No abstract

Proper functioning of the lymphatic system is required for normal immune responses, fluid balance, and lipid reabsorption. Multiple regulatory mechanisms are employed to ensure the correct formation and function of lymphatic vessels; however, the epigenetic modulators and mechanisms involved in this process are poorly understood. Here, we assess the regulatory role of mouse Dot1l, a histone H3 lysine (K) 79 (H3K79) methyltransferase, in lymphatic formation. Genetic ablation of Dot1l in Tie2(+) endothelial cells (ECs), but not in Lyve1(+) or Prox1(+) lymphatic endothelial cells (LECs) or Vav1(+) definitive hematopoietic stem cells, leads to catastrophic lymphatic anomalies, including skin edema, blood-lymphatic mixing, and underdeveloped lymphatic valves and vessels in multiple organs. Remarkably, targeted Dot1l loss in Tie2(+) ECs leads to fully penetrant lymphatic aplasia, whereas Dot1l overexpression in the same cells results in partially hyperplastic lymphatics in the mesentery. Genetic studies reveal that Dot1l functions in c-Kit(+) hemogenic ECs during mesenteric lymphatic formation. Mechanistically, inactivation of Dot1l causes a reduction of both H3K79me2 levels and the expression of genes important for LEC development and function. Thus, our study establishes that Dot1l-mediated epigenetic priming and transcriptional regulation in LEC progenitors safeguard the proper lymphatic development and functioning of lymphatic vessels.

Mechanistic understanding of germ cell formation at a genome-scale level can aid in developing novel therapeutic strategies for infertility. Germ cell formation is a complex process that is regulated by various mechanisms, including epigenetic regulation, germ cell-specific gene transcription, and meiosis. Gonads contain a limited number of germ cells at various stages of differentiation. Hence, genome-scale analysis of germ cells at the single-cell level is challenging. Conventional genome-scale approaches cannot delineate the landscape of genomic, transcriptomic, and epigenomic diversity or heterogeneity in the differentiating germ cells of gonads. Recent advances in single-cell genomic techniques along with single-cell isolation methods, such as microfluidics and fluorescence-activated cell sorting, have helped elucidate the mechanisms underlying germ cell development and reproductive disorders in humans. In this review, the history of single-cell transcriptomic analysis and their technical advantages over the conventional methods have been discussed. Additionally, recent applications of single-cell transcriptomic analysis for analyzing germ cells have been summarized.

Chronic graft-versus-host disease (cGVHD) remains a major obstacle impeding successful allogeneic hematopoietic cell transplantation (allo-HCT). MicroRNAs (miRs) play key roles in immune regulation during acute GVHD development. Preclinical studies to identify miRs that impact cGVHD pathogenesis are required to develop these as potential life-saving interventions. Using oligonucleotide array, we identified miR-31 that was significantly elevated in allogeneic T cells following HCT in mice. Using genetic and pharmacological approaches, we demonstrated a key role for miR-31 in mediating donor T-cell pathogenicity in cGVHD. Recipients of miR-31-deficient T cells displayed improved cutaneous and pulmonary cGVHD. Deficiency of miR-31 reduced T-cell expansion and Th17 differentiation, but increased generation and function of Tregs. MiR-31 facilitated Neuropilin-1 down-regulation, Foxp3 loss and IFNγ production in alloantigen-induced Tregs. Mechanistically, miR-31 was required for Hypoxia-inducible Factor 1α (HIF1α) upregulation in allogeneic T cells. Hence, miR-31-deficient CD4 T cells displayed impaired activation, survival, Th17 differentiation and glycolytic metabolism under hypoxia. Upregulation of Factor Inhibiting HIF1 (FIH1), a direct target of miR-31, in miR-31-deficient T cells was essential for attenuating T-cell pathogenicity. However, miR-31-deficienty CD8 T cells maintained intact glucose metabolism, cytolytic activity and graft-versus-leukemia response. Importantly, systemic administration of a specific inhibitor of miR-31 effectively reduced donor T-cell expansion, improved Treg generation, and attenuated cGVHD. Taken together, miR-31 is a key driver for T-cell pathogenicity in cGVHD but not for the anti-leukemia activity. Taken together, miR-31 is essential to drive cGVHD pathogenesis and represents a novel potential therapeutic target for controlling cGVHD.