Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction (DNA elimination). It is believed that the DNA to be eliminated is identified by their homology with ~28 nt small RNAs (scnRNAs) which are made by a RNAi-related mechanism. Detailed mechanisms as well as exact biological functions of the RNA-directed DNA elimination remain obscure. The goal of this specific project is to understand: (i) How a RNAi-related mechanism directs DNA elimination. (ii) How the mating types are determined as a consequence of DNA elimination. We recently revealed that scnRNAs are processed by Dicer protein Dcl1p and form complex with Argonaute protein Twi1p. The RNA helicase Ema1p facilitates interaction of scnRNA and chromatin and this interaction induces H3K9 and K27 methylations that are catalyzed by the histone methyltransferase Ezl1p. To understand how these proteins are connected to each other, their detailed functions and novel proteins interacting with them will be analyzed. One of the possible biological functions of the DNA elimination is mating type determination. Tetrahymena possesses seven mating types and the different mating types are thought to be determined by alternative DNA elimination of a single locus. But how mating types are determined is totally unknown. We aim to identify mating type determinants using microarray screening, proteomics and mating type transformation with a cDNA library. Then, we will analyze how expression of those molecules is controlled by DNA elimination. In diverse eukaryotes, RNA silencing mechanisms mediate heterochromatin formation and regulate many chromatin functions. Through our study, we could gain deeper insights not only into DNA elimination in a curious microbe, but also into how chromatin functions are epigenetically controlled by small RNAs in most of the eukaryotes. We are confident that this work will provide a big impact on RNA silencing and chromatin studies.