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Molecular and Cellular Biology, December 2007, p. 8510-8521, Vol. 27, No. 24
0270-7306/07/$08.00+0     doi:10.1128/MCB.01615-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

T-bet's Ability To Regulate Individual Target Genes Requires the Conserved T-Box Domain To Recruit Histone Methyltransferase Activity and a Separate Family Member-Specific Transactivation Domain{triangledown}

Megan D. Lewis,1 Sara A. Miller,2 Michael M. Miazgowicz,1 Kristin M. Beima,1 and Amy S. Weinmann1*

Department of Immunology,1 Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 981952

Received 31 August 2007/ Accepted 30 September 2007


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ABSTRACT
 
Appropriate cellular differentiation and specification rely upon the ability of key developmental transcription factors to precisely establish gene expression patterns. These transcription factors often regulate epigenetic events. However, it has been unclear whether this is the only role that they play in functionally regulating developmental gene expression pathways or whether they also participate in downstream transactivation events at the same promoter. The T-box transcription factor family is important in cellular specification events in many developmental systems, and determining the molecular mechanisms by which this family regulates gene expression networks warrants attention. Here, we examine the mechanism by which T-bet, a critical T-box protein in the immune system, influences transcription. T-bet is both necessary and sufficient to induce permissive histone H3-K4 dimethyl modifications at the CXCR3 and IFN-{gamma} promoters. A T-bet structure-function analysis revealed that the conserved T-box domain, with a small C-terminal portion, is required for recruiting histone methyltransferase activity to promoters. Interestingly, this function is conserved in the T-box family and is necessary, but not sufficient, to induce transcription, with an independent transactivation activity also required. The requirement for two separable functional activities may ultimately contribute to the stringent role for T-box proteins in establishing specific developmental gene expression pathways.


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INTRODUCTION
 
Lineage-restricted transcription factors are responsible for establishing the changing gene expression patterns that are required for the appropriate differentiation and functioning of each unique cell type of the body. Precisely establishing these gene expression networks during development and in response to environmental stimuli is absolutely critical for maintaining cellular identity and functional capability. The T-box transcription factor family is a key regulator of the cascade of gene expression events required for cellular specification during development (25). The original T-box family members were identified due to their critical role in embryonic development. In fact, several human genetic diseases are associated with diminished T-box protein function, and the homozygous deletion of T-box proteins such as Brachyury, Eomesodermin (Eomes), and Tbx6 results in a lethal embryonic phenotype in mouse systems (5, 7, 22, 25, 26, 28, 29). The importance of the T-box family in hematopoietic cell development has been more recently recognized with the discovery of T-box expressed in T cells (T-bet) in CD4+ T helper 1 (Th1) cells and the subsequent identification of the overlapping expression profile of Eomes in CD8+ T cells (27, 33).

The critical nature of T-bet in Th1-cell development has been well established in numerous studies, and at least part of its role in this process is due to its ability to directly regulate key lineage determinant genes such as IFN-{gamma} and CXCR3 (6, 9, 18, 19, 23, 33, 34). T-bet has been shown to bind directly to the promoter regions of these genes, and the expression of T-bet is required and sufficient to induce transcription. However, the mechanisms by which T-bet is able to regulate these transcriptional events are incompletely understood and have been the focus of many studies (1, 20, 35, 36).

T-bet, as well as other lineage determinant transcription factors, must be able to establish highly specific changes in gene expression patterns to allow for alternative cell fate choices during development. It has been hypothesized that, to accomplish this, many of the transcription factors that are important in these processes are involved in establishing chromatin states that are appropriate for the individual cell fate decisions. Examining the changing nature of the chromatin structure during lineage commitment has received a great deal of attention, especially at the Th1 and Th2 cytokine loci in the immune system (2, 3, 11, 12, 14, 31). GATA-3 and STAT6 have been shown to be involved in establishing a permissive chromatin state at the Th2 cytokine locus, and T-bet expression has been shown to correlate with permissive histone acetylation, as well as the induction of DNase hypersensitivity at the IFN-{gamma} locus in Th1 cells (4, 6, 8, 24, 40).

Additional mechanisms by which these critical lineage determinant factors regulate gene expression events have also been suggested. For example, T-bet has been shown to interact and effectively compete with the key Th2 transcription factor GATA-3 (16). This competition plays a role in the early decision to establish a Th1- or Th2-cell fate. In addition, T-bet has been shown to physically and/or functionally interact with RelA, NFAT1, HLX, and RUNX3 in different contexts to aid in the establishment of the gene expression patterns required for a Th1-cell fate decision (10, 15, 18, 21, 24). Collectively, the studies performed to date have suggested several regulatory mechanisms that function both at the level of establishing the chromatin environment of target genes and in downstream transactivation events. However, it has been unclear if T-bet is directly involved in epigenetic events as well as subsequent transactivation events at the same promoter.

To further address the mechanisms by which T-bet regulates target promoters, we recently identified promoters bound by T-bet by using a chromatin immunoprecipitation-genomic microarray (ChIP-chip) approach (6). In addition to IFN-{gamma} and CXCR3, T-bet is able to associate directly with a diverse array of target promoters in B, Th1, and NK cells. Surprisingly, T-bet's ability to associate with these target promoters is cell type independent, but its ability to functionally regulate target gene expression is highly context dependent (6). For instance, T-bet is absolutely required for CXCR3 and IFN-{gamma} expression in Th1 cells, but its role in the regulation of other targets, such as IL-2Rß and CCL3, is much more modest and varies depending on the cell type background. In addition, T-bet is able to bind to a subset of target promoters, such as those of CALM2 and JMJD1A, where no functional role of binding has been uncovered. It is also worth noting that another T-box family member, Brachyury, is able to associate with the T-bet target promoters, including that of the prototypic Th1 cytokine, gamma interferon (6). Collectively, these data suggest that the DNA binding event is not the key regulated event in determining the functional outcome of the association of the T-box family with target promoters but that, rather, additional context-dependent events are required for the target gene-specific activity of this family.

In this study, we examined the mechanism by which T-bet is able to regulate target gene activity. T-bet expression is both necessary and sufficient to establish the permissive histone H3-lysine 4 (H3-K4) dimethyl, but not the fully activated trimethyl, modification at the CXCR3 and IFN-{gamma} promoters. A T-bet structure-function analysis separated the functional recruitment of histone methyltransferase activity from an additional requirement for T-bet in subsequent transactivation events. Therefore, inducing a poised chromatin state is not the only role for T-bet at these target promoters; T-bet participates in downstream regulatory events as well. Surprisingly, other T-box family members are also able to recruit methyltransferase activity to the CXCR3 promoter, suggesting that there is a conserved role for this family in establishing a permissive chromatin structure during cellular differentiation in development.


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MATERIALS AND METHODS
 
Transient transfections. EL4 T cells were transfected using the Amaxa nucleofection system. Reagent V was used with the Nucleofector program O-17. For each transfection, 4 million cells were used. Following the transfection, cells were rested for 10 min in RPMI before being plated in RPMI-10% fetal bovine serum. Cell aliquots for RNA, Western, and ChIP analyses were harvested 14 to 22 h posttransfection. For Western analysis, glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz) or V5 epitope tag (Invitrogen) antibodies were utilized. Data from one experiment representative of at least three independent experiments are presented.

