Lactogenic Hormonal Induction of Long Distance Interactions between β-Casein Gene Regulatory Elements
2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês
10.1074/jbc.m109.032490
ISSN1083-351X
AutoresElena B. Kabotyanski, Monique Rijnkels, Courtneay Freeman-Zadrowski, Adam C. Buser, Dean P. Edwards, Jeffrey M. Rosen,
Tópico(s)RNA Research and Splicing
ResumoLactogenic 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 YY-1-HDAC1 complex was not supershifted by HDAC1 antibodies (7Chang L.K. Liu S.T. Nucleic Acids Res. 2000; 28: 3918-3925Crossref PubMed Scopus (89) Google Scholar) and suggest that a histone deacetylase other than HDAC1 is most likely associated with YY-1 resulting in transcriptional repression. One possible candidate could be HDAC3 that possesses histone deacetylase activity, represses transcription when tethered to a promoter, and binds the YY-1 transcription factor (8Yao Y.L. Yang W.M. Seto E. Mol. Cell. Biol. 2001; 21: 5979-5991Crossref PubMed Scopus (362) Google Scholar). Transcriptional enhancers function at a distance from their target genes to help facilitate the formation of stable transcription preinitiation complexes. However, the question of how these distant activators interact with their target genes still remains open. The similar dynamics of assembly of transcription factors, the co-activator p300 and RNA pol II, as well as histone acetylation 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) suggested that these two regulatory regions might communicate with each other through protein-protein interactions, forming a chromatin loop. It is possible that a looping structure between promoter and enhancer provides binding surfaces that increase preinitiation complex recruitment and enhance the transition from transcription initiation to elongation (9Sawado T. Halow J. Bender M.A. Groudine M. Genes Dev. 2003; 17: 1009-1018Crossref PubMed Scopus (150) Google Scholar). The chromosome conformation capture (3C) assay is a quantitative method that allows identification of physical interactions between chromatin segments of up to several hundred kilobases apart (10Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2581) Google Scholar, 11Tolhuis B. Palstra R.J. Splinter E. Grosveld F. de Laat W. Mol. Cell. 2002; 10: 1453-1465Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar, 12Dekker J. Trends Biochem. Sci. 2003; 28: 277-280Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 13Splinter E. Grosveld F. de Laat W. Methods Enzymol. 2004; 375: 493-507Crossref PubMed Scopus (97) Google Scholar, 14Vakoc C.R. Letting D.L. Gheldof N. Sawado T. Bender M.A. Groudine M. Weiss M.J. Dekker J. Blobel G.A. Mol. Cell. 2005; 17: 453-462Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). In this assay, chromatin fragments are fixed with formaldehyde, followed by digestion with restriction enzymes and ligation under dilute DNA concentrations, which promotes intramolecular ligation of cross-linked fragments over intermolecular ligation of random fragments. The cross-links are then reversed, and DNA is purified. Quantitative real time PCR is used to amplify DNA fragments containing novel ligation products using primers specific for the region under investigation (24Cope N.F. Fraser P. Cold Spring Harbor Protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2009Google Scholar). The development of 3C technology has contributed greatly to our understanding of long range chromatin architecture, and specifically, it has revealed that transcriptional regulatory DNA elements often form looped structures with proximal promoters to regulate gene expression. This method has been used to analyze the higher order chromatin structure at the β-globin (11Tolhuis B. Palstra R.J. Splinter E. Grosveld F. de Laat W. Mol. Cell. 2002; 10: 1453-1465Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar, 14Vakoc C.R. Letting D.L. Gheldof N. Sawado T. Bender M.A. Groudine M. Weiss M.J. Dekker J. Blobel G.A. Mol. Cell. 2005; 17: 453-462Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), TH2 cytokine loci (15Spilianakis C.G. Flavell R.A. Nat. Immunol. 