The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.
In order to establish whether there are differences in DNA-binding proteins between zona fasciculata (ZF) and zona reticularis (ZR) cells of the human adrenal cortex, we prepared nuclear extracts from separated ZF and ZR cells. The formation of DNA-protein complexes was studied using an element in the first intron of the type I and type II 3 -hydroxysteroid dehydrogenase genes (HSD3B1 and HSD3B2). Using the element in the HSD3B2 gene as a probe, a complex (C1) was formed with extracts from ZF cells but was formed only at a low level with ZR cell extracts. Another pair of complexes (C2/C3) was formed with both ZF and ZR cell extracts. The ZF-specific protein forming C1 was enriched by column chromatography on DEAE-Sepharose and carboxymethylSepharose. Oligonucleotide competition analysis on the enriched fraction gave results consistent with those obtained on the unfractionated material. A further enrichment was brought about by passing the protein over an oligonucleotide affinity column based on the HSD3B2 element. The protein bound to the column was identified as -enolase by mass spectrometry. Although -enolase is a glycolytic enzyme, it binds to specific DNA sequences and has been found to be present in nuclei of various cell types. We performed immunohistochemistry on sections of adult human adrenal cortex and found -enolase to be located in nuclei of ZF cells but to be predominantly cytoplasmic in ZR cells. Transfection of an -enolase expression vector into NCI-H295R human adrenocortical cells increased HSD3B2 promoter activity, suggesting a possible functional role for this protein in regulation of HSD3B2 expression.
Germ cell nuclear factor (GCNF) is a member of the nuclear receptor superfamily, which is expressed in the adult predominantly in the male and female germ cells. In the male, GCNF is expressed in spermatogenic cells. GCNF binds as a homodimer to direct repeat response elements of the consensus half-site sequence, AGGTCA, with 0 bp spacing (DR0). Using this information, a search of genomic databases was performed to identify candidate GCNF responsive, spermatogenic-specific, genes that contain DR0 sequences. The mouse protamine genes are the strongest candidates identified to date, as they are post-meiotically expressed in round spermatids and contain DR0 elements in their proximal promoters. Previous work has shown that both recombinant and endogenous GCNF bind to DR0 elements in the mouse protamine 1 and 2 (Prm 1 and Prm 2) promoters with high affinity and specificity. The present work shows that in transient transfection assays in GC-1 and JEG-3 cells, co-transfection of a GCNF-VP16 expression plasmid with reporter plasmids containing either the wild type Prm 1 or Prm 2 promoter established that GCNF-VP16 is able to regulate transcription from both promoters in a DR0-dependent manner. Wild type GCNF, in contrast, acts as a repressor of basal transcription on both the Prm 1 and Prm 2 promoters in a DR0-dependent manner. Furthermore, CREMtau activation of these promoters is also repressed by wild-type GCNF, indicating that GCNF also acts as a repressor of activated transcription. GCNF therefore defines a novel nuclear receptor-signaling pathway that may regulate a subset of genes involved in the terminal differentiation process of spermatogenesis, exemplified by the protamines.
Proper expression of the protamine genes is an important event in the terminal differentiation of the male gametes in mammals. Here we present evidence that the novel orphan member of the nuclear receptor gene superfamily, Germ Cell Nuclear Factor (GCNF), may play a role in the regulation of these genes. Previously, we have shown that high‐level expression of GCNF mRNA is restricted to spermatids (stages 1–8) in the adult male mouse, which makes it temporally and spatially available to regulate the mouse protamine genes. Furthermore, we have previously identified a sequence to which GCNF can bind, which consists of a direct repeat of the core halfsite AGGTCA with zero base pairs spacing the repeats (DR0). We have identified several genes that contain DR0 sequences in their 5′ promoter regions, including the protamines. The mouse protamine 1 and 2 (Prm1 and Prm2) genes therefore are potential target genes for GCNF regulation. We show that GCNF binds to one of the two DR0 sequences in the Prm1 promoter, and to the DR0 sequence in the Prm2 promoter in a specific manner. Furthermore, by using antibodies directed against GCNF, we detect endogenous GCNF in testis nuclear extracts and elutriated round spermatid nuclear extracts in Western blots. Additionally, by using these antibodies in gel‐shift assays, we show that this endogenous GCNF can bind to both the Prm1 and Prm2 promoters. This evidence supports the hypothesis that GCNF mediates a novel signaling pathway, two targets of which may be the Prm1 and Prm2 genes in spermatids. Mol. Reprod. Dev. 50:396–405, 1998. © 1998 Wiley‐Liss, Inc.
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