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Originally published In Press as doi:10.1074/jbc.M201739200 on March 23, 2002

J. Biol. Chem., Vol. 277, Issue 22, 20011-20019, May 31, 2002
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Interaction of PIMT with Transcriptional Coactivators CBP, p300, and PBP Differential Role in Transcriptional Regulation*

Parimal MisraDagger §, Chao QiDagger , Songtao YuDagger , Sejal H. ShahDagger , Wen-Qing CaoDagger , M. Sambasiva RaoDagger , Bayar Thimmapaya, Yijun ZhuDagger , and Janardan K. ReddyDagger ||

From the Departments of Dagger  Pathology and  Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611-3008

Received for publication, February 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PIMT (PRIP-interacting protein with methyltransferase domain), an RNA-binding protein with a methyltransferase domain capable of binding S-adenosylmethionine, has been shown previously to interact with nuclear receptor coactivator PRIP (peroxisome proliferator-activated receptor (PPAR)-interacting protein) and enhance its coactivator function. We now report that PIMT strongly interacts with transcriptional coactivators, CBP, p300, and PBP but not with SRC-1 and PGC-1alpha under in vitro and in vivo conditions. The PIMT binding sites on CBP and p300 are located in the cysteine-histidine-rich C/H1 and C/H3 domains, and the PIMT binding site on PBP is in the region encompassing amino acids 1101-1560. The N-terminal of PIMT (residues 1-369) containing the RNA binding domain interacts with both C/H1 and C/H3 domains of CBP and p300 and with the C-terminal portion of PBP that encompasses amino acids 1371-1560. The C-terminal of PIMT (residues 611-852), which binds S-adenosyl-L-methionine, interacts respectively with the C/H3 domain of CBP/p300 and with a region encompassing amino acids 1101-1370 of PBP. Immunoprecipitation data showed that PIMT forms a complex in vivo with CBP, p300, PBP, and PRIP. PIMT appeared to be co-localized in the nucleus with CBP, p300, and PBP. PIMT enhanced PBP-mediated transcriptional activity of the PPARgamma , as it did for PRIP, indicating synergism between PIMT and PBP. In contrast, PIMT functioned as a repressor of CBP/p300-mediated transactivation of PPARgamma . Based on these observations, we suggest that PIMT bridges the CBP/p300-anchored coactivator complex with the PBP-anchored coactivator complex but differentially modulates coactivator function such that inhibition of the CBP/p300 effect may be designed to enhance the activity of PBP and PRIP.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nuclear receptor superfamily consists of several ligand-regulated transcription factors that include the steroid and thyroid hormone receptors, vitamin D3 receptor, retinoic acid receptors, and the peroxisome proliferator-activated receptors (PPARs),1 among others (1-3). Liganded nuclear receptors participate in diverse biological processes by controlling gene expression patterns in a cell-specific manner. This is accomplished by interacting with specific response elements (DNA sequence) located in the promoter regions of target genes, by recruiting transcriptional cofactors that function in nucleosome remodeling, and by recruiting RNA polymerase II holoenzyme to initiate transcription (1, 2, 4, 5). In recent years many nuclear receptor-interacting cofactors termed coactivators have been identified (4, 5). These coactivators, which enhance transcriptional activation of nuclear receptors, include the p160/SRC-1 (steroid receptor coactivator-1) family with three members (SRC-1, TIF2/GRIP1/SRC2, and pCIP/ACTR/AIB1/RAC3/TRAM1/SRC-3) (6-12), CREB-binding protein (CBP) (13), adenovirus E1A-binding protein p300 (14), PPAR-binding protein (PBP) (TRAP220/DRIP205) (15, 16), PPARgamma coactivators PGC-1alpha and PGC-1beta (17), and the general coactivator PRIP/ASC-2/RAP250/TRBP/NRC (18-22), among others (23). They all contain one or more conserved LXXLL (where L is leucine and X is any amino acid) signature motifs, which have been found to be necessary and sufficient for ligand-dependent interactions with the activation function-2 (AF-2) domain present in the C-terminal nuclear receptor ligand binding domain (12, 24). There is increasing appreciation that while some coactivators, such as p160/SRC-1 family members, interact predominantly with nuclear receptors to enhance gene-specific transcription (2, 5), others (for example CBP/p300 and PBP) appear to interact with nuclear receptors and many other transcription factors (25-27). Nuclear receptors and their coactivators regulate the transcriptional activity of target genes that play central roles in key biological phenomena such as early development, cell proliferation, differentiation, apoptosis, metabolic homeostasis, and cancer (25).

The p160 family of nuclear receptor coactivators and CBP/p300 possess intrinsic histone acetyltransferase (HAT) activity in addition to their role of enhancing transcriptional activation in transient transfection assays (5, 10, 28, 29). These coactivators also recruit other proteins with HAT activity to form a multiprotein complex anchored by CBP/p300. This complex regulates transcription through remodeling chromatin by acetylating histones (30). Recently, coactivator-associated enhancement of transcription has also been shown to involve the recruitment of coactivator-associated proteins such as coactivator-associated arginine methyltransferase 1 (CARM1), a member of the S-adenosyl-L-methionine-dependent, protein arginine methyltransferase (PRMT) family (31). CARM1 catalyzes the methylation of arginine residues particularly in histone 3 (31). The PRMT family consists of at least five members (PRMT1, PRMT2, PRMT3, PRMT4/CARM1, and PRMT5) (32) of which CARM1/PRMT4 is known to enhance the function of the p160 family of coactivators by increasing nuclear receptor-dependent gene transcription. CARM1/PRMT4 does this by binding to and methylating the KIX domain of CBP/p300 in addition to its initially described role in histone 3 methylation (33). The KIX domain-methylated CBP/p300 fails to activate CREB-dependent genes. Therefore, the CBP/p300 is able to direct nuclear receptor-dependent gene transcription (33). From these observations it appears that histone acetylation, histone methylation, and selective coactivator methylation (namely CBP/p300 methylation) constitute novel regulatory mechanisms that involve the CBP/p300-anchored coactivator complex in nuclear receptor-mediated gene transcription.

