Project description:Histone post-translational modifications (PTM) greatly influence gene expression and are widely considered to regulate progression through development. However, the function of some PTMs remain elusive. H4K20 is sequentially methylated in concert with the cell cycle. In proliferating cells, SET8/KTM5A writes the monomethyl mark in G2/M phase, which then is converted to the di- and trimethylated states by SUV4-20H1/H2 (KMT5B/KMT5C) methyltransferases in the next G1 and S phase. In quiescent, differentiated cells, H4K20me2 represents the most abundant histone modification present in vertebrate chromatin. To address the function of H4K20 methyl states in development, we blocked the deposition of H4K20me2 and H4K20me3 by depleting the SUV4-20H1 in Xenopus embryos. This results in a severe ciliogenic defect in multiciliated cells (MCCs), as well as the repression of hundreds of cytoskeleton and cilium related genes. Further, we demonstrate that this defect can be rescued by wildtype, but not catalytically inactive SUV4-20h1, as well as by overexpressing PHF8, an H4K20me1-specific histone demethylase. Ciliogenic defects cannot be rescued by master regulators of ciliogenesis on the phenotypic or transcriptional levels. Taken together, this indicates that SUV4-20H1 plays a critical role in multiciliogenesis.
Project description:Histone post-translational modifications (PTM) greatly influence gene expression and are widely considered to regulate progression through development. However, the function of some PTMs remain elusive. H4K20 is sequentially methylated in concert with the cell cycle. In proliferating cells, SET8/KTM5A writes the monomethyl mark in G2/M phase, which then is converted to the di- and trimethylated states by SUV4-20H1/H2 (KMT5B/KMT5C) methyltransferases in the next G1 and S phase. In quiescent, differentiated cells, H4K20me2 represents the most abundant histone modification present in vertebrate chromatin. To address the function of H4K20 methyl states in development, we blocked the deposition of H4K20me2 and H4K20me3 by depleting the SUV4-20H1 in Xenopus embryos. This results in a severe ciliogenic defect in multiciliated cells (MCCs), as well as the repression of hundreds of cytoskeleton and cilium related genes. Further, we demonstrate that this defect can be rescued by wildtype, but not catalytically inactive SUV4-20h1, as well as by overexpressing PHF8, an H4K20me1-specific histone demethylase. Ciliogenic defects cannot be rescued by master regulators of ciliogenesis on the phenotypic or transcriptional levels. Taken together, this indicates that SUV4-20H1 plays a critical role in multiciliogenesis.
Project description:H4 lysine 20 dimethylation (H4K20me2) is the most abundant histone modification in vertebrate chromatin. It arises from sequential methylation of unmodified histone H4 proteins by the mono-methylating enzyme PR-SET7/KMT5A, followed by conversion to the dimethylated state by SUV4-20H (KMT5B/C) enzymes. We have blocked the deposition of this mark by depleting Xenopus embryos of SUV4-20H1/H2 methyltransferases. In the larval epidermis, this results in a severe loss of cilia in multiciliated cells (MCC), a key component of mucociliary epithelia. MCC precursor cells are correctly specified, amplify centrioles, but ultimately fail in ciliogenesis because of the perturbation of cytoplasmic processes. Genome-wide transcriptome profiling reveals that SUV4-20H1/H2-depleted ectodermal explants preferentially down-regulate the expression of several hundred ciliogenic genes. Further analysis demonstrated that knockdown of SUV4-20H1 alone is sufficient to generate the MCC phenotype and that its catalytic activity is needed for axoneme formation. Overexpression of the H4K20me1-specific histone demethylase PHF8/KDM7B also rescues the ciliogenic defect in a significant manner. Taken together, this indicates that the conversion of H4K20me1 to H4K20me2 by SUV4-20H1 is critical for the formation of cilia tufts.
