Identification, Characterization, and Heritability of Murine Metastable Epialleles: Implications for Non-genetic Inheritance.

Generally repressed by epigenetic mechanisms, retrotransposons represent around 40% of the murine genome. At the Agouti viable yellow (Avy) locus, an endogenous retrovirus (ERV) of the intracisternal A particle (IAP) class retrotransposed upstream of the agouti coat-color locus, providing an alternative promoter that is variably DNA methylated in genetically identical individuals. This results in variable expressivity of coat color that is inherited transgenerationally. Here, a systematic genome-wide screen identifies multiple C57BL/6J murine IAPs with Avy epigenetic properties. Each exhibits a stable methylation state within an individual but varies between individuals. Only in rare instances do they act as promoters controlling adjacent gene expression. Their methylation state is locus-specific within an individual, and their flanking regions are enriched for CTCF. Variably methylated IAPs are reprogrammed after fertilization and re-established as variable loci in the next generation, indicating reconstruction of metastable epigenetic states and challenging the generalizability of non-genetic inheritance at these regions.

METTL3 regulates heterochromatin in mouse embryonic stem cells.

METTL3 (methyltransferase-like 3) mediates the N6-methyladenosine (m6A) methylation of mRNA, which affects the stability of mRNA and its translation into protein1. METTL3 also binds chromatin2-4, but the role of METTL3 and m6A methylation in chromatin is not fully understood. Here we show that METTL3 regulates mouse embryonic stem-cell heterochromatin, the integrity of which is critical for silencing retroviral elements and for mammalian development5. METTL3 predominantly localizes to the intracisternal A particle (IAP)-type family of endogenous retroviruses. Knockout of Mettl3 impairs the deposition of multiple heterochromatin marks onto METTL3-targeted IAPs, and upregulates IAP transcription, suggesting that METTL3 is important for the integrity of IAP heterochromatin. We provide further evidence that RNA transcripts derived from METTL3-bound IAPs are associated with chromatin and are m6A-methylated. These m6A-marked transcripts are bound by the m6A reader YTHDC1, which interacts with METTL3 and in turn promotes the association of METTL3 with chromatin. METTL3 also interacts physically with the histone 3 lysine 9 (H3K9) tri-methyltransferase SETDB1 and its cofactor TRIM28, and is important for their localization to IAPs. Our findings demonstrate that METTL3-catalysed m6A modification of RNA is important for the integrity of IAP heterochromatin in mouse embryonic stem cells, revealing a mechanism of heterochromatin regulation in mammals.

m6A RNA methylation regulates the fate of endogenous retroviruses.

Endogenous retroviruses (ERVs) are abundant and heterogenous groups of integrated retroviral sequences that affect genome regulation and cell physiology throughout their RNA-centred life cycle1. Failure to repress ERVs is associated with cancer, infertility, senescence and neurodegenerative diseases2,3. Here, using an unbiased genome-scale CRISPR knockout screen in mouse embryonic stem cells, we identify m6A RNA methylation as a way to restrict ERVs. Methylation of ERV mRNAs is catalysed by the complex of methyltransferase-like METTL3-METTL144 proteins, and we found that depletion of METTL3-METTL14, along with their accessory subunits WTAP and ZC3H13, led to increased mRNA abundance of intracisternal A-particles (IAPs) and related ERVK elements specifically, by targeting their 5' untranslated region. Using controlled auxin-dependent degradation of the METTL3-METTL14 enzymatic complex, we showed that IAP mRNA and protein abundance is dynamically and inversely correlated with m6A catalysis. By monitoring chromatin states and mRNA stability upon METTL3-METTL14 double depletion, we found that m6A methylation mainly acts by reducing the half-life of IAP mRNA, and this occurs by the recruitment of the YTHDF family of m6A reader proteins5. Together, our results indicate that RNA methylation provides a protective effect in maintaining cellular integrity by clearing reactive ERV-derived RNA species, which may be especially important when transcriptional silencing is less stringent.

SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation.

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2-4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation.

Loss of Uhrf1 in neural stem cells leads to activation of retroviral elements and delayed neurodegeneration.

