Investigating the role of PRMT1 and hnRNPK in the spatial regulation of heterochromatin

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

PROJECT SUMMARY In virtually all cell types, heterochromatin is enriched at the nuclear periphery, where it contacts the nuclear lamina and forms lamina-associated domains (LADs). LADs tend to be transcriptionally inactive and enriched for repressive histone modifications. It has been suggested that one possible function of peripheral positioning is to sequester genes whose expression would be inappropriate in that cell type, thereby ensuring maintenance of cell identity. In support of this model, LAD organization has been linked to both human disease and normal development. However, due to the limited number of factors known to regulate LADs in human cells, it has been challenging to directly test these and other models for LAD function. To this end, our group completed a genome-wide screen to identify factors required for maintaining chromatin positioning at the nuclear periphery. The arginine methyltransferase PRMT1 emerged as a top hit from the screen, and subsequent work found that PRMT1 regulated the peripheral attachment of hundreds of LADs across the genome. Interestingly, we also observed a reduction in the heterochromatic mark H3K9me2 over a subset of PRMT1-sensitive LADs. Having characterized PRMT1 as a novel regulator of peripheral chromatin, we set out to elucidate the mechanism by which it maintains LAD positioning. Strikingly, a canonical PRMT1 target, the RNA-binding protein hnRNPK, also emerged as a top hit from our genome-wide screen. hnRNPK has previously been demonstrated to bind nuclear lamina components and heterochromatin, making it an attractive candidate for serving as a tether of peripheral chromatin. The goal of this proposal is to determine the mechanism by which PRMT1 and hnRNPK regulate peripheral chromatin organization and to elucidate the effect of disrupted LAD positioning on heterochromatin maintenance and transcriptional repression over time. I hypothesize that PRMT1-mediated methylation on hnRNPK allows hnRNPK to act as a physical bridge tethering heterochromatin to the nuclear lamina. I will first profile LAD positioning genome-wide following hnRNPK KD to determine if loss of hnRNPK recapitulates the impaired peripheral positioning of LADs observed upon PRMT1 KD. I will then determine using imaging and genomics approaches whether PRMT1 regulates the localization of hnRNPK to chromatin or to the lamina. Finally, I will specifically perturb arginine methylation on hnRNPK to test if this modification is required for maintenance of peripheral positioning. In parallel, these novel regulators will be used to disrupt LAD organization and investigate the impact on heterochromatin maintenance and transcriptional repression over time. Ultimately, the work proposed here will pave the way for future studies understanding how spatial chromatin organization contributes to normal development and human disease.