Nucleosome positioning and transcriptional regulation in Drosophila differentiated cells

多细胞动物的每个细胞只表达（转录）其所有基因的一半。一些基因在所有细胞中转录，一些在细胞类型的子集中转录，而一些仅在单个细胞类型中转录。细胞中正确的基因表达，在发育和分化中打开和关闭基因，对于正常的细胞功能，正常的生物体发育和终身健康至关重要。每个单个细胞的基因表达谱由其身份决定，并在发育和细胞分化过程中建立。基因组中所有基因的10-20%仅在睾丸中转录，在注定分化成精子的细胞中;所有基因的1-2%仅在肌肉细胞中转录。基因转录的调控涉及几个协同工作的过程-特别是DNA包装和序列特异性DNA结合因子，然后招募转录机器。细胞中的DNA与蛋白质一起包装形成染色质。基本的包装单位是核小体-蛋白质复合物，DNA链缠绕在其周围，形成串珠结构。每个核小体的精确位置不是由DNA序列直接决定的，并且在细胞类型之间变化。平均而言，在大多数研究的细胞类型中，核小体以典型模式定位在基因周围，特别是在基因转录开始的DNA区域（转录起始位点，TSS）。核小体的这种定位被认为对于允许结合特定DNA序列并调节相邻基因转录的蛋白质进入是重要的。平均而言，具有较高表达水平的基因具有更准确定位的核小体。然而，导致这种观点的研究使用了组织培养细胞或混合细胞群。我们还没有对分化的细胞进行研究，我们有大量的初步数据表明，在精母细胞（产生精子的细胞）中，许多基因转录水平极高，不遵循这些关于核小体定位的既定规则。这些数据对高基因表达和典型核小体定位必然相关的结论提出了质疑。他们还提出了精母细胞如何实现这些基因的高水平转录的问题，以及其他细胞类型是否也偏离了既定的规则。这些基因依赖于一个特定的转录因子复合体，称为睾丸减数分裂阻滞复合体（TMAC）;我们已经发现，当该复合物突变时，核小体在TMAC-靶TSS上的定位发生改变。在本项目中，我们有三个主要目的：1）确定核小体在精原细胞中TSS上的定位，精母细胞的前体细胞，以揭示核小体的定位如何随着基因在这一谱系中的开启和关闭而变化。我们将进一步确定特定核小体定位因子和转录调节因子在这些位点丢失的影响。我们将在这些细胞的基因组中绘制TMAC和其他调节因子的结合位点。2）为了检查我们在睾丸特异性基因表达中看到的核小体位置的不寻常模式是否也在其他细胞类型特异性基因表达程序中看到，或者在这种情况下是否使用了典型模式。在这两种情况下，我们将确定模式如何在分化中变化。我们将分析成年肌肉细胞中TSS周围的核小体位置，以及它们的直接前体细胞。3）为了了解在精母细胞中TSS缺乏典型核小体定位的情况下如何驱动高水平基因表达的分子细节，我们将分析负责睾丸特异性表达的DNA序列。我们将确定它们如何与TMAC相互作用，以及它们如何影响正常位置的局部核小体位置，以及何时将它们移动到基因组中的新位置。

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.