Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes - Kyono library

Recent genome-wide association studies have established that most complex disease-associated loci are found in noncoding regions where defining their function is nontrivial. In this study, we leverage a modular massively parallel reporter assay (MPRA) to uncover sequence features linked to context-specific regulatory activity. We screened enhancer activity across a panel of 198-bp fragments spanning over 10k type 2 diabetes- and metabolic trait-associated variants in the 832/13 rat insulinoma cell line, a relevant model of pancreatic beta cells. We explored these fragments’ context sensitivity by comparing their activities when placed up- or downstream of a reporter gene, and in combination with either a synthetic housekeeping promoter (SCP1) or a more biologically relevant promoter corresponding to the human insulin gene (INS). We identified clear effects of MPRA construct design on measured fragment enhancer activity. Specifically, a subset of fragments (n = 702/11,656) displayed positional bias, evenly distributed across up- and downstream preference. A separate set of fragments exhibited promoter bias (n = 698/11,656), mostly towards the cell-specific INS promoter (73.4%). To identify sequence features associated with promoter preference, we used Lasso regression with 562 genomic annotations and discovered that fragments with INS promoter-biased activity are enriched for HNF1 motifs. HNF1 family transcription factors are key regulators of glucose metabolism disrupted in maturity onset diabetes of the young (MODY), suggesting genetic convergence between rare coding variants that cause MODY and common T2D-associated regulatory variants. We designed a follow-up MPRA containing HNF1 motif-enriched fragments and observed several instances where deletion or mutation of HNF1 motifs disrupted the INS promoter-biased enhancer activity, specifically in the beta cell model but not in a skeletal muscle cell line, another diabetes-relevant cell type. Together, our study suggests that cell-specific regulatory activity is partially influenced by enhancer-promoter compatibility and indicates that careful attention should be paid when designing MPRA libraries to capture context-specific regulatory processes at disease-associated genetic signals.近年来的全基因组关联研究已证实，绝大多数与复杂疾病相关的基因座均位于非编码区域，而阐明这些区域的功能极具挑战性。本研究借助模块化大规模平行报告基因检测（modular massively parallel reporter assay, MPRA），挖掘与特定情境下调控活性相关的序列特征。我们以胰腺β细胞的相关模型——832/13大鼠胰岛素瘤细胞系为研究对象，针对超过10000个与2型糖尿病及代谢性状相关的变异位点所在的198碱基对片段库，筛选其增强子活性。我们通过将这些片段置于报告基因的上游或下游，并分别搭配合成持家启动子（SCP1）或更具生物学相关性的人胰岛素基因（INS）启动子，比较其活性差异，以此探究这些片段的情境敏感性。我们发现MPRA载体设计对所检测的片段增强子活性存在显著影响。具体而言，有702/11656个片段表现出位置偏好性，其上下游偏好的分布相对均匀。另有698/11656个片段表现出启动子偏好性，其中73.4%的片段更倾向于搭配细胞特异性的INS启动子。为挖掘与启动子偏好性相关的序列特征，我们结合562项基因组注释开展套索回归分析，发现表现出INS启动子偏好性的片段富含HNF1基序。HNF1家族转录因子是葡萄糖代谢的关键调控因子，其功能异常可导致青少年发病的成年型糖尿病（maturity onset diabetes of the young, MODY），这提示导致MODY的罕见编码变异与常见2型糖尿病相关的调控变异之间存在遗传趋同现象。我们设计了包含富集HNF1基序片段的后续MPRA实验，结果发现多个HNF1基序的缺失或突变会破坏其INS启动子偏好性的增强子活性，且该现象仅在β细胞模型中出现，而在另一项与糖尿病相关的骨骼肌细胞系中未观察到此效应。综上，本研究表明细胞特异性的调控活性在一定程度上受增强子-启动子相容性的影响，同时也提示在设计MPRA文库以捕获疾病相关遗传信号的情境特异性调控过程时，需格外留意载体设计的细节。