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. 2010 Feb 16;107(7):3135-40.
doi: 10.1073/pnas.0914142107. Epub 2010 Feb 1.

Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration

Andrea L Ferris  1 , Xiaolin Wu, Christina M Hughes, Claudia Stewart, Steven J Smith, Thomas A Milne, Gang G Wang, Ming-Chieh Shun, C David Allis, Alan Engelman, Stephen H Hughes
Affiliations

Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration

Andrea L Ferris et al. Proc Natl Acad Sci U S A. .

Abstract

Lens epithelium-derived growth factor (LEDGF) fusion proteins can direct HIV-1 DNA integration to novel sites in the host genome. The C terminus of LEDGF contains an integrase binding domain (IBD), and the N terminus binds chromatin. LEDGF normally directs integrations to the bodies of expressed genes. Replacing the N terminus of LEDGF with chromatin binding domains (CBDs) from other proteins changes the specificity of HIV-1 DNA integration. We chose two well-characterized CBDs: the plant homeodomain (PHD) finger from ING2 and the chromodomain from heterochromatin binding protein 1alpha (HP1alpha). The ING2 PHD finger binds H3K4me3, a histone mark that is associated with the transcriptional start sites of expressed genes. The HP1alpha chromodomain binds H3K9me2,3, histone marks that are widely distributed throughout the genome. A fusion protein in which the ING2 PHD finger was linked to the LEDGF IBD directed integrations near the start sites of expressed genes. A similar fusion protein in which the HP1alpha chromodomain was linked to the LEDGF IBD directed integrations to sites that differed from both the PHD finger fusion-directed and LEDGF-directed integration sites. The ability to redirect HIV-1 DNA integration may help solve the problems associated with the activation of oncogenes when retroviruses are used in gene therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Gene expression and integration site preferences. Top: The uppermost panel (Expression) represents >17,000 well-characterized mouse genes placed into bins of 100 according to the level of expression measured in the MEF-KO cells. The average level of expression for the genes in each bin is given on the y axis (log2 scale). Bottom: Distribution of H3K4me3 marks from the data of Barski et al. (30) plotted against bins of 100 genes according to the level of expression in human CD4+ cells. Upper Middle, Middle, Lower Middle: These three panels show, on the y axis, the number of integrations in each bin of 100 genes for LEDGF and the two CBD–IBD fusions. The genes were placed in bins according to their level of expression in MEF-KO cells, shown in the uppermost panel. The integration data have been normalized to a total of 10,000 integrations. The data for the ING2 PHD finger fusion were based on the number of integrations within 2.5 kb of a TSS for the 100 genes in each bin, so each point represents the number of total integrations in 0.5 Mb. For huLEDGF and the HP1α chromodomain fusions, which direct integrations throughout genes, we first enumerated the total number of integrations in all of the genes in each bin. Because the size of the genes varies (highly expressed genes tend to be smaller), the total target size in different bins also varies. To solve this problem, the data were renormalized on the basis of a total target size of 1 Mb in each bin. This means that the ING2 fusion data cannot be directly compared with the HP1α fusion and huLEDGF data (see text). Note that the various panels have different scales on the y axis.
Fig. 2.
Fig. 2.
ING2 PHD finger fusion directs tightly clustered integrations. The figure was prepared by marking integration sites on the University of California, Santa Cruz database of the mouse genome (mm9). The nucleotide positions are given at the top of each panel, and the transcript(s) from the gene are shown at the bottom. A scale bar for the DNA segment is shown at the top of the figure. The data from the two ING2–IBD 454 experiments (run A and run B) are shown separately in all panels. Integrations are shown as vertical bars. (A) Region of mouse chromosome 19 that contains the Malat1 gene, which contains both ING2–IBD-directed integrations. The approximate positions of the segment shown in B and C are marked at the top. (B) A small segment of Malat1 in which there were clustered ING2–IBD-directed integrations; (C) a second cluster in a different segment of the Malat1 gene where there were ING2–IBD-directed integrations at exactly the same sites in the two 454 experiments.
Fig. 3.
Fig. 3.
Distribution of integration sites near TSSs. (Upper) The y axis shows the percentage of the total integrations for the individual CBD–IBD fusions that occur within 100 bp intervals from a TSS. The TSS is in the middle, integrations upstream of a TSS are on the left (negative numbers), and those downstream are on the right (positive numbers). The ING2–IBD causes preferential integration near TSSs. The distribution of H3K4me3 marks in the genome of human CD4+ cells (30) is shown for comparison. (Lower) Data for the HP1α–IBD- and huLEDGF–IBD-directed integrations, which were indistinguishable from the baseline in the upper graph, shown on an expanded scale.
Fig. 4.
Fig. 4.
Genes that contain hotspots for CBD–IBD fusions. The format is the same as in Fig. 2. (A) The entire Fbxl11 gene. At this resolution, the multiple integration sites near the promoter appear as thick black lines. (B) A closer view of the region near the Fbxl11 TSS, showing the individual integration sites.
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