Quantitative PCR and RT-PCR. RNA was prepared using the QIAGEN RNeasy protocol with the DNase step included. A reverse transcription (RT) reaction was performed to convert the RNA into cDNA by using the Superscript first-strand synthesis system (Invitrogen). Quantitative PCR was then performed with the brilliant SYBR green quantitative PCR core reagent kit (Stratagene) and gene-specific primers. All gene-specific primers spanned an intronic region to distinguish DNA contamination. Beta-actin was examined as an expression control. The expression of each transfection sample was first normalized to beta-actin levels before being depicted as a ratio to the expression of the wild-type T-bet (comprising amino acids 1 to 530 [T-bet 1-530]). For ChIP samples, primers were designed based on the promoter region of the gene. Samples were run with a standard curve on either an Opticon or a Chromo 4 quantitative PCR machine (Bio-Rad).

ChIP assay. The ChIP assay was performed as previously described (6, 37). For the experiments involving transfected samples, cells from two individual transfections were combined, resuspended in a total volume of 30 ml of RPMI-10% fetal bovine serum, and then cross-linked for 10 min. For primary cells, CD4+ T cells were isolated from either wild-type BALB/c or T-bet–/– mice. Cells were activated overnight on plate-bound anti-CD3 and anti-CD28 before being subjected to Th1-skewing conditions for 6 days as previously described (6). The H3-K4 di- and trimethyl antibodies were from Upstate Biotechnology (now Millipore). PCR primers for the murine promoters were as follows: for the CXCR3 promoter, 5'-CTGCAAACAGCAGCTGAAGC-3' and 5'-GAAAGTGGTTGGTCTCTGGC-3'; for the IFN-{gamma} promoter, 5'-GGCTTCCTCACCACATTGGC-3' and 5'-GACTCCTTGGGCTCTCTGAC-3'; for the JMJD1A promoter, 5'-GCTTCGAGTCGTTGCTGAGA-3' and 5'-GAGCCTTGAACCGAGAAGAC-3'; and for the IL-4 promoter, 5'-CTTCAACCTAGCCCAGAACC-3' and 5'-GTAGGGTTGCCACTGGCTCT-3'. For ChIP assays, at least three independent experiments were performed, and results from a representative experiment are presented.

Construction of T-bet mutant expression plasmids. Murine T-bet and Tbx6 genes were cloned from cDNA generated from CD4+ Th1 cells, the Eomes gene was cloned from CD8+ T cells, the Brachyury gene was cloned from dendritic cells, and the SET7/9 gene was cloned from EL4 T cells. The cDNA was cloned into the pcDNA3.1/V5-HIS vector by using the TOPO TA expression kit (Invitrogen). The natural stop codon was deleted to allow for a C-terminal V5 epitope tag to be added. Deletion mutant constructs were cloned from the plasmids containing the wild-type sequence. All wild-type and mutant constructs were sequenced.

Methyltransferase activity assay. Whole-cell or nuclear extracts were prepared and incubated with a primary antibody for 1 h at 4°C. Protein G beads were then added, and the immunoprecipitation (IP) reaction mixtures were incubated for another 2 h. The IP reaction mixtures were then washed and resuspended in methyltransferase reaction buffer (20 mM Tris [pH 8], 200 mM NaCl, and 0.4 mM EDTA) with 20 µM S-adenosylmethionine (Sigma) and recombinant histone H3 (2.5 µg; Upstate Biotechnology) for 1.5 h at room temperature. Reaction mixtures were resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer for subsequent Western analysis.


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RESULTS
 
T-bet expression is required and sufficient to establish a poised chromatin state. Previous studies have demonstrated that T-bet is required for IFN-{gamma} and CXCR3 transcription in Th1 cells (6, 19, 33, 34). In addition, T-bet expression positively correlates with the permissive H3-K9 acetylation modification at these promoters (4, 6). To further address the role that T-bet plays in establishing a permissive chromatin environment at target promoters, we examined the requirement for T-bet to functionally induce the permissive H3-K4 methylation modifications in Th1 cells. CD4+ T cells were isolated from either wild-type or T-bet–/– mice and cultured under Th1 conditions. Shown in Fig. 1A are the results of a ChIP assay examining the H3-K4 methylation status at the CXCR3 and IFN-{gamma} promoters in wild-type and T-bet–/– Th1 cells. IFN-{gamma} and CXCR3 were expressed in wild-type Th1 cells, but the expression of both genes was almost completely absent in T-bet–/– Th1 cells (reference 6; also data not shown). The IFN-{gamma} and CXCR3 promoters both had high levels of the H3-K4 dimethyl modification in wild-type Th1 cells, but this modification was severely diminished in the T-bet–/– Th1 cells. We did not detect significant H3-K4 trimethylation in either wild-type or T-bet–/– cells (Fig. 1A). We also examined the JMJD1A and IL-4 promoters as controls. T-bet is able to bind to the JMJD1A promoter in Th1 cells; however, no functional role for T-bet at this gene has been demonstrated (6). In contrast to CXCR3 and IFN-{gamma}, the JMJD1A promoter showed high levels of H3-K4 di- and trimethylation in both wild-type and T-bet–/– Th1 cells, suggesting that T-bet is not required for establishing these modifications at this promoter. Also, the absence of T-bet expression modestly increased the dimethylation status at the IL-4 promoter, consistent with the findings that T-bet–/– cells have a bias towards expressing Th2 cytokines (33).


Figure 1
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  		FIG. 1. T-bet is required and sufficient for the induction of the permissive H3-K4 dimethyl modification at the CXCR3 and IFN-{gamma} promoters. (A) Shown are representative results from a ChIP experiment examining the H3-K4 dimethyl (H3K4me2) and trimethyl (H3K4me3) modifications at various promoters in CD4+ T cells isolated from either wild-type (WT) or T-bet–/– (knockout [KO]) mice and cultured under Th1-skewing conditions. A ChIP assay was performed with antibodies specific to the histone H3-K4Me2 and H3-K4Me3 modifications or a nonspecific immunoglobulin G (IgG) control as indicated below the graphs. Quantitative PCR analysis was performed with promoter-specific primers as indicated above each graph. The y axis of each graph represents the level of specific-antibody precipitation relative to the total-input control for each cell type. (B) EL4 T cells were transfected with either a control pcDNA3.1 (lanes 1 to 4) or a T-bet (lanes 5 to 8) expression vector. Cells were cross-linked and a ChIP analysis was performed using antibodies specific to histone H3-K4Me3 (lanes 1 and 5) or H3-K4Me2 (lanes 2 and 6) or a nonspecific IgG antibody control (lanes 4 and 8). An aliquot of the total chromatin input is also shown (lanes 3 and 7). Promoter-specific primers were utilized in the PCR portion of the assay, as indicated to the left of the gel images.

