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• W1979098148 abstract "Transcriptional regulation of steroidogenic acute regulatory protein (StAR) determines adrenal and gonadal cell steroidogenesis. Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the StAR promoter. MA-10 cells treated with 8-bromo-cAMP had increased acetylated histone H3 associated with the proximal (but not distal) StAR promoter, nascent StAR transcripts, and progesterone production within 15 min, whereas StAR mRNA increased at 30 min. At 360 min, steroidogenesis remained elevated, but mRNA, nascent RNA, and StAR promoter-associated H3 acetylation all declined. StAR promoter-associated H4 acetylation was unchanged by 8-bromo-cAMP treatment of MA-10 cells. In vivoanalysis of macaque and human granulosa cells showed that luteinization was associated with increased StAR promoter-associated H3 acetylation. We conclude that acetylation of H3 (but not H4) associated with the proximal promoter is associated with StAR gene transcription, that chromatin modification occurs in discrete regions of the promoter, that the initial steroidogenic response to 8-bromo-cAMP occurs prior to increased StAR mRNA accumulation, and that MA-10 cell StAR gene transcription and promoter-associated H3 acetylation are biphasic during a 6-h treatment period. The union of the chromatin immunoprecipitation assay with quantitative real-time polymerase chain reaction described and validated here should enhance the analysis of gene expression. Transcriptional regulation of steroidogenic acute regulatory protein (StAR) determines adrenal and gonadal cell steroidogenesis. Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the StAR promoter. MA-10 cells treated with 8-bromo-cAMP had increased acetylated histone H3 associated with the proximal (but not distal) StAR promoter, nascent StAR transcripts, and progesterone production within 15 min, whereas StAR mRNA increased at 30 min. At 360 min, steroidogenesis remained elevated, but mRNA, nascent RNA, and StAR promoter-associated H3 acetylation all declined. StAR promoter-associated H4 acetylation was unchanged by 8-bromo-cAMP treatment of MA-10 cells. In vivoanalysis of macaque and human granulosa cells showed that luteinization was associated with increased StAR promoter-associated H3 acetylation. We conclude that acetylation of H3 (but not H4) associated with the proximal promoter is associated with StAR gene transcription, that chromatin modification occurs in discrete regions of the promoter, that the initial steroidogenic response to 8-bromo-cAMP occurs prior to increased StAR mRNA accumulation, and that MA-10 cell StAR gene transcription and promoter-associated H3 acetylation are biphasic during a 6-h treatment period. The union of the chromatin immunoprecipitation assay with quantitative real-time polymerase chain reaction described and validated here should enhance the analysis of gene expression. steroidogenic acute regulatory protein chromatin immunoprecipitation polymerase chain reaction bromo human chorionic gonadotropin glyceraldehyde-3-phosphate dehydrogenase threshold cycle adrenocorticotropic hormone extracellular signal-regulated kinase The translocation of cholesterol from the relatively sterol-rich outer mitochondrial membrane to the relatively cholesterol-poor inner mitochondrial membrane is the rate-limiting step in steroid synthesis (1Christenson L.K. Strauss III, J.F. Biochim. Biophys. Acta. 2000; 1529: 175-187Crossref PubMed Scopus (161) Google Scholar). Steroidogenic acute regulatory protein (StAR)1 plays an integral role in this cholesterol translocation as evidenced by experiments of nature (2Lin D. Sugawara T. Strauss III, J.F. Clark B.J. Stocco D.M. Saenger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (853) Google Scholar) and mouse gene knockout studies (3Caron K.M. Soo S.C. Wetsel W.C. Stocco D.M. Clark B.J. Parker K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11540-11545Crossref PubMed Scopus (375) Google Scholar). In the absence of functional StAR protein, gonadal and adrenal steroidogenesis is markedly impaired and unmetabolized cholesterol accumulates as sterol esters in cytoplasmic lipid droplets. The conservation of StAR protein structure and expression patterns in steroidogenic tissues of piscine, avian, amphibian, and mammalian species testifies to the importance of this protein in steroid synthesis (4Bauer M.P. Bridgham J.T. Langenau D.M. Johnson A.L. Goetz F.W. Mol. Cell. Endocrinol. 