4.1 Introduction: “Inner Life of the Genome”

The size of the genome in one of the most well-studied prokaryotes, E.coli, is 4.6 million base pairs (approximately 1.1 mm if cut and stretched out).

Let’s do a rough calculation about how much DNA a eukaryotic cell has.

Assume

1. A somatic cell has 2 full copies of every chromosome. The total number of base pairs for ALL chromosomes combined is 6 X 10^{9 bp}.
2. We know the length of each base pair is 0.34nm (see DNA structure chapter!)

If you were to take the DNA of all the chromosomes, stretch it out, and lay them end to end then what will the total length of ALL the DNA in the cell be?

Hopefully, you got to a number of 2.0 meters! Now consider the size of the eukaryotic nucleus which ranges from 2- 10 microns! For a good frame of reference, a single grain of salt is 100 microns which is still 10 times larger than the upper range for nucleus size!

This results in an engineering problem that cells need to solve.

Problem 1: How does all of the DNA fit inside a bacterial cell and even more amazingly inside the nucleus of a eukaryotic cell?

At the same time consider all the different cells in the body. The human body contains approximately many different cell types, but each cell type shares the same genomic sequence. In spite of having the same genetic code, cells not only develop into distinct types from this same sequence but also maintain the same cell type over time and across divisions.

They are also specialized in their function- liver cells for example produce enzymes that help with detoxification but they do not produce antibodies, that is the job of a different cell type.

These cells have different expression patterns due to the temporal and spatial regulation of genes. (See video below for a good analogy).

This leads to the second problem to contend with- once the genome has been folded and all packaged up how can we open small sections of it so the machinery to read and express the information coded within the genes can gain access?

The epigenome (“epi” means above in Greek, so epigenome means above genome) is the set of chemical modifications or marks that influence gene expression and are transferred across cell divisions and, in some limited cases, across generations of organisms.

In this chapter, you will learn about the solution to the ‘Packaging Problem’ and how cells regulate access to genes.

The solution involves interactions of DNA with specific proteins, leading to the formation of a nucleoprotein complex called CHROMATIN. 

We will focus exclusively on Eukaryotic chromatin, although bacterial chromosomes (which are circular) also coil and supercoil with the help of proteins.

4.2 Types of Chromatin in Eukaryotic Cells

When we visualize chromosomes we usually think of the characteristic highly condensed structures (X-shaped).

DNA  in this form exists only transiently for a brief phase in Mitosis, where it is maximally condensed. These discrete packets are ideal for the job at hand which is to accurately separate and segregate to opposite poles of the cell.  The majority of the time a cell is in fact in interphase and not dividing.

During Interphase which can be further subdivided into Gap 1-G1, S-phase, and G2]  cells are transcribing (RNA) and translating (making proteins) sometimes on demand and depending on the needs of the cell.

Not all parts of the genome however are active. During interphase (Figure 4.1 below) chromatin exists in various states of condensation called heterochromatin and euchromatin respectively.

The transition between these chromatin forms involves changes in the amounts and types of proteins bound to the chromatin that can occur during gene regulation, i.e. when genes are turned on or off.

Experiments to be described later showed that active genes tend to be in the more dispersed euchromatin where enzymes of replication and transcription have easier access to the DNA.  Transcriptionally inactive genes are heterochromatic, obscured by additional chromatin proteins present in heterochromatin.

4.3 Chromatin Organization

The proteins that are associated with eukaryotic DNA to form chromatin are divided into Histones and Non-Histone proteins. Together they help condense the DNA. We will devote much of our time to Histones as they are responsible for the first and fundamental unit of packaging.

4.3.1 Histones

There are 5 major classes of histones: H1, H2A, H2B, H3, and H4. Histones are basic proteins containing many lysine and arginine amino acids.  Their positively charged side chains enable these amino acids to bind to the acidic, negatively charged phosphodiester backbone of double-helical DNA. About a gram of histones is associated with each gram of DNA.

Histones are among the most highly conserved proteins.  Very few amino acid differences distinguish a human histone from histones in a mouse, sea urchin, or yeast cell. For instance, the H4 in cows differs from H4 in peas by only 2 amino acids.  This eludes to the fundamental aspect of the role and function of these proteins.

Note that only eukaryotes (i.e., organisms with a nucleus and nuclear envelope) have histones. Prokaryotes, such as bacteria, do not.

4.3.2 First level of organization- The Nucleosome

How do the histones help organize DNA? Here looking at the experiments to identify and isolate these proteins becomes helpful. Investigators started by gently disrupting cell nuclei to extract the chromatin using salts. The salts helped “unfold” the chromatin and they were able to visualize what it looked like using an electron microscope.  The resulting image looked like “ beads on a string ”

Here the string is DNA, which is wrapped around nucleosome core particles, the beads. To determine what makes up this nucleosome (“bead”) scientists used enzymes called nucleases. Nucleases cut DNA by breaking phosphodiester bonds between nucleotides. The particular enzyme used is only able to cut exposed DNA (“the string” between the “beads” )

When this was carried out for a SHORT time, the nucleosome particles were released. Electrophoresis of DNA extracted from digests of nucleosome beads-on-a-string preparations generated DNA fragments about 200 base pairs in length.

