UBR2 mediates transcriptional silencing during spermatogenesis via histone ubiquitination.

Abstract

Ubiquitination of histones provides an important mechanism regulating chromatin remodeling and gene expression. Recent studies have revealed ubiquitin ligases involved in histone ubiquitination, yet the responsible enzymes and the function of histone ubiquitination in spermatogenesis remain unclear. We have previously shown that mice lacking the ubiquitin ligase UBR2, one of the recognition E3 components of the N-end rule proteolytic pathway, are infertile associated with meiotic arrest at prophase I. We here show that UBR2 localizes to meiotic chromatin regions, including unsynapsed axial elements linked to chromatin inactivation, and mediates transcriptional silencing via the ubiquitination of histone H2A. UBR2 interacts with the ubiquitin conjugating enzyme HR6B and its substrate H2A and promotes the HR6B-H2A interaction and the HR6B-to-H2A transfer of ubiquitin. UBR2 and ubiquitinated H2A (uH2A) spatiotemporally mark meiotic chromatin regions subject to transcriptional silencing, and UBR2-deficient spermatocytes fail to induce the ubiquitination of H2A during meiosis. UBR2-deficient spermatocytes are profoundly impaired in chromosome-wide transcriptional silencing of genes linked to unsynapsed axes of the X and Y chromosomes. Our findings suggest that insufficiency in UBR2-dependent histone ubiquitination triggers a pachytene checkpoint system, providing a new insight into chromatin remodeling and gene expression regulation.

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UBR2 mediates transcriptional silencing during
spermatogenesis via histone ubiquitination
Jee Young Ana,1, Eun-A. Kima,1, Yonghua Jianga,1, Adriana Zakrzewskaa, Dong Eun Kima, Min Jae Leeb,
Inhee Mook-Jungc, Yi Zhangd, and Yong Tae Kwona,2
aCenter for Pharmacogenetics and Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261;
bDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115;
University College of Medicine, Seoul 110-799, Korea; and
at Chapel Hill, Chapel Hill, NC 27599
cDepartment of Biochemistry and Biomedical Science, Seoul National
dDepartment of Biochemistry and Biophysics, University of North Carolina
Edited by Alexander Varshavsky, California Institute of Technology, Pasadena, CA, and approved November 6, 2009 (received for review September 9, 2009)
Ubiquitination of histones provides an important mechanism regu-
lating chromatin remodeling and gene expression. Recent studies
have revealed ubiquitin ligases involved in histone ubiquitination,
yettheresponsibleenzymesandthefunctionofhistoneubiquitina-
tionin spermatogenesisremainunclear. We havepreviouslyshown
thatmicelackingtheubiquitinligaseUBR2,oneoftherecognitionE3
components of the N-end rule proteolytic pathway, are infertile
associated with meiotic arrest at prophase I. We here show that
UBR2 localizes to meiotic chromatin regions, including unsynapsed
axial elements linked to chromatin inactivation, and mediates
transcriptional silencing via the ubiquitination of histone H2A.
UBR2 interacts with the ubiquitin conjugating enzyme HR6B and
its substrate H2A and promotes the HR6B–H2A interaction and
the HR6B-to-H2A transfer of ubiquitin. UBR2 and ubiquitinated
H2A (uH2A) spatiotemporally mark meiotic chromatin regions sub-
ject to transcriptional silencing, and UBR2-deficient spermatocytes
fail to induce the ubiquitination of H2A during meiosis. UBR2-
deficient spermatocytes are profoundly impaired in chromosome-
wide transcriptional silencing of genes linked to unsynapsed axes
oftheXandYchromosomes.Ourfindingssuggestthatinsufficiency
in UBR2-dependent histone ubiquitination triggers a pachytene
checkpointsystem,providinganewinsightintochromatinremodel-
ing and gene expression regulation.
chromatin inactivation ∣ H2A ∣ meiosis ∣ N-end rule ∣ ubiquitin
T
specific N-terminal amino acids of short-lived substrates as an es-
sential component of degradation signals called N-degrons (1, 2).
