Title: In Vivo Chromatin Remodeling Events Leading to Inflammatory Gene Transcription under Diabetic Conditions
Abstract: The transcription factor NF-κB (NF-κB) plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators including histone acetyltransferases (HATs) such as CBP/p300 and p/CAF. Evidence shows that high glucose (HG) conditions mimicking diabetes can activate the transcription of NF-κB-regulated inflammatory genes. However, the underlying in vivo transcription and nuclear chromatin remodeling events are unknown. We therefore carried out chromatin immunoprecipitation (ChIP) assays in monocytes to identify 1) chromatin factors bound to the promoters of tumor necrosis factor-α (TNF-α) and related NF-κB-regulated genes under HG or diabetic conditions, 2) specific lysine (Lys (K)) residues on histone H3 (HH3) and HH4 acetylated in this process. HG treatment of THP-1 monocytes increased the transcriptional activity of NF-κB p65, which was augmented by CBP/p300 and p/CAF. ChIP assays showed that HG increased the recruitment of NF-κB p65, CPB, and p/CAF to the TNF-α and COX-2 promoters. Interestingly, ChIP assays also demonstrated concomitant acetylation of HH3 at Lys9 and Lys14, and HH4 at Lys5, Lys8, and Lys12 at the TNF-α and COX-2 promoters. Overexpression of histone deacetylase (HDAC) isoforms inhibited p65-mediated TNF-α transcription. In contrast, a HDAC inhibitor stimulated gene transcription and histone acetylation. Finally, we demonstrated increased HH3 acetylation at TNF-α and COX-2 promoters in human blood monocytes from type 1 and type 2 diabetic subjects relative to nondiabetic. These results show for the first time that diabetic conditions can increase in vivo recruitment of NF-κB and HATs, as well as histone acetylation at the promoters of inflammatory genes, leading to chromatin remodeling and transcription. The transcription factor NF-κB (NF-κB) plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators including histone acetyltransferases (HATs) such as CBP/p300 and p/CAF. Evidence shows that high glucose (HG) conditions mimicking diabetes can activate the transcription of NF-κB-regulated inflammatory genes. However, the underlying in vivo transcription and nuclear chromatin remodeling events are unknown. We therefore carried out chromatin immunoprecipitation (ChIP) assays in monocytes to identify 1) chromatin factors bound to the promoters of tumor necrosis factor-α (TNF-α) and related NF-κB-regulated genes under HG or diabetic conditions, 2) specific lysine (Lys (K)) residues on histone H3 (HH3) and HH4 acetylated in this process. HG treatment of THP-1 monocytes increased the transcriptional activity of NF-κB p65, which was augmented by CBP/p300 and p/CAF. ChIP assays showed that HG increased the recruitment of NF-κB p65, CPB, and p/CAF to the TNF-α and COX-2 promoters. Interestingly, ChIP assays also demonstrated concomitant acetylation of HH3 at Lys9 and Lys14, and HH4 at Lys5, Lys8, and Lys12 at the TNF-α and COX-2 promoters. Overexpression of histone deacetylase (HDAC) isoforms inhibited p65-mediated TNF-α transcription. In contrast, a HDAC inhibitor stimulated gene transcription and histone acetylation. Finally, we demonstrated increased HH3 acetylation at TNF-α and COX-2 promoters in human blood monocytes from type 1 and type 2 diabetic subjects relative to nondiabetic. These results show for the first time that diabetic conditions can increase in vivo recruitment of NF-κB and HATs, as well as histone acetylation at the promoters of inflammatory genes, leading to chromatin remodeling and transcription. Certain key factors have been implicated in diabetes-induced accelerated atherosclerotic and inflammatory disease including hyperglycemia (1Ruderman N. Williamson J.R. Brownlee M. 