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Bad News for DNA Methylation Protocols.

Ema

Senior Member
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Midwest USA
A new paper showing that DNA methylation does not *cause* changes to gene expression. This should give anyone engaged in methylation "treatments" pause about the efficacy of such interventions. Even if one can improve methylation status, this study shows that it doesn't affect gene expression the way many people have postulated in the past.

Frequent lack of repressive capacity of promoter DNA methylation identified through genome-wide epigenomic manipulation

Abstract
It is widely assumed that the addition of DNA methylation at CpG rich gene promoters silences gene transcription. However, this conclusion is largely drawn from the observation that promoter DNA methylation inversely correlates with gene expression. The effect of forced DNA methylation on endogenous promoters has yet to be comprehensively assessed.

Here, we conducted artificial methylation of thousands of promoters in human cells using an artificial zinc finger-DNMT3A fusion protein, enabling assessment of the effect of forced DNA methylation upon transcription and histone modifications, and the durability of DNA methylation after the removal of the fusion protein. We find that DNA methylation is frequently insufficient to transcriptionally repress promoters.

Furthermore, DNA methylation deposited at promoter regions associated with H3K4me3 is rapidly erased after removal of the zinc finger-DNMT3A fusion protein.

Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3.

These findings suggest that promoter DNA methylation is not generally sufficient for transcriptional inactivation, with implications for the emerging field of epigenome engineering.
 

Ema

Senior Member
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4,729
Location
Midwest USA
Alterations of DNA methylation by glutathione depletion
Author links open overlay panelKhingkanLertratanangkoonaChun JWubNiramolSavarajbMary LThomasa
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https://doi.org/10.1016/S0304-3835(97)00300-5Get rights and content

Abstract
One of the most consistent findings in cancer cells is an overall decrease of 5-methylcytosine content in DNA. The causes that lead to this alteration are not known. We have shown in a recent study that the methyl-donor, methionine (Met), can easily be depleted and that O- and S-methylation can be impaired in response to glutathione (GSH) depletion. This is because mammalian cells are capable of resynthesizing GSH after GSH is depleted, and GSH turnover occurs at the expense of Met. An extensive utilization of Met for the resynthesis of GSH causes Met depletion and impairment in methylation. In the present study we now demonstrate that GSH depletion has a significant impact on DNA methylation. An i.p. dose of a model GSH-depleting hepatotoxin, bromobenzene (BB), caused a progressive impairment in genomic DNA methylation in the Syrian hamster. The administration of a single i.p. dose of Met labeled with [14CH3]Met to BB-treated hamsters at either 1, 3, 5.5 or 9 h after BB resulted in an increase of methyl-group incorporation into liver genomic DNA at 24 h after BB. With respect to the time points chosen for Met administration, methyl-group incorporation found in the BB+Met groups were 1-, 2-, 4- and 12-fold of the controls that received only Met. We further employed an in vitro methylation assay using specific bacterial SssI CpG methylase as the catalyzing enzyme to demonstrate that BB caused a progressive increase of unmethylated CpG sites in genomic DNA. Interestingly, the time response curve of global DNA methylation in vitro showed an identical pattern to that observed in the in vivo experiment. The results provide strong evidence that GSH-depleting agents significantly impair cytosine methylation. Thus, alterations in gene expression could result from a high dose and/or prolonged exposure to GSH-depleting agents, e.g. medications, chemotherapeutic agents and environmental toxins.
 

Hip

Senior Member
Messages
17,806
@Ema the methylation protocols of @Freddd and Richvank are not about DNA methylation.

In this 2010 pdf document by Rich Van Konynenburg, he says:
What are some things that might be expected if the methylation capacity were diminished, and are they observed in CFS?

• Overexpression of many genes because of lack of gene silencing by methylation—observed (71).
• Lowered synthesis of choline and creatine—abnormal ratio of choline to creatine observed in brain (72-75).
• Lowered synthesis of carnitine—deficit observed (76).
• Lowered synthesis of coenzyme Q-10—supplementation observed to be beneficial (77).
• Lowered synthesis of myelin basic protein—slow brain processing speed observed (78).

So it does look as if Rich thought decreased methylation would cause overexpression of many genes, which the above paper indicates does not in fact happen.



