Chromatin Modification and the Endothelial-specific Activation of the E-selectin Gene

Abstract

E-selectin plays a role in the binding and extravasation of leukocytes from the bloodstream. The E-selectin gene is rapidly and transiently expressed by endothelial cells activated by inflammatory stimuli. Despite the identification of factors critical for cytokine-induced activation of the E-selectin promoter, little is known about the mechanisms that restrict the gene expression to endothelial cells. We used in vivo approaches to characterize the E-selectin promoter in primary cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells. In endothelial cells specifically, nucleosomes are remodeled after tumor necrosis factor (TNF) α induction. Chromatin immunoprecipitation analysis demonstrated the binding of the p65 (RelA) component of nuclear factor-κB (NF-κB) to the endogenous E-selectin promoter after TNFα stimulation along with IκB kinase α. Multiple coactivators, including p300, steroid receptor coactivator-1, and p300/cAMP-response element-binding protein (CREB)-binding protein (CBP)-associated factor localize differentially to the E-selectin promoter. Additionally, TNFα induced localized histone hyperacetylation, phosphorylation, and methylation in the E-selectin gene specifically in endothelial cells. Post-induction repression of E-selectin expression is associated with recruitment of multiple deacetylases. Collectively, these studies suggest a model for the selective induction of the E-selectin gene in which the core promoter chromatin architecture is specifically modified in endothelial cells. E-selectin plays a role in the binding and extravasation of leukocytes from the bloodstream. The E-selectin gene is rapidly and transiently expressed by endothelial cells activated by inflammatory stimuli. Despite the identification of factors critical for cytokine-induced activation of the E-selectin promoter, little is known about the mechanisms that restrict the gene expression to endothelial cells. We used in vivo approaches to characterize the E-selectin promoter in primary cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells. In endothelial cells specifically, nucleosomes are remodeled after tumor necrosis factor (TNF) α induction. Chromatin immunoprecipitation analysis demonstrated the binding of the p65 (RelA) component of nuclear factor-κB (NF-κB) to the endogenous E-selectin promoter after TNFα stimulation along with IκB kinase α. Multiple coactivators, including p300, steroid receptor coactivator-1, and p300/cAMP-response element-binding protein (CREB)-binding protein (CBP)-associated factor localize differentially to the E-selectin promoter. Additionally, TNFα induced localized histone hyperacetylation, phosphorylation, and methylation in the E-selectin gene specifically in endothelial cells. Post-induction repression of E-selectin expression is associated with recruitment of multiple deacetylases. Collectively, these studies suggest a model for the selective induction of the E-selectin gene in which the core promoter chromatin architecture is specifically modified in endothelial cells. E-selectin is a member of the selectin family of adhesion molecules that play a central role in the binding and extravasation of leukocytes from the bloodstream into sites of inflammation (for review, see Refs. 1Kansas G.S. Blood. 1996; 88: 3259-3287Crossref PubMed Google Scholar and 2Pober J.S. Arthritis Res. 2002; 4: 109-116Crossref PubMed Scopus (152) Google Scholar). E-selectin gene expression is rapidly up-regulated upon exposure to inflammatory cytokines such as tumor necrosis factor α (TNFα) 1The abbreviations used are: TNF, tumor necrosis factor; HDAC, histone deacetylase; CHART PCR, chromatin accessibility real-time PCR; ChIP; chromatin immunoprecipitation; CRR, cytokine response region; HUVEC, human umbilical vein endothelial cell; p/CAF, p300/CBP-associated factor; IKK, IκB kinase; TBP, TATA-binding protein; RT, reverse transcription; SCR-1, steroid receptor coactivator; UASMC, umbilical artery smooth muscle cell.1The abbreviations used are: TNF, tumor necrosis factor; HDAC, histone deacetylase; CHART PCR, chromatin accessibility real-time PCR; ChIP; chromatin immunoprecipitation; CRR, cytokine response region; HUVEC, human umbilical vein endothelial cell; p/CAF, p300/CBP-associated factor; IKK, IκB kinase; TBP, TATA-binding protein; RT, reverse transcription; SCR-1, steroid receptor coactivator; UASMC, umbilical artery smooth muscle cell. (3Bevilacqua M.P. Stengelin S. Gimbrone Jr., M.A. Seed B. Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1658) Google Scholar). The increase in mRNA after the induction is due to an increase in the rate of gene transcription, which is maintained for several hours in the continuous presence of cytokine (4Read M.A. Neish A.S. Gerritsen M.E. Collins T. J. Immunol. 1996; 157: 3472-3479PubMed Google Scholar, 5Whelan J. Ghersa P. Hooft van Huijsduijnen R. Gray J. Chandra G. Talabot F. DeLamarter J.F. Nucleic Acids Res. 1991; 19: 2645-2653Crossref PubMed Scopus (202) Google Scholar). After removal of TNFα, there is a rapid post-induction transcriptional repression, with gene expression falling to basal levels within 24 h. Studies of the regulation of E-selectin gene expression have identified promoter elements that regulate inflammatory cytokine inducibility. Sequences within the first 160 bp upstream of the transcriptional start site, the cytokine response region (CRR), are necessary for efficient cytokine-inducible expression of the human E-selectin promoter in transient transfection assays. This promoter region contains binding sites for nuclear factor-κB (NF-κB) and activating transcription factor-2/c-JUN transcription factors as well as the architectural factor, high mobility group I(Y) (HMG-I(Y)) (6Kaszubska W. Hooft van Huijsduijnen R. Ghersa P. DeRaemy-Schenk A.M. Chen B.P. Hai T. DeLamarter J.F. Whelan J. Mol. Cell. Biol. 1993; 13: 7180-7190Crossref PubMed Google Scholar, 7Lewis H. Kaszubska W. DeLamarter J.F. Whelan J. Mol. Cell. Biol. 1994; 14: 5701-5709Crossref PubMed Google Scholar, 8Schindler U. Baichwal V.R. Mol. Cell. Biol. 1994; 14: 5820-5831Crossref PubMed Google Scholar, 9Whitley M.Z. Thanos D. Read M.A. Maniatis T. Collins T. Mol. Cell. Biol. 1994; 14: 6464-6475Crossref PubMed Scopus (177) Google Scholar). Mutations within any of these sites severely reduces or eliminates inducible expression from the E-selectin promoter. The ability of the transcriptional activators to stimulate E-selectin gene expression depends upon their specific spatial arrangement (10Meacock S. Pescini-Gobert R. DeLamarter J.F. Hooft van Huijsduijnen R. J. Biol. Chem. 1994; 269: 31756-31762Abstract Full Text PDF PubMed Google Scholar) and their ability to recruit a series of coactivators (11Sheppard K.A. Rose D.W. Haque Z.K. 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, 12Merika M. Williams A.J. Chen G. Collins T. Thanos D. Mol. Cell. 1998; 1: 277-287Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). NF-κB inhibition, mRNA degradation, and post-induction repression all play a role in the down-regulation of E-selectin gene expression (4Read M.A. Neish A.S. Gerritsen M.E. Collins T. J. Immunol. 1996; 157: 3472-3479PubMed Google Scholar, 13Kluger M.S. Johnson D.R. Pober J.S. J. Immunol. 1997; 158: 887-896PubMed Google Scholar, 14Chu W. Presky D.H. Swerlick R.A. Burns D.K. J. Immunol. 1994; 153: 4179-4189PubMed Google Scholar). Despite the identification of factors critical for cytokine-induced activation of the E-selectin promoter, little is known about the mechanisms that restrict the gene expression to endothelial cells. In fact, the activators that regulate the cytokine response region in the E-selectin promoter are broadly expressed, and the proximal promoter of the E-selectin gene is inducible in both endothelial and non-endothelial cells when it is used in promoter-reporter constructs and transient transfection assays. Mechanisms must exist that allow NF-κB to activate only specific genes in a given cell type. Because the higher order structure of chromatin represents an obstacle to the binding of transcription factors and the formation of preinitiation complexes (for review, see Ref. 15Adelman K. Lis J.T. Mol. Cell. 2002; 9: 451-452Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), we hypothesized that chromatin remodeling might be key in controlling the selective induction of many NF-κB-dependent inflammatory genes, including E-selectin (16Agalioti T. Lomvardas S. Parekh B. Yie J. Maniatis T. Thanos D. Cell. 2000; 103: 667-678Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 17Lomvardas S. Thanos D. Cell. 2002; 110: 261-271Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 18Rao S. Gerondakis S. Woltring D. Shannon M.F. J. Immunol. 