Methods of detecting additional epigenetic modifications

This 2018 German review subject was detecting DNA modifications that are derivatives of the much-studied 5-methlycytosine:

“The discovery of modified nucleobases arising from 5-methylcytosine (5mC) through consecutive oxidation to give 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) has stimulated intense research efforts regarding the biological functions of these epigenetic marks.

Recent findings revealed that 5hmC and 5fC are stable DNA modifications in the genome, thus suggesting that oxidized 5mC derivatives may function as epigenetic marks in their own right, exhibiting regulatory purposes and participating in DNA replication, transcription, repair, and recombination.

The bisulfite-sequencing method (BS-Seq) has widely been used as the gold standard in determining the methylation status with single-base resolution in genomic DNA. The BS-Seq method, however, has some severe drawbacks, such as:

  • Harsh reaction conditions which might cause undesired DNA damage,
  • Requirements for relatively large amounts of input DNA,
  • Dependence on PCR, and resulting short sequence reads, as well as
  • Reduced sequence complexity due to deamination of all nonmethylated cytosines and
  • Accompanied challenges for primer hybridization.

Most importantly, however, with BS-Seq it is not possible to discriminate between 5mC and 5hmC..Furthermore, since 5fC and 5aC undergo deamination similar to unmodified cytosine, they are indistinguishable from C under bisulfite conditions.” “Chemoselective labeling and site-specific mapping of 5-formylcytosine as a cellular nucleic acid modification” (click the PDF link)


A flying human tethered to a monkey

Ponder this drone photo of “a flying human tethered to a monkey” ground drawing made over 1,000 years ago as reported by National Geographic and excerpted by the Daily Star:
Flying human tethered to a monkey

Aren’t the geoglyph and its description pretty good expressions of our evolved condition? Especially since it’s the interpretation of people who lived more a millennium ago?

With so many information sources freely available now, one couldn’t successfully argue that they understood the world better than we do, though. The price paid for figuring things out today is our “flying human” time and efforts, without which we’re as ignorant as our “monkey.”

A few aspects of the current comprehension of the differences between our two pictured primates are in Genetic imprinting, sleep, and parent-offspring conflict:

“I remain skeptical of a tendency to ascribe most modern woes to incongruence between our evolved nature and western cultural practices. We did not evolve to be happy or healthy but to leave genetic descendants, and an undue emphasis on mismatch risks conflating health and fitness [genetic rather than physical fitness].”

Our “flying human” can make happiness and health choices that our “monkey” can’t:

Our genetic adaptations often try to fool us into doing things that enhance fitness at costs to our happiness.

Our genes do not care about us and we should have no compunction about fooling them to deliver benefits without serving their ends.

Contraception, to take one obvious example, allows those who choose childlessness to enjoy the pleasures of sexual activity without the fitness-enhancing risk of conception.”

Other aspects of each of our two pictured primates’ differences are illuminated in a reference to A study of DNA methylation and age:

“Aging is not and cannot be programmed. Instead, aging is a continuation of developmental growth, driven by genetic pathways.

Genetic programs determine developmental growth and the onset of reproduction. When these programs are completed, they are not switched off.

Aging has no purpose (neither for individuals nor for group), no intention. Nature does not select for quasi-programs. It selects for robust developmental growth.”

The epigenetic clock theory of aging cited the same author, and modified his point to say:

“The proposed epigenetic clock theory of ageing views biological ageing as an unintended consequence of both developmental programmes and maintenance programmes.”

Our “flying human” can make choices that aren’t available to our “monkey” concerning the structure, direction, and duration of our one precious life:

“What are you doing to reverse epigenetic processes and realize what you want? Do you have ideas and/or behaviors that interfere with taking constructive actions to change your phenotype?”

The Not-Invented-Here syndrome

I have high expectations of natural science researchers. I assume that their studies will improve over time, and develop methods and experiments that produce reliable evidence to inform us of human conditions.

My confidence is often unrealistic. Scientists are people, after all, and have the same foibles as the rest of us.

I anticipate that researchers will keep abreast of others’ work around the world. If other groups in their research areas are developing better methods and exploring hypotheses that discover better applications for humans, why not adopt them in the interest of advancing science?

That’s not what happened with this 2018 UK rodent study. The rat model some of the coauthors have built their reputations on depends on disturbing rat pregnancies by administering glucocorticoids. But both the rat model and a guinea pig model in Do you have your family’s detailed medical histories? demonstrated that physicians who disturb their pregnant human patients in this way may be acting irresponsibly toward their patients’ fetuses and their future generations.

