NCOR1—a new player on the field of T cell development

Nuclear receptor corepressor 1 (NCOR1) is a transcriptional corepressor that links chromatin‐modifying enzymes with gene‐specific transcription factors. Although identified more than 20 years ago as a corepressor of nuclear receptors, the role of NCOR1 in T cells remained only poorly understood. However, recent studies indicate that the survival of developing thymocytes is regulated by NCOR1, revealing an essential role for NCOR1 in the T cell lineage. In this review, we will briefly summarize basic facts about NCOR1 structure and functions. We will further summarize studies demonstrating an essential role for NCOR1 in controlling positive and negative selection of thymocytes during T cell development. Finally, we will discuss similarities and differences between the phenotypes of mice with a T cell‐specific deletion of NCOR1 or histone deacetylase 3 (HDAC3), because HDAC3 is the predominant member of the HDAC family that interacts with NCOR1 corepressor complexes. With this review we aim to introduce NCOR1 as a new player in the team of transcriptional coregulators that control T cell development and thus the generation of the peripheral T cell pool.


INTRODUCTION
Coactivator complexes are usually associated with histone acetyltransferases (HATs), which promote transcription. For reasons of clarity of the drawing, other components of the corepressor or coactivator complexes and additional interacting factors/complexes have been omitted. 9 Arrows from HDACs or HATs indicate deacetylation or acetylation of lysine residues on histone tails, respectively. (B) NCOR1 interacts also with other transcription factors (e.g. transcriptional repressors) and thus regulates their repressive activity. (C) Schematic drawing of the domain structure of NCOR1. The repressive function of NCOR1 is dependent on three repression domains (RD) located at the N-terminal part of NCOR1. RDs have been shown to actively recruit proteins of repressor complexes. Histone deacetylation is mediated by the deacetylase activation domain (DAD) and histone interaction domain (HID). These domains contain SANT-like motifs (named after their presence in Swi3, Ada2, NCOR1/SMRT and TFIIB), which promote the deacetylation of histones. Further, DAD is required for HDAC3 recruitment and HDAC3 enzymatic function. The C-terminal region of NCOR1 is composed of several nuclear receptor interacting domains (RIDs), which mediate recruitment and binding of NCOR1 to DNAbound unliganded NR. For more details about the functions of the various NCOR1 domains we refer the reader to recent reviews. 10,26 Figure 1 has been adapted from reference 10

BASIC FACTS ABOUT NCOR1
NCOR1 (encoded by the Ncor1 gene) was initially isolated in complex with unliganded thyroid hormone (TR) and retinoic acid receptors (RAR). 8 NCOR1 is essential for mediating the transcriptional repression by these nuclear receptors (NRs) in the absence of their ligands via its interaction with HDACs, in particular HDAC3, although interactions with other HDAC members have been shown.
Moreover, NCOR1 is also a corepressor of other NRs such as the peroxisome proliferator-activated receptors, liver X receptor, and estrogen-related receptor (for a detailed description of how NCOR1 regulates NR-mediated transcriptional repression we refer to other reviews 9-11 ). NCOR1 is part of larger multi-subunit complexes and not only associates with members of the NR transcription factor family but also with several other transcription factors including AP-1, 12 Pit-1, 13 MyoD, 14 MeCP2, 15 c-MYC, 16 CREB, 17 and with several BTB-ZF transcription factors such as BCL6, 18,19 PLZF, 20 Kaiso, 21 and MAZR. 22 This indicates that the activity of a broad spectrum of transcription factors depends on NCOR1 corepressor complexes ( Fig. 1A and B).
NCOR1 is a large protein of approximately 2450 amino acids with a molecular weight of 270 kDa and is closely related (approx. 40% amino acid sequence identity) to the corepressor SMRT (also known as NCOR2). 23-25 NCOR1 contains several functional domains that mediate its repressive activity and binding to NRs (Fig. 1C). 10,26 The Ncor1 gene locus encodes, due to alternative splicing, several NCOR1 protein isoforms that differ in the number of nuclear receptor interaction domains (RIDs), which in turn might alter the function and activity of the respective NCOR1 isoforms. [27][28][29] Interestingly, NCOR1 protein isoforms can exert opposite functions, as revealed in adipocytes. 28 Moreover, the generation of various NCOR1 isoforms is regulated by hormonal and metabolic signaling, highlighting the importance of the nutritional environment in regulating NCOR1 activity. 30 In humans, NCOR1 expression displays a broad tissue distribution, including brain, gastrointestinal tract, endocrine tissues, and reproductive organs (www.proteinatlas.org). 31,32 A search in the Imm-Gen database (www.immgen.org) 33 reveals that NCOR1 is also widely expressed in all immune cell lineages including T cells. 22,[34][35][36] is predominantly found in the nucleus; however, cytoplasmic localization of NCOR1 has been reported as well. [37][38][39][40][41] Despite the high homology to SMRT, NCOR1 has essential and nonredundant functions during embryonic development, because germline deletion of NCOR1 is embryonic lethal at E15. 5

