TRIM28 mediates chromatin modifications at the TCRαenhancer and regulates the development ofT and natural killer T cellsXiao-Fei Zhoua,1, Jiayi Yua,1, Mikyoung Changa, Minying Zhangb, Dapeng Zhoub,c, Florence Cammasd,2,and Shao-Cong Suna,c,3

Departments of aImmunology and bMelanoma Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030; cUniversity ofTexas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030; and dDepartment of Functional Genomics and Cancer, Institut deGénétique et de Biologie Moléculaire et Cellulaire, 67404 Illkirch, France

Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University HealthNetwork, Toronto, ON, Canada, and approved October 26, 2012 (received for review August 23, 2012)

T-cell receptor–α (TCRα) rearrangement in CD4+CD8+ double-positive immature thymocytes is a prerequisite for production of αβT cells and invariant natural killer T cells. This developmental eventis regulated by the TCRα enhancer (Eα), which induces chromatinmodification and recruitment of the recombination-activating pro-teins Rag1 and Rag2. However, the molecular mechanism underly-ing the activation and long-range action of Eα remains incompletelyunderstood. We show here that the chromatin-modifying factorTRIM28 is highly expressed in double-positive thymocytes and per-sistently phosphorylated at serine 473. TRIM28 binds to Eα andinduces histone 3 lysine 4 trimethylation in the Eα and distantregions of the TCRα locus, coupled with recruitment of Rag pro-teins. T-cell–conditional ablation of TRIM28 impaired TCRα generearrangement and compromised the development of αβ T cellsand invariant natural killer T cells. These findings establish TRIM28as a unique regulator of thymocyte development and highlight anepigenetic mechanism involving TRIM28-mediated active chromatinmodification in the TCRα locus.

iNKT | NKT | H3K4me3 | kap1

The development of αβ T cells involves sequential rearrange-ment of the T-cell receptor (TCR) gene segments (1). TCRβ

gene rearrangement occurs in the CD4−CD8− double-negative(DN) thymic precursor cells, in which the successfully rearrangedTCRβ chain forms a pre-TCR together with a surrogate TCRαchain (2). The pre-TCR transduces signals to stimulate the pro-liferation and survival of DN cells during their progression to theCD4+CD8+ double-positive (DP) stage and activates the rear-rangement of TCRα locus. DP thymocytes with successfully re-arranged TCRα genes express surface TCR complexes and undergopositive selection via recognition of self-MHC/peptide, leadingto lineage commitment toward CD4+CD8− or CD8+CD4− single-positive (SP) thymocytes. In addition, a subpopulation of DP thy-mocytes, which expresses an invariant TCRα chain and semivariantTCRβ chains, serves as precursors of invariant natural killer T(iNKT) cells that are characterized by recognition of glycolipidantigens presented by the MHC-like molecule CD1d (3, 4).The rearrangement of the TCRα locus initiates with the use

of relatively 3′ Vα gene segments and 5′ Jα gene segments (5).However, if the initial (or primary) TCRα gene rearrangementdoes not lead to successful positive selection of the DP thymocytes,subsequent (or secondary) Vα–Jα rearrangement occurs betweenprogressively more 5′ Vα and 3′ Jα gene segments to replace thefailed primary Vα–Jα rearrangement. Therefore, mature T cellsexpress TCRα genes with diverse Vα–Jα combinations, contribut-ing to the enormous diversity of the TCR repertoire. In contrast tothe diverse TCRα use of αβ T cells, the TCRα of iNKT cells isformed exclusively by the Vα14–Jα18 recombination (6, 7). Thus,impaired TCRα gene rearrangement often severely compromisesthe development of iNKT cells (8).TCRα rearrangement requires germ-line transcription at the

Vα and Jα loci and is regulated by the T early-alpha promoter

and TCRα enhancer (Eα) (9, 10). In particular, Eα functionsin cis to regulate the Vα–Jα rearrangement across the entireJα locus. Mice lacking Eα have a profound defect in TCRα re-combination and expression (11). Although they can producesome mature αβ T cells, these T cells have highly biased set of Vαgene segments (11). One important mechanism of Eα action isto induce long-range chromatin modifications across the Jα locusand part of the Vα region (12). The opening of the chromatinstructure enhances the accessibility of the recombination signalsequences by the recombination-activating protein Rag1 (5).Moreover, histone 3 lysine 4 trimethylation (H3K4me3) re-cruits Rag2 to the modified chromatin via interaction with theplant homeodomain finger of Rag2 (13, 14). However, the mo-lecular mechanisms that mediate Eα activation and its long-rangeaction in chromatin modification are incompletely understood.In particular, how the H3K4me3 is induced in the Eα and TCRαlocus of the DP thymocytes remains unclear.TRIM28 (tripartite motif protein 28), also named KAP1 or

