The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair

Abstract

The molecular mechanism by which foreign DNA integrates into the human genome is poorly understood yet critical to many disease processes, including retroviral infection and carcinogenesis, and to gene therapy. We hypothesized that the mechanism of genomic integration may be similar to transposition in lower organisms. We identified a protein, termed Metnase, that has a SET domain and a transposase/nuclease domain. Metnase methylates histone H3 lysines 4 and 36, which are associated with open chromatin. Metnase increases resistance to ionizing radiation and increases nonhomologous end-joining repair of DNA doublestrand breaks. Most significantly, Metnase promotes integration of exogenous DNA into the genomes of host cells. Therefore, Metnase is a nonhomologous end-joining repair protein that regulates genomic integration of exogenous DNA and establishes a relationship among histone modification, DNA repair, and integration. The data suggest a model wherein Metnase promotes integration of exogenous DNA by opening chromatin and facilitating joining of DNA ends. This study demonstrates that eukaryotic transposase domains can have important cell functions beyond transposition of genetic elements.

Fig. 1.

Metnase has pre-SET, SET, and transposase domains. (A) Metnase pre-SET domain aligned with the consensus pre-SET sequence and with human SUV39H1. (B) Metnase SET domain aligned with the consensus SET sequence and with SUV39H1. (C) Metnase transposase domain aligned with the transposase domain consensus and with planarian Mariner-9 transposase. (D) Metnase integrase domain aligned with the integrase domain consensus and with the HIV-1 integrase core domain. (E) Metnase expression in normal human tissues as measured by RT-PCR.

Fig. 2.

Histone methylation by Metnase. (A) Recombinant GST-Metnase transferred ³H-methyl groups from SAM to recombinant human histone H3 and total human histones as analyzed by scintillation counting. ³H levels (cpm) were subtracted from GST alone as a baseline control. (B) Recombinant GST-Metnase transferred ³H-methyl groups from SAM to recombinant human histone H3 and total human histones as analyzed by autoradiography. (C) Recombinant GST-Metnase in vitro transferred unlabeled methyl groups from SAM to pure recombinant human histone H3 lysines as detected by Western blot. (D) The transfer of unlabeled methyl groups from SAM to H3 K36 requires both SAM and histone H3.

Fig. 3.

Metnase promotes foreign DNA integration. (A) Western blot analysis of the protein expression of transfected Metnase. (B) Metnase promotes cis integration of foreign DNA. Human 293 cells were transfected with the pCDNA3.1-Metnase expression vector, which also carries neo. The number of G418-resistant colonies is a measure of integration. (C) Metnase promotes trans integration of foreign DNA. Human 293 cells were cotransfected with the pcDNA-Metnase expression vector and the plasmid, pBOS, which expresses a puromycin resistance cassette. The number of puromycin-resistant colonies is a measure of pBOS integration. (D) Metnase promotes integration of Moloney leukemia virus (MLV) DNA. pJ6-Metnase was cotransfected into human 293 cells with the MLV vector MSCV2.1, which carries neo, and MLV integration was scored as the number of G418-resistant colonies. (E) A single DSB does not enhance the trans integration of foreign DNA induced by Metnase. Inducing a DSB by using the restriction enzyme I-SceI did not increase the ability of Metnase to integrate foreign DNA. Metnase was expressed from the pcDNA vector and the hygromycin selectable marker was carried on a separate pRNA-U6 vector.

Fig. 4.

Metnase mediates foreign DNA integration. (A) siRNA was used to reduce the RNA and protein expression of Metnase. (B and C) Integration of plasmid (pBOS) and Moloney Leukemia Virus DNA (pMSCV) in control and Metnase knockdown cells.

Fig. 5.

Metnase enhances NHEJ. (A) NHEJ was measured by the ability of a linearized vector to have its free ends rejoined and transform bacteria to ampicillin resistance. Precise end joining was measured by the ability of the pBluescript plasmid linearized within a β-galactosidase gene to make functional β-galactosidase protein when rescued from human 293 cells with stably increased or decreased expression of Metnase. PCR analysis by using T7/SP6 primers of standardized concentrations of rescued plasmid showed an increased amount of end-joined plasmid when Metnase is overexpressed. (B) Mutations of residues known to be required for histone methylase or transposase function in Metnase reduced the ability of Metnase to stimulate NHEJ. (C) Western analysis shows that the Metnase mutants are all expressed well at the protein level. (D) Cell survival after γ-radiation in cells over- or underexpressing Metnase.