A pre-N-degron of a stable protein can be converted into an N-
degron through N-terminal modifications, such as deamidation,
oxidation, and arginylation (3–5). UBR1 and UBR2 are really
interesting new gene (RING) finger E3s that share size (200
kDa), sequence (46% identity), and enzymatic specificities to
type-1 (Arg, Lys, and His; basic) and type-2 (Phe, Leu, Trp, Tyr,
and Ile; bulky hydrophobic) destabilizing N-terminal residues
(3, 5, 6). Regardless of the similarity in biochemical properties,
deficiency of UBR1 and UBR2 causes completely different null
phenotypes in mammals. Mutations of human UBR1 cause
Johanson–Blizzardsyndrome(JBS),anautosomalrecessivedisor-
derwithmultipledevelopmentalabnormalitiesincludingexocrine
pancreatic insufficiency; knockout of mouse UBR1 also results
in metabolic abnormalities such as JBS-like exocrine pancreatic
defects (reviewed in ref. 2). UBR2−∕−male mice are infertile
associated with germ cell apoptosis and arrest of spermatocytes
at meiotic prophase I, whereas UBR2−∕−female mice show re-
duced fertility and partial lethality (5). Besides these functions,
theN-endrulepathwayplaysaroleinavarietyofprocesses,includ-
ing cardiovascular system, DNA repair, neurogenesis, olfaction,
sensingoxygenandnitric oxide,G-proteinsignaling, chromosome
segregation, peptide import, selective degradation of misfolded
proteins, and apoptosis (reviewed in ref. 2; 3–8).
he N-end rule pathway is a ubiquitin (Ub)-dependent proteo-
lytic system where E3 ligases called N-recognins recognize
Spermatogenesis involves one of the most dramatic chromatin-
remodeling processes, including synapsis and transcriptional
silencing. During the pachytene stage of meiosis, the X and Y
chromosomes are located into the nuclear periphery and form
the XY body, where they achieve partial synapsis in the pseu-
doautosomal region (PAR). In the XY body, the transcription
of genes linked to unsynapsed XY axes are silenced through a
process called meiotic sex chromosome inactivation (MSCI) (9).
Unpaired chromosomal regions are silenced during meiosis not
only in the sex chromosomes but also in autosomal chromosomes
through a pachytene checkpoint system called meiotic silenc-
ing of unsynapsed chromatin (MSUC) (9). Meiotic chromatin
silencing involves differential histone modifications, including
ubiquitination, phosphorylation, methylation and acetylation
(10). Ubiquitinated histone H2A (uH2A) represents the most
abundant ubiquitination substrate in mammals, comprising 5–
15% of total H2A. uH2A is also the major Ub conjugate in male
meiosis, which accumulates in the XY body and unsynapsed chro-
mosomal regions undergoing MSUC (11). However, E3 ligases
for H2A ubiquitination and the function of this modification
in meiosis remain unknown. In somatic cells, Ring1B, a Polycomb
group protein, mediates monoubiquitination of H2A, for chro-
matin inactivation processes, including X inactivation of female
somatic cells and transcriptional silencing of Hox genes (12–15).
Recently, 2A-HUB/hRUL138 has been shown to mediate H2A
monoubiquitination for transcriptional repression of specific che-
mokine genes (16). In yeast, the E2 enzyme Rad6 and the E3
ligase Bre1 promote H2B monoubiquitination (17). Rad6 and
its mammalian homologs, HR6A and HR6B, can function as
E2 for the N-end rule proteolytic pathway (3; reviewed in ref. 2).
In mammals, the human homolog of yeast Bre1, RNF20/RNF40,
mediates H2B monoubiquitination (18, 19).