This study didn’t find mechanisms that explained transgenerational epigenetic birth weight effects through the F2 generation:

“Although the phenotype is transmitted to a second generation, we are unable to detect specific changes in DNA methylation, common histone modifications or small RNA [including microRNA] profiles in sperm.

The inheritance mechanism for the paternally derived glucocorticoid-reprogrammed phenotype may not be linked with the specific germline DNA, sRNA and chromatin modifications that we have profiled here.”

The linked guinea pig model was developed specifically to inform physicians of the consequences through the F3 generation of disturbing human pregnancies with glucocorticoids:

“Antenatal exposure to multiple courses of sGC [synthetic glucocorticoid] has been associated with hyperactivity, impaired attention, and neurodevelopmental impairment in young children and animals. It is imperative that the long-term effects of antenatal exposure to multiple courses of sGC continue to be investigated since the use of a ‘rescue’ (i.e. a second) course of sGC has recently re-introduced the practice of multiple course administration.”

If a study’s purpose is to investigate potential mechanisms of epigenetic inheritance, why not adopt a model that better characterizes common human conditions, regardless of which research group initially developed it?

The prenatal stress model used in The lifelong impact of maternal postpartum behavior is one model that’s more representative of human experiences. Those researchers pointed out in Prenatal stress produces offspring who as adults have cognitive, emotional, and memory deficiencies that:

“Corticosterone-treated mice and rats exposed to chronic stress are models that do not recapitulate the early programming of stress-related disorders, which likely originates in the perinatal period.”

Animal models that chemically redirect fetal development also “do not recapitulate the early programming of stress-related disorders.”

Other than research that’s done to warn against disrupted development, how can animal studies like the current study help humans when their models don’t replicate common human conditions? This failure to use more relevant models has follow-on effects such as human intergenerational and transgenerational epigenetic inheritance being denigrated due to insufficient evidence.

Of course there’s insufficient human evidence! Researchers developed and sponsors funded animal study designs that ensured there wouldn’t be wide applicability to humans. Few derivative human studies were developed and funded as a result. “Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations”

Little evidence for mitochondrial DNA methylation

This 2018 Japanese rodent study used three different techniques to detect mitochondrial DNA methylation:

“Whilst 5-methylcytosine (5mC) is a major epigenetic mark in the nuclear DNA in mammals, whether or not mitochondrial DNA (mtDNA) receives 5mC modification remains controversial.

We used bisulfite sequencing, McrBC digestion analyses and liquid chromatography mass spectrometry, which are distinctly differing methods for detecting 5mC..we analysed mtDNAs from mouse ESCs [embryonic stem cells] and from mouse liver and brain tissues.

Taken together, we propose that 5mC is not present at any specific region(s) of mtDNA and that levels of the methylated cytosine are fairly low, provided the modification occurs. It is thus unlikely that 5mC plays a universal role in mtDNA gene expression or mitochondrial metabolism.”

Bisulfite sequencing infers the presence of CpG (CG above) and non-CpG (CH above) methylation through unconverted residues:

“Synthetic and native mtDNA gave similar patterns, suggesting that the resistance of cytosines to bisulfite conversion is not due to methylation.”

It seems that epigenetic changes to mitochondrial DNA occur primarily through histone modifications. Lysine acetylation is gnarly and dynamic is one paper that detailed aspects of this functionality in mitochondria. “Accurate estimation of 5-methylcytosine in mammalian mitochondrial DNA”

The epigenetic clock theory of aging

My 400th blog post curates a 2018 US/UK paper by two of the coauthors of Using an epigenetic clock to distinguish cellular aging from senescence. The authors reviewed the current state of epigenetic clock research, and proposed a new theory of aging:

“The proposed epigenetic clock theory of ageing views biological ageing as an unintended consequence of both developmental programmes and maintenance programmes, the molecular footprints of which give rise to DNAm [DNA methylation] age estimators.

It is best to interpret epigenetic age estimates as a higher-order property of a large number of CpGs much in the same way that the temperature of a gas is a higher-order property that reflects the average kinetic energy of the underlying molecules. This interpretation does not imply that DNAm age simply measures entropy across the entire genome.

