DNA methylation machinery and pathways

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The cellular responses to hypoxia have been well characterised by prevalent alterations in the expression of a vast number of genes. Many of these genes are controlled by hypoxia-inducible factors (HIFs), the master regulator for oxygen sensing and transcription factor for hypoxic-inducible genes’ expression. HIFs are heterodimers consisting of an oxygen-sensitive α-subunit and a constitutively expressed β –subunit (Bruick, 2003; Semenza, 2003). Under hypoxic conditions, the HIF- α-subunit and β –subunit can form an active HIF. The HIF can thereby bind to the hypoxia-response element (HRE) at the promoter of a plethora of target genes, leading to their transcriptional activation (Appelhoff et al., 2004; Palmer and Clegg, 2014)
Previous studies have found that both DNMTs and TETs can be regulated by HIF (Lai et al., 2018; Zhang et al., 2019). Study did by Watson (2013) showed that hypoxia induced pro-fibrotic changes in human cardiac fibroblast cells together with a concomitant global DNA hypomethylation and increased expression of DNMT1 and DNMT3B. The authors found that DNMT1 and DNMT3B gene promoter regions contain a consensus sequence for a HIF-1α-binding HRE, and proved the hypoxia-induced upregulation of DNMT enzymes is mediated by HIF-1α (Watson et al., 2013). Similarly, hypoxia-induced overexpression of TETs has been observed both in mice and in zebrafish (Wang et al., 2017). Lin and collaborators reported that hypoxia upregulated the expression of all three members of TET enzymes as well as the 5-hmC level in HepG2 cells. Knockdown of HIF-1α attenuated the hypoxia-induced expression of TET enzymes (Lin et al., 2017). These results indicated that hypoxia could control the expression of TET enzymes and regulate DNA methylation in a HIF-dependent manner.
Besides, one study in mice found that hypoxia induced genomic DNA hypomethylation and reduced the steady-state of SAM content both and . They also observed a surprisingly upregulated expression of methionine adenosyltransferase 2A (MAT2A), a key enzyme that is responsible for converting methionine to SAM in the one-carbon cycle. This overexpression is mediated through HIF-1α activation pathway, although it remains hard to explain how this upregulation in MAT2A leads to a decrease in SAM (Liu et al., 2011).
Interestingly, it has been demonstrated that the consensus HIF binding site sequence 5’-RCGTG-3’ contains a CpG dinucleotide, suggesting that HIF dependent gene regulation could be intrinsically affected by DNA methylation under hypoxia (Nanduri et al., 2017; Wenger et al., 2005). Moreover, evidence has also been found that TET may in return regulate HIF (Kao et al., 2016). For instance, one study demonstrated that Tet1 could stabilise HIF-α and enhance HIF-α transcription activity (Wang et al., 2017).
One of the most important strategies for cellular adaptation to hypoxic condition is the shift from mitochondrial oxidative phosphorylation to an anaerobic glycolytic pathway. A critical step in this shift is HIF-mediated activation of pyruvate dehydrogenase kinase-1 (PDK-1). This enzyme inactivates pyruvate dehydrogenase (PDH) which is the mitochondrial enzyme responsible for converting pyruvate to acetyl-CoA. In addition, lactate dehydrogenase A (LDHA), the key enzyme that converts pyruvate to lactate, is also activated by HIF. Like this, a reduction of delivery acetyl-CoA into the TCA cycle may thereby reduce the production of nicotinamide adenine dinucleotide (NADH) and inhibit electron transport chain (Palmer and Clegg, 2014) (Fig. 1-11). It can be thus hypothesised that hypoxic conditions may result in changes in levels of TCA metabolites, which are known to affect the activity of 2-OGDDs.
Figure 1-11 HIF-dependent regulation on glycolysis and TCA cycle. Hypoxic condition trigers a shift from mitochondria oxidative phosphorylation to anaerobic glycolysis pathway. Green arrows represent activating effects by HIF, red ones represent inhibiting effects. LDHA: lactate dehydrogenase A; GLUT: glucose transporter; PDH: pyruvate dehydrogenase; PDK1: PDK-1; SLC16A3: solute carrier family 16 member 3. Figure modified from Lin et al.(Lin et al., 2014).
The most important 2-OGDDs involved in hypoxic responses including PHD1-3 (prolyl hydroxylases of HIF α subunits) as well as TET enzymes. As 2-OGDDs, the activity of TETs inherently depends on the level of co-substrates: oxygen, Fe2+, 2-oxoglutarate as co-substrates. Thus hypoxia inducing the shortage of oxygen substrate can result in reducing TET catalytic activity which further contributes to DNA hypermethylation. This reduction in TET activity could occur independently of hypoxia-associated alterations in TET expression (Thienpont et al., 2016). On the other hand, the hypoxia-induced alteration in TCA metabolites like 2-oxoglutarate, fumarate and succinate may also affect the DNA methylation process through the interference of TETs activities (Lamadema et al., 2018).
