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

Abstract

Phosphorylation of the C terminus SQ motif that defines H2A.X variants is required for efficient DNA double-strand break (DSB) repair in diverse organisms but has not been studied in ciliated protozoa. Tetrahymena H2A.X is one of two similarly expressed major H2As, thereby differing both from mammals, where H2A.X is a quantitatively minor component, and from Saccharomyces cerevisiae where it is the only type of major H2A. Tetrahymena H2A.X is phosphorylated in the SQ motif in both the mitotic micronucleus and the amitotic macronucleus in response to DSBs induced by chemical agents and in the micronucleus during prophase of meiosis, which occurs in the absence of a synaptonemal complex. H2A.X is phosphorylated when programmed DNA rearrangements occur in developing macronuclei, as for immunoglobulin gene rearrangements in mammals, but not during the DNA fragmentation that accompanies breakdown of the parental macronucleus during conjugation, correcting the previous interpretation that this process is apoptosis-like. Using strains containing a mutated (S134A) SQ motif, we demonstrate that phosphorylation of this motif is important for Tetrahymena cells to recover from exogenous DNA damage and is required for normal micronuclear meiosis and mitosis and, to a lesser extent, for normal amitotic macronuclear division; its absence, while not lethal, leads to the accumulation of DSBs in both micro-and macronuclei. These results demonstrate multiple roles of H2A.X phosphorylation in maintaining genomic integrity in different phases of the Tetrahymena life cycle.

LIA4 Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

“…7). Detection of ␥H2AX in developing macronuclei was thought to result from double-strand breaks generated during DNA rearrangement (55). In support of this idea, we reported that ⌬LIA5 cells exhibited delayed or decreased accumulation of ␥H2AX.…”

“…As we could not detect evidence for IES excision, chromosome breakage, or the nuclear reorganization associated with these events in ⌬LIA4 conjugants, we were interested to assess whether other nuclear events were also perturbed. Accumulation of phosphorylated histone H2AX (␥H2AX) in the developing macronucleus has been reported (55). Because modification of this conserved histone variant occurs in response to DNA damage, the authors hypothesized that the ␥H2AX appears in developing macronuclei after doublestrand breaks are introduced upon IES excision.…”

TetrahymenaMeiotic Nuclear Reorganization Is Induced by a Checkpoint Kinase–dependent Response to DNA Damage

“…For Cna1p and ␥-H2A.X immunostaining, cells were fixed by mixing 20 l of partial Schaudin's fixative (saturated HgCl 2 , ethanol 2:1) with a 5-ml cell suspension. After two washes with methanol, cells suspended in methanol were dropped on a slide (see Song et al, 2007). For fluorescence in situ hybridization (FISH), 5 ml of cell suspension were centrifuged (3 min, 350 ϫ g) and 1 ml of Carnoy's fixative (methanol-chloroform-acetic acid, 6:3:2) was added to the pellet.…”

“…After maximal elongation, the MICs shortened again and transformed into condensed bivalents ( Figure 1). Immunostaining of phosphorylated histone H2A.X (␥-H2A.X) and recombination protein Rad51 has provided evidence that DSB formation and repair are ongoing in MICs during elongation (Loidl and Scherthan, 2004;Song et al, 2007;Mochizuki et al, 2008).…”

“…␥-H2A.X is considered to be an indicator of the occurrence of meiotic DSBs (Mahadevaiah et al, 2001;Song et al, 2007), but there are recent reports on DSB-independent H2A.X phosphorylation (Marti et al, 2006;Hanasoge and Ljungman, 2007;Ismail and Hendzel, 2008). ␥-H2A.X forms patches in elongating wild-type MICs (Figure 5a), whereas it is absent from MICs in the spo11⌬ mutant (Figure 5e; see also Mochizuki et al, 2008).…”