This project will contribute to DEEP in three ways: (1) Developing methods for high-level analysis of epigenomic data with a focus on DNA methylation data, (2) developing and offering a server comprising high-level data analysis software for the DEEP project. This server will be offered to the DEEP consortium and to the world-wide scientific community. (3) Collaborating with other DEEP subprojects on high-level analysis of data generated in DEEP.
The German epigenome programme DEEP comprises a coordinated interdisciplinary network of expert groups to map and functionally interpret reference epigenomes of selected human cells and tissues in normal and diseased states. The primary goal of the disease-oriented programme is to DEEPen the understanding of complex diseases such as inflammatory diseases of joint and bowel and metabolic diseases such as hepatosteatosis and adipositas. In total 17 subprojects will interact in three strongly interconnected scientific platforms to achieve this goal: Epigenomic data production, bioinformatics analysis and disease-oriented projects. The disease-oriented platforms will contribute expertise to isolate highly purified cell types/tissues for epigenomic mapping and will analyse cell type-/ tissue-associated aspects of the complex diseases. In the second platform, epigenomic sequencing units will generate primary epigenomic data that will be submitted to a central data collection unit and interpreted in close collaboration with central bioinformatics units responsible for high quality public data deposition and deep functional interpretation. A platform programme coordination & outreach will monitor the quality controlled data generation and foster interactions between bioinformatics and experimental analysis groups across platforms by implementing a joint workgroup structure.
In this subproject, we will provide central data management and storage. We will host raw sequencing data from different facilities and align them to reference genomes according to common standards. We will develop a browser to visualize results from further bioinformatic analyses. The central data storage will be tightly linked with the application server that is developed in SP1-1 (MPI-INF).
Within the DEEP consortium this subproject shall focus on the high-level analysis of histone modification data. This type of data still poses many new problems like the assessment of its technical and biological variability and the correlations among different modifications. Based on the data generated in the project, in conjunction with publicly available data, these problems shall be studied with the goal of developing the needed algorithms for comparison of modification patterns between different cellular states.
Within the DEEP consortium this subproject will provide the annotation of the small RNA sequencing data. We will use existing algorithms and further develop these algorithms to annotate the expression of known and novel non coding RNAs (ncRNAs). We will also computationally predict targets of small RNAs. We will in particular try to develop methods to predict ncRNA targets in the nucleus. These efforts will also analyse data from non-standard cloning and sequencing methods developed by the Chen lab at MDC.
DNA methylation in form of 5-methylcytosine and 5-hydroymethylcytosine (and further modifications) plays a crucial role in developmental gene control and genome stability in mammals and in human. Locus and genome wide specific changes in DNA methylation are observed in almost all complex human diseases. Because of their extreme stability and localized detection, DNA-modifications serve as excellent disease associated high resolution epigenetic landmarks. One of the major goals of DEEP is to produce high-resolution DNA methylation maps and to identify DNA-methylation changes as suitable biomarkers for larger clinical studies. The production of 70 complete whole genome bisulfite methylomes (20x coverage) will be performed at the two Methylome-NGS sequencing units at UDE and UdS. Both units will closely cooperate in protocol development and standardized data production. Supported by QIAGEN GmbH the units will develop novel strategies for multiplexed library assays and automated marker validation using 454 based deep bisulfite amplicon sequencing. In the validation studies B. Horsthemke (UDE) and J. Walter (UdS) will address question of allele specific effects and heterogeneity in epigenetic disease signatures. The sequencing unit UdS will add the expertise for hyMeDIP-Seq to determine 5-hydroxmethylcytosine in selected cell types.
The second major working task in this subproject is the generation of high resolution Chromatin accessibility maps for 70 isolated primary cell types. Chromatin accessibility is a major parameter in epigenome mapping. The DNAseI-seq work will exclusively be performed at UdS which is equipped for this task. A DNAseI pipeline will be established in one of the sequencing units at UdS. To establish the pipepline UdS will receive training/support through BLUEPRINT by H. Stunnenbergs lab. The DNAseI will use detailed and IHEC approved ENCODE protocols (J. Stams lab UWash). UdS will begin to establish the process pipeline in cell lines (IMR90) and two primary cell types Cd4+ lymphocytes (human) and mouse tissues (liver) starting from April 2012. Towards the end of 2012 the group should then be prepared to receive cell material from DEEP to start the reference mapping production. Quality approved DNAseI-libraries (following bar coded QC sequencing) will be sequenced at a coverage of a 20M reads/library. Data will always be produced in biological and technical duplicates. The computational mapping and evaluation of the DNAsI-seq data will be performed in close collaboration with the MPI-INF. BAM files will be submitted to the DCC and interrogated for peak detection/interpretation (e.g. in connection with DNA methylation) in collaboration with the MPI-INF who has a strong record in DNA feature extraction and with the MPI-MG in Berlin (M. Vingron) who posseses expertise in DNAseI, chromatin and TF-binding site mapping.