2003; 170: 3724-3731Crossref PubMed Scopus (105) Google Scholar, 19Weinmann A.S. Plevy S.E. Smale S.T. Immunity. 1999; 11: 665-675Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). A second potential mechanism for controlling chromatin structure is selective histone modification. The histone octamers around which genomic is be modified their and as to complexes that or expression of the region (for review, see Ref. W. 2003; PubMed Scopus Google Scholar). In the histone as an that a of genes that be expressed in a given cell in response to a T. Science. PubMed Scopus Google Scholar). studies of NF-κB-dependent gene expression suggest that histone play a role in the regulation of specific genes S. S. G. Immunol. 2002; PubMed Scopus Google Scholar, S. G. 2002; PubMed Scopus (177) Google Scholar, T. Chen G. Thanos D. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). we chromatin immunoprecipitation and analysis to that the induction of E-selectin gene expression is associated with localized hyperacetylation, phosphorylation, and methylation of as well as remodeling the promoter. the localized histone the in the E-selectin in smooth muscle that these chromatin are an of the expression of the E-selectin and umbilical vein endothelial cells from human umbilical and as Jr., M.A. Google Scholar). maintained in and endothelial umbilical artery smooth muscle cells from and maintained to the from the and and and and of D. M. of W. of cultures of to in and with human TNFα for the the and the a of used with the in with to and E-selectin the in in and a real-time are as the of E-selectin mRNA to the cells and to the of of the with and of the used for and CHART in a Chromatin as M. H. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar, H. R. 1997; 11: PubMed Scopus Google Scholar) with the Chromatin from of or of smooth muscle cells. for the with The cells with for to from a the of the by the and an The chromatin into to reverse and The of real-time The in Additionally, from chromatin real-time The in chromatin chromatin of chromatin with the Additionally, an from the chromatin used for a to the binding for in with of with and the from an with from the chromatin Additionally, of the chromatin by the of an genomic by real-time are as the of to the In chromatin are are only about the presence or of a protein a of Because a binding to be about the of a of the chromatin accessibility real-time S. E. Shannon M.F. J. Immunol. PubMed Scopus Google Scholar). or with TNFα for or The cells and in and in and The in and in of the with the of of with the of for The by the of and of K. the of from or cells used in real-time with the in as the of from the cells to the cells. TNFα of the E-selectin the of the human E-selectin promoter upon induction of the gene by TNFα, we primary cultures of which are known to be to inflammatory group and many demonstrated that upon exposure to TNFα, these cells rapidly the E-selectin adhesion the induction of E-selectin mRNA in endothelial real-time cells to human TNFα for and h. and used to the of E-selectin mRNA to the expression of of E-selectin gene expression after the of E-selectin mRNA levels to the and After of continuous exposure to TNFα, levels of E-selectin mRNA to the levels of cells. are with of the regulation of E-selectin gene expression (3Bevilacqua M.P. Stengelin S. Gimbrone Jr., M.A. Seed B. Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1658) Google Scholar, M.A. Neish A.S. Gerritsen M.E. Collins T. J. Immunol. 