FIRST, HOW & WHY DO CELLS WRITE EPIGENOMES?

DNA METHYLATION occurs throughout the GENOME, but it is predominately found at cytosine followed by guanine dinucleotides (CG), symmetrically across the double helix (illustrated right). This DNA "braille" is interpreted by TRANSCRIPTION FACTORS that carry out various roles in TRANSCRIPTION.  Surprisingly, unique tracts of DNA with an unexpectedly high amount of CG repeats are speckled across the GENOME, but usually right at the beginning of genes. Called "CG ISLANDS", we now understand that the DNA METHYLATION level at these ISLANDS is very important for regulating GENE TRANSCRIPTION. At GENE PROMOTERS, DNA METHYLATION restricts activating TRANSCRIPTION FACTOR access and causes gene silencing. 

Every time a cell replicates, it copies it's DNA. In order to pass on a properly regulated EPIGENOME and ensure continuity of cell type function, a copy of it's global DNA METHYLATION pattern or METHYLOME must be faithfully reproduced on this new strand of DNA. This methylation maintenance function is carried out by the enzyme DNA METHYLTRANSFERASE I (DNMTI) and co-factor S-adenosyl methionine (SAM), where a methyl group is added to the 5' carbon of cytosine, forming 5-METHYLCYTOSINE. 

DNA methylation is often considered the most stable EPIGENETIC MODIFICATION, and it can be remarkably stable. Smokers, for example maintain pro-cancer DNA METHYLATION marks for years after quitting smoking, where some chemicals in cigarettes actually methylate DNA at dangerous locations and compromise proper gene regulation. On even longer-time scales, researchers have observed some rare sites of DNA methylation to be maintained through germ cell development and to be passed on generation-after-generation. This means that some EPIGENETIC marks can be inherited transgenerationally.

In other forms of long-term stability, DNA  methylation  locks in a silent EXPRESSION state at promoters,  suppresses dangerous REPETITIVE ELEMENT expression, and is required for X-CHROMOSOME INACTIVATION and proper IMPRINTING. Loss of these functions is associated with several cancer and many forms of EPIGENETIC DISEASE.

Yet, DNA methylation can be amazingly dynamic. During development and throughout CELLULAR DIFFERENTIATION, segments and CG sites throughout the genome rise and fall in DNA methylation level. De novo methyltransferases DNMT3A and DNMT3B "write" new sites of methylation  and the TET family of proteins initiate the "erasing" or DNA DEMETHYLATION process. We now understand that some human pathologies originate in EPIGENETIC dysregulation of these enzymes themselves. For example, sometimes DNA methyltransferases are "hijacked" and overexpressed by cancer cells to silence cancer repressing parts of the GENOME. As a subset of EPIGENETIC and EPIGENOME ENGINEERING, user-defined DNA methylation editing opens up a new battlefront against these complex evasive cancer mechanisms. 

Extending out from the NUCLEOSOME core, the N-terminal TAIL of HISTONES is subjected to extensive modification. Histone modifications are wide ranging and include: acetylation, methylation, phosphorylation, sumoylation, and ubiquitination. Their collective distribution constitutes the HISTONE CODE of gene regulation. 

Some HISTONE MODS are relatively straightforward. For example, HISTONES are highly positively charged and naturally associate with the negative charge of DNA. By adding negatively charged acetyl groups to HISTONE TAILS, the interaction between DNA and HISTONES is locally weakened. The consequence is DNA becomes accessible for TRANSCRIPTION FACTOR READING. 

Although many of the other HISTONE MODS are capable of altering HISTONE charges, it's simplest to consider all HISTONE MODS as a code to be bound, interpreted, and manipulated by proteins. For example, the addition of 3 methyl groups at histone 3 at lysine 4 (H3K4me3) is generally associated with gene activation. Change that to lysine 27 and the gene is silent.  

There are typically multiple, often dozens of enzymes, within a particular eukaryotic organism capable of modifying histones. Each HISTONE MOD type has a particular enzyme or enzyme complex type. For example, acetyl groups are added by HISTONE ACETYL TRANFERASES (HATs) and are removed by HISTONE DEACETYLASES (HDACS). Methyl groups by HISTONE METHYLTRANSFERASES and on and on. In the context of EPIGENETIC ENGINEERING each EPIGENETIC MODIFYING ENZYME is a potential tool for adaptation. That is, researchers can take the best that nature has to offer and incorporate these enzyme tools into custom and DNA site-specific EPIGENETIC ENZYMES.

