Tunicate cytostatic factor TC14-3 induces a polycomb group gene and histone modification through Ca2+ binding and protein dimerization

When Phe65 of TC14-3 was mutated to an acidic amino acid, the resultant mutant protein failed to dimerize. The replacement of Thr69 with Arg69 made dimers unstable. When Glu106 was changed to Gly106, the resultant mutant protein completely lost Ca2+ binding. All these mutant proteins lacked cytostatic activity, indicating the requirement of protein dimerization and calcium for the activity. Polyandrocarpa Eed, a component of PcG, is highly expressed during budding, like TC14-3. When wild-type and mutant TC14-3s were applied in vivo and in vitro to Polyandrocarpa cells, only wild-type TC14-3 could induce Eed without affecting histone methyltransferase gene expression. Eed-expressing cells underwent trimethylation of histone H3 lysine27. PmEed knockdown by RNA interference rescued cultured cells from the growth-inhibitory effects of TC14-3.These results show that in P. misakiensis, the cytostatic activity of TC14-3 is mediated by PmEed and resultant histone modification, and that the gene expression requires both the protein dimerization and Ca2+-binding of TC14-3. This system consisting of a humoral factor, PcG, and histone methylation would contribute to the homeostatic regulation of cell growth and terminal differentiation of invertebrate multipotent cells.Cell and tissue homeostasis are among the most important features of living organisms. In vertebrates, various types of extracellular molecules act as cell growth regulators. For example, angiostatin and endostatin are potent inhibitors of endothelial cell proliferation and angiogenesis [1,2]. They contribute to our understanding of in vivo cell growth homeostasis and therapeutic control of tumor angiogenesis [3]. Among invertebrates, many species have multipotent cells that undergo cell growth and differentiation during regeneration and budding [4,5]. Therefore, many unique and interesting homeostatic factors are expected to exist in invertebrates. However, our understanding of such factors and global mechanisms