Sequence determinants of human gene regulatory elements

Abstract

DNA determines where and when genes are expressed, but the full set of sequence determinants that control gene expression is not known. To obtain a global and unbiased view of the relative importance of different sequence determinants in gene expression, we measured transcriptional activity of DNA sequences that are in aggregate ∼100 times longer than the human genome in three different cell types. We show that enhancers can be classified to three main types: classical enhancers1, closed chromatin enhancers and chromatin-dependent enhancers, which act via different mechanisms and differ in motif content. Transcription factors (TFs) act generally in an additive manner with weak grammar, with classical enhancers increasing expression from promoters by a mechanism that does not involve specific TF-TF interactions. Few TFs are strongly active in a cell, with most activities similar between cell types. Chromatin-dependent enhancers are enriched in forkhead motifs, whereas classical enhancers contain motifs for TFs with strong transactivator domains such as ETS and bZIP; these motifs are also found at transcription start site (TSS)-proximal positions. However, some TFs, such as NRF1 only activate transcription when placed close to the TSS, and others such as YY1 display positional preference with respect to the TSS. TFs can thus be classified into four non-exclusive subtypes based on their transcriptional activity: chromatin opening, enhancing, promoting and TSS determining factors – consistent with the view that the binding motif is the only atomic unit of gene expression.

Identifying critical transcriptional targets of the MYC oncogene using a novel competitive precision genome editing (CGE) assay

“…found in both publications (refs 13,14 ), and (2) display robust MYC binding at the gene promoter within open chromatin on the basis of ATAC-seq signal, and clear change in expression upon MYC silencing in colon cancer cells (ref. 15,16 ; Supplementary Fig. 2).…”

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

“…The choice to focus on the range -350 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 [30]. 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 [21,27,29,[32][33][34]36]. 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 [27][28][29], and because episomal plasmids provide an isolated context that minimises 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 [27,[30][31][32][33][34], except for one recent study that tested combinations of synthetic elements [29].…”

“…We used plasmid-based MPRAs because they are highly scalable [27][28][29], and because episomal plasmids provide an isolated context that minimises 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 [27,[30][31][32][33][34], except for one recent study that tested combinations of synthetic elements [29]. 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.…”

Compatibility logic of human enhancer and promoter sequences

“…S6g). Consistent with these factors playing a functional role, previous studies have found that adding GABPA or YY1 motifs to promoters increases gene expression in various reporter assays and cell types [34][35][36][37] . Together, these analyses suggest that P2 promoters can best be distinguished from P1 promoters by the presence of certain transcription factors including GABPA and YY1, rather than canonical core promoter motifs.…”