Identification of Tension Sensing Motif of Histone H3 inSaccharomyces cerevisiaeand Its Regulation by Histone Modifying Enzymes

Abstract

To ensure genome stability during cell division, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies before segregation. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. In budding yeast, the residue Gly44 of histone H3 is critical for retaining the conserved Shugoshin protein Sgo1p at the pericentromeres for monitoring the tension status during mitosis. Studies carried out in this work showed that Lys42, Gly44, and Thr45 of H3 form the core of a tension sensing motif (TSM). Similar to the previously reported G44S mutant, K42A, G44A, and T45A alleles all rendered cells unable to respond to erroneous spindle attachment, a phenotype suppressed by Sgo1p overexpression. TSM functions by physically recruiting or retaining Sgo1p at pericentromeres as evidenced by chromatin immunoprecipitation and by in vitro pulldown experiments. Intriguingly, the function of TSM is likely regulated by multiple histone modifying enzymes, including the histone acetyltransferase Gcn5p, and deacetylases Rpd3p and Hos2p. Defects caused by TSM mutations can be suppressed by the expression of a catalytically inactive mutant of Gcn5p. Conversely, G44S mutant cells exhibit prominent chromatin instability phenotype in the absence of RPD3. Importantly, the gcn5 2 suppressor restores the tension sensing function in tsm 2 background in a fashion that bypasses the need of stably associating Sgo1p with chromatin. These results demonstrate that the TSM of histone H3 is a key component of a mechanism that ensures faithful segregation, and that interaction with chromatin modifying enzymes may be an important part of the mitotic quality control process.KEYWORDS histone H3; chromatin; mitosis; Shugoshin; Saccharomyces cerevisiae F AITHFUL partitioning of the genome duplicates in mitosis requires that the sister chromatids be engaged in bipolar attachment to mitotic spindle. Once all chromosomes are appropriately captured by the spindles, anaphase starts with the action of separase that cleaves the cohesin complex, thus separating the two sister chromosomes (Nasmyth 2002). Premature anaphase onset causes aneuploidy, a common trait associated with spontaneous abortion, birth defects, and cancer. Chromosome biorientation is a result of sister chromatid cohesion by the cohesin complex, stable attachment of spindles to kinetochores, and that the two sister kinetochores each attach to spindles emanating from different spindle pole bodies (Kschonsak and Haering 2015). Erroneous attachment of spindles to kinetochore activates the spindle assembly checkpoint (SAC), which prevents metaphase-to-anaphase transition so that errors can be corrected (Akera and Watanabe 2016). The two essential elements of biorientation are the spindle-kinetochore interaction and the tension between sister chromatids (Goshima and Yanagida 2000;Pinsky and Biggins 2005;Wang et al. 2014). The latter results from the physical cohesion of the sister chromatids that resi...

Tripartite Chromatin Localization of Budding Yeast Shugoshin Involves Higher-Ordered Architecture of Mitotic Chromosomes

“…Because the interaction between Sgo1p and TSM does not require any posttranslational modification (L uo et al 2010; L uo et al 2016), a non-epigenetic feature may distinguish the PC Sgo1p targets from other areas nearby. We felt that chromatin architecture would be a good candidate that dictates the (in)accessibility of the CEN/PC region to Sgo1p.…”

“…Our recent findings that Gcn5p acts as a negative regulator for tension sensing motif and Sgo1p functional interaction (L uo et al 2016; B uehl et al 2018) alludes to an intriguing possibility that Gcn5p, acetylated H3, or a downstream effector may prevent Sgo1p from binding to the chromosome arms. ChIP-seq data show a lack of correlation between Gcn5p and these Sgo1p-free valleys in mitosis (Figure 3), arguing against a direct, physical role of Gcn5p.…”

“…Alternatively, the homodimerization activity of Sgo1p, evidenced by yeast two-hybrid tests (M ishra et al 2017), may facilitate the growth of the Sgo1p domain from centromeres to pericentric regions where the cohesin complex resides. By binding to nucleosomes, cohesin may also help to make the tension sensing motif more accessible for Sgo1p before biorientation is established (F ernius and H ardwick 2007; K awashima et al 2010; L uo et al 2010; L uo et al 2016). Due possibly to the total pool size of Sgo1p, it only spreads to the first and nearest cohesin cluster.…”

A Failsafe for Sensing Chromatid Tension in Mitosis with the Histone H3 Tail inSaccharomyces cerevisiae

“…These histones not only provide a framework for packaging DNA within the nucleus, but also have important roles in a wide variety of cellular processes, including transcription (Zhang et al 1998;Berger 2007), nuclear import (Blackwell et al 2007), DNA replication (Ramachandran and Henikoff 2015), recombination (Hunt et al 2013), and the cell cycle (Megee et al 1995;Ng et al 2013). Contrary to the general perception that chromosomes are merely passive cargos during cell division, work from our lab and others has demonstrated an active role for histones in mitotic control and maintaining chromosome fidelity during cell division (Luo et al 2010(Luo et al , 2016Yamagishi et al 2010).…”

“…The budding yeast Saccharomyces cerevisiae lacks these heterochromatin marks, and therefore the centromeric Bub1p-mediated histone H2A S121 phosphorylation provides the major nucleation for the recruitment of Sgo1p (Fernius and Hardwick 2007;Kawashima et al 2010). We have reported that the tensionsensing motif (TSM), 42 KPGT, of histone H3 is critical to the pericentric recruitment of Sgo1p (Luo et al 2010(Luo et al , 2016. Point mutations within this motif diminish pericentric Sgo1p recruitment without significantly affecting its centromeric localization.…”