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Altered gene expression in Werner & Bloom syndromes is associated with sequences having G-quadruplex forming potential

ABSTRACT: The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE19205 | GEO | 2009/12/10

SECONDARY ACCESSION(S): PRJNA120813

REPOSITORIES: GEO

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Project description:The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA. Cell culture conditions and media Human fibroblast cell strains (WS: AG05229, AG12795, AG12797; BS: GM02932, GM03402, GM16891; RTS: AG18371, AG18375, AG05013; Normal/Wild-type: AG04054, AG06310, AG09975) were obtained from the Coriell Repository (Camden, NJ), from donors matched for gender and of similar ages, and were at similar passage levels. Cells were cultured in MEM supplemented with Earle’s salts, 20% fetal bovine serum, 1x penicillin/streptomycin, and 1x fungizone in 3% O2 at 37oC and harvested for RNA extraction during active growth and at ~ 80% confluence. GeneChip microarray expression Total RNA from the 12 fibroblast cell strains was isolated by extraction with TRIzol (Invitrogen) and purified using the RNeasy system (Qiagen). Total RNA was amplified by in vitro transcription using the Ovation RNA Amplification System V2 (NuGen). The resultant cDNA was fragmented and labeled using the FL-Ovation cDNA Biotin Module V2 (NuGen), and then purified using QIAquick columns (Qiagen), as specified by the Ovation System manual. Labeled probe was hybridized to Affymetrix U133A 2.0 GeneChips, and ultimately scanned using an Axon GenePix array scanner. Statistical analysis of microarray expression experiment The output files were normalized by Robust Multiarray Average (RMA), using the R package GCRMA and gene expression levels were log2-transformed.

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.

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Altered gene expression in Werner & Bloom syndromes is associated with sequences having G-quadruplex forming potential

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