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Originally published In Press as doi:10.1074/jbc.M605774200 on September 8, 2006

J. Biol. Chem., Vol. 281, Issue 49, 37345-37352, December 8, 2006
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GATA-1-mediated Transcriptional Repression Yields Persistent Transcription Factor IIB-Chromatin Complexes*Formula

Melissa L. Martowicz1, Jeffrey A. Grass, and Emery H. Bresnick2

From the Molecular and Cellular Pharmacology Program, University of Wisconsin School of Medicine, Madison, Wisconsin 53706

Received for publication, June 16, 2006 , and in revised form, September 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The hematopoietic GATA factors GATA-1 and GATA-2, which have distinct and overlapping roles to regulate blood cell development, are reciprocally expressed during erythropoiesis. GATA-1 directly represses Gata2 transcription, and reduced GATA-2 synthesis promotes red blood cell development. Gata2 repression involves "GATA switches" in which GATA-1 displaces GATA-2 from Gata2 regulatory regions. We show that extragenic GATA switch sites occupied by GATA-2 associate with as much RNA polymerase II (Pol II) and basal transcription factors as present at the active Gata2 promoters. Pol II bound to GATA switch sites in the active locus was phosphorylated on serine 5 of the carboxyl-terminal domain, indicative of elongation competence. GATA-1-mediated displacement of GATA-2 from GATA switch sites reduced Pol II recruitment to all sites except the far upstream –77-kb region. Surprisingly, TFIIB occupancy persisted at most sites upon repression. These results indicate that GATA-2-bound extragenic regulatory elements recruit Pol II, GATA-1 binding expels Pol II, and despite the persistent TFIIB-chromatin complexes, Pol II recruitment is blocked.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
All blood cell types are derived from a common precursor, the hematopoietic stem cell. The process of hematopoiesis requires highly regulated transcriptional mechanisms, as relatively small changes in transcription factor levels can dictate distinct cell fates (1, 2). The requirement for stringent transcriptional regulation of the hematopoietic transcription factor Gata2 appears to be critical, as deregulated GATA-2 expression either blocks or promotes hematopoiesis (3–9).

During early hematopoiesis, GATA-2 expressed in multipotent hematopoietic precursors maintains proliferation and/or survival (10, 11). Gata2 null mice are embryonic lethal at embryonic days 10–11 due to anemia (10). GATA-2 is also expressed outside the hematopoietic system in the developing central nervous system, fetal liver, fetal heart, mast cells, megakaryocytes, and endothelial cells and is involved in pituitary and urogenital development (10–15). GATA-1 is less broadly expressed, being restricted to the erythroid, megakaryocytic, and eosinophil lineages, as well as the Sertoli cells in testis (16–20). GATA-1 is essential for erythropoiesis, megakaryocyte maturation, and eosinophil production (17, 18, 21, 22).

GATA-2 levels decline as GATA-1 levels rise during erythropoiesis (7, 21, 23, 24). GATA-1 represses Gata2 transcription by displacing GATA-2 from sites at –77, –3.9, –2.8, –1.8, and +9.5 kb relative to the hematopoietic-specific 1S Gata2 promoter (25–27). GATA-1-mediated displacement of GATA-2 or a "GATA switch" is tightly coupled to broad histone deacetylation of the locus, transcriptional repression from the two Gata2 promoters (1S and 1G), and loss of GATA-2 protein (25). DNase I hypersensitive sites (HSs)3 have been mapped to the –77, –3.9, –2.8, and –1.8-kb regions of the Gata2 locus. The GATA switch abrogates the strong –1.8-kb HS and reduces the intensity of the weak –2.8 HS (26). The GATA factor coregulator FOG-1 (28) is required for the GATA switch (29) and Gata2 repression (29, 30), but not for Gata2 transcription (29). FOG-1 interacts directly with the nucleosome remodeling and deacetylase chromatin remodeling complex, which is an important mediator of repression (31).

It is instructive to compare the structure/function of GATA factor complexes at the GATA-1-activated beta-globin locus (32, 33) and the GATA-1-repressed Gata2 locus. Pol II occupancy at the far upstream beta-globin locus control region (LCR) is restricted to the four HSs (33). Intergenic transcripts were detected in the vicinity of the HS-bound Pol II (33, 34), and the transcripts are abrogated by Pol II elongation blockade with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (33). GATA-1 increases Pol II occupancy at the HSs (32), and GATA-1 is crucial for Pol II recruitment to the betamajor promoter. Based on GATA-1-mediated Pol II recruitment to the beta-globin LCR and the finding that GATA-1-mediated repression of Gata2 is associated with reduced Pol II at the 1S and 1G promoters (25), we investigated whether Pol II is recruited and dynamically regulated at Gata2 HSs.

