FUNCTIONAL DISSECTION OF THE HISTONE DEMETHYLASE JMJD3 IN B CELL LYMPHOPOIESIS

Histone H3 lysine-27 trimethylation (H3K27me3) is an epigenetic mark that exerts a critical role in heritable gene repression. Levels of H3K27me3 at genomic target sites are tightly controlled by the opposing action of H3K27me3-specific methylases and demethylases, respectively. Modulation of H3K27me3 levels influence cell proliferation, survival and differentiation. In mammalian cells, Polycomb group protein Enhancer of Zeste Homologue 2 (Ezh2) catalyzes H3K27 trimethylation as component of the Polycomb Repressive Complex 2. Deposition of H3K27me3 at target genes is reversed by the action of H3K27me3 demethylases. The Jumonji-C containing proteins JMJD3/KDM6B and UTX/KDM6A are the only known enzymes involved in H3K27me3 demethylation. Jmjd3 has been previously shown to play an important role in mediating macrophage driven inflammatory responses and in in regulating somatic cell reprogramming and cellular senescence. In immune cells including B cells expression of Jmjd3 is tightly controlled. Whereas basal Jmjd3 levels are detected throughout B cell development, strong upregulation of demethylase expression is observed after stimulation through respectively the B cell antigen receptor, Toll-like receptors and members of the TNF receptor superfamily. To study the role of JMJD3 in B lymphocyte development and activation, I generated Jmjd3 conditional knock-out mice (JMJD3fl). Analysis of B cell-specific Jmjd3 KO mice revealed multiple defects linked to the lack of demethyalse activity. Jmjd3 regulated the size of the B cell progenitor pool acting primarily on the pre-B cell compartment that was reduced in mutant mice. Jmjd3 deficient animals showed also alterations in peripheral B cell development. An increase in the proportion and absolute number of splenic marginal zone B cells was associated to a reduction in peritoneal cavity B-1a B cells. In vivo BrdU labeling assays suggested a longer lifespan of Jmjd3 mutant mature B cells, which was not dependent on improved cell survival. Jmjd3 was dispensable for germinal center formation and T-cell dependent immune responses. Moreover, measurement of basal serum immunoglobulin titers excluded a major role for Jmjd3 in plasma cell homeostasis. In vitro stimulation assays revealed a selective defect of Jmjd3 mutant B cells to proliferate in response to the TLR4 ligand LPS, which was alleviated by IL-4 co-stimulation. Jmjd3 was critical to drive the first one-to-two cell divisions following LPS stimulation suggesting a critical role in the initial activation of the resting B cells. Upon stimulation with LPS, Jmjd3 mutant B cells showed neither specific defects in cell-cycle progression nor increased apoptosis. Candidate gene expression analyses, supported by RNA sequencing data, revealed a comprehensive control exerted by Jmjd3 on the expression of a substantial number of cell-cycle regulated genes including those cyclins, CDK inhibitors and factors involved in DNA replication and mitosis. Regulation of gene expression mediated by Jmjd3 was not associated with measurable changes in global H3K27me3 levels. All together these results identify Jmjd3 as an important regulator of B cell lymphopoiesis and a selective effector of B cell innate immune responses.