expression is mediated by long-range interactions with -regulatory elements.

Background: Tauopathies are a group of neurodegenerative diseases driven by abnormal aggregates of tau, a microtubule associated protein encoded by the gene. expression is absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that expression is controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding genetic risk factors.

Methods: We performed HiC, chromatin conformation capture (Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27Ac and CTCF in NPCs and neurons differentiated from human iPSC cultures. We nominated candidate cis-regulatory elements (cCREs) for in human NPCs, differentiated neurons, and pure cultures of inhibitory and excitatory neurons. We then assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in AD cases and controls.

Results: Using orthogonal genomics approaches, we nominated 94 cCREs for , including the identification of cCREs specifically active in differentiated neurons. Eleven regions enhanced reporter gene transcription in luciferase assays. Using CRISPRi, 5 of the 94 regions tested were identified as necessary for expression as measured by RT-qPCR and RNA-seq. Rare and predicted damaging genetic variation in both nominated and confirmed CREs was depleted in AD cases relative to controls (OR = 0.40, p = 0.004), consistent with the hypothesis that variants that disrupt enhancer activity, and thereby reduce expression, may be protective against neurodegenerative disease.

Conclusions: We identified both proximal and distal regulatory elements for and confirmed the regulatory function for several regions, including three regions centromeric to beyond the well-described H1/H2 haplotype inversion breakpoint. This study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.