4C-seq to show the effects of insertion of STITCH into MYC+30kb and NEUROG2-65kb positions on the chromatin conformation in human iPSCs and differentiated neural progenitor cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB35286 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR3650280.fastq.gz | Fastqsanger.gz | |||
| ERR3650290.fastq.gz | Fastqsanger.gz | |||
| ERR3650281.fastq.gz | Fastqsanger.gz | |||
| ERR3650282.fastq.gz | Fastqsanger.gz | |||
| ERR3650283.fastq.gz | Fastqsanger.gz |
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Project description:We reconstituted arrays of CTCF binding sites (L1, L2, L3, L4, R1, R2, and R3) and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We first inserted STITCH into the 30-kb downstream from MYC (STITCH+30kb) in human iPSCs, after one of the two alleles are deleted. We made deletion of each CTCF array, L (delL) and R (delR) in STITCH+30kb. We also inserted STITCH into the 65-kb downstream of NEUROG2 in human iPSCs and integrated a transgene consisting of tetR-KRAB followed by DNA encoding the 2A peptide and the puromycin resistant gene with piggyBac transposition into the genome in the STITCH/NEUROG2 clone (NEUROG2/KRAB). We performed 4C-seq (Circular chromatin conformation capture assay followed by deep-sequencing) from the flanking regions of the STITCH+30kb insertion site as viewpoints to see how STITCH impacts on the chromatin conformation in STITCH+30kb, delL, delR, and the intact clone (Hap). We also performed 4C-seq from the NEUROG2 promoter region in the NEUROG2/KRAB iPSCs and the differentiated neural progenitor cells with and without DOX.
2020-05-01 | E-MTAB-8492 | biostudies-arrayexpress
Project description:We reconstituted arrays of CTCF binding sites (L1, L2, L3, L4, R1, R2, and R3) and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We inserted STITCH into the 65-kb downstream of NEUROG2 in human iPSCs and integrated a transgene consisting of tetR-KRAB followed by DNA encoding the 2A peptide and the puromycin resistant gene with piggyBac transposition into the genome in the STITCH/NEUROG2 clone (NEUROG2/KRAB). We performed 4C-seq (Circular chromatin conformation capture assay followed by deep-sequencing) from the viewpoint designed at R3 of the inserted STITCH in the differentiated neural progenitor cells with and without DOX.
2020-05-01 | E-MTAB-8957 | biostudies-arrayexpress
Project description:4C-seq to show the effects of insertion of STITCH into NEUROG2-65kb positions on the chromatin conformation in neural progenitor cells differentiated from human iPSCs.
| PRJEB37852 | ENA
Project description:The proneural NEUROG2 is essential for neuronal commitment, cell cycle exit and neuronal differentiation. Characterizing genes networks regulated downstream of NEUROG2 is therefore of prime importance. To identify NEUROG2 early response genes, we combined gain of function in the neural tube with a global detection of modified transcripts using microarrays. We included in our study a mutant form of NEUROG2 (NEUROG2AQ) that cannot bind DNA and cannot trigger neurogenesis. Using this approach, we identified 942 genes modified at the onset of NEUROG2 activation. The global analysis of functions regulated by NEUROG2 allowed unmasking its rapid impact on cell cycle control. We found that NEUROG2 specifically represses a subset of cyclins acting at the G1 and S phases of the cell cycle, thereby impeding S phase re-entry. This repression occurs before modification of p27kip1, indicating that the decision to leave the cell cycle precedes the activation of this Cyclin-dependant Kinase Inhibitor. Moreover, NEUROG2 down-regulates only one of the D-type cyclins, cyclinD1, and maintaining cyclinD1 blocks the ability of the proneural to trigger cell cycle exit, without altering its capacity to trigger neuronal differentiation. The fact that NEUROG2 represses a subset but not all cell cycle regulators indicates that cell cycle exit is not an indirect consequence of neuronal differentiation but is precisely controlled by NEUROG2. Altogether our findings indicate that NEUROG2, by specifically repressing G1 and S cyclins, allows committed neuronal precursors to perform their last mitosis but blocks their re-entry in the cell cycle, thus favouring cell cycle exit. Stage HH10-11 embryos (11 to 15 somites) were electroporated with a control vector (pGIG-GFP), a NEUROG2-expressing vector (pCIGNEUROG2-GFP), or a NEUROG2AQ-expressing vector (pCIGNEUROG2AQ-GFP). For each biological replicate, neural tubes from 20 embryos were pooled for GFP+ cells collection. GFP+ cells were collected 6h later using FACS sorting (Epics Altra HSS cell sorter, Toulouse Rio platform) and processed for RNA probe preparation and hybridization on Affymetrix microarrays. For each experimental condition, four biological replicates were processed.
2012-05-04 | E-GEOD-37782 | biostudies-arrayexpress
Project description:To identify and purify NEUROG2-expressing cells and trace their short-term lineage, we engineered two NEUROG2-mCherry knock-in human embryonic stem cell (hESC) lines. Transcriptomic profiling of NEUROG2:mCherry knock-in hESC-derived cerebral organoids revealed an enrichment of neurogenic, oligodendrocyte precursor cell and extracellular matrix-associated gene transcripts in mCherry-high cells. Conversely, only neurogenic gene transcripts were enriched in mCherry-high cells from Neurog2:mCherry knock-in mouse cortices.
