Microglia are resident myeloid cells in the central nervous system (CNS) that control homeostasis and protect CNS from damage and infections. Microglia and peripheral myeloid cells accumulate and adapt tumor supporting roles in human glioblastomas that show prevalence in men. Cell heterogeneity and functional phenotypes of myeloid subpopulations in gliomas remain elusive. Here we show single-cell RNA sequencing (scRNA-seq) of CD11b⁺ myeloid cells in naïve and GL261 glioma-bearing mice that reveal distinct profiles of microglia, infiltrating monocytes/macrophages and CNS border-associated macrophages. We demonstrate an unforeseen molecular heterogeneity among myeloid cells in naïve and glioma-bearing brains, validate selected marker proteins and show distinct spatial distribution of identified subsets in experimental gliomas. We find higher expression of MHCII encoding genes in glioma-activated male microglia, which was corroborated in bulk and scRNA-seq data from human diffuse gliomas. Our data suggest that sex-specific gene expression in glioma-activated microglia may be relevant to the incidence and outcomes of glioma patients.

Charles Roberts, Peter Park, Bradley Bernstein and colleagues examine the consequences of SMARCB1 loss on enhancer landscapes in human rhabdoid tumors. They show that SMARCB1 is essential for the integrity and abundance of SWI/SNF complexes and facilitates their targeting to appropriate enhancers. SMARCB1 (also known as SNF5, INI1, and BAF47), a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex1,2, is inactivated in nearly all pediatric rhabdoid tumors3,4,5. These aggressive cancers are among the most genomically stable6,7,8, suggesting an epigenetic mechanism by which SMARCB1 loss drives transformation. Here we show that, despite having indistinguishable mutational landscapes, human rhabdoid tumors exhibit distinct enhancer H3K27ac signatures, which identify remnants of differentiation programs. We show that SMARCB1 is required for the integrity of SWI/SNF complexes and that its loss alters enhancer targeting—markedly impairing SWI/SNF binding to typical enhancers, particularly those required for differentiation, while maintaining SWI/SNF binding at super-enhancers. We show that these retained super-enhancers are essential for rhabdoid tumor survival, including some that are shared by all subtypes, such as SPRY1, and other lineage-specific super-enhancers, such as SOX2 in brain-derived rhabdoid tumors. Taken together, our findings identify a new chromatin-based epigenetic mechanism underlying the tumor-suppressive activity of SMARCB1.

Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation.

Most neurological diseases are associated with chronic inflammation initiated by the activation of microglia, which produce cytotoxic and inflammatory factors. Signal transducers and activators of transcription (STATs) are potent regulators of gene expression but contribution of particular STAT to inflammatory gene expression and STAT-dependent transcriptional networks underlying brain inflammation need to be identified. In the present study, we investigated the genomic distribution of Stat binding sites and the role of Stats in the gene expression in lipopolysaccharide (LPS)-activated primary microglial cultures. Integration of chromatin immunoprecipitation-promoter microarray data and transcriptome data revealed novel Stat-target genes including Jmjd3, Ccl5, Ezr, Ifih1, Irf7, Uba7, and Pim1. While knockdown of individual Stat had little effect on the expression of tested genes, knockdown of both Stat1 and Stat3 inhibited the expression of Jmjd3 and inflammatory genes. Transcriptional regulation of Jmjd3 by Stat1 and Stat3 is a novel mechanism crucial for launching inflammatory responses in microglia. The effects of Jmjd3 on inflammatory gene expression were independent of its H3K27me3 demethylase activity. Forced expression of constitutively activated Stat1 and Stat3 induced the expression of Jmjd3, inflammation-related genes, and the production of pro-inflammatory cytokines as potently as lipopolysacharide. Gene set enrichment and gene function analysis revealed categories linked to the inflammatory response in LPS and Stat1C + Stat3C groups. We defined upstream pathways that activate STATs in response to LPS and demonstrated contribution of Tlr4 and Il-6 and interferon-γ signaling. Our findings define novel direct transcriptional targets of Stat1 and Stat3 and highlight their contribution to inflammatory gene expression. Key Message Combined analysis of genomic Stat occupancy and transcriptome revealed novel Stat target genes in LPS-induced microglia. Jmjd3 transcription factor is a novel transcriptional target of Stat1 and Stat3. Stat1 and Stat3 cooperate with Jmjd3 to induce the expression of pro-inflammatory genes. Constitutively active Stat1 and Stat3 fully mimic the LPS-induced upregulation of inflammatory genes and secretion of cytokines.

The organization of DNA into chromatin is dynamic; nucleosomes are frequently displaced to facilitate the ability of regulatory proteins to access specific DNA elements. To gain insight into nucleosome dynamics, and to follow how dynamics change during differentiation, we used a technique called time-ChIP to quantitatively assess histone H3.3 turnover genome-wide during differentiation of mouse ESCs. We found that, without prior assumptions, high turnover could be used to identify regions involved in gene regulation. High turnover was seen at enhancers, as observed previously, with particularly high turnover at super-enhancers. In contrast, regions associated with the repressive Polycomb-Group showed low turnover in ESCs. Turnover correlated with DNA accessibility. Upon differentiation, numerous changes in H3.3 turnover rates were observed, the majority of which occurred at enhancers. Thus, time-ChIP measurement of histone turnover shows that active enhancers are unusually dynamic in ESCs and changes in highly dynamic nucleosomes predominate at enhancers during differentiation.

Activation of transcription requires alteration of chromatin by complexes that increase the accessibility of nucleosomal DNA. Removing nucleosomes from regulatory sequences has been proposed to play a significant role in activation. We tested whether changes in nucleosome occupancy occurred on the set of genes that is activated by the unfolded protein response (UPR). We observed no decrease in occupancy on most promoters, gene bodies, and enhancers. Instead, there was an increase in the accessibility of nucleosomes, as measured by micrococcal nuclease (MNase) digestion and ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing), that did not result from removal of the nucleosome. Thus, changes in nucleosome accessibility predominate over changes in nucleosome occupancy during rapid transcriptional induction during the UPR.