Gatekeeper Chromatin and Meiotic Commitment In Yeast

分化成新的细胞类型是动物、植物和许多其他真核生物发育的关键一步。 该项目将研究如何使用酵母作为模型系统来调节这一重要过程，并将揭示控制细胞命运决定的新机制。该项目还将扩大有才华的学生谁将追求职业生涯在科学上的磁铁高中暴露天才学生的科学发现的过程中，为大三和大四之间的大学生提供动手研究的机会，并培训研究生在假设驱动的分子遗传学研究。与多细胞生物中的分化程序类似，酵母中的孢子形成由转录级联控制。孢子形成的转录级联由早期、中期和晚期基因组组成。直到中间基因表达后才能形成孢子。ndt 80是激活中间启动子的转录因子，它在中间基因表达之前转录。NDT 80启动子可以被视为一个平台，其中产生开关样输出，使细胞进行配子发生。本实验室进行的研究表明，NDT 80启动子组装成一个类似于沉默的异染色质的状态，特别是在减数分裂发育过程中。这种异染色质状态（称为“看门染色质”）需要一种名为Sum 1的DNA结合阻遏蛋白，该蛋白招募一种名为Hst 1的组蛋白脱乙酰酶（Sir 2的近亲）。在减数分裂G2结束时（就在定型点之前），Sum 1在多个残基上被细胞周期蛋白依赖性激酶（CDK）和称为Ime 2的减数分裂特异性CDK样激酶磷酸化。一个中心的假设，将在这个项目中进行测试的是，磷酸化的Sum 1下调Hst 1沉默调节蛋白，触发异染色质的看门人染色质的过渡到一个状态，其中Sum 1是可交换的DNA。由此产生的Sum 1阻遏物的分数占用允许低水平NDT 80表达。当新翻译的Ndt 80蛋白竞争性地从DNA中置换Sum 1以启动正性自动调节环（Sum 1和Ndt 80竞争性地结合NDT 80启动子中的单个DNA元件）时，获得承诺。在这个项目中，这个“看门人染色质模型”的承诺将被测试。该项目的具体目标是：1）建立一种检测染色质中Sum 1蛋白交换的方法; 2）将联合收割机Sum 1磷酸位点突变体和类似物敏感性激酶结合起来，创建一种系统，该系统在NDT 80时同步经历关/开看门人染色质转换，以响应特定的磷酸化修饰，并使用该系统定义控制该转换的生化步骤。

The commitment to differentiate into new cell types is a crucial step in the development of animals, plants and many other eukaryotes. This project will study how this important process is regulated using yeast as the model system, and will uncover new mechanisms for controlling cell fate decisions. The project will also expand the pool of talented students who will pursue careers in science by exposing gifted students from a magnet high school to the process of scientific discovery; providing college students between their junior and senior year with hands-on research opportunities; and training graduate students in hypothesis-driven molecular genetic research. Similar to differentiation programs in multicellular organisms, sporulation in yeast is governed by a transcriptional cascade. The transcriptional cascade of sporulation consists of early, middle and late sets of genes. Commitment to sporulation is not acquired until middle genes are expressed. Ndt80 is the transcription factor that activates middle promoters, and it is transcribed just before middle genes are expressed. The NDT80 promoter can be viewed as a platform where a switch-like output is generated that commits cells to gametogenesis. Studies performed in this laboratory indicate that the NDT80 promoter is assembled into a state resembling silenced heterochromatin specifically during meiotic development. This heterochromatic state (referred to as "gatekeeper chromatin") requires a DNA-binding repressor protein named Sum1 that recruits a histone deacetylase named Hst1 (a close relative of Sir2). At the end of meiotic G2 (just prior to the commitment point) Sum1 is phosphorylated on multiple residues by a cyclin-dependent kinase (CDK), and a meiosis-specific CDK-like kinase named Ime2. A central hypothesis that will be tested in this project is that the phosphorylation of Sum1 downregulates the Hst1 sirtuin to trigger the transition of heterochromatic gatekeeper chromatin to a state in which Sum1 is exchangeable with DNA. The resulting fractional occupancy of the Sum1 repressor permits low-level NDT80 expression. Commitment is acquired when newly translated Ndt80 protein competitively displaces Sum1 from DNA to initiate a positive autoregulatory loop (Sum1 and Ndt80 competitively bind to a single DNA element in the NDT80 promoter). In this project, this "gatekeeper chromatin model" for commitment will be tested. The specific goals of the project are 1) To establish an assay to monitor the exchangeability of the Sum1 protein in chromatin and 2) to combine sum1 phosphosite mutants and analog-sensitive kinases to create a system that synchronously undergoes the off/on gatekeeper chromatin transition at NDT80 in response to specific phosphomodifications, and to use this system to define biochemical steps controlling this transition.