The data from Fig. 1A suggest that T-bet expression is required for the permissive H3-K4 dimethyl modification at CXCR3 and IFN-{gamma}, two target genes that require T-bet for expression. We next wanted to determine if T-bet expression alone is sufficient to induce the permissive H3-K4 methylation modification at these promoters. To address this question, we utilized an EL4 T-cell transfection system. Consistent with previously published results, we did not detect T-bet protein expression in EL4 cells by Western analysis (data not shown and reference 33), although a small amount of mRNA could be detected by RT-PCR (6). In addition, Eomes, Tbx6, and Brachyury transcripts were undetectable (data not shown). Therefore, this system provides a background of minimal endogenous T-box protein expression. We transfected EL4 T cells with either a control or a T-bet expression vector and performed a ChIP analysis to examine the histone modification status at the target gene promoters. As shown in Fig. 1B, a dramatic increase in the level of permissive H3-K4 dimethylation modification was observed at the CXCR3 and IFN-{gamma} promoters in the cells transfected with T-bet in comparison to those transfected with the pcDNA control. In contrast, no increase in H3-K4 trimethylation was observed. Consistent with the results from the primary Th1 cells, the JMJD1A promoter starts out in a fully permissive H3-K4 methylated state and T-bet expression does not alter it further. The methylation status of the IL-4 promoter is also not affected by the overexpression of T-bet. Overall, these data suggest that T-bet overexpression alone is sufficient to induce the H3-K4 dimethylated, but not trimethylated, state at the CXCR3 and IFN-{gamma} promoters. Taken together, the data indicate that T-bet is both required and sufficient to induce the poised H3-K4 dimethylation state at the target promoters that require T-bet for expression but that it does not significantly influence the chromatin environment at the promoters where T-bet association has no known functional effect.

The N- and C-terminal domains of T-bet contain transactivation potential. The experiments depicted in Fig. 1 indicate that T-bet is functionally involved in recruiting a histone methyltransferase activity to the CXCR3 and IFN-{gamma} promoters. These data lead to several questions concerning the mechanism by which T-bet regulates target promoters. Is T-bet's only function at the CXCR3 and IFN-{gamma} promoters to create a permissive chromatin environment so that other, more ubiquitously expressed transcription factors can then activate the genes? Or is T-bet also involved in subsequent transactivation events that are downstream and independent of the chromatin-mediated activity? To start to address these questions, we performed a T-bet structure-function analysis to determine if T-bet's role in creating a permissive chromatin environment could be separated from its ability to activate transcription. For these experiments, we monitored T-bet's activity at several known target genes (6). CXCR3 and IFN-{gamma} represent promoters that require T-bet for activity, while CCL3 represents a target that is modestly regulated by T-bet in a cell type-dependent manner. As a control, we also monitored CALM2 which, like JMJD1A, is bound by T-bet but for which no functional effect of binding has been observed to date (6).

Deletion constructs were made by eliminating portions of the N- and/or C-terminal domain of T-bet (Fig. 2). In all cases, the T-box DNA binding domain was retained to ensure that each mutant could associate with the endogenous T-box DNA binding elements found within the target gene promoters. Therefore, this experimental system takes into account the role that T-bet plays at the target promoters in the context of the normal chromatin environment at each gene, thus providing a way to examine both chromatin and transactivation events under natural conditions. After the transfection of EL4 T cells with these constructs, we monitored the endogenous target gene transcriptional activity by quantitative RT-PCR analysis, and as a control, the levels of expression of each mutant protein construct in the same transfected samples were also monitored by Western analysis (see Fig. 3 and 5). In addition, in independent experiments, we also used ChIP to monitor the ability of the mutant constructs to induce the H3-K4 dimethylation activity (see Fig. 4 and 6).


Figure 2
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  		FIG. 2. Schematic representation of the T-bet deletion constructs. All T-bet expression constructs were cloned into the pcDNA3.1 vector with a C-terminal V5 epitope tag for monitoring protein expression levels. In all cases, the T-box DNA binding domain, indicated in black, was contained within the constructs to allow for binding to the endogenous T-bet target genes. The initial N-terminal and final C-terminal amino acids for each mutant are indicated.


Figure 3
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  		FIG. 3. Transactivation potential is contained within both the N- and C-terminal domains of T-bet. EL4 T cells were transfected with either the control pcDNA3.1 expression vector or the T-bet C-terminal (A and B) or N-terminal (C and D) deletion constructs as indicated. Aliquots of the transfected samples were processed for RNA and quantitative RT-PCR analyses (A and C) and the evaluation of protein expression by Western analysis utilizing a V5 antibody ({alpha}V5) to examine construct expression or a glyceraldehyde-3-phosphate dehydrogenase antibody ({alpha}Gapdh) as a loading control (B and D). The quantitative RT-PCR analysis was performed with gene-specific primers to monitor the influence of the wild-type and mutant T-bet constructs on the expression of the endogenous target genes indicated at the bottom of the graph. The y axis indicates the level of expression of each deletion mutant relative to that of the wild-type T-bet 1-530 transfection after the normalization of values for all samples to beta-actin as a control. Results for the C-terminal (A and B) and N-terminal (C and D) deletion constructs from a representative experiment monitoring both target gene expression and the mutant protein expression levels in transfected cells are shown.


Figure 5
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  		FIG. 5. The loss of T-bet's N- and C-terminal domains results in a complete absence of transactivation potential. T-bet deletion constructs that had the N terminus deleted in conjunction with the progressive deletion of the C-terminal domain were utilized. (A and B) EL4 T cells were transfected with the T-bet mutant constructs as indicated in the legends to Fig. 3 and 4. (A) The ability of the mutant constructs to activate endogenous target gene expression was monitored by quantitative RT-PCR. (B) A Western analysis examining the levels of expression of the constructs in the transfected cells from panel A is shown. {alpha}V5, V5 antibody; {alpha}GAPDH, glyceraldehyde-3-phosphate dehydrogenase antibody.


Figure 4
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  		FIG. 4. T-bet's N- and C-terminal domains are dispensable for initiating the H3-K4 dimethyl (H3K4me2) modification at the CXCR3 promoter. (A) Shown are results from representative ChIP experiments examining the status of the H3-K4 dimethyl modification in response to T-bet deletion mutant overexpression. EL4 T cells were transfected with the control pcDNA3.1 vector (lanes 1 to 4) or one of the following T-bet expression constructs as indicated above the gel images: T-bet 1-530 (lanes 5 to 8), T-bet 1-468 (lanes 9 to 12), T-bet 1-402 (lanes 13 to 16), T-bet 1-331 (lanes 17 to 20), or T-bet 120-530 (lanes 21 to 24). Transfected samples were cross-linked, and ChIP assays were performed with an antibody specific to either H3-K4 trimethyl (H3K4me3; lanes 1, 5, 9, 13, 17, and 21), H3-K4 dimethyl (lanes 2, 6, 10, 14, 18, and 22), or a nonspecific IgG control (lanes 4, 8, 12, 16, 20, and 24). An aliquot of the total input chromatin from each transfection is also shown (lanes 3, 7, 11, 15, 19, and 23). Primers specific to the promoters of CXCR3, IFN-{gamma}, JMJD1A, or IL-4 were utilized as indicated to the left of the gel images. (B) A quantitative PCR analysis of the samples from panel A was performed as described in the legend to Fig. 1A.


