Marcus H. Stoiber

Diversity and dynamics of the Drosophila transcriptome

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.

Comparative analysis of the transcriptome across distant species.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a ‘universal model’ based on a single set of organism-independent parameters.

Genome-guided transcript assembly by integrative analysis of RNA sequence data

The identification of full length transcripts entirely from short-read RNA sequencing data (RNA-seq) remains a challenge in the annotation of genomes. Here we describe an automated pipeline for genome annotation that integrates RNA-seq and gene-boundary data sets, which we call Generalized RNA Integration Tool, or GRIT. Applying GRIT to Drosophila melanogaster short-read RNA-seq, cap analysis of gene expression (CAGE) and poly(A)-site-seq data collected for the modENCODE project, we recovered the vast majority of previously annotated transcripts and doubled the total number of transcripts cataloged. We found that 20% of protein coding genes encode multiple protein-localization signals and that, in 20-d-old adult fly heads, genes with multiple polyadenylation sites are more common than genes with alternative splicing or alternative promoters. GRIT demonstrates 30% higher precision and recall than the most widely used transcript assembly tools. GRIT will facilitate the automated generation of high-quality genome annotations without the need for extensive manual annotation.

De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing

Advances in single molecule sequencing technology have enabled the investigation of the full catalogue of covalent DNA modifications. We present an assay, Modified DNA sequencing (MoD-seq), that leverages raw nanopore data processing, visualization and statistical testing to directly survey DNA modifications without the need for a large prior training dataset. We present case studies applying MoD-seq to identify three distinct marks, 4mC, 5mC, and 6mA, and demonstrate quantitative reproducibility across biological replicates processed in different labs. In a ground-truth dataset created via in vitro treatment of synthetic DNA with selected methylases, we show that modifications can be detected in a variety of distinct sequence contexts. We recapitulated known methylation patterns and frequencies in E. coli, and propose a pipeline for the comprehensive discovery of DNA modifications in a genome without a priori knowledge of their chemical identities.

Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.

BasecRAWller: Streaming Nanopore Basecalling Directly from Raw Signal

All current nanopore basecalling applications begin with the segmentation of raw signal into discrete events, which are ultimately processed into called bases. We propose the basecRAWller algorithm, a pair of unidirectional recurrent neural networks that enables the calling of DNA bases in real time directly from the rawest form of nanopore output. This shift in nanopore basecalling provides a number of advantages over current processing pipelines including: 1) streaming basecalling, 2) tunable ratio of insertions to deletions, and 3) potential for streaming detection of modified bases. Key to the streaming basecalling capability is sequence prediction at a delay of less than 1/100th of a second, allowing future signal to continuously modulate sequence prediction. BasecRAWller is computationally efficient enabling basecalling at speeds faster than current nanopore instrument measurement speeds on a single core. Further, basecalling can be paused and resumed without any change in the resulting predicted sequence, transforming the potential applications for dynamic read rejection capabilities. The basecRAWller algorithm provides an alternative approach to nanopore basecalling at comparable accuracy and provides the community with the capacity to train their own basecRAWller neural networks with minimal effort.

Early transcriptional response pathways in Daphnia magna are coordinated in networks of crustacean specific genes

Natural habitats are exposed to an increasing number of environmental stressors that cause important ecological consequences. However, the multifarious nature of environmental change, the strength and the relative timing of each stressor largely limit our understanding of biological responses to environmental change. In particular, early response to unpredictable environmental change, critical to survival and fitness in later life stages, is largely uncharacterized. Here, we characterize the early transcriptional response of the keystone species Daphnia magna to twelve environmental perturbations, including biotic and abiotic stressors. We first perform a differential expression analysis aimed at identifying differential regulation of individual genes in response to stress. This preliminary analysis revealed that a few individual genes were responsive to environmental perturbations and they were modulated in a stressor and genotype‐specific manner. Given the limited number of differentially regulated genes, we were unable to identify pathways involved in stress response. Hence, to gain a better understanding of the genetic and functional foundation of tolerance to multiple environmental stressors, we leveraged the correlative nature of networks and performed a weighted gene co‐expression network analysis. We discovered that approximately one‐third of the Daphnia genes, enriched for metabolism, cell signalling and general stress response, drives transcriptional early response to environmental stress and it is shared among genetic backgrounds. This initial response is followed by a genotype‐ and/or condition‐specific transcriptional response with a strong genotype‐by‐environment interaction. Intriguingly, genotype‐ and condition‐specific transcriptional response is found in genes not conserved beyond crustaceans, suggesting niche‐specific adaptation.

