Chromatin Condensation

Although chromatin has been shown to condense in the presence of divalent or multivalent cations in vitro, its condensation is far from being an undirected and random process in vivo (De Frutos et al., 2001). Cellular processes depend on the maintenance of histone modification patterns across transcriptionally active and inactive loci. Therefore, chromatin condensation must be highly orchestrated to fulfil this requirement throughout the cell cycle (Valls et al., 2005; Kouskouti and Talianidis, 2005).

Regulating the degree of compaction is a key element in partitioning the eukaryotic genome into functionally distinct chromosomal domains. The underlying mechanisms that direct segments of the genome either into a compacted heterochromatic or an open euchromatic state are manifold and remain to be investigated.

By these means, histones represent integral and dynamic components of the transcription regulating machinery.

The densest condensation of chromatin occurs during the transition from interphase to mitosis with the formation of the pronounced x-shaped metaphase chromosome.

Initially, DNA topoisomerase II (Topo II) and condensin were proposed to be the major constituents driving condensation in mitosis (Earnshaw et al., 1985; Gasser et al., 1986; Saitoh et al., 1994; Strunnikov et al., 1995).

Condensins represent ring-shaped pentameric complexes composed of two proteins of the structural maintenance of chromosomes (SMC) family and three non-SMC subunits, namely a kleisin and two HEAT-repeat proteins. They are conserved across all eukaryotic species and key factors in mitotic chromosome condensation (Hirano, 2012; Hudson et al., 2009). The association of condensin with chromatin occurs at the N-terminal tail of histone H2A and is regulated by phosphorylation by the chromosomal passenger complex

(Swedlow and Hirano, 2003). Inactivation or depletion of condensin subunits has been shown to abolish the resolution of chromosome bridges in anaphase, resulting in severe defects in chromosome segregation (Hirano, 2012; Hudson et al., 2009). Formation of condensed chromosomes was still observed, however, with strongly reduced structural integrity (Gerlich et al., 2006; Hudson et al., 2003; Vagnarelli et al., 2006). Therefore, there is probably not a single, but rather multiple interlinked mechanisms driving condensation.

With cohesion and KIF4, other factors contributing to chromatin condensation have been identified. Cohesion is mediated by the cohesin complex, which is composed of two proteins from the SMC family, two proteins from the kleisin family and an accessory subunit. It is involved in resolving sister chromatids and alignment of chromosomes on the spindle at metaphase (Gimenez-Abian, et al. 2004; Lavoie et al., 2002; Nakajima et al., 2007; Shintomi and Hirano, 2009).

KIF4 is a DNA-binding kinesin motor protein which interacts with condensin and localizes to the arms of mitotic chromosomes. KIF4 and the condensin subunit SMC2 rely on each other for their localization on chromosome arms. Mitotic chromosomes in KIF4-deficient cells revealed a fat and short structure, a phenotype similarly seen in condensin-deficient cells, although their structural integrity is even more compromised (Samejima et al., 2012). A double mutant exhibited a total loss of structural integrity. This phenotype was partly rescued upon depletion of Topo II which suggests an opposing pathway for the enzyme. Condensin promotes lateral chromosome condensation by forming supercoiled loops of chromatin. Assumably, KIF4 clusters these loops together or, in combination with other proteins, forms supercoiled loops of its own to compact chromosomes further. Topo II could try to oppose this by untangling these loops in order to keep chromosome arms from becoming too long as they compact laterally (Samejima et al., 2012).

Modifying histones also represent a key element in partitioning the chromatin landscape into functionally distinct domains. Histone modifications function either by disrupting intra- and internucleosomal contacts or by affecting the interaction of chromatin with non-histone proteins. By these means, histones serve as integral and dynamic surfaces for a plethora of different PTMs, creating highly detailed, cell cycle-dependent binding interfaces for a multitude of histone modifiers and binders.

Depending on the type of modification, localized changes in charge can occur. Thus, acetylation and phosphorylation of histone tails can potentially alter histone-DNA or internucleosomal contacts, influencing chromatin structure by electrostatic mechanisms (Mersfelder and Parthun, 2006).

Histone acetylation is a crucial regulator of the degree of chromatin folding and condensation by promoting the formation of transcriptionally active euchromatin. Acetylation levels of lysine residues have been shown to increase globally upon entry into interphase and decrease in mitosis (Masumoto et al., 2005; Patzlaff et al., 2010; Vaquero et al., 2006; Wako et al., 2002; Wako et al., 2005). Hyperacetylation of histone tails was reported to disrupt the formation of higher-order chromatin structures, but also diminish histone-DNA interactions (Tse et al., 1998; Annunziato et al., 1988).