The DNA methylation status and DNA methylation-related enzymes can affect genome integrity. The possible mechanisms involved in DNA methylation-mediated alterations in genome stability include: i) regulating transcriptional activity; ii) maintaining the stability of repeat elements; ii) preventing the mutations and chromosomal rearrangement; iii) participating in DNA repair mechanisms that prevent and/or correct genetic errors (Zhou and Robertson, 2016).
DNA methylation at promoters and gene bodies regulates transcriptional activity in different ways. The predominant DNA methylation occurs at CpG sites in mammals. 80% of these CpG dinucleotides are methylated. Instead, CpG islands, which contain longer stretch of CpG rich region and typically located at gene promoters, often remain unmethylated (Bird and Wolffe, 1999). Hypermethylation of CpG islands in promoters generally linked to gene silencing, whereas hypomethylation permits active transcription (De Smet et al., 1999). About 70% mammalian genes possess CpG islands in promoter, including housekeeping genes, developmental genes, tumour suppressors and cell-cycle genes. Aberrant hypermethylation at promoters of these genes is often observed in cancer or other diseases (Das and Singal, 2004). In contrast to promoter methylation, gene body methylation is often associated with active transcription (Jones, 1999; Yang et al., 2014b). Besides, it has been observed that exons tend to be more methylated than introns, and that the transcriptional start sites (TSS) proximal region and transcription termination sites are devoid of methylation (Lister et al., 2009). These findings indicate a role of DNA methylation in transcriptional elongation and termination, and perhaps alternative splicing (Jeltsch, 2010).
The DNA methylation-mediated gene transcription can be achieved through i) directly prevention of transcription factor binding, or ii) indirectly through attracting of methyl-CpG binding domain proteins that recruit ‘repressor complexes’ thus leading to histone modifications and alterations in chromatin structure (Bird and Wolffe, 1999; Jones et al., 1998). The proposed mechanisms of DNA methylation mediated gene repression are illustrated in Fig. 1-12.
Figure 1-12 Mechanisms of DNA-methylation-mediated repression. (a)DNA methylation in the cognate DNA-binding sequences of some transcription factors (TF) can result ininhibition of DNA binding. By blocking activators from binding targets sites, DNA methylation directly inhibits transcriptional activation.(b)Methyl-CpG-binding proteins(MBPs) directly recognize methylated DNA and recruit co-repressor molecules to silence transcription and to modify surrounding chromatin.(c)In addition to their DNAmethyltransferase activities, DNMT enzymes are also physically linked to histone deacetylase (HDAC) and histone methyltransferase (HMT) activities. In this case, the addition of methyl groups to DNA is coupled to transcriptional repression and chromatin modification.(d)DNA methylation within the body of genes can also have adampening effect on transcriptional elongation. MBPs might be involved in inhibiting elongation, either directly or by their effects on the surrounding chromatin structure (Klose and Bird, 2006).
DNA methylation can also occur in the context of non-CpG sites, the gene-regulatory functions of this form of DNA methylation are less clear. However, increasing evidence was obtained in recent studies which showed that methylation at non-CpG sites also has effects on gene expression regulation (Patil et al., 2014). These findings highlight the need to uncover the originally negligent functions of non-CpG methylation.
The vast majority of vertebrate genomes are occupied by non-coding regions (in human, 98.5%), which are important contributors to genome/chromosome stability. Repeat elements comprise nearly half of these non-coding sequences. Interspersed repeats [ transposable elements (TEs)] and tandem repeats ( microsatellites, minisatellites, and satellites) are the two main groups of repeat elements (Ahmed and Liang, 2012). Evidence showed that both groups can be affected by DNA methylation, thus leading to the alterations in genome stability and disease onset.
TEs are discrete mobile DNA segments that capable of moving and integrating randomly within the genome, which is associated with genome instability. DNA methylation of TEs can preserve their stability trough silencing gene transcription. Besides, numerous TEs are key components of heterochromatin enriched at regions flanking centromeres and telomeres (Schueler and Sullivan, 2006). DNA methylation levels on these TEs are functionally related to the formation and stability of constitutive heterochromatin (Zhou and Robertson, 2016).
Microsatellite loci are repetitive sequences typically consisting of one to four nucleotide repeats, and they are particularly susceptible to mutations through length changes. DNA methylation appears to protect against microsatellite repeat instability (MSI) through both the DNA methylation induced transcriptional repression of genes involved in MSI, and the DNMT1 protein involved mismatch repair (MMR) (Putiri and Robertson, 2011).
DNA hypomethylation is generally associated with an increased frequency of gene rearrangements and chromosomal translocations as a consequence of increased homologous recombination (HR) (Zhou and Robertson, 2016). HR often occurs between DNA regions sharing high sequence identity. It occurs regularly during meiosis and also occasionally in somatic cells. Transcription increases the frequency of HR by exposing single stranded DNA and facilitates the homologous sequences approaching each other, whereas DNA methylation mediated transcriptional silencing inhibits HR (Domínguez-Bendala and McWhir, 2004). It is generally believed that HR is the basis for many mutational events. Thus, significantly increases mutation rates observed in extensive DNA hypomethylation condition could be potentially linked to the increasing rate of mitotic chromosomal recombination (Chen et al., 1998).

