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. 2010 Jul 2;285(27):21082-91.
doi: 10.1074/jbc.M110.125674. Epub 2010 Apr 19.

Repression of retrotransposal elements in mouse embryonic stem cells is primarily mediated by a DNA methylation-independent mechanism

Leah K Hutnick  1 , Xinhua Huang, Tao-Chuan Loo, Zhicheng Ma, Guoping Fan
Affiliations

Repression of retrotransposal elements in mouse embryonic stem cells is primarily mediated by a DNA methylation-independent mechanism

Leah K Hutnick et al. J Biol Chem. .

Abstract

In defense of deleterious retrotransposition of intracisternal A particle (IAP) elements, IAP loci are heavily methylated and silenced in mouse somatic cells. To determine whether IAP is also repressed in pluripotent stem cells by DNA methylation, we examined IAP expression in demethylated mouse embryonic stem cells (mESCs) and epiblast-derived stem cells. Surprisingly, in demethylated ESC cultures carrying mutations of DNA methyltransferase I (Dnmt1), no IAP transcripts and proteins are detectable in undifferentiated Oct4(+) ESCs. In contrast, approximately 3.6% of IAP-positive cells are detected in Oct4(-) Dnmt1(-/-) cells, suggesting that the previously observed increase in IAP transcripts in the population of Dnmt1(-/-) ESCs could be accounted for by this subset of Oct4(-) Dnmt1(-/-) ESCs undergoing spontaneous differentiation. Consistent with this possibility, a dramatic increase of IAP mRNA (>100-fold) and protein expression was observed in Dnmt1(-/-) ESC cultures upon induction of differentiation through the withdrawal of leukemia-inhibitory factor for 6 or more days. Interestingly, both mRNAs and proteins of IAP can be readily detected in demethylated Oct4(+) epiblast-derived stem cells as well as differentiated mouse embryo fibroblasts, neurons, and glia upon conditional Dnmt1 gene deletion. These data suggest that mESCs are a unique stem cell type possessing a DNA methylation-independent IAP repression mechanism. This methylation-independent mechanism does not involve Dicer-mediated action of microRNAs or RNA interference because IAP expression remains repressed in Dnmt1(-/-); Dicer(-/-) double mutant ESCs. We suggest that mESCs possess a unique DNA methylation-independent mechanism to silence retrotransposons to safeguard genome stability while undergoing rapid cell proliferation for self-renewal.

