Random autosomal monoallelic gene expression is a process in which transcription occurs stochastically from only one of two homologous alleles in a diploid cell independent of the genetic sequence or parental origin. The long-term goal of this project is to fully elucidate the regulation of random autosomal monoallelic expression and its relationship to development and disease. A complete understanding of this process will have significant implications when considering the genetic basis of both Mendelian and complex disorders as well as pathologies associated with haploinsufficiencies. Here, a series of Aims are proposed, that will utilize state-of-the-art molecular/genomic and cell biological approaches, to further define the underlying mechanism by which random autosomal monoallelic expression is initiated, maintained, and regulated upon differentiation. An RNA-seq screen will be performed to identify genes that exhibit random autosomal monoallelic expression upon the differentiation of mouse embryonic stem cells (ESCs) to different lineages/cell types. This will determine if the genes that are monoallelically expressed are differentiation program specific or if there is a single set of genes that become expressed monoallelically upon commitment of ESCs to multiple differentiation programs. In addition, the level of expression of the monoallelically expressed genes will be examined to identify genes whose expression follows the active allele dosage versus those that exhibit transcriptional compensation. In order to determine how random autosomal monoallelic expression is initiated and maintained stable ESC lines will be developed in which individual alleles are labeled with an MS2 or PP7 cassette such that the transcription site of each allele can be visualized in living cells. Using these cell lines the spaial and temporal dynamics of individual alleles will be studied upon the transition from bi-allelic to monoallelic expression. Circular chromosome conformation capture will be used to determine if the active and silent alleles associate with different regulatory elements and/or exist in differen gene hubs in 3D nuclear space. In addition, chromatin immunoprecipitation followed by allele-specific PCR covering an informative SNP will be used to determine if the active allele is bookmarked during mitosis to allow for the transmission of the monoallelic state during cell division. A series of experiments are proposed to provide insights into the mechanism by which monoallelic expression and transcriptional compensation are regulated. First, we will disrupt the ground state by introducing a third allele into monoallelically expressing cells and assess the impact on transcription of the endogenous active allele. In a second approach the CRISPR/dCas9 system will be used to independently manipulate the transcriptional status of each endogenous allele in monoallelic cells to assess the impact on transcription of the other allele and to see if the monoallelic state can be re-set. Together, the proposed studies will provide fundamental advances into our understanding of the relationship of random autosomal monoallelic expression to differentiation and will further define how it is established and regulated.
This project will provide significant advances toward understanding the establishment, maintenance, and regulation of random autosomal monoallelic expression upon the differentiation of mouse embryonic stem cells to different cell types. Our findings will have significant implications when considering pathologies associated with the deletion or mutation of one copy of a gene as well as single gene and complex multi-gene disorders.
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