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Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing

ABSTRACT: Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing

PROVIDER: PRJNA379274 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5345340_1.fastq.gz	Fastqsanger.gz
	SRR5345340_2.fastq.gz	Fastqsanger.gz
	SRR5345350_1.fastq.gz	Fastqsanger.gz
	SRR5345350_2.fastq.gz	Fastqsanger.gz
	SRR5345331_1.fastq.gz	Fastqsanger.gz

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Similar Datasets

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing [Exome-Seq]

Project description:Purpose: DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Methods: Whole-exome sequencing of extracted DNA was performed to confirm DNMT3A mutations. Library construction, exon capture, and sequencing was performed by Otogenetics (Atlanta, GA, USA). In brief, paired-end libraries were generated using the Illumina TruSeq DNA sample preparation kit. Exons were enriched using the Agilent Human All Exon V5 (51 Mb) capture system. Illumina HiSeq2500 was used for sequencing with a paired-end sequencing length of 100-125 bp and approximately 70 million reads per sample. Results: the DNMT3A mutations induced by K562 cells were transfected with a plasmid encoding Cas9, a CRISPR guide-RNA (gRNA) targeting the DNMT3A gene, and green fluorescent protein (GFP) followed by single-cell sorting. GFP-positive single cell clones were genotyped to confirm the presence of DNMT3A mutations. MT2-MT5 were verified by analysis of whole-exome sequencing data, and WTblk and WT1 were again confirmed to have no mutations in the DNMT3A gene. Conclusions: CRISPR/Cas9 gene editing allowed the generation of DNMT3A-mutated K562 cells that may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.

2018-01-09 | GSE96625 | GEO

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing [RNA-Seq]

Project description:Purpose: DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Methods: mRNA profiles of wild-type (WT) and DNMT3A mutated k562 cell lines were generated by deep sequencing, using Illumina HiSeq2500. Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality at the ends. Remaining sequence reads were then aligned to the human reference genome (hg19) using Tophat2. Gene read counts were measured using FeatureCounts and FPKM values were calculated with cufflinks. edgeR was used to identify differentially expressed genes between conditions, and topGO was used for annotation (Alexa, Rahnenfuhrer, and Lengauer, 2006). Sample comparison for differential gene expression was as follows: WTblk and WT1 versus MT2, MT3, MT4, and MT5. Gene enrichment set analysis (GSEA) was conducted with KEGG, Biocarta, and Reactome pathway datasets (Subramanian et al., 2005). Results: DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. Conclusions: CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.

2018-01-09 | GSE96634 | GEO

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing [MeDIP-Seq]

Project description:Purpose: DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Methods: Methylated DNA immunoprecipitation (MeDIP) was used to detect regional differences in DNA methylation between DNMT3A WT and MT clones. DNA extraction of WTblk, WT1, MT1, MT2, and MT4 cell lines was performed as previously described. 5-mC profiling, fragmentation, library preparation and sequencing were performed by Otogenetics (Atlanta, GA, USA). In brief, paired-end libraries were generated using the Illumina TruSeq DNA sample preparation kit. Illumina HiSeq2500 was used for sequencing with a paired-end sequencing length of 100-125 bp and approximately 40 million reads per sample. Results: We attempted to identify differentially methylated genes based on DNMT3A mutation status and found very few regions of differential methylation between WT and MT cell lines (Figure 3C), which did not exceed the number of regions expected to exhibit differential methylation by chance alone. These data suggest that regional methylation patterns do not differ between DNMT3A MT and WT cell lines in our gene-edited K562 cells. Conclusions: CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.

2018-01-09 | GSE96577 | GEO

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing.

Project description:DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.

| S-EPMC6728079 | biostudies-literature

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing

Project description:This SuperSeries is composed of the SubSeries listed below.

2018-01-09 | GSE96635 | GEO

Exome-seq of Jak2VF erythroid progenitors cells transduced with Dnmt3a-Cas9-GFP lentivirus vs. emp-Cas9-GFP controls 17 weeks post transplantation

Project description:Exome sequencing of erythroid progenitors from Dnmt3a-Cas9-GFP+/- vs. emp-Cas9-GFP+/- controls.

2018-10-29 | E-MTAB-7243 | biostudies-arrayexpress

ATAC-seq of Jak2VF LSK cells transduced with Dnmt3a-Cas9-GFP lentivirus vs. emp-Cas9-GFP controls 8 weeks post transplantation

Project description:Determination of chromatin accessibility in hematopoietic stem cell progenitors (LSK) of Jak2VF-emp-Cas9 and Jak2VF-Dnmt3a-Cas9 8 weeks post transplantation.

2018-10-29 | E-MTAB-6649 | biostudies-arrayexpress

RNA-seq of Jak2VF LSK cells transduced with Dnmt3a-Cas9-GFP lentivirus vs. emp-Cas9-GFP controls 8 weeks post transplantation

Project description:Determination of differentially expressed genes from in vivo hematopoietic stem cell progenitors (LSK) in Jak2VF-emp-Cas9 and Jak2VF-Dnmt3a-Cas9 8 weeks post transplantation.

2018-10-29 | E-MTAB-6644 | biostudies-arrayexpress

RNA-seq of Jak2VF LSK cells transduced with Dnmt3a-Cas9-GFP lentivirus vs. emp-Cas9-GFP controls one week and five weeks post methylcellulose plating

Project description:Determination of differentially expressed genes in culture-derived hematopoietic stem cell progenitors (LSK) between Jak2VF-emp-Cas9 and Jak2VF-Dnmt3a-cas9.

2018-10-29 | E-MTAB-6635 | biostudies-arrayexpress

Systematic profiling of DNMT3A variants reveals protein instability mediated by the DCAF8 E3 ubiquitin ligase adaptor

Project description:Clonal hematopoiesis is a prevalent age-related condition associated with greatly increased risk of hematologic malignancies. Mutations in DNA methyltransferase 3A (DNMT3A) are the most common drivers of this state and contribute to multiple types of hematologic malignancy. DNMT3A variants occur across the gene and some are associated with particular disease states, but the functional relevance and mechanisms of pathogenesis of the majority of the mutations is unknown. Here, we systematically investigated the methyltransferase activity and protein stability of 253 disease-associated DNMT3A mutations, finding that 74% were loss-of-function mutations. Nearly half of these variants exhibited reduced protein stability and, as a class, correlated with greater clonal expansion and AML development. To identify the mechanisms underlying the instability, we conducted a CRISPR screen and uncovered regulated destruction of DNMT3A mediated by the DCAF8 E3 ubiquitin ligase adaptor. We establish a new paradigm to classify novel variants that has prognostic and therapeutic significance for patients with hematologic disease.

2021-06-24 | GSE178798 | GEO

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