FACS. Chromatin immunoprecipitation (ChIP) and ChIP-chip arrays 3 4

Transcription

1 FACS Stem progenitor sorting for progenitor analysis Adult bone marrow cells were depleted with Miltenyi lineage depletion column (Miltenyi). For identification of CMPs, GMPs, and MEPs the cells were incubated with allophycocyanin (APC) anti c-kit (2B8, Pharmingen), phycoerythrin (PE) anti-fcgrii/iii (2.4G2, Pharmingen) and fluorescein-isothiocyanate (FITC) anti CD-34 (RAM34, Pharmingen). LSK cells among Lin cells were enumerated after staining with APC c-kit and Pacific Blue Sca-1 (E , Pharmingen). Stained cells were sorted using a MoFlo cell sorter (Dako). 1 Erg +85 venus expression in adult bone marrow and thymus The transgenic mouse line was created by microinjecting a construct containing the SV+40 promoter, the fluorescent protein Venus and the Erg +85 region into F1 mice. Foetal liver FACs was performed on foetal livers taken from E12.5 embryos. Thymic tissue was dissected from 6- week-old mice and double negative thymocytes were analysed by staining cells with CD8-biotin and CD4-biotin and streptavidin PerCP-cy5; CD25-APC and CD44-PB. Bone marrow FACs was performed on cells extracted by crushing the femurs of transgenic mice. Bone marrow cells underwent red blood cell lysis, using BD Pharm Lyse lysing buffer, before being stained or passed through as lineage depletion column, as required. FACS analysis was performed on a Beckman Coulter Cyan ADP and the data was analysed using FlowJo. Cell viability and cell cycle analysis 2 To analyse cell cycle, cultured cells were fixed in 70% ethanol for 12 hours at 20 C and incubated in 0.05% (v/v) TritonX-100 (Sigma), 0.1mg/ml DNAse-free RNAse and 50μg/ml Propidium Iodide (Sigma-Aldrich, Cat No: P-4864) for 4 hours at room temperature. The fluorescence emission was analyzed using 633nm laser by FACS Canto II. Apoptotic cells were quantified by staining cells with Annexin V-APC (BD pharmigen) followed by FACS analysis. Chromatin immunoprecipitation (ChIP) and ChIP-chip arrays 3 4 Proteins were cross-linked to DNA using formaldehyde (0.4 1%) and cells lysed. Chromatin was fragmented using a Bioruptor sonicator (Diagenode SA). Separate immunoprecipitates were produced using immunoglobulin raised against target proteins. DNA from protein-associated complexes and corresponding input samples was recovered using phenol-chloroform extraction and assayed by SYBR-green real time PCR under standard conditions or labelled for array hybridization. For quantitative analysis of individual regions of interest, immunoprecipitated chromatin was quantified by SYBR Green real time PCR analysis (Stratagene), and the levels of enrichment were normalized to that obtained with a control rabbit antibody. For ChIP-chip array hybridization, samples were labelled with Cy3 dye, mixed with Cy5-labeled control DNA, and resuspended in hybridization buffer with cot-1 DNA and blocking agent. 4,7 This mix was hybridized to 8 15K Agilent slides for 24 h in a hybridization oven in accordance with the manufacturer s instructions. The custom microarray was designed using high density ChIP probes from the Agilent earray database and printed by Agilent Technologies. On average, one 60 bp oligomer was selected for every 200 bp of repeat masked sequence to cover the ERG locus to its flanking genes. The array design has been deposited in ArrayExpress and can be found

2 under A-MEXP After hybridization, slides were scanned using a DNA microarray scanner (Agilent, Santa Clara, CA, USA). The Cy3foreground-background/Cy5foreground-background signal ratios were averaged and normalized by the median intensity across the array. The normalized values were computed as log2 values and plotted as such in Fig. 3A. The microarray design and results have been deposited in ArrayExpress and can be found under A-MEXP-1853 and E-MTAB-431, respectively. Analysis of oncomine microarray datasets Publically available microarray datasets were accessed via Oncomine and searched for data pertaining to ERG and T-acute lymphoblastic leukemia. Plots were generated by Oncomine and edited to show relevant data.