Results
UBR2 Localizes to Meiotic Chromatin Regions in Spermatocytes,
Including Unsynapsed Axes Linked to Transcriptional Silencing. We
tested whether UBR2 localizes to meiotic chromatin by using
immunohistochemistry on spread chromosomes. UBR2 staining
was detected in specific regions of meiotic chromosomes with
dynamic changes in spatiotemporal localizations throughout
meiotic prophase (Fig. 1 and Fig. S1). The specificity of anti-
UBR2 antibody was verified by using a peptide competition assay
Author contributions: J.Y.A., E.-A. K., Y.J., M.J.L., I.M.-J., and Y.T.K. designed research;
J.Y.A., E.-A. K., Y.J., A.Z., D.E.K., and M.J.L. performed research; Y.Z. contributed new
reagents/analytic tools; J.Y.A., E.-A. K., Y.J., D.E.K., M.J.L., I.M.-J., Y.Z., and Y.T.K. analyzed
data; and J.Y.A., E.-A. K., Y.J., and Y.T.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1J.Y.A., E.-A. K., and Y.J. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: yok5@pitt.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0910267107/DCSupplemental.
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(Fig. S2). At the leptotene stage when spermatocytes enter
meiosis, UBR2 staining appeared as foci in the chromatin and
as segment-like staining along the newly emerging axial elements
(Fig. S1A). By the zygotene stage when homologous chromo-
somes are in the process of synapsing, UBR2 staining was
enriched on unsynapsed axial regions (Fig. 1A and C). As synapsis
of autosomal chromosomes expands throughout the axes, UBR2
staining gradually disappears from the axes that have achieved
synapsis (Fig. S1C and F) and, instead, became enriched on
the unpaired axes of the X and Y chromosomes that have already
established synapsis along the PAR at late zygotene. In the XY
body, UBR2 accumulated on unpaired XYaxes but was not de-
tected in the XY chromatin domain, a pattern that persisted
through diplotene (Fig. 1B, arrowhead). These results reveal
UBR2 as a chromatin-associated E3 ligase that marks MSCI.
By the late pachytene stage when homologous chromosomes
have completed synapsis and recombination, the number of
UBR2 foci surged throughout the entire chromatin except for
the XY chromatin domain (Fig. 1B). The intense UBR2 staining
in postsynaptic chromatin correlated, spatially and temporally, to
that of ubiquitinated histone H2A.
UBR2, in Combination with HR6B, Mediates Ubiquitination of Histone
H2A.GiventhatUBR2anduH2AmarkMSCI(Fig.1andFig.S1),
wespeculatedthatUBR2maypromoteH2Aubiquitinationaspart
of MSCI and MSUC. Loss-of-function experiments to determine
the role of UBR2 and its homolog UBR1 in H2A ubiquitination
in normally growing or DNA-damaged somatic cells were incon-
clusive, perhaps due to the presence of other E3 ligases (12–15,
20). Todeterminedirectly whether UBR2andUBR1 can mediate
H2A ubiquitination, we developed a synthetic-degron-based
affinity procedure that allows rapid purification of N-recognins
from mammalian tissues at low (75–150 mM) salt concentrations
(seeMethods),inwhichprotein–proteininteractionsarepreserved.
ByusingPhe-peptidebearingatype-2destabilizingN-terminalre-
sidueasanaffinityligand,wepartiallypurifiedendogenousUBR2
(as a mixture with UBR1) by 1,000–3,000 folds from rat testes
(Fig. 2Aa). An analogous procedure with Val-peptide bearing
a stabilizing N-terminal residue typically yielded no detectible N-
recognins (Fig. 2Ab).
Ub ligase requires E2 to promote protein ubiquitination.
Amongst approximately 50 mammalian E2s, HR6B and its close
homolog, HR6A, are the only E2s known to localize to meiotic
chromatin and involved in MSCI (10, 21). (The mammalian gen-
ome encodes two HR6 isoforms, HR6A and HR6B, with 95%
identity to each other, which we refer to as RAD6 when their
biochemical properties cannot be distinguished.) Anti-RAD6/
HR6B immunoblotting of precipitates indicated that RAD6
was copurified by Phe-peptide but not by Val-peptide (Fig. 2Ac),
and immunoprecipitation of endogenous UBR2 from captured
proteins brought down RAD6 (Fig. 2Ad). Thus, endogenous
UBR2 and HR6B/RAD6 form an E2–E3 complex in these
extracts.
