All tags Epigenetics Epigenetic Mechanisms in Early Mammalian Development Webinar

Epigenetic Mechanisms in Early Mammalian Development Webinar

Watch this on-demand webinar presented by Dr Maria-Elena Torres-Padilla 

Join Dr Torres-Padilla as she reviews the basic mechanisms underlying the earliest steps of mammalian development to understand early aspects of embryonic development, human reproduction and stem cell biology.

Webinar Topics:

  • Reprogramming and cell fate
  • Heterochromatin formation after fertilization
  • Nuclear organization and chromatin composition


About the Presenter:

Dr Maria-Elena Torres-Padilla completed her undergraduate studies at the Faculty of Sciences of the UNAM, Mexico and in 2002, obtained her Ph.D from the Institut Pasteur, Paris.

Between 2002 and 2006, she was a postdoctoral fellow at The Gurdon Institute, University of Cambridge, before working as a scientist with Laszlo Tora until 2008.

Maria-Elena currently leads a team studying Epigenetics and cell fate in early mammalian development at the IGBMC in Strasbourg, France.

Webinar Transcript:

Hello and welcome to today's webinar. Today's principal speaker is Maria-Elena Torres-Padilla, principal investigator at the IGBMC in Strasbourg, France. Maria-Elena did her undergraduate studies at the Faculty of Sciences of the UNAM, Mexico and obtained her PhD from the Institut Pasteur in Paris in 2002. She was a postdoctoral fellow at the Gurdon Institute, University of Cambridge, between 2002 and 2006 before working as a scientist with Laszlo Tora until 2008. Maria-Elena currently leads a team studying epigenetics and cell fate in early mammalian development. Joining Maria-Elena today will be Sonja Flott; Sonja has a PhD in Molecular Biology from the University of Dundee. Before joining Abcam, Sonja worked as a postdoctoral research associate at the Gurdon Institute in Cambridge, and has a diverse background in molecular biology and biochemistry. I will now handover to Maria-Elena who will start this webinar.

METP:    Thank you, Lucy. Welcome everyone. Today I am going to be talking about epigenetics and cell fate in early mammalian development. First of all, I would like to introduce the model system that I am going to be developing, which is the mouse pre-implantation embryo. This first slide shows you the very early stages of mouse embryogenesis that start at fertilization, from the oocyte and the sperm. After fertilization, of course, the zygote which is depicted here has to regain what we call developmental totipotency. That means that the zygote has to be able to generate all of the cells available in the organism. After this zygote starts dividing, the zygote starts forming a blastocyst after the third day of development. The blastocyst is the first time when you can actually distinguish two differentiated population of cells in the embryo. So the inner cell mass, which is depicted here in green, is actually pluripotent, whereas the trophectoderm, which is depicted here in red, is considered to be the first differentiated tissue in the embryo. So the mouse embryo is an excellent system to understand how the transitions of potency, from totipotency to pluripotency and differentiation are actually governed. Our main question is how chromatin regulates these transitions, and whether epigenetics has its role to play?

There is a number of things that happen in the embryo during these very early stages: one of the most important ones is that the embryo undergoes its activation of the genome for the first time. It is known that the oocyte, which is the mature gamete, is actually silenced transcriptionally. The oocyte carries a load of maternal transcripts that will be degraded by the 2-cell stage. The embryo is known to activate the genome by two different ways: the minor one that, of course, is here in the late zygote stage; and the major one, which is more robust than the first one, and it occurs at the 2-cell stage. That implies that there are a number of things occurring on the chromatin to allow this transcriptional activation. For example, acetylation of histones, recruitment of chromatin-associated proteins, but also phosphorylation of the RNA polymerase II, and this is believed to leave poor initiation of transcription.

