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Blood, Vol. 113, Issue 22, 5456-5465, May 28, 2009
The transcriptional program controlled by the stem cell leukemia gene Scl/Tal1 during early embryonic hematopoietic development
Blood Wilson et al.
113: 5456
Supplemental materials for: Wilson et al
Files in this Data Supplement:
- Table S1. List of 228 Peaks Bound by Scl in HPC-7 (PDF, 62.5 KB) -
All peaks are listed by chromosome number, start and end position in the mouse genome (build mm8 UCSC).
- Table S2. Motif discovery (PDF, 94.7 KB) -
Over-represented motifs (z-score >= 2.0 relative to background set of sequences; see materials and methods). Over-represented motifs were determined by searching i) an in-house library of 54 consensus motifs, ii) the JASPAR Core library (138 matrices) and iii) a subset of the TRANSFAC release 10.4 library (506 matrices of human and mouse origin out of a total of 816 matrices).
- Table S3. List of 228 peaks and associated genes (PDF, 75.9 KB) -
Peaks were analysed to determine if they were within a promoter region, intronic or between two flanking genes (see materials and methods). Peak co-ordinate, gene name and Ensembl ID are shown.
- Table S4. Primer sequences used for quantitative real-time PCR and cloning (PDF, 42.8 KB) -
Primers were designed using Primer3.
- Table S5. Summary of F0 transgenic embryo analysis (PDF, 16.7 KB) -
Shown is the total number of transgenic embryos generated for each construct together with the staining patterns observed. FL = fetal liver. Absence of fetal liver expression was confirmed in three SV-LacZ control embryos using histological sections.
- Table S6. Presence of GATA-binding motifs and -binding events within the nuclear targets of Scl (PDF, 98.5 KB) -
All peaks are listed by chromosome number, start and end position in the mouse genome (build mm8). The table lists the number of identified GATA motifs in all Scl bound regions localised to loci containing genes with “transcriptional regulation” annotation. Rows highlighted in grey indicate loci that contain at least one peak shown to have a Gata2 binding event with a false discovery rate (FDR) of less than 0.0009 (rounded down to 0); other FDRs are listed in the table.
- Figure S1. Nucleotide sequence alignments of the 11 Scl-bound regions cloned into LacZ reporter construct (JPG, 305 KB)
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E-box, GATA and Ets motifs are highlighted in yellow, green and red respectively.
- Figure S2. Expression profiles of candidate Scl target genes across 91 tissue samples profiled as part of the Genomics Institute of the Novartis Research Foundation BioGPS project (http://biogps.gnf.org) (JPG, 292 KB)
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The x-axis shows average raw expression values from Affymetrix microarray gene expression profiling experiments. Expression values below 50 indicate that a gene is likely not expressed in this tissue.
- Figure S3. Confirmation of novel targets of Scl binding in HPC-7 by real-time PCR (JPG, 75.7 KB)
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Real time PCR analysis of ChIP assays in HPC-7 cells performed with anti-Scl antibody. Levels of enrichment were normalised to IgG and compared to a negative control region (Runx1 +31kb). Primers used are described in Table S4.
- Figure S4. LacZ-staining patterns in individual Gfi1b +16kb transgenic embryos (JPG, 60.1 KB)
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Individual embryos are ordered based on the intensity of staining pattern from left to right. White arrowheads indicate the reproducible staining of the fetal liver seen in all three embryos. A close up view is shown for the left-most embryo to illustrate the low level fetal liver staining.
- Figure S5. Scl target genes identified to be involved in the focal adhesion kinase pathway and the MAP kinase pathway (JPG, 360 KB)
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The gene list of Scl candidate target genes was analysed using the DAVID tool (Database for Annotation, Visualisation and Integrated Discovery) for overrepresentation of components of biological pathways using KEGG (Kyoto Encyclopaedia of Genes and Genomes) annotation. Similar results were obtained using tools from the Panther database (data not shown) and the Webgestalt tools (bioinfo.vanderbilt.edu/webgestalt). (A) Map kinase signalling pathway: 11 genes are candidate Scl targets (Map4k4, Rap1a, Rac2, Prkacb, Map3k5, Rasa2, Rap1b, Mknk1, Mknk2, Stmn1, Dusp2). (B) Focal Adhesion Kinase pathway: 10 genes are candidate Scl targets (Vav1, Ccnd3, Rap1a, Rac2, Flt1, Pik3r1, Rap1b, Itgb3, Itga2b, Diap1).
- Figure S6. Scl-centric network showing confirmed Gata2 binding events (JPG, 340 KB)
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(A) Flow diagram showing presence of GATA motif within 39 of the 40 nuclear targets of Scl and confirmed Gata2 binding events with relevant false discovery rates. (B) Scl centric network diagram showing experimentally confirmed Gata2 binding events thus validating 32/39 of the computationally inferred links based on the presence of GATA motifs.
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