Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network

ABSTRACT: Contemporary high throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including chromatin immunoprecipitation sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF co-occupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous co-occupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmd-bound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional, newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide new insights into the transcriptional codes regulating muscle gene expression, and offer a generalizable approach for similar studies in other systems. Examination of Lmd occupancy to genomic DNA from sorted mesodermal cells

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Brian Busser

PROVIDER: E-GEOD-38402 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network.

Busser Brian W BW Huang Di D Rogacki Kevin R KR Lane Elizabeth A EA Shokri Leila L Ni Ting T Gamble Caitlin E CE Gisselbrecht Stephen S SS Zhu Jun J Bulyk Martha L ML Ovcharenko Ivan I Michelson Alan M AM

Proceedings of the National Academy of Sciences of the United States of America 20121126 50

Contemporary high-throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs, and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including ChI ...[more]

PMID: 23184988

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Project description:DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, we developed an experimental approach, called protein/DNA binding and high-throughput sequencing (PB-seq), that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin. We applied our methods to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB-seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF-bound and HSF-free DNA, and then detecting HSF-bound DNA by high throughput sequencing. We compared PB-seq binding profiles with ones observed in vivo by ChIP-seq, and developed statistical models to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. We found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity. We also investigated the extent to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase-seq data and the ChIP-chip profiles for many histone modifications and TFs, and found GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation to be the most predictive covariates. Lastly, we generated an unbiased model of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE. These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity. We performed an in vitro binding experiment with purified HSF and naked, sheared genomic Drosophila S2 DNA (PB-seq), to derive an accurate set of potential HSF binding sites in the Drosophila genome. HSF-bound DNA was specifically eluted and detected by high throughput sequencing. Drosophila HSF was N-terminally tagged with glutathione s-transferase and a tobacco etch virus (TEV) protease cleavage site. The C-terminus of the recombinant HSF was fused to the 3xFLAG epitope. Recombinant HSF was purified from E. coli with glutathione resin as previously described (PMID: 20078429) , with the following modifications: HSF-3xFLAG elution was achieved by addition of 6xHistidine tagged TEV protease and TEV protease was cleared from the HSF preparation using a Nickel-NTA column. We incubated 600pM HSF and 2500ng genomic DNA (sonicated to 100-600bp fragment size as previously described in PMID: 20844575) in 1500μl final volume of 1xHSF binding buffer and let it come to equilibrium for an hour at room temperature. We added 20μl ANTI-FLAG M2 affinity gel for 10 minutes, washed 8 times with 1xHSF binding buffer to remove unbound DNA; 3xFLAG peptide was added to a final concentration of 200ng/μl to specifically elute HSF and HSF-bound DNA. The mock IP was done in the absence of recombinant HSF.

Project description:Understanding the regulatory genome remains a significant challenge. Annotation of regulatory elements and identification of the transcription factors (TFs) targeting these elements are key steps in understanding how a given cell interprets its genetic blueprint. One goal of the modENCODE (model organism Encyclopedia of DNA Elements) project is to survey a diverse sampling of TFs, both DNA-binding and non-DNA binding factors, to provide a framework for the subsequent study of the mechanisms by which transcriptional regulators target the genome. Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of 84 TFs at various stages of development. This regulatory map reveals a variety of genomic targeting patterns, including factors with strong preferences toward proximal promoter binding, factors that target intergenic and intronic DNA, and factors with distinct chromatin state preferences. The data also suggest the existence of a partially self-contained Polycomb regulatory network, and highlight the importance of Trithorax-like (Trl) in maintaining hotspots of DNA binding throughout development. Furthermore, the data identify over 5,800 instances in which TFs target DNA regions with demonstrated enhancer activity. Regions of high TF co-occupancy are more likely to be associated with open enhancers used across cell types, while lower TF occupancy regions are associated with complex enhancers that are also regulated at the epigenetic level. A putative regulatory network generated based on these 84 regulators reveals hundreds of co-binding events, thousands of potential regulatory interactions, and distinct regulatory strategies at developmental and housekeeping genes. These data serve as a resource for the research community in the continued effort to dissect transcriptional regulatory mechanisms directing Drosophila development. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf This is a dataset generated by the Drosophila Regulatory Elements modENCODE Project led by Kevin P. White at the University of Chicago.

Dataset Information

Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network

Publications

Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network.

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