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.14 (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.

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.14 (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.

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