MOLECULAR AND CELLULAR BIOLOGY, Dec. 2007, p. 8510–8521 Vol. 27, No. 240270-7306/07/$08.00�0 doi:10.1128/MCB.01615-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

T-bet’s Ability To Regulate Individual Target Genes Requires theConserved T-Box Domain To Recruit Histone Methyltransferase

Activity and a Separate Family Member-SpecificTransactivation Domain�

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

Department of Immunology1 and Molecular and Cellular Biology Program,2 University of Washington, Seattle,Washington 98195

Received 31 August 2007/Accepted 30 September 2007

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

Lineage-restricted transcription factors are responsible forestablishing the changing gene expression patterns that arerequired for the appropriate differentiation and functioning ofeach unique cell type of the body. Precisely establishing thesegene expression networks during development and in responseto environmental stimuli is absolutely critical for maintainingcellular identity and functional capability. The T-box transcrip-tion factor family is a key regulator of the cascade of geneexpression events required for cellular specification during de-velopment (25). The original T-box family members were iden-tified due to their critical role in embryonic development. Infact, several human genetic diseases are associated with dimin-ished T-box protein function, and the homozygous deletion ofT-box proteins such as Brachyury, Eomesodermin (Eomes),and Tbx6 results in a lethal embryonic phenotype in mousesystems (5, 7, 22, 25, 26, 28, 29). The importance of the T-boxfamily in hematopoietic cell development has been more re-cently recognized with the discovery of T-box expressed in Tcells (T-bet) in CD4� T helper 1 (Th1) cells and the subse-quent identification of the overlapping expression profile ofEomes in CD8� T cells (27, 33).

The critical nature of T-bet in Th1-cell development hasbeen well established in numerous studies, and at least part of

its role in this process is due to its ability to directly regulatekey lineage determinant genes such as IFN-� and CXCR3 (6, 9,18, 19, 23, 33, 34). T-bet has been shown to bind directly to thepromoter regions of these genes, and the expression of T-bet isrequired and sufficient to induce transcription. However, themechanisms by which T-bet is able to regulate these transcrip-tional events are incompletely understood and have been thefocus of many studies (1, 20, 35, 36).

T-bet, as well as other lineage determinant transcriptionfactors, must be able to establish highly specific changes ingene expression patterns to allow for alternative cell fatechoices during development. It has been hypothesized that, toaccomplish this, many of the transcription factors that areimportant in these processes are involved in establishing chro-matin states that are appropriate for the individual cell fatedecisions. Examining the changing nature of the chromatinstructure during lineage commitment has received a great dealof attention, especially at the Th1 and Th2 cytokine loci in theimmune system (2, 3, 11, 12, 14, 31). GATA-3 and STAT6 havebeen shown to be involved in establishing a permissive chro-matin state at the Th2 cytokine locus, and T-bet expression hasbeen shown to correlate with permissive histone acetylation, aswell as the induction of DNase hypersensitivity at the IFN-�locus in Th1 cells (4, 6, 8, 24, 40).

Additional mechanisms by which these critical lineage de-terminant factors regulate gene expression events have alsobeen suggested. For example, T-bet has been shown to interactand effectively compete with the key Th2 transcription factorGATA-3 (16). This competition plays a role in the early deci-

* Corresponding author. Mailing address: Department of Immunol-ogy, University of Washington, Box 357650, 1959 NE Pacific St., Se-attle, WA 98195. Phone: (206) 616-7235. Fax: (206) 543-1013. E-mail:weinmann@u.washington.edu.

� Published ahead of print on 8 October 2007.

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sion to establish a Th1- or Th2-cell fate. In addition, T-bet hasbeen shown to physically and/or functionally interact withRelA, NFAT1, HLX, and RUNX3 in different contexts to aidin the establishment of the gene expression patterns requiredfor a Th1-cell fate decision (10, 15, 18, 21, 24). Collectively, thestudies performed to date have suggested several regulatorymechanisms that function both at the level of establishing thechromatin environment of target genes and in downstreamtransactivation events. However, it has been unclear if T-bet isdirectly involved in epigenetic events as well as subsequenttransactivation events at the same promoter.

To further address the mechanisms by which T-bet regulatestarget promoters, we recently identified promoters bound byT-bet by using a chromatin immunoprecipitation-genomic mi-croarray (ChIP-chip) approach (6). In addition to IFN-� andCXCR3, T-bet is able to associate directly with a diverse arrayof target promoters in B, Th1, and NK cells. Surprisingly,T-bet’s ability to associate with these target promoters is celltype independent, but its ability to functionally regulate targetgene expression is highly context dependent (6). For instance,T-bet is absolutely required for CXCR3 and IFN-� expressionin Th1 cells, but its role in the regulation of other targets, suchas IL-2R� and CCL3, is much more modest and varies depend-ing on the cell type background. In addition, T-bet is able tobind to a subset of target promoters, such as those of CALM2and JMJD1A, where no functional role of binding has beenuncovered. It is also worth noting that another T-box familymember, Brachyury, is able to associate with the T-bet targetpromoters, including that of the prototypic Th1 cytokine,gamma interferon (6). Collectively, these data suggest that theDNA binding event is not the key regulated event in deter-mining the functional outcome of the association of the T-boxfamily with target promoters but that, rather, additional con-text-dependent events are required for the target gene-specificactivity of this family.

