Lactogenic Hormonal Induction of Long Distance Interactions between β-Casein Gene Regulatory Elements

Abstract

Lactogenic hormone regulation of β-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of β-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately −6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the β-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, β-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription. Lactogenic hormone regulation of β-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of β-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately −6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the β-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, β-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription. The transcription of the β-casein milk protein gene is induced synergistically by the lactogenic hormones prolactin (Prl) 2The abbreviations used are: PrlprolactinPRprogesterone receptor3Cchromosome conformation captureChIPchromatin immunoprecipitationpolpolymerasep-polphosphorylated polMECmammary epithelial cellEGFepidermal growth factorhPR-Bhuman PR-BGAPDHglyceraldehyde-3-phosphate dehydrogenaseGRglucocorticoid receptorHChydrocortisone. and glucocorticoids together with local growth factors, cell-cell and cell-substratum interactions that activate specific transcription factors and change chromatin structure. These alterations in chromatin structure include nucleosome remodeling (1Xu R. Spencer V.A. Bissell M.J. J. Biol. Chem. 2007; 282: 14992-14999Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and post-translational modification of histones, both at the nucleosome level and at the level of larger chromatin domains. prolactin progesterone receptor chromosome conformation capture chromatin immunoprecipitation polymerase phosphorylated pol mammary epithelial cell epidermal growth factor human PR-B glyceraldehyde-3-phosphate dehydrogenase glucocorticoid receptor hydrocortisone. In a previous study (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar) using chromatin immunoprecipitation (ChIP) analysis, we examined the dynamics of recruitment of different transcription factors at the hormonally activated β-casein promoter proximal, as well as the more distal mouse β-casein enhancer, the latter located >6 kb upstream of the transcription start site (3Rijnkels M. Elnitski L. Miller W. Rosen J.M. Genomics. 2003; 82: 417-432Crossref PubMed Scopus (82) Google Scholar). For simplicity, these regulatory elements are referred to as the “proximal promoter” and “distal enhancer.” Hormonal stimulation of cells with Prl alone resulted in a rapid recruitment of Stat5 to the β-casein promoter and enhancer, and reciprocally the dissociation of YY-1 from the proximal promoter, but this was not sufficient to promote β-casein gene transcription. β-Casein gene transcription required treatment with both prolactin and glucocorticoids and the synergistic interaction of the glucocorticoid receptor and the LAP (liver-enriched activating protein) isoform of C/EBPβ followed by stable association of p300 and phosphorylated RNA polymerase (pol) II at the promoter and enhancer. Because C/EBPβ (LIP isoform, liver-enriched transcriptional inhibitory protein) interacts directly with YY-1 (4Bauknecht T. See R.H. Shi Y. J. Virol. 1996; 70: 7695-7705Crossref PubMed Google Scholar) and YY-1 interacts with several histone deacetylases (5Yang W.M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12845-12850Crossref PubMed Scopus (485) Google Scholar, 6Yang W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), it is possible that interactions between YY-1 and C/EBPβ isoform LIP are responsible for the repression of β-casein promoter in the absence of lactogenic hormones through the recruitment of histone deacetylases. However, ChIP assays using antibodies to HDAC1 did not reveal a correlation of HDAC1 association with the dynamics of YY-1 disassociation at the proximal promoter (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar). These data are consistent with results demonstrating that the was not by HDAC1 antibodies PubMed Scopus Google Scholar) and suggest that a histone HDAC1 is with YY-1 in transcriptional possible that histone transcription to a promoter, and the YY-1 transcription factor Y.L. W.M. Seto E. Mol. Biol. PubMed Scopus Google Scholar). at a from to the of stable transcription However, the of these with The dynamics of of transcription factors, the p300 and RNA pol as well as histone at the proximal promoter and the distal enhancer (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar) that these two regulatory regions with each through a chromatin is possible that a looping structure between promoter and enhancer provides that recruitment and the from transcription to T. J. M. 2003; PubMed Scopus Google Scholar). The chromosome conformation capture assay is a quantitative that of interactions between chromatin of to several J. M. PubMed Scopus Google Scholar, E. W. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Sci. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, E. W. PubMed Scopus Google Scholar, T. M. M.J. J. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). In this chromatin are with followed by with and DNA which of of The are and DNA is real time is used to DNA using specific for the Scholar). The of to of long range chromatin and it that transcriptional regulatory DNA elements with proximal to regulate gene expression. used to the chromatin structure at the E. W. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. M.J. J. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar), PubMed Scopus Google Scholar), the M. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar), and J.M. 2007; PubMed Scopus Google Scholar) to a the assay of the for the between and transcription. is to lactogenic hormone induction of the β-casein gene expression in the mammary J. Endocrinol. PubMed Scopus Google Scholar, J.M. PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar). The of the inhibitory of progesterone in mouse mammary epithelial and it that progesterone both induction and glucocorticoid of β-casein transcription through a of the progesterone receptor with the signaling in a manner interaction with the β-casein promoter W. J. Rosen J.M. Mol. Endocrinol. 2007; PubMed Scopus Google Scholar). treatment was to recruitment of to the enhancer and the interactions of the enhancer with pol and of of C. and D. In this study using ChIP we the dynamics of association with the β-casein gene proximal promoter and a correlation in YY-1 and dissociation from the promoter HC11 cells with Prl and using the we that treatment with Prl and induced a interaction between the β-casein gene proximal promoter and distal enhancer a chromatin loop structure. DNA looping was observed in nontreated cells or cells treated with or Prl for the time we that the synergistic interactions between steroid and peptide hormone-induced are required for the of these long range chromatin this we used the three-dimensional mammary acini in which primary of mammary epithelial cells from to were a of demonstrate that lactogenic hormone treatment of these primary three-dimensional the DNA loop between the β-casein promoter and enhancer, the results with the HC11 cell we the to primary from the virgin and lactating mouse mammary to that these long range chromatin interactions are mammary the long range interactions between the promoter and enhancer were inhibited by previous results (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar) and the results from this we suggest a model for the of a at the β-casein and we that of the active chromatin loop between the proximal promoter and distal enhancer is a step required to stable transcription. was by the and of and was used at a of was from and used at a of was from The was from was from DNA was from and was used at a of PR-B was from an and as W. J. Rosen J.M. Mol. Endocrinol. 2007; PubMed Scopus Google Scholar). For cells were with at a of HC11 mammary epithelial cells were in at and in with cells were for an this cells were with for progesterone receptor and in for were treated with hormones Prl and for of were from which were between and and cell were as by Full Text Full Text PDF PubMed Scopus Google Scholar). mammary the cell was in and and was to which were with of growth at a cells mammary were in for followed by of in and of in with hydrocortisone and prolactin For assays from mammary primary mammary epithelial were as by Yao E. Bissell M.J. Biol. 2007; PubMed Scopus Google Scholar). RNA was from or hormone-treated HC11 primary and mammary using For HC11 of was and cells were and by the through a several For primary of was directly to acini of and with For mammary of was in of using a The of RNA from mammary primary or HC11 cells was by at using a The was from of RNA by using the of β-casein expression was by real time as (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar). ChIP was as (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar). and were from and were by using real time with an The assay in HC11 cells was as T. M. M.J. J. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar) with were in of and with for at The was by to a of following at for and for were at for in of with of and for were by with at were with of and in of the The was which is and in of was to a of and the were at for was to the of to the was with of at The was by to and at for The were of and of DNA and at for was and were at to the from were with and at DNA was with and with DNA was with were in and treated with of for at Scholar) was to assays in primary three-dimensional acini and in from mammary acini from to with at for using was and the was by followed by for at used which the and (3Rijnkels M. Elnitski L. Miller W. Rosen J.M. Genomics. 2003; 82: 417-432Crossref PubMed Scopus (82) Google Scholar) to a that of possible in the in is used to for in between the used DNA was to with and DNA was as R. in and Scholar). for the assay were to the from the at promoter, and from site at and of the resulted in a In a distal was to as a control from site kb from β-casein promoter, with of the a were by and by The of were from the and DNA was using the and by DNA The are in real time was using as a for DNA The range of was for the and control by to time was with of DNA different using of a for for and for The of were by to a by of the control DNA In addition, were to to for in or were in were using the data for the of are in The assay was for at two different cell with using quantitative ChIP assays that YY-1 is to the β-casein promoter and β-casein expression in the absence of lactogenic hormones (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar). that this repression of interactions between YY-1 and