Silencing of proviruses in embryonic cells: efficiency, stability and chromatin modifications

Abstract

Embryonic stem cells repress retroviral infection through transcriptional silencing of proviral DNAs. We characterized two distinct mechanisms of silencing in embryonic mouse cells infected by Moloney murine leukaemia virus (MLV): a highly efficient one targeting the proline transfer RNA primer-binding site (PBSpro), and a less efficient one operating independently of the PBS. Rare virus-expressing populations were isolated, and the timing and efficiency of establishment of silencing were determined. Superinfection of the selected virus-expressing cells with a second virus carrying a distinguishable reporter revealed that the PBSpro-directed silencing was still largely intact, whereas the PBS-independent silencing was partially reduced. The timing and stability of silencing, and the associated chromatin modifications on newly established and endogenous proviruses were determined. The results indicate that epigenetic mechanisms with different specificity and efficiency are used to silence the exogenous retroviral sequences in embryonic cells.

Figure 1

Kinetics of silencing. Kinetics of viral silencing in ES cells using a GFP reporter virus. (A) Flow analysis of GFP-positive cells at different time points after infection (day 0) by wt (pro) and mutant (proB2) PBS virus in ES cells or differentiated cells. Representative experiment is shown (see Supplementary Fig S1A online for mean±s.d.). Arrows denote extent of silencing due to PBSpro-dependent (red) and PBSpro-independent mechanisms (purple). (B) Same assay on F9 scrambled pool and on ZFP809 KD F9 pool 9 ([7], see also Supplementary Fig S1C online). Note change of y axis from A. Averages±s.e.m. from three independent experiments are shown. For F9 EC cell line results see Supplementary Fig S1B online. (C) Flow analysis after FACS sorting of cells that escape restriction and express GFP. NIH/3T3 cells are shown as control. (D) Same analysis in F9 cells and F9 ZFP809 KD clone. Error bars show standard error of the mean (s.e.m.) for n=3 (C) or n=4 (D) biological replicates. See also Supplementary Fig S2 online for analyses of proviral DNA copy number and Supplementary Fig S3 online for ES-specific markers analysis. ES, embryonic stem; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; PBSpro, proline tRNA primer-binding site; wt, wild-type.

Figure 2

GFP reporter gene expression in cell populations after long-term selections or cell sorting. (A) F9 cells were infected first with VSV-G pseudotyped MLV containing neo^r reporter with PBSpro (WT) or PBSproB2 (mutant) constructs. Half the population was selected for 2 week using G418 (14dS) and half was grown without selection (noS). Both populations were then infected with GFP virus with PBSpro or PBSproB2 and analysed by flow cytometry. (B) Percent of GFP expressing cells after second infection with PBSpro or PBSproB2. Infection efficiency in each cell line normalized with NIH/3T3=100%. (C) Cells initially infected with mCherry viruses were sorted and then subjected to a second round of infections with the GFP viruses, and analysed by flow cytometry. Percent of GFP expressing cells after second infection with wt PBSpro or mutant PBSproB2 is shown. Infection efficiency in each cell line normalized with NIH/3T3=100%. Averages±s.e.m. for n=3 (B) or n=5 (C) biological replicates are shown. See Supplementary Fig S5 online for analyses of proviral DNA copy number and ES-specific marker. ES, embryonic stem; GFP, green fluorescent protein; MLV, murine leukaemia virus; PBSpro, proline tRNA primer-binding site; wt, wild-type.

Figure 3

ChIP analysis of infected F9 cells subjected to long-term selection. ChIP-based measurement of (A) H3K9me3 and (B) H3K27me3 at the viral PBS sequence of cells infected with MLV containing neo^r gene, with PBSpro or PBSproB2 and selected for 14 days or infected without selection. Averages±s.e.m. from three independent experiments are shown. For each experiment, relative enrichment values (% of input) are shown, normalized to the signal of negative control (Gapdh). Results for negative (Aprt) and positive (Polmrt, BMP2) control genes are shown. ChIP, chromatin immunoprecipitation; MLV, murine leukaemia virus; PBSpro, proline tRNA primer-binding site.

Figure 4

Differences between 3T3 and F9 cells in chromatin marks and DNA methylation levels. ChIP-based measurement of (A) H3Ac, (B) H3K9me2, (C) H3K9me3 and (D) H3K27me3 at the viral PBS sequence of cells infected with GFP reporter virus with PBSpro or PBSproB2. As the two cell types gave different background binding, the signal was first normalized to IgG control ChIP, and normalized signal of negative control α-crystalline (H3Ac) or the Gapdh promoters (H3K9me2/3, H3K27me3) was set to 1. Averages±s.e.m. from three independent experiments are shown. Negative and positive control genes gave the expected enrichment values (not shown). (E) Bisulfite sequencing analysis of the 5′LTR of the infecting virus was performed on NIH/3T3 and F9 cells; Oct4 was used as control. Percentages of methylated CpGs are shown from 10 to 15 cloned DNA molecules per cell and infection type (Supplementary Fig S6 online). ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; 5′LTR, 5′ long terminal repeat; PBSpro, proline tRNA primer-binding site.

Figure 5

Expression pattern and chromatin modifications of classes I, II and III ERVs. Quantitative RT–PCR analysis of (A) eMLVs (class I), (B) IAP (class II) and (C) MERV-L (class III) messenger RNA expression in embryonic and differentiated cells. Bars represent the mean±s.e.m. of three independently prepared samples, relative to three control genes (UBC, CYCA and Gapdh). ChIP-based measurement of (D) H3K9me3 and (E) H3K27me3 at the viral sequence of cells infected with MLV containing neo^r with PBSpro or PBSproB2 and selected for 14 days or infected without selection. Relative enrichment values (% of input) in all ChIP experiments were normalized to the relative enrichment at the Gapdh promoter. Negative and positive control genes gave the expected enrichment (not shown). ChIP, chromatin immunoprecipitation; MLV, murine leukaemia virus; ERV, endogenous retrovirus; PBSpro, proline tRNA primer-binding site; RT–PCR, reverse transcription PCR.