The CRISPR-Cas9 crATIC HeLa transcriptome: Characterization of a novel cellular model of ATIC deficiency and ZMP accumulation

Abstract

In de novo purine biosynthesis (DNPS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (EC 2.1.2.3)/inosine monophosphate cyclohydrolase (EC 3.5.4.10) (ATIC) catalyzes the last two reactions of the pathway: conversion of 5-aminoimidazole-4-carboxamide ribonucleotide [aka Z -nucleotide monophosphate (ZMP)] to 5-formamido-4-imidazolecarboxamide ribonucleotide (FAICAR) then to inosine monophosphate (IMP). Mutations in ATIC cause an untreatable and devastating inborn error of metabolism in humans. ZMP is an adenosine monophosphate (AMP) mimetic and a known activator of AMP-activated protein kinase (AMPK). Recently, a HeLa cell line null mutant for ATIC was constructed via CRISPR-Cas9 mutagenesis. This mutant, crATIC, accumulates ZMP during purine starvation. Given that the mutant can accumulate ZMP in the absence of treatment with exogenous compounds, crATIC is likely an important cellular model of DNPS inactivation and ZMP accumulation. In the current study, we characterize the crATIC transcriptome versus the HeLa transcriptome in purine-supplemented and purine-depleted growth conditions. We report and discuss transcriptome changes with particular relevance to Alzheimer's disease and in genes relevant to lipid and fatty acid synthesis, neurodevelopment, embryogenesis, cell cycle maintenance and progression, extracellular matrix, immune function, TGFβ and other cellular processes.

Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity

“…Interestingly, a CD36-PPARγ axis exists, which regulates fatty acid storage, triglyceride synthesis, glucose uptake, and fatty acid metabolism [74]. It is unclear as why crADSL, crATIC, and crGART would all be robustly expressing these genes, however the results reported here and previously [10,11] suggest a link between DNPS and the CD36-PPARγ axis.…”

“…Next, we assessed whether the GART substrate 5-PRA accumulates in the crGART cell line. We demonstrated previously that crADSL [10] and crATIC [11] cells accumulate substrate when cultured in adenine free media. crGART cells were cultured as previously described and metabolites extracted.…”

“…To get a better sense of which DEGs are due to DNPS deficiency rather than intermediate accumulation, we identified the significant DEGs unique to the crGART vs HeLa comparison. There were 1282 genes that changed significantly in the crGART vs HeLa comparison but did not change in the crADSL vs HeLa [10] and crATIC vs HeLa comparisons [11]. The list of genes was used to query PANTHER via the Gene Ontology web portal (www.geneontology.org).…”

“…HPLC-EC analysis was performed as previously described ( [10,14] and [11]). No DNPS intermediates were detected in adenine-supplemented or -starved cells.…”

“…The ADSL and ATIC mutants (crADSL and crATIC) accumulate DNPS pathway intermediates when cultured in purine free media. These intermediates, SAICAR and ZMP respectively, are regulators of transcription, and we have presented evidence for transcriptional regulation via DNPS deficiency and intermediate accumulation ([10] and [11]). We were unable to detect intermediate accumulation in the GART mutant (crGART).…”