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| Status |
Public on Apr 26, 2023 |
| Title |
DNA Binding Specificities of Fkh1, Fkh2, Hcm1, and Fhl1 Revealed by SELEX-seq |
| Organism |
synthetic construct |
| Experiment type |
Other
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| Summary |
Quantifying the preferences of DNA binding proteins is an essential step in determining how transcription factors (TFs) interact with their targets in the genome. High-throughput in vitro binding assays have been used to identify the inherent DNA preferences of TFs in a controlled environment isolated from confounding factors such as genome accessibility, DNA methylation, and TF binding cooperativity. Unfortunately, the limited variable region or sequencing depth typically utilized by many of the available experimental approaches makes it difficult to measure the preferences of moderate- to low-affinity binding sites, and impossible to measure small-scale differences between closely related homologs. The Forkhead box (FOX) family of TFs is known to play a crucial role in regulating a variety of key processes from proliferation and development, to tumor suppression and aging. By using the high-sequencing depth SELEX-seq approach to study all four FOX homologs in Saccharomyces cerevisiae, we have been able to precisely quantify the contribution and importance of nucleotide positions flanking the core of the binding sites. Essential to this process was the alignment of our SELEX-seq reads to a set of candidate core sequences determined by two newly developed strategies of alignment and reprioritization applied to enriched k-mers.
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| Overall design |
SELEX-seq was performed on Fkh1, Fkh2, Hcm1, and Fhl1 constructs as described in Cooper et al. (to be submitted). We provide sequencing results for both the intial library (R0) as well as the selected libraries after one (R1) or two (R2) rounds of selection. A test experiment was performed using just the Fkh1 construct with a shallower depth of sequencing. The final, large-scale experiments were performed on both all constructs. The initial libraries included a 16 bp randomized region flanked by two fixed adapters absent of any known binding site for the used transcription factors.
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| Web link |
https://doi.org/10.1093/nar/gkad372
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| Contributor(s) |
Rohs R, Cooper BH, Machado AC, Gan Y, Aparicio O |
| Citation(s) |
37177995 |
| |
| Submission date |
Jun 24, 2021 |
| Last update date |
Jul 26, 2023 |
| Contact name |
Remo Rohs |
| Organization name |
University of Southern California
|
| Department |
Quantitative and Computational Biology
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| Lab |
Rohs Lab
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| Street address |
1050 Childs Way
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90089 |
| Country |
USA |
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| Platforms (2) |
| GPL19424 |
Illumina NextSeq 500 (synthetic construct) |
| GPL19604 |
Illumina HiSeq 2500 (synthetic construct) |
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| Samples (13)
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| Relations |
| BioProject |
PRJNA741127 |
| SRA |
SRP325475 |
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