Patterns of recombination in snakes reveal a tug-of-war between PRDM9 and promoter-like features.

In some mammals, notably humans, recombination occurs almost exclusively where the protein PRDM9 binds, whereas in vertebrates lacking an intact PRDM9, such as birds and canids, recombination rates are elevated near promoter-like features. To determine whether PRDM9 directs recombination in nonmammalian vertebrates, we focused on an exemplar species with a single, intact PRDM9 ortholog, the corn snake (Pantherophis guttatus). Analyzing historical recombination rates along the genome and crossovers in pedigrees, we found evidence that PRDM9 specifies the location of recombination events, but we also detected a separable effect of promoter-like features. These findings reveal that the uses of PRDM9 and promoter-like features need not be mutually exclusive and instead reflect a tug-of-war that is more even in some species than others.

AGO2 silences mobile transposons in the nucleus of quiescent cells.

Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway, with essential roles in development and disease. Yet little is known about its regulation in vivo. Here we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells in which mTOR signaling is low, AGO2 accumulates in the nucleus, where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.

Patterns of recombination in snakes reveal a tug of war between PRDM9 and promoter-like features.

In vertebrates, there are two known mechanisms by which meiotic recombination is directed to the genome: in humans, mice, and other mammals, recombination occurs almost exclusively where the protein PRDM9 binds, while in species lacking an intact PRDM9, such as birds and canids, recombination rates are elevated near promoter-like features. To test if PRDM9 also directs recombination in non-mammalian vertebrates, we focused on an exemplar species, the corn snake (Pantherophis guttatus). Unlike birds, this species possesses a single, intact PRDM9 ortholog. By inferring historical recombination rates along the genome from patterns of linkage disequilibrium and identifying crossovers in pedigrees, we found that PRDM9 specifies the location of recombination events outside of mammals. However, we also detected an independent effect of promoter-like features on recombination, which is more pronounced on macro- than microchromosomes. Thus, our findings reveal that the uses of PRDM9 and promoter-like features are not mutually-exclusive, and instead reflect a tug of war, which varies in strength along the genome and is more lopsided in some species than others.

MLL3/MLL4 methyltransferase activities control early embryonic development and embryonic stem cell differentiation in a lineage-selective manner.

H3K4me1 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D) are critical for enhancer activation, cell differentiation and development. However, roles of MLL3/4 enzymatic activities and MLL3/4-mediated enhancer H3K4me1 in these processes remain unclear. Here we report that constitutive elimination of both MLL3 and MLL4 enzymatic activities prevents initiation of gastrulation and leads to early embryonic lethality in mice. However, selective elimination of MLL3/4 enzymatic activities in embryonic, but not extraembryonic, lineages leaves gastrulation largely intact. Consistent with this, embryonic stem cells (ESCs) lacking MLL3/4 enzymatic activities can differentiate toward the three embryonic germ layers but show aberrant differentiation to extraembryonic endoderm (ExEn) and trophectoderm. The failure in ExEn differentiation can be attributed to markedly reduced enhancer-binding of the lineage-determining transcription factor GATA6. Furthermore, we show that MLL3/4-catalyzed H3K4me1 is largely dispensable for enhancer activation during ESC differentiation. Together, our findings suggest a lineage-selective, but enhancer activation-independent, role of MLL3/4 methyltransferase activities in early embryonic development and ESC differentiation.

Regulation of endogenous retrovirus-derived regulatory elements by GATA2/3 and MSX2 in human trophoblast stem cells.

The placenta is an organ with extraordinary phenotypic diversity in eutherian mammals. Recent evidence suggests that numerous human placental enhancers are evolved from lineage-specific insertions of endogenous retroviruses (ERVs), yet the transcription factors (TFs) underlying their regulation remain largely elusive. Here, by first focusing on MER41, a primate-specific ERV family previously linked to placenta and innate immunity, we uncover the binding motifs of multiple crucial trophoblast TFs (GATA2/3, MSX2, GRHL2) in addition to innate immunity TFs STAT1 and IRF1. Integration of ChIP-seq data confirms the binding of GATA2/3, MSX2, and their related factors on the majority of MER41-derived enhancers in human trophoblast stem cells (TSCs). MER41-derived enhancers that are constitutively active in human TSCs are distinct from those activated upon interferon stimulation, which is determined by the binding of relevant TFs and their subfamily compositions. We further demonstrate that GATA2/3 and MSX2 have prevalent binding to numerous other ERV families - indicating their broad impact on ERV-derived enhancers. Functionally, the derepression of many syncytiotrophoblast genes after MSX2 knockdown is likely to be mediated by regulatory elements derived from ERVs - suggesting ERVs are also important for mediating transcriptional repression. Overall, this study characterizes the regulation of ERV-derived regulatory elements by GATA2/3, MSX2, and their cofactors in human TSCs, and provides mechanistic insights into the importance of ERVs in human trophoblast regulatory network.

