B cells can suppress autoimmunity by secreting interleukin-10 (IL-10). Although subpopulations of splenic B lineage cells are reported to express IL-10 in vitro, the identity of IL-10-producing B cells with regulatory function in vivo remains unknown. By using IL-10 reporter mice, we found that plasmablasts in the draining lymph nodes (dLNs), but not splenic B lineage cells, predominantly expressed IL-10 during experimental autoimmune encephalomyelitis (EAE). These plasmablasts were generated only during EAE inflammation. Mice lacking plasmablasts by genetic ablation of the transcription factors Blimp1 or IRF4 in B lineage cells developed an exacerbated EAE. Furthermore, IRF4 positively regulated IL-10 production that can inhibit dendritic cell functions to generate pathogenic T cells. Our data demonstrate that plasmablasts in the dLNs serve as IL-10 producers to limit autoimmune inflammation and emphasize the importance of plasmablasts as IL-10-producing regulatory B cells.

The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins.In eukaryotes, activation of gene expression involves the ordered assembly of transcriptional regulators, chromatinmodifying enzymes, RNA polymerase II, and associated general transcription factors onto cis-acting elements that are embedded in chromatin. Chromatin-remodeling enzymes play an integral role in gene activation by perturbing chromatin structure and making specific loci permissive for transcription. Molecular analysis of multiple gene activation events suggests that the temporal recruitment of transcription factors and chromatin-remodeling enzymes is gene specific and dictated by the interplay between specific activators and local chromatin structure (1,52,55).Two classes of enzymes have been shown to remodel chromatin structure either by catalyzing covalent modifications of histones or by hydrolyzing ATP to mobilize nucleosomes. Among the latter class of enzymes are the SWI/SNF chromatin-remodeling complexes. A distinguishing feature of this family is the presence of a bromodomain in the ATPase subunit, which promotes interaction with acetylated histones and links the activities of the two classes of chromatin remodelers in the regulation of gene expression (22). SWI/SNF enzymes physically interact with histone acetyltransferases (HATs), histone deacetylases (HDACs), and methyltransferases, showing the potential for coordination of chromatin-remodeling activities (reviewed in reference 53).Mammalian SWI/SNF chromatin-remodeling enzymes are multisubunit complexes that contain either the Brg1 or Brm ATPase subunits and can activate or repress expression of a subset of genes (39, 53). They function in cell cycle control, and some of the subunits are tumor suppressors (49). Diverse SWI/ SNF complexes exist that are distinguished by the particular ATPase, the presence of unique subunits, and tissue-specific ...

Autism spectrum disorder (ASD) comprises a range of neurodevelopmental disorders characterized by deficits in social interaction and communication as well as by restricted and repetitive behaviours. ASD has a strong genetic component with high heritability. Exome sequencing analysis has recently identified many de novo mutations in a variety of genes in individuals with ASD, with CHD8, a gene encoding a chromatin remodeller, being most frequently affected. Whether CHD8 mutations are causative for ASD and how they might establish ASD traits have remained unknown. Here we show that mice heterozygous for Chd8 mutations manifest ASD-like behavioural characteristics including increased anxiety, repetitive behaviour, and altered social behaviour. CHD8 haploinsufficiency did not result in prominent changes in the expression of a few specific genes but instead gave rise to small but global changes in gene expression in the mouse brain, reminiscent of those in the brains of patients with ASD. Gene set enrichment analysis revealed that neurodevelopment was delayed in the mutant mouse embryos. Furthermore, reduced expression of CHD8 was associated with abnormal activation of RE-1 silencing transcription factor (REST), which suppresses the transcription of many neuronal genes. REST activation was also observed in the brains of humans with ASD, and CHD8 was found to interact physically with REST in the mouse brain. Our results are thus consistent with the notion that CHD8 haploinsufficiency is a highly penetrant risk factor for ASD, with disease pathogenesis probably resulting from a delay in neurodevelopment.

Central nervous system (CNS) injury transforms naive astrocytes into reactive astrocytes, which eventually become scar-forming astrocytes that can impair axonal regeneration and functional recovery. This sequential phenotypic change, known as reactive astrogliosis, has long been considered unidirectional and irreversible. However, we report here that reactive astrocytes isolated from injured spinal cord reverted in retrograde to naive astrocytes when transplanted into a naive spinal cord, whereas they formed astrocytic scars when transplanted into injured spinal cord, indicating the environment-dependent plasticity of reactive astrogliosis. We also found that type I collagen was highly expressed in the spinal cord during the scar-forming phase and induced astrocytic scar formation via the integrin-N-cadherin pathway. In a mouse model of spinal cord injury, pharmacological blockade of reactive astrocyte-type I collagen interaction prevented astrocytic scar formation, thereby leading to improved axonal regrowth and better functional outcomes. Our findings reveal environmental cues regulating astrocytic fate decisions, thereby providing a potential therapeutic target for CNS injury.