In order to understand whether early epigenetic mechanisms instruct the long-term behavior of neural stem cells (NSCs) and their progeny, we examined Uhrf1 (ubiquitin-like PHD ring finger-1; also known as Np95), as it is highly expressed in NSCs of the developing brain and rapidly down-regulated upon differentiation. Conditional deletion of Uhrf1 in the developing cerebral cortex resulted in rather normal proliferation and neurogenesis but severe postnatal neurodegeneration. During development, deletion of Uhrf1 lead to global DNA hypomethylation with a strong activation of the intracisternal A particle (IAP) family of endogenous retroviral elements, accompanied by an increase in 5-hydroxymethylcytosine. Down-regulation of Tet enzymes rescued the IAP activation in Uhrf1 conditional knockout (cKO) cells, suggesting an antagonistic interplay between Uhrf1 and Tet on IAP regulation. As IAP up-regulation persists into postnatal stages in the Uhrf1 cKO mice, our data show the lack of means to repress IAPs in differentiating neurons that normally never express Uhrf1 The high load of viral proteins and other transcriptional deregulation ultimately led to postnatal neurodegeneration. Taken together, these data show that early developmental NSC factors can have long-term effects in neuronal differentiation and survival. Moreover, they highlight how specific the consequences of widespread changes in DNA methylation are for certain classes of retroviral elements.

Polygenic control of the wavy coat of the NCT mouse: involvement of an intracisternal A particle insertional mutation of the protease, serine 53 (Prss53) gene, and a modifier gene.

The Nakano cataract mouse (NCT) manifests a wavy coat for their first hair as a genetic trait. In this study, we explored the molecular genetic basis of the wavy coat. We revealed by crossing experiments that the wavy coat is controlled by a major gene on chromosome 7 of NCT, homozygosity of which is a prerequisite for developing the wavy coat, and by a gene on chromosome 9 with a minor effect to reinforce the manifestation of the trait. In humans, a polymorphism of the protease, serine 53 (PRSS53) gene on the homologous chromosome is known to be associated with curly scalp hair. We then investigated the Prss53 gene and discovered that NCT has an insertion of an intracisternal A particle element in the first intron of the gene. Nevertheless, the expression of the Prss53 is not altered in the NCT skin both in transcript and protein levels. Subsequently, we created C57BL/6J-Prss53em1 knockout mice and found that these mice manifest vague wavy coats. A portion of backcross and intercross mice between the C57BL/6J-Prss53em1 and NCT manifested intense or vague wavy coats. These findings demonstrate the polygenic nature of the wavy coat of NCT and Prss53 knockout mice and highlight the similarity of the trait to the curly hair of humans associated with the PRSS53 alteration.

Quiescence-induced LncRNAs trigger H4K20 trimethylation and transcriptional silencing.

A complex network of regulatory pathways links transcription to cell growth and proliferation. Here we show that cellular quiescence alters chromatin structure by promoting trimethylation of histone H4 at lysine 20 (H4K20me3). In contrast to pericentric or telomeric regions, recruitment of the H4K20 methyltransferase Suv4-20h2 to rRNA genes and IAP elements requires neither trimethylation of H3K9 nor interaction with HP1 proteins but depends on long noncoding RNAs (lncRNAs) that interact with Suv4-20h2. Growth factor deprivation and terminal differentiation lead to upregulation of these lncRNAs, increase in H4K20me3, and chromatin compaction. The results uncover a lncRNA-mediated mechanism that guides Suv4-20h2 to specific genomic loci to establish a more compact chromatin structure in growth-arrested cells.

Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice.