The next step in the multistep transcriptional activation process involves participation of the TRAP/DRIP/ARC mediator complex anchored by the coactivator PBP (4, 5, 26). While CBP/p300 and the p160 family of cofactors that form the initial multiprotein complex function by exhibiting HAT and arginine methyltransferase activities, there is limited functional information about the coactivator PBP and other proteins that form the TRAP/DRIP/ARC complex. This complex facilitates interaction with RNA polymerase II complexes of the basal transcriptional machinery, but how this is accomplished remains unclear (4, 5, 16, 26, 34, 35). PBP lacks HAT activity, and it is uncertain if the other members of the TRAP/DRIP/ARC complex in mammals possess enzymatic activity of any kind. Disruption of the PBP and CBP/p300 genes in the mouse results in embryonic lethality around E11.5 days, indicating that disruption of these pivotal anchoring coactivators affects the function of many nuclear receptors and possibly other transcription factors (27, 36-40). The recently identified coactivator designated PRIP/ASC2/RAP250/NRC/TRBP has been shown to interact with several nuclear receptors and also with CBP/p300 and TRAP130 of the TRAP/DRIP/ARC complex (18-22). Thus, PRIP appears to serve as a bridge between the initial histone acetyltransferase-histone methyltransferase complex of CBP/p300 and p160 coactivators and the downstream TRAP/DRIP/ARC complex. Disruption of this PRIP gene in the mouse leads to embryonic lethality around E13.5 days, implying that PRIP (like CBP/p300 and PBP) is also critical for embryonic development and survival.2 We have previously isolated a PRIP-interacting protein, designated PIMT, using the yeast two-hybrid approach with PRIP as bait. We found that PIMT enhances the nuclear receptor coactivator function of PRIP (41). PIMT, which has an invariant GXXGXXI segment found in K-homology motifs and many RNA-binding proteins, binds RNA (41). PIMT also has a nine amino acid VVDAFCGVG methyltransferase motif I and binds S-adenosyl-L-methionine, the methyl donor for the methyltransfer reaction, suggesting that it may be a putative RNA methyltransferase (41). We report here that PIMT interacts with CBP, p300, and PBP but not with SRC-1 and PGC-1alpha , suggesting that PIMT functions as a link between the two major multiprotein complexes anchored by CBP/p300 and PBP, respectively. Of considerable interest is that while PIMT enhances the PRIP and PBP-mediated nuclear receptor transcriptional activity, it inhibits the CBP/p300-mediated nuclear receptor function.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Constructions-- pCDNA3.1-PIMT, pCDNA3.1-PIMT (aa 1-369), pGEX-PIMT (aa 577-852), pGEX-PIMT (aa 326-852), pCMV-3XFLAG-PIMT, pCMX-PBP, pCDNA3.1-PRIP, pCDNA3.1-SRC1, pCMV-p300, 3XPPRE-Luc, and pCMV-PPARgamma 1 have been described (41). pCDNA3.1-PGC1alpha -FLAG was a generous gift from Dr. Bruce M. Spiegelman. pCDNA3.1-CBP-HA was constructed by subcloning the HindIII-NotI fragment containing the entire coding region of CBP from pRC/RSV-CBP.HA into pCDNA3.1. Sequences encoding peptide fragments of entire CBP, p300 and PBP fused to glutathione S-transferase (GST) (hereafter GST-CBP, GST-p300, and GST-PBP) were generated by subcloning respective PCR fragments amplified from the appropriate plasmids into the correct reading frame of pGEX4T1 (Amersham Biosciences). GST-PIMT (aa 1-334), pCDNA3.1-PIMT (aa 370-610), and pCDNA3.1 PIMT (aa 611-852) were generated by subcloning PCR fragments into the pGEX and pCDNA3.1, respectively. Sequences of all clones were verified by sequencing.

Construction of Adenovirus PIMT-- To construct recombinant adenovirus-PIMT (Ad/PIMT), the full-length PIMT coding region was cloned by PCR from plasmid pCMV-PIMT (41) and inserted into the BamHI/XhoI site of pFastBac HTc (Invitrogen). The entire coding region of PIMT with the hexahistidine affinity tag was cut with RsrII/XhoI and transferred to the SalI site of pShuttle vector (Quantum Biotechnologies, Inc.) by blunting the ends. The recombinant Ad/PIMT was generated as described previously (42). The adenoviral construct of Ad/LacZ was the generous gift of Dr. W. El-Deiry (University of Pennsylvania, Philadelphia) and has been described previously (43).

GST Pull-down Assays-- GST fusions or GST alone was expressed in Escherichia coli BL21 (DE3) bound to glutathione-Sepharose 4B beads (Amersham Biosciences) and incubated with [35S]methionine-labeled coactivator/coactivator-binding protein expressed by in vitro translation by using the TNT-coupled transcription-translation system (Promega). Briefly, the binding assays were carried out by incubating 10 µl of [35S]methionine-labeled coactivator/coactivator-binding protein for 2 h at 4 °C with the immobilized GST fusion protein in GST binding buffer (180 mM KCl, 20 mM Tris-HCl, pH 7.9, 1 mM EDTA, 0.05% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). Beads were washed four times with 1 ml of binding buffer containing 0.1% Nonidet P-40, and bound protein was eluted by boiling for 2 min in 20 µl of SDS sample buffer, was analyzed by SDS-PAGE, and was subjected to autoradiography.

Immunoprecipitation and Western Blotting-- COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and transfected with pCMV-PIMT-FLAG plasmid encoding FLAG-tagged PIMT along with one of the coactivator expression plasmids. After 72 h cells were harvested and lysed at 4 °C by vortexing in lysis buffer (100 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1% Nonidet P-40, 1 mM EDTA) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin). Lysates were clarified by centrifugation at 18,000 × g for 20 min and immunoprecipitated at 4 °C for 2 h with anti-CBP, anti-p300, anti-PBP (Santa Cruz Biotechnology), or anti-PRIP, respectively, followed by precipitation with 50 µl of protein A/G-Sepharose (Sigma) overnight at 4 °C. After four washes with lysis buffer (described above), the immunoprecipitates were eluted by boiling for 5 min in Laemmli sample buffer. Resulting immunoprecipitates were electrophoresed in 10% SDS-PAGE, transferred onto a nitrocellulose membrane, immunoblotted with anti-FLAG monoclonal antibody M2 (Sigma), and detected using ECL chemiluminescence (Amersham Biosciences).

