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

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC & Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

Single nucleus multiomics identifies ZEB1 and MAFB as candidate regulators of Alzheimer's disease-specific cis-regulatory elements.

Cell type-specific transcriptional differences between brain tissues from donors with Alzheimer's disease (AD) and unaffected controls have been well documented, but few studies have rigorously interrogated the regulatory mechanisms responsible for these alterations. We performed single nucleus multiomics (snRNA-seq plus snATAC-seq) on 105,332 nuclei isolated from cortical tissues from 7 AD and 8 unaffected donors to identify candidate cis-regulatory elements (CREs) involved in AD-associated transcriptional changes. We detected 319,861 significant correlations, or links, between gene expression and cell type-specific transposase accessible regions enriched for active CREs. Among these, 40,831 were unique to AD tissues. Validation experiments confirmed the activity of many regions, including several candidate regulators of APP expression. We identified ZEB1 and MAFB as candidate transcription factors playing important roles in AD-specific gene regulation in neurons and microglia, respectively. Microglia links were globally enriched for heritability of AD risk and previously identified active regulatory regions.

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

Background: Tauopathies are a group of neurodegenerative diseases driven by abnormal aggregates of tau, a microtubule associated protein encoded by the MAPT gene. MAPT expression is absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression is controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT 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 MAPT 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 MAPT 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 MAPT, 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 MAPT 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 MAPT enhancer activity, and thereby reduce MAPT expression, may be protective against neurodegenerative disease. Conclusions: We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT 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.

Long-read genome sequencing for the molecular diagnosis of neurodevelopmental disorders.

Exome and genome sequencing have proven to be effective tools for the diagnosis of neurodevelopmental disorders (NDDs), but large fractions of NDDs cannot be attributed to currently detectable genetic variation. This is likely, at least in part, a result of the fact that many genetic variants are difficult or impossible to detect through typical short-read sequencing approaches. Here, we describe a genomic analysis using Pacific Biosciences circular consensus sequencing (CCS) reads, which are both long (>10 kb) and accurate (>99% bp accuracy). We used CCS on six proband-parent trios with NDDs that were unexplained despite extensive testing, including genome sequencing with short reads. We identified variants and created de novo assemblies in each trio, with global metrics indicating these datasets are more accurate and comprehensive than those provided by short-read data. In one proband, we identified a likely pathogenic (LP), de novo L1-mediated insertion in CDKL5 that results in duplication of exon 3, leading to a frameshift. In a second proband, we identified multiple large de novo structural variants, including insertion-translocations affecting DGKB and MLLT3, which we show disrupt MLLT3 transcript levels. We consider this extensive structural variation likely pathogenic. The breadth and quality of variant detection, coupled to finding variants of clinical and research interest in two of six probands with unexplained NDDs, support the hypothesis that long-read genome sequencing can substantially improve rare disease genetic discovery rates.