FIG. 4.

(A) SCL +19 core enhancer is active in both blood and bone cell lines. (i) Stable transfection assay in 416B cells, an SCL-expressing cell line derived from a multipotent hematopoietic progenitor. (ii) Stable transfection assay in MC3T3-E1 cells, a preosteoblastic cell line. The luciferase activities are given as increase over the activity of the basic (SV/luc) vector. Each bar is the mean of the relative luciferase activity from at least two experiments performed in triplicate ± standard deviation. (B) Fli1, Elf1, and GATA2 bind the SCL +19 core enhancer in both blood and bone cell lines. The expression profiles of selected Ets and GATA factors were assessed by RT-PCR in (i) 416B and (ii) MC3T3-E1 cell lines. Chromatin immunoprecipitation assays were performed with anti-Ets1, -Ets2, -Fli1, -Elf1, -GATA2, -acetylated histone H3 (K9A), and control immunoglobulin G antibodies in the (iii) 416B and (iv) MC3T3-E1 cell lines. The level of enrichment with each antibody was normalized to the levels obtained with control immunoglobulin G and plotted as the increase over the level of enrichment at a control region (the promoter region of the α-fetoprotein gene). Histone acetylation status (K9A) was used as a surrogate marker of chromatin accessibility. The SCL +19 region is accessible in both cell lines and is bound by Fli1, Elf1, Erg, and GATA2 in 416B cells and Fli1, Elf1, and GATA2 in MC3T3-E1 cells. Histone acetylation and transcription factor binding at the SCL +19 region are lower in MC3T3-E1 cells than in 416B cells, in line with the lower expression of endogenous SCL in the former.