To determine whether UBR2 and RAD6 cooperatively med-
iate the ubiquitination of H2A, we performed in vitro ubiquitina-
tionassays by using free H2A. Human HR6B alone was able to
Fig. 1.
staining is enriched on unsynapsed axial regions. (B) At diplotene, the num-
ber of UBR2 foci surge throughout the entire chromatin except for the XY
chromatin domain (arrowhead). (C and D) Enlarged images for UBR2 locali-
zation to unsynapsed axial regions of autosomes at zygotene (C) and the sex
chromosomes at early pachytene (D) Arrow, PAR. [Scale bar (A and B), 10 μm;
(C and D), 5 μm.]
Localization of UBR2 to meiotic chromosomes. (A) At zygotene, UBR2
Fig. 2.
(A) Partial purification of UBR2 from rat testes by using degron-bearing
peptides (a). UBR2 interacts with RAD6 in the testes (B–D). Protein samples
prepared by Phe-peptide (E3-F) or Val-peptide (E3-V) were subjected to im-
munoblotting for UBR2 (B), RAD6 (C), and anti-UBR2 immunoprecipitation
and anti-RAD6 immunoblotting (D). F, Phe-peptide; V, Val-peptide. (B) In vitro
ubiquitination assay (20 μL) with 100 ng E3-F prepared from rat testes, 30 ng
HR6B, and 1 μg H2A in the presence of Ub activating reagents, including 1 μg
FLAG-Ub and 100 ng E1, followed by immunoblotting for uH2A. (C) Same as
(B) except that H2A ubiquitination was detected by using anti-FLAG immu-
noblotting. (D) In vitro ubiquitination assay with 100 ng E3-F prepared from
þ∕þ (wt) and UBR2−∕−mouse tissues (ko) and 2 μg free H2A. (E) Same as (B)
except that 50 ng HR6B and 5 μg nucleosome (nucleo.) were used in the pre-
sence or absence of 0.05 mM Ub aldehyde (alde.).
UBR2, in combination with HR6B, mediates ubiquitination of H2A.
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mediate ubiquitination of H2A (Fig. 2B, lane 2), consistent with
a previous observation that E2-14K/RAD6 can mediate E3-
independent ubiquitination of histones in vitro (22). HR6B-
dependent H2A ubiquitination was mainly restricted to monou-
biquitination, except for weak bands that appear to be H2A
conjugated with two Ub molecules (Fig. 2B, lane 2; Fig. 2C, lane
2). Importantly, E3 samples prepared with Phe-peptide (E3-F)
promoted significantly not only monoubiquitination and diubi-
quitination (Fig. 2B, lanes 3 vs. 2) but also polyubiquitination
(Fig. 2C, lanes 3 vs. 2). In contrast, protein samples prepared
in parallel with Val-peptide (E3-V) did not promote and, more-
over, often inhibited H2A ubiquitination (Fig. 2C, lane 4); under
the same conditions, UbcH8 and UbcH10 did not show detectible
H2A ubiquitination activity either in the presence or absence of
E3-F. To determine whether UBR1and UBR2 are responsible for
H2A ubiquitination activity of E3-F, in vitro H2A ubiquitination
assays were performed with E3-F, in which UBR1 and/or UBR2
were immunodepleted. Fig. S3A shows that immunodepletion of
UBR1 and UBR2 from E3-F reduces significantly H2A ubiqui-
tination. E3-F showed a moderate activity for H2Aubiquitination
of chicken oligonucleosome (Fig. 2E, lanes 3 vs. 1) but was
markedly increased in the presence of Ub aldehyde, an inhibitor
of deubiquitinating enzymes (DUBs) (Fig. 2E, lanes 4 vs. 3),
suggesting that E3-F-dependent H2A polyubiquitination may
be controlled by DUBs. To determine whether UBR2−∕−testes
contain reduced E3 activity for H2A, E3-F was prepared after
combining various tissues from wild-type and UBR2−∕−mice.
