Changing the epigenome

Can we change our inherited promoter methylation? [1]

A study asked if men with a family history of type 2 diabetes and compared promoter methylation before and after a six month exercise program that resulted in changes in the promoter methylation in muscle itself. [1].

• Fifteen men with (FH⁺) and 13 men without (FH⁻) a first-degree FH of T2D participated in the study. were included in this study
• Subjects were asessed with oral glucose.
• All were healthy but sedentary. Some were smokers.
• All FH⁺ and FH⁻ men participated in a 6-month supervised exercise intervention consisting of mainly endurance exercise: 1-h spinning class and two sessions of 1-h aerobic class per week led by a certified instructor.
• methylation was assessed in muscle biopsies. .
• Nine monozygotic twin pairs discordant for T2D were identified from the Swedish Twin Registry. They underwent clinical examinations, and muscle biopsies were taken in fasting state. .

non diabetic vs diabetic

These seem to be traditional CpG sites of metylation. cg00796424 seems to refer to he position in the HOXC11 gene. HOXC11 is a transcription factor.

entified 134 individual genes that changed in the degree of DNA methylation after exercise in all men independent of FH status (Supplementary Table 6). Of these 134 genes, 115 showed decreased and 19 genes showed increased methylation after exercise. Exercise-induced changes in methylation of four selected genes are illustrated in Fig. 2A. The expression of these four genes correlates negatively with DNA methylation (Fig. 2B–E). To functionally test if promoter DNA methylation of these genes is associated with reduced expression, we produced reporter gene constructs in which the human pro

3. Epigenetic changes in general pathways…

The top KEGG pathways of genes, which are differentially methylated in skeletal muscle after exercise. KEGG pathways of genes, which exhibit decreased (A) and increased (B) methylation in skeletal muscle of all men (n = 28) after a 6-month exercise intervention with the expected number of genes (white), the observed number of genes (black), and the total number of genes in the pathway in parentheses. The P values were
adjusted for multiple testing.

It would seem that the hypothesis is that exercise does not do a thing to change the methylation status of any set of genes.

Calcium signalling pathways if very ambiguous. Does this include the sacroplasmic reticulum and the dihydropuridine/ryanodine receptor(RYR) complex that makes our muscles contract? Note the increase in synthesis and degradation of ketone bodies. Note the increase in purine metabolism. SERCA is an ATPase that sequesters calcium back into the carcoplasmic reticulum.

What does it mean that 178genes in this pathway are methylated less? Are the promoters methylated less?

Note that the purine metabolism also appears on this list. The glycine, serine, and theronine metabolism pathway seems to be the most deviant from the expected.

This post is simply not going to dive deeper into the Nitert study. [1] Making sense of all of the genes involved in glycine, serine, etc amino acid metabolism is a little bit too much.

Of calorie restriction…

DNMT3a, that’s it, in the hippocampus of calorie restricted mice

That exercise decreased the methylation of some undisclosed genes in the KEGG calcium signalling pathway [1] is extremely thought provoking. The epigenetics of calorie restriction is perhaps more studied and maybe even easier to understand the mechanism(s). It should be noted that calorie restriction had no influence on age related decline in Dnmt3a protein levels in the mouse hippocampus. [2]

Acetylation and deacetylation of histones….[3]

This review made some good points but stopped short of elineating a mechanism by which we can change our epigenomes.

• Calorie restriction, a reduction in intake of fats and carbohydrates, decreasersthe availability of ATP for cellular functions This increases the ratio of AMP to ATP. AMP kinase (AMPK) is activated by increases in the AMP/ATP ratio. AMPK in turn phosphorylates other enzymes thereby activating them. Sirtuins, enzymes that remove acetyl groups from proteins, are targets of AMPK phosphorylatrion. Sirtuins have an addition layer of regulation: NAD⁺. NAD⁺ builds up when the mitochondia electron transport chain consumes more NADH than the TCA cycle can produce from the metabolism of fats and carbohydrates.
• AMPK plays an important role in histone lysine acetylation. Lysine has a positive charge facilitating the winding of negatively charged DNA. AMPK is able to phosphorylate the H2BS36 site and activate specific histone acetyl transferases (HATs). thereby enhancing acetylation of histones.
• In the nucleus AMPK is able to phosphorylate and inactivate histone deacetylases HDAC4 and HDAC5, thus shifting the balance to histone acetylation and release of DNA, This review gave anecdotal examples of CR increasing the metylation of select genes.

Are say muscle related genes clustered to one part of the chromosome such that Let’s look at the slow skeletal muscle/cardiac isoform of tropnin C.