In this study, we examined the mechanism by which T-bet isable to regulate target gene activity. T-bet expression is bothnecessary and sufficient to establish the permissive histoneH3-lysine 4 (H3-K4) dimethyl, but not the fully activated tri-methyl, modification at the CXCR3 and IFN-� promoters. AT-bet structure-function analysis separated the functional re-cruitment of histone methyltransferase activity from an addi-tional requirement for T-bet in subsequent transactivationevents. Therefore, inducing a poised chromatin state is not theonly role for T-bet at these target promoters; T-bet participates indownstream regulatory events as well. Surprisingly, other T-boxfamily members are also able to recruit methyltransferase activityto the CXCR3 promoter, suggesting that there is a conserved rolefor this family in establishing a permissive chromatin structureduring cellular differentiation in development.

MATERIALS AND METHODS

Transient transfections. EL4 T cells were transfected using the Amaxa nucleo-fection system. Reagent V was used with the Nucleofector program O-17. Foreach transfection, 4 million cells were used. Following the transfection, cells wererested 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 hposttransfection. For Western analysis, glyceraldehyde-3-phosphate dehydroge-nase (Santa Cruz) or V5 epitope tag (Invitrogen) antibodies were utilized. Datafrom one experiment representative of at least three independent experimentsare presented.

Quantitative PCR and RT-PCR. RNA was prepared using the QIAGENRNeasy protocol with the DNase step included. A reverse transcription (RT)reaction was performed to convert the RNA into cDNA by using the Superscriptfirst-strand synthesis system (Invitrogen). Quantitative PCR was then performedwith the brilliant SYBR green quantitative PCR core reagent kit (Stratagene)and gene-specific primers. All gene-specific primers spanned an intronic regionto distinguish DNA contamination. Beta-actin was examined as an expressioncontrol. The expression of each transfection sample was first normalized tobeta-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 wererun with a standard curve on either an Opticon or a Chromo 4 quantitative PCRmachine (Bio-Rad).

ChIP assay. The ChIP assay was performed as previously described (6, 37).For the experiments involving transfected samples, cells from two individualtransfections 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 beforebeing subjected to Th1-skewing conditions for 6 days as previously described (6).The H3-K4 di- and trimethyl antibodies were from Upstate Biotechnology (nowMillipore). PCR primers for the murine promoters were as follows: for theCXCR3 promoter, 5�-CTGCAAACAGCAGCTGAAGC-3� and 5�-GAAAGTGGTTGGTCTCTGGC-3�; for the IFN-� promoter, 5�-GGCTTCCTCACCACATTGGC-3� and 5�-GACTCCTTGGGCTCTCTGAC-3�; for the JMJD1A pro-moter, 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 threeindependent experiments were performed, and results from a representativeexperiment are presented.

Construction of T-bet mutant expression plasmids. Murine T-bet and Tbx6genes were cloned from cDNA generated from CD4� Th1 cells, the Eomes genewas cloned from CD8� T cells, the Brachyury gene was cloned from dendriticcells, and the SET7/9 gene was cloned from EL4 T cells. The cDNA was clonedinto the pcDNA3.1/V5-HIS vector by using the TOPO TA expression kit (In-vitrogen). The natural stop codon was deleted to allow for a C-terminal V5epitope tag to be added. Deletion mutant constructs were cloned from theplasmids containing the wild-type sequence. All wild-type and mutant constructswere sequenced.

Methyltransferase activity assay. Whole-cell or nuclear extracts were pre-pared and incubated with a primary antibody for 1 h at 4°C. Protein G beadswere then added, and the immunoprecipitation (IP) reaction mixtures wereincubated for another 2 h. The IP reaction mixtures were then washed andresuspended in methyltransferase reaction buffer (20 mM Tris [pH 8], 200 mMNaCl, and 0.4 mM EDTA) with 20 �M S-adenosylmethionine (Sigma) andrecombinant histone H3 (2.5 �g; Upstate Biotechnology) for 1.5 h at roomtemperature. Reaction mixtures were resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer for subsequent Westernanalysis.

