SOHLH2/SOHLH1 heterodimer regulates the <i>Sohlh1</i> promoter through a restricted upstream region.

<p>(A) Reporter assay using the pGL3-Basic vector containing the −1036 bp mouse <i>Sohlh1</i> 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 <i>Sohlh1</i> 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 <i>Sohlh1</i> 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. <i>P</i> values were calculated by Student's <i>t</i>-test. *<i>P</i><0.05. SOHLH2 and SOHLH1 regulate the activity of the mouse <i>Sohlh1</i> promoter through a region −154 to −321 bp upstream from its translational start site.</p

A model of <i>Sohlh1</i> gene regulation by the SOHLH2/SOHLH1/SP1 complex.

<p>(A) The SOHLH2 homodimer, SP1, or an unknown Factor X turns on weak <i>Sohlh1</i> 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 <i>Sohlh1</i> promoter. (C) The <i>Sohlh1</i> gene is highly upregulated through its auto-regulatory mechanism, which involves the SOHLH2/SOHLH1/SP1 complex.</p

The three <i>Sohlh1</i>-promoter E-boxes are not equal in regulating <i>Sohlh1</i>.

<p>Reporter assays using the pGL3-Basic vector with various mutations of the E-boxes in the <i>Sohlh1</i> 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. <i>P</i> values were calculated by Student's <i>t</i>-test. *<i>P</i><0.05.</p

SOHLH2 and SOHLH1 form homodimers.

<p>(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.</p

Binding of SOHLH1 and SP1 to the <i>Sohlh1</i> promoter region <i>in vivo</i>.

<p>Binding of SOHLH1 and SP1 to the <i>Sohlh1</i> promoter region <i>in vivo</i> was examined by ChIP assay using P8 testes. The <i>Sohlh1</i> promoter region (−371 to −284) and the control region far upstream of the <i>Sohlh1</i> 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. <i>P</i> values were calculated by Student's <i>t</i>-test. *<i>P</i><0.03.</p

Transcriptional synergy between SP1 and the SOHLH proteins.

<p>(A) Reporter assays using the pGL3-Basic vector with the −1036 bp intact <i>Sohlh1</i> 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 <i>Sohlh1</i> 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. <i>P</i> values were calculated by Mann-Whitney test. *<i>P</i><0.05. (B) Reporter assays using the pGL3-Basic vector containing the −1036 bp <i>Sohlh1</i> 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 <i>Sohlh1</i> promoter (black bar), which was arbitrarily set at 1. <i>P</i> values were calculated by Welch's <i>t</i>-test. *<i>P</i><0.05. Error bars represent the S.E.M. of the means of 3–8 separate experiments done in triplicate.</p

Interaction of SP1 to the SOHLH proteins.

<p>(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.</p

Conserved regulatory regions of the mouse and rat <i>Sohlh1</i> gene.

<p>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 <i>Sohlh1</i> promoter region of the chimpanzee and human (not shown).</p

<i>Gtsf1l</i> and <i>Gtsf2</i> expression in male germ-cell development.

<p>(A) RT-PCR of <i>Gtsf1l</i>, <i>Gtsf2</i>, and <i>Gapdh</i> mRNAs using total RNA from the whole embryo at E7.5 and from the testes at various time points from E13.5 to 8 weeks. P: postnatal day. (B-K) Frozen sections of adult testis were immunostained with anti-GTSF1L and anti-GTSF2 antibodies (green), revealing GTSF1L (B-F) and GTSF2 (G-K) protein expression in the seminiferous tubules at developmental stages I-III (B, G), IV-VI (C, H), VII-VIII (D, I), IX-X (E, J), and XI-XII (F, K). Nuclei were stained with DAPI (blue). Scale bar: 100 μm.</p

GTSF1L and GTSF2 have two N-terminal CHHC-type Zn-finger domains.

<p>(A) Domain architecture of mouse UPF0224-family proteins. The N-terminal region has two CHHC Zn-finger domains. The black lines indicate the regions used to raise rabbit antibodies against GTSF1L and GTSF2. (B) Alignment of the amino-acid residues of the mouse UPF0224-family proteins using CLUSTAL-X. The first and second CHHC Zn-finger domains are indicated in blue and red, respectively. Asterisks indicate amino-acid residues that are conserved among GTSF1, GTSF1L, and GTSF2. Pluses indicate amino-acid residues that are conserved between GTSF1L and GTSF2.</p