Steroid Hormone Receptor-mediated Histone Deacetylation and Transcription at the Mouse Mammary Tumor Virus Promoter
2001; Elsevier BV; Volume: 276; Issue: 35 Linguagem: Inglês
10.1074/jbc.c100315200
ISSN1083-351X
AutoresLynn A. Sheldon, Matthias Becker, Catharine L. Smith,
Tópico(s)interferon and immune responses
ResumoAcetylation of lysines in histones H3 and H4 N-terminal tails is associated with transcriptional activation and deacetylation with repression. Our studies with the mouse mammary tumor virus (MMTV) promoter in chromatin show significant levels of acetylation at promoter proximal and distal regions prior to transactivation. Upon activation with glucocorticoids or progestins, promoter proximal histones become deacetylated within the region of inducible nuclease hypersensitivity. The deacetylation lags behind the initiation of transcription, indicating a role in post-activation regulation. Our results indicate a novel mechanism by which target promoters are regulated by steroid receptors and chromatin modification machinery. Acetylation of lysines in histones H3 and H4 N-terminal tails is associated with transcriptional activation and deacetylation with repression. Our studies with the mouse mammary tumor virus (MMTV) promoter in chromatin show significant levels of acetylation at promoter proximal and distal regions prior to transactivation. Upon activation with glucocorticoids or progestins, promoter proximal histones become deacetylated within the region of inducible nuclease hypersensitivity. The deacetylation lags behind the initiation of transcription, indicating a role in post-activation regulation. Our results indicate a novel mechanism by which target promoters are regulated by steroid receptors and chromatin modification machinery. histone deacetylase mouse mammary tumor virus long terminal repeat estrogen receptor glucocorticoid receptor progesterone receptor chromatin immunoprecipitation fetal bovine serum polymerase chain reaction hormone response element dexamethasone nucleosome The association of histone acetylation with transactivation, and deacetylation with repression, was first suggested in 1964 by Allfreyet al. (1Allfrey V. Faulkner R.M. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Scopus (1712) Google Scholar). Such associations are now well documented in numerous studies (2Wolffe A.P. Guschin D. J. Struct. Biol. 2000; 129: 102-122Crossref PubMed Scopus (290) Google Scholar, 3Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1535) Google Scholar, 4Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2365) Google Scholar), including several with steroid hormone-regulated promoters (5Chen H., J., L.R. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 6DiRenzo J. Shang Y. Phelan M. Sif S. Meyers M. Kingston R.E. Brown M. Mol. Cell. Biol. 2000; 20: 7541-7549Crossref PubMed Scopus (192) Google Scholar, 7Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar). Acetylation of specific lysine residues in histone N-terminal tails decreases their net positive charge, which has been proposed to cause an electrostatic repulsion between the histones and the negatively charged phosphate backbone of DNA (8Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar) that results in a more open chromatin conformation. There are, however, data that conflict with this simple model. Mutskov et al. (9Mutskov V. Gerber D. Angelov D. Ausio J. Workman J. Dimitrov S. Mol. Cell. Biol. 1998; 18: 6293-6304Crossref PubMed Scopus (118) Google Scholar) have shown that hyperacetylated and hypoacetylated histone tails can associate almost equally well with DNA in chromatin at physiological salt concentrations. Mizuguchi et al. (10Mizuguchi G. Vassilev A. Tsukiyama T. Nakatani Y. Wu C. J. Biol. Chem. 2001; 276: 14773-14783Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) show that hyperacetylation of histones alone does not stimulate transcription from the adenovirus E4 promoter. An alternative model proposes that the pattern and nature of histone tail modification provides a “code” that can be recognized by specific factors that then associate with the tail, thereby determining the functional consequence of the histone modification (11Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6498) Google Scholar). The two models are not mutually exclusive.Circumstantial evidence suggests that transactivation of some promoters is associated with deacetylation. Treatment with histone deacetylase (HDAC)1 inhibitors, which results in hyperacetylation of histones, can decrease rather than increase transactivation at some promoters or result in no change at others (12Van Lint C. Emiliani S. Verdin E. Gene Expr. 1996; 5: 245-253PubMed Google Scholar). The HDAC inhibitor sodium butyrate inhibits transactivation of the ovalbumin promoter by estrogen receptors (ERs) (13McKnight G.S. Hager L. Palmiter R.D. Cell. 1980; 22: 469-477Abstract Full Text PDF PubMed Scopus (91) Google Scholar), and of the tyrosine aminotransferase promoter (14Plesko M. Hargrove J.L. Granner D.K. Chalkley R. J. Biol. Chem. 1983; 258: 13738-13744Abstract Full Text PDF PubMed Google Scholar) and the MMTV-LTR by glucocorticoid receptors (GRs) (15Bresnick E.H. John S. Berard D.S. LeFebvre P. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3977-3981Crossref PubMed Scopus (114) Google Scholar). Furthermore, Deckert and Struhl (16Deckert J. Struhl K. Mol. Cell. Biol. 2001; 21: 2726-2735Crossref PubMed Scopus (177) Google Scholar) have recently shown that in Saccharomyces cerevisiae both acetylated and deacetylated histones H3 and H4 can be associated with transcriptionally active promoters.Our results demonstrate that at the MMTV-LTR there is a significant level of basal acetylation at the promoter when it is inactive that decreases during hormone activation. This differs from what has generally been found at mammalian gene promoters characterized thus far (7Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar, 17Cheung P., G., T.K. Cheung W.L. Sassone-Corsi P. Denu J.M. Allis C.D. Mol. Cell. 2000; 5: 905-915Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar), but it has been described at some yeast promoters (Refs. 16Deckert J. Struhl K. Mol. Cell. Biol. 2001; 21: 2726-2735Crossref PubMed Scopus (177) Google Scholar,18Reinke H. Gregory P.D. Horz W. Mol. Cell. 2001; 7: 529-538Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, and others). The experiments we describe significantly advance our understanding of the role of deacetylation in transcriptional regulation at the MMTV-LTR. The results suggest a mechanism by which steroid hormone-targeted promoter activity is regulated by the acetylation state of histones in addition to other proteins found at the promoter in response to stimuli that induce transactivation.RESULTSTo better understand the mechanisms that underlie chromatin remodeling and transactivation by steroid hormone receptors, we investigated the acetylation status of histones H3 and H4 in the MMTV promoter in response to steroid hormone receptor activation. The MMTV-LTR has six well defined positioned nucleosome families, designated nucleosomes (Nuc) A–F. NucA overlaps the TATA region and the transcription start site, and NucF is the farthest 5′ to the transcription start site (−1 kilobase) (Fig.1A) (22Fragoso G. John S. Roberts M.S. Hager G.L. Genes Dev. 1995; 9: 1933-1947Crossref PubMed Scopus (155) Google Scholar, 23Fragoso G. Hager G.L. Methods Companion Methods Enzymol. 1997; 11: 246-252Crossref Scopus (33) Google Scholar). Four GR binding sites or hormone response elements (HREs) are located in the NucB region (Fig. 1 A). A nuclease hypersensitive site develops across NucB in response to glucocorticoid treatment, which indicates chromatin reorganization or remodeling at this nucleosome (24Payvar F. DeFranco D. Firestone G.L. Edgar B. Wrange O. Okret S. Gustafsson J.A. Yamamoto K.R. Cell. 1983; 35: 381-392Abstract Full Text PDF PubMed Scopus (437) Google Scholar, 25Richard-Foy H. Hager G. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (448) Google Scholar, 26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar). Just 5′ to NucB there are two HREs in the NucC region, and nuclease hypersensitivity extends into this nucleosome (26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar, 27Fletcher T.M. Ryu B.-W. Baumann C.T. Warren B.S. Fragoso G. John S. Hager G.L. Mol. Cell. Biol. 2000; 20: 6466-6475Crossref PubMed Scopus (79) Google Scholar). Nuclease hypersensitive sites in promoters are generally associated with histone acetylation and with transcriptionally active promoters (28Hebbes T.R. Thorne A.W. Crane-Robinson C. EMBO J. 1988; 7: 1395-1402Crossref PubMed Scopus (701) Google Scholar, 29Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (481) Google Scholar, 30Urnov F.D. Wolffe A.P. Mol. Endocrinol. 2001; 15: 1-16Crossref PubMed Scopus (47) Google Scholar).We determined the acetylation level of histones H3 and H4 at NucB and NucF, located inside and outside the hypersensitive site, respectively (25Richard-Foy H. Hager G. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (448) Google Scholar). A difference in the acetylation status of histones at these two nucleosomes can indicate whether any observed changes are associated with ATP-dependent chromatin remodeling. The mouse adenocarcinoma cell line 1470.2 was treated with 100 nm Dex for 1 h. Nuclei were isolated and subjected to micrococcal nuclease digestion. Acetylation status was determined using ChIPs assays with antibodies to acetylated lysines in H3 (Lys-9 and -14) and H4 (Lys-5, -8, -12, and -16). There is a significant level of basal acetylation of histone N-terminal tails H3 and H4 at both NucB and NucF prior to treatment with Dex (Fig. 1 B, −Dex). Dex treatment results in a decrease in H3 and H4 acetylation at NucB to about 50% of that seen in the untreated cells (Fig. 1, C andD). In striking contrast, at both H3 and H4 acetylation of NucF is not decreased but is somewhat increased by hormone treatment, indicating that deacetylation is associated with NucB and the nuclease hypersensitive site. It is unlikely that the decrease in acetylation is due to loss or sliding of NucB, as it was shown that GR-induced chromatin remodeling at the MMTV promoter does not