Cdkn1a and Id1 are direct target genes of SCL in immature hematopoietic cells. (A) Cells with an LT-HSC phenotype were purified by flow cytometry from Scl+/+ and Scl+/− bone marrows. After reverse transcription, gene expression was assessed by quantitative PCR array for the indicated cell cycle–related genes and stem cell genes. Data shown are representative of 2 independent experiments. (B) Gene expression in LT-HSCs from Scl+/+ and Scl+/− bone marrows was determined by quantitative RT-PCR. mRNA levels of the indicated genes were first normalized using Hprt as an internal control and second compared with expression levels in the Scl+/+ LT-HSC population, which was set as 1 (illustrated here as the dotted line; mean ± SD of 4 replicates after normalization; 2 experiments). (C) Diagram illustrating the positions of primers used for ChIP in panel D for the indicated murine genes. Note the presence of E boxes in the vicinity of the primers. Black box represents TAL1 E boxes (supplemental Figure 4A)42 ; open box, other E boxes. (D) Chromatin extracts from lineage-depleted bone marrow cells were immunoprecipitated with an anti-murine SCL antibody or species-matched IgG (control, shown as dotted line). Immunoprecipitated promoter sequences were analyzed by quantitative PCR (mean ± SD of 4 replicates; 2 experiments). β-actin downstream sequences serve as negative controls for PCR amplification.
