Dataset Information

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 approximately 14% of the predicted factors within the fly genome and provide an important new community resource for the identification of cis-regulatory modules.

SUBMITTER: Noyes MB

PROVIDER: S-EPMC2377422 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Noyes Marcus B MB Meng Xiangdong X Wakabayashi Atsuya A Sinha Saurabh S Brodsky Michael H MH Wolfe Scot A SA

Nucleic acids research 20080310 8

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 D ...[more]

PMID: 18332042

Similar Datasets

Project description:We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis - GRaMS (Growth Rate Modeling of Specificity) - that estimates bacterial growth rates as a function of the quality of the recognition sequence. We benchmark these different methods of motif discovery using Zif268, a well characterized C2H2 zinc finger transcription factor on both a 28bp randomized library for the standard B1H method and on 6bp randomized library for the CV-B1H method for which forty-five different experimental conditions were tested: 5 time points and three different IPTG and 3-AT concentrations. We find that GRaMS analysis is robust to the different experimental parameters whereas other analysis methods give widely varying results depending on the conditions of the experiment. Finally, we demonstrate that the CV-B1H assay can be performed in liquid media, which produces recognition models that are similar in quality to sequences recovered from selection on solid media. All selections were performed using the bait transcription factor Zif268. 45 different B1H selections were performed on plates and the selected sites were multiplexed and sequenced in a single lane. A single selection was performed in liquid media and sequenced along with other multiplexed sets of sites in a separate lane. For the experiments performed on plates, the of the selections assays was varied (4, 8, 12, 18, 24 hours). The stringency of the selections was also varied by changing the concentration of IPTG (0, 10, 50 uM) and 3-AT (0.5, 1, 2 mM). The initial randomized library used for all selections was also sequenced in a single lane. The randomized portion of each transcription factor binding site is 6bp long and is immediately 5' of the constant sequence, GCGG, which is the consensus binding site for finger 1 of Zif268. Upstream of the randomized binding site is a 4bp randomized region referred to as the key region. The purpose of the key region is to detect possible PCR 'jackpotting.' The single liquid media experiment was run for 4 hours using 50uM IPTG and 5mM 3-AT. Four selections were performed using a prey library with a 28bp long randomized region and not fixed consensus site region. These selections were performed at either 36 or 48 hours using 10uM IPTG and 0, 2, or 5 mM 3-AT.

Project description:In many metazons, such as humans and Drosophila, homeodomain proteins comprise the second largest family of sequence specific transcription factors. In Drosophila, homeodomains play an important role in development. Many homeodomain proteins display a high level of homology across metazons, presumably due to importance of their functional roles. We comprehensively characterized the DNA binding preferences of all 84 Drosophila homeodomain transcription factors which contain a single DNA binding domain. Previously, we employed a bacterial one hybrid (B1H) assay to select for 20 to 40 high affinity transcription factor binding sites [Noyes et al. (2008). Cell. 133(7):1277-1289]. In this system, E. coli are transfected with two plasmids. One plasmid encodes the DNA binding domain of a homeodomain fused to two zinc finger domains and the omega subunit of RNAP. The other plasmid is drawn from a library of prey plasmids which contain a 10bp randomized transcription factor binding site (TFBS) region in the promoter of the reporter gene His3. The E. coli strain used is a His3 homolog and omega subunit knock out strain. If a transcription factor has high affinity for a TFBS, more His3 will be produced, leading to the production of more histidine and an increase in the growth rate. If the transcription factor does not bind with sufficient affinity to the TFBS, little or no histidine will be produced resulting in little or no growth. The stringency of the B1H system can be tuned using the chemicals IPTG and 3-AT. IPTG induces production of the chimeric transcription factor, and 3-AT is a competitive inhibitor of the enzyme encoded by His3. One of the advantages of the B1H system is that the transcription factor does not have to be purified and that many experiments can be easily conducted in parallel. In this study, in stead of picking 20 to 40 colonies and sequencing their TFBSs, we used high-throughput Illumina sequencing to sequence the selected sites of all of the colonies growing on a plate. This provided quantitative data regarding the growth rate of cells possessing each selected TFBS variant, which is a function of the affinity of the transcription factor for the binding site. With this quantitative data, we can build more accurate models of transcription factor binding.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets