S
UPERCOILING AND PROTEIN BINDING ARE REQUIRED FOR THE COMPACTION OF CHROMOSOMES1.2.2.1 SU P E R C O I L I N G I S T HE P RI MA R Y M E C HA N I SM F O R T H E S E C O N DA RY L E VE L ORG A NI ZAT IO N OF B A CT E RI A L G EN O M ES
Bacteria, like all organisms, must package their genome into a small cellular volume while enabling factors to access the DNA to perform vital cell processes such as DNA replication and gene expression (Ishihama, 2009). For example, the E. coli chromosome has a contour length, the length at maximum physically possible extension, of ~1,600 µm and is compacted ~1000-fold within a rod-shaped cell only ~1 µm in diameter and ~2-4 µm in length (Reyes-Lamothe, Wang, & Sherratt, 2008; Zimmerman, 2006). A substantial level of bacterial genome compaction is achieved by the introduction of negative supercoiling and binding of proteins that can isolate topological domains and bend DNA (Azam & Ishihama, 1999; Dame, 2005; D. Jackson et al., 2012; Reyes-Lamothe et al., 2008; Thanbichler, Wang, & Shapiro, 2005).
The principle mechanism by which bacterial chromosomes are condensed is through DNA supercoiling (D. Jackson et al., 2012; Reyes-Lamothe et al., 2008). The chromosomes of most bacteria are negatively supercoiled, with the mechanisms of regulation and influences on cellular processes best understood in E. coli and Salmonella enterica. Negative supercoils are introduced into the bacterial chromosomes by the ATP dependent gyrase enzymes (gyrA and gyrB genes) and relaxed by topoisomerase I (topA gene), IV (parC and parE genes),
10 and III (topB gens) (Rovinskiy, Agbleke, Chesnokova, Pang, & Higgins, 2012; Zechiedrich et al., 2000). Thus, the global supercoiled state of DNA within a bacterial cell is set by the relative abundance and activities of these counteracting enzymes (Snoep, Van Der Weijden, Andersen, Westerhoff, & Jensen, 2002). Evidence suggests that the introduction of negative supercoiling gives rise to the smallest unit of bacterial chromosome organization through the formation of non- constrained, independent supercoiled domains (Postow, Hardy, Arsuaga, & Cozzarelli, 2004). In E. coli, these supercoiled domains have been estimated to have an average size of ~10Kb (Postow et al., 2004; R. A. Stein, Deng, & Higgins, 2005).
The negative supercoiling of bacterial chromosomes makes a significant contribution to the compaction of bacterial nucleoids, but it does not account for the total level of compaction observed (Dame, 2005; Dillon & Dorman, 2010; Postow et al., 2004; Thanbichler et al., 2005; Zimmerman, 2006). Unlike eukaryotes, bacteria do not contain histone proteins. However, a number of bacterial Nucleoid Associated Proteins (NAPs) have been identified that are thought to behave as histone homologues. These NAPs exhibit varying degrees of DNA binding, bending, looping and dimerization properties in vitro (Azam & Ishihama, 1999; Dame, 2005; Dorman, 2013; Thanbichler et al., 2005). Four of the classical NAPs have been investigated in great detail: the heat-stable nucleoid- structuring protein (H-NS), the heat-stable protein (HU), the factor for inversion stimulation (Fis), and the integration host factor (IHF). The DNA binding and bending properties of these proteins are thought to function in place of eukaryotic histones playing a role in the isolation of topological domains and compaction of the nucleoid (Dorman, 2013). However, studies also indicate that in vivo the role of the NAPs could be more in the regulation of cellular processes, such as gene expression, rather than architectural (Dame, 2005; Grainger, Hurd, Goldberg, & Busby, 2006).
In addition, the recently characterized non-classical NAPs (i.e. SeqA, SlmA, and MatP) that exhibit macrodomain-specific DNA binding properties (reviewed in (Dame, Kalmykowa, & Grainger, 2011)), may represent alternative candidates that facilitate the secondary and tertiary level organization of bacterial nucleoids (see section 1.2.1.1 for further discussion).
