SUPPLEMENTAL MATERIALS AND METHODS
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1 SUPPLEMENTAL MATERIALS AND METHODS ChIP-Seq analysis ChIPed DNA sample libraries were analyzed using Illumina parallel sequencing. Peak detection of enriched binding regions was performed using FindPeaks 1 with an estimated false discovery rate < 0.05 as the selection criterion for enriched regions. The identified peaks were mapped onto mouse genome using UCSC mus musculus reference genome version 8 (build 2006). Any peaks that are overlapping ( 1 base pair) with repetitive genomic regions or controls were discarded. Peaks from biological replicates were consolidated with their genomic coordinates merged if they overlap at least one base pair on the genome. Characterization and summary of the whole-genome occupancy pattern of peaks were performed using the Cis-regulatory Element Annotation System (CEAS) annotation tool. 2,3 ChIP-chip analysis ChIP-chip data on enriched regions and selected transcription start sites was analyzed using MA2C 4 which adjusts for sequence GC content bias. Raw intensity measures were normalized using default parameter setting. Peak detection was performed using 300bp sliding window with coverage constraint of having at least 3 probes. Final set of peaks were determined if the difference in intensity values of chipped sample and input has a significance level of p-value < Probewise averaging of all peaks was empirically estimated from all data points. An object-oriented implementation for analyzing multiple ChIP-chip experiments based on MA2C analysis is available at author s website: Analysis of Hoxa9-ER gene expression profiles Gene expression values were calculated using a robust multi-array average (RMA). 5 Temporal differential gene expression analysis was performed using EDGE. 6 The significance measures (p-values) of individual genes were adjusted for multiple hypothesis testing using Benjamini-Hochberg method. 7 A gene was deemed significant if its temporal expression change satis.es a composite criterion: 1) with adjusted p-value < 0.05; and 2) having 1.5-fold change (median expression of three replicates) compared to controls at any of the three time points (namely 72, 96, 120 hours). For genes with multiple probesets, we selected only one probeset with the highest significance. In consistency with the use of mouse genome mm8 in ChIP-Seq, genes were annotated with NetAffx Mouse430_2 annotation matrix (release 25, UCSC mm8 / NCBI build 36). When no information of official gene symbol is available, the sequence identifiers (e.g., GenBank accession numbers) are used. Clustering of Hoxa9-ER significant genes All significant genes were clustered into four groups based on their temporal patterns using Self- Organizing Maps 8 in a similar way as Huang, et al. 9 Prior to clustering, the expression of each gene was normalized to have zero mean and unit variance. A hexagonal topology of 2 by 2 prototypes was initiated and each gene was subject to the network 400 iterations. The L 2distance measure was used as distance metric. The centroid and standard deviation (SEM) of each cluster was computed and plotted in Fig. S2E. The significant genes were mapped to closest H/M peaks using CisGenome. 10 De novo motif discovery analysis For de novo motif discovery, enriched regions were distilled to a centered peak core sequence of 200 bp,
2 and overlapping peaks were collapsed into a single non-redundant core sequence prior to analysis. Subsequently, this dataset included 800 independent sequences subjected to the GADEM algorithm, an extension of MEME. 11 Three duplicate discovery runs were performed and combined into one de novo motif set; STAMP 12 was used to annotate identified motifs with the TRANSFAC 11.3 database, using E=1E-5 threshold and information content trimming >0.4; this resulted in 15/31 matches. Motifs were validated independently using multiple alternative approaches including Weeder, MEME, and CisGenome, 10,13 15 which yielded comparable results to de novo motif discovery using GADEM. Motif enrichment analysis MEME suite (Find Individual Motif Occurrences) was used to analyze enrichment of the experimentally determined Hoxa9/Meis1 consensus motif ATGATTTATGGC (Hoxa9-Meis1-Pbx1) and Meis1 consensus TGTC motif. Comprehensive search of known transcription factor binding motifs was performed for transcription factors included in Genomatix proprietary Mat Base Matrix Family Library (Version 8.2, January 2010) that includes a total of 727 motifs (170 motif families) and their corresponding position weight matrices. The DNA sequences of length 200bp from the center of each H/M peak were scanned for presence of any known transcription factor binding motif. A transcription factor binding motif is considered to be statistically significantly enriched in the H/M peaks if the number of sequences in which the motif is found to be present is significantly higher than its expected whole-genome occurrences according to standard z-test (z-score >2.81; p-value < 0.005). Evaluation of regulatory potentials of H/M peaks The base pair-wise average regulatory potential (RP) scores 16 of all H/M