Establishment of the haemopoietic transcriptional programme: From systems approaches to molecular mechanisms

Our genes control how our body develops from one fertilized egg cell and all cells in our body contain the same set of genes. This cell rapidly divides and develops into a large variety of distinct cell types that make up the various organs in our body. All these cells express different genetic programs, meaning that not all of our genes are always active in every cell type. This cell-type-specific gene activation pattern is governed by another layer of control (on top of the layer of the genes) that tells cells which genes to switch on and off, thereby deciding which cell type develops. This additional control layer is called the 'epigenetic' layer and consists of two components: (1) a genome-wide network through which genes regulate each other to generate the appropriate gene expression patterns; (2) the DNA packing apparatus. Each cell contains one meter of DNA, and to be able to fit it into the nucleus, it is densely compacted by so called chromatin proteins such that inactive genes are highly compact and their DNA hidden, whereas active genes are in areas of reduced compaction. To activate an inactive, compact gene, protein complexes, so called 'transcription factors' push chromatin aside or modify it, so that genes become accessible to the factors that activate them. Studies in the past years focused on one gene at a time and led to the discovery of the transcription factors and chromatin components that control their activity. We learned to extract the tune that individual genes play but failed to hear the symphony. Our understanding of how all the genes in mammals are orchestrated to switch on and off in the right order is still superficial. Moreover, much of what we know is based on studies from cell lines, which represent fixed cell types or are cancer cells, and from simpler organisms, such as yeast. The situation in mammals is much more complex because building an organism from a fertilized egg involves turning one cell type into another (so called 'differentiation') in a precise hierarchical order which requires tight coordination of the activity of all the genes. In other words, building an organism is like building a house: we have to put the individual components together in a precise order and not start with the roof before the cellar. This proposal will use blood cell development in the mouse as a model to investigate the dynamics of cell differentiation in mammals. We will study all genes of a given cell type and use a sophisticated in vitro system based on embryonic stem cells where we can generate and purify different blood cell types. We then will identify which transcription factors and chromatin components regulate which genes at the different developmental stages and study at which level and when they are expressed. Until recently such global or 'systems biology' studies were beyond reach since the technology was lacking. However, with the latest technology we can determine the entire DNA sequence of one cell type in a very short time. This technology has been modified to study epigenetic changes at all genes and can now be used to identify what distinguishes genes of one cell type from those of another. However, one feature of such experiments is that they produce enormous amounts of data and require specialist knowledge to make sense of them. This is achieved by bioinformaticians developing new computer programs and mathematical modelers running simulations to predict the integrated, 'collective' behavior of genes. To this end we have formed an interdisciplinary consortium consisting of experimental researchers and computational biologists who will collaborate to understand how thousands of genes work together to generate specific cell types. The ultimate aim of these studies is to be able to understand how individual development is encoded in the DNA-sequence and to predict how changes in the DNA sequence impact on developmental processes.

A pivotal challenge for post-genomic research is to understand in a system-wide fashion how networks of transcription factors (TFs) and chromatin components regulate cell fate decisions in an integrated manner. While recent genome-wide studies have offered a first glimpse onto the complexity of transcription factor-DNA interactions in specific cell types, we know very little about the dynamical relationships between different network states during development. We also do not know how the ordered, bidirectional interplay between TFs and specific chromatin states leads to the stable expression of lineage specific genetic programs. This proposal will use the differentiation of haemopoietic cells from mouse embryonic stem cells as a model to investigate the molecular mechanisms and dynamics of cell differentiation in a system-wide fashion. To this end we have formed a consortium consisting of experimental researchers and computational biologists who will identify the genome-wide dynamics of TF assembly during development. We will perform experiments examining global DNA methylation and chromatin alterations, obtain mechanistic insights by manipulating the trans-regulatory environment and verify conclusions by examining specific genes in more detail. In doing so, we will address the following general questions: 1) What is the molecular basis of the hierarchical action of key regulators of haematopoietic development, 2) How are known key regulators integrated into wider transcriptional networks with a particular emphasis on the dynamical nature of network state transitions 3.) How do these transcription factors interact with the chromatin template and how do they regulate chromatin accessibility and chromatin modification? 4) Can we decipher the genomic regulatory blueprint for development of a mammalian organ system?

Our work will have a tremendous impact not only on our immediate research field but also far beyond. The studies proposed here will lead to a better understanding of the biology of haemopoietic stem cells, and therefore have the potential to benefit all future therapeutic approaches utilizing these cells. Moreover, we will pioneer technological and conceptual advances that will be vital for exploiting post-genomic datasets in translational settings in biotechnology and medicine.. Through its substantial involvement with the human genome project as well as other post-genomic activities, UK-plc has heavily invested into the early phase of genomic and post-genomic biology. To ensure eventual translation of these early efforts into improved 'health and wealth' benefits, it is now vital to build on the early investment through strategic funding of research consortia that not only produce world-class science but also function as hubs for knowledge generation, integration and distribution. To achieve maximum impact we plan the following activities: (i) We will make our system-wide datasets and network models publicly available. It will be impossible for us to examine all sub-aspects of these huge data sets, they will therefore serve as paradigms for future studies of normal as well as aberrant differentiation. This will benefit anybody who studies normal or aberrant cell fate decisions in academia, industry or the clinic. (ii) We will generate network models and develop computational tools that will be highly relevant to scientists studying other developmental/differentiation pathways both in academia and industry. (iii) We will organize a workshop where the scientific community and members from industry will be invited to discuss our and their results and new developments in the field. (iv) One significant potential outcome of our work is the identification of transcription factor combinations that may be used to drive the production of HSCs from ES cells or in vitro expansion of cord blood derived HSCs. HSCs are not only of tremendous interest for regenerative medicine applications but would also provide a very attractive source of in vitro cell types for drug development and toxicity screening assays and may thus be of significant commercial benefit. We will make our expertise available to members from industry and academia who wish to explore this possibility. (v) Knowledge of gene networks in stem cells may instigate the development of new strategies to induce stem cells to cross lineage barriers or even trigger the reversal of lineage restrictions in terminally differentiated cells thus renewing stem cell populations with regenerative capacity. The generation of iPS cells, after all, was only possible after the elucidation of the gene regulatory network maintaining pluripotency. (vi) Specifically, we will gather information that will be vital to understand how embryonic endothelial cells can become haemopoietic cells and which transcription factors are required for this process. In addition, our studies of global chromatin accessibility will identify regions within the genome of endothelial cells that are amenable to reprogramming. This will benefit regenerative medicine. (vii) Through our international collaborations there will be a significant knowledge transfer into the UK (viii) Last, but not least, our work will enhance the skills base in the UK. Future advances in biology and medicine will depend on building a skills base consisting of researchers which will be capable of thinking both in molecular terms as well as in system-wide terms, and researchers working in this consortium will be exposed to the forefront of research in this field. In addition, both Leeds and Cambridge run MSc courses on Bioinformatics and Computational Biology respectively and this grant will enable to offer rotation projects to these students.