Chromatin and nuclear signaling Background
Chromatin is the combination of DNA and other proteins that make up the contents of the nucleus. The primary protein component of chromatin is histones which act to compactly package the DNA. Chromatin is only found in eukaryotic cells, prokaryotic cells have a very different organisation of their DNA which is not normally referred to as chromatin. The primary functions of chromatin are to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene expression and DNA replication.
Despite all cells having identical genetic material, phenotypic variation occurs during development. This can be attributed to multiple factors that modulate gene expression without changing the underlying DNA sequence. Over the past decade, considerable progress has been gamed regarding the elucidation of these mechanisms. To understand how these mechanisms work, it is important to know about the organization of DNA within the cell and the factors that contribute to its structure and function.
The DNA that makes up the human genome is roughly 2 m in length if stretched end-to-end, while a human cell is on average only about 10 um in diameter. Eukaryotic cells are able to confine enormous lengths of genomic DNA within the tiny volume of the nucleus—only about 10 μm in diameter. The efficient packaging of the genome is made possible by the coexistence of the DNA with an approximately equal mass of specialized structural proteins. The resulting complex of DNA and proteins is known as chromatin and incorporates multiple hierarchical levels of spatial organization.
Most of the protein mass in chromatin consists of histone octamers. Each histone octamer consists of four pairs of the histone proteins H2A, H2B, H3, and H4, and acts as a little spool that holds about 1.7 turns of negatively supercoiled DNA. The wrapping of DNA around each histone octamer engages approximately 147 bp of DNA and, together with an additional stretch of naked DNA known as linker DNA, gives rise to a tethered bead called nucleosome.
The nucleosome represents the first level in the spatial organization of chromatin. The crystal structure of the nucleosome has been solved to a resolution of 2.8 Å. The length of linker DNA connecting adjacent nucleosomes varies between roughly 10 and 100 bp depending on the organism and on the position of the nucleosomes along the genomic DNA. An array of linked nucleosomes gives rise to a bead-on-a-string conformation, with an effective diameter of roughly 10 nm. This conformation can in turn self-assemble in vitro to form a fiber with a diameter of approximately 30 nm.
The 30-nm chromatin fiber represents the second level in the spatial organization of chromatin. The formation of this fiber was found to be consistent with several possible arrangements of nucleosomes, including a two-start zigzag ribbon and an interdigitated solenoid structure. Different types of nucleosome arrangement may even coexist within the same fiber. However, the extent to which the 30-nm chromatin fiber actually exists in vivo remains a matter of debate. It is possible that multiple adjacent 30-nm fibers blend to form a polymer melt consisting of interdigitated bead-on-a-string conformations in vivo.
The additional folding of chromatin beyond the 30-nm fiber is often referred to as the higher order spatial organization of chromatin. However, additional folding of the chromatin fiber has been shown to result in the formation of loops. Some loops are believed to play important roles in the regulation of gene expression, for example by enabling long-range interactions between DNA regulatory elements and promoters.
Collections of random and regulatory loops may further fold to produce domains with genomic lengths on the order of 1 Mbp. Interactions between chromatin fibers within a domain should be relative frequent, whereas interactions across different domains should be less likely. Chromatin domains have been described by means of various denominations and classifications, such as topological domains, fractal globules, lamina-associated domains, nucleolus-associated domains, and topologically associating domains. Domains are also thought to shift dynamically between different subchromosomal compartments based on the level of expression or other activity, so that domains in the same compartment are more likely to interact than domains in different compartments.
The spatial subdivision of chromosomes into domains is believed to accommodate an interchromatin compartment, which is a network of channels providing enough space between the domains for protein complexes to interact with DNA loops emanating from the periphery of the domains, i.e., the perichromatin region. Chromosomes undergo severe gross morphological changes throughout each cell cycle and it is during mitosis that the segregation of properly folded chromosomes plays a key role in maintaining genomic stability. This process is necessary in order for a perfect copy of the genome to be passed from mother cell to daughter cell.
Moreover, organization within chromosome territories has also been found to be non-random. RNA transcripts as well as RNA processing machinery are concentrated outside of chromosome territories, and in agreement with this observation, protein-coding genes show preferential localization to the periphery of chromosome territories. Furthermore, transcriptional status has been suggested to influence nuclear localization. For example, sharing of transcriptional machinery or regulatory elements is thought to cause genes to co-localize at certain nuclear regions, and in yeast, genes have been reported to relocate to the nuclear periphery or nuclear pores upon activation.
