Regulation of the transcription cycle by co-ordinate interaction of ATP-dependent chromatin remodelling and histone post-translational modifications.

体内所有细胞的发育和特性是由一套编码在基因中的指令决定的，这些指令存在于细胞核的DNA中。所有单元格都包含相同的信息。人体内各种各样的细胞类型，每一种都有不同的功能，是通过改变这些信息的读取或“表达”的方式来实现的。在真核生物中，例如人类，DNA被折叠并压缩成可管理的单位，方法是像线一样缠绕由称为组蛋白的蛋白质组成的卷轴，形成染色质。这些可管理的单位被称为核小体，为细胞提供了额外的信息，因为其核心的组蛋白可以通过加入醋酸盐、磷酸盐、甲基或其他化学基团来选择性地修饰。这些组蛋白翻译后修饰(HPTM)可以作为所谓的表观遗传“标记”，在基因组中编码额外的信息。这些标记可以起到锚定或招募酶复合体的作用，这些酶复合体可以沉默或允许基因表达。通过改变组蛋白翻译后修饰的分布以及它们被读取或解码的能力，基因可以在细胞中选择性地关闭或打开，控制细胞的发育和功能。我们试图了解这种情况是如何发生的，因为许多人类疾病，如癌症和淋巴瘤，都是由基因表达改变或紊乱引发的。通过了解这些基因调控的表观遗传机制，可以开发治疗疾病的新疗法。在我们的研究中，我们使用了人类细胞系和“模式生物”黑腹果蝇(果蝇)。尽管乍一看可能看起来并非如此，但果蝇和人类是从共同的祖先进化而来的，因此有许多共同的设计原则。一个有用的类比是将高性能赛车与儿童卡丁车进行比较。尽管更复杂，但控制的基本要素--转向和刹车--是相同的。以同样的方式，果蝇使用许多与人类相同的机制来控制基因表达。因此，我们可以用果蝇作为人类的替身，所谓的“模式生物”。这很有用，因为它允许我们进行对人类不可能或不道德的实验，例如故意删除或改变基因，以确定它们在发育中的作用。在我们的工作中，我们使用了苍蝇品系，在这些品系中，我们可以标记、改变或删除(“敲除”)建立和解释表观遗传标记的蛋白质复合体，以确定它们在基因调控中的功能。在这项研究中，我们将确定组蛋白翻译后修饰如何影响名为NURF的关键表观遗传调节因子的分布。我们将确定NURF如何改变核小体的位置。通过改变核小体的位置，NURF可以影响RNA聚合酶(表达基因的酶)与DNA的相互作用。为了做到这一点，我们将使用一种名为染色质免疫沉淀(芯片)的技术来寻找NURF所针对的果蝇基因组区域，并通过使用DNA测序仪确定它们的DNA序列来识别这些区域，DNA测序仪可以一次性对数百万个DNA片段进行测序。我们将研究NURF招募是否与组蛋白修饰的存在相关。随后，我们将使用相同的CHIP-Seq技术来确定NURF在这些区域的招募是否能够影响RNA聚合酶的活性，方法是使用相同的CHIP-Seq技术来定位RNA聚合酶在含有或不含有NURF调节器的细胞中的分布以及NURF可以结合的组蛋白修饰。

The development and identity of all cells in the body is determined by a set of instructions encoded in genes, found in DNA in the cell nucleus. All cells contain the same information. The immense variety of cell types in the human body, each with distinct functions is achieved by changing the way this information is read, or "expressed". In eukaryotes, such as humans, DNA is folded and compacted into manageable units by wrapping like thread around a spool composed of proteins called histones, to form chromatin. These manageable units, called nucleosomes, offer an additional level of information to the cell as the histone proteins at their core can be selectively modified by the incorporation of acetate, phosphate, methyl or other chemical groups. These histone post-translational modifications (HPTMs) can act as so-called epigenetic "marks" to encode additional information in the genome. These marks can function to tether or recruit enzyme complexes that either silence or allow gene expression. By varying the distribution of histone post-translational modifications, and their ability to be read or decoded, genes can be selectively turned off or on in cells, controlling the development and function of cells. We seek to understand how this occurs as many human diseases, such as cancers and lymphomas, are triggered by altered or disordered gene expression. By understanding these epigenetic mechanisms of gene regulation, new therapies to cure disease can be developed. In our research, we use both human cell lines and the "model organism" Drosophila melanogaster (fruit flies). Although at first glance it may not seem so, Drosophila and humans have evolved from a common ancestor and thus share many design principals. A useful analogy is to compare a high-performance racing car and a child's go-kart. Although one is more sophisticated, the basic elements of control - steering and brakes - are the same. In the same way, Drosophila uses many of the same mechanisms to control gene expression as humans. As such, we can use Drosophila as a stand-in for humans, a so-called "model organism". This is useful as it allows us to do experiments that are impossible or unethical in humans, for example deliberately deleting or altering genes to determine their role in development. In our work we use fly strains in which we can tag, alter or delete ("knock-out") the protein complexes that establish and interpret epigenetic marks to determine their function in gene regulation. In this study we will determine how the distribution of a key epigenetic regulator called NURF is affected by histone post-translational modifications. We will determine how NURF then alters the positions of nucleosomes. By changing the position of nucleosomes NURF can affect the interaction of RNA polymerase (the enzyme that "expresses" genes) with DNA. To do this we will use a technique called chromatin immunoprecipitation (ChIP) to fish-out regions of the Drosophila genome to which NURF is targeted and identify these by determining their DNA sequence using a DNA sequencer that can sequence millions of DNA fragments in one go. We will examine whether NURF recruitment correlates with the presence of histone-modifications. Subsequently we will determine whether NURF recruitment to these regions is able to affect the activities of RNA polymerase by using the same technique of ChIP-Seq to map the distribution of RNA polymerase on genes in cells that contain or lack the NURF regulator and the histone modifications to which NURF can bind.