Stefan Bonn

Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development

Chromatin modifications are associated with many aspects of gene expression, yet their role in cellular transitions during development remains elusive. Here, we use a new approach to obtain cell type–specific information on chromatin state and RNA polymerase II (Pol II) occupancy within the multicellular Drosophila melanogaster embryo. We directly assessed the relationship between chromatin modifications and the spatio-temporal activity of enhancers. Rather than having a unique chromatin state, active developmental enhancers show heterogeneous histone modifications and Pol II occupancy. Despite this complexity, combined chromatin signatures and Pol II presence are sufficient to predict enhancer activity de novo. Pol II recruitment is highly predictive of the timing of enhancer activity and seems dependent on the timing and location of transcription factor binding. Chromatin modifications typically demarcate large regulatory regions encompassing multiple enhancers, whereas local changes in nucleosome positioning and Pol II occupancy delineate single active enhancers. This cell type–specific view identifies dynamic enhancer usage, an essential step in deciphering developmental networks.

A systematic screen for protein–lipid interactions in Saccharomyces cerevisiae

Protein–metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein–lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid‐binding fingerprints of 172 proteins, including 91 with predicted lipid‐binding domains. We identified 530 protein–lipid associations, the majority of which are novel. To show the data sets biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live‐cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid‐binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids.

DNA methylation changes in plasticity genes accompany the formation and maintenance of memory

The ability to form memories is a prerequisite for an organisms behavioral adaptation to environmental changes. At the molecular level, the acquisition and maintenance of memory requires changes in chromatin modifications. In an effort to unravel the epigenetic network underlying both short- and long-term memory, we examined chromatin modification changes in two distinct mouse brain regions, two cell types and three time points before and after contextual learning. We found that histone modifications predominantly changed during memory acquisition and correlated surprisingly little with changes in gene expression. Although long-lasting changes were almost exclusive to neurons, learning-related histone modification and DNA methylation changes also occurred in non-neuronal cell types, suggesting a functional role for non-neuronal cells in epigenetic learning. Finally, our data provide evidence for a molecular framework of memory acquisition and maintenance, wherein DNA methylation could alter the expression and splicing of genes involved in functional plasticity and synaptic wiring.

Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP

This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type–specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type–specific nuclear marker. Tissue-specific chromatin is extracted, sheared by sonication and used for ChIP-seq or other analyses. The key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of FACS of nuclei, yielding very high purity. The protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. The full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d.

cis-Regulatory networks during development: a view of Drosophila

Understanding how regulatory networks initiate, maintain and synchronise transcriptional states remains a fundamental goal of developmental biology. Complex patterns of spatio-temporal gene expression are generated through the combined inputs of signalling and transcriptional networks converging on cis-regulatory modules (CRMs). Detailed studies in Drosophila, using transgenic reporter assays and mutagenesis analysis, have dissected the regulatory logic of a number of CRMs. These data have recently been complemented by genome-wide maps of transcription factor binding, revealing an unprecedented view of CRM occupancy and network complexity. The synthesis of data for three well-characterised Drosophila developmental networks reveals emerging themes at both a CRM and a cis-regulatory network level.

HDAC inhibitor-dependent transcriptome and memory reinstatement in cognitive decline models

Aging and increased amyloid burden are major risk factors for cognitive diseases such as Alzheimers disease (AD). Effective therapies for these diseases are lacking. Here, we evaluated mouse models of age-associated memory impairment and amyloid deposition to study transcriptome and cell type-specific epigenome plasticity in the brain and peripheral organs. We determined that aging and amyloid pathology are associated with inflammation and impaired synaptic function in the hippocampal CA1 region as the result of epigenetic-dependent alterations in gene expression. In both amyloid and aging models, inflammation was associated with increased gene expression linked to a subset of transcription factors, while plasticity gene deregulation was differentially mediated. Amyloid pathology impaired histone acetylation and decreased expression of plasticity genes, while aging altered H4K12 acetylation-linked differential splicing at the intron-exon junction in neurons, but not nonneuronal cells. Furthermore, oral administration of the clinically approved histone deacetylase inhibitor vorinostat not only restored spatial memory, but also exerted antiinflammatory action and reinstated epigenetic balance and transcriptional homeostasis at the level of gene expression and exon usage. This study provides a systems-level investigation of transcriptome plasticity in the hippocampal CA1 region in aging and AD models and suggests that histone deacetylase inhibitors should be further explored as a cost-effective therapeutic strategy against age-associated cognitive decline.

