American Society of Hematology

Figure 3

$Figure 3. Nuclear localization studies in cells homozygous for WT or LCR mutant β-globin loci. (A-E) Relocalization of the WT and mutant β-globin loci away from the nuclear periphery during erythroid differentiation. Fetal liver cells from at least 6 homozygous WT or homozygous mutant fetuses were fractionated by flow cytometry and probed by DNA FISH (green), followed by immunostaining of LaminB1 (red) to define the nuclear periphery and counterstaining of nuclei by DAPI staining (blue). Scale bar indicates 2 μm. Representative images from WT and Δ1-2 cells of fraction 2 (A,C) and fraction 4 (B,D) are shown. Images are single z-sections, so the diameter of the nucleus varies depending on the level of the slice. (E) Percentage of β-globin loci located away from the nuclear periphery in immature (fraction 2) and mature (fraction 4) erythroid cells. In fraction 2, no significant differences between WT and LCR HS mutants are present. In contrast, in fraction 4, significantly more WT alleles are located away from the nuclear periphery than for each of LCR HS-deleted lines (*P < .05). Values also vary significantly between Δ1,4 and all other mutants (P < .05) and between the ΔLCR and Δ1-2 mutations (bracketed and shown by asterisks). More than 100 loci were analyzed for each data point. Error bars represent the SEM. (F-I) Association of WT and β-globin loci to foci of hyperphosphorylated Pol II. (F-H) Fetal liver cells from at least 6 homozygous WT or homozygous mutant fetuses were fractionated by flow cytometry and probed by DNA FISH (green), followed by immunostaining of phospho-Pol II (red) to define transcription factories and counterstaining of nuclei by DAPI staining (blue). Scale bar indicates 2 μm. Representative images from WT (F) and Δ1-2 (G) cells are shown. Both alleles are shown in their respective z-sections. (H) Percentage of β-globin loci overlapping with TFys in mature erythroid cells (fraction 4). Association frequency of the ΔLCR and Δ2-3 mutant alleles with TFys differs significantly from all other genotypes (*P < .05). WT, Δ1-2, and Δ1,4 did not differ significantly from each other. More than 50 loci were analyzed for each data point. Error bars represent the SEM. (I) Recruitment of hyperphosphorylated Pol II to the β-globin gene in erythroid cells from mice homozygous for LCR HS deletions normalized to WT. ChIP was performed on WT and LCR mutant chromatin using a phosphorylated form of PolII. Three different chromatin preparations were analyzed by quantitative RT-PCR of the β-major globin start site (β-Start) and normalized to actin. Values for each mutant line were normalized to the WT line (*P < .05 relative to WT).$

Nuclear localization studies in cells homozygous for WT or LCR mutant β-globin loci. (A-E) Relocalization of the WT and mutant β-globin loci away from the nuclear periphery during erythroid differentiation. Fetal liver cells from at least 6 homozygous WT or homozygous mutant fetuses were fractionated by flow cytometry and probed by DNA FISH (green), followed by immunostaining of LaminB1 (red) to define the nuclear periphery and counterstaining of nuclei by DAPI staining (blue). Scale bar indicates 2 μm. Representative images from WT and Δ1-2 cells of fraction 2 (A,C) and fraction 4 (B,D) are shown. Images are single z-sections, so the diameter of the nucleus varies depending on the level of the slice. (E) Percentage of β-globin loci located away from the nuclear periphery in immature (fraction 2) and mature (fraction 4) erythroid cells. In fraction 2, no significant differences between WT and LCR HS mutants are present. In contrast, in fraction 4, significantly more WT alleles are located away from the nuclear periphery than for each of LCR HS-deleted lines (*P < .05). Values also vary significantly between Δ1,4 and all other mutants (P < .05) and between the ΔLCR and Δ1-2 mutations (bracketed and shown by asterisks). More than 100 loci were analyzed for each data point. Error bars represent the SEM. (F-I) Association of WT and β-globin loci to foci of hyperphosphorylated Pol II. (F-H) Fetal liver cells from at least 6 homozygous WT or homozygous mutant fetuses were fractionated by flow cytometry and probed by DNA FISH (green), followed by immunostaining of phospho-Pol II (red) to define transcription factories and counterstaining of nuclei by DAPI staining (blue). Scale bar indicates 2 μm. Representative images from WT (F) and Δ1-2 (G) cells are shown. Both alleles are shown in their respective z-sections. (H) Percentage of β-globin loci overlapping with TFys in mature erythroid cells (fraction 4). Association frequency of the ΔLCR and Δ2-3 mutant alleles with TFys differs significantly from all other genotypes (*P < .05). WT, Δ1-2, and Δ1,4 did not differ significantly from each other. More than 50 loci were analyzed for each data point. Error bars represent the SEM. (I) Recruitment of hyperphosphorylated Pol II to the β-globin gene in erythroid cells from mice homozygous for LCR HS deletions normalized to WT. ChIP was performed on WT and LCR mutant chromatin using a phosphorylated form of PolII. Three different chromatin preparations were analyzed by quantitative RT-PCR of the β-major globin start site (β-Start) and normalized to actin. Values for each mutant line were normalized to the WT line (*P < .05 relative to WT).

This Feature Is Available To Subscribers Only