1996; 157: 3472-3479PubMed Google Scholar, 5Whelan J. Ghersa P. Hooft van Huijsduijnen R. Gray J. Chandra G. Talabot F. DeLamarter J.F. Nucleic Acids Res. 1991; 19: 2645-2653Crossref PubMed Scopus (202) Google Scholar). the induction of E-selectin gene expression with the binding of we used to the presence of the chromatin in used for real-time to the of the A with the cytokine region to as the of the to an of the transcription start to as the the the is the chromatin the real-time to the and about the of factor binding the of The are as the of in the that in the the The from the TATA-binding protein and that is the after of TNFα and by is the and is to basal levels by This with the expression binding is the any NF-κB a in E-selectin and after NF-κB is to be a critical of E-selectin expression J. Ghersa P. Hooft van Huijsduijnen R. Gray J. Chandra G. Talabot F. DeLamarter J.F. Nucleic Acids Res. 1991; 19: 2645-2653Crossref PubMed Scopus (202) Google Scholar, 7Lewis H. Kaszubska W. DeLamarter J.F. Whelan J. Mol. Cell. Biol. 1994; 14: 5701-5709Crossref PubMed Google Scholar, 8Schindler U. Baichwal V.R. Mol. Cell. Biol. 1994; 14: 5820-5831Crossref PubMed Google Scholar, 9Whitley M.Z. Thanos D. Read M.A. Maniatis T. Collins T. Mol. Cell. Biol. 1994; 14: 6464-6475Crossref PubMed Scopus (177) Google Scholar, M.A. M.Z. Williams A.J. Collins T. J. 1994; PubMed Scopus Google Scholar). there that NF-κB in vivo with the promoter of the E-selectin the we that the p65 component of the NF-κB to the with after TNFα of p65 E-selectin mRNA and to be with levels falling the the of the region after of is the after to be the E-selectin promoter as p65 levels are or to that are known to with such as Additionally, studies for and Multiple to the E-selectin coactivators have in NF-κB-dependent gene expression (11Sheppard K.A. Rose D.W. Haque Z.K. 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). We used analysis to histone coactivators with the E-selectin promoter and element-binding protein (CREB)-binding protein histone with for binding to histone of recruitment to the E-selectin promoter. is the region of the E-selectin promoter in after with TNFα In is and levels with induction of E-selectin p300, the steroid receptor a member of the family of coactivators, is the region after TNFα p300/CBP-associated factor a recruitment from that of or the region any there is a of of the after for after TNFα The in both and of the coactivators to the promoter suggest that and p/CAF, in the regulation of the E-selectin The the E-selectin and of the are of including phosphorylation, and methylation (for review, see Ref. W. 2003; PubMed Scopus Google Scholar). The in a in a that an of the is or the E-selectin promoter in after TNFα we used specific for modified histone in and multiple the region promoter and region of the E-selectin gene known to with genes K. U. S. PubMed Scopus Google Scholar, G. Google Scholar). specific for and and and a of localized the promoter and of the E-selectin gene and a increase in histone the with of the region the In both is in the and promoter of the E-selectin a localized is to that the and which the modified E-selectin chromatin analysis with that sites and of histone is in the and the promoter and a of the E-selectin gene of histone that these to have of and and are the and after and is to that the of to from the of the gene to the gene to be within the promoter, and and analysis of high and the in to be the E-selectin and to the promoter of inflammatory genes S. S. G. Immunol. 2002; PubMed Scopus Google Scholar). E-selectin in the promoter and of the gene This is post-induction and for the and of the E-selectin gene to any that histone is a localized of the E-selectin to a role for the IκB kinase α in the of chromatin in genes K.A. A.S. 2003; PubMed Scopus Google Scholar, S. 2003; PubMed Scopus Google Scholar). analysis an recruitment to the promoter. The kinase specifically associated with the region of the E-selectin promoter the and by in a to that of p65 as a we used an kinase in the NF-κB (for review, see Ref. Immunol. 2003; PubMed Scopus Google Scholar) and to levels any This that there is in the recruitment of to the E-selectin promoter. a the E-selectin methylation is an of transcriptional repression, and of a (for review, see Ref. K. D. 2003; 19: Full Text Full Text PDF PubMed Scopus Google Scholar). role histone methylation plays in the regulation of the E-selectin specific histone used in assays. of methylation of the E-selectin gene in endothelial cells methylation as a for chromatin by the protein protein and is with which Studies have that is only in genes, is in both and genes H. R. A.J. J. J. T. 2002; PubMed Scopus Google Scholar). We that upon of with TNFα, the is it the within the promoter region This that