Control of Inflammatory Response by Tissue Microenvironment.

Inflammation is an essential defense response but operates at the cost of normal functions. Whether and how the negative impact of inflammation is monitored remains largely unknown. Acidification of the tissue microenvironment is associated with inflammation. Here we investigated whether macrophages sense tissue acidification to adjust inflammatory responses. We found that acidic pH restructured the inflammatory response of macrophages in a gene-specific manner. We identified mammalian BRD4 as a novel intracellular pH sensor. Acidic pH disrupts the transcription condensates containing BRD4 and MED1, via histidine-enriched intrinsically disordered regions. Crucially, decrease in macrophage intracellular pH is necessary and sufficient to regulate transcriptional condensates in vitro and in vivo, acting as negative feedback to regulate the inflammatory response. Collectively, these findings uncovered a pH-dependent switch in transcriptional condensates that enables environmental sensing to directly control inflammation, with a broader implication for calibrating the magnitude and quality of inflammation by the inflammatory cost.

Canonical BAF complex activity shapes the enhancer landscape that licenses CD8+ T cell effector and memory fates.

CD8+ T cells provide host protection against pathogens by differentiating into distinct effector and memory cell subsets, but how chromatin is site-specifically remodeled during their differentiation is unclear. Due to its critical role in regulating chromatin and enhancer accessibility through its nucleosome remodeling activities, we investigated the role of the canonical BAF (cBAF) chromatin remodeling complex in antiviral CD8+ T cells during infection. ARID1A, a subunit of cBAF, was recruited early after activation and established de novo open chromatin regions (OCRs) at enhancers. Arid1a deficiency impaired the opening of thousands of activation-induced enhancers, leading to loss of TF binding, dysregulated proliferation and gene expression, and failure to undergo terminal effector differentiation. Although Arid1a was dispensable for circulating memory cell formation, tissue-resident memory (Trm) formation was strongly impaired. Thus, cBAF governs the enhancer landscape of activated CD8+ T cells that orchestrates TF recruitment and activity and the acquisition of specific effector and memory differentiation states.

BRD9 regulates interferon-stimulated genes during macrophage activation via cooperation with BET protein BRD4.

Macrophages induce a number of inflammatory response genes in response to stimulation with microbial ligands. In response to endotoxin Lipid A, a gene-activation cascade of primary followed by secondary-response genes is induced. Epigenetic state is an important regulator of the kinetics, specificity, and mechanism of gene activation of these two classes. In particular, SWI/SNF chromatin-remodeling complexes are required for the induction of secondary-response genes, but not primary-response genes, which generally exhibit open chromatin. Here, we show that a recently discovered variant of the SWI/SNF complex, the noncanonical BAF complex (ncBAF), regulates secondary-response genes in the interferon (IFN) response pathway. Inhibition of bromodomain-containing protein 9 (BRD9), a subunit of the ncBAF complex, with BRD9 bromodomain inhibitors (BRD9i) or a degrader (dBRD9) led to reduction in a number of interferon-stimulated genes (ISGs) following stimulation with endotoxin lipid A. BRD9-dependent genes overlapped highly with a subset of genes differentially regulated by BET protein inhibition with JQ1 following endotoxin stimulation. We find that the BET protein BRD4 is cobound with BRD9 in unstimulated macrophages and corecruited upon stimulation to ISG promoters along with STAT1, STAT2, and IRF9, components of the ISGF3 complex activated downstream of IFN-alpha receptor stimulation. In the presence of BRD9i or dBRD9, STAT1-, STAT2-, and IRF9-binding is reduced, in some cases with reduced binding of BRD4. These results demonstrate a specific role for BRD9 and the ncBAF complex in ISG activation and identify an activity for BRD9 inhibitors and degraders in dampening endotoxin- and IFN-dependent gene expression.

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells.

Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and self-assemble through its low-complexity (LC) domain. The ability of RNA-binding proteins like EWSR1 to self-assemble or phase separate in cells has raised questions about the contribution of this process to EWS-FLI1 activity. We examined EWSR1 and EWS-FLI1 activity in Ewing sarcoma cells by siRNA-mediated knockdown and RNA-seq analysis. More transcripts were affected by the EWSR1 knockdown than expected and these included many EWS-FLI1 regulated genes. We reevaluated physical interactions between EWS-FLI1, EWSR1, and RNA Pol II, and employed a cross-linking based strategy to investigate protein assemblies associated with the proteins. The LC domain of EWS-FLI1 was required for the assemblies observed to form in cells. These results offer new insights into a protein assembly that may enable EWS-FLI1 to bind its wide network of protein partners and contribute to regulation of gene expression in Ewing sarcoma.

TDP-43 regulates transcription at protein-coding genes and Alu retrotransposons.

The 43-kDa transactive response DNA-binding protein (TDP-43) is an example of an RNA-binding protein that regulates RNA metabolism at multiple levels from transcription and splicing to translation. Its role in post-transcriptional RNA processing has been a primary focus of recent research, but its role in regulating transcription has been studied for only a few human genes. We characterized the effects of TDP-43 on transcription genome-wide and found that TDP-43 broadly affects transcription of protein-coding and noncoding RNA genes. Among protein-coding genes, the effects of TDP-43 were greatest for genes <30 thousand base pairs in length. Surprisingly, we found that the loss of TDP-43 resulted in increased evidence for transcription activity near repetitive Alu elements found within expressed genes. The highest densities of affected Alu elements were found in the shorter genes, whose transcription was most affected by TDP-43. Thus, in addition to its role in post-transcriptional RNA processing, TDP-43 plays a critical role in maintaining the transcriptional stability of protein-coding genes and transposable DNA elements.

Intrinsically disordered RGG/RG domains mediate degenerate specificity in RNA binding.

RGG/RG domains are the second most common RNA binding domain in the human genome, yet their RNA-binding properties remain poorly understood. Here, we report a detailed analysis of the RNA binding characteristics of intrinsically disordered RGG/RG domains from Fused in Sarcoma (FUS), FMRP and hnRNPU. For FUS, previous studies defined RNA binding as mediated by its well-folded domains; however, we show that RGG/RG domains are the primary mediators of binding. RGG/RG domains coupled to adjacent folded domains can achieve affinities approaching that of full-length FUS. Analysis of RGG/RG domains from FUS, FMRP and hnRNPU against a spectrum of contrasting RNAs reveals that each display degenerate binding specificity, while still displaying different degrees of preference for RNA.