Alternatively, PIMT-binding proteins were immunoprecipitated from HEK293 cells that were infected with Ad/PIMT or Ad/LacZ. HEK293 cells were cultured to 95% confluence in 150-cm plates. The cells were at first incubated with 10 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with Ad/PIMT or Ad/LacZ respectively at a multiplicity of infection of 100. After 1.5 h, 35 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum was added. Infected HEK293 cells were harvested 24 h after infection. The cell lysates were immunoprecipitated with anti-His tag (Santa Cruz Biotechnology) followed by precipitation with protein A/G-Sepharose as described above and immunoblotted with anti-CBP, anti-p300, anti-PRIP, and anti-PBP. The antigen-antibody complexes were then detected using the alkaline phosphatase-conjugated substrate kit (Bio-Rad).

Immunofluorescence-- COS-1 cells were transfected with pCMV-PIMT-FLAG along with one of the coactivator expression plasmids, namely CBP, p300, and PBP using Polyfect (Qiagen). After 48 h transfection, the cells were washed and fixed in 1% formaldehyde and washed twice with phosphate-buffered saline, pH 7.4, after which autofluorescence was quenched with 50 mM ammonium chloride in phosphate-buffered saline. Cells were washed with phosphate-buffered saline, permeabilized with 0.1% Triton X-100, and then blocked with 0.2% fish skin gelatin. Cells were incubated with the primary antibody anti-FLAG (Sigma) along with one of the antibodies (anti-CBP, anti-p300, or anti-PBP) followed by incubation with secondary antibodies. Fluorescence microscopy and digital image collection were performed by using an Olympus (New Hyde Park, NY) microscope and a photometrix cooled charge-coupled device camera driven by DELTAVISION software from Applied Precision (Seattle).

Transactivation Assay-- CV1 cells (for CBP transactivation) and HEK293 cells (for p300 and PBP transactivation) were plated in Dulbecco's modified Eagle's medium with 10% fetal bovine serum without antibiotics in six well plates and cultured for 24 h before transfection. Transfections were carried out with LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions for 5 h. Each transfection contains 20 eta g of PPAR expression vector, 1.5 µg of luciferase reporter plasmid DNA, 0.1 µg of beta -galactosidase expression vector, and 1 µg of appropriate expression vector. Cell extracts were analyzed 24 h after transfection for luciferase and beta -galactosidase activities (Tropix).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PIMT Interacts with CBP, p300, PBP, and PRIP but Not with PGC1 and SRC-1-- Previously, we have shown that PIMT interacts with PRIP (41). To determine whether PIMT interacts with other coactivators we used bacterially generated GST fusion of truncated PIMT containing the RNA methyltransferase domain encompassing aa 577-852 (hereafter, PIMTC) (Fig. 1A) and tested its direct in vitro interaction with in vitro translated CBP, p300, PBP, PGC-1, and SRC-1. As shown in Fig. 1B, CBP, PBP, PRIP, and p300 interacted with immobilized GST-PIMTC, but PGC-1 and SRC-1 failed do so.


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  		Fig. 1.   PIMT interacts with coactivators CBP, p300, PBP, and PRIP but not with SRC-1 and PGC-1 in vitro. A, physical map of PIMT showing RNA binding, methyltransferase domains and also the three PIMT segments, PIMTA (aa 1-334), PIMTB (aa 326-852), and PIMTC (aa 577-852) used for GST-PIMT fusions. B, in vitro interaction of the C-terminal of PIMT (aa 577-852) containing an RNA methyltransferase domain with different coactivators. GST pull-down assays using GST-Sepharose beads bound with purified E. coli expressed GST-PIMTC and [35S]methionine-labeled full-length CBP, PBP, PRIP, SRC-1, PGC1, and p300 reveals interaction of CBP, PBP, PRIP, and p300 with PIMT, but no interaction of this segment with SRC-1 and PGC-1alpha . C, GST pull-down assay using GST-PIMTA and GST-PIMTB confirms the lack of interaction of SRC-1 and PGC-1alpha with the remaining two fragments of PIMT.

Having confirmed that PGC-1alpha and SRC-1 failed to interact with truncated PIMT (PIMTC), we then tested the interaction of GST-PIMTA (aa 1-334) and GST-PIMTB (aa 326-852) with in vitro translated [35S]methionine-labeled full-length SRC-1 and PGC-1alpha (Fig. 1C). We failed to detect any binding of SRC-1 and PGC-1alpha with these PIMT fragments. We also used full-length in vitro translated PIMT and investigated its in vitro interaction with various SRC-1- and PGC-1alpha -truncated proteins expressed as GST fusions. In these assays full-length PIMT did not interact with these two coactivators (data not shown).

Mapping of the CBP and PIMT Domains Required for Interaction-- In order to define the interacting domain(s) of CBP on PIMT, we prepared GST fusion proteins containing various peptides of CBP and determined the ability of each of these to interact with [35S]methionine-labeled in vitro translated full-length PIMT. GST and GST-CBP fusion proteins (shown schematically in Fig. 2A) were bacterially expressed, purified, coupled to glutathione-Sepharose beads, and used for in vitro binding assays. CBP2 peptide (aa 301-600) containing the cysteine-histidine-rich 1 (C/H1) region and CBP7 peptide (aa 1801-2100) containing the cysteine-histidine-rich 3 (C/H3) region interacted significantly with full-length PIMT (Fig. 2B). The extent of interaction of PIMT with CBP2 peptide is decreased when a pull-down assay was done at the higher salt concentration of 250 mM KCl, indicating weak interaction of PIMT with peptide containing the C/H1 domain compared with that of peptide containing the C/H3 domain (data not shown). Using further truncated CBP2 and CBP7 fragments, we narrowed down the PIMT binding sites on CBP to two regions encompassing amino acids 301-400 (Fig. 2B, CBP2A) and 1801-1900 (Fig. 2B, CBP7A), respectively.


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  		Fig. 2.   Identification of CBP- and PIMT-interacting domains. A, physical map of CBP depicting various domains: NR, nuclear hormone receptor domain; C/H1, cysteine-histidine-rich domain 1; C/H3, cysteine-histidine-rich domain 3; K/X, phospho-CREB binding domain. PIMT interacts with the C/H1 and C/H3 regions of CBP as shown in B and C below. B, different fragments of GST-CBP were immobilized on GST-Sepharose and incubated with [35S]methionine-labeled in vitro translated full-length PIMT. Full-length PIMT binds to peptides CBP2 (aa 301-600) and CBP7 (aa 1801-2100). The binding sites of PIMT on CBP have been further narrowed down to the regions encompassing the C/H1 (aa 301-400) and C/H3 (aa 1801-1900) domains. C, identification of PIMT domains that interact with CBP. [35S]methionine-labeled in vitro translated PIMTN, PIMTM, and PIMTC fragments were incubated with GST-CBP2A and GST-CBP7A. PIMTN binding sites on CBP appear to be located in regions of aa 301-400 and aa 1801-1900, whereas the PIMTC binding domain on CBP is located in the region encompassing aa 1801-1900. PIMTM did not bind with these GST-CBP fragments.

