Supplementary MaterialsS1 Fig: Histone marks are constant between biological replicates and validated by ChIP-qPCR. promoters, in XY (blue) or XX (pink) supporting cells at E10.5 (left) and E13.5 (right) (outliers N-Acetyl-L-aspartic acid excluded). *** represents p 0.0001 as determined by students t test. (C&D) Venn diagrams depicting quantity of overlapping bivalent promoters between Sertoli cells (blue) and pregranulosa cells (pink) at E10.5 (C) and E13.5 (D).(TIF) pgen.1007895.s002.tif (490K) GUID:?B2353203-FAA0-4472-9B15-0F72E328464B S3 Fig: Up-regulation of testis or ovary pathway genes is associated with loss of H3K27me3. (A) Bar graphs denoting gene expression log intensity values from Nef et al, 2005, for select genes in XY (blue) and XX (pink) supporting cells at E13.5. *** represents p 0.0001 as determined by students t test. Values represent imply SEM. (B) A closer look at H3K27me3 ChIP-seq songs at in E13.5 pregranulosa cells (top) and Sertoli cells (bottom) shows loss of H3K27me3 at the promoter of in XX but not XY cells.(TIF) pgen.1007895.s003.tif (330K) GUID:?00C7D5D2-2344-43C7-B596-D9569511D932 S4 Fig: Repressed ovary pathway genes retain bivalent marks in adult Sertoli cells. (A) ChIP-qPCR for H3K27me3 (reddish), H3K4me3 (green) and IgG (grey) at the promoter of several ovary-specific genes in purified Sertoli cells from adult ( 2m/o) males. Each qPCR was performed on 3 biological replicates, each replicate contained purified Sertoli cells from 1C2 adult males. (B) ChIP-re-ChIP on adult testes for H3K27me3 followed by either H3K4me3, or a no-antibody control, performed on two impartial replicates, with testes from 1C2 adult males. Values represent imply SEM.(TIF) pgen.1007895.s004.tif (410K) GUID:?D4C05D4F-70B6-4498-AF57-3DA472C80953 S5 Fig: H3K27me3 spreads over repressed loci in Sertoli cells. UCSC genome browser songs of example repressed genes in XY supporting cells where H3K27me3 deposition (reddish) is confined to narrow locations at E10.5 (top rows) and spreads upstream and N-Acetyl-L-aspartic acid downstream from the TSS, and within the gene body at E13.5 (bottom rows). Collapsed H3K27me3 monitors are symbolized in pubs above monitors.(TIF) pgen.1007895.s005.tif (347K) GUID:?473C0478-D143-402A-951B-93851C3BDE4F S6 Fig: Pregranulosa-determining genes with vital assignments in ovary advancement are targets of PcG repression in Sertoli cells. Heatmap of H3K27me3 enrichment amounts on the promoters of pregranulosa-promoting genes in Sertoli cells, which range from high (light crimson) to low (light green). Beliefs signify log2 enrichment normalized to H3. A nearer go ENG through the genes with 4 H3K27me3 enrichment are proven in the proper column. Genes with known assignments in ovary advancement are bolded.(TIF) pgen.1007895.s006.tif (709K) GUID:?79DD1085-DD96-4299-B07B-920EA918D996 S7 Fig: The Wnt pathway is targeted for H3K27me3-mediated repression in Sertoli cells. (A&B) Gene Ontology useful evaluation using GREAT of pregranulosa-specific promoters and N-Acetyl-L-aspartic acid flanking locations designated by H3K27me3 demonstrates the Wnt signaling pathway is definitely significantly targeted for repression in Sertoli cells (A), and that the developmental processes most highly displayed are those associated with the formation of the reproductive system, in particular the female reproductive and urogenital system (B). (C) Genome internet browser songs showing ChIP-seq profiles for H3K4me3 (green) and H3K27me3 (reddish). Promoters highlighted in blue. Black boxes symbolize significant enrichment when compared to flanking areas as determined by HOMER.(TIF) pgen.1007895.s007.tif (1.2M) GUID:?2CC0538B-5263-4718-9DDA-34EC4671F940 S8 Fig: Sex reversal is rescued in double knockout XY gonads. (A&B) XY gonads are stained with the pregranulosa cell marker FOXL2 (green), Sertoli cell marker SOX9 (reddish), and vasculature and germ cell marker PECAM (blue). Loss of in E13.5 XY gonads prospects to reduction of SOX9+ Sertoli cells, gain of FOXL2+ pregranulosa cells, and testis cords are lost (A). DKO gonads do not have FOXL2+ pregranulosa cells, and testis wire formation is definitely rescued (B). XY gonads develop as ovaries (C). DKO gonads develop as testes (D).(TIF) pgen.1007895.s008.tif (775K) GUID:?BDA51276-91E6-4343-8C02-B7007D8D676F S9 Fig: CBX2 targets for repression in adult testes. ChIP-qPCR for CBX2 adult testes from 2m/o mice (2 males/experiment). * represents p 0.01 while determined by college students t test when compared to the negative control could save testis development in mutants. We display that manifestation and testis development were rescued in XY mice. Furthermore, we display that CBX2 directly binds the downstream Wnt signaler (is definitely transiently indicated in XY progenitor cells from ~E10.5-E12.5, soon after the gonad is first formed [6, 7]. main function is definitely to upregulate its downstream target . upregulation and subsequent maintenance through prospects to Sertoli cell differentiation and establishment of the testis pathway . In the absence of a Y chromosome, the canonical Wnt signaling molecules and become upregulated in XX progenitor cells (Vainio, 1999, Chassot, 2008). The subsequent downstream stabilization of -catenin  together with upregulation of additional transcription factors such as , prospects to the.