Twist ChIP-seq
High resolution mapping of Twist to DNA in Drosophila embryos:  Efficient functional analysis and evolutionary conservation
We performed a ChIP-seq assay of the transcription factor Twist in the early Drosophila melanogaster embryo (1-3 hours old), where Twist has a role in dorsal-ventral patterning.  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 SELEX (in vitro).  Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50bp 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 ±150bp surrounding Twist occupied summits, enrichment of GA and CA-repeat sequences near Twist occupied summits, and a very general depletion of A/T rich sequences from input chromatin that extends to ChIP-Seq data from multiple genomes and laboratories.  Our results show that high resolution in vivo binding data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic.  Because it was alluded to in the main text of our study, though not a main focus, we also include in this submission a Dorsal ChIP-seq experiment done in Drosophila melanogaster embryos of the same stage (1-3 hours).  This study was published in Genome Research ID# GENOME/2009/104018.  Ozdemir A, Fisher-Aylor KI, Pepke S, Samanta M, Dunipace L, McCue K, Zeng L, Ogawa N, Wold BJ, Stathopoulos A. 2011.  High resolution mapping of Twist to DNA in Drosophila embryos:  Efficient functional analysis and evolutionary conservation.
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 binding, and testing the functional significance of Twist occupied regions and motifs within regions.