Introduction
The journey of DNA discovery spans several significant milestones over the past century and a half. Although DNA's initial discovery dates back 145 years, it was the groundbreaking works of Avery, MacLeod, McCarty, Watson, and Crick around 70 years ago that truly revolutionized our understanding of this molecule of life. Since then, research on DNA has burgeoned, culminating in the complete sequencing of the human genome in the "Human Genome Project" by 2003. However, DNA's script—the genotype—is not always translated into visible traits, or phenotypes, mainly due to the complex regulatory mechanisms governing gene expression. One critical facet of this regulation is epigenetics, a field that explores inheritable chemical changes affecting genes without altering the DNA sequence itself. This article delves into the pivotal role of DNA methylation, a key epigenetic mechanism, in gene regulation and human diseases.
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DNA Methylation
Five-Methylcytosine: "The Fifth Base"
DNA methylation involves adding a methyl group to the fifth carbon of cytosine, primarily at CpG dinucleotides (Cytosine linked to Guanine by a phosphate). This process is mediated by DNA methyltransferases (DNMTs) and uses S-adenosyl methionine (SAM) as the methyl group donor. Maintenance DNMT1 and de novo methyltransferases DNMT3A and DNMT3B play major roles in preserving and establishing these epigenetic marks, respectively. These enzymes ensure that established methylation patterns are inherited during DNA replication, providing a stable epigenetic marker for gene regulation. DNMT1 works primarily in differentiated cells, whereas DNMT3A and DNMT3B are crucial during embryonic development.
Fig. 1 Generation of DNA methyl modifications (Liyanage V. R. B., et al. 2014).
Five-Hydroxymethylcytosine: "The Sixth Base"
Beyond 5-methylcytosine (5mC), other methylation marks like 5-hydroxymethylcytosine (5hmC) exist. These marks are dynamically regulated by TET enzymes, which oxidize 5mC to 5hmC. 5hmC plays critical roles in the central nervous system and embryonic development and has been linked to active transcriptional states. The active demethylation of DNA involves BER pathways, where oxidized forms such as 5hmC, 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) act as intermediates that are eventually replaced by unmodified cytosine through a sequential enzymatic process. TET enzymes, in conjunction with necessary cofactors, ensure this dynamic regulation, highlighting the complexity and importance of DNA methylation beyond simple gene silencing.
Role of DNA Methylation in Biological Processes
Gene Expression Regulation
DNA methylation is traditionally associated with gene repression, especially when present at promoter regions. Methylation of CpG islands within promoters typically prevents transcription factor binding, thereby hindering gene expression. Moreover, methylation can recruit methyl-binding proteins (MBPs) which form repressive chromatin complexes. For example, MeCP2 binds to methylated DNA and recruits histone deacetylases (HDACs) to establish a more compact chromatin structure, inhibiting transcription.
Interestingly, recent insights reveal a more nuanced role for DNA methylation and its variants. For instance, 5hmC is predominantly found in active chromatin regions, suggesting it may facilitate gene expression rather than repress it. This highlights a context-dependent role of DNA methylation in gene regulation.
RNA Splicing and Genome Organization
In addition to its role in transcription, DNA methylation influences RNA splicing and genome organization. Intragenic methylation, particularly within exons, can impact alternative splicing by affecting spliceosome recruitment and exon recognition. Proteins like MeCP2 and CTCF are central to this process. MeCP2, for instance, modulates splicing by recruiting HDACs to methylated exons, influencing exon inclusion.
CTCF, a well-known insulator protein, also plays a crucial role in methylation-dependent splicing and genome organization. Hypomethylation of CTCF binding sites allows CTCF to pause RNA polymerase II, thereby aiding exon inclusion in genes like CD44. Beyond splicing, CTCF mediates chromatin loops and long-range genomic interactions, contributing to the overall three-dimensional structure of the genome.
Genomic Imprinting and X-Chromosome Inactivation
DNA methylation is essential for genomic imprinting, a process where genes are expressed in a parent-of-origin-specific manner. Methylation of imprint control regions (ICRs) and differentially methylated regions (DMRs) silences one allele, allowing for monoallelic expression. Proteins like CTCF and the maternal effect gene ZFP57, which recruits DNMT1 to specific alleles, play critical roles in this process.
Another area where DNA methylation is vital is X-chromosome inactivation (XCI), a process ensuring dosage compensation between male (XY) and female (XX) mammals. During XCI, one X chromosome in each female cell is randomly silenced through extensive methylation, forming a condensed structure known as the Barr body. XIST, a noncoding RNA, coats the inactive X chromosome and recruits PRC2 and DNMTs to establish and maintain the silenced state.
DNA Methylation and Human Diseases
Alterations in DNA methylation patterns have profound implications for human health. Abnormal DNA methylation is linked to a variety of diseases, including cancer, neurodevelopmental disorders, and imprinting-related syndromes.
Cancer
Aberrant DNA methylation is a hallmark of cancer. Hypomethylation can lead to genomic instability and activation of oncogenes, while hypermethylation of tumor suppressor genes' promoters silences their expression, contributing to tumorigenesis. For example, hypermethylation of the MLH1 gene promoter, a mismatch repair gene, is commonly observed in colorectal cancer, leading to microsatellite instability and cancer progression.
Neurodevelopmental Disorders
Epigenetic modulation is crucial for brain development and function. MeCP2 mutations are strongly associated with Rett syndrome, a neurodevelopmental disorder characterized by severe cognitive and motor deficits. The dual role of MeCP2 in binding 5mC and 5hmC and regulating gene expression underscores the importance of precise methylation control in neural processes. Moreover, disruptions in TET enzyme function and subsequent alterations in 5hmC levels are also linked to neurodevelopmental disorders, indicating the significance of dynamic DNA methylation in brain health.
Imprinting Disorders
DNA methylation defects underlie various imprinting disorders such as Prader-Willi syndrome and Angelman syndrome, both characterized by intellectual disability and developmental anomalies. These disorders stem from abnormal methylation in imprinted regions, disrupting parent-specific gene expression patterns. For instance, loss of maternal-specific methylation at the 15q11-13 region leads to Prader-Willi syndrome, while loss of paternal-specific methylation in the same region causes Angelman syndrome.
Conclusion
DNA methylation, through the addition of methyl groups to cytosine residues by DNMTs, plays a pivotal role in regulating gene expression, alternative splicing, and genome organization. The interplay between DNA methylation and histone modifications adds an additional layer of complexity to epigenetic regulation. Moreover, the discovery of 5hmC and its role in active demethylation has expanded our understanding of the dynamic nature of DNA methylation. As research continues to uncover the intricate mechanisms of DNA methylation, its significance in development, disease, and therapeutic applications becomes increasingly evident.
References
- Liyanage V. R. B., et al. DNA modifications: function and applications in normal and disease states. Biology. 2014, 3(4): 670-723.
- Dahm R. Friedrich Miescher and the discovery of DNA. Developmental Biology. 2005, 278(2): 274-288.
- Dahl C., et al. Advances in DNA methylation: 5-hydroxymethylcytosine revisited. Clinica Chimica Acta. 2011, 412(11-12): 831-836.
- Kohli R. M., Zhang Y. TET enzymes, TDG and the dynamics of DNA demethylation. Nature. 2013, 502(7472): 472-479.
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