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The polycomb group protein L3MBTL1 represses a SMAD5-mediated hematopoietic transcriptional program in human pluripotent stem cells.

Perna F, Vu LP, Themeli M, Kriks S, Hoya-Arias R, Khanin R, Hricik T, Mansilla-Soto J, Papapetrou EP, Levine RL, Studer L, Sadelain M, Nimer SD - Stem Cell Reports (2015)

Bottom Line: Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells.We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation.Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: pernaf@mskcc.org.

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Regulation of SMAD5 by L3MBTL1 Occurs in Erythroid Cells(A) KD of L3MBTL1 induces early expression of the erythroid-specific marker GlyA in iPSC-derived CD34+ cells. Representative flow cytometry analysis of iPSCs after 2 days in Epo-induced culture.(B) Expression levels of EKLF, NF-E2, LMO2, EPOR, and fetal globin gene were assessed in iPSC-derived erythroid progeny by qRT-PCR. Data were normalized by GAPDH expression. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(C) K562 erythroleukemia cells were retrovirally transduced to express L3MBTL1-HA (blue bars) or empty MIGR1 control (gray bars), crosslinked with 1% formaldehyde, and immunoprecipitated with an anti-SMAD5 antibody or IgG antibody. Primers covering the SMAD-binding motifs (Adelman et al., 2002) across the upstream enhancer, the proximal promoter, and the intronic enhancer of the EKLF were utilized. Data were normalized by 10% input. The data represent the mean ± SD of the three independent experiments.(D) EKLF, NF-E2, GATA-1, LMO2, EPOR, and BCL11A expression levels were evaluated by qRT-PCR in L3MBTL1-KD CB CD34+ cells after 7 days of erythroid-supporting culture (100 ng/ml SCF and 6 U/ml EPO) compared to controls. GAPDH served as a housekeeping gene control. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(E) Fetal globin levels were evaluated in L3MBTL1-KD CB CD34+ cells by western blot. Tubulin served as the loading control.(F) Overexpression of L3MBTL1 decreases globin gene mRNA levels in cord blood CD34+ cells, as shown by qRT-PCR. Cells were cultured for 3 days in liquid culture that supports erythroid differentiation. GAPDH served as the housekeeping gene. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(G) Overexpression of L3MBTL1 dramatically decreases protein expression levels of gamma globin in K562 cells, as shown by western blot assay. Tubulin served as the loading control. See also Figure S4.
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fig4: Regulation of SMAD5 by L3MBTL1 Occurs in Erythroid Cells(A) KD of L3MBTL1 induces early expression of the erythroid-specific marker GlyA in iPSC-derived CD34+ cells. Representative flow cytometry analysis of iPSCs after 2 days in Epo-induced culture.(B) Expression levels of EKLF, NF-E2, LMO2, EPOR, and fetal globin gene were assessed in iPSC-derived erythroid progeny by qRT-PCR. Data were normalized by GAPDH expression. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(C) K562 erythroleukemia cells were retrovirally transduced to express L3MBTL1-HA (blue bars) or empty MIGR1 control (gray bars), crosslinked with 1% formaldehyde, and immunoprecipitated with an anti-SMAD5 antibody or IgG antibody. Primers covering the SMAD-binding motifs (Adelman et al., 2002) across the upstream enhancer, the proximal promoter, and the intronic enhancer of the EKLF were utilized. Data were normalized by 10% input. The data represent the mean ± SD of the three independent experiments.(D) EKLF, NF-E2, GATA-1, LMO2, EPOR, and BCL11A expression levels were evaluated by qRT-PCR in L3MBTL1-KD CB CD34+ cells after 7 days of erythroid-supporting culture (100 ng/ml SCF and 6 U/ml EPO) compared to controls. GAPDH served as a housekeeping gene control. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(E) Fetal globin levels were evaluated in L3MBTL1-KD CB CD34+ cells by western blot. Tubulin served as the loading control.(F) Overexpression of L3MBTL1 decreases globin gene mRNA levels in cord blood CD34+ cells, as shown by qRT-PCR. Cells were cultured for 3 days in liquid culture that supports erythroid differentiation. GAPDH served as the housekeeping gene. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(G) Overexpression of L3MBTL1 dramatically decreases protein expression levels of gamma globin in K562 cells, as shown by western blot assay. Tubulin served as the loading control. See also Figure S4.

Mentions: We, and others, have suggested that L3MBTL1 plays an important role in erythroid differentiation of human CD34+ HSPCs (Aziz et al., 2013; Perna et al., 2010). We found that downregulation of L3MBTL1 occurs during normal erythroid differentiation, in a manner similar to that seen during mesodermal differentiation (Figure 1A). We first assessed the effect of L3MBTL1-KD in iPSC-derived CD34+ cells undergoing erythroid differentiation, and observed a marked acceleration in the expression of the erythroid-specific marker GlyA (after 2 days in EPO-induced culture) by flow cytometry in the L3MBTL1-KD cells (Figure 4A). This was followed by a significant increase in the expression of several erythroid-specific transcription factors, especially EKLF (Figure 4B). We also confirmed this phenotype in β-thalassemic iPSCs (Papapetrou et al., 2011), which we lentivirally infected to express shRNAs targeting L3MBTL1 (Figure S4A). Similar to the CB-iPSC line, knocking down L3MBTL1 in thalassemic erythroid progeny also increased EKLF expression (Figure S4B) and increased fetal globin gene expression compared to controls (Figure S4C).


