|
| Status |
Public on Jan 26, 2022 |
| Title |
CRISPR activation screen to identify X-chromosomal Xist activators in male embryonic stem cells |
| Organisms |
Mus musculus; Synthetic plasmid |
| Experiment type |
Other
|
| Summary |
As the master regulator of X-chromosome inactivation (XCI), the Xist RNA is expressed nearly ubiquitously in female mice. Xist is only absent in the germ line and at the pluripotent state. Xist is generally assumed to be expressed “by default” in females, while being actively repressed in the few tissues where it is silent. Whether activating mechanisms also contribute remained largely unknown. Through a pooled CRISPR screen we identify the GATA family of transcription factors as potent direct activators of Xist. We describe a GATA-responsive regulatory element (RE79), located ~100 kb upstream of the Xist promoter. In cell lines derived from the two extraembryonic lineages, XEN and TS cells, where imprinted Xist expression is maintained, RE79 is bound by different sets of GATA factors expressed in those tissues. Here we use a CRISPR activation screen targeting X-chromosomal genes on differentiating XO mouse embryonic stem cells to identify putative Xist activators.
|
| |
|
| Overall design |
A CRISPRa-SunTag screen for Xist was performed in differentiating mESCs (E14-STN) carrying a deletion of the Tsix-promoter to sensitize the male cell line for Xist upregulation. The library contained 8773 gDNAs targeting 757 X-chromosomal genes and 23 autosomal controls, as well as 200 non-targeting guides. After 2 days of differentiation via LIF-withdrawal, the cells were stained for Xist RNA using a FlowFISH protocol and the top 15% bins of Xist-expression was sorted.
|
| |
|
| Contributor(s) |
Ravid Lustig L, Kumar AS, Schwämmle T, Noviello G, Dunkel I, Weigert R, Pacini G, Buschow R, Ghauri A, Stötzel M, Meissner A, Schulz EG |
| Citation(s) |
37932452 |
| |
| Submission date |
Jan 19, 2022 |
| Last update date |
Nov 16, 2023 |
| Contact name |
Till Schwämmle |
| E-mail(s) |
Till.schwaemmle@molgen.mpg.de
|
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Department |
Otto-Warburg-Laboratory
|
| Lab |
Schulz Lab
|
| Street address |
Ihnestrasse 63
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platforms (2) |
| GPL21103 |
Illumina HiSeq 4000 (Mus musculus) |
| GPL30924 |
Illumina HiSeq 4000 (Synthetic plasmid) |
|
| Samples (7)
|
|
| This SubSeries is part of SuperSeries: |
| GSE194018 |
A role for the GATA transcription factor family in X-chromosome inactivation as direct potent Xist activators |
|
| Relations |
| BioProject |
PRJNA798744 |
Less...
More...