2000; 168: 119-125Crossref PubMed Scopus (98) Google Scholar).Studies of StAR gene expression (mRNA) in gonadal and adrenal cells revealed that the steroidogenic capacity of these cells is tightly linked to the abundance of StAR transcripts (5Clark B.J. Combs R. Hales K.H. Hales D.B. Stocco D.M. Endocrinology. 1997; 138: 4893-4901Crossref PubMed Scopus (93) Google Scholar). Analysis of StAR promoter function in human (6Christenson L.K. Johnson P.F. McAllister J.M. Strauss III, J.F. J. Biol. Chem. 1999; 274: 26591-26598Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Christenson L.K. Osborne T.F. McAllister J.M. Strauss III, J.F. Endocrinology. 2001; 142: 28-36Crossref PubMed Scopus (67) Google Scholar, 8Sugawara T. Kiriakidou M. McAllister J.M. Kallen C.B. Strauss III, J.F. Biochemistry. 1997; 36: 7249-7255Crossref PubMed Scopus (151) Google Scholar, 9Sugawara T. Saito M. Fujimoto S. Endocrinology. 2000; 141: 2895-2903Crossref PubMed Scopus (94) Google Scholar), domestic animal (10Rust W. Stedronsky K. Tillmann G. Morley S. Walther N. Ivell R. J. Mol. Endocrinol. 1998; 21: 189-200Crossref PubMed Scopus (52) Google Scholar, 11LaVoie H.A. Garmey J.C. Veldhuis J.D. Endocrinology. 1999; 140: 146-153Crossref PubMed Scopus (79) Google Scholar), and rodent (12Reinhart A.J. Williams S.C. Clark B.J. Stocco D.M. Mol. Endocrinol. 1999; 13: 729-741PubMed Google Scholar, 13Clark B.J. Soo S.C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocrinol. 1995; 9: 1346-1355Crossref PubMed Google Scholar, 14Silverman E. Eimerl S. Orly J. J. Biol. Chem. 1999; 274: 17987-17996Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 15Wooton-Kee C.R. Clark B.J. Endocrinology. 2000; 141: 1345-1355Crossref PubMed Scopus (98) Google Scholar) cells revealed the importance of a variety of transcription factor response elements (SF-1, C/EBPβ, SREBP-1a, GATA-4, DAX-1, and Sp-1) within the first 250 bases of the promoter proximal to the TATA box that influence basal and/or hormone-dependent (cAMP) StAR gene transcription. Transcription factor binding and analysis of mutant promoter constructs confirmed a role for many of these factors in StAR gene activity (6Christenson L.K. Johnson P.F. McAllister J.M. Strauss III, J.F. J. Biol. Chem. 1999; 274: 26591-26598Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Christenson L.K. Osborne T.F. McAllister J.M. Strauss III, J.F. Endocrinology. 2001; 142: 28-36Crossref PubMed Scopus (67) Google Scholar, 8Sugawara T. Kiriakidou M. McAllister J.M. Kallen C.B. Strauss III, J.F. Biochemistry. 1997; 36: 7249-7255Crossref PubMed Scopus (151) Google Scholar, 9Sugawara T. Saito M. Fujimoto S. Endocrinology. 2000; 141: 2895-2903Crossref PubMed Scopus (94) Google Scholar, 10Rust W. Stedronsky K. Tillmann G. Morley S. Walther N. Ivell R. J. Mol. Endocrinol. 1998; 21: 189-200Crossref PubMed Scopus (52) Google Scholar, 11LaVoie H.A. Garmey J.C. Veldhuis J.D. Endocrinology. 1999; 140: 146-153Crossref PubMed Scopus (79) Google Scholar, 12Reinhart A.J. Williams S.C. Clark B.J. Stocco D.M. Mol. Endocrinol. 1999; 13: 729-741PubMed Google Scholar, 13Clark B.J. Soo S.C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocrinol. 1995; 9: 1346-1355Crossref PubMed Google Scholar, 14Silverman E. Eimerl S. Orly J. J. Biol. Chem. 1999; 274: 17987-17996Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 15Wooton-Kee C.R. Clark B.J. Endocrinology. 2000; 141: 1345-1355Crossref PubMed Scopus (98) Google Scholar, 16Reinhart A.J. Williams S.C. Stocco D.M. Mol. Cell. Endocrinol. 1999; 151: 161-169Crossref PubMed Scopus (76) Google Scholar).Covalent modifications of histones and remodeling of chromatin structure are thought to play a critical role in the regulation of gene transcription (17Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2367) Google Scholar, 18Kadonaga J.T. Cell. 1998; 92: 307-313Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). The highly basic N-terminal histone tails that project away from the core histone complex play a key role in higher order chromatin structure and in interactions of histones with other chromatin-associated regulatory proteins (19Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6793) Google Scholar, 20Hansen J.C. Tse C. Wolffe A.P. Biochemistry. 