DEV02747 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. The expression of Scmh1 and other PcG genes in the testes. (A) The expression of Scmh1 in the adult organs. The gel was stained with EtBr to verify the amounts loaded by examining the amount of 28S and 18S rRNA. (B) Scmh1, Phc1 and Phc2 gene expression in spermatogenesis by northern blot analysis. The gel was stained with EtBr to verify the amounts loaded by examining the amount of 18S rRNA.

• Supplemental Figure 2 -

  Fig. S2. Generation of the Scmh1 mutant allele. (A) Genomic organization of the wild-type Scmh1 gene encoding C-terminal region (top), targeting vector (middle) and knockout allele (bottom), are schematically represented. Exons are shown as filled boxes and exonic regions encoding SPM domain, which are replaced by neo cassette, are shown as a red box. Also shown are the diagnostic restriction sites, ApaI, A; BamHI, B; NotI, N; XhoI, X; SacI, S, the external probe used for southern blot analysis, and (a), (b) and (c) used for genotype polymerase chain reaction (PCR) primer. (B) Southern blot analysis. Genomic DNA was digested with SacI/XhoI and hybridized with the external probe shown in A. The 6.4 kb band corresponds to the wild-type and the 3.3 kb band to the Scmh1 KO allele. Genotypes are indicated. (C) Genotype PCR used with primer (a), (b) and (c) shown in A. The 0.6 kb band corresponds to the wild-type and the 1.4 kb band to the Scmh1 KO allele. (D) Northern blot analysis to confirm the absence of 3.5 kb Scmh1 transcripts in the Scmh1-mutant testes. (E) RT-PCR analysis for Scmh1 expression. (Top) Coding region of Scmh1 mRNA is schematically shown. Regions corresponding to a start codon (ATG), two mbt repeats and a SPM domain, and positions of respective primers are indicated. (Bottom) Representative results are shown of RT-PCR by using primer pairs 1/2 and 3/4 and for β-actin as a control. (F) Homeotic transformations of the axis in Scmh1^−/− mice. Skeletal system of wild-type (a,c,e,g,i,k) and Scmh1^−/− (b,d,f,h,j,l) mice is comparatively shown. (a,b) Lateral views of the cervical region. (c,d) Lateral views of the cervico-thoracic transitional zone. (e,f) Ventral views of the rib cages. (g,h) Ventral views of the xiphoid process of the sternum. (i,j) Ventral view of the skull. (k,l) Ventral views of hyoid bone and stylohyoid ligament. (G) The posterior transformations in Scmh1^−/− mice and its penetrance. Thirty-nine Scmh1^−/− mice were analyzed and the following parameters were scored. (a) Appearance of the ectopic bone seen in the craniodorsal region of the C1 vertebra or ectopic arch associated with the occipital bone. (b) Fusion of C1 and C2. (c) Appearance of cervical rib on C7. (d) Detachment of the seventh rib from the sternum. (e) Formation of the sacroiliac joint. (H) Cell proliferation on a 3T9 protocol. At 3-day intervals, the total numbers of wild-type (diamonds), Scmh1^+/− (triangles) and Scmh1^−/− (circles) MEFs per culture were determined. Bars indicate standard errors from the mean. (I) Proliferation of MEFs from respective genotypes at passage 9 (P9). (J) Northern blot analysis of p16 and p19^ARF transcripts and western blot analysis of p53 protein in P3, P5 and P6 wild-type and Scmh1^−/− MEFs. The gel was stained with EtBr to verify the amounts loaded by examining the amount of 28S rRNA. Proteins in each lane were visualized by coomassie brilliant blue (CBB) staining. (K) Proliferation of Scmh1/ARF (left) and Scmh1/p53 (right) double mutants at P9. The total number of wild-type (triangles), Scmh1^−/−/ARF^+/+ or Scmh1^−/−/p53^+/+ (diamonds), Scmh1^+/+/ARF^−/− or Scmh1^+/+/p53^−/− (circles) and Scmh1^−/−/ARF^−/− or Scmh1^−/−/p53^−/− (squares) MEFs per culture were determined.

• Supplemental Figure 3 -

  Fig. S3. Testicular phenotypes in Scmh1<^b>⁻<^/b>^/<^b>⁻ mice. Cross sections of testes from day 15 pp wild-type and Scmh1^−/− mice. Lower (a,d), moderate (b,e) and higher (c,f) magnification views were shown. (a-c) Views of normal seminiferous tubules in the wild-type testis. (d-f) In strongly affected Scmh1^−/− mice, most of the seminiferous tubules were histologically abnormal, most contained a few spermatogonia, whereas the numbers of Sertoli cells were unaffected. Degenerating pachytene spermatocytes and vacuoles were frequently seen, as indicated by large arrowheads and asterisks, respectively. Scale bars: 100 μm in (a) and (d), 50 μm in (b) and (e), and 10 μm in (c) and (f).

• Supplemental Figure 4 -

  Fig. S4. SCP3 distribution in Scmh1<^b>⁻<^/b>^/<^b>⁻ spermatocytes using spread spermatocytes preparation. Late pachytene spermatocytes remained in Scmh1^−/− testes and exhibited normal SCP3 distribution, including PAR of sex chromosomes. Panels are views of Scp3 (green), SCP3 (green)/BRCA1 (red) and DAPI (blue).

• Supplemental Figure 5 -

  Fig. S5. Normal MSCI in Scmh1^-<^/b>^/<^b>^- spermatocytes. (A) Relative (Scmh1^−/−/Scmh^+/+) levels of gene expression as determined by microarray analysis. Average ratios of gene expression for each point (15, 18 and 20 pp). (B) Cot-1 RNA FISH of wild-type and Scmh1^−/− pachytene spermatocytes. The XY chromatin domain enriched in γH2A.X was negative for Cot-1 RNA in Scmh1^−/− spermatocytes as well as the wild type. Upper and lower panels are views of wild-type and Scmh1^−/− spermatocytes, respectively. Left, middle and right panels are views of γH2A.X (green)/DAPI (blue), Cot1 (red) and γH2A.X (green)/Cot1 (red), respectively.

• Supplemental Figure 6 -

  Fig. S6. Subcellular localization of Scmh1 in U2OS osteosarcoma cells and female TS cell. (A) Subcellular localization of endogenous (a) and overexpressed (d,g,j) Scmh1 in U2OS cells were compared with that of Phc2 (b,e,h,k). Merged images are shown in panels (c), (f), (i) and (l). (a,b,c) Endogenous Scmh1 and Phc2. (d,e,f) Overexpressed full-length Scmh1 gene products were predominantly localized in the cytoplasm. (g,h,i) Overexpressed truncated Scmh1 gene products (SPM domain only) were predominantly localized in the cytoplasm and altered the localization of Phc2. (j,k,l) Overexpressed truncated Scmh1 gene products (mbt repeats only) were predominantly colocalized with endogenous Phc2 in the nuclei. (B) Exclusion of endogenous Scmh1 from the inactive X chromosome in the female TS cells. (Upper panels) Subnuclear localization of Scmh1 (a) was compared with Ezh2 (b) and TOPRO3 (c). Merged image is shown in (d). Note the exclusion of Scmh1 from the inactive X chromosome domain, which was demarcated by the accumulation of Ezh2. (Lower panels) Subnuclear localization of Rnf2 (e) was compared with Ezh2 (f) and TOPRO3 (g). Merged image is shown in (h).