Now, when I say that we are interested in chromatin, you can actually visualize a chromatin in a very simplistic way, as the way to organize the information that is stored in the DNA. So depending on how you organize this information you can generate different epigenomes, and there are a number of mechanisms that can regulate how this organized information is impacted. For example, the histones themselves that are present in the nucleosomes and the content of this histone, in particular the histone variants which can be of different sorts. For example, H3 has a specific variant and tissue a specific variant, but depending on which histones you put in these nucleosomes, you can give rise to different chromatin structure. Of course, the histones are modified and this also has a very important impact on chromatin function. The DNA itself can also be methylated and, again, this also regulates chromatin function. Lastly, chromatin remodeling whereby the chromatin remodelers can physically move the nucleosomes around. You can transit between a very closed chromatin state, which is a repressive environment characterized, for example, by K9 methylation of H3 and this leads to a condensed chromatin. The open state has acetylated histones activating marks, and this is more permissive to transcription. Anything that regulates histone modification, DNA methylation, but also chromatin remodelers can give rise to these two different chromatin states.

Now, one of the main questions that is in our interest is as following: Is the chromatin at the basis of the cell plasticity and epigenetic reprogramming that are introduced? One of the reasons why we think that it is the case is the following. Here you will see the different stages of early mammalian development, and what we think the plasticity does; so very plasticity at the beginning that is reduced upon development. If you look at the DNA staining of the nuclear representative of these different stages, you see by just looking at the DAPI that the chromatin is highly reorganized. This tells us that perhaps globally the chromatin is involved in regulating the cellular plasticity. In my lab we want to understand the following questions, so all of them are involved in the question on the top, which is how the structure of the chromatin is set up during development? I will be dividing my talk into two different subjects: the first one is to try to understand whether chromatin regulates cell fate, and the second one whether it regulates reprogramming? But before I come to these two questions, I will tell you a little bit on how we can address how the structure of the chromatin is initially set up.

Why do we need to study why the chromatin state is reorganized, and I think it is very simple and it's just because after fertilization a new chromatin state has to be established. The reason behind that is that the sperm is mostly packed in protamines and not in histones. So during fertilization these protamines, depicted here in red, have to be removed and the paternal DNA has to be assembled into a nucleosome configuration, de novo. That means that in the zygote where you can distinguish the male and the female pronucleus, you have a pronucleus that has to be assembled into chromatin and marked. On the other hand, you have the maternal chromatin that is inherited from the originals. We believe that this process of assembly and marking is very important in keeping with the reprogramming, and the developmental plasticity. A very easy way of understanding how this assembly process is occurring, is the following. We started by asking, when are the histones first incorporated in parental chromatin? The way you can do that is by using a histone, in this case, a histone H3.3 fused to a GFP, generated mRNA in vitro and microinject this in individual embryos. Then do a time-lapse analysis of the embryo, which is what you are going to see at the bottom. So you see two different cycles just after fertilization, and on the right side you will see the signal from the GFP.

What you will see is that you can actually time very nicely when the GFP starts being detected in the parental chromatin. This gives us an idea of what histones go to which parental chromatin, and under which time. What we have understood, what the components from the embryonic chromatin, one can ask the following question, which is, how does newly formed chromatin acquire its identity? In other words, one, you have the histones, what tells the DNA that it has to be silenced or it has to be active? To try to understand this we have focused on a heterochromatic region, which is the pericentromeric. The pericentromeric of the mouse chromosome is depicted here in light pink, and this is an important region to secure mitosis on segregation. The pericentromeric in the mouse is formed by what we call 'major satellites', which are tandem repeats of 234 base pairs. The question that we have been trying to address is how these tandem repeats become silenced after fertilization? I remind you, we think that this is essential so that the first mitosis can occur.

What we proposed a few years ago is that a specific incorporation of H3.3, so the histone variant is necessary for the formation of this chromatin. What we showed is that these major satellites become transcribed very early in development, during the first S-phase. That this leads perhaps to double-stranded RNA formation that could potentially help in tethering heterochromatin protein 1, together with an acquisition of K27 methylation, and this will lead, for the first time, to heterochromatinisation of pericentric chromatin. This leads to silencing and to the proper localization of the chromo centers at the 2-cell stage. It is important here that I mention that I am talking about K27 methylation and silencing marks, because the paternal chromatin is devoid of K9 methylation. One of the things that you might be surprised about, is that I am talking about silencing chromatin and, at the same time, I'm saying that this needs to be transcribed. So keeping with the models that were described for yeast and Arabidopsis we think that in mammalian cells heterochromatin also needs to be transcribed to be silenced.

