The genetic information that is required for all cellular processes is encoded in deoxyribonucleic acid (DNA). The size of genomic DNA varies between organisms, but it is in all cases large compared to the dimensions of the cell. Therefore, cells need to compact their DNA in order to fit it into the limited space. Prokaryotic cells usually contain a single circular chromosome that is condensed via DNA supercoiling and small architectural proteins, and the chromosome together with the proteins forms a nucleoid within the cytoplasm (Drlica and Rouviere-Yaniv, 1987).
Eukaryotes generally have a much larger genome than prokaryotes which imposes an even bigger challenge. For example, one copy of a human genome is made of three billion base pairs (bp) which extends to a total length of about two meters. However, the genome needs to be packed into a special organelle termed the nucleus with an average diameter of roughly 10 µm, in a way that still allows rapid accessibility of DNA at any given time. This compares to arranging 48 km of thread inside a basketball while ensuring that any part of the thread can be accessed within seconds (McGinty and Tan, 2015).
To overcome this problem, cells pack their DNA into a structure called chromatin.
The smallest building block of chromatin is the nucleosome which is formed by wrapping DNA around a protein scaffold (Kornberg, 1974). This organization does not only allow for the storage of the genetic information but also offers multiple layers of spatial and temporal organization necessary for the regulatory control for higher forms of life.
Although high condensation of chromatin is needed for storage, the accessibility of DNA is required for fundamental cellular processes such as transcription, DNA repair and replication.
1.1.1 Nucleosomes as building blocks of chromatin
The organization of DNA into nucleosomes is an elegant way for the cell to fit genetic information into the limited space of the eukaryotic nucleus. Nucleosomes are the basic
repeating unit of chromatin and consist of an octameric complex formed by histone core proteins around which 145 – 147 base pairs (bp) of DNA are wrapped (McGinty and Tan, 2015). Since the first high resolution structure of a Xenopus laevis nucleosome was solved in 1997 (Luger et al., 1997), many additional structures of nucleosomes from various organisms have been reported on their own or bound by additional factors, showing their high architectural conservation and underlining their paramount importance throughout eukaryotic life (Armache et al., 2011; Barbera et al., 2006; Luger et al., 1997; Makde et al., 2010; McGinty et al., 2014; Tan and Davey, 2011; Tsunaka et al., 2005; Vasudevan et al., 2010; White et al., 2001) (Figure 1).
Figure 1 | Assembly pathway and architecture of the nucleosome. Histones H2A and H2B form two dimers while histones H3 and H4 assemble into a tetramer before they bind together to form the octamer. 147 bp of DNA warp around the octamer in a left-handed superhelix. The dyad axis marks the pseudo-symmetry and the superhelical locations are numbered for the plus side and indicated for the minus side. PDB ID 3MVD.
The octameric centre of the nucleosome is formed by two copies of each of the four core histones H2A, H2B, H3 and H4 which share a common architecture named the histone fold (Arents et al., 1991). This fold is characterized by three α-helices connected by two loops. The two shorter outer α-helices pack against a central longer one. Histones also have N- and C-terminal extensions in addition to the central histone fold. Using this fold, two histones interact with each other in a ‘handshake’ motif. The octamer is assembled in a stepwise manner where H2A and H2B form two H2A-H2B heterodimers that bind with a tetramer formed by the two H3 and H4 histones. DNA is wound around the core octamer in a left-handed superhelix comprising 1.65 turns of DNA and the DNA-histone contacts are formed by ionic interactions, direct hydrogen bonds and hydrogen bonds through water molecules, as well as non-polar interactions (Luger et al., 1997;
McGinty and Tan, 2015). The two parallel DNA gyres surrounding the core are connected via a DNA stretch that follows a diagonal path on the side of the octamer where the two DNA ends exit the nucleosome. Thus, the nucleosome has a pseudo-twofold symmetry around a central axis or the dyad (Flaus et al., 1996).
The positions of the DNA around the nucleosome are termed superhelical location (SHL) with a new SHL after each full turn of the DNA duplex every 10 bp. The dyad axis is used as a central reference point with its SHL defined as 0 and the SHLs running from –7 to +7. A characteristic feature of the nucleosome is the acidic patch, a strongly negatively charged area on each disc face of the nucleosome formed by amino acid residues of the H2A-H2B heterodimers. The acidic patch is a common interaction site for various factors operating on chromatin and many structures of nucleosome-bound factors show interactions with the acidic patch surface (Armache et al., 2011; Barbera et al., 2006; Fang et al., 2016; Makde et al., 2010; McGinty et al., 2014).
The N- and C-termini of histones are unstructured and extend outwards from the central nucleosomal architecture. In this way, they form a flexible platform that is a target for post-translational modifications such as phosphorylation, acetylation, methylation and ubiquitylation. These modifications are installed, detected and interpreted by protein complexes of the chromatin reader and writer/eraser classes to alter chromatin organization and bring about downstream effects (Jenuwein and Allis, 2001; Prakash and Fournier, 2018).
1.1.2 Nucleosomes in transcription
Transcription of RNA polymerase II starts with the assembly of a pre-initiation complex on promoter DNA. However, access to promoter DNA is impaired by nucleosomes, which are arranged on the DNA in a ‘beads-on-a-string’-like fashion. The presence of nucleosomes can inhibit initiation (Almer and Horz, 1986; Almer et al., 1986; Knezetic and Luse, 1986; Lorch et al., 1987) and obstruct the path of transcribing RNA polymerase II (Brown et al., 1996; Carey et al., 2006).
In order for transcription initiation to occur, the nucleosomes occupying the promoter must be removed or shifted to free cis promoter elements (Henikoff, 2016;
Lorch and Kornberg, 2017). Indeed, several studies have shown that the nucleosomes are not evenly distributed across the genome and nucleosome-depleted regions (NDR) exist at promoters (Lantermann et al., 2010; Lee et al., 2007; Mavrich et al., 2008b; Schones et al., 2008). These regions coincide with the location of active RNA polymerase II and are flanked by specialized +1 and –1 nucleosomes on the downstream and upstream side of the NDR, respectively (Henikoff, 2016; Lorch and Kornberg, 2017).
Further downstream and upstream, the nucleosomes are arranged in a well-defined array that loses the phasing gradually with distance from the +1 and –1 nucleosome (Bai and Morozov, 2010). NDRs were shown to vary in size and, despite their name, can be occupied by ‘fragile’ nucleosomes that are subject to rapid disassembly and exchange as part of the additional regulatory step during transcription initiation (Kubik et al., 2015).
The architecture of gene promoters varies in different organisms. For instance, the transcription start site (TSS) in Saccharomyces cerevisiae is often located 10 – 15 bp inside the +1 nucleosome, which shields the access to this element (Albert et al., 2007). In contrast, human cells usually initiate transcription from a TSS about 60 bp upstream of the +1 nucleosome (Jiang and Pugh, 2009).
Despite these variations, NDRs are generally found throughout different species.
Eukaryotic cells have evolved a large toolset of specialized protein complexes, called chromatin remodellers, to regulate the positioning of nucleosomes at promoter regions and to establish the nucleosome-depleted region (Becker and Workman, 2013; Clapier et al., 2017).