them into the CP’s proteolytic tunnel. Meiosis-specific recruitment of fully assembled 26S
proteasome to chromatin was indicated by localization analysis of fully functional, constitutive
CP20S component a5 (a5Pup2-GFP; Fig. 4, A and
D, and fig. S8) (17) and RP component Rpn12
[GFP-Rpn12 or hemagglutinin (HA)–Rpn12;
Fig. 4, B and E, and fig. S9] (18). Low numbers
of CP20S and RP19S foci in premeiotic cells (t =
0 hours) was consistent with proteasome occupancy of many genome regions in vegetative
cells (Fig. 4, A and B) (19). Proteasome foci increased during premeiotic S phase and/or leptonema (t = 2 hours), with an intermittent dip or
steadily, reaching peak levels of 29.2 (± 14.4)
CP20S and 32.6 (± 16.6) RP19S foci, respectively.
Proteasome foci peaked 1 hour after pachynema
(Fig. 4, D and E) and were highest in nuclei
exhibiting fragmented Zip1 staining as typically
observed at pachytene exit. Thus, RP19S and/or
CP20S recruitment to chromatin occurs in two
waves, the first during leptonema and the second during pachytene exit. Both CP20S and RP19S
tended to localize to chromatin regions devoid
of Zip1 (Fig. 4, A and B), consistent with a proteasome role in displacing Zip1 from the corresponding chromosome regions.
CP20S recruitment to chromatin is controlled
by a subset of meiosis-specific factors. Efficient
recruitment of CP20S in a strain expressing catalytically inactive spo11-yf (13) suggest that meiotic DSBs are dispensable, at least for early CP20S
recruitment, whereas a5Pup2 foci were substantially reduced at all times in both zip1D and zip3D
(Fig. 4, C and D, and fig. S10). Polycomplexes in
zip3D and spo11-yf also frequently contained at
least one CP20S focus (>90%; Fig. 4C), indicat-
ing a potential Zip1 role in CP20S recruitment.
However, a role of synapsis itself in CP20S
recruitment is unlikely because spo11-yf and zip3D
show similar defects in SC assembly (2, 5). More
likely, Zip1 and Zip3 control CP20S recruitment
via their spatial and/or functional association
with coupled centromeres (2, 14, 16).
A conserved meiotic proteasome function was
garnered from examination of the Caenorhabditis
elegans germ line. Depletion of the a3Pre9 ortholog
PAS-3 disrupted both early and late SC morphogenesis, as suggested by increased abundance in zygotene nuclei of PCs containing both
axis protein HTP-3 and central element protein
SYP-1 (Fig. 4F and fig. S12) (20, 21). Moreover,
during pachynema, SYP-1 underwent premature
and excessive polarization [fig. S12A, (iii)], a distribution normally limited to diplonema (21).
Consistent with evolutionarily conserved functions along meiotic chromosomes, the CP20S
extensively associated with SYP-1–decorated,
pachytene chromosome squashes (Fig. 4G) [see
also accompanying paper (22)].
Meiosis-specific proteasome association with
chromosome axes was also detected in mouse
spermatocytes, in which CP20S signal overlaps with
SYCP3-stained chromosome axes (3, 22) at all
meiotic stages except leptonema (Fig. 4H), whereas staining was absent in nonmeiotic testicular
cells (fig. S13C).
Our findings support the following model:
Before the DSB-induced homology search, nonhomologous interactions can become stabilized
by promiscuous association with chromosomes
of SC proteins, including yeast Zip1 [Fig. 4I, (i)].
Following its recruitment, the proteasome displaces SC proteins, restricting the latter to centromeres [Fig. 4I, (ii)]. At this stage, the proteasome
may also ensure high DSB levels via its role in
axis morphogenesis (1) and/or via removal of
proteins that normally render DSB formation
by Spo11 reversible (1, 23). Nonhomologous centromere coupling could provide a structural barrier against precocious nonallelic pairing (3). This
pairing block is then destabilized by proteasome-mediated removal of SC proteins [Fig. 4I, (iii)].
Once integrated into the meiotic program, chromosomally tethered proteasome may have acquired lineage-specific functions and localization
patterns. Our model postulates independent
proteasome functions in homolog pairing, axis
morphogenesis, and DSB formation that in turn
control SC assembly and CO formation. The proteasome may also have distinct roles in the two
latter processes. Notably, proteasome effects cannot simply be attributed to DSB reduction or
failed Zip1 recruitment (supplementary text).
Interactions between the proteasome and
polyubiquitinated substrates are assumed to
be stochastic (8). Our findings suggest that the
proteasome is targeted to chromosomal sites by
appropriately modified substrates, analogous to
its association, e.g., with the endoplasmic reticulum or with sites of DNA damage (8, 17, 19, 24).
Notably, meiotic recombination sites in many
organisms are occupied by RING finger E3 ubiquitin and/or SUMO ligases (6). Moreover, the
ubiquitin-SUMO-proteasome system controls CO
formation and positioning (7, 22, 25). Our work
identifies chromosomal tethering of 26S proteasome during meiosis as an evolutionarily
410 27 JANUARY 2017 • VOL 355 ISSUE 6323 sciencemag.org SCIENCE
Fig. 3. The proteasome controls axis morphogenesis and
synapsis (33°C). (A) Zip1 and
Zip3-GFP localization in WT class
I, II, or III nuclear spreads; in
pre9D; and in the presence of
MG132 (bottom). Blue dotted
lines as described in Fig. 1B.
Bar, 1 mm. (B) Zip1 localization in
WT and pre9D cultures. For Zip1
classes, see (A). (C) Red1-
staining classes. Bar, 1 mm.
(D) Quantitation of Red1 classes
at t = 6 hours in PRE9 and pre9D
in ndt80D background. “Other”
indicates poorly spread nuclei.
n ≥ 3; error bars represent SD;
*P < 0.03, two-tailed t test.
(E) Zip3-GFP focus numbers in a
pdr5D culture treated with
dimethyl sulfoxide (mock; 37.4 ±
16.1) or with MG132 (34.8 ± 15.0)
in pre9D (38.3 ± 14.3) or in an
untagged WT strain (5.3 ± 3.1)
at the time of WT pachytene
(t = 6 hours). Error bars represent SD (see also fig. S7C). (F) Zip1 localization in PRE9 spo11D (n = 3) and pre9D spo11D (n = 3) nuclei. Arrows indicate
polycomplexes. Bar, 1 mm. (G) Quantitation of Zip1 localization in a spo11D background. #Polycomplexes (PC) were scored independently of patterns of
chromatin-associated Zip1. Fractions are averages from two cultures at t = 3.5 and 5 hours or t = 4 and 6 hours, respectively; error bars represent SD; *P <
0.03, two-tailed t test. For nucleus numbers, see table S1.