In contrast, the digestion of naked DNA (not associated with proteins) yielded a continuous smear of randomly sized fragments.

These results suggested that the binding of proteins to DNA in chromatin protects regions of the DNA from nuclease digestion so that the enzyme can attack DNA only at sites separated by approximately 200 base pairs.

This revealed the basic unit of chromatin and packing is a Nucleosome (the beads) consisting of ~ 200bp of DNA wound around a nucleosome core particle plus some adjacent linker DNA.

The length of the DNA extracted from the nucleosomes varies between organisms, ranging from 170-240 but the variation results entirely from the linker DNA length between the nucleosomes.

The length of DNA that is wrapped around the histone proteins that make up the core is always about 147 bp base pairs long.

The identity of the proteins that make up the core of the nucleosome came next and included familiar techniques of separating proteins and running them on SDS-PAGE gels. Revealing that the nucleosome core identified an octameric protein complex (two copies each of H2A, H2B, H3, and H4) with the 147 bp DNA wound around it.

Figure 4.2 Nucleosomes contain DNA wrapped around a protein core of eight histone molecules. Image Credit: Figures by Maria Nefeli Stefanidou (CC-BY-SA-ND)

Assembly of the Nucleosome

Nucleosomes have a common structure comprising two of the histones H2A, H2B, H3, and H4 that come together to form a histone octamer, which binds and wraps approximately 1.7 turns of DNA, or about 146 base pairs.

All core histones share a conserved structure known as the histone fold, which consists of three alpha-helices connected by 2 short loops.

The N-terminus of the proteins that do not participate in the fold are referred to as Histone Tails and play a crucial role in the regulation of chromatin structure.

The assembly of the nucleosome occurs in a step-wise process beginning with  H3 and H4 dimers, the dimers join to form a tetramer. The DNA begins to wrap around the tetramer and is then joined by the H2A. H2B dimers which ap the complex at the top and bottom. (Figure 4. 3) to form the completed nucleosome core particle.

Figure 4.3. Schematic Representation of Stepwise Assembly of the Nucleosome Core Particle, Figure by Maria Nefeli Stefanidou (CC-BY-SA-ND). The structure of nucleosome particles is based on Published NCP Structures (Luger, 2003). Structure Volume 12 Issue 12 Pages 2098-2100 (December 2004) DOI: 10.1016/j.str.2004.11.004

Before you continue you should

1. Watch the Lecture videos that cover the material above. (if you haven’t already)

Histone-DNA interactions:

The three-dimensional structure of the nucleosome using X-ray crystallography was solved by Dr. Karolin Luger which provided a deeper understanding of chromatin organization.

1. Electrostatic interactions of negatively charged DNA (phosphodiester backbone) with positively charged amino acids of histones.
2. Additional interactions occur via hydrogen bonds with bases of the DNA.

This association is consistent with the need to package any type of DNA without regard to sequence.

However, there are areas of the genome where nucleosomes position themselves preferentially. These include regions with A-T-rich sequences that are more accommodating of the ‘bend’ or ‘compression’ that occurs in the minor groove as the DNA wraps around the histone octamer.

4.3.3 30-nm Fibres and Higher-Order Chromatin

All chromosomal DNA starts as the “beads-on-string” structure or 10 nm fiber, however, in a living cell, this extended form of structure is not seen.  Nucleosomes are further coiled into a shorter, thicker fiber, termed the “30-nanometer fiber,” because it is approximately 30 nanometers in diameter.

Experimentally this structure can be observed after a high salt chromatin extraction (as shown in Figure 4.2)

The 10nm to 30nm step is believed to be aided by a fifth Histone H1, also known as ‘Linker histone’. In experiments when H1 is present the  30-nanometer fibers form readily. H1 binds DNA where the DNA joins and leaves the histone octamer and helps lock the DNA into place, acting as a clamp around the nucleosome.

Additionally, the tails of the core histones are important for the formation of 30nm fibers.

Non-Histone Proteins

Histone proteins assist in packaging DNA in interphase chromosomes into a pretty compact fiber, but the chromosome is not fully condensed yet!

There are at least five levels (orders) of chromatin structure leading up to the highly condensed mitotic chromosome (Figure 4.4). The 30nm fibers folded into 300 nm loops and eventually into the mitotic chromosome

Figure 4.4.  Higher orders of chromatin.  Image created for this book by Maria Nefeli Stefanidou (CC-BY-SA-ND)

Non-histone proteins like condensins, scaffold proteins, and cohesins play a role in further coiling and compacting the DNA until it resembles the metaphase chromosome.

Remember that the final metaphase chromosome shape is established and made only in preparation for metaphase. The bulk of the time the cell is in interphase and chromosomes do not resemble those discrete units we are used to thinking of when we think of chromosomes!

