Nucleosome positioning and transcriptional regulation in Drosophila differentiated cells

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Each cell in a multicellular animal expresses (transcribes) only about half of all its 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 its identity, and is established during development and cellular differentiation. 10-20% of all the genes in the genome are transcribed exclusively in testes, in cells destined to differentiate into sperm; 1-2% of all genes are transcribed exclusively in muscle cells. Regulation of gene transcription involves several processes working in concert - notably DNA packaging and sequence-specific DNA-binding factors, which then recruit the transcription machinery. The DNA in the cell is packaged with proteins to form chromatin. The fundamental packing unit is a nucleosome - protein complexes around which the DNA strand is wrapped, forming a beads-on-a-string structure. The precise position of each nucleosome is not determined by the DNA sequence directly and varies between cell-types. On average, in most studied cell-types, nucleosomes are found in a canonical pattern positioned around genes, particularly in the region of DNA where transcription of the gene starts (the transcription start site, TSS). This positioning of nucleosomes is thought important for allowing access of proteins that bind specific DNA sequences and regulate transcription of the adjacent gene. On average, genes that have higher expression levels have more accurately positioned nucleosomes. However, the studies that have led to this view have used tissue culture cells or mixed cell populations. Differentiated cells have not been examined.We have extensive preliminary data to indicate that many genes transcribed at extremely high levels in spermatocytes, the cells that give rise to sperm, do not follow these established rules regarding nucleosome positioning. These data cast doubt on the conclusion that high gene expression and canonical nucleosome positioning are necessarily linked. They also raise the question of how spermatocytes achieve a high level of transcription of these genes, and whether other cell-types also deviate from established rules. These genes rely on a specific transcription factor complex, termed testis meiotic arrest complex (TMAC); we have found that nucleosome positioning at TMAC-target TSSs is altered when this complex is mutated.In this project we have three main aims1) To characterise the nucleosome positioning at TSSs in spermatogonia, the precursors of spermatocytes to reveal how the positioning of nucleosomes changes as genes are turned on and off in this lineage. We will further determine the effect of loss of specific nucleosome positioning factors and transcriptional regulators at these sites. We will map the binding sites of TMAC and other regulators across the genome in these cells.2) To examine whether the unusual pattern of nucleosome position we see for testis-specific gene expression is also seen in other cell-type specific gene expression programmes, or whether the canonical pattern is used in this situation. In either case we will determine how the pattern changes in differentiation. We will analyse nucleosome position around TSSs in adult muscle cells, and their immediate precursor cells.3) To understand the molecular details of how high levels of gene expression are driven in the absence of canonical nucleosome positioning at TSSs in spermatocytes we will characterise the DNA sequences responsible for the testis-specific expression. We will determine how they interact with TMAC and how they influence local nucleosome position in their normal location and when we move them to a new position in the genome.