RESULTS

T-bet expression is required and sufficient to establish apoised chromatin state. Previous studies have demonstratedthat T-bet is required for IFN-� and CXCR3 transcription inTh1 cells (6, 19, 33, 34). In addition, T-bet expression positivelycorrelates with the permissive H3-K9 acetylation modificationat these promoters (4, 6). To further address the role thatT-bet plays in establishing a permissive chromatin environmentat target promoters, we examined the requirement for T-bet tofunctionally induce the permissive H3-K4 methylation modifi-cations in Th1 cells. CD4� T cells were isolated from eitherwild-type or T-bet�/� mice and cultured under Th1 conditions.Shown in Fig. 1A are the results of a ChIP assay examining theH3-K4 methylation status at the CXCR3 and IFN-� promotersin wild-type and T-bet�/� Th1 cells. IFN-� and CXCR3 wereexpressed in wild-type Th1 cells, but the expression of bothgenes was almost completely absent in T-bet�/� Th1 cells(reference 6; also data not shown). The IFN-� and CXCR3

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promoters both had high levels of the H3-K4 dimethyl modi-fication in wild-type Th1 cells, but this modification was se-verely diminished in the T-bet�/� Th1 cells. We did not detectsignificant H3-K4 trimethylation in either wild-type orT-bet�/� cells (Fig. 1A). We also examined the JMJD1A and

IL-4 promoters as controls. T-bet is able to bind to the JMJD1Apromoter in Th1 cells; however, no functional role for T-bet atthis gene has been demonstrated (6). In contrast to CXCR3and IFN-�, the JMJD1A promoter showed high levels of H3-K4di- and trimethylation in both wild-type and T-bet�/� Th1

FIG. 1. T-bet is required and sufficient for the induction of the permissive H3-K4 dimethyl modification at the CXCR3 and IFN-� promoters.(A) Shown are representative results from a ChIP experiment examining the H3-K4 dimethyl (H3K4me2) and trimethyl (H3K4me3) modificationsat various promoters in CD4� T cells isolated from either wild-type (WT) or T-bet�/� (knockout [KO]) mice and cultured under Th1-skewingconditions. A ChIP assay was performed with antibodies specific to the histone H3-K4Me2 and H3-K4Me3 modifications or a nonspecificimmunoglobulin G (IgG) control as indicated below the graphs. Quantitative PCR analysis was performed with promoter-specific primers asindicated above each graph. The y axis of each graph represents the level of specific-antibody precipitation relative to the total-input control foreach 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. Cellswere cross-linked and a ChIP analysis was performed using antibodies specific to histone H3-K4Me3 (lanes 1 and 5) or H3-K4Me2 (lanes 2 and6) 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-specificprimers were utilized in the PCR portion of the assay, as indicated to the left of the gel images.

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cells, suggesting that T-bet is not required for establishingthese modifications at this promoter. Also, the absence ofT-bet expression modestly increased the dimethylation statusat the IL-4 promoter, consistent with the findings that T-bet�/�

cells have a bias towards expressing Th2 cytokines (33).The data from Fig. 1A suggest that T-bet expression is re-

quired for the permissive H3-K4 dimethyl modification atCXCR3 and IFN-�, two target genes that require T-bet forexpression. We next wanted to determine if T-bet expressionalone is sufficient to induce the permissive H3-K4 methylationmodification at these promoters. To address this question, weutilized an EL4 T-cell transfection system. Consistent withpreviously published results, we did not detect T-bet proteinexpression in EL4 cells by Western analysis (data not shownand reference 33), although a small amount of mRNA could bedetected by RT-PCR (6). In addition, Eomes, Tbx6, andBrachyury transcripts were undetectable (data not shown).Therefore, this system provides a background of minimal en-dogenous T-box protein expression. We transfected EL4 Tcells with either a control or a T-bet expression vector andperformed a ChIP analysis to examine the histone modificationstatus at the target gene promoters. As shown in Fig. 1B, adramatic increase in the level of permissive H3-K4 dimethyl-ation modification was observed at the CXCR3 and IFN-�promoters in the cells transfected with T-bet in comparison tothose transfected with the pcDNA control. In contrast, noincrease in H3-K4 trimethylation was observed. Consistentwith the results from the primary Th1 cells, the JMJD1A pro-moter starts out in a fully permissive H3-K4 methylated stateand T-bet expression does not alter it further. The methylationstatus of the IL-4 promoter is also not affected by the overex-pression of T-bet. Overall, these data suggest that T-bet over-

expression alone is sufficient to induce the H3-K4 dimethyl-ated, but not trimethylated, state at the CXCR3 and IFN-�promoters. Taken together, the data indicate that T-bet is bothrequired and sufficient to induce the poised H3-K4 dimethyl-ation state at the target promoters that require T-bet for ex-pression but that it does not significantly influence the chro-matin environment at the promoters where T-bet associationhas no known functional effect.

