Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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. 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Jay Johnson

PROVIDER: E-GEOD-19205 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

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

Johnson Jay E JE Cao Kajia K Ryvkin Paul P Wang Li-San LS Johnson F Brad FB

Nucleic acids research 20091204 4

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 Saccharomyces 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 sgs1Delta mutants, sugg ...[more]

PMID: 19966276

Similar Datasets

Project description:Soft tissue sarcomas are a diverse set of fatal human tumors where few agents have demonstrable clinical efficacy, with the standard therapeutic combination of doxorubicin and ifosfamide showing only a 25-30% response rate in large multi-institutional trials. Although liposarcomas are the most common histological form of adult soft tissue sarcomas, research in this area is severely hampered by the lack of experimentally tractable in vitro model systems. To this end, here we describe a novel in vitro model for human pleomorphic liposarcoma. The cell line (LS2) is derived from a pleomorphic liposarcoma that utilizes Alternative Lengthening of Telomeres (ALT) mechanism of telomere maintenance, which may be particularly important in modulating the response of this tumor type to DNA damaging agents. We present detailed baseline molecular and genomic data, including genome wide copy number and transcriptome profiles, for this model compared to its parental tumor and a panel of liposarcomas covering multiple histologies. The model has retained essentially all of the detectable alterations in copy number that are seen in the parental tumor, and shows molecular karyotypic and expression profiles consistent with pleomorphic liposarcomas. We also demonstrate the utility of this model, together with two additional human liposarcoma cell lines, to investigate the relationship between topoisomerase 2A expression and the sensitivity of ALT-positive liposarcomas to doxorubicin. This model, together with its associated baseline data, provide a powerful new tool to develop treatments for this clinically poorly-tractable tumor, and to investigate the contribution that ALT makes to modulating sensitivity to DNA damaging chemotherapeutic agents such as doxorubicin. Affymetrix U133 2.0 expression analyses were performed for 30 liposarcoma tumors, including 2 recurrences of 2 tumors. In addition, analyses were performed for three replicates of a tumor-derived cell line, LS2, as well as the tumor from which it was derived (5228). Finally, the Affymetrix Human Genome 50K DNA Mapping Array was used to determine copy number profiles for two Passages of LS2, as well as tumor 5228.

Project description:OBJECTIVE: MEIS1, a HOX cofactor, collaborates with multiple HOX and NUP98-HOX fusion proteins to accelerate the onset of acute myeloid leukemia (AML) through largely unknown molecular mechanisms. MATERIALS AND METHODS: To further resolve these mechanisms, we conducted a structure-function analysis of MEIS1 and gene-expression profiling, in the context of NUP98-HOXD13 (ND13) leukemogenesis. RESULTS: We show, in a murine bone marrow transplantation model, that the PBX-interaction domain, the homeodomain, and the C-terminal domain of MEIS1, are all required for leukemogenic collaboration with ND13. In contrast, the N-terminal domain of MEIS1 is dispensable for collaboration with ND13, but is required for Flt3 upregulation, indicating additional roles for MEIS1 in induction of leukemia independent of alterations in Flt3 expression. Gene-expression profiling of a cloned ND13 preleukemic cell line transduced with wild-type or Meis1 mutant forms revealed deregulation of multiple genes, including a set not previously implicated as MEIS1 targets. Chromatin immunoprecipitation revealed the in vivo occupancy of MEIS1 on regulatory sequences of Trib2, Flt3, Dlk1, Ccl3, Ccl4, Pf4, and Rgs1. Furthermore, engineered overexpression of Trib2 complements ND13 to induce AML while Ccl3 potentiates the repopulating ability of ND13. CONCLUSION: This study shows that Meis1-induced leukemogenesis with ND13 can occur in the absence of Flt3 upregulation and reveals the existence of other pathways activated by MEIS1 to promote leukemia. Establishment of pre-leukemic bone marrow cell lines following transduction with ND13 have been previously described (Pineault, Abramovich et al. 2005). In brief, lines were established from BM cells previously treated with 5-fluorouracil (5-FU) from (C57Bl/6Ly-Pep3b x C3H/HeJ) F1 (PepC3) (Ly5.1+/Ly5.2+) mice freshly transduced with the ND13-GFP or ND13pac virus and maintained in liquid culture. To generate ND13+Meis1 or ND13+Meis1 mutant BM cell lines, the ND13 BM cells were transduced by co-cultivation on irradiated (4,000 cGy) E86 producers for Meis1-YFP (or Meis1 mutant forms), respectively, for a period of 2 days in the presence of 5 µg/ml of protamine sulfate. Three independent experiments were performed for each of the four different conditions included in the study.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets