The beads

were pelleted with centrifugation and washed three times. Retained proteins were

eluted from the resin with SDS loading buffer. Samples were subjected to SDS-PAGE

and interaction between bait and prey was detected by anti-MBP Western blots.

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Protease inhibitors and phosphatase inhibitors: 1 mM Phenylmethylsulfonyl fluoride

(PMSF), Complete EDTA free Protease inhibitor cocktail tablets (Roche, 11873580001),

0.5 mM Sodium orthovanadate, Phosphatase inhibitor cocktail 1 (Sigma, P2850) and

Phosphatase inhibitor cocktail 2 (Sigma, P5726) were used at concentrations

recommended by the manufacturers.

Immunoblotting: Proteins in testis extracts were separated by SDS-PAGE and

transferred onto PVDF membranes by wet blotting. Proteins on the PVDF membranes

were detected by antibodies diluted in blocking buffer (2,5% BSA, 0,02% NaN3 in TBS-

Tween 0.05%), following the blocking of the membranes in the blocking buffer for 60

min. Primary antibodies: guinea pig anti-HORMAD1 (1:5000) and anti-HORMAD2

(1:2500) were raised by us previously (Wojtasz et al. 2009), rabbit anti-HISTONE H3

(1:100000, Abcam, ab1791), mouse anti-GAPDH (1:2000, Santa Cruz, sc32233),

mouse anti-MBP antibody coupled with horse radish peroxidase (NEB, 1ug/ml).

Secondary antibodies: peroxidase coupled anti-rabbit IgG light chain (1:20000, Jackson

ImmunoResearch Europe Ltd., 211-032-171), goat anti-mouse IgG (1:20000, Jackson

ImmunoResearch Europe Ltd., 115-035-166), goat anti-guinea pig IgG (1:20000,

Jackson ImmunoResearch Europe Ltd., 106-035-003).

Supplemental Figure Legends

Supplemental Figure S1. Generation of Hormad2-/- mice.

A, Targeting construct, wt and modified Hormad2 genomic locus. Black boxes represent

exons (not to scale). Recombination at the homology arms (HA) of the targeting

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construct modifies intron 3 by introducing: 1) an additional exon (SA-IRES-LacZ-neo)

that contains a strong splice acceptor site (SA) and poly-adenylation site, 2) a

transcriptional unit that contains the strong housekeeping phosphoglycerine kinase

(PGK) promoter driving a Blasticidin resistance gene (BSD) as a selection marker. This

modification of intron 3 disrupts the Hormad2 ORF after the 64th codon

(Hormad2insertion). Recombination catalyzed by FLPe at FRT sites removes the SA-

IRES-LacZ-neo exon and the PGK-BSD gene, and restores the Hormad2 ORF

(Hormad2restored). Hormad2restored is a functional allele that can be disrupted by Cre-

mediated recombination between loxP sites (Hormad2deletion). Excision of exon 4 causes

a frameshift after the 64th codon. The positions of PCR-genotyping primers are

indicated. Red bars mark the 3` and internal Southern blot probes; the predicted length

of restriction fragments is indicated. B, Southern blot of DNA from wt (+/+),

Hormad2+/insertion (+/-), Hormad2insertion/insertion (-/-) mice derived from the embryonic stem

cell clone (ES12). DNA was digested with BamHI and MunI (left panel) or with EcoRV

(right panel). This line has a single integration of the targeting cassette in the Hormad2

locus. C, Western blot analysis of extracts prepared from testes of Hormad2deletion/deletion

(-/-) mouse and Hormad2+/deletion (+/-) and wt (+/+) litter mate controls (ES12 line). An

antibody raised against the C-terminus of HORMAD2 does not detect full length

HORMAD2 in Hormad2deletion/deletion (-/-) testes. The lower panel shows anti-Histone H3

western blot, which is used as a loading control.

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Supplemental Figure S2. Hormad2-/- spermatocytes are eliminated in stage IV

testis tubules.

