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| Status |
Public on Apr 29, 2024 |
| Title |
Mesoscale chromatin confinement facilitates target search of pioneer transcription factors in live cells [RNA-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
To dissect the molecular mechanisms of the transcription factor FOXA2 accessing nucleosomal targets and controlling cell fates, we combined live-cell single-molecule imaging, genome editing, chromatin binding, chromatin accessibility, and RNA sequence analysis. Using the hESC differentiation cellular system to anterior primitive streak (APS) and definitive endoderm (DE), we demonstrated by RNA-seq that FOXA2 is a lineage-determining transcription factor for DE formation and that its intrinsically disordered C-terminal domain (CTD) is essential for its normal function. Through ChIP-seq and ATAC-seq at multiple time points of exogenous expression, we also showed that the CTD is required for FOXA2 effectively accessing targets in closed chromatin and altering chromatin accessibility. Together with the single-molecule kinetics captured by fluorescence microscopy, this study uncovers new mechanistic insights on the activity of lineage-determining transcription factors in engaging chromatin and accessing nucleosomal targets.
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| Overall design |
We generated homozygous genome-edited human embryonic stem cell lines (H7 hESC) with deletion of the entire protein-coding region, or the CTD, or both the CTD and NTD at the FOXA2 locus. Wild-type and edited hESCs were differentiated to APS and DE for poly-A RNA-seq. We generated U2OS cell lines with inducible expression of FOXA1/2/3 and SOX2 full length and mutants with the Tet-on system. Cells were induced with Dox for 4 days for ChIP-seq and ATAC-seq.
RNA-Seq of poly-A enriched RNA in human H7 embryonic stem cells (undifferentiated, APS and DE stages), homozygous Halo-FOXA2 knock-in clone KI14, FOXA2 knock-out clones KO1, KO21, KO30, homozygous delta C-teminus FOXA2 clone dC11, and homozygous delta C- and N-termini clone dCdN10.
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| Contributor(s) |
Niu D, Cattoglio C, Deng W |
| Citation missing |
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| Submission date |
May 23, 2022 |
| Last update date |
Apr 30, 2024 |
| Contact name |
Wulan Deng |
| E-mail(s) |
denglabpku@outlook.com
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| Organization name |
Peking University
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| Department |
BIOPIC
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| Street address |
Beijing Haidian District Yiheyuan Road #5
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (11)
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| This SubSeries is part of SuperSeries: |
| GSE203650 |
Mesoscale chromatin confinement facilitates target search of pioneer transcription factors in live cells |
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| Relations |
| BioProject |
PRJNA841659 |
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