Folate And Methylation - (Mar/31/2005 )
Dear Urba,
it is a pleasure to have you here discussing these points and venting ideas out in the open.
I think this is the essence of a PhD and sometimes I feel up coming PhD students are not getting enough of this, at least from our lab!! More discussions like this should be done for sure.
The papers you have cited are very interesting, it is "black box" science using animal models, you antagonise the system with a nutrient defiency and look for a readout afterwards, what happens exactly to lead to the result can be unclear. There have been some inroads into the issue of folate and one particular paper I have read with great interest is the follwoing:
Mol Cell Biol. 2003 Aug;23(15):5293-300.
Transposable elements: targets for early nutritional effects on epigenetic gene
regulation.
Waterland RA, Jirtle RL.
Department of Radiation Oncology, Duke University Medical Center, Durham, NC
27710, USA.
Early nutrition affects adult metabolism in humans and other mammals,
potentially via persistent alterations in DNA methylation. With viable yellow
agouti (A(vy)) mice, which harbor a transposable element in the agouti gene, we
tested the hypothesis that the metastable methylation status of specific
transposable element insertion sites renders them epigenetically labile to early
methyl donor nutrition. Our results show that dietary methyl supplementation of
a/a dams with extra folic acid, vitamin B(12), choline, and betaine alter the
phenotype of their A(vy)/a offspring via increased CpG methylation at the A(vy)
locus and that the epigenetic metastability which confers this lability is due
to the A(vy) transposable element. These findings suggest that dietary
supplementation, long presumed to be purely beneficial, may have unintended
deleterious influences on the establishment of epigenetic gene regulation in
humans.
What is also observed, is a correlation with an increased incidence of developmental neural tube defects in the developing embryo in folate deficient mothers.
I personally enjoy the benchwork, but good benchwork can not be conducted without good theory behind it. I am glad to have this converstaion with you and would like to continue to do so in the future!
Nick
Dear Nick,
The Information You wrote is interesting too.
I was lucky to find something more about chicken and egg question - You formulated Apr 3, 2005:
Transcriptional Gene Silencing Promotes DNA Hypermethylation through a Sequential Change in Chromatin Modifications in Cancer Cells
Clare Stirzaker, Jenny Z. Song, Ben Davidson and Susan J. Clark
Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown, New South Wales, and Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia
Cancer Res. 2004 Jun 1;64(11):3871-7.
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DNA Methylation Precedes H3-K9 Histone Methylation.
To determine whether there was a difference in the H3 histone methylation state of the various GSTP1 constructs, we performed ChIP analysis using a polyclonal antibody generated to dimethylated H3-K9 on LNCaP cells and LNCaP cells transfected with different GSTP1 constructs. Fig. 4B summarizes the extent of H3-K9 methylation associated with the various GSTP1 constructs relative to the endogenous GSTP1 promoter from LNCaP cells. In the LNCaP cells, the endogenous GSTP1 promoter histones are methylated, i.e., the promoter region binds methylated histone H3-K9 antibodies, and this correlates with the promoter being extensively methylated and the gene silent. Conversely, the shuttle and HpaII-seeded shuttle, which are unmethylated and actively transcribed, are not associated with H3-K9 histone methylation. The Sp1 deletion, which is unmethylated but is transcriptionally silent, is also not associated with methylated H3-K9 histones. Interestingly, the Sp1 deletion HpaII-seeded construct was also not significantly associated with methylated H3-K9 histones, although the promoter region is hypermethylated. Increasing the number of doublings (25d to 36d) increased the level of DNA methylation (Fig. 3F) but did not increase the level of histone methylation (Fig. 4B) . Therefore, we conclude that DNA methylation of the GSTP1 promoter precedes histone methylation[/b].
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is the last the general rule???
About the main question
"I would like to ask in particular is can folate affect histone methylation as well?" - Mar 31, 2005
Methylation of DNA, and Histones uses the same source of methyl - SAM. The system that provides methyl is complex enough. It consists of:
[b]INPUT - Ser - supposed to be the main source of one carbon units ( dietary plus "branched" from glycolysis)
Gly, Met, Choline(liver cell...), His, Trp (degradation of these two gives 1-carbon units into folate system too)
The SYSTEM: part of the metabolism of Ser,Gly, Choline, His, Trp plus FOLATE metabolism and METHIONINE cycle
(this "part of the metabolism..." consists of nice company of enzymes and atractive vitamine B12 -methylcobalamine , cyanocobalamine....- bearing interesting stories about it's behavior....TOO)
OUTPUT: CH3-CpG in DNA, methyl on HISTONES......and lots of the oteher SAM acceptors.