A, Cryo-sections of testes from 12-week-old wt and Hormad2-/- mice. SYCP3 (AE

component) was detected by immunofluorescence (IF). Strong nuclear SYCP3 staining

is characteristic of spermatocytes (sc). Post-meiotic cell types, such as spermatids (st)

and sperm (sp) are not observed in Hormad2-/- testes. Tubules that lack spermatocytes

are also observed in Hormad2-/- testes (*).B, Nuclear cleaved PARP1 (apoptosis

marker) was detected by IF on cryo-sections of testis from 12-week-old Hormad2-/-

mouse. Most spermatocytes are eliminated and apoptotic spermatocytes (asc) are

found at the end of epithelial cycle stage IV-early stage V as identified by the presence

of intermediate spermatogonia (In), mitotic intermediate spermatogonia (m) and

spermatogonia B (SgB). Bars, 100 µm

Supplemental Figure S3. Chromosome axis formation and SC formation are

comparable in wt and Hormad2-/- meiocytes.

SYCP3 (chromosome axis), HORMAD1 (unsynapsed axis) and either a meiosis-specific

cohesin, STAG3 (A), or the SC transverse filament component SYCP1 (B) were

detected on nuclear spreads of pachytene stage spermatocytes (A) or oocytes (B) of wt

and Hormad2-/- mice at 22 dpp (A) or 17,5 dpc (B). C, SYCP3, HORMAD1 and MLH1

(future CO marker) were detected on nuclear spreads of a Hormad2-/- oocyte (17.5

dpc). MLH1foci formation occurs in oocytes in the pachytene stage. Nevertheless, SC

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formation is not completed in a small fractions of MLH1-positive wt (7.8% n=154) and

Hormad2-/- (7.4% n=162) oocytes. Bars, 10 µm.

Supplemental Figure S4. Recombination protein foci numbers are similar in wt

and Hormad2-/- meiocytes.

SYCP3, HORMAD1 and either RAD51 (A), DMC1 (B), RPA (C) or MLH1 (D) were

detected on nuclear spreads of zygotene (A and B) or pachytene (C) stage

spermatocytes of wt and Hormad2-/- mice (12-week-old) , and on nuclear spreads of

wt and Hormad2-/- oocytes (17.5 days post coitum) in pachytene stage (D). Bars in A-

D, 10 µm. E, Assessment of MLH1 foci distribution between bivalents in the absence of

HORMAD2. Wt and Hormad2-/- oocytes that completed SC formation were selected,

and MLH1 foci were counted on all bivalents. Total number of counted bivalents (n) and

percentages of bivalents with the indicated number of MLH1 foci in pachytene oocytes

at 17.5dpc are shown. The percentages of bivalents that have zero, one, two or three

MLH1 foci are not significantly different in Hormad2-/- and wt oocytes as indicated by

the Chi-squared test p values. F, Assessment of MLH1 focus distribution relative to

other MLH1 foci (cytological interference between MLH1 foci) in the Hormad2-/- mutant.

We measured distances between MLH1 foci along AEs of fully synapsed bivalents

(numbers of bivalents are indicated) that contained more than one MLH1 foci in wt and

Hormad2-/- oocytes at 17.5dpc. Cumulative plots of the frequency distribution of MLH1

inter-focus distances normalized to AE length of corresponding bivalents are shown.

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Kolmogorov-Smirnov test (p value shown) indicates that the distribution of normalized

MLH1 inter-focus distances are not significantly different in wt and Hormad2-/- oocytes.