Stabilin2 (Stab2) encodes a large transmembrane protein which is predominantly expressed in the liver sinusoidal endothelial cells (LSECs) and functions as a scavenger receptor for various macromolecules including hyaluronans (HA). In DBA/2J mice, plasma HA concentration is ten times higher than in 129S6 or C57BL/6J mice, and this phenotype is genetically linked to the Stab2 locus. Stab2 mRNA in the LSECs was significantly lower in DBA/2J than in 129S6, leading to reduced STAB2 proteins in the DBA/2J LSECs. We found a retrovirus-derived transposable element, intracisternal A particle (IAP), in the promoter region of Stab2DBA which likely interferes with normal expression in the LSECs. In contrast, in other tissues of DBA/2J mice, the IAP drives high ectopic Stab2DBA transcription starting within the 5' long terminal repeat of IAP in a reverse orientation and continuing through the downstream Stab2DBA. Ectopic transcription requires the Stab2-IAP element but is dominantly suppressed by the presence of loci on 59.7-73.0 Mb of chromosome (Chr) 13 from C57BL/6J, while the same region in 129S6 requires additional loci for complete suppression. Chr13:59.9-73 Mb contains a large number of genes encoding Krüppel-associated box-domain zinc-finger proteins that target transposable elements-derived sequences and repress their expression. Despite the high amount of ectopic Stab2DBA transcript in tissues other than liver, STAB2 protein was undetectable and unlikely to contribute to the plasma HA levels of DBA/2J mice. Nevertheless, the IAP insertion and its effects on the transcription of the downstream Stab2DBA exemplify that stochastic evolutional events could significantly influence susceptibility to complex but common diseases.

Mouse GTSF1 is an essential factor for secondary piRNA biogenesis.

The piRNA pathway is a piRNA-guided retrotransposon silencing system which includes processing of retrotransposon transcripts by PIWI-piRNAs in secondary piRNA biogenesis. Although several proteins participate in the piRNA pathway, the ones crucial for the cleavage of target RNAs by PIWI-piRNAs have not been identified. Here, we show that GTSF1, an essential factor for retrotransposon silencing in male germ cells in mice, associates with both MILI and MIWI2, mouse PIWI proteins that function in prospermatogonia. GTSF1 deficiency leads to a severe defect in the production of secondary piRNAs, which are generated from target RNAs of PIWI-piRNAs. Furthermore, in Gtsf1 mutants, a known target RNA of PIWI-piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective. Our findings indicate that GTSF1 is a crucial factor for the slicing of target RNAs by PIWI-piRNAs and thus affects secondary piRNA biogenesis in prospermatogonia.

Nxf1 natural variant E610G is a semi-dominant suppressor of IAP-induced RNA processing defects.

Endogenous retroviruses and retrotransposons contribute functional genetic variation in animal genomes. In mice, Intracisternal A Particles (IAPs) are a frequent source of both new mutations and polymorphism across laboratory strains. Intronic IAPs can induce alternative RNA processing choices, including alternative splicing. We previously showed IAP I∆1 subfamily insertional mutations are suppressed by a wild-derived allele of the major mRNA export factor, Nxf1. Here we show that a wider diversity of IAP insertions present in the mouse reference sequence induce insertion-dependent alternative processing that is suppressed by Nxf1CAST alleles. These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation. Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of Nxf1 genetic modifier activity for IAP insertion alleles. Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.

MVH in piRNA processing and gene silencing of retrotransposons.

VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.

Atrx promotes heterochromatin formation at retrotransposons.

More than 50% of mammalian genomes consist of retrotransposon sequences. Silencing of retrotransposons by heterochromatin is essential to ensure genomic stability and transcriptional integrity. Here, we identified a short sequence element in intracisternal A particle (IAP) retrotransposons that is sufficient to trigger heterochromatin formation. We used this sequence in a genome-wide shRNA screen and identified the chromatin remodeler Atrx as a novel regulator of IAP silencing. Atrx binds to IAP elements and is necessary for efficient heterochromatin formation. In addition, Atrx facilitates a robust and largely inaccessible heterochromatin structure as Atrx knockout cells display increased chromatin accessibility at retrotransposons and non-repetitive heterochromatic loci. In summary, we demonstrate a direct role of Atrx in the establishment and robust maintenance of heterochromatin.

KAP1 controls endogenous retroviruses in embryonic stem cells.

More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.

RNase L restricts the mobility of engineered retrotransposons in cultured human cells.

Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (NΔ385), but not a catalytically inactive RNase L mutant (R667A), impaired the mobility of engineered human LINE-1 (L1) and mouse intracisternal A-type particle retrotransposons in cultured human cells. Furthermore, WT RNase L, but not an inactive RNase L mutant (R667A), reduced L1 RNA levels and subsequent expression of the L1-encoded proteins (ORF1p and ORF2p). Consistently, confocal immunofluorescent microscopy demonstrated that WT RNase L, but not RNase L R667A, prevented formation of L1 cytoplasmic foci. Finally, siRNA-mediated depletion of endogenous RNase L in a human ovarian cancer cell line (Hey1b) increased the levels of L1 retrotransposition by ∼2-fold. Together, these data suggest that RNase L might function as a suppressor of structurally distinct retrotransposons.

Global profiling of DNA methylation erasure in mouse primordial germ cells.

Epigenetic reprogramming, characterized by loss of cytosine methylation and histone modifications, occurs during mammalian development in primordial germ cells (PGCs), yet the targets and kinetics of this process are poorly characterized. Here we provide a map of cytosine methylation on a large portion of the genome in developing male and female PGCs isolated from mouse embryos. We show that DNA methylation erasure is global and affects genes of various biological functions. We also reveal complex kinetics of demethylation that are initiated at most genes in early PGC precursors around embryonic day 8.0-9.0. In addition, besides intracisternal A-particles (IAPs), we identify rare LTR-ERV1 retroelements and single-copy sequences that resist global methylation erasure in PGCs as well as in preimplantation embryos. Our data provide important insights into the targets and dynamics of DNA methylation reprogramming in mammalian germ cells.

Env-less endogenous retroviruses are genomic superspreaders.

Endogenous retroviruses (ERVs) differ from typical retroviruses in being inherited through the host germline and therefore are a unique combination of pathogen and selfish genetic element. Some ERV lineages proliferate by infecting germline cells, as do typical retroviruses, whereas others lack the env gene required for virions to enter cells and thus behave like retrotransposons. We wished to know what factors determined the relative abundance of different ERV lineages, so we analyzed ERV loci recovered from 38 mammal genomes by in silico screening. By modeling the relationship between proliferation and replication mechanism in detail within one group, the intracisternal A-type particles (IAPs), and performing simple correlations across all ERV lineages, we show that when ERVs lose the env gene their proliferation within that genome is boosted by a factor of ∼30. We also show that ERV abundance follows the Pareto principle or 20/80 rule, with ∼20% of lineages containing 80% of the loci. This rule is observed in many biological systems, including infectious disease epidemics, where commonly ∼20% of the infected individuals are responsible for 80% of onward infection. We thus borrow simple epidemiological and ecological models and show that retrotransposition and loss of env is the trait that leads endogenous retroviruses to becoming genomic superspreaders that take over a significant proportion of their host's genome.

Epigenetic mechanism causes Wnt9b deficiency and nonsyndromic cleft lip and palate in the A/WySn mouse strain.

BACKGROUND: The heritable multifactorial etiology of human nonsyndromic cleft lip with or without cleft palate (CL ± P) is not understood. CL ± P occurs in 15% of neonates in the homozygous A/WySn mouse strain, with a multifactorial genetic etiology, the clf1 and clf2 variant genes. Clf1 acts as a mutant allele of Wnt9b but its coding sequence is normal. An IAP (intracisternal A particle) retrotransposon inserted near the Wnt9b gene is associated with clf1. METHODS: Transcription of noncoding sequence between the IAP and the Wnt9b gene was examined in A/WySn embryos. The levels of Wnt9b transcript and of an "IAP antisense" transcript initiated in the IAP and extending into the noncoding interval were assayed in A/WySn and C57BL/6J whole embryos or heads across embryonic days 8 to 12. Methylation of the 5' LTR of the IAP was examined in E12 A/WySn embryo heads. RESULTS: Mean Wnt9b transcript levels were lower in A/WySn than in C57BL/6J at all ages examined and lower in CL ± P embryos than in their normal littermates. The "IAP antisense" transcript was found in all A/WySn embryos and was highest in CL ± P embryos. The IAP at Wnt9b was generally unmethylated in CL ± P embryos and approximately 50% methylated in normal littermates. CONCLUSION: The clf1 mutation in A/WySn is a "metastable epiallele", in which stochastic deficiency in some individuals of DNA methylation of a retrotransposon uniquely inserted near the Wnt9b gene allows transcriptional activity of the retrotransposon and interference with transcription from Wnt9b. Methylation of metastable epialleles should be investigated in human nonsyndromic CL ± P.

