Abstract The concept “chromostasis” or chromatin homeostasis refers to a chromatin environment that suppresses cellular plasticity and genome instability. Regulation of gene expression depends on histone post-translational modifications (PTM), DNA methylation, histone variants, and effector proteins that not only influence the structure and function of chromatin, but also affect essential processes such as DNA repair capacity, and cellular proliferation. The histone code hypothesis predicts that crosstalk between PTMs controls direct specific and distinct DNA-templated programs such as transcription, replication and DNA repair. Although histone PTMs can be mutually exclusive in their functional role, they still can have a strong influence into each other. A clear example of this is the H3K36me3-H3K27me3 axis. While H3K36me3 is associated with active transcription, H3K27me3, which is produced by the Polycomb repressive complex 2 (PRC2), maintains gene repression. H3K36me3 and H3K27me3 also have an antagonistic role on DNA double strand break (DSB) repair pathway choice and consequently controls genome stability status. Furthermore, the crosstalk between these PTMs is evident since depletion of H3K36me3 greatly influences the cellular H3K27me3 levels, and viceversa. Nevertheless, how these two PTMs functionally interact to control gene transcription and genome stability is still unclear. Using naturally occurring mutations in histone H3 as a model system, we will address the role of histone methylation in chromostasis. Notably, mutations in the lysine 36 on histone H3 to a methionine (H3K36M), as found in head and neck disorders, lead to a global reduction of H3K36me2/3 levels. The role of H3K36M and its crosstalk with H3K27me3 and other PTMs, as well as its influence in the epigenome and maintenance of genome stability, is poorly understood. Here we aim to understand how the balance between H3K27me3 and H3K36me2/3 controls gene transcription and genome stability in human cells. To this end, we will determine how H3K36M impacts the epigenetic landscape and whether rewiring the underlying epigenetic mechanisms can be exploited to maintain genome integrity. Our new preliminary data reveals a novel connection between H3K36M, the Polycomb complexes PRC1 and PRC2, DNA methylation, and genome instability in human cells. Moreover, we found a novel epigenetic complex sequestered by H3K36M. Finally, our studies show that H3K27me3 levels determine the proficiency of DNA repair via homologous recombination (HR) and sensitivity to replication- dependent DSBs. In this proposal, we will elucidate the H3K36M-mediated mechanisms of gene regulation (aim 1), and the role of H3K36M and H3K27me3 in DNA repair and genome stability in vitro (aim 2) and in vivo (aim 3). Taken together, our studies will reveal critical epigenetic processes needed at a timely manner at the right genes, to avoid disruptions in “chromostasis” that could cause cellular transformation and deve...