Genomic structure of the gene for mouse germ-cell nuclear factor (GCNF). II. Comparison with the genomic structure of the human GCNF gene.

BACKGROUND: Germ-cell nuclear factor (GCNF, NR6AI) is an orphan nuclear receptor. Its expression pattern suggests it functions during embryogenesis, in the placenta and in germ-cell development. Mouse GCNF cDNA codes for a protein of 495 amino acids, whereas the four reported human cDNA variants code for proteins of 454 to 480 amino acids. Apart from this size difference, there is sequence conservation of up to 98.7%. To elucidate the genomic structure that gives rise to the different human GCNF mRNAs, the sequence information of the human GCNF locus is compared to the previously reported structure of the mouse locus. RESULTS: The genomic structures of the mouse and human GCNF genes are highly conserved. The comparison reveals that the shorter human protein results from skipping the 45 base-pair third exon. Three different human isoforms - GCNF-1, GCNF-2a and GCNF-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon. CONCLUSION: By homology with the mouse gene, 11 GCNF coding exons can be defined on human chromosome 9. All human GCNF cDNAs identified so far are, however, derived from mRNAs generated by splicing the fourth to the second exon. Although the genomic sequence is highly conserved, the analysis suggests that alternative splicing generates a higher complexity of human GCNF isoforms compared with the situation in the mouse.

Developmental expression of the estrogen receptor-related receptor gamma in the nervous system during mouse embryogenesis.

The ERR's (estrogen receptor-related receptors) are constitutive activators of the classical estrogen response element. In this report, we demonstrate that ERRgamma is highly expressed in the nervous system of the developing mouse embryo and that the adult pattern of expression of ERRgamma is, with few exceptions, established during embryogenesis. Transcripts are preferentially detected in already differentiating areas of the nervous system.

Differential expression of the estrogen receptor-related receptor gamma in the mouse brain.

To elucidate estrogen functions, the expression of the estrogen receptor-related receptor ERRgamma, a novel orphan nuclear receptor regulating transcription via estrogen responsive elements, has been localized by in situ hybridization in adult murine brain. ERRgamma transcripts were abundantly present in the isocortex, the olfactory system, cranial nerve nuclei and major parts of the coordination centers, e.g. reticular formation and major parts of the extrapyramidal motor systems. In addition, ERRgamma expression was detected in trigeminal ganglion neurons. ERRgamma distribution was clearly distinguished from that described for ERRalpha, for ERRbeta, and for estrogen receptors (ER) pointing at functional differences between ERRgamma and these receptors.

Alternative splicing and expression of the mouse estrogen receptor-related receptor gamma.

Estrogen receptor-related receptors (ERRs) are orphan members of the nuclear receptor superfamily, closely related to the estrogen receptor. With a PCR-based cloning strategy we have identified two cDNA isoforms from a neonatal mouse whole brain cDNA library encoding ERRgamma. We show that alternative splicing gives rise to different 5'-ends. The predicted peptide sequence of ERRgamma2 differs from ERRgamma1 by additional 23 N-terminal residues. The presence of identical isoforms in human suggests a pivotal function of ERRgamma in mammalia. Northern blot analysis reveals that ERRgamma is expressed as early as day 11 of embryonic development (E11). Whole mount in situ hybridization analysis shows major expression of ERRgamma in the central nervous system at E12.5. In the adult mouse a 5.7 kb transcript is detected in heart, brain, kidney, and skeletal muscle. Therefore, ERRgamma may be involved in the differentiation, as well as the maintenance of the differentiated properties in the brain.

Genomic structure of the gene for mouse germ cell nuclear factor (GCNF).

BACKGROUND: The germ cell nuclear factor (GCNF, also known as retinoid acid receptor-related testis-associated receptor, neuronal cell nuclear receptor or NR6A1) is an orphan receptor in the nuclear receptor superfamily found in mammals, amphibians and fish. The mouse Gcnf gene is expressed in the placenta and the developing nervous system and germ cells, and responds to retinoic acid. RESULTS: We have defined the intron-exon structure of the mouse Gcnf gene and found that it contains 11 exons. Exons 1-4 encode the 75 amino acid amino-terminal domain and exon 4 also encodes the core DNA-binding domain. The carboxy-terminal extension is encoded by exon 5, exons 6 and 7 encode the hinge region, and exons 7-11 encode the putative ligand-binding domain. Unusually, the two zinc-finger motifs in the DNA-binding domain are encoded by separate exons. CONCLUSIONS: The protein-coding region of GCNF is contained in 11 exons. The genomic structure of this nuclear receptor gene will be useful for further studies.