Alkbh1 and Tzfp repress a non-repeat piRNA cluster in pachytene spermatocytes.

Piwi proteins and Piwi-interacting small RNAs (piRNAs) have known functions in transposon silencing in the male germline of fetal and newborn mice. Both are also present in adult testes; however, their function here remains a mystery. Here, we confirm that most piRNAs in meiotic spermatocytes originate from clusters in non-repeat intergenic regions of DNA. The regulation of these piRNA clusters, including the processing of the precursor transcripts into individual piRNAs, is accomplished through mostly unknown processes. We present a possible regulatory mechanism for one such cluster, named cluster 1082B, located on chromosome 7 in the mouse genome. The 1082B precursor transcript and its 788 unique piRNAs are repressed by the Alkbh1 dioxygenase and the testis-specific transcription repressor Tzfp. We observe a remarkable >1000-fold upregulation of individual piRNAs in pachytene spermatocytes isolated from Alkbh1- and Tzfp-deficient murine testes. Repression of cluster 1082B is further supported by the identification of a 10-bp Tzfp recognition sequence contained within the precursor transcript. Downregulation of LINE1 and IAP transcripts in the Alkbh1- and Tzfp-deficient mice leads us to propose a potential role for the 1082B-encoded piRNAs in transposon control.

Phylogenetic and DNA methylation analysis reveal novel regions of variable methylation in the mouse IAP class of transposons.

BACKGROUND: Select retrotransposons in the long terminal repeat (LTR) class exhibit interindividual variation in DNA methylation that is altered by developmental environmental exposures. Yet, neither the full extent of variability at these "metastable epialleles," nor the phylogenetic relationship underlying variable elements is well understood. The murine metastable epialleles, Avy and CabpIAP, result from independent insertions of an intracisternal A particle (IAP) mobile element, and exhibit remarkably similar sequence identity (98.5%). RESULTS: Utilizing the C57BL/6 genome we identified 10802 IAP LTRs overall and a subset of 1388 in a family that includes Avy and CabpIAP. Phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. To characterize interindividual variation across clades, liver DNA from 17 isogenic mice was subjected to combined bisulfite and restriction analysis (CoBRA) for 21 separate LTR transposons (7 per clade). The lowest and highest mean methylation values were 59% and 88% respectively, while methylation levels at individual LTRs varied widely, ranging from 9% to 34%. The clade with the most conserved elements had significantly higher mean methylation across LTRs than either of the two diverged clades (p = 0.040 and p = 0.017). Within each mouse, average methylation across all LTRs was not significantly different (71%-74%, p > 0.99). CONCLUSIONS: Combined phylogenetic and DNA methylation analysis allows for the identification of novel regions of variable methylation. This approach increases the number of known metastable epialleles in the mouse, which can serve as biomarkers for environmental modifications to the epigenome.

Effects of dppa3 on DNA methylation dynamics during primordial germ cell development in mice.

DNA methylation is a central epigenetic event that regulates cellular differentiation, reprogramming, and pathogenesis. Genomewide DNA demethylation occurs in preimplantation embryos and in embryonic germ cell precursors called primordial germ cells (PGCs). We previously showed that Dppa3, also known as Stella and PGC7, protects the maternal genome from tet methylcytosine dioxygenase 3 (Tet3)-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in zygotes. Here, we demonstrated that retrotransposon genes, such as long interspersed nuclear element-1 (Line-1) and intracisternal A particle (IAP), showed higher 5mC levels in Dppa3-null PGCs. In contrast, oxidative bisulfite sequence analysis revealed that the amounts of 5hmC in Line-1 and IAP were slightly reduced in the Dppa3-deficient PGCs. From our findings, we propose that Dppa3 is involved in the Tet-mediated active demethylation process during reprogramming of PGCs.