To date, the most effective in vitro intervention against epigenetic ageing is achieved through expression of Yamanaka factors, which convert somatic cells into pluripotent stem cells, thereby completely resetting the epigenetic clock. In vivo, haematopoietic stem cell therapy resets the epigenetic age of blood of the recipient to that of the donor.

Future epidemiological studies should consider other sources of DNA (for example, buccal cells), because more powerful estimates of organismal age can be obtained by evaluating multiple tissues..other types of epigenetic modifications such as adenine methylation or histone modifications may lend themselves for developing epigenetic age estimators.”

I’ve previously curated four other papers which were referenced in this review:

The challenge is: do you want your quality of life to be under or over this curve?

What are you doing to reverse epigenetic processes and realize what you want? Do you have ideas and/or behaviors that interfere with taking constructive actions to change your phenotype?

If you aren’t doing anything, are you honest with yourself about the personal roots of beliefs in fate/feelings of helplessness? Do beliefs in technological or divine interventions provide justifications for inactions? “DNA methylation-based biomarkers and the epigenetic clock theory of ageing” (not freely available)

The lifelong impact of maternal postpartum behavior

This 2018 French/Italian/Swiss rodent study was an extension of the work done by the group of researchers who performed Prenatal stress produces offspring who as adults have cognitive, emotional, and memory deficiencies and Treating prenatal stress-related disorders with an oxytocin receptor agonist:

“Reduction of maternal behavior [nursing behavior, grooming, licking, carrying pups] was predictive of behavioral disturbances in PRS [prenatally restraint stressed] rats as well as of the impairment of the oxytocin and its receptor gene expression.

Postpartum carbetocin [an oxytocin receptor agonist unavailable in the US] corrected the reduction of maternal behavior induced by gestational stress as well as the impaired oxytocinergic system in the PRS progeny, which was associated with reduced risk-taking behavior.

Moreover, postpartum carbetocin had an anti-stress effect on HPA [hypothalamic-pituitary-adrenal] axis activity in the adult PRS progeny and increased hippocampal mGlu5 [type 5 metabotropic glutamate] receptor expression in aging.

Early postpartum carbetocin administration to the dam enhances maternal behavior and prevents all the pathological outcomes of PRS throughout the entire lifespan of the progeny..proves that the defect in maternal care induced by gestational stress programs the development of the offspring.

This chart from Figure 4 summarized the behavioral performance of aged adult male progeny in relation to the experimental variables of:

  1. Stress administered to the mothers three times daily every day during the second half of pregnancy up until delivery; and
  2. The effects on the mothers’ behavior of daily carbetocin administration during postpartum days 1 through 7.

The symbols denote which of these relationships had statistically significant effects:

  • “* p [Pearson’s correlation coefficient] < 0.05 PRS-Saline vs. CONT-Saline;
  • # p < 0.05 PRS-Carbetocin vs. the PRS-Saline group.”

There are many interesting aspects to this study. Ask the corresponding coauthor Dr. Sara Morley-Fletcher at for a copy.

One place the paper referenced the researchers’ previous studies was in this context:

“Postpartum carbetocin administration reversed the same molecular and behavioral parameters in the hippocampus, as does adult chronic carbetocin treatment, i.e. it led to a correction of the HPA axis negative feedback mechanisms, stress and anti-stress gene expression, and synaptic glutamate release. The fact that postpartum carbetocin administration [to the stressed mothers in this study] had the same effect [on the PRS infants in this study] as adult carbetocin treatment [to the PRS offspring in the previous study] indicates a short-term effect of carbetocin when administered in adulthood and a reprogramming (long-term) effect lasting until an advanced age when administered in early development.”

This group’s research seems to be constrained to treatments of F0 and F1 generations. What intergenerational and transgenerational effects would they possibly find by extending research efforts to F2 and F3 generations?

As the study may apply to humans:

The study demonstrated that stresses during the second half of pregnancy had lifelong impacts on both the mothers’ and offsprings’ biology and behavior. Studies and reviews that attribute similar human biological and behavioral conditions to unknown causes, or shuffle them into the black box of individual differences, should be recognized as either disingenuous or insufficient etiological investigations.

The study showed that prevention of gestational stress was a viable strategy. The control group progeny’s biology and behavior wasn’t affected by carbetocin administration to their mothers because neither they nor their mothers had experience-dependent epigenetic deficiencies.