We then determined the domain(s) of PIMT that are important for CBP interaction. Three different PIMT peptides (namely PIMTN (aa 1-369), PIMTM (aa 370-610), and PIMTC (aa 611-852)) were translated in vitro in the presence of [35S]methionine, and equal portions of these peptides were used to assess their binding to GST alone, GST-CBP2A, and GST-CBP7A in the in vitro pull-down assays (Fig. 2C). Both PIMTN (aa 1-369) and PIMTC (aa 611-852) interacted with CBP7A (aa 1801-1900), whereas only PIMTN interacted with CBP2A (aa 301-400) (Fig. 2C), indicating two independent domains of interaction of PIMTN on CBP. PIMTM did not bind these GST-CBP fusion peptides. We can conclude that PIMT has two independent binding sites on CBP, one in the region of aa 301-400 and the other in the region of aa 1801-1900. These CBP regions also interact with adenoviral E1A oncoprotein (44).

Mapping of the p300 and PIMT Domains Required for Interaction-- We also determined the interacting domains of PIMT and p300 using GST fusion proteins containing various peptides of p300 (Fig. 3). Of the GST-p300 fusion proteins (depicted schematically in Fig. 3, A and B), p300/2 (aa 301-597) and p300/5 (aa 1501-1800) interacted with full-length in vitro translated PIMT (Fig. 3B). Further analysis revealed that p300/2A and p300/5C interacted strongly with PIMT, indicating that the PIMT binding domain in p300 is in the region encompassing aa 301-920 and aa 1701-1800 (Fig. 3B). The results of the binding of PIMT peptides (PIMTN, PIMTM, and PIMTC) to p300 clearly established that the PIMT N and C domains interact(s) with p300 (Fig. 3C).


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  		Fig. 3.   Identification of p300 and PIMT domains that interact with each other. A, physical map of p300 showing NR, C/H1, C/H3, K/X, and HAT domains. PIMT interacts with the C/H1 and C/H3 regions of p300 based on the results shown in B and C below. B, schematic diagram of GST-p300 fusion peptides used in pull-down assays, and demonstration that full-length [35S]methionine-labeled PIMT interacts with p300/2A (aa 301-400) and p300/5C (aa 1701-1800). C, identification of PIMT domains that interact with p300. [35S]methionine-labeled in vitro translated PIMTN, PIMTM, and PIMTC fragments were incubated with GST-p300/2A and GST-p300/5C. PIMTN binding sites on p300 appear to be located in regions of aa 301-400 and aa 1801-1900, whereas the PIMTC binding domain on CBP is located in the region encompassing aa 1801-1900. PIMTM did not bind with these GST-CBP fragments.

Mapping of the PBP and PIMT Domains Required for Interaction-- In vitro GST pull-down analyses were performed with recombinant GST fusion proteins of PBP and [35S]methionine-labeled in vitro translated PIMT proteins. GST-PBP fragments were generated to map the regions required for interaction with PIMT (Fig. 4, A and B). Three overlapping fragments in the C-terminal of PBP (PBP5, PBP6, and PBP7 covering aa 740-1130, 981-1370, and 1360-1560, respectively) interacted with PIMT, but PIMT failed to interact with fragments containing the LXXLL region (PBP2, PBP3, and PBP4) and GST alone (Fig. 4B). To determine the domain(s) of PIMT involved in interaction with PBP, we used different truncated PIMT peptides in GST pull-down assays (Fig. 4C). The results show that PIMTN (aa 1-369) interacts with the extreme C-terminal of PBP in the region encompassing aa 1371-1560 (Fig. 4C, PBP11), whereas PIMTC (aa 611-852) interacts with PBP in the region encompassing aa 1101-1370 (Fig. 4C, PBP10), indicating two different interactive sites of PIMT for PBP.


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  		Fig. 4.   Identification of PBP- and PIMT-interacting domains. A, map of PBP depicting the LXXLL and PIMT-interacting regions. B, GST pull-down assays using various fragments of PBP. [35S]methionine-labeled full-length PIMT generated by in vitro translation was incubated with glutathione-Sepharose beads bound with purified E. coli expressed GST or different fragments of PBP. PIMT binds with three overlapping GST-PBP fragments, PBP5 (aa 740-1130), PBP6 (aa 980-1370), and PBP7 (aa 1371-1560). C, identification of PIMT domains that interact with PBP. [35S]methionine-labeled PIMTN (aa 1-369), PIMTM (aa 370-610), and PIMTC (aa 611-852) were allowed to interact with various GST-PBP fragments encompassing PBP region aa 981-1560. PIMTN binds with the PBP11 (aa 1371-1560), and PIMTC binds with PBP9 (aa 1101-1370). PIMTM did not bind with any GST-PBP fragment.

Interaction of PIMT with CBP, p300, PBP, and PRIP In Vivo (PIMT Complex)-- Given that PIMT interacts with the nuclear receptor coactivators CBP, p300, and PBP in vitro, we wanted to determine whether PIMT interacts with CBP, p300, and PBP in the context of intact cells. We used two different approaches. First, a vector encoding human PIMT with the C-terminal FLAG epitope was cotransfected separately along with one of the coactivators (namely CBP, p300, PBP, and PRIP) (used as positive control) into COS-1 cells derived from African Green Monkey Kidney (American Type Culture Collection, CRL1651). The potential complex between PIMT and each coactivator was immunoprecipitated separately using respective antibodies (i.e. anti-CBP, anti-p300, anti-PBP, and anti-PRIP), and the products were analyzed by immunoblot using anti-FLAG to demonstrate the presence of PIMT in the precipitates (Fig. 5A). The results showed that all antibodies precipitated PIMT, demonstrating that PIMT interacts in vivo with these nuclear receptor coactivators (Fig. 5A) and suggesting the existence of a PIMT-coactivator complex in vivo. Second, to pull down such a complex, we infected HEK293 cells with Ad/PIMT or Ad/LacZ and immunoprecipitated recombinant PIMT using anti-His antibodies. The immunoprecipitate obtained in this fashion contained CBP, p300, PBP, PRIP, as well as PIMT (Fig. 5B), indicating the existence of a complex of at least five proteins anchored by or containing PIMT as a component.


