Transcription Factors in the Development and Function of Group 2 Innate Lymphoid Cells

Group 2 innate lymphoid cells (ILC2s) are tissue-resident cells and are a major source of innate TH2 cytokine secretion upon allergen exposure or parasitic-worm infection. Accumulating studies have revealed that transcription factors, including GATA-3, Bcl11b, Gfi1, ROR , and Ets-1, play a role in ILC2 differentiation. Recent reports have further revealed that the characteristics and functions of ILC2 are influenced by the physiological state of the tissues. Specifically, the type of inflammation strongly affects the ILC2 phenotype in tissues. Inhibitory ILC2s, memory-like ILC2s, and ex-ILC2s with ILC1 features acquire their characteristic properties following exposure to their specific inflammatory environment. We have recently reported a new ILC2 population, designated as exhausted-like ILC2s, which emerges after a severe allergic inflammation. Exhausted-like ILC2s are featured with low reactivity and high expression of inhibitory receptors. Therefore, for a more comprehensive understanding of ILC2 function and differentiation, we review the recent knowledge of transcriptional regulation of ILC2 differentiation and discuss the roles of the Runx transcription factor in controlling the emergence of exhausted-like ILC2s. The concept of exhausted-like ILC2s sheds a light on a new aspect of ILC2 biology in allergic diseases

The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells

Members of the Runx family of transcriptional regulators are required for the appropriate expression of CD4 and CD8 at discrete stages of T cell development. The roles of these factors in other aspects of T cell development are unknown. We used a strategy to conditionally inactivate the genes encoding Runx1 or Runx3 at different stages of thymocyte development, demonstrating that Runx1 regulates the transitions of developing thymocytes from the CD4−CD8− double-negative stage to the CD4+CD8+ double-positive (DP) stage and from the DP stage to the mature single-positive stage. Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages, respectively. Runx1-deficient CD4+ T cells had markedly reduced expression of the interleukin 7 receptor and exhibited shorter survival. In addition, inactivation of both Runx1 and Runx3 at the DP stages resulted in a severe block in development of CD8+ mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells

Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis

Fujita, T, Kitaura, F, Oji, A, et al. Transgenic mouse lines expressing the 3xFLAG‐dCas9 protein for enChIP analysis. Genes Cells. 2018; 23: 318– 325. https://doi.org/10.1111/gtc.1257

Repression of interleukin-4 in T helper type 1 cells by Runx/Cbfβ binding to the Il4 silencer

Interferon γ (IFNγ) is the hallmark cytokine produced by T helper type 1 (Th1) cells, whereas interleukin (IL)-4 is the hallmark cytokine produced by Th2 cells. Although previous studies have revealed the roles of cytokine signaling and of transcription factors during differentiation of Th1 or Th2 cells, it is unclear how the exclusive expression pattern of each hallmark cytokine is established. The DNaseI hypersensitivity site IV within the mouse Il4 locus plays an important role in the repression of Il4 expression in Th1 cells, and it has been named the Il4 silencer. Using Cbfβ- or Runx3-deficient T cells, we show that loss of Runx complex function results in derepression of IL-4 in Th1 cells. Binding of Runx complexes to the Il4 silencer was detected in naive CD4+ T cells and Th1 cells, but not in Th2 cells. Furthermore, enforced expression of GATA-3 in Th1 cells inhibited binding of Runx complexes to the Il4 silencer. Interestingly, T cell–specific inactivation of the Cbfβ gene in mice led to elevated serum immunoglobulin E and airway infiltration. These results demonstrate critical roles of Runx complexes in regulating immune responses, at least in part, through the repression of the Il4 gene

Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbfβ for their development

The fetal liver is a major hematopoietic site containing progenitor cells that give rise to nearly all blood cells, including B-1 cells. Because the fetal liver is not a de novo site of hematopoietic stem cell (HSC) or progenitor-cell emergence, it must be seeded by yolk sac (YS)-derived erythromyeloid progenitors at embryonic day (E) 8.5-E10 and aorta-gonado-mesonephros (AGM)-derived HSCs at E10.5-E11.5. Although the B-1 progenitor cell pool in the fetal liver is considered to be of HSC origin, we have previously proposed that YS-derived B-1 progenitors may also contribute to this pool. Until now, it has been impossible to determine whether HSC-independent B-1 progenitor cells exist in the fetal liver. Here, we demonstrate the presence of transplantable fetal-liver B-1 and marginal zone B progenitor cells in genetically engineered HSC-deficient embryos. HSC-deficient YS and AGM tissues produce B-1 progenitors in vitro and thus may serve as sites of origin for the B-1 progenitors that seed the fetal liver. Furthermore, we have found that core-binding factor beta (Cbfβ) expression is required for fetal-liver B-1 progenitor cell maturation and expansion. Our data provide, to our knowledge, the first evidence for the presence of B-1 progenitor cells in the fetal liver that arise independently of HSCs and implicate Cbfβ as a critical molecule in the development of this lineage

