Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin

Abstract

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12-13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

Figure 1. Chromosome 19 locus linked to HPFH in a Maltese family

a) The Maltese HPFH pedigree. HbF levels are indicated as percentage of total Hb (%HbF). HPFH individuals are shown as half-filled symbols. b) LOD scores derived from genome-wide linkage analysis. The putative HPFH locus on chr. 19 is indicated by an arrow. pLOD = parametric LOD score; MPT = multi point test; cM = centiMorgan. c) Sequence analysis of KLF1. HPFH individuals were heterozygous for two mutations (arrows; Supplementary Table 1). The predicted effects of the mutations on KLF1 protein are shown below.

Figure 2. KLF1 target genes are downregulated in wt/KLF1 p.K288X HEPs

a) RNA isolated from HEPs derived from normal (wt/wt) and HPFH (wt/KLF1 p.K288X) family members was used for genome-wide expression analysis. Deregulated genes common between wt/wt and wt/KLF1 p.K288X and mouse wt/wt versus Klf1 null mutant erythroid progenitors (Supplementary Table 2) were used for cluster analysis. b) Validation of key target genes by qPCR. Expression levels of BCL11A were normalized using GAPDH as a reference. Expression levels of HBG1/HBG2 (HBG) were calculated as ratio to HBA1/HBA2 (HBA) expression. Medians are indicated by red lines in the box plots. Asterisk: p=0.0209.

Figure 3. Increased HBG1/HBG2 expression after knockdown of KLF1 in normal HEPs

a) HEPs derived from normal donors were transduced with shRNA-expressing lentiviruses. Cells were harvested five days after transduction, and nuclear extracts prepared. Top panels: KLF1 protein expression assessed by Western blot analysis. Bottom panels: BCL11A protein levels were reduced upon KLF1 knockdown. NPM1 served as a loading control. None = mock transduction; TRC = control non-specific shRNA; sh1 and sh2 = two independent shRNAs targeting KLF1. A non-specific band is indicated (<). b) RNA was isolated from HEPs five days after transduction with the indicated lentiviruses, and used in quantitative S1 nuclease protection assays to measure globin expression. Arrows indicate protected fragments diagnostic for HBA1/HBA2 (HBA), HBG1/HBG2 (HBG) and HBB mRNAs. c) Quantitation of data shown in b) by Phosphorimager analysis. d) Box plots of HBG/HBA ratios after sh1-mediated KLF1 knockdown in HEPs derived from three independent donors. Medians are indicated by red lines. Asterisk: p=0.0463. e) Box plots of qPCR analysis of AHSP, BCL11A and HBG expression after sh1/sh2-mediated KLF1 knockdown. AHSP is a known KLF1 target gene and serves as a positive control. Expression levels of AHSP and BCL11A were normalized using GAPDH as a reference. Expression levels of HBG were calculated as ratio to total β-like globin expression (HBG + HBB) expression. Medians are indicated by red lines. Circles: points outside the range of the error bars. Asterisk: p=0.020; double asterisks: p<0.003.

Figure 4. Expression of exogenous KLF1 in HPFH HEPs

a) HEPs derived from individual II-5 were transduced with lentiviral constructs expressing GFP, KLF1 truncated at amino acid 288 (TR) or full-length KLF1 (FL). Seven days after transduction, nuclear extracts were prepared and expression of BCL11A and KLF1 was assessed by Western blot. NPM1 served as a loading control. b) RNA was isolated from II-5 HEPs seven days after transduction with the indicated lentiviruses, and was used for quantitative S1 nuclease protection assays to measure globin mRNA expression. Arrows indicate protected fragments diagnostic for HBA1/HBA2 (HBA), HBG1/HBG2 (HBG) and HBB mRNAs. c) Quantitation of data shown in b) by Phosphorimager analysis.

Figure 5. KLF1 binds to the promoter of the BCL11A gene in vivo

a) Schematic drawings of the promoter areas of the BCL11A, HBB and RASSF1A genes. Positions of potential KLF1 binding sites (CACC boxes) and PCR primers used are indicated. Arrows indicate transcription start sites. b) ChIP analysis of KLF1 binding to the BCL11A promoter in human fetal liver cells and adult HEPs. The HBB promoter served as a positive control . RASSF1A was used as a negative control, and the unrelated CD71 antibody served as a control for the specificity of the KLF1 antibody. Asterisk: p<0.05; double asterisks: p<0.01. Error bars: standard deviation.