Compared with wild-type mice (Fig. 2D, lanes 6 vs. 4), E3-F from
UBR2−∕−mice showed markedly reduced E3 activity for ubiqui-
tination of free H2A (Fig. 2D, lanes 5 vs. 6). Likewise, E3-F pur-
ified from UBR2−∕−tissues produced reduced polyubiquitinated
histone species from nucleosome as determined by anti-FLAG
immunoblotting (Fig. S3B, lanes 3 vs. 2). Thus, UBR1 and
UBR2, in combination with HR6B, can mediate monoubiquitina-
tion and polyubiquitination of H2A in vitro.
In situ hybridization and immunostaining on testicular cross
sections revealed that UBR1 and UBR2 are prominently ex-
pressed in spermatogonia and spermatocytes, respectively (5).
While mutually exclusive tissue distribution of UBR1 and UBR2
explains why UBR2−∕−testes exhibit null phenotypes in meiosis,
we do not exclude the possibility that UBR1 also participates in
H2A ubiquitination during meiosis, as the localization of UBR1
to meiotic chromosomes has not been characterized extensively.
UBR2 Interacts with HR6B and H2A and Promotes the HR6B–H2A
Interaction and the Discharge of Ubiquitin from HR6B ∼ Ub. We
determined the interaction of HR6B with UBR2 and H2A by
using GST-pulldown assays with purified GST-HR6B, H2A, and
UBR2 (as a mixture with UBR1). Fig. 3A shows that HR6B
interacts with both UBR2 (Fig. 3A, lane 6) and its substrate
H2A (Fig. 3A, lane 4, long exposure). The HR6B–H2A interac-
tion appears to be intrinsically weak (Fig. 3A, lane 4) but can be
drastically enhanced by E3-F (Fig. 3A, lanes 6 vs. 4, third from
top), suggesting that the activity of E3-F in H2A ubiquitination
involves the enhanced interaction and/or affinity between E3-
bound HR6B and H2A. Likewise, the amount of UBR2 immo-
bilized by GST-HR6B was increased in the presence of H2A
(Fig. 3A, lanes 5 vs. 6 and lanes 8 vs. 9), indicative of a three-
way cooperative interaction between E3, E2, andsubstrate. These
results suggest that UBR2 binds both HR6B and H2A and
enhances the interaction of HR6B with H2A, promoting the
transfer of Ub from HR6B ∼ Ub to H2A.
We determined the interaction of UBR2 with HR6B and H2A
by using a ubiquitination-coupled binding assay, in which UBR2
(as a mixture with UBR1) immobilized on F-peptide-beads were
mixed with HR6B or H2A in the presence or absence of E1 and
Ub activating reagents (Fig. 3B). This assay can measure the
levels of HR6B and H2A in complex with E3s while histone is
being ubiquitinated. HR6B showed a robust retention (up to ap-
proximately 50%) to the F-peptide (Fig. 3B, lane 3) but not to
Fig. 3.
H2A, followed by immunoblotting for UBR2, H2A, and HR6B. Input: UBR2, 20%; H2A, 10%; HR6B, 10%. (B) Ubiquitination-coupled E3 binding assay. UBR2 (as a
mixture with UBR1) immobilized on F-peptide-beads were mixed with HR6B and/or H2A in the presence of E1 and Ub activating reagents, followed by im-
munoblotting of proteins (HR6B, H2A, and uH2A) retained by X-peptide. (C) A model for H2A ubiquitination in the XY body by the UBR2-HR6B complex.
