SUPPORTING INFORMATION for Regulation of gene expression by the BLM helicase correlates with the presence of G4 motifs Giang Huong Nguyen1,2, Weiliang Tang3, Ana I. Robles1, Richard P. Beyer4, Lucas T. Gray5, Judith A. Welsh1, Aaron J. Schetter1, Kensuke Kumamoto1,6, Xin Wei Wang1, Ian D. Hickson2,7, Nancy Maizels5, 3,8 1 Raymond J. Monnat, Jr. and Curtis C. Harris 1Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A; 2Department of Medical Oncology, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, U.K.; 3Department of Pathology, University of Washington, Seattle, WA U.S.A.; 4 Center for Ecogenetics and Environmental Health, University of Washington, Seattle, WA U.S.A.; 5Department of Immunology and Department of Biochemistry, University of Washington, Seattle, WA U.S.A.; 6Department of Organ Regulatory Surgery, Fukushima Medical University, Fukushima, Japan; 7Cellular and Molecular Medicine, Nordea Center for Healthy Aging, University of Copenhagen, Denmark; 8Department of Genome Sciences, University of WA, Seattle, WA U.S.A. SI Index: Supporting Information for this manuscript includes the following 19 items. A more detailed Materials and Methods section is followed by 18 Tables and Figures in order of their appearance in the manuscript text: 1) SI Materials and Methods 2) Figure S1. Study design and experimental workflow. 3) Figure S2. Immunoblot verification of BLM depletion from human fibroblasts. 4) Figure S3. PCA of mRNA and miRNA expression in BLM-depleted human fibroblasts. 5) Figure S4. qPCR confirmation of mRNA array data. 6) Table S1. BS patient and control detail. 7) Table S2A. Significantly differentially expressed mRNAs identified in BS compared with matched control human fibroblasts. 8) Table S2B. Significantly differentially expressed mRNAs identified in BLM-depleted compared with isogenic, nonspecific shRNA-treated control human 82-6 fibroblasts. 9) Table S2C. Common significantly differentially expressed mRNAs in BS and BLM-depleted cells. 10) Table S3. Gene Set Enrichment Analysis (GSEA) detail. 11) Table S4. Significantly differentially expressed miRNAs identified in BS or BLM-depleted fibroblasts. 12) Table S5. Experimentally validated targets of miRNAs significantly altered in BS or BLM-depleted cells. 13) Figure S5. qPCR confirmation of miRNA array data. 14) Figure S6. G4 motif frequencies and enrichment near transcription start sites and intron 1 boundaries of genes with altered expression in BLM-depleted cells. 15) Table S6. Hypergeometric test of miRNA targeting vs. significantly altered mRNA expression 16) Table S6a. Selected target genes differentially expressed in BS and BLM depleted cells 17) Table S7A. Differentially Expressed Genes in BS patient vs NM cells that have at least one G4 motif in each region 18) Table S7B. Differentially Expressed Genes in BLM-depleted vs NS-treated that have at least one G4 motif in each region 19) Table S8. G4 statistics for genes significantly altered in BS and BLM-depleted cells. 1. SI Materials and Methods: Cells and cell culture conditions. We analyzed mRNA and miRNA expression in primary fibroblast strains from BS patients and from matched primary human skin fibroblasts from healthy individuals (NM), as well as in primary human fibroblasts depleted for BLM protein by expression of a BLM-specific shRNA. Primary human skin fibroblast strains from BS patients (n=16, donor median age 11.5 years) or from control donors (n=15, donor median age 19 years) were obtained from the Coriell Cell Repositories (Camden, NJ, USA). Normal control fibroblasts (NM) were age and gender-matched to BS cases. Additional detail on these BS patient and NM fibroblasts is given in Table S1. Cells were cultured in Eagle's Minimum Essential Medium with Earle's salts, supplemented with 15% fetal bovine serum, non-essential amino acids, penicillin/streptomycin and 2mM L-glutamine (all supplied by Invitrogen, Carlsbad, CA, USA). All cells were cultured at 37°C in a humidified 5% CO2 incubator. Passage number was calculated based on the number of passages at purchase plus the number of passages performed prior to cell harvesting. The human primary fibroblast strain 82-6 was initiated from a foreskin fibroblast preparation as previously described (1), and grown in Dulbecco-modified Eagle medium (Mediatech, Manassas, VA, USA) supplemented with 10% fetal bovine serum (HyClone, o Logan, UT, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 C in a humidified 5% CO2 incubator. BLM depletion experiments. BLM protein was depleted from 82-6 primary human fibroblasts by lentiviral transduction with expression of a BLM-specific shRNA as previously described (2, 3). In brief, a BLM-specific shRNA (BLM-3: 5’-GTACTAAATGGCAATTTAA-3’) was cloned into the lentiviral vector pLKO.1 under control of the human U6 promoter (4). Depletions were performed by transducing 106 primary fibroblasts cells with a BLM-shRNA-expressing lentivirus at a multiplicity of infection (MOI) of 10 for 2 d, followed by puromycin selection (2.0 µg/mL) for 8 d. BLM protein depletion to ≥90% was verified on day 10 by Western blot analysis, where day 0 was the start of viral transduction (2)(Figure S2). Depletions were performed in triplicate, using either the BLM-specific shRNA or a scrambled shRNA with no known target sequence in the human genome (plasmid 1864, Addgene, Cambridge, MA), to provide controls for comparison with untransduced cells. RNA isolation and quality control. RNA was isolated from human diploid 82-6 primary fibroblast cultures when 75% confluent. Cells were washed in 1X PBS, scrapped off plates and collected as pellets that were stored at -80ºC. Total RNA was isolated using TRIZOL reagent, following the manufacturer’s protocol (Invitrogen). Total RNA samples were isolated using Qiagen’s miRNAeasy Mini Kit (Qiagen, Valencia, CA). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and only samples of high quality, defined by distinct 28S and 18S rRNA bands together with a RIN (RNA integrity number) >7, were used for further analyses. mRNA expression profiling. Whole genome transcript exon profiling of BS and NM fibroblasts obtained from the Coriell collection of human primary fibroblasts, and of BLM-depleted 82-6 primary human fibroblasts were performed using the Affymetrix GeneChip Human Exon 1.0 ST Array, which contains four probes/exon and ~40 probes/gene (Affymetrix, Santa Clara, CA). RNA labeling, hybridization, washing, and image acquisition were performed at the Laboratory of Molecular Technology (NCI-Frederick) using a standard Affymetrix labeling, hybridization and wash protocol. miRNA expression profiling. Profiling of miRNA expression in BS and NM primary fibroblasts was performed using a custom miRNA microarray chip (OSU-CCC version 4.0) that includes 898 probes to human and 704 probes to mouse mature or precursor miRNAs, spotted in duplicate (5). Briefly, total RNA (5µg) was converted to biotin-labeled complementary DNA, hybridized onto the chips, and processed by direct detection of the biotin-containing transcripts by streptavidin-Alexa 647 conjugate. Slides were subsequently scanned with the Axon 4000B Scanner (Molecular Device) and spot intensities were quantified with Genepix (version Pro 6.0.1.00). Profiling of miRNA expression in BLM- depleted primary human fibroblasts was performed using the Nanostring nCounter Human miRNA Expression Assay Kit (Nanostring, Seattle, WA). For these analyses we used 100 ng RNA and the processing protocol recommended by the manufacturer. In order to confirm miRNA expression differences we used qPCR. RNA samples were reverse- transcribed using an Applied Biosystems High-Capacity cDNA Archive Kit (Applied Biosystems/AB, Foster City, CA) prior to assay in triplicate using miRNA-specific Taqman Gene Expression Assays (ABI). Double-stranded cDNA for each sample was amplified for 40 cycles using the TaqMan Universal PCR Master Mix using the manufacturer’s protocol on the 7500HT Sequence Detection System (ABI). For quality control, any samples with either an 18S rRNA or RNAU66 cycle value >20, or a gene or miRNA cycle value >36, were considered to be of poor quality and removed. Alternatively, PCR reactions were run following a 12-round pre-amplification on a BioMark 48.48 Dynamic Array System (Fluidigm, South San Francisco, CA). The mRNA or miRNA readings were calculated using the comparative method (2-ΔCt), where Ct = threshold cycle and ΔCt = (Ct gene (or miRNA) – Ct 18S rRNA (or RNAU66)). Identification of differentially expressed mRNAs. Raw Human Exon 1.0 ST microarray expression data were pre-processed and normalized with Affymetrix® Expression Console™ Software using RMA normalization (affymetrix.com). From normalized data, genes with significant evidence for differential expression were identified using the Bioconductor limma package using a linear fixed effects model with adjustments for age and gender for the primary fibroblast data to calculate the contrast BS- NM (6, 7). For the BLM-depleted 82-6 normal human fibroblasts, a linear fixed effects model was used to calculate three contrasts, BLM-control, NS-control, and BLM-NS. The limma methodology calculates a p-value for each gene using a modified t-test in conjunction with an empirical Bayes method to moderate the standard errors of the estimated log-fold changes. This method of detecting differentially expressed genes draws strength