TIF1β, was originally identified as a transcriptional corepressor thatinteracts with KRAB (Krüppel-associated box) domain-containingzinc finger repressor proteins (15). It is generally thought thatTRIM28 represses transcription by recruiting chromatin-modifyingenzymes, such as the histone 3 lysine 9 (H3K9) methyltransferaseSETDB1 (16, 17) and the nucleosome-remodeling and histonedeacetylation complex. However, TRIM28 has also been shownto interact with other transcription factors and to promote chro-matin relaxation and target gene transcription (18–20). More recentgenomewide ChIP-sequencing studies have revealed the associationof some major TRIM28 target genes with transcriptionally active,instead of repressive, histone modifications, such as H3K4me3 (21).Some other target gene promoters of TRIM28 are enriched forthe active H3K4me3 and the repressive H3K27me3 marks (22).These findings suggest previously unappreciated complexity inTRIM28-mediated chromatin modification.In the present study, we provide genetic evidence that TRIM28

mediates active chromatin modification in the TCRα gene locusand has an important role in regulating TCRα rearrangement andT-cell development. TRIM28 binds to the Eα site and promotesH3K4me3 modification and Rag1/Rag2 recruitment to the Jα re-gion. Thus, ablation of TRIM28 in the T-cell lineage attenuates the

Author contributions: X.-F.Z., J.Y., and S.-C.S. designed research; X.-F.Z., J.Y., M.C., andM.Z. performed research; D.Z. and F.C. contributed new reagents/analytic tools; D.Z.analyzed data; and X.-F.Z., J.Y., and S.-C.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1X.-F.Z. and J.Y. contributed equally to this work.2Present address: Institut de Recherche en Cancérologie de Montpellier, Institut Nationalde la Santé et de la Recherche Médicale U896, Université Montpellier 1, Centre Régionalde Lutte contre le Cancer Val d’Aurelle Paul Lamarque, F-34298 Montpellier, France.

3To whom correspondence should be addressed. E-mail: ssun@mdanderson.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1214704109/-/DCSupplemental.

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expression of various Vα–Jα genes within the DP thymocyte pre-cursor cells, which is associated with impaired development ofiNKT cells and αβ T cells. These findings establish TRIM28 as acrucial regulator of thymocyte development and provide an ex-ample for how TRIM28-mediated active chromatin modificationregulates an important physiological process.

ResultsTRIM28 Is Constitutively Phosphorylated in Thymocytes and HighlyExpressed in DP Stage. To assess the role of TRIM28 in thymo-cyte development, we analyzed its expression in different stagesof thymocytes. Interestingly, although TRIM28 was expressed inall thymic stages, the CD4+CD8+ DP thymocytes expressed astrikingly higher level of TRIM28 mRNA (Fig. 1A) and protein(Fig. S1A). Recent studies demonstrate that phosphorylation ofTRIM28 at S473 or S824 inhibits its association with hetero-chromatin proteins and appears to induce chromatin relaxationand target gene activation (23–25). We found that TRIM28 wasconstitutively phosphorylated at S473, although not at S824, inthymocytes (Fig. 1B). The constitutive phosphorylation of TRIM28appeared to be maintained by in vivo exposure of the thymocytesto stimuli, as the S473 phosphorylation was rapidly lost upon invitro incubation (Fig. 1B). Consistent with the in vivo stimulationof thymocytes, through their pre-TCR and TCR, by self-antigensduring their development and maturation, we found that the S473phosphorylation of TRIM28 was strongly induced upon cross-linking of the TCR and CD28 surface receptors in vitro in thetotal thymocytes (Fig. 1C) and DP thymocytes (Fig. S1B).We next examined whether the constitutive TRIM28 phosphor-

ylation could result from signal transduction through the pre-TCRby using Rag1 KO mice, which have a blockade in thymocytedevelopment at the DN3 stage as a result of the inability to rear-range the TCRβ chain for the formation of pre-TCR. Treatment ofRag1 KO mice with an agonistic anti-CD3 antibody is known torescue the development of DP thymocytes and thought to mimicthe pre-TCR signaling (26, 27). In contrast to the normal thy-mocytes, the Rag1 KO thymocytes had only a background level ofTRIM28 S473 phosphorylation (Fig. 1D). Remarkably, however,injection of the Rag1 KO mice with anti-CD3 resulted in potentinduction of TRIM S473 phosphorylation (Fig. 1D), suggestingthe involvement of pre-TCR signal in the induction of TRIM28S473 phosphorylation.

