title: Quantitative proteomic discovery of dynamic epigenome changes that control human cytomegalovirus (HCMV) infection.
abstract: This work represents the first comprehensive quantitative analysis of global histone post-translational modifications (PTMs) from a virus infection, namely human cytomegalovirus (HCMV) infection. We used a nanoLC-MS/MS platform to identify and quantify the dynamic histone H3 and H4 PTMs expressed during HCMV replication in primary fibroblasts. Specifically, we examined the changes in histone PTMs over a 96 h time course to sample the immediate early (IE), early (E), and late (L) stages of viral infection. Several changes in histone H3 and H4 PTMs were observed, including a marked increase in H3K79me2 and H3K27me3K36me2, and a decrease in H4K16ac, highlighting likely epigenetic strategies of transcriptional activation and silencing during HCMV lytic infection. Heavy methyl-SILAC (hm-SILAC) was used to further confirm the histone methylation flux (especially for H3K79) during HCMV infection. We evaluated DOT1L (the H3K79 methyltransferase) mRNA levels in mock and HCMV-infected cells over a 96 h time course, and observed a significant increase in this methyltransferase as early as 24 hpi showing that viral infection up-regulates DOT1L expression, which drives H3K79me2. We then used shRNA to create a DOT1L knockdown cell population, and found that HCMV infection of the knockdown cells resulted in a 10-fold growth defect when compared with infected control cells not subjected to knockdown. This work documents multiple histone PTMs that occur in response to HCMV infection of fibroblasts, and provides a framework for evaluation of the role of epigenetic modifications in the virus-host interaction.
authors.affiliation: From the ‡Department of Molecular Genetics, Cleveland Clinic, Lerner Research Institute, Cleveland, Ohio 44195;
authors.forename: Christine M
authors.initials: CM
authors.lastname: O'Connor
authors.affiliation: §Department of Chemical Engineering, Imperial College London, London, UK, SW7 2AZ;
authors.forename: Peter A
authors.initials: PA
authors.lastname: DiMaggio
authors.affiliation: ¶Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544;
authors.forename: Thomas
authors.initials: T
authors.lastname: Shenk
authors.affiliation: ‖Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, 19104 bgarci@mail.med.upenn.edu.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
mesh_terms: D002460:Cell Line; D002853:Chromatography, Liquid; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003586:Cytomegalovirus Infections / Q000378:metabolism*; D004279:DNA, Viral / Q000032:analysis; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology*; D055785:Gene Knockdown Techniques; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008780:Methyltransferases / Q000235:genetics; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology
chemical_list: D004279:DNA, Viral; D006657:Histones; D014764:Viral Proteins; C473477:DOT1L protein, human; D008780:Methyltransferases; D011495:Histone-Lysine N-Methyltransferase