1. Introduction

Applying “Paired-Tag” (parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing) for joint epigenetic and gene expression profiling at single-cell resolution can improve our understanding of the epigenetic underpinnings of disease-affected tissues. Research has linked dysregulated gene expression to the development of conditions such as Alzheimer’s disease, and evidence suggests that epigenetic factors play a significant potentially targetable role in developing and maintaining disease-associated transcriptomic profiles.

A series of introductory articles have described the development and applications of i) Paired-Tag, first described in a Nature Methods article by Bing Ren at the University of California San Diego, and ii) Droplet Paired-Tag – a fast, accessible methodology – as described in a Nature Structural & Molecular Biology article from the same team.

Epigenome Technologies now provides optimized Paired-Tag kits and services to researchers in the epigenetics field under an exclusive license from the Ludwig Institute for Cancer Research. Paired-Tag remains the only commercially available technology for joint epigenetic and gene expression profiling in single cells.

Can this technology define the dynamic, cell-type-specific epigenomic landscapes of complex tissues and diseases and identify potential therapeutic targets? A recent application in the brains of Alzheimer’s disease patients, described in this fourth introductory article, suggests that Paired-Tag technology can open a world of possibilities.

2. Single-cell Epigenome Analysis of Alzheimer’s Disease Brains

A recent application of Paired-Tag sought to identify the main cell types involved in Alzheimer’s disease pathology and understand the molecular mechanisms underlying this devastating disease. Establishing Paired-Tag technology as fit-for-purpose in single-cell epigenetic/transcriptomic studies of Alzheimer’s disease represented the primary goal of this study, which involved demonstrating agreement with previous single-cell results, identifying differential histone modification dynamics within cell types, linking Alzheimer’s disease risk mutations to epigenetic states, and exploring the functional enrichment of Alzheimer’s disease risk in cell-specific epigenetic states.

The findings of this exciting study (as summarized below) confirmed these objectives and highlighted Paired-Tag’s potential to advance Alzheimer’s disease research by identifying epigenetic regulators of pathological or pathogenic cell states.

This study involved improving Paired-Tag technology to support an in-depth understanding of Alzheimer’s disease

• Revision of chemistry and oligonucleotide design
• The implementation of lectin beads improved the nuclei recovery rate while redesigned combinatorial oligonucleotides improved DNA library complexity
• Paired-Tag profiling of Alzheimer’s disease and healthy control cortices
• Generation of libraries employed over one million nuclei across twenty post-mortem human brains (10 disease/10 controls; matched for age, sex, and APOE status), while sequencing sufficient libraries supported a study of over 500,000 tagmented nuclei
• Application of droplet Paired-Tag to Alzheimer’s disease brain
• The microfluidic-compatible Droplet Paired-Tag protocol applied the newly developed oligonucleotides to three post-mortem brains, which demonstrated improved RNA library complexity and high concordance with Split&Pool Paired-Tag
• Generation of a pilot Alzheimer’s disease cellular epigenetics resource
• Paired-Tag analysis of the Alzheimer’s disease brain facilitated the creation of a human parietal cortex cellular transcriptomic and epigenetic atlas, which represents a potentially instrumental resource in Alzheimer’s disease research

Paired-Tag: Providing New Scientific Insight Relevant to Alzheimer’s Disease

Analysis of the Paired-Tag data revealed significant findings in Alzheimer’s disease research:

• Recapitulation of frontal cortex disease signatures in the parietal cortex
• The thousands of differentially expressed genes identified across multiple cell types included 420 in astrocytes and 1334 in specific excitatory neurons
• The scREAD database of Alzheimer’s disease single-cell differential gene expression verified that cellular differential expression signatures display preservation between the frontal and parietal cortex
• Identification of chromatin state shifts in Alzheimer’s disease within cell types
• Applying ChromHMM to aggregated signals within Alzheimer’s disease and healthy control brain cells identified signatures of weak and robust transcription, bivalency, and repression
• Microglial enhancers as a mediator of Alzheimer’s disease risk
• Application of linkage disequilibrium score regression to histone modification maps determined enrichment for both strongly- and weakly-active regions in microglia, suggesting that epigenetic determinants of enhancer states contribute to Alzheimer’s disease risk
• Annotation of genome-wide association study variants revealed regions in the MS4A locus that shift from strong bivalency in microglia into a repressed state in Alzheimer’s disease patients
• Alleles associated with MS4A downregulation correlate with lower sTREM2 and earlier onset of Alzheimer’s disease
• Epigenetic erosion in excitatory neurons via aberrant acetylation
• The hundreds of differentially active peaks identified across cell types included 76 in excitatory neurons
• Comparing region activity in terms of H3K27ac deposition via repressed regions from ENCODE cortical and cell line samples identified a signature of aberrant activation in excitatory neurons in Alzheimer’s disease absent in other cell types

Bring The Power of Paired-Tag to Your Research

Paired-Tag from Epigenome Technologies represents an exciting and commercially available means for joint epigenetic and gene expression profiling at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei. Overall, Paired-Tag methodology may enable quantum leaps forward in our understanding of development and significantly improve disease management.

Here, Paired-Tag generated detailed insights into the molecular mechanisms of Alzheimer’s disease through joint epigenetic and gene expression profiling at the single-cell level. This advancement marks a significant step forward and may open new avenues for therapeutic interventions by targeting epigenetic remodeling complexes or de-repressed genes.