Histone modifications have an important influence on the establishment and maintenance of cellular identity, development and the etiology of diseases. It is well known that combinations of various histone modification marks can alter chromatin structure and modulate gene expression in response to environmental and internal signals. However, a comprehensive mapping approach for several cell types is only now becoming possible due to major advances in the field of sequencing technology and computational algorithms. In this project we will contribute with our expertise in chromatin research, deep-sequencing technology and genome-wide data analysis to the generation of genome-wide histone modification maps in various mammalian cell types.
We will generate 70 reference epigenomes for mammalian cells, which are investigated by the various pillars of the consortium: Metabolic Disorders (adipocytes, monocytes/macrophages, hepatocytes) and Chronic Inflammation (CD4+, epithelial cells, fibroblast, inflammatory macrophages). The generated data will be an invaluable resource for comparative studies across tissues, treatments and organisms, and it will enable the identification of epigenetic markers. Together with information on the DNA methylome and the non-coding RNA content this will result in a comprehensive picture of the epigenetic variability at unprecedented resolution and provide a mechanistic understanding of epigenetic control and therapy.
Prof. Dr. Thomas Jenuwein
Max Planck Institute for Immunobiology and Epigenetics
The landscape and dynamics of the transcriptome represents a key element of the epigenome as it both comprises distinct epigenetic effector mechanisms (e.g. miRNA) and is at the same time affected by epigenetic changes (e.g. transcript level or splicing influenced by methylation or histone modification). The establishment of comprehensive RNA expression profiles, including information on expression levels, small and long ncRNAs, alternative splicing, allele specific expression patterns and mRNA editing is thus essential to our understanding of the molecular basis of a reference epigenome. This subproject will provide support in transcriptome analysis for the entire DEEP consortium. Here, the small RNA and poly A RNA fractions will be analyzed separately for 70 samples.
The main objective of this subproject is to enable a smooth project workflow through providing the necessary support mechanisms i) to ensure that all contractual commitments can be met in time, ii) to support quality control and risk management, iii) to safeguard the proper integration of project activities and results into the IHEC structure and framework, and iv) to ensure wide dissemination of project results.
For the latter, the coordinator Prof. Jörn Walter, as the scientific leader of the DEEP initiative and main contact person for the BMBF/DLR, will be closely supported by the professional management partner EURICE, a spin-off company of Saarland University specialized in international project management and dissemination. Together, this management team will coordinate all consortium activities and ensure a smooth and timely implementation of the DEEP workplan. Management tasks include both, project-internal issues, such as
- Project organisation & communication
- Scientific monitoring & controlling
- Workflow and Quality Monitoring
as well as externally-directed tasks like
- Dissemination (nationally & internationally)
- Interfacing with other initiatives
- Integration in IHEC
DEEP Programme Management will have three main elements:
that jointly ensure the stability and soundness of the network itself, link it to the external scientific community and allow for the integration new developments through associated projects, provide a national infrastructure for training and mentoring in epigenomic research, and proactively support IP management and exploitation.
Controlling comprises all active organizational and administrative support to the scientific coordinator and the entire DEEP consortium, including administrative documentation and support to the communication with the BMBF.
DEEP Training courses in bioinformatic data interpretation that are offered twice a year for both, consortium members as well as additional, external participants, will ensure high-quality data interpretation, streamline interactions between the affiliated laboratories and provide a much-needed curriculum for epigenomic research in Germany.
DEEP Exploitation support includes the negotiation of a consortium agreement, screening of exploitation interests of individual participants, identification of exploitation opportunities within the DEEP legal framework and IHEC policy, the elaboration of a comprehensive overall DEEP exploitation-strategy, and continuous support in all matters related to IP and exploitation.
Being a key partner of the IHEC umbrella, DEEP has a strong interest in a clear and effective communication of the IHEC vision and its achievements from a global perspective. Thus, DEEP will set up a central communication unit to directly support the communication activities of IHEC across Europe. In order to enhance the public’s understanding of the outcomes and relevance of Epigenomic research, and to increase the visibility of IHEC in particular, the major task of this communication unit will be to contribute to the development and implementation of a global communications strategy for IHEC as suggested by the IHEC white paper “A Global Communications Plan for IHEC”. This will be accomplished in close cooperation with the IHEC steering committee and will be the purpose of a dedicated, international IHEC communication group, in which this DEEP communication unit will function as the European representative.