Bcl2l11
Ac Ac Ac Ac In DP thymocytes, NCOR1 is recruited to the Bcl2l11 gene locus and represses the expression of BIM. The molecular identity of the transcription factor (TF) that recruits NCOR1 to the Bcl2l11 locus is not known. Loss of NCOR1 leads to hyperacetylation of the Bcl2l11 promoter and increased BIM expression. See text for more details that SMRT is not able to compensate for the loss of NCOR1. 42 Loss of NCOR1 affects many organs, tissues, and cell lineages. E13.5 Ncor1 -/embryos show severe anemia and edema, which suggests altered erythropoiesis as the ultimate cause of death at E15.5. 42 Additionally, NCOR1-deficient embryos have markedly reduced liver sizes and display a defect in CNS development between E12 and E15 as indicated by a size reduction of the developing thalamus. 42 In order to overcome embryonic lethality of germline NCOR1-deficient mice and to study the function of NCOR1 in selected tissues and cell types, several laboratories generated mice that either harbor a conditional

A BRIEF SUMMARY OF T CELL DEVELOPMENT
The two major subsets of conventional peripheral T cells, which express a TCR formed by the TCR-and TCR-chains, can be broadly divided into the MHC class II-restricted CD4 + Th cell lineage and the MHC class I-restricted CD8 + cytotoxic T cell lineage. Both T cell lineages develop in the thymus from a common progenitor in a fascinating biological process, which is also a great model system to study basic principles of cell fate decisions of progenitor cells to a particular lineage. Moreover, developing T cells are "educated" to distinguish between self and nonself, which is required for the establishment of immunologic tolerance. Based on the dynamic expression of the CD4 and CD8 coreceptor molecules one can define four major stages of T cell development ( Fig. 2A). Impressive progress made during the last 20 years allowed the identification and characterization of several subpopulations within the four major developmental stages and revealed detailed molecular and cellular insight into the regulation of T cell development. In this review, T cell development will be only briefly discussed. For more details, we refer the readers to other excellent reviews that provide a comprehensive discussion of major studies that shaped our current view on T cell development. 1,2 The earliest developmental stage in T cell differentiation is repre-