The N- and C-terminal domains of T-bet contain transacti-vation potential. The experiments depicted in Fig. 1 indicatethat T-bet is functionally involved in recruiting a histone meth-yltransferase activity to the CXCR3 and IFN-� promoters.These data lead to several questions concerning the mecha-nism by which T-bet regulates target promoters. Is T-bet’s onlyfunction at the CXCR3 and IFN-� promoters to create a per-missive chromatin environment so that other, more ubiqui-tously expressed transcription factors can then activate thegenes? Or is T-bet also involved in subsequent transactivationevents that are downstream and independent of the chromatin-mediated activity? To start to address these questions, weperformed a T-bet structure-function analysis to determine ifT-bet’s role in creating a permissive chromatin environmentcould be separated from its ability to activate transcription. Forthese experiments, we monitored T-bet’s activity at severalknown target genes (6). CXCR3 and IFN-� represent promot-ers that require T-bet for activity, while CCL3 represents atarget that is modestly regulated by T-bet in a cell type-depen-dent manner. As a control, we also monitored CALM2 which,like JMJD1A, is bound by T-bet but for which no functionaleffect of binding has been observed to date (6).

Deletion constructs were made by eliminating portions ofthe N- and/or C-terminal domain of T-bet (Fig. 2). In all cases,

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

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the T-box DNA binding domain was retained to ensure thateach mutant could associate with the endogenous T-box DNAbinding elements found within the target gene promoters.Therefore, this experimental system takes into account the rolethat T-bet plays at the target promoters in the context of thenormal chromatin environment at each gene, thus providing away to examine both chromatin and transactivation eventsunder natural conditions. After the transfection of EL4 T cellswith these constructs, we monitored the endogenous targetgene transcriptional activity by quantitative RT-PCR analysis,and as a control, the levels of expression of each mutant pro-tein construct in the same transfected samples were also mon-itored by Western analysis (see Fig. 3 and 5). In addition, inindependent experiments, we also used ChIP to monitor theability of the mutant constructs to induce the H3-K4 dimethyl-ation activity (see Fig. 4 and 6).

We first examined the requirement for T-bet’s C-terminaldomain in activating endogenous target gene expression. Theprogressive deletion of the C-terminal domain resulted in agraded loss of the activity of T-bet at CXCR3, IFN-�, andCCL3 (Fig. 3A and B). Consistent with previous results, norole for T-bet at CALM2 was observed. The T-bet constructcomprising amino acids 1 to 331 (T-bet 1-331) was not able toactivate transcription at any of the target promoters tested. Itis worth noting that the C-terminal deletion mutant proteinsbehaved in similar manners at all three functional targets, withthe gradual loss in activity. The data thus suggest that the Cterminus of T-bet is required for the transactivation potentialat CXCR3, IFN-�, and CCL3, with the possibility that severalregions contribute to functional activity.

We next examined the transactivation potential of T-bet’sN-terminal domain (Fig. 3C and D). In contrast to the C-terminal mutant proteins, the N-terminal deletion series elic-ited modest variations in responses at CXCR3, CCL3, andIFN-�. The loss of the first 51 amino acids resulted in a signif-icant decrease in transactivation potential at all three targetgenes. However, the expression of each deletion mutant pro-tein prior to that point resulted in modest, promoter-specificactivity differences. At the CXCR3 promoter, full activity wasretained until the first 51 amino acids were deleted, after whichthere was a sharp decrease in activity. In contrast, there was amodest loss in activity (twofold) at the IFN-� promoter withthe deletion of the first 40 amino acids. Interestingly, the de-letion of the first 28 amino acids consistently resulted in amodest increase in the activity at the CCL3 promoter, suggest-ing that a minor inhibitory activity may be present within thefirst 28 amino acids that influences some target genes but notothers. Perhaps consistent with this result, positive and nega-tive cell type-specific consequences were observed in the ab-sence of T-bet at CCL3 and RAD51 (6). Again, CALM2 wasnot affected by any of the deletion mutant proteins.

Separation of transactivation potential from the ability tofunctionally recruit histone methyltransferase activity. Wenext wanted to determine whether the role for T-bet in theregulation of these genes is strictly at the level of creating apermissive chromatin environment or if there are additional,independent transactivation activities. To address this ques-tion, we examined the ability of the deletion mutant proteins torecruit the histone H3-K4 dimethylation-specific methyltrans-ferase activity to the CXCR3 and IFN-� promoters. If T-bet’s

FIG. 3. Transactivation potential is contained within both the N- and C-terminal domains of T-bet. EL4 T cells were transfected with eitherthe 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 ofthe transfected samples were processed for RNA and quantitative RT-PCR analyses (A and C) and the evaluation of protein expression byWestern analysis utilizing a V5 antibody (�V5) to examine construct expression or a glyceraldehyde-3-phosphate dehydrogenase antibody(�Gapdh) as a loading control (B and D). The quantitative RT-PCR analysis was performed with gene-specific primers to monitor the influenceof 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 axisindicates the level of expression of each deletion mutant relative to that of the wild-type T-bet 1-530 transfection after the normalization of valuesfor 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 representativeexperiment monitoring both target gene expression and the mutant protein expression levels in transfected cells are shown.

