Dynamic Histone Acetylation/Deacetylation with Progesterone ...

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Molecular Endocrinology 21(4):843–856 Copyright © 2007 by The Endocrine Society doi: 10.1210/me.2006-0244

Dynamic Histone Acetylation/Deacetylation with Progesterone Receptor-Mediated Transcription Sayura Aoyagi and Trevor K. Archer Chromatin and Gene Expression Section, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 Histone acetylation is a highly dynamic posttranslational modification that plays an important role in gene expression. Previous work showed that promoter histone deacetylation is accompanied by progesterone receptor (PR)-mediated activation of the mouse mammary tumor virus (MMTV) promoter. We investigated the role of this deacetylation and found that this histone deacetylation is not a singular event. In fact, histone acetylation at the MMTV promoter is highly dynamic, with an initial increase in acetylation followed by an eventual net deacetylation of histone H4. The timing of increase in acetylation of H4 coincides with the time at which PR, RNA polymerase II, and histone acetyltransferases cAMP response element-binding protein (CREB)-binding protein and p300 are recruited to the MMTV promoter. The timing in which histone H4 deacetylation occurs (after PR and RNA polymerase II recruitment) and the limited effect that

trichostatin A and small interfering RNA knockdown of histone deacetylase (HDAC)3 have on MMTV transcription suggests that this deacetylation activity is not required for the initiation of PRmediated transcription. Interestingly, two HDACs, HDAC1 and HDAC3, are already present at the MMTV before transcription activation. HDAC association at the MMTV promoter fluctuates during the hormone treatment. In particular, HDAC3 is temporarily undetected at the MMTV promoter within minutes after hormone treatment when the histone H4 acetylation increases but returns to the promoter near the time when histone acetylation levels start to decline. These results demonstrate the dynamic nature of coactivator/corepressor-promoter association and histone modifications such as acetylation during a transcription activation event. (Molecular Endocrinology 21: 843–856, 2007)

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ture and allow subsequent recruitment of additional transcription factors as well as the members of the basal transcriptional machinery for transcription activation (2–5). Histone proteins are also targets of extensive posttranslational modifications such as acetylation, phosphorylation, ADP ribosylation, methylation, and ubiquitination (6–9). These modifications are thought to contribute to the changes in histone-histone and histone-DNA interactions that could lead to modulation of chromatin structure (10, 11). These modifications can also act as signals for recruitment and binding platforms for other chromatin-modifying factors and complexes that lead to overall changes in chromatin architecture (12, 13). In particular, histone acetylation has been studied extensively in the context of gene regulation. Histone acetylation is highly dynamic and occurs on lysine residues mainly within the N-terminal tail domains of histone proteins. Acetylation of positively charged lysine residues could alter the histone-DNA interactions, creating a more open chromatin architecture (14–16). Histone acetylation is catalyzed by the enzymatic activities of histone acetyltransferases (HATs) and removed by the actions of histone deacetylases (HDACs). The dynamic interplay between the two opposing activities is thought to regulate cellular histone acetylation levels as well as at local promoter and gene

UCLEAR RECEPTORS (NRs), such as the glucocorticoid and progesterone receptors (GRs and PRs), represent a large family of transcription factors that play an important role in growth, development, reproduction, homeostasis, and metabolism (1, 2). NRs respond to signaling ligands such as steroid hormones to regulate transcription of target genes by binding to their cognate hormone response elements within chromatin. Ligand-bound receptors recruit coactivators such as members of the p160 family of coactivators (steroid receptor coactivator-1, 2, and 3), cAMP response elementbinding protein (CREB)-binding protein (CBP), p300 as well as ATP-dependent chromatin remodeling complex, SWI/SNF (mating type switching defective/sucrose nonfermenting), to modify the promoter chromatin architecFirst Published Online January 16, 2007 Abbreviations: CAT, Chloramphenicol acetyltransferase; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; CREB, cAMP response element-binding protein; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor; HAT, histone acetyltransferase; HDAC, histone deacetylase; MMTV, mouse mammary tumor virus; NR, nuclear receptor; Pol II, polymerase II; PR, progesterone receptor; siRNA, small interfering RNA; TSA, trichostatin A. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 843

