Locus Specific and Global 5-hydroxymethylcytosine Detection and Quantification

Curious about the role of 5-hydroxymethylcytosine in the genesis and function of the epigenome? Watch and learn as Sriharsa explains the study of 5-mC and 5-hmC as markers of epigenetically-modified genomic DNA, and the best methods for differentially detecting these modified species.

Script

Pete:

Hello, how's it going? This is Pete Jozi, I'm an editor of EpiGenie, and today we welcome you to the BiteSizeBio web seminar, which is sponsored by New England Biolabs, the world leader in the production and supply of reagents for the life science industry. NEB now offers the largest selection of recombinant native enzymes for genomic research, and it continues to expand its product offerings to emerging areas including proteomics, drug discovery, and epigenetics. If you'd like more information, you can visit them at www.neb.com.

Pete:
Today's presentation titled locus specific and global 5-hydroxymethylcytosine detection and quantification, and it will be presented by Dr. Sriharsa Pradhan, who is the division head of RNA biology at NEB. AS always, we'll have a Q&A session after the end of the talk, so please submit, throughout the course of the talk if you have any questions, please submit them in the questions box, which will appear at the right hand side of your screen, and we'll consolidate them and tee them up to Sriharsa at the end of the presentation.

Pete:
So on that note, I'll turn things over to Sriharsa for the presentation.

Srihasa:
Thank you. So today's webinar is specifically focused on our newest reagent as well as some of the fine stuff we do at New England Biolabs to detect the global as well as local specific 5-hydroxymethylcytosine, which is the epigenetic mark in the mammalian genome. So let me introduce to what is epigenetics in a very layman version.

Srihasa:
So if you look at what epigenetics is, this is exactly the study of heritable changes in the phenotype of a cell or organism that are not dictated by the genome per se. Now, let me explain this to you in little bit more clarity here. So if you look into the molecular basis of epigenetics, what you see is a chromosome. And this chromosome, if you take a tweezer and try to drag a little bit from the side of the chromosome, you are unraveling the chromatin fiber. This chromatin fibers are essentially the nucleosome particles, which are like bead and strings, and they actually for the primary structure of the chromatin. This, if you unravel it, if you take out the proteins, what you see is a double-stranded DNA. And on the double-stranded DNA you will see various kinds of modification that occurs, and one of the predominant modifications is the methylation of the DNA as it is depicted as the ME here.

Srihasa:
So, apart from DNA methylation, if you look in the chromatin there is also histone component, or which I was referring as the nucleosomes here, and this histone and DNA, they together make the nucleosome, the basic subunit of the chromatin. And when DNA can be methylated or unmethylated, in two different forms it can exist. However, histone is a little bit more complicated. Histone has these beautiful tails that juts out from the nucleosome core particles, and these tails can be modified in a variety of ways, such as methylated, and it can be mono-dio-trimethylated, then acetylation can occur, so as some of the molecules could be removed by acetylation, which is known as deacetylation or phosphorylation. And what, for example, I am showing you a histone H3 tail here, and you can see that the various amino acids that are getting modified here, for example, lysine4 on H3 tail can be modified by a variety of enzymes, which is like set seven, set nine, and these enzymes can put specific methyl mark, and that methyl mark can be removed by LSD1, a type of demethylator.

Srihasa:
So essentially, the modification mark is very reversible, so histone can stay in a very dynamic state where for certain biological functions a modification can be put in, and certain functions it can be removed.

Srihasa:
However, if you look into, broadly ask the question, what's the difference between a genome versus epigenome, and why is it important? So if you look into a cell, you'll know that that genome is generally very consistent, however epigenome changes. What I mean by consistent is, essentially, the basic subunit of the genome, that's the nucleotide sequence, they don't really change, until and unless there's a mutation. But however, the epigenome can change, or the modification on the genome that can change with age, diet, various kinds of disease, your lifestyle, or environmental impact.

Srihasa:
So that leads us to understand ... put a huge number of biological interest here, because starting epigenetics can actually lead us to understand molecular basis of diseases. We can also think about in a more applied manner, where the biomarker identification could be the result of studying epigenomic changes or diagnostic development, as well as drug targeting.