There are also other reasons why theoretically the methylation protocol may benefit ME/CFS:


In this recent poll on ME/CFS treatments, the methylation protocol came out poorly, with only 4% reporting major improvements from methylation (by comparison 50% reported major improvements from the antiviral Valcyte).
 

Sushi

Moderation Resource Albuquerque
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19,934
Location
Albuquerque
So it does look as if Rich thought decreased methylation would cause overexpression of many genes, which the above paper indicates does not in fact happen.
Aside from the effect on gene expression, when I had my values checked for the nutrients important for methylation, I was out of range for 12 out of 13. After a year of supplementing I was only out of range on 5. My basic take on this has been that being in range for nutrients is better than not being in range, so I have continued with low level supplementation though I don't experience an obvious direct benefit.
 
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Hip

Senior Member
Messages
17,806
For me, I have the impression that low doses of B12 and folate help improve mood, and help keep depression at bay. Though these supplements don't seem to help with my ME/CFS symptoms. I take 10 mg of B12 methylcobalamin intranasally just once a week, plus 200 mcg of folinic acid daily.
 

Sushi

Moderation Resource Albuquerque
Messages
19,934
Location
Albuquerque
I have the impression that low doses of B12 and folate help improve mood, and help keep depression at bay. Though these supplements don't seem to help with my ME/CFS symptoms.
I inject 1 mg of hydrox B12 2 or 3 times a week. I do feel a boost the day after, though I don't know why--but I'll take the boost and not worry about why.
 

Learner1

Senior Member
Messages
6,305
Location
Pacific Northwest
In this recent poll on ME/CFS treatments, the methylation protocol came out poorly, with only 4% reporting major improvements from methylation (by comparison 50% reported major improvements from the antiviral Valcyte).
The items in your poll are not ice cream flavors, i.e., which is the most liked by the crowd? They are not equal in characteristics or impact.

As has been discussed by many researchers, there are subsets of patients. Younger, for example, with his infection and immune subsets. Naviaux, with the metabolomics study, where there was something like 30% the same, but the rest was unique from patient to patient.

I've worked with methylation protocols for 10 years, mapped out everyone in my family's SNPs and done annual nutrient testing on multiple family members, which demonstrated how methylation co factors change over time due to environmental factors. We were able to test out many of the theories we read about in real time and found that manipulating methylation can be quite powerful.

It is a LOT more than folate and B12.

B6, B2, B1, magnesium, potassium, molybdenum, serine, glycine, glutamine, cysteine, methionine, etc. all have roles, too.

Though, I learned a lot from RichvanK and Freddd, the problem many people have with trying methylation protocols is that, rather than starting with their own idiosyncrasies as a start, they take Freddd's protocol or Rich's Simplified Methylation Protocol or whatever some other patient on the Internet recommends, then start having symptoms and conclude that methylation doesn't work for them.

Methylation is important. It is essential to life. It drives many, many processes in our bodies, like neurotransmitter production, immune system function, DNA replication, and the development of cancers. Some of the cofactors are also used elsewhere. It is not a good assumption that we are all widgets and need the same amounts or ratios of methylation nutrients, or one risks getting into trouble of one kind or another.

Even with similar genes, as in my family, we've found some enormous differences in our needs over the past 10 years. Even in one single person, depending on outside environmental factors, needs can vary dramatically from year to year. There may be some underlying tendencies, but the actual needs vary a lot.

This is why only 4% of people think methylation works. They don't get tested, to find out what their individual needs are, they aren't attuned to these dynamics, and they get themselves out of balance and into trouble.

Over the past 10 years, I've had unbalanced methylation make me fatigued, depressed, anxious, headachy, sick feeling, etc. and seen how tweaking pieces of the protocol can make a dramatic difference with symptoms disappearing in as little as a couple of hours.

Methylation is a powerful tool. As a cancer survivor, I've found it can be dangerous if out of balance. It is worth understanding as well as we can - its too complex to know everything, but getting it under control can dramatically help symptoms.

It can help to reduce oxidative stress. It can help mitochondria to function better. It can get toxins out of our bodies that may be producing brain fog or impacting ATP production. It can improve our immune function, neurotransmitter function and DNA replication.

