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Transcriptome profiling of the cytarabine response in U2OS cells overexpressing DNMT3A R882C isoform (compared to DNMT3A wt and empty vector control)

ABSTRACT: Transcriptome profiling of the cytarabine response in U2OS cells overexpressing DNMT3A R882C isoform (compared to DNMT3A wt and empty vector control)

PROVIDER: PRJNA644377 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12158000_1.fastq.gz	Fastqsanger.gz
	SRR12158000_2.fastq.gz	Fastqsanger.gz
	SRR12158001_1.fastq.gz	Fastqsanger.gz
	SRR12158001_2.fastq.gz	Fastqsanger.gz
	SRR12158002_1.fastq.gz	Fastqsanger.gz

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Transcriptome profiling of the cytarabine response in U2OS cells overexpressing DNMT3A R882C isoform (compared to DNMT3A wt and empty vector control)

Project description:Purpose: The goal of this study was to investigate the effect of the cytarabine treatment on the transcriptional profiles of U2OS cells retrovirally expressing DNMT3A R882C isoform, compared to DNMT3A WT and empty vector controls Methods: U2OS DNMT3A WT, R882C, and empty vector cells were treated with 10uM cytarabine or vehicle control for 24 hours. Biological triplicates were generated, ie each replicate was performed on different day using a new batch of cells. Total RNA was collected and QC'd; mRNA was enriched using Poly-T oligo attached to magnetic beads and Illumina libraries were prepared and sequenced on Novaseq6000 (PE 150). Library construction and sequencing were performed by Novogene Corporation, Inc according to standard workflow. Results: Using a custom built pipeline in PartekFlow, the reads were aligned to human (build hg38) genome with the STAR aligner 2.6.1d and low quality alignments were filtered. About 25 million sequence reads per sample were mapped to the human genome. We identified 5714 transcripts/sample and low expression genes with counts less than 10 were filtered and the raw read counts were normalized using TMM model with an offset of 1 to avoid zero counts. Unsupervised hierarchical clustering was performed on the top 2000 genes with highest variance Conclusions: The study identifies gene networks involved in cytarabine response that are deregulated by presence of mutant DNMT3A.

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Project description:Despite the impact of DNMT3A mutation in acute myeloid leukemia has been emphasized, the precise molecular mechanisms in leukemogenesis are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant hematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are down-regulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells. DNMT3A mutant also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), leading to transcriptional silencing of PU.1. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. Taken together, our results highlight the functional role of DNMT3A mutation, forming the basis for leukemia development. GFP-labeled empty vector, DNMT3A wild-type (WT), R882H-transduced LSK cells derived from transplanted mice were utilized for compared the expression profiles (3 sorted empty vector-transduced LSK cells, 3 sorted DNMT3A WT-transduced LSK cells, and 3 sorted DNMT3A R882H-transduced LSK cells. Total RNA was extracted by TaKaRa NucleoSpin RNA XS according to the manufacturerâs protocol. Amplification and biotin labeling of fragmented cDNA was carried out from 3.67 ng of total RNA by using NuGen Ovation Pico WTA System V2 (NuGEN) and SureTag Complete DNA Labeling Kit (Agilent). Each 2 Î¼g of cDNA were hybridized to the Agilent SurePrint G3 Mouse Gene Expression 8x60K (Agilent) using Gene Expression Hybridization Kit (Agilent). After scanning, the signal intensity for each feature was measured by Agilent Feature Extraction (Agilent).

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