The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Transcriptome-wide Interrogation of the Functional Intronome by Spliceosome Profiling.

Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA sequencing (RNA-seq) provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-seq alone to reveal the full repertoire of spliced species. Here, we present "spliceosome profiling," a strategy based on deep sequencing of RNAs co-purifying with late-stage spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single-nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus, much as ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.

Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline.

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.

The RNase PARN-1 Trims piRNA 3' Ends to Promote Transcriptome Surveillance in C. elegans.

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and are essential for fertility in diverse organisms. An interesting feature of piRNAs is that, while piRNA lengths are stereotypical within a species, they can differ widely between species. For example, piRNAs are mainly 29 and 30 nucleotides in humans, 24 to 30 nucleotides in D. melanogaster, and uniformly 21 nucleotides in C. elegans. However, how piRNA length is determined and whether length impacts function remains unknown. Here, we show that C. elegans deficient for PARN-1, a conserved RNase, accumulate untrimmed piRNAs with 3' extensions. Surprisingly, these longer piRNAs are stable and associate with the Piwi protein PRG-1 but fail to robustly recruit downstream silencing factors. Our findings identify PARN-1 as a key regulator of piRNA length in C. elegans and suggest that length is regulated to promote efficient transcriptome surveillance.

The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing.

piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. We show that Rhino binds specifically to the heterochromatic clusters that produce piRNA precursors, and that binding directly correlates with piRNA production. Rhino colocalizes to germline nuclear foci with Rai1/DXO-related protein Cuff and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced and that rhino, cuff, and uap56 mutations increase expression of spliced cluster transcripts over 100-fold. LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. We therefore propose that Rhino anchors a nuclear complex that suppresses cluster transcript splicing and speculate that stalled splicing differentiates piRNA precursors from mRNAs.

Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

Dicer partner proteins tune the length of mature miRNAs in flies and mammals.

Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. Alternative splicing of the loquacious (loqs) mRNA generates three distinct Dicer partner proteins. To understand the function of each, we constructed flies expressing Loqs-PA, Loqs-PB, or Loqs-PD. Loqs-PD promotes both endo- and exo-siRNA production by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: a specific subset of miRNAs requires Loqs-PB. Surprisingly, Loqs-PB tunes where Dicer-1 cleaves pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The longer form of miR-307a represses glycerol kinase and taranis mRNA expression. The mammalian Dicer-partner TRBP, a Loqs-PB homolog, similarly tunes where Dicer cleaves pre-miR-132. Thus, Dicer-binding partner proteins change the choice of cleavage site by Dicer, producing miRNAs with target specificities different from those made by Dicer alone or Dicer bound to alternative protein partners.

The cellular EJC interactome reveals higher-order mRNP structure and an EJC-SR protein nexus.

In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here, we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major "canonical" EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction.

UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery.

piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.

The RNA-Binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria.

PIWI-interacting RNAs (piRNAs) silence transposons in Drosophila ovaries, ensuring female fertility. Two coupled pathways generate germline piRNAs: the ping-pong cycle, in which the PIWI proteins Aubergine and Ago3 increase the abundance of pre-existing piRNAs, and the phased piRNA pathway, which generates strings of tail-to-head piRNAs, one after another. Proteins acting in the ping-pong cycle localize to nuage, whereas phased piRNA production requires Zucchini, an endonuclease on the mitochondrial surface. Here, we report that Armitage (Armi), an RNA-binding ATPase localized to both nuage and mitochondria, links the ping-pong cycle to the phased piRNA pathway. Mutations that block phased piRNA production deplete Armi from nuage. Armi ATPase mutants cannot support phased piRNA production and inappropriately bind mRNA instead of piRNA precursors. We propose that Armi shuttles between nuage and mitochondria, feeding precursor piRNAs generated by Ago3 cleavage into the Zucchini-dependent production of Aubergine- and Piwi-bound piRNAs on the mitochondrial surface.

Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster.

