H2A.Z-Dependent Regulation of Cohesin Dynamics on Chromosome Arms

Abstract

Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation. Chromosomal integrity is essential for normal growth and development. In eukaryotes, there are three essential complexes of proteins that are central to chromosome dynamics. These are cohesin, condensin, and the Smc5/6 complex, known collectively as the structural maintenance of chromosomes (SMC) complex. Each complex shares a related architecture, and central to them are a heterodimer of SMC proteins: Smc1 and -3 in cohesin, Smc2 and -4 in condensin, and Smc5 and -6 in Smc5/6. These SMC proteins are large coiled-coil molecules with globular N and C termini containing Walker A and B ATP-binding motifs. They fold and interact at a flexible hinge, with ATP acting to hold the globular domains together. A kleisin protein bridges each heterodimer to form a putative ring-shaped structure, and each complex has specific additional non-SMC proteins that serve as regulators and effectors of function (1-3).Chromosome condensation and sister chromatid cohesion are the key roles for condensin and cohesin, respectively (1). However, defects in DNA repair have also been described for yeast strains harboring hypomorphic mutant alleles of condensin (4) and cohesin (5-7) subunits. In the case of cohesin, a role in DNA repair could stem from the fact that DNA repair by homologous recombination (HR) requires the sister chromatid to be in close proximity to the damaged chromatid. However, more recently cohesin in mammalian cells has been shown to also act as a transcriptional insulator in collaboration with CTCF (8, 9), and so the DNA repair function of cohesin may be more complex than a simple scaffolding mechanism. As its name suggests, deciphering Smc5/6 function has proved more elusive, though most studies have focused on a role for this complex in...

An acetyltransferase-independent function of Eso1 regulates centromere cohesion

“…Cohesin levels at the centromeres are higher than on chromosome arms (Outwin et al , 2009; Tapia-Alveal et al , 2014b). As a cohesin regulator, the most obvious reason for the premature separation of centromeres was the absence of cohesin at this locus.…”

“…This occurs spontaneously in null mutants and in hypomorphs after DNA damage or replication stress (Verkade et al , 1999; Harvey et al , 2004). The mitotic failure is due to the postanaphase retention of cohesin on chromosome arms but not centromeres (Outwin et al , 2009; Tapia-Alveal et al , 2014a, b), that is, a failure of the prophase pathway. Although the mechanism leading to cohesin retention is not yet clear, the mitotic defects can be overcome by ectopic expression of separase (Outwin et al , 2009) and the loss of the histone variant H2A.Z (Tapia-Alveal et al , 2014b); both of these scenarios significantly lower arm cohesin levels, although H2A.Z levels have little to no effect on centromeric cohesin.…”

“…The mitotic failure is due to the postanaphase retention of cohesin on chromosome arms but not centromeres (Outwin et al , 2009; Tapia-Alveal et al , 2014a, b), that is, a failure of the prophase pathway. Although the mechanism leading to cohesin retention is not yet clear, the mitotic defects can be overcome by ectopic expression of separase (Outwin et al , 2009) and the loss of the histone variant H2A.Z (Tapia-Alveal et al , 2014b); both of these scenarios significantly lower arm cohesin levels, although H2A.Z levels have little to no effect on centromeric cohesin. A similar dependence on Smc5/6 for cohesin removal has been found in S. cerevisiae during meiosis (Copsey et al , 2013) but not during mitosis (Jeppsson et al , 2014), in which the prophase pathway does not function.…”

Functional interplay between cohesin and Smc5/6 complexes

“…First, overexpression of the protease separase, which cleaves the kleisin subunit of cohesin, removes the arm cohesin complexes and suppresses the mitotic defects (Outwin, et al 2009). Second, in a screen for suppressors of the mitotic defects of smc6 mutants, we found that loss of the histone variant H2A.Z lowers arm cohesin levels by up to 50%, and this too allows mitosis to proceed (Tapia-Alveal, et al 2014). Importantly, in both scenarios the DNA damage and/or replication stress sensitivity of smc6 mutants is also suppressed, showing that the sensitivity is largely due to the mitotic defects.…”

“…One hint that cohesin and Smc5/6 may have overlapping functions comes from the observation that most combinations of hypomorphic alleles of cohesin and Smc5/6 genes in S. pombe are either synthetic lethal or confer severe mitotic defects (Tapia-Alveal, et al 2014; Tapia-Alveal, et al 2011; Verkade, et al 1999). At least in the case of cohesin, the cyclic nature of its association with chromosomes means that a delicate balance of cohesin dynamics is critical; either too much or too little sister chromatid cohesion is incompatible with accurate chromosome segregation.…”