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  		Fig. 5.   PIMT interacts with CBP, p300, PRIP and PBP In Vivo. A, cell lysates were prepared from COS-7 cells transfected with pCDNA3.1-FLAG-PIMT and one of the coactivator expression plasmids separately and immunoprecipitated with anti-CBP, anti-p300, anti-PRIP, or anti-PBP antibodies. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-FLAG-epitope-tagged protein. PIMT is coprecipitated by anti-CBP (lane 3), anti-p300 (lane 4), anti-PRIP (lane 5) and anti-PBP (lane 6) but not by preimmune serum (lane 2). Lane 1 represents cell lysate (1/20 input). B, HEK293 cells infected with Ad/LacZ or Ad/PIMT were lysed and immunoprecipitated with anti-His tag. The immunoprecipitates when subjected to immunoblotting with anti-CBP, anti-p300, anti-PBP, anti-PRIP, or anti-FLAG (for PIMT) reveal that PIMT exists as a complex with these coactivators.

PIMT Colocalizes with CBP, p300, and PBP in the Nucleus-- We have shown that PIMT interacts with CBP, p300, PBP, and PRIP both in vitro and in vivo. Previously, we demonstrated by immunofluorescence microscopy that PIMT and PRIP colocalize within the nucleus. To evaluate whether PIMT colocalizes with its newly identified binding partners, a plasmid containing three FLAG epitopes linked to the C-terminal portion of the PIMT protein was separately cotransfected into COS-1 cells along with one of the coactivator plasmids (i.e. CBP, p300, or PBP). Immunofluorescence (green) with anti-FLAG revealed that the expressed PIMT protein is localized in the nucleus (Fig. 6, A, D, and G). Localizations of CBP, p300, and PBP using anti-CBP, anti-p300, and anti-PBP, respectively, revealed that CBP, p300, and PBP are also localized in the nucleus (red fluorescence, Fig. 6, B, E, and H). When localized images of PIMT (green) were separately merged with localized images of CBP, p300, and PBP (red), an appreciable degree of overlapping localization (yellow) of PIMT with CBP (Fig. 6C), p300 (Fig. 6F), and PBP (Fig. 6I) have been noted. These results demonstrated colocalization of PIMT with coactivator CBP, p300, and PBP in the nucleus similar to the PIMT and PRIP colocalization described before (41).


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  		Fig. 6.   PIMT colocalizes with CBP, p300, and PBP. PIMT was expressed transiently in COS-7 cells by using three FLAG epitopes linked to the C-terminus of PIMT, and the FLAG-epitope of PIMT was visualized with Deltavision deconvolution microscopy using anti-FLAG antibodies (A, D, and G). CBP, p300, and PBP were visualized under deconvolution microscopy after immunofluorescence staining with respective antibodies (B, E, and H, respectively). Merging of the PIMT (A) and CBP (B); PIMT (D) and p300 (E); PIMT (G) and PBP (H) images revealed generally overlapping patterns (yellow) of PIMT with CBP (C), p300 (F), and PBP (I).

Differential Modulation of Transcriptional Activities of Coactivator CBP, p300, and PBP by PIMT-- To determine the functional relevance of the interaction of PIMT with CBP, p300, and PBP, we transiently overexpressed PIMT with CBP in CV1 cells and PBP and p300 in HEK293 cells along with PPARgamma . We monitored the transcriptional activity of PPARgamma with the expression of the thymidine kinase promoter-driven PPRE-linked reporter luciferase gene. When transfected individually, CBP, p300, and PBP consistently increased the transcription of the PPARgamma -mediated luciferase gene by approximately 2-fold in the case of CBP (Fig. 7A), 5-fold in the case of p300 (Fig. 7B), and 3-fold in the case of PBP (Fig. 7C) in the presence of the PPARgamma ligand BRL49653. Cotransfection of PIMT and CBP or p300 in this assay resulted in a repression of ligand-dependent reporter gene expression (Fig. 7, A and B), a phenomena that is well established in the case of adenoviral E1A oncoprotein-mediated transcriptional repression (14). Cotransfection of PIMT and PBP resulted in further enhancement (Fig. 7C) indicating a synergism of PIMT and PBP activities. Earlier, we showed that within the context of transient transfection PIMT also synergized PRIP action (41). These results demonstrate that PIMT differentially modulates the coactivator-mediated transcriptional activity, acting as a transcriptional repressor of CBP and p300 while functioning as an enhancer of PBP and PRIP coactivator function.


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  		Fig. 7.   PIMT represses CBP/p300-mediated transcriptional function but enhances PBP-mediated transactivation activity. Cells were cotransfected with 1.5 µg of PPRE-Luc, 20 ng of pCMV-PPARgamma , 0.1 µg of pCMVbeta , and 1 µg of additional indicated plasmids in the absence (solid bar) or presence (open bar) of 10-5 M BRL49653. Transfection without the indicated plasmid was compensated by the same amount of pCDNA3.1. Luciferase activity from the transfection without either exogenous CBP, p300, or PBP, and PIMT in the absence of ligand, was defined as the value 1. The results are the mean of three independent transfection experiments. A, CBP-mediated transfection analysis in CV1 cells. B and C, p300 and PBP-mediated transfection experiments, respectively, in HEK293 cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Coactivators play a central role in mediating nuclear receptor transactivation by functioning as at least two large multiprotein complexes, one anchored by CBP/p300 and the other by PBP (4, 5). The multistep model of transcription proposes that the acetylation-methylation functions of the initial CBP/p300-mediated complex leads to a transition from CBP/p300-dependent to a mediator-dependent stage of transcription involving the TRAP/DRIP/ARC complex of coactivators anchored by PBP (4, 33). The central importance of CBP/p300 and PBP in transcription is underscored by embryonic lethality observed in gene knockout studies in mice (36-40). One other coactivator of equal importance appears to be the recently cloned PRIP/ASC-2/RAP250/TRBP/NRC (18-22), which has not been identified as part of either the CBP/p300 or the PBP-anchored multiprotein coactivator complexes. However, disruption of the PRIP gene appears to result in embryonic lethality.2 The embryonic lethality of null mutation as well as PRIP's ability to interact with a variety of nuclear receptors and CBP (18-22) suggested that PRIP is as indispensable as CBP, p300, and PBP in mediating the transcriptional activity of nuclear receptors and other transcription factors (41). To elucidate the functional role of PRIP and to identify coactivator-binding protein(s), we used PRIP as bait in a yeast two-hybrid screen and identified PIMT as a PRIP-interacting protein capable of enhancing PRIP's coactivator function (41). Interestingly, PIMT binds to S-adenosyl-L-methionine and RNA, implying that PIMT may function as a putative methyltransferase (41). Truncated PIMT, without the methyltransferase domain, still showed its ability to enhance the PRIP-mediated transcriptional activity of PPARgamma and RXRalpha , suggesting that putative enzyme activity of PIMT may not be crucial to transcription under transient transcription conditions (41).

