William W. Fisher

Dynamic reprogramming of chromatin accessibility during Drosophila embryo development

BackgroundThe development of complex organisms is believed to involve progressive restrictions in cellular fate. Understanding the scope and features of chromatin dynamics during embryogenesis, and identifying regulatory elements important for directing developmental processes remain key goals of developmental biology.ResultsWe used in vivo DNaseI sensitivity to map the locations of regulatory elements, and explore the changing chromatin landscape during the first 11 hours of Drosophila embryonic development. We identified thousands of conserved, developmentally dynamic, distal DNaseI hypersensitive sites associated with spatial and temporal expression patterning of linked genes and with large regions of chromatin plasticity. We observed a nearly uniform balance between developmentally up- and down-regulated DNaseI hypersensitive sites. Analysis of promoter chromatin architecture revealed a novel role for classical core promoter sequence elements in directing temporally regulated chromatin remodeling. Another unexpected feature of the chromatin landscape was the presence of localized accessibility over many protein-coding regions, subsets of which were developmentally regulated or associated with the transcription of genes with prominent maternal RNA contributions in the blastoderm.ConclusionsOur results provide a global view of the rich and dynamic chromatin landscape of early animal development, as well as novel insights into the organization of developmentally regulated chromatin features.

DNA regions bound at low occupancy by transcription factors do not drive patterned reporter gene expression in Drosophila

In animals, each sequence-specific transcription factor typically binds to thousands of genomic regions in vivo. Our previous studies of 20 transcription factors show that most genomic regions bound at high levels in Drosophila blastoderm embryos are known or probable functional targets, but genomic regions occupied only at low levels have characteristics suggesting that most are not involved in the cis-regulation of transcription. Here we use transgenic reporter gene assays to directly test the transcriptional activity of 104 genomic regions bound at different levels by the 20 transcription factors. Fifteen genomic regions were selected based solely on the DNA occupancy level of the transcription factor Kruppel. Five of the six most highly bound regions drive blastoderm patterns of reporter transcription. In contrast, only one of the nine lowly bound regions drives transcription at this stage and four of them are not detectably active at any stage of embryogenesis. A larger set of 89 genomic regions chosen using criteria designed to identify functional cis-regulatory regions supports the same trend: genomic regions occupied at high levels by transcription factors in vivo drive patterned gene expression, whereas those occupied only at lower levels mostly do not. These results support studies that indicate that the high cellular concentrations of sequence-specific transcription factors drive extensive, low-occupancy, nonfunctional interactions within the accessible portions of the genome.

Regulation of Early Endosomal Entry by the Drosophila Tumor Suppressors Rabenosyn and Vps45

The small GTPase Rab5 has emerged as an important regulator of animal development, and it is essential for endocytic trafficking. However, the mechanisms that link Rab5 activation to cargo entry into early endosomes remain unclear. We show here that Drosophila Rabenosyn (Rbsn) is a Rab5 effector that bridges an interaction between Rab5 and the Sec1/Munc18-family protein Vps45, and we further identify the syntaxin Avalanche (Avl) as a target for Vps45 activity. Rbsn and Vps45, like Avl and Rab5, are specifically localized to early endosomes and are required for endocytosis. Ultrastructural analysis of rbsn, Vps45, avl, and Rab5 null mutant cells, which show identical defects, demonstrates that all four proteins are required for vesicle fusion to form early endosomes. These defects lead to loss of epithelial polarity in mutant tissues, which overproliferate to form neoplastic tumors. This work represents the first characterization of a Rab5 effector as a tumor suppressor, and it provides in vivo evidence for a Rbsn-Vps45 complex on early endosomes that links Rab5 to the SNARE fusion machinery.

Spatial expression of transcription factors in Drosophila embryonic organ development.

