Defining the contributions and interactions of paternal and maternal genomes during embryo development is critical to understand the fundamental processes involved in hybrid vigor, hybrid sterility, ...and reproductive isolation. To determine the parental contributions and their regulation during
Arabidopsis embryogenesis, we combined deep-sequencing-based RNA profiling and genetic analyses. At the 2–4 cell stage there is a strong, genome-wide dominance of maternal transcripts, although transcripts are contributed by both parental genomes. At the globular stage the relative paternal contribution is higher, largely due to a gradual activation of the paternal genome. We identified two antagonistic maternal pathways that control these parental contributions. Paternal alleles are initially downregulated by the chromatin siRNA pathway, linked to DNA and histone methylation, whereas transcriptional activation requires maternal activity of the histone chaperone complex CAF1. Our results define maternal epigenetic pathways controlling the parental contributions in plant embryos, which are distinct from those regulating genomic imprinting.
Display omitted
► The early
Arabidopsis embryonic transcriptome has a dominant maternal contribution ► The paternal contribution increases gradually during early embryogenesis ► Parental contributions are maternally controlled by the chromatin siRNA pathway ► Paternal activation requires maternal activity of histone chaperone complex CAF1
The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A ...spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.
Recent advances in the cost-efficiency of sequencing technologies enabled the combined DNA- and RNA-sequencing of human individuals at the population-scale, making genome-wide investigations of the ...inter-individual genetic impact on gene expression viable. Employing mRNA-sequencing data from the Geuvadis Project and genome sequencing data from the 1000 Genomes Project we show that the computational analysis of DNA sequences around splice sites and poly-A signals is able to explain several observations in the phenotype data. In contrast to widespread assessments of statistically significant associations between DNA polymorphisms and quantitative traits, we developed a computational tool to pinpoint the molecular mechanisms by which genetic markers drive variation in RNA-processing, cataloguing and classifying alleles that change the affinity of core RNA elements to their recognizing factors. The in silico models we employ further suggest RNA editing can moonlight as a splicing-modulator, albeit less frequently than genomic sequence diversity. Beyond existing annotations, we demonstrate that the ultra-high resolution of RNA-Seq combined from 462 individuals also provides evidence for thousands of bona fide novel elements of RNA processing-alternative splice sites, introns, and cleavage sites-which are often rare and lowly expressed but in other characteristics similar to their annotated counterparts.
In many cancer types, MYC proteins are known to be master regulators of the RNA‐producing machinery. Neuroblastoma is a tumor of early childhood characterized by heterogeneous clinical courses. ...Amplification of the MYCN oncogene is a marker of poor patient outcome in this disease. Here, we investigated the MYCN‐driven transcriptome of 20 primary neuroblastomas with and without MYCN amplification using next‐generation RNA sequencing and compared the results to those from an in vitro cell model for inducible MYCN (SH‐EP MYCN‐ER). Transcriptome sequencing produced 30–90 million mappable reads for each dataset. The most abundant RNA species was mRNA, but snoRNAs, pseudogenes and processed transcripts were also recovered. A total of 223 genes were significantly differentially expressed between MYCN‐amplified and single‐copy tumors. Of those genes associated with MYCN both in vitro and in vivo, 32% of MYCN upregulated and 37% of MYCN downregulated genes were verified either as previously identified MYCN targets or as having MYCN‐binding motifs. Pathway analyses suggested transcriptomal upregulation of mTOR‐related genes by MYCN. MYCN‐driven neuroblastomas in mice displayed activation of the mTOR pathway on the protein level and activation of MYCN in SH‐EP MYCN‐ER cells resulted in high sensitivity toward mTOR inhibition in vitro. We conclude that next‐generation RNA sequencing allows for the identification of MYCN regulated transcripts in neuroblastoma. As our results suggest MYCN involvement in mTOR pathway activation on the transcriptional level, mTOR inhibitors should be further evaluated for the treatment of MYCN‐amplified neuroblastoma.
What's new?
In neuroblastoma, amplification of the MYCN oncogene is associated with a poor prognosis. Now that next‐generation sequencing technologies are becoming more affordable, it may soon be possible to identify the mutations underlying individual tumors, and to use that information to develop individualised treatment strategies. In this study, the authors used “RNA deep sequencing” to examine the MYCN‐driven transcriptomes of a number of neuroblastomas. Their results suggest that mTOR inhibitors should be further evaluated for the treatment of MYCN‐amplified neuroblastoma.
Bidirectional promoters (BPs) are prevalent in eukaryotic genomes. However, it is poorly understood how the cell integrates different epigenomic information, such as transcription factor (TF) binding ...and chromatin marks, to drive gene expression at BPs. Single-cell sequencing technologies are revolutionizing the field of genome biology. Therefore, this study focuses on the integration of single-cell RNA-seq data with bulk ChIP-seq and other epigenetics data, for which single-cell technologies are not yet established, in the context of BPs.
