20. G. Ferreira, M. I. Miranda, V. De la Cruz, C. J. Rodríguez-Ortiz,
F. Bermúdez-Rattoni, Eur. J. Neurosci. 22, 2596–2604
21. J. Courtin et al., Nature 505, 92–96 (2014).
22. J. Debiec, V. Doyère, K. Nader, J. E. Ledoux, Proc. Natl. Acad.
Sci. U.S.A. 103, 3428–3433 (2006).
23. A. Vazdarjanova, B. L. McNaughton, C. A. Barnes, P. F. Worley,
J. F. Guzowski, J. Neurosci. 22, 10067–10071 (2002).
24. S. K. Barot, Y. Kyono, E. W. Clark, I. L. Bernstein, Proc. Natl.
Acad. Sci. U.S.A. 105, 20959–20963 (2008).
25. Q. Yuan, J. S. Isaacson, M. Scanziani, PLOS ONE 6, e20486
26. L. G. Reijmers, B. L. Perkins, N. Matsuo, M. Mayford, Science
317, 1230–1233 (2007).
27. R. Feil, J. Wagner, D. Metzger, P. Chambon, Biochem. Biophys.
Res. Commun. 237, 752–757 (1997).
28. T. Kawashima et al., Nat. Methods 10, 889–895 (2013).
29. C. R. Brewin, E. A. Holmes, Clin. Psychol. Rev. 23, 339–376
We thank K. Deisseroth (Stanford University) for the Arch T
3.0-EYFP cDNA, H. Hioki and T. Kaneko (Kyoto University) for the
STB vector, and H. Bito (Tokyo University) for the E-SARE cDNA.
All members of the Inokuchi laboratory supported and discussed
this study. This work was supported by the CREST program of JST;
Japan Society for the Promotion of Science KAKENHI grant
numbers JP23220009 (K.I.), JP23650208, JP25293136 (F.K.),
JP25430015 (A.M. W.), JP25860429, JP15K19194 (Y. T.), and
15K18344 (M. Nomoto); a Grant-in-Aid for Scientific Research on
Innovative Areas “Memory dynamism” [JP25115002 (K.I.) and
JP26115523 (A.M. W.)] and “Microendophenotypes of psychiatric
disorders” [JP15H01295 (A.M. W.)] from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT); MEXT-Supported
Program for the Strategic Research Foundation at Private
Universities S1311009 (F.K.); the Mitsubishi Foundation; the Uehara
Memorial Foundation; and the Takeda Science Foundation (support
to K. I.). All data necessary to understand and assess the conclusions
of this research are available in the supplementary materials.
Materials and Methods
Figs. S1 to S8
Tables S1 and S2
Data File S1
24 October 2016; accepted 4 January 2017
A SUMO-ubiquitin relay recruits
proteasomes to chromosome axes to
regulate meiotic recombination
H. B. D. Prasada Rao,1,2 Huanyu Qiao,1,2 Shubhang K. Bhatt,2 Logan R. J. Bailey,2
Hung D. Tran,2 Sarah L. Bourne,2 Wendy Qiu,2 Anusha Deshpande,2 Ajay N. Sharma,2
Connor J. Beebout,2 Roberto J. Pezza,3 Neil Hunter1,2,4,5*
Meiosis produces haploid gametes through a succession of chromosomal events, including
pairing, synapsis, and recombination. Mechanisms that orchestrate these events remain
poorly understood. We found that the SUMO (small ubiquitin-like modifier)–modification
and ubiquitin-proteasome systems regulate the major events of meiotic prophase in
mouse. Interdependent localization of SUMO, ubiquitin, and proteasomes along
chromosome axes was mediated largely by RNF212 and HEI10, two E3 ligases that are also
essential for crossover recombination. RNF212-dependent SUMO conjugation effected a
checkpointlike process that stalls recombination by rendering the turnover of a subset of
recombination factors dependent on HEI10-mediated ubiquitylation. We propose that
SUMO conjugation establishes a precondition for designating crossover sites via selective
protein stabilization. Thus, meiotic chromosome axes are hubs for regulated proteolysis
via SUMO-dependent control of the ubiquitin-proteasome system.