It is not a topic for some simplistic polling question, or some overly simplistic study saying its good or bad, but worth understanding and working with to find a balance that supports the body's needs, as there are many variables that can make a big difference in our symptoms.

I apologize for my rant here, but ME/CFS research has repeatedly identified methylation and its co factors and products as significant factors, and it is worth understanding that it must be individualized and cannot be some "one size fits all" solution.

Testing and customizing is the path to success. Shortcuts lead to trouble.
 

Sundancer

Senior Member
Messages
569
Location
Holland
In this recent poll on ME/CFS treatments, the methylation protocol came out poorly, with only 4% reporting major improvements from methylation (by comparison 50% reported major improvements from the antiviral Valcyte).

well, being a newbie here I still would like to answer that one ( just into the methylation-thing and having enormous steps forward, think here almost enough and restorative sleep ( after almost 3 years starting with no sleep, then followed by happy when I get 5 hours per night, while waking 2 or 3 times) I can exert more without triggering PEM, mood much better, mucous membranes getting more healthy, nails changing . This all in the first 3 months, with small doses of B12 and known shortages of minerals.

When the cause of ME is a viral infection, then of course, killing the little monsters is the best route to go. But for me I've never thought of viral infection being the crux. What I've read until now makes the probability of problems with the MTHFR gene very probably, looking at both my history and that of family members.

By now I know that most cases of ME are indeed related to viral infection, so it stands to reason that an antiviral protocol would help the largest part of patients in a poll

Even though we all have somewhat similar symptoms, the rootcause is not similar. Maybe the methylation is the rootcause in only a small proportion of patients, but for those patients it is an answer that may address their problems.

I know a guy from another forum who had all the signs of ME, he's been treating treating himself with the cutler protocol, a long, slow and tedious business. but by now he has regained much of his former self. From hardly being able to function he's back working ( not fulltime yet, but he's not finished chelating so still has hope that he'll recover more.) Years he's been working on it. That was his root.
 

Sundancer

Senior Member
Messages
569
Location
Holland
I apologize for my rant here, but ME/CFS research has repeatedly identified methylation and its co factors and products as significant factors, and it is worth understanding that it must be individualized and cannot be some "one size fits all" solution.

thanks! I did not know that and am diligently doing research right now, the individual take on it had already surfaced, i will do the test and tweak until I've found balance
 

Ema

Senior Member
Messages
4,729
Location
Midwest USA
Dr Ben Lynch, probably one of the most well-known figures in this field, tells us in this article why we shouldn't pay much attention to most individual SNPs...and why doing so is a wild goose chase that is unlikely to end well.

This new paper suggests that the field of epigenetics is also far, far ahead of itself.

I think there will always be people that have success with one treatment method or another. That isn't to say those people should change what they are doing if it is working for them, but we should also keep an eye on what scientific studies are revealing as well. People insisted that phrenology worked for a while too, and surely some people were helped by it over the years. But no one is doing it today for good reason.

I read The Gene by Dr Siddhartha Mukherjee last year. It was one of the best books I've ever read. His grasp on the current state of genetics research is impressive, to say the least. And when he writes that he wouldn't have any genetic testing at this point in time because we just don't know how to properly interpret 99% of that data, I believe him.

I also see this trend of turning people into "previvors" based on their genetics quite concerning and would rather that mindset didn't infect the chronic illness community as well. We have enough hurdles without making up extras based on faulty paradigms.
 

Hip

Senior Member
Messages
17,806
As has been discussed by many researchers, there are subsets of patients.

Where there are identifiable subsets, my poll selected them. For example, in the poll I gave instructions that people should only vote for Valcyte if they tested positive for a chronic active infection with herpesvirus, which Valcyte treats.



Methylation is important. It is essential to life. It drives many, many processes in our bodies, like neurotransmitter production, immune system function, DNA replication, and the development of cancers.

That sound a bit like the advertising spiel supplement manufacturers use to sell products. I prefer empirical evidence of efficacy rather than spin.

The methylation protocol itself is "spun" all over this forum, but few people stop to look for actual evidence of its efficacy.



