Germ Cell Nuclear Factor Is a Response Element-Specific Repressor of Transcription

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Germ cell nuclear factor (GCNF) is an orphan nuclear receptor that plays important roles in development and reproduction, by repressing the expression of essential genes such as Oct4, GDF9, and BMP15, through binding to DR0 elements. Surprisingly, whereas recombinant GCNF binds to DR0 sequences as a homodimer, endogenous GCNF does not exist as a homodimer but rather as part of a large complex termed the transiently retinoid-induced factor (TRIF). Here, we use evolutionary trace (ET) analysis to design mutations and peptides that probe the molecular basis for the formation of this unusual complex. We find that GCNF homodimerization and TRIF complex formation are DNA-dependent, and ET suggests that dimerization involves key functional sites on both helix 3 and helix 11, which are located on opposing surfaces of the ligand binding domain. Targeted mutations in either helix of GCNF disrupt the formation of both the homodimer and the endogenous TRIF complex. Moreover, peptide mimetics of both of these ET-determined sites inhibit dimerization and TRIF complex formation. This suggests that a novel helix 3-helix 11 heterotypic interaction mediates GCNF interaction and would facilitate oligomerization. Indeed, it was determined that the endogenous TRIF complex is composed of a GCNF oligomer. These findings shed light on an evolutionarily selected mechanism that reveals the unusual DNAbinding, dimerization, and oligomerization properties of GCNF.

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confidence: 99%

Evolutionary Trace-based Peptides Identify a Novel Asymmetric Interaction That Mediates Oligomerization in Nuclear Receptors

Morgan

Sattar

et al. 2005

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“…This high amino acid identity within the DBD and LBD across species indicates the functional conservation of GCNF protein in binding to its DNAresponse element and its putative ligand during evolution. Indeed, the DNA binding specificity of GCNF is conserved among mouse, human and Xenopus (3,(12)(13)(14)(15).…”

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confidence: 99%

“…GCNF can specifically bind to either a DR0 element, a direct repeat of the estrogen receptor half-site (AGGTCA) with zero base pair spacing between the half-sites, as a homodimer (3,12,13,(15)(16)(17), or an extended DR0 half-site (TCAAGGTCA) either as a monomer (12), a homodimer (13,17), or both a monomer and homodimer (17). Recently, two dimerization motifs in mouse GCNF, one located in the DBD including the adjacent TA box and the other in ␣-helix 3 of the LBD, have been characterized (17).…”

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confidence: 99%

The Embryonic Function of Germ Cell Nuclear Factor Is Dependent on the DNA Binding Domain

Lan

Chung

et al. 2002

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Germ cell nuclear factor (GCNF), an orphan nuclear receptor, is essential for mouse embryogenesis. GCNF specifically binds to a DR0 response element via its DNA binding domain (DBD) in vitro and functions as a repressor of gene transcription. To further study the role of GCNF during embryogenesis, we have employed a Cre/ loxP strategy and generated a line of GCNF mutant mice (GCNF lox/lox ) in which the 243-base pair DBD-encoding exon has been deleted in the germline. However, the ligand binding domain (LBD) of GCNF is still expressed at the mRNA and protein levels in the GCNF lox/lox mice. GCNF lox/lox mice die at 9.5-10.5 days postcoitum. The tailbuds of these mutant embryos protrude outside the yolk sac. Expression of Oct-4 in the somatic cells of GC-NF lox/lox embryos at 8.25 days postcoitum was not silenced as in the GCNF ؉/؉ embryos. Therefore, GCNF lox/ lox mice phenocopy the GCNF ؊/؊ mice. Our results indicate that the DBD is essential for the function of GCNF during early mouse embryogenesis, and that the LBD does not mediate any function independent of the DBD at this stage of embryonic development. Our results also suggest that GCNF is indeed a transcriptional factor that represses gene transcription mediated via its DBD.Germ cell nuclear factor (GCNF, 1 NR6A1) is a novel orphan member of the nuclear receptor superfamily as it is more distantly related to other members and forms a sixth and separate subbranch of the family (1, 2). GCNF was initially cloned by our laboratory using low stringency screening with a DNA binding domain (DBD) probe (3) and subsequently cloned by other laboratories and given other names, e.g. RTR (retinoid receptor-related testis-specific receptor (4)) and NCNF (neuronal cell nuclear factor (5)). To date, homologs of GCNF have been cloned from several other species including human, Xenopus, and zebrafish (2, 6 -8). The mouse GCNF gene contains 11 exons (9) and is located on chromosome 2, 2 while the human GCNF gene is located on chromosome 9 at the locus q33-34

“…RTR has been demonstrated to bind preferentially as a homodimer to DNA response elements consisting of the consensus sequence [AGGTCA] 2 (23)(24)(25)(26). However, little is still known about the transcriptional activity of RTR.…”

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confidence: 99%

RAP80, a Novel Nuclear Protein That Interacts with the Retinoid-related Testis-associated Receptor

Yan

Kim

Jetten

2002

In this study, we describe the characterization of a novel nuclear protein, referred to as RAP80. The RAP80 cDNA was cloned from a human testis cDNA library and encodes a 719-amino acid protein containing two potential CX 2 CX 11 HX 3 C-type zinc finger motifs at its carboxylterminal region. Analysis of its genomic structure revealed that the RAP80 gene covers more than 90 kb and consists of 15 exons and 14 introns. Fluorescence in situ hybridization mapped the RAP80 gene to human chromosome 5q35. RAP80 mRNA is expressed in many human tissues, but its expression is particularly high in testis. In situ hybridization showed that RAP80 is highly expressed in germ cells of mouse testis but is not differentially regulated during spermatogenesis. Confocal microscopy showed that RAP80 is localized to the nucleus, where it is distributed in a speckled pattern. Deletion analysis showed that a bipartite nuclear localization signal at the amino terminus is important in mediating nuclear transport of RAP80. Monohybrid analysis showed that RAP80 might function as an active repressor of transcription. Mammalian two-hybrid analysis demonstrated that RAP80 was able to interact with the retinoid-related testis-associated receptor (RTR), an orphan receptor that has been implicated in the control of embryonic development and spermatogenesis. Pulldown analysis showed that RAP80 and RTR physically interact in vitro. Deletion and point mutation analyses revealed that part of the hinge domain of RTR is required for this interaction. RAP80 is able to inhibit the interaction of RTR with the co-repressor N-CoR likely by competing with N-CoR for RTR binding. Our results suggest that RAP80 may be functioning as a modulator of RTR signaling.The nuclear receptor superfamily is composed of a large number of ligand-dependent transcription factors that include nuclear orphan receptors for which a ligand has not yet been identified (1-4). Nuclear receptors share a common domain structure that includes an amino-terminal domain, a DNAbinding domain (DBD), 1 hinge domain, and a ligand-binding domain (LBD). These domains are involved in the recognition of specific DNA response elements, receptor dimerization, nuclear localization, and ligand binding and contain repressor and transactivation functions. Repression and activation of transcription by nuclear receptors are mediated through interactions with co-repressor and co-activators, respectively. Nuclear receptors have been demonstrated to regulate many physiological processes, including embryonic development and cell growth and differentiation and have been implicated in a number of human diseases (5-8). Less, however, is known about the role of many nuclear orphan receptors, including the retinoidrelated, testis-associated receptor (RTR), also referred to as germ cell nuclear factor (9, 10). The orphan receptor RTR (named NR6A1 by the Nuclear Receptor Nomenclature Committee) has been cloned from several species, including mouse (9, 10), human (11-14), zebrafish (15), and Xenopus laevis (16). Duri...