Srihasa:
So essentially, what we believe is if you sequence your genome once, you know the nucleotide sequence, but to understand the epigenetic changes, which is accompanied with age, diet, disease, and lifestyle, you need to determine your epigenome multiple times over the lifespan of an organism

Srihasa:
So what is this modification of DNA? Now, we all know that there are four basic building blocks on DNA, which are four nucleotides, A, T, G, and C. And now, what happens to the C, or which is known as, which I made a figure here, is [inaudible 00:06:35] cytosine, this can actually modify by three enzymes that are present in most of the organisms like human, mouse, all kind of mammals. So these are called DNA-methyltransferase. These are essentially cytosine-specific methyltransferase, DNA-methyltransferase 1, 3, and 3b. So what these enzymes can do is essentially, they take a s-adenosylmethionine or AdoMet and put the methyl groups on the fifth carbon position, and the net product, AdoMet will be converted to AdoHis, and you get a methyl group right on the fifth carbon of the cytosine ring.

Srihasa:
Now, for the longest time we thought that this is a very stable mark and it really doesn't change very much, and this is our epigenetic blueprint. But very recently, about two or three years ago, there are some very nice scientific discoveries where people found a group of iron dependent enzymes. They are called 10,11 translocation factor 1, 2, and another one recently known was a 3. These all enzymes, they can actually detect this methyl group on the fifth carbon position and oxidate it to 5-hydroxymethylcytosine. And 5-hydroxymethylcytosine has come to the picture, essentially, as an epigenetic mark on the genome.

Srihasa:
So we are talking about the fifth base as methylcytosine, sixth base as hydroxymethylcytosine, and there is also latest observation that this residue can also further oxidize to form of a carboxyl group, but those two modifications are not in our current discussion, so we will focus our emphasis on understanding how to integrate
hydroxymethylcytosine on the genome.

Srihasa:
So what does the exact role of 5-hydroxymethylcytosine? I mean, we have enough of evidence now to speculate that this can be a part of the demethylation protocol during mammalian development or gene expression, so it could be a very transitory mark. So if anything is associated with methylation and demethylation, that will have a profound influence on the gene expression, so that will essentially dictate whether the gene should be transcription active or repressed.

Srihasa:
And now, as we know, that this 5-hydroxymethylcytosine molecule is actually very enriched in the brain. Basically about 30% of the methylcytosine in the brain is 5-hydroxymethylcytosine. So neuronal cells that are highly enriched in 5-hydroxymethylcytosine, and embryonic cells are about 10% of their methylcytosine is 5-hydroxymethylcytosine. So obviously they do play a significant role during function and development of either brain or the embryonic stem cell, and also we are aware that they hyrdoxymethylcytosine containing genes that are mutate in acute myeloid leukemia, so that could be a function down there, and several other diseases.

Srihasa:
That brings a very interesting phenomenon. All the hydroxymethylcytosine and the genes, would they be participating in particular allele biomarker for some of the disease and discovery phase? So maybe there is a role down there, but once again, this is very early in the science, and a lot more to be discovered in the future.

Srihasa:
So what, if anything, we have focused mostly in my group, as well as some of my collaborators, as well as other scientists, is, how do you detect 5-hydroxymethylcytosine? And once you detect, how do you quantify that? Because if you have a particular locus, you really need to know what percentage of your genomic DNA contains 5-hydroxymethylcytosine in that particular locus.

Srihasa:
So in the good old traditional method, people have used antibodies to pull down 5-methylcytosines and 5-hydroxymethylcytosine recently. But those technologies, they do not tell you the quantitative percentage. So we double up the protocol, which I'm going to describe here in a minute. Before that, that protocol is now packed into a kit format, of where essentially a scientist will pick up the kit and try to ask, what percentage of my locus has 5-hydroxymethylcytosine or 5-methylcytosine? It's a very user-friendly protocol. And it doesn't need any kind of extra equipment in the lab. It's a simple and robust method for identification and quantification of 5-HMC and 5-methylcytosine within a specific DNA locus, and this is highly reproducible. You can quantify it easily using a qPCR protocol compatible with existing PCR technique, as well as it is amenable for high support sequence analysis.