“Troponin is a central regulatory protein of striated muscle contraction, and together with tropomyosin, is located on the actin filament. Troponin consists of 3 subunits: TnI, which is the inhibitor of actomyosin ATPase; TnT, which contains the binding site for tropomyosin; and TnC, the protein encoded by this gene. The binding of calcium to TnC abolishes the inhibitory action of TnI, thus allowing the interaction of actin with myosin, the hydrolysis of ATP, and the generation of tension. Mutations in this gene”

• Glycerate kinase is an enzyme involved in glycolysis and sugar metabolism. BRCA associated protein 1
• Stabillin 1 acts as a scavenger receptor for acetylated low density lipoprotein. Binds to both Gram-positive and Gram-negative bacteria and may play a role in defense against bacterial infection. In the neighborhood, TLR9 …is a key component of innate and adaptive immunity. TLRs (Toll-like receptors) control host immune response against pathogens through recognition of molecular patterns specific to microorganisms.
• The BAP1 gene product is a a ubiquitinating enzyme that plays a key role in chromatin by mediating deubiquitination of histone H2A and HCFC1

This thought experiment will need to end here! While there is a tendency for genes with related functions, genes whose proteins with completely different functions may be clustered together.

The epigenetic clock of aging…[4]

Unnikrishnan and coauthors wrote an insightful review that addresses why some CpG sites may become hyper-methylated as we age and other hypo-methylated.

Reference	Age	Tissue/cell	Genomic coordinates
Humans*
Day et al. (2013)	30–90	Blood, brain, kidney, muscle	Hypermethylated CpGs are found primarily in CG-islands while hypomethylated sites are very rare in cG-islands in all tissues. For Brain and kidney, the trend was similar for shores. in blood and muscle, the shores were enriched in hypomethylated CpGs.
Raddatz et al. (2013)	18–75	Epidermis	Hypermethylated DMRs are enriched in promoters and hypomethylated DMRs are enriched in enhancers.
Steegenga et al. (2014)	30–43 & 52–66	PBMCs	Hypermethylated DMCGs predominantly present in CC-islands while hypomentylated DMCGs found in less dense CpG regions, e.g., shelves and single CpGs.
Bysani et al. (2017)	28–64	Liver	Enrichment of DMCGs observed at transcription start sites, 5′-untranslated regions, and first exons. Under-representation of DMCGs at the gene body, 3’-UTR, and intergenic regions. Significant enrichment of DMGCs observed at GC-islands and under-represented in shelfs.

TET and cysteine rich domains…[6-8]

The 2021 Dick and Chen review on TET proteins reviews links of these isoforms to in stress-induced neuroepigenetic and behavioral adaptations. The cysteine rich domain stabilizes TET DNA interactions. [6] The Liu review approaches TET isoforms from a phylogenetic perspective. [7] Many species have been edited out of the ribbon diagram showed below.

• CXXC is the Zn²⁺ finger motif found in DNA binding proteins that TET use to bind to the CpG islands. [6.7}
• Fe(II) is part of the catalytic center that includes cysteines and use His and Asp to bind Fe, [6,7] Nickel(II) binds to this motif. [7]. Vitamin C acts on Fe(II) and directly contacts with the catalytic domain to enhance the TET catalytic activity. [7]
• 2-OG binding site, 2-oxoglutaric acid is cofactor like Fe(II)
• Zn(II) binding sites are not only in Zn-fingers but also in Cys-rich and DSBH domains bringing flexible regions from two domains together to facilitate
  overall structure formation.
• DSBH double-stranded β-helix is part of the catalytic domain that catalyzes the conversion of 5mC to 5hmC (and 5fC and 5caC) in a α-ketoglutarate (α-KG) and Fe(II)-dependent manner. The cysteine rich domain is wound about this region. [7]
• Cys-rich domains are an inseparable part of the catalytic domain formed by DSBH and Zn-finger interactions. [7]
• Proline, glutamine rich, and unknow domains were mentioned in the Liu review as being unexplored.
  
  
  
  

The oxidative stress link [8-9]

A group of Chinese scientists were interested in a particular renal protein Klotho, whose expression is less in Chronic Kidney Disease. CKD was induced in male C57BL/6 mice of 6-8 weeks old received (right )unilateral ureteral obstruction UUO.
, i.e. hpoxia. By monitoring Klotho promoter methyulation and expression we are given mechanistic insight between redox environment and gene expression, a pivot point for life on this planet.

• Six Klotho CpG sites were found to be more methylated in CKD than control animals.
• Sodium hydrogen sulfide (NaHS) attenuated incrases in alpha smooth muscle actin, fribronectin and fibrosis in the UUO model of CKD versus the sham operated.
• UUO increased Klotho promoter methylation. NaHS attenuated this increases. Klotho pomoter methylation was correlated with the protein levels. UUO almost obliterated klotho protein levels.
• While UUO increased protein levels of DNMT1 and TET1-3, the most dramatic change was almost total inhibition of TET activity. NaHS restored some of this activity. Oddly UUO had no effecto n klotho 5-hydroxy methyl cytosine. NaHS doubled klotho 5-hmC.
• Switching to a cell culture system, hypoxia increased 5-mC in the klotho promoter. TET activity was inversely correlated with the time that the cells had undergone hypoxia. TET activity was inversely correlated with the percent O₂.
• Hypoxia for 72 hours increases expression of TET1-2 but not TET3. Hypoxia increased reactive oxygen species as measured by a fluorescent probe. Hypoxia decreased Fe²⁺ in the cells. Ascorbate somewhat restored the O₂ concentration dependent decrease in TET activity.
• Mice were injected with pimonidazole, a reagent used to detect hypoxia. UUO increased renal hypoxia as well as increasing hypoxia inducible factor 1 HIF-1α
  . NaHS decreased HIF-1α protein levels. Normoxia induces the degradation of HIF-1α. The speculation is that HIF-1α controls the transcription of TET genes.
  
