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Blood, Vol. 109, Issue 5, 1908-1916, March 1, 2007
The paralogous hematopoietic regulators Lyl1 and Scl are coregulated by Ets and GATA factors, but Lyl1 cannot rescue the early Scl–/– phenotype
Blood Chan et al.
109: 1908
Supplemental materials for: Chan et al, Vol 109, Issue 5, 1908-1916
Files in this Data Supplement:
- Figure S1. In vivo activity of the Lyl1 promoter transgenic cassette correlates with lacZ expression in Lyl1 lacZ knock-in mice (PDF, 160 KB) -
E11.5 embryos were collected from Lyl1 lacZ knock-in embryos1 and SV/lac/Lyl1P transgenic lines and analyzed by wholemount X-gal staining followed by histochemical analysis. For both lines, lacZ staining was observed in endothelium (ET), fetal liver (FL), dorsal aorta (DA), and endocardium (EC).
- Figure S2. The Lyl1 promoter transgenic cassette targets expression to cells with megakaryocyte morphology in vivo (PDF, 62.9 KB) -
E11.5 and E14.5 embryos were collected from SV/lac/Lyl1P transgenic lines and analyzed by wholemount X-gal staining. Histochemical analysis of fetal liver sections revealed expression of the transgene (blue) in cells with megakaryocyte morphology.
- Figure S3. Sequence alignment of the Lyl1 promoter region showing conserved motifs and specific mutations introduced to disrupt transcription factor binding (PDF, 270 KB) -
To mutate the GGCC motif, the region indicated by a hashed line was deleted. For all other mutations, specific residues were replaced with the sequences shown underneath the alignment.
- Figure S4. Chromatin immunoprecipitation assays of the Lyl1 and α-fetoprotein promoters (PDF, 23.4 KB) -
ChIP assays were analyzed by real-time PCR and normalized against the IgG control sample. Specific upstream factors bound to the Lyl1 promoter were not bound to the  -fetoprotein promoter. Moreover, none of the Ets or GATA factors analyzed in this study bound the Lyl1 promoter in NIH3T3 cells.
- Figure S5. Ectopic expression of SCL and Lyl1 in SCL–/– mouse ES cells (PDF, 93.1 KB) -
(A) Real-time PCR analysis demonstrates that SCL–/– ES cells transfected with MSCV-puro Flag-tagged SCL and Lyl1 constructs express comparable levels of SCL and Lyl1 mRNA, respectively. RNA was prepared from control-transfected and SCL- and Lyl1-transfected ES cells as well as SCL- and Lyl1-transfected day 5 embryoid bodies. (B) Ectopically expressed Flag-tagged SCL and Lyl1 proteins are present at comparable levels in ES cells transfected with the respective MSCV constructs. Flag-tagged SCL and Lyl1 were detected using a Flag-Cy3 monoclonal antibody (Sigma, St Louis, MO) at a dilution of 1:500. DAPI nuclear stain (blue) was included to indicate the location of ES cell colonies. There is some background staining from the underlying feeder layers. However, the clusters of ES cells are clearly visible as being Cy3 positive in SCL- and Lyl1-transfected SCL–/– ES cells but Cy3 dull to negative in the cells transfected with the empty vector control.
REFERENCE
1. Capron C, Lecluse Y, Kaushik AL, et al. The SCL relative LYL-1 is required for fetal and adult hematopoietic stem cell functions and B cell differentiation. Blood. 2006;107:4678-4686.
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