Sequence determinants of human gene regulatory elements

Abstract

DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF–TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression.

Super-enhancers require a combination of classical enhancers and novel facilitator elements to drive high levels of gene expression

“…Whereas R3 facilitates the activities of R1 and R2 to a similar degree regardless of Rm/R4 coincidence, Rm and R4 function in a context-dependent manner, each partially redundant to the other. Interestingly, Sahu and colleagues recently used a STARR-seq method to show that four out of the five MYC SE constituents have no detectable enhancer activity in HepG2 cells (Sahu et al, 2022). It is unclear whether these four non-functional elements are required for full SE activity, but it raises the possibility that facilitators could be a common feature of SEs.…”

Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome

“…The choice to focus on the range from À350 to +50 bp was motivated by our previous study of human promoters, which indicated that most of the relevant information for promoter function is generally contained within this range (van Arensbergen et al, 2017). This definition of promoters is longer than that of core promoters (which are usually only $100-bp long) as was used in most previous enhancer reporter assays (Ohler et al, 2002;Haberle et al, 2019;Inoue and Ahituv, 2015;Klein et al, 2020;Davis et al, 2020;King et al, 2020;Sahu et al, 2021). We considered this to be important because the extra regulatory information contained in those additional sequences may be relevant for interactions of the promoters with CREs.…”

“…In order to address this issue, we systematically tested the compatibility of thousands of combinations of candidate CREs (cCREs) and promoters using MPRAs. We used plasmid-based MPRAs because they are highly scalable (Inoue and Ahituv, 2015;van Arensbergen et al, 2019;Sahu et al, 2021) and because episomal plasmids provide an isolated context that minimizes confounding effects of variable chromatin environments and differences in 3D folding. However, so far, MPRAs have mostly been used to assess the activity of single elements, either as enhancers or as promoters (Inoue and Ahituv, 2015;van Arensbergen et al, 2017;Arnold et al, 2013;Klein et al, 2020;Davis et al, 2020;King et al, 2020), except for one recent study that tested combinations of synthetic elements (Sahu et al, 2021).…”

“…We used plasmid-based MPRAs because they are highly scalable (Inoue and Ahituv, 2015;van Arensbergen et al, 2019;Sahu et al, 2021) and because episomal plasmids provide an isolated context that minimizes confounding effects of variable chromatin environments and differences in 3D folding. However, so far, MPRAs have mostly been used to assess the activity of single elements, either as enhancers or as promoters (Inoue and Ahituv, 2015;van Arensbergen et al, 2017;Arnold et al, 2013;Klein et al, 2020;Davis et al, 2020;King et al, 2020), except for one recent study that tested combinations of synthetic elements (Sahu et al, 2021). To be able to dissect compatibility between enhancers and promoters systematically, we designed cloning strategies that allowed us to test thousands of pairwise cCRE-promoter combinations in different positions and orientations in a reporter plasmid.…”

A generalizable framework to comprehensively predict epigenome, chromatin organization, and transcriptome

“…The sequence patterns are generated from the attribution scores of DNA sequence toward enhancer activity in an investigated cell line. We observed that the generated sequence patterns matched with TF motifs such as CREB, JUN, YY, ETS, and IRF, which related to enhancer activity and were enriched in enhancers [39].…”