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Published April 2011 | Published + Supplemental Material
Journal Article Open

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 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.

Additional Information

© 2011 Cold Spring Harbor Laboratory Press. Received September 17, 2010; accepted in revised form January 4, 2011. Published in Advance March 7, 2011. We thank the Caltech Jacobs Genome Facility members I. Antoshechkin and L. Schaeffer for library building and DNA sequencing, as well as D. Trout, B. King, and H. Amrhein for primary sequence data processing and visualization. We are grateful to A. Mortazavi and A. Kirilusha (Caltech Biology) for software and discussion of analysis; M. Biggin and S. Celniker (Lawrence Berkeley Lab) for sharing unpublished data; and M. Levine (University of California at Berkeley) for antibodies. K.I.F.-A. was funded by a NSF pre-doctoral fellowship, and S.P. was funded by The Gordon and Betty Moore Foundation. Work at Lawrence Berkeley National Laboratory was conducted under Department of Energy contract DE-AC02-05CH11231. This work was funded by the Functional Genomics Resource Center of the Caltech Beckman Institute, NIH grant R01GM077668 (A.S.), NIH grant U54HG004576 (B.J.W.), and the Bren Chair (B.J.W).

Attached Files

Published - Ozdemir2011p13474Genome_Res.pdf

Supplemental Material - Ozdemir.Fisher.SupFigures.Revision2.pdf

Supplemental Material - SupplementalText_Ozdemir.doc

Supplemental Material - Supplemental_Table_1.xls

Supplemental Material - Supplemental_Table_2.pdf

Supplemental Material - Supplemental_Table_3_revised.pdf

Supplemental Material - Supplemental_Table_4.xls

Supplemental Material - Supplemental_Table_5.xls

Supplemental Material - Supplemental_Table_6.xls


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