Supplemental Figure S5. HORMAD2 is not required for the formation of SC

stretches and RAD51 and RPA foci in Hormad1-/- spermatocytes

SYCP3, and either SYCP1 (A), RAD51 (C) or RPA (E) were detected on nuclear

spreads of Hormad1-/- and Hormad1-/- Hormad2-/- spermatocytes in late-zygotene-like

(full or nearly full axis formation) (A) and early-zygotene (partial axis formation) (C and

E) stages. Bars, 10 µm. B, The frequency distribution of SC stretches in late-zygotene-

like spermatocytes. D and F, Quantification of foci numbers of RAD51 and RPA in early-

mid-zygotene Hormad1-/- and Hormad1-/- Hormad2-/- spermatocytes. Median foci

numbers are not significantly different in Hormad1-/- and Hormad1-/- Hormad2-/-

spermatocytes (Mann–Whitney test).

Supplemental Figure S6. Sex body formation is defective in Hormad2-/-

spermatocytes

A, Graph showing the frequency of Hormad2-/- spermatocytes in which the indicated

percentage of X chromosome axis is surrounded by γH2AFX-rich chromatin. Only

Hormad2-/- spermatocytes where the γH2AFX-rich chromatin overlaps with the X

chromosome axes were scored. B, SYCP3 (chromosome axis), a meiotic antigen

recognized by the anti-XLR antibody and HORMAD1 (unsynapsed axis) were detected

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by IF in nuclear spreads of wt and Hormad2-/- pachytene spermatocytes collected from

21-day-old mice. Matched exposure images of anti-XLR staining are shown. Three

different types of anti-XLR staining patterns were distinguished and the frequency of the

three types of staining-patterns in wt and Hormad2-/- spermatocytes are shown in C.

Anti-XLR staining patterns: anti-XLR stained chromatin surrounds completely (B top

row-wt; C, whole XY axes), or partially (B, middle row- Hormad2-/-; C, part of XY axes)

the HORMAD1 marked unsynapsed sex chromosome axes, or no anti-XLR stained

chromatin is detected (B, bottom row-Hormad2-/-; C, no stain). D, γH2AFX, MDC1 and

HORMAD1 were detected in nuclear spreads of wt and Hormad2-/- pachytene

spermatocytes. MDC1 accumulates in sex chromosome-associated γH2AFX-rich

chromatin in wt and Hormad2-/- pachytene spermatocytes (n=300 in both genotypes).

E, Images of RNA FISH of wt and Hormad2 -/- early pachytene spermatocytes showing

silencing of Utx in a wt spermatocyte (top), and expression of Utx in a Hormad2-/-

spermatocyte (RNA FISH signal is marked by arrow). DNA was detected by DAPI

staining, HORMAD1 and γH2AFX were detected by IF. Bars, 10 μm in B and D, and 5

μm in E.

Supplemental Figure S7. ATR and TOPBP1 localization to unsynapsed sex

chromosome axes is reduced in Hormad2-/- spermatocytes

Indicated proteins were detected in nuclear spreads of wt and Hormad2-/- early-

pachytene spermatocytes. A, Matched exposure images of TOPBP1 are shown in wt

and Hormad2-/- meiocytes at low (TOPBP1 low) and high exposures (TOPBP1 high).

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TOPBP1 staining is much lower on X and Y chromosome axes in the Hormad2-/-

spermatocytes than in the wt spermatocytes. Nevertheless, a cloud like staining of

TOPBP1 is observed in γH2AFX–rich chromatin in both the wt and the mutant

spermatocytes. In addition, punctate TOPBP1 staining can be observed along sex

chromosome axis in the Hormad2-/- spermatocyte. B Image of a nuclear spread of a

Hormad2-/- pachytene spermatocyte (top panel) and the zoomed in image of the sex

chromosomes in the same cell (lower panel). Note the punctate accumulation of ATR

along the sex chromosome axes in the vicinity of DMC1 foci. C, γH2AFX accumulates

on chromatin loops that are associated with regions of sex chromosome axis that are

rich in DMC1 foci. The DMC1 focus that is most distal to the PAR region marks the end

of the γH2AFX-rich chromatin domain (arrow) in the shown cell. p marks PAR region in