The following question is: Is that transcription mechanism all-important for the pericentric chromatin, or could these be a general mechanism for all the heterochromatic regions in the genome? So would a similar mechanism apply to other heterochromatic regions, and this is really to try to address how heterochromatin is established. So the question will be: Can we find transcription of other heterochromatic regions in the embryo? For this, what we did is to use repetitive elements in the genome as a model, because we know that they must, at some point, become heterochromatic until they are fully silenced in differentiated cells. What we did is to ask whether, for example, two major representatives of the repetitive elements in the genome, like Line L1 and IAP are transcribed during early development. What you are seeing here is RNA-FISH analysis studies to detect nascent transcription of first* stage and 8-cell stage embryos with a probe for Line in red and IAP in green.

As you can see, both the Line and the IAP elements are actively transcribed at the 2-cell stage, and their transcription decreases as development proceeds. So what I did suggest is that all the heterochromatic regions, apart from pericentric chromatin are actively transcribed. Now, of course, you would wonder how the transcription of these elements could be regulated to start with, so what is activating the transcription? One of the things that we became interested in addressing is whether they could be related to RNA? What we were able to show is that the small oligonucleotides that are depicted here in red, from elements like Line L1 can regulate transcription of these elements after fertilization. The experiment that we did to address that is the following. What we did is to microinject these RNA, as I said to you, in the zygote and then perform RNA-FISH at the 2-cell stage. This is what you're seeing here, RNA-FISH signals per line, and for IAP in the controls. What you can see is that when you inject the RNA depicted in this region, you can see an increased transcriptional activation of the line element, but not of the IAPs and this is quantified here in these squares that you see in the graph. If you inject a scrambled RNA you do not have any effect, so what it suggested to us is that these small RNAs are derived from the Line element, can also enhance the transcriptional activity. Suggesting that after fertilization, RNA could be an important regulator of the transcription of this element.

As I mentioned to you, we are interested in understanding how these newly-formed chromatin acquire its identity? Again, coming back to my diagram about the pericentromeric domains, one of the things that is interesting in the embryo is that if you look at the localization in an interfaced nucleus, it has quite a peculiar localization. For example, if you attain your somatic cell of interest with DAPI, you will see the very typical localization of the chromo centers, which contain these pericentromeric domains. In contrast, if you do the same staining in the zygote, you will see that the localization of the pericentromeric domains is in these rings that you can see. These you can actually also look, if you use a probe this time using DNA-FISH studies to localize the genomic regions of the major satellites. What you will see is that the major satellite probe forms these rings, and what you're seeing is a specific nucleus at the 2-cell stage on the left, and the reconstructed 3D analysis on the right.

So the fact that these regions are localized in this very peculiar way prompted us to ask, what are the spatial localizations of pericentric chromatin is important for silencing, and for development? The way we addressed that is the following. We used a zinc finger protein that was designed specifically to recognize the major satellite repeats, and we fused these to the emerin which is a nuclear membrane protein. What we expected, or we hoped for was to be able to displace the major satellite repeats instead of getting these rings that I showed you earlier, to the nuclear periphery. The way we did that, again, is by using this zinc finger fused to the GFP as a negative control, and a zinc finger to the emerin. We produced mRNA in vitro of each of these two constructs, and then injected it in the embryo to express these proteins. If you analyze the localization of the resulting proteins, this is what you see, so what you're seeing is two different 2-cell stage embryos on the top expressing the zinc finger GFP, where you can track the localization of the major satellite. At the bottom, you see the localization on the zinc finger emerin protein, which is at the expected localized internuclear periphery.