In this study, we have examined the ability of PIMT to interact with other coactivators such as CBP, p300, PBP, SRC-1, and PGC-1alpha in an attempt to further explore the role of PIMT in nuclear receptor signal transduction. Our results show that PIMT interacts with CBP, p300, and PBP under in vitro and in vivo conditions, but it failed to interact with SRC-1 and PGC-1alpha , implying selectivity of association. Using GST pull-down assays, we mapped the PIMT binding site(s) on CBP and p300 to their C/H1 and C/H3 domains. Further analysis revealed that the N-terminal of PIMT containing the RNA binding domain interacts with these two sites, whereas the C-terminal S-adenosyl-L-methionine binding domain of PIMT appears to interact with only the C/H3 domain. These two regions, namely C/H1 and C/H3, of CBP/p300 participate in the binding and activity of a variety of transcription factors (26, 45). In particular, a small domain of C/H3 binds diverse proteins including adenoviral E1A oncoprotein and the coactivator TIF-2/SRC-2 (45). The PIMT binding site on PBP was localized to region aa 1101-1560. Since this region is devoid of the LXXLL motif, it appears that binding of PIMT to PBP is not contingent upon the presence of this motif. Nevertheless, the PIMT binding site on PRIP was in the region (aa 773-927) of PRIP that has an LXXLL that is considered necessary for PRIP-nuclear receptor interaction (18, 41).

The in vivo interaction of PIMT with CBP, p300, and PBP was ascertained by immunoprecipitation using antibodies against specific coactivators and then immunoblotting with anti-FLAG to detect the presence of this epitope-tagged PIMT (Fig. 5). Alternatively, the presence of CBP, p300, PBP, and PRIP were detected by immunoblot analysis of immunoprecipitates obtained using anti-His antibodies to precipitate adenovirally expressed PIMT with hexahistidine. These approaches clearly established the presence in vivo of PIMT-coactivator complex containing at least four of what appear to be general coactivators. Furthermore, immunofluorescence studies with anti-FLAG revealed the presence of expressed epitope-tagged PIMT protein in a speckled pattern in nuclei. A merging of the distribution patterns of CBP, p300, or PBP with that of PIMT revealed overlapping colocalization of PIMT with these coactivators. From these studies it is reasonable to assume that the PIMT complex includes, in addition to PRIP, some components of HAT-methyltransferase containing the CBP/p300-coactivator complex as well as PBP, the major component of the TRAP/DRIP/ARC complex. Thus, PIMT and PRIP appear to serve as linkers between two major coactivator complexes involved in the multistep model of transcription (4, 5). However, it is plausible to consider the existence of one major coactivator complex (Fig. 8) and that different signals pass selectively through different components of this megacomplex to accomplish transcription factor-specific gene transcription instead of the sequential recruitment of two complexes. It is important to recall that SRC-1 with its histone acetyltransferase activity was found to be dispensable in generating the PPARalpha -dependent signal transduction in SRC-1-null mice. This implies a redundancy of the p160 family of coactivators for PPARalpha signaling despite the fact that SRC-1 is an essential component of the CBP/p300-coactivator complex (46). In contrast, partial blunting of steroid hormone responses was found in SRC-1-null mice (47), further attesting to the use of different coactivators for different circuits necessary for receptor-mediated signal transduction.


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  		Fig. 8.   A proposed model in which PIMT functions as a link between the CBP/p300-anchored coactivator complex with the PBP-anchored TRAP/DRIP/ARC complex. CBP/p300 and other proteins in this complex, such as members of the SRC family, possess intrinsic histone acetyltransferase activity, and CARM1 contains histone methyltransferase activity. The TRAP/DRIP/ARC complex anchored by PBP and other proteins such as PRIP with no known enzymatic activities facilitate the recruitment of general transcription factors (GTFs) and RNA polymerase II holoenzyme to initiate transcription of the ligand-bound nuclear receptors.

We found that PIMT functions as a strong enhancer of PBP coactivator function when the PPARgamma -mediated transcription function was assayed using transient transfection in the presence of PPARgamma ligand BRL49653. In contrast, our results also show that PIMT represses the CBP/p300 coactivator function. The integrator protein p300 was demonstrated to interact with PPARalpha and enhance its transcriptional activity (48). The repressor effect of PIMT on the CBP/p300 coactivator function is opposite of what we observed in this study with PBP and previously with PRIP (41) where PIMT augmented the coactivator functions of both PRIP and PBP. The mechanism by which PIMT exerts this coactivator-dependent differential transcriptional activity is not clear. It is possible that PIMT by virtue of its ability to bind CBP/p300 may be disrupting the initial coactivator complex formation in a manner analogous to that of E1A oncoprotein (44). E1A binding to CBP/p300 interferes with normal cellular transcription and cell cycle progression (14). However, the C/H3 domain where E1A binds is not required for the E1A inhibition of transcription. This is due to the E1A inhibition of the assembly of CBP-nuclear receptor-coactivator complex formation (44). PIMT also binds to CBP/p300 at this C/H3 site, and overabundance/overexpression of PIMT or lower concentrations of CBP/p300 may lead to disassembly or nonassembly of the CBP/p300-coactivator complex. Another possibility is that the RNA binding property of PIMT together with its putative methyltransferase activity, as suggested by its ability to bind S-adenosyl-L-methionine, may influence transcription or RNA processing machinery. PIMT reveals the presence of S-adenosyl-L-methionine binding site VVDAFCGVG, which is similar to the highly conserved methyltransferase motif I with a consensus hh(D/S)(L/P)FXGXG (where h represents a hydrophobic residue and X represents any amino acid) (41). The FCGVGGN present in PIMT corresponds with a single methylating consensus sequence (FGGRGGF) motif found in many methyl-accepting substrates including RNA and DNA methyltransferases (49, 50). One of the substrates happens to be the NPL3 gene product in yeast that is implicated in a wide variety of cellular processes including nuclear import, rRNA processing, association with and export of poly(A+) messenger RNA from the nucleus, and mRNA splicing (51-53). It would be important to examine if PIMT serves as a substrate for CARM1 and participates in mRNA processing during transcription. Further work is essential to characterize the biochemical functions of PIMT and also to ascertain its in vivo function by generating mice with a disrupted PIMT gene.

