Testis-specific activation of gene expression

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Each cell in a multicellular animal expresses (transcribes) a subset of all the available genes. Some genes are transcribed in all cells, some in a subset of cell types, while some are transcribed only in a single cell type. Correct gene expression in cells, both turning on and turning off genes in development and differentiation, is essential for normal cellular function, for normal organism development, and for lifelong health. The gene expression repertoire of each individual cell is determined by the cell's identity, and is established during development and cellular differentiation. Sperm are extremely specialised cells, capable of performing several unique biological functions including fertilisation of the egg. In humans, and most other animals, 10-20% of all the genes in the genome are transcribed exclusively in testes, in cells destined to differentiate into sperm, and this expression is critical for the formation and function of sperm, and thus for normal reproduction. Surprisingly, very little is known about the regulatory elements that drive testis-specific gene expression, despite how critically important this is for fertility.In this project we will use the fruit fly, Drosophila melanogaster, to investigate how transcription of testis-specifically expressed genes is activated. We have previously identified a set of proteins that work together as a complex to bind to DNA at the regulatory regions of target genes and activate their expression. Four of the proteins within this complex contain regions that we predict bind directly to DNA. We know that only a very short region of DNA sequence at the start site of testis-specific genes is needed for them to be active. However, the actual DNA sequences of these short regions bear little similarity with one another, when comparing between the over 1000 genes regulated by this complex. How is it that this complex recognises apparently very different stretches of DNA, binds to them, and causes activation of the adjacent gene, while ignoring other DNA sites?We will answer this question using a set of independent experimental approaches coupled with interlinked and integrative computational analyses.1) We will determine the DNA sequence binding preference for each of the DNA-binding proteins in our complex. This will allow us to look at where these sequences are found in the whole genome - we expect them to be enriched at the positions of testis-expressed genes. 2) We will identify all the sites in the genome at which each of complex proteins are found in cells in the testis - we expect them to be enriched at the positions of testis-expressed genes. 3) We will make mutant versions of the proteins that can still contribute to the complex but that can no longer bind DNA, and then identify all the sites in the genome at which these mutant proteins are found in cells in the testis, and what effect this has on gene expression - we expect binding at some sites to be lost, and that the genes associated with these sites will no longer be expressed. 4) The results of these experiments will generate a clearer understanding of the biological rules that determine testis-specific gene expression in response to our protein complex. To determine whether we actually understand these rules we will test the activity of a set of mutant versions of gene control regions that we predict should, or should not, activate gene expression.5) Finally, newly evolving genes are often expressed exclusively in testes, and their expression requires our regulatory protein complex. How do these genes gain this expression pattern? We will begin to test this by directing our complex to different regions of the genome and evaluating whether targeting it to a particular region is sufficient to activate expression of nearby DNA sequences.