Department of Biology, The University of York, York YO10 5DD, UK.
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
Gene. 2022 Aug 15;835:146533. doi: 10.1016/j.gene.2022.146533. Epub 2022 May 24.
Eukaryotic chromosomes are divided into domains with distinct structural and functional properties, such as differing levels of chromatin compaction and gene transcription. Domains of relatively compact chromatin and minimal transcription are termed heterochromatic, whereas euchromatin is more open and actively transcribed. Insulators separate these domains and maintain their distinct features. Disruption of insulators can cause diseases such as cancer. Many insulators contain tRNA genes (tDNAs), examples of which have been shown to block the spread of activating or silencing activities. This characteristic of specific tDNAs is conserved through evolution, such that human tDNAs can serve as barriers to the spread of silencing in fission yeast. Here we demonstrate that tDNAs from the methylotrophic fungus Pichia pastoris can function effectively as insulators in distantly-related budding yeast. Key to the function of tDNAs as insulators is TFIIIC, a transcription factor that is also required for their expression. TFIIIC binds additional loci besides tDNAs, some of which have insulator activity. Although the mechanistic basis of TFIIIC-based insulation has been studied extensively in yeast, it is largely uncharacterized in metazoa. Utilising publicly-available genome-wide ChIP-seq data, we consider the extent to which mechanisms conserved from yeast to man may suffice to allow efficient insulation by TFIIIC in the more challenging chromatin environments of metazoa and suggest features that may have been acquired during evolution to cope with new challenges. We demonstrate the widespread presence at human tDNAs of USF1, a transcription factor with well-established barrier activity in vertebrates. We predict that tDNA-based insulators in higher organisms have evolved through incorporation of modules, such as binding sites for factors like USF1 and CTCF that are absent from yeasts, thereby strengthening function and providing opportunities for regulation between cell types.
真核染色体被划分为具有独特结构和功能特性的域，例如染色质紧缩和基因转录的程度不同。相对紧凑的染色质和最小转录的域被称为异染色质，而常染色质则更为开放且活跃转录。绝缘子将这些域分隔开并保持其独特的特征。绝缘子的破坏会导致癌症等疾病。许多绝缘子包含 tRNA 基因（tDNAs），已经证明这些基因的例子可以阻止激活或沉默活性的传播。这种特定 tDNA 的特征在进化中是保守的，因此人类 tDNAs 可以作为裂殖酵母中沉默传播的屏障。在这里，我们证明了甲醇营养型真菌巴斯德毕赤酵母中的 tDNA 可以有效地作为远缘芽殖酵母中的绝缘子发挥作用。tDNA 作为绝缘子的功能关键是 TFIIIC，它也是 tDNA 表达所必需的转录因子。TFIIIC 除了 tDNAs 之外还结合其他基因座，其中一些具有绝缘子活性。尽管 TFIIIC 为基础的绝缘子的机制基础在酵母中已经得到了广泛的研究，但在后生动物中却在很大程度上没有被描述。利用公开的全基因组 ChIP-seq 数据，我们考虑了从酵母到人可能足以允许 TFIIIC 在后生动物更具挑战性的染色质环境中有效隔离的机制的程度，并提出了在进化过程中可能获得的特征，以应对新的挑战。我们证明了在人类 tDNAs 中广泛存在 USF1，这是一种在脊椎动物中具有良好屏障活性的转录因子。我们预测，高等生物中的 tDNA 为基础的绝缘子已经通过整合模块进化而来，例如结合因子（如 USF1 和 CTCF）的结合位点，这些因子在酵母中不存在，从而增强了功能并为不同细胞类型之间的调节提供了机会。