1998; 37: 17637-17641Crossref PubMed Scopus (215) Google Scholar, 21Luger K. Richmond T.J. Curr. Opin. Genet. Dev. 1998; 8: 140-146Crossref PubMed Scopus (406) Google Scholar). The reversible post-translational modifications of the histone N terminus include ADP-ribosylation, glycosylation, methylation, phosphorylation, and the best documented modification, acetylation (22Davie J.R. Spencer V.A. J. Cell. Biochem. Suppl. 1999; 32/33: 141-148Crossref Google Scholar, 23Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). Acetylation neutralizes the positive charge on lysines located at the N terminus of histones H3 and H4 and was originally thought to allow the proteins to dissociate from the negatively charged DNA, thereby allowing the DNA to interact with transcription factors and the transcriptional machinery (24Mizzen C.A. Allis C.D. Cell. Mol. Life Sci. 1998; 54: 6-20Crossref PubMed Scopus (186) Google Scholar). However, more recent studies indicate that acetylation, while facilitating transcription, can do so without displacement of the N-terminal tail domains from the DNA. Moreover, recent in vivo studies indicate that the histone N terminus is a highly structured domain that is primarily involved in protein-protein interactions. Acetylation of the histones increases the α-helical character of the N-terminal domains (25Wang X. Moore S.C. Laszckzak M. Ausio J. J. Biol. Chem. 2000; 275: 35013-35020Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This structural change may influence the interactions of histones with other chromatin proteins, ultimately leading to the destabilization of the higher order chromatin folding (26Wang X. He C. Moore S.C. Ausio J. J. Biol. Chem. 2001; 276: 12764-12768Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Acetylation and other post-translational modifications have been proposed to represent a “histone code” that might determine the sequence and nature of protein interactions that facilitate transcription and/or DNA replication (23Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). However, this interesting hypothesis has yet to be critically evaluated through experimentation.A link between histone acetylation and gene transcription has been suspected for many years (reviewed in Ref. 17Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2367) Google Scholar). The discovery that coactivator proteins possess histone acetyltransferase activity provided direct evidence for an important role for histone acetylation and transcriptional regulation (27Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1277) Google Scholar). It is postulated that DNA-binding proteins recruit transcriptional coactivators that acetylate the histones associated with the gene promoter, allowing access of and/or recruitment of other proteins (e.g. TATA-binding proteins, RNA polymerase II, etc.) to the DNA to promote transcription. Thus, histone acetylation could be thought of as an excellent marker of gene activity. Conversely, histone deacetylases are thought to be involved in the silencing of gene transcription.A recently developed method to identify remodeled chromatin using reversible formaldehyde cross-linking of proteins and DNA and antibodies to immunoprecipitate DNA associated with acetylated histones or chromatin associated with specific transcription factors has provided new insights into the early events of transcriptional regulation. However, the previously described chromatin immunoprecipitation (ChIP) assays have been largely qualitative or at best semiquantitative in nature, limiting the assay output to an all-or-nothing readout (28Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 29Sambucetti L.C. Fischer D.D. Zabludoff S. Kwon P.O. Chamberlin H. Trogani N. Xu H. Cohen D. J. Biol. Chem. 1999; 274: 34940-34947Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 