Functional biology of alternative promoters in the cerebral cortex - SUMMARY Alternative promoters are common in mammalian genomes, yet remain poorly understood. The activity of alternative promoters is thought to critical for regulating isoform selection and driving the diversity of expression patterns of genes and proteins. In addition to producing different protein isoforms and driving isoform-specific expression patterns, studies have shown that promoter and associated transcription start site (TSS) choice impacts splicing and untranslated region (UTR) use impacting mRNA localization, stability, and translation, highlighting the complex manners via which promoters contribute to gene expression. While we have known for over two decades that over half of human genes have alternative promoters, this complexity remains overlooked or ignored in most genetic, functional, and disease modeling studies. This is of particular concern with regards to brain development and function and neurodevelopmental and neurological disorders (NDDs). The brain overall, and cerebral cortex in particular, has been reported to have among the highest rates of isoform diversity and NDD associated genes feature multiple promoters and isoforms. Alternative promoters have been defined through comparative genomics, transcriptomics, and epigenomics. Most recently, single cell and long-read RNA sequencing technologies are being used to characterize accessibility, gene expression, and isoform use in healthy and diseased brain. Even with emerging maps of single cell accessibility and isoform activity, major questions remain regarding the cell-type and stage-specific specific activity, transcriptional regulatory mechanisms, and functional relevance of alternative promoters. While alternative promoters have the capacity to direct discrete transcriptional activity, a simple model of alternative promoter regulatory and isoform independence is not sufficient to explain the structure, function, and interaction landscapes of alternative promoters. Our preliminary data and other studies show that promoter-promoter interactions are common, that alternative promoters of the same gene can be co-active in the same single cells, and that many alternative promoters produce what are predicted to be nearly identical mRNA sequences beyond the TSS and 5’ UTR. Here, we will address the need for better understanding of alternative promoter biology in the mouse and human cerebral cortex through genomic and transcriptomic profiling paired with functional studies, including promoter modulation and deletion in vitro and in vivo. In Aim1, we will build an integrated cell-type and stage resolved map of alternative promoter and isoform use in mouse and human cortex. In Aims 2 and 3, we interrogate the sufficiency and necessity of NDD-linked alternative promoters, defining promoter interactions, assaying promoter function, and via studies of alternative promoter deletion mouse models. Overall, this work will address major questions regarding alternative promoters in the mammalian brain, building an alternative promoter informed gene regulatory model and yielding insights on alternative promoters and isoform expression of NDD risk genes.
