Understanding chromosomal regulatory mechanisms in proliferating and quiescent cells

Introduction

Condensin is a highly conserved hetero-pentameric protein complex in eukaryotes. It contains two structural maintenance of chromosome (SMC) subunits (Cut3 and Cut14 in S. pombe) and three non-SMC subunits (Cnd1, Cnd2 and Cnd3 in S. pombe). We have investigated this complex that is a central player in chromosome dynamics for years (Figure 1). Previously isolated ts mutants displayed severe defects in chromosome condensation and segregation during mitosis (Saka et al., 1994). Two other mutants (cnd2-1 and cut14-Y1) exhibited hypersensitivities to various DNA-damaging agents such as UV radiation, hydroxyurea, and camptothecin (Aono et al., 2002; Akai et al., 2011). We are studying on molecular mechanisms how condensin and other nuclear proteins precisely act on chromosome in proliferating and quiescent cells.

                                                  Figure 1. Condensin is a central player in chromosome dynamics

SMC hinge domain plays a critical role in condensin

A novel Cut14/SMC2 hinge mutant, cut14-Y1, displayed defects in DNA binding and annealing in vitro, and in DNA damage repair and chromosome segregation in vivo (Akai et al., 2011). This hinge mutation is critical for condensin function for segregation and DNA damage repair. This mutation is also rescued by the defect in RPA the major single strand DNA binding protein, suggesting that condensin hinge and RPA possess the opposing function (please see movie). The hinge domain is the site of DNA interaction, ATPase-dependent phosphorylation, and dynamic opening, and plays a critical role for segregation and damage repair (Akai et al., 2014, Figure 2). We presented a hypothesis that the ATPase domain provides the mobility of condensin along chromosome by releasing it from DNA.

Figure 2. Condensin SMC hinge domain is the site of DNA interaction, ATPase-dependent phosphorylation, and dynamic opening

Condensin HEAT subunits required for ploidy maintenance     

We recently found that condensin non-SMC subunits affect chromosome ploidy: mutants of non-SMC subunits containing the HEAT repeats could not maintain haploid state (Xu et al., 2015, Figure 3). Such condensin mutant cells become diploid, and do not form viable spores when crossed with haploid wild type. We plan to investigate this phenomenon in more detail by constructing an experimental system that we will be able to alter condensin mutant cells from the state of haploid to diploid so that the mechanism of ploidy change may be investigated in detail. Condensin may have other yet discovered important functions in the stressed cells.

Figure 3. Condensin non-SMC subunits affect chromosome ploidy (left: mutants of non-SMC subunits, right: mutation sites of cnd1 and cnd3 on conserved HEAT-repeat structure)

Transcription sites need condensin for chromosome segregation

We identified condensin-enriched sites along the all three chromosomes of S. pombe, using chromatin immunoprecipitation (ChIP) coupled with whole-genome DNA sequencing (ChIP-seq method) (Figure 4). During mitosis and interphase, condensin accumulates at RNA and protein-coding genes including heat-shock protein (Hsp) genes. This finding strongly suggests that chromosomal loci with abundant polymerases and/or transcribed RNA needs active condensin for proper chromosome segregation (Nakazawa et al., 2015). Condensin may remove an obstacle to chromosome segregation at transcription sites.

Figure 4. Condensin accumulation sites were mapped to fission yeast chromosomes by ChIP-sequencing method

Genetic suppressor analysis combined with WGS

The complex of Cut1/securin and Cut2/separase plays a fundamental role for chromosome segregation (e.g.Yanagida, 2009). Our recent results by genetic suppressor analysis combined with whole-genome sequencing (WGS) are quite intriguing as the complex is implicated in unexpected cell functioning that includes nutritional response mechanisms. This new finding may shed light on a linkage between chromosome segregation and nutritional metabolic regulations.