The Good Doc's notebook: manipulating epigenetics

Hi folks .

I was writing on a thread in the Breeders laboratory about epigenetic inheritence and experiments I've done on C. sativa with inhibitors of histone modification/demodification and DNA modification. I tried to start a new thread there to keep my notes together, but my post count is too low for me to have permission to initiate a thread. Alas. So, I am repeating some of that here and will expand on it as I dredge up memories and locate entries in what remains of my notebooks over the last 30 years of mad scientist stuff with the Noble Herb.

These first few posts are copied from the "On epigenetic heredity" thread at the Breeder's Lab.

Holler if you don't care for this sorts stuff here and I'll Cease And Disist.

Hola mi amigos,

Okey-doke, I'll get on it and report back. Sorry for the delay, I am freshly back from the rainforest in Xx Xxxxxx...mebbe a couple daze, max. Deal?

Hasta -> Rico

P.S. As a taster, chromosomes carry two types of information: 1) Dat stuff encoded in the DNA sequence and 2) dat stuff encoded in da pattern of DNA methylation and histone (protein) modification (acetylation, methylation, etc). Epigenetic phenomena arise, in part, from numero dos. These modifications to DNA and the proteins that give chromosomes their shape, alter the ability of the information encoded in DNA to be expressed. In general, some parts of chromosomes acquire modifications to DNA and histones that shut off expression of the DNA sequences. This tendency to be silent is heritable through multiple rounds of cell division. These patterns are STRIPPED AND RE-ESTABLISHED during the sexual process that gives rise to seeds. This is one reason why propagation through seeds yields variability in the traits we see. On the other hand, sexual reproduction rejuvinates a line and explains why clones fade through multiple rounds of propagation...this is a topic worth exploring in a different thread ifn there is any interest in this sorta thang.

(I could go on for daze like this if there is a "market" for the info. Otherwise I'll channel it into my books and teaching material for my gradual students.).

Originally Posted by zamalito
So if I understand this right. The use of methylation inhibitors allow you to identify which traits are the result of epigenetic phenomena. Then you can adjust your breeding techniques accordingly.

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Indeed, but with some caveats. Many traits are the result of complex interactions between alleles of genes (DNA sequence variation) and metastable gene expression states (epigenetic phenomena). The use of 5-azacytidine, for example, leads to depletion in DNA methylation which reverses (in part) the portion of the genome that is tightly packed (heterochromatic)...leading to expression of previously silent genes. If a trait is reproducibly lost when methylation is relieved, you might reasonably propose that the trait resulted from inactivation of some previously silent region. That region could be identified using modern molecular techniques, including cloning the methylated portion of the genome (using methyl-CpG binding proteins to select this portion of the genome), sequencing of the methylated sites (using hydroxylamine to identify the regions where "C" has become "5-methyl-C"), and whole genome scanning on chips. Lots of options...if only the funding were generally available to pursue this further. Thus sorta of study is in full force in other plants, though.

Originally Posted by zamalito
I'd love to hear more about not only your techniques and the the results but the hows and why's of both the results and the development of your experimental techniques.

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I will dig up my notebooks from those hoary old studies and supplement them with anecdotes, when appropriate. Much of this work was done by me and my fellow inner space fellows in the late 70s-early 80s and again (a bit) in the early 90s.

Science is very cool....

Hmm...looks like I don't have permission to start a thread here, alas.

I suppose I could use this thread to start recording my experiments in the 70s and 80s with inhibitors of DNA and chromatin modification in my breeding program.

To start off, I think I'll dump what I recall from my lysergically challenged synapses and reorganize it as I locate what remains of my notes and data (most is long lost).

IN DA BEGINNING...
Short-chain organic acids (sodium butyrate, mostly). We did not know what this was doing back then, except leading to heritable changes in phenotypic expression that was maintained during cloning but often lost when using seeds to propagate traits. Now we know that this is a weak inhibitor of HDACs (histone deacetylatases) and promotes re-activation of silenced genes by opening up chromatin. Active in the millimolar range...rather a lot when compared to modern HDAC inhibitors (e.g. Trichostatin A which is active in nanomolar concentrations... ~ a million fold more potent!). These were used to treat seeds and in sterile propagation media when cloning tissue. We isolated many morphological variants (leaf size and shape, colour, flower morphology, seed morphology) and variants with different tastes and smells. There were qualitiative differences in psychoactive properties. We did not use objective criteria to evaluate potency.