binding sites and their surrounding ±4 kb regions (extended H/M regions) were computed and shown as a function of distance from the center of the regions. More specifically, we compute the average RP score on a base pair basis across all extended H/M binding region. Comparison of H/M peaks and evolutionarily conserved elements The evolutionary conservation scores were obtained from UCSC phastcons17way 17 database for each enriched region and its adjacent regions extending up to 6 in width. A virtual peak is constructed by combining the left and right side of enriched region subject to the same width of enriched region. In a sliding widow fashion, six virtual peaks are constructed contiguously outwards from the center of the enriched region with no overlap with each other. A two samplet-test is performed comparing the average conservation score within the enriched region against that of each virtual peak. Script for running this analysis (using R) is available upon request. Mass spectroscopy To identify proteins co-immunoprecipitating with HA-tagged Hoxa9 and FLAG-tagged Meis1, immunoprecipitates were resolved on SDS-PAGE gels and stained with Colloidal Blue (Invitrogen) followed by destaining and cysteine reduction/carbamidomethylation (10 mm DTT+50 mmiodoacetamide). Macerated and dried gel slices were re-swollen and digested in ammonium bicarbonate buffer with trypsin (Promega). Peptides were extracted sequentially using acetonitrile/tfa gradient; extracts were pooled and concentrated prior to reverse phase chromatography (Aquasil C18,
3 Picofrit column, New Objectives). Eluted peptides were directly introduced into an ion-trap mass spectrometer (LTQ-XL, ThermoFisher) with a nano-spray (in MS/MS mode). Data were converted to mzxml format and searched against mouse IPI (v 3.50) + reverse database using X!Tandem with k-score plug-in (Global Proteome Machine). Proteins with >0.9 probability were selected (ProteinProphet29/30). Outputs were subjected to PeptideProphet and ProteinProphet analysis; proteins with a ProteinProphet probability of >0.9 were considered for further analysis. MS/MS spectra corresponding to proteins that were unique to the experimental sample were manually verified (Table S6). 18 REFERENCES 1. Robertson G, Hirst M, Bainbridge M, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods. 2007;4: Ji X, Li W, Song J, Wei L, Liu XS. CEAS: cis-regulatory element annotation system. Nucleic Acids Res. 2006;34:W Shin H, Liu T, Manrai AK, Liu XS. CEAS: cis-regulatory element annotation system.bioinformatics. 2009;25: Song JS, Johnson WE, Zhu X, et al. Model-based analysis of two-color arrays (MA2C).Genome Biol. 2007;8:R Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 2003;31:e Storey JD, Xiao W, Leek JT, Tompkins RG, Davis RW. Significance analysis of time course microarray experiments. Proc Natl Acad Sci U S A. 2005;102: Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series B. 1995;57: Kohonen T. Self-Organizing Maps. Series in Information Sciences. 1995; Huang J, Jennings NR, Fox J. An Agent-based Approach to Health Care Management. Int Journal of Applied Artificial Intelligence. 1995;9: Ji H, Jiang H, Ma W, Johnson DS, Myers RM, Wong WH. An integrated software system for analyzing ChIP-chip and ChIP-seq data. Nat Biotechnol. 2008;26: Li L. GADEM: a genetic algorithm guided formation of spaced dyads coupled with an EM algorithm for motif discovery. J Comput Biol. 2009;16: Mahony S, Benos PV. STAMP: a web tool for exploring DNA-binding motif similarities.nucleic Acids
4 Res. 2007;35:W Pavesi G, Mereghetti P, Mauri G, Pesole G. Weeder Web: discovery of transcription factor binding sites in a set of sequences from co-regulated genes. Nucleic Acids Res. 2004;32:W Bailey TL, Boden M, Buske FA, et al. MEME SUITE: tools for motif discovery and searching.nucleic Acids Res. 2009;37:W Bailey TL, Williams N, Misleh C, Li WW. MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res. 2006;34:W Taylor J, Tyekucheva S, King DC, Hardison RC, Miller W, Chiaromonte F. ESPERR: learning strong and weak signals in genomic sequence alignments to identify functional elements.genome Res. 2006;16: Siepel A, Bejerano G, Pedersen JS, et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 2005;15: Nesvizhskii AI, Vitek O, Aebersold R. Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat Methods. 2007;4:
5 Figure S1. Endogenous Hoxa9 and Meis1 protein levels in transformed mouse hematopoietic progenitor (MHP) cells Hoxa9 and Meis1 protein levels from whole cell lysates of MLL-AF9 transduced (MLL) and HA- Hoxa9/FLAG-Meis1 transduced (HM2) MHP cells were detected by western blot using anti-hoxa9 (Millipore) and anti-meis1 (Abcam) antibodies. Hoxa9 and Meis1 protein levels are shown to be comparable. β-actin, used as a loading control, was detected by an anti β-actin mouse monoclonal antibody (Sigma). Figure S2. Comparison of conservation scores in H/M peaks and the extended regions outside of H/M peaks, related to Fig. 3A For each extended region, an equal length of sequence segment was chosen successively outside of H/M sequences and their associated conservation was computed. Error bars are 2 SEM.
