DNA methylation

In eukaryote and prokaryote world, as a genetic material, DNA molecules stably transferred from generation to generation (a cell to its daughter cells, the parents to their offspring) in terms of the sequence context........ in most of the time.

Genome changes. A special type of modification in the base is "methylation".  DNA methylation draws scientists' eyeballs because it has been proved to play roles in GENE EXPRESSION.  The genome can be changed the activity without change the context.  And some of these marks changed in parents can be transmitted to offsprings (epigenetic effect) 

More than 20 types of epigenetic and DNA-damage modifications have been identified, most scientists have only been able to study one type, cytosine methylation.

===============================================================

Review

Nucleic Acid Modifications in Regulation of Gene Expression

by Kai Chen, Boxuan Simen Zhao, and Chuan He

Cell Chemical Biology, Volume 23, Issue 1, 21 January 2016, Pages 74–85
doi:10.1016/j.chembiol.2015.11.007

========************************************=========

Abstract

Nucleic acids carry a wide range of different chemical modifications. In contrast to previous views that these modifications are static and only play fine-tuning functions, recent research advances paint a much more dynamic picture. Nucleic acids carry diverse modifications and employ these chemical marks to exert essential or critical influences in a variety of cellular processes in eukaryotic organisms. This review covers several nucleic acid modifications that play important regulatory roles in biological systems, especially in regulation of gene expression: 5-methylcytosine (5mC) and its oxidative derivatives, and N6-methyladenine (6mA) in DNA; N6-methyladenosine (m6A), pseudouridine (Ψ), and 5-methylcytidine (m5C) in mRNA and long non-coding RNA. Modifications in other non-coding RNAs, such as tRNA, miRNA, and snRNA, are also briefly summarized. We provide brief historical perspective of the field, and highlight recent progress in identifying diverse nucleic acid modifications and exploring their functions in different organisms. Overall, we believe that work in this field will yield additional layers of both chemical and biological complexity as we continue to uncover functional consequences of known nucleic acid modifications and discover new ones.

========************************************=========

5-Methylcytosine Methylation in Higher Eukaryotes

The existence of cytosine methylation (5mC) in genomic DNA was first reported by Wyatt in 1951 (Wyatt, 1951).


5mC writer:

• establish 5mC (de novo): Dmnt3a, Dmnt3b, Dmnt3L,
• maintain C methylation from hemimethylated DNA at CpG site: Dmnt1

5mC effector/ reader, proteins that recognize 5mC and carry out subsequent actions:

• bind to methyl-CpG: MeCP1,MeCP2, MBD1, MBD2, MBD4.

Scheme of the Reversible Cytosine Methylation in DNA and Binding Proteins that Are Known To or Proposed To Bind Modified Cytosine Derivatives, Derived from Liyanage et al., 2014  (Figure 1. from the reference)


5mC eraser: not a simple action by one or few enzyme:

• TET proteins are methylcytosine dioxygenase that utilize dioxygen to oxidize 5mC==> 5hmC==>5fC==>5caC
• Both 5fC and 5caC can be recognized and excised by human thymine DNA glycosylase (TDG), followed by base excision repair (BER) to replace the modified cytosine with a normal cytosine.
• or, diluted through cell division (5hmC, 5fC, and 5caC ==> to the unmethylated stage) 

DNA methylation modulates the chromatin structure and affects cognate gene expression by maintaining various expression patterns across cell types (Cheng and Blumenthal, 2010 and De Carvalho et al., 2010).

in promoter   ===> suppression;
in gene body ===> possitive correlation with gene expression 


Two models for "Methylation induced gene repression (promoter):

1. "indirect” model: DNA methylation may recruit its reader proteins that act as transcription repressors, preventing transcriptional factors from accessing the promoter region.
2. "direct” model: DNA methylation may be a disruptor to interfere with the binding of certain transcription factors and thus prevent the activation of corresponding genes

 ## of course the world is not so simple........... the transcription regulation roles of DNA methylation typically synergize with various histone marks as the methyltransferases, demethylases, and readers of DNA methylation interact with various histone marks or histone modification enzymes.





DNA 5mC methylation was considered to be dynamic and reversible.

N6-methyladenine (6mA) found in Genome

In bacteria, 6mA serves as an important marker participating in DNA repair, replication, and cell defense (restriction–modification (R-M) systems,in which 6mA, 5mC and 4mC can be recognized by corresponding restriction endonucleases as a label to prevent the host genome from restriction digestion and further enable the degradation of unmethylated foreign DNA (Murray, 2002)) .

** 4mC modifications can be differentiated at base resolution using a revised TAB-seq protocol (4mC-Tet-assisted-bisulfite- sequencing, Yu et al., 2015, Nucleic Acids Res., 43, e148).

