S of LEDGF/p75. Both LEDGF/p75 and HRP-2 carry a
S of LEDGF/p75. Both LEDGF/p75 and HRP-2 carry a PWWP domain, recently identified as a chromatin reader recognizing epigenetic marks, such as methylated histone side-chains [6,24,25]. Previously, we demonstrated that swapping the PWWP-domain of LEDGF/ p75 with that of HRP-2 could rescue lentiviral replication and integration site selection in genes [6]. Here, integration distribution in LEDGF/p75 KD cells overexpressing HRP-2 was comparable to wild-type cellsSchrijvers et al. Retrovirology 2012, 9:84 http://www.retrovirology.com/content/9/1/Page 5 ofCD4+ T-cell MarksWTHRP-2 KDLEDGF/p75 KDLEDGF/p75 KD HRP-2 KD LEDGF/p75 KD + HRP-HeLa Marks Repression Histone methylation marks associated with transcriptional: ActivationWTHRP-2 KDLEDGF/p75 KDLEDGF/p75 KD HRP-2 KD LEDGF/p75 KD + HRP–0.2 -0.1 0.random0.0.Disfavored compared to MRCFavored compared to MRCFigure 4 LEDGF/p75 and HRP-2 depletion shifts integration towards random. A pooled score was determined for HIV-1 integration near epigenetic marks associated with transcriptional repression or activation for each cell line and plotted in a bar diagram. The X-axis represents deviation from random with positive values indicating favored integration compared to MRC, negative values disfavored integration compared to MRC. The pooled score is the average of the different ROC values – 0.5 (to indicate the deviation from random) obtained with individual markers (not shown) in a 10 kb window. For HeLaP4, H3K9me2, H3K9me3, H3K27me3, and H4K20me3 were pooled as associated with `repression’; H2BK5me1, H3K4me1, H3K36me3, H4K20me1 were pooled as associated with `activation’. For CD4+ T-cells, H3K9me2, H3K9me3, H3K27me2, H3K27me3, H3K79me3 and H4K20me3 were pooled as Fruquintinib chemical information PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 associated with `repression’; H2BK5me1, H3K4me1, H3K4me2, H3K9me1, H3K27me1, H3K36me3, H4K20me1 were pooled as associated with `activation’.(Figure 3 and Additional file 2 and 3), suggesting only subtle differences between LEDGF/p75 and HRP-2 for the interaction with the local chromatin. Although LEDGF/p75 and HRP-2 double KD shifted integration significantly out of transcription units, integration remained distinct from random in the double KD cells (Table 1). Multiple (mutually non-exclusive) reasons can be put forward to explain this observation. First, in these experiments we employed RNAi and even though the knockdown was potent (>90 and 84 on mRNA level for LEDGF/p75 and HRP-2 respectively), residual LEDGF/p75 and HRP-2 might account for the residual bias. The generation of a human double KO cell line, devoid of both LEDGF/p75 and HRP-2, will provide a more definite answer. Second, this result could suggest the presence of additional cellular co-factor(s) that affect PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28128382 targeting in the absence of LEDGF/p75 and HRP-2. Transcription factor IIS (TFIIS) for example harbors an IBD-like domain [6,8], although structurally moredistantly related to the IBD of LEDGF/p75 and HRP-2, but lacks a PWWP-domain. However, in the presence of LEDGF/p75, HRP-2 does not seem to play a role in HIV replication [6] or targeting (this work), suggesting alternate IBD containing tethers will probably only play a minor role in HIV replication or targeting in WT conditions, unless expression levels differ strongly, accrediting LEDGF/p75 as principal tether [6]. Third, the bias might reflect specific constraints of the viral IN, the local chromatin environment or the pre-integration complex itself. Although HIV integration favors weak palindromic sequences, our anal.
