Background: KDM6A is a demethylase encoded by a gene with female-biased expression due to escape from X inactivation. Its main role is to facilitate gene expression through removal of the repressive H3K27me3 mark, with evidence of some additional histone demethylase-independent functions. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in cancer. Methods: Kdm6a was knocked out using CRISPR/Cas9 gene editing in F1 male and female mouse embryonic stem cells (ES) derived from reciprocal crosses between C57BL6 x Mus castaneus. Diploid and allelic RNA-seq analyses were done to compare gene expression between wild-type and Kdm6a knockout (KO) clones. The effects of Kdm6a KO on sex-biased gene expression were investigated by comparing gene expression between male and female ES cells. Changes in H3K27me3 enrichment and chromatin accessibility at promoter regions of genes with expression changes were characterized by ChIP-seq and ATAC-seq followed by diploid and allelic analyses. Results: We report that Kdm6a KO in male and female embryonic stem (ES) cells derived from F1 hybrid mice cause extensive gene dysregulation, disruption of sex biases, and specific parental allele effects. Among the dysregulated genes are candidate genes that may explain abnormal developmental features of Kabuki syndrome caused by KDM6A mutations in human. Strikingly, Kdm6a knockouts result in a decrease in sex-biased expression and in preferential downregulation of the maternal alleles of a number of genes. Most promoters of dysregulated genes show concordant epigenetic changes including gain of H3K27me3 and loss of chromatin accessibility, but there was less concordance when considering allelic changes. Conclusions: Our study reveals new sex-related roles of KDM6A in the regulation of developmental genes, the maintenance of sex-biased gene expression, and the differential expression of parental alleles.

Breast cancer (BC) is the second leading cause of cancer deaths among women. DEK is a known oncoprotein that is highly expressed in over 60% of breast cancers and is an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To identify novel oncogenic functions of DEK, we performed RNA-Seq analysis on isogenic Dek-knockout and complemented murine BC cells. Gene ontology analyses identified gene sets associated with immune system regulation and cytokine-mediated signaling and differential cytokine and chemokine expression was confirmed across Dek-proficient versus Dek-deficient cells. By exposing murine bone marrow-derived macrophages (BMDM) to tumor cell conditioned media (TCM) to mimic a tumor microenvironment, we showed that Dek-expressing breast cancer cells produce a cytokine milieu, including up-regulated Tslp and Ccl5 and down-regulated Cxcl1, Il-6, and GM-CSF, that drives the M2 polarization of macrophages. We validated this finding in primary murine mammary tumors and show that Dek expression in vivo is also associated with increased expression of M2 macrophage markers in murine tumors. Using TCGA data, we verified that DEK expression in primary human breast cancers correlates with the expression of several genes identified by RNA-Seq in our murine model and with M2 macrophage phenotypes. Together, our data demonstrate that by regulating the production of multiple secreted factors, DEK expression in BC cells creates a potentially immune suppressed tumor microenvironment, particularly by inducing M2 tumor associated macrophage (TAM) polarization.

How multipotent progenitors give rise to multiple cell types in defined numbers is a central question in developmental biology. Epigenetic switches, acting at single gene loci, can generate extended delays in the activation of lineage-specifying genes and impact lineage decisions and cell type output. Here, we analyzed a timed epigenetic switch controlling expression of mouse Bcl11b, a transcription factor that drives T-cell commitment, but only after a multi-day delay. To investigate roles for this delay in controlling lineage decision making, we analyzed progenitors with a deletion in a distal Bcl11b enhancer, which extends this delay by ∼3 days. Strikingly, delaying Bcl11b activation reduces T-cell output but enhances innate lymphoid cell (ILC) generation in the thymus by redirecting uncommitted progenitors to the ILC lineages. Mechanistically, delaying Bcl11b activation promoted ILC redirection by enabling upregulation of the ILC-specifying transcription factor PLZF. Despite the upregulation of PLZF, committed ILC progenitors could subsequently express Bcl11b, which is also needed for type 2 ILC differentiation. These results show that epigenetic switches can control the activation timing and order of lineage-specifying genes to modulate cell type numbers and proportions.

SUMMARY: Antibody responses are determined by activated B cells bifurcating into plasmablasts (PBs) and germinal center B cells (GCBCs). Gene regulatory networks (GRNs) underlying human B cell fate choice remain uncharted. Temporally resolved single-cell multi-omics, computational modeling and CRISPR-based perturbations were used to assemble, simulate and test high-resolution GRNs underlying PB and GC fates. The results converged with orthogonal predictions of transcription factor (TF) action at single-nucleotide resolution, revealing dominant and reciprocal actions of IRF4 and its binding partners at simple and composite IRF motifs. Single-cell perturbation analysis of these TFs demonstrated multiple reciprocal negative feedback loops controlling the bifurcation. Additionally, IRF4 and BLIMP1, co-repressed the cell cycle regulators MYC and CCND2 . G0/G1 lengthening accelerated the switching of cells to an IRF4 ^hi BLIMP1 ^hi regulatory state and enhanced the probability of PB specification, thereby uncovering a self-reinforcing regulatory module that couples cell cycle dynamics to B cell fate choice.

The transcription factors IRF4 and BLIMP1 (PRDM1) promote plasma cell (PC) differentiation by repressing B cell identity genes while inducing the unfolded protein response, ER/Golgi biogenesis, and immunoglobulin secretion. How IRF4 and BLIMP1 partition and coordinate their genomic activities during plasma cell differentiation remains unresolved. Using naïve human B cells and a stepwise in vitro differentiation system, we performed CRISPR/Cas9 perturbations of IRF4 or PRDM1 in plasma cell precursors followed by single-cell RNA sequencing. Despite their mutually reinforced expression and shared recognition of related IRF-family motifs (ISREs and EICEs), loss of IRF4, but not of BLIMP1, yielded a stunted intermediate with incomplete silencing of B cell identity genes and defective induction of the secretory program. Multiome profiling, base-pair-resolution motif modeling, CUT&RUN, and DNA-binding assays identified non-conserved nucleotides within ISRE/EICE motifs that discriminate IRF4 from BLIMP1 binding. These findings reveal a motif-lexicon-dependent IRF4-BLIMP1 interplay, in which IRF4 first acts independently and then in concert with BLIMP1. This regulatory logic may also underlie programming of additional lymphocyte effector states.