We describe the localization and differential regulation of Pol II at Gata2 HSs. GATA-1-mediated repression abrogated Pol II occupancy at all sites of the locus except the –77-kb region and, to a lesser degree, the 1G promoter. TFIIB occupancy persisted at most GATA switch sites upon repression, whereas TATA-binding protein (TBP) occupancy paralleled that of P-Ser-5 Pol II. These results support a model in which GATA-2-bound extragenic regulatory elements recruit Pol II and GATA-1-mediated displacement of GATA-2 instigates repression via generation of TFIIB-chromatin complexes that fail to support subsequent Pol II recruitment.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cell Culture—G1E-ER-GATA-1 cells (25, 29, 35), which stably express GATA-1 as a fusion to the human estrogen receptor ligand-binding domain (supplemental Fig. S1), were maintained in Iscove's modified Dulbecco's medium (Invitrogen) containing 2% antibiotic-antimycotic (Invitrogen), 2 units/ml erythropoietin, 120 nM monothioglycerol (Sigma), 0.6% conditioned medium from a Kit ligand-producing Chinese hamster ovary cell line, 15% fetal bovine serum (Invitrogen), and 1 µg/ml puromycin. NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (Biofluids) containing 10% calf serum and 1% antibiotic-antimycotic.

Antibodies—Rabbit polyclonal IgG anti-TBP (S-I1, sc-273), anti-TFIIB (C-18, sc-225), and anti-Pol II (N-20, sc-899) antibodies were from Santa Cruz Biotechnology., Inc. Mouse monoclonal IgM anti-RNA Polymerase II H14 (MMS-134R), which recognizes the phosphoserine 5 version of Pol II, was from Covance Research Products. AffiniPure (Jackson ImmunoResearch) rabbit anti-mouse IgM, µ chain-specific antibody was used as the secondary antibody for the Phospho-Ser-5 Pol II ChIP analysis. Rabbit preimmune serum and mouse ascites fluid (IgM, {lambda} M2521 from murine myeloma; Sigma) were controls.

RNA Isolation and Reverse Transcriptase PCR—RNA was prepared from the same cultures used for ChIP. Total RNA was purified with TRIzol (Invitrogen). cDNA was prepared by annealing RNA (1 µg) with 250 ng of a 5:1 mixture of random and oligo(dT) primers at 68 °C for 10 min. This was followed by incubation with 100 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) combined with 10 mM dithiothreitol (Invitrogen), 20 units of RNasin (Promega), and 0.5 mM dNTPs at 42 °C for 1 h. Reactions were diluted to a final volume of 150 µl and heat inactivated at 98 °C for 5 min. Real-time PCR reactions (15 µl) contained 2 µl of cDNA, 7.5 µl of SYBR Green PCR master mix (Applied Biosystems), and the appropriate primers. Product accumulation was monitored by SYBR Green fluorescence. Control reactions lacking reverse transcriptase yielded very low or no signals. Relative expression levels were determined from a standard curve of serial dilutions of G1E-ER-GATA-1 cDNA samples. Forward and reverse primers for real-time RT-PCR (5'-3'): glyceraldehyde-3-phosphate dehydrogenase, TGCCCCCATGTTTGTGATG and TGTGGTCATGAGCCCTTCC; GATA-2-exon3/4, GCAGAGAAGCAAGGCTCGC and CAGTTGACACACTCCCGGC.

