Gatekeeper Chromatin and Meiotic Commitment In Yeast

分化为新细胞类型的承诺是动物、植物和许多其他真核生物发展的关键一步。该项目将研究如何使用酵母作为模型系统调节这一重要过程，并将揭示控制细胞命运决定的新机制。此外，还计划让“磁石高中”的优秀学生参与科学发现的过程，扩大从事科学事业的人才队伍。为大三到大四的学生提供动手研究的机会；训练研究生进行假设驱动的分子遗传学研究。类似于多细胞生物的分化程序，酵母的孢子形成是由转录级联控制的。产孢的转录级联由早、中、晚三组基因组成。直到中间基因表达后才获得产孢的承诺。Ndt80是激活中间启动子的转录因子，在中间基因表达前转录。NDT80启动子可以被视为一个平台，在这个平台上产生类似开关的输出，使细胞进入配子发生。本实验室进行的研究表明，NDT80启动子在减数分裂发育过程中特异性地组装成类似于沉默异染色质的状态。这种异色状态（被称为“看门人染色质”）需要一种名为Sum1的dna结合抑制蛋白来招募一种名为Hst1的组蛋白去乙酰化酶（Sir2的近亲）。在减数分裂G2结束时（就在承诺点之前），Sum1在多个残基上被周期蛋白依赖性激酶（CDK）和减数分裂特异性CDK样激酶Ime2磷酸化。本项目将测试的一个中心假设是，Sum1的磷酸化下调Hst1 sirtuin，从而触发异色看门人染色质向Sum1与DNA可交换状态的转变。由此产生的Sum1抑制因子的部分占用允许低水平的NDT80表达。当新翻译的Ndt80蛋白竞争性地从DNA中取代Sum1以启动一个正的自调节环（Sum1和Ndt80竞争性地结合到Ndt80启动子中的单个DNA元件上）时，承诺就获得了。在这个项目中，这个承诺的“看门人染色质模型”将被测试。该项目的具体目标是：1)建立一种检测方法来监测Sum1蛋白在染色质中的可交换性；2)将Sum1磷酸体突变体和类似物敏感激酶结合起来，创建一个系统，该系统可以在NDT80处同步经历关/开守门染色质转变，以响应特定的磷酸化二化，并使用该系统来定义控制这种转变的生化步骤。

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.