1. Epigenetics and Inheritance
AN Emerging Basic Field of Science at the epicenter of modern medicine
Part 2

2. Epigenetics
Epigenetics is generally defined “as relating to or arising from non-genetic influences on gene expression”.
Epi is the Greek prefix meaning upon, above, in addition to, or near. The work was coined by Conrad Waddington in the early 1940s to explain “the causal interactions between genes and their products, which bring the phenotype into being”.
A more current definition is the field of genetics which looks at how genes are variably expressed during the formation of an embryo and during the lifespan of the individual without a change in the DNA sequence.
Studies have shown that some of these epigenetic changes can be inherited.

3. Duplicated metaphase chromosome – 1400 nm diameter and 2 µm long
Sizes:
DNA – 2 nm diameter
DNA wrapped around a nucleosome made of histones (called a “bead on a string”). Eventually a long strand of these beads will form –11 nm diameter
“Beads on a string” (nucleosomes + DNA) coiled into a helical structure producing a chromatin fiber – 30 nm diameter
Further condensation of chromatin into loops, scaffolds, and domains – 700 nm diameter
Duplicated metaphase chromosome – 1400 nm diameter and 2 µm long
No changes were made in the image; however, sizes were added to the right side of the image.

4. Structure of a Chromosome
The highly condensed metaphase (mitotic) duplicated chromosome is easily stained and is used to show the location of genes on the p arm (above the centromere where the 2 duplicated chromosomes are attached) and on the q arm (below the centromere).
ghr.nlm.nih.gov/handbook/basics?show=all

5. Gene Expression DNA in the nucleus
From the mRNA precursors transcribed from the DNA, introns are spliced out and exons are spliced together to be expressed.
mRNA (messenger RNA)
composed of spliced together exons
mRNA is translated into Protein in the
cytoplasm by tRNA (transfer RNA)
at the site of the rRNA (ribosomal RNA)
Chart: Lynda Jones, MS, ONPRC

6. Mechanisms of Epigenetics
There are 3 known mechanisms of epigenetic:
1) DNA Methylation
2) Histone Modifications
3) RNA Interference (RNAi)
Methylation modifications of DNA and expressed proteins have been known for years.
In 1969, Griffith and Mayler suggested that these modifications may modulate gene expression.

7. Schematic of the Mechanisms of Epigenetic Regulation
There are 3 known mechanisms of epigenetic:
1) DNA Methylation. Genes can be switched off by adding a methyl (-CH3) group to the cytosine at a CpG site on the DNA causing condensation of the DNA. Genes can be switched on by removing the methyl group from the same site.
2) Histone Modifications. Genes can also be switched on or off by methylation (-CH3), acetylation (-COCH3), phosphorylation (-PO3 2-), ubiquitylation (-ubiquitin polypeptide), or sumoylation (-small ubiquitin-related modifier) of the histone tail proteins around which the DNA is wrapped. The particular histone modification determine whether the gene would be switched on or off.
3) Post-Transcriptional Gene Silencing by RNA Interference. RNAi (RNA interference), including microRNA (miRNA) and small interfering RNA (siRNA), silence gene expression by altering the transcribed mRNA before it can be translated into protein.

8. DNA Methylation
Methyl groups are almost always added to cytosine bases with the sequence: ---CG GC--- When the DNA is replicated, each of the new DNA double helices will have one old strand, complete with methyl groups, and one new strand, which is not methylated. There is a specific enzyme, DNA methytransferase I, which will replace the missing methyl group.
Blue methyl groups shown on red DNA at a CpG site.
Methylation of DNA at a cytosine is the best understood of the epigenomic modifications.
Methylation takes place on the cytosine at the CpG sites on the DNA strand. The “p” in CpG refers to the phosphodiester bond between the cytosine and the guanine showing they are next to each other on the same side of a DNA strand, in both single- and double-stranded DNA (writing C-G which would denote a base pairing across from each other in the double-stranded DNA).
Within the genome, 70 – 80% of the CpG dinucleotides are methylated. However, there are areas that contain an unusually high number of CpG dinucleotides, known as CpG islands, which are typically free of methylation.
Methylating the cytosines on both sides of the double-stranded DNA silences the gene by causing the DNA to condense. The DNA cannot be expressed since the genes have no access to the various enzymes involved in transcription of DNA.
Demethylation causes the DNA to unravel and genes to be expressed.
Theoretical & Computational Biophysics Group NIH Center for Macromolecular Modeling & Bioinformatics, University of Illinois at Urbana-Champaign.