involve either of these processes (26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar).To understand the kinetic relationship between transcription and deacetylation, a time course of both was carried out (Fig.2). Treatment of cells with Dex for as little as 15 min results in some deacetylation at NucB at histones H3 and H4; however, transcription has already reached its maximum. Deacetylation reaches its maximum at the 50% level after 30 min of treatment and persists after transcription decreases. By 15 min of treatment, histone acetylation at NucF is somewhat higher than that observed in untreated cells, as shown in Fig. 1, and does not change significantly at any time point (data not shown). These results indicate that deacetylation at NucB is associated with hormone-induce transcription but lags behind transcriptional activation, as deacetylation is still decreasing when transcription is maximal.Figure 2Time course of transcription and deacetylation at the MMTV-LTR. Nuclear run-on transcription assays were done on nuclei isolated from 1470.2 cells treated for 0, 15, 30, and 60 min with 100 nm Dex. For ChIP assays, 1470.2 cells were treated with Dex for the same times prior to formaldehyde cross-linking and processed as described in Fig. 1 legend. Relative levels of acetylated H3 (shaded bars) and acetylated H4 (hatched bars) are indicated on the left y axis. Levels of MMTV transcription (broken line) are expressed as fold induction with basal transcription (0 min) set to a value of 1 and are indicated on the right y axis. n = 3–6 for the ChIP analysis and 3–5 for the run-on analysis; error bars represent S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The progesterone receptor (PR) is closely related to the GR, binds to the same HREs, and can activate transcription from the MMTV-LTR. Like GR, stably expressed PR is able to induce hypersensitivity at NucB (19Smith C.L. Wolford R.G. O'Neill T. Hager G.L. Mol. Endocrinol. 2000; 14: 956-971Crossref PubMed Scopus (24) Google Scholar). To determine whether the deacetylation at histones H3 and H4 is specific to the GR, cell line 3017.1(19), which expresses both GR and PR, was treated for 1 h with either Dex or the synthetic progestin R5020, and ChIP assays were done (Fig.3). Deacetylation is observed at both histones H3 and H4 when either GR or PR is activated in this cell line. We see very little change at NucF after hormone addition, as we observed in the 1470.2 cells. These results suggest that at the MMTV-LTR, related steroid hormone receptors regulate chromatin remodeling and transcription by a common mechanism that involves histone deacetylation.Figure 3PR can mediate deacetylation of histones H3 and H4 at the MMTV promoter. 3017.1 cells were treated for 1 h with either 100 nm Dex or 30 nm R5020. It was shown previously that R5020 does not activate the GR in these cells at the concentration used in these experiments (19Smith C.L. Wolford R.G. O'Neill T. Hager G.L. Mol. Endocrinol. 2000; 14: 956-971Crossref PubMed Scopus (24) Google Scholar). A, after hormone treatment, ChIP assays with antibodies to acetylated histones H3 and H4 and PCR analysis of various fractions were done as described for Fig. 1. Control represents cells treated with vehicle only and shows the basal level of histone acetylation. “None” is the no antibody control. B andC, statistical analysis of the results from four independent experiments. Values from each experiment were normalized and expressed as described in Fig. 1 legend. Error barsrepresent S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONHistone acetylation within the proximal MMTV promoter region is significant prior to hormone activation, decreases as maximally activated transcription proceeds, and persists after the decline in transcription begins. The relatively high level of basal histone acetylation may facilitate the rapid onset of steroid hormone receptor-activated transcription and nuclease hypersensitivity at the MMTV promoter. Miziguchi et al. (10Mizuguchi G. Vassilev A. Tsukiyama T. Nakatani Y. Wu C. J. Biol. Chem. 2001; 276: 14773-14783Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) report that histone hyperacetylation at an in vitro assembled chromatin template is not sufficient to cause increased transcription but synergistically facilitates transcription induced by the binding of an activator and the activity of the ATP-dependent chromatin remodeling factor, NURF (nucleosome remodeling factor). Additionally, maximal histone deacetylation at the MMTV promoter is achieved just prior to the decrease in transcription, which suggests that deacetylation plays a role in the down-regulation of activated transcription, consistent with the generally observed correlation between deacetylation and transcriptional repression. HDAC inhibitors can, however, decrease rather than increase transcription at the MMTV promoter (15Bresnick E.H. John S. Berard D.S. LeFebvre P. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3977-3981Crossref PubMed Scopus (114) Google Scholar) but not in a time-frame consistent with repression. 