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. 2008 Mar;28(6):2059-65.
doi: 10.1128/MCB.01362-07. Epub 2008 Jan 14.

macroH2A1-dependent silencing of endogenous murine leukemia viruses

Lakshmi N Changolkar  1 , Geetika Singh, John R Pehrson
Affiliations

macroH2A1-dependent silencing of endogenous murine leukemia viruses

Lakshmi N Changolkar et al. Mol Cell Biol. 2008 Mar.

Abstract

We show that macroH2A1 histone variants are important for repressing the expression of endogenous murine leukemia viruses (MLVs) in mouse liver. Intact MLV proviruses and proviruses with deletions in env were nearly silent in normal mouse liver and showed substantial derepression in macroH2A1 knockout liver. In contrast, MLV proviruses with a deletion in the 5' end of pro-pol were expressed in normal liver and showed relatively low levels of derepression in knockout liver. macroH2A1 nucleosomes were enriched on endogenous MLVs, with the highest enrichment occurring on the 5' end of pro-pol. The absence of macroH2A1 also led to a localized loss of DNA methylation on the 5' ends of MLV proviruses. These results demonstrate that macroH2A1 histones have a significant role in silencing endogenous MLVs in vivo and suggest that specific internal MLV sequences are targeted by a macroH2A1-dependent silencing mechanism.

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Figures

FIG. 1.
FIG. 1.
macroH2A1 represses the expression of endogenous MLVs. The expression of specific endogenous MLV sequences in normal liver and macroH2A1 knockout liver was determined by real-time PCR. Representative samples of PCR products from normal (N) and knockout (KO) liver are shown directly below three of the sites that were probed. The P values for the two-tailed t test were 0.02 or less for all expression comparisons except for those at the beginning and end of gag (see Table S1 in the supplemental material for all P values). The MLV diagram is based on the annotation of RLTR4 in the Repbase Update (23) and alignment with Moloney MLV sequences.
FIG. 2.
FIG. 2.
Deletions in the 5′ end of pro-pol are associated with decreased macroH2A1-dependent repression. The deletions in specific endogenous MLV proviruses are shown. The 2-kb segment indicates the range of the deletions associated with reduced macroH2A1-dependent repression. The 600-bp segment indicates the region that is common to all of these deletions; note that the deletion in provirus no. 36 does not include this 600-bp segment and that this provirus shows a high level of macroH2A1-dependent repression (*). Relative expression was determined by real-time PCR. The statistical analyses of these comparisons are shown in Table S1 in the supplemental material. Proviruses were numbered sequentially according to their occurrence in the genome of C57BL/6 mice, genome build 37.1 (see Table S2 in the supplemental material).
FIG. 3.
FIG. 3.
Northern blot analyses of endogenous MLV expression. Total mouse liver RNA was used. The locations of the gag, pro-pol, and env hybridization probes are shown in gray on the diagram below the blots. Hybridization with a 28S rRNA or β-actin probe was used to show equal loading of the lanes. Band sizes were estimated from a standard curve constructed from an RNA standard. Lanes: KO, RNA from female macroH2A1 knockout liver; N, RNA from normal female liver.
FIG. 4.
FIG. 4.
macroH2A1 nucleosomes are enriched on endogenous MLVs. The relative concentration of macroH2A1 was determined by real-time PCR using DNA from macroH2A1 nucleosomes that were purified by thiol-affinity chromatography (10). A value of 1 indicates a macroH2A1 concentration equal to that of the bulk chromatin applied to the thiol-affinity procedure. The values shown are the averages determined from four independent preparations. The P values for a t test of the enrichment were <0.02 at all sites (see Table S1 in the supplemental material for specific P values and the standard deviation analysis).
FIG. 5.
FIG. 5.
DNA methylation of endogenous MLVs is reduced in macroH2A1 knockout liver. DNA isolated from mouse liver was digested to completion with HpaII and analyzed on Southern blots by using the gag, pro-pol, and env probes shown in Fig. 3. Lanes: N, DNA from normal mouse liver; KO, DNA from macroH2A1 knockout liver; M, DNA digested with MspI. Positions of marker bands are indicated between the blots. The hybridization seen between the M lane and the first N lane of the pol blot is spillover from coincidental hybridization of the probe to fragments in the marker lane.
FIG. 6.
FIG. 6.
Close proximity of derepressed MLV provirus to gene EG244556. The diagram shows the position of MLV provirus no. 36 relative to that of EG244556 on mouse chromosome 8. Provirus no. 36 is strongly derepressed in macroH2A1 knockout liver (Fig. 2), and EG244556 expression is increased 70% in knockout liver. Arrows indicate direction of transcription.
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