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level (17, 18). The majority of the literature has correlated histone acetylation with gene activation, and histone deacetylation with gene repression (19–21). Indeed, many of the transcription coactivators that are recruited to target genes by transcription activators such as NRs, which include factors such as CBP/p300, steroid receptor coactivator-1, pCAF, and TATA binding protein-associated factorII250, contain intrinsic histone acetyltransferase (HAT) activity, whereas many of the transcription corepressors complexes contain subunits with HDAC activities such as nucleosome remodeling and deacetylation/Mi-2, mSin3a, and nuclear receptor corepressor/ silencing mediator of retinoid and thyroid hormone receptor complexes (22–32). In addition acetylated lysine residues may act as signals for recruitment of ancillary chromatin-modifying factors as well as act as binding sites for proteins such as acetyltransferase p300, which contains bromo domains that recognize acetylated lysine residues (12, 13, 34–36). The mouse mammary tumor virus promoter (MMTV) has been particularly useful in evaluating the effects of chromatin remodeling with transcription activity mediated by NRs such as GR and PR (4, 37). MMTV promoter assumes a well-defined chromatin structure when stably integrated into the host genome. The promoter organizes into six phased nucleosomes termed A–F (38, 39). The A and B nucleosomes that are closest to the transcription start sites harbor the GR and PR binding site and sites of binding for other transcription factors that are necessary for efficient transcription (37, 39–41). Upon hormone treatment, the nucleosome B region becomes hypersenstitive to restriction enzymes (38). MMTV promoter stably integrated into T47D human breast cancer cells that express endogenous PR, but lack GR (T47D/2963.1 cells), adopts a chromatin structure that is constitutively hypersensitive to restriction enzymes over the nucleosome B region with hormone-independent binding of factors such as nuclear factor 1 (42). The 2963.1 cells contain approximately 10 copies of the MMTV-long terminal repeat per cell (42). Using the 2963.1 cells, previous work has demonstrated that histone H4 acetylation level decreased upon PR-mediated activation of MMTV in the nucleosome B and A region (43). A similar decrease in histone acetylation has been observed for PRand GR-mediated transcription of MMTV in mouse mammary adenocarcinoma cells (44, 45). To investigate the role of deacetylation of histone H4 in PR-mediated transcription, hormone treatment time course chromatin immunoprecipitation (ChIP) experiments were performed to correlate the timing of H4 deacetylation with other transcription activation events occurring at the MMTV promoter. The results of the time course ChIP experiments show that the acetylation status of histone H4 is highly dynamic where histone H4 undergoes an initial increase in acetylation, followed by the decrease in histone acetylation. Time course ChIP experiments also revealed that recruitment of PR, CBP, p300, and RNA polymerase II (Pol II), and the loss of histone deacetylases (HDACs) 1 and 3,

Aoyagi and Archer • Histone Acetylation and Transcription

which are already associated with the MMTV before hormone treatment, coincides with the time of increase in histone H4 acetylation, whereas the timing of HDAC reassociation at the MMTV promoter is coincident with the deacetylation of histone H4. Taken together, our results highlight the dynamic nature of histone posttranslational modifications such as acetylation and coactivator/corepressors association during a transcription activation event.

RESULTS Deacetylation of Histone H4 Lysine 5 and 8 upon PR-Mediated MMTV Transcription Previous work using 2963.1 cells demonstrated that histone H4 acetylation at nucleosomes B and A decreases upon PR-mediated activation of the stably integrated MMTV promoter with a chloramphenicol acetyltransferase (CAT) reporter (43). When these cells are treated with synthetic progesterone R5020, the activation of the MMTV promoter can be assessed by the expression of the CAT gene as monitored by the CAT assay or RT-PCR (supplemental Fig. 1 published as supplemental data on The Endocrine Societys Journals Online web site at http:// mend.endojournals.org and Fig. 6, respectively). To characterize the histone H4 deacetylation activity associated with PR-mediated transcription of MMTV, ChIP experiments were performed in 2963.1 cells using antibodies against acetylated lysine 5, 8, 12, and 16 of H4 before and after 1 h treatment with R5020 to identify the specific lysine residues that were deacetylated. The analysis of the ChIP experiments were performed by real-time PCR with primers specific to nucleosome F, B, a, and b regions of the MMTV promoter as indicated (Fig. 1A). Before hormone treatment, acetylation levels at each of the four lysine residues are relatively higher past the transcription start site (nucleosome a region) compared with those in the promoter (nucleosome F and B) (Fig. 1, B–E). When the 2963.1 cells were treated with R5020 for 1 h, decreases in acetylation levels of lysine 5 and 8 of histone H4 were observed localized to the region near the transcription start site (nucleosome B and a), compared with relatively little change in acetylation status at the nucleosome F and b region (Fig. 1, B and C). In contrast, the acetylation levels at lysines 12 and 16 of histone H4 remain relatively unchanged or show only small changes upon R5020 treatment, suggesting that hormone treatment led to site-specific deacetylation near the transcription start site (Fig. 1, D and E). Time Course of Changes in Acetylation of Histone H4 Lysine 5 and Recruitment of PR and RNA Pol II at MMTV To investigate the role histone H4 site-specific deacetylation may play in the MMTV activation process, 2963.1 cells were treated with R5020 for various times (as indicated in Fig. 2, x-axis), followed by a ChIP


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Fig. 1. PR-Dependent Activation of MMTV Transcription Is Coupled with Deacetylation of Lysine 5 and 8 of Histone H4 near the Transcription Start Site T47D/2963.1 cells were treated with ethanol (EtOH)(⫺R5020) or 10 nM R5020 for 60 min. The ChIP assay was performed using antibodies against lysine-specific acetylated H4 as indicated and nonspecific IgG (N.S. IgG) as background control. A, Primers used in the real-time PCR analysis. B–E, The graphs represent quantitation of the real-time PCR results of the ChIP assays using antibodies against indicated acetylated lysine residues of histone H4. The areas along the MMTV promoter with respect to the transcription start site probed by PCR analysis are represented on the x-axis. The error bars represent the SEM.

assay with antibody against acetylated lysine 5 of histone H4, the lysine residue that showed a decrease in acetylation upon 60 min of R5020 treatment (Fig. 1B). Interestingly, the time course ChIP experiment showed that before deacetylation, histone H4 undergoes an initial increase in acetylation that occurs within 5–15 min of R5020 treatment. This is followed by deacetylation starting at around 30 min and a net loss of acetylation at 60 min of R5020 treatment. This is most evident at the nucleosome a region. Similar increases in acetylation of H4 occurred at nucleosome B and b regions, followed by decline in the acetylation level (Fig. 2).

