Repression Of Transposable Elements Research Articles

Morc1 reestablishes H3K9me3 heterochromatin on piRNA-targeted transposons in gonocytes

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.

Genomic profiling of six human somatic histone H1 variants denotes that H1X accumulates at recently incorporated transposable elements.

Histone H1, a vital component in chromatin structure, binds to linker DNA and regulates nuclear processes. We have investigated the distribution of histone H1 variants in a breast cancer cell line using ChIP-Seq. Two major groups of variants are identified: H1.2, H1.3, H1.5and H1.0 are abundant in low GC regions (B compartment), while H1.4 and H1X preferentially localize in high GC regions (A compartment). Examining their abundance within transposable elements (TEs) reveals that H1X and H1.4 are enriched in recently-incorporated TEs (SVA and SINE-Alu), while H1.0/H1.2/H1.3/H1.5 are more abundant in older elements. Notably, H1X is particularly enriched in SVA families, while H1.4 shows the highest abundance in young AluY elements. Although low GC variants are generally enriched in LINE, LTR and DNA repeats, H1X and H1.4 are also abundant in a subset of recent LINE-L1 and LTR repeats. H1X enrichment at SVA and Alu is consistent across multiple cell lines. Further, H1X depletion leads to TE derepression, suggesting its role in maintaining TE repression. Overall, this study provides novel insights into the differential distribution of histone H1 variants among repetitive elements, highlighting the potential involvement of H1X in repressing TEs recently incorporated within the human genome.

Abstract A118: Chromatin accessibility profiling of human pancreatic tumors reveals epigenetic features of malignancy and rapid recurrence

Abstract Pancreatic ductal adenocarcinoma (PDAC) disease progression involves complex cell state transitions in both malignant and non-malignant populations within the tumor microenvironment. To uncover PDAC cell states and epigenetic features associated with clinical outcome, we studied 42 PDAC samples (35 primary tumors and 7 metastases) from 41 patients with single cell ATAC-seq, along with 10 sample-matched single cell RNA-seq profiles. Our work uncovered thousands of chromatin regions with altered accessibility in malignant cells relative to normal ductal and acinar cells. Numerous epigenetic changes were universal to all PDAC clusters regardless of patient-of-origin and occurred proximal to genes involved in glycolysis, regulation of MYC, hypoxic response, extracellular matrix remodeling, and repression of transposable elements. Topic modeling of high dimensional chromatin data revealed networks of co-accessible cis-regulatory regions active in malignant cells, as well as networks specific to basal-like and classical PDAC molecular subtypes. Furthermore, our epigenomic data also suggest alternate paths of lineage infidelity across the classical-basal continuum: while basal-like PDAC cells acquire chromatin accessibility mimicking that of skin or mammary epithelial tissue, several classical subtype tumors featured a subpopulation of malignant cells with increased accessibility of neural-associated chromatin regions genome-wide. Within the immune compartment, we also identified a cis-regulatory network with increased activity in T-cells sampled from rapidly recurring PDAC tumors. Motif analysis of this network revealed enrichment for SMAD and FOXP1 motifs, suggesting widespread modification of T-cells by TGF-β. To our knowledge, our work is the largest single-cell resolution study of chromatin state in human PDAC to date, revealing key epigenetic features associated with PDAC subtype and clinical outcome. Citation Format: Kevin MacPherson, Jason M. Link, Patrick Worth, Brett Sheppard, Andrew Fields, Colin Daniel, Andrew Nishida, Carl Pelz, Trent Waugh, Ethan Agritelle, John Muschler, Andrew Adey, Rosalie Sears. Chromatin accessibility profiling of human pancreatic tumors reveals epigenetic features of malignancy and rapid recurrence [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A118.

Maternally inherited siRNAs initiate piRNA cluster formation

PIWI-interacting RNAs (piRNAs) guide transposable element repression in animal germ lines. In Drosophila, piRNAs are produced from heterochromatic loci, called piRNA clusters, which act as information repositories about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a positive feedback loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed before cognate piRNAs are present remains unknown. Here, we report spontaneous de novo piRNA cluster formation from repetitive transgenic sequences. Cluster formation occurs over several generations and requires continuous trans-generational maternal transmission of small RNAs. We discovered that maternally supplied small interfering RNAs (siRNAs) trigger de novo cluster activation in progeny. In contrast, siRNAs are dispensable for cluster function after its establishment. These results reveal an unexpected interplay between the siRNA and piRNA pathways and suggest a mechanism for de novo piRNA cluster formation triggered by siRNAs.

Crosstalk between RNA m6A and DNA methylation regulates transposable element chromatin activation and cell fate in human pluripotent stem cells.

Transposable elements (TEs) are parasitic DNA sequences accounting for over half of the human genome. Tight control of the repression and activation states of TEs is critical for genome integrity, development, immunity and diseases, including cancer. However, precisely how this regulation is achieved remains unclear. Here we develop a targeted proteomic proximity labeling approach to capture TE-associated proteins in human embryonic stem cells (hESCs). We find that the RNA N6-methyladenosine (m6A) reader, YTHDC2, occupies genomic loci of the primate-specific TE, LTR7/HERV-H, specifically through its interaction with m6A-modified HERV-H RNAs. Unexpectedly, YTHDC2 recruits the DNA 5-methylcytosine (5mC)-demethylase, TET1, to remove 5mC from LTR7/HERV-H and prevent epigenetic silencing. Functionally, the YTHDC2/LTR7 axis inhibits neural differentiation of hESCs. Our results reveal both an underappreciated crosstalk between RNA m6A and DNA 5mC, the most abundant regulatory modifications of RNA and DNA in eukaryotes, and the fact that in hESCs this interplay controls TE activity and cell fate.

Loss of epigenetic suppression of retrotransposons with oncogenic potential in aging mammary luminal epithelial cells.

A primary function of DNA methylation in mammalian genomes is to repress transposable elements (TEs). The widespread methylation loss that is commonly observed in cancer cells results in the loss of epigenetic repression of TEs. The aging process is similarly characterized by changes to the methylome. However, the impact of these epigenomic alterations on TE silencing and the functional consequences of this have remained unclear. To assess the epigenetic regulation of TEs in aging, we profiled DNA methylation in human mammary luminal epithelial cells (LEps)-a key cell lineage implicated in age-related breast cancers-from younger and older women. We report here that several TE subfamilies function as regulatory elements in normal LEps, and a subset of these display consistent methylation changes with age. Methylation changes at these TEs occurred at lineage-specific transcription factor binding sites, consistent with loss of lineage specificity. Whereas TEs mainly showed methylation loss, CpG islands (CGIs) that are targets of the Polycomb repressive complex 2 (PRC2) show a gain of methylation in aging cells. Many TEs with methylation loss in aging LEps have evidence of regulatory activity in breast cancer samples. We furthermore show that methylation changes at TEs impact the regulation of genes associated with luminal breast cancers. These results indicate that aging leads to DNA methylation changes at TEs that undermine the maintenance of lineage specificity, potentially increasing susceptibility to breast cancer.

Modeling early germline immunization after horizontal transfer of transposable elements reveals internal piRNA cluster heterogeneity