The most interesting events- The Methyl trap in folate system, Ser, Met,His metabolism problems, B12 behavior, methyl defficient diets and consequencies of it - SAM concentration decrement - to 35%...., Methotrexate....
Theoretical conclusions: if something wrong with folate system this sometimes leads to "not enough of substrate SAM in neighbouring Methionine cycle, totally hypomethylated DNA and some hypermethylated genes promoters" and WHAT ABOUT Histone methylation? If Histone methylases are controled by methylation and expressed after hypomethylation of correponding gene coding this methylase - thats o.k - fits to the experimental observations about some methylation of histones (silencing suppressors) in tumorogenesis....(I don't know is it possible and true).
The same is expected about DNA phosphorylation...The leakage of acethylation seams to be not so horrifying - acethylation only amplifies demethylation effect in some cases. It is one of the possible SCENARIES.
This is simmilar to the one when using methyl defficient diet - theoretically, zero input of Choline, Met...SAM down to 35%...switched on Histone methylase, "phosphorylation machine"...silencing some tumour supressors too...
It's possible to derive using the drawings plus some logic, even probabilistic, simmilar scenaries to carcinogenes for example TPA,DMBA...But lot of facts is not obtained yet. How carcinogenes affect methylases? Mutations or deletings? How they change The SYSTEM behavior which depends on accompying systems too...? The Great PUZZLE !!!
What about the experiments? Which input signals must be and could be controled? How to control folate system responsible for purine pyrimidine bases synthesys too? Which histone points could be investigated? Predicted ten yaers ago and observed stochastics will appear again. What about the dynamics?
Some of experiments in this direction are published (about histones - very small information, much more about DNA methylation). So this question is actual and without doubt must be rised again and again, because of ...methyl defficient diets results too...but better to study this not only in respect of folate subsystem, but using more wide target - one carbon units system. The complex systems need the complex analysis. If such aproach is under Your discussion Dear Nick too, I'll try in future to describe some interesting facts and conclusions about 1-carbon unit metabolism till they reach DNA, because before planing the experiments real BRAIN STORM is needed. It's easy to loose years on it, and how to reach the solution with great but only some effort - the problem too.
Well, I'm running to caffe to play Sunday chess and maybe Majong later the same time hoping to solve something about OCU on the board too..
Good lucK.
K.Urba from Vilnius covered by snow...
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is the last the general rule???
Dear Urba,
The last is not the general rule, in fact it refutes the general rule that histone methylation precedes DNA methylation. It was an in-vitro test system that was used and may not reflect in-vivo situation.
I must thank you for reminding me to think outside the square. The folate cycle is only one part of the one carbon cycle and I should consider other factors as well, Serine metabolism and the like.
There is lots of brainstorming going on here in the lab, and designing experiments to answer the questions (yet to be determined) is the challenge!
have a good one Urba.
Nick
Dear Nick,
Thanks for usefull answer about Histones. Nobody can know everything, only the GOD.
I hope, the rough one carbon unit metabolism description I presented will be usefull for some students looking at discussions and will help for others better understand me. I've seen lots of articles without such a scetch and it made me sad and angry. I hope the students in future when became scientists consider and describe complexity together with possible reasons more clear. But even skilfull Japanese from Kyoto -great job done - www.genome.jp presented ONE CARBON POOL BY FOLATE but not full OCU metabolism.
I like Your style of moderation - precise and quick and rising problems, so I would like to share some remarks, ideas and facts fished myself from the ocean of information because I've calculated their's weigh and roads underwater!