4.4 Regulation of Chromatin Structure

To recap we have just seen that chromosomal DNA associates with histones, forming an organized complex known as chromatin. This accomplished the goal of allowing DNA to fit into a smaller volume within a eukaryotic cell’s nucleus. 

However, compaction of the DNA also limits the accessibility of the DNA to proteins (transcription factors- which we will learn about soon) that will transcribe the gene [Gene Expression].

For gene expression to occur,  the chromatin structure needs to move back and forth between condensed and decondensed forms.

Changes to chromatin structure can be brought about in 2 ways and they are not mutually exclusive adding layers of complexity to regulation!

1. Via Histone modifications
2. Via Chromatin remodeling complexes.

4.4.1 Histone Modifications

The amino acid residues of histone tails are subject to many post-translational modifications and the list is ever-growing. In particular, histones contain a large percentage of lysine and arginine (basic amino acids) residues and serines, and threonines.

The side chains of these residues are modified by the action of enzymes.

The most studied modifications include acetylation (adding acetyl groups) of lysines, phosphorylation (adding phosphate groups) of serines, and methylation (adding methyl groups) of lysines and arginines.

The addition of the groups is facilitated by enzymes (collectively known as epigenetic “writers”)  whilst de-modifying enzymes (or “erasers”) remove these marks. (see table below)

Figure 4.6 Enzymatic modifications of key amino acids within histone tails regulate chromatin structure. Image Credit:  Image created for this book by Maria Nefeli Stefanidou (CC-BY-SA-ND)

These opposing activities enable a highly dynamic regulation of gene expression as modifications can be added or removed depending on whether a particular writer or eraser is recruited to a specific location of the genome.

Modifications can occur at different amino acids on different histones and can create more than 100 unique potential changes in the histones.

Modifications also result in unique patterns or signatures that DNA-binding proteins recognize.

Proteins with BROMODOMAINS- recognize Acetylated Lysine residues.

Proteins with CHROMODOMAINS- recognize methylated residues.

To add to the complexity, these proteins and modifying enzymes are contained within a larger multiprotein complex and can recruit another ‘molecular machine’ the chromatin remodeling complexes. These complexes serve to further reposition nucleosomes as discussed below.

4.4.2 Chromatin Remodeling Complexes

• As the name suggests, these are protein complexes meaning they contain multiple different proteins working together.
• They ‘remodel’ chromatin- and alter chromatin structure by repositioning nucleosomes. The mechanisms include
  • sliding nucleosomes along DNA
  • evicting nucleosome components (such as H2A–H2B dimers)
  • ejecting full nucleosomes (creating nucleosome-free regions)
  • replacing with variant histone subunits.

There are many families of chromatin remodeling complexes with a variety of different names, and while they are diverse in protein composition they all have at least one enzymatic  ‘ATPase subunit which allows them to utilize the energy released from ATP hydrolysis to reposition nucleosomes. (1)

Additionally, as mentioned above they are often recruited to the chromatin by ‘reading’ the ‘marks’ left by histone modifiers OR contain proteins with histone-modifying activities and histone recognition activities.

Figure 4.7 Modes of Action of Chromatin Remodeling Complexes. Image created for this book by Maria Nefeli Stefanidou (CC-BY-SA-ND)

4.5 Links to Medicine

Given the intimate role chromatin structure plays in regulating gene expression, it is not surprising that changes in acetylation signaling resulting from misregulated HATs or HDACs can cause abnormal gene expression patterns and have been identified in numerous cancers. (2)

Some examples are

• Genetic alterations in HATs in hematological and solid cancers (3)
• Hyper-active HDACs in many cancers (4)
• Components of several chromatin-remodeling complexes are highly mutated in cancer.

Not surprisingly there are a wide variety of small-molecule inhibitors targeting acetylation signaling pathways in development for use as anti-cancer drugs (see table below).

Remember to:

1. Watch the Lecture videos that cover the material above. This will help to clarify or reinforce certain concepts if they are unclear.
2. Complete any associated practice exercises

References and Attributions

This chapter contains material taken from the following CC-licensed content and Public Domain content. Changes include rewording, removing paragraphs and replacing them with original material, and combining material.

References

(1) Clapier, C., Iwasa, J., Cairns, B. et al. Mechanisms of action and regulation of ATP-dependent chromatin-remodeling complexes. Nat Rev Mol Cell Biol 18, 407–422 (2017). https://doi.org/10.1038/nrm.2017.26

(2) Gong F, Chiu LY, Miller KM (2016) Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer. PLOS Genetics 12(9): e1006272. https://doi.org/10.1371/journal.pgen.1006272

(3) Di Cerbo V, Schneider R. Cancers with wrong HATs: the impact of acetylation. Briefings in functional genomics. 2013;12(3):231–43. pmid:23325510

(4) Gui CY, Ngo L, Xu WS, Richon VM, Marks PA. Histone deacetylase (HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(5):1241–6. pmid:14734806