Figure 5
Figure 5. Levels of expression of erythroid-specific genes in SCL mutant erythroid cells. (A top) Schematic representation of the progressive maturation of erythroid cells. The BFU-E, CFU-E, and proE stages are Ter119−, whereas basoE, polyChE, orthoChE erythroid cells, retic, and RBC are Ter119+. (Bottom) Total RNA was prepared from Ter119− erythroid progenitors (day 0 population) or Ter119+ erythroid cells (day 2 population) derived from day E12.5 embryos. The genes chosen for analysis and indicated on the left side of the figure are grouped into 2 categories (early and intermediate) according to their normal pattern of expression during erythroid differentiation from the early BFU-E stage to mature erythrocytes22,32 (and this study). Levels of gene expression during normal erythroid maturation are schematized by gray triangles. The y-axis represents the enrichment in cDNA sequences as quantitated by real-time PCR normalized to 18S ribosomal gene control sequences for each gene. Error bars indicate plus or minus 1 SD from at least 3 independent experiments (*P < .01). (B) Chromatin derived from Ter119− and Ter119+ populations purified from sclWt/Wt and sclRER/RER fetal liver cultured cells was immunoprecipitated with α-SCL antibodies and the scl + 40 enhancer region analyzed by real-time PCR. The y-axis represents enrichment over input DNA, normalized to a control sequence in the gapdh gene. N indicates the transcriptionally inactive −16 region of the scl locus.

Levels of expression of erythroid-specific genes in SCL mutant erythroid cells. (A top) Schematic representation of the progressive maturation of erythroid cells. The BFU-E, CFU-E, and proE stages are Ter119, whereas basoE, polyChE, orthoChE erythroid cells, retic, and RBC are Ter119+. (Bottom) Total RNA was prepared from Ter119 erythroid progenitors (day 0 population) or Ter119+ erythroid cells (day 2 population) derived from day E12.5 embryos. The genes chosen for analysis and indicated on the left side of the figure are grouped into 2 categories (early and intermediate) according to their normal pattern of expression during erythroid differentiation from the early BFU-E stage to mature erythrocytes22,32  (and this study). Levels of gene expression during normal erythroid maturation are schematized by gray triangles. The y-axis represents the enrichment in cDNA sequences as quantitated by real-time PCR normalized to 18S ribosomal gene control sequences for each gene. Error bars indicate plus or minus 1 SD from at least 3 independent experiments (*P < .01). (B) Chromatin derived from Ter119 and Ter119+ populations purified from sclWt/Wt and sclRER/RER fetal liver cultured cells was immunoprecipitated with α-SCL antibodies and the scl + 40 enhancer region analyzed by real-time PCR. The y-axis represents enrichment over input DNA, normalized to a control sequence in the gapdh gene. N indicates the transcriptionally inactive −16 region of the scl locus.

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