A systematic characterization of factors that regulate Drosophila segmentation via a bacterial one-hybrid system

Abstract

Specificity data for groups of transcription factors (TFs) in a common regulatory network can be used to computationally identify the location of cis-regulatory modules in a genome. The primary limitation for this type of analysis is the paucity of specificity data that is available for the majority of TFs. We describe an omega-based bacterial one-hybrid system that provides a rapid method for characterizing DNA-binding specificities on a genome-wide scale. Using this system, 35 members of the Drosophila melanogaster segmentation network have been characterized, including representative members of all of the major classes of DNA-binding domains. A suite of web-based tools was created that uses this binding site dataset and phylogenetic comparisons to identify cis-regulatory modules throughout the fly genome. These tools allow specificities for any combination of factors to be used to perform rapid local or genome-wide searches for cis-regulatory modules. The utility of these factor specificities and tools is demonstrated on the well-characterized segmentation network. By incorporating specificity data on an additional 66 factors that we have characterized, our tools utilize ∼14% of the predicted factors within the fly genome and provide an important new community resource for the identification of cis-regulatory modules.

Characterization of the DNA-binding Properties of the Mohawk Homeobox Transcription Factor

“…The IBS is composed of an inverted repeat (ACAnnTGT) that consists of two monomeric recognition motifs for each Iro-C homeodomain (ACA). The arrangement of the two monomeric recognition motifs appears to be flexible, as Drosophila ara has been shown to recognize an opposite inverted repeat conformation (TGTnnACA), but not a direct repeat ACAnnACA (17)(18)(19). The monomeric recognition motif of the Drosophila Mkx ortholog CG11617 has been characterized and is similar to Irx family members, except for a strong preference for a thymine (T) at position 1 of the binding motif (TnACA) (11).…”

“…The Mkx fragment was subcloned into the pB1H22-12 vector as a fusion to the Zif12 and the subunit of bacterial RNA polymerase. These selections were performed as described previously, where the recognition motif was compiled from the sequences of 10-bp library members recovered from the binding site selection (11,19). The web-based Motifsampler program was used to develop a position probability matrix for the Mkx-binding site (20,21).…”

“…A fragment of mouse Mkx including the homeodomain and adjacent conserved domain CD-A (amino acids 70 -161) was cloned into the pB1H22-12 vector, as a fusion with the subunit of RNA polymerase and the first and second zinc fingers of Zif268. This construct was co-transformed with a reporter plasmid that contains a region of random decamers adjacent to the binding site of the Zif268, and transformants were selected for their ability to grow under nutrient-deficient conditions (11,19).…”

In vitro DNA-binding profile of transcription factors: methods and new insights

“…PCR is used in SELEX and sequencing which may introduce bias to some sequences or artifacts. SELEX-seq may have difficulty finding the fine specificity difference between sequences with single nucleotide differences Noyes et al 2008). Some of these methods have certain special functions and can provide some additional information on DNA-TF binding specificity, which cannot be produced by dsDNA microarray and SELEX-seq.…”

Investigating giant (Gt) repression in the formation of partially overlapping pair‐rule stripes

“…This software received as input one or more position weight matrices (PWM), each one modeling binding sites for a specific factor, and a sequence where the binding sites were predicted. We used a Gt PWM available at the web site of the Berkeley Drosophila Transcription Network Project (http://bdtnp.lbl.gov/FlyNet/SearchChipper?first¼15; Li at al., 2008) and a DSTAT PWM available at http://labs.umassmed.edu/WolfeLab/ binding site database.html (Noyes et al, 2008).…”