Cooperative interactions between UBR2, HR6B, and H2A. (A) GST-pulldown assay (300 μl) with 200 ng GST-HR6B, 250 ng E3-F from rat testes, and 1 μg
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the V-peptide (Fig. S3C). In the presence of E1 and Ub acti-
vating reagents, H2A promoted the binding of HR6B∕HR6B ∼
Ub to E3 (Fig. 3B, lanes 8 vs. 6), which was also observed in
an independent GST-pulldown assay (Fig. 3A, lanes 9 vs. 8).
We also observed that a large portion (up to approximately
30%) of H2A binds to immobilized E3 (Fig. 3B, lane 4), and
the UBR2–H2A interaction is further enhanced when E3 is en-
gaged with E2 (Fig. 3B, lanes 5 vs. 4). In the presence of E1 and
Ub activating reagents, H2A molecules were massively converted
to polyubiquitin-conjugated species while they remain bound to
the E3–E2 complex (Fig. 3B, lane 8, bottom). Concurrently, the
steady-state level of E3-bound, unmodified H2A was markedly
depleted, as determined by immunoblotting with anti-H2A anti-
body that cannot detect ubiquitinated H2A, yet another evidence
for the E2-H2A transfer of Ub. Thus, the cooperative interaction
of UBR2, HR6B∕HR6B ∼ Ub, and H2A enhances the overall
affinity between each other, promoting the HR6B-H2A transfer
of Ub.
One notable property of HR6B is that the interaction of HR6B
to H2A is drastically accelerated when HR6B is charged with Ub
(Fig. 3A, lanes 7 vs. 4), suggesting that the HR6B-substrate affin-
ity is reduced once HR6B ∼ Ub transfers its cargo to H2A. This
Ub-dependent oscillation of the E2-substrate affinity is consistent
with a model (Fig.3C), in which HR6B ∼ Ub (with high affinity to
H2A) binds to H2A, and Ub-discharged HR6B (with low affinity
to H2A) dissociates from H2A ∼ Ub, in part explaining how an
E2–E3 pair mediates histone ubiquitination cycles while scanning
chromatin (see Discussion).
Chromosome-Wide Ubiquitination of H2A is Disrupted in UBR2-
Deficient Spermatocytes. To determine whether the inactivation
of UBR2 impairs the ubiquitination of H2A during meiosis, an
antibody to uH2A, which can detect both monoubiquitinated
and polyubiquitinated H2A (see Fig. 2E), was applied on spread
chromosomes from control and UBR2−∕−spermatocytes. In con-
trolcells,uH2Astainingwasfirstdetected atlate-zygotenemainly
on unsynapsed axes and as foci-like signals on the chromatin
domain (Fig. 4A). As spermatocytes enter pachytene, uH2A sig-
nalssurgedthroughouttheentirechromatin,withthehighestlevel
in the XY body (Fig. 4B and 4C, arrowheads). Overall, uH2A
showed remarkable similarity to UBR2 in temporal and spatial
localization on meiotic chromosomes at zygotene and pachytene,
exceptthattheUBR2stainingisalmostcompletelyexcludedfrom
the XY chromatin domain (Fig. 4C vs. Fig. S1D, arrowhead),
consistent with the model presented in Fig. 3C (see Discussion).
Importantly, the uH2A staining both in axes and the chromatin
domain was drastically diminished in UBR2−∕−spermatocytes
(Fig. 4, right panels) throughout the entire meiotic stages, which
was reproducibly observed in 154 nuclei from P16 testes. Down-
regulation of uH2A in mutant spermatocytes prior to the onset of
meiotic defects eliminates the possibility that H2Aubiquitination
is nonspecifically affected due to developmental arrest. These
results suggest that chromosome-wide ubiquitination of H2A is
disrupted in UBR2−∕−spermatocytes.
UBR2 Is Required for Transcriptional Silencing of Genes Linked to
Unsynapsed Axes of the Sex Chromosomes in Meiosis.As both UBR2
and uH2A mark meiotic chromatin inactivation (Figs. 1 and 4),
we determined whether UBR2-knockout would impair MSCI.
