title: Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis.
abstract: Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the -14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow.
authors.affiliation: From the Division of Pediatric Oncology, The Johns Hopkins University, Baltimore, Maryland 21231 and.
authors.forename: Wan Yee
authors.initials: WY
authors.lastname: Leong
authors.affiliation: From the Division of Pediatric Oncology, The Johns Hopkins University, Baltimore, Maryland 21231 and.
authors.forename: Hong
authors.initials: H
authors.lastname: Guo
authors.affiliation: From the Division of Pediatric Oncology, The Johns Hopkins University, Baltimore, Maryland 21231 and.
authors.forename: Ou
authors.initials: O
authors.lastname: Ma
authors.affiliation: the Department of Pediatric Hematology-Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Hui
authors.initials: H
authors.lastname: Huang
authors.affiliation: the Department of Pediatric Hematology-Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Alan B
authors.initials: AB
authors.lastname: Cantor
authors.affiliation: From the Division of Pediatric Oncology, The Johns Hopkins University, Baltimore, Maryland 21231 and afriedm2@jhmi.edu.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Friedman
mesh_terms: D000818:Animals; D050676:Core Binding Factor Alpha 2 Subunit / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D006098:Granulocytes / Q000166:cytology* / Q000378:metabolism; D006410:Hematopoiesis; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D015533:Transcriptional Activation; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D050676:Core Binding Factor Alpha 2 Subunit; D019000:Phosphotyrosine; D004247:DNA; D019061:src-Family Kinases; D006655:Histone Deacetylases
keywords: Runx1; Src; hematopoiesis; histone deacetylase (HDAC); myeloid cell; transcription factor; tyrosine kinase