Global genome analysis of the downstream binding targets of testis determining factor SRY and SOX9

Abstract

A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination.

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.

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.

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 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.

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).