5-Azacytidine (5-AC). 5-AC is a deoxycytidine nucleoside analogue that gets incorporated into DNA during DNA synthesis, replacing some of the "C" residues oin the genome with "5-AC". When incoporated at targets for cytidine DNA methyltransferases (DNA M-ases), mostly at 5'-CpG-3' sites but also some others, these create suicide substrates for DNA methylation. What happens is that the DNA M-ases try to transfer a methyl group from S-adenosylmethionine to the 5-AC and get stuck halfway through the reaction (the aza group is an electron sink and traps the intermediate in the transfer). This leads to formation of covalent complexes between the M-ases and the DNA, welding the M-ase to ther DNA and blocking subsequent methylation of other sites in the genome, thereby depleting the nucleus of M-ase activity. As a consequence, cytidine methylation is lost (mostly through DNA replication, but there is some evidence for DNA methylation removal by enzyme action as well in some tissues and in some developmental stages). A previously unrecognized secondary consequence of trapping the M-ase on the DNA is that it creates a non-replicatable DNA lesion (like welding a cow to some train tracks, thereby blocking a train from translocating down the tracks) that causes replication forks to stall and eventually break. These DNA breaks lead to mutations and genome rearrangements with profound GENETIC (not epigenetic) consequences...The take home point: 5_AC is a potent inhibitor of DNA methylation, but the experimental breeder must recognize that it can also cause genetic changes.

We used 5-AC and other analogues to create phenotypes that were shockingly similar in morphology and heritability to those caused by sodium butyrate (and, later, to Trichostatin A). An early conclusion from these studies is that whatever these inhibitors did to alter patters of gene expression, they were acting in the same overall "pathway". We now know that this pathway relates to heritable chromatin structures that establish stable patterns of gene expression.

To be continued...

Ok, then. Back on track. From here on out it is new content. Any of you all that have monkeyed with these tools should hump on in if you care to share!

Holler if you don't care for this sorts stuff here and I'll Cease And Disist.

Click to expand...

STFU with that nonsense... because of you, I feel like a noob again and have no clue wtf you are talking about Looks like it is time to go to the library..

Thank you for making me want to learn something new =]

Indica Sativa said:

STFU with that nonsense... because of you, I feel like a noob again and have no clue wtf you are talking about Looks like it is time to go to the library..

Thank you for making me want to learn something new =]

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Hey, thanks for dropping by the lab!

If you find any of the terms or concepts in this thread to be confusing, please pont at what you don't understand and I will do my best to help. I teach and run a research lab that specializes in these and related subjects and spend a lot of my time helping folks master these very cool topics.

Ok...back to work for me!

Fantastic thread drrico!

Before I begin my usual diving in 100%, obsession of knowledge - I've got one question.

Can the above types of work be done by a lamen/home-grower?

I can get a white lab coat if that helps - but access to an actual lab is not possible. ...and of course I'd rather obsess and spend all my free time learning about something I can actually do without visiting the local college.

Thanks for taking to time to help out drrico!

Klutter said:

Fantastic thread drrico!

Before I begin my usual diving in 100%, obsession of knowledge - I've got one question.

Can the above types of work be done by a lamen/home-grower?

I can get a white lab coat if that helps - but access to an actual lab is not possible. ...and of course I'd rather obsess and spend all my free time learning about something I can actually do without visiting the local college.

Click to expand...

Thanks!

You certainly can do these experiments at home. Some care must be taken to limit exposure to yourself and to other organisms. If you have experience growing shrooms, you should have no problems. If you are new to working with this sort of material, I can give you some general guidelines for practicing safe sorcery.

Some of the materials may be a bit difficult to source, but not unobtanium by any stretch of the imagination. Much easier than precursors for organic synthesis...

Indica Sativa said:

Thanks for taking to time to help out drrico!

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It is truly a pleasure. I live to serve (do you want fries with that?).

I believe that we live in a technocracy that threatens democratic processes by placing control in the hands of a small number of edumacated humans. Education is one way to help the people recover power. And I am especially tickled to be able to sow these seeds in the IcMag community!

Right...back to work...