In addition to prokaryotes, several eukaryotes have relatively abundant 6mA in genomic DNA. In 2015, three groups reported the presence of 6mA in three different eukaryotes (algae chlamydomonas, C elegans, Drosophila ) independently.

C.elegans genome was thought lacking DNA methylation. The discovery of 6mA, its demethylase NMAD-1, and potential methyltransferase DAMT-1 in C elegans changes this view ===> will 6mA serves as DNA methylation mark (instead of 5mC)?





Figure 2.

N6-Methylation on Adenine in Genomic DNA

(A) A brief overview of biological function of methyl groups in bacterial genomic DNA.

(B) High-throughput mapping of N6-methyladenine (6mA) in Chlamydomonas reinhardtii revealed a unique distribution pattern in the genome with complete depletion at transcription start sites (TSS) and high enrichment at the linker region between nucleosomes.

(C) In Caenorhabditis elegans, 6mA is installed by DAMT-1 and reversibly removed by NMAD-1. The “crosstalk” between 6mA and histone modification, particularly the histone H3 methylation, indicates critical roles that 6mA may play in gene expression regulation.

(D) 6mA in Drosophila melanogaster could be converted back to A by Tet homolog DMAD. Intriguingly, the 6mA level is correlated with the expression level of transposon, supporting the regulatory significance of 6mA in eukaryotes.



Evidence of 6mA function in other situation:

• In zebrafish, the knockdown of METTL3 (mRNA m6A writer) leads to smaller heads, eyes, and brain ventricles, and curved notochords (Ping et al., 2014)
• m6A is the most prevalent internal modification in mRNAs and long non-coding RNAs (lncRNAs) in higher eukaryotes (Wei et al., 1975).
• in the mammalian transcriptome, approximately three m6A marks exist per mRNA molecule and occur within a consensus motif of G(m6A)C (70%) or A(m6A)C (30%), but the methylation percentage at each site varies substantially (all from studies before 1990)

for mRNA,

• m6A “writer:
  a complex of methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms' tumor 1-associating protein (WTAP)
• m6A “eraser: (AlkB family proteins) the fat mass and obesity-associated protein (FTO) and ALKBH5
• m6A reader: YTHDF1 and YTHDF2

N6-Methyladenosine (m6A) in mRNA and Its Biological SignificanceThe reversible methylation and demethylation process occurs in the nucleus, catalyzed by methyltransferase complex and demethylases, respectively. The 6mA modification has profound effects on mRNA fate: it switches mRNA to active translation mode, and also accelerates its decay rate. Figure 3 from the reference

pseudouridine (Ψ) =====> epitranscriptomics ?

https://en.wikipedia.org/wiki/Epitranscriptomics

((here is a plenty info about discoverings and function of modified bases in RNA (mRNA, tRNA, rRNA, miRNA........))) ---beyond my interestings now.

Outlooks

• We now appreciate that DNA methylation, as a bona fide epigenetic marker, is not only inheritable and dynamic, but also involved in diverse regulatory processes.
• The recent discoveries of 6mA as a functional DNA mark in eukaryotic genomic DNA raise the possibility that 6mA plays regulatory roles complementary to 5mC.
• With a better understanding of 6mA methyltransferase and 6mA demethylase and discovery of potential reader proteins, DNA methylation looks to be a ubiquitous epigenetic marker in almost all kingdoms of life.
• internal m6A methylation in mRNA was shown to be reversible

====================================================================

DNA N6-methyladenine: a new epigenetic mark in eukaryotes?

Luo, GZ et al., 2015. Nature Reviews Molecular Cell Biology
16, 705–710  doi:10.1038/nrm4076

• Most eukaryotic 6mA research has focused on unicellular protists; 6mA accounts for ~0.4–0.8% of the total adenines in these genomes

• Recently, 6mA was detected by multiple approaches in the genomic DNA of two metazoans, C. elegans (0.01–0.4%) and D. melanogaster (0.001–0.07%)
• In prokaryotes, most of the 6mA sites are located within palindromic sequences

• 6mA is the most abundant internal modification in mRNAs and is widely conserved in eukaryotes. 

 

Figure 1 | Methods to detect N6 -methyladenine (6mA) in genomic DNA.
Left: an antibody against 6mA can sensitively recognize 6mA and enrich 6mA-containing DNA for subsequent next-generation sequencing (NGS); 6mA-sensitive restriction enzymes can specifically recognize either methylated or unmethylated adenines in their recognition-sequence motifs, and this can be captured by sequencing to determine the exact locations of 6mA.
Right: liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) can differentiate methylated adenine from unmodified adenine and quantify the 6mA/A ratio by normalization to a standard curve; single-molecule real-time (SMRT) sequencing can detect modified nucleotides by measuring the rate of DNA base incorporation (dashed arrow) during sequencing.