57 research outputs found
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 anticorrelation
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
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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
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
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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
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
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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
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