Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes - Tovar 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颇具挑战性。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），挖掘与语境特异性调控活性相关的序列特征。我们在832/13大鼠胰岛素瘤细胞系——一种胰腺β细胞的相关模型——中，针对覆盖10000余个2型糖尿病及代谢性状相关变异的198bp DNA片段库开展增强子活性筛选。通过比较片段分别置于报告基因上游、下游时的活性，以及与合成持家基因启动子（SCP1）或更具生物学相关性的人胰岛素基因（INS）启动子组合时的活性，我们探究了这些片段的语境依赖性。 我们发现MPRA构建体设计对所测得的片段增强子活性存在显著影响：其中702/11656个片段表现出位置偏好性，且上游与下游偏好的片段数量分布均匀；另有698/11656个片段表现出启动子偏好性，其中73.4%的片段偏好细胞特异性INS启动子。为挖掘与启动子偏好性相关的序列特征，我们基于562项基因组注释开展Lasso回归分析，发现具有INS启动子偏好活性的片段显著富集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起病的成人型糖尿病（MODY），这提示导致MODY的罕见编码变异与常见2型糖尿病相关调控变异之间存在遗传趋同现象。 我们后续设计了包含HNF1基序富集片段的MPRA实验，结果显示，在β细胞模型中，HNF1基序的缺失或突变会破坏INS启动子偏好的增强子活性，但在另一种糖尿病相关细胞类型——骨骼肌细胞系——中则未观察到此现象。综上，本研究表明细胞特异性调控活性在一定程度上受增强子-启动子兼容性影响，同时提示在构建MPRA文库以捕获疾病相关遗传信号的语境特异性调控过程时，需谨慎考量文库设计细节。