Determination of allele frequency in pooled DNA: comparison of three PCR-based methods.

Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).

Combinatorial expression of alpha- and gamma-protocadherins alters their presenilin-dependent processing.

ABSTRACT α- and γ-protocadherins (Pcdhs) are type I transmembrane receptors expressed predominantly in the central nervous system and located in part in synapses. They are transcribed from complex genomic loci, giving rise in the mouse to 14 α-Pcdh and 22 γ-Pcdh isoforms consisting of variable domains, each encompassing the extracellular region, the transmembrane region, and part of the intracellular region harboring the α- or γ-Pcdh-specific invariant cytoplasmic domain. Presenilin-dependent intramembrane proteolysis (PS-IP) of γ-Pcdhs and the formation of α/γ-Pcdh heteromers led us to investigate the effects of homo- and heteromer formation on γ- and putative α-Pcdh membrane processing and signaling. We find that upon surface delivery, α-Pcdhs, like γ-Pcdhs, are subject to matrix metallo-protease cleavage followed by PS-IP in neurons. We further demonstrate that the combinatorial expression of α- and γ-Pcdhs modulates the extent of their PS-IP, indicating the formation of α/γ-Pcdh heteromers with an altered susceptibility to processing. Cell-specific expression of α/γ-Pcdh isoforms could thus determine cell and synapse adhesive properties as well as intracellular and nuclear signaling by their soluble cytoplasmic cleavage products, α C-terminal fragment 2 (α-CTF-2) and γ-CTF-2.

De-regulation of gene expression and alternative splicing affects distinct cellular pathways in the aging hippocampus

Aging is accompanied by gradually increasing impairment of cognitive abilities and constitutes the main risk factor of neurodegenerative conditions like Alzheimers disease (AD). The underlying mechanisms are however not well understood. Here we analyze the hippocampal transcriptome of young adult mice and two groups of mice at advanced age using RNA sequencing. This approach enabled us to test differential expression of coding and non-coding transcripts, as well as differential splicing and RNA editing. We report a specific age-associated gene expression signature that is associated with major genetic risk factors for late-onset AD (LOAD). This signature is dominated by neuroinflammatory processes, specifically activation of the complement system at the level of increased gene expression, while de-regulation of neuronal plasticity appears to be mediated by compromised RNA splicing.

Oasis: online analysis of small RNA deep sequencing data

Summary: Oasis is a web application that allows for the fast and flexible online analysis of small-RNA-seq (sRNA-seq) data. It was designed for the end user in the lab, providing an easy-to-use web frontend including video tutorials, demo data and best practice step-by-step guidelines on how to analyze sRNA-seq data. Oasis’ exclusive selling points are a differential expression module that allows for the multivariate analysis of samples, a classification module for robust biomarker detection and an advanced programming interface that supports the batch submission of jobs. Both modules include the analysis of novel miRNAs, miRNA targets and functional analyses including GO and pathway enrichment. Oasis generates downloadable interactive web reports for easy visualization, exploration and analysis of data on a local system. Finally, Oasis’ modular workflow enables for the rapid (re-) analysis of data. Availability and implementation: Oasis is implemented in Python, R, Java, PHP, C++ and JavaScript. It is freely available at http://oasis.dzne.de. Contact: stefan.bonn@dzne.de Supplementary information: Supplementary data are available at Bioinformatics online.