Figure 6
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  		FIG. 6. Localization of the domain required for functional recruitment of methyltransferase activity to the CXCR3 and IFN-{gamma} promoters. (A) The ability of the double-truncation mutant proteins to induce the H3-K4 dimethyl (H3K4me2) modification was monitored by ChIP as described in the legend to Fig. 4. EL4 T cells were transfected with a pcDNA3.1 control vector (lanes 1 to 5), T-bet 120-468 (lanes 6 to 9), T-bet 120-402 (lanes 10 to 13), or T-bet 120-331 (lanes 14 to 17). The transfected samples were precipitated with antibodies specific to H3-K4 trimethyl (H3K4me3; lanes 1, 6, 10, and 14), H3-K4 dimethyl (lanes 2, 7, 11, and 15), H3-K9 acetyl (H3AcK9) (lane 3), or an IgG control (lanes 5, 9, 13, and 17). An aliquot of the total input chromatin is also shown (lanes 4, 8, 12, and 16). (B) Shown is the quantitative PCR analysis of the samples described in the legend to panel A. The construct symbol key is the same as that in Fig. 5. (C) A ChIP assay was performed to determine if the double-truncation mutant proteins retain the ability to bind to the IFN-{gamma} promoter. EL4 T cells were transfected with either a pcDNA3.1 control (lanes 1 to 3), T-bet 120-468 (lanes 4 to 6), T-bet 120-402 (lanes 7 to 9), T-bet 120-331 (lanes 10 to 12), or T-bet 1-530 (lanes 13 to 15). The transfected samples were processed for standard ChIP analysis and precipitated with either the V5 epitope tag antibody (lanes 1, 4, 7, 10, and 13) or an IgG control (lanes 3, 6, 9, 12, and 15). An aliquot of the input chromatin from each transfection is also shown (lanes 2, 5, 8, 11, and 14).

We first examined the requirement for T-bet's C-terminal domain in activating endogenous target gene expression. The progressive deletion of the C-terminal domain resulted in a graded loss of the activity of T-bet at CXCR3, IFN-{gamma}, and CCL3 (Fig. 3A and B). Consistent with previous results, no role for T-bet at CALM2 was observed. The T-bet construct comprising amino acids 1 to 331 (T-bet 1-331) was not able to activate transcription at any of the target promoters tested. It is worth noting that the C-terminal deletion mutant proteins behaved in similar manners at all three functional targets, with the gradual loss in activity. The data thus suggest that the C terminus of T-bet is required for the transactivation potential at CXCR3, IFN-{gamma}, and CCL3, with the possibility that several regions contribute to functional activity.

We next examined the transactivation potential of T-bet's N-terminal domain (Fig. 3C and D). In contrast to the C-terminal mutant proteins, the N-terminal deletion series elicited modest variations in responses at CXCR3, CCL3, and IFN-{gamma}. The loss of the first 51 amino acids resulted in a significant decrease in transactivation potential at all three target genes. However, the expression of each deletion mutant protein prior to that point resulted in modest, promoter-specific activity differences. At the CXCR3 promoter, full activity was retained until the first 51 amino acids were deleted, after which there was a sharp decrease in activity. In contrast, there was a modest loss in activity (twofold) at the IFN-{gamma} promoter with the deletion of the first 40 amino acids. Interestingly, the deletion of the first 28 amino acids consistently resulted in a modest increase in the activity at the CCL3 promoter, suggesting that a minor inhibitory activity may be present within the first 28 amino acids that influences some target genes but not others. Perhaps consistent with this result, positive and negative cell type-specific consequences were observed in the absence of T-bet at CCL3 and RAD51 (6). Again, CALM2 was not affected by any of the deletion mutant proteins.

Separation of transactivation potential from the ability to functionally recruit histone methyltransferase activity. We next wanted to determine whether the role for T-bet in the regulation of these genes is strictly at the level of creating a permissive chromatin environment or if there are additional, independent transactivation activities. To address this question, we examined the ability of the deletion mutant proteins to recruit the histone H3-K4 dimethylation-specific methyltransferase activity to the CXCR3 and IFN-{gamma} promoters. If T-bet's only role in influencing promoter activity was to create a permissive chromatin environment, then it would not be possible to uncouple T-bet's ability to recruit the methyltransferase activity from the transcriptional activation potential at the target genes. In contrast, the uncoupling of the two events would suggest that there are two or more independent steps that require T-bet to activate the target genes, ultimately creating a more stringent requirement for T-bet in the regulation of these promoters.

EL4 T cells were transfected with either the control pcDNA vector, wild-type T-bet, or the T-bet deletion mutant proteins (Fig. 4). ChIP analyses were then performed to examine the ability of the mutant proteins to induce the permissive H3-K4 dimethylation modification at the CXCR3 and IFN-{gamma} promoters. Surprisingly, the overexpression of most N- and C-terminal deletion mutant proteins resulted in similar increases in the permissive H3-K4 dimethyl modification at the CXCR3 promoter (Fig. 4A and B). There was a decrease in activity for T-bet 1-331, but it still retained the ability to induce the H3-K4 dimethyl modification to a significant degree (approximately 2.6-fold) (Fig. 4A and B). Therefore, despite the fact that several of these mutant proteins (e.g., T-bet 1-331 and T-bet 120-530) had severely impaired transcriptional regulatory activity, they were still able to establish a permissive chromatin structure. Similar to the results at the CXCR3 promoter, at the IFN-{gamma} promoter the majority of the T-bet deletion constructs, even those with significantly diminished transcriptional activity (e.g., T-bet 120-530 and T-bet 1-402), retained the capacity to induce the H3-K4 dimethyl modification. However, in contrast to its performance at the CXCR3 promoter, at the IFN-{gamma} promoter the T-bet 1-331 mutant protein was unable to significantly induce the H3-K4 dimethylation. Taken together, these data suggest that establishing a permissive chromatin environment is insufficient to upregulate IFN-{gamma} or CXCR3 transcription.

Differential domain requirement for recruiting methyltransferase activity to CXCR3 versus IFN-{gamma}. The data suggest that it is indeed possible to separate transactivation potential from T-bet's ability to induce epigenetic alterations at target promoters. It was somewhat surprising that eliminating either the N- or C-terminal domain of T-bet did not completely impair its ability to functionally recruit the methyltransferase activity to the CXCR3 promoter. This result led us to further refine the domain requirements for this activity. To accomplish this, T-bet mutant proteins with truncations of the N-terminal domain in conjunction with deletions of increasing portions of the C terminus were created (Fig. 2). We first monitored the transcriptional activity of these mutant proteins. Consistent with the requirement for activities in both the N- and C-terminal domains for transactivation potential, these truncation mutant proteins did not retain any significant transcriptional activity at CXCR3, IFN-{gamma}, or CCL3 (Fig. 5A and B).