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We recently showed that in vitro culture of monocytes under high glucose (HG) versus normal glucose (NG) conditions led to the activation of the transcription factor nuclear factor κB (NF-κB) and significantly increased the expression of several inflammatory chemokines and cytokines (9Guha M. Bai W. Nadler J. Natarajan R. J. Biol. Chem. 2000; 275: 17728-17739Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 10Shanmugam N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar). The potent inflammatory cytokine, tumor necrosis factor-α (TNF-α), chemokine, and monocyte chemoattractant protein-1 (MCP-1) were among the factors induced (9Guha M. Bai W. Nadler J. Natarajan R. J. Biol. Chem. 2000; 275: 17728-17739Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 10Shanmugam N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar). Furthermore, these factors were transcriptionally regulated by HG with NF-κB being a major regulatory factor. In addition, the involvement of multiple upstream signaling pathways such as oxidant stress, protein kinase C, and mitogen-activated protein kinases were demonstrated (9Guha M. Bai W. Nadler J. Natarajan R. J. Biol. Chem. 2000; 275: 17728-17739Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 10Shanmugam N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar). Recent studies, including our own, indicate that HG, AGEs, and the receptor of AGE ligand S100B can activate NF-κB in vitro in monocytes and lead to the expression of inflammatory genes such as TNF-α, MCP-1, interleukins, and cyclooxygenase-2 (COX-2) (9Guha M. Bai W. Nadler J. Natarajan R. J. Biol. Chem. 2000; 275: 17728-17739Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 10Shanmugam N. Reddy M.A. Guha M. Natarajan R. 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Lee V. Morcos M. Tritschler H. Ziegler R. Wahl P. Bierhaus A. Nawroth P.P. Diabetes Care. 1998; 21: 1310-1316Crossref PubMed Scopus (140) Google Scholar). However, very little is known about the specific molecular in vivo transcription mechanisms and nuclear chromatin remodeling events underlying the regulation under diabetic conditions of these NF-κB-regulated inflammatory genes implicated in monocyte activation and dysfunction. NF-κB is an inducible transcription factor that plays a pivotal role in regulating the expression of more than 100 genes including TNF-α, MCP-1, and COX-2 (15Leonardo M.J. Baltimore D. Cell. 1998; 58: 227-229Abstract Full Text PDF Scopus (1259) Google Scholar, 16Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1689) Google Scholar). NF-κB is a heterodimer that usually consists of 65- and 50-kDa subunits (p65 and p50) complexed to its inhibitor, IκB-α, in the cytoplasm (15Leonardo M.J. Baltimore D. Cell. 1998; 58: 227-229Abstract Full Text PDF Scopus (1259) Google Scholar, 16Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1689) Google Scholar). Upon cell stimulation by stimuli such as TNF-α, the IκB unit is phosphorylated, ubiquitinated, and degraded, thereby allowing free NF-κB heterodimer to be transported to the nucleus and participate in gene regulation (16Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1689) Google Scholar). p65 is a key transcriptionally active component of NF-κB. Our recent data shows that HG can increase nuclear p65 and decrease cytosolic IκB-α levels in monocytes (9Guha M. Bai W. Nadler J. Natarajan R. J. Biol. Chem. 2000; 275: 17728-17739Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The nuclear transactivation potential of NF-κB has been shown to require a number of different coactivators including CBP/p300, p/CAF, and SRC-1 (17Sheppard K.A. Rose D.W. Haque Z.A. Kurokawa R. Mcinerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar) These cofactors have histone acetyltransferase (HAT) activity and play key roles in the transcription machinery (18Cheung W.L. Briggs S.D. Allis C.D. Curr. Opin. Cell Biol. 2000; 12: 326-333Crossref PubMed Scopus (262) Google Scholar, 19Kurdistani S.K. Grunstein M. Nat. Rev. Mol. Cell Biol. 2003; 4: 276-284Crossref PubMed Scopus (555) Google Scholar). Histone acetylation has been shown to be associated with increased gene transcription (18Cheung W.L. Briggs S.D. Allis C.D. Curr. Opin. Cell Biol. 2000; 12: 326-333Crossref PubMed Scopus (262) Google Scholar, 19Kurdistani S.K. Grunstein M. Nat. Rev. Mol. Cell Biol. 2003; 4: 276-284Crossref PubMed Scopus (555) Google Scholar, 20Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1079Crossref PubMed Scopus (7709) Google Scholar, 21Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-439Crossref PubMed Scopus (1412) Google Scholar, 22Roth S.Y. Jenu J.M. Allis C.D. Annu. Rev. Biochem. 