TRIM28 Regulates T-Cell Development. To determine the functionof TRIM28 in T-cell development, we generated TRIM28 T-cell–specific KO (TKO) mice by breeding TRIM28-floxed mice withLck-Cre mice. We analyzed thymocyte development by using age-matched TRIM28+/+Lck-Cre (called WT) and TRIM28fl/flLck-Cre(called TKO) mice. The total cellularity of thymocytes was similarbetween TKO andWT littermate controls (Fig. 2A). However, theTKO mice had a significantly reduced frequency of CD4 SP cells,coupled with a significant increase in the proportion of CD4+

CD8+ DP thymocytes (Fig. 2 B and C). On the contrary, theTRIM28 deficiency did not significantly alter the percentage ofthe DN population (Fig. 2C) or the percentage of the DN sub-populations (Fig. S2A).Based on the level of surface TCRβ expression, thymocytes can

be further divided into three subpopulations: TCRβhi, TCRβint,and TCRβlow. Newly generated DP thymocytes form the TCRβlowsubpopulation. The subsequent TCRα rearrangement and for-mation of the αβ TCR complex lead to higher levels of TCRβsurface expression and transition to the TCRβint subpopulation.Following positive selections, DP cells further up-regulate theirsurface TCRβ to become the TCRβhi SP cells (2). The TKO thy-mocytes had a drastic accumulation of TCRβlow cells and con-comitant reduction in the proportion of TCRβint cells (Fig. 2D),a phenotype that was also revealed within the gated DP cellpopulation (Fig. 2E). On the contrary, the TRIM28 deficiencydid not alter the intracellular level of TCRβ expression (Fig. S2B).These data suggest that TRIM28 is required for the transition ofDP thymocytes from the TCRβlow to the TCRβint stages.

Fig. 1. Constitutive phosphorylation and expression profile of TRIM28 inthymocytes. (A) qPCR analyses of relative TRIM28 mRNA level in flow cyto-metrically sorted thymocyte subsets. Data are normalized to the expressionof a reference gene, β-actin, and presented as fold relative to the value ofthe DN sample. (B) Immunoblot analyses of phosphorylated (p-) TRIM28 infreshly isolated thymocytes that were rested for the indicated time periodsin vitro. Total (Pan-) TRIM28 was detected after stripping the phospho-immunoblots, and HSP60 was used as a loading control. γ-Irradiated WT andTKO thymocytes were included as positive and negative controls. (C) Im-munoblot analyses of phospho- and Pan-TRIM28 in thymocytes that wererested for 2 h and stimulated with anti-CD3 plus anti-CD28 for the indicatedtime periods. (D) Rag1 KO or WT mice were not treated (NT) or injected withanti-CD3 (75 μg) for 48 h, and thymocytes were isolated for phospho- andPan-TRIM28 immunoblot analyses.

Fig. 2. Impaired thymocyte development in TKO mice. (A) Mean ± SD valuesof total thymocyte numbers calculated based on multiple WT and TKO mice.(B and C) Flow cytometry analyses of the percentage of DN (CD4−CD8−), DP(CD4+CD8+), CD4 SP (CD4+CD8−), and CD8 SP (CD4−CD8+) cell populations inWT and TKO mice (6 wk old). Data are presented as a representative plot (B)and mean ± SD values (C) of multiple animals in three independent experi-ments. (D) Flow cytometry analyses of the percentage of TCRβlow, TCRβint, andTCRβhi populations within total thymocytes. Data are presented as a repre-sentative plot (Left) and mean ± SD values of multiple mice (Right). (E) Flowcytometry plot (Left) and summary graph (Right) showing percentage ofTCRβlow and TCRβint populations in DP thymocytes.

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TRIM28 Deficiency Attenuates TCRα Rearrangements and PerturbsTCRα Gene Use. As increased TCRβ surface expression and for-mation of the TCRβint thymocytes depends on successful TCRαrearrangement, we reasoned that TRIM28 might have a role inregulating the rearrangement of Vα–Jα gene segments. To test thispossibility, we performed quantitative real-time PCR (qPCR) toquantify mRNAs for different Vα–Jα genes in a DP thymic cellpopulation (CD4+CD8+TCRβintCD69−; Fig. 3A) that has notundergone positive selection (28). We randomly selected proximal(Vα19 and Jα50), central (Vα6, Vα8, Vα14, and Jα30), and distal

(Vα3, Jα18, and Jα2) regions to examine their transcript levels(Fig. 3B). The expression of most, although not all, of the rear-ranged TCRα transcripts was reduced in the TRIM28-deficientthymocytes (Fig. 3C). Consistently, the expression of TCRα con-stant region was also reduced in the TKO thymocytes (Fig. 3D). Incontrast, the expression of the TCRδ constant region, as well asthe expression of a nearby gene, Dad1, was comparable betweenthe WT and TKO thymocytes (Fig. 3D). The different TCRαgenes displayed distinct levels of defect; however, the differentlevels of TCRα rearrangement defect have no clear correlationswith the proximal vs. distal gene segments (Fig. 3C). Thus, TRIM28appears to promote the rearrangement of the proximal and distalTCRα gene segments.We next analyzed the TCRα expression by flow cytometry by

using several currently available TCRα antibodies, including thosedetecting Vα2, Vα3.2, Vα8.3, and Vα11. Consistent with the qPCRanalyses, the TRIM28-deficient thymocytes had a reduction in thesurface expression of these Vα chains (Fig. 4A), although the levelof the defect varied among the different TCRα clones, with themost dramatic defect being seen with TCRα3.2 and TCRα11.The defect of TKO mice in TCRα rearrangement prompted