We next examined the ability of the combination N- and C-terminal truncation mutant constructs to induce the H3-K4 dimethyl modification at the promoters. The T-bet 120-468 and T-bet 120-402 mutant proteins were able to induce a significant amount of H3-K4 dimethylation at the CXCR3 promoter (Fig. 6A and B). Once again, the ability of T-bet to functionally recruit methyltranferase activity to the CXCR3 promoter was retained by mutant constructs that were unable to activate transcription. A mutant protein containing only the T-box DNA binding domain was no longer able to induce the H3-K4 dimethylation. Therefore, the mutant data suggest that the T-box domain, with a small portion of either the N or C terminus perhaps for stabilization, is required to recruit the histone methyltransferase activity to the CXCR3 promoter.

Consistent with the more stringent requirement for the functional recruitment of methyltransferase activity to the IFN-{gamma} promoter, the combination N- and C-terminal truncation mutant proteins were more impaired in their ability to recruit methyltransferase activity to this promoter than to the CXCR3 promoter (Fig. 4 and 6). The T-bet 120-468 mutant protein did retain some activity, even though it was unable to activate IFN-{gamma} transcription. However, in contrast to its activity at the CXCR3 promoter, the T-bet 120-402 mutant construct did not significantly induce the dimethyl modification and was at the level of the mutant protein containing the T-box alone. We confirmed that these truncation mutant constructs retained the ability to bind to the IFN-{gamma} promoter similarly to full-length T-bet by ChIP, with the affinity of only the T-box-alone construct (T-bet 120-331) appearing to be slightly less than that of the full-length protein (Fig. 6C).

Divergent T-box family members can recruit methyltransferase activity to the CXCR3 promoter. The data from the T-bet deletion series suggest that the crucial region required for functionally recruiting histone methyltransferase activity to the CXCR3 promoter resides in the T-box domain itself. The T-box domain is highly conserved in this transcription factor family (25). Therefore, if the T-box contains the critical features required for recruiting methyltransferase activity, it is possible that other family members may have this ability as well. To test this hypothesis, we examined whether a closely related T-box family member, Eomes, and the more distantly related family members Tbx6 and Brachyury are able to recruit histone methyltransferase activity to the CXCR3 promoter.

We first confirmed that other T-box family members are able to associate with the T-bet target promoters being examined in this study. Consistent with previously published results, Brachyury is able to bind to the same promoters as T-bet (Fig. 7A) (6). We have also confirmed that Eomes is able to associate with these promoter regions as well (data not shown). The binding of multiple family members to the same target promoters is likely due to the highly conserved nature of the T-box DNA binding domain in this family.


Figure 7
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  		FIG. 7. The ability to recruit histone methyltransferase activity to target genes is a conserved function for the T-box family. (A) Brachyury (Brach.) is able to associate with the T-bet target genes. Shown is a ChIP analysis performed with the human 721 B-cell line. The chromatin was precipitated with an antibody specific to either Brachyury (lane 1), T-bet (lane 2), or a nonspecific IgG control (lane 4). The total input chromatin is also shown (lane 3), and the promoter-specific primers utilized in the PCR are indicated to the left of the gel image. (B) EL4 T cells were transfected with either a pcDNA3.1 control vector (lanes 1 to 4), Tbx6 (lanes 5 to 8), Eomes (lanes 9 to 12), or Brachyury (lanes 13 to 16) as indicated. The ability of the T-box family members to induce the H3-K4 dimethyl (H3K4me2) modification was monitored by ChIP. Transfected samples were precipitated with an antibody to H3-K4 trimethyl (H3K4me3; lanes 1, 5, 9, and 13), H3-K4 dimethyl (lanes 2, 6, 10, and 14), or a nonspecific IgG control (lanes 4, 8, 12, and 16), and the analysis of an aliquot of the total input chromatin is also shown (lanes 3, 7, 11, and 15). Promoter-specific primers were used in the PCR portion of the assay as indicated to the left of the gel image. (C and D) The ability of these T-box family members to regulate endogenous target gene expression was examined by quantitative RT-PCR (C), and a Western analysis to monitor protein expression levels was performed as indicated in the legend to Fig. 3 (D).

We next wanted to determine if the binding of individual T-box family members results in either conserved or variable functional consequences. To accomplish this, we examined both their ability to induce the H3-K4 dimethyl modification and their transactivation potential (Fig. 7B and C). As shown in Fig. 7B, Eomes, Tbx6, and Brachyury were all able to induce H3-K4 dimethylation at the CXCR3 promoter. Similar to T-bet, these T-box family members were unable to induce the fully permissive H3-K4 trimethylated state (Fig. 7B). These data suggest that, indeed, the ability to functionally recruit the H3-K4 dimethyltransferase activity is a conserved function for this family. In contrast to this conserved function, individual family members appear to have variable transactivation potentials. Despite the ability of Tbx6 to create a permissive chromatin environment at the CXCR3 promoter, it has less transactivation potential in this setting than either T-bet or Eomes (Fig. 7B and C). Both T-bet and Eomes are able to significantly activate CXCR3, with T-bet being the most efficient in this setting. Interestingly, despite the ability of Brachyury to bind to and induce the H3-K4 dimethyl modification at the CXCR3 promoter, it is completely unable to transactivate this gene (Fig. 7).

Consistent with the more stringent requirement for the recruitment of the methyltransferase activity to the IFN-{gamma} promoter, the closely related T-box family member Eomes is able to recruit methyltransferase activity to the IFN-{gamma} promoter but the more distantly related Tbx6 and Brachyury cannot (Fig. 7B). This characteristic also coincides with the ability of Eomes to upregulate IFN-{gamma} transcriptional activity, whereas Tbx6 and Brachyury are not able to activate this gene at all (Fig. 7C). This is not due to an inability of the T-box proteins to associate with the promoter because Brachyury does in fact have the ability to bind to IFN-{gamma} (Fig. 7A). It is also worth noting that Eomes does not appear to be able to transactivate IFN-{gamma} to the same extent as T-bet in this setting. These data suggest that although T-bet and Eomes show significant homology, they do in fact have differences that are important for their functional activities, and it appears that these differences are downstream of their conserved role in epigenetic events. Collectively, the data suggest that the T-box family has a conserved ability to functionally recruit histone-modifying activity but that the family members also have a degree of target selectivity in their mechanisms of activation.

