title: Silencing of ASXL1 impairs the granulomonocytic lineage potential of human CD34⁺ progenitor cells.
abstract: The ASXL1 gene encodes a chromatin-binding protein involved in epigenetic regulation in haematopoietic cells. Loss-of-function ASXL1 mutations occur in patients with a range of myeloid malignancies and are associated with adverse outcome. We have used lentiviral-based shRNA technology to investigate the effects of ASXL1 silencing on cell proliferation, apoptosis, myeloid differentiation and global gene expression in human CD34(+) cells differentiated along the myeloid lineage in vitro. ASXL1-deficient cells showed a significant decrease in the generation of CD11b(+) and CD15(+) cells, implicating impaired granulomonocytic differentiation. Furthermore, colony-forming assays showed a significant increase in the number of multipotent mixed lineage colony-forming unit (CFU-GEMM) colonies and a significant decrease in the numbers of granulocyte-macrophage CFU (CFU-GM) and granulocyte CFU (CFU-G) colonies in ASXL1-deficient cells. Our data suggests that ASXL1 knockdown perturbs human granulomonocytic differentiation. Gene expression profiling identified many deregulated genes in the ASXL1-deficient cells differentiated along the granulomonocytic lineage, and pathway analysis showed that the most significantly deregulated pathway was the LXR/RXR activation pathway. ASXL1 may play a key role in recruiting the polycomb repressor complex 2 (PRC2) to specific loci, and we found over-representation of PRC2 targets among the deregulated genes in ASXL1-deficient cells. These findings shed light on the functional role of ASXL1 in human myeloid differentiation.
authors.affiliation: LLR Molecular Haematology Unit, NDCLS, John Radcliffe Hospital, Oxford, UK.
authors.forename: Carwyn
authors.initials: C
authors.lastname: Davies
authors.forename: Bon Ham
authors.initials: BH
authors.lastname: Yip
authors.forename: Marta
authors.initials: M
authors.lastname: Fernandez-Mercado
authors.forename: Petter S
authors.initials: PS
authors.lastname: Woll
authors.forename: Xabier
authors.initials: X
authors.lastname: Agirre
authors.forename: Felipe
authors.initials: F
authors.lastname: Prosper
authors.forename: Sten E
authors.initials: SE
authors.lastname: Jacobsen
authors.forename: James S
authors.initials: JS
authors.lastname: Wainscoat
authors.forename: Andrea
authors.initials: A
authors.lastname: Pellagatti
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Boultwood
mesh_terms: D018952:Antigens, CD34 / Q000096:biosynthesis*; D016022:Case-Control Studies; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D048708:Cell Growth Processes / Q000235:genetics; D019070:Cell Lineage; D020868:Gene Silencing; D006801:Humans; D020014:K562 Cells; D022423:Myeloid Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D059467:Transcriptome
chemical_list: C473949:ASXL1 protein, human; D018952:Antigens, CD34; D034741:RNA, Small Interfering; D012097:Repressor Proteins