11 1.2.2.2 EF FI CI E NT P A C K AG ING OF E U K AR Y OT I C G EN O M ES I S
P R I MA R I LY A C C O MP L I S H E D B Y H I ST O N E P RO T E I NS
In eukaryotes, supercoiling makes a smaller contribution to chromosome compaction, instead nuclear DNA is associated with numerous proteins in a complex called chromatin. The basic building block of chromatin is the nucleosome (Olins & Olins, 1974; C. L. F. Woodcock, Safer, & Stanchfield, 1976; C. L. F. Woodcock, Sweetman, & Frado, 1976). Nucleosomes are composed of two copies each of the four core histone proteins (H2A, H2B, H3 and H4). The C-terminal two thirds of each histone protein come together to form a hydrophobic protein octamer core, around which approximately 147 bp of DNA is wound (Luger, Mäder, Richmond, Sargent, & Richmond, 1997) (Figure 1.2A). In most eukaryotes a fifth histone, histone H1, associates with the linker DNA providing partial protection from nuclease digestion for ~20 bp of DNA (Allan, Cowling, Harborne, Cattini, & Gould, 1981; Happel & Doenecke, 2009; Christopher L Woodcock & Ghosh, 2010). Together, the wrapping of DNA around nucleosomes and association of the linker histone H1 contribute significantly to the compaction of eukaryotic chromosomes.
The reversible, post-translational modification of histone proteins further influences the level of chromosome compaction and contributes to the epigenetic code. The N-terminal tails of the core histone proteins protrude out from the octamer making them the most accessible to post-translational modification (Figure 1.2A). Amino acid residues in the N-terminal histone tails and the nucleosome core can undergo specific and variable post-translational modification, including methylation, acetylation and phosphorylation (Lennartsson & Ekwall, 2009; Strahl & Allis, 2000). These post-translational modifications form part of a ‘histone code’ that can be interpreted by other proteins to bring about specific downstream events. This histone code can be transient or stable. In the latter case, if the modifications are heritable, they constitute a true ‘epigenetic code’ (Lennartsson & Ekwall, 2009; Turner, 2000). Not only does the post-translational modification of nucleosomes influence the accessibility and interpretation of the DNA, the dynamic positioning of nucleosomes along chromosomes can also affect cellular possesses such as transcription (Bai & Morozov, 2010; Dai et al., 2009; Schones et al., 2008; Wu et al., 2013).
The contribution that nucleosomes make to eukaryotic genome compaction is two-fold. In addition to the winding of DNA around the nucleosome, interactions between neighbouring nucleosomes facilitate the further compaction of chromatin,
12 giving rise to the debated 30-nm fibre (Dehghani, Dellaire, & Bazett-Jones, 2005; Maeshima, Hihara, & Eltsov, 2010; Tremethick, 2007; van Holde & Zlatanova, 2007). Early studies of native chromatin using electron microscopy (EM) led to the proposal of two models for the 30-nm chromatin fibre: 1) the one-start helix/solenoid model (Figure 1.2B and D), and 2) the two-start helix model (Figure 1.2C and E). The former involves interactions between adjacent nucleosomes that are connected by linker DNA that is bent to follow a superhelical path. This results in 6-8 nucleosomes, encompassing approximately 1,200 bp of DNA, per turn of the solenoid (G. Li & Reinberg, 2011; Widom & Klug, 1985; C L Woodcock & Dimitrov, 2001). By contrast, in the two-start helix model, adjacent nucleosomes are connected by straight linker DNA. The two-start model was proposed based on the interpretation of EM experiments of chromatin in low ionic strength buffers that appeared to have a Zig-Zag like nucleosome arrangement (G. Li & Reinberg, 2011; S. P. Williams et al., 1986; C L Woodcock & Dimitrov, 2001). The different structures observed for the 30-nm fibre may in part be due to the majority of studies on chromatin structure having been done in vitro using isolated chromosomes. Conducting molecular level studies on chromatin structure in vivo may shed light on this debate.