T-bet can associate with methyltransferase activity. T-box family members do not contain known methyltransferase activity domains. Consistent with this property, we could not detect any inherent methyltransferase activity for T-bet (data not shown). Therefore, one mechanism for the observed epigenetic changes is the direct recruitment of a methyltransferase complex to the target promoters. Alternatively, T-bet may be involved in an event upstream of the induction of the H3-K4 dimethyl modification that ultimately results in a cascade of events that is responsible for the observed epigenetic alterations. To begin to address the mechanism by which T-bet is able to induce the H3-K4 dimethyl modification at the target promoters, we examined whether T-bet can associate with methyltransferase activity in coimmunoprecipitation (co-IP) experiments. We transfected EL4 T cells with either a control pcDNA vector or the wild-type T-bet 1-530. As a positive control, we also performed a transfection with SET7/9, a known H3-K4 methyltransferase. Following immunoprecipitation with a V5 epitope tag antibody, the co-IP samples were monitored for methyltransferase activity. As shown in Fig. 8, an H3-K4 dimethyl-specific methyltransferase activity was present in the T-bet IP sample relative to the pcDNA control sample. Interestingly, we could not detect any H3-K4 trimethyl-specific activity in the T-bet co-IP sample (data not shown). These data suggest that T-bet can physically associate with a methyltransferase complex specific for the H3-K4 dimethyl activity. To further confirm this observation, we repeated the co-IP experiments with stimulated 721 B cells in order to examine endogenous T-bet expression levels. In the stimulated 721 B cells, H3-K4 dimethyl-specific methyltransferase activity was enriched in the T-bet-precipitated sample relative to that in a control antibody precipitation. Taken together, these data provide the possibility that T-bet's ability to functionally induce the H3-K4 dimethyl modification is by the direct recruitment of a methyltransferase complex to the target promoters.


Figure 8
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  		FIG. 8. T-bet can associate with histone methyltransferase activity. A co-IP experiment was performed, and histone methyltransferase activity was monitored. (A) EL4 T cells were transfected with either a pcDNA 3.1 control vector (lane 1), T-bet 1-530 (lane 2), or SET7/9 (lane 3). The T-bet 1-530 and SET7/9 constructs contain a C-terminal V5 epitope tag. Whole-cell extracts were made from the individual samples and precipitated with a V5 antibody (lanes 1 to 3). A methyltransferase activity assay was performed with the precipitated samples, which were subsequently processed for Western analysis to monitor H3-K4 dimethyl-specific methyltransferase activity. An amount of the recombinant histone H3 equal to that used in the methyltransferase reaction was also run as a control (–; lane 4). The membrane was probed with an antibody specific to histone H3-K4 dimethyl ({alpha}H3K4Me2) as shown. (B) The membrane was then stripped and reprobed with a V5-specific antibody ({alpha}V5) to confirm the precipitation of the transfected constructs. The minor band indicated by the asterisk represents cross-reactivity with the heavy chain. (C) A co-IP experiment to monitor methyltransferase activity was performed as described in panel A. 721 B cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin (P/I). Nuclear extracts were precipitated with either a control Cdk6 antibody (lane 1) or a T-bet-specific antibody (lane 2). The co-IP samples were subjected to a methyltransferase activity assay, and the Western analysis was performed with an H3-K4 dimethyl-specific antibody.


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DISCUSSION
 
In this study, we addressed the role for T-bet in the regulation of target genes. T-bet expression is both necessary and sufficient to induce the permissive H3-K4 dimethyl chromatin modification at the IFN-{gamma} and CXCR3 promoters. A structure-function analysis separated the requirement for T-bet in creating a poised chromatin state at target promoters from an independent, downstream transactivation potential that is also required to upregulate transcription. Surprisingly, T-bet's T-box domain appears to be critical for recruiting the methyltransferase activity. Consistent with a requirement for the T-box domain, three additional T-box family members, Eomes, Tbx6, and Brachyury, are also able to functionally recruit methyltransferase activity to the CXCR3 promoter, suggesting a conserved function for this family. Collectively, the data suggest that the T-box family has a conserved role in inducing permissive epigenetic modifications but that family member-specific transactivation events that occur downstream of these epigenetic modifications are also required to induce transcriptional activity at specific target promoters. This dual requirement for two separable functional activities may help to explain the specificity for T-box protein family member activity at very precise developmental time points.

The T-box protein family is absolutely critical in establishing gene expression patterns in numerous developmental systems (25). The DNA binding domain for this family is highly conserved for specific binding to T-sites found within developmentally important genes (38). The T-box proteins need to access their cognate DNA binding sites at very precise times during cellular specification events. At these cell fate decision checkpoints, it would not be surprising that the T-box proteins may be required to access DNA that is located in a nonpermissive chromatin state. Our findings suggest that the T-box domain is able both to bind DNA and to participate in the functional recruitment of permissive histone methyltransferase activity to the target genes. It is worth noting that although the T-box domain is highly conserved, there is some variability among family members. If the amino acids responsible for this interaction are not conserved in all family members, it is possible that the ability to functionally recruit methyltransferase activity will vary. Therefore, it will now be important to determine the exact amino acids required for this function. Nevertheless, this newly identified role for the T-box domain provides a means by which this family has the potential to alter the epigenetic status of its target genes during development. It also suggests that the T-box family can access its DNA binding sites even when they are not in a permissive chromatin environment. The ability of the T-box proteins to induce the H3-K4 dimethylated, but not the trimethylated, pattern suggests that they are important in creating a permissive, poised epigenetic state at the targets but that subsequent events are required to create a fully activated state.

It will now be important to determine the methyltransferase that is recruited by the T-box family. Our data suggest that T-bet can interact with a methyltransferase activity, but the identity of the complex is still unknown. Several methyltransferases are able to induce methylation at H3-K4 (30). Insight into the possible candidates can be gained from the observation that the H3-K4 dimethyl, but not trimethyl, state is induced at the target promoters. In addition, biochemically, we could detect only H3-K4 dimethyl-specific methyltransferase activity in association with T-bet-precipitated samples. These data suggest that the methyltransferase likely specifically creates the poised H3-K4 dimethylated state but that other, independent events are required to create the fully permissive H3-K4 trimethylated chromatin structure at these promoters.

It is possible that the T-box proteins recruit a single methyltransferase family. However, it is also equally possible that a conserved domain or common protein found in several methyltransferase complexes is responsible for the interaction (39). Perhaps the latter scenario is more likely, because the expression patterns for the methyltransferases are likely to vary significantly during development. The overlapping and exclusive expression patterns of the T-box and methyltransferase families may ultimately provide another layer of functional specificity during development.

It is intriguing that there is a more stringent requirement for additional domains within the T-box proteins to functionally recruit methyltransferase activity to the IFN-{gamma} promoter than to the CXCR3 promoter. This distinction does not appear to be due to the affinity of the T-box proteins to bind to the IFN-{gamma} promoter, as Brachyury can associate with this promoter but cannot induce the epigenetic alterations. This finding may reflect the biological need to tightly control IFN-{gamma} expression in both a development- and an activation-dependent manner, in contrast to the developmental regulation of CXCR3. It is possible that events associated with cellular activation serve to stabilize the interaction of a T-bet-methyltransferase complex at the IFN-{gamma} promoter through a specific N- or C-terminal domain not conserved in all T-box proteins. The required role for additional domains at select targets may help to establish a hierarchy for target gene selection, creating a scenario in which some genes are regulated by a broad range of T-box family members whereas others require a very specific T-box protein. This possibility is consistent with T-box protein target selection during development. In development, T-box protein expression patterns can overlap, yet there are some T-box target genes that are very specific to an individual family member whereas others are redundantly regulated by multiple family members (32). Therefore, it will now be important to carefully dissect the mechanisms that account for the similarities as well as the differences in the ability of the T-box proteins to induce target-specific epigenetic modifications.