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only role in influencing promoter activity was to create a per-missive chromatin environment, then it would not be possibleto uncouple T-bet’s ability to recruit the methyltransferaseactivity from the transcriptional activation potential at the tar-get genes. In contrast, the uncoupling of the two events wouldsuggest that there are two or more independent steps thatrequire T-bet to activate the target genes, ultimately creating amore stringent requirement for T-bet in the regulation of thesepromoters.

EL4 T cells were transfected with either the control pcDNAvector, wild-type T-bet, or the T-bet deletion mutant proteins(Fig. 4). ChIP analyses were then performed to examine theability of the mutant proteins to induce the permissive H3-K4dimethylation modification at the CXCR3 and IFN-� promot-ers. Surprisingly, the overexpression of most N- and C-terminaldeletion mutant proteins resulted in similar increases in thepermissive H3-K4 dimethyl modification at the CXCR3 pro-moter (Fig. 4A and B). There was a decrease in activity forT-bet 1-331, but it still retained the ability to induce the H3-K4dimethyl modification to a significant degree (approximately2.6-fold) (Fig. 4A and B). Therefore, despite the fact thatseveral of these mutant proteins (e.g., T-bet 1-331 and T-bet120-530) had severely impaired transcriptional regulatory ac-

tivity, they were still able to establish a permissive chromatinstructure. Similar to the results at the CXCR3 promoter, at theIFN-� 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 toinduce the H3-K4 dimethyl modification. However, in contrastto its performance at the CXCR3 promoter, at the IFN-� pro-moter the T-bet 1-331 mutant protein was unable to signifi-cantly induce the H3-K4 dimethylation. Taken together, thesedata suggest that establishing a permissive chromatin environ-ment is insufficient to upregulate IFN-� or CXCR3 transcrip-tion.

Differential domain requirement for recruiting methyltrans-ferase activity to CXCR3 versus IFN-�. The data suggest that itis indeed possible to separate transactivation potential fromT-bet’s ability to induce epigenetic alterations at target pro-moters. It was somewhat surprising that eliminating either theN- or C-terminal domain of T-bet did not completely impair itsability to functionally recruit the methyltransferase activity tothe CXCR3 promoter. This result led us to further refine thedomain requirements for this activity. To accomplish this, T-bet mutant proteins with truncations of the N-terminal domainin conjunction with deletions of increasing portions of the C

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 deletionmutant overexpression. EL4 T cells were transfected with the control pcDNA3.1 vector (lanes 1 to 4) or one of the following T-bet expression constructsas 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 eitherH3-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 specificto the promoters of CXCR3, IFN-�, JMJD1A, or IL-4 were utilized as indicated to the left of the gel images. (B) A quantitative PCR analysis ofthe samples from panel A was performed as described in the legend to Fig. 1A.

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terminus were created (Fig. 2). We first monitored the tran-scriptional activity of these mutant proteins. Consistent withthe requirement for activities in both the N- and C-terminaldomains for transactivation potential, these truncation mutantproteins did not retain any significant transcriptional activity atCXCR3, IFN-�, or CCL3 (Fig. 5A and B).

We next examined the ability of the combination N- andC-terminal truncation mutant constructs to induce the H3-K4dimethyl modification at the promoters. The T-bet 120-468and T-bet 120-402 mutant proteins were able to induce a sig-nificant amount of H3-K4 dimethylation at the CXCR3 pro-moter (Fig. 6A and B). Once again, the ability of T-bet tofunctionally recruit methyltranferase activity to the CXCR3promoter was retained by mutant constructs that were unableto activate transcription. A mutant protein containing only theT-box DNA binding domain was no longer able to induce theH3-K4 dimethylation. Therefore, the mutant data suggest thatthe T-box domain, with a small portion of either the N or Cterminus perhaps for stabilization, is required to recruit thehistone methyltransferase activity to the CXCR3 promoter.

Consistent with the more stringent requirement for the func-tional recruitment of methyltransferase activity to the IFN-�promoter, the combination N- and C-terminal truncation mu-tant proteins were more impaired in their ability to recruitmethyltransferase activity to this promoter than to the CXCR3promoter (Fig. 4 and 6). The T-bet 120-468 mutant protein didretain some activity, even though it was unable to activateIFN-� transcription. However, in contrast to its activity at the

CXCR3 promoter, the T-bet 120-402 mutant construct did notsignificantly induce the dimethyl modification and was at thelevel of the mutant protein containing the T-box alone. Weconfirmed that these truncation mutant constructs retained theability to bind to the IFN-� promoter similarly to full-lengthT-bet by ChIP, with the affinity of only the T-box-alone con-struct (T-bet 120-331) appearing to be slightly less than that ofthe full-length protein (Fig. 6C).

