The USTC co-opts an ancient machinery to drive piRNA transcription in C. elegans.

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and nonself nucleic acids and maintain genome integrity and are essential for fertility in a variety of organisms. In Caenorhabditis elegans, most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes have been shown to be required for piRNA biogenesis, the mechanism of piRNA transcription remains elusive. Here we used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) that is essential for piRNA biogenesis. The USTC contains piRNA silencing-defective 1 (PRDE-1), SNPC-4, twenty-one-U fouled-up 4 (TOFU-4), and TOFU-5. The USTC forms unique piRNA foci in germline nuclei and coats the piRNA cluster genomic loci. USTC factors associate with the Ruby motif just upstream of type I piRNA genes. USTC factors are also mutually dependent for binding to the piRNA clusters and forming the piRNA foci. Interestingly, USTC components bind differentially to piRNAs in the clusters and other noncoding RNA genes. These results reveal the USTC as a striking example of the repurposing of a general transcription factor complex to aid in genome defense against transposons.

Accessible Region Conformation Capture (ARC-C) gives high-resolution insights into genome architecture and regulation.

Nuclear organization and chromatin interactions are important for genome function, yet determining chromatin connections at high resolution remains a major challenge. To address this, we developed Accessible Region Conformation Capture (ARC-C), which profiles interactions between regulatory elements genome-wide without a capture step. Applied to Caenorhabditis elegans, ARC-C identifies approximately 15,000 significant interactions between regulatory elements at 500-bp resolution. Of 105 TFs or chromatin regulators tested, we find that the binding sites of 60 are enriched for interacting with each other, making them candidates for mediating interactions. These include cohesin and condensin II. Applying ARC-C to a mutant of transcription factor BLMP-1 detected changes in interactions between its targets. ARC-C simultaneously profiles domain-level architecture, and we observe that C. elegans chromatin domains defined by either active or repressive modifications form topologically associating domains (TADs) that interact with A/B (active/inactive) compartment-like structure. Furthermore, we discover that inactive compartment interactions are dependent on H3K9 methylation. ARC-C is a powerful new tool to interrogate genome architecture and regulatory interactions at high resolution.

The Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion.

Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene (also called MECOM), in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.

The DREAM complex promotes gene body H2A.Z for target repression.

The DREAM (DP, Retinoblastoma [Rb]-like, E2F, and MuvB) complex controls cellular quiescence by repressing cell cycle genes, but its mechanism of action is poorly understood. Here we show that Caenorhabditis elegans DREAM targets have an unusual pattern of high gene body HTZ-1/H2A.Z. In mutants of lin-35, the sole p130/Rb-like gene in C. elegans, DREAM targets have reduced gene body HTZ-1/H2A.Z and increased expression. Consistent with a repressive role for gene body H2A.Z, many DREAM targets are up-regulated in htz-1/H2A.Z mutants. Our results indicate that the DREAM complex facilitates high gene body HTZ-1/H2A.Z, which plays a role in target gene repression.

Transcriptional and post-transcriptional regulation of checkpoint genes on the tumour side of the immunological synapse.

Cancer is a disease of the genome, therefore, its development has a clear Mendelian component, demonstrated by well-studied genes such as BRCA1 and BRCA2 in breast cancer risk. However, it is known that a single genetic variant is not enough for cancer to develop leading to the theory of multistage carcinogenesis. In many cases, it is a sequence of events, acquired somatic mutations, or simply polygenic components with strong epigenetic effects, such as in the case of brain tumours. The expression of many genes is the product of the complex interplay between several factors, including the organism's genotype (in most cases Mendelian-inherited), genetic instability, epigenetic factors (non-Mendelian-inherited) as well as the immune response of the host, to name just a few. In recent years the importance of the immune system has been elevated, especially in the light of the immune checkpoint genes discovery and the subsequent development of their inhibitors. As the expression of these genes normally suppresses self-immunoreactivity, their expression by tumour cells prevents the elimination of the tumour by the immune system. These discoveries led to the rapid growth of the field of immuno-oncology that offers new possibilities of long-lasting and effective treatment options. Here we discuss the recent advances in the understanding of the key mechanisms controlling the expression of immune checkpoint genes in tumour cells.

