MECHANISTIC INSIGHTS INTO THE DEVELOPMENTAL-STAGE SPECIFIC ACTIVITY OF A UBIQUITOUSLY EXPRESSED TRANSCRIPTION FACTOR

All our cells originate from one single fertilised egg cell which divides many times and gives rise to all the complex cell types in our body. The most fascinating aspect of such developmental processes is the fact that the timing of how different cells form is always the same, thus ensuring that our organs develop in the right order and at the right time. The reason for this synchrony is that all information for our body plan is encoded in our DNA which contains instructions on when our genes should be expressed and when they should be silent. The result is that humans are built with one plan and primates are built by another. However, humans and primates are 99% identical in their DNA, the question therefore arises what is different between the two species. The answer most likely can be found in subtle changes of the order of expression of genes that regulate the expression of other genes, the so-called transcription factors. These proteins can "read" the genetic code and they work together with many other regulator proteins to make sure that a gene is expressed in the right cell and at the right time. In the last years it was found that there are different types of transcription factors: those which are only found in one cell type, and those who are present in many cell types. All types of factors work together to regulate specific genes. When cell-specific factors are missing, usually this cell type does not form. When a factor is missing that is present in every cell, the effects can be dramatic, and often the organism is unable to develop. In this proposal we wish to study a member of the latter class of transcription factors, Sp1. We have shown that without it, blood cell development gets more and more compromised and mature blood cells cannot form. Now we want to know, how Sp1 works, when exactly it is needed and with which other factors it cooperates.

However, not only the timing of expression of transcription factors can be different. Also the levels of how much of these regulators is present in a given cell is critical since small changes in the concentration of a factor that regulates other regulators will have a huge effect in further development. An important example of what differences in factor levels can make is seen in people with Down's syndrome who have an extra chromosome in each cell. They have too much of the products of these genes from this chromosome and their development is strongly perturbed. In this grant, we will change the levels of Sp1 and measure, how such changes impact of the expression of its target genes and how this, in turn, then impacts of how many cells of a given type are formed.
To this end, we will use a system based on mouse embryonic stem cells that mimics normal development in vitro and where we can generate enough cells to examine many different features. Our work will answer a number of fundamental questions on how transcription factors interact with each other and how the balance and timing of important regulator genes is controlled.

Development is regulated by the interplay of tissue-specific and ubiquitously expressed transcription factors, such as Sp1. Sp1 knock-out mice die in utero with multiple phenotypic aberrations. We have used the differentiation of mouse ES cells as a model to compare differentiation potential, global gene expression patterns and Sp1 target regions in Sp1 wild-type and deficient cells representing different stages of haematopoiesis. Sp1-/- cells progress through most embryonic stages of blood cell development but cannot complete terminal differentiation. For most Sp1 target and non-target genes, gene expression is unaffected by Sp1 inactivation. However, Cdx and multiple Hox genes are stage-specific targets of Sp1 and are down-regulated. As a consequence, genes involved in haematopoietic specification are progressively deregulated. The molecular mechanism of this deficiency is unclear.

Our work demonstrates that the early absence of active Sp1 sets a cascade in motion that culminates in a failure of terminal haematopoietic differentiation and emphasizes the role of ubiquitously expressed transcription factors for tissue-specific gene regulation. The current proposal will build on this work. We wish to dissect parts of the Sp1-responsive transcriptional network in a system-wide fashion, by studying (i) at which precise developmental time-point Sp1 is essentially required, (ii) which target genes directly respond to Sp1 presence or absence and how, (iii) how changing levels of Sp1 impact on down-stream target gene expression and last, but not least (iv) to which extent downstream effectors of Sp1 such as the Hox/Cdx genes can fully or partially rescue the Sp1 knock-out phenotypes. Our studies will deliver profound mechanistic insights into how a ubiquitously expressed transcription factor and tissue specific factors interact to drive developmental-stage-specific gene expression and how regulating the levels of Hox/Cdx genes drives differentiation forward.

Who will benefit from this research:
Our work will have an impact not only on our immediate research field but also far beyond. Currently the scientific community has embarked on a quest to not just study single genes within single cells, but to examine biological phenomena in a systems-wide fashion using high-throughput methodologies. The haematopoietic system is an excellent model to perform such studies because is already the subject of multiple systems-wide studies and multiple data-sets are already available that are waiting to be integrated into complex models.

If successful, our work will benefit a number of diverse research fields. This includes
(i) developmental biology, because so far the relevance of ubiquitously expressed transcription factors for the regulation of developmental-stage-specific gene expression is not well understood
(ii) stem cell research, because the molecular details of early haematopoietic specification are unclear
(iii) mathematical modelling and bioinformatics research, because our time-course data and those from a perturbed system will provide ample opportunity for mathematical modelling and developing novel methods for data integration.
(vi) leukaemia research, because Hox genes are important regulators of leukaemogenesis and understanding their regulation will be of utmost importance for the understanding of tumour cell behaviour.

How will they benefit from this research?
(i) We will make our system-wide data sets and network models publicly available.
(ii) We will generate data that will be highly relevant to scientists studying other developmental/differentiation pathways both in academia and industry.
(iii) One significant potential outcome of our work is the identification of how early regulators of haematopietic are regulated. Such knowledge may be used to drive the production of HSCs from ES cells which may be of significant commercial benefit. We will make our expertise available to members from industry and academia who wish to explore this possibility.
(iv) Through our international collaboration there will be a significant knowledge transfer into the UK
(v) 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 on this grant will be exposed to the forefront of research in this field. They will also have the opportunity to be trained at the Institute of Computational Biology.
(vi) Along the same lines, we are attracting students into this area. Birmingham has a number of studentship schemes, and one Wellcome Trust student has already been encouraged to participate in our research. Our data will also form the basis of Master's and PhD projects in the Cazier department.