2001; 70: 81-120Crossref PubMed Scopus (1624) Google Scholar). Acetylated histone proteins confer accessibility of the DNA template to the transcriptional machinery for gene expression. Histone deacetylases (HDACs), on the other hand, catalyze removal of acetyl groups on amino-terminal lysine residues and act as transcriptional repressors or silencers of genes (23de Ruijter A.J. van Gennip A.H. Caron H.N. Kemp S. van Kuilenburg A.B. Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2494) Google Scholar, 24Wade P.A. Hum. Mol. Genet. 2001; 10: 693-698Crossref PubMed Scopus (280) Google Scholar). The exchange of HATs for HDACs can mediate regulated gene expression. It is now clear that, apart from binding of transcription factors to their promoter DNA binding sites, transcriptional activation or repression is also linked to the recruitment of protein complexes that alter chromatin structure and architecture via enzymatic modifications of histone tails and/or nucleosome remodeling. Thus, although NF-κB and p65 seem to be involved in gene transcription under diabetic conditions, the specific in vivo regulation mechanisms at the level of chromatin are not known. In the present study, we hypothesized that diabetic conditions lead to chromatin remodeling and key alterations in the nuclear transcriptome that induce inflammatory gene transcription and enhanced monocyte activation. We have effectively used chromatin immunoprecipitation (ChIP) assays to demonstrate for the first time the occurrence of chromatin rearrangements at the promoters of inflammatory genes in vivo in monocytes under diabetic conditions. We noted that HG culture of monocytes that mimics diabetic conditions could specifically increase the recruitment of coactivator HATs such as CBP and p/CAF to the promoters of inflammatory genes and leading to the acetylation of nucleosomal factors histone H3 (HH3) and HH4 while also decreasing the association of HDACs. There was concomitant recruitment of NF-κB p65 to the promoters of key HG-induced NF-κB-dependent genes such as TNF-α and COX-2. Additionally, we demonstrated in vivo relevance by examining histone acetylation patterns in monocytes from diabetic patients. Our results reveal for the first time that in vivo chromatin remodeling occurs under diabetic conditions in cell culture and in patients. Materials—Anti-p65, anti-p50, and anti-CBP antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All others antibodies were from Upstate Biotechnology (Lake Placid, NY). Trichostatin (TSA) was from Sigma. 295TNF-αLuc plasmid was a gift from Dr. J. S. Economou (University of California, Los Angeles). p65 expression vector (hemagglutinin-tagged) was a gift from Dr. E. Zandi (University of Southern California). Expression vectors for CBP/p300 and p/CAF were gifts from Dr. B. Forman (Beckman Research Institute, Duarte, CA), HDAC1, HDAC4, HDAC5, and HDAC6 were from Dr. S. Schreiber (Harvard University, Boston, MA), and HDAC2 and HDAC3 were from Dr. Li Li (Wayne State University, Detroit, MI). Effectene transfection reagent and plasmid DNA isolation kits were from Qiagen (Valencia, CA), PCR reagents were from Applied Biosystems (Foster City, CA), and the luciferase assay system was from Promega (Madison, WI). Cell Culture—Human THP-1 monocytic cells were maintained as described (10Shanmugam N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar) in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, HEPES (10 mm), glutamine (2 mm), streptomycin (50 μg/ml), penicillin (50 units/ml), β-mercaptoethanol (50 μm), and 5.5 mm glucose (Normal glucose, NG) or 25 mm glucose (HG). DNA Transfections and Luciferase Assays—2 × 106 cells were placed in 6-well culture dishes in 1.5 ml of RPMI 1640 medium and transfected with 1 μg of the 295TNF-αLuc construct (9Guha M. Bai W. Nadler J. Natarajan R. J. Biol. Chem. 2000; 275: 17728-17739Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), 0.2 μg of p65 expression vector, and 3 μg of CBP/p300 or p/CAF plasmids as needed, using Effectene in 2% serum-containing medium overnight. Samples were balanced for total DNA content with control plasmid. Cells were then washed and cultured for 24 h in complete medium containing 10% fetal calf serum and either NG or HG. Cells were then washed with phosphate-buffered saline, lysed as