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Department of Medicine, Johns Hopkins Bayview Medical Center (P.M.Y., X.X.), Johns Hopkins University, Baltimore, Maryland 21224; Molecular Regulation and Neuroendocrinology Section, Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (Y.L.), Bethesda, Maryland 20892; and Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School (J.D.F.), Piscataway, New Jersey 08854
Address all correspondence and requests for reprints to: Dr. Paul M. Yen, Endocrinology Division, Department of Medicine, Johns Hopkins Bayview Medical Center, 4940 Eastern Avenue, Room B114, Baltimore, Maryland 21224. E-mail: pyen3{at}jhmi.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase) in a rat pituitary cell line that expresses TRs. We found that TRß, several associated coactivators (steroid receptor coactivator-1, glucocorticoid receptor interacting protein-1, and TR-associated protein 220), and RNA Pol II were rapidly recruited to thyroid hormone response elements as early as 15 min after T3 addition. When the four target genes were compared, we observed differences in the types and temporal patterns of recruited coactivators and histone acetylation. Interestingly, the temporal pattern of RNA Pol II was similar for three genes studied. Our findings suggest that thyroid hormone-regulated target genes may have distinct patterns of coactivator recruitment and histone acetylation that may enable highly specific regulation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recent chromatin immunoprecipitation studies (ChIP) have suggested that nuclear hormone receptors may recruit coactivators in distinct temporal patterns (9, 10, 11, 12). Currently, there has been only limited study of the recruitment of histone acetylation and coactivator to the promoters of endogenous target genes regulated by T3. One of us previously (10) studied the temporal recruitment of TR, SRCs, p300, and TRAP/Mediator complex to the dio1 and sarcoplasmic endoplasmic reticulum calcium-adenosine triphosphatase (SERCA) promoters in HeLa and GH3 cells, respectively. After T3 treatment, TR recruits p160/SRC proteins and p300 initially, which then rapidly induce histone acetylation, before recruitment of the TRAP/Mediator complex.
Recently, we performed cDNA microarray analyses to examine the regulation of target genes in mice that were in hypothyroid, euthyroid, and hyperthyroid states (13). Our findings showed that positively regulated target genes were regulated in three major patterns, and some, but not all, target genes exhibited basal repression. These pattern differences suggest that recruitment of coactivators to the TREs of target genes may vary in a gene-specific manner. To address this question, we examined and compared histone H3 and H4 acetylation and coactivator recruitment to TREs of four endogenous target cells in rat pituitary GH3 cells. Our findings suggest that some T3-regulated target genes have distinct patterns of histone acetylation and coactivator recruitment, which may account for differential regulation of gene expression by hormone.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase (Cyp7), SERCA, and phosphoenolpyruvate carboxylase (PEPCK). In the absence of T3, minimal unliganded TRß bound to the Cyp7 and SERCA promoters, a small amount bound to the GH promoter, and a larger amount bound to the PEPCK promoter (Fig. 1
). T3 treatment increased TRß recruitment by 15 min for all TREs (Fig. 1). The recruitment appeared to be cyclical, because there was another increase at approximately 60 min for Cyp7, SERCA, and PEPCK, then a larger increase at 105 min for all four target genes. Of note, each successive cycle increased and reached an apparent maximum after 105 min. The TRß recruitment at later time points was measured over wider intervals so we were not able to determine precisely the maxima of cycles beyond 2 h. With the exception of some small differences, TRß recruitment was similar for each of the TREs (Fig. 1).
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). In particular, we examined SRC-1 and glucocorticoid receptor-interacting protein 1 (GRIP-1)/SRC-2, which are members of the SRC/p160 family of coactivators for nuclear hormone receptors (14), and TRAP220, which is the key subunit of the TRAP/Mediator complex that interacts with nuclear hormone receptors (7, 8). SRC-1 and GRIP-1 both were recruited in a ligand-dependent manner, and each had a similar temporal recruitment pattern among the four target genes. SRC-1 had initial maximal recruitment at 15–30 min, again at 60–90 min, and then again at 2 h for all four target genes. In general, the level of SRC-1 recruitment increased with each successive cycle. GRIP-1 had initial maximal recruitment at 45 min, again at 75–90 min, and then again at 3.5 h. Similar to SRC-1, recruitment increased with each successive cycle. It is noteworthy that as time after T3 addition increased, the time of maximal recruitment during cycles diverged for these two related coactivators, such that by 2 h, SRC-1 showed maximal recruitment, whereas GRIP-1 had minimal recruitment.
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Distinct Profiles of Coactivator Recruitment
We plotted and analyzed the coactivator recruitment patterns for the TREs of each target gene at 15-min intervals over a 2-h span (Figs. 2
and 3). It can be readily appreciated that the overall temporal patterns of coactivators were different for each target gene and thus formed a distinct profile for each target gene. These findings suggest the amount of coactivators recruited to TREs as well as their overall temporal pattern can vary significantly among different target genes.
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). AcH3 could be detected on the GH, Cyp7, and PEPCK TREs. AcH3 could be detected within 15 min after T3 treatment on the GH TRE and peaked between 45 and 75 min before declining. There were small increases in AcH3 on PEPCK and Cyp7 observed 15 and 45 min after T3 treatment, respectively, with a subsequent decline to baseline by 120 min. In Fig. 4A, AcH3 on GH TRE was determined from 30 cycles of amplification, and on Cyp7 and PEPCK it was determined from 35 cycles amplification, to best demonstrate histone acetylation patterns. Thus, band intensities cannot be compared directly among genes in this figure. However, AcH3 for GH was significantly higher than those for Cyp7 and PEPCK when all were amplified for 30 cycles (data not shown).
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RNA Pol II Recruitment
We next examined the recruitment of RNA Pol II to the various TREs by ChIP assay (Fig. 5). RNA Pol II could be detected near the TREs of GH, Cyp7, and PEPCK. RNA Pol II recruitment was rapid; it occurred 10 min after T3 addition on all three target genes. Although the magnitude of recruitment varied among the three target genes, there was an initial increase and decline, with a second increase occurring between 45 and 60 min before a subsequent decline. Thus, in contrast to our observations for coactivator recruitment and H3 and H4 acetylation, RNA Pol II had a similar overall pattern among the three target genes studied (Fig. 6).
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DISCUSSION |
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The recruitment of coactivators and RNA Pol II as well as acetylation of histones occurred rapidly after T3 addition (within 15 min). These findings suggest that in a T3-responsive cell, some target genes are primed for rapid transcriptional response soon after T3 addition. Indeed, given that TRAP220 is recruited to the GH and SERCA (9) promoters after RNA Pol II recruitment, our findings suggest that at these promoters, the TRAP/Mediator complex functions by activating a prebound RNA Pol II basal transcription complex. Although this is the case for the four target genes studied, recent time-course studies using microarrays to study T3-mediated transcription in human fibroblasts suggest that not all target genes respond at the same rate (15). It is likely that some target genes may be regulated directly and others indirectly (i.e. require the induction of other cofactors before target gene activation).
Each target gene had a different pattern of coactivator recruitment in terms of the particular coactivators recruited and their recruitment periods and maximas. TRAP220 could not be detected on the PEPCK promoter, suggesting that it may play a less prominent role in the transcription of this target gene. These findings raise the possibility that modulation of coactivator concentration by cell type or pharmacological/hormonal treatment may affect the transcriptional response of individual target genes. Additionally, the temporal patterns of TRAP220 recruitment and histone acetylation varied among the TREs, whereas SRC-1 and GRIP-1 recruitment patterns were fairly similar among the TREs. These findings suggest that target genes may have distinct profiles for temporal recruitment of coactivators. Furthermore, they are consistent with a multistep pathway of T3-induced gene activation in which different coactivator complexes facilitate different functional steps (6, 9). In a complementary manner, it recently has been shown that glucocorticoid and progesterone receptors preferentially recruit different SRC coactivators on the mouse mammary tumor virus promoter (16). Taken together with our findings, these data suggest that both the HRE sequence as well as the liganded receptor determine which coactivators can be recruited to the HREs of target genes.