us to examine the Vα gene use in the peripheral CD4 and CD8T cells of these mutant animals. The use frequency of most of theTCRα clones analyzed was significantly reduced in the TRIM28-deficient CD4 T cells, although the frequency of another TCRαclone, TCRα8.3, was not significantly changed (Fig. 4B). The per-turbed TCRα use was also seen with CD8 T cells (Fig. 4B), sug-gesting that the TRIM28 deficiency impaired the TCRα repertoireof the CD8 SP even though it did not alter the number of thissubset of thymocytes. Skewed TCR use is associated with immu-nological disorders, such as infectious diseases and autoimmunity(29). In this regard, a recent work suggests that TKO mice displaylymphopenia and autoimmune symptoms (30). Consistent withthis recent study, we found that the TRIM28-deficient naive CD4T cells were hyporesponsive to TCR/CD28-stimulated IL-2 pro-duction (Fig. S3). It remains to be examined whether the alteredTCRα rearrangement contributes to the hyporesponsive phenotypeof the TRIM28-deficient T cells. Notwithstanding, these find-ings suggest an important role for TRIM28 in regulating TCRαgene rearrangement and maintaining normal TCRα repertoire.

TRIM28 TKO Mice Have Severe Defect in iNKT Development. In con-trast to the TCRα diversity of the αβ T cells, the TCRα of iNKTcells is formed exclusively by the Vα14–Jα18 recombination(6, 7). Thus, the development of iNKT cells is particularly sen-sitive to alterations in TCRα gene rearrangement (8). Importantly,the TKO mice had a drastic reduction in the frequency and ab-solute number of iNKT cells in the thymus, spleen, and liver (Fig.5 A and B). This result was not simply a result of the reduction inthe CD4 T-cell population, as the frequency of iNKT cells was stillconsiderably lower in the TKO mice when analyzed among theCD4+ T cells (Fig. S4).The development of iNKT cells requires positive selection

through recognition of self-glycolipid presented by the CD1dmolecule on cortical DP thymocytes (31). To examine whetherTRIM28 regulated iNKT development in a cell-intrinsic manneror through regulation of antigen presentation by DP thymocytes,we performed mixed bone-marrow chimera studies by transferringbone marrow cells derived from WT (B6.SJL) and TKO mice.Under these conditions, the SJL-derived WT thymocytes are ableto serve as antigen-presenting cells in TKO iNKT development.As SJL and TKO cells express CD45.1 and CD45.2 congenitalmarkers, respectively, we were able to monitor the developmentof iNKT cells specifically derived from the TKO bone marrow.Despite the presence of normal antigen-presenting thymocytes,the TKO thymocytes were still defective in iNKT generation(Fig. 5C). This defect was not caused by lower cell populationderived from the TKO bone marrow, as the chimeric mice evencontained a higher frequency of the TKO-derived cells in thethymus and spleen (Fig. S5). These results suggest a cell-intrinsicrole for TRIM28 in regulating iNKT development.

Fig. 3. Defective TCRα rearrangements in TKO mice. (A) Strategy for sortingCD4+CD8+TCRβintCD69− progenitor population. (B) Schematic map of se-lected Vα (TRAV) and Jα (TRAJ) region for qPCR analyses, including proximal,central, and distal Vα segments and Jα segments. (C) qPCR analyses ofrearranged Vα–Jα segments in WT and TKO thymic progenitor population.Data are representative of three independent experiments with four mice ineach genotype per experiment. (D) qPCR analyses of the constant region ofTCRδ (TRDC) and TCRα (TRAC) as well as a nearby gene, Dad1.

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iNKT Defect of TRIM28 TKO Mice Can Be Rescued by PrearrangedVα14–Jα18 TCRα Transgene. Following successful Vα14–Jα18 recom-bination, immature iNKT cells undergo a multistage maturationprocess that can be monitored based on their surface expres-sion of CD44 and NK1.1 markers (32, 33). The TRIM28 de-ficiency did not affect the iNKT maturation from CD44low

NK1.1− stage to the mature CD44highNK1.1+ stage (Fig. S6 Aand B). However, the TKO mice had drastically reduced numbersof all stages of iNKT cells, including the most immature precursorcells (Fig. S6C). Apoptosis analyses did not reveal appreciabledifferences between the WT and TKO iNKT cells (Fig. S7). Thesefindings suggest that TRIM28 may regulate the early stages of

Fig. 4. Impaired TCRα use in TRIM28-deficientthymocytes and peripheral T cells. (A) Flow cytom-etry analysis of the percentage of thymocytesexpressing specific TCRα chains. Data are presentedas a representative plot (Left) and summary graphs(Right) of multiple mice. (B) Flow cytometry analysisof the percentage of CD4 and CD8 peripheral T cellsexpressing specific TCRα chains. Data are presentedas a representative plot for CD4 T cells (Left) andsummary graphs of CD4 and CD8 T cells (Right) ofmultiple mice.