The polycomb group protein L3MBTL1 represses a SMAD5-mediated hematopoietic transcriptional program in human pluripotent stem cells.

Perna F, Vu LP, Themeli M, Kriks S, Hoya-Arias R, Khanin R, Hricik T, Mansilla-Soto J, Papapetrou EP, Levine RL, Studer L, Sadelain M, Nimer SD - Stem Cell Reports (2015)

Regulation of SMAD5 by L3MBTL1 Occurs in Erythroid Cells(A) KD of L3MBTL1 induces early expression of the erythroid-specific marker GlyA in iPSC-derived CD34+ cells. Representative flow cytometry analysis of iPSCs after 2 days in Epo-induced culture.(B) Expression levels of EKLF, NF-E2, LMO2, EPOR, and fetal globin gene were assessed in iPSC-derived erythroid progeny by qRT-PCR. Data were normalized by GAPDH expression. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(C) K562 erythroleukemia cells were retrovirally transduced to express L3MBTL1-HA (blue bars) or empty MIGR1 control (gray bars), crosslinked with 1% formaldehyde, and immunoprecipitated with an anti-SMAD5 antibody or IgG antibody. Primers covering the SMAD-binding motifs (Adelman et al., 2002) across the upstream enhancer, the proximal promoter, and the intronic enhancer of the EKLF were utilized. Data were normalized by 10% input. The data represent the mean ± SD of the three independent experiments.(D) EKLF, NF-E2, GATA-1, LMO2, EPOR, and BCL11A expression levels were evaluated by qRT-PCR in L3MBTL1-KD CB CD34+ cells after 7 days of erythroid-supporting culture (100 ng/ml SCF and 6 U/ml EPO) compared to controls. GAPDH served as a housekeeping gene control. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(E) Fetal globin levels were evaluated in L3MBTL1-KD CB CD34+ cells by western blot. Tubulin served as the loading control.(F) Overexpression of L3MBTL1 decreases globin gene mRNA levels in cord blood CD34+ cells, as shown by qRT-PCR. Cells were cultured for 3 days in liquid culture that supports erythroid differentiation. GAPDH served as the housekeeping gene. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(G) Overexpression of L3MBTL1 dramatically decreases protein expression levels of gamma globin in K562 cells, as shown by western blot assay. Tubulin served as the loading control. See also Figure S4.
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fig4: Regulation of SMAD5 by L3MBTL1 Occurs in Erythroid Cells(A) KD of L3MBTL1 induces early expression of the erythroid-specific marker GlyA in iPSC-derived CD34+ cells. Representative flow cytometry analysis of iPSCs after 2 days in Epo-induced culture.(B) Expression levels of EKLF, NF-E2, LMO2, EPOR, and fetal globin gene were assessed in iPSC-derived erythroid progeny by qRT-PCR. Data were normalized by GAPDH expression. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(C) K562 erythroleukemia cells were retrovirally transduced to express L3MBTL1-HA (blue bars) or empty MIGR1 control (gray bars), crosslinked with 1% formaldehyde, and immunoprecipitated with an anti-SMAD5 antibody or IgG antibody. Primers covering the SMAD-binding motifs (Adelman et al., 2002) across the upstream enhancer, the proximal promoter, and the intronic enhancer of the EKLF were utilized. Data were normalized by 10% input. The data represent the mean ± SD of the three independent experiments.(D) EKLF, NF-E2, GATA-1, LMO2, EPOR, and BCL11A expression levels were evaluated by qRT-PCR in L3MBTL1-KD CB CD34+ cells after 7 days of erythroid-supporting culture (100 ng/ml SCF and 6 U/ml EPO) compared to controls. GAPDH served as a housekeeping gene control. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(E) Fetal globin levels were evaluated in L3MBTL1-KD CB CD34+ cells by western blot. Tubulin served as the loading control.(F) Overexpression of L3MBTL1 decreases globin gene mRNA levels in cord blood CD34+ cells, as shown by qRT-PCR. Cells were cultured for 3 days in liquid culture that supports erythroid differentiation. GAPDH served as the housekeeping gene. The data represent the mean ± SD of the three independent experiments. ∗∗p < 0.01 by Student’s t test.(G) Overexpression of L3MBTL1 dramatically decreases protein expression levels of gamma globin in K562 cells, as shown by western blot assay. Tubulin served as the loading control. See also Figure S4.
Mentions: We, and others, have suggested that L3MBTL1 plays an important role in erythroid differentiation of human CD34+ HSPCs (Aziz et al., 2013; Perna et al., 2010). We found that downregulation of L3MBTL1 occurs during normal erythroid differentiation, in a manner similar to that seen during mesodermal differentiation (Figure 1A). We first assessed the effect of L3MBTL1-KD in iPSC-derived CD34+ cells undergoing erythroid differentiation, and observed a marked acceleration in the expression of the erythroid-specific marker GlyA (after 2 days in EPO-induced culture) by flow cytometry in the L3MBTL1-KD cells (Figure 4A). This was followed by a significant increase in the expression of several erythroid-specific transcription factors, especially EKLF (Figure 4B). We also confirmed this phenotype in β-thalassemic iPSCs (Papapetrou et al., 2011), which we lentivirally infected to express shRNAs targeting L3MBTL1 (Figure S4A). Similar to the CB-iPSC line, knocking down L3MBTL1 in thalassemic erythroid progeny also increased EKLF expression (Figure S4B) and increased fetal globin gene expression compared to controls (Figure S4C).

Bottom Line: Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells.We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation.Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: pernaf@mskcc.org.

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Related in: MedlinePlus