The transition from transcription initiation to elongation involves phosphorylation of the large subunit (Rpb1) of RNA polymerase II on the repetitive carboxyl-terminal domain. The elongating hyperphosphorylated Rpb1 is subject to ubiquitination, particularly in response to UV radiation and DNA-damaging agents. By using computer modeling, we identified regions of Rpb1 and the adjacent subunit 6 of RNA polymerase II (Rpb6) that share sequence and structural similarity with the domain of hypoxia-inducible transcription factor 1␣ (HIF-1␣) that binds von Hippel-Lindau tumor suppressor protein (pVHL). pVHL confers substrate specificity to the E3 ligase complex, which ubiquitinates HIF-␣ and targets it for proteasomal degradation. In agreement with the computational model, we show biochemical evidence that pVHL specifically binds the hyperphosphorylated Rpb1 in a proline-hydroxylationdependent manner, targeting it for ubiquitination. This interaction is regulated by UV radiation.

Human renal clear cell carcinoma (RCC) is frequently associated with loss of the von Hippel-Lindau (VHL)tumor suppressor (pVHL), which inhibits ubiquitylation and degradation of the alpha subunits of hypoxiainducible transcription factor. pVHL also ubiquitylates the large subunit of RNA polymerase II, Rpb1, phosphorylated on serine 5 (Ser5) within the C-terminal domain (CTD). A hydroxylated proline 1465 within an LXXLAP motif located N-terminal to the CTD allows the interaction of Rpb1 with pVHL. Here we report that in RCC cells, pVHL regulates expression of Rpb1 and is necessary for low-grade oxidative-stress-induced recruitment of Rpb1 to the DNA-engaged fraction and for its P1465 hydroxylation, phosphorylation, and nondegradative ubiquitylation. Egln-9-type prolyl hydroxylases, PHD1 and PHD2, coimmunoprecipitated with Rpb1 in the chromatin fraction of VHL ؉ RCC cells in response to oxidative stress, and PHD1 was necessary for P1465 hydroxylation while PHD2 had an inhibitory effect. P1465 hydroxylation was required for oxidativestress-induced Ser5 phosphorylation of Rpb1. Importantly, overexpression of wild-type Rpb1 stimulated formation of kidney tumors by VHL ؉ cells, and this effect was abolished by P1465A mutation of Rpb1. These data indicate that through this novel pathway involving P1465 hydroxylation and Ser5 phosphorylation of Rbp1, pVHL may regulate tumor growth.pVHL is the main tumor suppressor for which loss of activity is causatively linked to renal clear cell carcinoma (RCC), the most malignant and common form of kidney cancer. The VHL gene is mutated or hypermethylated in about 40 to 70% of sporadic RCC. Hereditary loss of pVHL function in von Hippel-Lindau (VHL) disease also results in highly vascularized RCC and capillary tumors of other organs, such as hemangioblastoma of the central nervous system and retinal angioma (19,20). A body of experimental evidence, based on a subcutaneous xenograft model system, supports the idea that accumulation of the alpha subunit of the hypoxia-inducible transcription factor (HIF) HIF-2␣ and induction of HIF target gene products, resulting from the loss of pVHL-mediated ubiquitylation, are necessary and sufficient to promote growth of RCC tumors (22,23). HIF activation has also been demonstrated as an early tumorigenesis event in kidneys from VHL patients (32). Biochemically, pVHL is the substrate-recognizing component of a multiprotein E3 ubiquitin ligase complex containing elongins C and B, Cullin 2, and the RING-H2 finger protein Rbx-1 (for a review, see reference 19). pVHL-dependent ubiquitylation of HIF-␣s is preceded by hydroxylation of conserved proline residues located within LXXLAP motifs (16,17) by the O 2 -, Fe(II)-, and oxyglutarate-regulated Egl-9-type proline hydroxylases (PHDs) (7). Thus, an important aspect of pVHL's tumor suppressing activity is the prevention of HIF-␣ accumulation, which in turn suppresses induction of the HIF target genes. Clearly, however, pVHL activity is not limited to regulation of HIFs. Other targets of pVHL-associated E...

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is induced by hypoxia in oxygensensitive cells of the carotid body and pheochromocytomaderived PC12 cells. TH is also regulated by the von Hippel-Lindau tumor suppressor protein (pVHL). Here, we report that induction of TH gene expression involves activation of the hypoxia-inducible transcription factors (HIFs) that interact with a specific hypoxia-responsive element (HRE) in the proximal region of the TH promoter. We also show that some of the effects of pVHL on activity of the TH promoter are mediated through HIFs. Low levels of pVHL are associated with decreased HIFa ubiquitination, increased accumulation of HIFa proteins, increased binding of HIFs to the HRE within the TH promoter, and increased activity of a TH promoterreporter construct. In contrast, high levels of pVHL repress HIF accumulation and inhibit its activity in hypoxic cells. These results indicate that HIFs may play an important role in regulation of TH gene expression in oxygen-sensitive cells and also in the development of hypercatecholaminemia in pheochromocytoma tumors.

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The chemokine receptor CCR5 constitutes a major co-receptor for the R5 strains of HIV-1, and a mutant allele of the CCR5 gene, especially in the homozygous form ∆32/∆32, confers resistance against infection by the virus. The frequency of the ∆32 allele was determined in blood donors from 16 provinces, covering the entire territory of Poland. Among 861 individuals 182 (21.1%) were carriers of the mutated allele; 7 of them (0.8 %) were homozygotes ∆32/∆32, and 175 (20.3%) were heterozygotes ϩ/∆32, resulting in a 10.9% frequency of the ∆32 allele. The highest frequencies of the mutated allele were found in the eastern and western provinces, and the lowest frequencies of the ∆32 allele were detected in the provinces in the center of the country. This pattern of distribution may reflect the migration of the population from the eastern territories of Poland to the western part of the country after World War II.