We observed that recruitment of TRß, SRC-1, GRIP-1, and TRAP220 as well as histone acetylation near the TREs occurred rapidly after T3 addition. Previously, we used microarray analyses to examine the regulation of target genes in mice that were in hypothyroid, euthyroid, and hyperthyroid states (13). Our findings showed that positively regulated target genes were regulated in three major patterns, and some, but not all, target genes exhibited basal repression. The low recruitment of TRß on the Cyp7 and SERCA TREs in the absence of T3 raises the possibility that corepressors may be poorly recruited to these positively regulated target genes and thus may exhibit little basal repression in vivo. Additional studies are needed to determine whether this is indeed the case.
Acetylation of H4 appeared to be more dynamic and robust than that of H3. Although the functional consequences of these events are not known, they raise the possibility that T3-induced changes in H4 acetylation may contribute more to transcription than those in H3 acetylation. A recent study also showed an increase in H4 acetylation by T3 using an in vitro artificial chromatin template (17). Currently little is known about T3 regulation of H3 acetylation in target genes. Moreover, it is possible that changes in acetylation of other histones also may contribute to the regulation of these target genes. Recently, histone methylation has been shown to participate in chromatin changes that occur during hormone-induced transcription (18, 19). Our studies measured histone acetylation; however, acetylation/methylation of specific lysine residues in histones may be important for the requisite chromatin changes that facilitate T3-mediated transcription.
In three of the target genes, there were two peaks of H4 acetylation, and in one target gene, there was only one peak (Cyp7, PEPCK, and SERCA vs. GH). These data highlight the variability in the patterns of overall histone acetylation among target genes. In contrast, the RNA Pol II recruitment pattern was surprisingly similar among the three genes at least for the first 90 min after T3 addition. These latter findings suggest that differences in the coactivator and acetylated histone patterns can still lead to convergent patterns for RNA Pol II recruitment and, presumably, T3-mediated transcriptional activation of the target genes. The recruitment of RNA Pol II to the promoters probably serves as an indirect marker of active transcription. Ideally, the patterns of RNA Pol II and coactivator recruitment will need to be correlated with the transcriptional rate at various time points. Two previous studies showed increased T3-mediated transcription rates of GH in pituitary GC cells and SERCA in GH3 cells within 1–2 h after T3 addition (10, 20).
Recently, several groups used ChIP assays to show that liganded nuclear hormone receptors and associated coactivators were recruited to HREs in a cyclical manner (9, 10, 11, 12). In general, this recruitment correlated with histone acetylation and transcriptional activation, although differences in the length of the cycle period for recruitment of estrogen receptor and coactivators were observed (9, 11, 12). The reason for these different observations is not known; however, it is possible that differences in the frequency of CHIP assay measurements, immunoprecipitating antibodies, reaction conditions, or cell strains or, perhaps, cell cycle variation may contribute to these different observations. In this connection, we recently used Western blotting to show that the amount and expression pattern of endogenous TRß in GH3 cells varied during the cell cycle, which, in turn, could have an impact on transcriptional activity (21). Recent studies also have suggested the potential role of proteosomal degradation of nuclear hormone receptors and cofactors in the temporal patterns seen in ChIP assays (12, 22, 23). Finally, there may be other factors that may account for the observed temporal changes in coactivator recruitment. Although cyclical recruitment of TRs and coactivators to TREs (on and off DNA) is one explanation for our ChIP data, sliding models, in which TR/coactivator complexes move along DNA, or looping models, in which TR/coactivator complexes bind to distant TREs and connect to RNA Pol II with exchange of factors along DNA, are also possible (24). It also is possible that protein-protein interactions may induce conformational changes in TR coactivator complexes or mask epitopes and thereby alter antibody detection of protein/DNA complexes in ChIP assays.
