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| Status |
Public on Feb 06, 2012 |
| Title |
Protein/DNA in vitro Binding seq (PB-seq) for Drosophila HSF and genomic Drosophila DNA |
| Organism |
Drosophila melanogaster |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
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.
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| Contributor(s) |
Guertin MJ, Martin AL, Siepel A, Lis JT |
| Citation(s) |
22479205 |
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| Submission date |
Oct 03, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Joseph Guertin |
| E-mail(s) |
[email protected]
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| Organization name |
University of Connecticut
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| Department |
Center for Cell Analysis and Modeling
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| Lab |
Molecular Genomics
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| Street address |
400 Farmington Ave
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030 |
| Country |
USA |
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| Platforms (2) |
| GPL9061 |
Illumina Genome Analyzer II (Drosophila melanogaster) |
| GPL13304 |
Illumina HiSeq 2000 (Drosophila melanogaster) |
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| Samples (6)
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| Relations |
| SRA |
SRP008661 |
| BioProject |
PRJNA147109 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE32570_RAW.tar |
816.6 Mb |
(http)(custom) |
TAR (of SAM) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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