Lia1p, a novel protein required during nuclear differentiation for genome-wide DNA rearrangements in Tetrahymena thermophila.

Extensive genome-wide rearrangements occur during somatic macronuclear development in Tetrahymena thermophila. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins’ localization to differentiating somatic nuclei, and here we demonstrate that one, LIA1, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of LIA1 (LIA1) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the Tetrahymena genome. Differentiation of the somatic genome during development from a totipotent germ line involves extensive genetic reprogramming. Genes are activated or silenced as necessary, and chromosomal domains are established to enforce these actions. The unique nuclear dualism of the ciliated protozoan Tetrahymena thermophila provides an ideal context in which to examine such chromosomal reorganization during development. This organism, like other ciliates, contains two structurally and functionally distinct nuclei within the cytoplasm of a single cell (see reference 30). The diploid micronucleus, while transcriptionally silent during vegetative growth, harbors the germ line genome for genetic propagation during the next sexual generation (14). The polyploid macronucleus contains 45 to 50 copies of the genome and is the site of all somatic gene expression. During the vegetative life cycle, the micronucleus divides mitotically, while the polyploid macronucleus divides via a poorly understood amitotic mechanism (reviewed in reference 18). It is during the sexual stages that these different nuclei are formed. Conjugation of cells of complementary mating types induces both meiosis of micronuclei and loss of parental somatic macronuclei and the genomes within. The zygotic genome, the combination of meiotic products derived from the micronucleus of each mating partner, then replicates and undergoes differentiation to give rise to the new germ line and somatic nuclear precursors. The new germ line genome is preserved transcriptionally silent within micronuclei while the new somatic genome becomes transcriptionally active within developing macronuclear anlagen. Specific chromatin modifications are established within the macronuclear chromosomes