Transcriptional regulation by the E7 oncoproteins of human papillomavirus type 16; interaction with chromatin-modifying enzymes

Infection of human cells with papillomaviruses of the "high-risk" type, e.g. HPV-16, leads to a deregulation of both cell cycle control and cell differentiation, an activity that involves two viral genes, referred to as E6 and E7. Cell cycle deregulation and dedifferentiation are linked to specific changes in cellular gene expression, and it was shown that the E7 oncoprotein can modulate the transcription of various target genes. A major goal of the current project is to determine the role of chromatin-modifying enzymes, which appear to play an important role in cellular growth control, in transcriptional regulation by the E7 oncoprotein of HPV-16. In particular, we propose to investigate if the known human histone deacetylases (HDAC) and histone acetyltransferases (HAT) are involved in transcriptional regulation by E7. In a first step, the physical interaction of E7 with both classes of enzymes will be studied. By biochemical analyses, we will investigate whether E7 can sequester chromatin-modifying enzymes from cellular extracts; to this end, we will determine the ability of E7-associated proteins to either acetylate or deacetylate histones in vitro. We will also study the interaction of E7 with cloned members of the HDAC protein family. In a second step, we will determine if E7-dependent activation of the genes encoding cyclin E and cyclin A depends on functional interactions with chromatin-modifying enzymes. We will also determine the influence of members of the HDAC protein family on transcriptional activity of E7-responsive genes, including the cyclin E and cyclin A genes, and determine if E7 can influence transcriptional regulation by the HDAC proteins. We also intend to identify cellular genes, which are subject to transcriptional repression by E7. To this end, we will set up a transformation assay with E7 in chicken fibroblasts and determine alterations in cellular gene expression that are induced by E7. To study transcriptional repression by E7, we will first express E7 in the chicken fibroblast cell transformation system and monitor the effect of E7 on transformation sensitive genes, which have been established through studies of cell transformation by Myc. Given the functional homology between the cellular c- myc oncogene and HPV-16 E7, we will experimentally address the hypothesis that Myc and E7 share the ability to repress a subset of cellular genes. We will also investigate if E7 shares with Myc the ability to specifically block trans-activation by transcription factors of the C/EBP family.

The aim of the project was to determine regulatory mechanisms by which the E7 oncoprotein of HPV-16 contributes to tumorigenesis. Special emphasis of the project was on E7-dependent changes in the transcriptional activity of certain cell cycle regulatory genes, in particular, cyclin E, which represents one of the most important switches which need to be activated to allow passage through the restriction point. The experiments have shown that inhibitors of histone deacetylases can block the E7-dependent activation of the cyclin E gene. We also found that this effect depends on binding sites for the transcription factors E2F and SP1. In further studies we found that histone deacetylases (HDAC) also play a role in tumor suppression, in a process referred to as "cellular senescence". We found, by comparing histone deacetylases from normal and senescent cells, that histone deacetylase 2 is present in different complexes in senescent cells, when compared to normal cells. These results suggest that cellular senescence may be regulated in a HDAC2-dependent fashion. In further experiments, we analyzed the ability of E7 to break senescence. In the course of these experiments, we found that cellular senescence is associated with distinct changes in the glycolytic pathway. We found that pyruvate kinase is present in senescent cells in a special (tetrameric) conformation which is characterised by very high substrate affinity. This change results in a high throughput of glucose to pyruvate and disables the cell to provide new nucleotide precursors from glycolysis. These changes in the metabolism are associated with growth arrest, a known hallmark of cellular senescence. In further studies we found that breaking the senescent phenotype by HPV- 16 E7 leads to a resumption of cell proliferation and this is accompanied by a shifting of pyruvate kinase from the high affinity (tetrameric) form to the low affinity (dimeric) form. We also identified acid glucosidase as a new E7- binding cellular protein and found that, surprisingly, E7 acts as an allosteric activator of acid glucosidase, leading to the mobilization of cellular glycogen stores. These results suggest a model in which the transforming activity of the E7 oncogene involves distinct alterations of the cellular carbohydrate metabolism besides its effects on the transcription of various cellular genes.