T-bet Binding to Newly Identified Target Gene Promoters Is Cell Type-independent but Results in Variable Context-dependent Functional Effects
2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês
10.1074/jbc.m513613200
ISSN1083-351X
AutoresKristin M. Beima, Michael M. Miazgowicz, Megan D. Lewis, Pearlly S. Yan, Tim H-M. Huang, Amy S. Weinmann,
Tópico(s)RNA Research and Splicing
ResumoRecently developed target gene identification strategies based upon the chromatin immunoprecipitation assay provide a powerful method to determine the localization of transcription factor binding within mammalian genomes. However, in many cases, it is unclear if the binding capacity of a transcription factor correlates with an obligate role in gene regulation in diverse contexts. It is therefore important to carefully examine the relationship between transcription factor binding and its ability to functionally regulate gene expression. T-bet is a T-box transcription factor expressed in several hematopoietic cell types. By utilizing a chromatin immunoprecipitation assay coupled to genomic microarray technology approach, we identified numerous promoters, including CXCR3, IL2Rβ, and CCL3, that are bound by T-bet in B cells. Most surprisingly, the ability of T-bet to associate with the target promoters is not dependent upon the cell type background. Several of the promoters appear to be functionally regulated by T-bet. However, we could not detect a functional consequence for T-bet association with many of the identified promoters in overexpression studies or an examination of wild type and T-bet-/- primary B, CD4+, and CD8+ T cells. Thus, there is a high variability in the functional consequences, if any, that result from the association of T-bet with individual target promoters. Recently developed target gene identification strategies based upon the chromatin immunoprecipitation assay provide a powerful method to determine the localization of transcription factor binding within mammalian genomes. However, in many cases, it is unclear if the binding capacity of a transcription factor correlates with an obligate role in gene regulation in diverse contexts. It is therefore important to carefully examine the relationship between transcription factor binding and its ability to functionally regulate gene expression. T-bet is a T-box transcription factor expressed in several hematopoietic cell types. By utilizing a chromatin immunoprecipitation assay coupled to genomic microarray technology approach, we identified numerous promoters, including CXCR3, IL2Rβ, and CCL3, that are bound by T-bet in B cells. Most surprisingly, the ability of T-bet to associate with the target promoters is not dependent upon the cell type background. Several of the promoters appear to be functionally regulated by T-bet. However, we could not detect a functional consequence for T-bet association with many of the identified promoters in overexpression studies or an examination of wild type and T-bet-/- primary B, CD4+, and CD8+ T cells. Thus, there is a high variability in the functional consequences, if any, that result from the association of T-bet with individual target promoters. Upon exposure to pathogenic stimuli, an intricately coordinated immune response composed of specialized cells is required for host defense. In order for these tightly regulated cellular responses to occur, individual hematopoietic cell types must carry out very precise and coordinated alterations in their gene expression patterns to up-regulate specific functional capabilities. A great deal of research has been undertaken to better address the molecular mechanisms by which critical, lineage-restricted transcription factors contribute to this process in the immune system, but much still remains unknown.T-box expressed in T cells (T-bet) is a member of the T-box transcription factor family and plays a critical role in the generation of CD4+ T helper 1 (Th1) cells (1Szabo S.J. Kim S.T. Costa G.L. Zhang X. Fathman C.G. Glimcher L.H. Cell. 2000; 100: 655-669Abstract Full Text Full Text PDF PubMed Scopus (2692) Google Scholar). Th1 cell responses are critical for the clearance of altered self-cells such as those infected by pathogens or cancerous cells. Overexpression of T-bet in Th2 or naive CD4+ T cells commits nature solidifying the critical of them to a Th1 phenotype (1Szabo S.J. Kim S.T. Costa G.L. Zhang X. Fathman C.G. Glimcher L.H. Cell. 2000; 100: 655-669Abstract Full Text Full Text PDF PubMed Scopus (2692) Google Scholar). Further T-bet in Th1 development, T-bet-/- mice have a profound defect in generating a Th1 cell response (2Szabo S.J. Sullivan B.M. Stemmann C. Satoskar A.R. Sleckman B.P. Glimcher L.H. Science. 