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| Status |
Public on Jan 27, 2020 |
| Title |
Context-dependent gene regulation by homeodomain transcription factor complexes revealed by shape-readout deficient proteins |
| Organism |
Drosophila melanogaster |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
Other
Expression profiling by high throughput sequencing
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| Summary |
Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.
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| Overall design |
SELEX-seq was performed on different combinations of the three homeodomain transcription factors Deformed, Homothorax and Extradenticle for both wild-type and shape-readout-impaired mutant protein. In addition, ATAC-seq and ChIP-seq was performed in D.melanogaster 3rd instar imaginal wing discs against both wild-type and shape-readout mutant Extradenticle, as well as Homothorax and Antennapedia TFs. Intrinsic DNA Shape (Zhou, PNAS, 2015) was correlated with TF binding affinity to identify position-specific shape readout (minor groove readout). Amino acid mutations abolishing the identified shape readout were subsequently used in vitro and in vivo to probe the degree to which binding mechanism are recapitulated in vivo. Further Hi-C and RNA-seq data was used to dissect the regulatory network of HD TF complexes and identify complex-specific gene function.
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| Contributor(s) |
Mann RS, Bussemaker HJ, Kribelbauer JF |
| Citation(s) |
32053778 |
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| Submission date |
Jan 24, 2019 |
| Last update date |
Jun 03, 2020 |
| Contact name |
Harmen J Bussemaker |
| Organization name |
Columbia University
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| Department |
Biological Sciences
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| Lab |
Bussemaker Lab
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| Street address |
1212 Amsterdam Avenue, MC 2441
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| City |
New York City |
| State/province |
New York |
| ZIP/Postal code |
10027 |
| Country |
USA |
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| Platforms (2) |
| GPL13304 |
Illumina HiSeq 2000 (Drosophila melanogaster) |
| GPL19132 |
Illumina NextSeq 500 (Drosophila melanogaster) |
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| Samples (33)
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| Relations |
| BioProject |
PRJNA516909 |
| SRA |
SRP181908 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE125604_RAW.tar |
2.2 Gb |
(http)(custom) |
TAR (of BED, BW, HIC) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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