Quantitative Chromatin Immunoprecipitation (ChIP) Assay—Real-time PCR quantitative ChIP analysis was conducted as described (36). Cells were cross-linked with 1% formaldehyde. In Figs. 1, 3, 4, and 5, cells were grown in medium containing 15% fetal bovine serum and treated with or without 1 µM tamoxifen for 48 h. In Fig. 2, cells were treated for 16 h with 0, 8, and 200 nM beta-estradiol; beta-estradiol and tamoxifen activate ER-GATA-1 similarly. Immunoprecipitated DNA was analyzed by real-time PCR (ABI Prism 7000; PE Applied Biosystems). Primers were designed by PRIMER EXPRESS 1.0 (PE Applied Biosystems) to amplify 50- to 150-bp amplicons and were based on GenBankTM accession number AB009272 [GenBank] and sequences in Ensembl (www.ensembl.org/Mus_musculus/geneview?geneENSMUSG00000015053). Samples from three or more independent ChIP experiments were analyzed. Product was measured by SYBR green fluorescence in 15-µl reactions with 1 µl of sample. The amount of product was determined relative to a standard curve of input chromatin. Dissociation curves showed that PCR reactions yielded homogeneous products.

Forward and Reverse Primers for Quantitative ChIP Assay—Primer sequences are included in supplemental Table S1.

Protein Analysis—Immunoprecipitations of endogenous TBP and TFIIB were performed using TrueBlot (eBioscience) to eliminate the IgG heavy chain, according to the manufacturer's instructions with minor modifications. Whole-cell lysates were prepared from 0.5 x 107 untreated G1E-ER-GATA-1 cells in 0.5 ml of Nonidet P-40 lysis buffer (150 mM NaCl, 1% Nonidet P-40, and 50 mM Tris, pH 8.0) containing 2 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 20 µg/ml leupeptin by incubating on ice for 5 min, douncing 20 times, incubating on ice for 5 min, and centrifuging at 13,000 x g for 30 min at 4 °C. Lysates (4 mg) were precleared with 50 µl of anti-rabbit IgG beads (eBioscience) for 30 min at 4 °C, followed by incubation with either 2 µg of anti-TBP or TFIIB antibody for 1 h at 4°C. Immune complexes were adsorbed to 50 µl of anti-rabbit IgG beads by incubating for 1 h at 4°C. Immune complexes were washed five times with lysis buffer. Samples were eluted in 100 µl of SDS sample buffer (50 mM Tris, pH 6.8, 50 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), and 20 µl of eluted material was resolved on a 10% SDS-PAGE with 10 µl of whole cell lysate (1 x 105 cells). Lysates were prepared by boiling 1 x 106 cells for 10 min in SDS sample buffer. Proteins were analyzed by Western blotting. Anti-TBP and anti-TFIIB were added at a dilution of 1:500 in 5% dry milk/TBST (150 mM NaCl, 0.1% Tween 20, and 10 mM Tris-HCl, pH 7.9). Rabbit IgG TrueBlot was added at a dilution of 1:1000 in 5% dry milk/TBST. Immunoreactivity was detected via chemoluminescence with ECL Plus (Amersham Biosciences).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Cell Type-specific Pol II Occupancy at Extragenic HSs of the Active Gata2 Locus Is Abrogated upon Repression—GATA switches at the –77, –3.9, –2.8, –1.8, and +9.5-kb regions of the Gata2 locus result in broad histone deacetylation of the locus and transcriptional repression (25–27). As Pol II occupies extragenic regulatory regions of thebeta-globin locus and GATA-1 increases Pol II occupancy at these sites (32, 33), it is possible that GATA factor-mediated Pol II recruitment to extragenic elements is a fundamental step in GATA factor-mediated transcriptional regulation. Thus, we tested whether Pol II localizes to the GATA switch sites of the endogenous Gata2 locus.

High resolution quantitative ChIP analysis was conducted to determine where Pol II resides at the Gata2 locus (Fig. 1A). Treatment of GATA-1-null G1E cells stably expressing conditionally active ER-GATA-1 (G1E-ER-GATA-1) with tamoxifen for 24 and 48 h strongly repressed Gata2 transcription from both 1S and 1G promoters (Fig. 1B). Pol II occupied the promoters, the open reading frame, and the four extragenic GATA switch sites (–77, –3.9, –2.8, and –1.8 kb) at the transcriptionally active Gata2 locus (Fig. 1C). Pol II occupancy overlapped with all previously mapped ER-GATA-1 and GATA-2 occupancy sites (27), except the –12-kb region, in which Pol II but no ER-GATA-1 occupancy was detected. The –12-kb region lacks known regulatory elements. To determine whether GATA switches regulate Pol II occupancy, occupancy was analyzed at the repressed locus 48 h post-tamoxifen treatment. Pol II occupancy was nearly abolished at the proximal GATA switch sites (–3.9, –2.8, and –1.8 kb), the 1S promoter, and the open reading frame. Analysis of Pol II occupancy at the –2.8-kb region and the 1S promoter with a distinct Pol II antibody yielded similar results (supplemental Fig. S2). However, Pol II occupancy persisted at the –77-kb region, and ~50% of the Pol II remained at the 1G promoter (Fig. 1D). The persistent Pol II at the –77-kb region post-ER-GATA-1 occupancy indicates that ER-GATA-1 occupancy is insufficient to displace Pol II from sites bound by ER-GATA-1. Taken together with the GATA-1 activity to recruit Pol II to the beta-globin locus, GATA-1 regulates Pol II occupancy in a context-dependent manner. Furthermore, the nearly quantitative loss of Pol II occupancy at the 1S promoter with partial retention of Pol II at the 1G promoter, despite repression of both promoters (Fig. 1B, data not shown), suggests the 1S and 1G promoters are differentially regulated. Both promoters are transcribed in erythroid cells, whereas the 1G promoter has broader activity in certain non-hematopoietic cell types (13, 37).


