Sox transcription factor function and redundancy in the central nervous system

During the early development of complex multicellular organisms such as humans, cells must adopt particular fates in order to generate the variety of tissues and organs necessary to build the embryo. Early in development, specific sets of cells gain the ability to subsequently develop the various cell types of the nervous system. Once specified, this cell population will divide in an undifferentiated state, known as neural stem cells, to generate sufficient cells that can subsequently be directed to make neurons and other cell types necessary to build a nervous system. Not only are these neural stem cells important for normal development, they may also be isolated or generated from other cell types and grown in the laboratory. It is hoped that neural stem cells will in the future provide a route for the treatment of human neurological disorders that are currently intractable.

Underpinning the developmental choices cells make and their maintenance of the stem cell state are sets of proteins known as transcription factors (TFs) that act in the cell nucleus to control the specific sets of genes that define the neural state. One such class of TFs important in neural stem cells are known as Sox proteins. While there has been considerable work aimed at addressing how Sox proteins act to control the stem cell state in mammals, this work is complicated by the fact that three closely related proteins are present in neural cells at the same time and compensate for each other when mutations are made. This makes it difficult to understand how these proteins function and this is an important issue since they play such a crucial role in stem cell biology.

The fruit fly, Drosophila melanogaster, is a model system widely used in the laboratory to study basic aspects of the genetics and development of complex multicellular animals. In general, the fly offers a much simpler system for studying basic biological processes since it is easy to maintain, easy to manipulate genetically and does not raise concerns about excessive animal use in experimental work. Over the years it has been established that many of the cell fate choices fly cells make are governed by sets of regulatory proteins that are very closely related to mammalian proteins performing similar roles. In the case of Sox proteins acting in the nervous system, we have shown the fly offers a simpler experimental system that still shares some of the complexity shown by mammalian proteins. Instead of three Sox proteins, the fly has only two, and we have shown that mouse and human Sox proteins are able to efficiently function in the fly.

Sox proteins function by controlling sets of genes that define the phenotype of a cell and our recent work has shown that Sox proteins in fly and mouse neural stem cells control many of the same genes. However, despite a considerable amount of work on both mammalian and fly Sox proteins we still have a very poor mechanistic understanding of how they act to regulate their target genes. If we are to generate and manipulate neural stem cells in that lab for therapeutic uses, it is important we fully understand the roles Sox proteins play, particularly since they are now often used to produce and maintain stem cells.

We will perform a detailed analysis of both fly and mammalian Sox proteins in the Drosophila model to understand more fully how they recognize the specific genes they control in the nucleus, how related Sox proteins act together and are able to compensate for each others loss and explore exactly why important cells types such as neural stem cells need to express closely related Sox proteins. Although our work is performed in the fly, the fact that Sox function is so similar in fly and mouse means that what we learn will be relevant to human biology.

SoxB1 proteins are crucial regulators of cell fate in the vertebrate CNS and play key roles in ES and iPS cells. Sox1, 2 & 3 are widely coexpressed in the vertebrate CNS where they act redundantly to specify neural fate and maintain neural stem cells. The unusually high degree of genetic redundancy shown by SoxB1 proteins complicates their analysis in vertebrates and while there has been considerable SoxB1 genomic analysis, there are many unanswered questions relating to how they control regulatory networks. Drosophila, which has two SoxB1 genes with redundant functions in the developing CNS, offers an tractable system for studying conserved aspects of SoxB1 biology. Mammalian SoxB1 genes rescue fly mutant phenotypes, and fly and mouse SoxB1 proteins regulate very similar sets of target genes in neural cells. We will address important facets of conserved SoxB1 biology in the fly: characterizing how SoxB1 proteins bind and interact at defined enhancers of CNS expressed genes; generating nucleotide level binding maps of SoxB1 proteins in a single neural cell type; directly addressing the ability of fly and mammalian SoxB1 proteins to functionally compensate in vivo; and testing the hypothesis that SoxB1 redundancy has been maintained over evolution to provide robustness to regulatory networks defining neural stem cells. We will analyse identified CRMs in vivo, including the use of specific ChIP and re-ChIP assays, to characterise SoxB1 binding interactions. We will generate nucleotide level SoxB1 binding data when prologues are expressed singly or together and relate binding to chromatin state via genome wide ChIP in a single cell type, using these data to define binding sequence contexts. By CRISPR/Cas9 engineering, we replace SoxB1 coding sequences with fly and mammalian paralogues to fully assess functional redundancy. We will test SoxB1 target gene expression in stressed animals with altered SoxB1 gene dosage to assess how redundancy contributes to robustness.

Who will benefit
As described in the previous section, academic researchers are obvious direct beneficiaries of the research proposed in this application. As we note, the impact of our findings are likely to cut across a much wider community than fly biologists, with researchers working in areas such as mammalian CNS development, evolutionary biology, stem cell biology and human disease biology likely to benefit from our work. Over the past decade we have collaborated with many individual researchers and research groups to perform genomics experiments. This includes providing training and helping researchers perform genomics experiments. By maintaining research in leading edge genomic approaches we maintain this valuable facet of our laboratories outreach. Staff directly employed on the grant will develop analytical and experimental skills as well as more generally applicable skills such as scientific writing and public presentation. We alluded to how indirect benefits may accrue more widely in the biomedical research community, including biotechnology companies pioneering the use of iPS cells. More nebulously, we believe our continuing development of genomics approaches, particularly our focus on developing methods for exploring the genome in vivo, helps maintain the profile of UK genomics research in the international arena.

How will they benefit
Obviously, academic researchers benefit by reading our papers or accessing our data, including those close to the biology of Sox function as well as those with more peripheral interests, for example in general aspects of transcriptional regulation. The provision, via public repositories, of both the raw and processed datasets we generate will be beneficial to the computational biology/bioinformatics community. In terms of training, maintaining an active fly genomics research group allows us to continue to offer hotel space for visiting researchers from Cambridge and elsewhere in the UK. We believe it is important, particularly for graduate students and postdocs, that researchers are exposed to genomics technologies and that they have hands-on experience of generating and analysing such data. All of our own graduate students and postdocs are competent in all areas of genomics, and our core staff are constantly learning new methods on the job. These benefits accrue over the period of the grant as well as after the end date, the data are available for as long as the public repository. The longer term benefits, particularly to biotechnology, are more difficult to quantify but certainly the provision of trained researchers is a potential immediate benefit. How our insights impact on our understanding of mammalian stem cell biology and CNS development will remain to be seen but Drosophila has an excellent track record in this regard.

What will be done
We publish our data and present results at local, national and international scientific meetings, ensuring exposure to our work. We publicise our work within to the wider research community via traditional routes and social media. We are constantly exploring the establishment of collaborative work: of the current or recently finished research grants held by the PI, 3 are international collaborations, 4 are collaboration within Cambridge and 1 is a single PI grant. To help project the results of our fly work to the wider community we present at fly and developmental biology meetings, we participate in the local East of England Stem Cell community and the recently established Physics of Medicine initiative in Cambridge.