Chromatin Chapter 2: Modifications and the Histone Code

As mentioned, histones can be modified as well (this was mentioned briefly in the DNA repair posts).  Poly(ADP)-ribosylation causes the histones to bind less tightly to the DNA via repulsive negative charges.  This modification can be found at sites of DNA under repair.  Ubiquitination of H2A and H2B marks the proteins for degradation.  Phosphorylation tends to compact chromatin, but the mechanism by which this is done is unknown.  Finally, acetylation via HATs (histone acetyl transferases) on lysine and arginine residues in the core histones neutralizes the positive charge of the histones and causes them to repel from the DNA, leading to enhanced transcriptional activity. 

Modification	Effect
Poly(ADP) Ribosylation	Histones repel DNA, opening it for DNA repair
Ubiquitination	Degradation of H2A and H2B
Phosphorylation	Chromatin compaction
Acetylation	Histones repel DNA, opening it for transcription and DNA repair

Naively, one would consider the most important aspect of gene regulation to be the primary DNA sequence.  Nonetheless, chromatin plays a part in both transcription and replication, and the importance of this regulation is becoming more and more apparent.  One of the primary mechanisms by which replication and transcription are regulated via chromatin is from acetylation of histones.  Acetylation is performed by histone acetyl transferases (HATs), of which there are two kinds: A-HATs that mediate transcription-related acetylation, and B-HATs that modulate replication-related acetylation.  The most frequently observed acetylation events occur on histones H3 and H4, with A-HAT acetylating lysines 8 and 16 of H4 and 14 of H3; B-HAT acetylates lysines 5 and 12 of H4 and 9 of H3.

During DNA replication (when B-HATs are most active); histone acetylation is linked to chromatin maturation of the nascent DNA strand.  p48 is a histone escort that is in complex with a B-HAT, (Hat1p).  Chromatin assembly factor (CAF1) displaces B-HAT to form the chromatin assembly complex.  Histone de-acetylase (HD1) displaces CAF1 in the complex and removes acetylation events to mature the chromatin.  Acetylation events that remain on histones are recognized by specific proteins with specific domains, called bromodomains, that recognize and bind the acetylated lysine residues.

In contrast to histone acetylation, methylation via histone methyl transferases (such as human SUV39H1) mediates gene repression.  Gene activity is modulated by the balance of methylation and acetylation.  As mentioned, there are proteins that bind acetylated histones via bromodomains; similarly, there are proteins with chromodomains that bind methylated histones.  Two such proteins are Swi6 and HP1 (heterochromatin protein 1). 

It is important to note that actively transcribed genes are complexed with nucleosomes.  However, the chromatin in the region of these active genes tend to have acetylated histones and are also tend to be missing a couple nucleosomes from the promoter region.  Histone H1, the linker histone, is also missing from regions of the gene, and little DNA methylation is detected.  The DNA at actively transcribed genes is also more susceptible to nuclease attack and digestion.   

The Histone Code

Data considering differential gene regulation based on histone methylation and acetylation lead to the development of the histone code hypothesis.  The histone code is considered the pattern of markings on histones that cover the DNA.  Specific markings of these histones indicate newly-replicated chromatin, damaged DNA, and transcriptionally active or repressed regions.  Reader complexes recognize the marks on the chromatin and perform a function, while writer complexes such as HATs and methyltransferases mark the nucleosomes.  Write-read cycles spread chromatinization along DNA by consisting of DNA reader/writer complexes that bind a modified histone and create a marking on a nearby histone that then propagates chromatin modifications.  Barrier DNA sequences block these reader-writer complexes from expanding beyond a set boundary.   

Chromatin Chapter 1: DNA Organization, the Nucleosome, and Histones

Chromatin is one of those things that I never really paid any attention until I suddenly realized how important and interesting it is.  How is it that so much information (DNA) can be stored in such a small space (the nucleus)?  How is it that this information is used when it is so tightly packaged?  It may sound nerdy, but I’m still in awe at how important chromatin really is.  This next set of posts will explore the basics of chromatin and begin to touch on the effects it has on genes and cells.  I’ve already mentioned how it affects DNA repair, but the process has even more far-reaching effects.

DNA Organization

Chemically, DNA and RNA are composed of sugar phosphate backbones with nitrogenous bases attached.  The sugar comes in the form of ribose (in the case of RNA) or deoxyribose (in the case of DNA).  Deoxyribose lacks the 2’ hydroxyl group on ribose.  The ribose / deoxyribose sugars are connected via a phosphate linkage (PO₄) via the 3’ and 5’ hydroxyl groups.  The 1’ hydroxyl group is where the nitrogenous base attached.  Purines contain a purine ring and come in the form of adenine and guanine.  Pyrimidines consist of cytosine, uracil, and thymine.  The ribose / deoxyribose connected to the base and the phosphate are considered nucleic acids.

Nearly everyone knows the famous story of Watson and Crick and their discovery of the structure of DNA.  They hypothesized (correctly so) that DNA consists of a double-helix held together by the hydrogen bonds formed by the nitrogenous bases (adenine to thymine; guanine to cytosine).  This double helix is antiparrallel, right-handed, and has polarity: the 5’ end is attached to a phosphate group, while the 3’ end consists of a free hydroxyl group.  The helix turns once every 10.5 bases at a total distance of 36 Å, with a 3.4-Å rise per base and a width of 20 Å.  The entire helix is negatively charged due to the phosphate groups that connect the sugars. 

DNA does not exist in the cell as a free-floating molecule.  Instead, it is shaped and organized by chromatin, the makeup of the chromosomes consisting of the DNA itself and the attached proteins.  In the case of humans, unraveled DNA measures about two meters in length, but cell nuclei are, at most, 10 μm.  Therefore, the cell must attain a 10,000-fold compaction while still performing all the requirements for the cell.  To accomplish this, the cell uses the chromatin hierarchy, composed of five orders of organization.

The Nucleosome

The first order of chromatin packaging is the nucleosome, the most basic organization mechanism used to compact the DNA.  The nucleosome packages 147 bp of DNA wrapped on “beads” of eight histone proteins (making an octamer). These octamers are positioned at intervals on the DNA, and,  if the DNA is spread, the  nucleosomes attached to the DNA look like beads on a string.  The nucleosome consists of histones H2A, H2B, H3, and H4, and nucleosomes are attached to each other by linker histone H1.  Linker and nucleosomal histones are made throughout S phase, when new DNA is synthesized and must be compacted.   Histones can be modified in several ways to affect the structure and dynamics of the DNA.  The proteins have largely been conserved through evolutionary history but variants do exist.  These variants are synthesized mostly during interphase and insert into mature chromatin via chromatin remodeling complexes.  One of these variants, H2A.Z,  limits chromatin condensation; another variant H2A.X is involved in DSB response.  H3.3 can be found in long-term active chromatin.  In general, these variants are involved in changes in chromatin that remain for long periods of time in the cell. 

In addition to histones, a number of other proteins bind DNA and are included in the chromatin.  Namely, the high mobility group proteins (HMGs), polymerases, and DNA repair enzymes interact with the DNA and the chromatin. 

The DNA itself is wrapped around the nucleosome 1.75 times, with about 60 bp of DNA between nucleosomes and associated with linker histone H1.  The nucleosomes contain groves that fit the DNA between H2B and H4 and H4 and H3.  The octamer itself exists as two H2A/H2B dimers and one H4-H3-H3-H4 tetramer.