Lang, W. H. | Search bioRxiv

bioRxiv2026

BRD4 recruitment desilences transcription without erasure or depletion of repressive chromatin

How genes are desilenced within mesoscale repressive chromatin is a crucial yet poorly understood phenomenon. Prevailing models posit that methylation of lysine-9 of histone H3 (H3K9me3) engages heterochromatin protein 1 (HP1) to drive chromatin compaction and transcriptional silencing. The erasure of this repressive mark and its replacement with acetyl/acyl groups recruits positive factors such as BRD4/BET to elicit gene transcription. We report that in Friedreichs ataxia, a synthetic gene regulator (SynGR1) drives transcription across repressive chromatin without removal or replacement of H3K9me3 or HP1. By selectively recruiting BRD4/BET into repressive GAA-repeats in frataxin (FXN), SynGR1 creates a paradoxical state where gene transcription and repressive chromatin co-exist. Contrary to convention, we find that BRD4 readily partitions into phase separated HP1 condensates in vitro and into HP1 puncta in patient-derived cells, thus presenting a mechanistic explanation for desilencing transcription without the dispersal of mesoscale repressive chromatin. Epigenetic drugs that gate sequential steps in transcription, synergistically stimulate FXN expression while concomitantly increasing, rather than eliminating, repressive H3K9me3 and HP1 levels. More broadly, this study highlights the dynamic nature of repressive chromatin and the context-dependence of epigenetic marks in regulating gene expression.

cell biology↗

bioRxiv2024

Reconciling competing models on the roles of condensates and soluble complexes in transcription factor function

Phase separation explains the exquisite spatial and temporal regulation of many biological processes, but the role of transcription factor-mediated condensates in gene regulation is contentious, requiring head-to-head comparison of competing models. Here, we focused on the prototypical yeast transcription factor Gcn4 and assessed two models for gene transcription activation, i.e., mediated via soluble complexes or transcriptional condensates. Both models rely on the ability of transcription factors and coactivators to engage in multivalent interactions. Unexpectedly, we found that propensity to form homotypic Gcn4 condensates does not correlate well with transcriptional activity. Contrary to prevailing models, binding to DNA suppresses Gcn4 phase separation. Notably, the ability of Gcn4 to form soluble complexes with coactivator subunit Med15 closely mirrored the propensity to recruit Med15 into condensates, indicating that these properties are intertwined and cautioning against interpretation of mutational data without head-to-head comparisons. However, Gcn4 variants with the highest affinity for Med15 do not function as well as expected and instead have activities that reflect their abilities to phase separate with Med15. These variants therefore indeed form cellular condensates, and those attenuate activity. Our results show that transcription factors can function as soluble complexes as well as condensates, reconciling two seemingly opposing models, and have implications for other phase-separating systems. Highlights- Homotypic phase separation propensities of Gcn4 variants do not predict in vivo activities well. - DNA binding leads to solubilization of GCN4 condensates, which is reversed by Med15 association. - The abilities to co-phase separate and form soluble complexes with Med15 are highly intertwined. - Variants with high affinities for Med15 form condensates that attenuate function.

biophysics↗