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。在接近酵母模型中抑制的分子机制时，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。每一个都有一个共同的特征，紧密排列的二聚体核小体，和一个独特的特征，量化这些二聚体之间的DNA连接体长度。基于使结构成核的特定阻遏子和与组蛋白尾部相互作用的辅阻遏子，我假设我们定义的三种结构域中的每一种都有特定的结构。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，定义抑制因子，共抑制因子和染色质之间拟议相互作用的动力学和稳定性，以及体内和体外组装的结构域的形态学和物理化学研究。Haber发现的重组增强子在结构上对两种酵母单倍体细胞类型III号染色体左臂的重组潜力进行了差异控制。它促进了MATa位点与hml - α在交配型互转换中的相互作用。在本论文的第二部分，我们通过鉴定与重组增强子相互作用的蛋白质，以及研究诱导同步交配类型转换过程中这三种元素的结构，来解决这三种元素之间的远程通信机制。我们还将研究沉默位点，以解决染色质结构看似矛盾的问题，这种结构阻碍转录，但却完全适合重组或转座子整合。提出的研究采用广泛的实验方法来解决基因调控中的两个基本问题，即广泛抑制结构域的染色体组织和染色质结构域之间的远程通信。

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.