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| Status |
Public on Nov 09, 2020 |
| Title |
DOT1L-interacting protein AF10 (MLLT10) is a barrier to somatic cell reprogramming |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Epigenetic regulators have important roles during embryonic development as well as somatic cell reprogramming. We previously showed that inhibition of DOT1L, the histone H3 lysine 79 methyltransferase, increases the efficiency of reprogramming via regulation of lineage specific genes. However, the role of DOT1L-interacting proteins in reprogramming remains unknown. In this study, DOT1L interactors were identified using the BioID method in which a promiscuous BirA ligase (BirA*) was employed to biotinylate DOT1L-proximal proteins. The resulting interaction candidates were investigated for their effects on reprogramming. Candidate genes were knocked-down in human fibroblasts via shRNAs followed by reprogramming. Our results indicated that knock-down of AF10 (MLLT10), significantly increased the iPSC generation efficiency, suggesting that it acts as a barrier to reprogramming similar to DOT1L. This finding was verified by CRISPR/Cas9 mediated knockout of AF10. Overexpression of AF10 reversed the effect of AF10 knockout and decreased reprogramming efficiency. To determine how AF10 silencing changes the gene expression, RNA-sequencing was performed on human fibroblasts undergoing reprogramming. AF10 suppression resulted in downregulation of fibroblast-specific genes and accelerated the activation of pluripotency-related genes. Our analysis also demonstrated that silencing of AF10 results in gene expression changes similar to DOT1L inhibition during reprogramming. Taken together, this study uncovered AF10 as a novel barrier to reprogramming and contributed to our understanding of epigenetic mechanisms that maintain cell identity.
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| Overall design |
Examination of CRISPR-Cas9 mediated AF10 knockout effect on gene expression profile of dH1f cells 6 days after OSKM transduction. 3 biological replicates from each of reprogramming fibroblasts that express control sgRNA or sgRNA targeting AF10 were prepared.
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| Contributor(s) |
Uğurlu-Çimen D, Sevinç K, Önder T |
| Citation(s) |
34215314 |
| |
| Submission date |
Nov 08, 2020 |
| Last update date |
Jul 14, 2021 |
| Contact name |
Kenan Sevinç |
| E-mail(s) |
ksevinc15@ku.edu.tr
|
| Organization name |
Koç University
|
| Department |
School of Medicine
|
| Street address |
Rumelifeneri Yolu, Sariyer
|
| City |
Istanbul |
| ZIP/Postal code |
34450 |
| Country |
Turkey |
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| Platforms (1) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA675353 |
| SRA |
SRP291572 |
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