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In this study, we focus on different modes of regulation of STRA13, a human ortholog of the mouse basic helix-loop-helix transcriptional factor, previously identified by us as a new von Hippel-Lindau tumor suppressor gene (VHL) target. The gene was overexpressed in VHL-deficient cell lines and tumors, specifically clear cell renal carcinomas and hemangioblastomas. Introduction of wild type VHL transgene into clear cell renal carcinoma restored low level expression of STRA13. Overexpression was also detected in many common malignancies with an intact VHL gene, suggesting the existence of another, VHL-independent pathway of STRA13 regulation. Similar to many other von HippelLindau tumor-suppressor protein (pVHL) targets, the expression of STRA13 on the mRNA level was hypoxiasensitive, indicating oxygen-dependent regulation of the gene, presumably through the pVHL/hypoxia-inducible factor 1 (HIF-1) pathway. The yeast two-hybrid screening revealed interaction of the STRA13 protein with the human ubiquitin-conjugating enzyme (UBC9) protein, the specificity of which was confirmed in mammalian cells. By adding the proteasome inhibitor acetylleucinyl-leucinyl-norleucinal, we demonstrated that the 26 S proteasome pathway regulates the stability of pSTRA13. Co-expression of STRA13 and UBC9 led to an increase of the pSTRA13 ubiquitination and subsequent degradation. These data established that UBC9/STRA13 association in cells is of physiological importance, presenting direct proof of UBC9 involvement in the ubiquitindependent degradation of pSTRA13. Hypoxia treatment of mammalian cells transiently expressing STRA13 protein showed that stability of pSTRA13 is not affected by hypoxia or VHL. Thus, STRA13, a new pVHL target, is regulated in cells on multiple levels. We propose that STRA13 may play a critical role in carcinogenesis, since it is a potent transcriptional regulator, abundant in a variety of common tumors.Functional inactivation of the von Hippel-Lindau tumor-suppressor protein (pVHL) 1 causes a hereditary cancer syndrome characterized by the predisposition to develop tumors of kidney, central nervous system, retina, pancreas, and adrenal gland. Recent studies highlighted pVHL as a key regulator of cellular responses to oxygen deprivation. pVHL regulates activity of the hypoxia-inducible factor 1 (HIF-1), consisting of an HIF-1␣/HIF-1␤ heterodimer. Under normoxic conditions, the ␣ subunit interacts with the pVHL-elongin B-elongin C complex and is rapidly degraded (1). Hypoxia or pVHL deficiency stabilizes HIF-1␣, which results in activation of a set of genes including vascular endothelial growth factor (VEGF) (2, 3), glucose transporter GLUT-1 (3), glycolytic enzymes (reviewed in Ref. 4), transforming growth factor-␣ (5), and transforming growth factor-␤1 (6). We have recently expanded the list of hypoxia-sensitive pVHL targets. Using the RNA differential display technique, we identified and characterized two novel hypoxia-responsive VHL targets, carbonic anhydrases 9 and 12 (7). Here we describe a third gene down...

Heritable inactivation of specific regions of the genome is a widespread, possibly universal phenomenon for gene regulation in eukaryotes. Self-perpetuating, clonally inherited chromatin structure has been proposed as the explanation for such phenomena as position-effect variegation (PEV) and control of segment determination and differentiation in flies, X-chromosome inactivation and parental imprinting in mammals, gene silencing by paramutation in maize and silencing of the mating-type loci in yeasts. We have now found that the clr4 gene, which is essential for silencing of centromeres and the mating-type loci in Schizosaccharomyces pombe, encodes a protein with high homology to the product of Su(var)3-9, a gene affecting PEV in Drosophila. Like Su(var)3-9p, Clr4p contains SET and chromo domains, motifs found in proteins that modulate chromatin structure. Site-directed mutations in the conserved residues of the chromo domain confirm that it is required for proper silencing and directional switching of the mating type, like SET domain. Surprisingly, RNA differential display experiments demonstrated that clr4+ can mediate transcriptional activation of certain other loci. These results show that clr4 plays a critical role in silencing at mating-type loci and centromeres through the organization of repressive chromatin structure and demonstrate a new, activator function for Clr4p.

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Our findings suggest that Fus1 achieves its protective role in inflammation, autoimmunity, and cancer via the regulation of mitochondrial calcium and calcium-coupled parameters.