A nonessential HP1-like protein affects starvation-induced assembly of condensed chromatin and gene expression in macronuclei of Tetrahymena thermophila.

Gene expression in eukaryotes relies upon the accessibility of the DNA template to a variety of components in the transcription apparatus, which is controlled in turn by a locus-specific chromatin structure. Chromatin of higher eukaryotes is often discussed in terms of two cytologically distinct domains: euchromatin and heterochromatin. Heterochromatin is classically distinguished from euchromatin by its paucity of genes, its highly condensed chromatin structure throughout the cell cycle, its late replication in S phase, and its enrichment in repetitive DNA sequences (25, 32). However, genes have been identified that reside within heterochromatic regions (21, 27), and the expression of some of these genes is dependent upon their placement in heterochromatin. Furthermore, recent evidence has shown that heterochromatin plays important roles in long-range intra- and interchromosomal interactions that affect nuclear organization (8, 10), chromosome segregation (9, 33, 46), and locus-controlled patterns of transcriptional activity (42). Heterochromatin and euchromatin represent two different structural environments, and each has profound effects on gene expression (55, 58). A large body of genetic evidence suggests that heterochromatin interferes with the expression of normally euchromatic genes. In yeast cells, for example, mitotically transmissible silencing of silent mating-type loci depends upon nearby sequences, as well as on histone and nonhistone chromosomal components (44). Sequences not related to mating type become transcriptionally repressed when placed at or near this specialized chromatin environment, and similar positional silencing occurs near telomeres (24). Likewise in Drosophila melanogaster, heterochromatic position-effect variegation (PEV) is characterized by mitotically transmissible silencing and position-dependent gene silencing (55, 57, 58). Silencing by pericentric heterochromatin has also been observed in transgenic mice (18). Thus, the underlying mechanism leading to positional silencing appears to exist in yeast, flies, and mammals. PEV in flies is influenced by modifiers (both enhancers and suppressors), and these gene products have been proven important in dissecting the composition of heterochromatin. One of the PEV modifier genes, Su(var)205, encodes HP1, which is perhaps the best-studied structural protein associated with heterochromatin (11, 12). A loss-of-function mutation in Su(var)205 leads to increased expression of euchromatic genes that suffer from position effect (i.e., White), while overproduction of HP1 results in decreased expression of these genes (12). Opposite results have been obtained for heterochromatin-positioned genes (i.e., Light), suggesting that genes localized in heterochromatin differ in their regulatory requirements (26). Conservation of HP1 throughout evolution (50) and the lethality of HP1 null alleles (13) suggest that HP1 is important for cell viability and development in flies. In support of this, Swi6p, a chromodomain-containing protein from fission yeast, while not essential, is required to maintain transcriptional repression at the silent mating-type loci and centromeres (2, 15, 38). Recent studies of Δswi6 cells have shown a reduced spore viability, along with altered expression of some meiotic genes, suggesting that proteins of this general family play an important yet poorly understood role in gene expression (37). Like other ciliates, Tetrahymena spp. exhibit nuclear dimorphism, wherein each cell contains both a germ line micronucleus and a somatic macronucleus (22, 23). Even though these nuclei coexist as neighbors in a common cytoplasm, they differ considerably in function and provide a useful model for studying the function of HP1-like proteins. Recently, we reported the identification of an HP1-like protein, Hhp1p, that is absent from transcriptionally silent micronuclei but is modestly enriched in the condensed chromatin regions (referred to as chromatin bodies) that punctuate transcriptionally active macronuclei (30). Here, we report the disruption of all somatic (macronuclear) copies of the endogenous HHP1 genes in Tetrahymena thermophila. Our results demonstrate that, unlike Drosophila HP1, Hhp1p is not essential. Although no phenotypic difference is observed in vegetative cells lacking Hhp1p, a clear phenotypic difference is observed when cells are starved for nutrients. During a shift to nongrowth (starvation) conditions, the survival rate of cells lacking Hhp1p (ΔHHP1) is reduced relative to wild-type cells, morphological changes in chromatin body size fail to occur, and the activation of two starvation-induced genes is markedly reduced. These results suggest a model wherein the loss of Hhp1p leads to aberrant assembly of the condensed chromatin in macronuclei during the transition from growth to nongrowth conditions. We suspect that failure to properly assemble condensed chromatin domains leads to the reduced expression of starvation-induced genes that reside in and utilize condensed chromatin in a positive fashion to bring about their expression.