MOLECULAR MECHANISMS OF TRANSCRIPTIONAL REPRESSION

转录抑制在真核生物的基因调控中起着广泛的作用。基因座控制、沉默、性染色体剂量补偿、印记和其他表观遗传效应涉及染色质作为抑制手段。在接近酵母模型中的阻遏的分子机制中，我们已经确定了几个酵母结构域的核小体组织：两个HM位点，两个a细胞特异性基因和重组增强子。这些二聚体中的每一个都是共同的特征，即紧密堆积的二聚体核小体，以及独特的特征，即这些二聚体之间的DNA的量化接头长度。 基于特定的阻遏物，核的结构和与组蛋白尾部相互作用的辅阻遏物，我假设特定的架构，为每一个我们已经定义的三种类型的域。该提案的前半部分涉及通过定量测定每个独特菌株的染色质组成来测试模型结构，定义阻遏物，共阻遏物和染色质之间拟议相互作用的动力学和稳定性，以及在体内和体外组装的结构域的形态学和物理化学研究。由哈伯发现的重组增强子，其结构特征由我们的实验室，差异控制重组潜力的两种酵母单倍体细胞类型中的染色体III的左臂。它促进了MATa基因座和HMLalpha在交配型相互转换中的相互作用。在本建议的第二部分中，我们解决了这三个元素之间的远程通信的机制，通过识别与重组增强子相互作用的蛋白质和研究诱导的同步交配型转换过程中的所有三个元素的结构。我们还将研究沉默基因座，以解决染色质结构的看似矛盾，排除转录，但完全适合重组或转座子整合。拟议的研究采取了广泛的实验方法，在基因调控的两个基本问题，广泛的抑制域和染色质域之间的长距离通信的染色体组织。

Transcriptional repression serves widely in gene regulation in eukaryotes. Locus control, silencing, sex chromosome dosage compensation, imprinting and other epigenetic effects involve chromatin as a means of repression. In approaching the molecular mechanism of repression in the yeast model, we have determined the nucleosomal organization of several yeast domains: the two HM loci, two a-cell specific genes, and the recombination enhancer. Each of these as a common feature, close packed dimeric nucleosomes, and a unique feature, quantized linker length of DNA between these dimers. Based on the specific repressors that nucleate the structures and co-repressors that interact with histone tails, I postulate specific architecture for each of the three types of domains we have defined. The first half of this proposal deals with testing the model structures by quantitative determination of the composition of chromatin for each unique strain, definition of the kinetics and stabilities of proposed interactions between repressors, co-repressors and chromatin, and morphological and physicochemical study of the domains assembled in vivo and in vitro. The recombination enhancer discovered by Haber and characterized structurally by our laboratory, differentially controls the recombination potential of the left arm of chromosome III in the two yeast haploid cell types. It facilitates interactions between the MATa locus and HMLalpha in mating type interconversion. In the second part of this proposal, we address the mechanisms of long range communication among these three elements by identification of proteins that interact with the recombination enhancer and study of the structures of all three elements during induced synchronous mating type switching. We will also study the silent loci to address the seeming paradox of a chromatin structure that precludes transcription yet is perfectly appropriate for recombination or transposon integration. The proposed research takes a wide range of experimental approach to two fundamental questions in gene regulation, the chromosomal organization of extensive repressed domains and long range communication between chromatin domains.