2C. L. Smith, unpublished observations.. It is thus possible that the HDAC inhibitors target a non-histone protein in which acetylation is inhibitory to transcriptional initiation. Acetylation of the non-histone coactivator protein ACTR (activator of thyroid and retinoic acid receptors) coincides with inhibition of ER-mediated transcription (5Chen H., J., L.R. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar).The dynamic pattern of histone acetylation we observed at the MMTV promoter is different from two other systems examined in kinetic detail. Experiments with the estrogen-responsive cathepsin D and the pS2 promoters (5Chen H., J., L.R. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 7Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar) show low levels of histone acetylation prior to treatment with estradiol, which then rise and reach a peak just prior to the maximum of transcription at 60 min. Elevated levels of acetylation then persist to some degree as transcription declines and increases again. Reinke et al. (18Reinke H. Gregory P.D. Horz W. Mol. Cell. 2001; 7: 529-538Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) describe a transient hyperacetylation at the PHO8 promoter in yeast that occurs prior to chromatin remodeling and transcriptional activation. However, unlike our observation at the MMTV-LTR, levels of acetylation did not drop below those observed prior to activation. The variety of acetylation patterns and the dynamics of timing observed in these promoter systems suggests that different genes utilize histone and non-histone protein acetylation in distinct ways to regulate transcriptional activity. The mechanisms by which acetylation functions in transcriptional regulation are likely more complex than simply electrostatic repulsion between acetylated histones and DNA, or a histone code that directs non-histone proteins to the promoter, and are not yet fully understood. The association of histone acetylation with transactivation, and deacetylation with repression, was first suggested in 1964 by Allfreyet al. (1Allfrey V. Faulkner R.M. Mirsky A.E. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 786-794Crossref PubMed Scopus (1712) Google Scholar). Such associations are now well documented in numerous studies (2Wolffe A.P. Guschin D. J. Struct. Biol. 2000; 129: 102-122Crossref PubMed Scopus (290) Google Scholar, 3Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1535) Google Scholar, 4Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2365) Google Scholar), including several with steroid hormone-regulated promoters (5Chen H., J., L.R. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 6DiRenzo J. Shang Y. Phelan M. Sif S. Meyers M. Kingston R.E. Brown M. Mol. Cell. Biol. 2000; 20: 7541-7549Crossref PubMed Scopus (192) Google Scholar, 7Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar). Acetylation of specific lysine residues in histone N-terminal tails decreases their net positive charge, which has been proposed to cause an electrostatic repulsion between the histones and the negatively charged phosphate backbone of DNA (8Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar) that results in a more open chromatin conformation. There are, however, data that conflict with this simple model. Mutskov et al. (9Mutskov V. Gerber D. Angelov D. Ausio J. Workman J. Dimitrov S. Mol. Cell. Biol. 1998; 18: 6293-6304Crossref PubMed Scopus (118) Google Scholar) have shown that hyperacetylated and hypoacetylated histone tails can associate almost equally well with DNA in chromatin at physiological salt concentrations. Mizuguchi et al. (10Mizuguchi G. Vassilev A. Tsukiyama T. Nakatani Y. Wu C. J. Biol. Chem. 2001; 276: 14773-14783Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) show that hyperacetylation of histones alone does not stimulate transcription from the adenovirus E4 promoter. An alternative model proposes that the pattern and nature of histone tail modification provides a “code” that can be recognized by specific factors that then associate with the tail, thereby determining the functional consequence of the histone modification (11Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6498) Google Scholar). The two models are not mutually exclusive. Circumstantial evidence suggests that transactivation of some promoters is associated with deacetylation. Treatment with histone deacetylase (HDAC)1 inhibitors, which results in hyperacetylation of histones, can decrease rather than increase transactivation at some promoters or result in no change at others (12Van Lint C. Emiliani S. Verdin E. Gene Expr. 1996; 5: 245-253PubMed Google Scholar). The HDAC inhibitor sodium butyrate inhibits transactivation of the ovalbumin promoter by estrogen receptors (ERs) (13McKnight G.S. Hager L. Palmiter R.D. Cell. 1980; 22: 469-477Abstract Full Text PDF PubMed Scopus (91) Google Scholar), and of the tyrosine aminotransferase promoter (14Plesko M. Hargrove J.L. Granner D.K. Chalkley R. J. Biol. Chem. 1983; 258: 13738-13744Abstract Full Text PDF PubMed Google Scholar) and the MMTV-LTR by glucocorticoid receptors (GRs) (15Bresnick E.H. John S. Berard D.S. LeFebvre P. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3977-3981Crossref PubMed Scopus (114) Google Scholar). Furthermore, Deckert and Struhl (16Deckert J. Struhl K. Mol. Cell. Biol. 2001; 21: 2726-2735Crossref PubMed Scopus (177) Google Scholar) have recently shown that in Saccharomyces cerevisiae both acetylated and deacetylated histones H3 and H4 can be associated with transcriptionally active promoters. Our results demonstrate that at the MMTV-LTR there is a significant level of basal acetylation at the promoter when it is inactive that decreases during hormone activation. This differs from what has generally been found at mammalian gene promoters characterized thus far (7Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar, 17Cheung P., G., T.K. Cheung W.L. Sassone-Corsi P. Denu J.M. Allis C.D. Mol. Cell. 2000; 5: 905-915Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar), but it has been described at some yeast promoters (Refs. 16Deckert J. Struhl K. Mol. Cell. Biol. 2001; 21: 2726-2735Crossref PubMed Scopus (177) Google Scholar,18Reinke H. Gregory P.D. Horz W. Mol. Cell. 2001; 7: 529-538Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, and others). The experiments we describe significantly advance our understanding of the role of deacetylation in transcriptional regulation at the MMTV-LTR. The results suggest a mechanism by which steroid hormone-targeted promoter activity is regulated by the acetylation state of histones in addition to other proteins found at the promoter in response to stimuli that induce transactivation. RESULTSTo better understand the mechanisms that underlie chromatin remodeling and transactivation by steroid hormone receptors, we investigated the acetylation status of histones H3 and H4 in the MMTV promoter in response to steroid hormone receptor activation. The MMTV-LTR has six well defined positioned nucleosome families, designated nucleosomes (Nuc) A–F. NucA overlaps the TATA region and the transcription start site, and NucF is the farthest 5′ to the transcription start site (−1 kilobase) (Fig.1A) (22Fragoso G. John S. Roberts M.S. Hager G.L. Genes Dev. 1995; 9: 1933-1947Crossref PubMed Scopus (155) Google Scholar, 23Fragoso G. Hager G.L. Methods Companion Methods Enzymol. 1997; 11: 246-252Crossref Scopus (33) Google Scholar). Four GR binding sites or hormone response elements (HREs) are located in the NucB region (Fig. 1 A). A nuclease hypersensitive site develops across NucB in response to glucocorticoid treatment, which indicates chromatin reorganization or remodeling at this nucleosome (24Payvar F. DeFranco D. Firestone G.L. Edgar B. Wrange O. Okret S. Gustafsson J.A. Yamamoto K.R. Cell. 1983; 35: 381-392Abstract Full Text PDF PubMed Scopus (437) Google Scholar, 25Richard-Foy H. Hager G. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (448) Google Scholar, 26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar). Just 5′ to NucB there are two HREs in the NucC region, and nuclease hypersensitivity extends into this nucleosome (26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar, 27Fletcher T.M. Ryu B.-W. Baumann C.T. Warren B.S. Fragoso G. John S. Hager G.L. Mol. Cell. Biol. 2000; 20: 6466-6475Crossref PubMed Scopus (79) Google Scholar). Nuclease hypersensitive sites in promoters are generally associated with histone acetylation and with transcriptionally active promoters (28Hebbes T.R. Thorne A.W. Crane-Robinson C. EMBO J. 1988; 7: 1395-1402Crossref PubMed Scopus (701) Google Scholar, 29Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (481) Google Scholar, 30Urnov F.D. Wolffe A.P. Mol. Endocrinol. 2001; 15: 1-16Crossref PubMed Scopus (47) Google Scholar).We determined the acetylation level of histones H3 and H4 at NucB and NucF, located inside and outside the hypersensitive site, respectively (25Richard-Foy H. Hager G. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (448) Google Scholar). A difference in the acetylation status of histones at these two nucleosomes can indicate whether any observed changes are associated with ATP-dependent chromatin remodeling. The mouse adenocarcinoma cell line 1470.2 was treated with 100 nm Dex for 1 h. Nuclei were isolated and subjected to micrococcal nuclease digestion. Acetylation status was determined using ChIPs assays with antibodies to acetylated lysines in H3 (Lys-9 and -14) and H4 (Lys-5, -8, -12, and -16). There is a significant level of basal acetylation of histone N-terminal tails H3 and H4 at both NucB and NucF prior to treatment with Dex (Fig. 1 B, −Dex). Dex treatment results in a decrease in H3 and H4 acetylation at NucB to about 50% of that seen in the untreated cells (Fig. 1, C andD). In striking contrast, at both H3 and H4 acetylation of NucF is not decreased but is somewhat increased by hormone treatment, indicating that deacetylation is associated with NucB and the nuclease hypersensitive site. It is unlikely that the decrease in acetylation is due to loss or sliding of NucB, as it was shown that GR-induced chromatin remodeling at the MMTV promoter does not involve either of these processes (26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar).To