BackgroundA fraction of all genomes is composed of transposable elements (TEs) whose mobility needs to be carefully controlled. In gonads, TE activity is repressed by PIWI-interacting RNAs (piRNAs), a class of small RNAs synthesized by heterochromatic loci enriched in TE fragments, called piRNA clusters. Maintenance of active piRNA clusters across generations is secured by maternal piRNA inheritance providing the memory for TE repression. On rare occasions, genomes encounter horizontal transfer (HT) of new TEs with no piRNA targeting them, threatening the host genome integrity. Naïve genomes can eventually start to produce new piRNAs against these genomic invaders, but the timing of their emergence remains elusive.ResultsUsing a set of TE-derived transgenes inserted in different germline piRNA clusters and functional assays, we have modeled a TE HT in Drosophila melanogaster. We have found that the complete co-option of these transgenes by a germline piRNA cluster can occur within four generations associated with the production of new piRNAs all along the transgenes and the germline silencing of piRNA sensors. Synthesis of new transgenic TE piRNAs is linked to piRNA cluster transcription dependent on Moonshiner and heterochromatin mark deposition that propagates more efficiently on short sequences. Moreover, we found that sequences located within piRNA clusters can have different piRNA profiles and can influence transcript accumulation of nearby sequences.ConclusionsOur study reveals that genetic and epigenetic properties, such as transcription, piRNA profiles, heterochromatin, and conversion efficiency along piRNA clusters, could be heterogeneous depending on the sequences that compose them. These findings suggest that the capacity of transcriptional signal erasure induced by the chromatin complex specific of the piRNA cluster can be incomplete through the piRNA cluster loci. Finally, these results have revealed an unexpected level of complexity that highlights a new magnitude of piRNA cluster plasticity fundamental for the maintenance of genome integrity.

Methylation of lysine 36 on histone H3 is required to control transposon activities in somatic cells.

Transposable elements constitute a substantial portion of most eukaryotic genomes and their activity can lead to developmental and neuronal defects. In the germline, transposon activity is antagonized by the PIWI-interacting RNA pathway tasked with repression of transposon transcription and degrading transcripts that have already been produced. However, most of the genes required for transposon control are not expressed outside the germline, prompting the question: what causes deleterious transposons activity in the soma and how is it managed? Here, we show that disruptions of the Histone 3 lysine 36 methylation machinery led to increased transposon transcription in Drosophila melanogaster brains and that there is division of labour for the repression of transposable elements between the different methyltransferases Set2, NSD, and Ash1. Furthermore, we show that disruption of methylation leads to somatic activation of key genes in the PIWI-interacting RNA pathway and the preferential production of RNA from dual-strand piRNA clusters.

Comparative analysis reveals epigenomic evolution related to species traits and genomic imprinting in mammals

DNA methylation is an epigenetic modification that plays a crucial role in various regulatory processes, including gene expression regulation, transposable element repression, and genomic imprinting. However, most studies on DNA methylation have been conducted in humans and other model species, whereas the dynamics of DNA methylation across mammals remain poorly explored, limiting our understanding of epigenomic evolution in mammals and the evolutionary impacts of conserved and lineage-specific DNA methylation. Here, we generated and gathered comparative epigenomic data from 13 mammalian species, including two marsupial species, to demonstrate that DNA methylation plays critical roles in several aspects of gene evolution and species trait evolution. We found that the species-specific DNA methylation of promoters and noncoding elements correlates with species-specific traits such as body patterning, indicating that DNA methylation might help establish or maintain interspecies differences in gene regulation that shape phenotypes. For a broader view, we investigated the evolutionary histories of 88 known imprinting control regions across mammals to identify their evolutionary origins. By analyzing the features of known and newly identified potential imprints in all studied mammals, we found that genomic imprinting may function in embryonic development through the binding of specific transcription factors. Our findings show that DNA methylation and the complex interaction between the genome and epigenome have a significant impact on mammalian evolution, suggesting that evolutionary epigenomics should be incorporated to develop a unified evolutionary theory.

Histone H3K27me3 methylation regulates the expression of secreted proteins distributed at fast-evolving regions through transcriptional repression of transposable elements

The fine-tuned expression dynamics of the effector genes are pivotal for the transition from vegetative growth to host colonization of pathogenic filamentous fungi. However, mechanisms underlying the dynamic regulation of these genes remain largely unknown. Here, through comparative transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses of the methyltransferase PoKmt6 in rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae), we found that PoKmt6-mediated H3K27me3 deposition was enriched mainly at fast-evolving regions and contributed to the silencing of a subset of secreted proteins (SP) and transposable element (TE) families during the vegetative growth of P. oryzae. Intriguingly, we observed that a group of SP genes, which were depleted of H3K27me3 modification, could also be silenced via the H3K27me3-mediated repression of the nearby TEs. In conclusion, our results indicate that H3K27me3 modification mediated by PoKmt6 regulates the expression of some SP genes in fast-evolving regions through the suppression of nearby TEs.

