High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation

Abstract

Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identified previously studied Twist-dependent CRMs and robustly predicted new CRM activity in transgenesis, with newly identified Twist-occupied regions supporting diverse spatiotemporal patterns (>74% positive, n = 31). Some, but not all, candidate CRMs require Twist for proper expression in the embryo. The Twist motifs most favored in genome ChIP data (in vivo) differed from those most favored by Systematic Evolution of Ligands by EXponential enrichment (SELEX) (in vitro). Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50 bp of the ChIP summit and, of these, CACATG was most prevalent. Mutagenesis experiments demonstrated that different Twist E-box motif types are not fully interchangeable, suggesting that the ChIP-derived consensus (CABVTG) includes sites having distinct regulatory outputs. Further analysis of position, frequency of occurrence, and sequence conservation revealed significant enrichment and conservation of CABVTG E-box motifs near Twist ChIP-seq signal summits, preferential conservation of 6150 bp surrounding Twist occupied summits, and enrichment of GA-and CA-repeat sequences near Twist occupied summits. Our results show that high resolution in vivo occupancy data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic.

Complex interactions between cis-regulatory modules in native conformation are critical for Drosophila snail expression

“…More recently, however, additional CRMs have been identified sharing similar spatiotemporal profiles to previously characterized CRMs, including one that shares close similarity with the sna expression pattern (Ozdemir et al, 2011;Perry et al, 2010). Another recent study presumably labeled this CRM as a 'shadow' enhancer because it is located at a distance from the snail gene, whereas the proximally located CRM was defined as the primary acting enhancer (Perry et al, 2010).…”

“…sna 1 /sna 18 ) to viability, whereas the other group was limited to assaying early gastrulation defects presumably because a large deficiency background was used; (2) our deletions were guided by our own Twist ChIP-seq data (Ozdemir et al, 2011), effectively guiding definition of the distal CRM as a larger region (~2.0 kb), (3) a spacer sequence (i.e. ampicillin resistance cassette) was not put in place of deletions in our constructs, which allowed us to assay whether native spacing is important; (4) the sna-coding sequence, which may possibly influence cis-regulatory mechanism or stability of transcripts, was left intact within our reporter constructs; and (5) the other group did not assay the gastrulation defects associated with the distal CRM delete large transgene but relied on cDNA rescue data conducted previously (Hemavathy et al, 2004).…”

“…For the most part, these secondary CRMs were identified as a result of ChIP-chip and ChIP-seq analyses as regions of occupancy located at a distance from genes of interest, up to 10 kb or more (e.g. Li et al, 2008;Ozdemir et al, 2011;Sandmann et al, 2007;Zeitlinger et al, 2007). These newly identified CRMs have been described as being redundant to previously identified promoter-proximal located CRMs and, most recently, it has been proposed that they function to provide robustness to environmental or genetic perturbation (Frankel et al, 2010;Perry et al, 2010).…”

Twist1 mediates repression of chondrogenesis by β-catenin to promote cranial bone progenitor specification

“…In Drosophila, Twist consensus sites differ substantially from canonical E-box sites (Ozdemir et al, 2011). We took an unbiased approach to identifying binding sites for Twist1 in the Sox9 locus and mined ChIP-seq (ChIP followed by sequencing) data from a human mammary epithelial cell line (Casas et al, 2011).…”

Drosophilapoised enhancers are generated during tissue patterning with the help of repression

“…A target gene was assigned to DV enhancers only if the enhancer's expression domain overlapped and resembled the gene's expression domain (Supplemental Material). For this purpose, published mRNA in situ hybridization data from the Berkeley Drosophila Genome Project (BDGP) (Tomancak et al 2002(Tomancak et al , 2007Hammonds et al 2013) database were used for enhancers identified by Kvon et al (2012) and Ozdemir et al (2011). For some of these enhancers, no target gene was identified with confidence, and thus those enhancers were not included in mRNA-seq analysis shown in Supplemental Figure S1 (Supplemental Material).…”