Reactivation of fetal γ-globin is of outstanding demand in patients with β-hemoglobinopathies. B-Cell/Lymphoma 11A (BCL11A) is a well-known repressor of γ-globin, and its expression is directly activated by Kruppel-Like Factor 1 (KLF1). KLF1 is a major regulator of human fetal to adult hemoglobin switching and reduced expression of KLF1 due to mutations is associated with hereditary persistence of fetal hemoglobin (HPFH). Analysis of the HPFH phenotype has led to the proposal that KLF1 has a dual role in γ-globin suppression, through its preferential activation of the β-globin gene and as a key activator of expression of the BCL11A repressor protein. To study regulation of erythropoiesis and globin expression by KLF1 and BCL11a in an in vivo model, we used mice carrying a human β-globin locus transgene with combinations of Klf1 haploinsufficiency, and Bcl11a floxed and EpoRCre knockin alleles. We measured hematological parameters of the mutant mice. With the exception of a small reduction in MCV (mean corpuscular volume), parameters of Klf1wt/ko animals were similar to those observed in the control animals. Bcl11acko/cko animals displayed a small but significant reduction of HCT (Hematocrit), RBC (red blood cell count) and HGB (hemoglobin) values. The reductions in these values were more pronounced in the Klf1wt/ko::Bcl11acko/cko animals. In addition, Klf1wt/ko::Bcl11acko/cko mice displayed small but significantly increased values for MCV, MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration). We observed a higher concentration of erythropoietin in Bcl11acko/cko and compound Klf1wt/ko::Bcl11acko/cko animals suggesting a mild compensated anemia. To extend these observations, we analyzed embryonic blood and fetal livers at day E18.5, just prior to birth. Flow cytometry analysis of E18.5 blood revealed no difference in the CD71+Ter119+ population in peripheral blood of Bcl11acko/cko embryo's. This percentage was increased in Klf1wt/ko blood samples and was highest in blood from the compound Klf1wt/ko::Bcl11acko/cko embryos. Similar results were obtained following flow cytometry of E18.5 fetal liver cells. Consequently, the percentage of mature CD71−/Ter119+ cells in fetal liver and peripheral blood of E18.5 Klf1wt/ko::Bcl11acko/cko embryos was significantly lower than that observed in Klf1wt/ko, Bcl11acko/cko and control embryos. Analysis of Klf1wt/ko, Bcl11acko/cko and Klf1wt/ko::Bcl11acko/cko mutant embryos demonstrated increased expression of mouse embryonic α - and β-like globins during fetal development. Expression of human γ-globin remained high in Bcl11acko/cko embryos during fetal development, and this was further augmented in Klf1wt/ko::Bcl11acko/cko embryos. After birth, expression of human γ-globin and mouse embryonic globins decreased in Bcl11acko/cko and Klf1wt/ko::Bcl11acko/cko mice, but the levels remained much higher than those observed in control animals. We find that haploinsufficiency for KLF1 delays γ- to β-globin switching leading to a ∼2-fold increase in the γ/(γ+β) ratio at E14.5 and E18.5. Part of this increase can be explained by diminished BCL11A expression in embryos with KLF1 insufficiency. Collectively, these data support the proposed role of the KLF1-BCL11A axis in γ-globin regulation. In conclusion, our results suggest that haploinsufficiency for KLF1 prolongs reticulocyte maturation, and that this phenotype is further exacerbated in combination with BCL11A deficiency. Despite this, the impact on erythropoiesis is modest and none of the compound mutant mice suffer from overt anemia even at prenatal stages when the demand for erythroid expansion is high. Collectively, our data support an important role for the KLF1-BCL11A axis in erythroid maturation and developmental regulation of globin expression and importantly in the absence of BCL11A, KLF1 still preferentially activates the β-globin gene.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.