Project Summary Epigenetic modifications are essential chemical modifications that play critical roles in gene regulation, development, and diseases. Therefore, understanding how epigenetic changes between species occur and how they affect gene regulation has potential to advance our knowledge of regulatory evolution. However, the details of epigenetic evolution are sparse, and how epigenetic evolution correlates with phenotype evolution is even less understood. The proposed research will address this gap of knowledge by integrating novel data on DNA methylation with primate brain evolution. Studies of human brains have demonstrated that distinctive brain cell types have substantially different DNA methylation and gene expression, and analyses without separating these cell types can yield misleading results. Additionally, comparative studies of primates and other mammals have shown that the anatomical and cellular structure of brain regions evolve at varying rates as a result of differences in neurodevelopmental events linked to overall brain size. DNA methylation is a key molecular mechanism to record and affect development, and shows difference between brain regions. Therefore, the proposed research will test a novel hypothesis that DNA methylation of distinctive cell types in human and non-human primate brains shows variation consistent with brain size evolution. Moreover, validation studies will be performed for specific candidate genes and genomic regions that show DNA methylation and gene expression difference related to brain region differences and species differences. Specifically, evolutionary histories will be constructed for DNA methylation (Specific Aim 1) and gene expression (Specific Aim 2) from two major subclasses of neurons (excitatory and inhibitory neurons) as well as oligodendrocytes (a major non-neuronal cell) of brains from diverse anthropoid primates, including humans, apes, and monkeys. Highly divergent brain regions in terms of function and anatomy (e.g., prefrontal cortex and the pons) will be compared to connect changes at the phenotypic level to molecular changes. Some of these candidate genes will be further investigated in deeper histological resolution (Specific Aim 3). This study will generate novel data to expand our understanding of epigenetic evolution of brains, and to infer functionally important positions of noncoding genomic regions. Furthermore, it will also provide knowledge on how epigenome changes during evolution and how epigenome evolution correlates with phenotype, which is a fundamental yet currently little understood topic.