Mechanisms of transcriptional regulation through chromatin in the commitment of the yeast cells

真核细胞中的基因表达涉及蛋白质与包装到核小体中的 DNA 的相互作用，核小体是染色质的主要结构单位，由组蛋白八聚体和 147 个 DNA 碱基对组成。由于核小体限制反式作用因子进入其同源 DNA 位点，因此核小体定位已被提议作为转录调控的机制。在这项研究中，我们重点关注核小体定位在 a 细胞特异性基因 BAR1、早期减数分裂基因 HOP1 以及诱导基因 PHO5 和 SNZ1 的调节中的作用。我们实施了一种新策略，通过将短的、多样化的核小体破坏序列引入精确的基因组区域来测试定位核小体在抑制中的功能意义。分别可形成B'构象和Z-DNA的poly dA·poly dT和poly d(CG)·poly d(GC)插入片段长度的增加导致表达增加。对基因组染色质结构的分析表明，启动子区域的核小体位置因较长的聚 dA·聚 dT 和聚 d(CG)·聚 d(CG) 序列而不稳定。这些结果与核小体定位在 BAR1 和 PHO5 的正确表达中起着重要作用的观点一致。我们还表明，核小体定位在决定 HOP1 和 SNZ1 基因的启动子可及性方面起着重要作用。此外，我们强烈表明 Isw2 和 Itc1 是 Isw2 染色质重塑复合物的重要组成部分，用于在 BAR1 和 HOP1 中定位核小体。最后，我们证明定位的核小体可阻止 Hap1 激活剂在体内结合。这些结果支持核小体定位具有限制转录因子进入的调节功能的机制。我们的结果还表明，DNA 结构的内在特性使其成为研究不同细胞和生物体中局部染色质功能以及人工改变体内基因表达的有用工具。较少的

Gene expression in eukaryotic cells involves the interactions of proteins with DNA packaged into nucleosomes, which are the primary structural units of chromatin, comprising a histone octamer and 147 base pairs of DNA. Since the nucleosome limits the access of trans-acting factors to their cognate DNA sites, nucleosome positioning has been proposed as a mechanism of transcriptional regulation. In this study, we focused roles of nucleosome positioning in regulation of a-cellspecific gene BAR1, an early meiotic gene HOP1, and inducible genes PHO5 and SNZ1. We implemented a new strategy to test the functional significance of the positioned nucleosomes in repression by introducing short, diverse nucleosome-disrupting sequences into a precise genomic region. Increased lengths of poly dA・poly dT and poly d(CG)・poly d(GC) inserts, which can form the B' conformation and Z-DNA, respectively, caused increased expression. Analyses of the genomic chromatin structure revealed that the nucleosome po … More sitions in the promoter regions are destabilized by longer poly dA・poly dT and poly d(CG)・poly d(CG) sequences. These results are consistent with the idea that nucleosome positioning plays an essential role in proper expression of BAR1 and PHO5. We also showed that nucleosome positioning plays important roles in determining promoter accessibility of HOP1 and SNZ1 genes. In addition, we showed that Isw2 and Itc1 are essential components of the Isw2 chromatin remodeling complex to position nucleosomes in BAR1 and HOP1. Finally, we demonstrated that a positioned nucleosome prevents Hap1 activator binding in vivo. These results strongly support the mechanism in which nucleosome positioning has a regulatory function to limit the access of transcription factors. Our results also illustrate that the intrinsic properties of DNA structure make them useful tools for inquiring into local chromatin function in diverse cells and organisms as well as for artificial alteration of gene expression in vivo. Less