With both Tbx6 and Eomes possessing some transactivation potential at target genes such as CXCR3, it is difficult to rule out the possibility that a difference in DNA binding affinity is at least partly responsible for the differential transactivation potentials of these two family members in relation to T-bet. That is, it is likely that some DNA binding affinity differences are inherent in the T-box family, and it is possible that these differences result in at least some of the variability in the observed transactivation potentials. However, the fact that Brachyury has no transactivation activity at the CXCR3 promoter despite the ability to bind to the promoter and induce the H3-K4 dimethyl modification suggests that small differences in binding affinity are not solely responsible for the differences observed in transactivation potential. These data thus suggest that the ability of the T-box family to bind and create a permissive chromatin environment is not the sole activity responsible for family member-specific transcriptional potential but that, rather, other independent functional domains determine the transactivation potential at specific target genes downstream of the induction of epigenetic changes.

The dual requirement for the functional recruitment of a histone methyltransferase and for a subsequent transactivation activity to induce transcription may also help to explain the stringent and specific requirement for individual family members at a given target gene (13). This may add another layer of specificity to the regulation of gene expression that would not be present otherwise. It is interesting that target genes that are modestly modulated by T-bet and Eomes, such as IL-2Rß, do not require T-bet for the epigenetic modification at the promoter in Th1 cells (6, 17). This finding leads to the speculation that the role of a T-box family member at a specific gene may be dependent upon the number of events that it is required to regulate at that given gene and in that unique cell type setting. The current data now provide specific transcriptional regulatory events to examine to address these questions. Ultimately, these answers will provide insight into the very precise requirements for individual T-box family members in the regulation of target genes during cellular differentiation processes.


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ACKNOWLEDGMENTS
 
We thank Kerri Mowen for the methyltransferase activity assay protocol and Steve Smale for critical review of the manuscript.

This work was supported by a Leukemia and Lymphoma Society Special Fellowship (A.S.W.) and the NIH grant AI061061 (A.S.W.). M.M.M. was supported by a predoctoral training grant from the NCI (CA09537), and S.A.M. was supported by a grant from the NIGMS (T32 GM07270).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Immunology, University of Washington, Box 357650, 1959 NE Pacific St., Seattle, WA 98195. Phone: (206) 616-7235. Fax: (206) 543-1013. E-mail: weinmann{at}u.washington.edu Back