Divergent T-box family members can recruit methyltrans-ferase activity to the CXCR3 promoter. The data from theT-bet deletion series suggest that the crucial region requiredfor functionally recruiting histone methyltransferase activity tothe CXCR3 promoter resides in the T-box domain itself. TheT-box domain is highly conserved in this transcription factorfamily (25). Therefore, if the T-box contains the critical fea-tures required for recruiting methyltransferase activity, it ispossible that other family members may have this ability aswell. To test this hypothesis, we examined whether a closelyrelated T-box family member, Eomes, and the more distantlyrelated family members Tbx6 and Brachyury are able to recruithistone methyltransferase activity to the CXCR3 promoter.

We first confirmed that other T-box family members are ableto associate with the T-bet target promoters being examined inthis 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 asso-ciate with these promoter regions as well (data not shown).The binding of multiple family members to the same targetpromoters is likely due to the highly conserved nature of theT-box DNA binding domain in this family.

We next wanted to determine if the binding of individualT-box family members results in either conserved or variablefunctional consequences. To accomplish this, we examinedboth their ability to induce the H3-K4 dimethyl modificationand their transactivation potential (Fig. 7B and C). As shownin Fig. 7B, Eomes, Tbx6, and Brachyury were all able to induceH3-K4 dimethylation at the CXCR3 promoter. Similar to T-bet, these T-box family members were unable to induce thefully permissive H3-K4 trimethylated state (Fig. 7B). Thesedata suggest that, indeed, the ability to functionally recruit theH3-K4 dimethyltransferase activity is a conserved function forthis family. In contrast to this conserved function, individualfamily members appear to have variable transactivation poten-tials. Despite the ability of Tbx6 to create a permissive chro-matin environment at the CXCR3 promoter, it has less trans-activation potential in this setting than either T-bet or Eomes(Fig. 7B and C). Both T-bet and Eomes are able to significantlyactivate CXCR3, with T-bet being the most efficient in thissetting. Interestingly, despite the ability of Brachyury to bind toand induce the H3-K4 dimethyl modification at the CXCR3promoter, it is completely unable to transactivate this gene(Fig. 7).

Consistent with the more stringent requirement for the re-cruitment of the methyltransferase activity to the IFN-� pro-moter, the closely related T-box family member Eomes is ableto recruit methyltransferase activity to the IFN-� promoter butthe more distantly related Tbx6 and Brachyury cannot (Fig.7B). This characteristic also coincides with the ability of Eomesto upregulate IFN-� transcriptional activity, whereas Tbx6 andBrachyury are not able to activate this gene at all (Fig. 7C).

FIG. 5. The loss of T-bet’s N- and C-terminal domains results in acomplete absence of transactivation potential. T-bet deletion con-structs that had the N terminus deleted in conjunction with the pro-gressive deletion of the C-terminal domain were utilized. (A and B)EL4 T cells were transfected with the T-bet mutant constructs asindicated in the legends to Fig. 3 and 4. (A) The ability of the mutantconstructs to activate endogenous target gene expression was moni-tored by quantitative RT-PCR. (B) A Western analysis examining thelevels of expression of the constructs in the transfected cells from panelA is shown. �V5, V5 antibody; �GAPDH, glyceraldehyde-3-phosphatedehydrogenase antibody.

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This is not due to an inability of the T-box proteins to associatewith the promoter because Brachyury does in fact have theability to bind to IFN-� (Fig. 7A). It is also worth noting thatEomes does not appear to be able to transactivate IFN-� to thesame extent as T-bet in this setting. These data suggest thatalthough T-bet and Eomes show significant homology, they doin fact have differences that are important for their functionalactivities, and it appears that these differences are downstreamof their conserved role in epigenetic events. Collectively, thedata suggest that the T-box family has a conserved ability tofunctionally recruit histone-modifying activity but that the fam-

ily members also have a degree of target selectivity in theirmechanisms of activation.

T-bet can associate with methyltransferase activity. T-boxfamily members do not contain known methyltransferase ac-tivity domains. Consistent with this property, we could notdetect any inherent methyltransferase activity for T-bet (datanot shown). Therefore, one mechanism for the observed epi-genetic changes is the direct recruitment of a methyltrans-ferase complex to the target promoters. Alternatively, T-betmay be involved in an event upstream of the induction of theH3-K4 dimethyl modification that ultimately results in a cas-

FIG. 6. Localization of the domain required for functional recruitment of methyltransferase activity to the CXCR3 and IFN-� promoters.(A) The ability of the double-truncation mutant proteins to induce the H3-K4 dimethyl (H3K4me2) modification was monitored by ChIP asdescribed 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-bet120-402 (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 samplesdescribed 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 thedouble-truncation mutant proteins retain the ability to bind to the IFN-� 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). Thetransfected 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).