To assess how the timing of initial increase and the subsequent decrease in acetylation of lysine 5 of H4 correlate with other events that occur at the MMTV promoter, similar time course ChIP experiments were conducted to monitor the timing of PR and RNA Pol II recruitment upon hormone treatment. Both PR and RNA Pol II are recruited to the MMTV promoter (nucleosome B) and reporter (nucleosome a and b), respectively, within 5–15 min of R5020 treatment (Fig. 3, A and B), coinciding with the time at which acetylation of histone H4 lysine 5 increases (Fig. 2). As expected, very little association of PR and RNA Pol II were observed within the reporter (nucleosome b) and nucleo-

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Fig. 2. PR-Mediated MMTV Transcription Is Associated with an Initial Transient Increase in Acetylation, Followed by Deacetylation of Lysine 5 of Histone H4 T47D/2963.1 cells were treated with 10 nM R5020 for indicated times (x-axis). The ChIP assay was performed using antibodies against acetylated lysine 5 of histone H4 and nonspecific IgG (N.S. IgG) as background control. The graphs represent quantitation of the real-time PCR results. The areas along the MMTV promoter probed by PCR analysis are indicated above the graphs. The error bars represent the SEM.

some F region, respectively, upon hormone treatment. Unlike acetylation of lysine 5 of histone H4, which starts to decline past 30 min of hormone treatment, both PR and RNA Pol II remain bound to the MMTV at 60 min after R5020 treatment (Fig. 2). We did not observe the cyclic association of the receptor and RNA Pol II with the MMTV promoter as described for estrogen receptor (ER) regulation of the pS2 gene. Perhaps this is because transcription was not synchronized with the use of ␣-amanitin before hormone treatment in our experiments thereby measuring the time course of PR and Pol II association of a more heterogenous population of cells (46). Timing of Recruitment of HDACs and HATs to the MMTV Promoter upon Hormone Treatment Histone acetylation is a highly dynamic posttranslational modification that is catalyzed by HATs and removed by HDACs. In the presence of hormone, PR has been shown to interact with p300 and CBP and


recruit the HAT enzymatic activities to its target promoters (47, 48). Therefore, the timing of recruitment of CBP and p300 was monitored to determine whether it correlates with the increase in histone H4 acetylation. 2963.1 cells were treated with R5020 for various times as indicated, and ChIP assay was performed with antibodies against p300 and CBP (Fig. 4, A and B, respectively). The ChIP samples were analyzed by real-time PCR using primers specific to the MMTV regions as indicated in the figure legends and the coding region of a house-keeping gene glyceraldehyde-3phosphate dehydrogenase (GAPDH), which was used as a negative control. The time-course ChIP assay indicates that both p300 and CBP were recruited to the MMTV promoter within 5–15 min of hormone treatment (Fig. 4, A and B), which coincides with the timing of increase in acetylation of lysine 5 of histone H4 (Fig. 2). This is in contrast to CBP and p300 association at the GAPDH-coding region where the ChIP signals are negligible or very limited, demonstrating that what is observed at the MMTV promoter is not a systematic experimental fluctuation that may result within an experiment. Whereas the acetylation level of lysine 5 of histone H4 starts to decrease around 30 min after R5020 treatment, resulting in the net loss of acetylation by 60 min, both p300 and CBP remain bound to MMTV over the 60-min hormone treatment (Fig. 4, A and B). To monitor the time point at which the HDACs are recruited to the promoter after hormone treatment to cause the decrease in histone H4 acetylation, time course ChIP experiments were conducted using antibodies against HDAC1 and HDAC3, two HDACs commonly associated with NR activity (4, 49). Surprisingly, both HDAC1 and HDAC3 are already present at the MMTV before R5020 treatment (Fig. 5, A and B, time 0). This is not a general phenomenon because GAPDH has negligible levels of HDAC1 and HDAC3 association. The HDAC association at the MMTV promoter fluctuates during the 60-min R5020 treatment time course. In particular, HDAC3 is temporarily undetectable from the MMTV promoter after R5020 treatment (⬃5 min), which coincides with the timing of increased histone H4 lysine 5 acetylation, but returns to the promoter close to the time when histone acetylation levels start to decline (30–60 min) (Figs. 5B and 2). These fluctuations in HDAC1 and HDAC3 association at the MMTV promoter during the 60-min R5020 time course are not due to changes in the cellular levels of these proteins as demonstrated by the Western blot analysis (Fig. 5C). It is also possible that the apparent loss of HDAC3 from the MMTV promoter at 5 min of hormone treatment is due to the masking of the epitope by the incoming components of transcription machinery. However, this is unlikely because we observe the recruitment of RNA Pol II, p300, and CBP and, most likely, along with it, the components of the transcription machinery at 5 min of hormone treatment, which then remain at the promoter during the 60-min hormone time course. If the loss of HDAC3 signal observed from the MMTV is due to the epitope



Fig. 3. PR and RNA Pol II Are Recruited to the MMTV Promoter upon R5020 Treatment and Remain Associated with the Promoter over the 60-Min Hormone Treatment Time Course T47D/2963.1 cells were treated with 10 nM R5020 for indicated times (x-axis). The ChIP assay was performed using antibodies against either PR (panel A) or RNA Pol II (panel B). Nonspecific IgG (N.S. IgG) was used as background control. The graphs represent quantitation of the real-time PCR results. The areas along the MMTV promoter probed by PCR analysis are indicated above the graphs. Real-time PCR analysis of nucleosome b (for panel A) and nucleosome F (for panel B) were performed as negative control. The error bars represent the SEM.

