Title: Integrated single-cell sequencing reveals epigenetic regulators in model of human neural tube morphogenesis

Abstract:

Development is governed not only by genome sequence but by the epigenetic information layered upon it. DNA methylation, chromatin accessibility, and transcription are deeply interconnected, yet resolving how they influence one another requires measuring them within the same cell. Here I present scDyMAT-seq, a single-cell method that simultaneously quantifies DNA methylation on both strands at individual CpG dyads, chromatin accessibility, and the transcriptome from the same cell. The method combines GpC methyltransferase labeling of accessible chromatin with MspJI digestion and enzymatic conversion of unmethylated cytosines for a strand-specific methylation readout, avoiding bisulfite-induced degradation. Applied to HEK293T cells, scDyMAT-seq recovered expected regulatory features, including greater methylation depletion at more accessible regions defined by DNase I hypersensitivity, establishing the method as a tool for integrated single-cell profiling.

I then applied scDyMAT-seq to understand how regulatory state is reorganized when human neural tube morphogenesis fails. Neural tube defects are among the most common human birth defects and are strongly influenced by epigenetic regulation. However, human neurulation occurs after implantation and is inaccessible to direct study. Using a three-dimensional human stem-cell model of neural tube morphogenesis carrying a HES1 mutation, I found that mutant structures fail to specify significant neural ectoderm, forming a narrowed, discontinuous, and branched tube. Differential methylation and accessibility between the parental line and HES1 mutant revealed a regulatory shift at the neural plate border.  Altogether, this work introduces a single-cell method for jointly profiling dyad-resolved DNA methylation, chromatin accessibility, and transcription, and demonstrates its application to a human model of neural tube development and disease, allowing us to understand how epigenetic regulation is disrupted in neural tube defects.