Tcf1 transcription factor is a member of the Tcf/Lef family which consists of four members in vertebrates. Its orthologs in invertebrates include dTCF/pangolin in Drosophila and POP-1 in C. elegans (Arce et al. 2006; Cadigan 2012). Tcf1 was molecularly cloned in 1991 and found to be most abundantly expressed in T lymphocytes. In 1996, Lef1, a close homologue of Tcf1, was found to interact with β-catenin which is posttranslationally regulated by the Wnt signaling pathway. Since then, Tcf1, together with other members of Tcf/Lef family, is considered a Wnt effector transcription factor (Klaus and Birchmeier 2008). However, research efforts over the past two-and-half decades have revealed that Tcf1 interacts with several other transcription factors and cofactors, in addition to β-catenin, to cooperatively regulate gene transcription (Steinke and Xue 2014).
Tcf1 Structure and Functional Domains
The β-BD domain is on the N-terminus of full-length Tcf1 protein and mediates interaction between Tcf1 and the coactivator β-catenin. β-catenin protein is stabilized by signals derived from Wnt ligands, prostaglandins, and maybe T cell receptors; however, the significance of Tcf1-β-catenin interaction in T cells is yet to be fully resolved (Xue and Zhao 2012). Due to differential promoter usage and alternative splicing, Tcf1 protein is expressed in multiple isoforms, with a group of isoforms lacking the β-BD domain. These Tcf1 short isoforms are suggested to function as dominant negative inhibitors of the full-length Tcf1 protein, and their physiological roles have not yet been elucidated.
Located between the β-BD and HMG domains in Tcf1 is a region known as context-dependent regulatory domain (CRD). Unlike the β-BD and HMG domains, the primary sequence of CRD is less conserved among Tcf/Lef family members, and CRD may have both transcriptional activation and suppression activity depending on cell/gene context. The CDR and part of HMG domain mediate interaction with transducing-like enhancer of split (TLE) corepressors (known as Groucho in Drosophila), and TLEs recruit HDACs by direct interaction to repress gene expression. Recently, Tcf1 has been found to have intrinsic HDAC activity, which is mapped in the CRD (Xing et al. 2016). This surprising finding suggests that Tcf1 can directly bridge transcriptional and epigenetic regulations. The Tcf1 HDAC activity can be coupled with its DNA-binding capacity and can also be recruited by other transcription factors (such as Runx), thus enhancing efficiency and flexibility in target gene regulation.
Tcf1 Regulates T Cell Development in a Stage-Specific Manner
More recent studies have revealed more stage-specific roles of Tcf1, and its intricate interplay with its close homolog, Lef1. Notch signaling is essential for directing HSCs to the T cell lineage, and directly upregulates Tcf1 in ETPs (De Obaldia and Bhandoola 2015). Significantly, forced expression of Tcf1 in HSCs is sufficient to activate at least a portion of the T cell transcriptional program including Gata3 and Bcl11b. Early deletion of Tcf1 results in several severe, yet incomplete, blocks during T cell development. Whereas early deletion of Lef1 does not show a detectable impact, combined deletion of Tcf1 and Lef1 completely blocks DN to DP maturation (Yu et al. 2012). Within the DN stage, Tcf1 and Lef1 promote β-selection at the CD25+CD44− DN3 stage, although they are not absolutely required for initiating rearrangements of the TCRβ locus. In addition, Tcf1 functions as a tumor suppressor in early thymocytes (Fig. 2), because Tcf1-deficient mice develop thymic lymphomas which phenotypically resemble T cell acute lymphoblastic leukemia (T-ALL) in humans. In most T cell-related functions, Tcf1 and Lef1 exhibit functional redundancy. An interesting twist in the context of thymocyte transformation is that Tcf1 directly restrains Lef1 expression in early thymocytes, and the thymic lymphomas are rarely observed in Tcf1- and Lef1-double deficient mice. Importantly, an ETP subtype of T-ALL is associated with diminished Tcf1 expression, and two cases of ETP-T-ALL were found to have monoallelic TCF7 gene deletions (Yu et al. 2012).
