NDY1/KDM2B, EZH2 and H3K27 tri-methylation are required for immediate-early gene transcription.

<p><b>A and B</b>. HFFs were lentivirally or retrovirally transduced with the indicated constructs and they were subsequently infected with HCMV (MOI 0.5). The cells were fixed 5 hours later and the percentage of IE1-expressing cells was measured by FACS analysis. The bars show the percentage of IE1-positive cells (mean ± SD). <b>C</b>. Comparison of IE1 expression in HCMV-infected HFFs, transduced with pLKO.1, pLKO.1-shEZH2 or pLKO.1-shNDY1/KDM2B, prior to the infection. Cells were infected with HCMV (MOI 0.5). Western blots of cell lysates harvested at the indicated time points, were probed with anti-IE1 or anti-actin (loading control) antibodies. <b>D</b>. HFFs were infected with HCMV (MOI 0.5 PFU/cell) before and after a 30 minute pretreatment with the EZH2 inhibitor DZNep. The Western blotting shows the expression of EZH2 and IE1 in untreated and DZNep-pretreated cells at 24 hours from the start of the infection. <b>E</b>. HFFs were infected with HCMV (MOI 0.5 PFU/cell) before and after a 30-minute pretreatment with the EZH2 inhibitor DZNep. The infected cells were monitored by light microscopy 5 days later. In addition, they were stained for IE1 and counterstained with DAPI at 5 hours post-infection, and they were analyzed by epifluoerescence microscopy. Bar = 100 µm. <b>F</b>. The progeny virus harvested from the DZNep-treated and untreated cells as in D, 5 days after infection, was titrated by standard viral plaque assays. The bars show the viral titers (mean ± SD).</p

The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection

<div><p>Earlier studies had suggested that epigenetic mechanisms play an important role in the control of human cytomegalovirus (HCMV) infection. Here we show that productive HCMV infection is indeed under the control of histone H3K27 trimethylation. The histone H3K27 methyltransferase EZH2, and its regulators JARID2 and NDY1/KDM2B repress GFI1, a transcriptional repressor of the major immediate-early promoter (MIEP) of HCMV. Knocking down EZH2, NDY1/KDM2B or JARID2 relieves the repression and results in the upregulation of GFI1. During infection, the incoming HCMV rapidly downregulates the GFI1 mRNA and protein in both wild-type cells and in cells in which EZH2, NDY1/KDM2B or JARID2 were knocked down. However, since the pre-infection levels of GFI1 in the latter cells are significantly higher, the virus fails to downregulate it to levels permissive for MIEP activation and viral infection. Following the EZH2-NDY1/KDM2B-JARID2-independent downregulation of GFI1 in the early stages of infection, the virus also initiates an EZH2-NDY1/ΚDM2Β-JARID2-dependent program that represses GFI1 throughout the infection cycle. The EZH2 knockdown also delays histone H3K27 trimethylation in the immediate early region of HCMV, which is accompanied by a drop in H3K4 trimethylation that may contribute to the shEZH2-mediated repression of the major immediate early HCMV promoter. These data show that HCMV uses multiple mechanisms to allow the activation of the HCMV MIEP and to prevent cellular mechanisms from blocking the HCMV replication program.</p></div

Primers set used for ChIP analysis.

<p>Primers set used for ChIP analysis.</p

GFI1 mRNA and protein are degraded rapidly during HCMV infection and their degradation is followed by upregulation of EZH2, JARID2 and NDY1/KDM2B and downregulation of JMJD3.

<p><b>A</b>. The GFI1 mRNA levels in lysates of HFFs harvested at the indicated time points after HCMV infection were monitored by real time RT-PCR. <b>B</b>. The GFI1 protein levels in the same cells were monitored by western blotting. <b>C</b>. HCMV infection promotes the degradation of GFI1 at the RNA level. HFFs were mock-infected or infected with HCMV after treatment with Actinomycin D (5 µg/ml). GFI1 mRNA levels in cell lysates harvested at the indicated time points after exposure to the virus were measured by real time RT-PCR. <b>D</b>. Proteasomal inhibition blocks the rapid downregulation of GFI1 in HCMV-infected cells. HFFs were mock-infected or infected with HCMV after pretreatment with MG132 (10 µM). GFI1 protein levels in cell lysates harvested at the indicated time points after exposure to the virus were measured by western blotting. <b>E</b>. GFI1 protein levels in HFFs transduced with the indicated constructs and harvested before, and 2 hours after HCMV infection, were measured by western blotting. <b>F</b>. The expression of EZH2, NDY1/KDM2B, JARID2 and JMJD3 was measured by real time RT-PCR in lysates of HFFs, harvested at the indicated time points after HCMV infection.</p

Infection by HCMV depends on the downregulation of GFI1, a repressor of immediate-early gene transcription.

<p>Model summarizing the data on the interaction between the virus and the host. (Panel A) This panel describes the infection of wild type HFFs. The incoming virus rapidly degrades GFI1 to allow the activation of the MIEP of HCMV, and viral infection. In addition, virus infection alters the expression of NDY1/KDM2B, EZH2, JARID2 and JMJD3. The solid lines from these molecules to GFI1 indicate that they actively repress GFI1 both before and after infection, although due to HCMV-induced changes in their expression, the repression is enhanced after infection. (Panel B, Left) The repression of GFI1 in uninfected cells was blocked by the knockdown of NDY1/KDM2B, EZH2 or JARID2 and by the overexpression of JMJD3, resulting in significant up-regulation of GFI1 (dotted lines). (Panel B, Right) describes the infection of HFFs in the left side of panel B. The virus continues to degrade GFI1. However, the degradation of GFI1 by the virus is insufficient to downregulate it to levels that allow the activation of the MIEP and viral infection.</p

Lentiviral and retroviral constructs used.