described previously (10Shanmugam N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar), and stored at -70 °C for determination of luciferase activity. ChIP Assays—These were performed according to Boyd and Farnham (25Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1999; 19: 8393-8399Crossref PubMed Scopus (147) Google Scholar) with some modifications. Briefly, cells were cross-linked with 1% formaldehyde for 30-60 min, washed twice with cold phosphate-buffered saline, resuspended in lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1, 1× protease inhibitor mixture (Roche Applied Science)) and sonicated one to three times for 30 s each at 40% maximum setting of the sonicator (Branson Sonifier, model 250) followed by centrifugation for 10 min. One-tenth of the total lysate was used for total genomic DNA as “Input DNA” control. Supernatants were collected and diluted in buffer (1% Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.1, 1× protease inhibitor mixture) followed by immunoclearing with 1 μg of sheared salmon sperm DNA, 10 μl of rabbit IgG, and 20 μl of protein-A-Sepharose (Upstate Biotechnology) for 1 h at 4 °C. Immunoprecipitation was performed for 15 h at 4 °C with 2-5 μg each of specific antibodies. Precipitates were washed sequentially for 10 min each in TSE I buffer (0.1% SDS, 1% Triton X-100, 2mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.1), TSE II (TSE I with 500 mm NaCl), and buffer III (0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, 10 mm Tris-HCl, pH 8.1). Precipitates were then washed twice with TE buffer (10 mm Tris-HCl, pH 7.5, 0.1 mm EDTA) and extracted twice with 1% SDS containing 0.1 m NaHCO3. Eluates were pooled and heated at 65 °C for at least 6 h to reverse the formaldehyde cross-linking. DNA fragments were purified with Qiagen Qiaquick spin kit. For PCR, 1 μl from a 50-μl DNA extraction was used. PCR primers correspond to sequences within the promoter regions as follows: TNF-α, forward (5′-CCCTCCAGTTCTAGTTCTATC-3′) and reverse (5′-GGGGAAAGAATCATTCAACCAG-3′); COX-2, forward (5′-CAAGGCGATCAGTCCAGAAC-3′) and reverse (5′-GGTAGGCTTTGCTGTCTGAG-3′); interleukin-6 (IL-6), forward (5′-TTGCGATGCTAAAGGACG-3′) and reverse (5′TGTGGAGAAGGAGTTCATAGC-3′); MCP-1, forward (5′-GCCTTTGCATATATCAGACA-3′) and reverse (5′-CAGGCTTGTGCCGAGATGTTC-3′). The corresponding PCR products sizes were 371, 464, 257, and 323 bp, respectively. RT-PCR—Total RNA was extracted as described previously (10Shanmugam N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar, 12Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). cDNA was generated with 0.6 μg of RNA using random hexamers and Maloney murine leukemia virus RT using a GeneAmp RNA PCR kit. 1/20th fraction was used in multiplex RT-PCRs with cytokine-specific primers paired with Quantum 18 S RNA internal standards (10Shanmugam N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar, 12Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The primers used to amplify TNF-α cDNA were 5′-ATGGCCACACTGACTCTCCT-3′ and 5′-TAGATGGGCTCATACCAGGG-3′ (product: 346 bp) from the coding region; and for COX-2, 5′-CAGCACTTCACGCATCAGTT-3′ and 5′-TCTGGTCAATGGAAGCCTGT-3′ (product: 756 bp). PCRs were performed for 25-30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 40 s in GeneAmp 9700 PCR machine (Applied Biosystems). PCR products were fractioned on 2% agarose gels and quantitated as described (10Shanmugam N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar, 12Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Results are expressed as relative changes in intensity after normalization for 18 S RNA levels. Isolation of Human Peripheral Blood Monocytes (PBMC)—50-60 ml of blood from adult volunteers with established type 1 or type 2 diabetes, and from normal healthy donors, was collected in the presence of anti-coagulant in accordance with an approved Institutional Review Board protocol. Monocytes were isolated from the blood according to our reported procedure (12Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). ChIP assays were then performed with antibodies to acetylated H3K9 and H3K14 as described earlier. p65-dependent Transcription from the TNF-α Promoter Is Augmented by CBP and p/CAF and Further Enhanced by HG—Evidence shows that