1. Epigenetic Dynamism and Diversity in the Developing Human Brain

The development of the human brain involves dynamic alterations at multiple epigenetic levels in the diverse types of cells present; however, we currently lack a description of these processes at the single-cell level in prenatal human brain tissues. Researchers from the laboratories of Mercedes F. Paredes (University of California, San Francisco) and Chongyuan Luo (University of California, Los Angeles) sought to fill this knowledge gap by describing the genome-wide reorganization that occurs at the epigenomic and structural (3D chromatin conformation) levels during normal development of human hippocampus (HPC) and dorsal prefrontal cortex (PFC) – regions of the brain involved in learning and cognition (Kolb et al. and Rubin et al.). Heffel et al. carried this epic task out by evaluating chromatin conformation and DNA methylation profiles from the same single nuclei generated by applying the single-nucleus methyl-3C sequencing (snm3C-seq3) technique (Lee et al. and Tian et al.) and integrating orthogonal multimodal chromatin tracing and RNA/protein imaging.

Snm3C-seq3 libraries are bisulfite-converted 3C libraries, using single-nucleus sorting (and distribution into multiple 384-well plates) to enable integration of randomly-primed cell barcodes. The resulting libraries produce both contact maps (read pairs joining two chromosomal regions in close proximity) and CpG methylation under each contact. The obligate sorting step necessitates large input cell numbers, but provides the option to sort for specific cell subpopulations (e.g., NeuN+ or Sox9+). While the snm3C-seq3 technique supports multimodal epigenetic analysis in single cells, particular care is needed for cluster annotation using established cell-type-specific DNA methylation patterns. Heffel et al. utilize co-embedding of snm3C and snRNA datasets using Harmony to force nearby clusters together, a strategy which appears to work well in the case of brain cell types. Methods which capture native RNA, such as parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing or “Paired-Tag” from Epigenome Technologies 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 further leaps forward in our understanding of development and significantly improve disease management (and more!).

The new findings from the Paredes and Luo labs, reported recently in Nature (Heffel et al.), now provide a single-cell 3D multimodal resource that supports the ongoing study of the gene regulatory networks controlling brain development and the dissection of neuropsychiatric risk loci. Epigenome Technologies now breaks down this fascinating study in the first of a series of blog articles.

2. 3D Multi-omic Analysis in the Developing Human Brain

• Heffel et al. first identified 139 cell populations organized into ten major groups across developmental stages by combining CG/non-CG methylation and chromatin conformation data
• Excitatory neurons displayed distinct epigenomic profiles in the PFC and HPC (agreeing with spatially separated in situ neurogenesis), while inhibitory neurons/non-neuronal cell types shared broad epigenomic profiles
• DNA methylation and chromatin conformation profiles could separate neurons and neural progenitor-derived glial cells by developmental stages; however, non-neural cell types displayed similar epigenomic patterns during development
• Shared CG methylation patterns at cell-type marker genes and computational integration of cells from different age groups defined the developmental trajectories of PFC and HPC cells
• Interestingly, the remodeling of CG and non-CG methylation in the HPC preceded remodeling in the PFC

3. Exploring the Temporal Order of DNA Methylation and Chromatin Conformation Dynamics

• Pseudotime analysis supports the exploration of the temporal dynamics of chromatin conformation and DNA methylation in the diverse cell populations present during brain tissue development
• This includes cortical radial glia-derived cells, which differentiate down both glial (astrocytes, oligodendrocyte progenitor cells, and oligodendrocytes) and neuronal (excitatory neurons) lineages
• Overall, cell-type marker genes became depleted of gene body non-CG methylation in neurons in the adult brain
• While most genes gain non-CG methylation during neuronal maturation, cell-type marker genes are specifically protected from mCH accumulation
• A subsequent focus on radial glia-to-astrocyte differentiation supported the identification of rapid chromatin conformation remodeling predominantly occurring during mid-gestation, followed by a prolonged maturation of the CG methylome extending into the adult brain
• However, analysis of additional cell types revealed the temporal separation of CG methylation and chromatin interaction dynamics in most cell-type differentiation trajectories (with nuanced cell-type-specific patterns)

Identifying a Specific Chromatin Conformation in Neurons

• Chromatin conformation capture techniques produce 3D genome architecture snapshots at multiple scales
• A and B compartments are detected through long-range interactions, while chromatin domains/loops are detected by short-range interactions
• Analysis of chromatin conformation profiles revealed that profiles from single brain cells ranged from mainly containing short-range interactions to containing a substantial number of long-range interactions
• Neuronal cell types displayed mostly short-range interactions, while glial and non-neural cell types mainly displayed long-range interactions
• Neuron and astrocyte differentiation involved distinct global chromatin conformation remodeling events
• The differentiation of neural progenitors – depleted in long-range interactions but not enriched with short-range interactions – involved an increased proportion of short-range interactions in neurons and long-range interactions in astrocytes

The Power of Multimodal Analysis in Single Cells

This first blog of the series describes how joint epigenetic analyses – DNA methylation and chromatin conformation – in the same single cell help to describe the development of the human brain – highlighting dynamic shifts when moving from progenitors to neuronal/glial populations during perinatal development and the temporal separation of DNA methylation and chromatin conformation reconfigurations – but also provides a multimodal resource that supports the ongoing study of gene regulatory dynamics.

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.