Physical and functional interaction between SET1/COMPASS complex component CFP-1 and a Sin3S HDAC complex in C. elegans.

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.

Comprehensive Analysis of Correlations in the Expression of miRNA Genes and Immune Checkpoint Genes in Bladder Cancer Cells.

Personalised medicine is the future and hope for many patients, including those with cancers. Early detection, as well as rapid, well-selected treatment, are key factors leading to a good prognosis. MicroRNA mediated gene regulation is a promising area of development for new diagnostic and therapeutic methods, crucial for better prospects for patients. Bladder cancer is a frequent neoplasm, with high lethality and lacking modern, advanced therapeutic modalities, such as immunotherapy. MicroRNAs are involved in bladder cancer pathogenesis, proliferation, control and response to treatment, which we summarise in this perspective in response to lack of recent review publications in this field. We further performed a correlation-based analysis of microRNA and gene expression data in bladder cancer (BLCA) TCGA dataset. We identified 27 microRNAs hits with opposite expression profiles to genes involved in immune response in bladder cancer, and 24 microRNAs hits with similar expression profiles. We discuss previous studies linking the functions of these microRNAs to bladder cancer and assess if they are good candidates for personalised medicine therapeutics and diagnostics. The discussed functions include regulation of gene expression, interplay with transcription factors, response to treatment, apoptosis, cell proliferation and angiogenesis, initiation and development of cancer, genome instability and tumour-associated inflammatory reaction.

Stable Caenorhabditis elegans chromatin domains separate broadly expressed and developmentally regulated genes.

Eukaryotic genomes are organized into domains of differing structure and activity. There is evidence that the domain organization of the genome regulates its activity, yet our understanding of domain properties and the factors that influence their formation is poor. Here, we use chromatin state analyses in early embryos and third-larval stage (L3) animals to investigate genome domain organization and its regulation in Caenorhabditis elegans At both stages we find that the genome is organized into extended chromatin domains of high or low gene activity defined by different subsets of states, and enriched for H3K36me3 or H3K27me3, respectively. The border regions between domains contain large intergenic regions and a high density of transcription factor binding, suggesting a role for transcription regulation in separating chromatin domains. Despite the differences in cell types, overall domain organization is remarkably similar in early embryos and L3 larvae, with conservation of 85% of domain border positions. Most genes in high-activity domains are expressed in the germ line and broadly across cell types, whereas low-activity domains are enriched for genes that are developmentally regulated. We find that domains are regulated by the germ-line H3K36 methyltransferase MES-4 and that border regions show striking remodeling of H3K27me1, supporting roles for H3K36 and H3K27 methylation in regulating domain structure. Our analyses of C. elegans chromatin domain structure show that genes are organized by type into domains that have differing modes of regulation.

Extreme HOT regions are CpG-dense promoters in C. elegans and humans.

Most vertebrate promoters lie in unmethylated CpG-dense islands, whereas methylation of the more sparsely distributed CpGs in the remainder of the genome is thought to contribute to transcriptional repression. Nonmethylated CG dinucleotides are recognized by CXXC finger protein 1 (CXXC1, also known as CFP1), which recruits SETD1A (also known as Set1) methyltransferase for trimethylation of histone H3 lysine 4, an active promoter mark. Genomic regions enriched for CpGs are thought to be either absent or irrelevant in invertebrates that lack DNA methylation, such as C. elegans; however, a CXXC1 ortholog (CFP-1) is present. Here we demonstrate that C. elegans CFP-1 targets promoters with high CpG density, and these promoters are marked by high levels of H3K4me3. Furthermore, as for mammalian promoters, high CpG content is associated with nucleosome depletion irrespective of transcriptional activity. We further show that highly occupied target (HOT) regions identified by the binding of a large number of transcription factors are CpG-rich promoters in C. elegans and human genomes, suggesting that the unusually high factor association at HOT regions may be a consequence of CpG-linked chromatin accessibility. Our results indicate that nonmethylated CpG-dense sequence is a conserved genomic signal that promotes an open chromatin state, targeting by a CXXC1 ortholog, and H3K4me3 modification in both C. elegans and human genomes.

