Chromatin remodelling drives immune cell-fibroblast communication in heart failure.

Chronic inflammation and tissue fibrosis are common responses that worsen organ function, yet the molecular mechanisms governing their cross-talk are poorly understood. In diseased organs, stress-induced gene expression changes fuel maladaptive cell state transitions1 and pathological interaction between cellular compartments. Although chronic fibroblast activation worsens dysfunction in the lungs, liver, kidneys and heart, and exacerbates many cancers2, the stress-sensing mechanisms initiating transcriptional activation of fibroblasts are poorly understood. Here we show that conditional deletion of the transcriptional co-activator Brd4 in infiltrating Cx3cr1+ macrophages ameliorates heart failure in mice and significantly reduces fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1+ cells identified a large enhancer proximal to interleukin-1ß (IL-1ß, encoded by Il1b), and a series of CRISPR-based deletions revealed the precise stress-dependent regulatory element that controls Il1b expression. Secreted IL-1ß activated a fibroblast RELA-dependent (also known as p65) enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL-1ß neutralization improved cardiac function and tissue fibrosis in heart failure. Systemic IL-1ß inhibition or targeted Il1b deletion in Cx3cr1+ cells prevented stress-induced Meox1 expression and fibroblast activation. The elucidation of BRD4-dependent cross-talk between a specific immune cell subset and fibroblasts through IL-1ß reveals how inflammation drives profibrotic cell states and supports strategies that modulate this process in heart disease and other chronic inflammatory disorders featuring tissue remodelling.

A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer.

Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene, Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the three Klf4 enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site in Klf4 enhancer E2 rescued enhancer function and Klf4 expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities of Klf4 enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.

Chromatin Remodeling Drives Immune-Fibroblast Crosstalk in Heart Failure Pathogenesis.

Chronic inflammation and tissue fibrosis are common stress responses that worsen organ function, yet the molecular mechanisms governing their crosstalk are poorly understood. In diseased organs, stress-induced changes in gene expression fuel maladaptive cell state transitions and pathological interaction between diverse cellular compartments. Although chronic fibroblast activation worsens dysfunction of lung, liver, kidney, and heart, and exacerbates many cancers, the stress-sensing mechanisms initiating the transcriptional activation of fibroblasts are not well understood. Here, we show that conditional deletion of the transcription co-activator Brd4 in Cx3cr1-positive myeloid cells ameliorates heart failure and is associated with a dramatic reduction in fibroblast activation. Analysis of single-cell chromatin accessibility and BRD4 occupancy in vivo in Cx3cr1-positive cells identified a large enhancer proximal to Interleukin-1 beta (Il1b), and a series of CRISPR deletions revealed the precise stress-dependent regulatory element that controlled expression of Il1b in disease. Secreted IL1B functioned non-cell autonomously to activate a p65/RELA-dependent enhancer near the transcription factor MEOX1, resulting in a profibrotic response in human cardiac fibroblasts. In vivo, antibody-mediated IL1B neutralization prevented stress-induced expression of MEOX1, inhibited fibroblast activation, and improved cardiac function in heart failure. The elucidation of BRD4-dependent crosstalk between a specific immune cell subset and fibroblasts through IL1B provides new therapeutic strategies for heart disease and other disorders of chronic inflammation and maladaptive tissue remodeling.

Time-resolved crystallography captures light-driven DNA repair.

Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.