The budding yeast heterochromatic protein Sir3 modulates genome-wide gene expression through transient direct contacts with euchromatin (RNA-Seq)

The SIR complex (Silent Information Regulator) is the building block of heterochromatic structures that silence underlying genes. It is well established that the silenced state is epigenetically inherited but it is not known how the SIR complex is maintained through cell divisions in optimal or variable growth conditions. The biological function of heterochromatin located in subtelomeric regions is also unclear since heterochromatin coverage appears to be limited to a few kbps near chromosome ends and the expression of subtelomeric genes is only marginally affected in the absence of the SIR complex. We use a three-pronged approach to address these questions. First, Nanopore-MetID, an in vivo foot printing technique similar to DamID that uses nanopore sequencing technology, identified over a thousand new transient contacts between Sir3 and euchromatic genes that are not detectable by ChIP-seq and revealed a previously undocumented low-density mode of Sir3 binding to subtelomeric regions that extends 15kbps downstream of subtelomeric SIR nucleation sites. Second, our measurements of genome-wide Sir3 exchange rates after exit from starvation show that heterochromatin is a highly dynamic structure in optimal growth conditions. Third, “spike-in” RNA-seq time course experiments in the same conditions reveal that Sir3 modulates global mRNA levels in correlation with fluctuations in nutrient availability. We now propose that subtelomeric regions serve as Sir3 hubs from which Sir3 is brought over to distal sites down the chromosome arm where it transiently contacts euchromatic genes in its path. We hypothesize that contacts between Sir3 and actively transcribed genes facilitate the removal of stalled transcription complexes and allow for optimal genome-wide transcription, which gives wt cells a competitive advantage over sir3Δ cells when nutrients are limited.沉默信息调节因子复合物（Silent Information Regulator）是介导内源基因沉默的异染色质结构的基本组成单元。已有研究证实，基因沉默状态可通过表观遗传方式稳定传递，但目前仍未明确在最优或可变生长条件下，SIR复合物如何在细胞分裂过程中得以维持。位于亚端粒区域的异染色质的生物学功能同样尚不明确：一方面，异染色质的覆盖范围似乎仅局限于染色体末端附近数kb的区域；另一方面，在缺失SIR复合物的情况下，亚端粒基因的表达仅受到轻微影响。为此，我们采用三管齐下的研究策略解答上述问题。其一，Nanopore-MetID——一种类似DamID的体内足迹技术，依托纳米孔测序技术搭建——鉴定出了千余个此前未被ChIP-seq检测到的Sir3与常染色质基因间的瞬时接触位点，并揭示了一种此前未被记录的Sir3结合亚端粒区域的低密度模式：该模式可延伸至亚端粒SIR成核位点下游15kb处。其二，我们对饥饿退出后全基因组范围内Sir3的交换速率进行了定量测定，结果表明在最优生长条件下，异染色质是一种高度动态的结构。其三，在相同条件下开展的spike-in RNA测序时间序列实验显示，Sir3可调控全局mRNA水平，且其调控效应与营养可利用性的波动密切相关。我们据此提出，亚端粒区域可作为Sir3的储存枢纽，Sir3由此被转运至染色体臂远端位点，并在行进路径中与常染色质基因形成瞬时结合。我们进一步假设，Sir3与活跃转录基因间的结合可促进停滞转录复合物的解离，从而实现最优的全基因组转录效率，这使得野生型细胞在营养受限条件下相较于sir3Δ缺失细胞具有显著的竞争优势。