Genome-Wide Identification and Analysis of Human Neuronal Activity-Dependent Enhancers in Evolution and Disease

Abstract

Neuronal activity-responsive gene expression plays a key role in the proper development, refinement, and plasticity of neural circuits, and the dysregulation of these gene responses have been implicated in developmental and cognitive disorders. Thus, identification and molecular characterization of the cis-regulatory DNA elements driving this response in neurons has the promise to profoundly advance our understanding of neurological disease mechanisms. The transcription factor cAMP/Ca2+-responsive element binding protein (CREB) is an important stimulus-responsive factor in many cell types of the body. CREB and its transcriptional co-activators are important for gene regulation in the brain and play a critical role in cognitive development, neuronal survival, and learning and memory. In the following studies we performed mechanistic analysis in human and mouse neurons to understand genome-wide binding of CREB and its neuronal co-activator, CREB Regulated Transcriptional Co-activator 1 (CRTC1, a.k.a. TORC1) at non-coding regulatory elements in response to neuronal depolarization. In addition, we investigated post-translational modifications of CRTC1 in response to BDNF treatment to better understand the biochemical basis of this co-factor’s activation in mouse neurons. Using human induced pluripotent stem cell (iPSC)-derived neurons we hoped to explore CREB-associated regulatory element function that cannot be studied in rodent models. While transcriptional and epigenomic analysis of human post-mortem brain samples has allowed for recent advances in identifying neuronal cis-regulatory regions of the genome, these approaches cannot address the dynamic, experience-dependent nature of their function in neuronal gene expression programs. We have identified human and mouse neuronal activity-dependent regulatory elements using ChIP-seq for both histone modifications and transcription factors in human pluripotent stem cell derived neuronal cultures, and thus provide a resource map of neuronal activity responsive regulatory elements across the human and mouse genomes. We identified human neuronal stimulus-dependent binding sites for CREB and CRTC1, including those that contain SNPs associated with neurological disorders, such as the schizophrenia-associated locus of CACNB2. Other CREB-associated regulatory elements potentially control expression of primate-specific transcripts and could be important sites of genetic divergence. Further functional investigation of these sequences will allow us to understand their role in human disease and evolution.