Meiosis halves the chromosome comple- ment via two successive rounds of cell division. Accurate segregation of homol- ogous chromosomes (homologs) during the first meiotic division requires their
connection by chiasmata—the conjunction of
crossing over and sister-chromatid cohesion (1).
Chiasma formation is the culmination of an
elaborate series of interdependent events that
include programmed recombination and the
pairing and synapsis of homologs. Each homol-
og comprises two sister chromatids, which are
organized into arrays of chromatin loops con-
nected to a common core or axis. Pairing and
synapsis are promoted by homologous recom-
bination, which occurs in physical and func-
tional association with these axes. As meiosis
progresses, axes align and become connected
along their lengths to form synaptonemal com-
The SUMO (small ubiquitin-like modifier)–
modification (SMS) and ubiquitin-proteasome
(UPS) systems are key regulators of cellular pro-teostasis (2, 3) and are implicated in various aspects of meiotic prophase (4–9). However, their
roles remain poorly characterized, especially in
mammalian meiosis. To obtain cytological evidence that the SMS and UPS regulate axis-associated events, we analyzed the localization of
SUMO, ubiquitin, and proteasomes along surface-spread chromosomes from mouse spermatocytes
(Fig. 1 and figs. S1 to S3).
The SUMO1 and SUMO2/3 isoforms local-
ized to axes during zygonema, as chromosomes
underwent synapsis, forming punctate patterns
of ~200 immunostaining foci (Fig. 1, A and B,
and fig. S1). Superresolution structured illumi-
nation microscopy (SIM) revealed that SUMO
was present on both unsynapsed and synapsed
axes; axial, supra-axial (extending into adjacent
chromatin), and SC central-region staining could
be discerned (Fig. 1A and fig. S1). General axis
staining disappeared after synapsis completed
and cells entered pachynema (Fig. 1, A and B,
and fig. S1). Subsequently, SUMO accumulated
on centromeric heterochromatin and the XY
chromatin (sex body) (10). Prominent axis stain-
ing of ubiquitin was also detected during zygo-
nema but persisted throughout pachynema (Fig.
1, A and B, and fig. S2). SIM revealed that most
ubiquitin foci localized to axes, but SC central-
region staining was occasionally discerned (Fig. 1A
and fig. S2). Ubiquitin also accumulated along axes
of the sex chromosomes during zygonema and
early pachynema, before spreading to the entire
XY chromatin (11). Prominent staining of cen-
tromeric heterochromatin was not seen for ubi-
quitin, but general chromatin staining became
apparent after mid-pachynema (Fig. 1, A and B,
and fig. S2). Abundant recruitment of protea-
somes along axes also occurred during zygo-
nema (Fig. 1, A and B, and fig. S3) and persisted
throughout pachynema and diplonema, when
chromosomes desynapsed. By SIM, proteasome
foci were largely axis associated, but less fre-
quent SC central-region staining was also seen.
Sex body, centromeric, and general chromatin
staining were not detected. This subchromosomal
recruitment of proteasomes to meiotic chromo-
some axes, which appears to be an evolutionarily
conserved feature of meiosis [see accompanying
paper (12)], predicts that axis-associated ubiqui-
tin should include chains linked through lysine
48; this inference was confirmed by using linkage-
specific ubiquitin antibodies (fig. S2) (13).
These immunostaining results suggest that
the SMS and UPS regulate axis-associated events
via protein degradation. To test this, we exploited short-term culture of testis cell suspensions
(14) and chemical inhibitors of SUMO conjugation (2-DO8) (15), ubiquitin activation (PYR41)
(16), or proteasomal degradation (MG132) (Fig.
2 and figs. S4 to S6). All three inhibitors caused
dramatic increases in large extra-chromosomal
1Howard Hughes Medical Institute, University of California,
Davis, CA 95616, USA. 2Department of Microbiology and
Molecular Genetics, University of California, Davis, CA 95616,
USA. 3Cell Cycle and Cancer Biology Research Program,
Oklahoma Medical Research Foundation, Oklahoma City, OK
73104, USA. 4Department of Molecular and Cellular Biology,
University of California, Davis, CA 95616, USA. 5Department
of Cell Biology and Human Anatomy, University of California,
Davis, CA 95616, USA.
*Corresponding author. Email: firstname.lastname@example.org