2002; 295: 338-342Crossref PubMed Scopus (968) Google Scholar). Taken together, the data strongly indicate that T-bet plays a critical role in Th1 development and cellmediated immunity.The importance of T-bet in generating a productive immune response is not limited to its role in Th1 development. Several studies have highlighted the role of T-bet in B, NK, NKT, DC, and CD8+ T cell development and function (3Peng S.L. Szabo S.J. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5545-5550Crossref PubMed Scopus (364) Google Scholar, 4Townsend M.J. Weinmann A.S. Matsuda J. Saloman R. Farnham P.J. Biron C.A. Gapin L. Glimcher L.H. Immunity. 2004; 20: 477-494Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 5Lugo-Villarino G. Maldonado-Lopez R. Possemato R. Penaranda C. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7749-7754Crossref PubMed Scopus (215) Google Scholar, 6Sullivan B.M. Juedes A. Szabo S.J. von Herrath M. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15818-15823Crossref PubMed Scopus (319) Google Scholar). For example, immunoglobulin isotype class switching is defective in T-bet-/- mice, and this defect appears to be B cell intrinsic and not solely due to the deficient Th1 response (3Peng S.L. Szabo S.J. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5545-5550Crossref PubMed Scopus (364) Google Scholar). In addition, NK and NKT cells intrinsically require T-bet to develop into functionally mature cells (4Townsend M.J. Weinmann A.S. Matsuda J. Saloman R. Farnham P.J. Biron C.A. Gapin L. Glimcher L.H. Immunity. 2004; 20: 477-494Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). In contrast to CD4+ T cells, the requirement for T-bet in CD8+ T cells is less pronounced (2Szabo S.J. Sullivan B.M. Stemmann C. Satoskar A.R. Sleckman B.P. Glimcher L.H. Science. 2002; 295: 338-342Crossref PubMed Scopus (968) Google Scholar). Data using dominant negative constructs suggest that the less stringent requirement for T-bet in CD8+ T cells is at least in part due to the expression of another closely related T-box family member, eomesodermin (Eomes), in these cells (7Pearce E.L. Mullen A.C. Martins G.A. Krawczyk C.M. Hutchins A.S. Zediak V.P. Banica M. DiCioccio C.B. Gross D.A. Mao C.A. Shen H. Cereb N. Yang S.Y. Lindsten T. Rossant J. Hunter C.A. Reiner S.L. Science. 2003; 302: 1041-1043Crossref PubMed Scopus (749) Google Scholar). At least in some cases, Eomes can act in a functionally redundant manner with T-bet activity. Taken together, the current data suggest that T-bet is a very important transcription factor in several immune cell types, and there may be some cellular specificity to the role it plays. However, the molecular mechanism responsible for these apparent cell type differences, including whether they result from direct or indirect effects, is poorly understood.To begin to address the role that T-bet plays in functionally distinct cell types, it is first necessary to identify the genes that are directly regulated by T-bet in different cellular settings. Recent advances in target gene identification strategies based upon the chromatin immunoprecipitation (ChIP) 2The abbreviations used are: ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP assay coupled to genomic microarray technology; IFN, interferon; IL, interleukin; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P/I, phorbol 12-myristate 13-acetate and ionomycin; Eomes, eomesodermin 2The abbreviations used are: ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP assay coupled to genomic microarray technology; IFN, interferon; IL, interleukin; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P/I, phorbol 12-myristate 13-acetate and ionomycin; Eomes, eomesodermin assay coupled to genomic microarray technology (chip) now make it possible to identify, in