Abstract
Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present “TurboCas,” a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 4929-4944.e8 |
| Journal | Molecular cell |
| Volume | 84 |
| Issue number | 24 |
| DOIs | |
| State | Published - Dec 19 2024 |
Funding
Research in the Shilatifard laboratory is supported by National Cancer Institute Outstanding Investigator Award grant R35CA197569 to A.S. We would like to thank Brianna Monroe for illustrations. We would like to thank Caila Ryan and Oliver Vickman for initial technical assistance. We would like to thank Rachel Rodrigues and Jon Van Vranken from the Harvard TCMP for assistance with the proteomics sample preparation, experimental design, and protocols. We would like to thank Basil Baby Mattamana (Aarohan M) and Raju Gajjela from the Northwestern Proteomics Core for the proteomics analysis of RNA Pol II/CycT1 IP samples. RNA Pol II/CycT1 IP Proteomics services were performed by the Northwestern Proteomics Core Facility (funding: NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center, instrumentation award (S10OD025194) from NIH Office of Director, and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569). We would like to acknowledge Dr. Sarah Gold, members of the Shilatifard laboratory, and Dr. Basar Cenik for their insights and suggestions. Research in the Shilatifard laboratory is supported by National Cancer Institute Outstanding Investigator Award grant R35CA197569 to A.S.H. We would like to thank Brianna Monroe for illustrations. We would like to thank Caila Ryan and Oliver Vickman for initial technical assistance. We would like to thank Rachel Rodrigues and Jon Van Vranken from the Harvard TCMP for assistance with the proteomics sample preparation, experimental design, and protocols. We would like to thank Basil Baby Mattamana (Aarohan M) and Raju Gajjela from the Northwestern Proteomics Core for the proteomics analysis of RNA Pol II/CycT1 IP samples. RNA Pol II/CycT1 IP Proteomics services were performed by the Northwestern Proteomics Core Facility (funding: NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center, instrumentation award ( S10OD025194 ) from NIH Office of Director , and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569 ). We would like to acknowledge Dr. Sarah Gold, members of the Shilatifard laboratory, and Dr. Basar Cenik for their insights and suggestions.
Keywords
- BRD4
- FOS
- FUBP3
- MYC
- chromatin-binding proteins
- dCas9
- gene regulation
- heat shock
- proximity labeling
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology