Integration of retroviral DNA into host cell DNA is a defining feature of retroviral replication. HIV integration is known to be favored in active transcription units, which promotes efficient transcription of the viral genes, but the molecular mechanisms responsible for targeting are not fully clarified. Here we used pyrosequencing to map 40,569 unique sites of HIV integration. Computational prediction of nucleosome positions in target DNA indicated that integration sites are periodically distributed on the nucleosome surface, consistent with favored integration into outward-facing DNA major grooves in chromatin. Analysis of integration site positions in the densely annotated ENCODE regions revealed a wealth of new associations between integration frequency and genomic features. Integration was particularly favored near transcription-associated histone modifications, including H3 acetylation, H4 acetylation, and H3 K4 methylation, but was disfavored in regions rich in transcription-inhibiting modifications, which include H3 K27 trimethylation and DNA CpG methylation. Statistical modeling indicated that effects of histone modification on HIV integration were partially independent of other genomic features influencing integration. The pyrosequencing and bioinformatic methods described here should be useful for investigating many aspects of retroviral DNA integration.

Krüppel-associated box domain-zinc finger proteins (KRAB–ZFPs) are tetrapod-specific transcriptional repressors encoded in the hundreds by the human genome. In order to explore their as yet ill-defined impact on gene expression, we developed an ectopic repressor assay, allowing the study of KRAB–mediated transcriptional regulation at hundreds of different transcriptional units. By targeting a drug-controllable KRAB–containing repressor to gene-trapping lentiviral vectors, we demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases (kb) away from their DNA binding sites, with an efficiency which is generally higher for promoters located within 15 kb or less. Silenced promoters exhibit a loss of histone H3-acetylation, an increase in H3 lysine 9 trimethylation (H3K9me3), and a drop in RNA Pol II recruitment, consistent with a block of transcriptional initiation following the establishment of silencing marks. Furthermore, we reveal that KRAB–mediated repression is established by the long-range spreading of H3K9me3 and heterochromatin protein 1 β (HP1β) between the repressor binding site and the promoter. We confirm the biological relevance of this phenomenon by documenting KAP1–dependent transcriptional repression at an endogenous KRAB–ZFP gene cluster, where KAP1 binds to the 3′ end of genes and mediates propagation of H3K9me3 and HP1β towards their 5′ end. Together, our data support a model in which KRAB/KAP1 recruitment induces long-range repression through the spread of heterochromatin. This finding not only suggests auto-regulatory mechanisms in the control of KRAB–ZFP gene clusters, but also provides important cues for interpreting future genome-wide DNA binding data of KRAB–ZFPs and KAP1. The regulation of gene activity by transcription factors is crucial to the function of all cells. Here, we studied the mechanisms of action of the largest family of gene regulators encoded by the human genome, the so-called KRAB–containing zinc finger proteins (KRAB–ZFPs), which in concert with their universal cofactor KAP1 act as transcriptional repressors. For this, we used two parallel approaches. First, by targeting an ectopic KRAB domain to hundreds of different genes, we found that KRAB/KAP1 can repress promoters located several tens of kilobases from the repressor DNA docking site. We further could show that KRAB induces such long-range effects by mediating the spread of repressive chromatin marks along the body of the gene, resulting in a block of transcriptional initiation at the promoter. In a second set of experiments, we analyzed an endogenous KRAB–ZFP gene cluster, where we could also document KAP1–dependent heterochromatin spreading and transcriptional repression. Together, these results support a model whereby KRAB–ZFPs and KAP1 can mediate long-range transcriptional repression through the spread of silencing chromatin marks. This study thus provides insight into KRAB/KAP1–induced gene regulation at KRAB–ZFP gene clusters, and will further help interpret genome-wide studies of KRAB–ZFPs and KAP1 DNA binding patterns.

Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV) integrates preferentially within active transcription units, whereas murine leukemia virus (MLV) integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN) coding region into HIV (to make HIVmIN) caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN) further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag) displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I–hypersensitive sites (i.e., +/− 1 kb), and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins. A required step in the replication cycle of retroviruses is the integration of a DNA copy of the viral genome into a host cell chromosome. Recent studies have shown that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) favor integration near different chromosomal features. HIV preferentially targets active genes, while MLV prefers integration near start sites of gene transcription. The authors investigated integration-target site–selection by HIV derivatives substituted with segments of MLV to determine which viral proteins are responsible for integration-targeting preferences. They found that the viral integrase protein is the dominant determinant of integration-site selection, probably through its tethering to cellular proteins bound near preferred genomic regions. In addition, components of the viral structural polyprotein, Gag, appear to be involved in targeting. These findings provide a functional map of the viral proteins involved in directing integration-site selection.