BackgroundSite-specific transcription factors (TFs) bind DNA regulatory elements to control expression of target genes, forming the core of gene regulatory networks. Despite decades of research, most studies focus on only a small number of TFs and the roles of many remain unknown.ResultsWe present a systematic characterization of spatiotemporal gene expression patterns for all known or predicted Drosophila TFs throughout embryogenesis, the first such comprehensive study for any metazoan animal. We generated RNA expression patterns for all 708 TFs by in situ hybridization, annotated the patterns using an anatomical controlled vocabulary, and analyzed TF expression in the context of organ system development. Nearly all TFs are expressed during embryogenesis and more than half are specifically expressed in the central nervous system. Compared to other genes, TFs are enriched early in the development of most organ systems, and throughout the development of the nervous system. Of the 535 TFs with spatially restricted expression, 79% are dynamically expressed in multiple organ systems while 21% show single-organ specificity. Of those expressed in multiple organ systems, 77 TFs are restricted to a single organ system either early or late in development. Expression patterns for 354 TFs are characterized for the first time in this study.ConclusionsWe produced a reference TF dataset for the investigation of gene regulatory networks in embryogenesis, and gained insight into the expression dynamics of the full complement of TFs controlling the development of each organ system.

Long-read, whole-genome shotgun sequence data for five model organisms.

Single molecule, real-time (SMRT) sequencing from Pacific Biosciences is increasingly used in many areas of biological research including de novo genome assembly, structural-variant identification, haplotype phasing, mRNA isoform discovery, and base-modification analyses. High-quality, public datasets of SMRT sequences can spur development of analytic tools that can accommodate unique characteristics of SMRT data (long read lengths, lack of GC or amplification bias, and a random error profile leading to high consensus accuracy). In this paper, we describe eight high-coverage SMRT sequence datasets from five organisms (Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, Arabidopsis thaliana, and Drosophila melanogaster) that have been publicly released to the general scientific community (NCBI Sequence Read Archive ID SRP040522). Data were generated using two sequencing chemistries (P4C2 and P5C3) on the PacBio RS II instrument. The datasets reported here can be used without restriction by the research community to generate whole-genome assemblies, test new algorithms, investigate genome structure and evolution, and identify base modifications in some of the most widely-studied model systems in biological research.

Presenilin-Based Genetic Screens in Drosophila melanogaster Identify Novel Notch Pathway Modifiers

Presenilin is the enzymatic component of γ-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimers disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for γ-tubulin in the pathway.

Functional Evolution of cis-Regulatory Modules at a Homeotic Gene in Drosophila

It is a long-held belief in evolutionary biology that the rate of molecular evolution for a given DNA sequence is inversely related to the level of functional constraint. This belief holds true for the protein-coding homeotic (Hox) genes originally discovered in Drosophila melanogaster. Expression of the Hox genes in Drosophila embryos is essential for body patterning and is controlled by an extensive array of cis-regulatory modules (CRMs). How the regulatory modules functionally evolve in different species is not clear. A comparison of the CRMs for the Abdominal-B gene from different Drosophila species reveals relatively low levels of overall sequence conservation. However, embryonic enhancer CRMs from other Drosophila species direct transgenic reporter gene expression in the same spatial and temporal patterns during development as their D. melanogaster orthologs. Bioinformatic analysis reveals the presence of short conserved sequences within defined CRMs, representing gap and pair-rule transcription factor binding sites. One predicted binding site for the gap transcription factor KRUPPEL in the IAB5 CRM was found to be altered in Superabdominal (Sab) mutations. In Sab mutant flies, the third abdominal segment is transformed into a copy of the fifth abdominal segment. A model for KRUPPEL-mediated repression at this binding site is presented. These findings challenge our current understanding of the relationship between sequence evolution at the molecular level and functional activity of a CRM. While the overall sequence conservation at Drosophila CRMs is not distinctive from neighboring genomic regions, functionally critical transcription factor binding sites within embryonic enhancer CRMs are highly conserved. These results have implications for understanding mechanisms of gene expression during embryonic development, enhancer function, and the molecular evolution of eukaryotic regulatory modules.

