Tcf1 and Lef1 have versatile functions in regulating T cell development and differentiation, but intrinsic requirements for these factors in regulatory T (T reg) cells remain to be unequivocally defined. Specific ablation of Tcf1 and Lef1 in T reg cells resulted in spontaneous multi-organ autoimmunity that became more evident with age. Tcf1/Lef1-deficient T regs showed reduced protection against experimentally induced colitis, indicative of diminished immuno-suppressive capacity. Transcriptomic analysis revealed that Tcf1 and Lef1 were responsible for positive regulation of a subset of T reg–overrepresented signature genes such as Ikzf4 and Izumo1r. Unexpectedly, Tcf1 and Lef1 were necessary for restraining expression of cytotoxic CD8+ effector T cell–associated genes in T reg cells, including Prdm1 and Ifng. Tcf1 ChIP-seq revealed substantial overlap between Tcf1 and Foxp3 binding peaks in the T reg cell genome, with Tcf1-Foxp3 cooccupancy observed at key T reg signature and cytotoxic effector genes. Our data collectively indicate that Tcf1 and Lef1 are critical for sustaining T reg suppressive functions and preventing loss of self-tolerance.

T cell identity is established during thymic development, but how it is maintained in the periphery remains unknown. Here we show that ablating Tcf1 and Lef1 transcription factors in mature CD8+ T cells aberrantly induces genes from non-T cell lineages. Using high-throughput chromosome-conformation-capture sequencing, we demonstrate that Tcf1/Lef1 are important for maintaining three-dimensional genome organization at multiple scales in CD8+ T cells. Comprehensive network analyses coupled with genome-wide profiling of chromatin accessibility and Tcf1 occupancy show the direct impact of Tcf1/Lef1 on the T cell genome is to promote formation of extensively interconnected hubs through enforcing chromatin interaction and accessibility. The integrative mechanisms utilized by Tcf1/Lef1 underlie activation of T cell identity genes and repression of non-T lineage genes, conferring fine control of various T cell functionalities. These findings suggest that Tcf1/Lef1 control global genome organization and help form intricate chromatin-interacting hubs to facilitate promoter-enhancer/silencer contact, hence providing constant supervision of CD8+ T cell identity and function.

In eukaryotes, deposition of the histone variant H2A.Z into nucleosomes through the chromatin remodeling complex, SWR1, is a crucial step in modulating gene transcription. Recently, H2A.Z has been shown to control the expression of responsive genes, but the underlying mechanism of how H2A.Z responds to physiological stimuli is not well understood. Here, we reveal that, in Neurospora crassa, H2A.Z is a negative regulator of catalase-3 gene, which is responsible for resistance to oxidative stress. H2A.Z represses cat-3 gene expression through direct incorporation at cat-3 locus in a SWR1 complex dependent pathway. Notably, loss of H2A.Z or SWR1 subunits leads to increased binding of a transcription factor, CPC1, at cat-3 locus. Additionally, introduction of plasmids containing gene encoding H2A.Z or SWR1 complex subunits into wild-type strains decreased CAT-3 expression, indicating that H2A.Z counteracts the positive effect of CPC1 to achieve low level cat-3 expression under non-inductive condition. Furthermore, upon oxidative stress, H2A.Z is rapidly evicted from cat-3 locus for the recruitment of CPC1, resulting in robust and full cat-3 gene expression in response to external stimuli. Collectively, this study strongly demonstrates that H2A.Z antagonizes the function of transcription factor to regulate responsive gene transcription under normal conditions and to poise for gene full activation under oxidative stress.

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