Figure 1.
Representative examples SRY downstream direct binding target gene promoters for (A) Tcf21, (B) Atn1, and (C) Higd2a.
The positive hybridization is specific to SRY ChIP-DNA signal and negative hybridization to the non-immune IgG ChIP-DNA signal. Hybridization signals are the average of three biological replicates of ChIP assays. Hybridization signals below the statistical significance of p<1×10−7 was not considered. The localization of SRY response element motif is indicated for each promoter as a horizontal line under the bar. PCR primers were designed from the position indicated by two arrows. The PCR gel identifies PCR product size with (Markers), genomic DNA (Input), IgG ChIP (IgG) and SRY ChIP (aSRY). Data represent ChIP-PCR assays from three different experiments and biological replicates.
Table 1.
Direct downstream binding targets of SRY during male sex determination.
Figure 2.
ChIP-PCR confirmation of Sox9 as a downstream direct target of SRY.
Sox9 hybridization signals were not detected as the binding of SRY to Sox9 promoter is shown to occur at the TESCO region located at −7K upstream of the transcription start site. Non immune IgG was used as a negative control for each assay. ChIP DNA from IgG represented negative control throughout the experiment. PCR was conducted on 200 ng DNA amplified by whole genome amplification kit (Sigma). Data represent ChIP-PCR assay from three different experiments and biological replicates.
Figure 3.
Representative examples of SOX9 binding to the target promoter.
(A) Slc22a7, (B) Fxc1, and (C) Enam. The positive hybridization is specific to SOX9 ChIP-DNA signal (bar above the horizontal line) and negative hybridization (bar below the horizontal line) to the non-immune IgG ChIP-DNA signal. Hybridization signals are the average of three biological replicates of ChIP assays. Hybridization signals below the statistical significance of p<1×10−7 was not considered. The localization of SRY response element motif is indicated for each promoter as a horizontal line under the bar. PCR Primers were designed from the position indicated by two arrows. The PCR gel identifies PCR product size with (Markers), genomic DNA (Input), IgG ChIP (IgG) and SRY ChIP (aSRY). Data represent ChIP-PCR assays for three different experiments and biological replicates.
Table 2.
Direct downstream binding targets of SOX9 during male sex determination.
Figure 4.
SRY (A) and SOX9 (B) DNA sequence binding motifs.
The y-axis indicates the base and size performance for binding, and x-axis the base pair sequence for the motif.
Figure 5.
SRY (A) and SOX9 (B) direct binding target gene expression profiles for genes with a statistically (p<0.05) significance change in expression between the developmental periods.
Microarray analysis of embryonic day E13, E14 and E16 testis data previously described [26] was used to construct the expression profiles of the selected genes.
Figure 6.
Functional gene categories for SRY and SOX9 direct binding target genes, number of representative genes per functional category listed are indicated.
Table 3.
Cellular Pathways Enrichment for Sox9 and Sry.
Figure 7.
Gene network of shortest connections to cellular processes for 71 direct downstream gene targets of SRY, as obtained by global literature analysis using Pathway Studio 7.0 (Ariadne Genomics, Inc., Rockville, MD).
Figure 8.
Gene network of shortest connections to cellular processes for 109 direct downstream gene targets of SOX9, as obtained by global literature analysis using Pathway Studio 7.0 (Ariadne Genomics, Inc., Rockville, MD).