Srihasa:
So now let's look into the method. So how does it work, basically? If I'm telling you so many things, then I will tell you how it exactly works. So one of the things is that if you look into a genomic DNA sample, you don't just have one strand of DNA. You will have multiple copy numbers of the DNA in your 50 nanogram or 100 nanogram of the samples. And that DNA, in a particular site, for example CCG, this site could be hydroxymethylated, could be methylated, or unmethylated. So you can only have three forms here. The essential CPG, as you know, most of the mammalian methylation occurs in CPG dinucleotide residues, so these could be in unmethylated, methylated, or hydroxymethylated format.

Srihasa:
Now, in the second step, what we do, we use an enzyme called a phase-specific enzyme, which is called beta-glucosyltransferase. It is from T4 phase, so it is know as T4 beta-glucosyltransferase, and we provide a substrate. So this glucosyltransferase will use the UDP-glucose as a substrate and put a sugar residue on the hydroxymethylcytosine and make it glucosylated. So now your hydroxymethylcytosine residue is now glucosinated hydroxymethylcytosine. And this enzyme is there on T4 phase and it regularly converts all the hydroxylmethylcytosine to glucosylhydroxymethylcytosine. I'll talk a little bit about the enzymes in the second phase of my talk.

Srihasa:
So now, this particular reaction part you are actually going to divide into three tubes. One, two, and three. And you can also decide have a control, should you wish to have, so the control reaction without [inaudible 00:13:49], you can put only the substrate here, and you can set them in an identical manner with 4, 5, and 6 reaction. For simplicity, I'm just going to focus on your experimental samples here.

Srihasa:
So the three tubes of reaction now you are using the isoschizomer, a restriction enzyme called MSP1 or HAPA2 14:!1], or you can have it on control, uncut control. Now these enzymes, what they do, they recognize the CCGG sites to digest it, but the have differential specificity when the DNA is methylated or hydroxymethylated.

Srihasa:
For example, all your uncut control DNA will have no sites missing, because all the sites will remain intact, because you're not putting any enzymes. But in HAPA2 DNA, HAPA2 enzymes when you put, it'll only cut the unmethylated faction of the genome. So it cuts everything that is CCGG, it'll leave out anything that has a methyl group or a glucosyl group, so this is essentially the hydroxyl group.

Srihasa:
However, MSP1, which will cut away the methyl group as well as the unmethyl group, it'll leave away the glucosyl group, so now what you have essentially done in this part of the reaction is that you have actually left anything that was hydroxymethylated to start with, so anything that is hydroxymethylated is now remain intact. Anything that is hydroxymethylated plus methylated is intact in the second part, and this is your hundred percent of the reaction. Now you can actually go for the quantitative piece here to find the percentage hydroxymethylated here, or percentage all methylization, which is 5-methylc and 5-hydroxymethylc, and this is the total input, this is your hundred percent.

Srihasa:
And if you are looking at your favorite gene and asking a question, I wanna do in a quick way to know whether my gene is hydroxymethylated or not, this reaction is simple. You just do an endpoint piece here as I am showing in the left panel here. So this is a typical result using the kit. So essentially on the top we are writing about different kind of reaction components here, so these will be the glucosyltransferase blocks insert added into the tube minus means it is not present in the tube, and you can see if you're asking a question, "Does my test brain DNA have a particular locus methylated or not?", you can just run an endpoint piece here. If you see the piece of your product here, then you know that site is hydroxymethylated. Obviously, then, if you do HAPA2, and you can say it is methylated, this is 5-methylcytosine, as well as 5-hydroxymethylcytosine.

Srihasa:
And what you could appreciate here is that the different DNA samples will ... That particular locus has different amount of hydroxymethylcytosine. As you see, the liver has lesser than brain DNA, whereas heart DNA has as good as brain DNA, so these are pretty enriched DNA for hydroxylethylcytosine, it is predominant, which the spleen doesn't have.

Srihasa:
So this is just a positive and negative reaction where you do an endpoint piece here, but if you want to know exactly what percentage of the sites are methylated or hydroxymethylated, then you have to rely on the quantitative piece here. And this is, again, the presentative data based on this particular endpoint piece here type of the samples. So you can see again, brain DNA has pretty high amount of 5-hydroxymethylcytosine, heart DNA has, and as you can see, spleen was very little here, and so in the quantitative piece here it is almost non-existent, very close to the background.

