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| Status |
Public on Aug 04, 2021 |
| Title |
Genome wide profiling of NEUROG3 occupancy in human pancreatic endocrine progenitors [CUT&RUN] |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Despite this critical role in islet cell development, the precise function and downstream genetic programs regulated directedly by NEUROG3 remain elusive. We therefore mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (iPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. To this aim, we generated a novel hiPSC line (NEUROG3-HA-P2A-Venus), where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique, an alternative method to CHIP-seq allowing transcription factor profiling from a low cell number, to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) differentiated from this hiPSC line. To optimize the stringency and relevance of NEUROG3 binding sites, we focused our analysis on regions identified both with HA and NEUROG3 antibodies and integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs and their NEUROG3-dependence. Mapping of NEUROG3 genome occupancy in PEPs uncovers an unexpectedly broad, direct control of the endocrine gene regulatory network (GRN) and raises novel hypotheses on how this master regulator controls islet and beta cell differentiation.
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| Overall design |
The hiPS SB AD3.1 cell line (Van de Bunt et al, Islets, 8(3), 83-95 (2016) has been edited using the CRISPR/Cas9 technology to C-terminally tag NGN3 with a 3HA epitope and P2A-3NLS-Venus. The generated NEUROG3-HA-P2A-Venus#31 clone was differentiated to pancreatic endocrine progenitors (PEP, day 13, end of stage 5) according to the protocol described by Petersen et al, Stem cell reports:1-37 (2017). Venus-positive cells were purified at PEP stage (day 13) by FACS and NEUROG3-bound genes isolated by CUT&RUN (Hainer and Fazzio, Current Protocols 126 (1):e85 (2019), using antibodies recognizing either NEUROG3 or the 3HA tag. CUT&RUN with anti-H3K27me3 and H3K4me3 antibodies was done to follow chromatin state. Donkey anti sheep and rabbit-anti-mouse IgG were used as negative controls.
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| Contributor(s) |
Schreiber V, Ye T, Gradwohl G, Jiménez S, Mercier R, Jost B |
| Citation(s) |
34352411 |
| |
| Submission date |
Apr 12, 2021 |
| Last update date |
Sep 08, 2021 |
| Contact name |
Valerie SCHREIBER |
| E-mail(s) |
schreibv@igbmc.fr
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| Organization name |
IGBMC
|
| Street address |
1 rue Laurent Fries
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| City |
Illkirch |
| ZIP/Postal code |
67400 |
| Country |
France |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (6)
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| This SubSeries is part of SuperSeries: |
| GSE179264 |
Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network |
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| Relations |
| BioProject |
PRJNA721460 |
| SRA |
SRP314556 |
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