Chakraborty Sangita A, Kazi Abid A, Khan Tamreen M, Grigoryev Sergei A
Department of Biochemistry and Molecular Biology and Hershey Medical Center, Hershey, Pennsylvania 17033-0850
Hershey Medical Center, Hershey, Pennsylvania 17033-0850.
Genetics. 2014 Nov;198(3):1015-29. doi: 10.1534/genetics.114.169508. Epub 2014 Sep 3.
Eukaryotic gene expression occurs in the context of structurally distinct chromosomal domains such as the relatively open, gene-rich, and transcriptionally active euchromatin and the condensed and gene-poor heterochromatin where its specific chromatin environment inhibits transcription. To study gene silencing by heterochromatin, we created a minichromosome reporter system where the gene silencer elements were used to repress the URA3 reporter gene. The minichromosome reporters were propagated in yeast Saccharomyces cerevisiae at a stable copy number. Conduction of gene silencing through nucleosome arrays was studied by placing various repeats of clone-601 DNA with high affinity for histones between the silencer and reporter in the yeast minichromosomes. High-resolution chromatin mapping with micrococcal nuclease showed that the clone-601 nucleosome positioning downstream of the HML-E gene silencing element was not significantly altered by chromatin silencing. Using URA3 reporter assays, we observed that gene silencing was conducted through arrays of up to eight nucleosomes. We showed that the shorter nucleosome repeat lengths, typical of yeast (167 and 172 bp), were more efficient in conducting silencing in vivo compared to the longer repeats (207 bp) typical of higher eukaryotes. Both the longer and the shorter repeat lengths were able to conduct silencing in minichromosomes independently of clone-601 nucleosome positioning orientations vs. the silencer element. We suggest that the shorter nucleosome linkers are more suitable for conducting gene silencing than the long repeats in yeast due to their higher propensity to support native-like chromatin higher-order folding.
真核基因表达发生在结构上不同的染色体结构域的背景下，例如相对开放、富含基因且转录活跃的常染色质，以及浓缩且基因贫乏的异染色质，其特定的染色质环境会抑制转录。为了研究异染色质介导的基因沉默，我们创建了一个微型染色体报告系统，其中基因沉默元件用于抑制URA3报告基因。微型染色体报告基因在酿酒酵母中以稳定的拷贝数进行增殖。通过在酵母微型染色体的沉默子和报告基因之间放置对组蛋白具有高亲和力的克隆601 DNA的各种重复序列，研究了通过核小体阵列进行的基因沉默。用微球菌核酸酶进行的高分辨率染色质图谱分析表明，HML-E基因沉默元件下游的克隆601核小体定位在染色质沉默过程中没有显著改变。使用URA3报告基因检测，我们观察到基因沉默是通过多达八个核小体的阵列进行的。我们表明，与高等真核生物典型的较长重复序列（207 bp）相比，酵母典型的较短核小体重复长度（167和172 bp）在体内进行沉默更有效。较长和较短的重复长度都能够在微型染色体中进行沉默，而与克隆601核小体相对于沉默元件的定位方向无关。我们认为，较短的核小体连接体比较长的重复序列更适合在酵母中进行基因沉默，因为它们更倾向于支持类似天然的染色质高级折叠。
