Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells

Using the relative expression levels of two SNP alleles of a gene in the same sample is an effective approach for identifying cis-acting regulatory SNPs (rSNPs). In the current study, we established a process for systematic screening for cis-acting rSNPs using experimental detection of AI as an initial approach. We selected 160 expressed candidate genes that are involved in cancer and anticancer drug resistance for analysis of AI in a panel of cell lines that represent different types of cancers and have been well characterized for their response patterns against anticancer drugs. Of these genes, 60 contained heterozygous SNPs in their coding regions, and 41 of the genes displayed imbalanced expression of the two cSNP alleles. Genes that displayed AI were subjected to bioinformatics-assisted identification of rSNPs that alter the strength of transcription factor binding. rSNPs in 15 genes were subjected to electrophoretic mobility shift assay, and in eight of these genes (APC, BCL2, CCND2, MLH1, PARP1, SLIT2, YES1, XRCC1) we identified differential protein binding from a nuclear extract between the SNP alleles. The screening process allowed us to zoom in from 160 candidate genes to eight genes that may contain functional rSNPs in their promoter regions

SDS-PAGE profile of purified recombinant proteins.

<p>SDS-PAGE (10%) of purified recombinant proteins from <i>E.coli</i> BL-21 transformed with pGEX-<i>WsCPR1</i> and pGEX-<i>WsCPR2</i>. Lane 1; Standard protein markers, Lane 2&3; Cell lysate (CL) of WsCPR1 and WsCPR2 expressing cells remained after incubation with GST-beads, Lane 4; Purified recombinant GST-fused WsCPR1, Lane 5; Purified recombinant GST fused WsCPR2, Lane 6; Purified WsCPR1 after removal of GST using thrombin and Lane 7; Purified CPR2 after removal of GST.</p

Three dimensional models and conserved residue prediction for WsCPR1 and WsCPR2.

<p>4A & 4D: Cartoon display of the 3-D structures of WsCPR1 <i>and</i> WsCPR2 as predicted by Phyre² using crystal structure of <i>Rattus norvagicus</i> (PDB ID: 1J9Z) as template. 4B & 4E: Predicted ligand (shown in green) binding sites as predicted by 3DLigandSite Web Server. 4C & 4F: Conserved residue analysis of <i>WsCPR1 and WsCPR2</i> were performed using Consurf and Conseq web servers. Residue conservation from variable to conserve is shown in blue (1) to violet (9). The residues involved in substrate binding and active site are shown in the center core of the structure.</p

List of primers used in the study.

*<p>Primers marked with star were provided with the kits.</p

Southern blot analysis of genomic DNA.

<p>A&B: Southern blot analysis of total DNA using <i>WsCPR1</i> and <i>WsCPR2</i> as a probe. Total DNA (30 µg) isolated from <i>Withania somnifera</i> was digested with the indicated restriction enzymes, The digested samples were electrophoresed on 0.8% agarose gel, blotted onto nylon membrane and subjected to hybridisation using DIG-labelled ORF of <i>WsCPR1</i> and <i>WsCPR2</i> as probes. First lane contains molecular markers with indicated molecular weight on the left side.</p

Phylogenetic analysis of deduced amino acid sequences.

<p>Phylogeny of <i>WsCPRs</i> was inferred using the Neighbor-joining method using MEGA 5 software. A total of 33 protein sequences used for analysis were from following plant species: <i>Withania somnifera</i> (WsCPR1: HM036710, WsCPR2: GU808569), <i>Petunia hybrida</i> (CPR1: DQ099544, CPR2: DQ099545); <i>Petroselinum crispum</i> (CPR1: AF024635, CPR2: AF024634); <i>Gossipium hirsutum</i> (CPR1: FJ719368, CPR2: FJ719369); <i>Populus trichocarpa</i> (CPR1: XM_002307300, CPR2: XM_002329600, CPR3: AF302498); <i>Arabidopsis thaliana</i> (CPR1: X66016, CPR2: X66017); <i>Capsicum annuum</i> (EU616557); <i>Vigna radiata</i> (P37116); <i>Vicia sativa</i> (Z26252); <i>Stevia rebaudiana</i> (DQ269454); <i>Ricinus communis</i> (XM_002514003); <i>Pisum sativum</i> (AF002698); <i>Picrorhiza kurroo</i>a (JN968968); <i>Artemisia annua</i> (EF104642); <i>Papaver somniferum</i> (U67185); <i>Taxus cuspidate</i> (AY571340); <i>Taxus chinensis</i> (AY959320); <i>Perilla frutescens</i> (GQ120439); <i>Ophiorrhiza pumila</i> (AB086169); <i>Medicago truncatula</i> (XM_003610061); <i>Lotus japonicas</i> (AB433810); <i>Catharanthus roseus</i> (Q05001); <i>Centaurium erythraea</i> (AY596976); <i>Zea mays</i> (CAC83301); <i>Triticum aestivum</i> (AGC27711) and <i>Eschscholzia californica</i> (U67186). All CPRs were grouped into two clusters where the WsCPR1 and WsCPR2 confined to their corresponding cluster like other CPRs.</p

Tissue-specific real-time expression analysis.

<p>Quantitative assessment of the expression of <i>WsCPR1</i> and <i>WsCPR2</i> in different tissues of <i>Withania somnifera.</i> Data were compared and analysed with analysis of variance (<i>ANOVA</i>). Values are means, with standard errors indicated by bars, representing three independent biological samples, each with three technical replicates. Differences were scored as statistical significance at *<i>p</i><0.05 and **<i>p</i><0.01 levels.</p

Time course effect of elicitor treatments on withanolides accumulation.

<p>10A: Effect of methyl jasmonate (MeJA) treatment on withanolides accumulation at different time intervals. HPLC analysis demonstrated the change in three key withanolides of withanolide A (WS-1), withanone (WS-2) and withaferin A (WS-3) at 6, 12, 24 and 48 h after treatments of micro-shoots with 0.1 mM MeJA. WS-3 was observed to be enhanced more with respect to WS-1 while WS-2 was detected in sample harvested after 48 h. All values obtained were means of triplicate with standard errors. Time course accumulation of WS-1 and WS-3 was statistically significant at <i>p</i><0.01 level. 10B: Effect of salicylic acid (SA) on withanolide accumulation at different time interval. The WS-3 level was also up-regulated in salicylic acid treated samples but WS-1 was enhanced more in comparison to methyl jasmonate (MeJA) treated samples. All values obtained were means of triplicate with standard errors. Time course accumulation of WS-1 and WS-3 was statistically significant at <i>p</i><0.001 level.</p