Regulation of Neuroepigenetic Mechanisms

Neuroepigenetic Mechanisms
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Neuroepigenetic mechanisms alter gene expression in a highly regulated manner, allowing for neuronal plasticity.

Neuroepigenetics is a newly-emerging field in neurobiology that is altering the general understanding of the traditional views of heritable epigenetic mechanisms. From a classical viewpoint, epigenetic mechanisms are involved in the heritable modifications of chromatin and histone structures which ultimately result in the variable expression of genes. However, following the recent discovery of these mechanisms in the highly differentiated, post-mitotic neurons of the brain, it became necessary to reconsider the heritability of these epigenetic mechanisms in neurons. While these “plastic” epigenetic mechanisms are important for gene expression in the central nervous system (CNS), it is of equal importance to consider the strategies adopted by cells to regulate these mechanisms. With this in mind, a lot of current research examines the two major epigenetic mechanisms, DNA methylation and histone acetylation, as well as the processes through which these mechanisms are regulated.[1] In addition, the numerous neuroepigenetic mechanisms described have also been implicated in several neurological and neurodegenerative diseases such as Rett’s Syndrome.[2]

Regulation of DNA methylation

In general, DNA methylation is the epigenetic process by which cytosine bases of CpG dinucleotides are methylated at the C5 region by DNA methyltransferases (DNMT) to generate 5-methyl cytosine (5mc). The methylation of these CpG islands typically leads to the inhibition of transcriptional activity in these DNA regions.[3] Thus, the regulation of DNMTs plays a significant role in the expression of genes, even in neurons. While this process of methylation has been well-studied over the years, the inverse demethylation of 5 methyl-cytosine bases is still controversial as an epigenetic mechanism. Yet, a great deal of research has been put into this demethylation process, and studies have shown evidence of its relation to the regulation of DNA methylation-related decrease in transcriptional activity.

DNA methyltransferases (DNMT)

The regulation of DNMTs and its relation to gene expression in the nervous system have been implicated in numerous studies over the past several years. One such study, conducted by Chestnut and his colleagues on NSC34 cell cultures as well as mice models, showed that the mechanisms through which DNMTs are regulated are also involved in the neurodegeneration and apoptosis processes of motor neurons. In particular, the evidence showed that the upregulation of DNMTs resulted in the onset of neuronal apoptotic mechanisms following DNA methylation, whereas the inhibition of DNMT activity through the application of RG108 and procainamide blocked this methylation and motor neuron apoptosis and degeneration. Thus, this study elucidates the importance of the regulation of DNMTs and DNA methylation activity in the motor neurons as a form of defense against neurodegeneration.[4]

Tet Protein Family

Several studies have shown that the major regulatory mechanisms with regards to DNA demethylation involve dioxygenase enzymes from the ten-eleven translocation (Tet) protein family. These studies have implicated the Tet1, Tet2 and Tet3 proteins as being responsible for the regulation of DNA methylation through the conversion of 5mC into 5-hydrooxymethylcytosine (5hmC).[5,6] This is then followed by the deamination of 5hmC to acquire 5-hydroxyuracil (5hmU), leaving a 5hmU-G mismatched region that is recognized and eliminated through the activity of many glycosylases. This damaged region is then repaired through base excision repair mechanisms, causing the reversion to the original unmodified C-G region.[5-8] Before the glycosylase-excision, it has also been shown that 5hmC can further oxidise to generate 5-formylcytosine (5fC) and 5 carboxylcytosine (5caC).[5,6]

Effect of Tet1 and Tet2 proteins on DNA methylation
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Expression of wild-type Tet proteins in U2OS cells causes the generation of 5hmC, while this does not occur in cells expressing the Fe(II)-binding mutants of Tet1 or Tet2. 2 days following transfection, the cells were co-stained with Flag and 5hmC antibodies; the nuclei of these cells were also counterstained by DAPI.[6]

In addition to the general description of this pathway, these studies have shown that the Tet1 gene is dependent on neuronal activity and is also important for the regulation of gene expression in adult brains. For example, in a study involving the overexpression of the Tet1 protein in the hippocampus, the results displayed evidence of the increased expression of several genes associated with memory through the reduction of DNA methylation levels.[5] Another study also displayed the importance of Tet1 in the hydroxylation of 5mC in adult mouse brains in vivo in causing the increased expression of genes in the dentate gyrus.[8]

Regulation of Histone Modifications

The post-translational modifications of the histones are another major epigenetic mechanism that influences gene expression. Histones are proteins in the nuclei of cells that serve as structural supports for chromatin, forming DNA-histone complexes known as nucleosomes. The modifications made to the histones alter the ability of the transcriptional machinery to access the DNA, thus regulating gene expression in an activity-dependent manner.[3] There are numerous processes through which histones can be post-translationally affected; these processes include the acetylation, methylation and phosphorylation of specific residues on the histones such as lysine, arginine and serine.[3,9] There is evidence for a significant amount of cross-regulation between histone modifications within as well as between histones; for example, the acetylation of a lysine residue prevents it from being concurrently methylated.[10] Of all these different mechanisms, the regulation of acetylation processes has been well documented and will be the focus of this section.

