Acute Activation of Genes Through Transcriptional Condensates Impact Non-target Genes in a Chromatin Domain

Abstract

AbstractTranscription activation of genes by estrogens is driven by enhancers, which are often located within the same Topologically Associating Domain (TAD) as non-targeted promoters. We investigated how acute enhancer-driven activation affects neighbouring non-target genes within the same TAD. Using single-molecule RNA FISH (smFISH), we tracked the transcription ofTFF1(enhancer-targeted) andTFF3(non-targeted) during estrogen stimulation. We observed mutually exclusive expression patterns:TFF1expression peaked at 1 hour, whileTFF3reached its peak at 3 hours, afterTFF1’s activation had diminished. Chromatin looping data indicated that the enhancer loops withTFF1but notTFF3, suggesting thatTFF3upregulation is not due to direct enhancer-promoter interactions. CRISPR deletion of theTFF1enhancer and 1,6-hexanediol (HD) exposure revealed that theTFF1enhancer:promoter undergo Liquid-Liquid Phase Separation (LLPS), which sequesters the transcriptional machinery and inhibitsTFF3expression. As estrogen signalling wanes or LLPS is disrupted,TFF1expression declines whileTFF3expression increases. Our findings reveal that enhancer-driven activation can indirectly influence neighbouring genes, highlighting a dynamic shift in gene expression as signalling progresses.