The initiation of cellular differentiation involves alterations in gene expression that depend on chromatin changes, at the level of both higher-order structures and individual genes. Consistent with this, chromatin-remodelling enzymes have key roles in differentiation and development. The functions of ATP-dependent chromatin-remodelling enzymes have been studied in several mammalian differentiation pathways, revealing cell-type-specific and gene-specific roles for these proteins that add another layer of precision to the regulation of differentiation. Recent studies have also revealed a role for ATP-dependent remodelling in regulating the balance between proliferation and differentiation, and have uncovered intriguing links between chromatin remodelling and other cellular processes during differentiation, including recombination, genome organization and the cell cycle.

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

Myogenin is required not for the initiation of myogenesis but instead for skeletal muscle formation through poorly understood mechanisms. We demonstrate in cultured cells and, for the first time, in embryonic tissue, that myogenic late genes that specify the skeletal muscle phenotype are bound by MyoD prior to the initiation of gene expression. At the onset of muscle specification, a transition from MyoD to myogenin occurred at late gene loci, concomitant with loss of HDAC2, the appearance of both the Mef2D regulator and the Brg1 chromatin-remodeling enzyme, and the opening of chromatin structure. We further demonstrated that ectopic expression of myogenin and Mef2D, in the absence of MyoD, was sufficient to induce muscle differentiation in a manner entirely dependent on Brg1. These results indicate that myogenin specifies the muscle phenotype by cooperating with Mef2D to recruit an ATP-dependent chromatin-remodeling enzyme that alters chromatin structure at regulatory sequences to promote terminal differentiation.

Skeletal muscle differentiation requires the coordinated activity of transcription factors, histone modifying enzymes, and ATP-dependent chromatin remodeling enzymes. The type II protein arginine methyltransferase Prmt5 symmetrically dimethylates histones H3 and H4 and numerous nonchromatin proteins, and prior work has implicated Prmt5 in transcriptional repression. Here we demonstrate that MyoD-induced muscle differentiation requires Prmt5. One of the first genes activated during differentiation encodes the myogenic regulator myogenin. Prmt5 and dimethylated H3R8 (histone 3 arginine 8) are localized at the myogenin promoter in differentiating cells. Modification of H3R8 required Prmt5, and reduction of Prmt5 resulted in the abrogation of promoter binding by the Brg1 ATPase-associated with the SWI/SNF chromatin remodeling enzymes and all subsequent events associated with gene activation, including increases in chromatin accessibility and stable binding by MyoD. Prmt5 and dimethylated H3R8 were also associated with the myogenin promoter in activated satellite cells isolated from muscle tissue, further demonstrating the physiological relevance of these observations. The data indicate that Prmt5 facilitates myogenesis because it is required for Brg1-dependent chromatin remodeling and gene activation at a locus essential for differentiation. We therefore conclude that a histone modifying enzyme is necessary to permit an ATP-dependent chromatin remodeling enzyme to function.Gene expression is a highly regulated process that frequently requires coordinated function between transcription factors and chromatin remodeling enzymes. These enzymes are divided into two classes: ATP-dependent remodelers that hydrolyze ATP and alter nucleosome structure and histone modifiers that covalently modify specific histone residues posttranslationally. The activation of skeletal muscle differentiation is regulated by members of the basic helix-loop-helix family of tissue-specific transcription factors, including MyoD, Myf5, Mrf4, and myogenin, as well as by members of the Mef2 family of transcriptional regulators, which act cooperatively with basic helix-loop-helix proteins (8,34,43). Numerous chromatin remodeling enzymes have been shown to both positively and negatively affect myogenic gene expression. These include histone acetyl transferases; types I, II; and III histone deacetylases; histone lysine methyltransferases; and members of the SWI/SNF family of ATP-dependent remodeling enzymes (17,47,50). The relationships between the different classes of chromatin remodeling enzymes during myogenesis have been largely unexplored.The protein arginine methyltransferases (PRMTs) are an additional class of enzymes that can be linked to histone modification and gene regulation during skeletal muscle differentiation. This family has 9 members (Prmt1 to Prmt9), six of which have been shown to generate asymmetric (type I) or symmetric (type II) dimethylarginine and to affect a range of cellular processes through arginine methylation of substrate...