Srihasa:
So this is what the power of FMR 5-hydroxymethylcytosine and 5-methylcytosine analysis kit. And obviously, the kit works very well on CPZ methylated DNA, which is essentially in the human, mouse, and any kind of vertebrate DNA that you are talking about. We have used this kit, and we have looked into various kinds of gene body methylization as well an answer element as well as the promoters and all different region we try to ask the question, does methylation and hydroxymethylation correlate in a gene body?

Srihasa:
So this is from the human brain DNA. These are the two genes called VANGL1 and EGFR genes, and this is the genomic map of these genes. And you can see the black boxes are exons, and their introns. And there's on the top, the numbers of nucleotides are annotated on the top. And you can see that gene body methylation is very prevalent in this. So these are about nine steps of quantitative PCR reaction primer we're integrating throughout the gene body, and as you can see that the blue bars are 5-methylcytosine and the green bars are 5-hydroxymethylcytosine, and you can see if you look into the translation start sites, set one and set two ... Sorry, transcription start sites, you do not see any type of methylation or hydroxymethylation, but in the gene body you see a nice enrichment of both the marks.

Srihasa:
And the same thing with EGFR. You can see a nice enrichment of 5-methylcytosine as well as 5-hydroxymethylcytosine in the gene body. But once again, at the transcription start site, you do not see anything happening. There is absolutely very little amount of modified bases.

Srihasa:
Now, where else can you use this kind of technology? Okay, if you are using antibodies to do a whole genome analysis of 5-hydroxymethylcytosine or 5-methylcytosine by using immunoprecipitation technique or hMeDIP as it's known as, you can validate arrays. But you see a lot of pics that are coming up with your antibody than you want to really validate, then you can use this tip. As a result of this type of application, we actually looked into human embryonic stem cells, and we found that enhancer elements are highly enhanced in 5-hydroxymethylcytosine. This is essentially hMeDIP data. And that corresponds to a lot of activation marks, like H3K4-monomethyl, H3K27-acetyl marks, so we wanted to know, do the enhancer elements really are enriched in 5-hydroxymethylcytosine?

Srihasa:
So we picked up various hits from this kind of genomic analysis, and these are the four examples from here, and we took the control region where there is no hit, and the chromosome one and the ID numbers are here. As you can see, MSP-1 give a nice big presence of beta-glucosyltransferase in our test case one, two, three, and four, indicating that all the enhancer elements are truly enhanced in 5-hydroxymethylcytosine, whereas control regions have absolutely very close to background.

Srihasa:
So this is another example. If you are using a hMeDIP protocol, you want to try to validate it, this is another way to go for that.

Srihasa:
So using this kit, there is numerous publications now, and here are a couple of publications that I love absolutely. So the technology that I spoke to you just now is essentially covered in the Journal of Biological Chemistry 2011 publication on July 15th, so would you need more information, you can actually look into this particular paper, where it is in detail. And apart from my lab, this particular technology is used in UK in Wolf Reik's lab, Richard Meehan's lab, and in UCLA Steve Jacobsen's lab. There are papers in natural genome research as well as genome biology describing this technology and its application for various biological studies, whether it is in mouse embryonic stem cells or human embryonic stem cells.

Srihasa:
So with this, let's try to think about from the locus-specific to the global. Some of you have 20 different samples. You really want to know, do I want to go for locus-specific? First and foremost, you can do a quick check whether your DNA has 5-hydroxymethylcytosine or not, because every tissue, every sample could have a different sealed amount of 5-hydroxymethylcytosine. So for this we have doubled up the protocol, and also all these enzymes are available at New England Biolab, and this is a very straightforward, simple protocol I am going to run you through, and I will show you how we have utilized that through about 60 different genome analyses, or rather 60 different sample analyses.

Srihasa:
So in the central about this particular protocol, you have an enzyme called beta-glucosyltransferase, which will glucosylate 5-hydroxymethylcytosine. Now, this is the enzyme I was talking to you is from T4 phase, beta-glucosyltransferase. This is your typical 5-hydroxymethylcytosine on the left, on the right with the enzyme, if you put uridine diphosphoglucose you can see that the glucose residue is actually transferred to this hydroxyl group. So this is the 5-glucosylhydroxymethylcytosine. And if this residue could be tagged with the fluorophore or with a radioactive level, then you will be able to quantitate. And for all of my work in the lab we have used radioactive UDP-glucose to transfer the radioactive glucose, and you can measure that by using simply some counter. And once again, this paper is very recently published in biochemistry, and this is the reference on the bottom, and I'm going to describe a few things from that paper now.