30Bennett M.K. Ngo T.T. Athanikar J.N. Rosenfeld J.M. Osborne T.F. J. Biol. Chem. 1999; 274: 13025-13032Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 31Parekh B.S. Maniatis T. Mol. Cell. 1999; 3: 125-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The studies described here demonstrate for the first time that acetylation of histone H3 (but not histone H4) associated with the proximal region of the StAR promoter is associated with the transcriptional activity of that gene. We also describe a sensitive and reproducible method for quantitation of promoter activity (i.e. histone acetylation) linking the ChIP assay to quantitative real-time PCR analysis of the promoter element in the StAR gene. This marriage of methodologies will allow quantification of the activity of multiple genes under in vivo conditions.DISCUSSIONThere has been a concerted effort by several laboratories to understand the hormonal/cAMP-dependent regulation of StAR gene expression to unlock the mysteries surrounding regulation of steroidogenesis. Our experiments illustrate for the first time that increased StAR promoter-associated histone H3 acetylation is associated with StAR gene activation as indicated by accumulation of nascent StAR transcripts and processed StAR mRNA. These studies also demonstrate for the first time the hormonal (hCG) regulation of gene promoter activity (i.e. acetylation) in a whole animal model system, whereby periovulatory monkey granulosa cells were collected before and after an ovulatory dose of gonadotropin. Finally, these experiments describe for the first time the combination of the ChIP procedure and quantitative real-time PCR, allowing for a sensitive, reproducible, and unbiased measure of protein association with gene promoter elements under in vivo conditions. Previously, ChIP assays have been based on semiquantitative analysis usually involving the examination of PCR products at a fixed cycle number, followed by densitometry of the radioactive band or ethidium bromide-stained DNA, or they were purely qualitative in nature (28Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 29Sambucetti L.C. Fischer D.D. Zabludoff S. Kwon P.O. Chamberlin H. Trogani N. Xu H. Cohen D. J. Biol. Chem. 1999; 274: 34940-34947Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 30Bennett M.K. Ngo T.T. Athanikar J.N. Rosenfeld J.M. Osborne T.F. J. Biol. Chem. 1999; 274: 13025-13032Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 31Parekh B.S. Maniatis T. Mol. Cell. 1999; 3: 125-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The experimental method described here greatly improves the quantitative nature of the ChIP procedure and should allow for a more accurate assessment of differences in protein-DNA interaction and the monitoring of heterogeneity of cellular responses within a natural chromatin environment. Using a nested PCR strategy, the sensitivity and specificity of this assay can be increased.Tropic hormone-stimulated steroidogenesis is dependent on the expression and function of the StAR protein. In these studies, we show for the first time that acetylation of H3 associated with the StAR promoter is temporally linked to the cAMP-dependent increases in StAR gene expression in MA-10 mouse Leydig cells. Conversely, 8-Br-cAMP treatment of MA-10 cells failed to influence the acetylation of H4 associated with the StAR gene promoter. Differential acetylation of the core histones is not a unique attribute of the StAR promoter. Indeed, several recent studies using the same antibodies employed in our ChIP analysis have exhibited differential association of acetylated H3 or H4 with other gene promoters under conditions known to activate the gene (29Sambucetti L.C. Fischer D.D. Zabludoff S. Kwon P.O. Chamberlin H. Trogani N. Xu H. Cohen D. J. Biol. Chem. 1999; 274: 34940-34947Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 30Bennett M.K. Ngo T.T. Athanikar J.N. Rosenfeld J.M. Osborne T.F. J. Biol. Chem. 1999; 274: 13025-13032Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). ChIP analysis of the p21waf1gene promoter was recently shown to be associated with increased H3 acetylation, whereas H4 acetylation did not change