First - Met cycle directly controls The Polyamine synthesis too giving SAM to it, perhaps Ornithine DC is the best discussed enzyme. Nucleosomas consists of histones and some polyamine giving positive charge...So, really the name of this topic maybe could be changed into DNA METHYLATION, HISTONE (-polyamine), CHROMATIN STUDY because the first three ones are too close on the pathways all depending on SAM and too close and important in reality. Plus, DNA hypomethylation, theoretically, could be the consequence of increased ODC activity leading to increased synthesys of polyamine giving much "more bricks" for tumour nucleosoma building....When I "measure" only DNA methylation and look at Histones I obtain different results depending on the reason - is ODC increased activity switched on.
Second, when i was reading about Serine metabolism I was lucky to pay attention to the little message:
K.Snell, G,Weber. Enzymic disbalance in serine metabolism in rat tumours. // Abstracts of lectures, symposia and free comunications. 14 th international cancer congress. Budapest, 1986, August 21-27, Akademiai kyado, vol.2, p.523. The authors published interesting results - in lots of tumours (hepatomas, carcinomas, limphosarcomas) they detected very low activity of Ser-dehidratase and Ser-aminotransferase, the normal or even increased activity of Ser synthesys enzymes and increased activity of Ser turn into Gly! They concluded that this promotes more intensive purine-pyrimidine bases synthesis in cancer cell. Right idea, but not fully spreaded on the other important cell growth regulating processes. I've tried to obtain more conclusions from any Ser catabolism repression. Conclusion I've found - SAM level falls down and DNA hypomethylation must occure too (after 9 weaks it is ireversable using SAM for rat livers). Later I called to Mr. Snell - he was at Surrey U.K., but I can't convince him about importance of ...his result and at that moment he had no support to continue research in this direction. I' would like to see You judging this guestion and maybe prooving experimentaly. And what about Ser catabolism repression, folate system intensive job, and Histone methylation in cancer cell? Do this form sharp 5metFolate trap? These very complex and difficult questions, I hope and wish Your lab will solve . OCU biological meaning problem seems to be of extremly importance.
Sincerely Yours Kestutis Urba
hi Urba,
I am sorry for not being so quick off the mark this time round, I was attending an epigenetics conference in Canberra, and was really excited with all the work presented there, I was amazed with the calibre of speakers, the who's who of epigenetics, many results and data they presented included unpublished stuff and this is the way to move sicence forward, open and sharing of ideas.
But you have a very interesting point with regard to serine metabolism. I am not too sure if this has been done, but it would certianly make sense to knockdown folate AND serine levels and look for a methyultaion readout. One of the problems with science today is funding.....if it is not sexy no one will fund it. I believe folic acid in diet is sexy as it is widely known to be an essential vitamin that our bodies are unable to synthesise and the link between folate and neural tube defects......serine metabolism on the other hand is not as sexy.
We are trying to get a grant together to hopefully look at these questions in the mouse. I would love to be able to formulate a question, jump into some experiments and answer the questions. I have to say there are a lot of questions arisen from my recent conference.
N
Hi Nick,
I was sure - if Nick is offline for a longer time he's at the interesting conference somwhere and this was one more evidence that my theories are close to the reality. Thanks for information about Canberra discussions - I've found abstracts in www.pi.csiro.au/markoliphant-conf/ and the important statements, for example - "...Loss of acetylated Lys16 and trimethylated Lys20 residues of histone H4 are a common hallmark of human cancer...". E.Ballestar, Madrid.
I'm trying to predict "in theory" before in vitro and in vivo experiments folate influence on methylation and more wide: changes in OCU system influence to methylation. One important step when investigating folate system -in theory- was published in
Nijhout HF, Reed MC, Budu P, Ulrich CM.
Free Full Text A mathematical model of the folate cycle: new insights into folate homeostasis.
J Biol Chem. 2004 Dec 31;279(53):55008-16. Epub 2004 Oct 20.
PMID: 15496403 [PubMed - indexed for MEDLINE]
But this mathematical model does'nt consists folate surrounding and, it seems to me, that biochemical cell subsystems need much more complicated math if someone seeks precision, because of complicated feedbacks, stochastics and all the time there rises the question: are the equations enough adeqate to reality? For presented model, maybe, not yet, but the first step, of course, was useful.
I prefere the other way - so called scenaries of changes on metabolic pathways. Not pretending to unreal precise given by math of differential equations, but working using some probabilistic or even fuzzy logic. This means -someone must drive on the pathways, knowing the main probable moments of it by heart -following some flows and trucks crashes- like Serine catabolism disorders in cancer cell - Snell's effect.