masking by the transcription machinery, one would expect it to remain undetectable during the 60-min hormone treatment time course. Histone H4 Deacetylation Is Not Required for the Activation of MMTV Transcription The timing in which histone H4 deacetylation occurs (after PR and RNA Pol II recruitment) suggests that this deacetylation activity is not required for the activation of PR-mediated transcription of MMTV. To test this idea, the 2963.1 cells were first treated with a HDAC inhibitor TSA for 30 min before and during R5020 treatment for 4 h. The trichostatin A (TSA) treatment was kept at a relatively short time of 30 min and hormone treatment was limited to 4 h to circumvent the pleiotropic effects downstream of histone deacetylase inhibition such as changes in cofactor

levels and apoptosis (43, 50, 51). Treatment of 2963.1 cells with 100 or 500 ng/ml of TSA for 30 min induced increase in the acetylation level of cellular histone H4 as indicated by the Western blot of acid-extracted histones from nuclei (Fig. 6A, left panel) and remained hyperacetylated over the 4-h R5020 treatment (TSA still present) (Fig. 6A, right panel). ChIP experiments performed with antibodies against acetylated lysine 5 of histone H4 demonstrated that treatment with TSA caused a slight decline in acetylation at nucleosome a, which is consistent with previous observations (43, 44). However, 30 min TSA pretreatment not only prevented the deacetylation of histone H4 that occurs after 1 h of R5020 at MMTV but led to a slight increase in acetylation, consistent with the presence of HATs CBP and p300 (Fig. 6B). To assess whether histone H4 deacetylation is required for the activation of MMTV activity, 2963.1



Fig. 4. p300 and CBP Are Recruited to the MMTV Promoter within 5–15 Min after Hormone Treatment and Remains Associated with the Promoter over the 60-Min R5020 Treatment Time Course T47D/2963.1 cells were treated with EtOH (⫺R5020) or 10 nM R5020 for indicated times (x-axis). The ChIP assay was performed using antibodies against either (A) p300 or (B) CBP. Nonspecific IgG (N.S. IgG) was used as background control. The graphs represent quantitation of the real-time PCR results. The areas along the MMTV promoter probed by PCR analysis are indicated above the graphs. Real-time PCR analysis of the GAPDH gene was performed as negative control. The error bars represent the SEM.

cells were pretreated with 100 or 500 ng/ml of TSA followed by treatment with R5020 for 4 h. RNA was harvested, RT-PCRs performed and analyzed by real-time PCR using primers specific for the CAT reporter that is under the control of the MMTV promoter. The RT-PCR experiments show that pretreatment with TSA, which inhibits the deacetylation of histone H4 that occurs after 1 h of R5020 treatment, does not inhibit the activation of MMTV but, in fact, leads to a slight increase in transcript levels (Fig. 6C). Given that TSA is not a specific inhibitor of HDACs 1 and 3 but a general inhibitor of class I and II HDACs and may have consequences on a variety of biological processes commonly associated with the usage of inhibitors (52), we specifically targeted HDAC3 for knockdown by the use of small interfering RNA (siRNA) because HDAC3 showed closer correlation to histone H4 acetylation status upon hormone treatment than

HDAC1. The Western blot demonstrates that siRNA treatment leads to efficient knockdown of HDAC3 compared with treatment of cells with lamin siRNA control (Fig. 7A). After knockdown of HDAC3 by siRNA, the 2963.1 cells were treated with R5020 for 4 h, and transcription driven by the MMTV promoter was assessed by RT-PCR. As shown in Fig. 7B, knockdown of HDAC3 leads to an increase in MMTVCAT reporter transcript (Fig. 7B). The same trend holds true for some of the endogenous genes CCAAT enhancer binding protein-␤, human zincfinger transcription factor, and FK506 binding protein 5 that are known to be regulated by PR (53, 54); all show an increase in transcript levels upon HDAC3 knockdown (Fig. 7C). These experiments decouple the observed deacetylation of histone H4 from the activation of MMTV transcription mediated by the PR but suggest a role for the regulation of levels of transcripts generated after initiation of transcription.



Fig. 5. HDAC1 and HDAC3 Are Already Present at the MMTV Promoter before R5020 Treatment and Show Dynamic Changes in Association at the Promoter over the 60-Min R5020 Treatment Time Course T47D/2963.1 cells were treated with 10 nM R5020 for indicated times (x-axis). The ChIP assay was performed using antibodies against either (A) HDAC1 or (B) HDAC3. Nonspecific IgG (N.S. IgG) was used as background control. The graphs represent quantitation of the real-time PCR results. The areas along the MMTV promoter probed by PCR analysis are indicated above the graphs. Real-time PCR analysis of the GAPDH gene was performed as negative control. The error bars represent the SEM. C, Cellular HDAC1 and HDAC3 levels were evaluated by Western blot analysis of whole-cell extracts prepared from cells treated with R5020 for indicated times. Antibody against ␣-tubulin was used as loading control.