Also complicating the issue of cofactor/DNA interactions on target genes are recent fluorescent recovery after photobleaching studies examining the binding of green fluorescent protein fusions of glucocorticoid receptor and GRIP-1 as well as estrogen receptor and SRC-1 to tandem enhancer element arrays. These studies show that they all have very rapid half-maximal times for fluorescent recovery (<10 sec), suggesting that there is a rapid exchange of nuclear receptors and their coactivators on HREs (1, 13, 20). Our current ChIP data and those of others show a cyclical recruitment of coactivator to HREs that occurs over minutes rather than seconds (25, 26, 27). It is possible that the fluorescent recovery after photobleaching data reflect events occurring in individual cells, whereas the ChIP data represent events taking place in a population of cells. In contrast, as mentioned above, it is possible that mechanisms other than mere cofactor/DNA association and dissociation may be measured by ChIP assays.
The present models of nuclear hormone receptor action show that two major coactivator complexes, SRCs and TRAP/Mediator, are involved in ligand-dependent transcription by nuclear hormone receptors (1, 2). For SRC complexes, components such as p300/CBP and p300/CBP-associated factor have been implicated in histone acetylation. Additionally, it is possible that acetylation of SRCs may play a role in exchanging components in the complexes (28). For TRAP/Mediator complexes, several subunits are homologous with yeast mediator proteins and thus may be involved in the recruitment and/or activation of RNA Pol II to the minimal promoter (7, 8). The SRCs and TRAP220 interact with similar subdomains within the ligand-binding domains of nuclear hormone receptors, so it has been proposed that there may be a reciprocal sequential recruitment of these cofactors (9, 10, 11, 12). We observed peak TR recruitment at 105 min, followed shortly thereafter by peak SRC-1 recruitment at 120 min, consistent with the idea that TRß may be the major recruitment partner for SRC-1. However, we did not observe this sequential pattern for coactivators, although it is possible that the exchange was too rapid to be detected by ChIP assays. Our findings thus raise the possibility of very rapid exchange or simultaneous recruitment of multiple coactivator complexes to the TREs.
In summary, we have observed distinct temporal profiles of TRß and coactivator recruitment and histone acetylation in the four endogenous target genes in a TH-responsive cell line. The precise nature of the coordinated recruitment of TR, coactivators, and histone acetylation resulting in RNA Pol II recruitment and transcription needs additional elucidation. However, our findings demonstrate that variable and distinct recruitment patterns of coactivator complexes occur on individual target genes regulated by thyroid hormone, which, in turn, may help determine the specificity and strength of the hormonal response of target genes.
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MATERIALS AND METHODS |
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ChIP Assay
Rat pituitary GH3 cells were grown to 90% confluence in phenol red-free DMEM supplemented with 10% charcoal-dextran-stripped fetal bovine serum (FBS) for at least 3 d. After the addition of 10–6 M T3 at various time intervals, ChIP assays were performed according to the manufacturer’s protocol (Upstate Biotechnology, Inc.) with some minor modifications. Briefly, cells were cross-linked by adding formaldehyde directly to the culture medium to a final concentration of 1% and were incubated for 10 min at room temperature. Cells then were washed twice with ice-cold PBS and collected by scraping in 5 ml ice-cold PBS containing a protease inhibitor mixture (Roche, Indianapolis, IN). Cells then were resuspended in 300 µl sodium dodecyl sulfate lysis buffer containing protease inhibitor mixture and incubated on ice for 10 min. The lysates were sonicated three times for 10 sec each time (Misonix, Farmingdale, NY) to reduce DNA fragment length to approximately 500-2000 bp and then were subjected to centrifugation for 10 min to remove debris. Supernatants were collected and diluted 5-fold in ChIP dilution buffer containing protease inhibitor mixture as described above. Two hundred microliters of this chromatin solution was saved to quantitate the amount of input DNA present in different samples before immunoprecipitation. The rest of the chromatin solutions were immunocleared with 60 µl salmon sperm DNA/protein A agarose slurry for 1 h at 4 C with agitation if rabbit antibodies were used. Alternatively, chromatin solutions were immunocleared with 60 µl protein A/G plus agarose slurry (Santa Cruz Biotechnology, Inc.) with 2 µg sheared salmon sperm DNA if antibodies were generated from species other than rabbit.