  

Gu and coauthors speculated that H₂S decreased ROS generated by hypoxic mitochondria. An alternative explanation is that prevented oxidation of cysteines involved in coordinating Fe and/or just maintaining the structure of he catalytic domain. In a way these two mechanisms are complementary. Zhang and coautors published a follow up study of sorts linking TET2 to the expression of the endothelial blood brain barrier tight junction protein ZO-1. [9] The results are just not as tight but perhaps equally important in the pathology of brain aging.

Changing the epigenome?

The Nitert exercise study of men with a family history of T2D suggests it is changeable. [1] The literature review of changing methylation association with aging versus caloric restriction kind of went no where. Digging into the structure of TET proteins revealed a lot of cysteines. The next question was if oxidative stress can regulate TET enzyme activity. The answer is yes. Perhaps we cannot change the methylation patterns we inherited from our parents of every single gene in our bodies seems kind of nonsense. It seems so much more likely that genes off the histones actively being transcribed are fair game for epigenetic changes due to changes in oxygen tensions in the environment. Or maybe the blood flow is improved to his muscles. Our thought experiment with the cardiac/slow skeletal muscle troponin C gene revealed some genes associated with response to bacteria infections. Perhaps a man engaged in exercise has changes in oxygen tension. Too much contraction might use up O₂. Why should the TLR9 and stabillin 1 genes be potentially influenced by a guy exercising? There is more to understand.

References

1. Nitert MD, Dayeh T, Volkov P, Elgzyri T, Hall E, Nilsson E, Yang BT, Lang S, Parikh H, Wessman Y, Weishaupt H, Attema J, Abels M, Wierup N, Almgren P, Jansson PA, Rönn T, Hansson O, Eriksson KF, Groop L, Ling C. Impact of an exercise intervention on DNA methylation in skeletal muscle from first-degree relatives of patients with type 2 diabetes. Diabetes. 2012 Dec;61(12):3322-32. PMC free article
2. Chouliaras L, van den Hove DL, Kenis G, Dela Cruz J, Lemmens MA, van Os J, Steinbusch HW, Schmitz C, Rutten BP. Caloric restriction attenuates age-related changes of DNA methyltransferase 3a in mouse hippocampus. Brain Behav Immun. 2011 May;25(4):616-23 PubMed, Sci-Hub free paper
3. Zhai J, Kongsberg WH, Pan Y, Hao C, Wang X, Sun J. Caloric restriction induced epigenetic effects on aging. Front Cell Dev Biol. 2023 Jan 13;10:1079920. PMC free article
4. Unnikrishnan A, Freeman WM, Jackson J, Wren JD, Porter H, Richardson A. The role of DNA methylation in epigenetics of aging. Pharmacol Ther. 2019 Mar;195:172-185. PMC free article
5. Zhang Q, Liu X, Gao W, Li P, Hou J, Li J, & Wong J (2014). Differential regulation of the ten-eleven translocation (TET) family of dioxygenases by O-linked beta-N-acetylglucosamine transferase (OGT). The Journal of Biological Chemistry 289(9), 5986–5996. 10.1074/jbc.M113.524140. [PMC free article]
6. Dick A, Chen A. The role of TET proteins in stress-induced neuroepigenetic and behavioural adaptations. Neurobiol Stress. 2021 Jun 11;15:100352. PMC free article
7. Dongyang Liu and others, Function determinants of TET proteins: the arrangements of sequence motifs with specific codes, Briefings in Bioinformatics, Volume 20, Issue 5, September 2019, Pages 1826–1835 free paper
8. Gu Y, Chen J, Zhang H, Shen Z, Liu H, Lv S, Yu X, Zhang D, Ding X, Zhang X. Hydrogen sulfide attenuates renal fibrosis by inducing TET-dependent DNA demethylation on Klotho promoter. FASEB J. 2020;34:11474–11487. Sci-Hub free paper
9. Wang L, Mao B, Fan K, Sun R, Zhang J, Liang H, Liu Y. ROS attenuates TET2-dependent ZO-1 epigenetic expression in cerebral vascular endothelial cells. Fluids Barriers CNS. 2022 Sep 8;19(1):73. PMC free article