Fig. 5. Defective iNKT development and its rescue by a rearranged Vα14–Jα18 TCRα transgene in TRIM28-deficient mice. (A and B) Flow cytometry analysisof iNKT cells in the indicated organs of WT and TKO mice, based on staining with anti-TCRβ and PBS57-loaded CD1d tetramer. Data are presented as arepresentative plot (A) and summary graphs (B). (C) Lethally irradiated SJL mice were reconstituted with a mixture (1:1 ratio) of bonemarrow cells isolated fromthe SJL (CD45.1+) and TKO (CD45.2+) mice. The chimeric mice were subjected to flow cytometry for analyzing iNKT cells in the indicated organs. Data arepresented as mean ± SD values of the percentage or absolute numbers of iNKT cells. (D and E) Flow cytometry analysis of iNKT cells, based on TCRβ and CD1d-tetramer staining, in the indicated organs of WT-Vα14-Tg and TKO-Vα14-Tg mice. Data are presented as a representative plot (D) or summary graphs (E).

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iNKT cell development through mediating TCRα rearrangement.To examine this possibility, we crossed the TKO mice with trans-genic (Tg) mice (Vα14-Tg) expressing a rearranged Vα14–Jα18TCRα gene under the control of endogenous Vα11 promoter andIg enhancer (34). The iNKT development in these Tg mice involvesnormal process of TCRβ rearrangement, but it bypasses therequirement of TCRα rearrangement. Thus, if TRIM28 in-deed controls iNKT cell development through mediating TCRαrearrangement, the iNKT defect in the TKO mice should be atleast partially rescued by the Vα14–Jα18 transgene. Indeed, wefound that, when crossed with the Vα14-Tg mice, the TKO miceno longer displayed the significant defect in iNKT cell production(Fig. 5 D and E). These results suggest that TRIM28 regulatesiNKT cell development via mediating TCRα rearrangement.

TRIM28 Binds to Eα and Induces Chromatin Modification in Jα Locus.During TCRα rearrangement, Rag1 and Rag2 bind to a focalregion of active chromatin encompassing TCRα joining (Jα)segments to initiate recombination (35, 36). ChIP assays revealedlittle or no binding of TRIM28 to the Jα segments (Fig. 6A). In-terestingly, however, a strong association of TRIM28 with the Eαwas observed in the same assay (Fig. 6A). Eα is a highly con-served region of approximately 200 bp located downstream ofthe TCRα constant region (11). Eα induces chromatin modifi-cation across the entire Jα locus, which promotes the recruitmentof Rag proteins (12). The activation of Eα involves H3K4me3and H3-Ac modifications, although the underlying mechanismhas remained unclear (35–37). Interestingly, our ChIP assaysrevealed a dramatic defect of Eα H3K4me3 in TKO thymocytes,whereas another active chromatin mark, H3-Ac, was not af-fected by the TRIM28 deficiency (Fig. 6B). The TRIM28 de-ficiency also did not appreciably affect the H3K9me3 orH3K27me3 modifications (Fig. S8). Therefore, TRIM28 mayregulate Eα activity by inducing H3K4me3.To examine whether TRIM28 regulates the long-range effect

of Eα in chromatin modification, we analyzed the H3K4me3 inthe Jα locus of WT and TRIM28-deficient DP thymocytes. In WTDP thymocytes, H3K4me3 was found to be associated with severalJα gene segments (Fig. 6C). Importantly, the TRIM28 deficiencyseverely inhibited the H3K4me3 in most of the Jα gene segments.These findings provide an important insight into the molecular

mechanism by which TRIM28 regulates the activity of Eα andmediates the long-range effect of Eα across the Jα locus.Eα-mediated chromatin modification is crucial for recruiting

Rag1 and Rag2 to the Jα region, which in turn is required forVα–Jα recombination (5, 13, 14). In particular, Rag2 is recruitedto the Jα region via its association with H3K4me3 of Jα region,whereas the Rag1 recognizes the recombination signal sequencesthat become accessible upon chromatin opening (5, 13, 14). Indeed,we detected binding of Rag1 and Rag2 to several Jα segments, andthis binding was diminished in the TRIM28-deficient thymocytes(Fig. 6D). V(D)J gene segment recombination is restricted inG0/G1 stage, and periodic degradation of Rag2 at G1-to-S tran-sition is critical to protect mouse from lymphoid malignancies (38).Cell cycle is thus important for regulating the expression levelof Rag2 and the TCRα gene rearrangement (38). Attenuation ofRag2 binding to Jα in TRIM28 KO mice could be because ofchanging of cell cycle progression. Because the TRIM28-deficientperipheral T cells are hyporesponsive to TCR/CD28-stimulatedproliferation and have a defect in the expression of some cellcycle-related genes (30), we analyzed the cell cycle of the WT andTKO DP thymocyte progenitor cells. The TRIM28 deficiencydid not appreciably alter the cell cycle progression of these DPthymocyte progenitor cells (Fig. S9). Consistently, loss of TRIM28also did not alter the expression level of the Rag2 or Rag1 (Fig.6E). These results, together with the findings that TRIM28 medi-ates H3K4me3 modification at the Jα locus, suggest that TRIM28regulates the recruitment of Rag1 and Rag2 to the Jα locus viaan epigenetic mechanism.