3 Table S1. Antibodies for ChIP Target Manufacturer, product number Acetyl K9/K14 histone H3 Millipore, SCL LYL1 LMO2 ERG FLI1 GATA2 GATA3 ETV2 hhex Santa Cruz, sc12984x Abcam, ab R & D Systems, AF2726 Santa Cruz, sc354x Abcam, ab15289 Santa Cruz, sc9008x Santa Cruz, sc9009x Abcam, ab65825 Santa Cruz, sc15128x

4 Table S2. Antibodies for flow cytometry Target Anti-mouse c-kit-pe-cy7 Anti-mouse c-kit-apc Anti-mouse Sca1-Pacific blue Anti-mouse CD34-FITC Manufacturer, clone number e-bioscience, 2B8 Pharmingen, 2B8 Pharmingen, E Pharmingen, RAM34 Anti-FcgRII/III-PE Pharmingen, 2.4G2 Anti-mouse Ter119-APC Anti-mouse Mac1-APC Anti-mouse Gr1-pacific blue Anti-mouse B220-APC changed from BD Anti-mouse CD3-FITC Anti- mouse CD4-APC BD Pharmingen, TER-119 BD Pharmingen, ICRF44 e-bioscience, RB6-8C5 e-bioscience, RA3-6B2 e-bioscience, 17A2 e-bioscience, GK1.5 Anti- mouse CD8-FITC e-bioscience, Anti-mouse CD4-biotin BD Pharmingen, RM4-4 Anti-mouse CD8-biotin changed from BD ebioscience, Anti-mouse CD44-pacific blue Anti-mouse CD25-APC BD Pharmingen, IM7 BD Pharmingen, PC61 Anti- human CD34-PE BD Pharmingen, 581 Anti- human CD38-FITC Anti- human CD7-FITC Anti- human CD1a-APC BD Pharmingen, HIT2 BD Pharmingen, M-T701 BD Pharmingen, HI149

5 Table S3. PCR primers Target Sequence Purpose herg DP F-TTCCAGAAGTTTTCGTTTC R-AGGGACATGAGAGAAGAG ChIP herg PP F-CACATCCAGGCACCGTTAGG R-CTACTCCAACCATCCGCAAGG ChIP herg +85 F-ACAACACCACTCCGCATTG R-TGAACACTCGTTACAAGACTAATC ChIP hlmo2 neg F-GAAATAAATATCTCCACTGTCCTG R-CTATCTGCCTATCTCTCATCTATC ChIP herg mrna F-GCTGCTCAACCATCTCCTTCC R-AAGGCGGCTACTTGTTGGTC qrt-pcr merg mrna F-CTCTCACATCTCCACTAC R-GGCTCATAAGGTAAATCTG qrt-pcr h β-actin F-AGCACTGTGTTGGCGTACAG R-AGAGCTACGAGCTGCCTGAC qrt-pcr m β-actin F-CCTAAGGCCAACCGTGAAAAG R-TCTTCATGGTGCTAGGAGCCA qrt-pcr merg +85 F-GCTGTGGTCCACTCTTTCC R-CGGCAAGTTCTGTCCAACC ChIP m αfp F-ACAAGTGACCCCTGCTCTGT R-CCTGTTTAAGGGATGCCTGTT ChIP Notch HD domain F-GGCTGAGTTTCTTTAGAGTC R-CCTCCCCTGAGGTTACACCT Mutation analysis Notch HD domain F-GAGTGTCCCATTGCGGGGCT R-TGCAGAGGTCAGAAAGTGTT Mutation analysis Notch PEST domain F-TACCAGGGCCTGCCCAACAC R-GCCTCTGGAATGTGGGTGAT Mutation analysis Notch PEST F-AAGGACCTCAAGGCACGGAG Mutation domain R-GAGGTGTGGCTGTGATGGTG analysis

6 Figure S1. ERG mrna expression in acute leukemias (A) ERG expression in T-ALL grouped by initial vs/ relapse samples ( 5 ; reporter _x_at). (B) ERG expression in a panel of acute leukemias grouped by diagnosis vs/ relapse sample. ( 6 ; reporter 913_at). (C) ERG expression in a panel of T-ALL cell lines grouped by PTEN mutation status ( 7 ; reporter _x_at).

7 Figure S2. ERG expression in T-ALL xenografts and Molt4 cell line

8 Figure S3. AcH3 ChIP-chip across the human ERG locus in CD4 and CD8 T cells Upper panel is a Vista plot showing conservation between human and mouse across the ERG locus. Lower panels (red) show AcH3 ChIP-chip across the same region.

9 Figure S4. Binding of 10 haematopoietic transcription factors to the merg locus in the HPC7 stem/progenitor cell line ChIP-seq traces from data generated by Wilson et al, 8 uploaded to the UCSC genome browser showing overlapping binding at the Erg +85 enhancer.