The polycomb group protein L3MBTL1 represses a SMAD5-mediated hematopoietic transcriptional program in human pluripotent stem cells.
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.
Affiliation: Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: firstname.lastname@example.org.Show MeSH
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Mentions: The generation of iPSC lines has provided opportunities to understand the fundamental processes of human cell fate decisions in the context of tissue regeneration and human disease. We first observed spontaneous downregulation of L3MBTL1 expression in a human iPSC line generated from cord blood cells (iCBCs) (Figures S1A–S1C) upon mesodermal differentiation, suggesting that decreased L3MBTL1 expression is required for mesoderm specification (Figure 1A). To investigate the role of L3MBTL1 on the specification of hematopoiesis during iPSC differentiation, we knocked down L3MBTL1 using H1P-hygro-EGFP+ lentiviral vectors that express small hairpin RNAs (shRNAs) directed against L3MBTL1 (Figure 1B). The subcloned green fluorescent protein (GFP)+ colonies showed a marked decrease in L3MBTL1 expression and retained stem cell morphology and stem-cell-related cell surface markers, including TRA-1-81, KLF4, OCT4, and NANOG expression (Figures S1D–S1G). Expression of mesoderm-specific transcription factors (T, TAL1, LMO2, and RUNX1) was consistently increased in the KD cells, while key markers for endoderm and ectoderm decreased (Figure 1C). We observed downregulation of SOX2 even though the iPSCs maintained self-renewal properties. While consistent with a previous report (Wang et al., 2012), we did find upregulation of SOX3 (data not shown), which also could explain the self-renewal phenotype. Functional assessment of clonogenic hematopoietic progenitors (CFUs) revealed that the lack of L3MBTL1 induced an increase in hematopoietic progenitor cell generation (Figure 1D). The hematopoietic colonies derived from the L3MBTL1-KD cells were also larger in size (data not shown). CD45−CD31+CD34+ cells, which represent a primitive bipotent endothelial-like precursor population that is exclusively responsible for hematopoietic fate (Wang et al., 2004), were also much more abundant within the L3MBTL1-KD cells compared to controls (Figure 1E).
Affiliation: Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address: email@example.com.