The histone chaperone SPT2 regulates chromatin structure and function in Metazoa.

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

Widespread transposon co-option in the Caenorhabditis germline regulatory network.

The movement of selfish DNA elements can lead to widespread genomic alterations with potential to create novel functions. We show that transposon expansions in Caenorhabditis nematodes led to extensive rewiring of germline transcriptional regulation. We find that about one-third of Caenorhabditis elegans germline-specific promoters have been co-opted from two related miniature inverted repeat transposable elements (TEs), CERP2 and CELE2. These promoters are regulated by HIM-17, a THAP domain-containing transcription factor related to a transposase. Expansion of CERP2 occurred before radiation of the Caenorhabditis genus, as did fixation of mutations in HIM-17 through positive selection, whereas CELE2 expanded only in C. elegans. Through comparative analyses in Caenorhabditis briggsae, we find not only evolutionary conservation of most CERP2 co-opted promoters but also a substantial fraction that are species-specific. Our work reveals the emergence and evolutionary conservation of a novel transcriptional network driven by TE co-option with a major impact on regulatory evolution.

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.

DREAM represses distinct targets by cooperating with different THAP domain proteins.

The DREAM (dimerization partner [DP], retinoblastoma [Rb]-like, E2F, and MuvB) complex controls cellular quiescence by repressing cell-cycle and other genes, but its mechanism of action is unclear. Here, we demonstrate that two C. elegans THAP domain proteins, LIN-15B and LIN-36, co-localize with DREAM and function by different mechanisms for repression of distinct sets of targets. LIN-36 represses classical cell-cycle targets by promoting DREAM binding and gene body enrichment of H2A.Z, and we find that DREAM subunit EFL-1/E2F is specific for LIN-36 targets. In contrast, LIN-15B represses germline-specific targets in the soma by facilitating H3K9me2 promoter marking. We further find that LIN-36 and LIN-15B differently regulate DREAM binding. In humans, THAP proteins have been implicated in cell-cycle regulation by poorly understood mechanisms. We propose that THAP domain proteins are key mediators of Rb/DREAM function.

periodicDNA: an R/Bioconductor package to investigate k-mer periodicity in DNA.

Periodic occurrences of oligonucleotide sequences can impact the physical properties of DNA. For example, DNA bendability is modulated by 10-bp periodic occurrences of WW (W = A/T) dinucleotides. We present periodicDNA, an R package to identify k-mer periodicity and generate continuous tracks of k-mer periodicity over genomic loci of interest, such as regulatory elements. periodicDNA will facilitate investigation and improve understanding of how periodic DNA sequence features impact function.

Distinctive regulatory architectures of germline-active and somatic genes in C. elegans.

RNA profiling has provided increasingly detailed knowledge of gene expression patterns, yet the different regulatory architectures that drive them are not well understood. To address this, we profiled and compared transcriptional and regulatory element activities across five tissues of Caenorhabditis elegans, covering ∼90% of cells. We find that the majority of promoters and enhancers have tissue-specific accessibility, and we discover regulatory grammars associated with ubiquitous, germline, and somatic tissue-specific gene expression patterns. In addition, we find that germline-active and soma-specific promoters have distinct features. Germline-active promoters have well-positioned +1 and -1 nucleosomes associated with a periodic 10-bp WW signal (W = A/T). Somatic tissue-specific promoters lack positioned nucleosomes and this signal, have wide nucleosome-depleted regions, and are more enriched for core promoter elements, which largely differ between tissues. We observe the 10-bp periodic WW signal at ubiquitous promoters in other animals, suggesting it is an ancient conserved signal. Our results show fundamental differences in regulatory architectures of germline and somatic tissue-specific genes, uncover regulatory rules for generating diverse gene expression patterns, and provide a tissue-specific resource for future studies.

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.

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.

Comparative Epigenomics Reveals that RNA Polymerase II Pausing and Chromatin Domain Organization Control Nematode piRNA Biogenesis.

