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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    • "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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    ABSTRACT: Basic helix-loop-helix (bHLH) transcription factors recognize the canonical E-box (CANNTG) to regulate gene transcription; however, given the prevalence of E-boxes in a genome, it has been puzzling how individual bHLH proteins selectively recognize E-box sequences on their targets. TWIST is a bHLH transcription factor that promotes epithelial-mesenchymal transition (EMT) during development and tumor metastasis. High-resolution mapping of TWIST occupancy in human and Drosophila genomes reveals that TWIST, but not other bHLH proteins, recognizes a unique double E-box motif with two E-boxes spaced preferentially by 5 nucleotides. Using molecular modeling and binding kinetic analyses, we found that the strict spatial configuration in the double E-box motif aligns two TWIST-E47 dimers on the same face of DNA, thus providing a high-affinity site for a highly stable intramolecular tetramer. Biochemical analyses showed that the WR domain of TWIST dimerizes to mediate tetramer formation, which is functionally required for TWIST-induced EMT. These results uncover a novel mechanism for a bHLH transcription factor to recognize a unique spatial configuration of E-boxes to achieve target specificity. The WR-WR domain interaction uncovered here sets an example of target gene specificity of a bHLH protein being controlled allosterically by a domain outside of the bHLH region. © 2015 Chang et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & Development 03/2015; 29(6):603-616. DOI:10.1101/gad.242842.114 · 10.80 Impact Factor
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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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    ABSTRACT: Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. We addressed this question during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes. We defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, we investigated the regulatory logic of these enhancers. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole set of trichome enhancers. These enhancers harbor remarkable features with respect to their functional architectures, including a weak or non-existent clustering of Svb binding sites. The in vivo function of each site relies on its intimate context, notably the flanking nucleotides. Two additional cis-regulatory motifs, present in a broad diversity of composition and positioning among trichome enhancers, critically contribute to enhancer activity. Our results show that Svb directly regulates a large set of terminal effectors of the remodeling of epidermal cells. Further, these data reveal that trichome formation is underpinned by unexpectedly diverse modes of regulation, providing fresh insights into the functional architecture of enhancers governing a terminal differentiation program.
    Genome biology 08/2013; 14(8):R86. DOI:10.1186/gb-2013-14-8-r86 · 10.81 Impact Factor
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    • "Although many hundreds of Twi-binding sites have been identified in genome-wide studies, the number of functional sites (i.e., those that actually modulate rates of target gene transcription in response to changing levels of Twi) is probably considerably smaller (Ozdemir et al., 2011). "
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    ABSTRACT: A central challenge of developmental and evolutionary biology is to explain how anatomy is encoded in the genome. Anatomy emerges progressively during embryonic development, as a consequence of morphogenetic processes. The specialized properties of embryonic cells and tissues that drive morphogenesis, like other specialized properties of cells, arise as a consequence of differential gene expression. Recently, gene regulatory networks (GRNs) have proven to be powerful conceptual and experimental tools for analyzing the genetic control and evolution of developmental processes. A major current goal is to link these transcriptional networks directly to morphogenetic processes. This review highlights three experimental models (sea urchin skeletogenesis, ascidian notochord morphogenesis, and the formation of somatic muscles in Drosophila) that are currently being used to analyze the genetic control of anatomy by integrating information of several important kinds: 1) morphogenetic mechanisms at the molecular, cellular and tissue levels that are responsible for shaping a specific anatomical feature, 2) the underlying GRN circuitry deployed in the relevant cells, and 3) modifications to gene regulatory circuitry that have accompanied evolutionary changes in the anatomical feature. © 2013 Wiley Periodicals, Inc.
    genesis 06/2013; 51(6). DOI:10.1002/dvg.22380 · 2.02 Impact Factor
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