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HIV integration:LEDGF/p75-modulated targets and LEDGF/p75 regulation

English title HIV integration:LEDGF/p75-modulated targets and LEDGF/p75 regulation
Applicant Ciuffi Angela
Number 120553
Funding scheme Project funding
Research institution Institut de Microbiologie - CHUV Faculté de Biologie et Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Experimental Microbiology
Start/End 01.06.2008 - 31.05.2010
Approved amount 186'000.00
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Keywords (10)

retrovirus; HIV; integration; integrase; LEDGF/p75; transcription; PSIP1; regulation; ChIPSeq; cancer

Lay Summary (English)

Lead
Lay summary
During their life cycle, retroviruses integrate into the host genome and then use the host cellular machinery to ensure the transcription of their genome and the production of viral proteins, necessary to the production of new infectious viral particles. The site of integration has important consequences for both the virus (i.e. viral transcription) and the host cell (i.e. insertional mutagenesis). Thus, the identification of host proteins involved in these steps should contribute to a better understanding of the biology of retroviruses, as well as provide additional targets for therapeutic approach and increase the safety of retroviral-based gene therapy vectors. We have recently shown that a cellular protein, LEDGF/p75, was in part responsible for directing the human immunodeficiency virus (HIV) integration into transcription units, and that LEDGF/p75-modulated genes, as identified by transcriptional profiling, were favored targets for integration. Data so far suggest that only lentiviral integrases (IN) (the enzyme catalyzing the integration reaction process) interact with LEDGF/p75, suggesting that other retroviruses use different cellular factors to help them integrate at specific genomic sites. The present project aims at:(i)identifying genes whose expression is modulated by LEDGF/p75, to gain insight into the cellular role of this protein, and(ii)investigating the mechanisms involved in the regulation of LEDGF/p75 expression. LEDGF/p75 target genes will be characterized by using chromatin immunoprecipitation (ChIP) with anti-LEDGF/p75 antibodies followed by sequencing of the captured DNA. Target genes so identified will be contrasted with those identified previously by expression arrays using partial knock-downs of LEDGF/p75. Target genes identified by both techniques will be used to perform bioinformatics analysis to propose consensus binding site(s) for LEDGF/p75, and/or DNA binding proteins that are binding partners of LEDGF/p75. Regulation of LEDGF/p75 expression will be investigated using luciferase reporter gene technology and electrophoretic mobility shift assays. Sequence analysis around the transcriptional start site of LEDGF/p75 reveals the absence of a TATA box or an initiator sequence, therefore we will first determine the minimal promoter necessary to allow LEDGF/p75 transcription.These investigations will help to better characterize the cellular role of LEDGF/p75, more particularly its DNA binding consensus or its cellular partners, as well as understand the mechanisms involved in the regulation of its expression.
Direct link to Lay Summary Last update: 21.02.2013

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