C/EBPβ isoform LIP through the recruitment of histone deacetylases. However, we ChIP assays using antibodies to we did not correlation of HDAC1 association with the dynamics of YY-1 disassociation at the proximal we an of the transcriptional that histone W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, Y.L. W.M. Seto E. Mol. Biol. PubMed Scopus Google Scholar). ChIP assays using to in HC11 cells treated with Prl and a in chromatin association at the proximal promoter in the and These results a correlation with the rapid disassociation of YY-1 from the proximal promoter followed stimulation The dissociation of YY-1 in cells treated with both hormones was observed by in a time Stat5 was in HC11 cells treated with lactogenic Stat5 and YY-1 with a in association with the β-casein Stat5 YY-1 and These data that YY-1 β-casein gene expression in the absence of prolactin through association with and that lactogenic hormones by this In previous we were to an level of phosphorylated RNA polymerase II the distal enhancer of the β-casein gene that is located between −6 and kb upstream of the start site of transcription (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar). The association of II at both regulatory regions the of a long range interaction between the β-casein promoter and enhancer recruitment of RNA pol II and the transcription of at the distal enhancer. Because of these RNA we to DNA looping we assay in HC11 cells and treated with and in for different of of are required to the results of the we a control that possible in control was used for of by quantitative real time of for the we the range of required for to the between the of and a that interactions between regions, two of DNA were as in the promoter and enhancer and in the promoter and a distal which is kb from the promoter in HC11 cells and cells treated with and Prl for and The were using both and were confirmed by DNA The assay with both for was in and For of different we real time in a range for each DNA The range of was by Using this a in the of interactions between promoter and enhancer in cells treated with and Prl nontreated cells was observed In no in the of interactions was These data suggest that the interactions between the promoter and enhancer of the β-casein gene are specific and are induced of HC11 cells with and In HC11 cells treated with we observed a in to both promoter and enhancer (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar). directly by recruitment was at both regulatory regions of β-casein no for at the distal enhancer. the that the of both hormones is not required for communication through DNA we assays in HC11 cells treated with or Prl and with alone for or no in interactions between the promoter and enhancer as with cells. results were for HC11 cells treated with Prl alone for and and a but in was at For both no in the of interactions was not These results with the in β-casein mRNA in HC11 cells with and Prl or in (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar), and that stimulation with both the steroid and peptide hormones is required for these interactions between the promoter and enhancer. the interaction between the promoter and enhancer of the β-casein gene observed following lactogenic hormone stimulation in HC11 cells in primary three-dimensional mammary acini and it was mammary the we the for in the three-dimensional mammary acini in which primary are a of For these we primary from to J. Bissell M.J. J. Sci. Full Text PDF PubMed Scopus Google Scholar), for primary the cells that to epithelial cells in to the mammary is to of and glucocorticoids and of β-casein mRNA L. C. L. Google Scholar). to and β-casein gene primary were in for followed by treatment with Prl and for an at the level of β-casein gene expression stimulation of primary with quantitative was mRNA from treated and control three-dimensional acini these approximately a of β-casein mRNA expression was observed in hormone-treated as with cultures. to lactogenic hormone induction of β-casein mRNA in primary three-dimensional acini was by in long range chromatin we these primary followed by quantitative real time In three-dimensional acini treated with and we observed a in the of interactions between promoter and enhancer as with cultures. the no in the of interactions was These data with in HC11 cells and suggest that lactogenic hormones regulate long range chromatin interactions between the promoter and enhancer of the β-casein gene in primary mouse three-dimensional cultures. the between the β-casein promoter and enhancer were mammary we to primary from the mammary of virgin and lactating of a of β-casein mRNA expression was observed in the mammary of lactating as with virgin we in primary from these in Using quantitative real time PCR, we an in the of interactions between promoter and enhancer in from as with virgin no in the of interactions was These results were in two in the of interactions between promoter and enhancer to and the expression of β-casein mRNA was observed lactating to virgin not progesterone β-casein induction in a progesterone we human PR-B HC11 which this by and we treated these cells with or and with or for used HC11 cells treated with hormones in the as a control for β-casein RNA was and using specific to β-casein and β-Casein mRNA were by quantitative In cells treated with and Prl for we observed a induction of β-casein mRNA with