Establishment of the haemopoietic transcriptional programme: From systems approaches to molecular mechanisms
Lead Research Organisation:
University of Manchester
Department Name: Cancer Research UK Manchester Institute
Abstract
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.
Technical Summary
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?
Planned Impact
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.
Publications
Thambyrajah R
(2016)
New insights into the regulation by RUNX1 and GFI1(s) proteins of the endothelial to hematopoietic transition generating primordial hematopoietic cells.
in Cell cycle (Georgetown, Tex.)
Thambyrajah R
(2015)
GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1
in Nature Cell Biology
Thambyrajah R
(2018)
HDAC1 and HDAC2 Modulate TGF-ß Signaling during Endothelial-to-Hematopoietic Transition
in Stem Cell Reports
Vijayabaskar MS
(2019)
Identification of gene specific cis-regulatory elements during differentiation of mouse embryonic stem cells: An integrative approach using high-throughput datasets.
in PLoS computational biology
Villa F
(2021)
CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.
in EMBO reports
Wareing S
(2012)
ETV2 expression marks blood and endothelium precursors, including hemogenic endothelium, at the onset of blood development.
in Developmental dynamics : an official publication of the American Association of Anatomists
Wareing S
(2012)
The Flk1-Cre-mediated deletion of ETV2 defines its narrow temporal requirement during embryonic hematopoietic development.
in Stem cells (Dayton, Ohio)
Description | All blood cells originate from stem cells that reside in the bone marrow. Healthy stem cells and their progeny churn out trillions of blood cells every day. However, in certain diseases such as cancers of the blood, stem cells can become dysfunctional and need to be replaced by healthy cells. It is therefore of vital importance to understand, how the blood system develops in the first place. Cells with the ability to give rise to blood develop very early in the embryo and are then maintained for life. Blood cell development is regulated by our genes and by the way their activity is controlled at different stages of embryo development. Importantly, this process involves not just one gene, but thousands of them. Funded by a larger and longer grant from the BBSRC, a consortium of researchers from the Universities of Cambridge (Bertie Göttgens), Leeds (David Westhead) and Manchester (Valerie Kouskoff and Georges Lacaud) led by Constanze Bonifer at the University of Birmingham have now examined six different stages of blood cell development in fine detail, by collecting information on the activity of thousands of different genes, and they have identified the factors that control their activity. This study, published in Developmental Cell, shows the computational analysis of such "big data" and show experiments that reveal so far unknown regulators of blood cell development. Moreover, by uncovering which factors regulate the majority of blood cell specific genes, the Manchester group could harness this knowledge to define the minimum requirements to generate blood cells from another cell type. To reach out to the scientific community and the interested public, the Leeds group generated a website with browsing capabilities that allows unlimited data access (www.haemopoiesis.leeds.ac.uk). In our work we examined how cells develop towards blood by collecting "multi-omics" data, from measuring gene activity, changes in the structure of their chromosomes and the interaction of regulatory factors with the genes themselves. Our research shows in unprecedented detail how blood cell development is controlled by vast networks of interacting genes. In addition, our new publicly available data resource will help many researchers to study additional regulators. This knowledge is essential, if we want to understand how blood cells can become malignant, but also for generating stem cells for regenerative medicine. |
Exploitation Route | We provided a very powerful ressource to identify and study genes implicated in blood cell development and malignancies. |
Sectors | Education Healthcare Pharmaceuticals and Medical Biotechnology |
URL | http://www.bioinformatics.leeds.ac.uk/labpages/hematopoiesis/ |
Description | Mechanistic Insights into Priming and Early gene activation processes in the Haematopoietic system |
Amount | £300,000 (GBP) |
Funding ID | BB/F000499/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2007 |
End | 10/2010 |
Title | DamID pipeline |
Description | Bioinformatic pipeline to analyse DamID sequences and perform peaks detection |
Type Of Material | Technology assay or reagent |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | manuscripts |
Title | Mouse mutant for RUNX1 isoforms |
Description | Deletion or replacement of RUNX1 distal isoform of RUNX1 |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Provided To Others? | No |
Impact | manuscripts under preparation |
Title | Mouse reporter for RUNX1 isoforms expression |
Description | Despite the functional significance of Runx1, the relative and specific activities of its 2 promoters remain largely uncharacterized. To investigate these activities, we introduced 2 reporter genes under the control of the proximal and distal promoters in embryonic stem cell and transgenic mouse lines. |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Provided To Others? | No |
Impact | Our study reveals that both in vitro and in vivo the proximal Runx1 isoform marks a hemogenic endothelium cell population, whereas the subsequent expression of distal Runx1 defines fully committed definitive hematopoietic progenitors. Interestingly, hematopoietic commitment in distal Runx1 knockout embryos appears normal. Altogether, our data demonstrate that the differential activities of the 2 Runx1 promoters define milestones of hematopoietic development and suggest that the proximal isoform plays a critical role in the generation of hematopoietic cells from hemogenic endothelium. Identification and access to the discrete stages of hematopoietic development defined by the activities of the Runx1 promoters will provide the opportunity to further explore the cellular and molecular mechanisms of hematopoietic development. |
Title | RUNX1 binding sites in haemogenic endothelium |
Description | We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE. |
Type Of Material | Database/Collection of data |
Year Produced | 2014 |
Provided To Others? | Yes |
Impact | Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE.This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development. |
URL | http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE55335 |
Title | chromatin, transcription factors binding during hematopoietic developemnt |
Description | dymanic study of the regulation of expression upon haematopoietic development. |
Type Of Material | Database/Collection of data |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | Publication as a ressource in Dev. cell |
Description | Collaboration with Dr Valerie Kouskoff/ lola BBSRC |
Organisation | University of Manchester |
Department | Cancer Research UK Manchester Institute |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Scientific collaboration |
Collaborator Contribution | Scientific collaboration |
Impact | publication, datasets |
Start Year | 2010 |
Description | Collaboration with Prof B. gottgens |
Organisation | University of Cambridge |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Scientific collaboration |
Collaborator Contribution | Scientific collaboration |
Impact | publications, datasets |
Start Year | 2010 |
Description | Collaboration with Prof C. Bonifer/lola |
Organisation | University of Birmingham |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Scientific collaboration |
Collaborator Contribution | Scientific collaboration |
Impact | Publications, collaboration funded by BBSRC award |
Start Year | 2007 |
Description | Collaboration with Prof D. Westhead/ Lola |
Organisation | University of Leeds |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Scientific collaborations |
Collaborator Contribution | Scientific collaborations |
Impact | publications, datasets |
Start Year | 2010 |
Description | Chairing CHO 2013 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | share information and instigate discussion share information |
Year(s) Of Engagement Activity | 2013 |
Description | Colloquium 2013 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | share information, education share information |
Year(s) Of Engagement Activity | 2013 |
Description | Seminar NIG |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | share information, education share information |
Year(s) Of Engagement Activity | 2013 |
Description | seminar Jutendo |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | share information, education share information |
Year(s) Of Engagement Activity | 2013 |