Homeotic hotspot in the human genome for eye and brain disease

项目总结 经典的同源异型突变(如果蝇Ubx)会破坏胚胎发育， 将一种组织类型转化为另一种组织类型。我们已经发现了分子基础 影响眼睛或大脑的四种人类X连锁疾病-Basr综合征，中心凹 发育不全、视网膜色素变性和脊髓小脑性共济失调，这些疾病具有同源性或 退化基础。每种疾病都是由一个大的常染色体DNA插入引起的 在SOX3附近的同一个Xq27回文位点，该片段编码了一个有效的反式- 与SRY(睾丸决定因素)同源的调控因子。预测了XQ插入 破坏染色质结构，在新定义的组织中异位激活SOX3 并列的增强子，并通过功能获得(GOF)改变细胞命运(顺势)。我们 提出SOX3在BASR中将视网膜色素上皮(RPE)转化为神经视网膜， 对SCAX5中的小脑浦肯野细胞重新编程，并触发光感受器退化 在RP24中。使用一种新的Palins聚合酶链式反应方法，我们定义了断点和候选 每次插入的增强剂。我们还发现&gt；10进一步的Xq27疾病会影响 眼睛、大脑或其他器官--包括可能具有类似机制的未侦破病例。 我们将定义新的Xq27回文插入并测试我们的疾病假设 染色质和发育水平的致病机制，使用患者来源的IPSCs， 三维染色质相互作用分析(Hi-C)，体外分化，系列scRNA-seq图谱； 和[2] (Homol 信息丰富的小鼠转基因，包括二进制CRISPR/Cas9 HPRT跳入 OGY促进集成)我们首创的GOF系统-具有Sox3HA表达 在RPE、视杆细胞、浦肯野细胞或其他组织中通过已建立的Cre驱动程序激活，在 构成(XY)或马赛克(XX)图案，并以持续或Dox诱导的方式。

PROJECT SUMMARY Classical homeotic mutations (e.g. Drosophila Ubx) disrupt embryonic development, transforming one tissue type into another. We have discovered the molecular basis of four human X-linked disorders affecting the eye or brain – BASR syndrome, foveal dysgenesis, retinitis pigmentosa and spinocerebellar ataxia, which have a homeotic or degenerative basis. Each disorder is caused by insertion of a large autosomal DNA segment at the same Xq27 palindromic site near SOX3, which encodes a potent trans- cription factor homologous to SRY (testis determinant). The Xq insertions are predicted to disrupt chromatin architecture, activating SOX3 ectopically in tissues defined by newly juxtaposed enhancers, and altering cell fate (homeosis) via a gain-of-function (GOF). We propose that SOX3 changes retinal pigment epithelia (RPE) into neuroretina in BASR, reprograms cerebellar Purkinje cells in SCAX5, and triggers photoreceptor degeneration in RP24. Using a novel palinsert PCR assay, we defined the breakpoints and candidate enhancers for each insertion. We also identified >10 further Xq27 disorders affecting the eye, brain or other organs – including unsolved cases with a likely similar mechanism. We will define new Xq27 palindrome insertions and test our hypothesis for disease pathogenesis at chromatin and developmental levels, using [1] patient-derived iPSCs, 3D chromatin interaction assays (Hi-C), in vitro differentiation, serial scRNA-seq profiles; and [2] (homol informative mouse transgenes, including the binary CRISPR/Cas9 Hprt HoP-In ogy promoted integration) GOF system we pioneered – with Sox3HA expression activated in RPE, rods, Purkinje cells, or other tissues via established Cre drivers, in a constitutional (XY) or mosaic (XX) pattern, and in a sustained or Dox-inducible manner.