Ligand-dependent Enhancer Activation Indirectly Modulates Non-target Promoters in a Chromatin Domain

Transcription activation of genes by estrogen 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 of TFF1 (enhancer-target gene) and TFF3 (non-target gene) during estrogen stimulation. We observed mutually exclusive expression patterns: TFF1 expression peaked at 1 hour, while TFF3 reached its peak at 3 hours, after TFF1 activation had diminished. Chromatin looping data indicated that the enhancer loops with TFF1 but not TFF3 , suggesting that TFF3 upregulation is not due to direct enhancer-promoter interactions. CRISPR deletion of the enhancer, affected TFF1 transcription more acutely than TFF3. 1,6-hexanediol (HD) exposure suggested that the TFF1 enhancer:promoter undergo a potential ERα-mediated condensate formation, which sequesters the transcriptional machinery and inhibits TFF3 expression. As estrogen signalling fades at 3h, TFF1 expression declines while TFF3 expression increases. Our findings reveal that enhancer-driven activation can indirectly repress neighbouring genes within the same TAD, highlighting a dynamic shift in gene expression as signalling progresses.