DISCUSSION Histone acetylation is a highly dynamic posttranslational modification that is catalyzed by HATs and removed by HDACs. The balance of the two opposing activities is thought to determine the overall level of acetylation status of histones. At the local promoter and gene level, HATs and HDACs are recruited by activators/repressors in the context of coactivators and corepressors, respectively, to regulate the transcriptional output of target genes (17, 18). Previous work

demonstrated that PR-mediated transcription of MMTV in T47D/2963.1 human breast cancer cells is associated with histone deacetylation after 1 h of hormone treatment (43). Because histone deacetylation is generally correlated with transcription repression, we initiated a detailed characterization of this deacetylation event. We analyzed the changes in acetylation of specific lysine residues K5, K8, K12, and K16 on the histone H4 Nterminal tail domain after 1 h of hormone treatment. The deacetylation occurred primarily at lysine residues 5 and 8 of histone H4 near the transcription start site whereas



Fig. 6. Histone Deacetylase Inhibitor TSA Does Not Inhibit PR-Mediated MMTV Transcription Activation A, T47D/2963.1 cells were treated with indicated amounts of TSA for 30 min. Histones were acid extracted from isolated nuclei. The histone H4 acetylation levels were analyzed by Western blot analysis with the antibodies indicated on the left. The Western blots presented are representatives of two independent experiments each. B, T47D/2963.1 cells were treated with or without 500 ng/ml TSA for 30 min, followed by EtOH or 10 nM R5020 for 1 h. ChIP assays were performed using antibodies against acetylated lysine 5 of histone H4. The ChIP assay was analyzed by real-time PCR using primers specific to the nucleosome a region of the MMTV. The error bars represent the SEM. C, T47D/2963.1 cells were treated with indicated amounts of TSA for 30 min followed by 10 nM R5020 or EtOH (⫺R5020) treatment for 4 h. Total RNA was harvested and analyzed by real-time RT-PCR with primers specific for MMTV or GAPDH as control. The level of MMTV transcripts as determined by real-time PCR was normalized to that of GAPDH, and the value for untreated control (⫺TSA, ⫺R5020) was set to 1. The error bars represent the SEM.

the acetylation levels at lysines 12 and 16 remained unchanged. The acetylation levels of all four lysine residues were relatively high near the transcription start site (nucleosome a) compared with promoter regions (nucleosome B and F) (Fig. 1, B–E). This is consistent with observations made in yeast where acetylation of histones H3 and H4 were found to be enriched near the 5⬘-end of coding regions using a genome-wide mapping technique (55). In addition, perhaps the level of acetylation past the transcription site reflects the open nature of the MMTV promoter in this cell line. Previous characterization of the MMTV promoter in 2963.1 cells revealed that, although the promoter is hormone inducible, there is some basal transcriptional activity along with constitutive binding of factors such as NF-1, as determined by footprinting assays (42). Interestingly, when we sought to determine the timing in which histone H4 deacetylation occurs after

hormone treatment, the results indicated that histone acetylation level at the MMTV during transcription activation is much more dynamic than a single deacetylation event observed at 1 h after hormone treatment. Time course hormone treatment between 0 and 60 min followed by ChIP assay for acetylated lysine 5 of histone H4 revealed that MMTV activation is accompanied by the initial increase in acetylation, within 5–15 min of hormone treatment (Fig. 2). This is followed by the decrease in acetylation over the rest of the 60-min hormone treatment time course. As a followup to these observations, it would be of interest to correlate the kinetics of MMTV transcription with the dynamic changes in levels of histone acetylation. The increase in the histone acetylation level coincided with PR and RNA Pol II association at the promoter (Fig. 3). PR has been shown to interact with HATs p300 and CBP in a ligand-dependent manner



Fig. 7. siRNA Knockdown of HDAC3 Leads to an Increase in PR-Mediated Transcription 2963.1 cells were transfected with siRNA against lamin A/C control or HDAC3 for 72 h. The cells were then treated with 10 nM R5020 or EtOH (⫺R5020) for 4 h. A, Cellular levels of HDAC3 and lamin A/C were determined by Western blot analysis of whole-cell extracts derived from siRNA and hormone-treated cells. Antibody against ␤-actin was used as loading control. B, Total RNA harvested from siRNA and hormone-treated cells was analyzed by real-time RT-PCR with primers specific for MMTV or GAPDH as control. The level of MMTV transcripts as determined by real-time PCR was normalized to that of GAPDH, and the value for untreated control (lamin siRNA, ⫺R5020) was set to 1. The error bars represent the SEM. C, Total RNA harvested from siRNA and hormone-treated cells was analyzed by real-time RT-PCR with primers specific for the indicated PR-regulated genes or GAPDH as control. The level of transcripts as determined by real-time PCR was normalized to that of GAPDH, and the value for untreated control (⫺lamin siRNA, ⫺R5020) was set to 1. The error bars represent the SEM. CEBP/␤, CCAAT enhancer binding protein ␤.

and recruit the HAT activities to target genes (47, 48). In accordance with this idea, our work has demonstrated that CBP and p300 are recruited to the MMTV promoter at the same time as PR (Fig. 4). The kinetics of PR recruitment to the MMTV promoter is consistent with previous studies performed in the same parental cell line (T47D) where PR was shown to be recruited at the MMTV within 20 min of hormone treatment (the earliest time point tested), and remained at the promoter over the 60 min of hormone treatment time course (48). Next, we performed ChIP experiments to determine the time point at which HDAC activities were recruited to the MMTV that leads to the deacetylation of histone H4 between 30–60 min of hormone treatment. We monitored the association of HDAC1 and HDAC3, two HDACs that are commonly associated with NR activities (4, 49). Surprisingly, we found

that both HDAC1 and HDAC3 were already present at the MMTV before hormone treatment, in comparison with GAPDH, which has nearly undetectable levels of associating HDAC1 and HDAC3 (Fig. 5). Because histone acetylation levels are governed by the balancing act of HAT and HDAC activities, it is possible that the HDACs present at the promoter before hormone treatment are representatives of untargeted HDACs that are in place to maintain a basal level of histone acetylation. Given that the level of acetylation of histones near the transcription start site is relatively high compared with regions more upstream, such as nucleosome F, it is likely that there are untargeted HATs associated near this region that, together with HDAC1 and HDAC3, maintain a steady state level of histone acetylation. Because our ChIP experiments show little or no association of these HATs at the MMTV before

