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Series GSE19205 Query DataSets for GSE19205
Status Public on Dec 10, 2009
Title Altered gene expression in Werner & Bloom syndromes is associated with sequences having G-quadruplex forming potential
Organism Homo sapiens
Experiment type Expression profiling by array
Summary 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.
 
Overall design 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 Earleā€™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.
 
Contributor(s) Johnson JE, Cao K, Ryvkin P, Wang L, Johnson FB
Citation(s) 19966276
Submission date Nov 25, 2009
Last update date Dec 06, 2018
Contact name Jay Johnson
E-mail(s) johe@mail.med.upenn.edu
Phone 215-573-6529
Organization name University of Pennsylvania
Department Pathology and Laboratory Medicine
Lab F. Brad Johnson
Street address 422 Curie Blvd
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
 
Platforms (1)
GPL571 [HG-U133A_2] Affymetrix Human Genome U133A 2.0 Array
Samples (12)
GSM476001 Normal_fibroblast_AG06310
GSM476002 Normal_fibroblast_AG04054
GSM476003 Normal_fibroblast_AG09975
Relations
BioProject PRJNA120813

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE19205_RAW.tar 21.3 Mb (http)(custom) TAR (of CEL)
Processed data included within Sample table

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