understand the kinetic relationship between transcription and deacetylation, a time course of both was carried out (Fig.2). Treatment of cells with Dex for as little as 15 min results in some deacetylation at NucB at histones H3 and H4; however, transcription has already reached its maximum. Deacetylation reaches its maximum at the 50% level after 30 min of treatment and persists after transcription decreases. By 15 min of treatment, histone acetylation at NucF is somewhat higher than that observed in untreated cells, as shown in Fig. 1, and does not change significantly at any time point (data not shown). These results indicate that deacetylation at NucB is associated with hormone-induce transcription but lags behind transcriptional activation, as deacetylation is still decreasing when transcription is maximal.The progesterone receptor (PR) is closely related to the GR, binds to the same HREs, and can activate transcription from the MMTV-LTR. Like GR, stably expressed PR is able to induce hypersensitivity at NucB (19Smith C.L. Wolford R.G. O'Neill T. Hager G.L. Mol. Endocrinol. 2000; 14: 956-971Crossref PubMed Scopus (24) Google Scholar). To determine whether the deacetylation at histones H3 and H4 is specific to the GR, cell line 3017.1(19), which expresses both GR and PR, was treated for 1 h with either Dex or the synthetic progestin R5020, and ChIP assays were done (Fig.3). Deacetylation is observed at both histones H3 and H4 when either GR or PR is activated in this cell line. We see very little change at NucF after hormone addition, as we observed in the 1470.2 cells. These results suggest that at the MMTV-LTR, related steroid hormone receptors regulate chromatin remodeling and transcription by a common mechanism that involves histone deacetylation.Figure 3PR can mediate deacetylation of histones H3 and H4 at the MMTV promoter. 3017.1 cells were treated for 1 h with either 100 nm Dex or 30 nm R5020. It was shown previously that R5020 does not activate the GR in these cells at the concentration used in these experiments (19Smith C.L. Wolford R.G. O'Neill T. Hager G.L. Mol. Endocrinol. 2000; 14: 956-971Crossref PubMed Scopus (24) Google Scholar). A, after hormone treatment, ChIP assays with antibodies to acetylated histones H3 and H4 and PCR analysis of various fractions were done as described for Fig. 1. Control represents cells treated with vehicle only and shows the basal level of histone acetylation. “None” is the no antibody control. B andC, statistical analysis of the results from four independent experiments. Values from each experiment were normalized and expressed as described in Fig. 1 legend. Error barsrepresent S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To better understand the mechanisms that underlie chromatin remodeling and transactivation by steroid hormone receptors, we investigated the acetylation status of histones H3 and H4 in the MMTV promoter in response to steroid hormone receptor activation. The MMTV-LTR has six well defined positioned nucleosome families, designated nucleosomes (Nuc) A–F. NucA overlaps the TATA region and the transcription start site, and NucF is the farthest 5′ to the transcription start site (−1 kilobase) (Fig.1A) (22Fragoso G. John S. Roberts M.S. Hager G.L. Genes Dev. 1995; 9: 1933-1947Crossref PubMed Scopus (155) Google Scholar, 23Fragoso G. Hager G.L. Methods Companion Methods Enzymol. 1997; 11: 246-252Crossref Scopus (33) Google Scholar). Four GR binding sites or hormone response elements (HREs) are located in the NucB region (Fig. 1 A). A nuclease hypersensitive site develops across NucB in response to glucocorticoid treatment, which indicates chromatin reorganization or remodeling at this nucleosome (24Payvar F. DeFranco D. Firestone G.L. Edgar B. Wrange O. Okret S. Gustafsson J.A. Yamamoto K.R. Cell. 1983; 35: 381-392Abstract Full Text PDF PubMed Scopus (437) Google Scholar, 25Richard-Foy H. Hager G. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (448) Google Scholar, 26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar). Just 5′ to NucB there are two HREs in the NucC region, and nuclease hypersensitivity extends into this nucleosome (26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar, 27Fletcher T.M. Ryu B.-W. Baumann C.T. Warren B.S. Fragoso G. John S. Hager G.L. Mol. Cell. Biol. 2000; 20: 6466-6475Crossref PubMed Scopus (79) Google Scholar). Nuclease hypersensitive sites in promoters are generally associated with histone acetylation and with transcriptionally active promoters (28Hebbes T.R. Thorne A.W. Crane-Robinson C. EMBO J. 1988; 7: 1395-1402Crossref PubMed Scopus (701) Google Scholar, 29Hebbes T.R. Clayton A.L. Thorne A.W. Crane-Robinson C. EMBO J. 1994; 13: 1823-1830Crossref PubMed Scopus (481) Google Scholar, 30Urnov F.D. Wolffe A.P. Mol. Endocrinol. 2001; 15: 1-16Crossref PubMed Scopus (47) Google Scholar). We determined the acetylation level of histones H3 and H4 at NucB and NucF, located inside and outside the hypersensitive site, respectively (25Richard-Foy H. Hager G. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (448) Google Scholar). A difference in the acetylation status of histones at these two nucleosomes can indicate whether any observed changes are associated with ATP-dependent chromatin remodeling. The mouse adenocarcinoma cell line 1470.2 was treated with 100 nm Dex for 1 h. Nuclei were isolated and subjected to micrococcal nuclease digestion. Acetylation status was determined using ChIPs assays with antibodies to acetylated lysines in H3 (Lys-9 and -14) and H4 (Lys-5, -8, -12, and -16). There is a significant level of basal acetylation of histone N-terminal tails H3 and H4 at both NucB and NucF prior to treatment with Dex (Fig. 1 B, −Dex). Dex treatment results in a decrease in H3 and H4 acetylation at NucB to about 50% of that seen in the untreated cells (Fig. 1, C andD). In striking contrast, at both H3 and H4 acetylation of NucF is not decreased but is somewhat increased by hormone treatment, indicating that deacetylation is associated with NucB and the nuclease hypersensitive site. It is unlikely that the decrease in acetylation is due to loss or sliding of NucB, as it was shown that GR-induced chromatin remodeling at the MMTV promoter does not involve either of these processes (26Fragoso G. Pennie W.D. John S. Hager G.L. Mol. Cell. Biol. 1998; 18: 3633-3644Crossref PubMed Scopus (55) Google Scholar). To understand the kinetic relationship between transcription and deacetylation, a time course of both was carried out (Fig.2). Treatment of cells with Dex for as little as 15 min results in some deacetylation at NucB at histones H3 and H4; however, transcription has already reached its maximum. Deacetylation reaches its maximum at the 50% level after 30 min of treatment and persists after transcription decreases. By 15 min of treatment, histone acetylation at NucF is somewhat higher than that observed in untreated cells, as shown in Fig. 1, and does not change significantly at any time point (data not shown). These results indicate that deacetylation at NucB is associated with hormone-induce transcription but lags behind transcriptional activation, as deacetylation is still decreasing when transcription is maximal. The progesterone receptor (PR) is closely related to the GR, binds to the same HREs, and can activate transcription from the MMTV-LTR. Like GR, stably expressed PR is able to induce hypersensitivity at NucB (19Smith C.L. Wolford R.G. O'Neill T. Hager G.L. Mol. Endocrinol. 2000; 14: 956-971Crossref PubMed Scopus (24) Google Scholar). To determine whether the deacetylation at histones H3 and H4 is specific to the GR, cell line 3017.1(19), which expresses both GR and PR, was treated for 1 h with either Dex or the synthetic progestin R5020, and ChIP assays were done (Fig.3). Deacetylation is observed at both histones H3 and H4 when either GR or PR is activated in this cell line. We see very little change at NucF after hormone addition, as we observed in the 1470.2 cells. These results suggest that at the MMTV-LTR, related steroid hormone receptors regulate chromatin remodeling and transcription by a common mechanism that involves histone deacetylation. DISCUSSIONHistone acetylation within the proximal MMTV promoter region is significant prior to hormone activation, decreases as maximally activated transcription proceeds, and persists after the decline in transcription begins. The relatively high level of basal histone acetylation may facilitate the rapid onset of steroid hormone receptor-activated transcription and nuclease hypersensitivity at the MMTV promoter. Miziguchi et al. (10Mizuguchi G. Vassilev A. Tsukiyama T. Nakatani Y. Wu C. J. Biol. Chem. 2001; 276: 14773-14783Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) report that histone hyperacetylation at an in vitro assembled chromatin template is not sufficient to cause increased transcription but synergistically facilitates transcription induced by the binding of an activator and the activity of the ATP-dependent chromatin remodeling factor, NURF (nucleosome remodeling factor). Additionally, maximal histone deacetylation at the MMTV promoter is achieved just prior to the decrease in transcription, which suggests that deacetylation plays a role in the down-regulation of activated transcription, consistent with the generally observed correlation between deacetylation and transcriptional repression. HDAC inhibitors can, however, decrease rather than increase transcription at the MMTV promoter (15Bresnick E.H. John S. Berard D.S. LeFebvre P. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3977-3981Crossref PubMed Scopus (114) Google Scholar) but not in a time-frame consistent with repression. 2C. L. Smith, unpublished observations.. It is thus possible that the HDAC inhibitors target a non-histone protein in which acetylation is inhibitory to transcriptional initiation. Acetylation of the non-histone coactivator protein ACTR (activator of thyroid and retinoic acid receptors) coincides with inhibition of ER-mediated transcription (5Chen H., J., L.R. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar).The dynamic pattern of histone acetylation we observed at the MMTV promoter is different from two other systems examined in kinetic detail. Experiments with the estrogen-responsive cathepsin D and the pS2 promoters (5Chen H., J., L.R. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 7Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar) show low levels of histone acetylation prior to treatment with estradiol, which then rise and reach a peak just prior to the maximum of transcription at 60 min. Elevated levels of acetylation then persist to some degree as transcription declines and increases again. Reinke et al. (18Reinke H. Gregory P.D. Horz W. Mol. Cell. 2001; 7: 529-538Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) describe a transient hyperacetylation at the PHO8 promoter in yeast that occurs prior to chromatin remodeling and transcriptional activation. However, unlike our observation at the MMTV-LTR, levels of acetylation did not drop below those observed prior to activation. The variety of acetylation patterns and the dynamics of timing observed in these promoter systems suggests that different genes utilize histone and non-histone protein acetylation in distinct ways to regulate transcriptional activity. The mechanisms by which acetylation functions in transcriptional regulation are likely more complex than simply electrostatic repulsion between acetylated histones and DNA, or a histone code that directs non-histone proteins to the promoter, and are not yet fully understood. Histone acetylation within the proximal MMTV promoter region is significant prior to hormone activation, decreases as maximally activated transcription proceeds, and persists after the decline in transcription begins. The relatively high level of basal histone acetylation may facilitate the rapid onset of steroid hormone receptor-activated transcription and nuclease hypersensitivity at the MMTV promoter. Miziguchi et al. (10Mizuguchi G. Vassilev A. Tsukiyama T. Nakatani Y. Wu C. J. Biol. Chem. 2001; 276: 14773-14783Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) report that histone hyperacetylation at an in vitro assembled chromatin template is not sufficient to cause increased transcription but synergistically facilitates transcription induced by the binding of an activator and the activity of the ATP-dependent chromatin remodeling factor, NURF (nucleosome remodeling factor). Additionally, maximal histone deacetylation at the MMTV promoter is achieved just prior to the decrease in transcription, which suggests that deacetylation plays a role in the down-regulation of activated transcription, consistent with the generally observed correlation between deacetylation and transcriptional repression. HDAC inhibitors can, however, decrease rather than increase transcription at the MMTV promoter (15Bresnick E.H. John S. Berard D.S. LeFebvre P. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3977-3981Crossref PubMed Scopus (114) Google Scholar) but not in a time-frame consistent with repression. 2C. L. Smith, unpublished observations.. It is thus possible that the HDAC inhibitors target a non-histone protein in which acetylation is inhibitory to transcriptional initiation. Acetylation of the non-histone coactivator protein ACTR (activator of thyroid and retinoic acid receptors) coincides with inhibition of ER-mediated transcription (5Chen H., J., L.R. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). The dynamic pattern of histone acetylation we observed at the MMTV promoter is different from two other systems examined in kinetic detail. Experiments with the estrogen-responsive cathepsin D and the pS2 promoters (5Chen H., J., L.R. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 7Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1431) Google Scholar) show low levels of histone acetylation prior to treatment with estradiol, which then rise and reach a peak just prior to the maximum of transcription at 60 min. Elevated levels of acetylation then persist to some degree as transcription declines and increases again. Reinke et al. (18Reinke H. Gregory P.D. Horz W. Mol. Cell. 2001; 7: 529-538Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) describe a transient hyperacetylation at the PHO8 promoter in yeast that occurs prior to chromatin remodeling and transcriptional activation. However, unlike our observation at the MMTV-LTR, levels of acetylation did not drop below those observed prior to activation. The variety of acetylation patterns and the dynamics of timing observed in these promoter systems suggests that different genes utilize histone and non-histone protein acetylation in distinct ways to regulate transcriptional activity. The mechanisms by which acetylation functions in transcriptional regulation are likely more complex than simply electrostatic repulsion between acetylated histones and DNA, or a histone code that directs non-histone proteins to the promoter, and are not yet fully understood. We thank our readers, Allan Munck, Gordon Hager, Steve Fiering, and Aniko Naray Fejes-Toth, and members of our laboratories for critiques and helpful discussions in the course of the work. We also thank Gordon Hager for his encouragement and support.
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