A Transcriptional Switch Governs Fibroblast Activation in Heart Disease

In diseased organs, stress-activated signaling cascades alter chromatin, triggering maladaptive cell state transitions. Fibroblast activation is a common tissue stress response that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains obscure. Pharmacologic BET inhibition alleviates cardiac dysfunction, providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here, we leverage single-cell epigenomic interrogation of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch underlying fibroblast activation. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed novel DNA elements whose accessibility dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer regulating the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program, and was required for TGFβ-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFβ-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction, and also demonstrate its upregulation upon activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide new trans- and cis- targets for treating fibrotic disease. 2 replicates for each sample were run for both single cell RNA and ATAC seq (2x Sham, 2x TAC, 2x TAC JQ1, 2xTAC JQ1 withdrawn). 2 replicates for each sample were run for PRO seq (2x Unstimulated, 2x TGFb, 2x TGFb + siRNA Ctrl, 2x TGFb + siRNA Meox1). 1 replicate for each sample was ran for 4C (1x Unstimulated, 1x TGFb). 2 replicates for MEOX1 ChIPseq. 3 replicates for each sample for bulkRNAseq (3x Sham, 3x TAC, 3x TAC JQ1). 8 PROseq samples (2x Unstimulated, 2x TGFb, 2x TGFb + siRNA Ctrl, 2x TGFb + siRNA Meox1). 6 MEOX1 ChIPseq samples (3x for Unstim and 3x for TGFb-treated conditions). 4 H3K27ac ChIPseq samples (3x for Unstim and 3x for TGFb-treated conditions).