Recently, structural proteins such components of the nuclear lamina and the nuclear pore complex have been found to interact with chromatin at specific genomic loci and are therefore thought to play a role in creating chromatin structure. In vitro differentiation prompts a small number of genes to alter their association with the nuclear lamina, and these nuclear rearrangements are accompanied by changes in gene expression. Additionally, insulator elements have been implicated in nuclear organization and this role is conserved from yeast to mammals.
Roles of Histones in Chromatin
Chromatin, repeating units of histone octamers each wrapped with 147 bp of DNA, is organized into increasingly complex structures until the completely folded metaphase chromosome is created. Histones are highly basic proteins with two domains, a globular core and a “highly dynamic N-terminal tail” and are some of the most conserved proteins across evolutionary boundaries. The globular core is responsible for histone/histone and histone/DNA binding while the protruding tails are heavily modified by a variety of posttranslational modifications: acetylation, methylation, phosphorylation, ubiquitination, sumoylation and ADP-ribosylation. These tail modifications have been associated with a variety of cellular processes such as DNA synthesis, transcriptional control, DNA repair, chromatin assembly and chromosome condensation.
Following DNA replication during S-phase, naked DNA is an available target for every DNA binding protein in the cell. Thus, replication-dependent chromatin assembly is a vital process within the cell that establishes a silent, closed chromatin conformation that helps to prevent aberrant gene expression. Replication-independent chromatin assembly can also occur, which is tightly associated with DNA repair.
The majority of chromatin assembly occurs immediately following DNA synthesis. This is the most important step in chromosome building as it establishes proper histone/DNA interactions that allow higher order chromatin packaging and segregation later in the cell cycle. Chromatin assembly occurs in a two-step manner with the H3/H4 tetramer being deposited onto the DNA first, followed by the addition of two H2A/H2B heterodimers. Interestingly, solutions of DNA and H3/H4 have been found to form complexes that appear similar to chromatin; whereas solutions lacking either H3 or H4 are unable to form any chromatin-like structures. H2A/H2B dimers are transported from the cytoplasm to the nucleus as cells progress through the G1/S transition. The assembly of the H2A/H2B dimers into the maturing nucleosome is likely facilitated by their binding affinity for DNA/H3/H4 complexes being higher than their affinity for naked DNA.
Experimental methods for studying chromatin
The higher-order spatial organization of chromatin has long been studied experimentally by means of visualization methods based on high-resolution microscopy. Among such methods are super-resolution fluorescence microscopy, electron microscopy, soft X-ray microscopy, and fluorescence in situ hybridization (FISH). The latter technique is particularly attractive because it can be used to measure, in a fairly direct manner, the spatial distance between specific locations inside a single intact cell. Measuring such distances for different pairs of genomic loci over many cells allows one to infer the 3D conformation of a particular domain of interest. Unfortunately these methods are very laborious and do not simultaneously provide the spatial resolution and the measurement throughput necessary to discern and locate many chromatin fibers and their interactions within large regions of the interphase nucleus.
During the past decade, however, increasingly higher resolution and throughput have been achieved by a number of sophisticated experimental techniques, including 4C, 5C, GCC, and Hi-C. These techniques are all based on the original method of chromosome conformation capture (3C) .The difference between this method and microscopy is that 3C does not attempt to capture directly the 3D spatial organization of chromatin. Instead, 3C seeks to measure the frequency of interactions between different fragments of genomic DNA in intact nuclei. Depending on the specific variant of 3C, interactions can be probed within or across particular domains, or within the whole genome.
1. Cremer M, von Hase J, Volm T, et al. Non-random radial higher-order chromatin arrangements in nuclei of diploid human cells[J]. Chromosome research, 2001, 9(7): 541-567.
2. Lanctôt C, Cheutin T, Cremer M, et al. Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions[J]. Nature Reviews Genetics, 2007, 8(2): 104-115.
3. Brickner J H, Walter P. Gene recruitment of the activated INO1 locus to the nuclear membrane[J]. PLoS biol, 2004, 2(11): e342.
4. Dorman C J. H-NS, the genome sentinel[J]. Nature Reviews Microbiology, 2007, 5(2): 157-161.
5. Holliday R. Epigenetics: an overview[J]. Developmental genetics, 1994, 15(6): 453-457.
6. Richmond T J, Davey C A. The structure of DNA in the nucleosome core[J]. Nature, 2003, 423(6936): 145-150.
7. Peterson C L, Laniel M A. Histones and histone modifications[J]. Current Biology, 2004, 14(14): R546-R551.
8. Felsenfeld G. Chromatin[J]. 1978.
9. Li B, Carey M, Workman J L. The role of chromatin during transcription[J]. Cell, 2007, 128(4): 707-719.