Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1 + SiglecF + transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo . Human orthologues of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.

Background: Gene regulatory networks control tissue homeostasis and disease progression in a cell-type specific manner. Ubiquitously expressed chromatin regulators modulate these networks, yet the mechanisms governing how tissue-specificity of their function is achieved are poorly understood. BRD4, a member of the BET (Bromo- and Extra-Terminal domain) family of ubiquitously expressed acetyl-lysine reader proteins, plays a pivotal role as a coactivator of enhancer signaling across diverse tissue types in both health and disease, and has been implicated as a pharmacologic target in heart failure. However, the cell-specific role of BRD4 in adult cardiomyocytes remains unknown. Methods: We combined conditional mouse genetics, unbiased transcriptomic and epigenomic analyses, and classical molecular biology and biochemical approaches to understand the role of BRD4 in adult cardiomyocyte homeostasis. Results: Here, we show that cardiomyocyte-specific deletion of Brd4 in adult mice leads to acute deterioration of cardiac contractile function with mutant animals demonstrating a transcriptomic signature enriched for decreased expression of genes critical for mitochondrial energy production. Genome-wide occupancy data show that BRD4 enriches at many downregulated genes (including the master co-activators Ppargc1a , Ppargc1b , and their downstream targets) and preferentially co-localizes with GATA4, a lineage determining cardiac transcription factor not previously implicated in regulation of adult cardiac metabolism. BRD4 and GATA4 form an endogenous complex in cardiomyocytes and interact in a bromodomainindependent manner, revealing a new functional interaction partner for BRD4 that can direct its locus and tissue specificity. Conclusions: These results highlight a novel role for a BRD4-GATA4 module in cooperative regulation of a cardiomyocyte specific gene program governing bioenergetic homeostasis in the adult heart.

BGB is a cofounder of, consultant to, and shareholder in Tenaya Therapeutics. SMH is an executive and officer of and shareholder in Amgen. SMH is a cofounder of and shareholder in Tenaya Therapeutics. NSG is a founder and science advisory board member of and equity holder in Gatekeeper, Syros, Petra, C4 Therapeutics, B2S Life Sciences, and Soltego. The Gray laboratory receives or has received research funding from Novartis,

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