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

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Genome Research (Impact Factor: 14.63). 03/2011; 21(4):566-77. DOI: 10.1101/gr.104018.109
Source: PubMed


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 ±150 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.

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    • "expression at the cellular level to impart heterogeneity. Genomicsbased studies conducted in Drosophila have determined that Twist modulates transcriptional regulatory networks with complex logical outputs by binding to over 2000 cis-regulatory elements (Sandmann et al., 2007; Ozdemir et al., 2011). Twist1 targets identified in MSCs also map to critical nodes within multiple gene regulatory networks, which explains in part how changes in its expression reliably predicts donor-to-donor differences in the biological properties of MSC populations . "
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    • "To determine whether the double E-box motif is evolutionarily conserved across species, we performed the same HOMER analysis on the previously published Drosophila Twist ChIP-seq data (Ozdemir et al. 2011). Indeed, we found that Drosophila Twist also preferentially bound the double E-box motif where two E-boxes are separated by exactly 5-nt spacing with an 11-fold enrichment over the calculated random occurrence frequency (P-value = 1 × 10 −35 ) (Fig. 2A). "
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    • "Furthermore, besides turnover of some motifs, svbF7, blue and yellow motifs are often embedded within short-sized islands of high evolutionary conservation, when compared to neighboring sequences (Figure 8). Similar strong evolutionary conservation was also noticed for the binding site of Twist [62] and its partner TFs [15], although these studies did not examine evolution of the detailed pattern of motif positioning. These data therefore suggest that despite diverse arrangements of motifs, patterns of evolutionary conservation likely represent the signature of functional constraints that locally shape the architecture of individual enhancers. "
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