American Society of Hematology

Figure 2

$Figure 2. Expression and elongation studies in cells homozygous for WT or LCR mutant β-globin loci. (A-D) Expression of α- and β-globin from WT and mutant alleles during erythroid differentiation by primary transcript RNA FISH. Fetal liver cells from at least 6 homozygous WT or homozygous mutant fetuses were fractionated by flow cytometry and probed for α-globin (red) and β-major primary transcripts (green) by RNA FISH, followed by counterstaining of nuclei by DAPI staining (blue). Scale bar indicates 2 μm. Representative images from WT mice of fraction 3 (A) and fraction 4 (B) are shown. (C) Ratio of β-globin to α-globin nascent transcripts. Values are the ratio of β-globin to α-globin alleles with bursts of active transcription (foci of nascent transcripts) shown as a percentage. Solid bars represent values for fraction 3 and hatched bars are fraction 4. More than 100 cells were analyzed for each data point. (D) β-globin mRNA per active allele. To obtain an estimate of the relative mRNA production from each burst of active transcription, we compared the effect of each mutation on mRNA production (Table 2) with its effect on bursts of transcription (Figure 2C). Values are the ratio of the percentage of WT β-globin mRNA accumulation for each mutation to the percentage of WT alleles associated with foci of nascent transcripts. Solid bars represent data using fraction 3 and hatched bars are fraction 4. Values are divided by 1.02 so that WT yields 100%. (E-F) Recruitment of elongation components to the β-globin locus in erythroid cells from mice homozygous for LCR HS deletions normalized to WT. ChIP was performed on WT and LCR mutant chromatin using Abs to FACT component SPT16 (E) and DSIF component SPT5 (F). Three different chromatin preparations were analyzed by quantitative RT-PCR of the β-major globin start site (β-Start) and exon-2 of β-major (β-Ex2) regions and normalized to necdin, which is not transcribed in these cells. Values for each mutant line were normalized to the WT line (*P < .05 and **P < .001 relative to WT). The same data without normalization to WT is shown in supplemental Figure 1A and B. Error bars represent the SEM.$

Expression and elongation studies in cells homozygous for WT or LCR mutant β-globin loci. (A-D) Expression of α- and β-globin from WT and mutant alleles during erythroid differentiation by primary transcript RNA FISH. Fetal liver cells from at least 6 homozygous WT or homozygous mutant fetuses were fractionated by flow cytometry and probed for α-globin (red) and β-major primary transcripts (green) by RNA FISH, followed by counterstaining of nuclei by DAPI staining (blue). Scale bar indicates 2 μm. Representative images from WT mice of fraction 3 (A) and fraction 4 (B) are shown. (C) Ratio of β-globin to α-globin nascent transcripts. Values are the ratio of β-globin to α-globin alleles with bursts of active transcription (foci of nascent transcripts) shown as a percentage. Solid bars represent values for fraction 3 and hatched bars are fraction 4. More than 100 cells were analyzed for each data point. (D) β-globin mRNA per active allele. To obtain an estimate of the relative mRNA production from each burst of active transcription, we compared the effect of each mutation on mRNA production (Table 2) with its effect on bursts of transcription (Figure 2C). Values are the ratio of the percentage of WT β-globin mRNA accumulation for each mutation to the percentage of WT alleles associated with foci of nascent transcripts. Solid bars represent data using fraction 3 and hatched bars are fraction 4. Values are divided by 1.02 so that WT yields 100%. (E-F) Recruitment of elongation components to the β-globin locus in erythroid cells from mice homozygous for LCR HS deletions normalized to WT. ChIP was performed on WT and LCR mutant chromatin using Abs to FACT component SPT16 (E) and DSIF component SPT5 (F). Three different chromatin preparations were analyzed by quantitative RT-PCR of the β-major globin start site (β-Start) and exon-2 of β-major (β-Ex2) regions and normalized to necdin, which is not transcribed in these cells. Values for each mutant line were normalized to the WT line (*P < .05 and **P < .001 relative to WT). The same data without normalization to WT is shown in supplemental Figure 1A and B. Error bars represent the SEM.

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