C. Bars, 10 µm.

Supplemental Figure S8. Accumulation of ATR activity on unsynapsed chromatin

in Spo11-/- meiocytes is defective in the absence of HORMAD2

Indicated proteins were detected in nuclear spreads of oocytes that were collected from

newborn mice of the indicated genotypes. Oocytes with fully formed chromosome axes

are shown. The ATR and γH2AFX images are matched exposure images. A shows the

image of a Hormad2+/+ Spo11-/- oocyte that contains a γH2AFX-rich pseudo-sex body

(top panel), and the image of a Hormad2-/- Spo11-/- oocyte that contains no pseudo-

sex body (bottom panel). B shows that ATR foci are associated with axes in the vicinity

of RPA foci both in Hormad2+/+ Spo11-/- and in Hormad2-/- Spo11-/- oocytes. C shows

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that γH2AFX accumulates on chromatin in both Hormad2+/+ Dmc1-/- and Hormad2-/-

Dmc1-/- oocytes. Bars, 10 µm.

Supplemental Figure S9. HORMAD2 is required for the elimination of Spo11-/-

oocytes

NOBOX (postnatal oocyte marker) was detected by IF on cryo-sections of ovaries from

6-week-old mice. DNA was detected by DAPI. NOBOX marked oocytes are readily

observed in wt, Hormad2-/- and Hormad2-/- Spo11-/- ovaries. Examples of primordial

(pd) and secondary (s) follicles are marked. Oocyte numbers are strongly reduced in

the SC-defective Spo11-/- mutant, and in the SC- and DSB repair-defective

Hormad2+/+Dmc1-/- and Hormad2-/- Dmc1-/- mutants. Bars, 100 µm.

Supplemental References:

Daniel K, Lange J, Hached K, Fu J, Anastassiadis K, Roig I, Cooke HJ, Stewart AF, Wassmann K, Jasin M et al. 2011. Meiotic homologue alignment and its quality surveillance are controlled by mouse HORMAD1. Nat Cell Biol 13: 599‐610. 

Harlow E, Lane D. 1999. Using antibodies : a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 

Ichijima Y, Ichijima M, Lou Z, Nussenzweig A, Camerini‐Otero RD, Chen J, Andreassen PR, Namekawa SH. 2011. MDC1 directs chromosome‐wide silencing of the sex chromosomes in male germ cells. Genes Dev 25: 959‐971. 

Lallemand Y, Luria V, Haffner‐Krausz R, Lonai P. 1998. Maternally expressed PGK‐Cre transgene as a tool for early and uniform activation of the Cre site‐specific recombinase. Transgenic Res 7: 105‐112. 

Mahadevaiah SK, Costa Y, Turner JM. 2009. Using RNA FISH to study gene expression during mammalian meiosis. Methods Mol Biol 558: 433‐444. 

Rodriguez CI, Buchholz F, Galloway J, Sequerra R, Kasper J, Ayala R, Stewart AF, Dymecki SM. 2000. High‐efficiency deleter mice show that FLPe is an alternative to Cre‐loxP. Nat Genet 25: 139‐140. 

Suzumori N, Yan C, Matzuk MM, Rajkovic A. 2002. Nobox is a homeobox‐encoding gene preferentially expressed in primordial and growing oocytes. Mech Dev 111: 137‐141. 

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Testa G, Schaft J, van der Hoeven F, Glaser S, Anastassiadis K, Zhang Y, Hermann T, Stremmel W, Stewart AF. 2004. A reliable lacZ expression reporter cassette for multipurpose, knockout‐first alleles. Genesis 38: 151‐158. 

Wojtasz L, Daniel K, Roig I, Bolcun‐Filas E, Xu H, Boonsanay V, Eckmann CR, Cooke HJ, Jasin M, Keeney S et al. 2009. Mouse HORMAD1 and HORMAD2, two conserved meiotic chromosomal proteins, are depleted from synapsed chromosome axes with the help of TRIP13 AAA‐ATPase. PLoS Genet 5: e1000702. 