Late deletion of Tcf1 and Lef1 at the DP stage reveals their novel roles in two aspects during T cell development, that is, promoting CD4+ T cell lineage choice and establishing CD8+ T cell identity. In these processes, Tcf1 and Lef1 show clear functional redundancy because ablating either factor shows little or very modest effects. The CD4+ versus CD8+ lineage choice by DP thymocytes is orchestrated by a defined transcriptional network. Gata3, Tox, Myb, and Thpok direct DP thymocytes to the CD4+ lineage, and Runx3 and Mazr are essential for generation of CD8+ lineage T cells (Collins et al. 2009). Among these, Thpok and Runx3 have mutually antagonistic effects. Unlike germline deletion of Tcf1, late inactivation of both Tcf1 and Lef1 using CD4-Cre does not significantly diminish total thymic cellularity or the mature T cell compartment that contains CD4+ and CD8+ T cells (Steinke et al. 2014). Late deletion of Tcf1/Lef1 does not affect the total numbers of mature T cells, but greatly diminishes CD4+ T cells and concomitantly increases CD8+ T cells. Further functional analyses reveal that CD4+ T cells undergo lineage redirection to a CD8+ T cell fate in the absence of Tcf1 and Lef1, similar to what has been observed in the context of Gata3 or Thpok deficiency. Mechanistically, Tcf1 and Lef1 act upstream of Thpok, partly by binding to a “general T lymphoid element” in the Thpok gene locus. Because Tcf1/Lef1 deficiency does not affect the expression of Gata3, Tox, and Myb in DP thymocytes, Tcf1 and Lef1 constitute an independent pathway but converge on Thpok to promote CD4+ lineage choice (Fig. 2).
Tcf1 and Lef1 are not required for DP thymocytes to choose the CD8+ T cell fate; however, Tcf1/Lef1-deficient CD8+ T cells show aberrant expression of the CD4 coreceptor and other CD4+ lineage-associated genes including Foxp3 and Rorc (Xing et al. 2016). Repression of the Cd4 gene in CD8+ T cells is known to be mediated by Runx1 and Runx3, which bind to a well-characterized Cd4 silencer located in intron 1 of the Cd4 gene (Collins et al. 2009). Tcf1 interacts with Runx3, binds to the Cd4 silencer, and cooperates with Runx3 in Cd4 gene silencing (Steinke et al. 2014). Beyond the Cd4 gene locus, Tcf1 and Lef1 have a much broader role in establishing a proper histone acetylation landscape in CD8+ T cells, and this role is ascribed to their intrinsic HDAC activity (Xing et al. 2016). The CD4+ lineage-associated genes are marked with increased levels of acetylation of lysines 9 and 27 in H3 histone in Tcf1/Lef1-deficient CD8+ T cells, which can be partly rectified by wild-type Tcf1, but not a Tcf1 mutant with compromised HDAC activity. Therefore, Tcf1 and Lef1 establish CD8+ T cell identity by broadly repressing lineage-inappropriate genes (Fig. 2). It remains to be elucidated how Tcf1/Lef1 HDAC activity is regulated to allow Tcf1 and Lef1 to function as transcriptional activators.
Tcf1 Regulates Mature T Cell Differentiation during Immune Responses
After exiting the thymus, mature CD4+ or CD8+ T cells migrate to and take up residence in secondary lymphoid tissues. Upon encountering cognate antigens, the mature T cells are activated and undergo differentiation processes to exert effector functions. CD4+ and CD8+ T cells are functionally distinct lymphocytes. CD4+ T cells provide versatile help tailored to specific pathogen types, whereas CD8+ T cells are cytotoxic cells that kill infected or transformed target cells. Tcf1 remains highly expressed in naïve T cells, but exhibits quite different expression patterns depending on the types and/or stages of cellular immune responses.
Tcf1 is also involved in regulation of other CD4+ helper T cell functions (Steinke and Xue 2014). In response to infection by helminth and other extracellular pathogens, activated CD4+ T cells differentiate into T helper 2 (Th2) cells, which produce IL-4, IL-5, and IL-13 cytokines. Tcf1 promotes Th2 responses by directly activating Gata3, the master regulator of Th2 differentiation. IL-17-producing T helper 17 (Th17) cells contribute to protection of mucosa but are also implicated in induction of tissue inflammation and autoimmune diseases. Tcf1 is intrinsically required for restraining IL-17A production in thymocytes as well as activated CD4+ T cells. Regulatory T cells (Tregs) are vital in maintaining self-tolerance and curtailing excessive immune responses. Tcf1 interacts with Foxp3, the master regulator of Tregs, and appears to negatively regulate the immunosuppressive function of Tregs. Collectively, Tcf1 adopts pivotal, yet diverse, regulatory functions in multiple CD4+ T cell responses.
