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| Status |
Public on Sep 30, 2022 |
| Title |
Identification and characterization of repressive domains in Drosophila transcription factors |
| Organism |
Drosophila melanogaster |
| Experiment type |
Other
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| Summary |
All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA-binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied and their repressive domains (RDs) are typically unknown, as are the RDs’ properties and the co-repressors (CoRs) they recruit. Here, we develop the high-throughput next-generation-sequencing-based method Repressive-Domain (RD)-seq to systematically identify RDs in complex libraries. Screening more than 200,000 fragments covering the coding sequences of all transcription-related proteins in Drosophila melanogaster, we identify 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs that are required for RD function, as demonstrated by motif mutagenesis, and are conserved between fly and human. Moreover, we show that RDs which contain one of five distinct repressive motifs interact with and depend on different CoRs, including Groucho, CtBP, Sin3A or Smrter. Overall, our work constitutes an invaluable resource and advances our understanding of repressors, their sequences, and the functional impact of sequence-altering mutations.
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| Overall design |
Examination of transcription factor's repressive domains activity between GFP-neg and GFP-pos cells through repressive domain sequencing (RD-seq). A Gal4-DBD-fused candidate library consisting of over 200,000 150 bp-long DNA fragments coding for 50 AA was generated. This library was tested in RD-seq screens with two different reporter cell lines in which GFP expression is driven by the zfh1-DSCP or the ent1-rps12 enhancer-promoter pair.
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| Contributor(s) |
Klaus L, de Almeida BP, Vlasova A, Nemčko F, Schleiffer A, Bergauer K, Hofbauer L, Rath M, Stark A |
| Citation(s) |
36545802 |
| Submission date |
Jul 01, 2022 |
| Last update date |
Dec 31, 2022 |
| Contact name |
Bernardo P de Almeida |
| E-mail(s) |
bernardo.almeida@imp.ac.at
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| Organization name |
Research Institute of Molecular Pathology (IMP)
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| Lab |
Stark Lab
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| Street address |
Campus-Vienna-Biocenter 1
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| City |
Wien |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platforms (1) |
| GPL22106 |
NextSeq 550 (Drosophila melanogaster) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA854946 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE207374_RAW.tar |
39.8 Mb |
(http)(custom) |
TAR (of TSV, TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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