Epigenomic Profiling of Brain: Technology Development and Mechanistic Study

Abstract

Epigenetic regulation plays a critical role in brain development and mediates the long-term effects of environmental exposures on neuronal function. However, the cellular complexity of the brain and the limited sensitivity of conventional chromatin profiling methods present major challenges for understanding how environmental perturbations alter gene regulation. The overall goal of this dissertation was to investigate environmentally induced epigenomic changes in the brain and to develop new technologies for chromatin profiling in low-input and single-cell samples. The first part of this work examined the effects of maternal immune activation (MIA) on the adult mouse frontal cortex. Epidemiological studies have identified maternal infection during pregnancy as a significant risk factor for neurodevelopmental and psychiatric disorders in offspring. To explore the molecular consequences of maternal immune activation, we utilized a mouse model in which pregnant dams were infected with the mouse-adapted influenza A/WSN/33 (H1N1) virus. A cross-fostering design was employed to distinguish prenatal and postnatal effects of MIA. Histone modifications (H3K27ac and H3K4me3) and transcriptomic changes were profiled in neuronal nuclei isolated from the frontal cortex of adult offspring. The results revealed widespread epigenomic and transcriptomic alterations associated with both prenatal and postnatal MIA exposure. Prenatal MIA preferentially affected regulatory programs involved in forebrain and telencephalon development, whereas postnatal MIA was associated with pathways related to axonogenesis and synapse organization. Differential regulatory elements were enriched for genetic loci associated with neuropsychiatric disorders, supporting a model in which MIA contributes to disease susceptibility through epigenetic reprogramming of neuronal regulatory networks. The second part of this dissertation focused on the development of ChIPinDrop, a droplet-based single-cell chromatin immunoprecipitation sequencing (scChIP-seq) platform. ChIPinDrop integrates droplet microfluidics with ChIPmentation to perform chromatin digestion, immunoprecipitation, barcoding, and tagmentation within a streamlined workflow. The method was optimized through improvements in droplet generation, in-droplet immunoprecipitation, barcode bead design, droplet fusion, and library preparation. Species-mixing experiments demonstrated high barcode specificity, while profiling of multiple human cell lines showed that single-cell chromatin profiles could distinguish distinct cellular populations. The optimized workflow generated pseudobulk profiles that recapitulated known histone modification patterns observed in reference datasets. In preliminary sample for GM12878, the workflow yielded an average of 1,876 unique reads per cell. Preliminary application to mouse brain tissue further demonstrated the feasibility of extending the platform to complex biological samples. Together, these studies advance both the biological understanding of environmentally induced epigenomic alterations and the technological capabilities available for their investigation. The findings provide insight into the epigenetic mechanisms linking maternal immune activation to neurodevelopmental outcomes and establish a foundation for future single-cell studies of chromatin regulation in complex tissues and disease models.