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 to the and in gene regulation P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1689) Google Scholar). is a key transcriptionally of shows that HG can increase nuclear and 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 of NF-κB been to a of coactivators including and R. S. D. T. Mol. Cell. Biol. 1999; PubMed Google Scholar) These have histone activity and key in the transcription S.D. Biol. 2000; PubMed Scopus Google Scholar, M. Rev. Mol. 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The of for can regulated gene expression. is from of transcription factors to transcriptional activation or is also to the recruitment of protein that chromatin and of histone NF-κB and to be involved in gene transcription under diabetic conditions, the specific in vivo regulation mechanisms at the of chromatin are not In the we that diabetic conditions lead to chromatin remodeling and key in the nuclear that inflammatory gene transcription and monocyte We have used chromatin immunoprecipitation (ChIP) assays to for the first time the of chromatin at the promoters of inflammatory genes in vivo in monocytes under diabetic conditions. We that HG culture of monocytes that diabetic conditions could increase the recruitment of such as and p/CAF to the promoters of inflammatory genes and leading to the acetylation of factors histone H3 (HH3) and HH4 also the of was concomitant recruitment of NF-κB to the promoters of key genes such as TNF-α and we demonstrated in vivo by histone acetylation in monocytes from diabetic results for the first time that in vivo chromatin remodeling under diabetic conditions in cell culture and in and were from were from was from was a from J. S. of expression was a from of for CBP/p300 and p/CAF were from and were from S. and and were from and were from were from and the was from THP-1 were as N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar) in and glucose or glucose and were in culture in of and with 1 of the (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), of expression and of CBP/p300 or p/CAF as in were for with were and for in and or were with as N. Reddy M.A. Guha M. Natarajan R. Diabetes. 2003; 52: 1256-1264Crossref PubMed Scopus (438) Google Scholar), and at for of ChIP were to and P.J. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar) with were with for with in inhibitor and to three for at of the by for of the was used for as were and in 2 inhibitor by with 1 of of and of for 1 at was for at with of specific were for in with and 1 were with and with were and at for at to reverse the were with 1 from a was to the as TNF-α, and reverse and reverse and reverse MCP-1, and reverse The products were and was as 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). was with of and a was used in with with 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 used to TNF-α were and from the and for and were for at for for and for in products were on and as 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). are as relative in for of of blood from with type 1 or type 2 and from normal was in the of in with an were from the blood to our 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 with to acetylated and as from the TNF-α by and p/CAF and by shows that NF-κB can with and its and also p/CAF R. S. D. T. Mol. Cell. Biol. 1999; PubMed Google Scholar, T. Science. PubMed Scopus Google Scholar, H. M.J. S. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), to and gene expression. is also that the activity of not that of CPB, is to transcription R. S. D. T. Mol. Cell. Biol. 1999; PubMed Google Scholar). HG can the expression of genes such as TNF-α, we first HG can and p65-mediated transcriptional activation of the TNF-α We THP-1 with a with or expression for p65, CBP, or p/CAF for and the under or HG conditions for in show that TNF-α transcription is on Furthermore, shows that this is increased under HG conditions Furthermore, CBP, and p/CAF could p65-mediated increase in activity under conditions and these are not stimulated by HG versus conditions and Interestingly, the transcriptional of as well as effects of CBP, or p/CAF and are under HG conditions. These results for the first time that HG also that HG can significantly increase the expression of key inflammatory genes NF-κB ChIP to to the of ChIP is a to in vivo of transcription factors and to chromatin in to an P.J. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar, V. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We used this to the of the NF-κB transcription at the promoters of key NF-κB-regulated genes in THP-1 in to we the recruitment of NF-κB and subunits and the and specific for of these the ChIP gene were and by specific NF-κB on the promoters of MCP-1, and of these genes are known to be regulated at in by THP-1 for in or HG were to ChIP first with an to of specific in the chromatin which of these ligand factors to were by was to a of the to from end used as which key κB increase in the relative of TNF-α shows increased by on the by HG treatment little or is in the of HG is in the of with to with that or to an on TNF-α gene is with of is in the of in the In addition, we also the promoters of NF-κB genes that are regulated by HG, MCP-1, and is in vivo to these gene promoters in to HG of in of to the TNF-α and COX-2 shows that HG treatment of THP-1 can increase in the (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, gene expression in is by of with and p/CAF R. S. D. T. Mol. Cell. Biol. 1999; PubMed Google Scholar). However, this not been in an in vivo or in to We therefore a time of HG treatment of THP-1 and with to p65, CBP, and acetylated at Lys9 and by with the or COX-2 with the TNF-α shows several 1) recruitment to the TNF-α as as HG treatment and 2) HG the recruitment of at a time as HG also the recruitment of p/CAF and this at these events also with the acetylation of The shows that is not by These results indicate for the first time that HG can histone acetylation in the TNF-α gene in vivo and that a TNF-α gene activation by HG and acetylated histone We also ChIP assays to at the human COX-2 which is also regulated by NF-κB 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, A. K. K. T. J. 1994; PubMed Scopus Google Scholar). shows the time of HG effects and that CBP, and are in a to the COX-2 to TNF-α is also with The is the transcriptional is to the COX-2 under conditions with time of HG the of bound bound Interestingly, is to in various and S. J. 2003; PubMed Scopus Google Scholar) and can gene These results that HG conditions can in HDAC to in vivo recruitment of transcription factors and and histone acetylation at inflammatory gene in in increased gene expression. Evidence shows that TNF-α can its gene expression. shows that TNF-α and recruitment and also the acetylation of HH3 at the TNF-α to the of Furthermore, TNF-α effects are augmented under HG conditions. of on HH3 and HH4 the of specific of histone acetylation at lysine residues for the of gene expression versus We therefore to which specific of HH3 and HH4 are acetylated at the TNF-α in to we ChIP assays with specific to HH3 acetylated at lysine (Lys (K)) or or to HH4 acetylated at Lys5, Lys8, or histone involved in gene shows that HG to a increase in the acetylation of Lys9 and on HH3 at the with Lys9 effects being more Furthermore, these events to p65, CBP, and p/CAF is by that can in and T. Science. PubMed Scopus Google Scholar) and that the activity of p/CAF is for R. S. D. T. Mol. Cell. Biol. 1999; PubMed Google Scholar). at and to be and also to HG, that acetylation at these not be to the transcriptional of by Interestingly, HH4 with to be acetylated at in the from the is to that this would be with genes. We the of acetylated on the COX-2 in to shows that also acetylation is increased in to HG in a to and p65, were in acetylation and acetylation the promoters. HH3 and HH4 on the TNF-α to be more acetylated with COX-2 Furthermore, the was to be acetylated at the COX-2 in the and acetylated by the and were not acetylated at the COX-2 These results indicate a of regulation at the of these results several genes have NF-κB on specific gene expression can be by the of histone acetylation as well as of chromatin factors in p65-mediated by acetylation is regulated by HATs, which and which the from the lysine residues of of of transcription factors from thereby leading to of gene transcription. that histone acetylation is to transcription in our we with or expression THP-1 show that this p65-mediated TNF-α activity the the HDAC inhibitor TSA, or HG, could activity in However, and were more potent in the or of or p65-mediated and is a specific inhibitor of multiple T. Science. PubMed Scopus Google Scholar, M.S. J.R. A. P.A. R. Nature. 1998; Scopus Google Scholar) and the of is to transcription and histone acetylation the of a the in vivo p65-mediated gene transcription and specific histone THP-1 were first with for ChIP assays were to the of acetylation of at and at the TNF-α and COX-2 promoters. The results in show that treatment acetylation at these Interestingly, at the to be more to than on COX-2 could be a of acetylation or the in of the specific and with these promoters. We also to TNF-α and COX-2 gene expression under these conditions. shows that TSA, to HG, could TNF-α and COX-2 expression. as in the relative is in the in of the These results that histone acetylation at these specific lysine gene expression. and in from with shows that hyperglycemia can lead to the transcription of inflammatory genes with NF-κB being of the key transcription factors shows that in HG can increase histone acetylation. Circulating monocytes in vivo in diabetic individuals would be continuously exposed to hyperglycemic conditions. the in vivo of our in THP-1 we histone acetylation in blood monocytes from