Epigenetic Regulation

Active and Repressed Chromatin
In eukaryotic cells, the DNA is packaged with protiens to form chromatin. The protein component consists of a Histone octomer (H2A, H2B, H3, & H4).

The chromatin structure plays a huge role in the controlling of which genes are expressed in the cell at a given time. There are two main forms of chromatin that exist in the cell: (1) open euchromatin and (2) Closed Heterochromatin.

Histones as an activation switch
The histone proteins are important in determining which state the chromatin will exist in the cell at any time. The chromatin can either continue to be closed in a repressed state or loosened to open in a more active state. This is done through modifications on the histone proteins.

There are two major histone modifications that directly relate to the state of the chromatin: (1) Acetylation and (2) Methylation.

(1) Acetylation


 * The addition of the negatively charged acetyl groups in the Histone proteins causes a neutralization of the protein to occur. The neturalization causes the interaction between histones to weaken, essentially causing the chromatin to loosen.


 * Acetylation of histones are done through Histone Acetyl Transferases (HATs) and the removal of acetyl groups is done through Histone Deacetylases (HDACs).

(2) Methylation




 * The next histone modification is done through the addition of methyl groups. The addition of these modifications are done through Histone Methyl transferases (HMTs) and they are removed by Histone Demethylases (HDMs).


 * The addition of a methyl group is not as clear cut as the addition of an acetyl group. Depending on which amino acid the methyl group is added, a different effect on the chromatin can occur. For instance, methylation on H3K9 (Histone 3 Lysine 9) causes the addition of the accessory protein HP1. Addition of HP1 causes a repression effect on the chromatin. Another example of methylation is on H3K4 which casues the association of the CHD1 protein causing an activation of the chromatin.

Readers, Writers, and Erasers
The idea of histone modification was easily explained through what is called the 'Histone Code Hypothesis'. The Histone Code Hypothesis explains of three different groups of proteins who play a key role in histone modification: (1) Writers, (2) Readers and, (3) Erasers.

(1) Writers


 * Writers are proteins that specifically add the histone modifications. These would include: HATs and HMTs.

(2) Readers


 * Readers are proteins that identify and respond to the modifications placed by the writer proteins. They have specific domains that recognize specific modifications. For instance, the Bromo domain recognizes Acetyl markers while the Chromo and Tudor Domains recognize methyl markers.

(3) Erasers

Erasers are proteins that works to remove the modifications placed by writers. These include the HDACs and the HDMs.

Chromatin Immunoprecipitation (ChiP)
ChiP analysis allows to study the site of TF binding to DNA across the entire genome.

Mechanism:
 * 1) The DNA and all proteins all washed in Formaldehyde
 * 2) *This causes cross-linking between all close molecules
 * 3) *If the protein is close to the DNA it will cross link keeping it in place
 * 4) Shear the DNA to about 300-500bp in length
 * 5) Use a specific antibody towards the protein of interest
 * 6) *This will then bind to the Protein and DNA complex
 * 7) Precipitate the protein
 * 8) *It will bring with it the DNA that it was interacting with
 * 9) Wash away the protein
 * 10) Use a high throughput sequencing method to sequence the DNA that was caught and compare it to the genome to see where it is.



DNA methylation and CpG Islands
Along with modifications done on histones, modifications can be done on the DNA themselves.



What makes this modification more on the side of epigenetics is the fact that these DNA methylation modifications are heritable to the daughter cells.

One of the most significant DNA methylation events in vertebrates occurs at the Cytosines of a gene's promoter. It has been found that methylation of the cytosine to 5-methylcytosine in the promoter region causes a repression effect.

Example: Developing Human RBCs
 * Remember that all cells have the same genomic content within their nucleus. Therefore all cells have the potential to produce any proteins that are found within the code of the genome. If this is the case, why do certain proteins only become translated in certain cells while absent in all other cells? Why does the globin protein become coded in RBC's and not in muscle cells? This is due to the metylation that occurs at the genes promoters. It was found that in RBC's the promoter for the globin gene were unmethylated while the promoter for the gene in muscle cells were heavily methylated. Methylation of the promoters caused the inactivation of the gene while unmethylated promoters were able to become transcribed.


 * What is also interesting is that methylation patterns in the genome change during the development of the organism. A perfect example of this occuring is in the production of various isoforms of the globin protein during human development. Depending on the specific development stage, a human needs a certain type of globin isoform to work with the oxygen levels available at the time of development. Two of the earliest activating globin isoforms are e-globin and y-globin proteins. e-globin was found to occur specific during the embryonic stage of development while the y-globin isoform occured during the fetal stage of development. It was discovered that during the embryonic stage, the promoter for e-globin is unmehtylated while the promoter for y-globin is heavily methylated. It was also discovered that during the fetal stage of development that the methylation patterns were switched where the y-globin promoter was unmethylated and the e-globin promoter was methylated.

Blocking of Transcription
It is known that methylation of the DNA causes a repression effect to occur. This is done in two ways: (1) methylation causes a blocking in TF's from binding to enhancers and (2) methylated cytosines are able to recruit proteins to tighten the nucleosome.

(1) TF blocking


 * Transcription factors have specific affinities to the enhancer seqeunces which they interact. The seqeunce can be disturbed with the addition of methylcytosines. Addition of these disturbances causes a blockage in the ability of TF's to bind enhancers ultimately stopping transcription.

(2) Recruitment of proteins


 * Methylcytosines can act as markers for specific proteins that interact cause various effects to the chromatin structure. For instance, methylcytosine can be recgonized by the particular protein MeCP2. MeCP2 is a reader protein (If we use Histone Code hypothesis terminology) that can recruit HDACs and HMT's which cause a tightening of the nucleosome.

Inheritance of DNA methylation
What makes DNA methylation such a powerful epigenetic model is the ability to inherit the DNA methylation marks from the parental cells. Another enzyme recruited by MeCP2 is called DNA methyltransferase-3 (Dnmt3). This methyltransferase has the ability to continue to methylate previously unmethylated cytosines on the DNA.

The ability to inherit th DNA methylation patterns from the parental cells is done through DNA methyltransferase-1 (Dnmt1). This enzyme recgonizes the methylctyosines on one strand of DNA and places the methylation mark on the newly developing strand opposite to it.