Centromeric Histone H3 Is Essential for Vegetative Cell Division and for DNA Elimination during Conjugation in Tetrahymena thermophila

Abstract

The Tetrahymena thermophila CNA1 gene encodes the centromeric H3, Cna1p. Green fluorescent protein (GFP)-tagged Cna1p localizes in micronuclei in dots whose number and behavior during mitosis and conjugation are consistent with centromeres. During interphase, Cna1p-GFP localizes in peripheral dots, suggesting centromeres are associated with the nuclear envelope. Newly synthesized Cna1p-GFP enters micronuclei in mitosis and accumulates in the nucleoplasm. Its deposition at centromeres starts at early S phase and continues through most of S phase. CNA1 is required for vegetative cell growth. Knockdown of CNA1 genes in the somatic macronucleus results in micronuclear DNA loss and delayed chromosome segregation during mitosis. During conjugation, Cna1p-GFP disappears from the centromeres in the developing macronucleus, consistent with centromeric sequences being internal eliminated sequences. Surprisingly, zygotic CNA1 is required for efficient elimination of germ line-specific sequences during development of the new macronuclei but not for the RNA interference pathway, through which sequences are targeted for elimination. Zygotically expressed Cna1p localizes in the spherical structures in which the later stages of DNA elimination occur, and these structures cannot be formed in the absence of zygotic CNA1, suggesting that, in addition to functioning in centromeres, Cna1p may also play a role in organizing the formation of the DNA elimination structures.

Phosphorylation of the SQ H2A.X Motif Is Required for Proper Meiosis and Mitosis in Tetrahymena thermophila

“…Soon after conjugation starts, the MIC moves away from the macronuclear pocket where it resides during interphase and begins to elongate into a teardrop shape (early stage II), then a spindle-shape (late stage II) (45), and then a head-neck-trunk crescent (stage III), followed by bidirectional elongation to become fully elongated in stage IV, where the five bivalent chromosomes are in parallel arrangement, with telomeres at one end of the crescent (45) and the centromeres at the other end (19). MICs stain with the anti-␥-H2A.X MAb beginning at early stage II, when they have just started to elongate (Fig.…”

“…The diploid MIC contains five pairs of chromosomes, divides mitotically, and is transcriptionally inactive during vegetative growth. In contrast, the MAC is transcriptionally active, contains ϳ225 acentric chromosomes (13,19,24), each in ϳ45 copies, which are derived by fragmentation, telomere addition, and endoreplication from the MIC chromosomes during the sexual process of conjugation. MACs divide by amitosis, a process in which chromosomes assort randomly without condensing or attaching to a mitotic spindle.…”

“…Because it precedes the meiotic divisions, the crescent stage is thought to be analogous to most of the prophase of meiosis I. However, while crescents in Tetrahymena exhibit some features of meiotic prophase found in other organisms, such as bouquet-like clustering of both telomeres (45) and centromeres (19), synaptonemal complexes (SCs) have not been detected (45,88), making it difficult to correlate the different stages of micronuclear meiotic prophase to the key events in meiosis such as homologous chromosome pairing and recombination. Thus, different mechanisms could be used in Tetrahymena.…”

The Tetrahymena meiotic chromosome bouquet is organized by centromeres and promotes interhomolog recombination

“…Notably, centromeres assemble at the opposite pole (Fig. 1J), leading to an extreme bouquet organisation with chromosome arms stretched in parallel between the two poles and homologous regions juxtaposed (Cui and Gorovsky, 2006;Mochizuki et al, 2008). The clustering of centromeres at the tip of the elongated nucleus suggests that either a centromere-proximal region or the centromere itself either drives elongation or attaches chromosomes to the elongating nucleus.…”

The smooth and stable operation of centromeres

“…Up to now, centromeres have been identified in a wide variety of organisms (Allshire and Karpen, 2008;Vagnarelli et al, 2008), and appear to affect various cellular activities, including asymmetric cell division (Chang et al, 2006), genome elimination (Cui and Gorovsky, 2006;Ravi and Chan, 2010), genotoxic stress responses (Major et al, 1999), ageing (Maehara et al, 2010) and speciation (Ventura et al, 2007). This may be due to the fact that centromeres are so fundamental that they invariably affect different cellular aspects.…”