DiscussionIn this study, we demonstrated an important role for TRIM28 inregulating TCRα rearrangement. TRIM28 was regulated duringthymocyte development, characterized by a sharp elevation at theDP thymocyte stage. In the DP thymic progenitor population,TRIM28 associated with the Eα and induced chromatin modi-fication across the Jα locus and was required for optimal re-cruitment of Rag1 and Rag2 to the Jα region. The expression ofmost, although not all, of the Vα–Jα genes was substantially re-duced in the preselection DP thymocytes of TKO mice. Whilethis manuscript was being prepared for submission, another groupreported a role for TRIM28 in regulating gene expression inthymocytes, although how TRIM28 regulates thymocyte deve-lopment has remained unclear (39). The present study demon-strates a unique function of TRIM28 in the control of Vα–Jαrecombination. The impaired TCRα rearrangement in the TKOmice had the most dramatic effect on iNKT cells. This is consistentwith the dependence of iNKT cells on a single TCRα chain (Vα14–Jα18), as opposed to the highly diverse TCRα repertoire of αβT cells. The iNKT deficiency was not caused by a blockade inits maturation, but apparently by the attenuated Vα14–Jα18 re-combination, as it could be rescued by a rearranged Vα14–Jα18transgene in the TKO mice.The TRIM28 deficiency also affected the development of αβ

T cells, although the effect was less dramatic than that on theiNKT cells. The less dramatic influence of TRIM28 deficiencyon αβ T cells reflects the nature of TCRα gene rearrangement.The rearrangement of TCRα genes is a persistent and multiroundprocess that involves progressive recombination of numerousVα and Jα segments. If a primary rearrangement does not leadto successful positive selection of the DP thymocytes, secondaryrearrangement proceeds to recombine an alternative pair ofVα–Jα segments. As the αβ T cells have a diverse TCRα reper-toire, they are likely less sensitive to partially impaired TCRαrearrangement than the iNKT cells. Our data suggest differentialsensitivities of the CD4 and CD8 SP thymocytes to the impairedTCRa rearrangement, as the frequency of CD4 SP thymocytes,but not that of the CD8 SP thymocytes, was significantly reducedin the TKO mice. Of course, the differential effect of TRIM28deficiency on CD4 and CD8 SP thymocyte development mayalso be attributed to additional functions of TRIM28 (30, 39).However, it is clear that the perturbation in TCRα rearrangement

Fig. 6. TRIM28 binds to Eα and regulates active chromatin modification andRag recruitment in the Jα locus. DP thymocytes were isolated from 3-wk-oldWT or TKO mice and subjected to ChIP or immunoblot assays. (A) ChIPanalyses using anti-TRIM28 or control IgG antibodies to measure the bindingof TRIM28 to Jα segments or Eα region in WT thymocytes. (B) ChIP analyses ofthe association of H3K4me3 and H3-AC to the Eα of WT or TKO thymocytes.(C and D) ChIP assays measuring the association of H3K4me3 (C) and Rag1 orRag2 (D) to the indicated Jα segments of WT or TKO mice. (E) Immunoblotanalyses of the indicated proteins in the cytoplasmic or nuclear extracts ofWT or TKO thymocytes. The nuclear Rag2 was quantified by densitometrybased on different animals (each circle represents an animal) and presentedas ratio to HDAC1 (Right).

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in the TKO mice affected the TCR repertoire of the CD4 andCD8 T cells. The thymocytes and the peripheral CD4 and CD8T cells of TKO mice had drastically reduced frequency in severalTCRα genes. Given the association of skewed TCR use withimmunological disorders, such as infectious diseases and auto-immunity (29), this finding has important implications. In thisregard, a recent work suggests that TKO mice display lymphopeniaand autoimmune symptoms (30). The TRIM28-deficient naiveCD4 T cells are also hyporesponsive to TCR/CD28-stimulatedIL-2 production (ref. 30 and the present study). AlthoughTRIM28 has a role in regulating TGF-β expression, it remainsan intriguing possibility that some of the peripheral immuneabnormalities of the TKO mice might be contributed by theTCRα rearrangement defect.It is generally thought that TRIM28 promotes the formation

of repressive chromatin by recruiting chromatin-modificationand remodeling factors; however, recent genome-wide ChIP-sequencing studies led to the surprising finding that some of themajor TRIM28-target genes are associated with active chromatinmarks, such as H3K4me3 (21). The role of TRIM28 in chromatinrelaxation and gene induction appears to require its phosphoryla-tion at specific serine residues, S473 or S824 (23–25). Interestingly,we found that TRIM28 is constitutively phosphorylated at S473.The present study identified the Eα as a binding target of TRIM28associated with the active chromatin mark H3K4me3 and H3-Acin DP thymocytes. TRIM28 was required for the formation of

H3K4me3, but not H3-Ac, a finding that supports the genome-wide ChIP-sequencing study and establishes a link between thispositive chromatin-modifying function of TRIM28 and an im-portant biological process: TCRα rearrangement. As such, thepresent study not only demonstrates a unique function of TRIM28in the regulation of thymocyte development, but also significantlyexpands our understanding of how TRIM28 regulates chromatinmodification and gene expression through regulating differenttypes of histone methylation.

