Non-coding/epigenetic regulation

项目3 -概要 青少年特发性脊柱侧凸（AIS）影响全球约3%的人口，估计花费数 每年仅在美国的手术上就花费数十亿美元。AIS的原因在很大程度上仍然未知。而 已经发现导致脊柱侧凸综合征（与其他症状相关）的基因突变， 鉴定引起非综合征/孤立性AIS（仅AIS，无任何其他症状）的突变， 不太成功。几项全基因组关联研究（GWAS）已经确定了AIS相关的单个 核苷酸多态性（SNPs）在邻近有希望的候选基因的非编码区，这表明 基因调控序列，如增强子，在AIS中的作用。在我们的初步结果中，我们表明， 敲除位于PAX 1基因座的AIS GWAS相关区域附近的脊髓增强子， 已知参与脊柱发育，与一个弯曲的尾巴表型有关，类似于Pax 1基因 敲除和亚型突变。有趣的是，弯曲的尾巴表型在女性中更明显， 与我们在该区域观察到的女性特异性AIS GWAS相关。AIS的另一个障碍 遗传学的一个重要特征是，直到最近，还没有一种特定的组织被广泛认为是导致AIS的畸变。 通过我们所有三个项目沿着与其他调查人员进行的工作， 细胞外基质（ECM），即基质体，被发现与AIS的发病机制密切相关。 在这里，我们将使用总RNA-seq来表征这些组织中表达的非编码RNA。此外，本发明还提供了一种方法， 在软骨基质体上使用组合的单细胞RNA/ATAC-seq来鉴定基因和调节基因， 以单细胞方式与AIS相关联的元素。此外，我们将进行H3 K4 me 3（一个标志，为积极的 启动子）和H3 K27 ac（活性启动子和增强子的标记）Hi-ChIP，一种使用 染色质构象与染色质免疫沉淀（ChIP）结合，以捕获特异性 染色体相互作用，以确定这些AIS相关的调控元件的靶基因。我们 基因组数据集也将输入项目1（人类）和项目2（斑马鱼），提供候选基因 和调节元件以筛选AIS相关的突变/表型。为了从功能上描述AIS- 我们将在细胞系和小鼠中使用调节元件测定， 小鼠敲除以表征与AIS相关的基因调控序列。这些序列 将从GWAS中选择，无论是从文献还是从项目1（人类）中，全基因组测序都是如此。 由项目1（人类）进行的AIS个体和来自我们的斑马鱼的导致AIS的近基因 项目2（斑马鱼）。我们敲除Pax 1增强子的初步结果已经证明了 这种方法。结合起来，我们的工作将提供一个基因和调控元件的基因组百科全书， 可能与AIS相关，并可作为基因调控功能表征的模型。 与脊柱侧凸、肌肉骨骼和其他人类疾病的其他亚型有关的元素。

PROJECT 3 - SUMMARY Adolescent idiopathic scoliosis (AIS) affects ~3% of the population worldwide and is estimated to cost several billion dollars annually in surgeries alone in the US. The causes of AIS remain largely unknown. While mutations in genes leading to syndromic scoliosis (associated with other symptoms) have been discovered, the identification of mutations causing non-syndromic/isolated AIS (only AIS without any other symptoms), have been less successful. Several genome-wide association studies (GWAS) have identified AIS-associated single nucleotide polymorphisms (SNPs) in noncoding regions adjacent to promising candidate genes, suggesting a role for gene regulatory sequences, such as enhancers, in AIS. In our preliminary results, we show that the knockout of a spinal cord enhancer near an AIS GWAS associated region residing in the PAX1 locus, a gene known to be involved in spinal development, is associated with a kinky tail phenotype, similar to the Pax1 gene knockout and hypomorphic mutation. Interestingly, the kinky tail phenotype is more apparent in females, fitting with the female specific AIS GWAS association that we observed for this region. Another hurdle in AIS genetics is that until recently there were no specific tissue/s whose aberration was widely known to cause AIS. Through work carried out by all three of our projects along with other investigators, the the cartilage extracellular matrix (ECM), i.e. matrisome, was found to be strongly implicated in the pathogenesis of AIS. Here, we will use total RNA-seq to characterize the noncoding RNAs expressed in these tissues. In addition, using combined single-cell RNA/ATAC-seq on the cartilage matrisome to identify the genes and regulatory elements associated with AIS in a single cell manner. In addition, we will carry out H3K4me3 (a mark for active promoters) and H3K27ac (a mark for active promoters and enhancers) Hi-ChIP, a technique that uses chromatin conformation in combination with chromatin immunoprecipitation (ChIP) to capture specific chromosomal interactions, to identify the target genes of these AIS-associated regulatory elements. Our genomic datasets will also feed into Project 1 (Human) and Project 2 (Zebrafish) providing candidate genes and regulatory elements to screen for AIS-associated mutations/phenotypes. To functionally characterize AIS- associated regulatory elements, we will use regulatory element assays in cell lines and mice combined with mouse knockouts to characterize gene regulatory sequences that are associated with AIS. These sequences will be selected from GWAS, both from the literature and Project 1 (Human), whole-genome sequencing on individuals with AIS carried out by Project 1 (Human) and near genes shown to cause AIS in zebrafish from our Project 2 (Zebrafish). Our preliminary results for the Pax1 enhancer knockout already attest for the potential of this approach. Combined, our work will provide a genomic encyclopedia of genes and regulatory elements that could be associated with AIS and serve as a model for the functional characterization of gene regulatory elements involved in additional subtypes of scoliosis, musculoskeletal and other human disease.