under conditions that regulate the cell cycle-dependent expression of p21waf1. Similarly, the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase promoters also exhibit preferential acetylation of H3 under conditions known to activate these genes (30Bennett M.K. Ngo T.T. Athanikar J.N. Rosenfeld J.M. Osborne T.F. J. Biol. Chem. 1999; 274: 13025-13032Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In contrast to these observations, viral infection stimulates both H3 and H4 acetylation of the interferon-β gene promoter (31Parekh B.S. Maniatis T. Mol. Cell. 1999; 3: 125-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), whereas four estrogen-responsive genes (pS2,EB1, c-myc, and CTD) were shown to exhibit preferential acetylation of H4 versus H3 following estrogen treatment of the cells (23Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar, 24Mizzen C.A. Allis C.D. Cell. Mol. Life Sci. 1998; 54: 6-20Crossref PubMed Scopus (186) Google Scholar, 25Wang X. Moore S.C. Laszckzak M. Ausio J. J. Biol. Chem. 2000; 275: 35013-35020Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 26Wang X. He C. Moore S.C. Ausio J. J. Biol. Chem. 2001; 276: 12764-12768Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar).The acetylation of H3 associated the StAR promoter was region-specific in that 8-Br-AMP stimulation caused hyperacetylation of only the proximal promoter. Moreover, the cAMP analog did not promote H3 acetylation in a context where the StAR gene is silent. These observations demonstrate the apparent regional selectivity of chromatin modification as well as underscore the association between histone acetylation and gene activity. However, because these experiments examined only a small region of the proximal promoter, we cannot exclude the possibility of acetylation of histones bound to other portions of the StAR gene or address the significance of such modifications regarding StAR gene transcription.Treatment of MA-10 cells with the protein kinase A agonist 8-Br-cAMP increased steroid output by these cells within 15 min. The ∼30-fold increase in progesterone output at the 15-min time point preceded the increase in StAR mRNA by 15 min, suggesting that the increased steroid synthesis either was independent of StAR mRNA accumulation or was the result of a post-transcriptional/translational event (e.g. translational regulation or phosphorylation of the StAR protein). Previous studies with MA-10 cells demonstrated that inhibition of transcription with actinomycin D blocked StAR mRNA expression and new protein production and caused a 75–80% decline in steroidogenic output by these cells at all time points (30–240 min) after exposure to hCG or dibutyryl-cAMP (5Clark B.J. Combs R. Hales K.H. Hales D.B. Stocco D.M. Endocrinology. 1997; 138: 4893-4901Crossref PubMed Scopus (93) Google Scholar). The inability to completely inhibit steroidogenesis is consistent with the notion that StAR function is modulated by a co- or post-translational modification, as originally suggested by Orme-Johnson and co-workers (40Pon L.A. Hartigan J.A. Orme-Johnson N.R. J. Biol. Chem. 1986; 261: 13309-13316Abstract Full Text PDF PubMed Google Scholar). Indeed, these investigators demonstrated that phosphorylation of a mitochondrial protein, later identified as StAR, was temporally associated with the acute effects of ACTH/hCG/dibutyryl-cAMP on steroidogenesis in adrenal, Leydig, and luteal cells (40Pon L.A. Hartigan J.A. Orme-Johnson N.R. J. Biol. Chem. 1986; 261: 13309-13316Abstract Full Text PDF PubMed Google Scholar, 41Pon L.A. Epstein L.F. Orme-Johnson N.R. Endocr. Res. 1986; 12: 429-446Crossref PubMed Scopus (65) Google Scholar, 42Pon L.A. Orme-Johnson N.R. Endocrinology. 