The RUNX family genes are the mammalian homologs of the Drosophila genes runt and lozenge, and members of this family function as master regulators of de®nitive hematopoiesis and osteogenesis. The RUNX genes encode the a subunit of the transcription factor PEBP2/CBF. The b subunit consists of the non-RUNX protein PEBP2b. We found that RUNX1/AML1, which is essential for hematopoiesis, is continuously subjected to proteolytic degradation mediated by the ubiquitin±proteasome pathway. When PEBP2b is present, however, the ubiquitylation of RUNX1 is abrogated and this causes a dramatic inhibition of RUNX1 proteolysis. Heterodimerization between PEBP2b and RUNX1 thus appears to be an essential step in the generation of transcriptionally competent RUNX1. Consistent with this notion, RUNX1 was barely detected in PEBP2b ±/± mouse. CBF(PEBP2)b± SMMHC, the chimeric protein associated with inv(16) acute myeloid leukemia, was found to protect RUNX1 from proteolytic degradation more ef®ciently than PEBP2b. These results reveal a hitherto unknown and major role of PEBP2b, namely that it regulates RUNX1 by controlling its turnover. This has allowed us to gain new insights into the mechanism of leukemogenesis by CBFb±SMMHC. Keywords: AML1/PEBP2b/proteolytic degradation/ Runx1/ubiquitylation IntroductionThe Runt domain transcription factor, PEBP2/CBF, is a heterodimeric transcription factor which is one of the major targets of transforming growth factor-b (TGF-b) and bone morphogenetic protein (BMP) (Hanai et al., 1999;Zhang et al., 2000). It is involved in the regulation of gene expression in a variety of biological activities, most notably hematopoiesis and osteogenesis (Ito, 1997(Ito, , 1999. Its b subunit, denoted as PEBP2b/CBFb, is homologous to the Drosophila Runt-binding proteins, Brother and Big brother. It does not bind to DNA, but the a subunit of PEBP2, which is homologous to the Drosophila gene products Runt and Lozenge, does have DNA-binding properties.The a subunit is encoded by RUNX, the mammalian homolog of the runt gene from Drosophila. RUNX contains an evolutionarily conserved 128 amino acid region termed the Runt domain, which is required both for DNA binding and heterodimerization with the b subunit (Kagoshima et al., 1993). In mammals, three a subunit genes exist, namely RUNX1, RUNX2 and RUNX3 (also referred to, respectively, as PEBP2aB, PEBP2aA and PEBP2aC, or CBFA2, CBFA1 and CBFA3, or AML1, AML3 and AML2) (Ito, 1999). RUNX1, which is frequently altered by the chromosome translocations associated with human leukemia (Look, 1997), is essential for inducing de®nitive hematopoiesis. It is also critical in regulating hematopoietic cell-speci®c genes in a variety of blood cells (Ito, 1997(Ito, , 1999. RUNX2 is essential for the generation and maturation of osteoblasts (Komori et al., 1997;Otto et al., 1997) and it plays pivotal roles in regulating the expression of bone-speci®c genes such as osteocalcin and osteopontin (Ducy et al., 1996). Haploinsuf®ciency of RUNX2 causes cleidocranial dysplasia, a human autosom...

Genes involved in the transforming growth factor ␤ (TGF-␤) signaling pathway are frequently altered in several types of cancers, and a gastric tumor suppressor RUNX3 appears to be an integral component of this pathway. We reported previously that apoptosis is notably reduced in Runx3 ؊/؊ gastric epithelial cells. In the present study, we show that a proapoptotic gene Bim was transcriptionally activated by RUNX3 in the gastric cancer cell lines SNU16 and SNU719 treated with TGF-␤. The human Bim promoter contains RUNX sites, which are required for its activation. Furthermore, a dominant negative form of RUNX3 comprised of amino acids 1 to 187 increased tumorigenicity of SNU16 by inhibiting Bim expression. In Runx3 ؊/؊ mouse gastric epithelium, Bim was down-regulated, and apoptosis was reduced to the same extent as that in Bim ؊/؊ gastric epithelium. We confirmed comparable expression of TGF-␤1 and TGF-␤ receptors between wild-type and Runx3 ؊/؊ gastric epithelia and reduction of Bim in TGF-␤1 ؊/؊ stomach. These results demonstrate that RUNX3 is responsible for transcriptional up-regulation of Bim in TGF-␤-induced apoptosis.

Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.

The RUNX family represents a small group of heterodimeric transcription factors that master-regulate osteogenesis and hematopoiesis in mammals. Their genetic defects cause human diseases such as cleidocranial dysplasia (CCD) and acute myelogenous leukemia. However, the mechanism(s) regulating their functions are still poorly understood. Here, we report a novel observation that suggests that the osteogenesisassociated homologue RUNX2 is negatively regulated by the phosphorylation of two conserved serines (S104 and S451) in two distinct functional aspects. The phosphorylation of S104 could abolish the heterodimerization of RUNX2 with the partner subunit, PEBP2β, which enhances the metabolic stability of RUNX2. On the other hand, the phosphorylation of S451 resides within the C-terminal transcription inhibition domain of RUNX2 and hence is implicated in its functional mobilization. One CCD mutation, S104R of RUNX2, appears to mimic the phosphorylation-dependent inhibition of heterodimerization, thereby rendering RUNX2 metabolically unstable.