Gatekeeper Chromatin and Meiotic Commitment In Yeast

承诺分化成新的细胞类型是动物、植物和许多其他真核生物发展的关键一步。这个项目将研究如何使用酵母作为模型系统来调节这一重要的过程，并将发现控制细胞命运决定的新机制。该项目还将通过让来自磁石高中的有天赋的学生接触科学发现的过程，为三年级和四年级的大学生提供实践研究机会，以及培训研究生进行假设驱动的分子基因研究，来扩大在科学领域从事职业生涯的有才华的学生的人数。与多细胞生物体中的分化程序类似，酵母中的产孢子是由转录级联控制的。孢子形成的转录级联由早、中、晚三组基因组成。在表达中间基因之前，不会获得对产孢量的承诺。Ndt80是激活中间启动子的转录因子，在中间基因表达之前被转录。NDT80启动子可以被视为一个平台，在这里产生开关样的输出，使细胞参与配子发生。在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被组装成类似于沉默的异染色质的状态。这种异染色质状态(称为“看门人染色质”)需要一个名为Sum1的DNA结合抑制蛋白，它招募一个名为Hst1的组蛋白脱乙酰酶(Sir2的近亲)。在减数分裂G2末期(就在承诺点之前)，Sum1在多个残基上被一个细胞周期蛋白依赖性激酶(CDK)和一个称为IME2的减数分裂特异的CDK样激酶所磷酸化。本项目将检验的一个中心假设是，Sum1的磷酸化下调Hst1 sirtuin，从而触发异染色质守门人染色质向Sum1可与DNA交换的状态的转变。由此产生的Sum1抑制子的部分占位允许低水平的NDT80表达。当新翻译的Ndt80蛋白竞争性地取代DNA中的Sum1启动正自动调节环(Sum1和Ndt80竞争性地与NDT80启动子中的单个DNA元件结合)时，就获得了承诺。在这个项目中，这一承诺的“守门人染色质模型”将得到检验。该项目的具体目标是：1)建立一种方法来监测Sum1蛋白在染色质中的互换性；2)结合Sum1亚磷酸盐突变体和模拟敏感激酶，创建一个在NDT80处同步经历关闭/开启门卫染色质转变的系统，并使用该系统来定义控制这一转变的生化步骤。

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.