Simmilar approach of scenaries was used by B.Vogelstein, when he presented his famous model of cancer in eighties - some consequance of events...among them - total hypomethylation in early step of carcinogenesis. But this was done more at the general biological level, and we need this one at the molecular.
Well, what are possible theoretical conclusions.
1. Folate intake low ( possible to reach this using MTX for blocking folate membrane transport too) , Met resynthesis suffers, not enough of SAM for DNA methylation...In Canberra's conference abstracts this statment there was stressed one more time - not methylases events yields DNA hipomethylation. This fits. But interesting are numbers and dynamics of this. And I never seen information about folate and histones methylation. This is the front line, dear Nick ,You attacking. In reality, every men when he eats something green or drinks beer obtains folate, but in some cases something happens....only 70% folate body needs...
2. Disregulation of folate sistem. What about the damages of folate system enzymes? Is it possible to swich out some of them and study methylation?
Extremly enteresting and not so complicated to solve on theoretical model of pathways.
Those questions are of great importance, but first this must be solved under condition that everything is O.K. about Serine, Choline (if liver)...His,Trp input systems and Met cycle
3. If You are lucky to catch folate effects on histone methylation- I wish, someone will try to do some experiment with
Serine - the main donor for DNA CpG methyl, because folate system without Ser turn to Gly like the soup without the soult and spoon. There are some impportant differencies theoretically: to disregulate folate system, or Serine catabolism, but one of results the same - DNA hypomethyaltion. I've explained these in preprint lying under the dust somewhere in Institutes of Vilnius and Moscow fifteen years ago.
Recently I've read some article about milk protein caseine influence on serine dehydratase activity.
Kanamoto R, Fujita K, Kumasaki M, Imai S, Kotaru M, Saeki T, Iwami K. Related Articles, Links
Free Full Text Inverse correlation between the nitrogen balance and induction of rat liver serine dehydratase (SDH) by dietary protein.
Biosci Biotechnol Biochem. 2004 Apr;68(4):888-93.
PMID: 15118319 [PubMed - indexed for MEDLINE] plus total 33 publications
Maybe theses facts could be usefull in some experiments to control Serine flows? Theoretically - this is the top.
( I've collected more interesting "crashes" and disregulations in OCU system, not only for Serin. Maybe later.)Thanks.
4. It was found, that the Cancer cell and embryo cell has lots of similarities (for example B12...). Tumour growth in some way could be considered to be the mistaken disregulated embryo growth without normal conditions. If there is some data about folate and neural tube development, this is important to understanding of the malignant growth too.
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Now, some ideas about The funding. Serine is formed from sugars when finally 6 carbon sceleton splits to 3 carbon.
I'm trying to remember, maybe theses words were from "Peter Paul and Marry" in sixties: "roses are red...sugar is sweet my love but not as sweet as you...". So, I've conclude, that Serine problem is sweet attractive too, if presented in such a poetry. The money rich now is Norway, because of the oil in the Norhtern sea. When asking The google about Norway methylation I've found that those brave people study methyl problems not only in petrol, but DNA too.
They support attractive projects if they are really important and have the future. Your project of course is.
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My problem now is the... wood. It's cold in the street. Even my relative have no understanding about the manuscript of the book "Carcinogenes, methylation disorders and a malignant growth" I'm improving and hope print next year, because there are only few scientists to discuss this in Lithuania. The President of academy of sciences is strong professor and scientist Biochemist and told me years ago to pay more attention on histones. I don't want to ask him some wood, because our country is still poor. I would like to ask You, Dear Nick, please, wright me some words - like some recomendation not for bureocracy but for people surrounding me about importance of methylation studies, that I must continue and finish this work. The sculptor Arthur gave me the key to the room I've renovated and computer for 24 hours (I need 25) and shared the last wood. Sorry, but life is life. Good luck.
Sincerely Yours,
Kestutis Urba
Dear Kestutis,
Wow, I am speechless with your rigour and thought into this problem.
I would be happy to write some words for you, but I would like to ask more about the words you would like me to write.