<p>Lentiviral and retroviral constructs used.</p

Primer sets used in Real-Time PCR.

<p>Primer sets used in Real-Time PCR.</p

The knockdown of GFI1 partially rescues the ability of HCMV to infect HFF cells in which EZH2 was knocked down.

<p><b>A</b>. HFFs were transduced with a TRIPZ-shEZH2 construct and with the lentiviral vector pLKO.1, or with a pLKO.1-based shGFI1construct, as indicated. Cells transduced with TRIPZ-shEZH2 and pLKO.1 (third lane), or TRIPZ-shEZH2 and pLKO.1-shGFI1 (last lane) were treated with Doxycycline, to induce shEZH2. A1. Western blotting showing the expression of EZH2 and GFI1 in HFFs transduced with the indicated constructs, in the presence or absence of Doxycycline prior to HCMV infection. A2. Western blotting, monitoring the expression of IE1 in the cells shown in F1, upon infection with HCMV. Lysates were harvested at 5 days from the start of HCMV infection. A3. Titration of the progeny virus obtained 7 days after HCMV infection of the cells shown in A1. The bars show the number of plaques per ml (mean ± SD).</p

NDY1/KDM2B, EZH2 JARID2 and JMJD3 control the expression of GFI1, a direct repressor of the HCMV MIEP, by regulating histone H3K27 tri-methylation in the GFI1 promoter.

<p><b>A</b>. (Upper panel). Schematic diagram of the major immediate-early promoter of HCMV, showing the relative location of the binding sites of the indicated transcriptional regulators (activators and repressors). (Lower panel). The expression of the indicated transcriptional regulators in HFFs in which EZH2, NDY1/KDM2B, or JARID2 were knocked down, or JMJD3 was overexpressed via transduction with the indicated constructs, was measured by real time RT-PCR. The bars show the relative expression of GFI1 (mean ± SD) in the cells transduced with these constructs. The western blot in the inset shows that the GFI1 protein, the only transcriptional regulator whose expression at the RNA level was induced by these constructs, is also upregulated. <b>B</b>. The knock down of NDY1/KDM2B, EZH2 or JARID2 enhances the binding of GFI1 to the HCMV promoter. HFFs were transduced with shEZH2, shNDY1/KDM2, shJARID2 or the empty lentiviral vector and they were subsequently infected with HCMV. ChIP assays addressing the binding of GFI1 on the two known GFI1 binding sites in the HCMV promoter or in exon 1 of the immediate-early region were carried out using lysates harvested from these cells 1 hour post-infection The bars show the fold increase in GFI1 binding (mean ± SD) in the shEZH2, shNDY1/KDM2B and shJARID2-transduced cells relative to the cells transduced with the empty vector. <b>C</b>. GFI1 is a direct repressor of the HCMV MIE promoter. HEK 293T cells transduced with the indicated lentiviral or retroviral constructs were transfected with an HCMV MIEP-EGFP reporter in which the HCMV MIEP was either wild type or mutated in the two known GFI1 binding sites. The activity of the HCMV MIEP was monitored by both fluorescence microscopy (upper panel) and fluorescence densitometric analyses (lower panel). Bars in the lower panel show the relative EGFP fluorescence in the indicated cells (mean ± SD). <b>D</b>. The knockdown of NDY1/KDM2B, EZH2 and JARID2 decrease the abundance of histone H3K27me3 in a negative regulatory domain of the GFI1 promoter (site # 1). ChIP analyses addressing the abundance of H3K27me3 at five different sites within the GFI1 promoter in HFFs transduced with the indicated constructs. The p16^Ink4a locus was used as the positive control. The upper panel shows the position of the five selected sites, relative to the transcription start site in the GFI1 promoter (arrow). The bars in the lower panel show the fold change in the abundance of H3K27me3 (mean ± SD) at these sites, and in the p16^Ink4a locus. NRE: Negative Regulatory Element.</p

The abundance of H3K4me3 and H3K27me3 at the HCMV MIEP in shControl and shEZH2 HFFs, at the pre-immediate early stage of the infection.

<p>A. Graphical illustration of the MIEP of HCMV. Open boxes represent regions probed by ChIP for association with histone H3K27me3 and histone H3K4me3. <i>Enh</i> stands for Enhancer and <i>crs</i> stands for cis-repressive sequence. B: HFFs were transduced with either the pLKO.1 empty vector or the shEZH2 lentiviral construct. A western blot of cell lysates was probed with the EZH2 antibody. C-E. HFFs transduced with a lentivirus construct of shEZH2 or with the empty vector, were infected with HCMV (MOI 0.5 PFUs/cell). ChIPs using antibodies against H3K27me3 and H3K4me3 were analysed by qPCR using primers specific for the indicated loci (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004136#s4" target="_blank">materials and methods</a>), at 0.5 h, 1.5 h and 3 h.p.i. The results are given as mean <u>+</u> SD for triplicate Real Time PCRs. The asterisks indicate statistical significance. * indicates p<0.05 and ** indicates p<0.01.</p