 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 26, 2024 |
| Title |
Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput |
| Organism |
Homo sapiens |
| Experiment type |
Other
Expression profiling by high throughput sequencing
|
| Summary |
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe “signaling-to-transcription network” mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
|
| |
|
| Overall design |
Various phosphosite mutants were introduced into TPR Jurkat cells via in vitro transcription of sgRNA and ABE8e (base editor) complex. To produce purely edited cell populations, single cell clones were isolated from the corresponding bulk edited cell populations by diluting 0.8 cells per well in 96-well plates. Once isolated, these cells were grown until confluence, then individually genotyped through extraction of its gDNA and PCR amplification of the edited genomic region of interest. Once validated, 4-8 single cell clones were then mixed together to avoid clone-specific effects. Transcriptional profiling was completed via single-cell RNA on five selected phosphosite mutants.
|
| |
|
| Contributor(s) |
Kennedy P, Myers S, Alarcón S |
| Citation(s) |
38684783 |
| |
| Submission date |
Sep 27, 2023 |
| Last update date |
May 28, 2024 |
| Contact name |
Samuel Myers |
| Organization name |
La Jolla Institute for Immunology
|
| Lab |
Myers
|
| Street address |
9420 Athena Circle
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
|
| Samples (10)
|
| GSM7808188 |
HEK3, scRNA gene expression |
| GSM7808189 |
LCP2splice, scRNA cell surface protein barcode |
| GSM7808190 |
LCP2splice, scRNA gene expression |
| GSM7808191 |
MAPK1_Y187C, scRNA cell surface protein barcode |
| GSM7808192 |
MAPK1_Y187C, scRNA gene expression |
| GSM7808193 |
PHLPP1_S118P, scRNA cell surface protein barcode |
| GSM7808194 |
PHLPP1_S118P, scRNA gene expression |
|
| Relations |
| BioProject |
PRJNA1021559 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE244164_Samples_hashtags_barcodesequences.txt.gz |
360 b |
(ftp)(http) |
TXT |
| GSE244164_barcodes.tsv.gz |
2.4 Mb |
(ftp)(http) |
TSV |
| GSE244164_feature_reference.csv.gz |
2.0 Kb |
(ftp)(http) |
CSV |
| GSE244164_features.tsv.gz |
288.6 Kb |
(ftp)(http) |
TSV |
| GSE244164_matrix.mtx.gz |
614.0 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
Less...
More...