In summary, recent studies have highlighted the critical importance of histone acetylation, histone methylation, and coactivator methylation in regulating gene transcription (2, 4, 5, 31-33). Our results strongly implicate the involvement of RNA-binding proteins such as PIMT and RNA methyltransferases in transcription.

    ACKNOWLEDGEMENTS

We thank Travis Harrison for his expert technical assistance with Delta Vision Microscopy and Dr. Robert Costa for his advice about generating adenoviral expression vector.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM23750 (to J. K. R.), CA84472 (to M. S. R.), R01 CA74403 (to B. T.), K08 ES00356 and CA 88898 (to Y. Z.) and the Joseph L. Mayberry, Sr. Endowment Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by the Dr. Reddy Research Foundation, Miyapur, Hyderabad 500050 India.

|| To whom correspondence should be addressed: Dept. of Pathology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611-3008. Tel.: 312-503-8144; Fax: 312-503-8249; E-mail: jkreddy@northwestern.edu.

Published, JBC Papers in Press, March 23, 2002, DOI 10.1074/jbc.M201739200

2 C. Qi and J. K. Reddy, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated response element; SRC-1, steroid receptor coactivator-1; PBP, PPAR-binding protein; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; TRAP, thyroid hormone receptor-associated protein(s); DRIP, vitamin D receptor-interacting protein(s); ARC, activator-recruited cofactor; CARM1, coactivator-associated arginine methyltransferase-1; PRMT1, protein arginine methyltransferase1; PRIP, PPAR-interacting protein; PIMT, PRIP-interacting protein with methyltransferase domain; RXR, retinoid-X receptor for 9-cis-retinoic acid; GST, glutathione S-transferase; aa, amino acid(s); HAT, histone acetyltransferase; Ad, adenovirus; C/H, cysteine-histidine-rich domain; HEK, human embryonic kidney.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schultz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995) Cell 83, 835-839[CrossRef][Medline] [Order article via Infotrieve]
2. McKenna, N. J., Lanz, R. B., and O'Malley, B. W. (1999) Endocr. Rev. 20, 321-344[Abstract/Free Full Text]
3. Brivanlou, A. H., and Darnell, J. E., Jr. (2002) Science 295, 813-818[Abstract/Free Full Text]
4. Malik, S., and Roeder, R. G. (2000) Trends Biochem. Sci. 25, 277-283[CrossRef][Medline] [Order article via Infotrieve]
5. Rosenfeld, M. G., and Glass, C. K. (2001) J. Biol. Chem. 276, 36865-36868[Free Full Text]
6. Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1995) Science 270, 1354-1357[Abstract/Free Full Text]
7. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403-414[CrossRef][Medline] [Order article via Infotrieve]
8. Zhu, Y., Qi, C., Calandra, C., Rao, M. S., and Reddy, J. K. (1996) Gene Expr. 6, 185-195[Medline] [Order article via Infotrieve]
9. Voegel, J. J., Heine, M. J. S., Zechel, C., Chambon, P., and Gronemeyer, H. (1996) EMBO J. 15, 3667-3675[Medline] [Order article via Infotrieve]
10. Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L., Nakatani, Y., and Evans, R. M. (1997) Cell 90, 569-580[CrossRef][Medline] [Order article via Infotrieve]
11. Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X. Y., Sauter, G., Kallioniemi, O. P., Trent, J. M., and Meltzer, P. S. (1997) Science 277, 965-968[Abstract/Free Full Text]
12. Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 677-684[CrossRef][Medline] [Order article via Infotrieve]
13. Chrivia, J. C., Kwok, R. P. S., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993) Nature 365, 855-859[CrossRef][Medline] [Order article via Infotrieve]
14. Eckner, R., Ewen, M. E., Newsome, D., Gerdes, M., DeCaprio, J. A., Lawrence, J. B., and Livingston, D. M. (1994) Genes Dev. 8, 869-884[Abstract/Free Full Text]
15. Zhu, Y., Qi, C., Jain, S., Rao, M. S., and Reddy, J. K. (1997) J. Biol. Chem. 272, 25500-25506[Abstract/Free Full Text]
16. Yuan, C. X., Ito, M., Fondell, J. D., Fu, Z. Y., and Roeder, R. G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7939-7944[Abstract/Free Full Text]
17. Lin, J., Puigserver, P., Donovan, J., Tarr, P., and Spiegelman, B. M. (1998) Cell 92, 829-839[CrossRef][Medline] [Order article via Infotrieve]
18. Zhu, Y., Kan, L., Qi, C., Kanwar, Y. S., Yeldandi, A. V., Rao, M. S., and Reddy, J. K. (2000) J. Biol. Chem. 275, 13510-13516[Abstract/Free Full Text]
19. Lee, S-K., Anzick, S. L., Choi, J-E, Bubendorf, L., Guan, X-Y., Jung, Y-K., Kallioniemi, O. P., Kononen, J., Trent, J. M., Azorsa, D., Jhun, B-H., Cheong, J. H., Lee, Y. C., Meltzer, P. S., and Lee, J. W. (1999) J. Biol. Chem. 274, 34283-34293[Abstract/Free Full Text]
20. Mahajan, M., and Samuels, H. H. (2000) Mol. Cell. Biol. 20, 5048-5063[Abstract/Free Full Text]
21. Caira, F., Antonson, P., Pelto-Huikko, M., Treuter, E., and Gustafsson, J-A. (2000) J. Biol. Chem. 275, 5308-5317[Abstract/Free Full Text]