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hormone treatment, it is unlikely to be that of CBP and p300 but, rather, another yet to be identified HAT such as PCAF. When the 2963.1 cells were treated with hormone, within about 5 min HDAC1 and HDAC3 were undetectable from the MMTV, but between 15–30 min of hormone treatment both HDAC1 and HDAC3 were present at the MMTV, which is particularly evident at nucleosome a. The temporary loss of detection of HDAC1 and HDAC3 and the recruitment of CBP and p300 at this early time point (5–15 min) is reflected in the increase in acetylation of histone lysine 5 of histone H4. The return of HDAC1 and HDAC3 coincide with the time of decrease in acetylation of H4 (Figs. 5 and 2) as evidenced by the overlap in timing between the start of decline in histone acetylation (15–30 min of hormone treatment) and the return of HDAC3 (15 min) to the promoter. There is a delay in histone deacetylation after HDAC recruitment; however, this may be attributed to the competing activities of HATs p300 and CBP that are also present at the promoter. Our data suggest that the HAT activity is eventually overtaken by the HDAC activity, as demonstrated by the eventual decrease in acetylation of histone H4 (Fig. 2). The changes in the relative activities of the HAT and HDAC activities may be modulated by events such as posttranslational modifications, e.g. phosphorylation and acetylation of HDAC1 and/or HDAC3, which have been shown to regulate their activities (56–58). Interestingly, recent work has shown that HDAC3, in the context of the nuclear receptor corepressor complex, preferentially deacetylates lysine residues 5 and 8 of histone H4 (59), which are the two lysine residues that undergo deacetylation at 60 min of hormone treatment (Fig. 1, B and C), supporting the role HDAC3 may play in the deacetylation of histone H4 during PR-mediated transcription of MMTV. The timing of histone H4 deacetylation that occurs after RNA Pol II recruitment suggests that histone deacetylation is not necessary for the initiation of PRmediated transcription. Therefore we predicted that inhibition of histone H4 deacetylation by the use of HDAC inhibitor TSA will not inhibit the activation of MMTV. 2963.1 cells were treated with TSA for 30 min followed by 4 h of hormone treatment and RT-PCR analysis of MMTV-CAT transcription. Inhibition of histone H4 deacetylation by the use of TSA led to a modest increase in mRNA generation and did not inhibit the PR-mediated MMTV activity as predicted, confirming that the deacetylase activity is not required for the activation of MMTV transcription (Fig. 6C). Our results are in contrast to past studies analyzing the effect of TSA and butyrate on MMTV activity where the use of these HDAC inhibitors at equivalent concentrations prevented MMTV activation (43, 60, 61). However it is important to note that in these studies the cells were exposed to HDAC inhibitors for a longer period of time (as long as 24 h) when many of the secondary effects of inhibiting HDAC activity, such as changes in expression levels of coactivators and in-


duction of apoptosis, have been shown to occur (43, 50, 51). Our study also differs from a previous study that examined the inhibition of GR-mediated MMTV activity by short time treatment of TSA (5–60 min) and attributed the TSA inhibition of MMTV to a mechanism that involves acetylation of nonhistone proteins that are required for basal transcription and inhibits not only GR-mediated MMTV activation, but the non-receptor-mediated basal level of transcription as well (44). These dissimilarities may be a result of the differences in the requirement for the activation of MMTV in c127i mouse mammary adenocarcinoma cells in contrast to T47D mammary cancer cell lines used in our assays. In addition, even though they are closely related steroid hormone receptors, GR-mediated, rather than PR-mediated, activation of MMTV may involve slightly different mechanisms. Indeed, the timing of histone deacetylation occurs at a faster time point (starting at around 5 min) after hormone treatment (dexamethasone) in their study, possibly indicating the differential role the deacetylation activity plays in the two experimental systems (44). A recent study has demonstrated that HDAC1 serves as a coactivator for GR-mediated activation of MMTV where hormone activation of GR leads to the acetylation of HDAC1, suppressing its deacetylase activity and allowing transcription to occur from the promoter (58). In addition, the authors have shown that siRNA knockdown of HDAC1 inhibits transcription from the MMTV promoter, demonstrating the requirement of the HDAC1 protein in GR-mediated activation of MMTV (58). In agreement with this work, in addition to HDAC3, we have observed HDAC1 association at the MMTV promoter in our system. However, the timing of HDAC3 association seems to correlate more tightly with acetylation status of lysine 5 of histone H4 in our studies. In addition to using TSA, general inhibitor of HDAC class I and II activity, we specifically knocked down the HDAC3 protein and examined its effects on PRmediated transcription. Both the transcripts from MMTV, as well as some of the endogenous PR-regulated genes, showed increase in mRNA production as determined by RT-PCR (Fig. 7). This suggests that perhaps, HDAC3, in addition to maintaining a specific promoter histone acetylation level before hormone treatment, plays a role in modulating the level of transcripts generated upon hormone treatment and/or required for steps further downstream of transcription initiation. For example, histone deacetylation maybe part of the process to attenuate transcription of MMTV, which may be an important process to return the promoter back to a state where it is poised for activation by additional hormonal signal. The ER-mediated transcription of the pS2 gene has been shown to involve recruitment of the nucleosome remodeling and deacetylation corepressor complex to presumably attenuate the cycle of productive transcription before commencement of subsequent cycles of transcription output, and it is possible that histone


