PUM1 mediates the posttranscriptional regulation of human fetal hemoglobin

Abstract

The fetal-to-adult hemoglobin switching at about the time of birth involves a shift in expression from γ-globin to β-globin in erythroid cells. Effective re-expression of fetal γ-globin can ameliorate sickle cell anemia and β-thalassemia. Despite the physiological and clinical relevance of this switch, its posttranscriptional regulation is poorly understood. Here, we identify Pumilo 1 (PUM1), an RNA-binding protein with no previously reported functions in erythropoiesis, as a direct posttranscriptional regulator of β-globin switching. PUM1, whose expression is regulated by the erythroid master transcription factor erythroid Krüppel-like factor (EKLF/KLF1), peaks during erythroid differentiation, binds γ-globin messenger RNA (mRNA), and reduces γ-globin (HBG1) mRNA stability and translational efficiency, which culminates in reduced γ-globin protein levels. Knockdown of PUM1 leads to a robust increase in fetal hemoglobin (∼22% HbF) without affecting β-globin levels in human erythroid cells. Importantly, targeting PUM1 does not limit the progression of erythropoiesis, which provides a potentially safe and effective treatment strategy for sickle cell anemia and β-thalassemia. In support of this idea, we report elevated levels of HbF in the absence of anemia in an individual with a novel heterozygous PUM1 mutation in the RNA-binding domain (p.(His1090Profs∗16); c.3267_3270delTCAC), which suggests that PUM1-mediated posttranscriptional regulation is a critical player during human hemoglobin switching.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

Figures

Figure 1.

EKLF upregulates PUM1 during erythroid terminal differentiation, PUM1 knockdown robustly increases the levels of γ-globin protein and HbF without altering β-globin, and PUM1 overexpression decreases the levels of γ-globin protein. (A) RNA sequencing analysis of Eklf+/+ and Eklf−/− murine Extensively Self Renewing Erythroblasts (ESRE) shows Pum1 transcript levels during expansion and differentiation (n=3). (B) The human PUM1 gene sequence with transcriptional start at position +1 (hg19; chr1 [p35.2]). Transcription factor–binding motifs for EKLF, p300, histone marks H3K4me3 and H3K4me2, and RNA Pol II binding are shown. (C) Western blot analysis of HUDEP2 cell extracts harvested after infection with either PUM1 or non-target control (NTC) lentiviral short hairpin RNAs (shRNAs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. Quantitation of the indicated proteins by western blot analysis of HUDEP2 cell extracts upon PUM1 knockdown is shown on the right (n = 3). (D) HPLC analysis of Hb in HUDEP2 cells after infection with either PUM1 or NTC lentiviral shRNAs was performed to quantitate the percentages of HbF and adult hemoglobin (HbAo) upon erythroid terminal differentiation (day 10). (E) Results of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay show the extent of PUM1 knockdowns after lentiviral transduction of the 3 PUM1 shRNAs in adult human primary HSPCs (n = 2). (F) Western blot analysis of adult human primary HSPC extracts harvested on day 11 of erythroid differentiation after infection with either PUM1 or NTC lentiviral shRNAs. GAPDH was used as the loading control. Quantitation of the indicated proteins by western blot analysis of HUDEP2 cell extracts upon PUM1 knockdown is shown on the right (n = 2). (G) Western blot analysis of erythroleukemia cell line K562 cells with and without PUM1 overexpression (OE). GAPDH was used as the loading control. Quantitation of the indicated proteins by western blot analysis of K562 cell extracts upon PUM1 overexpression is shown on the right (n = 3). (H) Flow cytometry analysis of adult primary human HSPCs at day 7 of erythroid differentiation using human CD71 and glycophorin A (hGlyA) antibodies shows that PUM1 knockdown does not impair the progression of erythroid differentiation. Gates are drawn based on unstained and single-color controls. Population percentages within each gate are indicated. Data were analyzed by using a two-sided Student t test. Bar graphs show mean ± standard deviation (SD). ∗P < .05; ∗∗P < .005; ∗∗∗P < .0005. FITC, fluorescein isothiocyanate; NS, not significant; PE, phycoerythrin.

Figure 2.

PUM1 binds to γ-globin mRNA and decreases γ-globin (HBG1) mRNA stability and translational efficiency, and the blood from an individual harboring a novel heterozygous mutation in PUM1 RNA-binding domain displays elevated percentages of HbF and F cells. (A) qRT-PCR analysis of RNA levels of the indicated transcripts in the immunoprecipitated eluates after RIP of PUM1 in HUDEP2 cells (n = 4). Bar graphs show mean ± standard error of the mean. (B) qRT-PCR analysis of 5-EU incorporated mRNAs of indicated genes at 0-hour pulse and 24-hour chase time points after incorporation in HUDEP2 cells infected with either PUM1 or NTC lentiviral shRNAs showed an increase in the stability of HBG1 but not HBG2 or HBB globins at the 24-hour time point upon PUM1 knockdown (n = 2). Bar graphs show mean ± SD. (C) Polysome fraction analysis in HUDEP2 cells infected with either PUM1 or NTC lentiviral shRNAs showing relative distribution of HBG1 mRNA in each of the 14 fractions. Fraction 8 corresponds to the monosome fraction (80S), whereas fractions 10 to 14 correspond to the polysome fractions. A decrease in the distribution of HBG1 mRNA in the monosomal fraction with a corresponding increase in the polysomal fraction was observed upon PUM1 knockdown, suggesting an increase in translational efficiency in these samples. (D) Ratio of the polysomes (mRNA pooled from fractions 10 to 12) to the monosomes (mRNA in fraction 8) in the polysome fraction analysis in panel C is shown (n = 2). Bar graphs show mean ± SD. (E) Left: DNA sequencing chromatogram of the blood from a patient with PUM1-associated developmental disability, ataxia, and seizure (PADDAS) who has a novel heterozygous mutation (c.3267_3270delTCAC) and normal (parent) blood. Right: The structure of the RNA-binding domain of PUM1 bound to RNA. The region altered by the mutation p.(His1090Profs∗16) is shown in yellow and magenta. (F) HPLC analysis of Hb. Red arrows indicate HbF levels. (G) F cells were stained by using a modified Kleihauer-Betke procedure in the blood of a patient with PADDAS and normal (parent) blood. Red arrows indicate the F cells. Data were analyzed by using a two-sided Student t test. Scale bar represents 30 μm. ∗P < .05; ∗∗P < .005.