The most prominent candidate for a modification directly modulating chromatin compaction is acetylation of lysine 16 of histone H4 (Shia et al., 2006b; Shogren-Knaak et al., 2006). With 80% of all H4 molecules being acetylated at lysine 16, it is the most abundant acetylation site in S. cerevisiae (Clarke et al., 1993; Smith et al., 2003), while most of the yeast genome exists in a decondensed state (Lohr et al., 1977; Smith et al., 2003).

Native chemical ligation has been used to produce nucleosome arrays harboring histone H4 acetylated at residue K16. These arrays subsequently exhibited unfolding of chromatin on a global scale, equivalent to a deletion of the entire N-terminal domain of histone H4 (Shogren-Knaak et al., 2006). This behaviour was also observed in nucleosome arrays containing histones enzymatically acetylated at H4 K16 (Robinson et al., 2008).

The histone H4 tail forms an α-helix over a short stretch of residues, centered around lysine 16, while the rest of the tail remains unstructured (Yang and Arya, 2011). In its unmodified state, the basic residues lysine 16, arginine 19, lysine 20 and arginine 23 of the α-helix face in one common direction.Their side chains are believed to allow a strong binding of the tail to the H2A/H2B acidic patch of an adjacent nucleosome (Luger and Richmond, 1998). Upon acetylation of K16, the orientation of lysine 16 is changed and the α-helix becomes destabilized. This results in a reduced interaction between the H4 tail and the acidic patch, which eventually fails to stabilize the packaging of chromatin and thus leads to chromatin decondensation (Yang and Arya, 2011).

Histone phosphorylation has been associated with a multitude of cellular processes, including transcriptional regulation, apoptosis, cell cycle progression, DNA repair, chromosome condensation, and developmental gene regulation (Cheung et al., 2000; Cruickshank et al., 2010; Houben et al., 2007; Johansen and Johansen, 2006;

Kouzarides, 2007; Loomis et al., 2009).

Global phosphorylation levels exhibit an opposite behaviour to acetylation levels by decreasing in interphase and rising upon entry into mitosis (Gurley et al., 1978; Perez-Cadahia et al., 2009; Sawicka and Seiser, 2014).

This anticorrelating interrelationship between these two types of modifications throughout the cell cycle aroused suspicion about a cross-talk. Predominantly, phosphorylation of serine and threonine residues on particular histone tails was reported to be involved in chromatin condensation during mitosis and meiosis.

Apart from phosphorylation of threonine 119 of histone H2A being linked to regulation of chromatin structure and function in mitosis (Aihara, 2004), the most extensively studied phosphorylation site influencing chromatin compaction during mitosis is H3 S10. It was identified as the major phosphorylation site on histone H3 (Paulson and Taylor, 1982) and as conserved hallmark of mitotic chromosomal condensation across different mammalian cell lines (Hendzel et al., 1997).

Immunofluorescence studies clearly demonstrate the temporal and spatial relationship between chromosome condensation and H3 S10 phosphorylation. Dephosphorylation at this residue sets on during anaphase and is completed within telophase, even before detectable traces of chromosome decondensation can be found (Hendzel et al., 1997; Maile et al., 2004). These observations encourage the suggestion that histone H3 S10 phosphorylation is important for chromosome condensation and segregation. However, it is necessary to point out that there is evidence that H3 S10 phosphorylation is only necessary for the initiation of condensation rather than for maintaining it (van Hooser et al., 1998).

As the very first, an evident cross-talk between H3 S10 phosphorylation and H4 K16 acetylation driving

mitotic chromatin condensation. This phosphorylation recruits the chromosome passenger complex, whose kinase Aurora B (Ipl1 in yeast) then establishes the phosphorylation of H3 S10. In the following, this modification draws in the deacetylase Hst2 of the sirtuin histone deacetylase (SIR) family, which removes the acetylation of H4 K16 (Wilkins et al., 2014) (Figure 1.6).

Figure 1.6: Mitotic chromatin condensation driven by a cascade of histone modifications.

Schematic overview of cross-talk between H3 S10 phosphorylation and H4 K16 acetylation which drives chromatin condensation in S. cerevisiae. Taken from Wilkins et al., 2014.

Thereby, the well-documented interaction between the H4 tail and the acidic patch can occur, which drives compaction of mitotic chromosomes (Luger et al., 1977; Schalch et al., 2005; Shia et al., 2006b; Shogren-Knaak et al., 2006; Wilkins et al., 2014).