Auto-regulation of the Sohlh1 gene by the SOHLH2/SOHLH1/SP1 complex: implications for early spermatogenesis and oogenesis

Abstract

Tissue-specific basic helix-loop-helix (bHLH) transcription factor proteins often play essential roles in cellular differentiation. The bHLH proteins SOHLH2 and SOHLH1 are expressed specifically in spermatogonia and oocytes and are required for early spermatogonial and oocyte differentiation. We previously reported that knocking out Sohlh2 causes defects in spermatogenesis and oogenesis similar to those in Sohlh1-null mice, and that Sohlh1 is downregulated in the gonads of Sohlh2-null mice. We also demonstrated that SOHLH2 and SOHLH1 can form a heterodimer. These observations led us to hypothesize that the SOHLH2/SOHLH1 heterodimer regulates the Sohlh1 promoter. Here, we show that SOHLH2 and SOHLH1 synergistically upregulate the Sohlh1 gene through E-boxes upstream of the Sohlh1 promoter. Interestingly, we identified an SP1-binding sequence, called a GC-box, adjacent to these E-boxes, and found that SOHLH1 could bind to SP1. Furthermore, chromatin-immunoprecipitation analysis using testes from mice on postnatal day 8 showed that SOHLH1 and SP1 bind to the Sohlh1 promoter region in vivo. Our findings suggest that an SOHLH2/SOHLH1/SP1 ternary complex autonomously and cooperatively regulates Sohlh1 gene transcription through juxtaposed E- and GC-boxes during early spermatogenesis and oogenesis.

Figure 1. SOHLH2 and SOHLH1 form homodimers.

(A) Western blots of lysates of HEK293 cells overexpressing SOHLH2, FLAG-SOHLH2, and SOHLH2-Myc, using anti-SOHLH2, anti-FLAG, and anti-Myc antibodies. (B) Western blots of lysates of HEK293 cells overexpressing SOHLH1, FLAG-SOHLH1, and SOHLH1-Myc, using anti-SOHLH1, anti-FLAG, and anti-Myc antibodies. (C) Lysates of HEK293 cells overexpressing FLAG-SOHLH2, SOHLH2-Myc, or SOHLH2 were immunoprecipitated with anti-FLAG agarose beads and subjected to western blotting using an anti-Myc antibody. Arrow: an SOHLH2-Myc band. Pre-immunoprecipitation lysates were used as input. (D) Lysates of HEK293 cells overexpressing FLAG-SOHLH1, SOHLH1-Myc, or SOHLH1 were immunoprecipitated with anti-FLAG agarose beads and subjected to western blotting using an anti-Myc antibody. Arrow: an SOHLH1-Myc band. Pre-immunoprecipitation lysates were used as input. Anti-FLAG agarose was loaded to indicate the IgG heavy chain (dashed arrow) and IgG light chain (arrowhead) bands.

Figure 2. SOHLH2/SOHLH1 heterodimer regulates the Sohlh1 promoter through a restricted upstream region.

(A) Reporter assay using the pGL3-Basic vector containing the −1036 bp mouse Sohlh1 promoter (200 ng), a pCAG-Sohlh2 expression vector (0 to 40 ng), a pCAG-Sohlh1 expression vector (0 to 40 ng), and a pRL-CMV normalization vector (0.1 ng). Results show the Sohlh1 promoter-driven firefly luciferase activity relative to CMV promoter-driven Renilla luciferase activity. (B) Reporter assays using pGL3-Basic vectors containing various lengths of the mouse Sohlh1 promoter (200 ng), a pCAG-Sohlh2 expression vector (200 ng), a pCAG-Sohlh1 expression vector (200 ng), and a pRL-CMV normalization vector (0.1 ng). Results show the firefly/Renilla luciferase activity relative to that obtained with the −1036 bp promoter fragment, which was arbitrarily set at 1. Error bars represent the S.E.M. of the means of 3–5 separate experiments done in triplicate. P values were calculated by Student's t-test. *P<0.05. SOHLH2 and SOHLH1 regulate the activity of the mouse Sohlh1 promoter through a region −154 to −321 bp upstream from its translational start site.

Figure 3. Conserved regulatory regions of the mouse and rat Sohlh1 gene.

The underlined E- and GC-box sequences are conserved between the mouse and rat. These E- and GC-box sequences are also found in the Sohlh1 promoter region of the chimpanzee and human (not shown).