9. Enzymes Involved in DNA Methylation
DNA methyltransferase (DNMT1)
DNA methyltransferase 2 (DNMT2)
DNA methyltransferase 3 (DNMT3)
There are five known enzymes which methylate DNA. All are DNA methyltransferase (DNMT) enzymes found in mammalian cells and include DNMT 1, 2, 3A, 3B, and 3L.
Once each cell’s fate has been decided during development of the embryo, this epigenetic code must be maintained for the lifespan of the organism.
When the cell divides, DNA methyltransferase (DNMT1) enzymes are active in adding the proper methyl groups to the new strands of DNA by searching out the CpG base pairs where only one strand has a methyl group (called hemimethylated DNA) and adding a new methyl group on the daughter DNA at the CpG site.
The function of DNA methyltransferase 2 (DMNT2) is thought to catalyze methylation of tRNA.
During the development of the embryo, the DNA methyltransferase 3 (DNMT3) family of enzymes is active in methylating the DNA at de novo CpG sites, which means they add methyl groups to CpGs where they were not present before.

10. DNA Methyltransferase 1 (DNMT 1)
Several structures have helped to explain how DNA methyltransferases perform their jobs. On the left, Protein Data Bank (PDB) entry 3pt6 includes DNMT1 and a small piece of DNA and shows the enzyme wrapping around the DNA and probing the edges of the bases in the DNA. This structure was solved using DNA with no methyl groups, which would normally not be a substrate of the enzyme. The structure shows that the enzyme recognizes the unmethylated bases on both sides, and holds the DNA away from the active site (in green).
There is currently no structure of DNMT1 with a half-methylated DNA, but on the right, we can get an idea of how it might act by looking at a bacterial methyltransferase (Hha1). The DNA binds deeper in the active site, and flips out the cytosine base (in red) that will be methylated.

11. Histone Modifications
During different stages of the cell cycle, the histone charge is modified by:
a) methylation
b) acetylation
c) phosphorylation
d) ubiquitylation
e) sumyolation.
Histones are proteins around which DNA wraps to from nucleosomes. This organization provides a means by which the DNA takes us less space in the nucleus.
Histone modifications are added to sections of the histone that protrude through the DNA into the cytoplasm and are called histone tails.

12. Two Nucleosomes Linked by a DNA Strand
Eight histone proteins – 2 each of H2A, H2B, H3, and H4 - cluster.
Since the DNA is a long strand of dinucleotides and would not fit into the cell nucleus as is, corralling the DNA is accomplished by wrapping base pairs of DNA 1.67 times around the 8 histones in a left-handed super-helical turn to form a nucleosome.

13. Nucleosome Formation
Histone proteins H2A, H2B, H3, and H4 are shown in their alpha helical shape.
A dimer is formed of H3-H4 and 2 dimers then form the H3-H4 tetramer. Similarly, the H2A-H2B dimers form and then combine to form a H2A-H2B tetramer.
The two tetramers then form the histone octamer around which approximately base pairs of DNA winds to form a nucleosome.
In order for DNA to be repaired and for transcription to occur, the nucleosome must unravel to expose the DNA. The nucleosome structure is regulated by numerous modifications found on both the N-terminus and C-terminus of the histone “tails” which condense and uncondense (unravel) the DNA. DNA can be expressed when in the uncondensed form.
No Changes have been made.