deacetylation we have observed during PR-mediated transcription is part of a similar mechanism (46). In budding yeast, histone modifications such as acetylation have been reported to be transient and return to its original state as a way to terminate response to transcription stimulus and to return and maintain proper chromatin structure (62). Similar mechanisms can be envisioned to play a role in PR-mediated transcription. In addition, the deacetylation activity maybe targeted toward nonhistone proteins. There are numerous proteins that have been shown to be acetylated, including factors of the basal transcriptional machinery as well as coactivators such as activator of thyroid hormone receptor (also known as SRC3) (63– 65). Perhaps deacetylation of one or several of these factors is necessary for steps such as attenuation of transcription. Evaluating these various possibilities for the role of deacetylation activity observed during PRmediated transcription for both MMTV and endogenous genes will be a subject for future studies. Our results demonstrate that PR-mediated activation of MMTV in T47D/2963.1 cells is accompanied by dynamic changes in histone acetylation that is governed by the changes in HAT and HDAC association at the promoter. We did not observe the cyclic fluctuation of coactivators and basal transcription machinery association at the MMTV upon PR-mediated transcription that has been described for the ER-mediated transcription (46). This may reflect the mechanistic differences in ER- vs. PR-mediated transcription but may also be due to differences in the experimental protocols used in the two studies. In our study, transcription was not synchronized with the use of ␣-amanitin before hormone treatment, thereby measuring the time course of coactivator/corepressor recruitment within a more heterogeneous population of cells compared with studies performed for ER-mediated activation of the pS2 gene (46). Nevertheless, our studies demonstrate the dynamic changes in histone modifications and coactivators/corepressors during a transcriptional activation to be a common theme for both ER- and PR-mediated transcription, and perhaps for other NRs as well (46, 66–68). Understanding the precise coordination of recruitment and release of these cofactors is likely to be key in dissecting the mechanisms of transcriptional activator-mediated transcription.

MATERIALS AND METHODS Cell Culture 2963.1 cells were derived from human T47D breast cancer cells by stable transfection of the chimeric bovine papilloma virus-based construct pJ83d, carrying the MMTV long terminal repeat attached to the bacterial CAT gene (42). Cells were grown at 37 C with 5% CO2 in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) supplemented with 10 mM HEPES and 2 mM glutamine (Invitrogen).


ChIP Assay For lysine-specific acetylated histone H4 and PR ChIP assays, 2963.1 cells (2 ⫻ 106) were seeded in 10-cm diameter tissue culture plates and treated the next day with 10 nM R5020 for times indicated in the figure legends. The cells were then fixed with 1% formaldehyde at 37 C for 10 min. Cells were collected by centrifugation in PBS containing protease inhibitors. The ChIP assays were performed according to the Upstate Biotechnology (Charlottesville, VA) protocol with minor modifications. Immunoprecipitation of chromatin was performed overnight at 4 C with antibodies against acetylated lysine 5 (Abcam, Cambridge, MA), lysine 8 (Upstate), lysine 12 (Upstate), lysine 16 (Abcam), or PR (H-190, Santa Cruz Biotechnology, Santa Cruz, CA) or normal serum Ig (IgG) (Santa Cruz Biotechnology). For RNA Pol II, HDAC1, HDAC3, P300, and CBP ChIP assays, 2963.1 cells (1.2 ⫻ 107) were seeded in 15-cm diameter tissue culture plates and treated the next day with 10 nM R5020 for times indicated in the figure legends. The cells were then fixed with 1% formaldehyde at 37 C for 10 min. Cells were collected by centrifugation in PBS containing protease inhibitors. Nuclei were isolated as previously described (69) and subjected to the ChIP assay following the protocol from Upstate Biotechnology. Immunoprecipitation was performed overnight with antibodies against RNA Pol II (N-20), HDAC1 (H-51), HDAC3 (H-99), p300 (C-20), CBP (A-22), or IgG, all from Santa Cruz Biotechnology. For all ChIP assays, after immunoprecipitation, 60 ␮l of salmon sperm DNA-protein A agarose was added for 1 h at 4 C to capture the immune complexes. The agarose beads were washed, chromatin extracted, proteinDNA cross-links reversed, and proteins digested by proteinase K as indicated in the Upstate ChIP assay protocol. DNA was purified by QIAquick PCR purification kit (QIAGEN, Valencia, CA) and analyzed by real-time PCR analysis using Stratagene Mx3000p instrument and Stratagene SYBR green QPCR master mix (Stratagene, La Jolla, CA) with the following primers. Nucleosome F (5⬘-TTC GTG CTC GCA GGG CT-3⬘ and 5⬘-CCT ATT GGA TTG GTC TTA TTG G-3⬘); Nucleosome B (5⬘-GGT TAC AAA CTG TTC TTA AAA CGA GGA T-3⬘ and 5⬘-CAG AGC TCA GAT CAG AAC CTT TG-3⬘); Nucleosome a (5⬘-TCT GAG CTT GGC GAG ATT TTC-3⬘ and 5⬘-GAA CGG TCT GGT TAT AGG TAC ATT GA-3⬘); Nucleosome b (5⬘-TTC CAT GAG CAA ACT GAA ACG T-3⬘ and 5⬘-TGT AGA AAC TGC CGG AAA TCG T-3⬘); and GAPDH (5⬘-TCG GAG TCA ACG GAT TTG G-3⬘ and 5⬘-GGC AAC AAT ATC CAC TTT ACC AGA GT-3⬘). The data presented are average of three independent experiments with SEM indicated.

