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 如ChIP-seq和ATAC-seq，已经在许多不同的细胞中鉴定了数百万个推定的调控元件， 类型和组织。此外，大规模并行报告基因分析（MPRA）使我们能够测试数千个 以高通量的方式研究调控序列及其变体的功能活性。此外，本发明还提供了一种方法， 基于慢病毒的MPRAs（lentiMPRAs）使得能够测试候选序列的调节活性 在难以转染的细胞中和通过基因组整合在染色质环境中具有高再现性。虽然这些 检测显著扩展了我们对调控元件的了解，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 表观基因组修饰决定它不存在。在这里，我们将开发一种新的技术，crMPRA (CUT& RUN MPRA），其结合了两种单独的技术，lentiMPRA和靶下切割和释放 使用核酸酶（CUT & RUN）以高通量方式同时分析调节活性， 蛋白质结合和数千个序列的表观遗传修饰。我们将利用lentiMPRA 不仅用于测试数千个候选序列的调节活性，而且还用于丰富基因组， 数千个特定序列的整合，这样就可以测定蛋白质结合和表观遗传 通过CUT & RUN进行修改。在目标1中，我们将通过利用已经被发现的序列来开发crMPRA。 先前通过lentiMPRA（调节活性）和ChIP-seq（蛋白结合和表观遗传标记）进行表征 在肝癌HepG2细胞中。我们将使用这些序列来构建MPRA文库并表征 他们通过lentiMPRA的调节活性。我们还将同时对特定TF进行CUT & RUN (e.g. EP300、FOXA 2、HNF 4A）和表观遗传标记（例如H3K27ac、H3K27me3）。对于目标2，我们将进一步测试 通过crMPRA的转录因子结合位点扰动文库，这些扰动如何影响调控 活性、蛋白结合和表观遗传修饰，并分析功能相关性或独立性 这些国家之间。因此，这项新技术将使我们能够加深对监管的理解 包括TF结合，组蛋白修饰和转录激活在内的几个水平的编码，以及其如何在转录过程中发挥作用。 基因的改变会导致人类疾病。

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.