The transcription factor C/EBP-beta and its role in ovarian function; evidence for direct involvement in the ovulatory process

The tightly regulated expression of p53 contributes to genomic stability, and transcription of the p53 gene is induced prior to cells entering S phase, possibly as a mechanism to ensure a rapid p53 response in the event of DNA damage. We have previously described the cloning of an additional 1000 bp of upstream p53 sequences that we have demonstrated play a role in the regulated expression of p53. As described in an earlier report, we preliminarily identified that a member of the CAAT/ enhancer-binding protein (C/EPB) family of transcription factors may play a role in regulating p53. Here we have demonstrated that a particular C/EBP␤ isoform, C/EBP␤-2, efficiently binds to the p53 promoter and induces its expression in a fashion that reflects the pattern of p53 expression seen as cells are induced to enter S phase and is absent from cells that are defective in proper p53 regulation. We conclude from these findings that C/EBP␤-2 plays a central role in the regulating of p53 transcription during the transition into S phase.

Section: Discussionmentioning

confidence: 99%

C/EBPβ Participates in Regulating Transcription of the p53 Gene in Response to Mitogen Stimulation

Boggs

Reisman

2007

“…C/EBP␤ plays a key role during differentiation of a number of cell types, including adipocytes (9 -11), hepatocytes (12), and cells of the hematopoietic system (13,14). C/EBP␤ also plays a role in mammary gland development (15) and in ovulation (16,17). C/EBP␤ has been shown to localize in areas of pericentric heterochromatin in 3T3-F442A predipocytes upon GH treatment, as well as during the induction of adipocyte differentiation of 3T3-F442A 2 and 3T3-L1 cells (22).…”

Section: Discussionmentioning

confidence: 99%

“…The N-terminal half of LAP contains the transactivation domain, whereas LIP lacks this transactivation domain and acts as an inhibitor of transcription (8). C/EBP␤ plays an important role during differentiation of a number of cell types (9 -14), and is also implicated in mammary gland development (15) and in ovulation (16,17). The participation of C/EBP␤ in a wide variety of physiological events suggests that multiple features of its regulation may contribute to diverse biological outcomes.…”

mentioning

confidence: 99%

Subnuclear Localization of C/EBPβ Is Regulated by Growth Hormone and Dependent on MAPK

Pilipuk

Galigniana

Schwartz

2003

Localization of transcription regulatory proteins in the nucleus is dynamically regulated, and may alter nucleoplasmic concentrations and/or assembly of multimolecular transcription regulatory complexes, which ultimately regulate gene expression. Since growth hormone (GH) regulates multiple transcription factors including C/EBP␤, the effect of GH on the subcellular localization of C/EBP␤ was examined in 3T3-F442A preadipocytes. Indirect immunofluorescence shows that C/EBP␤ is diffusely distributed in nuclei of quiescent cells. . Indirect immunofluorescence using antibodies specific for C/EBP␤ phosphorylated on the conserved MAPK site shows that GH also rapidly induces a punctate pattern of staining for the phosphorylated C/EBP␤. In addition, phosphorylated C/EBP␤ colocalizes to pericentromeric heterochromatin. The satellite DNA present in heterochromatin contains multiple C/EBP binding sites. DNA binding analysis shows that C/EBP␤, C/EBP␦, and C/EBP␣ (p42 and p30 forms) can bind to satellite DNA as homo-or heterocomplexes in vitro. Importantly, GH rapidly and transiently increases binding of endogenous C/EBP␤ from 3T3-F442A cells to satellite DNA. Further, the GH-promoted nuclear relocalization of C/EBP␤ to pericentromeric heterochromatin was prevented by the MEK inhibitor U0126. This observation suggests that GH-dependent MAPK activation plays a role in the regulation of nuclear relocalization of C/EBP␤. Nuclear redistribution introduces a new level of transcriptional regulation in GH action, since GHmediated phosphorylation and nuclear redistribution of C/EBP␤ may be coordinated to achieve spatial-temporal control of gene expression.Chromatin is organized into higher order fibers in interphase chromosomes, which localize into discrete territories, providing an excellent scenario for establishing spatio-temporal regulation of gene transcription (1, 2). Transcriptional regulatory proteins are generally restricted to several hundred discrete foci throughout the nucleoplasm (3, 4). Interestingly, foci rich in some transcription factors such as E2F-1 or Oct1 show only minor overlap with sites of transcription or RNA polymerase II (3, 4), whereas steroid hormone receptors and RNA polymerase II show significant colocalization (5, 6). Thus, the function of the transcription factor-rich foci in the nucleus that do not contain RNA polymerase II or nascent RNA is unclear. A simple explanation is that foci represent nonchromatin structures that function in the regulation of nucleoplasmic concentrations of gene regulatory factors and/or the assembly of multimolecular transcription factor complexes. This subnuclear

“…If truly so, C/EBP␣ should be considered as potential substitute for C/EBP␤ in the C/EBP␤-deficient mouse ovary (60 -63). It has been shown that the ovaries of the latter null mice do not ovulate (60,64) despite normal production of gonadal steroids and expression of steroidogenic enzymes, probably including StAR. 3 Like C/EBP␤, GATA-4 had not been described previously as a potential regulator of genes encoding steroidogenic enzymes, or their accessory proteins.…”

Section: Fig 10mentioning

confidence: 99%

CCAAT Enhancer-binding Protein β and GATA-4 Binding Regions within the Promoter of the Steroidogenic Acute Regulatory Protein (StAR) Gene Are Required for Transcription in Rat Ovarian Cells

Silverman

Eimerl

Orly

1999

171

127

Steroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the ؊1002/؉6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshly prepared cells. FSH inducibility determined over a 6-h incubation was 10 -40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR promoter constructs: a non-consensus binding sequence (؊81/؊72) for CCAAT enhancer-binding protein ␤ (C/EBP␤) and a consensus motif for GATA-4 binding (؊61/؊66). Western analyses showed that GATA-4 is constitutively expressed in the granulosa cells, while all isoforms of C/EBP␤ were markedly inducible by FSH. Site-directed mutations of both binding sequences practically ablated both basal and hormone-driven chloramphenicol acetyltransferase activities to less than 5% of the parental ؊96/؉6 construct. Unlike earlier notions, elimination of potential binding sites for steroidogenic factor-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBP␤ and GATA-4 represent a novel combination of transcription factors capable of conferring an acute response to hormones upon their concomitant binding to the StAR promoter.