Isolation of Histones Subconfluent 2963.1 cells were treated with TSA (Sigma, St. Louis, MO) and hormone as indicated in the figure legends. Nuclei were isolated as described previously (33). Acid-soluble proteins were isolated from nuclei in 100 ␮l of 0.4 N H2SO4 at 4 C for 1 h. After centrifugation for 5 min at 14,000 rpm, histones were precipitated from the supernatant in 1 ml of acetone placed at ⫺20 C overnight. After centrifugation, the proteins were resuspended in 50 ␮l of H2O. Histone proteins (5 ␮g) were electrophoresed using 14% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes at 300 mA for 3 h at 4 C. The membranes were incubated with antibodies specific to unmodified histone H4 (Upstate), pan-acetylated histone H4 (Upstate), and acetylated lysine 5 (Abcam) of histone H4 as indicated in the figures. The data presented are representative of two independent experiments.

854



Western Blot Analysis Subconfluent 2963.1 cells were treated as indicated in the figure legends. The cells were lysed buffer X [100 mM Tris-Cl (pH 8.5), 250 mM NaCl, 1% (wt/vol) Nonidet P-40, and 1 mM EDTA] with protease inhibitor cocktail (Sigma). Proteins were electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene diflouride membranes (Invitrogen). The membranes were incubated with antibodies against HDAC1 (H-51), HDAC3 (H-99), Lamin A/C (H-110) (Santa Cruz Biotechnology), ␣-tubulin (Sigma), and ␤-actin (Abcam).

2. 3. 4. 5.

RNA Isolation and RT-PCR 2963.1 cells were seeded in six-well plates (5 ⫻ 105/well) and grown overnight. The cells were treated with or without TSA followed by 10 nM R5020 as indicated in the figure legends. For siRNA experiments, after 72 h of transfection, the cells were treated with 10 nM R5020 for 4 h. Total RNA was isolated using Trizol reagent following manufacturer protocol (Invitrogen). 2 ␮g of total RNA was used for the reverse transcriptase reaction according to First strand synthesis protocols (Invitrogen). PCR analysis was performed by realtime PCR with the following primers: MMTV-Nucleosome A (5⬘-AGT CCT AAC ATT CAC CTC TTG TGT GT-3⬘ and 5⬘ACC CTC TGG AAA GTG AAG GAT AAG T-3⬘) and GAPDH (sequence listed above); CCAAT enhancer binding protein-␤ (5⬘-CGT GCC CGC TGC AGT T-3⬘ and 5⬘-CTC GCA GTT TAG TGG TGG TAA GTC-3⬘); human zincfinger transcription factor (5⬘-CGC TCC ATT ACC AAG AGC TCA T-3⬘ and 5⬘-CGA TCG TCT TCC CCT CTT TG-3⬘); and FK506 binding protein 5 (5⬘-CTG CAG AGA TGT GGC ATT CAC T-3⬘ and 5⬘-TCC AGA GCT TTG TCA ATT CCA A-3⬘). After normalizing PR regulated gene transcript levels were normalized by that of GAPDH, the level of transcription in the absence of R5020 and TSA treatment or HDAC3 siRNA was set to 1. The data presented are average of three independent experiments with SEM indicated. HDAC3 siRNA Knockdown

6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17.

2963.1 cells were seeded in six-well plates (1 ⫻ 10 /well) and grown overnight. Lamin A/C control siRNA or HDAC3 siRNA (100 pmol per well) (Dharmacon, Lafayette, CO) was transfected using Lipofectamine 2000 reagent (Invitrogen) as per manufacturer’s protocol for 72 h. 5

18. 19.

Acknowledgments 20. We thank Drs. Serena Dudek, Bonnie Deroo, and Pratibha Hebbar for critical reviews of the manuscript.

Received June 12, 2006. Accepted January 8, 2007. Address requests for reprints and all correspondence to: Dr. Trevor K. Archer, Chromatin and Gene Expression Section, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, 111 Alexander Drive, P.O. Box 12233 (MD D4-01), Research Triangle Park, North Carolina 27709. E-mail: [email protected]. This research was supported by the Intramural Research Program of the National Institutes of Health and National Institute of Environmental Health Sciences.

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OCTOBER 9–11, 2007—San Miguel Island, Azores 8th Workshop on Resistance to Thyroid Hormone and Action A forum for the presentation and discussion of the most recent basic and clinical achievements related to Resistance to Thyroid Hormone and Action Ponta Delgada, San Miguel, Azores/Portugal Tel: ⫹351-296209430 Fax: ⫹351-296209448 E-mail: [email protected] Website: www.8thIWRTH.org