Technologies for simultaneous characterization of regulatory activity and protein binding

项目摘要 基因调控元件的突变是人类疾病的主要原因。大规模的基因组分析，如 作为CHIP-SEQ和ATAC-SEQ，已经在许多不同的细胞中确定了数百万个假定的调控元件 类型和组织。此外，大规模平行的报告分析(MPRA)使我们能够测试数千 以高通量的方式对其功能活性的调节序列及其变体进行分析。此外, 基于慢病毒的MPRA(LentiMPRA)已经能够测试用于调节活性的候选序列 在难以转化的细胞和染色质背景下通过基因组整合具有高度的重复性。而这些 化验极大地扩展了我们对监管元素的知识，这些技术可以同时 分析特定序列的调节功能和转录因子、辅因子和 决定它的表观基因组修饰不存在。在这里，我们将开发一种新技术，crMPRA (Cut&Run MPRA)，它结合了两种不同的技术，lentiMPRA和裂解下的目标和释放 使用核酸酶(Cut&Run)同时高通量地分析调节活性， 数千个序列的蛋白质结合和表观遗传修饰。我们将利用lentiMPRA 既用于测试数千个候选序列的调节活性，也用于丰富基因组 一个特定序列的数千个整合，这样它就可以被检测蛋白质结合和表观遗传学 通过切割和运行进行修改。在目标1中，我们将通过利用以下序列开发crMPRA 先前通过lentiMPRA(调节活性)和CHIP-SEQ(蛋白质结合和表观遗传标记)来表征 在肝细胞癌HepG2细胞中。我们将使用这些序列来构建MPRA文库并鉴定 他们通过lentiMPRA进行的监管活动。我们还将同时对特定的TF进行切割和运行 (如EP300、FOXA2、HNF4a)和表观遗传标记(如H3K27ac、H3K27me3)。对于目标2，我们将进一步测试 通过crMPRA构建转录因子结合位点扰动文库这些扰动如何影响调控 活性、蛋白质结合和表观遗传修饰，并分析功能相关性或独立性 在这些州之间。因此，这项新技术将使我们能够增加对监管的理解 几个水平上的代码，包括TF结合、组蛋白修饰和转录激活，以及它是如何 改变可能会导致人类疾病。

Project Summary Mutations in gene regulatory elements are a major cause of human disease. Large-scale genomic assays, such as ChIP-seq and ATAC-seq, have identified millions of putative regulatory elements across many different cell types and tissues. Furthermore, massively parallel reporter assays (MPRAs), have allowed us to test thousands of regulatory sequences and their variants for their functional activity in a high-throughput manner. In addition, lentivirus-based MPRAs (lentiMPRAs) have enabled the testing of candidate sequences for regulatory activity with high reproducibility in hard to transfect cells and in chromatin context via genomic integration. While these assays have significantly expanded our knowledge of regulatory elements, technologies that can simultaneously analyze both the regulatory function of a specific sequence and the transcription factors, cofactors and epigenomic modifications that determine it do not exist. Here, we will develop a novel technology, crMPRA (CUT&RUN MPRA), that combines two separate techniques, lentiMPRA and cleavage under targets and release using nuclease (CUT&RUN) to simultaneously analyze in a high-throughput manner the regulatory activity, protein binding and epigenetic modification of thousands of sequences. We will take advantage of lentiMPRA both for testing thousands of candidate sequences for their regulatory activity, but also to enrich the genome with thousands of integrations of a specific sequence, such that it could be assayed for protein binding and epigenetic modifications via CUT&RUN. In Aim 1, we will develop crMPRA, by taking advantage of sequences that were previously characterized via lentiMPRA (regulatory activity) and ChIP-seq (protein binding and epigenetic marks) in hepatocellular carcinoma HepG2 cells. We will use these sequences to build an MPRA library and characterize them for their regulatory activity via lentiMPRA. We will also simultaneously carry out CUT&RUN on specific TFs (e.g. EP300, FOXA2, HNF4A) and epigenetic marks (e.g. H3K27ac, H3K27me3). For Aim 2, we will further test a transcription factor binding site perturbation library via crMPRA how these perturbations affect regulatory activity, protein binding and epigenetic modification, and analyze the functional correlation or independency between these states. As such, this novel technology will allow us to increase our understanding of the regulatory code at several levels including TF binding, histone modification, and transcriptional activation, and how its alteration can lead to human disease.