{triangledown} Published ahead of print on 8 October 2007. Back


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REFERENCES
 
    1
  1. Afkarin, M., J. R. Sedy, J. Yang, N. G. Jacobson, N. Cereb, S. Y. Yang, T. L. Murphy, and K. M. Murphy. 2002. T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells. Nat. Immunol. 3:549-557.[CrossRef][Medline]
    2. 2
  2. Agarwal, S., and A. Rao. 1998. Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity 9:765-775.[CrossRef][Medline]
    3. 3
  3. Ansel, K. M., I. Djuretic, B. Tanasa, and A. Rao. 2006. Regulation of Th2 differentiation and IL4 locus accessibility. Annu. Rev. Immunol. 24:607-656.[CrossRef][Medline]
    4. 4
  4. Avni, O., D. Lee, F. Macian, S. J. Szabo, L. H. Glimcher, and A. Rao. 2002. T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nat. Immunol. 3:643-651.[Medline]
    5. 5
  5. Baala, L., S. Briault, H. C. Etchevers, F. Laumonnier, A. Natiq, J. Amiel, N. Boddaert, C. Picard, A. Sbiti, A. Asermouh, T. Attie-Bitach, F. Encha-Razavi, A. Munnich, A. Sefiani, and S. Lyonnet. 2007. Homozygous silencing of T-box transcription factor EOMES leads to microcephaly with polymicrogyria and corpus callosum agenesis. Nat. Genet. 39:454-456.[CrossRef][Medline]
    6. 6
  6. Beima, K. M., M. M. Miazgowicz, M. D. Lewis, P. S. Yan, T. H.-M. Huang, and A. S. Weinmann. 2006. T-bet binding to newly identified target gene promoters is cell-type independent, but results in variable functional effects. J. Biol. Chem. 281:11992-12000.[Abstract/Free Full Text]
    7. 7
  7. Chapman, D. L., and V. E. Papaioannou. 1998. Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391:695-697.[CrossRef][Medline]
    8. 8
  8. Chen, G.-Y., H. Osada, L. F. Santamaria-Babi, and R. Kannagi. 2006. Interaction of GATA-3/T-bet transcription factors regulates expression of sialyl Lewis X homing receptors on Th1/Th2 lymphocytes. Proc. Natl. Acad. Sci. USA 103:16894-16899.[Abstract/Free Full Text]
    9. 9
  9. Cho, J. Y., V. Grigura, T. L. Murphy, and K. Murphy. 2003. Identification of cooperative monomeric Brachyury sites conferring T-bet responsiveness to the proximal IFN-{gamma} promoter. Int. Immunol. 15:1149-1160.[Abstract/Free Full Text]
    10. 10
  10. Djuretic, I. M., D. Levaon, V. Negreanu, Y. Groner, A. Rao, and K. M. Ansel. 2007. Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells. Nat. Immunol. 8:145-153.[CrossRef][Medline]
    11. 11
  11. Fields, P. E., S. T. Kim, and R. A. Flavell. 2002. Changes in histone acetylation at the IL-4 and IFN{gamma} loci accompany Th1/Th2 differentiation. J. Immunol. 169:647-650.[Abstract/Free Full Text]
    12. 12
  12. Fields, P. E., G. R. Lee, S. T. Kim, V. V. Bartsevich, and R. A. Flavell. 2004. Th2-specific chromatin remodeling and enhancer activity in the Th2 cytokine locus control region. Immunity 21:865-876.[CrossRef][Medline]
    13. 13
  13. Goering, L. M., K. Hoshijima, B. Hug, B. Bisgrove, A. Kispert, and D. J. Grunwald. 2003. An interacting network of T-box genes directs gene expression and fate in zebrafish mesoderm. Proc. Natl. Acad. Sci. USA 100:9410-9415.[Abstract/Free Full Text]
    14. 14
  14. Hatton, R. D., L. E. Harrington, R. J. Luther, T. Wakefield, K. M. Janowski, J. R. Oliver, R. L. Lallone, K. M. Murphy, and C. T. Weaver. 2006. A distal conserved sequence element controls Ifng gene expression by T cells and NK cells. Immunity 25:717-729.[CrossRef][Medline]
    15. 15
  15. Hwang, E. S., J. H. Hong, and L. H. Glimcher. 2005. IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508. J. Exp. Med. 202:1289-1300.[Abstract/Free Full Text]
    16. 16
  16. Hwang, E. S., S. J. Szabo, P. L. Schwartzberg, and L. H. Glimcher. 2005. T helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3. Science 307:430-433.[Abstract/Free Full Text]
    17. 17
  17. Intlekofer, A. M., N. Takemoto, E. J. Wherry, S. A. Longworth, J. T. Northrup, V. R. Palanivel, A. C. Mullen, C. R. Gasink, S. M. Kaech, J. D. Miller, L. Gapin, K. Ryan, A. P. Russ, T. Lindsten, J. S. Orange, A. W. Goldrath, R. Ahmed, and S. L. Reiner. 2005. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat. Immunol. 6:1236-1244.[CrossRef][Medline]
    18. 18
  18. Lee, D. U., O. Avni, L. Chen, and A. Rao. 2004. A distal enhancer in the interferon-g (IFN-g) locus revealed by genome sequence comparison. J. Biol. Chem. 279:4802-4810.[Abstract/Free Full Text]
    19. 19
  19. Lord, G., R. M. Rao, H. Choe, B. M. Sullivan, A. H. Lichtman, F. W. Luscinskas, and L. H. Glimcher. 2005. T-bet is required for optimal pro-inflammatory CD4+ T cell trafficking. Blood 106:3432-3439.[Abstract/Free Full Text]
    20. 20
  20. Matsuda, J. L., T. C. George, J. Hagman, and L. Gapin. 2007. Temporal dissection of T-bet functions. J. Immunol. 178:3457-3465.[Abstract/Free Full Text]
    21. 21
  21. Mehta, D. S., A. L. Wurster, A. S. Weinmann, and M. J. Grusby. 2005. NFATc2 and T-bet contribute to T-helper-cell-subset-specific regulation of IL-21. Proc. Natl. Acad. Sci. USA 102:2016-2021.[Abstract/Free Full Text]
    22. 22
  22. Mori, A. D., and B. G. Bruneau. 2004. Tbx5 mutations and congenital heart disease: Holt-Oram syndrome revealed. Curr. Opin. Cardiol. 19:211-215.[CrossRef][Medline]
    23. 23
  23. Mullen, A. C., F. A. High, A. S. Hutchins, H. W. Lee, A. V. Villarino, D. M. Livingston, A. L. Kung, N. Cereb, T.-P. Yao, S. Y. Yang, and S. L. Reiner. 2001. Role of T-bet in commitment of Th1 cells before IL-12-dependent selection. Science 292:1907-1910.[Abstract/Free Full Text]
    24. 24
  24. Mullen, A. C., A. S. Hutchins, F. A. High, H. W. Lee, K. J. Sykes, L. A. Chodosh, and S. L. Reiner. 2002. Hlx is induced by and genetically interacts with T-bet to promote heritable Th1 gene induction. Nat. Immunol. 3:652-658.[Medline]
    25. 25
  25. Naiche, L. A., Z. Harrelson, R. G. Kelly, and V. E. Papaioannou. 2005. T-box genes in vertebrate development. Annu. Rev. Genet. 39:219-239.[CrossRef][Medline]
    26. 26
  26. Packham, E. A., and J. D. Brook. 2003. T-box genes in human disorders. Hum. Mol. Genet. 12:R37-R44.[Abstract/Free Full Text]
    27. 27
  27. Pearce, E. L., A. C. Mullen, G. A. Martins, C. M. Krawczyk, A. S. Hutchins, V. P. Zediak, M. Banica, C. B. DiCioccio, D. A. Gross, C. A. Mao, H. Shen, N. Cereb, S. Y. Yang, T. Lindsten, J. Rossant, C. A. Hunter, and S. L. Reiner. 2003. Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science 302:1041-1043.[Abstract/Free Full Text]
    28. 28
  28. Rashbass, P., L. A. Cooke, B. G. Herrmann, and R. S. P. Beddington. 1991. A cell autonomous function of Brachyury in T/T embryonic stem cell chimeras. Nature 353:348-351.[CrossRef][Medline]
    29. 29
  29. Russ, A. P., S. Wattler, W. H. Colledge, S. A. J. R. Aparicio, M. B. L. Carlton, J. J. Pearce, S. C. Barton, M. A. Surani, K. Ryan, M. C. Nehls, V. Wilson, and M. J. Evans. 2000. Eomesodermin is required for mouse trophoblast development and mesoderm formation. Nature 404:95-99.[CrossRef][Medline]
    30. 30
  30. Ruthenburg, A. J., C. D. Allis, and J. Wysocka. 2007. Methylation of lysine 4 on histone H3: intricacy of writing and reading a single epigenetic mark. Mol. Cell 25:15-30.[CrossRef][Medline]
    31. 31
  31. Shnyreva, M., W. M. Weaver, M. Blanchette, S. L. Taylor, M. Tompa, D. R. Fitzpatrick, and C. B. Wilson. 2004. Evolutionarily conserved sequence elements that positively regulate IFN-gamma expression in T cells. Proc. Natl. Acad. Sci. USA 101:12622-12627.[Abstract/Free Full Text]
    32. 32
  32. Showell, C., O. Binder, and F. L. Conlon. 2004. T-box genes in early embryogenesis. Dev. Dyn. 229:201-218.[CrossRef][Medline]
    33. 33
  33. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100:655-669.[CrossRef][Medline]
    34. 34
  34. Szabo, S. J., B. M. Sullivan, C. Stemmann, A. R. Satoskar, B. P. Sleckman, and L. H. Glimcher. 2002. Distinct effects of T-bet in Th1 lineage commitment and IFN{gamma} production in CD4 and CD8 T cells. Science 295:338-342.[Abstract/Free Full Text]
    35. 35
  35. Tong, Y., T. M. Aune, and M. Boothby. 2005. T-bet antagonizes mSin3a recruitment and transactivates a fully methylated IFN-g promoter via a conserved T-box half-site. Proc. Natl. Acad. Sci. USA 102:2034-2039.[Abstract/Free Full Text]
    36. 36
  36. Usui, T., J. C. Preiss, Y. Kanno, Z. J. Yao, J. H. Bream, J. J. O'Shea, and W. Strober. 2006. T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription. J. Exp. Med. 203:755-766.[Abstract/Free Full Text]
    37. 37
  37. Weinmann, A. S., P. S. Yan, M. J. Oberley, T. H.-M. Huang, and P. J. Farnham. 2002. Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis. Genes Dev. 16:235-244.[Abstract/Free Full Text]
    38. 38
  38. Wilson, V., and F. L. Conlon. 2002. The T-box family. Genome Biol. 3:3008.1-3008.7.
    39. 39
  39. Wysocka, J., T. Swigut, T. A. Milne, Y. Dou, X. Zhang, A. L. Burlingame, R. G. Roeder, A. H. Brivaniou, and C. D. Allis. 2005. WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development. Cell 121:859-872.[CrossRef][Medline]
    40. 40
  40. Yamashita, M., M. Ukai-Tadenuma, M. Kimura, M. Omori, M. Inami, M. Taniguchi, and T. Nakayama. 2002. Identification of a conserved GATA3 response element upstream proximal from the interleukin-13 gene locus. J. Biol. Chem. 277:42399-42408.[Abstract/Free Full Text]

Molecular and Cellular Biology, December 2007, p. 8510-8521, Vol. 27, No. 24
0270-7306/07/$08.00+0     doi:10.1128/MCB.01615-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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