Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau ( VHL ) tumor suppressor. Roles for noncoding cis -regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profi les, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specifi c aspects of malignancy. Superenhancer profi ling identifi ed ZNF395 as a ccRCCspecifi c and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivo . VHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α-HIF1β heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter-enhancer interactions. Subtype-specifi c driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression. SIGnIFICAnCE:Comprehensive epigenomic profi ling of ccRCC establishes a compendium of somatically altered cis -regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL , a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specifi c promoter-enhancer complexes. Cancer Discov; 7(11); 1284-305.

The E2 transactivator of bovine papillomavirus type‐1 is unable to activate minimal promoters in vivo that contain only E2 binding sites and a TATA box. This block can be overcome by over‐expression of human TATA binding protein (TBP) or by the addition of either SP1 binding sites or an initiator element to the promoter, suggesting that the binding of TFIID may normally be a rate‐limiting step for activation by E2. Surprisingly, purified E2 and TBP bind co‐operatively to DNA in vitro when the sites are closely spaced. E2 does not affect the on rate of association but reduces the off rate. The E2 region responsible for this effect is located in the hinge region that links the classic transactivation and DNA binding domains. We demonstrate that the TBP stabilizing domain contributes in vivo to co‐operativity with co‐expressed TBP and to activation of the major late minimal promoter (MLP) containing E2 sites. In contrast, promoters with SP1 sites are activated to wild‐type levels by such a mutant. This promoter specificity is also evident in vitro. A truncated E2 mutant, lacking the classic transactivation domain but containing the TBP stabilizing domain, stimulates transcription of the MLP in vitro, but does not activate promoters with SP1 sites. In conclusion, our results show that the E2 transactivation domain has a modular structure. We have identified one domain which probably acts at an early step in the assembly of the pre‐initiation complex and which is involved in reducing the dissociation rate of bound TBP in vitro. The classic N‐terminal activation domain of E2 might affect one or several step(s) in the assembly of the preinitiation complex occurring after the binding of TFIID.

The E2 proteins of papillomaviruses (PV) bind to the coactivator CBP/p300 as do many other transcription factors, but the precise role of CBP/p300 in E2-specific functions is not yet understood. We show that the E2 protein of human PV type 8 (HPV8) directly binds to p300. Activation of HPV8 gene expression by low amounts of HPV8 E2 was stimulated up to sevenfold by coexpression of p300. The interaction between E2 and p300 may play a role in differentiation-dependent activation of PV gene expression, since we can show that the expression level of p300 increases during keratinocyte differentiation. Surprisingly, sequence-specific binding of E2 to its recognition sites within the regulatory region of HPV8 is not necessary for this cooperation, indicating that E2 can be recruited to the promoter via protein-protein interaction. HPV8 E2 binds via its N-terminal activation domain (AD), its C-terminal DNA binding domain (DBD), and its internal hinge region to p300 in vitro. Transient-transfection assays revealed that the AD is necessary and sufficient for cooperative activation with p300. However, we provide evidence that the interaction of the hinge and the DBD of HPV8 E2 with p300 may contribute. Our data suggest an important role of p300 in regulation of HPV8 gene expression and reveal a new mechanism by which E2 may be recruited to a promoter to activate transcription without sequence specific DNA binding.Papillomaviruses (PV) infect the basal cells of the skin or the mucosa, causing proliferative lesions like warts or dysplasias. PV require differentiating keratinocytes to replicate their DNA. The expression of the structural proteins is restricted to some of the most-differentiated keratinocytes. This cell tropism is due to the involvement of transcription factors specifically expressed in these cells. In addition to ubiquitously expressed and keratinocyte-specific cellular transcription factors, viral gene expression is also modulated by the viral E2 protein. The E2 protein binds via its carboxy (C)-terminal DNA binding domain (DBD) and dimerization domain to the 12-bp palindromic sequence ACCN 6 GGT mostly located within the regulatory region, also called the long control region or noncoding region (NCR) of the PV genome. In the case of bovine PV type 1 (BPV1), the long control region contains 12 E2 binding sites, which mediate a strong activation of several BPV1 promoters by E2 (48-50). Human PV (HPV) types infecting the genital mucosa contain four E2 binding sites in conserved positions. Here, only a moderate activation by E2 could be detected, and E2 repressed HPV gene expression in most cases. This repression is mediated by binding to two promoter-proximal E2 binding sites, as revealed by transient transfections of cervical carcinoma cell lines and immortalized skin keratinocytes (10,52,58,59). Repression of HPV gene expression by the E2 activator protein occurs when E2 binding sites are overlapping with the binding sites for cellular transcription factors necessary for promoter activity. In contrast to nat...