Histone Acetyl Transferases

Histone acetyl transferases (HATs) are proteins that are responsible for the acetylation of lysine residues on the N-terminal tails of histones. This transfer of the acetyl group generally results in the increased transcription of the surrounding DNA following the relaxation of the chromatin due to the weakening of the electrostatic bonds between the normally positive histone tails and the negatively charged DNA. There are two major HATs that have been extensively researched and described in scientific literature: the cyclic adenomonophosphate response element-binding (CREB) binding protein (CBP) and its closely homologous relative, p300.[3,11] The regulation of the HAT activity of CBP can occur through various signalling pathways that regulate its state of phosphorylation, such as cyclic adenosine monophosphate (cAMP) and nuclear calcium pathways. The regulation of these enzymes is also affected by the activity of arginine methyltransferase I and the presence of cell metabolites such as nicotinamide adenine dinucleotide + (NAD+) and acetate.[11,12] Studies have also shown that HATs, such as Tip60, are capable of auto-regulation through mechanisms involving auto-acetylation.11 In addition to this, there is evidence implicating the dysregulation and mutation of the CBP gene in patients with Rubinstein-Taybi Syndrome.[13]

Histone Deacetylase (HDACs)

While the acetylation of the lysine residues of histone tails results in the increase of gene transcription and expression, these acetyl groups can also be removed by histone deacetlyases (HDACs) in a reversal process to induce gene silencing. HDACs induce the enhancement of the DNA-histone interaction, which prevents the transcriptional machinery from accessing the DNA and its regulatory regions. There are four classes of HDACs (I-IV) that differ in their functionality and DNA sequence; of these, the functions of HDAC I and II classes have been extensively researched and shown to be involved in neural progenitor differentiation at various stages of CNS development.[14,15] This regulation of transcription has proven to be essential for certain processes in the brain; studies have shown that the activity of HDAC1 of the HDAC I class is involved in the differentiation of neural progenitors into glial cells, while the increased activity of HDAC2 of the same HDAC class causes the differentiation of these progenitor cells into neurons.[15]

HDACs can be regulated through numerous mechanisms, such as post-translational modifications and protein-protein interactions, as well as through the activity of proteins on HDAC genes. The major regulator of HDAC comes in the form of HDAC inhibitors (HDACi), which are capable of accumulating acetylated histones and generally increasing gene expression. Several studies have also shown that HDACs can be negatively regulated by several such HDACi such as trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA).[16]

Epigenetic Regulation of Histones
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Histone acetylation of lysine residues on the histone tails are regulated by acetylation and deacetylation mechanisms catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. HDACs can be inhibited by HDAC inhibitors which results in the net increase in histone acetylation levels and a relaxed chromatin conformation that is capable of activating transcription. On the other hand, chromatin with a tight conformation is transcriptionally inactive.[14]

Neuroepigenetic Mechanisms of Neurological Diseases

The Impact of Rett's Syndrome
A touching and inspirational video of a case of Rett's Syndrome. As neuroscience students, what could we do to help?

The staple neurological diseases with regards to epigenetic mechanisms are Rett’s Syndrome and Rubinstein-Taybi Syndrome. Numerous studies have characterized the various neuroepigenetic aspects of the diseases and these diseases will form the basis of this section. However, neuroepigenetic mechanisms have also been implicated in numerous other neurological diseases, such as Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS).[2]

Rett’s Syndrome

Rett’s Syndrome is a monogenic neurodevelopmental disorder that is clinically presented with involves defects in motor skills, cognitive abilities and communicative prowess. Rett’s Syndrome is caused by mutations in the X-linked MeCP2 gene, which encodes for the methyl-CpG-binding protein (MeCP2).[17] While a great deal has yet to be elucidated with regards to this function of the MeCP2 protein and its mutant forms, it is known that MeCP2 associates with transcriptional repressor complexes such as HDAC to suppress gene expression in neurons.[18] However, recent studies have also implicated the MeCP2 proteins in the activation of genes as well; one such study using murine models showed the association between MeCP2 and CREB1 in the activation of a target gene in the hypothalamus.[19]

Rubinstein-Taybi Syndrome

Rubinstein-Taybi Syndrome (RTS) is a genetic disorder, stemming from the mutation of one allele of the CBP gene, which leads to cognitive complications and dysfunction. In current scientific literature, there is evidence that CBP activity is necessary for the regular differentiation of cortical precursor cells into neurons, astrocytes, etc. One of the regulatory mechanisms of CBP function is its phosphorylation at the hands of atypical protein kinase C ζ, which allows CBP to promote histone acetylation at the promoter regions of neural genes responsible for precursor differentiation. However, cbp haploinsufficiency and genetic knockdown, which was analyzed using genetically variable mice models, interrupts this differentiation function of CBP, thus, providing a possible model for the onset of the cognitive deficits shown in RTS patients.[13]

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