2004; 5: 1017-1027Crossref PubMed Scopus (352) Google Scholar), the prostate-specific antigen (16Wang Q. Carroll J.S. Brown M. Mol. Cell. 2005; 19: 631-642Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar), and snail genes (17Palmer M.B. Majumder P. Green M.R. Wade P.A. Boss J.M. Cancer Res. 2007; 67: 6113-6120Crossref PubMed Scopus (28) Google Scholar) to list a few examples. Thus, the 3C assay has now become one of the essential tools for studying the relationship between nuclear organization and transcription. Progesterone is known to repress lactogenic hormone induction of the β-casein gene expression in the mammary gland during pregnancy (18Kuhn N.J. J. Endocrinol. 1969; 45: 615-616Crossref PubMed Scopus (24) Google Scholar, 19Rosen J.M. O'Neal D.L. McHugh J.E. Comstock J.P. Biochemistry. 1978; 17: 290-297Crossref PubMed Scopus (76) Google Scholar, 20Deis R.P. Delouis C. J. Steroid Biochem. 1983; 18: 687-690Crossref PubMed Scopus (27) Google Scholar). The mechanism of the inhibitory action of progesterone has been investigated in mouse mammary epithelial cells, and it has been demonstrated that progesterone inhibits both PRL induction and glucocorticoid potentiation of β-casein transcription through a direct cross-talk of the progesterone receptor (PR) with the Prl/Stat5 signaling pathway in a manner dependent on PR interaction with the β-casein promoter (21Buser A.C. Gass-Handel E.K. Wyszomierski S.L. Doppler W. Leonhardt S.A. Schaack J. Rosen J.M. Watkin H. Anderson S.M. Edwards D.P. Mol. Endocrinol. 2007; 21: 106-125Crossref PubMed Scopus (63) Google Scholar). Progesterone treatment was also shown to induce recruitment of PR to the enhancer and disrupted the interactions of the enhancer with Stat5, GR, C/EBPβ, p300, pol II, and acetylation of H3 causing sustained repressive modifications of chromatin. 3A. C. Buser and D. P. Edwards, unpublished observations. In this study using ChIP assays, we first determined the dynamics of HDAC3 association with the β-casein gene proximal promoter and demonstrated a correlation in YY-1 and HDAC3 dissociation from the promoter after treating HC11 cells with Prl and HC. By using the 3C assay, we next demonstrated that hormonal treatment with Prl and HC induced a physical interaction between the β-casein gene proximal promoter and distal enhancer forming a chromatin loop structure. No DNA looping was observed in nontreated cells or cells treated with HC or Prl alone. Thus, for the first time we have demonstrated that the synergistic interactions between steroid receptors and peptide hormone-induced signal transduction pathways are required for the formation of these long range chromatin interactions. To test this hypothesis further, we used the three-dimensional mammary acini culture model, in which primary cultures of mammary epithelial cells (MECs, derived from mice 14 to 18 days pregnant) were grown on a layer of extracellular matrix (Matrigel). We demonstrate that lactogenic hormone treatment (insulin-hydrocortisone-prolactin) of these primary three-dimensional cultures induces the DNA loop between the β-casein promoter and enhancer, thus confirming the results obtained with the HC11 cell line. Additionally, we applied the 3C analysis to primary MECs isolated from the virgin and lactating mouse mammary glands to confirm that these long range chromatin interactions are regulated during normal mammary gland development. Finally, the long range interactions between the promoter and enhancer were inhibited by progestin/PR. Based on our 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 study, we suggest a model for the assembly of a multiprotein complex at the β-casein gene, and we propose that hormone-dependent formation of the active chromatin loop between the proximal promoter and distal enhancer is a key step required to achieve stable transcription. Prolactin was kindly provided by the National Hormone and Pituitary Program (NIDDK, National Institutes of Health, Bethesda) and was used at a concentration of 1 μg/ml. Hydrocortisone (H-4001) was purchased from Sigma and used at a concentration of 1 μg/ml. R5020 (promegestrone; 17α,21-dimethyl-19-norpregna-4,9-diene-3,20-one) was obtained from PerkinElmer Life Sciences. The protease inhibitor mixture was purchased from Roche Applied Science. HindIII was obtained from Invitrogen. T4 DNA ligase was purchased from New England Biolabs (Ipswich, MA) and was used at a concentration of 4000 Weiss units/reaction. Human PR-B was expressed from an adenovirus vector and prepared as described previously (21Buser A.C. Gass-Handel E.K. Wyszomierski S.L. Doppler W. Leonhardt S.A. Schaack J. Rosen J.M. Watkin H. Anderson S.M. Edwards D.P. Mol. Endocrinol. 