The landscape of RNA polymerase II transcription initiation in C. elegans reveals promoter and enhancer architectures.

RNA polymerase transcription initiation sites are largely unknown in Caenorhabditis elegans. The initial 5' end of most protein-coding transcripts is removed by trans-splicing, and noncoding initiation sites have not been investigated. We characterized the landscape of RNA Pol II transcription initiation, identifying 73,500 distinct clusters of initiation. Bidirectional transcription is frequent, with a peak of transcriptional pairing at 120 bp. We assign transcription initiation sites to 7691 protein-coding genes and find that they display features typical of eukaryotic promoters. Strikingly, the majority of initiation events occur in regions with enhancer-like chromatin signatures. Based on the overlap of transcription initiation clusters with mapped transcription factor binding sites, we define 2361 transcribed intergenic enhancers. Remarkably, productive transcription elongation across these enhancers is predominantly in the same orientation as that of the nearest downstream gene. Directed elongation from an upstream enhancer toward a downstream gene could potentially deliver RNA polymerase II to a proximal promoter, or alternatively might function directly as a distal promoter. Our results provide a new resource to investigate transcription regulation in metazoans.

H4K20me1 contributes to downregulation of X-linked genes for C. elegans dosage compensation.

The Caenorhabditis elegans dosage compensation complex (DCC) equalizes X-chromosome gene dosage between XO males and XX hermaphrodites by two-fold repression of X-linked gene expression in hermaphrodites. The DCC localizes to the X chromosomes in hermaphrodites but not in males, and some subunits form a complex homologous to condensin. The mechanism by which the DCC downregulates gene expression remains unclear. Here we show that the DCC controls the methylation state of lysine 20 of histone H4, leading to higher H4K20me1 and lower H4K20me3 levels on the X chromosomes of XX hermaphrodites relative to autosomes. We identify the PR-SET7 ortholog SET-1 and the Suv4-20 ortholog SET-4 as the major histone methyltransferases for monomethylation and di/trimethylation of H4K20, respectively, and provide evidence that X-chromosome enrichment of H4K20me1 involves inhibition of SET-4 activity on the X. RNAi knockdown of set-1 results in synthetic lethality with dosage compensation mutants and upregulation of X-linked gene expression, supporting a model whereby H4K20me1 functions with the condensin-like C. elegans DCC to repress transcription of X-linked genes. H4K20me1 is important for mitotic chromosome condensation in mammals, suggesting that increased H4K20me1 on the X may restrict access of the transcription machinery to X-linked genes via chromatin compaction.

MMpred: functional miRNA--mRNA interaction analyses by miRNA expression prediction.

BACKGROUND: MicroRNA (miRNA) directed gene repression is an important mechanism of posttranscriptional regulation. Comprehensive analyses of how microRNA influence biological processes requires paired miRNA-mRNA expression datasets. However, a review of both GEO and ArrayExpress repositories revealed few such datasets, which was in stark contrast to the large number of messenger RNA (mRNA) only datasets. It is of interest that numerous primary miRNAs (precursors of microRNA) are known to be co-expressed with coding genes (host genes). RESULTS: We developed a miRNA-mRNA interaction analyses pipeline. The proposed solution is based on two miRNA expression prediction methods - a scaling function and a linear model. Additionally, miRNA-mRNA anti-correlation analyses are used to determine the most probable miRNA gene targets (i.e. the differentially expressed genes under the influence of up- or down-regulated microRNA). Both the consistency and accuracy of the prediction method is ensured by the application of stringent statistical methods. Finally, the predicted targets are subjected to functional enrichment analyses including GO, KEGG and DO, to better understand the predicted interactions. CONCLUSIONS: The MMpred pipeline requires only mRNA expression data as input and is independent of third party miRNA target prediction methods. The method passed extensive numerical validation based on the binding energy between the mature miRNA and 3' UTR region of the target gene. We report that MMpred is capable of generating results similar to that obtained using paired datasets. For the reported test cases we generated consistent output and predicted biological relationships that will help formulate further testable hypotheses.