Crosstalk
I've mentioned that Trichostatin A and 5-AC can create similar heritable changes in phenotype. This might lead one to conclude that they operate in similar ways, but this is not strictly true. Furthermore, there is a bit of "chicken or egg" about all this. Here is the central issue:

Epigenetic phenomena are largely the output of changes in chromatin structure. Chromatin is the nucleoprotein material that makes up chromatin, composed of the double-stranded DNA that encodes the genes and the proteins that bind this DNA and organize it into a dense bundle within the nucleus. Chromatin is organized into two different types of chromosome structure: Euchromatin and Heterochromatin. Euchromatin mostly contains the expressed (or expressible) part of the genome. Heterochromatin largely contains the non-expressed portion of the genome, though it can be activated by enzymes and DNA binding proteins that create domains of euchromatin-like chromatin within heterochromatic regions of the genome.

The tendency of a heterochromatic region to remain heterochromatic (and, thereby, non-expressed) is modulated by DNA methylation and by histone modification. DNA methylation comes in two primary flavors: de novo methylation in which non-methylated DNA becomes methylated and maintenance methylation in which methylated DNA is propagated during DNA synthesis and cell division so that the methylation pattern is inherited (much like a DNA sequence is inherited....). Methylated DNA is methylated on BOTH DNA chains, typically at palindromic or quasi-palindromic recognition sequences (e.g. the dinucleotide CpG is present on both strands since CpG is the reverse complement to CpG...therefore 5-methyl-C is present on both strands at the CpG site and the site is said to be HOMO-methylated). When DNA is synthesized by the semi-conservative mechanism (most common form of DNA replication...ask Meselson and Stahl!), the newly synthesized DNA beomes HEMI-methylated. Hemi-methylated DNA is the preferred substrate for the maintenance DNA methyltransferase activity and the unmethylated newly synthesized strand is rapidly methylated, thereby maintaining the pattern of DNA methylation. This allows faithful segregation of the DNA methylation pattern to the daughter cells following mitosis (cell division). In contrast, unmethylated DNA is maintained in the unmethylated state, since unmethylated CpG (etc) is a poor substrate for the maintenance DNA methylatransferase. So then, how is the pattern of DNA methylation established?

Unmethylated DNA may become methylated by a de novo methylatransferase activity if there is an appropriate "signal" for methylation in the chromosome. The nature of this signal remains elusive, but some features have been identified. Long concatemeric arrays of duplicated DNA sequence elements often recruit de novo methylation. This has been the bane of many transgenic studies in gene therapy and biotechnology (though we have the solution to that!) in which manipulated DNA sequences are introduced into the genome of an organism and frequently integrate in a multicopy array that is expressed at first and becomes nonexpressible over time as it becomes inactivated by DNA methylation, histone modification and heterochromatin formation. This aspect of methylation appears to be an "immigration control" function that protects the genome from molecular parasites such as viruses and transposons. In fact, many of the stable heterochromatic regions of chromosomes such as at the centromeres look to be the relics of ancient invasions by molecular parasites that became inactivated through heterochromatin formation and subsequently drafted into service to play important roles in DNA segregation in mitosis (spindle attachment sites) and in phenotypic variegation (the topic of these nuggets I'm tossing you all, ifn you like em).

Another feature of chromosomes that influences DNA methylation is the modification state of histones. Histones are proteins that assemble with DNA into chromatin. The tendency of chromatin to adopt a "euchromatic" and expressible state or a "heterochromatic" and unexpressed state is a feature of the modification state of the histones. For example, acetylation of one histone protein is associated with euchromatin. Enzymes that actively deacetylate this histone (so-called HDACs) drive the chromatin into a heterochromatic configuration (this is a simplification...please forgive me...). That is why HDAC inhibitors like Trichostatin A maintain euchromatin and can lead to loss of heterochromatin when acetylase activity is robust. The recruitment of deacetylases to these sites appears to be co-ordinated by DNA methylation and by methyl-DNA binding proteins.

Therefore, there is substantial crosstalk between DNA methylation and histone modification. Futhermore, both de novo and maintenance methyl transferase activity seems to be modulated by histone modification states. In other words, heterochromatin tends to recruit DNA methyl transferases to maintain heterochromatin. This positive regulatory feedback loop creates a so-called BISTABLE switch state that is heritable. In one configuration, genes are expressd, in the other, genes are repressed. This state may be propagated for many generations, unperturbed, UNLESS you toos in methylation of acetylation inhibtors or push the organism throiugh a reproductive cycle.