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A

Targeting Construct SA-IRES-LacZ neo BSDPGK

EcoRV

FRT FRTE3 loxP E4 loxP E5 E6 E7

3’HABamHI5’HA

E3E2E1 E4 E5 E6 E7 E8

BamHIEcoRV

WT locusMunI

MunI

3’probe

10,7 kb

Hormad2 insertion

E1 E2 E3

EcoRV

neo BSDPGK

FRT loxP E4 loxP E5

BamHI

E6 E7 E8

BamHI

MunI

3’probeInternal probe

EcoRV

SA-IRES-LacZ

FRT

13,4 kb 8,1 kb

FLPe

Hormad2 deletion

E1 E2 E3 FRT loxP E5 E6 E7 E8

E1 E2 E3 FRT loxP E4 loxP E5 E6 E7 E8

Hormad2 restored

Cre

Southern Blot Genotyping

Southern Blot Genotyping

H2lox5 H2ox3PCR Genotyping Primers

PCR Genotyping Primers H2lox5

H2lox3

H2lox5

H2lox3H2FRT

H2FRT

H2FRT

H2lox3PCR Genotyping Primers

PCR Genotyping Primers

+/+ +/- -/- +/+ +/- -/-

10kb

8kb

-

-

3’ probe

Internal probe

B C

Hormad2 deletion

+/+ +/- -/-

anti-HORMAD2

anti-Histone H3

35 KDa-

17 KDa-

10kb -

8kb -

Hormad2 insertion

Hormad2 insertion

6kb -

5kb -

4kb -

3kb -

2kb -

Wojtasz_Fig.S1

ASYCP3

Hor

mad

2 -/

-H

orm

ad2

+/+

DNA (DAPI) DNA SYCP3

B

Hor

mad

2 -/

-

cleaved PARP1DNA (DAPI) DNA cleaved PARP1

st

scsp

st

scsp

st

scsp

*sc

* ** * *** *

sc

sc

sc

sc

sc

sc

sc sc

asc

SgB In

m

asc

SgB In

m

asc

SgB In

m

*

Wojtasz_Fig.S2

A

B SYCP3 HORMAD1 SYCP1

Hor

mad

2 +

/+H

orm

ad2

-/-

SYCP3 STAG3 HORMAD1

Hor

mad

2 -/

-H

orm

ad2

+/+

SYCP3 HORMAD1 STAG3

SYCP3 SYCP1 HORMAD1

C SYCP3 HORMAD1 MLH1

Hor

mad

2 -/

-

SYCP3 MLH1 HORMAD1

Wojtasz_Fig.S3

A B

Hor

mad

2 -/

-H

orm

ad2

+/+

Hor

mad

2 -/

-H

orm

ad2

+/+

SYCP3 HORMAD1 DMC1

CSYCP3 HORMAD1 RPA SYCP3 HORMAD1 MLH1

HORMAD1 MLH1 SYCP3

D

SYCP3 RAD51 HORMAD1

SYCP3 DMC1 HORMAD1

SYCP3 RPA HORMAD1

Hor

mad

2 -/

-H

orm

ad2

+/+

Hor

mad

2 -/

-H

orm

ad2

+/+

SYCP3 HORMAD1 RAD51

E

% o

f biv

alen

ts a

t p

achy

tene

100

4.1 4.5

66.2 66.6

P=0.5035

P=0.8079

P=0.5540

80

60

40

20

0

24.2 23.2

P=0.56055.5 5.7

0 MLH1 foci

1 MLH1 focus

2 MLH1 foci

3 MLH1 foci

Hormad2 -/- (n=1460)

Hormad2 +/+ (n=1400)

F

100

80

60

40

20

0

Hormad2 -/- (n=123)

Hormad2 +/+ (n=138)

P=0.991

0.2 0.4 0.6 0.8 1.0

% o

f int

er-f

ocus

dis

tanc

es

Normalized inter-focus distances

5 10 15 20 250

2

8

4

6

10

# of

cel

ls (n

=50

)