6 Figure S3. Hoxa9/ Meis1 binding regions show enhancer activity in luciferase assays (A) Twenty-two novel Hoxa9/ Meis1 binding regions of ~1,000 bp in length were cloned into ptal-luc luciferase reporter and electroporated into K-562 cells. Enhancer activity of the Hoxa9/ Meis1 binding
7 regions was compared to a known Runx1 conserved noncoding element (Runx1_chr16(+23)) (Nottingham et al. 2007) and two control regions randomly selected in the genome as a negative control. Data is presented as the Mean + SD in triplicate normalized to ptal-luc. (B) Comparison of ChIP-chip signal intensity in Hoxa9 / Mesi1 binding regions that were scored positively and negatively for enhancer activity in the presence and absence of 4-OHT. (C) Granular level details of ChIP-Chip signal intensity at 8 Hoxa9 / Meis1 binding regions that show enhancer activity.
8 Figure S4. Conditional transformation by Hoxa9-ER and identification of Hoxa9 regulated target genes, related to Fig. 4 (A) Hoxa9-ER cells cease dividing within 96 hours of 4-OHT withdrawal and show increased expression of the myeloid/monocytic differentiation markers Gr-1 and Mac-1 by flow cytometry (B). By day 6, the
9 majority of cells showed macrophage morphology, while myeloblast morphology was maintained in cells that were cultured in continuous 4-OHT (C). (D) Cascade of significant changes in gene expression secondary effects following 4-OHT withdrawal. (E) Cluster centroids of significant genes in Hoxa9-ER profiling. Four clusters of genes with significant changes in expression level after 4-OHT withdrawal. Genes are clustered into one of the four clusters based on their temporal dynamics. For each cluster, the centroid and SEM are shown with the total number of genes in that cluster (blue). (F) through (K) Heatmaps of genes with proximate Hoxa9 and/or Meis1 binding sites that are significantly differentially expressed over the 120 hrs period post 4-OHT withdrawal (FDR P<0.05 and median fold change >1.5) compared to controls. Genes are organized into 5 location categories according to the relative distance from their genomic features to the nearest Hoxa9/Meis1 binding sequences based on RefSeq gene annotation: (F) Sequences labeled as TSS located within 20k bp 5 to the transcription start site (G) Sequences labeled as 5 UTRs are the regions between the transcription and coding start sites. (H) Differentially regulated genes with intronic binding sites (I) sequences labeled 3 UTRs are defined as the regions between the coding and transcription termination sites. (J) Sequences labeled as TES are located within 20k bp to the transcription end site. Within each location category, genes are grouped based on their cluster designation (Fig. S4E and Table S2) and listed in descending order in their differential statistics. Data are normalized across samples such that the expression value of each individual gene has zero mean and standard deviation of one.
10 Figure S5. De novo motif discovery results, related to Fig. 5A and Fig. 5B (A) STAMP logos of de novo identified motifs that are enriched in H/M peaks. (B) Co-occurrence matrix of de novo motifs. (C) Bubble plot of co-occurrence of de novo motifs. The size of the bubbles indicates the magnitude of co-localization between two motifs.
11 Figure S6. Spatial distribution of de novo identified motifs, Related to Fig. 5A and Fig. 5B The histogram and density of de novo motif spatial distribution was computed with respect to the center of the H/M peaks.
12 Figure S7. Related to Fig. 5, Fig. 6, and Discussion (A) Motifs of Hoxa-Meis1-Pbx, STAT, C/EBPα, CREB, and ETS show complementary spatial distribution patterns. (B) Comparative motif enrichment analysis show distinct motif enrichment profiles in sequences bound by H/M (green), H/M + C/EBPα (blue), and C/EBPα alone (red).
13 Figure S8. Related to Fig. 7 and Discussion ChIP experiments showing Meis1, Pu.1, C/ebpα, Stat5a/b, P300, CBP, histone H3 and histone H4 acetylation association with Hoxasomes is dependent on Hoxa9 as evidenced by drop in ChIP signal following 4-OHT withdrawal. See Experimental Procedures and Fig. 2 legend.
14 Figure S9. Examples of Hoxa9/Meis1 binding sites carrying epigenetic signature of enhancers, related to Fig. 7 and Discussion Four H/M bindings sites near or at promoter region of Dnajc10, Cd34, Foxp1, and Flt3 are shown with epigenetic signatures of CBP, H3 acetyl, H4 acetyl, P300, and RNA pol II. For each epigenetic mark, chipchip results of 4-OHT (+) and 4-OHT withdrawal ( ) are shown.
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