In response to acute viral or intracellular bacterial infection, activated CD8+ T cells differentiate into cytotoxic effectors that express cytotoxic molecules including granzymes and perforin, in addition to IFN-γ and proinflammatory cytokines such as tumor necrosis factor. The CD8+ effectors are responsible for eliminating infected target cells and thus clearing the pathogen. A small portion of CD8+ effectors survives a contraction phase and transitions to form a memory CD8+ T cell pool. Memory CD8+ T cells provide long-term, enhanced protection against the same antigen. Tcf1 expression is greatly diminished in CD8+ effectors but partially restored in memory CD8+ T cells. Consistent with its expression pattern, Tcf1 appears to be dispensable for generation of functional CD8+ effectors but is essential for maintaining the longevity of memory CD8+ T cells. Tcf1-deficient memory CD8+ T cells show progressive loss over time, partly owing to diminished expression of Eomes in the absence of Tcf1. In addition, Tcf1 is required for generation of secondary effectors by memory CD8+ T cells upon encounter with the same antigen (Xue and Zhao 2012). The differential requirements for Tcf1 in primary and secondary CD8+ effectors suggest that Tcf1 may program the transcriptome and epigenome of naïve and antigen-experienced CD8+ T cells in different ways, and the underlying mechanisms merit in-depth investigation.
Some viruses, including human immunodeficiency virus (HIV), cannot be completely cleared by the infected hosts, leading to chronic infection where viral replication persists. In this context, antigen-specific CD8+ T cells initially acquire effector functions but gradually become less functional, a process known as CD8+ T cell exhaustion. The exhausted CD8+ T cells express multiple inhibitory receptors such as PD-1, and PD-1 blockage has proven to be an effective approach to revitalize exhausted CD8+ T cells to better control viral replication. Several recent studies have uncovered an important subset of exhausted CD8+ T cells that expresses Tcf1, Bcl6, and the Tfh-characteristic CXCR5 in patients with chronic infections and experimental mouse models (He et al. 2016a; Im et al. 2016; Leong et al. 2016). The Tfh-like CXCR5+CD8+ T cells have self-renewal capability, show stronger proliferative capacity in response to PD-1 blockage therapy, and are more effective in curtailing viral replication, compared with their CXCR5−CD8+ T cell counterparts. Importantly, Tcf1 is essential for generating the Tfh-like CXCR5+CD8+ T cells and maintaining the pool size of antigen-specific exhausted CD8+ T cells. Furthermore, forced expression of Bcl6 or ablation of Blimp1 increases the abundance of the Tfh-like CXCR5+CD8+ T cells. These observations suggest that the same regulatory circuit, that is, Tcf1-mediated induction of Bcl6 and repression of Blimp1, is utilized in both CD4+ Tfh cells and CXCR5+CD8+ T cells (Fig. 3).
Tcf1 Has Broad Regulatory Functions beyond T Lymphocytes
Beyond T lymphocytes, Tcf1 is expressed in several other blood lineage cells and non-blood cell types, albeit at lower abundance. Hematopoietic stem cells (HSCs) can give rise to all blood lineages following well-defined differentiation steps, and at the same time regenerate themselves via a self-renewal mechanism. Tcf1 and Lef1 are expressed in HSCs and contribute to regulation of HSC regenerative fitness, as measured in serial transplantation assays (Yu et al. 2016). Leukemic stem cells (LSCs) are considered the counterpart of HSCs in leukemia, responsible for initiation, propagation, and relapse of the disease. LSCs are more dependent on Tcf1 and Lef1 than HSCs in their self-renewal capacity, and in a murine chronic myeloid leukemia (CML) model, genetic targeting of Tcf1 and Lef1 synergizes with conventional tyrosine kinase inhibitor therapy (Yu et al. 2016). The differential requirements for Tcf1/Lef1 in HSCs and LSCs suggest that these factors might be potential therapeutic targets in treating leukemia.