Figure 1
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  		FIGURE 1.
Dynamic regulation of Pol II occupancy at extragenic GATA switch sites of the endogenous Gata2 locus. A, murine Gata2 locus organization. B, real-time PCR quantitation of Gata2 mRNA levels in untreated and tamoxifen-treated (24 or 48 h) G1E-ER-GATA-1 cells. C and D, quantitative ChIP analysis of Pol II chromatin occupancy at the Gata2 locus in untreated (transcriptionally active) (C) and tamoxifen-treated (48 h) (transcriptionally repressed) (D) G1E-ER-GATA-1 cells. The coordinate +1 reflects the first nucleotide of the Gata2 1S exon. E and F, quantitative ChIP analysis of Pol II phosphorylated at Ser-5 (P-Ser-5) in untreated (E) and tamoxifen-treated (F) G1E-ER-GATA-1 cells. The asterisks in panel E define the external limits of the regions analyzed. Preimmune and ascites signals were analyzed with all primer sites in all experiments and averaged 0.001 and 0.002, respectively. G, Pol II and P-Ser-5 occupancy in untreated and tamoxifen-treated G1E-ER-GATA-1 cells at the RPII215 promoter. H, quantitative ChIP analysis of Pol II chromatin occupancy at the Gata2 locus (left panel) and RPII215 promoter (right panel) in 3T3 cells. All graphs represent data from at least three independent experiments (mean ± S.E.).

 


Figure 2
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  		FIGURE 2.
GATA switch site- and promoter-bound Pol II are similarly sensitive to changes in ER-GATA-1 activity. The murine Gata2 locus is depicted at the top. The gray circles, open bars, and gray bars represent hypersensitive sites, untranslated, and translated exons, respectively. Quantitative ChIP analysis of Pol II occupancy in untreated andbeta-estradiol-treated (8 and 200 nM, 16 h) G1E-ER-GATA-1 cells (mean ± S.E., at least three independent experiments).

 
Extragenic Pol II might localize to the promoters via long-range Pol II transfer (38) or tracking (39) or might generate regulatory transcripts (40). RNA transcripts are detected at beta-globin upstream HSs bound by Pol II (33, 34) and at abundant intergenic sites within genomes (41). To determine whether Pol II at Gata2 extragenic sites is transcriptionally competent, we tested whether extragenic Pol II is phosphorylated at Ser-5 of the CTD (RNA Pol II carboxyl-terminal domain). Serine 5 phosphorylation is a key step in the transition of stalled to elongating Pol II (42, 43). P-Ser-5 was measured by quantitative ChIP analysis with the H14 antibody (44) (Fig. 1E). The distribution of P-Ser-5 and total Pol II overlapped at all sites of the active locus except the –12-kb region, at which P-Ser-5 was absent. P-Ser-5 persisted at the –77-kb region and partially at the 1G promoter upon repression. As expected, P-Ser-5 was lost from exon 3 within the open reading frame (Fig. 1F). Pol II occupancy at the RPII215 promoter was unaffected by tamoxifen treatment, demonstrating the specificity of reduced Pol II occupancy at the Gata2 locus and indicating that reduced Pol II occupancy is not a consequence of cellular differentiation (Fig. 1G). The persistence of P-Ser-5 at the –77-kb site and partial retention at the 1G promoter suggested that Pol II remained in an elongation-competent form at restricted subregions of the repressed locus. Other examples in which P-Ser-5 occupancy precedes activation include paused Pol II at the 5'-end of the inactive hsp70 gene in Drosophila melanogaster (45) and at the mammalian c-myc locus (46–48). At the inactive peroxisome proliferator-activated receptor {gamma}2 locus, Pol II associated with the basal transcriptional machinery does not initiate transcription until the SWI/SNF complex is recruited (49).