Srihasa:
So UDP-glucose by beta-glucosyltransferase is a very fast reaction. It's a very efficient enzyme. It transfers in an amazing rate. It puts 77 molecules of glucose from UDP glucose on a T4-gt DNA. So the turnover number is 77. That means every enzymes put about 77 molecules of glucose in a minute of the reaction. So you can understand the reaction is extremely fast, and so you can saturate the reaction very, very quickly.

Srihasa:
So first thing, what do we do, we make a standard curve. So what does standard curve ... We put a different amount of hydroxymethylcytosine, and this is essentially a DNA, the DNA is, we'll call it T4-gp DNA. GP stands for glucosyltransferase mutant DNA, so this DNA, all the Cs are hydroxymethylC. And we do a linear ... You'll see the reaction is very linear despite increasing amount of hydroxymethylcytosine and keeping the radioactive UDP glucose and enzyme constant, you can see the reaction never tapers down, even if the HMC concentration has gone to 20 millimolar at this point.

Srihasa:
And then what you can do is that you can use that standard curve, and you have all your DNA samples, as you can see we have tried a series of DNA samples, and you can actually quantitate how much of your DNA has hydroxymethylcytosine. Total percentage of hydroxymethylcytosine in cytosine, or you can check it on ... You know. Once again, this paper is just published, and anybody can download, it is openly accessible paper from Biochemistry website.

Srihasa:
And you can see we have looked into mouse embryo, mouse embryonic stem cell, ES cell, cell culture, cell line, different tissue on mouse, brain, liver, kidney, heart, lungs, different cell lives from human origin, U-87, 293, colorectal cancer, cell line. Then again, human normal lung tissue, breast tissue, colon, liver tissue, as well as the tumors from the corresponding tissue: lung, breast, colon, liver, and metastatic colon tissue. So you can see that this is a fantastic technique that works on a variety of tissue, and also we are trying to integrate CR chain, couple of plants here, arabidopsis, soybean, rice, and various kinds of other cells' genomic DNA, such as dogs, as well as monkey, and et cetera, et cetera.

Srihasa:
So this is a very versatile technique if you want to look into genome-wide 5-hydroxymethylcytosine, and it doesn't take a long time. In probably about one afternoon you should be able to do both the standard reaction and a variety of genomic DNA side by side.

Srihasa:
So this is another figure from the paper. We were also looking at signs here. In Biolabs we do a lot of basic research pertaining to this area, because we understand that what many of our fellow scientists do in their labs in the academic atmosphere or in a clinical atmosphere, we need to understand them, so we also try to do a lot of what I feel basic research here, and one of the work was to find out if there's a difference of 5-hydroxymethylcytosine in normal tissue versus cancer tissue.

Srihasa:
Now, what I am showing you here is, in essence, basically these are matched tissues. So normal lung versus tumor lung. This is from the same patient, so you have matched versus normal. Breast, again, matched versus tissue here. Colon matched tissue and liver tissue. As you can see, generally when there's a tumor you see a decrease in 5-hydroxymethylcytosine, and this is across the board, and we are very surprised and also not very surprised because of the fact that many of the cancer cells have low amount of 5-hydroxymethylcytosine. But this could be something that we have to understand how the role of 5-hydroxymethylcytosine, or what are the roles it plays in various kinds of tumor and cancers.

Srihasa:
So apart from these two published, we are very excited that we have some of the very new enzymes that are coming up, which can also integrate and determine 5-hydroxymethylcytosine. Obviously, many of this stuff is on the development now, and one of the enzymes we are very excited is called AbaS-1, and this is an enzyme which 5-hydroxymethylcytosine specific enzyme. It recognizes 5-hydroxymethylcytosine and excites the DNA. And some of the research is already published in Nucleic Acid Research, just initial characterization in 2011, this is a publication by Wang, H. et al.