In the mouse bone marrow, there is a small fraction (<1%) of cells that expresses high levels of Tcf1 as marked by a Tcf1-EGFP reporter allele. Further fractionation of the Tcf1-GFP+ BM cells identified a Thy1.2−IL-7Rα− CD25−α4β7+ subset, which has the ability to generate all three groups of innate lymphoid cells (ILCs) both in vivo and on clonal levels (Yang et al. 2015). This subset is hence termed early innate lymphoid progenitors (EILPs). ILCs do not express rearranged antigen receptors but functionally resemble helper T cells in cytokine production (De Obaldia and Bhandoola 2015). The ILC1 group contains the conventional natural killer (NK) cells and produces IFN-γ; ILC2 and ILC3 groups produce IL-4 and IL-17, respectively. Whereas Tcf1 and Lef1 appear to be redundant for NK cell development, Tcf1 alone is essential for ILC2 development and functions. On the other hand, Tcf1 deficiency specifically affected NKp46+ ILC3 cells. Collectively, Tcf1 has pivotal roles in ILC lineage specification in the BM, as well as ILC development and functions in peripheral tissues and secondary lymphoid organs.
Besides blood lineage cells, Tcf1 (in particular the short isoforms) is expressed in epithelial cells in the intestine and mammary gland. Germline-targeted Tcf1-deficient mice develop polyp-like intestinal neoplasms and mammary gland adenomas. In this context, Tcf1 is proposed to provide negative feedback to Tcf4-β-catenin activity in epithelial cells, exerting a tumor-suppressive role (Roose et al. 1999). Tcf1 is also detected in β cells in pancreatic islets (Campbell et al. 2016). Compared with nondiabetic lean human subjects, Tcf1 expression in islets is decreased in nondiabetic obese human subjects and further reduced in type 2 diabetic patients. In mice, Tcf1 deficiency is associated with decreased glucose tolerance and insulin response. Single nucleotide polymorphisms in the TCF7 locus have been associated with type 1 diabetes, highlighting Tcf1 as a therapeutic target in preserving the function of pancreatic β cells.
Tcf1 exerts regulatory roles in a stage-specific manner during development and antigen response of T lymphocytes, and is also pivotal in non-T lineage cells. In each cell context, the epigenomic landscape likely determines how Tcf1 works with other factors to regulate cell type-specific target genes. Given the newly discovered HDAC activity embedded in Tcf1, it is of interest to determine how Tcf1 contributes to shape the epigenomic landscape as well. Meanwhile, transcriptomic profiling will reveal a conserved transcriptional circuitry controlled by Tcf1 across different cell types. As a transcription factor, Tcf1 binds to approximately 8000 genomic locations in naïve CD8+ T cells, as determined by ChIP-Seq. However, only a fraction of Tcf1-associated genes were differentially expressed in Tcf1-deficient CD8+ T cells (Xing et al. 2016). What is the functional significance for all other genomic occupancy by Tcf1? It has been shown that Lef1 bends DNA by 130 degrees upon binding to target locations. Thus, Tcf1 and Lef1 may contribute to chromatin folding/looping and building appropriate three-dimensional conformation; this structural effect may not directly affect gene expression at the steady state, but may affect how the associated gene loci respond to incoming signals. It remains underinvestigated how Tcf1 per se is regulated. The Tcf7 gene locus is flanked by a super enhancer in naïve T cells, and this super enhancer is decommissioned in effector CD8+ T cells, but reactivated in memory CD8+ T cells, consistent with Tcf1 expression patterns during CD8+ T cell responses (He et al. 2016b). Additionally, the Tcf7 promoter is enriched with CpG islands, suggesting that this gene is dynamically regulated by transcriptional and epigenetic means. Covalent modification of Tcf1 orthologs, that is, acetylation of dTCF/pangolin and POP-1, has been described (Cadigan 2012), and whether mammalian Tcf1 is subject to similar regulation is an open question. In a way, Tcf1 can serve as a model molecule that helps reveal novel concepts in bridging transcriptional and epigenetic regulations.
- He B, Xing S, Chen C, Gao P, Teng L, Shan Q, et al. CD8+ T cells utilize highly dynamic enhancer repertoires and regulatory circuitry in response to infections. Immunity. 2016b;45:1341–54. doi. 10.1016/j.immuni.2016.11.009