We performed integrative analyses of novel human single-cell RNA-seq (scRNA-seq) data with bulk ChIP-seq and other epigenetics data. scRNA-seq data revealed distinct transcription states of BPs that were previously not recognized. We find associations between these transcription states to distinct patterns in structural gene features, DNA accessibility, histone modification, DNA methylation and TF binding profiles.
Our results suggest that a complex interplay of all of these elements is required to achieve BP-specific transcriptional output in this specialized promoter configuration. Further, our study implies that novel statistical methods can be developed to deconvolute masked subpopulations of cells measured with different bulk epigenomic assays using scRNA-seq data.
The intestinal mucosa is characterized by complex metabolic and immunological processes driven highly dynamic gene expression programs. With the advent of next generation sequencing and its ...utilization for the analysis of the RNA sequence space, the level of detail on the global architecture of the transcriptome reached a new order of magnitude compared to microarrays.
We report the ultra-deep characterization of the polyadenylated transcriptome in two closely related, yet distinct regions of the mouse intestinal tract (small intestine and colon). We assessed tissue-specific transcriptomal architecture and the presence of novel transcriptionally active regions (nTARs). In the first step, signatures of 20,541 NCBI RefSeq transcripts could be identified in the intestine (74.1% of annotated genes), thereof 16,742 are common in both tissues. Although the majority of reads could be linked to annotated genes, 27,543 nTARs not consistent with current gene annotations in RefSeq or ENSEMBL were identified. By use of a second independent strand-specific RNA-Seq protocol, 20,966 of these nTARs were confirmed, most of them in vicinity of known genes. We further categorized our findings by their relative adjacency to described exonic elements and investigated regional differences of novel transcribed elements in small intestine and colon.
The current study demonstrates the complexity of an archetypal mammalian intestinal mRNA transcriptome in high resolution and identifies novel transcriptionally active regions at strand-specific, single base resolution. Our analysis for the first time shows a strand-specific comparative picture of nTARs in two tissues and represents a resource for further investigating the transcriptional processes that contribute to tissue identity.
Alternative splicing is an important cellular mechanism that can be analyzed by RNA sequencing. However, identification of splicing events in an automated fashion is error-prone. Thus, further ...validation is required to select reliable instances of alternative splicing events (ASEs). There are only few tools specifically designed for interactive inspection of ASEs and available visualization approaches can be significantly improved.
Here, we present Manananggal, an application specifically designed for the identification of splicing events in next generation sequencing data. Manananggal includes a web application for visual inspection and a command line tool that allows for ASE detection. We compare the sashimi plots available in the IGV Viewer, the DEXSeq splicing plots and SpliceSeq to the Manananggal interface and discuss the advantages and drawbacks of these tools. We show that sashimi plots (such as those used by the IGV Viewer and SpliceSeq) offer a practical solution for simple ASEs, but also indicate short-comings for highly complex genes.
Manananggal is an interactive web application that offers functions specifically tailored to the identification of alternative splicing events that other tools are lacking. The ability to select a subset of isoforms allows an easier interpretation of complex alternative splicing events. In contrast to SpliceSeq and the DEXSeq splicing plot, Manananggal does not obscure the gene structure by showing full transcript models that makes it easier to determine which isoforms are expressed and which are not.
Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in ...human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project--the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.
An inadequate host response to the intestinal microbiota likely contributes to the manifestation and progression of human inflammatory bowel disease (IBD). However, molecular approaches to ...unravelling the nature of the defective crosstalk and its consequences for intestinal metabolic and immunological networks are lacking. We assessed the mucosal transcript levels, splicing architecture and mucosa-attached microbial communities of patients with IBD to obtain a comprehensive view of the underlying, hitherto poorly characterised interactions, and how these are altered in IBD.
Mucosal biopsies from Crohn's disease and patients with UC, disease controls and healthy individuals (n=63) were subjected to microbiome, transcriptome and splicing analysis, employing next-generation sequencing. The three data levels were integrated by different bioinformatic approaches, including systems biology-inspired network and pathway analysis.
Microbiota, host transcript levels and host splicing patterns were influenced most strongly by tissue differences, followed by the effect of inflammation. Both factors point towards a substantial disease-related alteration of metabolic processes. We also observed a strong enrichment of splicing events in inflamed tissues, accompanied by an alteration of the mucosa-attached bacterial taxa. Finally, we noted a striking uncoupling of the three molecular entities when moving from healthy individuals via disease controls to patients with IBD.
Our results provide strong evidence that the interplay between microbiome and host transcriptome, which normally characterises a state of intestinal homeostasis, is drastically perturbed in Crohn's disease and UC. Consequently, integrating multiple OMICs levels appears to be a promising approach to further disentangle the complexity of IBD.
PDF
