Abstract
PWWP domains are involved in the chromatin attachment of several proteins. They bind to both DNA and proteins and their interaction with specific histone methylation marks define them as a new class of histone code readers. The lens epithelium derived growth factor (LEDGF/p75) contains an N-terminal PWWP domain necessary for its interaction with chromatin but also a C-terminal domain which interacts with several proteins, such as lentiviral integrases. These two domains confer a chromatin-tethering function to LEDGF/p75 and in the case of lentiviral integrases, this tethering participates in the efficiency and site selectivity of integration. Although proteins interacting with LEDGF/p75 C-terminal domain have been extensively studied, no data exist about partners of its PWWP domain regulating its interaction with chromatin. In this study, we report the identification by yeast-two-hybrid of thirteen potential partners of the LEDGF PWWP domain. Five of these interactions were confirmed in mammalian cells, using both a protein complementation assay and co-immunoprecipitation approaches. Three of these partners interact with full length LEDGF/p75, they are specific for PWWP domains of the HDGF family and they require PWWP amino acids essential for the interaction with chromatin. Among them, the transcription activator TOX4 and the splicing cofactor NOVA1 were selected for a more extensive study. These two proteins or their PWWP interacting regions (PIR) colocalize with LEDGF/p75 in Hela cells and interact in vitro in the presence of DNA. Finally, single round VSV-G pseudotyped HIV-1 but not MLV infection is inhibited in cells overexpressing these two PIRs. The observed inhibition of infection can be attributed to a defect in the integration step. Our data suggest that a regulation of LEDGF interaction with chromatin by cellular partners of its PWWP domain could be involved in several processes linked to LEDGF tethering properties, such as lentiviral integration, DNA repair or transcriptional regulation.
Highlights
The PWWP domain is a 70–135 amino acid sequence containing the Pro-Trp-Trp-Pro (PWWP) motif, which is conserved between more than 60 eukaryotic proteins characterized for their DNA or chromatin interaction [1]
(by PCR and Gateway BP reaction), these entry clones were further introduced in different destination vectors. cDNA sequences coding for other PWWP domains used in this study and obtained form different origins were cloned into the pDonR207 plasmid: aa 1–99 of human HRP2
The N-terminal part, shared between the p52 and p75 forms, contains a PWWP domain, resistant to trypsin digestion [32] and involved in the selectivity of LEDGF interaction with chromatin [38]. This domain has been shown to interact to histone H3 trimethylated on lysine 36 (H3K36me3) [6,8,9], a property conserved with other PWWP domains [1,4,5,7]
Summary
The PWWP domain is a 70–135 amino acid sequence containing the Pro-Trp-Trp-Pro (PWWP) motif, which is conserved between more than 60 eukaryotic proteins characterized for their DNA or chromatin interaction [1]. DNA interaction was originally shown for the DNMT3b PWWP domain [10,11] and recent structural studies of several PWWP domains have revealed the presence of a positively charged surface enriched in basic residues and involved in this DNA binding property [12,13,14,15,16]. PWWP domains have been characterized as new histone code readers. They recognize methylated histones, a property conserved with other members of the Tudor domain ‘‘Royal family’’ such as the Chromo, MBT or tudor domains [21,22]. Histone-PWWP affinities are weak but the PWWP-nucleosome complexes are stabilized by additional PWWP-DNA interactions, as shown with the LEDGF PWWP domain [8,9]
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