a more global manner, the promoters that are bound by a transcription factor within the context of the normal chromatin environment of a cell. Therefore, ChIP-based techniques provide a very compelling starting point to uncover the gene expression networks with the potential to be directly regulated by a transcription factor.One assumption that is commonly made when interpreting data from ChIP-based assays is that the detected binding of a transcription factor at a promoter results in an obligate functional role for that factor in the regulation of the associated gene. Another common assumption made when examining a large data set from ChIP-chip studies is that if a select target gene is regulated then all genomic loci bound by the transcription factor are also regulated in a similar manner. However, transcription factor family members, such as the T-box family, often have highly homologous DNA binding domains but diverge in the domains required for functional activity. This presents the real possibility that divergent family members may have the ability to associate with similar genomic loci, but they differ in their ability to regulate gene expression. In addition, the requirements for the activity of a transcription factor in the context of complex promoter structures in distinct cellular settings or activation conditions is often unclear. Therefore, a rigorous analysis examining both the ability of a transcription factor to bind to a locus and cause a functional consequence on gene expression in diverse cellular settings is important. This will aid in the understanding of a particular transcription factor family if the ability to associate with a target sequence is in fact the key regulated event in its ability to influence gene transcription or rather that binding is more promiscuous, and other downstream events are required for more tightly controlled functional activity. In this study, we examine the correlation between binding and functional activity in several distinct cell types to begin to address this question for the T-box transcription factor family member T-bet.Utilizing a ChIP-based target gene approach, we have identified numerous promoter regions specifically bound by T-bet in B cells. Interestingly, T-bet has the ability to bind to the same subset of promoter regions in NK and T cells, suggesting that binding, at least for these targets, is independent of the cellular setting. However, upon examination of the functional consequence for T-bet binding to these promoters, the data indicate that some targets may be differentially regulated dependent upon the cell type background. Surprisingly, a significant number of target genes are not affected by either the overexpression or absence of T-bet, regardless of the ability of T-bet to associate with their promoter. In some of these cases, it is possible that either T-bet is not the functional family member at these promoters or there is functional redundancy in the activity of T-bet with other T-box family members or regulatory events. Indeed, Brachyury, another T-box family member present in the B cell line utilized for these studies, is able to associate with the same promoter regions that are bound by T-bet. Thus, the data demonstrate that the association of T-bet with a target promoter can result in several different outcomes, including obligatory, modest context-dependent, or no detectable regulatory role. The mechanism for these variable outcomes may involve the differential requirement for T-bet at distinct steps in transcriptional activity at each promoter. In support of this possibility, histone H3K9 acetylation is dependent upon T-bet expression only at the promoters that require T-bet for gene expression.EXPERIMENTAL PROCEDURESChromatin Immunoprecipitation—The chromatin immunoprecipitation procedure was performed as described previously (8Weinmann A.S. Farnham P.J. Methods. 2002; 26: 37-47Crossref PubMed Scopus (299) Google Scholar, 9Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H.-M. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (391) Google Scholar). Briefly, cells were cross-linked in 1% formaldehyde followed by nuclei isolation. Nuclei were sonicated and precleared with pansorbin cells (Calbiochem) prior to immunoprecipitation with specific or control antibodies overnight at 4 °C. Following wash and elution steps, cross-links were reversed at 65 °C for 4 h. For samples to be used in the microarray analysis, two consecutive immunoprecipitations were performed with the same antibody before the cross-links were reversed. The DNA in the immunoprecipitated samples was purified by proteinase K digestion followed by phenol/chloroform extraction and DNA precipitation. Samples were then either analyzed by standard PCR with promoterspecific primers or amplified by ligation-mediated PCR for microarray analysis. The T-bet and Brachyury antibodies used in these studies were from Santa Cruz Biotechnology; the histone H3 acetyl-K9 antibody was from Upstate Biotechnology, Inc., and the IgG antibody was from MP Biomedicals.For the ChIP-chip analysis, a human promoter microarray from NimbleGen Systems, Inc., was co-hybridized with either a T-bet-precipitated and total input control or an IgG-precipitated sample with the total input control. The IgG-precipitated/total microarray was utilized to eliminate nonspecific signals because of either nonspecific precipitation or ligation-mediated PCR amplification artifacts. Putative positive promoters were those that contained multiple consecutive and positive oligonucleotide probe signals. Only promoter regions that were confirmed in a subsequent standard ChIP analysis are shown in this paper because we did not perform an exhaustive analysis with the arrays, but rather we used them as a screening tool. The standard ChIP experiments shown as validation for the targets are representative of at least three independent experiments. In all ChIP experiments, IFNγ was first examined as a positive control and IL-4 as a negative control.Primary Cell Isolation—All mouse studies were performed with approval and by IACUC guidelines. CD4+ T cells, CD8+ T cells, and B cells were isolated from BALB/c wild type and T-bet-/- mice. CD8+ T cells and B cells were isolated from spleen and lymph nodes using Miltenyi microbeads (CD8α+ T cell isolation kit catalog number 130-090-859 or CD43 microbeads catalog number 130-049-801, respectively). CD8+ T cells were stimulated with plate-bound αCD28 (clone 37.51; Pharmingen) and αCD3 (clone 145-2C11; Pharmingen) in the presence of IL-2 (10 ng/ml) and IL-12 (10 ng/ml) for 3 days. B cells were either left unstimulated or stimulated with IL-12 (10 ng/ml), IL-18 (10 ng/ml), and αCD40 (10 μg/ml) for 12 h. CD4+ T cells were isolated from spleen and lymph nodes using the MagCollect mouse CD4+ T cell isolation protocol (MAGM202; R & D Systems). CD4+ T cells were activated with plate-bound αCD3 and αCD28 in the presence of αIL-4 (generously provided by NCI, National Institutes of Health) overnight and then cultured in Th1 conditions (IL-2 at 10 ng/ml and IL-12 at 5 ng/ml). Cells were split 3 days after cytokine addition to a density of 2 × 106 cells/well and cultured for another 3 days in Th1 conditions before harvest. In all experiments, cell purity was monitored following purification by flow cytometry.RT-PCR—RNA from primary CD4+ T cells, CD8+ T cells, or B cells was isolated using the Qiagen RNA purification protocol with the optional DNase step included. RT-PCR primers were designed to span at least one intronic region. RT-PCR was carried out using the EZ rTth polymerase kit (Applied Biosystems).Transient Transfection—Transient transfection analysis of EL4 cells was performed using the AMAXA nucleofection system. 4 million cells per transfection were resuspended in nucleofection solution V. Setting O-17 was used for nucleofection with the standard protocol. The T-bet pcDNA3 expression construct was provided by Christopher Wilson.Western Blot Analysis—Whole cell lysates from equal cell numbers of the human 721 B cell line, YT NK cell line, and Jurkat T cell line were loaded. The human 721 B cell line was kindly provided by Bill Sugden, and the YT NK cell line was kindly provided by Paul Sondel. Membranes were hybridized with the T-bet-specific antibody (Santa Cruz Biotechnology) before being stripped and reprobed with an antibody against GAPDH (Santa Cruz Biotechnology) as a loading control.RESULTST-bet Associates with the IFNγ Promoter in B Cells—T-bet is expressed in B cells in response to several stimuli (1Szabo S.J. Kim S.T. Costa G.L. Zhang X. Fathman C.G. Glimcher L.H. Cell. 2000; 100: 655-669Abstract Full Text Full Text PDF PubMed Scopus (2692) Google Scholar, 10Liu N. Ohnishi N. Ni L. Akira S. Bacon K.B. Nat. Immun. 2003; 4: 687-693Crossref Scopus (251) Google Scholar), but very little is known about the genes that are directly regulated by T-bet in this cellular setting. To begin to address the molecular role that T-bet plays in B cell responses, it is necessary to identify the genes that are direct T-bet targets in B cells. We utilized the human 721 B cell line for our studies. Low levels of T-bet are constitutively expressed in either unstimulated or IFNγ-stimulated 721 B cells (Fig. 1A). To identify T-bet targets, we utilized a ChIP-chip approach to provide a screening method to identify the DNA regions that are bound by T-bet in the context of the cellular environment of IFNγ-stimulated B cells.To ensure the feasibility of a ChIP-based approach for this study, a preliminary standard ChIP experiment utilizing a T-bet-specific antibody was performed in the B cells (Fig. 1B). IFNγ is a well characterized T-bet target gene in T cells (2Szabo S.J. Sullivan B.M. Stemmann C. Satoskar A.R. Sleckman B.P. Glimcher L.H. Science. 2002; 295: 338-342Crossref PubMed Scopus (968) Google Scholar, 11Cho J.Y. Grigura V. Murphy T.L. Murphy K. Int. Immunol. 2003; 15: 1149-1160Crossref PubMed Scopus (59) Google Scholar, 12Shnyreva M. Weaver W.M. Blanchette M. Taylor S.L. Tompa M. Fitzpatrick D.R. Wilson C.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12622-12627Crossref PubMed Scopus (119) Google Scholar). We examined if T-bet can also interact with the IFNγ promoter in B cells. As shown in Fig. 1B, T-bet is able to bind to the IFNγ promoter in B cells as demonstrated by the specific enrichment of the IFNγ promoter in the T-bet-precipitated chromatin in comparison to the nonspecific IgG antibody control. In contrast, no enrichment of the IL-4 promoter is detected above the nonspecific antibody background levels. To ensure this signal is specific for T-bet, we also performed the same experiment in cells that do not express T-bet. We did not observe any enrichment of the IFNγ promoter in the absence of T-bet expression in either unstimulated Jurkat T cells (Fig. 1A and Fig. 3A) or HeLa cells (data not shown). Taken together, the data suggest that T-bet has the ability to bind to the IFNγ promoter in B cells, and a ChIP-based target gene identification approach is feasible.FIGURE 3Examination of cell type binding specificity of T-bet. A ChIP experiment examining either unstimulated Jurkat T cells (A), P/I-stimulated Jurkat T cells (B), or YT NK cells (C) was performed. The chromatin was precipitated with a T-bet-specific antibody (lane 1) or an IgG control antibody (lane 3) with a standardized aliquot of the total (lane 2) also shown. Primers specific to the promoter regions of the indicated genes are shown to the left of the gel images.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Identification of Novel Promoter Regions Bound by T-bet in B Cells—After performing the initial controls, we undertook a ChIP-chip analysis to identify novel promoter regions bound by T-bet in B cells. Briefly, IFNγ-stimulated 721 B cells were cross-linked with formaldehyde followed by nuclei isolation and sonication. Specific T-bet-DNA complexes were isolated in two sequential immunoprecipitation steps with the T-bet-specific antibody. In parallel, a nonspecific IgG antibody precipitation was performed as a control. Following reversal of cross-links, the precipitated DNA was purified and amplified by ligation-mediated PCR. To identify the promoter regions that are specifically associated with T-bet, the T-bet-precipitated sample was co-hybridized with a total input control on a human promoter microarray. In parallel, the IgG-precipitated sample was subjected to the same analysis to subtract false positives because of nonspecific precipitation of DNA fragments. This analysis was performed as an initial screen only, and promoters were confirmed in standard ChIP assays utilizing promoter-specific primers to ensure that all targets presented in this study have been validated in this nonexhaustive screen.The data in Fig. 2 show the validation of numerous promoter regions that are specifically bound by T-bet in B cells. Also shown is one of the false positives from this screening, CCR1, which is not enriched in the T-bet-precipitated chromatin in comparison to the IgG control. The STAT1 promoter, which has been shown previously to be bound by T-bet (13Lovett-Racke A.E. Rocchini A.E. Choy J. Northrop S.C. Hussain R.Z. Ratts R.B. Sikder D. Racke M.K. Immunity. 