1. Single-cell Joint Epigenetic Analyses in the Developing Brain

In the first of this series of blogs, we described how Heffel et al. evaluated chromatin conformation and DNA methylation from the same single nuclei (Heffel et al.) by applying the single-nucleus methyl-3C sequencing (snm3C-seq3) technique (Lee et al. and Tian et al.). While this paper employed snm3C-seq3 to support multimodal epigenetic analysis in single cells, parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing or “Paired-Tag” from Epigenome Technologies represents an exciting commercially available means to generate joint epigenetic and gene expression profiles at the single-cell resolution and detect histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP–seq assays. Applying Paired-Tag technology may enable quantum leaps forward in our understanding of development and significantly improve disease management (and more!).

2. Epigenetic Dynamism and Diversity in the Developing Human Brain

The development of the brain involves dynamic alterations at multiple epigenetic levels in diverse cell types, and single-cell analytical tools have helped us to understand these processes. Research from the Mercedes F. Paredes (University of California, San Francisco) and Chongyuan Luo (University of California, Los Angeles) labs aimed to describe genome-wide reorganization at the epigenomic and chromatin conformation levels during the development of the hippocampus (HPC) and dorsal prefrontal cortex (PFC), which are involved in learning and cognition (Kolb et al. and Rubin et al.).

In the second of a series of blog articles, Epigenome Technologies reports on how the authors integrated multimodal chromatin and RNA imaging, evaluated regulatory programs active in the developing brain, and reported on how single-cell 3D multiomics represents a powerful means of dissecting neuropsychiatric risk loci.

3. Multimodal chromatin and RNA imaging in human brain tissue

• The team validated the enrichment of short-range interactions in mid-gestational neurons via the chromatin tracing and RNA multiplexed error-robust fluorescence in situ hybridization (MERFISH) platforms (Su et al., Bintu et al., and Boettiger et al.), supporting the imaging of 3D organization, gene expression, and nuclear architectural proteins
• The sequential and uniform labeling of 354 genomic loci supported the imaging of chromosome 14 at 250-kb resolution in hippocampal neurons
• RNA MERFISH analysis evaluated the same cells with a probe panel targeting nearly 300 genes that display cell-type-specific expression in the developing hippocampus
• Unbiased clustering of integrated MERFISH and DNA methylation profiles identified cell types
• Average cell-type-specific distance matrices from chromosome 14 imaging critical chromosomal organization features reported by chromatin conformation capture techniques
• Imaging quantification of spatial distance between loci revealed differences between neurons and non-neuronal cells
• Neuronal cells possessed genomic regions separated by short distances, which revealed a compact spatial distance indicative of a high interaction frequency, while distal genomic regions displayed more considerable distances indicative of low interaction frequencies
• Meanwhile, progenitors/non-neuronal cell types displayed the opposite pattern
• Imaging confirmed the enrichment of short-range interactions in neurons and long-range interactions in non-neurons and validated the emergence of short-range interactions at the expense of long-range interactions during radial glia differentiation into excitatory neurons
• Imaging of nuclear architectural proteins/post-translational modifications revealed that spatial distances of distal regions correlated with nuclear volume, while loci separated by short genomic distances correlated with H3K9me3 levels

4. DNA methylation and chromatin conformation data dissect neuropsychiatric risk loci

• The team localized heritability signals of neuropsychiatric disorders across developmental stages and cell types by integrating DNA methylation and chromatin conformation data
• Significantly greater enrichment of heritability in loop-connected DMRs supported the utility of chromatin loops in locating causal variants
• Of 190 putative causal schizophrenia loci, 111 and 81 loci contain at least one putative causal single nucleotide polymorphism (SNP) that overlapped with a DMR or loop-connected DMR
• They observed a strong correlation between the odds ratio of overlapping with a putative causal SNP and the enrichment of polygenic heritability across cell types
• The rs500102 putative causal variant for schizophrenia overlaps with a loop-connected DMR in excitatory neurons
• This variant represents an expression quantitative trait locus of RORB in brain tissue
• The region where rs500102 localizes connects by a loop domain to the RORB promoter in excitatory neurons
• The loop domain associates with the cell-type-specific reduction of DNA methylation in the RORB gene body and the region surrounding rs500102
• This robust example demonstrates the utility of single-cell multiomic profiling in generating hypotheses regarding the function of variants associated with genome-wide association studies

• An assessment of the developmental dynamics of enrichment for neuropsychiatric disorder heritability in neuronal cells revealed similar patterns for DMRs and loop-connected DMRs during development
• Loop-connected DMRs display higher overall enrichment of heritability
• Enriched polygenic heritability for schizophrenia and bipolar disorder increases from neuroprogenitors to early post-mitotic to post-mitotic neurons in late-gestational brains for excitatory and inhibitory cells
• Meta-analysis of excitatory/inhibitory cells revealed an increase in the enrichment of schizophrenia/bipolar disorder between neuroprogenitors and neurons in the infant brain, followed by a decrease in the adult brain
• These results indicate that the genetic risk of schizophrenia and bipolar disorder more strongly affects post-mitotic neurons than the neuroprogenitor population in developing human brain

5. The Power of Multimodal Analysis in Single Cells

This second blog article of the series further highlights the utility of multimodal epigenetic analysis – DNA methylation and chromatin conformation – in single cells as a means to understand human brain development. In addition to describing multimodal chromatin and RNA imaging, the authors reveal how their data can be applied to further our appreciation of the regulatory programs associated with the developing human brain and provides a resource to explore the genetic and epigenetic mechanisms associated with the development of brain diseases.

Paired-tag represents a complementary analytic platform, creating joint epigenetic and gene expression profiles at single-cell resolution and detecting histone modifications and RNA transcripts in individual nuclei. This advance was first developed by a team guided by Bing Ren at the University of California San Diego; now, Epigenome Technologies provides optimized Paired-Tag kits and services to researchers in the epigenetics field under an exclusive license from the Ludwig Institute for Cancer Research.

For more on how single-cell DNA methylation and chromatin conformational analyses can help to describe the developing human brain, see Nature, November 2014.