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Title: An investigation of the cellular functions of LEDGF/p75
Author: Hughes, Siobhan Marie
ISNI:       0000 0004 2690 6396
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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Lens epithelium-derived growth factor (LEDGF) is a critical co-factor of lentiviral integration that accounts for the characteristic bias of the retroviral genus towards integration within active transcription units of the host genome. It’s well-characterised role in viral replication notwithstanding, cellular functions of LEDGF are poorly understood. In this study gene expression profiling and proteomic analysis have been used to elucidate cellular functions of LEDGF. Gene expression profiling of mouse embryonic fibroblasts (MEFs) derived from Psip1-/- (LEDGF-null) embryos revealed that less than 200 genes were significantly up- or down-regulated compared to wild type (WT) MEFs. The changes in gene expression of a subset of affected genes were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. However, these changes could not be reversed by re-introduction of LEDGF into knockout MEFs. Furthermore, in vitro knockout of Psip1 did not reproduce the effects of in utero knockout, suggesting that LEDGF ablation per se does not account for the observed perturbations in gene expression. Collectively, these data strongly indicate that in spite of its apparent ubiquitous association with active transcription units, LEDGF does not play an essential nor direct role in the regulation of gene expression. Tandem affinity purification (TAP) of a cTAP-tagged truncation mutant of LEDGF (residues 326-530) identified the heterodimeric S-phase kinase, termed cell division cycle 7 (Cdc7):activator of S-phase kinase (ASK), as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with LEDGF from human cells, and endogenous Cdc7 and LEDGF proteins displayed nuclear co-localisation in G1 and S phases of the cell cycle. Moreover, Cdc7:ASK was enriched on chromatin when ectopically co-expressed with LEDGF in human cells, suggesting that LEDGF may affect chromatin binding of the kinase. Truncation analyses identified the integrase-binding domain (IBD) of LEDGF as essential and minimally sufficient for the interaction with Cdc7:ASK. Reciprocally, the interaction required auto-phosphorylation of the kinase and the presence of fifty C-terminal residues of ASK. LEDGF potently stimulated the kinase activity of Cdc7:ASK, increasing phosphorylation of one of its substrates, mini-chromosome maintenance 2 (MCM2), in vitro by more than 10-fold. The effect strictly depended on the interaction, as LEDGF mutants unable to bind Cdc7:ASK also failed to promote its kinase activity. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyper-active kinase. These results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7:ASK kinase activity, imposed by the C-terminus of ASK. While more work is required to fully elucidate the natural cellular functions of LEDGF, this study has provided some insight into its role in cell biology. In particular, understanding the role of LEDGF in Cdc7:ASK kinase regulation and the implications of this interaction during S phase of the cell cycle are promising avenues of investigation highlighted in this study.
Supervisor: Cherepanov, Peter ; Weber, Jonathan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral