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The WRN protein accumulates at tRNA and G-rich regions during S phase in HEK293 cells.

ABSTRACT: Werner Syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The mutated gene responsible for WS encodes a DNA helicase/exonuclease protein believed to affect different aspects of transcription, replication, and DNA repair. In this study, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify regions of the genome bound by the WRN protein in Human Embryonic Kidney (HEK) 293 cells. We found 409 such genomic sites, one third of which corresponded to tRNA genes and another third of which were within RNA polymerase II transcribed genes that tended to contain guanine-rich sequences. We confirmed the specificity of several sites using WRN-depleted HEK293 cells by ChIP followed by quantitative PCR. Expression profiling of the HEK293 cells and RT-PCR analyses indicated that the transcription of the genes (including tRNAs) bound by WRN were not changed. Instead, we found a greater accumulation of WRN protein at these sites during S phase. These results suggest that the WRN protein accumulates at specific genomic sites during S phase of the cell cycle in HEK293 cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE68714 | GEO | 2016/05/01

SECONDARY ACCESSION(S): PRJNA283489

REPOSITORIES: GEO

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Project description:Our focus of research, the Werner protein, is one of disease-associated RecQ helicases. Its deficiency results in the Werner syndrome (WS), characterized by accelerated rate of mutation and the organism's inability to resolve a variety of genome-damaging events. Mutations in the WRN gene (also referred to as RECQ3 or RECQL2) don't appear to increase a person's susceptibility to DNA damage, and they alone do not cause the development of the disease. Instead they accelerate and exacerbate the disease that could develop even without this sensitizing mutation due to the damaging environmental agents. An important consequence of understanding the mechanism of a pathology is the ability to treat it more effectively, and even to prevent it in some cases. Aging research is extremely important considering the changing demographics worldwide, increased overall longevity and rise in elderly population. Understanding of the mechanisms that underlie aging and development of age-related disease due to genomic aberration can lead us to improved treatment and alleviated burden of age-related disease. Activity of proteins such as WRN suggests that the complex process of ageing is controlled by a conserved number of genes. However, mutations in a series of proteins do not suggest that their normal function is involved in promoting longevity. We are interested in understanding how normal function of this protein stalls senescence. In order to determine these mechanisms we study protein function in normal and diseased states with the use of proteomics. Resources that are currently available to us include modern analytical instrumentation, support from computational sciences, and access to rare biological samples. Our work is aimed at defining consistently associated proteins and post-translational modifications of WRN that are required or are causal for its function in human cells. This knowledge will increase our understanding of WS pathogenesis and provide insight into novel strategies to alter the progression of WS or associated diseases such as ASCVD, cancer, diabetes, and osteoporosis.

Project description:Werner syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS is believed to be involved in different aspects of transcription, replication, and/or DNA repair. We generated a mouse model with a deletion in the helicase domain of the murine WRN homologue that recapitulates most of the WS phenotypes including an abnormal hyaluronic acid excretion, higher reactive oxygen species (ROS) levels, increased genomic instability and cancer incidence resulting in a 10-15% decreased life span expectancy. In addition, WS patients and Wrn mutant mice show hallmarks of a metabolic syndrome including premature visceral obesity, hypertriglyceridemia, insulin-resistant diabetes type 2 and associated cardiovascular diseases. In this study, we compared the expression profile of liver tissues from 9 months old Wrn mutant and wild type animals. Gene set enrichment analysis of the microarray data indicated that Wrn mutant mice exhibited down-regulation of genes normally decreased in several transgenic mouse models of hepatoma and during caloric restriction. Wrn mutant mice also altered the expression of genes involved in inflammation as well as in glutathione and xenobiotic metabolisms. These results indicate that Wrn mutant mice respond to the observed oxidative stress by altering the necessary pathways to survive. Vitamin C supplementation rescued the life span expectancy of Wrn mutant mice and reversed several age-related abnormalities in adipose, cardiac, and liver tissues, genomic integrity and inflammatory status. Finally, gene set enrichment analyses revealed that vitamin C decreased genes normally up regulated in human WS fibroblasts and cancers and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Experiment Overall Design: Microarray analyses were performed on the liver tissues of 9 months old mice. Four independent biological replicates of this experiment (wild type vs Wrn mutant or wild type vs vitamin C treated mutant mice) were carried out with a dye swap on two replicates of each genotype.

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The WRN protein accumulates at tRNA and G-rich regions during S phase in HEK293 cells.

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