CHROMATIN REMODELING

CHROMATIN REMODELING is epigenetic mechanism defined as the incorporation, positioning, and removal of NUCLEOSOMES, as well as the restructuring of HISTONE components (i.e. histone types )

and/or HISTONE-DNA interactions using energy (ATP).  Four main families of chromatin remodeler complexes exist based on sequence similarity and structure. These include SWI/SNF, chromodomain helicase DNA binding, ISWI, and INO80 complexes. 

Highlighting connections with other EPI-MODS, many CHROMATIN REMODELING COMPLEXES contain multiple EPIGENETIC EDITING activities. For example, the Nucleosome remodeling and Deacetylation complex (NuRD) possesses CHROMATIN REMODELING activity and HISTONE DEACETYLASE activity via components HDAC1 and HDAC2.

DNA LOOPING

DNA from GENES located millions of DNA letters away in linear distance is often found together in 3D space within cells. This is because DNA can LOOP back on itself. Yes, DNA is looped around HISTONES to aid in DNA packaging and to regulate TRANSCRIPTION, but it can also be looped in even more amazing ways. DNA LOOPING occurs most frequently in re-occurring chromosome domains called "Topological Associated Domains." These domains are flanked by CTCF binding domains and DNA itself is EXTRUDED or REELED out into loops using CONDENSIN proteins. 

DNA LOOPING even works across chromosomes, where active DNA regions are looped together in 3D space, and repressed regions together as well. Looping, importantly coordinates interactions between GENE PROMOTERS and ENHANCERS, thereby providing yet another layer of transcription regulation. 

Lastly, DNA looping is integrated among all of the other epigenetic mechanisms. Free looping, for example requires HISTONE MODIFICATION and CHROMATIN REMODELING to coordinate NUCLEOSOME ejection. 

Epigenetic state building is combinatorical and hierarchical. That is, many EPI-MODS are found exclusively together and many are never found together. Unique combinations are required for unique DNA structural or EXPRESSION outcomes. For example, an active gene would have unmethylated GENE PROMOTER DNA and associated HISTONES decorated with acetyl groups. To repress this gene, HDACs would remove the acetyl groups and this mark might be replaced with 2 methyl groups at lysine 9 of HISTONE 3 (H3K9me2). To transition to complete silencing, H3K9me2 can be interpreted to recruit DNMT to methylate the surrounding DNA. The combination of HISTONE MODS and DNA METHYLATION can be subsequently used to recruit CHROMATIN REMODELERS to pack in more NUCLEOSOMES and densely lock a gene down. At this point the gene is inaccessible for TRANSCRIPTION. In the context of EPIGENETIC ENGINEERING (up next), it's important to consider that some EPI-MOD changes, therefore, may alter other EPI-MODS or require multiple types in a sequence. Below, EPIGENETIC RNA enters this integrated picture. 

Everything about "Cellular Epi-writing" so far has been about how cells use the components of the EPIGENOME code like HISTONE modification to regulate TRANSCRIPTION of RNA. Researchers were surprised when GENES were discovered to only constitute 1% of our GENOME, but that nearly the entire thing was actually TRANSCRIBED. This extra RNA, at least some of it, functions   epigenetically.

Given the messenger role the classic mRNA plays, one could argue that any mRNA that codes for EPIGENETIC modifying capacity (i.e. codes for an epigenetic modifying enzyme such as DNMT), is itself EPIGENETIC. However, in this EPIGENETIC RNA context we'll be focusing on RNA that influences the EPIGENOME through direct association. 

Two primary classes of ncRNAs will be introduced here: long-noncoding RNAs (lncRNAs) and microRNAs (miRNAs).  As implied by their names, miRNAs (typically 21-25 bp) are smaller than lncRNAs (>200 bp). After TRANSCRIPTION, both are processed  like typical mRNAs (5′ capped, spliced, and polyadenylated), but then they can take on peculiar functions which are still being uncovered.

miRNAs are best charaterized for their ability to bind to mRNA and cause TRANSCRIPTION inhibition through the SILENCING RNA (siRNA) pathway in the cytoplasm. However, growing bodies of evidence imply miRNA to have nuclear functions as well. In particular, miRNAs have the ability to both activate target GENE PROMOTERS via assisted recruitment of RNA polymerase. Similarly, some lncRNAs can operate to control gene expression via CHROMATIN REMODELING COMPLEX interactions. LncRNAs can also be transcribed antisense from a protein-coding gene and can function in cis- to regulate local GENE EXPRESSION. Accumulating evidence

now supports a common theme in which lncRNAs create functional scaffolds to orchestrate the activity of a number CHROMATIN REMODELING COMPLEXES (see above figure).