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Hemoglobin switching in Klf1wt/Nan mice
Figure 6. Expression of human and mouse globins in bone marrow and spleen of adult control and Klf1wt/Nan animals. Expression of mouse and human globins was determined by reverse transcriptase quantitative PCR. Expression ratios of individual globins in Klf1wt/Nan samples were calculated relative to those observed for the control samples (Klf1wt/wt). Note logarithmic scale of the y-axis. N=3 for each group. *P<0.05; error bars indicate standard deviations.
gene. Two mechanisms may contribute to this phenome- non. Firstly, decreased proliferation and differentiation of fetal erythroblasts delays the replacement of primitive by definitive erythroid cells. Since we isolated RNA from populations of cells, the ratio of primitive/definitive cells will have an impact on the globin levels measured. Secondly, although by E16.5 adult globins are quantita- tively the dominant globins in Klf1wt/Nan embryos, the embryonic and fetal globin genes retained expression in adult spleen and bone marrow. Qualitatively, this persist- ent expression is another phenotypic similarity with CDA-IV patients. However, quantitatively there is a major difference. While in CDA-IV patients HbF levels of up to 37% of total hemoglobin have been reported,20 even the most highly expressed embryonic globin in adult Klf1wt/Nan mice, mz, contributes only 0.3% to the total amount of α-like globins. KLF1 activates expression of BCL11A14,40 and LRF,41 two transcriptional repressors directly involved in hemoglobin switching.42,43 RT-qPCR analysis of BCL11A and LRF expression indicates that reduced expression of these two factors contributes to the sustained expression of the embryonic/fetal genes in Klf1wt/nan erythroid cells (Online Supplementary Figure S2). We note that expression of BCL11A is also significantly reduced in Klf1wt/ko cells with little effect on expression of the embryonic/fetal genes.36 Mechanistically, this sug- gests that the repressor proteins are expressed well above the critical threshold level in mice, and a reduction to 40- 60% of normal expression would still be sufficient for quantitative silencing of the embryonic/fetal genes.
The impaired proliferation of E12.5 fetal liver-derived Klf1wt/Nan erythroblasts was initially surprising because a lack of KLF1 increased proliferation, likely due to impaired spontaneous differentiation.9 Whereas erythropoiesis in Klf1ko/ko mice is severely affected during terminal differen- tiation, erythropoiesis in Klf1wt/nan mice is much less affec- ted with respect to terminal differentiation. The presence of KLF1Nan not only results in the reduced expression of KLF1 target genes, but also induces expression of genes not normally regulated by KLF1.26,27,44 The combined effects of deregulation of canonical KLF1 target genes and ectopic gene expression likely underlie the observed line- age commitment infidelity and impaired proliferation and differentiation of Klf1wt/nan erythroblast cultures. Defective
growth of erythroid progenitor cultures derived from a CDA-IV patient has been reported,45,46 indicating that impaired proliferation of erythroid progenitors is another hallmark that CDA-IV patients and Klf1wt/Nan mice have in common. In contrast, adult Klf1wt/Nan mice expressed main- ly adult-type globin genes, as opposed to adult CDA-IV patients who maintain expression of embryonic and fetal globins at substantial levels.20-22,45
Importantly, the dominant effect of KLF1Nan is illustrat- ed by comparison with Klf1wt/ko mice which do not display deregulated expression of mouse embryonic globins in E14.5 yolk sac and fetal liver and adult bone morrow36 (Online Supplementary Figure S3). We found that the effects of KLF1Nan on developmental regulation of globin expression are very consistent, but surprisingly subtle. KLF1Nan affects the dynamics of progression from primi- tive to definitive erythropoiesis during mouse develop- ment. Compared to control embryos, definitive erythro- cytes emerge at a later stage as the dominant cell type in the circulation of Klf1wt/Nan embryos. We propose that this is at least in part due to the reduced expansion and differ- entiation capacity of the fetal liver progenitors, since cul- tured Klf1wt/Nan E12.5 fetal liver cells display growth and differentiation defects. Consistent with impaired ery- throid diffe-rentiation, we observed aberrant expression of erythroid flow cytometry markers (CD71, Ter119, CD9). Furthermore, we found misexpression of myeloid markers, in particular the megakaryocyte marker CD41, indicating that Klf1wt/Nan erythroid progenitors display lineage infidelity. This is akin to the previously reported aberrantly activated megakaryocyte program in Klf1ko/ko erythroid cells.39,47
Collectively, our data support the notion that KLF1CDA and KLF1Nan present with similar but also variant-specific phenotypes. Thus, our study further highlights the need to investigate the effects of individual KLF1 variants in detail.1 The recently developed human adult erythroid progenitor cell lines HUDEP-248 and BEL-A49 could be combined with CRISPR-mediated homology-directed recombination50 to investigate the impact of individual human KLF1 variants on the molecular control of erythro- poiesis, with a view to increase understanding of the broad spectrum of human red blood cell disorders caused by KLF1 variants.1
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