Materials and MethodsTRIM28-floxed mice (40) were crossed with Lck-Cre mice [provided by JunjiTakeda, Osaka University, Osaka, Japan (41)] to generate age-matchedTRIM28+/+Lck-Cre (called WT) and TRIM28fl/flLck-Cre (called TKO) mice. Vα14Tg mice expressing the rearranged Vα14–Jα18 TCRα chain (34) were crossedwith the TKO mice to generate WT-Vα14Tg and TKO-Vα14Tg littermates.B6.SJL (CD45.1+) and Rag1 KOmice were from Jackson Laboratory. All animalexperiments were done in accordance with protocols approved by the In-stitutional Animal Care and Use Committee of the University of Texas MDAnderson Cancer Center.

ACKNOWLEDGMENTS. We thank J. Takeda for Lck-Cre mice; the NationalInstitutes of Health (NIH) Tetramer Core Facility for CD1d tetramers; andthe personnel from the flow cytometry, DNA analysis, and animal facilitiesat MD Anderson Cancer Center for technical assistance. This work wassupported by US NIH Grants AI057555, AI064639, and GM84459.

1. Bassing CH, Swat W, Alt FW (2002) The mechanism and regulation of chromosomal V(D)Jrecombination. Cell 109(suppl):S45–S55.

2. Fehling HJ, von Boehmer H (1997) Early alpha beta T cell development in the thymusof normal and genetically altered mice. Curr Opin Immunol 9(2):263–275.

3. Gapin L, Matsuda JL, Surh CD, Kronenberg M (2001) NKT cells derive from double-positive thymocytes that are positively selected by CD1d. Nat Immunol 2(10):971–978.

4. Dao T, et al. (2004) Development of CD1d-restricted NKT cells in the mouse thymus.Eur J Immunol 34(12):3542–3552.

5. Shih HY, Hao B, Krangel MS (2011) Orchestrating T-cell receptor α gene assemblythrough changes in chromatin structure and organization. Immunol Res 49(1-3):192–201.

6. Van Kaer L, Joyce S (2005) Innate immunity: NKT cells in the spotlight. Curr Biol 15(11):R429–R431.

7. Bendelac A, Savage PB, Teyton L (2007) The biology of NKT cells. Annu Rev Immunol25:297–336.

8. Hager E, Hawwari A, Matsuda JL, Krangel MS, Gapin L (2007) Multiple constraintsat the level of TCRalpha rearrangement impact Valpha14i NKT cell development.J Immunol 179(4):2228–2234.

9. Abarrategui I, Krangel MS (2006) Regulation of T cell receptor-alpha gene recombinationby transcription. Nat Immunol 7(10):1109–1115.

10. Abarrategui I, Krangel MS (2009) Germline transcription: A key regulator of accessi-bility and recombination. Adv Exp Med Biol 650:93–102.

11. Sleckman BP, Bardon CG, Ferrini R, Davidson L, Alt FW (1997) Function of the TCRalpha enhancer in alphabeta and gammadelta T cells. Immunity 7(4):505–515.

12. Hawwari A, Bock C, Krangel MS (2005) Regulation of T cell receptor alpha geneassembly by a complex hierarchy of germline Jalpha promoters. Nat Immunol 6(5):481–489.

13. Liu Y, Subrahmanyam R, Chakraborty T, Sen R, Desiderio S (2007) A plant homeo-domain in RAG-2 that binds Hypermethylated lysine 4 of histone H3 is necessary forefficient antigen-receptor-gene rearrangement. Immunity 27(4):561–571.

14. Matthews AG, et al. (2007) RAG2 PHD finger couples histone H3 lysine 4 trimethy-lation with V(D)J recombination. Nature 450(7172):1106–1110.

15. Urrutia R (2003) KRAB-containing zinc-finger repressor proteins. Genome Biol 4(10):231.

16. Schultz DC, Ayyanathan K, Negorev D, Maul GG, Rauscher FJ, 3rd (2002) SETDB1:A novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that con-tributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins.Genes Dev 16(8):919–932.

17. Iyengar S, Farnham PJ (2011) KAP1 protein: An enigmatic master regulator of thegenome. J Biol Chem 286(30):26267–26276.