NCOR1 AND THE REGULATION OF T CELL DEVELOPMENT AND T CELL FUNCTION
As discussed earlier, NCOR1 interacts with members of the BTB-ZF transcription factor family, which are key regulators of T cell development and function, 77,78 suggesting that NCOR1 might also have important functions in T cells. Indeed, a very early study indicated already that NCOR1 is essential for proper T cell development. It has been shown that fetal E14.5 DN thymocytes isolated from Ncor1 -/embryos are severely impaired in their progression to the DP stage after 3 days in in vitro fetal thymic organ cultures (FTOC). Of note, a large number of dead cells were observed in Ncor1 -/-FTOCs, suggesting impaired survival in the absence of NCOR1. 42 To further study the role of NCOR1 during T cell development, two laboratories recently generated mice with a T cell-specific deletion of NCOR1. Wang et al. used the Lck-Cre (NCOR1-cKO Lck ) as well as the Cd4-Cre (NCOR1-cKO Cd4 ) strains to delete NCOR1, 35 whereas our laboratory used the Cd4-Cre deleter strain. 36 Both studies show that total thymocyte numbers are not significantly changed; however, the percentages as well as the numbers of SP thymocytes are reduced in NCOR1-cKO Cd4 and NCOR1-cKO Lck mice. As a consequence, this also leads to reduced numbers of peripheral T cells. Further, the generation of BM chimeric mice indicated that the reduction in the SP cell population is due to a T cell-intrinsic defect. 35 35 Further, NCOR1-deficient thymocytes up-regulate also the expression of EGR2, CD127, and BCL-2 during positive selection, 36 which are key molecules that mediate the survival of DP cells during the progression toward the SP stage. 82 This also indicates that the reduction of NCOR1-cKO Cd4 SP cells is not due an impaired expression of these molecules. Rather, the expression of the pro-apoptotic protein BIM that antagonizes the pro-survival function of BCL-2 in DP and SP thymocytes is dramatically increased in NCOR1-cKO Cd4 and NCOR1-cKO Lck mice. Further, in NCOR1-ckO Lck mice, cleaved caspase 3-positive DP thymocytes are enhanced by 2-fold whereas there is no increase in the expression of the pro-apoptotic factors FASL, TRAIL, TNF-, and BAK in activated or nonactivated DP thymocytes in vitro, 35 indicating a role for NCOR1 specifically in controlling BIM-dependent apoptosis. These findings also suggest that BIM-mediated thymocyte apoptosis as well as accelerated BIM-dependent negative selection contribute to the reduction of cells undergoing positive selection and increased cell death of SP thymocytes in the absence of NCOR1. 35,36 Indeed, by using a RIP-OVA mouse model that mimics negative selection of thymocytes activated by endogenously expressed antigens, Wang et al. revealed an enhanced negative selection in NCOR1-cKO Lck mice, most likely due to enhanced BIM expression levels. 35 In contrast, we showed that negative selection as determined by the SEB-mediated clonal deletion of V 8 + CD4SP cells was normal in NCOR1-cKO Cd4 mice. 36 35 Similarly, there is a reduction of FOXP3 + regulatory T (T reg ) cells in the absence of NCOR1. 35,36 Interestingly, Wang et al. observed a small relative increase in the percentage of FOXP3 + T reg cells within the peripheral CD4 + T cell population in NCOR1-cKO Lck mice, 35 whereas we observed in NCOR1-cKO Cd4 mice a mild relative reduction of the percentage of FOXP3 + T cells within the CD4SP subset as well as the peripheral CD4 + T cell population. 36 The reason for the inconsistent results regarding the fractions of FOXP3 + T reg cells remains currently unknown; however, a recent study revealed that NCOR1 together with HDAC3 is required for optimal suppressive activity of regulatory T (T reg ) cells. 34 Mice with a T reg cellspecific deletion of HDAC3 develop lethal autoimmunity because their T reg cells exhibit a severely impaired suppressive activity. It is known that the optimal enzymatic function of HDAC3 requires binding to the DAD domain present in NCOR1 and SMRT complexes 55 ; therefore, the contribution of NCOR1/SMRT-HDAC3 complexes in maintaining T reg cell function was investigated. Indeed, the disruption of NCOR1/SMRT-HDAC3 interaction in mice expressing NCOR1/SMRT with mutated DAD variants led to the generation of FOXP3 + T reg cells with a strongly impaired ability to inhibit T cell proliferation. 34 Together, these data indicated an important role for NCOR1 also in the development of T reg cells and that the association of HDAC3 with NCOR1 (and/or the related factor SMRT) is crucial for an optimal suppressive activity of peripheral T reg cells.