Transposable elements shape the evolution of mammalian development.

Transposable elements (TEs) promote genetic innovation but also threaten genome stability. Despite multiple layers of host defence, TEs actively shape mammalian-specific developmental processes, particularly during pre-implantation and extra-embryonic development and at the maternal-fetal interface. Here, we review how TEs influence mammalian genomes both directly by providing the raw material for genetic change and indirectly via co-evolving TE-binding Krüppel-associated box zinc finger proteins (KRAB-ZFPs). Throughout mammalian evolution, individual activities of ancient TEs were co-opted to enable invasive placentation that characterizes live-born mammals. By contrast, the widespread activity of evolutionarily young TEs may reflect an ongoing co-evolution that continues to impact mammalian development.

Endogenous Retroviruses Drive Lineage-Specific Regulatory Evolution across Primate and Rodent Placentae.

In mammals, the placenta mediates maternal-fetal nutrient and waste exchange and acts in an immunomodulatory way to facilitate maternal-fetal tolerance. The placenta is highly diverse across mammalian species, yet the molecular mechanisms that distinguish the placenta of human from other mammals are not fully understood. Using an interspecies transcriptomic comparison of human, macaque, and mouse late-gestation placentae, we identified hundreds of genes with lineage-specific expression-including dozens that are placentally enriched and potentially related to pregnancy. We further annotated the enhancers for different human tissues using epigenomic data and demonstrate that the placenta and chorion are unique in that their enhancers display the least conservation. We identified numerous lineage-specific human placental enhancers and found they highly overlap with specific families of endogenous retroviruses (ERVs), including MER21A, MER41A/B, and MER39B that were previously linked to immune response and placental function. Among these ERV families, we further demonstrate that MER41A/B insertions create dozens of lineage-specific serum response factor-binding loci in human, including one adjacent to FBN2, a placenta-specific gene with increased expression in humans that produces the peptide hormone placensin to stimulate glucose secretion and trophoblast invasion. Overall, our results demonstrate the prevalence of lineage-specific placental enhancers which are frequently associated with ERV insertions and likely facilitate the lineage-specific evolution of the mammalian placenta.

KDM1A maintains genome-wide homeostasis of transcriptional enhancers.

Transcriptional enhancers enable exquisite spatiotemporal control of gene expression in metazoans. Enrichment of monomethylation of histone H3 lysine 4 (H3K4me1) is a major chromatin signature of transcriptional enhancers. Lysine (K)-specific demethylase 1A (KDM1A, also known as LSD1), an H3K4me2/me1 demethylase, inactivates stem-cell enhancers during the differentiation of mouse embryonic stem cells (mESCs). However, its role in undifferentiated mESCs remains obscure. Here, we show that KDM1A actively maintains the optimal enhancer status in both undifferentiated and lineage-committed cells. KDM1A occupies a majority of enhancers in undifferentiated mESCs. KDM1A levels at enhancers exhibit clear positive correlations with its substrate H3K4me2, H3K27ac, and transcription at enhancers. In Kdm1a-deficient mESCs, a large fraction of these enhancers gains additional H3K4 methylation, which is accompanied by increases in H3K27 acetylation and increased expression of both enhancer RNAs (eRNAs) and target genes. In postmitotic neurons, loss of KDM1A leads to premature activation of neuronal activity-dependent enhancers and genes. Taken together, these results suggest that KDM1A is a versatile regulator of enhancers and acts as a rheostat to maintain optimal enhancer activity by counterbalancing H3K4 methylation at enhancers.

KRAB zinc finger protein diversification drives mammalian interindividual methylation variability.

Most transposable elements (TEs) in the mouse genome are heavily modified by DNA methylation and repressive histone modifications. However, a subset of TEs exhibit variable methylation levels in genetically identical individuals, and this is associated with epigenetically conferred phenotypic differences, environmental adaptability, and transgenerational epigenetic inheritance. The evolutionary origins and molecular mechanisms underlying interindividual epigenetic variability remain unknown. Using a repertoire of murine variably methylated intracisternal A-particle (VM-IAP) epialleles as a model, we demonstrate that variable DNA methylation states at TEs are highly susceptible to genetic background effects. Taking a classical genetics approach coupled with genome-wide analysis, we harness these effects and identify a cluster of KRAB zinc finger protein (KZFP) genes that modifies VM-IAPs in trans in a sequence-specific manner. Deletion of the cluster results in decreased DNA methylation levels and altered histone modifications at the targeted VM-IAPs. In some cases, these effects are accompanied by dysregulation of neighboring genes. We find that VM-IAPs cluster together phylogenetically and that this is linked to differential KZFP binding, suggestive of an ongoing evolutionary arms race between TEs and this large family of epigenetic regulators. These findings indicate that KZFP divergence and concomitant evolution of DNA binding capabilities are mechanistically linked to methylation variability in mammals, with implications for phenotypic variation and putative paradigms of mammalian epigenetic inheritance.

