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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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L3MBTL1 Transcriptionally Represses SMAD5(A) GEP of undifferentiated L3MBTL1-KD iPSCs, compared to controls, based on microarray analysis. We utilized independent clones of iPSCs (generated from cord blood CD34+ cells), which we independently infected with lentiviral vectors expressing shRNAs against L3MBTL1.(B) mRNA expression levels of several upregulated and downregulated genes, identified by microarray analysis, was confirmed by qPCR. Data were normalized by GAPDH expression and shown as shRNA versus control. The data represent the mean ± SD of the three independent experiments.(C) GSEA for the combined set of SMAD and hematopoietic stem cells genes compared with the differentially expressed genes in L3MBTL1-KD iPSCs.(D) Expression levels of phospho SMAD1/5, total SMAD5, phospho SMAD2, total SMAD2, and HHEX were evaluated in L3MBTL1-KD and control iPSCs by western blot. Tubulin served as the loading control.(E) iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with anti-L3MBTL1 or IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter and actin (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(F) L3MBTL1-KD and control iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with an anti-H3K27 tri-methyl antibody or an IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test. See also Figure S3.
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fig3: L3MBTL1 Transcriptionally Represses SMAD5(A) GEP of undifferentiated L3MBTL1-KD iPSCs, compared to controls, based on microarray analysis. We utilized independent clones of iPSCs (generated from cord blood CD34+ cells), which we independently infected with lentiviral vectors expressing shRNAs against L3MBTL1.(B) mRNA expression levels of several upregulated and downregulated genes, identified by microarray analysis, was confirmed by qPCR. Data were normalized by GAPDH expression and shown as shRNA versus control. The data represent the mean ± SD of the three independent experiments.(C) GSEA for the combined set of SMAD and hematopoietic stem cells genes compared with the differentially expressed genes in L3MBTL1-KD iPSCs.(D) Expression levels of phospho SMAD1/5, total SMAD5, phospho SMAD2, total SMAD2, and HHEX were evaluated in L3MBTL1-KD and control iPSCs by western blot. Tubulin served as the loading control.(E) iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with anti-L3MBTL1 or IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter and actin (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(F) L3MBTL1-KD and control iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with an anti-H3K27 tri-methyl antibody or an IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test. See also Figure S3.

Mentions: To investigate the mechanisms underlying the observed effects of L3MBTL1 on stem cell biology, we utilized an unbiased, genome-wide approach to analyze the gene expression profile (GEP) of two independent clones of undifferentiated L3MBTL1-KD iPSCs. We found increased expression of several BMP/SMAD target genes, as well as downregulation of negative regulators of BMP signaling (Figures 3A and 3B). ID2 and ID3 were among the upregulated genes, which are direct targets of BMP4 and regulate HSPC fate decisions (Hong et al., 2011; van Galen et al., 2014). HHEX also was upregulated; it is known to regulate globin gene expression during ontogeny and its promoter contains a 71 nucleotide BMP-responsive element (BRE) (Zhang et al., 2002). We also found increased expression of ZFP36L2, an RNA-binding protein required for BFU-E self-renewal (Zhang et al., 2013) (data not shown). Expression of SMAD7, which inhibits TGFβ-related signaling (Nakao et al., 1997), was downregulated, as were LEFTY1 and LEFTY2, which function as extracellular antagonists of Nodal signaling (Yeo and Whitman, 2001).


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)

L3MBTL1 Transcriptionally Represses SMAD5(A) GEP of undifferentiated L3MBTL1-KD iPSCs, compared to controls, based on microarray analysis. We utilized independent clones of iPSCs (generated from cord blood CD34+ cells), which we independently infected with lentiviral vectors expressing shRNAs against L3MBTL1.(B) mRNA expression levels of several upregulated and downregulated genes, identified by microarray analysis, was confirmed by qPCR. Data were normalized by GAPDH expression and shown as shRNA versus control. The data represent the mean ± SD of the three independent experiments.(C) GSEA for the combined set of SMAD and hematopoietic stem cells genes compared with the differentially expressed genes in L3MBTL1-KD iPSCs.(D) Expression levels of phospho SMAD1/5, total SMAD5, phospho SMAD2, total SMAD2, and HHEX were evaluated in L3MBTL1-KD and control iPSCs by western blot. Tubulin served as the loading control.(E) iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with anti-L3MBTL1 or IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter and actin (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(F) L3MBTL1-KD and control iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with an anti-H3K27 tri-methyl antibody or an IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test. See also Figure S3.
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fig3: L3MBTL1 Transcriptionally Represses SMAD5(A) GEP of undifferentiated L3MBTL1-KD iPSCs, compared to controls, based on microarray analysis. We utilized independent clones of iPSCs (generated from cord blood CD34+ cells), which we independently infected with lentiviral vectors expressing shRNAs against L3MBTL1.(B) mRNA expression levels of several upregulated and downregulated genes, identified by microarray analysis, was confirmed by qPCR. Data were normalized by GAPDH expression and shown as shRNA versus control. The data represent the mean ± SD of the three independent experiments.(C) GSEA for the combined set of SMAD and hematopoietic stem cells genes compared with the differentially expressed genes in L3MBTL1-KD iPSCs.(D) Expression levels of phospho SMAD1/5, total SMAD5, phospho SMAD2, total SMAD2, and HHEX were evaluated in L3MBTL1-KD and control iPSCs by western blot. Tubulin served as the loading control.(E) iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with anti-L3MBTL1 or IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter and actin (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test.(F) L3MBTL1-KD and control iPSCs were crosslinked with 1% formaldehyde and immunoprecipitated with an anti-H3K27 tri-methyl antibody or an IgG antibody (as a nonspecific control). Plotted values are relative enrichments (y axis) to 10% input and measured for sites in the SMAD5 promoter (x axis). The data represent the mean ± SD of the three independent experiments. ∗p < 0.05 by Student’s t test. See also Figure S3.
Mentions: To investigate the mechanisms underlying the observed effects of L3MBTL1 on stem cell biology, we utilized an unbiased, genome-wide approach to analyze the gene expression profile (GEP) of two independent clones of undifferentiated L3MBTL1-KD iPSCs. We found increased expression of several BMP/SMAD target genes, as well as downregulation of negative regulators of BMP signaling (Figures 3A and 3B). ID2 and ID3 were among the upregulated genes, which are direct targets of BMP4 and regulate HSPC fate decisions (Hong et al., 2011; van Galen et al., 2014). HHEX also was upregulated; it is known to regulate globin gene expression during ontogeny and its promoter contains a 71 nucleotide BMP-responsive element (BRE) (Zhang et al., 2002). We also found increased expression of ZFP36L2, an RNA-binding protein required for BFU-E self-renewal (Zhang et al., 2013) (data not shown). Expression of SMAD7, which inhibits TGFβ-related signaling (Nakao et al., 1997), was downregulated, as were LEFTY1 and LEFTY2, which function as extracellular antagonists of Nodal signaling (Yeo and Whitman, 2001).

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.

Show MeSH
Related in: MedlinePlus