1988; 123: 1942-1948Crossref PubMed Scopus (66) Google Scholar). In a more direct analysis of the role of StAR phosphorylation, Arakaneet al. (43Arakane F. King S.R. Du Y. Kallen C.B. Walsh L.P. Watari H. Stocco D.M. Strauss III, J.F. J. Biol. Chem. 1997; 272: 32656-32662Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) demonstrated that mutation of the human StAR protein at serine 195 or 194 in mouse StAR reduced StAR-dependent pregnenolone production by ∼50%. Although the initial increase in steroidogenesis is evidently not the result of increased StAR mRNA levels, the subsequent large increase in steroid production occurring after 15 min of exposure to 8-Br-cAMP is linked to the accumulation of StAR mRNA.It is notable that StAR gene transcription as monitored by levels of nascent StAR transcripts declined between 180 and 360 min of 8-Br-cAMP stimulation despite the continued presence of 1 mm8-Br-cAMP in the culture fluid. The concomitant decline in H3 acetylation between 180 and 360 min suggests that H3 acetylation may be required for cAMP-dependent StAR gene expression. We have recently found that cAMP activates the ERK signaling cascade in ovarian cells, resulting in a reduction of StAR protein levels and inhibition of steroidogenesis (44Seger R. Hanoch T. Rosenberg R. Dantes A. Merz W.E. Strauss III, J.F. J. Biol. Chem. 2001; 276: 13957-13964Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). These findings suggest that a strong tropic signal elicits a concomitant counterbalancing response that limits the magnitude or duration of the cellular reaction to the stimulus. It is possible that the ERK signaling pathway reduces StAR gene transcription through repressors of StAR gene expression such as DAX-1 (45Zazopoulos E. Lalli E. Stocco D.M. Sassone-Corsi P. Nature. 1997; 390: 311-315Crossref PubMed Scopus (360) Google Scholar) or possibly by promoting recruitment of histone deacetylases to the proximal StAR promoter, reversing the chromatin modifications that support transcription.Substantial differences in H3 acetylation of the StAR gene promoter between rhesus monkey non-luteinized and luteinized granulosa cells compared with control and 8-Br-cAMP-treated MA-10 cells were detected in this study. This observation is likely due to differences in basal expression of the StAR gene in these two different cell types. The StAR gene is essentially silent in non-luteinized granulosa cells as evidenced by low/non-detectable StAR mRNA (33Chaffin C.L. Dissen G.A. Stouffer R.L. Mol. Hum. Reprod. 2000; 6: 11-18Crossref PubMed Scopus (73) Google Scholar) and consistent with the detection of low levels of H3 acetylation in the proximal StAR promoter. This may be the result of expression of the StAR gene in a limited number of granulosa cells within the periovulatory follicle. The luteinization process stimulated by in vivoadministration of gonadotropins induces transcription of StAR in the full cohort of granulosa cells. This dramatic change in the number of transcriptionally active cells accounts for the large differences between the non-luteinized and luteinized granulosa cells. In contrast, MA-10 cells exhibit basal StAR gene transcription under control conditions; and therefore, the induction of StAR promoter activity following exposure to 8-Br-cAMP is limited.The development of the ChIP technique to assay protein-DNA interactions in an in vivo context was a major advance in the study of transcriptional regulation. We have now joined this method with quantitative real-time PCR, allowing the procedure to yield quantitative observations regarding protein (i.e.histone)-DNA interaction at specific sites within gene promoters. This method should permit investigators to quantitate changes in gene activity in tissues where transcriptional responses occur dyssynchronously in component cells. With the recent development of new fluorescent dyes for probe labeling, it is possible to examine multiple targets in a single PCR, expanding the capacity of the ChIP/quantitative real-time PCR technique to examine the in vivo protein-DNA interactions. For example, comparisons of proximal and distal StAR promoter chromatin could be made simultaneously, eliminating variation in results due to loading differences. Alternatively, simultaneous examination of multiple target genes using different fluorescent probes could be accomplished. Finally, with immunoprecipitating antibodies that specifically interact with transcription factors/coactivators in a chromatin environment, the ChIP/quantitative real-time PCR technique could be used to identify the specific factors involved and the order in which these factors associate with the gene promoter