KRAB-zinc finger protein gene expansion in response to active retrotransposons in the murine lineage.

The Krüppel-associated box zinc finger protein (KRAB-ZFP) family diversified in mammals. The majority of human KRAB-ZFPs bind transposable elements (TEs), however, since most TEs are inactive in humans it is unclear whether KRAB-ZFPs emerged to suppress TEs. We demonstrate that many recently emerged murine KRAB-ZFPs also bind to TEs, including the active ETn, IAP, and L1 families. Using a CRISPR/Cas9-based engineering approach, we genetically deleted five large clusters of KRAB-ZFPs and demonstrate that target TEs are de-repressed, unleashing TE-encoded enhancers. Homozygous knockout mice lacking one of two KRAB-ZFP gene clusters on chromosome 2 and chromosome 4 were nonetheless viable. In pedigrees of chromosome 4 cluster KRAB-ZFP mutants, we identified numerous novel ETn insertions with a modest increase in mutants. Our data strongly support the current model that recent waves of retrotransposon activity drove the expansion of KRAB-ZFP genes in mice and that many KRAB-ZFPs play a redundant role restricting TE activity.

Dual histone methyl reader ZCWPW1 facilitates repair of meiotic double strand breaks in male mice.

Meiotic crossovers result from homology-directed repair of DNA double-strand breaks (DSBs). Unlike yeast and plants, where DSBs are generated near gene promoters, in many vertebrates DSBs are enriched at hotspots determined by the DNA binding activity of the rapidly evolving zinc finger array of PRDM9 (PR domain zinc finger protein 9). PRDM9 subsequently catalyzes tri-methylation of lysine 4 and lysine 36 of Histone H3 in nearby nucleosomes. Here, we identify the dual histone methylation reader ZCWPW1, which is tightly co-expressed during spermatogenesis with Prdm9, as an essential meiotic recombination factor required for efficient repair of PRDM9-dependent DSBs and for pairing of homologous chromosomes in male mice. In sum, our results indicate that the evolution of a dual histone methylation writer/reader (PRDM9/ZCWPW1) system in vertebrates remodeled genetic recombination hotspot selection from an ancestral static pattern near genes towards a flexible pattern controlled by the rapidly evolving DNA binding activity of PRDM9.

PiRNAs Rise to Rescue Koalas.

PIWI-interacting small RNAs (piRNAs) establish sequence-specific adaptive restriction of resident genomic parasites to guard genome integrity. In this issue of Cell, Yu, Koppetsch, et al. describe an innate piRNA-response that specifically fragments the viral RNA genome in the germline of recently invaded koalas. This first line of defense might ensure survival until adaptive immunity develops.

The Arms Race Between KRAB-Zinc Finger Proteins and Endogenous Retroelements and Its Impact on Mammals.

Nearly half of the human genome consists of endogenous retroelements (EREs) and their genetic remnants, a small fraction of which carry the potential to propagate in the host genome, posing a threat to genome integrity and cell/organismal survival. The largest family of transcription factors in tetrapods, the Krüppel-associated box domain zinc finger proteins (KRAB-ZFPs), binds to specific EREs and represses their transcription. Since their first appearance over 400 million years ago, KRAB-ZFPs have undergone dramatic expansion and diversification in mammals, correlating with the invasions of new EREs. In this article we review our current understanding of the structure, function, and evolution of KRAB-ZFPs and discuss growing evidence that the arms race between KRAB-ZFPs and the EREs they target is a major driving force for the evolution of new traits in mammals, often accompanied by domestication of EREs themselves.

The KRAB-zinc-finger protein ZFP708 mediates epigenetic repression at RMER19B retrotransposons.