The ModERN Resource: Genome-Wide Binding Profiles for Hundreds of Drosophila and Caenorhabditis elegans Transcription Factors

The model organism Encylopedia of Regulatory Elements (modERN) project was designed to generate genome-wide binding profiles for the majority of transcription... To develop a catalog of regulatory sites in two major model organisms, Drosophila melanogaster and Caenorhabditis elegans, the modERN (model organism Encyclopedia of Regulatory Networks) consortium has systematically assayed the binding sites of transcription factors (TFs). Combined with data produced by our predecessor, modENCODE (Model Organism ENCyclopedia Of DNA Elements), we now have data for 262 TFs identifying 1.23 M sites in the fly genome and 217 TFs identifying 0.67 M sites in the worm genome. Because sites from different TFs are often overlapping and tightly clustered, they fall into 91,011 and 59,150 regions in the fly and worm, respectively, and these binding sites span as little as 8.7 and 5.8 Mb in the two organisms. Clusters with large numbers of sites (so-called high occupancy target, or HOT regions) predominantly associate with broadly expressed genes, whereas clusters containing sites from just a few factors are associated with genes expressed in tissue-specific patterns. All of the strains expressing GFP-tagged TFs are available at the stock centers, and the chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center and also through a simple interface (http://epic.gs.washington.edu/modERN/) that facilitates rapid accessibility of processed data sets. These data will facilitate a vast number of scientific inquiries into the function of individual TFs in key developmental, metabolic, and defense and homeostatic regulatory pathways, as well as provide a broader perspective on how individual TFs work together in local networks and globally across the life spans of these two key model organisms.

OpenHiCAMM: High-Content Screening Software for Complex Microscope Imaging Workflows

Summary High-content image acquisition is generally limited to cells grown in culture, requiring complex hardware and preset imaging modalities. Here we report an open source software package, OpenHiCAMM (Open Hi Content Acquisition for μManager), that provides a flexible framework for integration of generic microscope-associated robotics and image processing with sequential workflows. As an example, we imaged Drosophila embryos, detecting the embryos at low resolution, followed by re-imaging the detected embryos at high resolution, suitable for computational analysis and screening. The OpenHiCAMM package is easy to use and adapt for automating complex microscope image tasks. It expands our abilities for high-throughput image-based screens to a new range of biological samples, such as organoids, and will provide a foundation for bioimaging systems biology.

Exploiting regulatory heterogeneity to systematically identify enhancers with high accuracy

Identifying functional enhancers elements in metazoan systems is a major challenge. For example, large-scale validation of enhancers predicted by ENCODE reveal false positive rates of at least 70%. Here we use the pregrastrula patterning network of Drosophila melanogaster to demonstrate that loss in accuracy in held out data results from heterogeneity of functional signatures in enhancer elements. We show that two classes of enhancer are active during early Drosophila embryogenesis and that by focusing on a single, relatively homogeneous class of elements, over 98% prediction accuracy can be achieved in a balanced, completely held-out test set. The class of well predicted elements is composed predominantly of enhancers driving multi-stage, segmentation patterns, which we designate segmentation driving enhancers (SDE). Prediction is driven by the DNA occupancy of early developmental transcription factors, with almost no additional power derived from histone modifications. We further show that improved accuracy is not a property of a particular prediction method: after conditioning on the SDE set, naïve Bayes and logistic regression perform as well as more sophisticated tools. Applying this method to a genome-wide scan, we predict 1,640 SDEs that cover 1.6% of the genome, 916 of which are novel. An analysis of 32 novel SDEs using wholemount embryonic imaging of stably integrated reporter constructs chosen throughout our prediction rank-list showed >90% drove expression patterns. We achieved 86.7% precision on a genome-wide scan, with an estimated recall of at least 98%, indicating high accuracy and completeness in annotating this class of functional elements. Significance Statement We demonstrate a high accuracy method for predicting enhancers genome wide with > 85% precision as validated by transgenic reporter assays in Drosophila embryos. This is the first time such accuracy has been achieved in a metazoan system, allowing us to predict with high-confidence 1640 enhancers, 916 of which are novel. The predicted enhancers are demarcated by heterogeneous collections of epigenetic marks; many strong enhancers are free from classical indicators of activity, including H3K27ac, but are bound by key transcription factors. H3K27ac, often used as a one-dimensional predictor of enhancer activity, is an uninformative parameter in our data.