Immunoprecipitation was performed overnight with agitation at 4 C with specific antibodies. Immunoprecipitated chromatin complexes were collected with 60 µl salmon sperm DNA/protein A agarose slurry for 2 h at 4 C with agitation if antibodies were generated by rabbit. Immune chromatin complexes were collected with 60 µl protein A/G plus agarose slurry (Santa Cruz Biotechnology, Inc.) with 2 µg sheared salmon sperm DNA if antibodies were generated from species other than rabbit. Precipitates then were washed sequentially in low-salt immune complex wash buffer, high-salt immune complex wash buffer, and LiCl immune complex wash buffer for 3–5 min. Beads were washed twice in Tris-EDTA buffer and extracted twice with 1% sodium dodecyl sulfate and 0.1 M NaHCO3. Pooled eluates as well as saved chromatin solution for quantitating the amount of input DNA from above were heated at 65 C for 4 h in 0.2 M NaCl solution to reverse the formaldehyde cross-linking. After incubation at 45 C for 1 h of 10 µM EDTA in 40 µM Tris-HCl (pH 6.5) and 20 µg proteinase K, DNA fragments were purified with phenol/chloroform/isoamyl alcohol (25:24:1) and ethanol precipitation, and reconstituted in 50 µl H20. Five microliters of DNA solution was analyzed by PCR with 30–40 cycles of amplification. For PCR detection of the immunoprecipitated TRE promoter region, the following primers were used: rat GH: forward, 5'-CTTGGAGAGGCTCTGTTGCC; reverse, 5'-TGGGCTCGAGGGTGCTGGAC; rat CYP7: forward, 5'-AGT TCCATACAGTTCGCGTCC-3'; reverse, 5'-ACAGTGGGTCTGACTAGAC-3'; rat SERCA: forward, 5'-GGCTAAGGAGTGATGAGGCCTAAG-3'; reverse, 5'-ATCCACCTGCCTGTTAACCTGG-3'; and rat PEPCK: forward, 5'-CTTCTCATGACCTTTGGCCG-3'; reverse, 5'-CGGTTTGGAACTGACCTAACAC-3'. The promoter and surrounding regions amplified were: GH, –529 to 148; Cyp7, –515 to –66; SERCA, –349 to 148; and PEPCK, –458 to 128; they spanned previously characterized TREs (29, 30, 31, 32, 33).
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FOOTNOTES |
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1 Y.L. and X.X. are coequal first authors. 
Abbreviations: AcH3, Acetylated histone H3; CBP, cAMP response element binding protein-binding protein; ChIP, chromatin immunoprecipitation; Cyp7, cholesterol 7-hydroxylase; FBS, fetal bovine serum; GRIP, glucocorticoid receptor interacting protein 1; HRE, hormone response element; PEPCK, phosphoenolpyruvate carboxykinase; Pol II, polymerase II; SERCA, sarcoplasmic endoplasmic reticulum calcium-adenosine triphosphatase; SRC, steroid receptor coactivator; TR, thyroid hormone receptor; TRAP, TR-associated protein; TRE, thyroid hormone response element.
Received for publication February 24, 2005. Accepted for publication October 20, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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on responsive promoters is an integral feature of estrogen signaling. Mol Cell 11:695–707[CrossRef][Medline] is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators. J Biol Chem 276:35684–35692-coactivator complexes in living cells. Mol Cell Biol 21:4404–4412-hydroxylase gene promoter. J Biol Chem 269:17502–17507NURSA Molecule Pages Link:
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, Cyp7; 
, PEPCK; 
, GH.