ABSTRACT This Diversity Supplement proposal aims to support Vanessa Lopez, an incoming Postdoctoral Fellow, in extending the objectives of the Parent R00 grant. The focus of this supplement is on elucidating the impact of disrupting the Top2a-PRC2-H3K27me3 pathway on brain’s epigenetic state at a single-cell level. This goal aligns closely with the Parent R00's objectives and will further the aims of the Parent R00, particularly Specific Aim 2. The Parent R00 has uncovered a novel toxicological pathway implicating Top2a in sensing environmental toxicants during vertebrate development and leading to social behavior deficits in zebrafish and mouse. Additionally, it has highlighted the role of PRC2 and H3K27me3 in this pathway, especially in regulating autism risk genes. The proposed research aims to delve deeper into this pathway using single-cell multimodal omics sequencing and computational analysis. Bulk sequencing methods used in the Parent R00 provided valuable insights but lacked cell-type-specific information crucial for understanding complex epigenetic regulations of brain function. To overcome this limitation, the proposed Diversity Supplement will employ cutting-edge single-cell multimodal omics technologies, including Paired-Tag and Paired-seq, to map the single-cell transcriptome and epigenome of wild-type larval zebrafish brains as well as in zebrafish brains depleted of Top2a. This will enable the characterization of cell- type-specific epigenetic changes induced by Top2a inhibition. Specific Aim 1 will apply Paired-Tag to profile single-cell transcriptome and H3K27me3 in wild-type and Top2a knockout zebrafish brains to identify cell-type-specific changes. Specific Aim 2 will deploy Paired-seq to assess single-cell transcriptome and chromatin accessibility to identify cis-regulatory elements (cCREs) regulated by the Top2a-PRC2-H3K27me3 pathway and discover toxicant-responsive CREs targeting autism risk genes. Collaboration with a mentoring team consisting of experts in single-cell multimodal omics analysis, environmental toxicology, and autism research will provide Ms. Lopez with a unique skill set to conduct her research and advance her career to becoming an independent researcher. Overall, this Diversity Supplement aims to bridge knowledge gaps in understanding the molecular mechanisms underlying epigenetic toxicity and its implications for neurodevelopmental disorders like autism.