Genetics, Histone Code
Epigenetics is the study of making chemical modifications to DNA. Our DNA has a determined nucleotide sequence that cannot be changed. However, this genetic code can undergo chemical modification through epigenetic mechanisms. DNA strands wrap around proteins called histones, which are composed into structures called nucleosomes. There are four types of histones, named: H2A, H2B, H3, and H4. Octomers of two of each type of histone form nucleosomes. These nucleosomes are wrapped together in a spiral structure called a solenoid. Additional H1 proteins are associated with each nucleosome as links to maintain the overall chromatin structure. There are two states of chromatin: euchromatin, which is open and amenable to transcription, and heterochromatin, which is a compact DNA-protein structure that cannot be transcribed. Chemical modifications to these histones result in the conversion of DNA from its euchromatin state to its heterochromatin state and vice versa. The “histone code” is a hypothesis which states that DNA transcription is largely regulated by post-translational modifications to these histone proteins. Through these mechanisms, a person’s phenotype can change without changing their underlying genetic makeup, controlling gene expression.
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There are different types of modifications that exist, such as methylation and acetylation. In methylation, a methyl group (CH3) gets added to a specific nucleotide sequence. Acetylation involves adding an acetyl group (CH3CO) to a specific nucleotide sequence. These modifications can be either activating or repressing for a particular gene, depending on where the alterations occur.
There are regions upstream of transcription start sites on the promoter regions of DNA called “CpG islands,” which are a collection of cytosine and guanine dinucleotides. The cytosine molecule is the site of DNA methylation. After methylation, a transcription factor that usually binds to that specific promoter will be unable to recognize the sequence that it normally binds to since the modification conceals it. Therefore, DNA methylation turns off transcription of specific genes by acting at the promoter region.
On the other hand, chemical modifications of histones cause conversation of chromatin from euchromatin to heterochromatin states and vice versa. Each histone is a protein with an N terminus (the amino end) and a C terminus (the carboxy end). These histone tails protrude from the surface of the chromatin and comprise about 25 to 30% of the mass of the histone, providing a greater surface area for chemical modifications to occur on. Both of these tails contain many lysine amino acids, which are the primary sites of histone methylation and acetylation. Histone methylation, in which a methyl group gets added to a lysine, is generally repressive and supports the heterochromatin state. Histone acetylation, in which the mechanism is the addition of an acetyl group to a lysine, is activating and supports the euchromatin state.
The acetylation and methylation processes are carried out by specific proteins which exist in all of our cells. Histone acetyltransferases (HAT) transfer acetyl groups from acetyl coenzyme A to the lysine molecules. This transfer eliminates the natural positive charge of the histone proteins and therefore reduces its interaction with the negatively charged DNA phosphates, ultimately leading to a euchromatin state and turning on gene expression. Histone deacetylases (HDACs) function to remove acetyl groups from the lysine, leading to a heterochromatin state and turning off gene expression.
Histone methylation is not as straightforward as acetylation. The effect of histone methylation depends on the location of the modified position, and the number of methyl groups added.
There has been increasing interest in epigenetic research due to the possible effects it can have on modifying gene expression in vivo.
Epigenetic Editing Approaches
Gene editing involves directly altering the sequence of a gene. All epigenetic modification techniques must contain a DNA binding molecule and an active epigenetic modification molecule. These modifications first require DNA to be cut using an endonuclease to make a double-stranded break and then allow the two ends of the DNA to repair. During this repair process, the DNA in the vicinity of the cut undergoes replacement by a new sequence using a template delivered into the target cell along with an endonuclease. Three of the most widely studied DNA binding molecules are zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), and deactivated Cas9 (dCas9).
Zinc Finger Proteins (ZFP)
Zinc finger proteins are fused to a DNA cleavage domain to make zinc finger nucleases. These nucleases cause double-stranded breaks at a very specific targeted DNA sequence, which can then disrupt that specific sequence due to errors during the non-homologous repair end-joining pathway, thereby permanently disrupting the targeted gene. The zinc finger nucleases are highly individualized for their respective DNA sequences, and a new one must be engineered for each specific site, making this process too laborious and expensive.
Transcription Activator-Like Effectors (TALE)
These proteins are produced by a plant pathogen Xanthomonas that is capable of altering plant gene expression. They can be modified to have endonuclease activity by combining the TALE with a DNA cleavage domain, making a transcription activator-like effector nuclease. They work similarly to zinc finger nucleases, and their clinical application is also limited due to the necessity of creating new proteins for each target site.
Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/ Cas9
CRISPR arrays are regions of DNA in bacterial genomes that are used to store genetic information of infecting viruses so that the bacteria can transcribe the sequence into RNA upon reinfection with that specific virus to eventually cleave the homologous region in the viral genome. One of the endonucleases used by this system is called CRISPR-associated protein 9, or Cas9. Cas9 can be guided to any chosen DNA sequence by changing the sequence of the guide RNA to match the DNA sequence of interest.
Currently Under Investigation
One technique currently under investigation is the generation of hematopoietic stem and progenitor cells (HSPCs) that lack the CCR5 receptor to provide HIV resistant T cells in an HIV infected individual. Another technique is silencing the HER2/Neu oncogene using a zinc finger fusion protein via histone methylation. Clinical trials for using CRISPR gene editing for cancers such as melanoma, synovial sarcoma, and multiple myeloma are also currently ongoing at the University of Pennsylvania.
Current FDA Approved Drugs
Azacytidine and decitabine are the two FDA approved drugs that inhibit DNA methyltransferases and result in reduced DNA methylation. Azacytidine was approved for the treatment of myelodysplastic syndromes by the FDA in 2004, and decitabine received approval for the treatment of myelodysplastic syndromes in 2006.
Histone Deacetylase Inhibitors
Histone deacetylation leads to the repression of a specific gene. Drugs that inhibit deacetylation, known as histone deacetylase inhibitors (HDACi’s), lead to decreased histone deacetylation and ultimately increased gene expression. There are four HDACi’s currently approved by the FDA. Vorinostat obtained approval in 2006 for the treatment of cutaneous t-cell lymphoma. Romidepsin is a natural HDACi isolated from bacterial fermentation of Chromobacterium violaceum and was approved in 2009 for the treatment of cutaneous t-cell lymphoma. Belinostat received approval in 2014, and panobinostat approval came in 2015, both under the FDA’s accelerated approval program, for the treatment of relapsed peripheral t-cell lymphoma and relapsed multiple myeloma, respectively. Many other HDACi’s are currently under investigation.
Gene therapy refers to a process of introducing a gene, known as a transgene, and is presently being studied for various disorders, such as disorders of the eyes, hemophilia, cystic fibrosis, sickle cell disease, and thalassemia, and as cancer therapy.
Ocular Gene Therapy
Leber’s congenital amaurosis 2 (LCA2) is an autosomal recessive early-onset retinal degeneration caused by mutations in the RPE65 gene. Mutations in this gene result in the defective formation of the visual pigments including rhodopsin and cone opsin leading to dysfunctional photoreceptor function and loss of vision. Gene therapy for vision loss associated with this condition called voretigene neparvovex has been developed, which consists of an adeno-associated virus combined with the RPE65 gene, was investigated and subsequently, FDA approved in December 2017 for the treatment of biallelic RPE65 mutation-associated retinal dystrophy. It directly delivers a normal copy of the RPE65 gene into retinal cells and is the first gene therapy approved by the FDA for an inherited disorder.
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