of and it is that these to a the to be the of the gene after a specific that to the chromatin in the E-selectin to as regulate gene expression by as to factors and the This is by remodeling We the role of these complexes by an that both and the of the The that a is the in endothelial cells after the only to after of TNFα both the and region the of the that we a the accessibility of genomic by the of from a to that of an S. E. Shannon M.F. J. Immunol. PubMed Scopus Google Scholar). that we chromatin from after to Chromatin from both and cells by real-time along the and promoter of the E-selectin gene we the accessibility of the E-selectin gene in umbilical artery smooth muscle cells which In from after we to only of the from the E-selectin promoter as with chromatin In in from of the after This that the E-selectin gene in endothelial cells is to is by in smooth muscle cells. The and after with TNFα for and In the upstream of the transcription start site, including the CRR, upon with TNFα for only The levels for the by and by the and a to after in all of the promoter any in the accessibility of the and in the of the region upon exposure to TNFα that the E-selectin gene remodeling upon TNFα that the nuclear from and are to the chromatin of a in the of of the gene is in and This is from the cell of for E-selectin the of of the gene in response to TNFα in or This is in to the exposure of E-selectin to in that the cell in chromatin we for E-selectin are in and a Post-induction of E-selectin Multiple transcription is in (4Read M.A. Neish A.S. Gerritsen M.E. Collins T. J. Immunol. 1996; 157: 3472-3479PubMed Google Scholar). regulation of the E-selectin we the of binding to the E-selectin is to the after of TNFα and is is of the transcription start the and are any This in the recruitment of to the E-selectin are in complexes with and with including J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the we identified as to the E-selectin gene of a used to We to recruitment to the of E-selectin after TNFα induction. This is the which we to binding to the of the promoter Collectively, to a E-selectin gene Chromatin in the E-selectin is for the expression of E-selectin in endothelial chromatin from primary umbilical artery smooth muscle cells. In the umbilical these cells are in to the be induced by inflammatory cytokines to We first it to any of in the E-selectin in response to TNFα in umbilical vein smooth muscle cells. We to any in histone or in these cells we to nucleosomes This histone is associated with chromatin A.J. P. J.F. T. PubMed Scopus Google Scholar, M. D. S. K. T. PubMed Scopus Google Scholar). in smooth muscle cells that the E-selectin gene in these cells is in a mechanisms that and protein In we that the induction of E-selectin gene expression in endothelial cells by an inflammatory cytokine is associated with localized hyperacetylation, phosphorylation, and methylation of are by recruitment and chromatin the localized histone the in is smooth muscle cells. of we a model for the induction of E-selectin gene expression in endothelial cells that of the in In the model of the promoter of the a protein is the and in the it by as by the of A or to be with the of real-time PCR, a the for as a of be by a in of in both the and Additionally, the histone in the promoter region of the gene are with remodeling by the of the nucleosomes are in the promoter, the of the gene is in a This is with a of a in the proximal promoter region of the E-selectin gene U. Baichwal V.R. Mol. Cell. Biol. 1994; 14: 5820-5831Crossref PubMed Google Scholar). Additionally, the rapid of the induction of E-selectin are with an which is in to genes that have induction and NF-κB In cells in the E-selectin nucleosomes are is associated with the of the promoter of the gene in cells complexes nucleosomes and be for the rapid of nucleosomes after TNFα induction. after TNFα the is and the and p65 of NF-κB are the along with A and and of the model it a of that the NF-κB is the primary of NF-κB in endothelial cells and that regulate E-selectin expression T. Read M.A. Neish A.S. M.Z. Thanos D. Maniatis T. J. 9: PubMed Scopus Google Scholar). is to the transcriptional start there is localized histone and methylation and in the promoter of the gene The coactivators and are of the transcription start A and and of the family of coactivators stimulate NF-κB-dependent transcription (11Sheppard K.A. Rose D.W. Haque Z.K. Kurokawa R. McInerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. 