14. Histone Modification Sites Nucleosome Showing Histone Tails Protruding Through the DNA
DNA shown in red and orange
Histones shown in light blue, dark blue, green, and yellow.
Methylation, acetylation, phosphorylation, and ubiquitylation of histone tails at specific residues mediate gene expression through charge changes.
Histone tail modifications are another mechanism of epigenetics.
As the DNA winds around the histone proteins forming a nucleosome, part of the histone extends beyond the DNA into the cytoplasm. These histone tails provide a site for histone modification.
Histone modifications include methylation, acetylation, phosphorylation, ubiquitylation, and sumyolation – mostly on the lysine, serine, and arginine amino acids of the tail.
Histone tail protruding through the DNA

15. Acetylation of H4 Histone Tail
Acetylation of the lysine (K) residues at the N terminus of the histone protein eliminates the positive charges, thereby reducing the affinity between histones and DNA.  This makes RNA polymerase and transcription factors easier to access the promoter region.  Therefore, in most cases, histone acetylation enhances transcription while histone deacetylation represses transcription.
Histone acetylation is catalyzed by histone acetyltransferases (HATs) and histone deacetylation is catalyzed by histone deacetylases (HD).
Since up to 25% of the amino acids in histones can be basic amino acids, they are highly positively charged. During acetylation, phosphorylation, ubiquitylation, and sumyolation, this shift in charge can cause the DNA and histone to dislodge so genes can be expressed. Methylation does not change the charge and usually causes the gene to be suppressed.
These modifications have been implicated in cell cycle, differentiation, development, disease, and a variety of key processes that dictate cellular function. For instance, loss of histone acetylation and methylation has been shown to accompany cancer cells.

16. Histone Phosphorylation
Histone phosphorylation (-PO4-2) has divergent roles.
Sometimes it is involved in chromatin condensation associated with mitosis and meiosis as when phosphorylation (ph) of serine (S) on histone at the 10th amino acid position (H3S10ph) and the 28th amino acid position (H3S28ph) occur.
Sometimes phosphorylation is involved in chromatin relaxation by influencing demethylation and acetylation of adjacent amino acid residues on the histone.
Public domain.

17. Ubiquitylation
Ubiquitin is a 76-amino acid polypeptide. Ubiquitin universally regulates many activities in the body in an array of different configurations. When added to a histone tail in ubiquitylation , it can either suppress or enhance gene expression depending on which amino acid in H2A or H2B it is attached.
Ubiquitin was discovered as a covalent modifier of histone H2A nearly 30 years ago, and has since emerged as one of the cell's most broadly utilized protein modifications.

18. Sumyolation
Small Ubiquitin-related Modifiers (SUMOs) are around 100 amino acids in length, just slightly larger than ubiquitin. Sumyolation is a reversible post- translational modification of lysine residues on histone proteins by SUMOs. Sumyolation mediates gene silencing through recruitment of histone deacetylase and heterochromatin protein 1.
SUMOs are crucial in a variety of biological processes including transcription and protein stabilization.

19. Histone Modification http://nanoweb.ucsd.edu/~arya/research.html
In the histone tail, lysine (K), serine (S), and arginine (R) are the amino acids involved in histone
modification.
Most of the research on histone modification has been conducted on the N-terminus (amino, or -NH2, end of the histone protein beginning with amino acid #1) of histones H2A, H2B, H3, and H4.
Much less is known about C-terminus (carboxy, or –COOH, end) of histones H2A and H2B.
The C-terminus of H2A is very short consisting of 15 amino acids beyond the globular domain. It has two parts: a) amino acids (8 amino acids) pass between the strand of DNA wrapped around the nucleosome and b) amino acids (7 amino acids) protrude from the nucleosome.
The C-terminus tail of the H2B histone amino acid residues arginine (R) 119 (amino acid #119) and threonine (T) 122 (amino acid #122) play a direct role in controlling the ubiquitylation of proline (P) in amino acid position #1 on the N-terminus of H2B (H2BP1ub) and methylation of lysine (K) in amino acid position #4 on H3 (H3K4me) methylation by cross talk. Cross talk is where a histone modification on one tail influences the modification on another tail.