When spermatocytes enter the pachytene stage, the X and Y
chromosomes condense to form the sex body, where the chroma-
tin linked to unpaired XY axes is transcriptionally silenced. We
employed quantitative real-time PCR to examine the expression
of XY-linked genes in 4-week-old þ∕þ and UBR2−∕−testes.
Fig. S4A shows that UBR2 inactivation results in marked upreg-
ulation for the genes (MeCP2, Hprt, Hdac6, and Rbmy) known to
be silenced at pachytene, but not for those (Ube2a, Ube1x, and
Pctk1; indicated by asterisks) not subject to MSCI (23), indicating
a strong correlation between UBR2 function and MSCI. To char-
acterize the role of UBR2 in silencing at the chromosome level,
we performed microarray-based analysis of approximately 22;000
probe sets by using þ∕þ and UBR2−∕−testes at P17 when meiosis
has advanced to late pachytene (the accession no. GSE18824 at
the Gene Expression Omnibus database (http://www.ncbi.nlm.
nih.gov/geo/)). When the data were sorted for individual chromo-
somes, the average mRNA levels of genes encoded by autosomal
chromosomes were very similar in two groups (Fig. 5A). Strik-
ingly, UBR2−∕−testes showed overall upregulation of 542
X-specific and 15 Y-specific probes when compared to þ∕þ
testes (Fig. 5A; Table S1), indicative of chromosome-wide failure
in MSCI. Consistent with these results is previous Northern blot
analysis, in which five (Fthl17, Nxf2, Taf2q, Tktl1, and Rbmy) out
of seven randomly selected XY-linked genes (Fsc1, Fthl17, Nxf2,
Pramel3, Taf2q, Tktl1, and Rbmy) are upregulated in 6-week-old
UBR2−∕−testes compared to þ∕þ controls (5). These results
suggest that insufficiency in UBR2-dependent histone ubiquitina-
tion causes defects in transcriptional silencing of genes linked to
the sex chromosomes.
Transcriptional silencing of XY-linked genes is associated
with physical exclusion of proteins involved in transcription,
such as RNA polymerase II, from the chromatin. We deter-
mined whether the exclusion of RNA polymerase II from XY
is impaired in UBR2−∕−mice using immunostaining on spread
Fig. 4.
ubiquitination of H2A. Immunostaining of uH2A (red) was performed for
meiotic chromosomes from control and UBR2−∕−spermatocytes at zygotene
(A), early pachytene (B), and midpachytene (C). The X–Y pair is indicated by
arrowheads. The majority of UBR2−∕−spermatocytes are arrested at or before
early pachytene (5). The pachytene nuclei from UBR2−∕−mice shown in this
study are typical for those surviving spermatocytes. (Scale bar, 10 μm.)
UBR2-deficient spermatocytes are impaired in chromosome-wide
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chromosomes. In contrast to þ∕þ spermatocytes at pachytene
wherein RNA polymerase II was found to be excluded from
the XY chromosomes, the majority of UBR2−∕−spermatocytes
showed a uniform distribution of RNA polymerase II throughout
the entire nucleus (Fig. S4C). We next determined whether
UBR2 is required for XY pairing and XY body formation. Fluor-
escent in situ hybridization (FISH) analysis using probes for the X
and Y chromosomes showed that in the majority of UBR2−∕−
spermatocytes at the pachytene-like stage, X–Y pairing normally
occurred within the DAPI-rich sex body (Fig. 5B). Moreover, H-
and E-stained sections of seminiferous tubules from 4-week-old
þ∕þ and UBR2−∕−mice revealed that UBR2−∕−spermatocytes
contained distinct XY bodies at the nuclear periphery (Fig. S4B).
Although we do not exclude the possibility of subtle defects in the
mutants, these results suggest that XY pairing and XY body
formation do not strictly require UBR2. Collectively, our results
suggest that UBR2 is required for MSCI but is not critical for XY
pairing and XY body formation.
