MAPT expression is mediated by long-range interactions with cis-regulatory elements [ATAC-seq]. MAPT expression is mediated by long-range interactions with cis-regulatory elements [ATAC-seq]

Tau is encoded by MAPT and abnormal aggregates of tau are a hallmark of a group of neurodegenerative diseases called tauopathies. MAPT is lowly expressed in neural progenitor cells (NPCs), but it is more highly expressed in oligodendrocytes, astrocytes, and neurons that derive from NPCs. This expression switch at differentiation suggests that MAPT expression is controlled by transcription factors and cis-regulatory elements specific to these differentiated cell types, including neurons. Overall design: We performed HiC and three-dimensional chromatin conformation capture (Capture-C), single-nucleus multiomics (RNA-seq and ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27Ac and CTCF in neurons differentiated from human iPSC cultures. From these methods, we nominated candidate cis-regulatory elements (cCREs) for MAPT in human NPCs, NPCs differentiated to neurons, and pure inhibitory or excitatory neuron cultures. We then functionally assessed nominated regions using luciferase assays and CRISPR dCas9-KRAB inhibition experiments to assess the effect of nominated regions on MAPT expression.

Tau蛋白由MAPT基因编码，Tau蛋白的异常聚集是一类被称为tau蛋白病（tauopathies）的神经退行性疾病的标志性病理特征。MAPT在神经前体细胞（neural progenitor cells, NPCs）中呈低表达状态，但在由神经前体细胞分化而来的少突胶质细胞、星形胶质细胞以及神经元中表达水平显著升高。这种分化过程中的表达转换现象提示，MAPT的表达受到这些分化细胞类型（包括神经元）特异性的转录因子和顺式调控元件的调控。
实验整体设计：本研究对人诱导多能干细胞（human induced pluripotent stem cell, iPSC）诱导分化得到的神经元开展了多项组学与表观基因组学实验，包括HiC技术、三维染色质构象捕获（Capture-C）、单核多组学分析（RNA测序（RNA-seq）与转座酶可及性测序（ATAC-seq））、批量ATAC-seq，以及针对H3K27乙酰化修饰（H3K27Ac）和CTCF蛋白的染色质免疫共沉淀测序（ChIP-seq）。基于上述实验手段，我们分别在人神经前体细胞、向神经元分化的神经前体细胞以及纯抑制性神经元或兴奋性神经元培养体系中，筛选得到了MAPT的候选顺式调控元件（candidate cis-regulatory elements, cCREs）。随后，我们通过荧光素酶报告基因实验与CRISPR dCas9-KRAB抑制实验对候选区域进行功能验证，以评估这些区域对MAPT表达的调控作用。