A disease-associated mutation that weakens ZAP70 autoinhibition enhances responses to weak and self-ligands.
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ABSTRACT:
SUBMITTER: Shen L
PROVIDER: S-EPMC8009134 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals.
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Project description:BackgroundPrimary immunodeficiency is a life-threatening genetic disease that appeared to have an increased incidence in Manitoba Mennonites. Determining the genetic basis of this immunodeficiency was an essential step for providing early and appropriate medical intervention.MethodsInitially, DNA from probands affected with primary immunodeficiency and their family members was assessed for linkage to genes previously associated with immunodeficiency. Candidate genes were sequenced to identify the causative mutation. The frequency of the mutation among first and second degree relatives, as well as apparently unrelated community members was analyzed using a PCR-based assay.ResultsA previously described c.1624-11G>A mutation in ZAP70 was identified as the causative mutation in all affected probands that were analyzed. Among 125 study participants of Mennonite descent, 79 genotyped as normal, 39 were carriers and seven were affected. None of 115 non-Mennonite random individuals carried the mutation, whereas one of ten random DNA samples from individuals who self-identified as Mennonite was a carrier.ConclusionsIn collaboration with the target community, we have developed a robust screening test for determining ZAP70 genotype. Early identification of affected individuals has provided an opportunity for timely clinical intervention, while carrier identification has allowed for genetic counselling of at risk couples.
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Project description:Purpose: Next-generation sequencing (NSG) has revolutionized system-based analysis of cellular pathways. The goals of this study are to compare Phf6R274X mice BM LSK cells transcription profiling (RNA-seq) Method: BM LSK cells mRNA profiles of Phf6R274X mice and control were generated by deep sequencing, in triplicate, using illumine GAIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Using an optimized data analysis workflow, we identified 613 genes up-regulated and 133 genes down-regulated (P < 0.05) in the BM LSK cells from Phf6R274X and control mice. Altered expression of 7 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes may contribute to analyze PHF6R274X mutation function. Conclusion: Our study represents the first detailed analysis of BM LSK cell transcriptomes with PHF6R274X mutation. Our results suggested that Phf6R274X mutation enhanced the regeneration capacity of hematopoietic cells mainly by promoting the activity of cell proliferation and differentiation-related signaling pathways.
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