Functional cooperation between CREM and GCNF directs gene expression in haploid male germ cells

Abstract

Cellular differentiation and development of germ cells critically depend on a coordinated activation and repression of specific genes. The underlying regulation mechanisms, however, still lack a lot of understanding. Here, we describe that both the testis-specific transcriptional activator CREMtau (cAMP response element modulator tau) and the repressor GCNF (germ cell nuclear factor) have an overlapping binding site which alone is sufficient to direct cell type-specific expression in vivo in a heterologous promoter context. Expression of the transgene driven by the CREM/GCNF site is detectable in spermatids, but not in any somatic tissue or at any other stages during germ cell differentiation. CREMtau acts as an activator of gene transcription whereas GCNF suppresses this activity. Both factors compete for binding to the same DNA response element. Effective binding of CREM and GCNF highly depends on composition and epigenetic modification of the binding site. We also discovered that CREM and GCNF bind to each other via their DNA binding domains, indicating a complex interaction between the two factors. There are several testis-specific target genes that are regulated by CREM and GCNF in a reciprocal manner, showing a similar activation pattern as during spermatogenesis. Our data indicate that a single common binding site for CREM and GCNF is sufficient to specifically direct gene transcription in a tissue-, cell type- and differentiation-specific manner.

Figure 1.

Transcriptional modulatory function of CREMτ and GCNF. Transient transfection experiments were performed in HEK293 cells. (A) Expression vectors for CREMτ and GCNF were co-transfected with a luciferase reporter vector carrying an mGPDH testis-specific promoter fragment from −62 to −37 upstream of the minimal prolactin promoter (CRE/NR-PRL-Luc). Optionally, cells were stimulated with TSA. (B) Expression vectors for the viral activator domain VP16 (VP16 only), full-length GCNF or a fusion construct of full-length GCNF and VP16 (GCNF-VP16) were co-transfected with the CRE/NR-PRL-Luc reporter. Promoter activities are presented relative to CRE/NR-PRL-Luc activities, normalized to the total protein concentration of the cell extract ± SD. Each construct was tested in five independent transfection experiments with three culture dishes per experiment.

Figure 2.

CREMτ and GCNF bind to the CRE/NR site. For ChIP analysis, expression plasmids for CREMτ, GCNF and the CRE/NR-PRL-Luc reporter were co-transfected in HepG2 cells. Chromatin was fixed by formaldehyde and preserved as input control or immunoprecipitated with antibodies against (A) acetylated histone H3 (α-acetyl H3), (B) Flag-CREMτ (α-Flag), (C) HA-GCNF (α-HA) or (D) non-specific antibody (α-ns IgG). Immunoprecipitated DNA was quantified by quantitative PCR and normalized to input controls of the same samples.

Figure 3.

Point mutations within the CRE/NR site are critical for CREMτ and GCNF binding. In vitro-translated Flag-tagged CREMτ (A) or in vitro-translated HA-tagged GCNF (B) was incubated with the labelled CRE/NR site. For competition experiments, a 50-fold molar excess of wild-type or mutated unlabelled CRE/NR site was added (for sequences see ‘Material and Methods’ section). Antibodies used for complex interference experiments are indicated. Specific protein–DNA complexes are indicated by arrows, non-specific complexes are indicated by asterisks, and supershift complexes are indicated by arrowheads. (C and D) Expression plasmids for CREMτ, GCNF and reporter CRE/NR-4C-PRL-Luc (carrying the point mutation 4G → 4C) or reporter CRE/NR-5T-PRL-Luc (carrying the point mutation 5G → 5T) were transfected into HEK293 cells. Promoter activities were determined as described in Figure 1.

Figure 4.

CREM and GCNF interact physically. (A) Fusion proteins of GST with full length and the indicated deletion mutants of CREMτ were bacterially expressed and coupled to glutathione sepharose beads. Bound beads were incubated with radioactively labelled GCNF to assay protein–protein interactions between CREM and GCNF. (B) A fusion protein of GST with the indicated GCNF fragments were coupled to sepharose beads and incubated with radioactively labelled CREMτ. (C) Expression plasmids of Flag-tagged CREM and HA-tagged GCNF were transfected in HepG2 cells and immunoprecipitated (IP) with anti-Flag antibodies. Immunopreciptiated fractions were analysed by western blotting (WB) using anti-HA antibodies to detected HA-tagged GCNF and anti-Flag antibodies to detected Flag-tagged CREMτ. (D) Cell experiments were performed as described in figure legend of Figure 1A. Additionally, a GCNF deletion fragment containing solely the DNA binding domain of GCNF (DBD-GCNF) was co-transfected demonstrating that this fragment is sufficient for repressor function.

Figure 5.

CREMτ and GCNF regulate post-meiotically expressed target genes. Expression plasmids for CREMτ, GCNF and promoter constructs for the post-meiotically expressed target genes endozepine-like peptide (ELP) (A), pyruvate dehydrogenase 2 (Pdha2) (B), proacrosin (C), SP-10 (D) or testis-specific angiotensin converting enzyme (t-ACE) (E) were co-transfected in HepG2 cells. Promoter activities are presented relative to unstimulated luciferase activities, normalized to the total protein concentration of the cell extract ± SD.

Figure 6.

Testis-specific expression of a CRE/NR-driven transgene in vivo. A transgenic mouse line was constructed carrying the CRE/NR site upstream of a heterologous core promoter driving the expression of LacZ. (A) Several tissues from transgene-carrying and non-transgene-carrying mice were prepared and LacZ expression was studied by quantitative PCR. LacZ expression levels were normalized to β-actin expression levels. In a second normalization step, the normalized LacZ expression of transgenes were normalized to those of wild type littermates in a given tissue. Expression levels were expressed relative to testis expression rates. Data are shown from one representative pair of transgene-carrying versus non-transgene-carrying siblings and analyses are performed in triplicates ± SD. Testis of transgene-carrying mice (B–D) and wild-type littermate (E) were prepared and LacZ activity was monitored in histological sections. LacZ activity was detectable in spermatids (arrows) but neither in earlier steps of germ cell development (arrowheads) nor in other somatic tissues (asterisks). LacZ activity was also absent in non-transgene-carrying wild type littermates (E). Sections were counterstained with nuclear fast red and bars represent 50 µm.