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins.

The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases.

Microglia play a pivotal role in the maintenance of brain homeostasis but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Alzheimer's disease (AD) and in microglia surrounding neuritic ß-amyloid (Aß)-plaques in the brains of people with AD. The APOE pathway mediated a switch from a homeostatic to a neurodegenerative microglia phenotype after phagocytosis of apoptotic neurons. TREM2 (triggering receptor expressed on myeloid cells 2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia had lost their tolerogenic function. Our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target that could aid in the restoration of homeostatic microglia.

Mbd3/NURD complex regulates expression of 5-hydroxymethylcytosine marked genes in embryonic stem cells.

Numerous chromatin regulators are required for embryonic stem (ES) cell self-renewal and pluripotency, but few have been studied in detail. Here, we examine the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We find that Mbd3 and Brg1 antagonistically regulate a common set of genes by regulating promoter nucleosome occupancy. Furthermore, both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC): Mbd3 colocalizes with Tet1 and 5hmC in vivo, Mbd3 knockdown preferentially affects expression of 5hmC-marked genes, Mbd3 localization is Tet1-dependent, and Mbd3 preferentially binds to 5hmC relative to 5-methylcytosine in vitro. Finally, both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo. Together, our results identify an effector for 5hmC, and reveal that control of gene expression by antagonistic chromatin regulators is a surprisingly common regulatory strategy in ES cells.

Adaptation to P element transposon invasion in Drosophila melanogaster.

Transposons evolve rapidly and can mobilize and trigger genetic instability. Piwi-interacting RNAs (piRNAs) silence these genome pathogens, but it is unclear how the piRNA pathway adapts to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo. Paternally inherited P element transposons thus escape silencing and trigger a hybrid sterility syndrome termed P-M hybrid dysgenesis. We show that P-M hybrid dysgenesis activates both P elements and resident transposons and disrupts the piRNA biogenesis machinery. As dysgenic hybrids age, however, fertility is restored, P elements are silenced, and P element piRNAs are produced de novo. In addition, the piRNA biogenesis machinery assembles, and resident elements are silenced. Significantly, resident transposons insert into piRNA clusters, and these new insertions are transmitted to progeny, produce novel piRNAs, and are associated with reduced transposition. P element invasion thus triggers heritable changes in genome structure that appear to enhance transposon silencing.

Perspectives on ENCODE.

The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community.

Integration of high-resolution promoter profiling assays reveals novel, cell type-specific transcription start sites across 115 human cell and tissue types.

Accurate transcription start site (TSS) annotations are essential for understanding transcriptional regulation and its role in human disease. Gene collections such as GENCODE contain annotations for tens of thousands of TSSs, but not all of these annotations are experimentally validated nor do they contain information on cell type-specific usage. Therefore, we sought to generate a collection of experimentally validated TSSs by integrating RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression (RAMPAGE) data from 115 cell and tissue types, which resulted in a collection of approximately 50 thousand representative RAMPAGE peaks. These peaks are primarily proximal to GENCODE-annotated TSSs and are concordant with other transcription assays. Because RAMPAGE uses paired-end reads, we were then able to connect peaks to transcripts by analyzing the genomic positions of the 3' ends of read mates. Using this paired-end information, we classified the vast majority (37 thousand) of our RAMPAGE peaks as verified TSSs, updating TSS annotations for 20% of GENCODE genes. We also found that these updated TSS annotations are supported by epigenomic and other transcriptomic data sets. To show the utility of this RAMPAGE rPeak collection, we intersected it with the NHGRI/EBI genome-wide association study (GWAS) catalog and identified new candidate GWAS genes. Overall, our work shows the importance of integrating experimental data to further refine TSS annotations and provides a valuable resource for the biological community.

Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals.