The IHEC communication support will be guided by J. Walter, who is member of the IHEC Scientific Steering Committee and thus directly involved in key decision-making processes within IHEC, and will be implemented by the professional communication division of EURICE. In addition to its expertise in the professional management of international projects, EURICE has particularly established a strong track record in the development and realization of communication concepts for EU-funded and other international projects. As the managing team of this subproject J. Walter and EURICE will be responsible for a consistent IHEC communication management on European level.
This subproject will perform a genome-wide characterization of epigenetic alterations in steatotic livers, analyze their disease relevance for non-alcoholic fatty liver disease, and define the role of cytokines released by tissue macrophages for epigenetic alterations and disease progression. To reach this overall goal the following experimental strategies will be: (i) Human hepatocytes will be isolated from resected tissue of patients with and without non-alcoholic fatty liver disease (NAFLD) and will be delivered to our partners for analysis of the epigenome. (ii) An established in vitro protocol for induction of steatotic hepatocytes will be applied to study if similar epigenetic alterations are obtained in vitro compared to the in vivo situation. If this is the case the in vitro system will be used for further mechanistic studies. (iii) If epigenetic alterations with consequences for expression of the respective genes will be observed we will study their functional relevance. This will include siRNA knockdown and overexpression of candidate genes in primary human hepatocytes, followed by analysis of possible consequences for a steatotic phenotype, including a comprehensive analysis of lipid expression spectra. (iv) The P62 mouse represents a genetic animal model for non-inflammatory steatosis. In this subproject, we will isolate mouse hepatocytes using recently established standard protocols (Godoy et al., 2009) for analysis of the epigenome in order to study if this mouse model recapitulates some of the mechanisms relevant for human NAFLD. In addition, we will quantify the degree of steatosis using recently established imaging techniques for mouse livers (Hoehme et al., 2010). (v) We will establish co-cultures of hepatocytes and liver tissue macrophages (Kupffer cells) to study of secreted cytokines contribute to state of chronic inflammation and disease relevant epigenetic alterations. Together, this will path the way to novel therapies of NAFLD and to avoid its progression to steatohepatitis.
SP4-2 Epigenetics of steatosis: The role of Kupffer cells in the inflammatory response towards steatohepatitis
The phenotype of a fatty liver is a risk factor for the development of hepatocellular carcinoma (HCC), which represents the third leading cause of cancer-related mortality worldwide. A development from benign steatosis (first hit) towards steatohepatitis (second hit) thereby represents a critical step. We will employ p62 transgenic mice as a genetic animal model for a non-inflammatory steatosis. We will perform comprehensive epigenetic analyses of steatotic mice compared to healthy wild-type animals. In order to induce an inflammatory phenotype and to set a second hit, an endotoxemic model will be employed. Epigenetic and gene expression analyses will be performed in the different models and will be correlated to pathophysiological features. The role of Kupffer cells, the resident macrophages of the liver, in the progression from steatosis towards steatohepatitis will be studied by their depletion and subsequent characterization of differences in the epigenetic programme as well as regarding pathophysiological features. The latter will be assessed comprising metabolic alterations as well as inflammatory activation and tumor initiation events. A correlation of epigenetic programmes with inflammatory and tumor-promoting signalling routes will be determined.
SP4-3 Comparative epigenomic profiling of human adipose tissue cells in normal and diabesity samples
The project aims to interpret and test the epigenetic differences between small and large adipocytes, between visceral and subcutaneous adipocytes, between monocytes and adipose tissue macrophages (ATMs), and between these cells in normal and extreme obese individuals. Additionally, in a pilot study the Regensburg group already identified miRNA patterns in human plasma diabesity samples within the LipidomicNet project and plans to validate 40 ranked miRNAs in a cohort of diabesity patients with obesity, insulin resistance, type-2 diabetes and metabolic syndrome. We will also evaluate the effect of putative inducers of brown adipose tissue development on the epigenetic profile of appropriate precursor cells. We expect that these studies contribute to our understanding of the basis of adipocyte hyperplasia and hypertrophy as well as ATM formation in diabesity, the different roles of visceral and subcutaneous fat in disease, the role of inflammation and to the development of epigenetic biomarkers and drugs.
