BIOCHEMICAL FUNCTIONS OF ADENOVIRUS ONCOGENES

点击翻译按钮获取中文摘要

DESCRIPTION (Adapted from Applicant's Abstract): The adenovirus E1A 243R oncoprotein is a multifunctional transcriptional regulator that expresses diverse functions encoded in multiple domains. Of special interest is the transcriptional repression function that maps to E1A N-terminal protein sequences important for induction of cell cycle progression and cellular transformation. Our primary goal is to understand molecular mechanism and cellular targets of E1A transcriptional repression. Our long term goal is to understand how E1A repression regulates progression of the cell cycle. We have developed an in vitro transcription-repression system in which E1A 1-80, a recombinant protein containing the N-terminal 80 amino acids, is used as a prototype repressor to specifically repress transcription of E1A-repressible genes. Repression requires two small regions within the E1A N-terminus and does not appear to require promoter elements upstream of the TATA box. The first specific aim is to extend these findings to other E1A-repressible promoters, including those of the medically significant erbB2 protooncogene and HIV-1. The second specific aim is to continue well developed studies strongly implicating TBP (TFIID) as a direct cellular target of E1A repression and showing that E1A 1-80 can block interaction between TBP and TFIIB. We will: (i) define further the role of TFIID and TFIIB in E1A repression by analyzing interaction between the E1A repression domain and TBP using a collection of E1A and TBP alanine scanning mutants; (ii) probe the dual function of E1A in blocking TBP interaction with TATA-box DNA and with TFIIB; (iii) use purified preinitiation complexes (PICs) to ask whether E1A inhibits recruitment of TBP, TFIIB, or other GTFs to the promoter, and (iv) analyze the ability of TBP and TFIIB to overcome E1A repression in vivo. The third specific aim is to continue studies on the role of cellular protein p300 in E1A repression. We will ask whether: (i) E1A repression in vitro can be overcome by purified p300; (ii) E1A repression can be functionally separated from p300 binding by use of E1A alanine scanning mutants; and (iii) E1A repression can be fully reversed by overexpression or cell microinjection of p300 and TBP (or TFIID). The fourth specific aim is to use a reconstituted in vitro transcription system to study in greater detail the interactions between the E1A repression domain and its cellular targets TFIID, TFIIB, and p300. This system permits experiments to elucidate differences between E1A repressible and nonrepressible promoters. Finally, as long term goal, we propose to use subtractive cDNA libraries and mRNA Differential Display to identify cellular genes whose expression is modulated early after expression of the E1A N-terminal domain in quiescent cells. Functional assays will be used to identify Quiescence maintaining genes, i.e. potential tumor suppressor genes that may be targets for E1A repression, and (ii) up-regulated genes that are potential master switches in the growth cycle.