Epigenetic information can be inherited through the mammalian germline and represents a plausible transgenerational carrier of environmental information. To test whether transgenerational inheritance of environmental information occurs in mammals, we carried out an expression profiling screen for genes in mice that responded to paternal diet. Offspring of males fed a low-protein diet exhibited elevated hepatic expression of many genes involved in lipid and cholesterol biosynthesis and decreased levels of cholesterol esters, relative to the offspring of males fed a control diet. Epigenomic profiling of offspring livers revealed numerous modest (∼20%) changes in cytosine methylation depending on paternal diet, including reproducible changes in methylation over a likely enhancer for the key lipid regulator Ppara. These results, in conjunction with recent human epidemiological data, indicate that parental diet can affect cholesterol and lipid metabolism in offspring and define a model system to study environmental reprogramming of the heritable epigenome.

Epigenetic and chromosomal features drive transposon insertion in Drosophila melanogaster.

Transposons are mobile genetic elements prevalent in the genomes of most species. The distribution of transposons within a genome reflects the actions of two opposing processes: initial insertion site selection, and selective pressure from the host. By analyzing whole-genome sequencing data from transposon-activated Drosophila melanogaster, we identified 43 316 de novo and 237 germline insertions from four long-terminal-repeat (LTR) transposons, one LINE transposon (I-element), and one DNA transposon (P-element). We found that all transposon types favored insertion into promoters de novo, but otherwise displayed distinct insertion patterns. De novo and germline P-element insertions preferred replication origins, often landing in a narrow region around transcription start sites and in regions of high chromatin accessibility. De novo LTR transposon insertions preferred regions with high H3K36me3, promoters and exons of active genes; within genes, LTR insertion frequency correlated with gene expression. De novo I-element insertion density increased with distance from the centromere. Germline I-element and LTR transposon insertions were depleted in promoters and exons, suggesting strong selective pressure to remove transposons from functional elements. Transposon movement is associated with genome evolution and disease; therefore, our results can improve our understanding of genome and disease biology.

FAVOR: functional annotation of variants online resource and annotator for variation across the human genome.

Large biobank-scale whole genome sequencing (WGS) studies are rapidly identifying a multitude of coding and non-coding variants. They provide an unprecedented resource for illuminating the genetic basis of human diseases. Variant functional annotations play a critical role in WGS analysis, result interpretation, and prioritization of disease- or trait-associated causal variants. Existing functional annotation databases have limited scope to perform online queries and functionally annotate the genotype data of large biobank-scale WGS studies. We develop the Functional Annotation of Variants Online Resources (FAVOR) to meet these pressing needs. FAVOR provides a comprehensive multi-faceted variant functional annotation online portal that summarizes and visualizes findings of all possible nine billion single nucleotide variants (SNVs) across the genome. It allows for rapid variant-, gene- and region-level queries of variant functional annotations. FAVOR integrates variant functional information from multiple sources to describe the functional characteristics of variants and facilitates prioritizing plausible causal variants influencing human phenotypes. Furthermore, we provide a scalable annotation tool, FAVORannotator, to functionally annotate large-scale WGS studies and efficiently store the genotype and their variant functional annotation data in a single file using the annotated Genomic Data Structure (aGDS) format, making downstream analysis more convenient. FAVOR and FAVORannotator are available at https://favor.genohub.org.

Building integrative functional maps of gene regulation.

Every cell in the human body inherits a copy of the same genetic information. The three billion base pairs of DNA in the human genome, and the roughly 50 000 coding and non-coding genes they contain, must thus encode all the complexity of human development and cell and tissue type diversity. Differences in gene regulation, or the modulation of gene expression, enable individual cells to interpret the genome differently to carry out their specific functions. Here we discuss recent and ongoing efforts to build gene regulatory maps, which aim to characterize the regulatory roles of all sequences in a genome. Many researchers and consortia have identified such regulatory elements using functional assays and evolutionary analyses; we discuss the results, strengths and shortcomings of their approaches. We also discuss new techniques the field can leverage and emerging challenges it will face while striving to build gene regulatory maps of ever-increasing resolution and comprehensiveness.