Impaired tissue homing by the Ikzf3N159S variant is mediated by interfering with Ikaros function

AIOLOS, encoded by IKZF3, is a member of the IKZF family of proteins that plays an important role in regulating late B-cell differentiation. Human individuals heterozygous for the AIOLOS p.N160S variant displayed impaired humoral immune responses as well as impaired B and T cell development. We have previously reported that a mouse strain harboring an Ikzf3N159S allele that corresponds to human IKZF3N160S recapitulated immune-deficient phenotypes, such as impaired B cell development and loss of CD23 expression. In this study, we investigated the effect of the Ikzf3N159S variant and found that B1a cell development was impaired in Ikzf3N159S/N159S mice. In addition, CD62L expression was severely decreased in both B and T lymphocytes by the Ikzf3N159S mutation, in a dose-dependent manner. Mixed bone marrow chimera experiments have revealed that most immunodeficient phenotypes, including low CD62L expression, occur in intrinsic cells. Interestingly, while Ikzf3N159S/N159S lymphocytes were still present in the spleen, they were completely outcompeted by control cells in the lymph nodes, suggesting that the capacity for homing or retention in the lymph nodes was lost due to the Ikzf3N159S mutation. The homing assay confirmed severely decreased homing abilities to lymph nodes of Ikzf3N159S/N159S B and T lymphocytes but selective enrichment of CD62L expressing Ikzf3N159S/N159S lymphocytes in lymph nodes. This finding suggests that impaired CD62L expression is the major reason for the impaired homing capacity caused by the Ikzf3N159S mutation. Interestingly, an excess amount of Ikaros, but not Aiolos, restored CD62L expression in Ikzf3N159S/N159S B cells. Together with the loss of CD62L expression due to Ikaros deficiency, the AiolosN159S mutant protein likely interferes with Ikaros function through heterodimerization, at least in activating the Sell gene encoding CD62L expression. Thus, our results revealed that AiolosN159S causes some immunodeficient phenotypes via the pathogenesis referred to as the heterodimeric interference as observed for AiolosG158R variant

Runx proteins regulate Foxp3 expression

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes

Differential Requirement of Cd8 Enhancers E8I and E8VI in Cytotoxic Lineage T Cells and in Intestinal Intraepithelial Lymphocytes

CD8 expression in T lymphocytes is tightly regulated by the activity of at least six Cd8 enhancers (E8I-E8VI), however their complex developmental stage-, subset-, and lineage-specific interplays are incompletely understood. Here we analyzed ATAC-seq data on the Immunological Genome Project database and identified a similar developmental regulation of chromatin accessibility of a subregion of E8I, designated E8I-core, and of E8VI. Loss of E8I-core led to a similar reduction in CD8 expression in naïve CD8+ T cells and in IELs as observed in E8I−/− mice, demonstrating that we identified the core enhancer region of E8I. While E8VI−/− mice displayed a mild reduction in CD8 expression levels on CD8SP thymocytes and peripheral CD8+ T cells, CD8 levels were further reduced upon combined deletion of E8I-core and E8VI. Moreover, activated E8I-core−/−E8VI−/− CD8+ T cells lost CD8 expression to a greater degree than E8I-core−/− and E8VI−/− CD8+ T cells, suggesting that the combined activity of both enhancers is required for establishment and maintenance of CD8 expression before and after TCR activation. Finally, we observed a severe reduction of CD4 CTLs among the TCRβ+CD4+ IEL population in E8I-core−/− but not E8VI−/− mice. Such a reduction was not observed in Cd8a−/− mice, indicating that E8I-core controls the generation of CD4 CTLs independently of its role in Cd8a gene regulation. Further, the combined deletion of E8I-core and E8VI restored CD4 CTL subsets, suggesting an antagonistic function of E8VI in the generation of CD4 CTLs. Together, our study demonstrates a complex utilization and interplay of E8I-core and E8VI in regulating CD8 expression in cytotoxic lineage T cells and in IELs. Moreover, we revealed a novel E8I-mediated regulatory mechanism controlling the generation of intestinal CD4 CTLs

Direct Recruitment of Polycomb Repressive Complex 1 to Chromatin by Core Binding Transcription Factors

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.Stem Cell and Regenerative Biolog

Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element

Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRβ antigen receptor locus (Tcrb). Association between distal Vβ gene segments and the highly expressed DβJβ clusters, termed the recombination center (RC), is independent of enhancer function and recruitment of V(D)J recombinase. Instead, we find that tissue-specific folding of Tcrb relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Vβ array to the RC. The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts contacts between distal Vβ gene segments and significantly alters Tcrb repertoires. Our findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers