CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [Hi-C]. CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant [Hi-C]

Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ~664 kb from the ONECUT1 promoter, as coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes (T2D) associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease. Overall design: lab: Danwei Huangfu, MSKCC award: 1U01DK128852-01 accession: 4DNESOGBAIC7 condition: pancreatic precursor cells - H1 dataset_label: in situ Hi-C on PDX1-GFP idCAS9-KRAB H1 human embryonic stem cells differentiated to pancreatic progenitor cells (PP) experiment type: in situ Hi-C url: https://data.4dnucleome.org/experiment-set-replicates/4DNESOGBAIC7/ number_of_experiments: 2 experiment: in situ Hi-C on PDX1-eGFP dCAS9-KRAB H1 differentiated to endoderm of foregut with Arima - A1, A2 - 4DNEXMHLGGWC experiment: in situ Hi-C on PDX1-eGFP dCAS9-KRAB H1 differentiated to endoderm of foregut with Arima - A1, A2 - 4DNEXUDU96VW Series supplementary files: ****** workflow_run: https://data.4dnucleome.org/workflow-runs-awsem/3e86c39e-47f3-4656-ac56-ab63aa493af7/ genome_assembly GRCh38 derived_from: 4DNFI823LSII.chrom.sizes, 4DNFITG6TM26.pairs.gz, 4DNFINK7ALPW.pairs.gz, 4DNFIU1RS39P.txt, description: This is an output file of the Hi-C processing pipeline file_type: contact list-combined file_format: pairs file_name: 4DNFIEAQ6DIR.pairs.gz ****** workflow_run: https://data.4dnucleome.org/workflow-runs-awsem/3e86c39e-47f3-4656-ac56-ab63aa493af7/ genome_assembly GRCh38 derived_from: 4DNFI823LSII.chrom.sizes, 4DNFITG6TM26.pairs.gz, 4DNFINK7ALPW.pairs.gz, 4DNFIU1RS39P.txt, description: This is an output file of the Hi-C processing pipeline file_type: contact matrix file_format: mcool file_name: 4DNFIYC4AJW5.mcool ****** workflow_run: https://data.4dnucleome.org/workflow-runs-awsem/4a5f3a30-c608-4367-953b-e5300001f22b/ genome_assembly GRCh38 derived_from: 4DNFIYC4AJW5.mcool, description: Boundaries calls on Hi-C contact matrices file_type: boundaries file_format: bed file_name: 4DNFINRZXAQF.bed.gz ****** workflow_run: https://data.4dnucleome.org/workflow-runs-awsem/4a5f3a30-c608-4367-953b-e5300001f22b/ genome_assembly GRCh38 derived_from: 4DNFIYC4AJW5.mcool, description: Diamond insulation scores calls on Hi-C contact matrices file_type: insulation score-diamond file_format: bw file_name: 4DNFI4F6P2UE.bw ****** workflow_run: https://data.4dnucleome.org/workflow-runs-awsem/d04948a9-1b41-47f2-80b0-8e3a74eb90af/ genome_assembly GRCh38 derived_from: 4DNFIYC4AJW5.mcool, 4DNFI7MCA4R6.bedGraph.gz, description: Compartments signals on Hi-C contact matrices file_type: compartments file_format: bw file_name: 4DNFI9C517H3.bw ******BED files: HiC-Pro (3.1.0) alignment, HiTC (1.34.0), HiCDC+(1.5.2), TopDom, 50 kb file_format: bed file_name: 4DNFIJI7XHPW.bed.gz