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Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). after TNFα the transcriptional start of the promoter of the gene have in the of histone the promoter of the gene as well as in the activation is as a of and both the methylation and the of have or and The of the and the for these are is and the of the activation is by the the is for the higher of The localized of the region of the promoter NF-κB binding sites in or be to protein factors in a After is is the This is higher that is after that multiple to the CRR, multiple for This that are in repression of with in studies that of expression of the gene M.Z. Thanos D. Read M.A. Maniatis T. Collins T. Mol. Cell. Biol. 1994; 14: 6464-6475Crossref PubMed Scopus (177) Google Scholar, M.A. M.Z. Williams A.J. Collins T. J. 1994; PubMed Scopus Google Scholar). these in a of the of the chromatin of the E-selectin gene be the of histone modification. the cultures of primary human endothelial cells and smooth muscle cells levels of modified in to with cell M. E. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). Additionally, levels of modified in endothelial cells by exposure to of the histone induced in response to TNFα are localized in the E-selectin to the in genes, such as the localized that the induction of the gene in response to Maniatis T. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of is the that the and are is the promoter, and mRNA levels have to basal This suggest that of histone studies have demonstrated that transcription of the E-selectin gene is after TNFα exposure (4Read M.A. Neish A.S. Gerritsen M.E. Collins T. J. Immunol. 1996; 157: 3472-3479PubMed Google Scholar, 13Kluger M.S. Johnson D.R. Pober J.S. J. 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The of the histone kinase in endothelial cells is have a role for in the of after NF-κB activation K.A. A.S. 2003; PubMed Scopus Google Scholar, S. 2003; PubMed Scopus Google Scholar). and in the E-selectin promoter the that might be the histone of and be several and that are to transcriptional activation or a specific for the protein which have in transcriptional repression and the and of A.J. P. J.F. T. PubMed Scopus Google Scholar, M. D. S. K. T. PubMed Scopus Google Scholar, U. S. PubMed Scopus Google Scholar). a of methylation in a of inducible inflammatory genes S. G. 2002; PubMed Scopus (177) Google Scholar). This is by a rapid of levels of methylation after activation by a of methylation with post-induction transcriptional In the E-selectin gene there are levels of and only a of of methylation the after TNFα after transcriptional in the methylation of the E-selectin promoter after induction of the of histone be or is associated with genes, is with both and that histone methylation the promoter region of the a that is with studies G. G. D. U. S. PubMed Scopus Google Scholar, R. A.J. T. Biol. PubMed Scopus Google Scholar). This the This is with studies that methylation is a of transcription F. R.A. K. Mol. Cell. 2003; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). from these studies is that in a cell that there are nucleosomes that are upon gene induction. This to in is chromatin arrangement in endothelial there of the chromatin or to to the In a of both and from E-selectin differentially G. 2000; PubMed Scopus Google Scholar). The endothelial cells E-selectin in the basal or after by both basal and inducible expression of This of E-selectin be the of in chromatin accessibility within the endothelial that restrict of the gene to endothelial we have for the first the histone and chromatin arrangement for a of the histone of the T. Chen G. Thanos D. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and the K.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 2002; 110: Full Text Full Text PDF PubMed Scopus Google Scholar) have is the first to cell in histone and modification. mechanisms for cell expression and to only of genes are expressed in response to activation of a transcription We and for in and