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Bloom syndrome protein modulates protein-coding gene and miRNA expression as a function of G4 DNA content

ABSTRACT: Bloom syndrome is a rare autosomal recessive genetic instability and cancer predisposition syndrome caused by loss of function mutations in the BLM RECQ helicase gene. To ask if some of the distinctive pathological features of Bloom syndrome might reflect altered gene expression, we analyzed global mRNA and miRNA expression in fibroblasts from 16 patients and 15 matched normal controls, and in control primary diploid fibroblasts depleted of the BLM protein. We document significant differential expression of both protein-coding genes and miRNAs with well-characterized cancer associations in BLM-deficient cells. Differences in expression correlated significantly with G4 motifs, which are associated with potential to form G-quadruplex structures. These results indicate that BLM helicase may modulate gene expression by regulating the in vivo stability of G-quadruplex structures, and identify sets of genes and miRNAs whose expression, when altered, may drive the pathogenesis of Bloom syndrome and associated cancers.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE54502 | GEO | 2014/06/12

SECONDARY ACCESSION(S): PRJNA236736

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.

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Bloom syndrome protein modulates protein-coding gene and miRNA expression as a function of G4 DNA content

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