Srihasa:
We are very confident that the enzyme actually works well, because we have some unpublished data on E-14 embryonic stem cell DNA from a mouse, and we are excited with this enzyme, and we will very soon be able to publish some of the information that we have obtained on a genome-wide scale using this particular enzyme on mouse E-14 cell lines.

Srihasa:
So in summary, I think NEB has a tremendous amount of basic as well as applied research trying to look into epigenome and develop quick kits and reagents so that we can do science that will help the scientific community.

Srihasa:
And finally, I would like to thank the people who have helped me to do this work, as well as [inaudible 00:30:59] Shannon Kinney in my lab as well as Hang Gyeong Chin. They did the work on developing the JVC paper where they have studied the local specific work, along with Romas, who developed the kit, and Ted, who spearheaded some of the projects here. And I'm very grateful to Steven E. Jacobsen's lab for a lot of knee-deep data and continuous collaboration on some of the epigenetic studies, and the NEB marketing communication department for making the beautiful slides, organizing this wonderful talk, and thank you all for being a part of this webinar, and I appreciate if there is any questions, obviously if I cannot answer you any questions of if you need to know more information, you should be able to send an email to NEB in our website. I sincerely hope that we will be able to attend to your question. So I will take any questions now.

Pete:
Okay, Srihasa, you're in for a treat. We have some good ones from the audience. So to kick things off, we have one question that is kind of technical and may be related to the method of sample extraction and the impact on 5-hydroxymethylcytosine. The question is, how stable are 5-methylcytosine and 5-hydroxymethylcytosine during cell lysis and DNA extraction.

Srihasa:
Okay, that's an excellent question, actually. 5-methylcytosine is extremely stable. There is no doubt about that. And 5-hydroxymethylcytosine is also stable, but in a strong oxidative condition it can be further oxidized to 5-formylcytosine and 5-carboxycytosine. Now, does it happen in the environment? I doubt. But if you look into the biological pathway, it can be further oxidized by this tech class of protein. So 5-hydroxymethylcytosine can be further oxidized with 5-formyl or carboxylC. But if a scientist is just trying to get the cell sample and extract the DNA, I think it is fairly stable, because we have not seen any difference in our one year, two year old samples, so I would say yeah, it is pretty stable for lab conditions.

Pete:
Okay. This group in particular is pretty keen. They've seen cytosolic and nuclear deaminases, particularly in cytosine deaminase, that can deaminate 5-hydroxymethylcytosine and 5-methylcytosine to 5-hydroxymethylthymidine and thymidine. So they were curious if you've ever seen these derivatives in the process in other types of analysis like mass spec or-

Srihasa:
Yep, okay. What we have done is that we are using HPLC mass specs, so basically when we take the DNA, we try to make the mononucleocyte and we try to separate them in microbore HPLC and shoot them to mass spec. We have actually not seen too many things. As long as you store your DNA in a 3CDTF minus 20 degrees. That's where we store our DNA, at least. To my knowledge, we have not seen any kind of serious amount of oxidative product coming out of that in a span of six months to one year.

Srihasa:
And possibly that could be some chemical reaction going on, but that's probably so low it's probably not going to be significantly impact the scientific result. But yes, it's something one should be concerned about. But if you are looking at a DNA sample which is stored for 10, 20 years, and now trying to look into 5-hydroxymethylcytosine, you would think it would be in a damage right then and there.

Pete:
Okay, great, moving on. So here's a more general question. We had one from the audience. What's the nature of the tumor the lung, breast, colon, and liver cells that you used for in the analysis that you presented here?

Srihasa:
Well, that's a question probably I can't even answer, because most of the DNA samples we get are often from commercial source, because we are not an IH lab here, or we don't have the expertise of a cancer specialist with microdissecting the DNA and giving. But what I could just say that they are match versus the tumor. And I can't tell you the stage here, but in general, if the scientist wants to know whether the cancer cells have a much lesser amount of DNA hydroxymethylcytosine, I would say that's a pretty safe bet. But I'm collaborating now with a human genetics department in a cancer institute. I should be able to get validated DNA samples based on stage of the tumor, and that would be very exciting. So I would suggest scientists to look forward to some of our published papers in the future.