Interactions and Feedbacks in E-Cadherin Transcriptional Regulation.

Epithelial tissues rely on the adhesion between participating cells to retain their integrity. The transmembrane protein E-cadherin is the major protein that mediates homophilic adhesion between neighbouring cells and is, therefore, one of the critical components for epithelial integrity. E-cadherin downregulation has been described extensively as a prerequisite for epithelial-to-mesenchymal transition and is a hallmark in many types of cancer. Due to this clinical importance, research has been mostly focused on understanding the mechanisms leading to transcriptional repression of this adhesion molecule. However, in recent years it has become apparent that re-expression of E-cadherin is a major step in the progression of many cancers during metastasis. Here, we review the currently known molecular mechanisms of E-cadherin transcriptional activation and inhibition and highlight complex interactions between individual mechanisms. We then propose an additional mechanism, whereby the competition between adhesion complexes and heterochromatin protein-1 for binding to STAT92E fine-tunes the levels of E-cadherin expression in Drosophila but also regulates other genes promoting epithelial robustness. We base our hypothesis on both existing literature and our experimental evidence and suggest that such feedback between the cell surface and the nucleus presents a powerful paradigm for epithelial resilience.

Checkpoint Genes at the Cancer Side of the Immunological Synapse in Bladder Cancer.

Immune checkpoint inhibitors have revolutionized cancer therapy, but not all cancers respond to the currently available drugs, and even within cancers considered responsive to such modality, response rates range between 15 and 40%, depending on the cancer type, the line of treatment, and yet unknown clinical/molecular factors. Coordinated expression of checkpoint proteins was shown to occur on T cells, probably allowing fine-tuning of the signal transmitted to the cell. We performed a bioinformatic analysis of the expression of putative checkpoint mRNAs at the cancer side of the immunological synapse from the bladder cancer tumorgenome atlas (TCGA) database. Fifteen mRNAs, corresponding to both coinhibitory and costimulatory checkpoints, were shown to be expressed above a designated threshold. Of these, seven mRNAs were found to be coexpressed: CD277, PD-1L, CD48, CD86, galectin-9, TNFRSF14 (HVEM), and CD40. The expression of 2 of these mRNAs-BTN3A1 (CD277) and TNFRSF14 (HVEM)-was positively correlated with overall survival in the TCGA database. All these seven mRNA share putative binding sites of a few transcription factors (TFs). Of these, the expression of the TF BACH-2 was positively correlated with the expression of checkpoint mRNAs from the network. This suggests a joint transcriptional regulation on the expression of checkpoint mRNAs at the bladder tumor side of the immunological synapse.

Chromatin accessibility dynamics across C. elegans development and ageing.

An essential step for understanding the transcriptional circuits that control development and physiology is the global identification and characterization of regulatory elements. Here, we present the first map of regulatory elements across the development and ageing of an animal, identifying 42,245 elements accessible in at least one Caenorhabditis elegans stage. Based on nuclear transcription profiles, we define 15,714 protein-coding promoters and 19,231 putative enhancers, and find that both types of element can drive orientation-independent transcription. Additionally, more than 1000 promoters produce transcripts antisense to protein coding genes, suggesting involvement in a widespread regulatory mechanism. We find that the accessibility of most elements changes during development and/or ageing and that patterns of accessibility change are linked to specific developmental or physiological processes. The map and characterization of regulatory elements across C. elegans life provides a platform for understanding how transcription controls development and ageing.

An Alternative STAT Signaling Pathway Acts in Viral Immunity in Caenorhabditis elegans.