Measuring Transposable Element Activity in Adult Drosophila Ovaries.

Transposons are genetic elements that use various mechanisms of transposition to move around the genome, thus posing a risk to genomic integrity. Repression of transposable elements (TEs) involves the complex PIWI pathway and several proteins associated with heterochromatinization. All players of TE repression are indispensable for proper reproductive fitness, as loss-of-function mutations in these genes result primarily in sterility and impaired reproductive development. When investigating the function of novel genes with similar phenotypes, elevated transposon expression in reproductive tissues can be a marker for involvement in the aforementioned processes. Here, we present a protocol for investigating TE levels in adult Drosophila ovaries, from dissection to data analysis.

Co-option of the piRNA pathway to regulate neural crest specification.

Across Metazoa, Piwi proteins play a critical role in protecting the germline genome through piRNA-mediated repression of transposable elements. In vertebrates, activity of Piwi proteins and the piRNA pathway was thought to be gonad specific. Our results reveal the expression of Piwil1 in a vertebrate somatic cell type, the neural crest. Piwil1 is expressed at low levels throughout the chicken neural tube, peaking in neural crest cells just before the specification event that enables epithelial-to-mesenchymal transition (EMT) and migration into the periphery. Loss of Piwil1 impedes neural crest specification and emigration. Small RNA sequencing reveals somatic piRNAs with sequence signatures of an active ping-pong loop. RNA-seq and functional experiments identify the transposon-derived gene ERNI as Piwil1’s target in the neural crest. ERNI, in turn, suppresses Sox2 to precisely control the timing of neural crest specification and EMT. Our data provide mechanistic insight into a novel function of the piRNA pathway as a regulator of somatic development in a vertebrate species.

Transposable element repression using piRNAs, and its relevance to endogenous viral elements (EVEs) and immunity in insects.

The piRNA system controls transposable element (TE) mobility by transcriptional gene silencing and post-transcriptional gene silencing. Dispersed in insect genomes, piRNA clusters contain TE copies, from which they produce piRNAs (specific small RNAs). These piRNAs can both target the nascent transcripts produced by active TE copies and directly repress them by heterochromatinization. They can also target mature transcripts and cleave them following amplification by the so-called 'ping-pong' loop mechanism. Moreover, piRNA clusters contain endogenous viral elements (EVEs), from which they produce piRNAs. The current idea is that these piRNAs could participate in the antiviral response against exogenous viral infection. In this review, we show that among insects, to date, this antiviral response by the piRNA system appears mainly restricted to mosquitoes, but this could be due to the focus of most studies on arboviruses.

DNA methylation-free Arabidopsis reveals crucial roles of DNA methylation in regulating gene expression and development

A contribution of DNA methylation to defense against invading nucleic acids and maintenance of genome integrity is uncontested; however, our understanding of the extent of involvement of this epigenetic mark in genome-wide gene regulation and plant developmental control is incomplete. Here, we knock out all five known DNA methyltransferases in Arabidopsis, generating DNA methylation-free plants. This quintuple mutant exhibits a suite of developmental defects, unequivocally demonstrating that DNA methylation is essential for multiple aspects of plant development. We show that CG methylation and non-CG methylation are required for a plethora of biological processes, including pavement cell shape, endoreduplication, cell death, flowering, trichome morphology, vasculature and meristem development, and root cell fate determination. Moreover, we find that DNA methylation has a strong dose-dependent effect on gene expression and repression of transposable elements. Taken together, our results demonstrate that DNA methylation is dispensable for Arabidopsis survival but essential for the proper regulation of multiple biological processes.

The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

Reversing epigenetic repression of transposable elements for improving tumor immunogenicity.

Reversing epigenetic repression of transposable elements for improving tumor immunogenicity.