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cade of events that is responsible for the observed epigeneticalterations. To begin to address the mechanism by which T-betis able to induce the H3-K4 dimethyl modification at the targetpromoters, we examined whether T-bet can associate withmethyltransferase activity in coimmunoprecipitation (co-IP)experiments. We transfected EL4 T cells with either a controlpcDNA vector or the wild-type T-bet 1-530. As a positivecontrol, we also performed a transfection with SET7/9, aknown H3-K4 methyltransferase. Following immunoprecipita-tion with a V5 epitope tag antibody, the co-IP samples weremonitored for methyltransferase activity. As shown in Fig. 8,an H3-K4 dimethyl-specific methyltransferase activity waspresent in the T-bet IP sample relative to the pcDNA controlsample. Interestingly, we could not detect any H3-K4 trim-ethyl-specific activity in the T-bet co-IP sample (data notshown). These data suggest that T-bet can physically associatewith a methyltransferase complex specific for the H3-K4 di-methyl activity. To further confirm this observation, we re-peated the co-IP experiments with stimulated 721 B cells inorder to examine endogenous T-bet expression levels. In thestimulated 721 B cells, H3-K4 dimethyl-specific methyltrans-

ferase activity was enriched in the T-bet-precipitated samplerelative to that in a control antibody precipitation. Taken to-gether, these data provide the possibility that T-bet’s ability tofunctionally induce the H3-K4 dimethyl modification is by thedirect recruitment of a methyltransferase complex to the targetpromoters.

DISCUSSION

In this study, we addressed the role for T-bet in the regula-tion of target genes. T-bet expression is both necessary andsufficient to induce the permissive H3-K4 dimethyl chromatinmodification at the IFN-� and CXCR3 promoters. A structure-function analysis separated the requirement for T-bet in cre-ating a poised chromatin state at target promoters from anindependent, downstream transactivation potential that is alsorequired to upregulate transcription. Surprisingly, T-bet’s T-box domain appears to be critical for recruiting the methyl-transferase 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

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 wasprecipitated with an antibody specific to either Brachyury (lane 1), T-bet (lane 2), or a nonspecific IgG control (lane 4). The total input chromatinis 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 weretransfected 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) asindicated. The ability of the T-box family members to induce the H3-K4 dimethyl (H3K4me2) modification was monitored by ChIP. Transfectedsamples 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 anonspecific 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-boxfamily members to regulate endogenous target gene expression was examined by quantitative RT-PCR (C), and a Western analysis to monitorprotein expression levels was performed as indicated in the legend to Fig. 3 (D).

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methyltransferase activity to the CXCR3 promoter, suggestinga conserved function for this family. Collectively, the datasuggest that the T-box family has a conserved role in inducingpermissive epigenetic modifications but that family member-specific transactivation events that occur downstream of theseepigenetic modifications are also required to induce transcrip-tional activity at specific target promoters. This dual require-ment for two separable functional activities may help to explainthe specificity for T-box protein family member activity at veryprecise developmental time points.

The T-box protein family is absolutely critical in establishinggene expression patterns in numerous developmental systems(25). The DNA binding domain for this family is highly con-served for specific binding to T-sites found within developmen-

tally important genes (38). The T-box proteins need to accesstheir cognate DNA binding sites at very precise times duringcellular specification events. At these cell fate decision check-points, it would not be surprising that the T-box proteins maybe required to access DNA that is located in a nonpermissivechromatin state. Our findings suggest that the T-box domain isable both to bind DNA and to participate in the functionalrecruitment of permissive histone methyltransferase activity tothe target genes. It is worth noting that although the T-boxdomain is highly conserved, there is some variability amongfamily members. If the amino acids responsible for this inter-action are not conserved in all family members, it is possiblethat the ability to functionally recruit methyltransferase activitywill vary. Therefore, it will now be important to determine theexact amino acids required for this function. Nevertheless, thisnewly identified role for the T-box domain provides a means bywhich this family has the potential to alter the epigenetic statusof its target genes during development. It also suggests that theT-box family can access its DNA binding sites even when theyare not in a permissive chromatin environment. The ability ofthe T-box proteins to induce the H3-K4 dimethylated, but notthe trimethylated, pattern suggests that they are important increating a permissive, poised epigenetic state at the targets butthat subsequent events are required to create a fully activatedstate.

It will now be important to determine the methyltransferasethat is recruited by the T-box family. Our data suggest thatT-bet can interact with a methyltransferase activity, but theidentity of the complex is still unknown. Several methyltrans-ferases are able to induce methylation at H3-K4 (30). Insightinto the possible candidates can be gained from the observa-tion that the H3-K4 dimethyl, but not trimethyl, state is in-duced at the target promoters. In addition, biochemically, wecould detect only H3-K4 dimethyl-specific methyltransferaseactivity in association with T-bet-precipitated samples. Thesedata suggest that the methyltransferase likely specificallycreates the poised H3-K4 dimethylated state but that other, in-dependent events are required to create the fully permissiveH3-K4 trimethylated chromatin structure at these promoters.

