AML1/RUNX1 increases during G1 to S cell cycle progression independent of cytokine-dependent phosphorylation and induces cyclin D3 gene expression.
AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G(2)/M phase cells compared with G(1) cells. In addition, G(1) arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G(1) phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBFbeta-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G(1) to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations altering pathways regulating variation in AML1 stability potentially contribute to leukemic transformation.
Bernardin-Fried
Florence
F
Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland 21231, USA.
Kummalue
Tanawan
T
Leijen
Suzanne
S
Collector
Michael I
MI
Ravid
Katya
K
Friedman
Alan D
AD
D000199:Actins / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D050676:Core Binding Factor Alpha 2 Subunit; D056743:Cyclin D3; D016213:Cyclins / Q000096:biosynthesis* / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D016193:G1 Phase; D016195:G2 Phase; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008898:Mimosine / Q000494:pharmacology; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D012313:RNA / Q000737:chemistry; D016196:S Phase; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation
D000199:Actins; C534696:CCND3 protein, human; C534697:Ccnd3 protein, mouse; D050676:Core Binding Factor Alpha 2 Subunit; D056743:Cyclin D3; D016213:Cyclins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C493728:RUNX1 protein, human; C493726:Runx1 protein, mouse; D014157:Transcription Factors; D008898:Mimosine; D012313:RNA; D004247:DNA