If extragenic Pol II at the active Gata2 locus is functionally important, one would expect Pol II to be absent from the locus in cells that never express Gata2. We tested whether Pol II occupied the Gata2 locus in murine 3T3 fibroblasts, which do not express Gata2 (data not shown). Pol II did not occupy the –77, –3.9, –2.8, and –1.8-kb regions or the promoters (Fig. 1H), whereas Pol II occupied the constitutively active RPII215 promoter in 3T3 cells (Fig. 1H, right panel). Thus, Pol II occupancy at the Gata2 locus is cell-type specific.

A previous analysis of the beta-globin locus demonstrated that lower ER-GATA-1 activity is sufficient to occupy GATA motifs at the HSs of the LCR versus the betamajor promoter (50). As ER-GATA-1 occupancy at the LCR initiates factor recruitment and histone modifications, which are followed by ER-GATA-1 occupancy at the promoter and additional molecular events, the differential usage of GATA motifs appears to underlie the multistep activation mechanism. Similarly, Pol II loss at the GATA switch sites might not occur concomitantly, but rather Pol II at certain sites might be preferentially sensitive to ER-GATA-1. We tested whether Pol II at the HSs, sites of ER-GATA-1 occupancy, exhibits greater sensitivity to ER-GATA-1 versus Pol II at the promoters that are not occupied by ER-GATA-1. G1E-ER-GATA-1 cells were treated with 0, 8, or 200 nM beta-estradiol for 16 h. The concentration-dependent reductions in Pol II occupancy at the sites were similar, indicating that Pol II occupancy at GATA switch sites and sites lacking GATA factors is equally sensitive to ER-GATA-1 under the experimental conditions (Fig. 2). ER-GATA-1 activation resulted in slightly greater Pol II retention at the 1G promoter versus other sites, consistent with the partial Pol II retention at the repressed 1G promoter following a 48-h treatment with 1 µM tamoxifen (Fig. 1D).

GATA-1-mediated Repression Yields Persistent TFIIB-Chromatin Complexes—Although the basal transcription factors TFIID and TFIIB are crucial for recruiting Pol II to promoters (51–54), the role of these factors in recruiting intergenic Pol II is unknown. We tested whether intergenic Pol II colocalizes with TFIIB and the TFIID component TBP and, if so, whether Pol II and basal transcription factor occupancy are differentially regulated. Using a highly specific TFIIB antibody (Fig. 3A), quantitative ChIP analysis at the Gata2 locus in untreated G1E-ER-GATA-1 cells revealed TFIIB occupancy at extragenic sites at least as high if not higher than at the 1S and 1G promoters (Fig. 3B). TFIIB occupancy overlapped with P-Ser-5 occupancy and was not detected at numerous other Gata2 sites (Fig. 3B). The low enrichments at extragenic sites away from the promoters resembled the low enrichments at the inactive Necdin promoter (Fig. 3B, center panel), whereas the active RPII215 promoter yielded strong enrichments (Fig. 3B, right panel).

Because Pol II occupancy at multiple regions of the Gata2 locus is abrogated upon repression, we asked whether ER-GATA-1 binding to the chromatin similarly affects TFIIB occupancy. By contrast with Pol II, TFIIB occupancy was largely constant throughout the locus (compare left panels in Fig. 3, B and C) with one exception, the –1.8-kb region in which TFIIB occupancy was abrogated. Following the GATA switch, only low level ER-GATA-1 (and GATA-1) occupancy is detected at the –1.8-kb region, whereas considerably higher occupancy occurs at other GATA switch sites, and the –1.8-kb HS is lost (26). TFIIB occupancy at the Necdin and RPII215 promoters was unaffected by ER-GATA-1 (Fig. 3C, center and right panels). As expected from the lack of Pol II, TFIIB was not detected at Gata2 in 3T3 cells (Fig. 3D) but was detected at the RPII215 promoter (Fig. 3D, right panel).


