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Blood, Vol. 113, Issue 23, 5783-5792, June 4, 2009
Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors
Blood Landry et al.
113: 5783
Supplemental materials for: Landry et al
Files in this Data Supplement:
- Table S1. Primers to generate reporter constructs (PDF, 45.5 KB)
- Table S2. Whole-mount B-Gal staining pattern of candidate regulatory elements of LMO2 in transgenic assays (PDF, 63.2 KB)
- Table S3. Histological B-Gal staining pattern of candidate hematopoietic specific elements of LMO2 in transgenic assays (PDF, 103 KB)
- Table S4. Summary of functional validation of candidate regulatory elements identified by comparative genomics (PDF, 56 KB)
- Table S5. Transcription factor binding site analysis of Lmo2 hematopoietic specific elements that are conserved between human and mouse (PDF, 45.5 KB)
- Figure S1. Stable transfections of 416B cells with LMO2 pP enhancer constructs (JPG, 55.6 KB)
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Stable transfections of 416B cells with the minimal proximal promoter pP combined with 14 candidate regulatory elements of LMO2 tested in transgenic analysis. Normalised luciferase activities are depicted as fold induction over pP. Stable transfections were performed in at least two biological replicates and assayed in triplicates, and the error bars represent the combined standard deviations. The −75, −70, −64, and −25 enhancers increase the activity of the proximal promoter between 4 and 10 fold. The highest activity is conferred by the element −25, which in combination with pPex and −12 is able to drive robust B-Gal expression to fetal liver in transgenic analysis.
- Figure S2. Stable transfections of MS1, 416B, and MEL with hematopoietic specific LMO2 pPex multi-enhancer constructs (JPG, 55.9 KB)
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Stable transfections of MS1, 416B, and MEL cell lines, representing endothelial, myeloid, and erythroid progenitors, respectively. Normalised luciferase activities are depicted for single experiments performed in triplicates, and the error bars represent the standard deviations. (A) Stable transfection of LMO2 erythroid element −75 combined with the extended proximal promoter pPex. Enhancer activity is absent in MS1 cells, but present in hematopoietic cell lines 416B and MEL, with strongest activity in MEL cells. (B) Stable transfection of LMO2 hematopoietic progenitor elements −25/−12 combined with the extended proximal promoter pPex show high and specific activity in 416B cells.
- Figure S3. Conserved E-boxes, GATA, and ETS sites are present in the −75, −70, and −25 sequence elements but absent in combination in the +1 and 7 enhancers (JPG, 872 KB)
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Multiple sequence alignments of the region flanking the conserved E-boxes of the −75 (A), −70 (B), −25 (C), +1 (D), and +7 (E) elements. Predicted conserved transcription factor binding sites are boxed when present in all aligned species and marked by a dashed box when only conserved in eutherian mammals. An exception to this demarcation is a boxed GATA site in the −25 element which is not conserved at the same position in human but instead is present 33bp downstream (also boxed). The chicken sequence is not shown for the −25 enhancer as the region is not conserved in birds.
- Figure S4. Lmo2 candidate distal regulatory elements identified by comparative genomics show high Regulatory Potential scores (JPG, 43.5 KB)
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The cloned DNA-fragments of the 14 candidate Lmo2 distal regulatory elements identified by our comparative genomic approach as well as the distal (dp) and proximal (pPex) promoter fragments are depicted as a UCSC custom track (magenta). Of note, all these candidate elements are characterized by peaks of Regulatory Potential (RP) scores higher than 0.1, as defined by the ESPERR (Evolutionary and Sequence Pattern Extraction through Reduced Representation) procedure 50(blue track at bottom of diagram). Moreover, genomic intervals predicted to contain erythroid cis-regulatory modules (RP score greater than 0.05 and a conserved GATA1 consensus binding site 43) are indicated by black boxes and matched with a subset of Lmo2 candidate regulatory elements. Interestingly, endothelial elements (pPex, +1, +7) were correctly not predicted as potential erythroid elements whereas the functionally validated erythroid element −75 and Gata2-bound element −25 were.
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