NF-κB p65 can interact with CBP and its structural homolog p300, and also p/CAF (17Sheppard K.A. Rose D.W. Haque Z.A. Kurokawa R. Mcinerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar, 20Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1079Crossref PubMed Scopus (7709) Google Scholar, 26Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar), to stimulate NF-κB-dependent transactivation and gene expression. There is also evidence that the HAT activity of p/CAF, but not that of CPB, is required to coactivate p65-dependent transcription (17Sheppard K.A. Rose D.W. Haque Z.A. Kurokawa R. Mcinerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar). Since HG can augment the expression of NF-κB-dependent genes such as TNF-α, we first examined whether HG can affect CBP/p300-, p/CAF-, and p65-mediated transcriptional activation of the TNF-α promoter. We transfected THP-1 cells with a minimal hTNF-α promoter (295TNF-αLuc) with or without expression vectors for p65, CBP, p300 or p/CAF for 16 h and then cultured the cells under NG (5.5 mm) or HG (25 mm) conditions for 24 h. Luciferase activities in Fig. 1A show that TNF-α transcription is dependent on p65. Furthermore, Fig. 1B shows that this is increased under HG conditions (second bar set). Furthermore, CBP, p300, and p/CAF could each clearly enhance p65-mediated increase in TNF promoter activity under NG conditions and these are slightly, but not significantly, stimulated by HG versus NG conditions (third, fourth, and fifth bar sets). Interestingly, the transcriptional activities of p65 (second bar set) as well as coactivator effects of CBP, p300, or p/CAF (sixth, seventh, and eighth bar sets) are clearly enhanced under HG conditions. These results suggest for the first time that HG may enhance coactivator HAT recruitment. They also further support earlier observations that HG can significantly increase the expression of key inflammatory genes via NF-κB activation. ChIP Assays to Evaluate p65 Binding to the Promoters of Inflammatory Genes—The ChIP assay is a powerful technique to determine true in vivo binding of transcription factors and other nucleosomal proteins to chromatin in response to an agonist (25Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1999; 19: 8393-8399Crossref PubMed Scopus (147) Google Scholar, 27Orlando V. Trends Biochem. Sci. 2000; 25: 99-104Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). We used this assay to determine the status of the NF-κB transcription complex at the promoters of key NF-κB-regulated genes in THP-1 cells in response to HG. Specifically we evaluated the recruitment of NF-κB p65 and p50 subunits and the HAT coactivators, CBP and p/CAF, using antibodies specific for each of these factors. After the ChIP protocol, gene promoter regions were amplified and analyzed by semiquantitative PCRs using specific primer pairs around NF-κB binding regions on the promoters of TNFα, MCP-1, COX-2, and IL-6. All of these genes are known to be regulated at least in part by NF-κB. THP-1 cells cultured for 3 days in NG or HG were subjected to ChIP first with an antibody (Ab) to p65. Enrichment of specific DNA sequences in the chromatin immunoprecipitates, which indicates association of these ligand factors to DNA strands within intact chromatin, were visualized by PCR amplification. PCR was designed to amplify a 486-bp region of the hTNF-α promoter (-453 to +33) (24 bp from either end used as primers), which contains key κB binding elements. An increase in the relative amount of amplified TNF-α promoter-specific PCR product indicates binding. Fig. 2A clearly shows selective increased occupancy by p65 on the TNFα promoter by 48-h HG treatment (over 5-fold). Very little or no binding is seen in NG. Important controls demonstrate the specificity of HG effects, since there is no amplified band in the absence of Ab (lane 1), with mannitol (MANN, band similar to NG), with primers that amplify GAPDH promoter, or to an open reading frame exoncoding region on TNF-α gene (EXON). Control amplification is with total input DNA (Input DNA) (lower panel of Fig. 2A). There is no change in the amplification of input DNA in all the cases. In addition, we also examined the promoters of other NF-κB target genes that are regulated by