Figure 3
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  		FIGURE 3.
TFIIB occupancy persists upon repression at most Gata2 locus sites. A, Western blot analysis of endogenous TFIIB in whole cell lysate (Input) from untreated G1E-ER-GATA-1 cells and after immunoprecipitation with preimmune (PI) or anti-TFIIB antibodies. B and C, quantitative ChIP analysis of TFIIB chromatin occupancy in untreated (transcriptionally active) (B) and tamoxifen-treated (48 h) (transcriptionally repressed) (C) G1E-ER-GATA-1 cells. The left, center, and right panels of B and C depict TFIIB occupancy at the Gata2 locus, Necdin promoter, and RPII215 promoter, respectively. D, quantitative ChIP analysis of TFIIB at the Gata2 locus (left panel) and the RPII215 promoter (right panel) in 3T3 cells. The graphs represent data from at least three independent experiments (mean ± S.E.).

 
The persistence of TFIIB occupancy at sites in which Pol II occupancy declines to an undetectable level raises the question of whether other basal transcription factors remain in the TFIIB-chromatin complex. As the interaction of TBP with TFIIB is important for Pol II recruitment (54), we tested whether TBP colocalized with Pol II and TFIIB. Quantitative ChIP analysis with an anti-TBP antibody validated by Western blotting (Fig. 4A) revealed TBP occupancy at the –77-kb region, the HSs, and the promoters of the active Gata2 locus in G1E-ER-GATA-1 cells (Fig. 4B, left panel). No TBP occupancy was detected at the inactive Necdin promoter (Fig. 4B, right panel). TBP occupancy persisted at the –77-kb region upon repression, remained ~2-fold greater than the control antibody at the 1G promoter, and was abrogated at other sites (Fig. 4C, left panel). The P-Ser-5 and TBP distributions within the active and repressed locus overlapped, both before and after repression. By contrast to ER-GATA-1-mediated Gata2 repression, ER-GATA-1-mediated beta-globin locus activation involved dynamic changes in TFIIB occupancy. ER-GATA-1 strongly induced TFIIB (Fig. 4D), TBP (Fig. 4E), and Pol II (Fig. 4F) occupancy at the betamajor promoter. Thus, despite persistent TFIIB occupancy upon Gata2 repression and the established TFIIB activity to interact directly with Pol II (52), apparently TFIIB lost the capacity to efficiently recruit Pol II following ER-GATA-1 occupancy. Although we are unaware of a scenario in which TFIIB complexes exist on chromatin in the absence of TBP, potential explanations for this result include: the failure to cross-link TBP to the repressed chromatin; direct TFIIB-DNA interactions, because TFIIB has sequence-specific DNA binding activity (55, 56); or TFIIB tethering to the chromatin via protein-protein interactions.


Figure 4
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  		FIGURE 4.
TBP colocalizes with Pol II phosphorylated at Ser-5 at the transcriptionally active and repressed Gata2 locus. A, Western blot analysis of endogenous TBP in whole cell lysate (Input) from untreated G1E-ER-GATA-1 cells and after immunoprecipitation with preimmune (PI) or anti-TBP antibodies. B and C, quantitative ChIP analysis of TBP chromatin occupancy at the Gata2 locus in untreated (transcriptionally active) (B) and tamoxifen-treated (48 h) (transcriptionally repressed) (C) G1E-ER-GATA-1 cells. The plots on the right of panels B and C depict TBP occupancy at the Necdin promoter. D–F, quantitative ChIP analysis of TFIIB (D), TBP (E), and Pol II (F) at the betamajor promoter in untreated and tamoxifen-treated (48 h) G1E-ER-GATA-1 cells. The graphs represent data from at least three independent experiments (mean ± S.E.).

 
GATA-1 Antagonism of Pol II Recruitment with TFIIB Retention, a Common Repression Mechanism—We tested whether GATA-1-mediated abrogation of Pol II recruitment with TFIIB retention occurs at additional GATA-1-repressed loci. GATA-1 represses c-Myb and c-Kit genes via direct interactions with promoter and intronic regulatory elements, respectively (57, 58). Quantitative ChIP analysis revealed that ER-GATA-1-mediated repression of c-Myb and c-Kit involves strongly reduced Pol II occupancy (Fig. 5A) at their promoters with little to no change in TFIIB occupancy (Fig. 5B). Thus, reduced Pol II recruitment with persistent TFIIB-chromatin interactions at extragenic sites and promoters is a hallmark of repression at three GATA-1-repressed genes.

