RUI: Analysis of the Role of CHD1 in Chromatin Structure and Transcription

染色质重塑因子利用ATP的能量来调节染色质的结构，从而调节转录、复制和修复等过程。虽然在这些atp酶的体外作用机制方面已经取得了重大进展，但它们在体内的活性仍然未知。PI的长期目标是了解CHD1染色质重塑和组装因子是如何在体内被调节和靶向的。黑腹果蝇是研究这种不寻常蛋白质作用的理想模型系统。智力优势：本研究的目的是利用果蝇遗传学的强大工具来研究CHD1（一种染色体结构域、解旋酶和dna结合结构域蛋白）在体内的活性。CHD1是一种不同寻常的atp酶，它在体外具有核小体重塑和核小体组装活性。这些实验解决了一个特定的假设，即CHD1在全球范围内对活性基因上的染色质进行重组，这种活动导致转录抑制。这一假设将通过以下三个具体目标来解决：(1)通过检查CHD1突变体的功能获得和功能丧失的表型来研究CHD1的体内功能。(2)确定每个保守蛋白结构域的相对贡献。(3)研究CHD1 atp酶活性的增加和减少对染色体结构和RNA聚合酶II的影响。PI将采用遗传和细胞生物学方法的组合来分析突变表型，染色体形态和转录。这些成熟的技术适用于本科研究，并被PI广泛应用于相关染色质重塑因子Brahma和ISWI的研究中。PI的初步结果和专业知识使她处于一个独特的位置，以实现这些特定的目标，其结果将为CHD1的功能和调控提供重要的见解，CHD1是一种高度保守但知之甚少的atp酶。更广泛的影响：这项研究将主要由克莱蒙特麦肯纳学院、斯克里普斯学院和皮策学院的本科生进行。在联合科学系的前三年，PI亲自在实验室培训了15名本科生（13名女性和2名男性），其中包括两名拉丁裔第一代大学生。学生平均在研究实验室呆两年。在毕业的pi研究生中，100%在研究生院或医学院继续接受科学教育。在这项研究的三年里，PI将亲自在研究实验室培训另外12-15名本科生，为他们提供技能，帮助他们在科学事业上取得成功。PI在一个独特的部门（联合科学部），由三个顶级文科院校共享。斯克里普斯学院是一所女子学院，而克莱蒙特·麦肯纳学院和皮策学院在最具种族多样性的文理学院中分别排名第五和第七。这种多元化的学生群体使PI能够鼓励来自历史上代表性不足的群体的理科生。除了为本科生提供研究机会外，本研究还将允许本科生参加除南加州本科生研究会议外的年度果蝇研究会议。PI的一名本科生是该实验室最近发表在《遗传学》杂志上的论文的合著者。这项RUI研究将为PI和她的学生提供资源，以继续他们目前使用现代分子遗传技术对染色质重塑蛋白的竞争性研究。

Chromatin remodeling factors use the energy of ATP to modulate the structure of chromatin, thereby regulating processes such as transcription, replication, and repair. While significant advances have been made in regards to the mechanism of action of these ATPases in vitro, much remains unknown regarding their activities in vivo. The PI's long-term goal is to understand how the CHD1 chromatin remodeling and assembly factor is regulated and targeted in vivo. Drosophila melanogaster is an ideal model system in which to study the actions of this unusual protein.Intellectual merit: The goal of this research is to use the powerful tools of Drosophila genetics to investigate the in vivo activities of CHD1, a chromodomain, helicase, and DNA-binding domain protein. CHD1 is an unusual ATPase in that it possesses both nucleosome remodeling and nucleosome assembly activities in vitro. These experiments address the specific hypothesis that CHD1 functions globally to reassemble chromatin on active genes, an activity that results in the inhibition of transcription. This hypothesis will be addressed by pursuing three specific aims (1) Investigate the in vivo function of CHD1 by examining phenotypes of both gain of function and loss of function chd1 mutants. (2) Determine the relative contributions of each of the conserved protein domains. (3) Examine the consequences of both gain and loss of CHD1 ATPase activity on chromosome structure and RNA Polymerase II. The PI will employ a combination of genetic and cell biological approaches to analyze mutant phenotypes, chromosome morphology, and transcription. These well-established techniques are amenable to undergraduate research, and have been extensively used by the PI in her studies of related chromatin remodeling factors Brahma and ISWI. The PI's preliminary result and expertise place her in a unique position to accomplish these specific aims, the results of which should provide significant insights into the function and regulation of CHD1, a highly conserved yet poorly understood ATPase.Broader impacts: The research will be carried out primarily by undergraduate science students from Claremont McKenna, Scripps, and Pitzer Colleges. During her first three years in the Joint Science Department, the PI personally trained 15 undergraduates in the laboratory (13 women and two men), including two Latina first-generation college students. Students spend an average of two years in the research laboratory. Of the PIs research students who have graduated, 100% are continuing their science education in graduate or medical schools. Over the three years of this study, the PI will personally train an additional 12-15 undergraduates in the research laboratory, providing them with skills to help them succeed in their careers in science. The PI is in a unique department (The Joint Science Department) that is shared by three top-tier liberal arts institutions. Scripps is an all-women college, while Claremont McKenna and Pitzer College are the 5th and 7th most ethnically diverse liberal arts colleges in the top tier. This diverse student body allows the PI to encourage science students from historically underrepresented groups. In addition to providing research opportunities for undergraduates, this research will allow undergraduates to attend the Annual Drosophila Research Conference in addition to the Southern California Conference for Undergraduate Research. One of the PI's undergraduate research students is a co-author on the lab's most recent publication in Genetics. This RUI research will provide resources for the PI and her students to continue their current, competitive research into chromatin remodeling proteins using modern molecular genetic techniques.