Identification of piRNA disease associations using deep learning

Piwi-interacting RNAs (piRNAs) play a pivotal role in maintaining genome integrity by repression of transposable elements, gene stability, and association with various disease progressions. Cost-efficient computational methods for the identification of piRNA disease associations promote the efficacy of disease-specific drug development. In this regard, we developed a simple, robust, and efficient deep learning method for identifying the piRNA disease associations known as piRDA. The proposed architecture extracts the most significant and abstract information from raw sequences represented in a simplicated piRNA disease pair without any involvement of features engineering. Two-step positive unlabeled learning and bootstrapping technique are utilized to abstain from the false-negative and biased predictions dealing with positive unlabeled data. The performance of proposed method piRDA is evaluated using k-fold cross-validation. The piRDA is significantly improved in all the performance evaluation measures for the identification of piRNA disease associations in comparison to state-of-the-art method. Moreover, it is thus projected conclusively that the proposed computational method could play a significant role as a supportive and practical tool for primitive disease mechanisms and pharmaceutical research such as in academia and drug design. Eventually, the proposed model can be accessed using publicly available and user-friendly web tool athttp://nsclbio.jbnu.ac.kr/tools/piRDA/.

Functional Analysis of Individual piRNAs in Aedes aegypti Cells and Embryos Using Antisense Oligonucleotides.

In insects, PIWI-interacting (pi)RNAs fulfill versatile regulatory functions inside and outside the germline, including posttranscriptional repression of transposable elements and regulation of gene expression. Canonically, piRNAs act-and have been studied-as a conglomerate of several thousand sequences that cooperatively silence target RNAs. Interestingly, however, an increasing number of studies have demonstrated that individual piRNAs can have profound biological activity as a unique piRNA sequence. Prime examples are the tapiR1 and 2 piRNAs, which mediate target RNA degradation in the developing embryo of Aedes mosquitoes. To study such outstanding individual piRNA species, we describe here a method to interfere with RNA target silencing using antisense oligonucleotides in cell culture as well as in mosquito pre-blastoderm embryos. Although the method has been established for Aedes mosquitoes, it can likely be adapted for use in other invertebrate species as well.

Complex Genetic Interactions between Piwi and HP1a in the Repression of Transposable Elements and Tissue-Specific Genes in the Ovarian Germline.

Insertions of transposable elements (TEs) in eukaryotic genomes are usually associated with repressive chromatin, which spreads to neighbouring genomic sequences. In ovaries of Drosophila melanogaster, the Piwi-piRNA pathway plays a key role in the transcriptional silencing of TEs considered to be exerted mostly through the establishment of H3K9me3 histone marks recruiting Heterochromatin Protein 1a (HP1a). Here, using RNA-seq, we investigated the expression of TEs and the adjacent genomic regions upon Piwi and HP1a germline knockdowns sharing a similar genetic background. We found that the depletion of Piwi and HP1a led to the derepression of only partially overlapping TE sets. Several TEs were silenced predominantly by HP1a, whereas the upregulation of some other TEs was more pronounced upon Piwi knockdown and, surprisingly, was diminished upon a Piwi/HP1a double-knockdown. We revealed that HP1a loss influenced the expression of thousands of protein-coding genes mostly not adjacent to TE insertions and, in particular, downregulated a putative transcriptional factor required for TE activation. Nevertheless, our results indicate that Piwi and HP1a cooperatively exert repressive effects on the transcription of euchromatic loci flanking the insertions of some Piwi-regulated TEs. We suggest that this mechanism controls the silencing of a small set of TE-adjacent tissue-specific genes, preventing their inappropriate expression in ovaries.

Size does matter: piRNA and miRNA targeting

Piwi-interacting RNAs (piRNAs) guide PIWI proteins to bind and cleave RNAs. Originally, piRNAs were described as a system for transposable element repression. Recently, Anzelon, Choudhury, Hughes et al. uncovered the structural basis for piRNA targeting, whereby they are recognized in a manner distinct from targeting by miRNAs.