I would be happy to have a look at your manuscript and offer ideas and thoughts on it. It has become clear from the recent conference I attended, Epigenetics seems to touch every field in biology just like genomics has recently. Heritable modifications independent of DNA sequence is now becoming an important issue for evolution to stem cell biology to nuclear reprogramming.
It is also becoming clear that RNAi is playing an important role in all these processes, and I have had this question in my mind, can RNA be methylated, it turns out it can be methylated as demonstrated by the abstract by Ming-Bo Wang.
This is a very exciting field of research that can be investigated in all model systems, a major focus has been on the humble plant, Arabidopsis. The organisers of this conference have a strong interest in plants. Some of the models presented from arabidopsis can not be extended to mammalian systems as some components are missing, no doubt these will be discovered in due time. But I have to say, although a humble student still, the field of epigenetics is very exciting indeed. If your colleagues and mentor's can not see this, just tell them to look at the popular journals, Nature, Science and Cell and you will find a large number of papers relating to RNAi, Epigenetics and related papers.
I hope your manuscript gets published Kestutis, Australia is much warmer than Vilnius
Nick
Hi Nick,
I'm still in research of wooden plants this evening - now using the saw...the good way for the new ideas..
First, question - how to make this DNA methylation - chromatin forum having maximum usefullness and attractivness. This is the exceptional point where biochemical pathways meets biological processes and changes. May be one of the most important. I must study more deep the structure of question and directions necessary for discusion in the field I've got some understanding - molecular oncology. Our discusion about folate is a good training too and experience for me. Then we'll discuss the topics needed to solutions.
Epigenetics is the great idea, but i'm working on the methodological problem of happy end of (h)epigenetics too. This means positive solutions of it's problems and some modfication of this approach really connected to (re) programing You mentioned.
I'm going to study some ideas about His metabolism and methylation to be found before when having no google, only library, but folate system appears very important intermediate carier of process again!
Kestutis Urba
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P.S.The life problems, sorry for this, appeared much more horrifying than hepigenetical paradoxes. They are:
sculpture Arthur (to hew marble is my hobby) gave me the keys and computer for 24 hours per day. But there appeared some advisers having no understanding about existence of CELL,NATURE,SCIENCE who want to see the room I renovated used half an hour per day and locked. Arthur made very famous sculpture: this makes the joy for all the tourists - stone washing mashine with a white plastic window - real not stone age centrifuge...He has understanding about lots of things...and he needs some support - to say for "advisers" -for example "according to the opinion of much more better educated moderator and his friends, than honourable advisors obtained, not reaching the universities, the presentation of CARCINOGENES, METHYLATION DISORDERS AND A MALIGNANT GROWTH (carcinogenesis theory) with a chapter X- The general model of carcinogenesis initiation (this is from manuscript I've improved in 1999 and now working again ) have the chance to find some experimentator's interest with some possible consequencies". I've used some mosaic approach building the theory - solving the puzzle having lots of single facts. The sculpture understands mosaic's building beuty but some other not! So, dear Nick, if You write some real moderator's opinion in reply - then Arhur has the strong position to explain for surrounding people what the hell I'm doing! When some of them dranky - only little problems, if not...life is life.
Some people tell - there is no profit from this research job, better go to business...I don't want to sell anything...may be, it will be easier to explain them after our discussions too...this is maybe sounds like some SOS for manuscript, but I think if anyone reads this discussion, let replies too, please, expressing opinion about possible value of the theorethical models building like some we've discussed: Snell's effect on Serine catabolism tying to DNA hypomethylation too, and then stressing the other facts s about carcinogenes who gives this effect constructing logical chain of events, starting from chemical materials and ending on DNA hypomethylation!
Hi all,
for better understanding people uses drawings, Biochemists uses them very often. I.ve tried to present One carbon units metabolism with all the inputs - some image I've made. The answer of computer was: u r not allowed...
So I present this poor "image":
ONE CARBON UNITS metabolism
Glycolysis
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,
.
Ser
,
,
FOLATE syst. oooo , oooooo Met cycle . .......... SAM xxxxxxxxx. DNA methylation
, , ,
. . Gly
His Trp ,
Betaine
.
Choline
How to publish normal drawing?
soon be finished -K.urba