ABSTRACT The human Brainspan data was created to identify all transcripts involved in neural development and to help understand of how specific risk genes affect human brain development. In addition, these data will have important clinical relevance for translational medicine; these data can help discern which risk alleles associated with psychiatric and neurological disorders influence transcription and alternative splicing across different regions and developmental stages. Also, most Brainspan samples were processing for whole-genome sequencing (WGS) and/or DNA methylation analysis, which enables direct comparisons of single basepair changes, copy number variation, and RNA editing events in the developing human brain. As such, Brainspan data holds biologically and clinically important data on the genetic and molecular mechanisms underlying the development and increased disease susceptibility of the human brain. To expand upon this resource, we aim to create a matched profile of the human brains RNA modification landscape (epitranscriptome), for both methyl-6-adenosine (m6A) and 5-methyl-cytosine (5mC). We will profile the developmental trajectory of the RNA modifications and their activity in non-coding regions and impact on splicing, RNA editing, AU-rich regulation of transcripts, and association with DNA methylation changes (epigenetics). Finally, we will also test the impact of these modifications from patient-derived iPS cells that will be grown and assayed over five time points. This will be accomplished over five years, and across 1,075 samples, across the Mason and Sestan labs, with collaborators at the Broad institute available to help with assays and access to GTEx data from adult brains with m6A profiles. We will achieve these goals across three main aims. (1) Create a neuro-developmental map for epitranscriptome sites and levels, with an emphasis on m6A and m5C, for 35 brains from four time periods, and five regions of the brain, chosen based on their large differences seen in the BrainSpan data and prior implication in neurological development. (2) Detail the inter-individual variation in epitranscriptome levels and their epigenetic regulation using m6A variation with the changes in expression levels, and then link epigenetic changes to altered gene expression and m6A regulation. (3) We will delineate the epitranscriptome changes in autism brains and manifestation in patient-derived iPS cells, including an examination of epitranscriptome variation across 30 banked Autistic brain samples and testing of the impact on disruption of the readers and writers of RNA regulation (on induced pluripotent stem cells). These will represent the first-ever epitranscriptome maps from primary tissue of Autism brains and help guide future studies that examine the dysregulation of Autism gene expression networks and epitranscriptome states.

NARRATIVE Chemical modifications of DNA and RNA (e.g. epigenome and epitranscriptome) helps mediate the dynamic responses of genes to the environment, guides cell delineation during development, and drives key decision points in brain development, yet these essential molecular mechanisms have barely been studied in in developing human brains. Moreover, the ability to test the readers, writers, and erasers of RNA methylation is now feasible with CRISPR-based systems, which we propose to use here to pinpoint the most critical sites of RNA modifications during neuronal differential, in the context of the large epigenetic and epitranscriptome atlas built here, called (EpiBrain).