Control of insulator function and higher order genome organisation by the chromatin remodeling enzyme NURF

体内所有细胞的发育都是由DNA中的基因编码的一组指令决定的。所有单元格都包含相同的信息。人体中细胞类型的巨大变化，每种细胞都有不同的功能，这是通过改变这些信息的读取或“表达”方式来实现的。在真核生物中，正确发育所需的大量DNA被压缩成可管理的单位，就像绕在蛋白质线轴上的线一样，形成称为核小体的结构。除了压缩DNA之外，核小体还提供额外水平的所谓表观遗传信息。通过改变核小体的位置，可以调节基因控制元件的进入，并关闭或打开基因，从而改变细胞的身份和功能。此外，核小体是基因组组织中更高层次的基本单位，通过核小体，通常遥远的基因调控元件之间的功能相互作用得以稳定，从而允许基因表达的正确模式。因此，核小体组织的这种变化可以深刻地影响基因组组织和基因调控。在我们的实验室中，我们研究核小体组织是如何改变染色质重塑酶。这些大的蛋白质复合物使用ATP，细胞能量转移分子，改变核小体的位置，允许基因组结构的大规模重组（“重塑”）。我们试图了解这些复合物是如何工作的，因为许多人类疾病是由改变或紊乱的基因表达和全球基因组组织引发的。通过了解基因组组织和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“模式生物”果蝇（果蝇）。虽然不是立即显而易见的，但果蝇和人类是从一个共同的祖先进化而来的，因此分享了许多设计原则。一个有用的类比是将高性能赛车和儿童卡丁车进行比较。虽然一个更复杂，控制的基本要素-转向和刹车-是相同的。类似地，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，一种所谓的“模式生物”。这是有用的，因为它允许我们在人类身上做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，我们使用果蝇品系，我们可以标记，改变或删除（“敲除”）建立和调节基因组组织的蛋白质复合物，以确定它们在基因调控中的功能。在这项研究中，我们将确定一个关键的染色质重塑酶的功能称为cDNAF基因组组织。果蝇提供了一个特别强大的系统来表征BNF，因为BNF只存在于多细胞真核生物中，排除了在更简单的模式生物如酵母中的研究。在我们的研究中，我们将确定BMPF如何调节对高阶染色质组织至关重要的基因组相互作用。为此，我们将使用一种称为染色体构象捕获（ChIA-PET）的技术的变体，从细胞核中钓出BMPF复合物。然后，我们可以共同纯化通过BNF在物理上接近的基因组区域，并通过确定它们的DNA序列来识别它们。通过在这些位点上基因切除BMPF和辅因子蛋白，我们将确定BMPF在建立这些调控基因组相互作用中的功能重要性。该方法将提供高阶调控基因组相互作用的高分辨率视图，但需要数百万个细胞来执行，从而生成在许多细胞上平均的相互作用的快照。为了在短时间内捕获单细胞中重塑基因组相互作用的动态，我们还将部署先进的显微镜技术，允许活细胞中单个重塑分子和绝缘分子的成像。

The development of all cells in the body is determined by a set of instructions encoded in genes, found in DNA. 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, the large amount of DNA required for correct development is compacted into manageable units by wrapping like thread around a protein spool to form structures called nucleosomes. In addition to compacting DNA, nucleosomes provide additional levels of so-called epigenetic information. By varying nucleosome position, access to gene control elements can be regulated and genes turned off or on, changing cell identity and function. Moreover, nucleosomes are the fundamental units in higher orders of genome organisation through which functional interactions between often-distant gene regulatory elements are stabilized, allowing correct patterns of gene expression. As such changes in nucleosome organisation can profoundly affect genome organisation and gene regulation. In our laboratory we study how nucleosome organisation is altered by chromatin remodeling enzymes. These large protein complexes use ATP, the cells energy transfer molecule, to change nucleosome position, allowing the wholesale reorganization ("remodeling") of genome architecture. We seek to understand how these complexes work as many human diseases are triggered by altered or disordered gene expression and global genome organization. By understanding the epigenetic mechanisms underlying genome organisation and gene regulation, new therapies to cure disease can be developed. In our research, we use the "model organism" Drosophila melanogaster (fruit flies). Although not immediately be apparent, Drosophila and humans have evolved from a common ancestor and thus share many design principles. A useful analogy is to compare a high-performance racing car and a child's go-kart. While one is more sophisticated, the basic elements of control - steering and brakes - are the same. Similarly, 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 regulate genome organisation to determine their function in gene regulation. In this study we will determine functions of a key chromatin remodeling enzyme called NURF in genome organisation. Drosophila provides an especially powerful system to characterize NURF, as NURF is only present in multicellular eukaryotes precluding studies in simpler model organisms like yeast. In our research we will determine how NURF regulates genome interactions critical for higher order chromatin organisation. To do this we will use variants of a technique called chromosome conformation capture (called ChIA-PET) to fish-out NURF complexes from nuclei. We can then co-purify regions of the genome brought into close physically proximity by NURF and identify these by determining their DNA sequence. Through genetic ablation of NURF and co-factor proteins at these sites we will determine the functional importance of NURF in establishing these regulatory genome interactions. This method will provide a high-resolution view of higher order regulatory genome interactions but requires many millions of cells to perform thus generating a snapshot of interactions averaged over many cells. To capture the dynamics of remodeler genome interactions in single cells over short time scales genome interactions we will also deploy advanced microscopy techniques that allow imaging of single remodeler and insulator molecules in live cells.