# of SYCP1 stretches

5 10 15 20 250

2

8

4

6

10

# of

cel

ls (n

=43

)

# of SYCP1 stretches

A B

Hor

mad

1-/-

Hor

mad

2 -/

-

SYCP3 SYCP1 SYCP1 SYCP3

Hor

mad

1-/-

Hor

mad

2+/+

E

Hor

mad

1-/-

Hor

mad

2 -/

-H

orm

ad1-

/-H

orm

ad2+

/+H

orm

ad1-

/-H

orm

ad2

-/-

Hor

mad

1-/-

Hor

mad

2+/+

SYCP3 RPA

Num

ber

of R

PA fo

ci

per

cel

l

n.s

Hormad1-/-Hormad2+/+

(n=29)

Hormad1-/-Hormad2-/-

(n=31)

150

100

50

0

Hormad1-/-Hormad2+/+

Hormad1-/-Hormad2-/-

20

40

60

80

0Hormad1-/-Hormad2+/+

(n=30)

Hormad1-/-Hormad2-/-

(n=30)

Num

ber

of R

AD

51 fo

ci

per

cel

l

n.s

C SYCP3 RAD51 RAD51 SYCP3

RPA SYCP3

D

F

Wojtasz_Fig.S5

E

Hor

mad

2 -/

-H

orm

ad2

+/+

HORMAD1MDC1γH2AFXγH2AFX MDC1

HORMAD1Hormad2 +/+

Hormad2 -/-

whole XYaxes

part of XYaxes

no stain

100%

12%

0% 0%

28%

60%

D

A

Hor

mad

2 -/

-

SYCP3 HORMAD1XLR

Hor

mad

2 +

/+

SYCP3 HORMAD1 XLR

HORMAD1 γH2AFXDNA (DAPI) Utx RNADNA HORMAD1

γH2AFX

Hor

mad

2 -/

-H

orm

ad2

+/+

% o

f the

sp

erm

atoc

ytes

% of X chromosome axiswithin γH2AFX domain

(n=167)

C

B

5.6%

27.7%

21.5%

26.5%

11.9%

6.8%

% o

f the

pac

hyte

ne s

per

mat

ocyt

es

0

6

12

18

24

30

0

20

40

60

80

100

100

90-9

970

-90

40-7

010

-40

0-10

Wojtasz_Fig.S6

A γH2AFX TOPBP1

HORMAD1TOPBP1 low

Hor

mad

2 +

/+

γH2AFXHORMAD1

Hor

mad

2 -/

-

B

Hor

mad

2 -/

-

ATR

TOPBP1 high

SYCP3 DMC1 SYCP3 ATR DMC1

ATR DMC1

Hor

mad

2 -/

-

CγH2AFX DMC1

HORMAD1γH2AFX HORMAD1DMC1

p p pp

Wojtasz_Fig.S7

A

B

γH2AFX HORMAD1 SYCP3SYCP3 HORMAD1

RPA ATRRPASYCP3 ATR

γH2AFX SYCP3SYCP3C

Hor

mad

2 -/

- D

mc1

-/-

Hor

mad

2 +

/+ D

mc1

-/-

γH2AFX

Hor

mad

2 -/

- S

po1

1 -/

-H

orm

ad2

+/+

Sp

o11

-/-

γH2AFX

Hor

mad

2 +

/+ D

mc1

-/-

Hor

mad

2 -/

- D

mc1

-/-

Wojtasz_Fig.S8

Hor

mad

2 -/

- D

mc1

-/-

Hor

mad

2 +

/+ D

mc1

-/-

Hor

mad

2 +

/+ S

po1

1 -/

-

NOBOX

pd

s

pd

pd

s s

s s

pd

Hor

mad

2 -/

- S

po1

1 -/

-

s

pd pd

pd

pd

Hor

mad

2 -/

-

s

pd

pd

DNA NOBOXs

pd

DNA (DAPI)

Hor

mad

2 +

/+

s

pd

Wojtasz_Fig.S9