PPM1D Mutations Silence NAPRT Gene Expression and Confer NAMPT Inhibitor Sensitivity in Glioma
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ABSTRACT:
PROVIDER: PRJNA554122 | ENA |
REPOSITORIES: ENA
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Project description:NAPRT-mediated deamidated biosynthesis of nicotinamide adenine dinucleotide (NAD), an essential electron carrier for hundreds of biochemical reactions in all living cells, mediates the diet-microbe-host NAD metabolic interaction in mice. To study the function of this metabolic pathway in vivo, we recently generated a mutant mouse strain (NAPRT KO) in which a 21-nt fragment that encodes 7 amino acids in the catalytic domain of NAPRT was deleted in all cells using the CRISPR/Cas9-mediated gene editing technology. Our results showed that NAPRT is highly expressed in the gut epithelial cells, where it functions to maintain the cellular NAD pool for an efficient response to stress-induced acute NAD depletion. Consistently, NAPRT deficiency impairs DNA repair, sensitizes mice to AOM-induced gut DNA damage. Here we profiled transcriptomic changes in the colon of WT and NAPRT KO mice at different time points after injection of AOM.
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Project description:Human nontransformed retinal pigment epithelia RPE-PPM1D-T2 cells carrying a truncating mutation in exon 6 of the PPM1D were exposed to ionising radiation (3 Gy) and subsequently were grown in semisolid media for 8 weeks. Six spheroid clones (RPE-PPM1D-T2-SA clones 1-6) were recovered and then were cultivated in adherent conditions. DNA was isolated from asynchronically growing parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA-1 to 6 cells and was subjected to whole exome sequencing. DNA sequencing libraries were prepared using KAPA EvoPlus Kit (Roche) and were sequenced on the NovaSeq 6000 system using NovaSeq S1 Reagent Kit v1.5, 200 cycles (Illumina) with mean coverage >35 DNA samples, respectively. DNA fastq files were mapped to the hg19 reference using Novoalign (novoalign_2.08.03). PCR duplicates were removed from the BAM files using Picard Tools (picard-tools 1.129), and variant calling was performed using GATK HaplotypeCaller (3.8). Copy number variations (CNV) were analyzed using CNVkit version 0.7.4. Areas with median coverage >20 were included in the analysis. RNA fastq files were mapped to the hg19 reference using STAR (STAR-2.5.2b). The PCR duplicates were removed using Picard Tools (picard-tools 1.129).
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