|
| Status |
Public on Feb 12, 2022 |
| Title |
dCas9 Fusion to Computer Designed PRC2 Inhibitor Reveals Functional TATA Box in Distal Promoter Region [ATAC-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
The critical process in development, bifurcation of cellular fates, requires epigenetic H3K27me3 marks propagated by PRC2 complex. However, the precise chromatin loci of functional H3K27me3 marks are not yet known. Here we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB) that competes with EZH2 and thereby disrupts PRC2 function, to dCas9 (EBdCas9) to direct PRC2 inhibition at a precise locus using gRNA. We targeted EBdCas9 to 4 different genes ( TBX18, p16, CDX2 and GATA3 ) and observed epigenetic remodeling at a single nucleosome resulting in gene activation. Remarkably, while traditional TATA box is located 30bp upstream of TSS, we identified a functional TATA box, >500bp of TSS, normally repressed by PRC2 complex. Deletion of this TATA box eliminates EBdCas9 dependent TBP recruitment and transcriptional activation. Targeting EBdCas9 to CDX2 and GATA3 results in trophoblast trans-differentiation. EBdCas9 technology may provide a broadly applicable tool for epigenetic regulation at a single locus to control gene expression
|
| |
|
| Overall design |
WTC EBdCas9 induced cells and WTC NCdCas9 induced cells were transfected with TBX18 g6 RNA for 2 days and were harvested on day 3 for ATACseq analysis.
hESC Elf 1 cells were either induced with EB using Dox for 3 days or not induced and harvest for ATAC seq analysis.
|
| |
|
| Contributor(s) |
Ng WI, Ruohola-Baker H |
| Citation(s) |
35235780 |
| |
| Submission date |
Jan 29, 2022 |
| Last update date |
Apr 19, 2022 |
| Contact name |
Hannele Ruohola-Baker |
| E-mail(s) |
hannele@u.washington.edu
|
| Organization name |
Institute for Stem Cell and Regenerative Medicine
|
| Street address |
850 Republican Street
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
|
| Samples (4)
|
|
| This SubSeries is part of SuperSeries: |
| GSE195555 |
dCas9 Fusion to Computer Designed PRC2 Inhibitor Reveals Functional TATA Box in Distal Promoter Region |
|
| Relations |
| BioProject |
PRJNA801854 |
Less...
More...