Non-coding/epigenetic regulation

项目3-摘要 青少年特发性脊柱侧凸(AIS)影响全球约3%的人口，据估计 仅在美国，每年仅外科手术就有数十亿美元。AIS的原因在很大程度上仍不清楚。而当 已发现导致综合征性脊柱侧弯(与其他症状有关)的基因突变， 确定导致非综合征/孤立AIS的突变(仅AIS没有任何其他症状)，有 没那么成功。几项全基因组关联研究(GWAS)已经确定了与AIS相关的单个 与有希望的候选基因相邻的非编码区的核苷酸多态(SNPs)，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 位于PAX1基因座的AIS GWA相关区域附近的脊髓增强子被敲除 已知与脊柱发育有关，与尾巴扭曲表型有关，类似于Pax1基因 基因敲除和亚型突变。有趣的是，扭结的尾巴表型在雌性中更明显，符合 与我们在该地区观察到的女性特定的AIS Gwas关联。AIS的另一个障碍 遗传学是，直到最近，还没有特定的组织/S的异常被广泛认为是导致AIS的原因。 通过我们所有三个项目与其他研究人员共同开展的工作，软骨 细胞外基质(ECM)，即基质，被发现与AIS的发病密切相关。 在这里，我们将使用总RNA-SEQ来表征在这些组织中表达的非编码RNA。此外, 联合应用单细胞RNA/ATAC-seq在软骨基质上鉴定基因及其调控 以单元格方式与AIS相关联的元素。此外，我们还将进行H3K4me3(活动标志 启动子)和H3K27ac(活性启动子和增强剂的标志)Hi-Chip，这是一种使用 染色质构象结合染色质免疫沉淀(CHIP)捕获特异性 染色体相互作用，以确定这些AIS相关调控元件的靶基因。我们的 基因组数据还将提供给项目1(人类)和项目2(斑马鱼)提供候选基因 以及用于筛查AIS相关突变/表型的调控元件。要从功能上描述AIS- 相关的调节元件，我们将在细胞系和小鼠中使用调节元件分析，并结合 小鼠基因敲除，用于表征与AIS相关的基因调控序列。这些序列 将从文献和项目1(人类)中选择，全基因组测序 由项目1(人类)进行的AIS个体及其附近的基因显示导致斑马鱼AIS来自我们的 项目2(斑马鱼)。我们对Pax1增强子基因敲除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 与脊柱侧弯、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.