DNA binding, protein interaction and differential expression of the human germ cell nuclear factor.

The mouse germ cell nuclear factor (mGCNF) is an orphan nuclear receptor implicated in diverse biological processes, including gametogenesis, embryonic development and embryonal carcinoma cell differentiation. We have examined the binding and regulation of the human orthologue, hGCNF, expressed in the teratocarcinoma-derived cell line NTera-2/clone D1 (NT2/D1). Binding of GCNF to the direct repeat of the sequence -AGGTCA- (DR-0) is conserved in mammalia. The formation of interspecies dimers of the in vitro synthesized proteins suggests that cellular GCNF binding is mediated by homodimers. Both the mouse and the human protein bind in concert with cellular factors to DNA. Treatment of NT2/D1 cells with all-trans retinoic acid (atRA) is accompanied first by an up-regulation followed later by a down-regulation of hGCNF and its mRNA. Temporary up-regulation in NT2/D1 cells after treatment with atRA suggests that hGCNF is important for human neural determination and differentiation.

Retinoids induce differential expression and DNA binding of the mouse germ cell nuclear factor in P19 embryonal carcinoma cells.

The mouse germ cell nuclear factor (GCNF), a member of the nuclear receptor superfamily, is highly expressed during gametogenesis and in the developing nervous system. The in vitro translated protein binds as a homodimer to the direct repeat (DR) of the sequence -AGGTCA- (DR-0). In this report, we characterize a DR-0 binding activity in P19 cell extracts that is induced by retinoids. This induction is concentration dependent and specific for embryonal carcinoma cells. The cellular protein binds with the same specificity as in vitro expressed GCNF, but migrates as a slower complex, indicating interaction with partner proteins. Because antisera directed against GCNF recognize this complex, we propose that GCNF is part of the binding activity. Combining in vitro translated GCNF and extracts of non-expressing cells shows that such interactions can be formed posttranslationally. Northern analysis demonstrates a concentration dependent induction of GCNF mRNA by retinoic acid. A time course shows that the level of GCNF binding is transiently elevated, later downregulated, and not detectable in differentiated cells. We propose that GCNF regulation is an important step during determination of embryonal carcinoma cells.

Unique expression pattern of a novel mosaic receptor in the developing cerebral cortex.

Recently, a new type of transmembrane protein with a unique combination of protein domains was characterized from human, rabbit and chicken. This protein exhibits features of the low-density lipoprotein receptor family and shows homology to the receptor of the neuropeptide head activator isolated from hydra. To study the temporal and spatial pattern of expression of this unusual new receptor we have isolated a murine homolog and, in accordance with its human counterpart, named it mSorLA. Northern blot analysis revealed the highest abundance of mSorLA transcripts in the adult brain, lower levels in a variety of other organs and expression during embryogenesis. In situ hybridization showed predominant localization in neurons of the cortex, the hippocampus and the cerebellum. During embryonic development mSorLA displayed a unique pattern of expression in the cerebral cortex, where a subpopulation of neurons was labeled before final differentiation. Transcripts of mSorLA were also detected outside the central nervous system in regions active in morphogenesis.

The germ cell nuclear factor mGCNF is expressed in the developing nervous system.

Using a reinoic acid receptor hybridization probe, we have isolated a mouse embryonic cDNA that encodes the germ cell nuclear factor (mGCNF). The in vitro translated protein binds specifically to the direct repeat of the sequence-AGGTCA-which is characteristic of a subclass of nuclear receptors, albeit with a spacing of zero that is unique among the receptors. Northern analysis shows embryonic expression after mouse gastrulation and during early organogenesis, as well as expression in the adult testis. The message level decreases during embryonic development. Whereas there are two transcripts in the testis, only the larger one is found in embryos. In situ analysis of embryos at days 6.5-10.5 of gestation shows that, in the early postimplantation period, high transcript levels are found in ectodermal cells and in the primitive streak. During further development the expression becomes more restricted. Mainly cells of the developing nervous system are expressing the receptor. Our findings imply that GCNF is involved in two apparently disparate developmental events.

Characterization of the human germ cell nuclear factor gene.

A cDNA clone encoding the germ cell nuclear factor, GCNF, a member of the nuclear receptor superfamily has been isolated from the human embryonal carcinoma cell line NT2/D1. Sequencing of this clone reveals an open reading frame encoding a 476 amino acid protein. A comparison of the amino acid sequence of the human GCNF with its mouse homologue shows only six amino acid exchanges in the whole protein and a deletion in the amino-terminal region. Northern blot analysis demonstrates that the expression in the testis is conserved.