Age-related gene expression in luminal epithelial cells is driven by a microenvironment made from myoepithelial cells

Luminal epithelial cells in the breast gradually alter gene and protein expression with age, appearing to lose lineage-specificity by acquiring myoepithelial-like characteristics. We hypothesize that the luminal lineage is particularly sensitive to microenvironment changes, and age-related microenvironment changes cause altered luminal cell phenotypes. To evaluate the effects of different microenvironments on the fidelity of epigenetically regulated luminal and myoepithelial gene expression, we generated a set of lineage-specific probes for genes that are controlled through DNA methylation. Culturing primary luminal cells under conditions that favor myoepithelial propogation led to their reprogramming at the level of gene methylation, and to a more myoepithelial-like expression profile. Primary luminal cells’ lineage-specific gene expression could be maintained when they were cultured as bilayers with primary myoepithelial cells. Isogenic stromal fibroblast co-cultures were unable to maintain the luminal phenotype. Mixed-age luminal-myoepithelial bilayers revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. We provide evidence that the luminal epithelial phenotype is exquisitely sensitive to microenvironment conditions, and that states of aging are cell non-autonomously communicated through microenvironment cues over at least one cell diameter.

Abstract A24: Age- and lineage-dependent gene expression is maintained by microenvironment imposed epigenetic states in human mammary epithelial cells

Normal healthy tissues show changing patterns of gene expression as a consequence of aging, and there is a functional cost to these changes that can be relevant to disease pathology. The most obvious age-related disease in breast is cancer, with the large majority of newly diagnosed breast cancer occuring in women over 50. We have shown that breast gene expression changes that occur with age have functional consequences in the epithelial progenitor and differentiated cells, i.e, a decline of the myoepithelial lineage, loss of luminal cell specificity, and accumulation of differentiation defective multipotent progenitor cells. We have hypothesized that these tissue-level changes make older epithelia more susceptible to transformation. Because gene expression patterns reflect the wiring and response diagrams of cells, it is of central importance to understand the origins of age-related transcriptomes. In our studies, early passage normal human mammary epithelial cells (HMEC) show age-dependent functional and molecular hallmarks consistent with in vivo, suggesting that the underlying gene expression patterns are metastable. DNA methylation is a stable, but malleable, form of epigenetic regulation that may underpin these biologically metastable states. Genome-wide analysis of primary epithelia was used to identify a set of genes that exhibit age- and lineage-specific expression that was inversely correlated with promoter CpG methylation. Chemical perturbation of DNA methylation in pre-menopausal HMEC resulted in a biochemical phenocopy of more advanced age. Tissue-mimetic cultures were used to demonstrate that lineage-specific gene expression and methylation in luminal cells were imposed by distinct microenvironments. Optimal maintenance of the luminal phenotype required direct contact with the apical surface of myoepithelial cells. Mimetic tissues built with HMEC that differed in chronological donor age revealed that age-dependent gene expression and methylation patterns are communicated between the two different lineages, as exposure of pre-menopausal luminal cells to a post-menopausal microenvironment imposed transcriptional patterns in luminal cells consistent with post-menopause. These data demonstrate that lineage- and age-dependent phenotypes in HMEC are maintained by microenvironment-imposed metastable epigenetic states. Note: This abstract was not presented at the conference. Citation Format: Masaru Miyano, Marcus Stoiber, Martha Stampfer, Ben Brown, Mark A. LaBarge. Age- and lineage-dependent gene expression is maintained by microenvironment imposed epigenetic states in human mammary epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A24.