title: H3K27 Demethylation at the Proviral Promoter Sensitizes Latent HIV to the Effects of Vorinostat in Ex Vivo Cultures of Resting CD4+ T Cells.
abstract: Histone methyltransferase inhibitors (HMTis) and histone deacetylase inhibitors (HDACis) are reported to synergistically induce the expression of latent human immunodeficiency virus type 1 (HIV-1), but studies have largely been performed with cell lines. As specific and potent HMTis directed at EZH1 (enhancer of zeste 2 Polycomb repressive complex 2 subunit 1)/EZH2 are now in human testing, we wished to rigorously test such an inhibitor in a primary resting T-cell model of HIV latency. We found that GSK343, a potent and selective EZH2/EZH1 inhibitor, reduced trimethylation of histone 3 at lysine 27 (H3K27) of the HIV provirus in resting cells. Remarkably, this epigenetic change was not associated with increased proviral expression in latently infected resting cells. However, following the reduction in H3K27 at the HIV long terminal repeat (LTR), subsequent exposure to the HDACi suberoylanilide hydroxamic acid or vorinostat (VOR) resulted in increases in HIV gag RNA and HIV p24 antigen production that were up to 2.5-fold greater than those induced by VOR alone. Therefore, in primary resting CD4(+) T cells, true mechanistic synergy in the reversal of HIV latency may be achieved by the combination of HMTis and HDACis. Although other cellular effects of EZH2 inhibition may contribute to the sensitization of the HIV LTR to subsequent exposure to VOR, and to increase viral antigen production, this synergistic effect is directly associated with H3K27 demethylation at nucleosome 1 (Nuc-1). Based upon our findings, the combination of HMTis and HDACis should be considered for testing in animal models or clinical trials.
  
  
  OBJECTIVE
  Demethylation of H3K27 mediated by the histone methyltransferase inhibitor GSK343 in primary resting T cells is slow, occurring over 96 h, but by itself does not result in a significant upregulation of cell-associated HIV RNA expression or viral antigen production. However, following H3K27 demethylation, latent viral expression within infected primary resting CD4(+) T cells is synergistically increased upon exposure to the histone deacetylase inhibitor vorinostat. Demethylation at H3K27 sensitizes the HIV promoter to the effects of an HDACi and provides a proof-of-concept for the testing of combination epigenetic approaches to disrupt latent HIV infection, a necessary step toward the eradication of HIV infection.
authors.affiliation: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Tripathy
authors.affiliation: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Mary E M
authors.initials: ME
authors.lastname: McManamy
authors.affiliation: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Brandon D
authors.initials: BD
authors.lastname: Burch
authors.affiliation: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Nancie M
authors.initials: NM
authors.lastname: Archin
authors.affiliation: Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA dmargo@med.unc.edu.
authors.forename: David M
authors.identifier: http://orcid.org/0000-0001-5714-0002
authors.initials: DM
authors.lastname: Margolis
mesh_terms: D000704:Analysis of Variance; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D047369:Chromatin Immunoprecipitation; D000071221:Enhancer of Zeste Homolog 2 Protein; D004797:Enzyme-Linked Immunosorbent Assay; D006678:HIV / Q000187:drug effects* / Q000502:physiology; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D015151:Immunoblotting; D007191:Indazoles / Q000494:pharmacology*; D008745:Methylation / Q000187:drug effects; D063151:Polycomb Repressive Complex 2 / Q000037:antagonists & inhibitors; D011401:Promoter Regions, Genetic / Q000235:genetics; D011533:Proviruses / Q000235:genetics; D011728:Pyridones / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D000077337:Vorinostat
chemical_list: C586265:GSK343; D006657:Histones; D006877:Hydroxamic Acids; D007191:Indazoles; D011728:Pyridones; D034741:RNA, Small Interfering; D000077337:Vorinostat; C103523:EZH1 protein, human; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2