2024-12-19 | GSE277092 | GEO
Project description:The proneural NEUROG2 is essential for neuronal commitment, cell cycle exit and neuronal differentiation. Characterizing genes networks regulated downstream of NEUROG2 is therefore of prime importance. To identify NEUROG2 early response genes, we combined gain of function in the neural tube with a global detection of modified transcripts using microarrays. We included in our study a mutant form of NEUROG2 (NEUROG2AQ) that cannot bind DNA and cannot trigger neurogenesis. Using this approach, we identified 942 genes modified at the onset of NEUROG2 activation. The global analysis of functions regulated by NEUROG2 allowed unmasking its rapid impact on cell cycle control. We found that NEUROG2 specifically represses a subset of cyclins acting at the G1 and S phases of the cell cycle, thereby impeding S phase re-entry. This repression occurs before modification of p27kip1, indicating that the decision to leave the cell cycle precedes the activation of this Cyclin-dependant Kinase Inhibitor. Moreover, NEUROG2 down-regulates only one of the D-type cyclins, cyclinD1, and maintaining cyclinD1 blocks the ability of the proneural to trigger cell cycle exit, without altering its capacity to trigger neuronal differentiation. The fact that NEUROG2 represses a subset but not all cell cycle regulators indicates that cell cycle exit is not an indirect consequence of neuronal differentiation but is precisely controlled by NEUROG2. Altogether our findings indicate that NEUROG2, by specifically repressing G1 and S cyclins, allows committed neuronal precursors to perform their last mitosis but blocks their re-entry in the cell cycle, thus favouring cell cycle exit.
2012-05-05 | GSE37782 | GEO
Project description:To generate an unbiased view of changes to the retinal gene network in Neurog2 retinal mutants as well as understanding the effects of Mash1 ectopic expression in a Neurog2 retinal mutant, we generated and compared the E12.5 transcriptomes from Neurog2 heterozygote, Neurog2 mutant and Neurog2 mutant;Mash1 knock-in mice. A pair of E12.5 retinas from each biologic replicate underwent FACS sorting to isolate GFP+ cells. GFP+ cells were used to produce libraries for high throughput sequencing (n = 3 biologic replicates/genotype). Reads were aligned with BWA and Bowtie programs to the mm10 genome. Aligned reads were then analyzed for differentially expressed transcripts using the CuffDiff program in the Galaxy online bioinformatics package (www.usegalaxy.org).
2018-08-30 | GSE111666 | GEO
Project description:Humans exemplify gyrencephalic species with folded cerebral cortices, contrasting with lissencephalic mammals such as mice. Here we investigated how proneural genes Neurog2 and Ascl1 control cortical folding by regulating neurogenic patterns. Cortical neural progenitor cells (NPCs) stratify into four pools (proneural negative, Neurog2+, Ascl1+, double+) that are distributed evenly in mouse cortices and modular in gyrencephalic macaque cortices and pseudo-folded human cerebral organoids. Each pool has distinct developmental potentials, transcriptomes, epigenomes, and gene regulatory networks. Neurog2-Ascl1 form a bistable toggle switch double+ NPCs to prevent lineage commitment observed in single+ NPCs. Neurog2 and Ascl1 act redundantly to control neurogenic timing, with NPCs precociously depleted in Neurog2-/-;Ascl1-/- cortices. Finally, selective killing of Neurog2/Ascl1 double+ NPCs using Neurog2/Ascl1 split-Cre;Rosa-DTA transgenics breaks neurogenic symmetry in mice by locally disrupting Notch signaling, leading to cortical folding. Our findings suggest that Neurog2/Ascl1 double+ NPCs are Notch-ligand expressing ‘niche’ cells that regulate neurogenic continuity and cortical gyrification.
2021-05-26 | GSE84120 | GEO
Project description:Neuroendocrine prostate cancer (NEPC) presents a formidable clinical challenge owing to its aggressive progression and resistance to conventional therapies. A key driver of NEPC is the overexpression of MYCN, a well-established oncogene associated with neuroendocrine tumors. However, efforts to directly inhibit the N-Myc protein encoded by this gene have resulted in limited success, thereby hindering therapeutic advancements. To overcome this obstacle, we conducted unbiased genome-wide screening using isogenic prostate cancer cell lines to identify the synthetic vulnerabilities of MYCN. Among the identified candidates, NEUROG2 emerged as a significant candidate. Neurog2 is a proneural transcription factor (PTF) known for its role in developmental processes and trans-differentiation of adult cells. Our findings demonstrate that Neurog2 depletion does not affect non-malignant cells but significantly suppresses the growth of MYCN-overexpressing cells and tumors in orthotopic NEPC models. Furthermore, our observations indicate that Neurog2-driven modulation of PTFs potentially contribute to NEPC development. Thus, targeting Neurog2 holds promise as an effective therapeutic strategy for MYCN-overexpressing NEPC.
2025-04-29 | GSE255592 | GEO
Project description:Neuroendocrine prostate cancer (NEPC) presents a formidable clinical challenge owing to its aggressive progression and resistance to conventional therapies. A key driver of NEPC is the overexpression of MYCN, a well-established oncogene associated with neuroendocrine tumors. However, efforts to directly inhibit the N-Myc protein encoded by this gene have resulted in limited success, thereby hindering therapeutic advancements. To overcome this obstacle, we conducted unbiased genome-wide screening using isogenic prostate cancer cell lines to identify the synthetic vulnerabilities of MYCN. Among the identified candidates, NEUROG2 emerged as a significant candidate. Neurog2 is a proneural transcription factor (PTF) known for its role in developmental processes and trans-differentiation of adult cells. Our findings demonstrate that Neurog2 depletion does not affect non-malignant cells but significantly suppresses the growth of MYCN-overexpressing cells and tumors in orthotopic NEPC models. Furthermore, our observations indicate that Neurog2-driven modulation of PTFs potentially contribute to NEPC development. Thus, targeting Neurog2 holds promise as an effective therapeutic strategy for MYCN-overexpressing NEPC.
2025-04-29 | GSE255591 | GEO