INVESTIGATING THE ROLE OF RSC4 ACETYLATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 对于每个真核生物来说，一个复杂的问题是如何调控染色质重塑的过程，染色质重塑是调节整个基因组中DNA可及性的关键功能。利用加州大学旧金山分校的质谱仪，我们发现RSC4是RSC重塑复合体的一个重要亚单位，在K25的N端被Gcn5乙酰化。标准生长条件下RSC的纯化表明，RSC4的大部分是乙酰化的。WT和突变(K25R)RSC复合物的体外比较表明，它们在重塑核小体、结合DNA和识别乙酰化方面具有相似的能力。由于乙酰化似乎不会改变RSC的酶性质，我们正在采用几种活体方法来了解乙酰化在其中重要的生物过程和了解乙酰化的功能后果。具体地说，我们将使用质谱学来确定乙酰化受到调控的条件，并结合基因突变来确定负责这一调控的基因。这些研究首次在染色质重塑复合体的背景下表征了乙酰化的功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A complex problem for every eukaryotic organism is how to regulate the process of chromatin remodeling, a critical function to mediate DNA accessibility throughout the genome. Using the UCSF Mass Spec Facility, we have found that Rsc4, an essential sub unit of the RSC remodeling complex, is acetylated, by Gcn5, on K25 at its N-terminus. Purification of RSC under standard growth conditions suggests that most of Rsc4 is acetylated. In vitro comparisons of WT and mutant (K25R) RSC complexes reveal similar capabilities to remodel nucleosomes, bind DNA, and recognize acetylation. Since acetylation does not appear to alter the enzymatic properties of RSC, we are taking several in vivo approaches to understand the biological processes in which acetylation is important and to understand the functional consequences of the acetylation. Specifically, we will use mass spectrometry to identify conditions where acetylation is regulated and, combined with genetic mutations, identify the genes responsible for this regulation. These studies are the first to characterize the function of acetylation in the context of a chromatin remodeling complex.