Relationships between chromatin features and genome regulation

Regulation of gene expression is an essential process for all living organisms. Transcriptional regulation, associated with chromatin, is governed by: (1) DNA sequence, which creates regulatory sites (promoters, enhancers and silencers), where sequence motifs and features (e. g. CpG) can attract transcription factors (TFs) and influence chromatin structure or RNA polymerase II (Pol II) binding, initiation and elongation; (2) non-sequence, epigenetic factors - histone modifications, TF binding, chromatin remodelling (histone placement, eviction and reconstitution), and non-coding RNA regulation. These factors interact with each other, creating a complex network of interactions. In this thesis I describe computational studies of heterochromatin factors in regulation of gene and repeat expression, an analysis of active regulatory elements, and global analyses of big datasets in C. elegans. I first show that a team of heterochromatin factors - HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 - collaborates with piRNA and nuclear RNAi pathways to silence repetitive elements and protect the germline. I also found that the TACBGTA motif is particularly enriched on repeats and heterochromatin factors binding sites, and that repeat elements are derepressed in the soma during normal C. elegans ageing. I then describe the work on active regulatory regions. I show that CFP-1/CXXC1 binds CpG dense, nucleosome depleted promoters and, along SET-2, is required for H3K4me3 deposition at these loci. Using expression profiling I determined that the majority of CFP-1 binding targets are not significantly mis-regulated in cfp-1 mutants, but are weakly upregulated in bulk analyses. I also show that CFP-1 functionally interacts with the Sin3S/HDAC complex. In cfp-1 mutant I observed both loss and gain of SIN-3 binding, depending on chromatin context. Finally, I performed a data driven study on a large collection of ChIP-seq profiles using non-parametric sparse factor analyses (NSFA) and compared it to other, unsupervised machine learning algorithms. This study uncovered interactions and structure in genomic datasets. In addition, I present a collection of computational tools and methods I developed to facilitate processing, storage, retrieval, annotation, and analyses of large datasets in genomics

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.This work was supported by the Wellcome Trust [101863]

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

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.We are grateful to D. Fay for providing the 5× outcrossed lin-35 strain, and Robert Horvitz for antibodies. I.L., M.A.C., P.S., A.A., and J.A. were supported by Wellcome Trust Senior Research Fellowships to J.A. (054523 and 101863). J.A. also acknowledges support by core funding from the Wellcome Trust and Cancer Research UK. J.M.G. and S.S. were supported by National Institutes of Health (NIH) R01 grant GM34059. Part of this work was supported by NIH National Human Genome Research Institute (NHGRI) grant U01 HG004270 to the modENCODE consortium headed by J.D. Lieb.This is the final version of the article. It first appeared from CSH Press via http://dx.doi.org/10.1101/gad.255810.11

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

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

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

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.The work was supported by Wellcome Trust Senior Research Fellowships to JA (054523 and 101863), a Wellcome Trust PhD fellowship to JJ (097679), a Sir Robert Edwards Scholarship from Churchill College, an English Speaking Union Graduate Scholarship, and funding from the Cambridge Trust to MS, a Medical Research Council DTP studentship to JS, and a Thouron award to CW. This study was also supported by the European Sequencing and Genotyping Infrastructure (funded by the EC, FP7/2007-2013) under Grant Agreement 26205 (ESGI) as part of the transnational access program. We thank Drs. Hans Lehrach and Marie-Laure Yaspo for generous support of the ESGI project, Dr. Marc Sultan for setting up sequencing technology platforms, and Mathias Linser and the rest of the sequencing team of the Department of Vertebrate Genomics at the Max Planck Institute for Molecular Genetics for technical assistance. We also acknowledge core support from the Wellcome Trust (092096) and Cancer Research UK (C6946/A14492)

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