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

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

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

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

ZNF410 represses fetal globin by devoted control of CHD4/NuRD [preprint]

Major effectors of adult-stage fetal globin silencing include the transcription factors (TFs) BCL11A and ZBTB7A/LRF and the NuRD chromatin complex, although each has potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here through CRISPR screening we discover ZNF410 to be a novel fetal hemoglobin (HbF) repressing TF. ZNF410 does not bind directly to the γ-globin genes but rather its chromatin occupancy is solely concentrated at CHD4, encoding the NuRD nucleosome remodeler, itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for ZNF410’s effects on γ-globin repression. Knockout of ZNF410 reduces CHD4 by 60%, enough to substantially de-repress HbF while avoiding the cellular toxicity of complete CHD4 loss. Mice with constitutive deficiency of the homolog Zfp410 are born at expected Mendelian ratios with unremarkable hematology. ZNF410 is dispensable for human hematopoietic engraftment potential and erythroid maturation unlike known HbF repressors. These studies identify a new rational target for HbF induction for the β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved transcription factor with singular devotion to regulation of a chromatin subcomplex

Scr and KLF1 shRNA-treated UCB erythroblasts have similar erythroid differentiation profiles on differentiation day 8 (DD8).

<p><b>(A) Efficiency of KLF1 knockdown.</b> Top panel: KLF1 mRNA amount was measured in scramble (Scr) and KLF1 shRNA-treated cells by qRT-PCR. The amount of KLF1 mRNA in scramble-treated cells was set to 100 for each sample. Cyclophilin A mRNA was used as the internal standard for qRT-PCR. N = 19 for Scr shRNA, N = 3 for KLF1 shRNA-V1 and N = 16 for KLF1 shRNA-V2; error bars = standard error, * = p-value<0.001. Bottom panel: Representative Western blot analysis in Scr and KLF1 shRNA treated UCB erythroblasts. Actin was used as a loading control. For this sample, residual KLF1 mRNA = 13% and residual KLF1 protein = 42%. <b>(B) KLF1 knockdown does not adversely affect erythroid differentiation.</b> Representative flow cytometry plots indicate that the majority of erythroid cells are double positive for CD71 and CD235a in both scramble shRNA-infected and KLF1 shRNA-V2-infected cells on DD8. N = 3. Student’s t-test showed no significant difference in the number of double positive cells between Scr and KLF1 shRNA-treatment groups. Residual KLF1 mRNA = 13–25%. <b>(C) KLF1 knockdown does not affect erythroid maturation.</b> At day 8 and at day 12 of differentiation, cytospin slides of Scr or KLF1 shRNA treated cells were prepared, and erythroid maturation was scored blind to treatment group. 100 cells from each slide were scored based on their morphology and designated as basophilic, polychromatic or orthochromatic erythroblasts. N = 5, error bars = standard error. Student’s t-test showed no significant differences in the number of erythroblasts of each type between Scr and KLF1 shRNA treatment groups. Residual KLF1 mRNA = 6–24%.</p

KLF1 regulates β-globin, total globin and BCL11A expression in UCB erythroblasts.

<p>The amount of KLF1, β-globin, γ-globin and BCL11A mRNA was measured by qRT-PCR and normalized to Cyclophilin A mRNA. The fold change in expression of these genes in KLF1 shRNA-treated samples was calculated by setting the value in Scr shRNA controls to 100. Each point on the Pearson’s correlation plots represents a biological replicate, i.e. cells obtained from a different umbilical cord blood sample. Three of the samples were infected with the K1V1 shRNA, and the remaining samples (the majority) were infected with the K1V2 shRNA. <b>(A) A strong, positive correlation was observed between the amounts of KLF1 and β-globin mRNA</b> (N = 19, r² = 0.65, Prob>F = 0.0001); <b>(B) The amount of total globin (γ+β) expression positively correlates with residual KLF1 mRNA</b> (N = 19, r² = 0.54, Prob>F = 0.0003) <b>(C) There is a modest positive correlation between the amounts of KLF1 and BCL11A mRNA</b> (N = 17, r² = 0.27, Prob>F = 0.032). <b>(D) There is no linear relationship between the amounts of KLF1 and γ-globin mRNA</b> (N = 19, r² = 0.16, Prob>F = 0.095).</p

γ-globin mRNA amounts with modest and robust KLF1 knockdown.

<p>UCB samples were divided into two groups based on the amount of residual KLF1 mRNA after knockdown (see supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146802#pone.0146802.g002" target="_blank">Fig 2B</a>). The first group consists of samples which showed robust KLF1 knockdown (<20% residual KLF1; N = 10). The second group had modest knockdown of KLF1 (20–60% residual KLF1, N = 9). The primers for γ-globin collectively measure ^Aγ- and ^Gγ-globin mRNA. <b>A) Absolute amounts of γ-globin mRNA are increased above normal only in the presence of a threshold amount of KLF1.</b> Within each group, the fold change in γ-globin expression in KLF1 shRNA-treated cells was calculated by setting the amount of γ-globin mRNA in Scr shRNA-treated controls to 100. Cyclophilin A mRNA was used as the internal standard for qPCR. Error bars = standard error, * = p-value<0.005, NS = not significant. <b>(B) γ-globin is increased relative to total (γ+β)-globin with any amount of KLF1 knockdown.</b> The amount of γ-globin mRNA in Scr and KLF1 shRNA-treated cells was measured by qRT-PCR and reported as a fraction of total (γ+β)-globin mRNA. In each group the fold change in γ-globin expression in KLF1 shRNA-treated cells was calculated by setting the amount of γ-globin in Scr shRNA-treated controls to 100. Error bars = standard error, * = p-value<0.01.</p

KLF1 is enriched at the locus control region (LCR) and β-globin promoter in umbilical cord blood (UCB) erythroblasts.

<p>CD34+ cells were enriched from umbilical cord blood units, expanded for eight days, and <i>in vitro</i> differentiated for eight days (DD8). Chromatin immunoprecipitation (ChIP) assays were performed with DD8 erythroblasts. Chromatin was incubated with either a KLF1 antibody or non-specific antibody (IgG) as a negative control (black or white bars, respectively). Precipitated chromatin was subjected to quantitative PCR (qPCR) to determine the amount of precipitated DNA from 5’HS3 and 5’HS2 in the LCR, and from the ε-, γ- and β-globin promoters. The primers for the γ-globin promoter collectively measure the ^Aγ- and ^Gγ-globin genes. The insulin promoter was used as a negative control. Pr = promoter, N = 2, error bars = standard error, * = p-value<0.05 compared to non-specific antibodies. β-globin locus not drawn to scale.</p

Molecular analysis of the erythroid phenotype of a patient with BCL11A haploinsufficiency

The BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G.T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the g-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient's cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of g-globin expression in b-hemoglobinopathy patients. Our data strongly support this approach