Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability,Reduced Histone Deacetylase (HDAC) Binding,and Increased DNA Affinity,and Activated Runx1 Favors Granulopoiesis

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.     

CHIP functions an E3 ubiquitin ligase of Runx1

Runx1 is a key factor in the generation and maintenance of hematopoietic stem cells. Improper expression and mutations in Runx1 are frequently implicated in human leukemia. Here, we report that CHIP, the carboxyl terminus of Hsc70-interacting protein, also named Stub1, physically interacts with Runx1 through the TPR and Charged domains in the nucleus. Over-expression of CHIP directly induced Runx1 ubiquitination and degradation through the ubiquitin-proteasome pathway. Interestingly, we found that CHIP-mediated degradation of Runx1 is independent of the molecular chaperone Hsp70/90. Taken together, we propose that CHIP serves as an E3 ubiquitin ligase that regulates Runx1 protein stability via an ubiquitination and degradation mechanism that is independent of Hsp70/90.     

Analysis of HDAC1-mediated regulation of Runx2-induced osteopontin gene expression in C3h10t1/2 cells

Histone deacetylases (HDACs) deacetylate lysine residues of histone and non-histone proteins and thereby regulate the cell-cycle, gene expression, and several other processes. We have analyzed the effects of HDAC1 on Runx2-mediated regulation of osteopontin (OPN) promoter activation and gene expression in mesenchymal progenitor C3h10t1/2 cells and show that co-expression of HDAC1 along with Runx2 results in down-regulation of Runx2-induced OPN mRNA expression during both the proliferation and differentiation stages of C3h10t1/2 cells. Luciferase assay results revealed that HDAC1 efficiently down-regulated Runx2-stimulated OPN promoter activity in a dose-dependent manner whereas TSA relieved the HDAC1-mediated repression and up-regulated the Runx2-induced OPN promoter activity and mRNA expression. In vivo HDAC1 co-localized and physically interacted with Runx2 and associated with the OPN promoter. Thus, HDAC1 not only plays a critical role in regulation of Runx2-stimulated expression of osteogenic genes, like OPN, but also regulate the proliferation and differentiation stages of mesenchymal progenitor cells, such as C3h10t1/2.