Simultaneous Single-cell Epigenetic and Transcriptomic Analysis via Paired-Tag Highlights the Postnatal Impact of Prenatal Vaping

By Stuart P. Atkinson

Composite Scientific Image built from two figures of Chen et al. — Composite figure from Chen et al. Left: (a) Overview of experiment: maternal exposure to e-cigarette smoke during gestation, followed by single-nucleus RNA + CUT-n-Tag profiling. (b) RNA embedding of resulting nuclear expression profiles. Right: (a) Differential peaks, per cell type, for H3K4me1 and H3K27me3. (b) genomic annotation of called peaks for H3K4me1 and H3K27me3. (c) Overlap of exposed-vs-control differential peaks within ExN and InN.

Epigenetics: Linking Prenatal Vaping to Abnormal Brain Development in Offspring

The use of E-cigarettes or vaping during pregnancy can adversely affect offspring brain development (Lauterstein et al. and McGrath-Morrow et al.) thanks to the ability of the many harmful associated substances contained in vaping liquids/aerosols to pass through the placenta and induce neurotoxicity in offspring (Archie et al. and Nguyen et al.). A previous study from Charles Wang (Loma Linda University) employed single-nucleus RNA-sequencing and assay for transposase accessible chromatin-sequencing technology to reveal how prenatal vaping negatively impacted neuronal differentiation by altering chromatin accessibility at the promoters of genes critical to neuron development in offspring (Chen et al.); however, they hoped to venture further and explore the implications of additional layers of epigenetic modifications such as histone modifications - induced by prenatal vaping during early rat brain development.

A new study from the Wang lab, reported recently in Communications Biology Paired-Tag (parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing) from Epigenome Technologies (Zhu et al. and Protocol.io) to simultaneously analyze RNA transcription and histone modifications (permissive H3K4me1 and repressive H3K27me3) profiles in the same single nuclei isolated from the postnatal day 7 rat prefrontal cortex following prenatal exposure to vaping (Chen et al.). Importantly, the methylation of H3K4 and H3K27 histone lysine residues at the same gene promoters (forming so-called bivalent domains) plays a critical role in regulating neuronal development by maintaining the balance between active and inactive gene expression during neurogenesis. Meanwhile, the prefrontal cortex participates in higher-order brain functions such as cognition, learning, memory, reward, and addiction (Spinella, 2003) and remains vulnerable to stimuli such as nicotine, a primary constituent of vaping liquids/aerosols (Goriounova and Mansvelder).

Excitingly, this new study now highlights the negative impact of prenatal vaping on offspring and highlights Paired-Tag technology as a robust means of linking induced changes to histone modification profiles to gene expression alterations in the same single cell and exploring the effects of maternal vaping on brain development in offspring.

Figure 1 from Chen, et al. Single-cell embeddings of Paired-Tag dataset for two targets, H3K4me1 and H3K27me3. (Top) RNA-only, H3K4me1-only, and combined embedding of anti-H3K4me1 Paired-Tag. (Bottom) RNA-only, H3K27me3-only, and combined embedding of anti-H3K27me3 Paired-Tag.

Paired-Tag: The Simultaneous Analysis of Single-cell Epigenetic and Transcriptomic Profiles Reveals How Prenatal Vaping Negatively Impacts Neuronal Cells in Offspring

The authors first simultaneously profiled H3K4me1 and H3K27me3 patterning and transcriptomes from the same single nuclei using Paired-Tag; briefly, they exposed pregnant rats to E-cigarette vapor or control air from day 4 to 20 of gestation and then harvested the prefrontal cortex from the brains of postnatal day 7 offspring. Excitatory neurons identified by H3K4me1 or H3K27me3 profiles displayed similar expression of selected neuron markers, while differentially expressed genes between excitatory and inhibitory neurons from H3K4me1 and H3K37me3 datasets revealed consistent enrichment in biological processes such as neuron differentiation and development. Of the 1,306 differentially expressed genes, prenatal vaping impacted 205 of them in a cell-type-specific manner; furthermore, changes in H3K4me1 and H3K27me3 profiles at promoter regions accompanied alterations in excitatory or inhibitory neuron-specific gene expression. Overall, the Paired-Tag data provided evidence that prenatal vaping induced the altered expression of genes involved in neuronal processes in the prefrontal cortex of offspring by altering H3K4me1 and H3K27me3 profiles at gene promoters.

A detailed analysis of the single-cell transcriptional data from excitatory and inhibitory neurons suggested that prenatal vaping induced a broad effect on gene networks and pathways associated with neuron development in offspring, although these effects differed in males and females. The subsequent analysis of H3K4me1 and H3K27me3 in the prefrontal cortex of offspring revealed that prenatal vaping modulated profiles in most cell types captured; significantly, prenatal vaping dynamically altered H3K4me1 and H3K27me3 profiles associated with a large number of genes in excitatory and inhibitory neurons, which then promoted the altered expression of specific gene expression during neuronal development. A detailed analysis of the changing H3K4me1/H3K27me3 bivalent profile during brain development in response to prenatal vaping focused on excitatory neurons. Bivalent histone domains comprise overlapping regions of H3 lysine 27 methylation and H3 lysine 4 methylation, which prepare the developmental genes for rapid expression in a cell type or tissue-specific manner during differentiation and development (Bernstein et al., Macrae et al., and Yu et al.). Overall, the data suggested that prenatal vaping modulated the transition from H3K27me3 to H3K4me1 to orchestrate neuron-specific gene expression in the prefrontal cortex of the developing rat brain. Previous studies had revealed a requirement for the significant loss of H3K27me3 levels at promoters at many neural genes for the transition from transcriptionally inactive to active (Bernstein, Meissner, and Lander) and had highlighted a lack of impact of H3K4me1 (Rada-Iglesias et al.).

Composite scientific figure from figures 3 and 4 of Chen et al. — Composite figure from Chen et al. Top: (b) Differential expression within ExN and InN in mice pre-natally exposed to e-cigarette smoke. (c) Heatmap of average expression across selected genes in male and female mice (both exposed and control). Bottom: Heatmaps of average gene activation and repression as measured by H3K4me1 (d) and H3K27me3 (e) in ExN and InN, across both exposure and sex.

Paired-Tag: Linking Epigenetic Alterations to Gene Expression Changes in the Same Single Cell

In this exciting new study, Paired-Tag technology from Epigenome Technologies helped define the alterations induced by prenatal vaping in single cells of the developing brain in offspring; in detail, this approach permitted the authors to directly link vaping-induced alterations of bivalent histone modification patterning at gene promoters to altered gene expression in the same excitatory/inhibitory neuron. Whether this same mechanism impacts human offspring remains a question for future research.

Paired-Tag technology from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with comparable efficiency to single-nucleus RNA-seq/ChIP-seq assays while avoiding the need for cell sorting. The implementation of Paired-Tag technology may enable these researchers to define precisely how prenatal vaping by humans can affect brain development in offspring. Of note, Paired-Tag experiments were carried out with the assistance provided by Epigenome Technologies, following a previously described protocol.