Frontiers of retrovirology: complex retroviruses, retroelements and their hosts location:Montpellier, France date:21-23 September 2009
Integration of human immunodeficiency virus type 1 (HIV-1) displays a strong bias towards active genes. Knockdown or knockout of the specific integrase interaction partner LEDGF/p75 (Lens Epithelium Derived Growth Factor) abolishes this integration pattern, supporting the hypothesis that LEDGF/p75 tethers the HIV pre-integration complex to specific sites in the chromatin. We employed DamID-technology to identify the chromatin binding sites of LEDGF/p75. This method is based on the stable low-level expression of the bacterial EcoDam fused with the DNA-binding protein of interest, in our case LEDGF/p75. EcoDam is a bacterial DNA-methylase that methylates adenosines in the 5’-GATC-3’ recognition sequence. Endogenous adenosine methylation is absent in eukaryotic cells. LEDGF/p75-EcoDam expression in HeLa cells results in adenosine methylation adjacent to the endogenous chromatin binding sites of LEDGF/p75. Following purification of the genomic DNA, DpnI digestion and adaptor ligation, methylated DNA fragments were amplified and analyzed via DNA tiling array technology. Micro-array analysis was performed on ENCODE-chips covering ± 1% of the human genome which is highly annotated in regard to epigenetic modifications, transcription factor binding sites, etc. LEDGF/p75 binding regions were defined using the MAT algorithm (Model-Based Analysis of Tiling arrays) allowing correlations with the HIV 1 integration pattern and epigenetic modifications. No consensus binding site was found. LEDGF/p75 primarily binds downstream of the transcription start site of active transcription units which correlates with an enrichment of HIV 1 integration sites. Our results however are at odds with the previously proposed binding of LEDGF/p75 to stress response elements. Correlation analysis of LEDGF/p75 binding sites with more than 200 genomic features revealed an association with active chromatin markers, such as H3 and H4 acetylation, H3K4 mono-methylation and RNA polymerase II binding. Interestingly, some associations did not correlate with HIV-1 integration indicating that not all LEDGF/p75 complexes on the chromosome are amenable to HIV 1 integration.