Global epigenetic reprogramming is vital to purge germ cell-specific epigenetic features to establish the totipotent state of the embryo. This process transpires to be carefully regulated and is not an undirected, radical erasure of parental epigenomes. The TRIM28 complex has been shown to be crucial in embryonic epigenetic reprogramming by regionally opposing DNA demethylation to preserve vital parental information to be inherited from germline to soma. Yet the DNA-binding factors guiding this complex to specific targets are largely unknown. Here, we uncover and characterize a novel, maternally expressed, TRIM28-interacting KRAB zinc-finger protein: ZFP708. It recruits the repressive TRIM28 complex to RMER19B retrotransposons to evoke regional heterochromatin formation. ZFP708 binding to these hitherto unknown TRIM28 targets is DNA methylation and H3K9me3 independent. ZFP708 mutant mice are viable and fertile, yet embryos fail to inherit and maintain DNA methylation at ZFP708 target sites. This can result in activation of RMER19B-adjacent genes, while ectopic expression of ZFP708 results in transcriptional repression. Finally, we describe the evolutionary conservation of ZFP708 in mice and rats, which is linked to the conserved presence of the targeted RMER19B retrotransposons in these species.

Ten things you should know about transposable elements.

Transposable elements (TEs) are major components of eukaryotic genomes. However, the extent of their impact on genome evolution, function, and disease remain a matter of intense interrogation. The rise of genomics and large-scale functional assays has shed new light on the multi-faceted activities of TEs and implies that they should no longer be marginalized. Here, we introduce the fundamental properties of TEs and their complex interactions with their cellular environment, which are crucial to understanding their impact and manifold consequences for organismal biology. While we draw examples primarily from mammalian systems, the core concepts outlined here are relevant to a broad range of organisms.

Anthropoid primate-specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length.

Pregnancy and parturition are intricately regulated to ensure successful reproductive outcomes. However, the factors that control gestational length in humans and other anthropoid primates remain poorly defined. Here, we show the endogenous retroviral long terminal repeat transposon-like human element 1B (THE1B) selectively controls placental expression of corticotropin-releasing hormone (CRH) that, in turn, influences gestational length and birth timing. Placental expression of CRH and subsequently prolonged gestational length were found in two independent strains of transgenic mice carrying a 180-kb human bacterial artificial chromosome (BAC) DNA that contained the full length of CRH and extended flanking regions, including THE1B. Restricted deletion of THE1B silenced placental CRH expression and normalized birth timing in these transgenic lines. Furthermore, we revealed an interaction at the 5' insertion site of THE1B with distal-less homeobox 3 (DLX3), a transcription factor expressed in placenta. Together, these findings suggest that retroviral insertion of THE1B into the anthropoid primate genome may have initiated expression of CRH in placental syncytiotrophoblasts via DLX3 and that this placental CRH is sufficient to alter the timing of birth.

A lncRNA-like Role for LINE1s in Development.

Transposable elements (TEs) have profoundly affected the evolution of transcriptional and chromatin profiles in mammalian genomes. In a recent paper, Percharde et al. (2018) identify a lncRNA-like function for LINE1 transposable elements in regulating gene expression to facilitate embryonic stem cell self-renewal and preimplantation development.

The Role of KRAB-ZFPs in Transposable Element Repression and Mammalian Evolution.

Kruppel-associated box zinc-finger proteins (KRAB-ZFPs) make up the largest family of transcription factors in humans. These proteins emerged in the last common ancestor of coelacanth and tetrapods, and have expanded and diversified in the mammalian lineage. Although their mechanism of transcriptional repression has been well studied for over a decade, the DNA-binding activities and the biological functions of these proteins have been largely unexplored. Recent large-scale ChIP-seq studies and loss-of-function experiments have revealed that KRAB-ZFPs play a major role in the recognition and transcriptional silencing of transposable elements (TEs), consistent with an 'arms race model' of KRAB-ZFP evolution against invading TEs. However, this model is insufficient to explain the evolution of many KRAB-ZFPs that appear to domesticate TEs for novel host functions. We highlight some of the mammalian regulatory innovations driven by specific KRAB-ZFPs, including genomic imprinting, meiotic recombination hotspot choice, and placental growth.

A placental growth factor is silenced in mouse embryos by the zinc finger protein ZFP568.

Insulin-like growth factor 2 (IGF2) is the major fetal growth hormone in mammals. We identify zinc finger protein 568 (ZFP568), a member of the rapidly evolving Kruppel-associated box-zinc finger protein (KRAB-ZFP) family linked primarily to silencing of endogenous retroelements, as a direct repressor of a placental-specific Igf2 transcript (designated Igf2-P0) in mice. Loss of Zfp568, which causes gastrulation failure, or mutation of the ZFP568-binding site at the Igf2-P0 promoter causes inappropriate Igf2-P0 activation. Deletion of Igf2 can completely rescue Zfp568 gastrulation phenotypes through late gestation. Our data highlight the exquisite selectivity with which members of the KRAB-ZFP family repress their targets and identify an additional layer of transcriptional control of a key growth factor regulating fetal and placental development.