This is why only 4% of people think methylation works. They don't get tested, to find out what their individual needs are, they aren't attuned to these dynamics, and they get themselves out of balance and into trouble.

In Rich Van Konynenburg's own informal study on 30 ME/CFS patients using his simplified methylation protocol, he found that 27% of them achieved major improvements from methylation after three months. Rich did not select any subsets as far as I can tell, he just selected 30 patients willing to participate in his trial.

He says that in his trial: "27% reported so much improvement that they now felt essentially well! Several who had not worked in over 5 years were able to resume full-time employment without difficulty."

So Konynenburg's study did not do any testing to find out what patient's individual needs are, yet still reported huge improvements from 27%.

Whereas the forum poll only found 4% had major improvements ("major" being defined in a particular way on the poll).

This discrepancy in results might be explained by the often temporary nature of the benefits of vitamin B12 or methylation (see my next post below).



I apologize for my rant here, but ME/CFS research has repeatedly identified methylation and its co factors and products as significant factors, and it is worth understanding that it must be individualized and cannot be some "one size fits all" solution.

I have not seen any research on PubMed to show that, only "research" from fringe characters like Ben Lynch, who promotes methylation as the cure for everything, but never seems to have any empirical evidence to back up his claims. I don't trust people who are all marketing spiel, and no evidence. It's the duty of any scientist to provide evidence.
 
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Hip

Senior Member
Messages
17,806
well, being a newbie here I still would like to answer that one ( just into the methylation-thing and having enormous steps forward, think here almost enough and restorative sleep ( after almost 3 years starting with no sleep, then followed by happy when I get 5 hours per night, while waking 2 or 3 times) I can exert more without triggering PEM, mood much better, mucous membranes getting more healthy, nails changing . This all in the first 3 months, with small doses of B12 and known shortages of minerals.

If you look at the poll, I defined a "major improvement" in a particular way.

Have the vitamin B12 supplements you are taking resulted in a "major improvement" by the definition in the poll?

Basically, a "major improvement" is defined as one where a patient moves up 1 level on the ME/CFS severity scale of very severe, severe, moderate, mild and remission. For example, if after treatment a patient moves up from severe to moderate, or moves up from mild to remission, those types of 1-level improvements are classed as "major" in my poll.



Note that the vitamin B12 treatment for ME/CFS was in use before Rich Van Konynenburg developed his methylation protocol for ME/CFS. Vitamin B12 may benefit ME/CFS by mechanisms unrelated to methylation — such the mechanisms I listed in the above post.

Some ME/CFS patients report that vitamin B12 / methylation helps, then stops working after a while. See these threads for example:

b12 doesn't work anymore, need help
Any advice? Methylation stopped working
Methylation Stopped working



So the often temporary nature of the vitamin B12 / methylation benefits could help explain why the forum poll showed poor results from methylation: the protocol may help initially, but then its effects may disappear.
 
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Learner1

Senior Member
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6,305
Location
Pacific Northwest
B12 works every day in our bodies.

Again, its not a popularity contest. We have biochemistry that works (or doesn't work) in our bodies, whether we understand it or not.

Having adequate nutrients and methylation working properly is a foundation to health. It may not cure you, but it is extremely difficult to be healthy without having these in place.

And, rather than attacking people as fringe characters, you might do well to meet them, understand what they are doing, and ask questions. Dr. Lynch and his team have compiled a great deal of research and summarized it into a very helpful body of knkeledge, which has grown and evolved over time.

As I stated, I've worked with 10 years of data in testing out how the various biochemical pathways work relating to methylation, with the guidance of experts, and have found that dramatic positive and negative changes can be made by manipulating the many variables.

Glutathione production and recycling and methylation are pieces to this puzzle, though not sell understood. Many ME/CFS studies have identified them as such., including Naviaux's metabolomics study.
 

Learner1

Senior Member
Messages
6,305
Location
Pacific Northwest
Treatment of ME/CFS is fringe science.

Most big medical discoveries started out as fringe concepts, too. I find it wise to quietly test the theories and research one reads about and interpolate between some good work that may not be a complete body of knowledge. And, as I'm not a doctor, I have found that following mavericks who are more educated than I with ideas that differ from the status quo to be extremely valuable in solving the health problems that my family and I have had.