The study demonstrated a biological and behavioral cure for the PRS offspring by changing their stressed mothers’ behaviors during a critical period of their development. The above excerpt characterized improving the mothers’ behaviors as a long-term cure for the PRS descendants, as opposed to the short-term cure of administering carbetocin to the PRS children when they were adults.

What long-term therapies may be effective for humans who had their developmental trajectories altered by their mothers’ stresses during their gestation, or who didn’t get the parental care they needed when they needed it? “Reduced maternal behavior caused by gestational stress is predictive of life span changes in risk-taking behavior and gene expression due to altering of the stress/anti-stress balance” (not freely available)

Prenatal stress produces offspring who as adults have cognitive, emotional, and memory deficiencies

This 2018 French/Italian/Swiss rodent study used the prenatally restraint stressed (PRS) model to create problems that could be resolved by various chemicals:

“S 47445 is a positive modulator of glutamate AMPA-type receptors, possessing neurotrophic and enhancing synaptic plasticity effects as well as pro-cognitive and anti-stress properties.

Most of studies examining the antidepressant effects of new molecules are carried out using behavioral tests performed in unstressed animals.

Corticosterone-treated mice and rats exposed to chronic stress are models that do not recapitulate the early programming of stress-related disorders, which likely originates in the perinatal period. The PRS rat model is characterized by a prolonged corticosterone response to stress and by abnormal behavior.

All the behavioral alterations induced by PRS..were corrected by chronic S 47445 administration at both doses.”

The paper included a section comparing S 47445 to ketamine:

“Ketamine, however, causes severe cognitive impairment and psychotomimetic [mimics the symptoms of psychosis, reference not freely available] effects that are direct consequences of the prolonged inhibition of NMDA receptors in cortical and hippocampal interneurons, and seriously limit the chronic administration of the drug in the clinical setting. [reference not freely available]

S 47445 by inducing a direct activation of AMPARs displayed an antidepressant activity without the adverse effect of ketamine. Indeed, contrary to ketamine, S 47445 presented no psychotomimetic effects and induced no occurrence of spontaneous epileptic seizures. [reference freely available] Moreover, S 47445 also presented pro-cognitive properties.”

Compare the above with this April 2018 Chicago Tribune story that had opinions with no linked references:

“..ketamine, an anesthetic used to sedate both people and animals before surgery. It’s also a notorious street drug, abused by clubgoers seeking a trancelike, hallucinatory high. But in recent years, numerous studies have found that ketamine can be an effective and speedy treatment for people with depression.”

Which coverage better informed us?

Treating prenatal stress-related disorders with an oxytocin receptor agonist was performed by several of this paper’s coauthors. One of this paper’s references to it was:

“We have already reported that depolarization-evoked glutamate release in the ventral hippocampus is negatively correlated with risk-taking behavior of PRS rats, and that such correlation can be corrected by chronic treatment with monoaminergic/melatoninergic antidepressants or oxytocin receptor agonist. Thus, an impairment of glutamatergic transmission in the ventral hippocampus lies at the core of the pathological phenotype of PRS rats.”

Looking at the above graphic of the experimental design, I’m not sure why the term perinatal (occurring during or pertaining to the phase surrounding the time of birth) was used in the paper’s title and content to describe the stress period. The pregnant females were stressed three times every day during the second half of pregnancy up until delivery, so the prenatal (previous to birth) term was more applicable.

So, how does this study help humans?

One takeaway is to avoid stressing pregnant mothers-to-be if her children will be expected to become adults without cognitive, emotional, and behavioral problems.

The study demonstrated one way prenatal events cause lifelong effects. The PRS model provides another example of why it’s useless to ask adult humans to self-report the causes of epigenetic problems in their lives when these originated before birth, during infancy, or in early childhood well before humans develop the cognitive capability to recognize such situations. It’s incomprehensible that this unreliable paradigm is still given significant weight in stress studies, especially when the experimental designs:

“ not recapitulate the early programming of stress-related disorders, which likely originates in the perinatal period.”

Also, the relevant difference between humans and PRS rats is that we can ourselves individually change our responses to experiential causes of ongoing adverse effects. Standard methodologies can only apply external treatments such as those mentioned above. “The reduction in glutamate release is predictive of cognitive and emotional alterations that are corrected by the positive modulator of AMPA receptors S 47445 in perinatal stressed rats” (not freely available) Thanks to coauthors Stefania Maccari and Dr. Jerome Mairesse for providing a copy.