Pete:
Okay, great. And here's a more general biological question as well: Are there any studies that you know of that provide a functional leak between 5-HMC enhanced in the genome and their physiological implications? So, for instance, is there any work out there that ties 5-HMC to mechanisms like transcription or any other regulatory processes?

Srihasa:
Right, okay. So that paper from Straud which is in General Biology, that talks about 5-HMC and the enhancer elements. That's one. And also we know it's in the gene body, especially if you look into Wolf Reik's paper. So to me, it looks like that could be two mechanisms why 5-hydroxymethylcytosine is there. One is, it's somewhat regulating the transcription.

Srihasa:
Now, whether it is going through a demethylation pathway, which is published, there's a paper in Cell now on the APOBEC media to demethylation via 5-hydroxymethylcytosine in the brain. But once again, this is such a new science. I mean, even the reasons are not there. I appreciate this question, but to give a concrete answer now is going to very have ... Whatever papers have been published, it does show that there is a role in transcription, and based on where it is increased, if you look into the histone mark, which are four transcripts on the active gene that actually falls with hydroxymethylation media enrichment protocol.

Srihasa:
So my guess, as well as the scientific community that are working on, we strongly believe that it is a transcriptional control mechanism in the cell.

Srihasa:
Now, could it be a disease where the demethylation plays a role? Maybe. But once again, everything goes back to transcription, and I'm pretty sure it has a significant role in transcription, as well as development. Maybe some diseases like Alzheimer's, but I would not put my money down there until unless I see some papers.

Pete:
Okay, great. And just with respect to the PCT kit for the 5-HMC detection you have, just from a technical standpoint, what is the lowest input amount of sample that's required to run that assay?

Srihasa:
In my lab, we typically use between 15 to 100 nanogram of the total DNA. For qPCR you can go to 10 nanogram, 20 nanogram. We have been very fortunate to get around 10 nanogram we get very reproducible result. But if you are looking into how much DNA should digest, I guess if you have half a microgram of DNA available, you should be able to do all hundred different things. You can run the QPCR in triplicate or quadruplet, and if you are just looking for a yes or no answer, the endpoint PCS, I think 100 nanogram DNA is probably way more than enough.

Pete:
Okay, great. Now, we have one follow-up question to one that I asked you early around cytidine deaminases and the impact on hydroxymethylcytosine, and that is, within the context of cancer, is the decrease of 5-hydroxymethylcytosine in tumors gonna be due to the increased expression of cytidine deaminases and deamination? So do you you think it's a cause, a close correlate, or a potential effect at other things like the increased expression of cytidine deaminases? This comes from the same researcher that had an inquiry around if you had looked into hydroxylthymidine with other means like mass spec in these types of studies.

Srihasa:
Okay. I mean, I guess the cytidine deaminase question is this ... This is the same scientist, obviously, okay. So the answer is that any time you have a higher expression of the mRNA, and if that correlates with the protein and the substrate is there, it can actually move very, very quickly. So you can get higher amount of the product down there.

Srihasa:
But once again, it's like a chicken and egg story of which one is coming faster, whether the hydroxymethylcytosine is coming faster or the deamination, what is that, the enzymes are expressed in a strong manner. I think, once again, one has to look into the tissue sample in a more detailed manner to understand that, but cancer is a heterogeneous disease, again, I mean it's just ... Each cells are very different in their differentiations stage, so I'm afraid I would not be able to speculate too much into that, because there is no science to explain that. But it could be just a speculative answer here.

Pete:
Gotcha. Okay. So we had some great questions from the audience. And Srihasa, thank you so much for taking the time not only to present but to have a really lively Q&A. So we'd like to thank, also, our sponsors, again, New England Biolabs. And thanks of course to the audience for taking the time out of your day. We all know that you're each very busy, so thank you for taking the time to listen. And if you've enjoyed this seminar, you'd like to view the recording of this session, you can do so by visiting the seminar's page on bitesizedbio.com, and that recording should be available within the next 24 hours, and there you can also peruse through all the other webinars that they have archived there. And be sure to check back often, 'cause they're adding many throughout the year.

Pete:
So, 'til next time, good luck with your research, and goodbye from all of us at Bite Sized Bio and EpiGenie. Take care.

 

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