Point at anything you wish to have clarified or argue about or whatever. If you remain interested in this sort of thing, please let me know and I'll keep on typing. If you find it boring, let me know and I'll try to make it mo betta!

To be continued...

Welcome back to the lab!

Here's the next notebook installment. This one has to do with sexual reproduction and its effect on epigentic inheritance.

Epigenetic metastability and the "reset" button
So why would a humble plant (or animal) bother with epigenetic regulation of gene expression? It all comes down to sex, stem cells, differentiation and the development of a multicellular body plan with different cell types. To a first approximation, every cell in a multicellular organism contains the same genes. Even so, not every cell EXPRESSES all the same genes. Furthermore, some genes are very important for growth at early stages of development, but are harmful to the organism if expressed later in development. One very important way to regulate gene expression, especially when it is important to shut down a gene and keep it quiet through many rounds of cell division and even after cell division ceases, is to methylate DNA, recruit 5-methyl-C binding proteins, modify histones and create domains of heterochromatin. As I've previously explained, it is heterochromatin that locks DNA into a heritable non-expression state that mimics mutation but it FULLY REVERSIBLE by treatment with certain chemicals or....

...wait for it...

...SEX.

So what's love got to do, got to do with it? Well, when a male Canna and a female Canna love each other very much and get down, they make babies called seeds. Seeds contain all the genetic information to create a new plant. Initially, seeds are programmed to promote germination and then a nascent plant with embryonic leaves (cotyledons) is produced. As any grower knows, these little plants hardly resemble the ripe adults we are so fond of (especially the ladies). As the plant develops and matures, all manner of different structures are formed. Some of these structures are the product of stem cells that retain a vegetative pattern of growth, others are the product of stem cells that initially program development of flowers but eventually terminally differentiate and are consumed. When one examines the patterns of gene expression and DNA methylation in these different tissues, it is evident that any given plant has specific patterns but that any two plants may not. Some of these differences are genetic, but others are epigenetic and can be readily demonstrated by treating clones, independently, with DNA methylation inhibitors. These treatments yield genetically identical but phenotypically distinct sibling clones that BREED TRUE when propagated clonally. In other words, clones develop similarly and have similar DNA methylation patterns but when one clone is treated with 5-AC and another is left alone, the phenotypes of the clones diverge and the new phenotype in the treated clone may be propagated asexually by cloning this altered plant. The stable change in phenotype is correlated with a stable change in DNA methylation patterns.

Now, put these clones through a sexual cycle and you recover gametes (e.g. pollen if male) that is genetically identical AND epigenetically identical.

Jigga-wah?!

Yup. You see, the sexual cycle has steps that lead to both active and passive stripping of DNA modification (and, we imagine, histone modification...though we do not have data for that just yet). This resets the genome to the ground state of genetic "normalcy". This ground state is essential if early development is to occur properly. It is one reason why cloning animals through nuclear transplant uses a "reprogramming" cell to reset methylation patterns...though that particular approach is incomplete and leads to problems like, uh, cancer and death (see Dolly the sheep, for example).

Sex does amazing things for rejuvenating a genome. Its the pause that refreshes. While plants are particularly forgiving about clonal propagation, most sexual species eventually senesce and die without resetting the genome's methylation state (and purging it of mutations that accumulate through asexual reproduction and DNA synthesis...see Mueller's ratchet for an example or ask me to explain the important role of homologous recombination in purging genomes of deletrious mutations. But I digress...). So epigenetic tweaks are of limited lifespan: you can not propagate them infinitely through clonal growth and they can not be propagated at all (well...there are some exceptions...) by sexual reproduction. So seeding out your plant will strip the effect even if you self it.

Having fun yet? Please holler out or toss some 'spect or hurl tomatos...its getting lonely writing these essays to an empty room...fer instance, somebody may wish to know how all this stuff figures into cloning plants that have already entered into flowering...

To be continued...?

so these people who have moms of a vast variety both sativa dominant,indica dominant,and both in varied genes lose potency over the generations of clones made?
How would one ensure the quality and vigor if going about things like this?
Clone of a clone of a clone of a clone of a clone=less of a plant?