22. Ko, L., Cardona, G. R., and Chin, W. W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6212-6217[Abstract/Free Full Text]
23. Jung, D-J., Na, S-Y., Na, D. S., and Lee, J. W. (2002) J. Biol. Chem. 277, 1229-1234[Abstract/Free Full Text]
24. Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997) Nature 387, 733-736[CrossRef][Medline] [Order article via Infotrieve]
25. Goodman, R. H., and Smolik, S. (2000) Genes Dev. 14, 1553-1577[Free Full Text]
26. Vo, N., and Goodman, R. H. (2001) J. Biol. Chem. 276, 13505-13508[Free Full Text]
27. Crawford, S. E., Qi, C., Misra, P., Stellmach, V., Rao, M. S., Engel, J. D., Zhu, Y., and Reddy, J. K. (2002) J. Biol. Chem. 277, 3585-3592[Abstract/Free Full Text]
28. Spencer, T. E., Jenster, G., Burcin, M. M., Allis, C. D., Zhou, J. X., Mizzen, C. A., McKenna, N. J., Onate, S. A., Tsai, S. Y., Tsai, M-J., and O'Malley, B. W. (1997) Nature 389, 194-198[CrossRef][Medline] [Order article via Infotrieve]
29. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-959[CrossRef][Medline] [Order article via Infotrieve]
30. Blanco, J. C., Minucci, S., Lu, J., Yang, X-J., Walker, K., Chen, H., Evans, R. M., Nakatani, Y., and Ozato, K. (1998) Genes Dev. 12, 1638-1651[Abstract/Free Full Text]
31. Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S-M., Schurter, B. T., Aswad, D. W., and Stallcup, M. R. (1999) Science 284, 2174-2177[Abstract/Free Full Text]
32. Koh, S. S., Chen, D., Lee, Y-H., and Stallcup, M. R. (2001) J. Biol. Chem. 276, 1089-1098[Abstract/Free Full Text]
33. Xu, W., Chen, H., Du, K., Asahara, H., Tini, M., Emerson, B. M., Montminy, M., and Evans, R. M. (2001) Science 294, 2507-2511[Abstract/Free Full Text]
34. Rachez, C., Lemon, B. D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A. M., Erdjument-Bromage, H., Tempst, P., and Freedman, L. P. (1999) Nature 398, 824-828[CrossRef][Medline] [Order article via Infotrieve]
35. Naar, A. M., Beaurang, P. A., Zhou, S., Abraham, S., Solomon, W., and Tjian, R. (1999) Nature 398, 828-832[CrossRef][Medline] [Order article via Infotrieve]
36. Yao, T-P., Oh, S. P., Fuchs, M., Zhou, N-D., Ch'ng, L-E., Newsome, D., Bronson, R. T., Li, E., Livingston, D. L., and Eckner, R. (1998) Cell 93, 361-372[CrossRef][Medline] [Order article via Infotrieve]
37. Oike, Y., Takakura, N., Hata, A., Kaname, T., Akizuki, M., Yamaguchi, Y., Yasue, H., Araki, K., Yamamura, K., and Suda, T. (1999) Blood 93, 2771-2779[Abstract/Free Full Text]
38. Kung, A. L., Rebel, V. I., Bronson, R. T., Ch'ng, L-E, Siefff, C. A., Livingston, D. M., and Yao, T-P. (2000) Genes Dev. 14, 272-277[Abstract/Free Full Text]
39. Zhu, Y., Qi C., Jia, Y., Nye, J. S., Rao, M. S., and Reddy, J. K. (2000) J. Biol. Chem. 275, 14779-14782[Abstract/Free Full Text]
40. Ito, M., Yuan, C-X., Okano, H. J., Darnell, R. B., and Roeder, R. G. (2000) Mol. Cell 5, 683-693[CrossRef][Medline] [Order article via Infotrieve]
41. Zhu, Y., Qi, C., Cao, W-Q., Yeldandi, A. V., Rao, M. S., and Reddy, J. K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 10380-10385[Abstract/Free Full Text]
42. Yu, S., Cao, W-Q., Kashireddy, P., Meyer, K., Jia, Y., Hughes, D. E., Tan, Y., Feng, J., Yeldandi, A. V., Rao, M. S., Costa, R. H., Gonzalez, F. J., and Reddy, J. K. (2001) J. Biol. Chem. 276, 42485-42491[Abstract/Free Full Text]
43. Mitchell, K. O., and El-Deiry, W. S. (1999) Cell Growth & Differ. 10, 223-230[Abstract/Free Full Text]
44. Kurokawa, R., Kalafus, D., Ogliastro, M-H., Kioussi, C., Xu, L., Torchia, J., Rosenfeld, M. G., and Glass, C. K. (1998) Science 279, 700-703[Abstract/Free Full Text]
45. Lin, C. H., Hare, B. J., Wagner, G., Harrison, S. C., Maniatis, T., and Fraenkel, E. (2001) Mol. Cell 8, 581-590[CrossRef][Medline] [Order article via Infotrieve]
46. Qi, C., Zhu, Y., Pan, J., Yeldandi, A. V., Rao, M. S., Maeda, N., Subbarao, V., Pulikuri, S., Hashimoto, T., and Reddy, J. K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1585-1590[Abstract/Free Full Text]
47. Xu, J., Qiu, Y., DeMayo, F. J., Tsai, M. J., and O'Malley, B. W. (1998) Science 279, 1922-1925[Abstract/Free Full Text]
48. Dowell, P., Ishmael, J. E., Avram, D., Peterson, V. J., Nevrivy, D. J., and Leid, M. (1997) J. Biol. Chem. 272, 33435-33443[Abstract/Free Full Text]
49. Kagan, R. M., and Clarke, S. (1994) Arch. Biochem. Biophys. 310, 417-427[CrossRef][Medline] [Order article via Infotrieve]
50. Gary, J. D., and Clarke, S. (1998) Prog. Nucleic Acid Res. Mol. Biol. 61, 65-131[Medline] [Order article via Infotrieve]
51. Bossie, M. A., DeHoratius, C., Barcelo, G., and Silver, P. (1992) Mol. Biol. Cell 3, 875-893[Abstract]
52. Lei, E. P., Krebber, H., and Silver, P. A. (2001) Genes Dev. 15, 1771-1782[Abstract/Free Full Text]
53. McCulloch, V., Seidel-Rogol, B. L., and Shadel, G. S. (2002) Mol. Cell. Biol. 22, 1116-1125[Abstract/Free Full Text]

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