Emerging insights into a role for Topoisomerase IIB and Methyl-CpG binding protein 2 in regulating neuronal transcription

The mammalian brain has unparalleled diversity of cell types with distinct molecular, morphological, connectional and functional properties. The specialization of these cells occurs during development through a series of spatially and temporally controlled changes in gene expression that are critical for proper assembly and function. Neurons in particular have complex and elaborate gene regulatory systems which allow specific combinations of genes to be expressed at distinct levels and at discrete developmental stages, giving rise to heterogenous neuronal subtypes. Identifying unique neuronal gene regulatory factors that contribute to the dynamic changes in gene expression are critical to understanding the molecular mechanism that underlie proper brain development and function. Recent progress has been made to characterize the gene regulatory programs that govern the establishment and organization of the neuronal transcriptome across development. A signature feature of the neuronal transcriptome that has provided considerable insight into the distinct gene regulatory events required for proper neuronal development is the expression of the very long genes (e.g. \u3e 100 kilobases). These genes encode proteins enriched for functions in neuronal physiology, including signaling molecules, receptors, ion channels and cell adhesion molecules, and many long genes have been identified as disrupted in neurodevelopmental disorders. Notably, two transcriptional regulators, the Methyl-CpG binding protein 2 (MeCP2) and Topoisomerase IIB (TOP2B), were recently identified as being able to tune the expression of long genes in the neurons. MeCP2, the protein mutated in Rett syndrome, can function as a transcriptional repressor that binds methylated DNA within long genes and prevents the overexpression of long genes. In contrast, TOP2B functions to resolve topological constraints, such as supercoiling, by creating transient double-strand breaks in the DNA, and has been shown to facilitate the expression of very long genes. Therefore, defining the mechanism underlying how these seemingly antagonistic transcriptional regulators may co-operate to regulate the expression of long genes is paramount. Moreover, elucidating this mechanism may provide key insights towards understanding the unique gene-regulatory landscape in neurons during development and discerning how dysregulation may lead to neurodevelopmental disorders. Here, I investigate the role of MeCP2 and TOP2B during the transcription of long gene in neurons. Using biochemical approaches, I demonstrate a functional interaction between MeCP2 and TOP2B and map the sequences that mediate the TOP2B-MeCP2 interaction. The role of TOP2B during transcription and the precise mechanism by which TOP2B facilitates gene expression is largely unknown. To address the gap in knowledge, I adapted and implemented a unique genomic approach called etoposide-mediated topoisomerase immunoprecipitation sequencing (eTIP-seq) to profile TOP2B activity genome-wide. Through this analysis, I identify a length-dependent enrichment of TOP2B activity within long genes, including at key regulatory sites, downstream of promoters and at intragenic enhancers. To investigate the role of MeCP2 in regulating TOP2B activity, I conducted experiments in which I manipulated the levels of MeCP2 and assessed changes in TOP2B activity. I demonstrate that MeCP2 negatively regulates TOP2B activity at long genes that are repressed by MeCP2. Finally, I highlight outstanding questions regarding the functional significance of the interaction between MeCP2 and TOP2B that may be relevant to Rett syndrome pathology. Future studies can address these open questions and build upon our current understanding of this neuronal transcriptional regulatory mechanism