Figure 4. The three Sohlh1-promoter E-boxes are not equal in regulating Sohlh1.

Reporter assays using the pGL3-Basic vector with various mutations of the E-boxes in the Sohlh1 promoter, along with pCAG-Sohlh1 and pCAG-Sohlh2 expression vectors (200 ng each). Results show the firefly/Renilla luciferase activity relative to that of the −1036 bp intact promoter, which was arbitrarily set at 1. Error bars represent the S.E.M. of the means of 3–6 separate experiments done in triplicate. P values were calculated by Student's t-test. *P<0.05.

Figure 5. Transcriptional synergy between SP1 and the SOHLH proteins.

(A) Reporter assays using the pGL3-Basic vector with the −1036 bp intact Sohlh1 promoter (200 ng), a pRL-CMV normalization vector (0.1 ng), and expression vectors (pCAG-Sohlh2 (40 ng), pCAG-Sohlh1 (40 ng), and pCAG-Sp1 (40 ng)). Results show the Sohlh1 promoter-driven firefly luciferase activity relative to that of CMV promoter-driven Renilla luciferase. The white dashed line indicates the sum of the individual transcriptional activities of SOHLH2, SOHLH1, and SP1. The black dashed line indicates the sum of the transcriptional activities of SOHLH2+SOHLH1 and SP1. P values were calculated by Mann-Whitney test. *P<0.05. (B) Reporter assays using the pGL3-Basic vector containing the −1036 bp Sohlh1 promoter with various mutations in the E-boxes and/or GC-box (200 ng), a pRL-CMV normalization vector (0.1 ng), and expression vectors (pCAG-Sohlh2 (40 ng), pCAG-Sohlh1 (40 ng), and pCAG-Sp1 (40 ng)). Results show the firefly/Renilla luciferase activity relative to that of the intact −1036 bp Sohlh1 promoter (black bar), which was arbitrarily set at 1. P values were calculated by Welch's t-test. *P<0.05. Error bars represent the S.E.M. of the means of 3–8 separate experiments done in triplicate.

Figure 6. Interaction of SP1 to the SOHLH proteins.

(A) Lysates of HEK293 cells overexpressing SP1 and FLAG-SOHLH1 or SOHLH1 were immunoprecipitated with anti-FLAG agarose beads and analyzed by western blotting using anti-SOHLH1 or anti-SP1 antibodies. Arrowhead: FLAG-SOHLH1. Dashed arrow: SOHLH1. (B) Lysates of HEK293 cells overexpressing SP1 and FLAG-SOHLH2 or SOHLH2 were immunoprecipitated with anti-FLAG agarose beads and analyzed by western blotting using anti-SOHLH2 or anti-SP1 antibodies. Arrowhead: FLAG-SOHLH2. Dashed arrow: SOHLH2. Arrow: IgG heavy chain. Pre-immunoprecipitation lysate was used as input.

Figure 7. Binding of SOHLH1 and SP1 to the Sohlh1 promoter region in vivo.

Binding of SOHLH1 and SP1 to the Sohlh1 promoter region in vivo was examined by ChIP assay using P8 testes. The Sohlh1 promoter region (−371 to −284) and the control region far upstream of the Sohlh1 promoter (−8946 to −8834) were quantitated by Real-time PCR from chromatin fractions immunoprecipitated with anti-SOHLH1 antibody, anti-SP1 antibody, or control rabbit IgG. Fold enrichment represents the quantity of the region immunoprecipitated with anti-SOHLH1 (A) or anti-SP1 (B) relative to that immunoprecipitated with control rabbit IgG. Values are expressed as means ±S.E.M. of three technical replicates. P values were calculated by Student's t-test. *P<0.03.

Figure 8. A model of Sohlh1 gene regulation by the SOHLH2/SOHLH1/SP1 complex.

(A) The SOHLH2 homodimer, SP1, or an unknown Factor X turns on weak Sohlh1 transcription. (B) The resulting small amounts of SOHLH1 form the SOHLH2/SOHLH1/SP1 complex due to affinity. This complex is recruited to the E- and GC-box regions in the Sohlh1 promoter. (C) The Sohlh1 gene is highly upregulated through its auto-regulatory mechanism, which involves the SOHLH2/SOHLH1/SP1 complex.