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Table of contents :

I. Fast development of aquaculture industry
II. Fish nutrition, aquafeeds and the bottleneck in aquaculture
III. Dietary carbohydrates: promising substitutes for FM and FO as well as challenging risks for arnivorous fish
IV. Epigenetics, a potential mechanism in phenotypic adaptation
V. General objectives of the present thesis project
1.1 Overview of epigenetics
1.1.1 DNA methylation
1.1.2 Histone modifications
1.1.3 Nucleosome remodelling
1.1.4 Non-coding RNA
1.2 DNA methylation
1.2.1 DNA methylation machinery and pathways
1.2.2 DNA methylation dynamics during gametogenesis and embryogenesis
1.2.3 DNA methylation regulation by nutrients, oxidative stress and hypoxia
1.2.4 Global DNA hyper and hypomethylation and the related consequences
1.2.5 Non-CpG methylation and its functional highlights
1.2.6 Potential roles of 5-hmC, 5-fC and 5-caC
1.3 Epigenetics and metabolic programming
1.3.1 Concept of metabolic programming
1.3.2 Programmed phenotypes and suspected epigenetic mechanisms: selected examples from
invertebrates up to mammals
1.3.3 Metabolic programming for fish nutrition in aquaculture
1.4 Rainbow trout as a model to study epigenetics and programming:-understanding the low dietary
carbohydrates utilisation and glucose-intolerant phenotype
1.4.1 Embryonic development of rainbow trout
1.4.2 Dietary carbohydrate utilisation and glucose metabolic pathways in trout
1.4.3 Hypotheses to explain the poor dietary carbohydrate use in rainbow trout
2.1 Question I: How many DNA methylation-related genes are preserved in trout genome, and what are their evolutionary origins?
2.2 Question II: What are the nutritional factor(s) triggering the hepatic global DNA hypomethylation in trout after feeding a high-carbohydrates/low protein diet? And what are the detail mechanisms involved in this demethylation process?
2.3 Question III: Can DNA hypomethylation phenotype and glucose metabolism be modified through metabolic programming strategy?
3.1 Flowchart of thesis design
3.2 Part 1 Evolutionary history of DNA methylation related genes in chordates: new insights from
multiple whole genome duplications -Publication 1
3.3 Part 2: Factors triggering hepatic global DNA hypomethylation phenotype in trout fed a high
carbohydrate-low protein diet and the detail mechanisms – Publication 2
3.4 Part 3: Metabolic programming effects of hypoxia during early development and/or high
carbohydrate dietary stimulus at first feeding on epigenetic landscapes and glucose metabolism in trout juveniles
3.4.1 Acute hypoxia & high-carbohydrates diets as stimuli Short-term effects: Exposure to an acute hypoxic stimulus during early life affects the expression of glucose metabolism-related genes at first-feeding in trout – Publication 3 Long-term effects: Long-term programming effect of embryonic hypoxia exposure and high-carbohydrate diet at first feeding on glucose metabolism in juvenile rainbow trout – Publication
3.4.2 Chronic hypoxia & high-carbohydrate diets as stimuli: Programming of the glucose metabolism
in rainbow trout juveniles after chronic hypoxia at hatching stage combined with a high dietary
carbohydrate: Protein ratios intake at first-feeding – Publication 5
4.1 Part I Further investigation about the roles of DNA methylation /demethylation related genes in trout
4.1.1 Conservation of the DNA methylation-related enzymes in trout (even for the paralogs) but neo-/sub-functionalisation
4.1.2 Evolution of the expression territories of dnmt, tet and tdg genes
4.2 Part II Hepatic global DNA hypomethylation after feeding a high carbohydrate/low protein diet in trout: causes and beyond
4.2.1 New insights on dietary factors that induced global non-CpG hypomethylation in the liver of
4.2.2 Limitations and obstacles in the experimental design of the present study
4.3 Part III Programming of glucose metabolism in trout: achievement and challenges
4.3.1 How to ‘program’ fish: selection of the type of stimulus and the ‘’windows of metabolic plasticity’
4.3.2 Metabolic programming of trout with hypoxia: early hypoxic history induced alteration in glucose metabolism in juvenile trout- highlights of the effects on glucose transporter related genes (the gluts)
4.3.3 Mechanisms of programming: potential epigenetic mechanisms linked to the long-term
programming effects
5.1 What new knowledge did we obtain to explain the poor carbohydrate utilisation in trout?
5.2 Is it possible to improve the dietary carbohydrate utilisation of trout with metabolic programming
5.3 What are the potential impacts of the presnet thesis on aquaculture?


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