Across metazoans, innate immunity is vital in defending organisms against viral infection. In mammals, antiviral innate immunity is orchestrated by interferon signaling, activating the STAT transcription factors downstream of the JAK kinases to induce expression of antiviral effector genes. In the nematode Caenorhabditis elegans, which lacks the interferon system, the major antiviral response so far described is RNA interference (RNAi), but whether additional gene expression responses are employed is not known. Here we show that, despite the absence of both interferon and JAK, the C. elegans STAT homolog STA-1 orchestrates antiviral immunity. Intriguingly, mutants lacking STA-1 are less permissive to antiviral infection. Using gene expression analysis and chromatin immunoprecipitation, we show that, in contrast to the mammalian pathway, STA-1 acts mostly as a transcriptional repressor. Thus, STA-1 might act to suppress a constitutive antiviral response in the absence of infection. Additionally, using a reverse genetic screen, we identify the kinase SID-3 as a new component of the response to infection, which, along with STA-1, participates in the transcriptional regulatory network of the immune response. Our work uncovers novel physiological roles for two factors in viral infection: a SID protein acting independently of RNAi and a STAT protein acting in C. elegans antiviral immunity. Together, these results illustrate the complex evolutionary trajectory displayed by innate immune signaling pathways across metazoan organisms.IMPORTANCE Since innate immunity was discovered, a diversity of pathways has arisen as powerful first-line defense mechanisms to fight viral infection. RNA interference, reported mostly in invertebrates and plants, as well as the mammalian interferon response and JAK/STAT pathway are key in RNA virus innate immunity. We studied infection by the Orsay virus in Caenorhabditis elegans, where RNAi is known to be a potent antiviral defense. We show that, in addition to its RNAi pathway, C. elegans utilizes an alternative STAT pathway to control the levels of viral infection. We identify the transcription factor STA-1 and the kinase SID-3 as two components of this response. Our study defines C. elegans as a new example of the diversity of antiviral strategies.

A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress.

Repetitive sequences derived from transposons make up a large fraction of eukaryotic genomes and must be silenced to protect genome integrity. Repetitive elements are often found in heterochromatin; however, the roles and interactions of heterochromatin proteins in repeat regulation are poorly understood. Here we show that a diverse set of C. elegans heterochromatin proteins act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. Mutants in genes encoding HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 also show functionally redundant sterility, increased germline apoptosis, DNA repair defects, and interactions with small RNA pathways. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting cep-1/p53, endogenous meiotic double strand breaks, or the expression of MIRAGE1 DNA transposons. Functional redundancy among factors and pathways underlies the importance of safeguarding the genome through multiple means.

SeqPlots - Interactive software for exploratory data analyses, pattern discovery and visualization in genomics.

Experiments involving high-throughput sequencing are widely used for analyses of chromatin function and gene expression. Common examples are the use of chromatin immunoprecipitation for the analysis of chromatin modifications or factor binding, enzymatic digestions for chromatin structure assays, and RNA sequencing to assess gene expression changes after biological perturbations. To investigate the pattern and abundance of coverage signals across regions of interest, data are often visualized as profile plots of average signal or stacked rows of signal in the form of heatmaps. We found that available plotting software was either slow and laborious or difficult to use by investigators with little computational training, which inhibited wide data exploration. To address this need, we developed SeqPlots, a user-friendly exploratory data analysis (EDA) and visualization software for genomics. After choosing groups of signal and feature files and defining plotting parameters, users can generate profile plots of average signal or heatmaps clustered using different algorithms in a matter of seconds through the graphical user interface (GUI) controls. SeqPlots accepts all major genomic file formats as input and can also generate and plot user defined motif densities. Profile plots and heatmaps are highly configurable and batch operations can be used to generate a large number of plots at once. SeqPlots is available as a GUI application for Mac or Windows and Linux, or as an R/Bioconductor package. It can also be deployed on a server for remote and collaborative usage. The analysis features and ease of use of SeqPlots encourages wide data exploration, which should aid the discovery of novel genomic associations.