The Localization Pattern of UBR2 to Unsynapsed Axes is Distinct and
Independent from the BRCA1-Dependent MSCI Pathway. Recent
studies revealed that meiotic chromatin inactivation involves a
histone phosphorylation pathway, in which BRCA1 is accumu-
lated on unsynapsed XYaxes where it recruits ATR, which in turn
phosphorylates the histone H2AX, an essential histone modifica-
tion for MSCI and possibly MSUC as well (9). To determine
whether UBR2 functionally interacts with this histone phos-
phorylation, we compared the spatiotemporal localizations of
UBR2, BRCA1, and ATRon meiotic chromosomes. These MSCI
proteins accumulated on unpaired axes of autosomes and sex
chromosomes in a temporally synchronized manner (Figs. 6
and S5A–C). Intriguingly, they showed distinct spatial local-
ization patterns along the unsynapsed axes, in particular in
autosomes, in that the UBR2 staining on unsynapsed axes was
virtually excluded from the synaptic folk, whereas the axial
staining of BRCA1 and ATR was relatively weaker in the region
proximal to the telomere (Figs. 6B and S5B). Thus, histone ubi-
quitination and phosphorylation may be differentially regulated
along the chromosomal axes as illustrated in Fig. S5D.
We determined whether the recruitment of UBR2 and BRCA1
to meiotic chromosomes requires each other’s function. The
staining patterns of BRCA1 and ATR were not altered signif-
icantly in UBR2−∕−spermatocytes at zygotene through pachytene
(Fig. S6A–C). Moreover, phosphorylated H2AX (γH2AX) nor-
mally localized to the XY body of pachytene-like UBR2−∕−sper-
matocytes (Fig. S6D). Next, we examined the impact of BRCA1
deficiency on UBR2 localization by using spermatocytes from
BRCA1Δ11∕Δ11p53þ∕−mice (24), which produce a truncated
BRCA1 polypeptide lacking the exon 11-encoded amino acids
in the background of p53 haploinsufficiency. Fig. S7 shows that
the UBR2 staining on meiotic chromosomes is not significantly
altered in BRCA1-deficient spermatocytes. These results suggest
that the recruitments of UBR2 and BRCA1/ATR to unsynapsed
axes are mutually independent and that UBR2-dependent his-
tone ubiquitination is not essential for the function of BRCA1-
dependent histone phosphorylation pathway during MSCI.
Discussion. In this study we demonstrate that UBR2, the major
component of the N-end rule proteolytic pathway (reviewed in
ref. 2; 3, 5), plays a critical role in chromatin inactivation and
chromosome-wide transcriptional silencing during meiosis via
ubiquitination of histone H2A. Our results also provide mo-
lecular basis for several seemingly unrelated observations that
UBR2−∕−mice are infertile associated with meiotic arrest and
germ cell apoptosis (5) and that HR6B−∕−mice are infertile as-
sociated with impaired transcriptional silencing of some of
XY-linked genes (10; see also references therein). Notably, the
Fig. 6.
autosomes is distinct from that of BRCA1. (A and C) Localization of UBR2
(green) and BRCA1 (red) on unsynapsed axes of the autosomes at zygotene
(A) and the sex chromosomes at pachytene (C). (B) Enlarged images for
chromosomal regions indicated by the insets in (A). [Scale bar (A and C),
5 μm; (B) 1 μm.]
The localization pattern of UBR2 to unsynapsed axial regions of
Fig. 5.
somes in male meiosis. (A) Microarray analysis of control and UBR2−∕−testes
at P16. (B) Chromosome painting for X (red, arrowhead) and Y (green, arrow-
head) coupled with SCP3 immunostaining (green) in control and UBR2−∕−
spermatocytes. Dense peripheral DAPI staining indicates the sex body
(arrowheads). (Scale bar, 10 μm.)
UBR2 is required for transcriptional silencing of the X and Y chromo-
1916
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www.pnas.org/cgi/doi/10.1073/pnas.0910267107An et al.