Thanks brain,
Peace

BTW it's really cool to have a techie so to speak on how our world/hobby/medicine operates,comprehending it is on another level totally and up to everyone else to just learn it if so inclined........well babble on good sir,I at least am enjoying the read.

Peace

First off thank you. I understand only small portions of what you've said, but thank you.

Second, what do we do with this? Can you give me a real world example of what you've done to Mj with these techniques. I think its cool to experiment, but I want to know the why before the what.

Thanks

dotol, borre la chotiaera del segundo post....

y ojala siga en el area, pq yo tambien y se ve que usted tiene mucho que ensenar.


sorry for the spanish dudes, heh.

thenef said:

so these people who have moms of a vast variety both sativa dominant,indica dominant,and both in varied genes lose potency over the generations of clones made?
How would one ensure the quality and vigor if going about things like this?
Clone of a clone of a clone of a clone of a clone=less of a plant?

Thanks brain,
Peace

Click to expand...

Hola thenef!

It is true that some mothers will fade over time...I have had such luck. It is not so much that they reduce THC production as they generally get less healthy...more spindly...slower growing...and toss off clones that are more susceptible to disease and environmental stress and nutrient problems. While indica and sativa clones suffer suchlike, the hybrids are slower to fade as are some of the landrace outcrosses (hybrid vigor, you all!). The fade of clonally propagated plants results from accumulated mutations (I've picked up some cool colour variegated strains that way) and from "forgetfulness" of the epigenetic inheritance machinery.

In general, clonally propagated strains remain stable longer if you reduce growth in your mothers and refrain from cloning clones of clones of clones of...in fact, if you could clone from frozen tissue reliably, you could avoid most of the problems I mentioned. I do that with other orgainsms, but have not been successful with Canna popsickles...alas...

Thanks for popping in! Please keep hurling questions this way.

pumpkin2006 said:

First off thank you. I understand only small portions of what you've said, but thank you.

Second, what do we do with this? Can you give me a real world example of what you've done to Mj with these techniques. I think its cool to experiment, but I want to know the why before the what.

Thanks

Click to expand...

Hi punkin, thanks for poking your nose under the tent! It was getting lonely in here.

RE: What is the use of all this gibber-jabber? Fair question!

"Variation is the spice of life."

One reason why we enjoy such a plethora of varieties of herb to sample is that folks have identified traits that they have selected from strains that already exist and have used selective breeding to emphasize the good aspects of these strains and minimize the bad under their preferred means of cultivation and environments. This is the tip of the iceberg. If we accelerate evolution by introducing mutations (genetics) or epimutations (epigenetics), we can create/discover/uncover new traits that appeal to us.

For example, I have uncovered a chocolate-cherry phenotype by epigenetic manipulation that I have not been able to isolate through mutagenesis (in some other thread, some other time, I will discuss our experiments with mutagens....). This one was fun to grow and hold, but we never managed to capture this phenotype in a background that was robust to our conditions and met our goals for yield. I also isolated several colour variants with interesting variegated patterns that were lovely to look at. I have also isolated indica variants that produced sativa type highs and sativa variants that produced indica type highs. These variants maintained the phenotype through several cycles of cloning, but could not be transmitted through seed.

So the short answer to your question is that manipulation of epigentics leads to variation and phenotypes that I have not been able to capture through genetics. While genetic traits are more stable and easier to archive, epigenetic traits offer additional avenues to seek interesting phenotypes and are as easy or easier to isolate as mutations. From work in my lab, I know that some traits are ONLY accessible through epigenetics...I view this as one of the New Frontiers of Cannagen and fully expect that breeders will be exploiting this soon. There are already patents granted and big, highly funded, agricultural projects based on this approach in other plants...

Keep it green and have fun for gosh sakes!

Revegged_Clone said:

dotol, borre la chotiaera del segundo post....

y ojala siga en el area, pq yo tambien y se ve que usted tiene mucho que ensenar.


sorry for the spanish dudes, heh.

Click to expand...

Hola Revegged_Clone,

¡Utilizo solamente inglés pobre para explicar cosas porque asi es como me criaron! Soy del campo…

I am happy to help and will try to speak as well as I can...it is not my first nature; I play with words for fun!