Piwi-interacting RNAs (piRNAs) are important for genome regulation across metazoans, but their biogenesis evolves rapidly. In Caenorhabditis elegans, piRNA loci are clustered within two 3-Mb regions on chromosome IV. Each piRNA locus possesses an upstream motif that recruits RNA polymerase II to produce an ∼28 nt primary transcript. We used comparative epigenomics across nematodes to gain insight into the origin, evolution, and mechanism of nematode piRNA biogenesis. We show that the piRNA upstream motif is derived from core promoter elements controlling snRNA transcription. We describe two alternative modes of piRNA organization in nematodes: in C. elegans and closely related nematodes, piRNAs are clustered within repressive H3K27me3 chromatin, while in other species, typified by Pristionchus pacificus, piRNAs are found within introns of active genes. Additionally, we discover that piRNA production depends on sequence signals associated with RNA polymerase II pausing. We show that pausing signals synergize with chromatin to control piRNA transcription.

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.

Morphogenetic degeneracies in the actomyosin cortex.

One of the great challenges in biology is to understand the mechanisms by which morphogenetic processes arise from molecular activities. We investigated this problem in the context of actomyosin-based cortical flow in C. elegans zygotes, where large-scale flows emerge from the collective action of actomyosin filaments and actin binding proteins (ABPs). Large-scale flow dynamics can be captured by active gel theory by considering force balances and conservation laws in the actomyosin cortex. However, which molecular activities contribute to flow dynamics and large-scale physical properties such as viscosity and active torque is largely unknown. By performing a candidate RNAi screen of ABPs and actomyosin regulators we demonstrate that perturbing distinct molecular processes can lead to similar flow phenotypes. This is indicative for a 'morphogenetic degeneracy' where multiple molecular processes contribute to the same large-scale physical property. We speculate that morphogenetic degeneracies contribute to the robustness of bulk biological matter in development.

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.

Genome organization at different scales: nature, formation and function.

Since the discovery of chromosome territories, it has been clear that DNA within the nucleus is spatially organized. During the last decade, a tremendous body of work has described architectural features of chromatin at different spatial scales, such as A/B compartments, topologically associating domains (TADs), and chromatin loops. These features correlate with domains of chromatin marking and gene expression, supporting their relevance for gene regulation. Recent work has highlighted the dynamic nature of spatial folding and investigated mechanisms of their formation. Here we discuss current understanding and highlight key open questions in chromosome organization in animals.

Repressive Chromatin in Caenorhabditis elegans: Establishment, Composition, and Function.

Chromatin is organized and compacted in the nucleus through the association of histones and other proteins, which together control genomic activity. Two broad types of chromatin can be distinguished: euchromatin, which is generally transcriptionally active, and heterochromatin, which is repressed. Here we examine the current state of our understanding of repressed chromatin in Caenorhabditis elegans, focusing on roles of histone modifications associated with repression, such as methylation of histone H3 lysine 9 (H3K9me2/3) or the Polycomb Repressive Complex 2 (MES-2/3/6)-deposited modification H3K27me3, and on proteins that recognize these modifications. Proteins involved in chromatin repression are important for development, and have demonstrated roles in nuclear organization, repetitive element silencing, genome integrity, and the regulation of euchromatin. Additionally, chromatin factors participate in repression with small RNA pathways. Recent findings shed light on heterochromatin function and regulation in C. elegans, and should inform our understanding of repressed chromatin in other animals.

Broad Chromatin Domains: An Important Facet of Genome Regulation.

Chromatin composition differs across the genome, with distinct compositions characterizing regions associated with different properties and functions. Whereas many histone modifications show local enrichment over genes or regulatory elements, marking can also span large genomic intervals defining broad chromatin domains. Here we highlight structural and functional features of chromatin domains marked by histone modifications, with a particular emphasis on the potential roles of H3K27 methylation domains in the organization and regulation of genome activity in metazoans.

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.

aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity.

The conserved polarity effector proteins PAR-3, PAR-6, CDC-42, and atypical protein kinase C (aPKC) form a core unit of the PAR protein network, which plays a central role in polarizing a broad range of animal cell types. To functionally polarize cells, these proteins must activate aPKC within a spatially defined membrane domain on one side of the cell in response to symmetry-breaking cues. Using the Caenorhabditis elegans zygote as a model, we find that the localization and activation of aPKC involve distinct, specialized aPKC-containing assemblies: a PAR-3-dependent assembly that responds to polarity cues and promotes efficient segregation of aPKC toward the anterior but holds aPKC in an inactive state, and a CDC-42-dependent assembly in which aPKC is active but poorly segregated. Cycling of aPKC between these distinct functional assemblies, which appears to depend on aPKC activity, effectively links cue-sensing and effector roles within the PAR network to ensure robust establishment of polarity.

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.