Histones are proteins that both form the fundamental unit of DNA packaging and regulate genomic activity. In cancer, the balance between canonical histones and various functional histone variants is swayed, playing a key role in supporting cancer progression and proliferation. However, it is currently not fully understood how histone homeostasis is controlled and altered in cancer.
The expression of canonical histones is highly regulated, occuring within specialized nuclear foci called Histone Locus Bodies (HLBs), which have been suggested to be formed by phase separation of the NPAT protein. We have identified in cancer mutation databases a recurring NPAT mutation which appears independently in several cancer types. We reproduced this mutation, along with a set of other protein-truncating mutations, into a human cell line, allowing us to study their effects in a neutral environment. We hypothesize that in cancer, specific NPAT mutations may affect HLBs, leading to altered nuclear organization and perturbed histone homeostasis.
To test this hypothesis, we propose to build on our labs’ joint expertise and characterize the effects of these mutations using microscopic and genomic approaches. These include histone DNA oligoFISH, histone RNA FISH, NPAT immunofluorescence, RNA-Seq and Hi-C. Together these will provide us with a comprehensive view of the consequences of NPAT dysfunction on both nuclear organization and gene expression. If successful, this study will establish a foundation for studying the mechanisms controlling the balance of replication dependent-histones and histone variants in cancer.