T CELL DEVELOPMENT IN THE ABSENCE OF NCOR1 OR HDAC3-SIMILAR OR DIFFERENT PHENOTYPES?
As described earlier, NCOR1 controls the survival at distinct developmental stages. Because HDAC3 is the main HDAC member associated with NCOR1 corepressor complexes, one might expect that loss of HDAC3 in T cells leads to similar phenotypes. Indeed, several studies showed that HDAC3 is an important regulator of T cell development as well as of T reg cell functions. 34,[84][85][86][87][88] Early deletion of HDAC3 in common lymphoid progenitor cells (CLPs) using the Cd2-iCre transgene (HDAC3-cKO iCd2 ) or in DN thymocytes using the Lck-Cre deleter strain (HDAC3-cKO Lck ) leads to a developmental block at the DP stage due to a defect in positive selection, 85,87 87 Similarly, HDAC3-cKO Lck DP thymocytes show increased Rorc mRNA expression. 85 NCOR1-cKO Cd4 and NCOR1-cKO Lck mice also exhibit defects in positive selection 35,36 ; however, ROR-t expression was properly down-regulated in signaled NCOR1-cKO Cd4 thymocytes during positive selection. 36 Further, positively selected NCOR1-cKO Cd4 SP thymocytes up-regulate EGR2 and express slightly elevated levels of CD127 and BCL-2, 36 whereas HDAC3-cKO iCd2 semi-mature TCR hi CD24 + CD4SP thymocytes do not up-regulate CD127 and EGR2 and consequently do not induce BCL-2 expression. 87 In NCOR1-cKO Lck and NCOR1-cKO Cd4 mice the defect in positive selection translates into reduced numbers of SP thymocytes compared to WT mice and as a consequence also in reduced peripheral T cell numbers. Strikingly, Cd4-Cre mediated targeting of HDAC3 during late T cell development (HDAC3-cKO Cd4 ) does not alter the numbers or percentages of SP thymocytes, indicating a normal progression of positive selection. 86 Nevertheless, peripheral T cell numbers are severely reduced in HDAC3-cKO Cd4 mice. However, the majority of peripheral T cells are recent thymic emigrants due to a defect in post-thymic T cell maturation, as indicated also by the impaired up-regulation of the maturation marker CD55 in the absence of HDAC3. 86,87 Similarly, peripheral T cell numbers are reduced in NCOR1-cKO Cd4 mice; however, CD55 expression levels are normal on peripheral NCOR1-cKO Cd4 T cells indicating that NCOR1, unlike HDAC3, is dispensable for post-thymic T cell maturation. 36 Although HDAC3 is the main HDAC family member associated with NCOR1 corepressor complexes, the comparison of NCOR1-deficient and HDAC3-deficient phenotypes suggests that NCOR1 and HDAC3 act in different pathways, and raises the question why the phenotypes observed upon deletion of NCOR1 and HDAC3 are quite distinct from each other. To explain the differences between the phenotypes, several possibilities are conceivable. First of all, different Cre deleter strains were used for targeting NCOR1 or HDAC3 during T cell development, potentially accounting for some of the differences observed.
Moreover, we noted that NCOR1 protein is still detectable in NCOR1-cKO Cd4 DP thymocytes (despite an efficient genomic deletion of the floxed Ncor1 alleles) and completely absent only from the SP thymocyte stage on. 36 In contrast, in HDAC3-cKO iCd2 mice no HDAC3 protein is detectable from the DN3 stage on. 87 Thus, residual NCOR1 protein present in NCOR1-cKO Cd4 DP thymocytes might recruit HDAC3 to some target genes and facilitates thereby, for example, the downregulation of ROR-t expression prior to positive selection. Further, it is also conceivable that the NCOR1-related factor SMRT takes over some of the functions and thus might compensate for the loss of NCOR1. Finally, although HDAC3 is an integral part of NCOR1 corepressor complexes, 9,10 transcriptional regulation mediated by NCOR1 corepressor complexes might be not exclusively dependent on HDAC3 interactions. The observation that NCOR1 promotes deacetylation of the Bcl2l11 promoter region to restrain BIM expression, 35 whereas BIM expression remains unaffected in the absence of HDAC3, 87 suggests the involvement of other HDAC family members. Additional studies that compare T cell development in mice where the same Cre deleter strain is used to delete NCOR1 or HDAC3 are required to resolve these issues. These studies should also include a careful examination of residual NCOR1 and HDAC3 protein levels in various subsets upon deletion of the Ncor1 and Hdac3 alleles as well as RNA-seq and ChIP-seq approaches to dissect NCOR1-and HDAC3-dependent transcriptional networks during T cell development.

CONCLUDING REMARKS
Although NCOR1 was identified more than 20 years ago, and despite the characterization of many developmental and cellular processes regulated by NCOR1, there is still room to discover novel and important functions for NCOR1 and NCOR1-mediated processes. As briefly summarized in this review, NCOR1 is a crucial regulator of T cell development by controlling DP and SP thymocyte survival. Thus, the recently published studies on the role of NCOR1 in T cells added a previously unknown function for NCOR1, that is, the regulation of cell survival and apoptosis, to the growing list of biological processes that are controlled by NCOR1 in a cell type-specific manner.
It would not be surprising if it turns out that NCOR1, which is a new player on the field of T cell development, has also other important roles throughout a T cell's life.

DISCLOSURES
The authors declare no conflicts of interest.