增强子功能注释是解析组织特异性转录调控、筛选疾病相关非编码变异以开展研究的重要首要步骤。然而，在生理相关实验体系中实现无偏倚的增强子发现仍是一项重大挑战。为发掘与糖尿病相关的调控元件，我们在人类多能干细胞（human pluripotent stem cell, hPSC）胰腺分化系统中开展了CRISPR干扰（CRISPR interference, CRISPRi）筛选。在筛选发现的增强子中，我们重点关注了距ONECUT1启动子约664 kb的远程增强子——因ONECUT1的编码突变会导致胰腺发育不全与新生儿糖尿病。在hPSCs中纯合敲除该增强子，会使ONECUT1基因表达近乎完全丧失，并损害胰腺分化进程。该增强子包含一个经精细定位确认的2型糖尿病（type 2 diabetes, T2D）相关变异（rs528350911），该变异会破坏GATA基序。将该风险变异引入hPSCs后，编辑等位基因上关键胰腺转录因子（GATA4、GATA6与FOXA2）的结合水平显著降低，同时伴随ONECUT1转录水平小幅下降，证实了该风险变异在代谢疾病中的因果作用。本研究通过对该远程增强子的表征，拓展了我们对胰腺发育过程中转录调控的认知，并凸显了在疾病相关场景中开展增强子发现研究，对理解单基因与复杂疾病的应用价值。
整体实验设计：
实验室：Danwei Huangfu，纪念斯隆凯特琳癌症中心（MSKCC）
资助编号：1U01DK128852-01
登录号：4DNESOGBAIC7
实验条件：胰腺前体细胞 - H1
数据集标签：PDX1-GFP dCAS9-KRAB 原位Hi-C（in situ Hi-C）
实验对象：诱导至胰腺祖细胞（pancreatic progenitor cells, PP）的H1人类胚胎干细胞
实验类型：原位Hi-C（in situ Hi-C）
数据链接：https://data.4dnucleome.org/experiment-set-replicates/4DNESOGBAIC7/
实验数量：2
实验1：采用Arima法诱导PDX1-eGFP dCAS9-KRAB H1细胞至前肠内胚层的原位Hi-C实验（A1、A2组），登录号4DNEXMHLGGWC
实验2：采用Arima法诱导PDX1-eGFP dCAS9-KRAB H1细胞至前肠内胚层的原位Hi-C实验（A1、A2组），登录号4DNEXUDU96VW
系列补充文件：
******
工作流运行链接：https://data.4dnucleome.org/workflow-runs-awsem/3e86c39e-47f3-4656-ac56-ab63aa493af7/
基因组组装版本：GRCh38
来源文件：4DNFI823LSII.chrom.sizes、4DNFITG6TM26.pairs.gz、4DNFINK7ALPW.pairs.gz、4DNFIU1RS39P.txt
文件描述：Hi-C处理流程的输出文件
文件类型：接触列表-合并文件（contact list-combined）
文件格式：pairs
文件名：4DNFIEAQ6DIR.pairs.gz
******
工作流运行链接：https://data.4dnucleome.org/workflow-runs-awsem/3e86c39e-47f3-4656-ac56-ab63aa493af7/
基因组组装版本：GRCh38
来源文件：4DNFI823LSII.chrom.sizes、4DNFITG6TM26.pairs.gz、4DNFINK7ALPW.pairs.gz、4DNFIU1RS39P.txt
文件描述：Hi-C处理流程的输出文件
文件类型：接触矩阵（contact matrix）
文件格式：mcool
文件名：4DNFIYC4AJW5.mcool
******
工作流运行链接：https://data.4dnucleome.org/workflow-runs-awsem/4a5f3a30-c608-4367-953b-e5300001f22b/
基因组组装版本：GRCh38
来源文件：4DNFIYC4AJW5.mcool
文件描述：Hi-C接触矩阵的边界调用结果
文件类型：边界文件（boundaries）
文件格式：bed
文件名：4DNFINRZXAQF.bed.gz
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工作流运行链接：https://data.4dnucleome.org/workflow-runs-awsem/4a5f3a30-c608-4367-953b-e5300001f22b/
基因组组装版本：GRCh38
来源文件：4DNFIYC4AJW5.mcool
文件描述：Hi-C接触矩阵的Diamond绝缘评分调用结果
文件类型：绝缘评分-Diamond（insulation score-diamond）
文件格式：bw
文件名：4DNFI4F6P2UE.bw
******
工作流运行链接：https://data.4dnucleome.org/workflow-runs-awsem/d04948a9-1b41-47f2-80b0-8e3a74eb90af/
基因组组装版本：GRCh38
来源文件：4DNFIYC4AJW5.mcool、4DNFI7MCA4R6.bedGraph.gz
文件描述：Hi-C接触矩阵的区室化信号结果
文件类型：区室文件（compartments）
文件格式：bw
文件名：4DNFI9C517H3.bw
******
BED文件：采用HiC-Pro (3.1.0)、HiTC (1.34.0)、HiCDC+(1.5.2)、TopDom进行比对分析，分辨率为50 kb
文件格式：bed
文件名：4DNFIJI7XHPW.bed.gz