Plants regenerated from tissue culture contain stable epigenome changes in rice.

Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. DOI:http://dx.doi.org/10.7554/eLife.00354.001

Noncoder : a web interface for exon array-based detection of long non-coding RNAs

Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called 'noncoder'. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de

Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome

Transposable elements (TEs) have no longer been totally considered as “junk DNA” for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C(chromosomeconformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r=0.9, P<2.2×1016; IMR90 fibroblasts: r = 0.94, P < 2.2 × 1016) and also have a significant positive correlation withsomeremote functional DNA elements like enhancers and promoters (Enhancer: hESC: r=0.997, P=2.3×10−4; IMR90: r=0.934, P=2×10−2; Promoter: hESC: r = 0.995, P = 3.8 × 10−4; IMR90: r = 0.996, P = 3.2 × 10−4). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes

DNA methylation profiling of the human major histocompatibility complex: A pilot study for the Human Epigenome Project

The Human Epigenome Project aims to identify, catalogue, and interpret genome-wide DNA methylation phenomena. Occurring naturally on cytosine bases at cytosine-guanine dinucleotides, DNA methylation is intimately involved in diverse biological processes and the aetiology of many diseases. Differentially methylated cytosines give rise to distinct profiles, thought to be specific for gene activity, tissue type, and disease state. The identification of such methylation variable positions will significantly improve our understanding of genome biology and our ability to diagnose disease. Here, we report the results of the pilot study for the Human Epigenome Project entailing the methylation analysis of the human major histocompatibility complex. This study involved the development of an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, discovery of methylation variable positions, epigenotyping by matrix-assisted laser desorption/ionisation mass spectrometry, and development of an integrated public database available at http://www.epigenome.org. Our analysis of DNA methylation levels within the major histocompatibility complex, including regulatory exonic and intronic regions associated with 90 genes in multiple tissues and individuals, reveals a bimodal distribution of methylation profiles (i.e., the vast majority of the analysed regions were either hypo- or hypermethylated), tissue specificity, inter-individual variation, and correlation with independent gene expression data

Sex-biased mouse liver lincRNAs: validation and impact of mouse knockout models that perturb hepatic growth signaling

Sexual dimorphism has a broad impact on human health and disease, including autoimmune diseases, cardiovascular diseases, and liver diseases, specifically hepatocellular carcinoma. Pituitary growth hormone (GH) secretion has a major impact on sex differences in the liver. Previous studies show that signal transducer and activator transcription factor 5b (STAT5b) impacts male-biased gene expression. Somatostatin (SST) inhibits the release of GH, and this in turn impacts STAT5b activity and GH regulation feedback. Long intergenic noncoding RNAs (lincRNAs), which do not encode proteins, have various roles in diverse biological processes including cell differentiation, chromatin remodeling, and gene regulation. In the present study, we discovered lincRNAs in mouse liver using RNA-seq datasets, and identified male and female-biased lincRNAs. Moreover, we validated the sex-specificity of lincRNAs by performing real time PCR (qPCR) and analyzing UCSC genome browser screen shots by comparing male and female expression patterns. We validated the sex-specificities of 22 lincRNAs. Moreover, we investigated whether pituitary GH secretion impacts the regulation of these sex-biased lincRNAs in the same manner as seen in sex-biased protein coding RefSeq genes. qPCR experiments investigated the impact of GH secretion by using SST and STAT5b knockout mouse models, and hypophysectomized, and intermittent (pulsatile) GH-treated mice. Our results show that pituitary GH secretion impacts the regulation of sex-biased lincRNAs. The last part of this thesis investigates the molecular and functional role of sexbiased lincRNAs by using The Database for Annotation, Visualization, and Integrated Discovery (DAVID)

Grammar-Based Interactive Genome Visualization

Visualization is an indispensable method in the exploration of genomic data. However, the current state of the art in genome browsers – a class of interactive visualization tools – limit the exploration by coupling the visual representations with specific file formats. Because the tools do not support the exploration of the visualization design space, they are difficult to adapt to atypical data. Moreover, although the tools provide interactivity, the implementations are often rudimentary, encumbering the exploration of the data. This thesis introduces GenomeSpy, an interactive genome visualization tool that improves upon the current state of the art by providing better support for exploration. The tool uses a visualization grammar that allows for implementing novel visualization designs, which can display the underlying data more effectively. Moreover, the tool implements GPU-accelerated interactions that better support navigation in the genomic space. For instance, smoothly animated transitions between loci or sample sets improve the perception of causality and help the users stay in the flow of exploration. The expressivity of the visualization grammar and the benefit of fluid interactions are validated with two case studies. The case studies demonstrate visualization of high-grade serous ovarian cancer data at different analysis phases. First, GenomeSpy is being used to create a tool for scrutinizing raw copy-number variation data along with segmentation results. Second, the segmentations along with point mutations are used in a GenomeSpy-based multi-sample visualization that allows for exploring and comparing both multiple data dimensions and samples at the same time. Although the focus has been on cancer research, the tool could be applied to other domains as well

ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing

ChIA-PET Tool can be used to process long-range chromatin interaction data. Results are visualized on a user-friendly genome browser

A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells

Wepreviously reported the association of elevated levels of themultifunctional transcription factor, CCCTC binding factor (CTCF), in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecularmechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs) within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell deathwere observed only in breast cancer cells depleted of CTCF.We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1,WT1, EGR1, and c-Myc) was generally increased in non- breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis. © 2013 Neoplasia Press, Inc