Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus

Abstract

A complex landscape of genomic regulatory elements underpins patterns of metazoan gene expression, yet it has been technically difficult to disentangle composite regulatory elements within their endogenous genomic context. Expression of the Sox2 transcription factor (TF) in mouse embryonic stem cells (mESCs) depends on a distal regulatory cluster of DNase I hypersensitive sites (DHSs), but the contributions of individual DHSs and their degree of independence remain a mystery. Here, we comprehensively analyze the regulatory architecture of the Sox2 locus in mESCs using Big-IN to scarlessly deliver payloads ranging up to 143 kb, permitting deletions, rearrangements and inversions of single or multiple DHSs, and surgical alterations to individual TF recognition sequences. Multiple independent mESC clones were derived for each payload, extensively sequence-verified, and profiled for expression of Sox2 specifically from the engineered allele. We find that a single core DHS comprising a handful of key TF recognition sequences is sufficient to sustain significant expression in mESCs, though its contribution is modulated by additional DHSs. Moreover, their overall activity is influenced by specific DHS order and/or orientation effects. We built a highly predictive model for locus regulation which includes nonlinear components indicating both synergy and redundancy among. Our results suggest that composite regulatory elements and their influence on gene expression can be resolved to a tractable set of sequence features using synthetic regulatory genomics.

Episomal editing of synthetic constructs in yeast using CRISPR

“…To test the editing efficiency with CREEPY, we used an episomal construct containing 143-kb wild-type mouse Sox2 ( mSox2 ) fragment, which includes the coding sequence and distal regulatory clusters such as DNase I hypersensitive sites (DHSs) and CTCF-binding sites (Fig. 3a) 24 . First, we tested episomal editing by deleting a single CTCF site, CTCF8, in the mSox2 construct (Fig.…”

“…To test episomal editing, we used CREEPY to delete CTCF8, CTCF13, CTCF17 and CTCF25 in the mSox2 assembly, generating a construct with four CTCF sites removed in two steps. Beyond this, with CREEPY, over 60 constructs with deletions, inversions and surgical alterations of DHSs and CTCF of mSox2 , were built in yeast and delivered to mESCs in order to study its regulatory architecture 24 . In practice, we also tried another single-plasmid system, pYTK-Cas9, built with the yeast tool kit (Supplementary Figure 7a) 37 .…”

“…We first compare the efficiency of CRISPR/Cas9 for targeting episomes and chromosomes. With CREEPY optimized for episomal editing, we achieve simplex and multiplex editing, as demonstrated by engineering of a 143-kb mSox2 assembly containing the mouse Sox2 gene and regulatory regions 24 . Sox2 is a Yamanaka factor essential for maintaining stem cell pluripotency 25 .…”

Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters

“…3E). Under pileup, elements tested (in the same genomic position reference frame as the pileup, Data S4 for positions) are indicated both from this study (top: 500 bp regions peak from ArchR pipeline; bottom: PCR-amplified tested sequences, Methods), and two previous studies quantifying reporter activity, Zhou et al (61), and Brosh et al (8). Gray regions were not found to be significantly active.…”

“…Fine regulatory changes of target genes, caused by even single nucleotide changes to individual enhancers, can both give rise to disease (1-3) as well as drive novelty across evolution (1,4). Genetic methods have identified an extensive list of developmentally important genes in model systems (5,6), yet how the transcription of these genes is regulated by enhancers, and specifically how DNA sequence encodes the requisite functional information, remains incompletely understood even for the best-studied examples (7)(8)(9)(10). More broadly, biochemical marks correlated with enhancer status have now nominated over one million putative cis-regulatory elements (CREs) in the mouse and human genomes (11).…”