It is possible that the T-box proteins recruit a single meth-yltransferase family. However, it is also equally possible that aconserved domain or common protein found in several meth-yltransferase complexes is responsible for the interaction (39).Perhaps the latter scenario is more likely, because the expres-sion patterns for the methyltransferases are likely to vary sig-nificantly during development. The overlapping and exclusiveexpression patterns of the T-box and methyltransferase fami-lies may ultimately provide another layer of functional speci-ficity during development.

It is intriguing that there is a more stringent requirement foradditional domains within the T-box proteins to functionallyrecruit methyltransferase activity to the IFN-� promoter thanto the CXCR3 promoter. This distinction does not appear to bedue to the affinity of the T-box proteins to bind to the IFN-�promoter, as Brachyury can associate with this promoter butcannot induce the epigenetic alterations. This finding may re-flect the biological need to tightly control IFN-� expression inboth a development- and an activation-dependent manner, incontrast to the developmental regulation of CXCR3. It is pos-sible that events associated with cellular activation serve to

FIG. 8. T-bet can associate with histone methyltransferase activity.A co-IP experiment was performed, and histone methyltransferaseactivity was monitored. (A) EL4 T cells were transfected with either apcDNA 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-terminalV5 epitope tag. Whole-cell extracts were made from the individualsamples and precipitated with a V5 antibody (lanes 1 to 3). A meth-yltransferase activity assay was performed with the precipitated sam-ples, which were subsequently processed for Western analysis to mon-itor H3-K4 dimethyl-specific methyltransferase activity. An amount ofthe recombinant histone H3 equal to that used in the methyltrans-ferase reaction was also run as a control (�; lane 4). The membranewas probed with an antibody specific to histone H3-K4 dimethyl(�H3K4Me2) as shown. (B) The membrane was then stripped andreprobed with a V5-specific antibody (�V5) to confirm the precipita-tion of the transfected constructs. The minor band indicated by theasterisk represents cross-reactivity with the heavy chain. (C) A co-IPexperiment to monitor methyltransferase activity was performed asdescribed in panel A. 721 B cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin (P/I). Nuclear extracts were pre-cipitated with either a control Cdk6 antibody (lane 1) or a T-bet-specific antibody (lane 2). The co-IP samples were subjected to amethyltransferase activity assay, and the Western analysis was per-formed with an H3-K4 dimethyl-specific antibody.

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stabilize the interaction of a T-bet–methyltransferase complexat the IFN-� promoter through a specific N- or C-terminaldomain not conserved in all T-box proteins. The required rolefor additional domains at select targets may help to establish ahierarchy for target gene selection, creating a scenario in whichsome genes are regulated by a broad range of T-box familymembers whereas others require a very specific T-box protein.This possibility is consistent with T-box protein target selectionduring development. In development, T-box protein expres-sion patterns can overlap, yet there are some T-box targetgenes that are very specific to an individual family memberwhereas others are redundantly regulated by multiple familymembers (32). Therefore, it will now be important to carefullydissect the mechanisms that account for the similarities as wellas the differences in the ability of the T-box proteins to inducetarget-specific epigenetic modifications.

With both Tbx6 and Eomes possessing some transactivationpotential at target genes such as CXCR3, it is difficult to ruleout the possibility that a difference in DNA binding affinity isat least partly responsible for the differential transactivationpotentials of these two family members in relation to T-bet.That is, it is likely that some DNA binding affinity differencesare inherent in the T-box family, and it is possible that thesedifferences result in at least some of the variability in theobserved transactivation potentials. However, the fact thatBrachyury has no transactivation activity at the CXCR3 pro-moter despite the ability to bind to the promoter and inducethe H3-K4 dimethyl modification suggests that small differ-ences in binding affinity are not solely responsible for thedifferences observed in transactivation potential. These datathus suggest that the ability of the T-box family to bind andcreate a permissive chromatin environment is not the soleactivity responsible for family member-specific transcriptionalpotential but that, rather, other independent functional do-mains determine the transactivation potential at specific targetgenes downstream of the induction of epigenetic changes.

The dual requirement for the functional recruitment of ahistone methyltransferase and for a subsequent transactivationactivity to induce transcription may also help to explain thestringent and specific requirement for individual family mem-bers at a given target gene (13). This may add another layer ofspecificity to the regulation of gene expression that would notbe present otherwise. It is interesting that target genes that aremodestly modulated by T-bet and Eomes, such as IL-2R�, donot require T-bet for the epigenetic modification at the pro-moter in Th1 cells (6, 17). This finding leads to the speculationthat the role of a T-box family member at a specific gene maybe dependent upon the number of events that it is required toregulate at that given gene and in that unique cell type setting.The current data now provide specific transcriptional regula-tory events to examine to address these questions. Ultimately,these answers will provide insight into the very precise require-ments for individual T-box family members in the regulation oftarget genes during cellular differentiation processes.

ACKNOWLEDGMENTS

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

This work was supported by a Leukemia and Lymphoma SocietySpecial 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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