The Molecular Basis of Signal Processing at Gene Regulatory Elements PDF Download

Author: Nathaniel Dixon Tippens
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Languages : en
Pages : 0

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Book Description
Gene regulation is the central process that translates genetic code into complex cellular responses. However, we lack a full and quantitative understanding of the molecular basis for information integration across genomic space and time, especially in human genetics and disease. RNA Polymerase II (Pol2) has many features of a key signal integration complex: its regulation is highly conserved across most eukaryotes, it occupies diverse types of genomic regulatory elements, and it exhibits multi-state regulation and behaviors. It engages and transcribes not only gene promoters but also distal enhancers, suggesting possible roles beyond messenger RNA (mRNA) production. Pol2 progresses through unbound, initiating, paused, elongating, and terminating stages during the transcription cycle, and is thus uniquely situated to integrate information across diverse regulatory sites and states, as well as across relatively long molecular timescales and distances. To better understand and compare Pol2 behavior at regulatory elements, we sequenced nascent RNAs at single-molecule resolution to identify initiation, capping, and pause sites genome-wide. We find distinct sequence-specified pause classes with different capping profiles. Transcription start sites (TSSs) are predominantly found in dense clusters (transcription initiation domains, or TIDs) that show remarkably little change upon heat shock, demonstrating how large regulatory changes can occur by modulating pause release from pre-established TSS architectures. Within TIDs, TSSs appear to communicate through arrays of phased nucleosomes. These TSS ensembles define promoters and enhancers, unravel complex histone modification patterns, and indicate Pol2 may integrate information between regulatory elements. To explore the function of Pol2 at distal enhancers, we performed massively parallel reporter assays and found that gene distal TSSs are robust predictors of enhancer activity with higher resolution and specificity than histone modifications. We show that enhancer units are precisely delineated by TSSs, validate that these boundaries are sufficient to capture full activity, and confirm that core promoter sequences are required for enhancer function. Consistent with prior studies, most human promoters do not show distal enhancer activity in episomal assays, suggesting important functional differences between promoters and enhancers despite similar architectures. Finally, we dissect TIDs and find that the strongest unit within the TID is the best predictor of episomal enhancer activity. Together, these results define fundamental regulatory units of promoters and enhancers, and their behavior within TIDs. Together these data suggest Pol2 function may distinguish enhancers from promoters. Recent experiments in Drosophila demonstrate that enhancers exhibit much shorter Pol2 pausing than promoters, which could explain the lower H3K4me3 levels at enhancers. In this model, H3K4me3 levels are determined by SET1 binding to the paused Pol2 complex and would thus be sensitive to Pol2 pause behavior. For example, promoters' GC-rich sequences may stabilize Pol2 pause complexes for longer times, resulting in H3K4me3 accumulation. Positive feedback between H3K4me3 and the TAF3 subunit of the pre-initiation complex further exaggerates this effect by driving local Pol2 initiation nearby, resulting in the larger TIDs observed at promoters compared with enhancers. Finally, stable Pol2 pausing at promoters provides the time required to find and receive activation signals from enhancers. Some promoters may bypass this long pause checkpoint via frequent or direct recruitment of transcriptional activators, at the cost of reduced enhancer responsiveness. This framework situates Pol2 as a key signal processing complex at gene regulatory elements.