Associated Industrial partner
Dr. Günter Müller
Sanofi-Aventis Deutschland GmbH, R&D Diabetes
The goal of the DEEP network is to characterize epigenetic alterations in metabolic and inflammatory disorders with a focus on inflammatory cells, hepatocytes and adipocytes. Obesity, the most prevalent of these, is expected to affect 1.2 billion people by 2030. Indeed, the recent surge in obesity is thought to depend primarily on epigenetic reprogramming in early development. To date, little is known about the acute plasticity of our epigenomes towards circadian inputs, nor about the limits of epigenome control. This project aims to make a comprehensive investigation of circadian epigenetic variation in murine adipocytes, charting nutrient- and obesity-dependency, while at the same time exposing the limits of heterochromatin control in vivo, examining an epigenetically sensitized mouse model of stochastic obesity. The expansive dataset will be a milestone for comprehensive epigenomic profiling in mammalian disease providing a critical reference for circadian-, nutrient-, and obesity- driven plasticity, and offering some first-ever insights into the limits of epigenome integrity.
The subproject will analyze the epigenome of effector/memory T-cells isolated from chronically inflamed human tissue. Epigenetic signatures, notably DNA methylation patterns are expected to highlight differentiation pathways that result in a pathological and presumably irreversible stage of chronically activated cells that differ from normal, protective memory cells by impaired susceptibility to internal control mechanisms and to current therapies. The data will allow to understand pathophysiological mechanisms, to identify key switches in the transcriptional regulation and to develop diagnostic tools as well as therapeutic strategies for re-programming.
The genetic risk map of chronic inflammatory bowel disease (IBD) with its two subentities Crohn´s disease (CD) and ulcerative colitis (UC) has elucidated a crucial role for the intestinal epithelium in addition to a chronic overactivation of immune cells. The epithelial lining undergoes a selective, long term remodeling and may react with dysplasia and malignant transformation. It has been shown that during the course of IBD intestinal epithelial cells (IEC) reacquire the ability to mount an inflammatory immune response towards the indigenous microflora; on the other hand IBD-associated genetic variants (NOD2, ATG16L1) may render the cells inert to cytoinvasive bacteria causing secondary defects in protective and regenerative programmes in IECs and lead to a secondary overactivation of the underlying mucosa-associated lymphocyte system. Interestingly, IBD is associated to the life style of Western industrialized civilizations. It appears likely that the rising incidence is caused by environmental triggers (e.g. hygiene, nutrition, antibiotics) that ultimately result in a disease phenotype on the background of a stable genetic population background.
We will define reference epigenomes of purified IECs from CD patients and healthy controls and will further follow up functional consequences of the obtained epigenetic signatures from the epigenome analysis in functional models. Intestinal mucosal reference epigenomes from two monozygotic twin pairs discordant for CD will serve as an independent start point in order to understand epigenetic alterations in identical, yet phenotypically distinct genomes. Along the generated hypotheses we will study IEC and mucosal epigenome changes along development and disease and we will use epigenome-modifying therapies in defined mouse models of IBD. We hypothesize that epigenetic imprints in IECs may provide a missing link between a genetic risk map and the individual disease manifestation and that these links could lead to novel biomarkers and therapeutic targets in IBD.
In most immune-mediated inflammatory diseases, persistence of inflammation and subsequent irreversible tissue damage rather than inflammation itself is the main problem. Rheumatoid arthritis (RA), a chronic autoimmune arthritis that leads to the progressive destruction of affected joints, is a prominent example for this concept, and it has been shown that fibroblast-like synoviocytes (FLS) contribute significantly to the pathogenesis of RA. These cells are a key part of the local immune system in the joints and integrate signals form different sources into a pathological tissue response. While responding to the stimuli in the RA synovium, RA-FLS undergo fundamental changes, and multiple lines of evidence suggest that this result is a stable activation, which is maintained even in the absence of continuous stimulation by inflammatory triggers. As a consequence of this stable FLS-activation, the disease process is perpetuated and might progress when inflammation ameliorates. The underlying mechanisms are not entirely clear, but recent data suggest that in addition to the chronic exposure of FLS to inflammatory cytokines, growth factors and the extracellular matrix (ECM), epigenetic modifications contribute significantly to the imprinting of an aggressive phenotype. These include a variety of different mechanisms and result in long-term alterations of their behavior in vitro and in vivo. While a number of distinct epigenetic changes have been reported in RA-FLS in the last years, a comprehensive picture of their epigenome is missing. In this project, we want to analyze the epigenome of invasive RA-FLS versus osteoarthritic and normal synovial fibroblasts as well as fibroblasts exposed to stimuli known to be involved in the stable activation of these cells in RA. Further analyses will include functional studies on the identified epigenetic pathways both in vitro and in animal studies of chronic destructive arthritis as well as evaluation of their therapeutic potential. Also, this project will be tightly embedded into the DEEP consortium by comparing the data with those from other conditions and by focusing on the crosstalk between FLS and T- cells.