2004; 21: 719-731Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), was identified in our screen, which suggests that we are in fact identifying true target promoters. In addition, CXCR3, which is functionally regulated by T-bet in Th1 cells (14Lord G. Rao R.M. Choe H. Sullivan B.M. Lichtman A.H. Luscinskas F.W. Glimcher L.H. Blood. 2005; 106: 3432-3439Crossref PubMed Scopus (199) Google Scholar), was uncovered in our analysis by virtue of the association of T-bet with its promoter. IL2Rβ, which is important in activated and memory T cell responses, was also identified and validated.FIGURE 2Confirmation analysis for direct T-bet target genes identified in B cells. Shown is a representative ChIP experiment performed in IFNγ-stimulated 721 B cells. Chromatin was precipitated with a T-bet-specific antibody (lanes 1 and 4) or an IgG control antibody (lanes 3 and 6). A standardized aliquot of the input chromatin (lanes 2 and 5) is also shown. Primers specific to the promoter regions for putative T-bet targets that were identified in the ChIP-chip screen were used for PCR analysis as indicated to the left of the gel images. IFNγ and IL-4 were examined as a positive and negative control, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interestingly, several of the promoter regions that are bound by T-bet in B cells, such as CXCR3 and PSGL1, are differentially expressed in Th1 relative to Th2 cells (15Rogge L. Bianchi E. Biffi M. Bono E. Chang S.Y. Alexander H. Santini C. Ferrari G. Sinigaglia L. Seiler M. Neeb M. Mous J. Sinigaglia F. Certa U. Nat. Genet. 2000; 25: 96-101Crossref PubMed Scopus (195) Google Scholar). Although these promoter regions may have been predicted because of the well characterized role for T-bet in Th1 development, it was somewhat surprising that T-bet was able to bind to these promoters in B cells. Several other validated target genes, such as RAD51, CALM2, and MAPK1, are expressed in a more ubiquitous pattern, and therefore, one may not have predicted that T-bet associates with these genes in B cells. In addition, the promoters for CCL3 and CCL3L1 are both bound by T-bet (Fig. 2). CCL3 and CCL3L1 are important proinflammatory chemokines that are known to be suppressive for human immunodeficiency virus infection (16Gonzalez E. Kulkarni H. Bolivar H. Mangano A. Sanchez R. Catano G. Nibbs R.J. Freedman B.I. Quinones M.P. Bamshad M.J. Murthy K.K. Rovin B.H. Bradley W. Clark R.A. Anderson S.A. O'Connell R.J. Agan B.K. Ahuja S.S. Bologna R. Sen L. Dolan M.J. Ahuja S.K. Science. 2005; 307: 1434-1440Crossref PubMed Scopus (945) Google Scholar, 17Verani A. Scarlatti G. Comar M. Tresoldi E. Polo S. Giacca M. Lusso P. Siccardi A.G. Vercelli D. J. Exp. Med. 1997; 185: 805-816Crossref PubMed Scopus (147) Google Scholar). This locus has been the site of gene duplication events throughout evolution, and a recent study suggested that the copy number of CCL3L1 impacts the susceptibility to human immunodeficiency virus infection (16Gonzalez E. Kulkarni H. Bolivar H. Mangano A. Sanchez R. Catano G. Nibbs R.J. Freedman B.I. Quinones M.P. Bamshad M.J. Murthy K.K. Rovin B.H. Bradley W. Clark R.A. Anderson S.A. O'Connell R.J. Agan B.K. Ahuja S.S. Bologna R. Sen L. Dolan M.J. Ahuja S.K. Science. 