to induce gene transcription. The translocation of cholesterol from the relatively sterol-rich outer mitochondrial membrane to the relatively cholesterol-poor inner mitochondrial membrane is the rate-limiting step in steroid synthesis (1Christenson L.K. Strauss III, J.F. Biochim. Biophys. Acta. 2000; 1529: 175-187Crossref PubMed Scopus (161) Google Scholar). Steroidogenic acute regulatory protein (StAR)1 plays an integral role in this cholesterol translocation as evidenced by experiments of nature (2Lin D. Sugawara T. Strauss III, J.F. Clark B.J. Stocco D.M. Saenger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (853) Google Scholar) and mouse gene knockout studies (3Caron K.M. Soo S.C. Wetsel W.C. Stocco D.M. Clark B.J. Parker K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11540-11545Crossref PubMed Scopus (375) Google Scholar). In the absence of functional StAR protein, gonadal and adrenal steroidogenesis is markedly impaired and unmetabolized cholesterol accumulates as sterol esters in cytoplasmic lipid droplets. The conservation of StAR protein structure and expression patterns in steroidogenic tissues of piscine, avian, amphibian, and mammalian species testifies to the importance of this protein in steroid synthesis (4Bauer M.P. Bridgham J.T. Langenau D.M. Johnson A.L. Goetz F.W. Mol. Cell. Endocrinol. 2000; 168: 119-125Crossref PubMed Scopus (98) Google Scholar). Studies of StAR gene expression (mRNA) in gonadal and adrenal cells revealed that the steroidogenic capacity of these cells is tightly linked to the abundance of StAR transcripts (5Clark B.J. Combs R. Hales K.H. Hales D.B. Stocco D.M. Endocrinology. 1997; 138: 4893-4901Crossref PubMed Scopus (93) Google Scholar). Analysis of StAR promoter function in human (6Christenson L.K. Johnson P.F. McAllister J.M. Strauss III, J.F. J. Biol. Chem. 1999; 274: 26591-26598Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Christenson L.K. Osborne T.F. McAllister J.M. Strauss III, J.F. Endocrinology. 2001; 142: 28-36Crossref PubMed Scopus (67) Google Scholar, 8Sugawara T. Kiriakidou M. McAllister J.M. Kallen C.B. Strauss III, J.F. Biochemistry. 1997; 36: 7249-7255Crossref PubMed Scopus (151) Google Scholar, 9Sugawara T. Saito M. Fujimoto S. Endocrinology. 2000; 141: 2895-2903Crossref PubMed Scopus (94) Google Scholar), domestic animal (10Rust W. Stedronsky K. Tillmann G. Morley S. Walther N. Ivell R. J. Mol. Endocrinol. 1998; 21: 189-200Crossref PubMed Scopus (52) Google Scholar, 11LaVoie H.A. Garmey J.C. Veldhuis J.D. Endocrinology. 1999; 140: 146-153Crossref PubMed Scopus (79) Google Scholar), and rodent (12Reinhart A.J. Williams S.C. Clark B.J. Stocco D.M. Mol. Endocrinol. 1999; 13: 729-741PubMed Google Scholar, 13Clark B.J. Soo S.C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocrinol. 1995; 9: 1346-1355Crossref PubMed Google Scholar, 14Silverman E. Eimerl S. Orly J. J. Biol. Chem. 1999; 274: 17987-17996Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 15Wooton-Kee C.R. Clark B.J. Endocrinology. 2000; 141: 1345-1355Crossref PubMed Scopus (98) Google Scholar) cells revealed the importance of a variety of transcription factor response elements (SF-1, C/EBPβ, SREBP-1a, GATA-4, DAX-1, and Sp-1) within the first 250 bases of the promoter proximal to the TATA box that influence basal and/or hormone-dependent (cAMP) StAR gene transcription. Transcription factor binding and analysis of mutant promoter constructs confirmed a role for many of these factors in StAR gene activity (6Christenson L.K. Johnson P.F. McAllister J.M. Strauss III, J.F. J. Biol. Chem. 1999; 274: 26591-26598Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Christenson L.K. Osborne T.F. McAll" @default.
• W1979098148 created "2016-06-24" @default.
• W1979098148 creator A5047403775 @default.
• W1979098148 creator A5048183362 @default.
• W1979098148 creator A5090491933 @default.
• W1979098148 date "2001-07-01" @default.
• W1979098148 modified "2023-10-14" @default.
• W1979098148 title "Quantitative Analysis of the Hormone-induced Hyperacetylation of Histone H3 Associated with the Steroidogenic Acute Regulatory Protein Gene Promoter" @default.
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