Estrogen-induced chromatin looping changes identify a subset of functional regulatory elements.

Transcriptional enhancers can regulate individual or multiple genes through long-range three-dimensional (3D) genome interactions, and these interactions are commonly altered in cancer. Yet, the functional relationship between changes in 3D interactions associated with regulatory regions and differential gene expression appears context-dependent. In this study, we used HiChiP to capture changes in 3D genome interactions between active regulatory regions of endometrial cancer cells in response to estrogen treatment and uncovered significant differential long-range interactions that are strongly enriched for estrogen receptor α (ER) bound sites (ERBS). The ERBS anchoring differential loops with either a gene's promoter or distal regions were correlated with larger transcriptional responses to estrogen compared to ERBS not involved in differential interactions. To functionally test this observation, CRISPR-based Enhancer-i was used to deactivate specific ERBS, which revealed a wide range of effects on the transcriptional response to estrogen. However, these effects are only subtly and not significantly stronger for ERBS in differential loops. In addition, we observed an enrichment of 3D interactions between the promoters of estrogen up-regulated genes and found that looped promoters can work together cooperatively. Overall, our work suggests that changes in 3D genome structure upon estrogen treatment identify some functionally important regulatory regions; however, these changes aren't required for a transcriptional response to E2 in endometrial cancer cells.

Cis-regulatory control of transcriptional timing and noise in response to estrogen.

Cis-regulatory elements control transcription levels, temporal dynamics, and cell-cell variation or transcriptional noise. However, the combination of regulatory features that control these different attributes is not fully understood. Here, we used single-cell RNA-seq during an estrogen treatment time course and machine learning to identify predictors of expression timing and noise. We found that genes with multiple active enhancers exhibit faster temporal responses. We verified this finding by showing that manipulation of enhancer activity changes the temporal response of estrogen target genes. Analysis of transcriptional noise uncovered a relationship between promoter and enhancer activity, with active promoters associated with low noise and active enhancers linked to high noise. Finally, we observed that co-expression across single cells is an emergent property associated with chromatin looping, timing, and noise. Overall, our results indicate a fundamental tradeoff between a gene's ability to quickly respond to incoming signals and maintain low variation across cells.