Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts.

Divya S Vinjamur,Parth Amin,Yousef N Alhashem,Joyce A Lloyd,David C Williams,Safa F Mohamad

doi:10.1371/journal.pone.0146802

Abstract

In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies.

Highlights

Krüppel-like factor 1 (KLF1 or EKLF, Erythroid Krüppel-like factor) is a transcription factor that regulates erythrocyte development, including β-like globin gene expression, and synthesis of heme, the cytoskeleton, and the cell membrane ([1,2,3] reviewed in [4])
Chromatin immunoprecipitation (ChIP) assays indicate that Krüppel-like transcription factor 1 (KLF1) is enriched in the β-globin locus control region (LCR) at 5’ DNase I hypersensitive sites, 5’HS3 and 5’HS2, and at the β-globin promoter in Umbilical cord blood (UCB) erythroblasts at differentiation day 8 (DD8) (Fig 1)
These results suggest that KLF1 directly regulates the β-globin gene, but probably has less direct impact on expression of the γ-globin gene in UCB erythroblasts

Summary

Introduction

Krüppel-like factor 1 (KLF1 or EKLF, Erythroid Krüppel-like factor) is a transcription factor that regulates erythrocyte development, including β-like globin gene expression, and synthesis of heme, the cytoskeleton, and the cell membrane ([1,2,3] reviewed in [4]). KLF1 binds to the β-globin locus control region (LCR) and gene promoters, and interacts with histone acetyl transferases (HATs) [7,8,9]. Occurring heterozygous KLF1 mutations can cause hereditary persistence of fetal hemoglobin (α2γ2) [12,13,14]. Heterozygous KLF1 mutations are relatively more common in a thalassemia endemic region in China, and apparently ameliorate the severity of βthalassemia by increasing HbF [15]. There was a case report of an individual who is KLF1 null. This patient required transfusions from the age of 6 months due to anemia, but displayed very elevated fetal hemoglobin (HbF) of >70% up to 1 year of age [14]. Lentiviral knockdown of KLF1 in cultured adult erythroblasts results in increased γ-globin and decreased BCL11A, a γ-globin repressor [12,16,17,18]

Methods

Results

Conclusion