HG, namely MCP-1, COX-2, and IL-6. p65 is again similarly recruited in vivo to these gene promoters in response to HG (Fig. 2B). Time Course of HG-induced in Vivo Recruitment of Chromatin Remodeling Factors to the TNF-α and COX-2 Promoters—Evidence shows that HG treatment of THP-1 cells can increase p65 levels in the nuclei (9Guha M. Bai W. Nadler J. Natarajan R. J. Biol. Chem. 2000; 275: 17728-17739Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Furthermore, NF-κB-dependent gene expression in other cells is enhanced by association of p65 with CBP and p/CAF (17Sheppard K.A. Rose D.W. Haque Z.A. Kurokawa R. Mcinerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar). However, this has not been observed in an in vivo context or in response to HG. We therefore did a time course of HG treatment of THP-1 cells and performed ChIPs with antibodies to p65, CBP, p/CAF, and acetyl-HH3 (Ab recognizing histones acetylated at Lys9 and Lys14) followed by PCR analyses with primers amplifying the TNF or COX-2 promoter regions containing NF-kB binding sites. Fig. 3A with the TNF-α promoter shows several interesting new results: 1) p65 recruitment to the TNF-α promoter occurs as early as 16 h after HG treatment and falls off only after 72 h; 2) HG clearly increases the recruitment of CBP at a similar time frame as p65; 3) HG also increases the recruitment of p/CAF and this appears sustained even at 72 h; 4) these events also coincide with the acetylation of HH3. The lowest panel shows that input DNA is not altered by HG. These results indicate for the first time that HG can induce histone acetylation in the TNF-α gene locus in vivo and provides novel evidence that a close relationship exists between TNF-α gene activation by HG and acetylated histone accumulation. We also conducted ChIP assays to evaluate changes at the human COX-2 promoter, which is also regulated by NF-κB (12Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 28Appleby S.B. Ristimaki A. Nielson K. Narko K. Hla T. Biochem. J. 1994; 302: 723-727Crossref PubMed Scopus (460) Google Scholar). Fig. 3B shows the time course (16-24 h) of HG effects and indicates that CBP, p/CAF, and p65 are recruited in a temporal sequence to the COX-2 promoter similar to TNF-α promoter. p50 is also recruited with similar kinetics. The intriguing new result is that, while the transcriptional repressor HDAC1 is associated to the COX-2 promoter under basal NG conditions (lane 1), however, with increasing time of HG treatment, the levels of bound HDAC1 steadily decreased while bound CBP levels increased. Interestingly, HDAC1 is reported to appear in complexes containing various corepressors like Sin3 and Mi-2-NuRD complex (23de Ruijter A.J. van Gennip A.H. Caron H.N. Kemp S. van Kuilenburg A.B. Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2494) Google Scholar) and hence can mediate gene repression. These new results suggest that HG conditions can elicit dynamic alterations in HDAC to HAT activities, in vivo recruitment of transcription factors and coactivators, and histone acetylation at inflammatory gene loci in vivo, culminating in increased gene expression. Evidence shows that TNF-α can auto regulate its own gene expression. Fig. 3C shows that TNF-α increases p65 and CBP recruitment and also the acetylation of HH3 at the TNF-α promoter similar to the actions of HG. Furthermore, TNF-α effects are augmented under HG conditions. Identification of Specific Lysines Acetylated on HH3 and HH4 —Emerging data indicates the importance of specific patterns of histone acetylation at lysine residues for the final outcome of gene expression versus silencing. We therefore wanted to determine which specific lysines of HH3 and HH4 are acetylated at the TNF-α promoter in response to HG. For these, we performed ChIP assays with specific Abs to HH3 acetylated at lysine (Lys (K)) 9 or Lys14 or Abs to HH4 acetylated at Lys5, Lys8, Lys12, or Lys16 (key histone modifications involved in gene transcription). Fig. 4A shows that HG leads to a time-dependent increase in the acetylation of Lys9 and Lys14 on HH3 at the TNF promoter region, with Lys9 effects being more intense. Furthermore, it appears that, temporally, these events occur parallel to p65, CBP, and p/CAF recruitment. This is supporte