nontreated cells. for cells treated with hormones in the of we observed a in the level of β-casein mRNA in β-casein mRNA was in cells with and treated with and Prl However, we observed a more in β-casein mRNA induction in cells both with and treated with hormones in the of These data that the progesterone receptor in a progestin-dependent manner induction of the β-casein gene expression in HC11 cells. the following this by progesterone the and long range interactions between the β-casein gene promoter and this we the assay in HC11 cells with and treated with hormones for and in the absence and of In the absence of the a in cells treatment with hormones as with nontreated cells. using quantitative real time PCR, we that this was which is that observed in cells However, in the of the of the was with nontreated no was at or following treatment and at no in the of interactions was for both in cells not results were with the assay in two transcriptional of a gene is to a that involves recruitment of transcription factors and remodeling of chromatin nucleosome remodeling with post-translational of histones, and of a that are from the activate that these chromatin alterations and RNA polymerase However, the by which distal in the recruitment of chromatin remodeling and pol which at the is not The model involves the of an chromatin by interactions between elements of the distal enhancer and gene promoter by DNA looping M. PubMed Scopus Google Scholar, J.M. W. Full Text PDF PubMed Scopus Google Scholar, W. Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. M. PubMed Scopus Google Scholar). The and of long range interactions were and the of as and of D. L. PubMed Scopus Google Scholar) and J. M. PubMed Scopus Google Scholar, E. W. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Sci. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, E. W. PubMed Scopus Google Scholar, T. M. M.J. J. Mol. Full Text Full Text PDF PubMed Scopus Google Scholar) the of this level of chromatin In study we the of regulation of β-casein gene expression in mammary epithelial cells. In previous (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar) using ChIP we the dynamics of of transcription factors and YY-1 at the hormonally activated β-casein proximal promoter and distal enhancer. In addition, we the recruitment of which is in β-casein expression and post-translational modification the dynamics of histone and the time of RNA pol II and phosphorylated RNA pol II at both regulatory regions of the hormonally activated β-casein These data with the of the and chromatin at both β-casein regulatory regions that are kb by using the we that the hormonally activated β-casein proximal promoter and distal enhancer with each through the interactions a chromatin These a model that to the signaling by lactogenic hormones are to regulate β-casein transcription. In the absence of lactogenic both β-casein regulatory regions a of of histone C. and D. a modification with chromatin structure M. T. J. Sci. 2003; PubMed Scopus Google Scholar). YY-1 to a in β-casein repression Mol. Biol. PubMed Google Scholar). The YY-1 site in the β-casein promoter is a which is by interactions with the LIP isoform of C/EBPβ (4Bauknecht T. See R.H. Shi Y. J. Virol. 1996; 70: 7695-7705Crossref PubMed Google Scholar). LIP directly with YY-1 to to promote a chromatin (5Yang W.M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12845-12850Crossref PubMed Scopus (485) Google Scholar, 6Yang W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). using we observed the disassociation of from the β-casein proximal promoter followed by stimulation with and Prl and which with the dynamics of YY-1 dissociation and a with Stat5 Stat5 induced by prolactin to responsible for the disassociation of both YY-1 and from the β-casein stimulation with Stat5 phosphorylated by and the it interacts with in both the β-casein promoter and enhancer. However, β-casein gene expression is following stimulation by both prolactin and the for between Stat5 and E. M. 1996; PubMed Scopus Google Scholar, Mol. Biol. PubMed Scopus Google Scholar, J. 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PubMed Google Scholar, Mol. Biol. 1997; PubMed Google Scholar), which histone in chromatin remodeling and the of RNA polymerase II to the promoter T. 1996; PubMed Scopus Google Scholar). observed of Stat5 and C/EBPβ and the recruitment of p300 in cells with Prl at both β-casein gene regulatory regions (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar). treatment of cells with both and Prl resulted in a and in p300 as with hormone These results that p300 is at the gene regulatory regions through the interactions with the primary transcription factors following DNA the of phosphorylated RNA polymerase II at the distal enhancer (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. 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The level of was that observed in cell and was not to the β-casein proximal promoter in the absence of study the of chromatin we assays in HC11 cells expressing following stimulation with and Prl in the and absence of In the absence of we observed a in the of interactions between the promoter and enhancer in expressing no were in the of These results that long range interactions between the β-casein promoter and enhancer induced by lactogenic hormones in a chromatin that transcription. The by which this to with