with diabetes relative to from these human subjects were to with to acetylated histone or as shows the results of these ChIP is from normal and diabetic with type 1 diabetes 1 and and with type 2 diabetes 2 and Interestingly, is that diabetic show acetylation at the TNF-α and for also showed acetylation at the COX-2 the acetylation was and could be in However, was or acetylation in the normal of the with shows that diabetic have increase in acetylation relative to at the and COX-2 promoters These results of and acetylation with gene at the promoters of inflammatory genes in diabetic a more of chromatin for transcription. These with diabetic monocytes in vivo and molecular mechanisms by which HG, AGEs, and diabetes can the expression of key inflammatory genes. acetylation been to be with increased gene transcription S.D. Biol. 2000; PubMed Scopus Google Scholar, M. Rev. Mol. Biol. 2003; PubMed Scopus Google Scholar, T. Science. PubMed Scopus Google Scholar, Mol. Biol. Rev. 2000; PubMed Scopus Google Scholar, Rev. PubMed Scopus Google Scholar) and with transcriptionally chromatin of the to the transcriptional for gene expression. S. J. 2003; PubMed Scopus Google Scholar, P.A. Mol. PubMed Scopus Google Scholar), on the of on lysine residues of and as transcriptional or of genes. The of for regulated gene expression. Evidence shows that of inflammatory genes plays a major role in the of several However, very little is known about the regulation of histone acetylation and chromatin in disease such as diabetes and in this of the histone is that and of transcription factors are not for the of of gene expression. at in the of multiple transcription factor events can the recruitment of chromatin such as HATs, histone and These and on histone in the of specific of or and the of the G. S. S. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, Biol. 1997; PubMed Scopus Google Scholar, S.D. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar). Furthermore, a more regulatory role in gene transcription is the that acetylation in histone of NF-κB been to a of including and R. S. D. T. Mol. Cell. Biol. 1999; PubMed Google Scholar). the activity of p/CAF to be more R. S. D. T. Mol. Cell. Biol. 1999; PubMed Google Scholar). In this we have demonstrated for the first time that HG culture of monocytes mimicking diabetic conditions can lead to the recruitment in vivo of the key of as well as and and also acetylation of HH3 and In we showed for the first time that HG to not increased recruitment of such as and p/CAF leading to histone acetylation also of specific These factors increased recruitment of to the promoters of key genes such as TNF-α and We used ChIP assays to these in the nuclear in a thereby that diabetic conditions can in vivo chromatin remodeling events to inflammatory genes. we key in vivo our ChIP assays for the first time that monocytes from diabetic have increased acetylation of HH3 at the TNF-α and COX-2 promoters relative to under diabetic conditions, chromatin the of inflammatory genes is in an for gene transcription. of histone in a cell under normal conditions and transcription the of of the histone PubMed Scopus Google Scholar, Science. PubMed Scopus Google Scholar). The results to the of the histone acetylation and transcription in the a of histone such as acetylated which that the chromatin specific NF-κB-regulated genes is in an activation and are acetylated NF-κB various NF-κB-regulated genes to to the The TNF-α to be more than COX-2 as by the of histone acetylation. was acetylated at the TNF-α in the acetylated at the COX-2 in the and of acetylation of specific H3 and the of the specific gene regulated by thereby nuclear regulatory events at the of The by which NF-κB activity is could this However, the mechanisms regulating the NF-κB and are not In and are in a with the protein or as of the Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and can be in the W. F. M.J. M.A. W. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, O. M.A. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar). The is to promoters its or with transcription the protein to by with the of G. S. S. Mol. Cell. 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The role of histone acetylation under diabetic conditions is not These results to our the first that HG conditions and diabetes can in vivo chromatin remodeling and histone to increased of key transcription factors that inflammatory and genes. of specific gene histone acetylation in diabetic monocytes a molecular of increased inflammatory in these is in these histone to specific transcription However, of recently the of various histone that lead to gene activation or the role and in in role in disease such as diabetes not been that inflammatory gene regulation is an of these our results could lead to in of the to diabetes factors at the We N. for results Q. for in human and for We are to A. D. for and to J. S. S. and for as