18. Seki Y, et al. (2010) TIF1beta regulates the pluripotency of embryonic stem cells ina phosphorylation-dependent manner. Proc Natl Acad Sci USA 107(24):10926–10931.

19. Chang CJ, Chen YL, Lee SC (1998) Coactivator TIF1beta interacts with transcriptionfactor C/EBPbeta and glucocorticoid receptor to induce alpha1-acid glycoproteingene expression. Mol Cell Biol 18(10):5880–5887.

20. Rambaud J, Desroches J, Balsalobre A, Drouin J (2009) TIF1beta/KAP-1 is a coactivatorof the orphan nuclear receptor NGFI-B/Nur77. J Biol Chem 284(21):14147–14156.

21. Iyengar S, Ivanov AV, Jin VX, Rauscher FJ, 3rd, Farnham PJ (2011) Functional analysisof KAP1 genomic recruitment. Mol Cell Biol 31(9):1833–1847.

22. Hu G, et al. (2009) A genome-wide RNAi screen identifies a new transcriptionalmodule required for self-renewal. Genes Dev 23(7):837–848.

23. Ziv Y, et al. (2006) Chromatin relaxation in response to DNA double-strand breaks ismodulated by a novel ATM- and KAP-1 dependent pathway. Nat Cell Biol 8(8):870–876.

24. Chang CW, et al. (2008) Phosphorylation at Ser473 regulates heterochromatin protein1 binding and corepressor function of TIF1beta/KAP1. BMC Mol Biol 9:61.

25. Lee DH, et al. (2012) Phosphoproteomic analysis reveals that PP4 dephosphorylatesKAP-1 impacting the DNA damage response. EMBO J 31(10):2403–2415.

26. Levelt CN, Mombaerts P, Iglesias A, Tonegawa S, Eichmann K (1993) Restoration of earlythymocyte differentiation in T-cell receptor beta-chain-deficient mutant mice by trans-membrane signaling through CD3 epsilon. Proc Natl Acad Sci USA 90(23):11401–11405.

27. Shinkai Y, Alt FW (1994) CD3 epsilon-mediated signals rescue the development ofCD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR beta chain expression.Int Immunol 6(7):995–1001.

28. Hu T, Simmons A, Yuan J, Bender TP, Alberola-Ila J (2010) The transcription factorc-Myb primes CD4+CD8+ immature thymocytes for selection into the iNKT lineage.Nat Immunol 11(5):435–441.

29. Miles JJ, Douek DC, Price DA (2011) Bias in the αβ T-cell repertoire: Implications fordisease pathogenesis and vaccination. Immunol Cell Biol 89(3):375–387.

30. Chikuma S, Suita N, Okazaki IM, Shibayama S, Honjo T (2012) TRIM28 prevents auto-inflammatory T cell development in vivo. Nat Immunol 13(6):596–603.

31. Bendelac A (1995) Positive selection of mouse NK1+ T cells by CD1-expressing corticalthymocytes. J Exp Med 182(6):2091–2096.

32. Benlagha K, Kyin T, Beavis A, Teyton L, Bendelac A (2002) A thymic precursor to theNK T cell lineage. Science 296(5567):553–555.

33. Pellicci DG, et al. (2002) A natural killer T (NKT) cell developmental pathway involvinga thymus-dependent NK1.1(-)CD4(+) CD1d-dependent precursor stage. J Exp Med195:835–844.

34. Bendelac A, Hunziker RD, Lantz O (1996) Increased interleukin 4 and immunoglobulinE production in transgenic mice overexpressing NK1 T cells. J Exp Med 184(4):1285–1293.

35. Ji Y, et al. (2010) Promoters, enhancers, and transcription target RAG1 binding duringV(D)J recombination. J Exp Med 207(13):2809–2816.

36. Ji Y, et al. (2010) The in vivo pattern of binding of RAG1 and RAG2 to antigen re-ceptor loci. Cell 141(3):419–431.

37. Pekowska A, et al. (2011) H3K4 tri-methylation provides an epigenetic signature ofactive enhancers. EMBO J 30(20):4198–4210.

38. Li Z, Dordai DI, Lee J, Desiderio S (1996) A conserved degradation signal regulatesRAG-2 accumulation during cell division and links V(D)J recombination to the cellcycle. Immunity 5(6):575–589.

39. Santoni de Sio FR, et al. (2012) KAP1 regulates gene networks controlling T-cell de-velopment and responsiveness. FASEB J.

40. Cammas F, et al. (2000) Mice lacking the transcriptional corepressor TIF1beta aredefective in early postimplantation development. Development 127(13):2955–2963.

41. Takahama Y, et al. (1998) Functional competence of T cells in the absence of gly-cosylphosphatidylinositol-anchored proteins caused by T cell-specific disruption ofthe Pig-a gene. Eur J Immunol 28(7):2159–2166.

20088 | www.pnas.org/cgi/doi/10.1073/pnas.1214704109 Zhou et al.

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