2005; 307: 1434-1440Crossref PubMed Scopus (945) Google Scholar). We also detected T-bet association with another chemokine that resides in this locus, CCL4 (data not shown). Thus, T-bet may play an important role throughout this locus.T-bet Binding Is Independent of Cell Type Background—It is interesting to note that several of the T-bet target promoters that we identified in B cells are either regulated by T-bet in other cell types or differentially expressed in Th1 versus Th2 development. Therefore, one may hypothesize that T-bet will be able to bind to a portion of these promoters in other cell types. To address whether there is cell type specificity to the association of T-bet with these newly identified target promoters, we examined T-bet binding to these promoters in T and NK cells.We first examined the human Jurkat T cell line. In resting cells, T-bet protein expression is absent; however, it is highly induced by phorbol 12-myristate 13-acetate and ionomycin (P/I) stimulation (Fig. 1A). Before testing whether T-bet could associate with the target promoters in the P/I-stimulated Jurkat T cells, we first performed an additional negative control to demonstrate that the enrichment of the novel target promoters in the T-bet-precipitated chromatin sample is in fact dependent upon T-bet expression. We performed a ChIP experiment in the absence of T-bet expression in resting Jurkat T cells (Fig. 1A and Fig. 3A). Importantly, the targets presented here are not enriched in the T-bet-precipitated chromatin in the absence of T-bet protein expression, in either resting Jurkat T cells (Fig. 3A) or HeLa cells (data not shown). These data suggest that the signal observed indeed is dependent upon T-bet expression.It is worth noting that several false positives were uncovered during the analysis of the resting Jurkat T cells. For these regions, enrichment of a strong signal was detected in the T-bet-precipitated chromatin in the absence of T-bet protein expression in either unstimulated Jurkat cells or HeLa cells (data not shown). These signals may have been due to cross-reactivity of the T-bet antibody with another protein. The identification of these false positives makes it clear that potential targets identified in a ChIP-chip experiment, a procedure that is highly dependent upon antibody specificity, should be screened using the same antibody in a cell type that does not express that protein. This added control will help to eliminate false positives that are because of antibody crossreactivity or impurities, which are common with most antibody preparations.We next examined the targets in P/I-stimulated Jurkat T cells to determine whether T-bet can associate with the same set of promoter regions in a T cell background. Most interestingly, all of the targets identified in the B cells are also bound by T-bet in the Jurkat T cells (Fig. 3B). This observation was somewhat surprising because we hypothesized that the chromatin environment generated in different cellular backgrounds may provide at least some specificity for the ability of T-bet to associate with a subset of the target promoters. To further address this question in another cellular background, we next performed a ChIP experiment in the human YT NK cell line. The YT NK cell line has a high level of constitutive T-bet expression (Fig. 1A) and was previously utilized to identify RUNX1 as a functionally regulated T-bet target gene (4Townsend M.J. Weinmann A.S. Matsuda J. Saloman R. Farnham P.J. Biron C.A. Gapin L. Glimcher L.H. Immunity. 2004; 20: 477-494Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). Similar to the results in the T cells, T-bet associates with the newly identified B cell target genes in the NK cell line as well (Fig. 3C). These results suggest that the cell type background does not play a large role in restricting the inherent ability of T-bet to bind to the promoters.T-bet Levels or Stimulation Conditions Can Influence Binding—Because of the lack of cell type-restricted binding for the T-bet targets identified in B cells, we next examined the cell type binding specificity for two T-bet targets, RUNX1 and CDK6, that were originally identified in a ChIP-chip screen in the YT NK cell line using a CpG island microarray (4Townsend M.J. Weinmann A.S. Matsuda J. Saloman R. Farnham P.J. Biron C.A. Gapin L. Glimcher L.H. Immunity. 2004; 20: 477-494Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar) (Fig. 4A). As we observed with the T-bet targets that were originally identified in B cells, T-bet is able to associate with the RUNX1 and CDK6 promoters in the T cell background as well (Fig. 4B). Once again, binding specificity does not appear to be restricted by the cell type background.FIGURE 4
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