rodents (fig. S9A), but SRY was not destabi-
lized in those species (fig. S9B) because the
degron was blocked from translation due to
preceding stop codons (fig. S9, A to D). In
mice, a different mechanism has evolved to
avoid degron translation through exonization
of distal retrotransposon-derived sequences
(Fig. 1C and fig. S3) and splicing to excise the
degron (fig. S10). The apparent replacement of
SRY-S by SRY-T to overcome degron activity
provides an example of Y-chromosomal gene
evolution by gain of function.
Our findings overturn the conclusion that
has prevailed for 30 years regarding the struc-
ture ofSryin the mouse, the most intensively
studied mammalian model of sex determi-
nation. Further study of SRY-T will establish
whether exon2 confers new functionalities on
SRY in addition to protein stability and will
provide a more complete understanding of how
the sex-determining cascade is activated in mice
compared with other mammalian species.
REFERENCES AND NOTES- P. Koopman, J. Gubbay, N. Vivian, P. Goodfellow,
R. Lovell-Badge,Nature 351 , 117–121 (1991). - A. H. Sinclairet al.,Nature 346 , 240–244 (1990).
- J. Gubbayet al.,Nature 346 , 245–250 (1990).
- J. Gubbayet al.,Proc. Natl. Acad. Sci. U.S.A. 89 , 7953–7957 (1992).
- S. Kurokiet al.,Science 341 , 1106–1109 (2013).
- S. Kurokiet al.,Genesis 53 , 387–393 (2015).
- I. Stévantet al.,Cell Rep. 22 , 1589–1599 (2018).
- J. Bowles, L. Cooper, J. Berkman, P. Koopman,Nat. Genet. 22 ,
405 – 408 (1999). - L. Zhaoet al.,Proc. Natl. Acad. Sci. U.S.A. 111 , 11768–11773 (2014).
- S. Kurokiet al.,PLOS Genet. 13 , e1007034 (2017).
- J. M. Stenmanet al.,Science 322 , 1247–1250 (2008).
- I. Korenet al.,Cell 173 , 1622–1635.e14 (2018).
- L. L. Washburn, K. H. Albrecht, E. M. Eicher,Genetics 158 ,
1675 – 1681 (2001). - Y. Itohet al.,BMC Res. Notes 8 , 69 (2015).
- M. Stevanović, R. Lovell-Badge, J. Collignon, P. N. Goodfellow,
Hum. Mol. Genet. 2 , 2013–2018 (1993). - E. Suttonet al.,J. Clin. Invest. 121 , 328–341 (2011).
- T. Kidokoroet al.,Dev. Biol. 278 , 511–525 (2005).
ACKNOWLEDGMENTS
We thank H. Kondo and K. Ichiyanagi for critical advice on the
manuscript; Y. Kanai for providingHSP-Srytransgenic mice; J. Kohyama
for help with capillary-based immunodetection; H. Miyachi for help with
animal experiments; and all members of the Tachibana laboratory fortechnical support.Funding:This work was supported by KAKENHI
from the Japan Society for the Promotion of Science (JSPS) grants
19K22388 (M.T.), 18H02419 (M.T.), 17H06424 (M.T.), 17H06423
(M.T.), 20H05364 (S.K.), and 19K16490 (S.M.) and by a JSPS Research
Fellowship for Young Scientists to S.M. and R.M.Author
contributions:S.M., S.K., and M.T. designed the experiments. S.M.,
S.K., R.M., N.O., and M.T. performed the experiments and analyzed the
data. R.M. contributed to the analyses of RNA-seq and CAGE-seq
data. S.M., P.K., and M.T. interpreted the data and wrote the
manuscript.Competing interests:The authors declare no competing
interests.Data and materials availability:All data are available in
the main text or the supplementary materials. The accession number
for the RNA-seq and CAGE-seq data generated in this study is
GSE151474. The sequence ofSry-Twas deposited in the GenBank
database under accession code LC532173.
SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/370/6512/121/suppl/DC1
Materials and Methods
Figs. S1 to S10
Tables S1 to S3
References ( 18 – 24 )
Data S1 and S2
MDAR Reproducibility Checklist27 March 2020; accepted 6 August 2020
10.1126/science.abb6430124 2 OCTOBER 2020•VOL 370 ISSUE 6512 sciencemag.org SCIENCE
Fig. 4. SRY-S carries a C-terminal degron.
(A) Specific C-terminal sequences of
SRY-S and SRY-T were named S18 (blue)
and T15 (red). (B) Lentivirus vector used
for protein stability analysis. BFP and
EGFP-fused polypeptides were translated
from the same transcript. (C) Protein-
destabilizing activity of S18 and T15 was
gauged by the EGFP/BFP ratio using flow
cytometry. (D) Western blot analysis of
EGFP-S18 (left) and nonfusion EGFP (right)
expressed in lentivirus-infected cells. Cells
were treated with the protein synthesis
inhibitor cycloheximide (CHX) for 12 hours
in the presence or absence of the proteasome
inhibitor MG132. Quantified protein levels
are indicated. Equal protein loading was
confirmed by Ponceau S staining. (E) V-2P
substitution abolished S18 degron activity.
(F) Editing of endogenousSryto generate
anSry-S:V394P allele on anSry-T–deficient
background (left).Sry-S:V394P;Sryexon2D
mice (n= 4 lines) developed as males
(right). (G) Coimmunostaining profiles of
SRY and NR5A1 in E11.5 gonads of the
indicated genotypes. Data are representative
of biological triplicate experiments.
(H) Quantitation of SRY in NR5A1+cells in
E11.5 gonads of the indicated genotypes.
Data are shown as means ± SD. Numbers
of analyzed cells are shown in parentheses
above each bar. ***P< 0.001, ns, not
significant, between the indicated groups,
ANOVA with Tukey’s post hoc test.
G HSRYSRY/NR5A1/DAPISry-S:V394P;
WT (XY) WT (XX) Sry exon2∆ (XY) Sry exon2∆ (XY)F+ Genome editingV394Ppoly Q Exon2∆Exon1
HMGpoly Q Exon2∆Exon1
HMGSry exon2∆Genome (Sry locus)Sry-S:V394P;
Sry exon2∆Sry exon2∆ (XY)Sry-S:V394P;
Sry exon2∆ (XY)STOP
V394P50 μmABDStability (EGFP/BFP)CBFPIRESEGFPEGFP-S18LT R PeptideLT RS18T15EHTPYQEHLSTALWLAVSWKGIHKCTGPPD EPFSRY-SSRY-T392395
HMGpoly Q1 377HMGpoly Q1 377EF1Percent cellsStability (EGFP/BFP)EGFP-T15Not infected
EGFPEGFP-S18 (V-2P)Percent cells
10 -1 100 101 10210 -1 100 101 102 10 -1 100 101 10220
040608010020
040608010020
0406080(^100) Not infected
EGFP
E
EGFP
CHX
MG132
++
EGFP-S181.00 0.17 0.59A L WSL A PEGFP1.00 1.56 1.35++Ponceau SSry-S:V394P;Sryexon2∆ (XY)Sryexon2WT (XY)WT (XX)∆ (XY)SRY intensityin Nr5a1 positive cells (A.U.)^050100
(92) ******
(102)
ns (P = 0.135)(80)(100)RESEARCH | REPORTS