Pathobiology and Treatment of the UBTF E210K Neuroregression Syndrome

UBTf(NM_014233.3：C.628G&gt；A，p.Glu210Lys)中反复发生的从头显性突变是最近发生的 与一种被称为UBTFE210K神经退行性综合征的独特神经疾病有关。UBTF 以两种主要亚型UBTF1和UBTF2存在。UBTF1通过以下途径调节核糖体RNA(RRNA)转录 RNA聚合酶1(Pol I)，而UBTF2通过RNA聚合酶2(Pol II)调节mRNA转录。 UBTFC.628G&gt；A导致UBTF1和UBTF2(E210K)的氨基酸变化相同。疾病的发病是 年龄在2.5-3岁，以全球运动、认知和行为功能障碍进展缓慢为特征。 UBTFE210K修饰的与rDNA启动子的结合，影响通过 Pol1特异性TBP-复合体SL1。由于UBTF1和UBTF2对DNA可获得性和基因组都有贡献 稳定性，UBTF E210K突变体与DNA结合亲和力的增加可以改变与DNA的相互作用 当基因关闭时出现的组蛋白染色质，可能会使潜在的DNA质量变差 受到保护。UBTF E210K成纤维细胞Pre-rRNA和18S rRNA、核仁表达增加 异常，DNA双链断裂(DSB)数量显著增加，细胞周期缺陷 进展和细胞凋亡。Ubtf-/-对小鼠早期胚胎致死作用及突变体的转基因表达 人UBTFE210K在果蝇神经元中也是致命的。目前还没有已知的治疗疾病的方法 来治疗这种疾病。此外，我们还没有(I)澄清增加表达之间的关系 在患者成纤维细胞中发现的rRNA、DNA双链断裂和细胞周期异常，(Ii)确定了其机制 核仁功能障碍与非核仁DNA损伤之间的联系，或(Iii)UBTF的完全特征 E210K哺乳动物模型系统。我们的总体目标是了解UBTF通过哪些机制 E210K会导致进行性细胞功能障碍，并开发一种治疗这种毁灭性疾病的方法。我们 假设Ubtf+/E210K小鼠将表现出进行性运动和认知障碍，核仁异常 以及与DNA损伤累积相关的神经细胞凋亡性丢失。接下来，我们将确定这些影响 UBTF E210K对rRNA转录、RNA加工和RNA Pol I转录延伸的影响。集成 ChIP-Seq和RNA-Seq分析将用于确定UBTFE210K对染色质的影响 结构和全基因组的基因表达。我们将量化UBTF E210K对rDNA、非rDNA、 和核仁结构。补骨脂素交联剂将用于确定活性基因与非活性基因的比例。 γH_2A.X的芯片序列图谱将被用来在全基因组水平上绘制DNA损伤的位置图。最后，我们 将测试UBTF E210K细胞和动物模型的临床前治疗，口服生物利用度和中心 神经系统渗透性POL I抑制剂。

Recurrent de novo dominant mutations in UBTF (NM_014233.3:c.628G>A, p.Glu210Lys) were recently associated with a distinct neurological disorder dubbed the UBTF E210K neuroregression syndrome. UBTF exists as two major isoforms, UBTF1 and UBTF2. UBTF1 regulates ribosomal RNA (rRNA) transcription by RNA polymerase 1 (Pol I), whereas UBTF2 regulates mRNA transcription by RNA polymerase 2 (Pol II). UBTF c.628G>A results in the same amino acid change in both UBTF1 and UBTF2 (E210K). Disease onset is at 2.5 to 3 yrs and characterized by slow progression of global motor, cognitive and behavioral dysfunction. UBTF E210K modified binding to the rDNA promoter that affects formation of the preinitiation complex by the Pol1-specific TBP-complex SL1. Since both UBTF1 and UBTF2 contribute to DNA accessibility and genomic stability, the increased binding affinity of the UBTF E210K mutant to DNA could alter the interchange with histone chromatin that occurs when genes are switched off, potentially leaving the underlying DNA poorly protected. UBTF E210K fibroblasts show increased expression of pre-rRNA and 18S rRNA, nucleolar abnormalities, markedly increased numbers of DNA double-strand breaks (DSBs), defective cell-cycle progression, and apoptosis. Ubtf-/- is early embryonic lethal in mice and transgenic expression of mutant human UBTF E210K in Drosophila neurons is also lethal. There are no known disease-modifying treatments for this disorder. Furthermore, we have not yet (i) clarified the relationships among increase expression of rRNA, DNA DSBs and cell-cycle abnormalities identified in patient fibroblasts, (ii) determined the mechanistic connections between nucleolar dysfunction and non-nucleolar DNA damage, or (iii) fully characterized a UBTF E210K mammalian model system. Our overall goals are to understand the mechanisms by which UBTF E210K causes progressive cellular dysfunction and develop a treatment for this devastating disorder. We hypothesize that Ubtf+/E210K mice will exhibit progressive motor and cognitive deficits, nucleolar abnormalities and apoptotic neuronal loss in association with accumulated DNA damage. Next, we will determine the effects of UBTF E210K on rRNA transcription, RNA processing, and RNA Pol I transcription elongation. Integrated analysis of ChIP-Seq and RNA-Seq will be used to determine the effects of UBTF E210K on chromatin structure and genome-wide gene expression. We will quantify the effects of UBTF E210K on rDNA, non-rDNA, and nucleolar structure. Psoralen cross-linking will be used to determine the ratio of active to inactive genes. ChIP-Seq mapping of γH2A.X will be used to map sites of DNA damage at the genome-wide level. Finally, we will test pre-clinical treatment of UBTF E210K cellular and animal models with orally-bioavailable and central nervous system penetrant Pol I inhibitors.