2007; 21: 106-125Crossref PubMed Scopus (63) Google Scholar). For adenovirus infection, cells were transduced with hPR-B at a concentration of 50 plaque-forming units/cell. HC11 mammary epithelial cells were grown in 150-mm dishes at 37 °C and 5% CO2 in RPMI media supplemented with 10% bovine calf serum (SAFC Biosciences), 2 mm glutamine (SAFC Biosciences), 50 μg/ml gentamycin (Sigma), 5 μg/ml bovine insulin (Sigma), 10 ng/ml murine EGF (SAFC Biosciences). After cells reached confluency, they were grown for an additional 3 days (at this point cells were infected with adenovirus for experiments involving progesterone receptor recruitment) and then incubated in priming media (0.5 m glutamine, 5 μg/ml insulin, 10% stripped donor horse serum, RPMI 1640 media) for 48 h. Cells were treated with hormones HC (1 μg/ml), Prl (1 μg/ml), and R5020 (10 nm) for various periods of time. Primary MECs were isolated from 12 mice, which were between 14 and 18 days pregnant, and single cell suspensions were generated as described by Welm et al. (22Welm B.E. Dijkgraaf G.J. Bledau A.S. Welm A.L. Werb Z. Cell Stem Cell. 2008; 2: 90-102Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). To generate mammary acini, the final cell pellet was resuspended in plating medium (F-12 Nutrient Mix medium containing 10% fetal bovine serum, 10 ng/ml EGF, 5 μg/ml insulin, 1 μg/ml hydrocortisone, and 50 μg/ml gentamycin) and was added to 60-mm tissue culture dishes, which were pre-coated with 360 μl of growth factor-reduced Matrigel, at a density 106 cells per dish. Isolated mammary MECs were maintained in plating medium for 6 days followed by 2 days of incubation in priming medium (F-12 Nutrient Mix/gentomycin medium containing 5 μg/ml insulin) and then 2 days of incubation in differentiation medium (F-12 Nutrient Mix/gentamycin with hydrocortisone (1 μg/ml), insulin (5 μg/ml), and prolactin (3 μg/ml)). For 3C assays performed from mammary gland tissue, primary mammary epithelial organoids were isolated as described by Fata et al. (23Fata J.E. Mori H. Ewald A.J. Zhang H. Yao E. Werb Z. Bissell M.J. Dev. Biol. 2007; 306: 193-207Crossref PubMed Scopus (142) Google Scholar). Total RNA was isolated from untreated- or hormone-treated HC11 cells, primary cultures, and mammary gland samples using TRIzol reagent (Invitrogen). For HC11 cells, 500 μl of TRIzol was added, and cells were harvested and homogenized by passing the solution through a pipette tip several times. For primary cultures, 500 μl of TRIzol was added directly to acini cultures grown on Matrigel after removal of media and rinsing with phosphate-buffered saline. For mammary gland samples, 30 mg of tissue was homogenized in 500 μl of TRIzol using a Polytron. The amount of total RNA extracted from either mammary gland tissue, primary cultures, or HC11 cells was measured by absorbance at 260 nm using a NanoDrop spectrophotometer. The cDNA was generated from 5 μg of total RNA by using the Superscript First-Strand Synthesis System (Invitrogen). Quantification of β-casein expression was performed by real time PCR as described previously (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 performed as described previously (2Kabotyanski E.B. Huetter M. Xian W. Rijnkels M. Rosen J.M. Mol. Endocrinol. 2006; 20: 2355-2368Crossref PubMed Scopus (69) Google Scholar). Anti-YY1 (c-20) (sc-281) and anti-HDAC3 (07-522) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Upstate (Charlottesville, VA), respectively. Results were quantified by using real time PCR with SYBR Green on an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The 3C assay in HC11 cells was performed as described previously (14Vakoc C.R. Letting D.L. Gheldof N. Sawado T. Bender M.A. Groudine M. Weiss M.J. Dekker J. Blobel G.A. Mol. Cell. 2005; 17: 453-462Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar) with minor modifications. Cells (3 × 107) were dissolved in 45 ml of priming media and cross-linked with formaldehyde (1% final) for 10 min at room temperature. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 m following incubation at room temperature for 5 min and then stored on ice for 15 min. Cells were centrifuged at 2000 rpm for 10 min, resuspended in 1 ml of ice-cold lysis buffer (10 mm Tris, pH 8.0, 10 mm NaCl, 0.2% Nonidet P-40) supplemented with 0.1 ml of protease inhibitor mixture, and incubated on ice for 15 min. Then nuclei were released by Dounce homogenization with 20 strokes (pestle B) on ice. After centrifugation at 5000 rpm, nuclei were washed with 0.5 ml of 1× restriction enzyme buffer and then resuspended in 125 μl of the same buffer. The nuclei suspension was then divided into five individual tubes (each tube contained 25 μl, which is ∼6 × 106 cells), centrifuged, and resuspended in 362 μl of 1× restriction enzyme buffer each. SDS was added to a final concentration of 0.1%, and the nuclei were incubated at 65 °C for 10 min. Triton X-100 was then added to the final concentration of 1% to sequester the SDS. Digestion was performed with 400 units of HindIII at 37 °C overnight. The restriction enzyme was inactivated by raising SDS concentration to 2% and incubating at 65 °C for 30 min. The reactions were diluted into 8 ml of ligation reaction buffer containing 1% Triton X-100, 50 mm Tris, pH 7.5, 10 mm MgCl2, 10 mm dithiothreitol, 0.1 mg/ml bovine serum albumin, 1 mm ATP, and 4000 units of T4 DNA ligase, and incubated at 16 °C for 2 h. Proteinase K (100 μg) was added, and samples were incubated overnight at 65 °C to reverse the cross-links. Samples from five individual reactions were extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1, v/v), combined together, and ethanol-precipitated. After centrifugation at 10,000 rpm, DNA was purified again with phenol/chloroform/isoamyl alcohol (25:24:1, v/v) and precipitated with ethanol. To remove extra salt, DNA was washed five times with 70% ethanol. Samples were dissolved in TE buffer, pH 8.0, and treated with 0.5 μg of RNase A for 15 min at 37 °C. A modified protocol (24Cope N.F. Fraser P. Cold Spring Harbor Protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2009Google Scholar) was adapted to perform 3C assays in primary three-dimensional acini cultures grown on Matrigel and in MECs isolated from mammary gland tissues. To release acini from Matrigel prior to fixation with formaldehyde, incubation at 37 °C for 1 h using Dispase reagent (BD Biosciences) was performed, and the reaction was stopped by adding 10 mm EDTA, followed by incubation for 30 min at room temperature. We used BAC RP23-457P20, which contains the αs1-, β-, and γ-casein genes (3Rijnkels M. Elnitski L. Miller W. Rosen J.M. Genomics. 2003; 82: 417-432Crossref PubMed Scopus (82) Google Scholar) to generate a PCR template that consists of all possible ligation products in the region under investigation in equimolar amounts. This template is used to normalize for differences in primer efficiency between the used primer pairs. BAC DNA (20 μg) was digested to completion with HindIII, and DNA was religated as described (25Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Siedman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., Hoboken, NJ2006: 21.11.1-21.11.20Google Scholar). Primers for the 3C assay were chosen close to the HindIII sites (119 bp from the HindIII at promoter, 5′-CAA CTA CAT GTT CCT CCA GCC AAG TGA-3′, and 79 bp from HindIII site at BCE, 5′-TTT GAG GCC TTC TTC TGC TCC TTC AG-3′), and amplification of the ligated product resulted in a 255-bp fragment. In addition a distal reverse primer was chosen to serve as a control (39 bp from HindIII site ∼112 kb from β-casein promoter, 5′-CAT GAG TGA TGC AAA AGC AAC TTG ATG-3′) with PCR amplification of the ligated product yielding a 211-bp fragment. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The bands of interest were excised from the gel, and DNA was purified using the QIAquick spin columns (Qiagen) and analyzed by DNA sequencing. The partial sequences are listed in supplemental Fig. S1. Quantitative real time PCR was performed on ABI Prism 7700 Sequence Detection System using SYBR Green as a marker for DNA amplification. The linear range of amplification was determined for the experimental 3C templates and control 3C template by making serial dilutions (from 1:10 to 1:2000). Real time PCR was performed with 5 μl of 3C DNA template (at different dilutions) using 40 cycles of a three-step amplification (94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s). The relative amounts of experimental 3C templates were determined by comparison to a standard curve generated by serial dilutions of the control BAC 3C DNA template. In addition, all PCRs were normalized to GAPDH to equalize for small differences in template amount or quality. All PCRs were run in triplicate. All differences reported were statistically significant (p < 0.05) using