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Figures

FIGURE 1.
FIGURE 1.
IAP protein is only detected in lysates of E18.5 Dnmt1−/− mutant brain. A, depiction of protein structural domains of the full-length IAP clone MIA14. A deletion series of the gag peptide domain, which most closely corresponds to the p73 peptide used to generate the original IAP antibody (45), was cloned to create GST fusion peptides. B, immunoblotting revealing IAP expression in control (Con) and E18.5 Nestin-Cre; Dnmt1 conditional mutant brain lysate (Mut) of the original p73 serum before purification (anti-p73, left blot) and after column purification using the IAP2-GST fusion peptide (anti-p73, middle blot). Our IAP2 polyclonal antibody also strongly recognizes the 73-kDa gag protein (anti-IAP2, right blot) from mutant brain lysate (32). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
FIGURE 2.
FIGURE 2.
IAP protein reactivation is tightly linked to the onset of DNA demethylation in MEFs. A, Dnmt1 2lox/2lox MEFs were infected with MSCV-Cre, and genomic demethylation was monitored using restriction enzyme digestion with methyl-sensitive HpaII followed be Southern blot analysis of IAP repetitive elements. Genomic demethylation appears 4 days after infection (DIV). B, immunoblotting shows that IAP protein expression appears 5 days postinfection with MSCV-Cre; thus, the IAP protein expression occurs after significant genome demethylation. The far right lane is a positive control for IAP proteins detected in the brain of E18.5 Nestin-Cre; Dnmt1 conditional knockouts (Brain E18.5 MUT) (29). C, IAP immunochemistry (red) colocalizes with MSCV-Cre-GFP-infected cells (green) once the genome becomes significantly demethylated after DNMT1 deletion. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.
FIGURE 3.
FIGURE 3.
Detection of IAP protein expression in demethylated somatic cells. A, left, immunoblotting using anti-IAP2 shows IAP protein in control and E18.5 Nestin-Cre; Dnmt1 conditional mutant brain lysates. Right, IAP immunoreactivity in E18.5 dorsal cortex of E18.5 Nestin-Cre; Dnmt1 mutant mice (29). B, left, Western blot analysis of IAP protein expression in control and Emx1-Cre; Dnmt1 cortical lysate from neonate P0 to 1.5 years old. Right, IAP immunoreactivity in the dorsal cortex (ctx) of 6-month-old Emx1-Cre; Dnmt1 mutant mice (9). C, immunocytochemistry for IAP protein in cultured N2a neuroblastoma cell lines. Western blot showing IAP expression in N2a cells but not in other types of neuroblastoma cells, including B104 and Hs 683 from ATCC. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.
FIGURE 4.
FIGURE 4.
Expression of IAP protein is suppressed in Dnmt1−/− mESCs and is only reactivated upon partial differentiation coupled with dramatic increases in IAP mRNAs. A, IAP protein is not expressed in undifferentiated Dnmt1−/− mESCs, although Dnmt1−/− cells possess less than 22% of normal genomic DNA methylation levels (28). Scale bar, 25 μm. B, upon induction of differentiation of Dnmt1−/− (c/c) mESCs in the absence of LIF treatment, IAP mRNA levels increase dramatically over the time course of 6–9 days in culture. No induction of IAP expression was observed in wild type control (J1) mESCs. C, IAP protein levels in the heterogeneous lysate of Dnmt1−/− cells were also detected after 6 days of LIF withdrawal, reaching a detectable threshold by Western blot. The blot on the left is a longer exposure to show detectable IAP expression at Day 6 of LIF withdrawal. D, IAP immunocytochemistry shows that only Oct4-negative cells express IAP protein in Dnmt1−/− ESCs. IAP-expressing cells were detected in a small population (under 10%) at Day 0–4 of differentiation but reached peak levels at Day 6–9 upon the induction of differentiation by LIF withdrawal. Scale bar, 50 μm. E, IAP FISH analysis reveals that both wild type (J1) and Dnmt1−/− (c/c) undifferentiated colonies at Day 0 of LIF withdrawal do not express IAP mRNA. Upon LIF withdrawal to induce differentiation, partially differentiated Dnmt1−/− cells migrating away from the colony express IAP RNA (arrowheads). Dnmt1−/− cells with undifferentiated colony morphology (arrow) do not express IAP mRNA. Scale bar, 25 μm. F, Northern blot analysis of the IAP gag coding region reveals the dramatic induction of IAP mRNA as LIF is withdrawn from culture. -Fold induction levels are assessed after normalization of 28 S rRNA bands from wild type (J1) and Dnmt1−/− (c/c) during the time course of differentiation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole. Error bars, S.E.
FIGURE 5.
FIGURE 5.
Hypomethylated EpiSCs rapidly induce IAP protein reactivation. A, photomicrographs of a representative colony 2 days after lentiviral cre recombinase delivery to Dnmt12lox/2lox EpiSCs. IAP immunoreactivity is present in Oct4-positive (blue) infected (GFP-positive, green) EpiSCs. Scale bar, 25 μm. B, 4 days after lentiviral cre recombinase delivery, colonies arising from infected EpiSCs show a homogenous population of Oct4/IAP/GFP-positive cells. Scale bar, 25 μm.
FIGURE 6.
FIGURE 6.
Inhibition of histone deacetylases and proteosome activities does not lead to IAP protein expression in undifferentiated Dnmt1−/− mESCs. A, HDAC inhibitor treatments of Dnmt1−/− mESCs in the presence of LIF were performed to question whether blocking histone deacetylation, thus promoting the active chromatin conformation, in Dnmt1−/− mESCs would lead to IAP protein expression in undifferentiated cells. Confocal images show that HDAC inhibitors do not reactivate IAP protein expression in Dnmt1−/− mESCs, suggesting that an alternative repressive mechanism is involved. Scale bar, 25 μm. B, cell counts show that HDAC inhibitors increase the number of IAP-positive colonies, which correlates with the known differentiating effect of HDAC inhibitors in cell culture. C, after 8 h of treatment with the proteosome inhibitor MG132, IAP protein was not visualized in Oct3/4-expressing Dnmt1−/− mESCs (confocal photomicrograph; scale bar, 25 μm). Error bars, S.E. DAPI, 4′,6-diamidino-2-phenylindole.
FIGURE 7.
FIGURE 7.
Clonal analysis of IAP expression in Dicer−/−; Dnmt1−/− mESCs. A, IAP protein reactivation was counted in individual clones after genotyping was performed. The vast majority of IAP-positive cells were Oct3/4-negative, thus indicating that only Dicer/Dnmt1 double mutant mESCs express IAP after spontaneous differentiation. Of note, two clones had a few cells expressing weak levels of IAP protein in Oct3/4-positive cells. The numbers on the x axis denote the clone number for the 3–6 individual clones with one of the following genotypes: Dnmt12lox; Dicer2lox (control); Dnmt11lox; Dicer2lox (Dnmt1−/− mutant cells); Dnmt11lox; Dicer1lox (Dnmt1−/−; Dicer−/− double mutant ESCs)). B, photomicrographs of clone 127, where differential levels of IAP are expressed. Strong IAP immunostaining is seen in differentiating (Oct3/4-negative) cells, whereas a weak perinuclear stain can be occasionally seen in few Oct3/4+ cells (arrow, bottom). Scale bar, 25 μm.
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References

    1. Bestor T. H. (2000) Hum. Mol. Genet. 9, 2395–2402 - PubMed
    1. Goll M. G., Bestor T. H. (2005) Annu. Rev. Biochem. 74, 481–514 - PubMed
    1. Robertson K. D., Wolffe A. P. (2000) Nat. Rev. Genet. 1, 11–19 - PubMed
    1. Jaenisch R., Bird A. (2003) Nat. Genet. 33, 245–254 - PubMed
    1. Klose R. J., Bird A. P. (2006) Trends Biochem. Sci. 31, 89–97 - PubMed

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