The work described herein demonstrates Pol II and basal transcription factor complexes at extragenic GATA switch sites and Gata2 promoters, which are dynamically regulated via GATA switches. Several lines of evidence suggest that these complexes are functionally important. First, Pol II, TFIIB, and TBP at the extragenic sites are at least as high as that present at the 1S and 1G promoters. Second, the complexes were only detected in erythroid cells. Third, the complexes are dynamically regulated in response to ER-GATA-1 chromatin occupancy. ER-GATA-1-mediated repression remodels the complexes such that Pol II and TBP are no longer detected, whereas TFIIB persists at most sites (Fig. 6). Complexes at certain sites are differentially regulated, e.g. TFIIB occupancy is selectively lost from the –1.8-kb region upon repression and complexes at the –77-kb region are insensitive to ER-GATA-1. Lastly, Pol II resides at the –77-kb site independent of Pol II occupancy at the 1S promoter of the repressed locus, strongly suggesting that Pol II detected at upstream sites does not reflect the artifactual cross-linking of promoter-bound Pol II to regions that reside in the vicinity of the promoters. This finding is analogous to our report that Pol II can exist at the beta-globin LCR independent of Pol II occupancy at the associated promoters (32, 33). Collectively, these results strongly suggest that Pol II has important functions at Gata2 extragenic sites. Such functions might include Pol II transfer to the promoters or the generation of functional transcripts. The results described herein, defining the structure and regulation of spatially distinct complexes at the endogenous locus, provide a strong foundation for further dissecting the underlying mechanisms.


Figure 5
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  		FIGURE 5.
ER-GATA-1 abrogates Pol II, but not TFIIB occupancy, at c-Myb and c-Kit promoters. A and B, quantitative ChIP analysis of Pol II (A) and TFIIB (B) occupancy at c-Myb and c-Kit promoters in untreated and tamoxifen-treated (48 h) G1E-ER-GATA-1 cells. The graphs represent data from at least three independent experiments (mean ± S.E.).

 


Figure 6
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  		FIGURE 6.
Summary of ER-GATA-1-mediated regulation of Pol II, TFIIB, and TBP occupancy at the Gata2 locus. The gray circles and the arrows represent Gata2 HSs and promoters, respectively. The table summarizes GATA factor, P-Ser-5 Pol II, TBP, and TFIIB occupancy at the HSs and promoters of the endogenous Gata2 locus in the transcriptionally active (left) and repressed (right) states. +, occupancy detected; –, occupancy not detected; small +, occupancy detected but at a lower level than at other occupied sites at the locus.

 
Regardless of the function of the extragenic Pol II, our mechanistic analysis revealed Pol II occupancy of the full ensemble of sites only at the active locus, whereas TFIIB-chromatin interactions persist at all but the –1.8-kb region upon repression. In this regard, basal transcription factors can occupy heterochromatin in Saccharomyces cerevisiae (59) and D. melanogaster (60) and can occupy repressed loci in mammalian mitotic chromatin (61). It is attractive to propose that once GATA-1 binding expels Pol II from the Gata2 locus, the persistent TFIIB complex retains an important function. It seems unlikely that TFIIB persistence reflects that of a poised transcriptionally competent promoter, as once GATA-1 represses Gata2 no evidence exists to suggest that Gata2 is subsequently reactivated. Of considerable interest is why the TFIIB complex persists at a locus that is not destined for reactivation and how the persistent complex lacks the capacity to efficiently recruit Pol II. We anticipate that the answers to these questions will have broad implications for understanding the dynamic disassembly of transcriptional complexes during development.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK55700 and DK68634. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1. Back

1 A predoctoral fellow of the American Heart Association. Back

2 To whom correspondence should be addressed: University of Wisconsin School of Medicine, 1300 University Ave., Madison, WI 53706. Tel.: 608-265-6446; Fax: 608-262-1257; E-mail: ehbresni{at}wisc.edu.

3 The abbreviations used are: HS, Dnase I-hypersensitive site; ChIP, chromatin immunoprecipitation; LCR, locus control region; P-Ser-5, RNA polymerase II phosphorylated at serine 5; Pol II, RNA polymerase II; TBP, TATA-binding protein; TFIIB, transcription factor IIB. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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