Chromatin Targeting Signals, Nucleosome Positioning Mechanism and Non-Coding RNA-Mediated Regulation of the Chromatin Remodeling Complex NoRC

Active and repressed ribosomal RNA (rRNA) genes are characterised by specific epigenetic marks and differentially positioned nucleosomes at their promoters. Repression of the rRNA genes requires a non-coding RNA (pRNA) and the presence of the nucleolar remodeling complex (NoRC). ATP-dependent chromatin remodeling enzymes are essential regulators of DNA-dependent processes, and this regulation occurs via the modulation of DNA accessibility in chromatin. We have studied the targeting of NoRC to the rRNA gene promoter; its mechanism of nucleosome positioning, in which a nucleosome is placed over the transcription initiation site; and the functional role of the pRNA. We demonstrate that NoRC is capable of recognising and binding to the nucleosomal rRNA gene promoter on its own and binds with higher affinity the nucleosomes positioned at non-repressive positions. NoRC recognises the promoter nucleosome within a chromatin array and positions the nucleosomes, as observed in vivo. NoRC uses the release mechanism of positioning, which is characterised by a reduced affinity for the remodeled substrate. The pRNA specifically binds to NoRC and regulates the enzyme by switching off its ATPase activity. Given the known role of pRNA in tethering NoRC to the rDNA, we propose that pRNA is a key factor that links the chromatin modification activity and scaffolding function of NoRC.活性与阻遏状态的核糖体RNA（rRNA）基因，其启动子区域具有特异性表观遗传标记与位置差异化的核小体。核糖体RNA基因的阻遏需要非编码RNA（pRNA）以及核仁重塑复合物（NoRC）的存在。ATP依赖型染色质重塑酶是DNA依赖过程的核心调控因子，其通过调控染色质内DNA的可及性实现调控功能。本研究围绕NoRC靶向核糖体RNA（rRNA）基因启动子的过程、将核小体精准排布于转录起始位点的核小体定位机制，以及pRNA的功能角色展开了系统性探究。本研究证实，NoRC可独立识别并结合携带核小体的核糖体RNA（rRNA）基因启动子，且对处于非阻遏性位置的核小体具有更高的结合亲和力。正如体内实验所观测到的，NoRC可识别染色质阵列中的启动子核小体并完成核小体的精准排布。NoRC采用释放型定位机制，该机制的典型特征为对重塑后的底物结合亲和力下降。pRNA可特异性结合NoRC，并通过抑制其ATP酶活性实现对该酶的调控。鉴于已有研究证实pRNA可将NoRC锚定至核糖体DNA（rDNA），本研究提出pRNA是连接NoRC染色质修饰活性与支架功能的关键因子。