the Student's t test. Representative experimental data for all the dilutions of one experiment are shown in supplemental Fig. S2. The 3C assay was performed for at least two different cell culture experiments with essentially similar results. We demonstrated previously using quantitative ChIP assays that YY-1 is bound to the β-casein promoter and represses 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). We hypothesized that this repression may occur because of interactions between YY-1 and C/EBPβ isoform LIP through the recruitment of histone deacetylases. However, when we performed ChIP assays using antibodies to HDAC1, we did not observe any correlation of HDAC1 association with the dynamics of YY-1 disassociation at the proximal promoter. Here we tested HDAC3, an ortholog of the yeast transcriptional regulator RPD3 that possesses intrinsic histone deacetylase activity (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, 8Yao Y.L. Yang W.M. Seto E. Mol. Cell. Biol. 2001; 21: 5979-5991Crossref PubMed Scopus (362) Google Scholar). ChIP assays performed using antibody to HDAC3 in HC11 cells treated with Prl and HC, showed a transient decrease in chromatin association (about 2-fold) at the proximal promoter in the first 30 min (Fig. 1, A and B). These results show a good correlation with the rapid disassociation of YY-1 from the proximal promoter followed hormonal stimulation (Fig. 1C). The maximal dissociation of YY-1 in cells treated with both hormones was observed by 15–30 min, in a time frame when Stat5 binding was maximal. Thus, in HC11 cells treated with lactogenic hormones, Stat5 and YY-1 (together with HDAC3) display a reciprocal relationship in their association with the β-casein promoter; when Stat5 binding increases, YY-1 and HDAC3 decreases. These data suggested that YY-1 represses β-casein gene expression in the absence of prolactin through its association with HDAC3 and that lactogenic hormones act by relieving this repression. In our previous experiments, we were able to detect an increased level of phosphorylated RNA polymerase II (p-pol II) within the distal enhancer of the β-casein gene that is located between −6 and −6.4 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 p-pol II at both regulatory regions suggested either the possibility of a long range interaction between the β-casein promoter and enhancer or, alternatively, independent recruitment of RNA pol II and perhaps even the transcription of short RNAs at the distal enhancer. Because detection of these small RNA transcripts has been problematic, we decided to first determine whether DNA looping might occur. Accordingly, we performed 3C assay in HC11 cells untreated and treated with hormones, singly and in combination for different periods of time. A number of experimental controls are required to validate the results of the 3C assay. First, we generated a BAC control template that represented all possible ligation products in equimolar amounts. This BAC control template was used for normalization of signal intensities obtained by quantitative real time PCR of cross-linked 3C templates (correction for the PCR amplification efficiency). Second, we determined the range of template concentration required for linear PCR product formation. Third, to compare the cross-linking frequencies between the 3C template of interest and a 3C template that represents interactions between unrelated regions, two sets of DNA primers were designed as follows: Bp290_Be5401, flanking restriction sites in the promoter and enhancer region (255-bp PCR product), and Bp290_Be474, in the promoter and a distal region, which is 112 kb from the promoter (211-bp PCR product) (Fig. 2A). We performed 3C analysis in untreated HC11 cells and cells treated with HC and Prl for 15 min, 1 h, and 24 h. The purified ligation products were amplified using both Bp290_Be5401 and Bp290_Be474 primer sets (Fig. 2B). Both PCR products were confirmed by DNA sequencing (supplemental Fig. S1). The 3C assay with both primers sets for PCR amplification was repeated in three independent experiments and revealed similar results. For accurate quantification of different ligation products, we performed real time PCR in a linear range for each DNA amplification reaction. The linear range of amplification was determined by serial dilutions (supplemental Fig. S2). Using this a
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