Now, the question is, is the zinc finger emerin protein able to tether that major satellite? The way we addressed that was by doing DNA-FISH followed by immunostaining with lamin. So what you're seeing are representative nuclei at 2-cell stage embryos, where the DNA-FISH signal for major satellite is in green, and the lamin in red. As you can see in the plot provided on the right, the major satellite FISH signal is around the nucleus throughout the nucleus in both non-injected, and the negative control in the middle panel. However, if you look at the distribution of the major satellite DNA in the zinc finger emerin group, you will see that it is mostly displaced towards a nuclear periphery. This suggests our approach is efficient in tethering the pericentric chromatin to the nuclear periphery. Next, we addressed whether that is important for silencing, and the way one can do that is, again, by performing RNA-FISH. As I said earlier, RNA-FISH can detect nascent transcription, so we can use a probe from the major satellite and in the representative nuclei that you are seeing, the yellow signal on the left represents the active major satellites which are known to have a basal transcription at the 2-cell stage.

If you then compare it to the negative control in the GFP zinc finger, you will see that they have a very similar transcription on the right. However, when you express the zinc finger emerin construct whereby the major satellite is tethered to the nuclear periphery, you will see that the transcriptional activity isn't produced. What we suggested was, is that the localization of the major satellite repeat is important for initial acquisition of heterochromatic signatures. Most importantly, the embryos are not really very happy with this manipulation, because if you compare it to the controls that develop to the blastocyst stage, they're 90 or 85 per cent. The embryos that experienced the zinc finger emerin, shown at the bottom, develop only with a rate of 50 per cent. This suggests that targeting endogenously the pericentromeric chromatin to the nuclear periphery impairs development.

As you can see, we are also very interested in following the dynamics of the heterochromatin, because we know by the localization it is important for their function. So what we have been doing is to try to establish a flexible method to visualize endogenous genomic regions. What we did for that is to use exquisite specificity on the TAL effectors, which are bacterial protein, in recognizing a target sequence. Instead of using a nuclear dye fused to them, which is now usually used for genome editing, what we did is to fuse a fluorescent protein, in this case a GFP or the cherry, or your protein of interest. We call that method TGV for Tale-directed Genome Visualization. We have adapted that method for a number of repetitive sequences, so the telomere for the minor satellite and for the major satellite. I will show you an example of the major satellite, because I have been talking about this regularly. So with the sign of direct sequence, which is depicted on the bottom right, it will be recognized by the TAL effector, and we've fused this to mClover which is the green fluorescent block. You can then experience this in the embryos, and what you will see is that on the left you have the male pronucleus and you can basically track the major satellite in vivo, both in the male pronucleus, as I said, on the left and the female pronucleus on the right in the movie that is starting again; and the red is for the H2B. So with this tool we can now go back to the embryo and track in living embryos chromatin dynamics.

As I mentioned earlier, this method is very flexible because you can also use it to distinguish specifically the parents of chromosomes. What I am showing you here is one of those examples that we used to distinguish the sequencing from mus spretus and mus musculus. What you see at the bottom is the hybrid ES cells that have been transfected with TAL effectors addressed to distinguish the major satellite of mus musculus in green, and mus spretus in red, and you see the comparative DNA-FISH signal on the right. You can also use these TAL effectors to track the heterochromatin dynamics of parental chromatin in vivo in ES cells, as you can see on the movie on the right. So this method is very flexible and you can use to track endogenous sequences in vivo. Now that I've told you a little bit about how we know about the structure of the chromatin and how it slowly acquires its identity, I am going to be developing on the question of how this could potentially regulate cell fate, and how it regulates reprogramming.

In particular, we have been interested in understanding what could be the impact of nuclear organization on lineage commitment and development? For this we have been focusing on the position of loci that are activated in pluripotent cells, as a result of reprogramming in the embryo. For example, Nanog and Oct 4 are transcription factors that are expressed specifically in the inner cell mass, so the pluripotent compartment, but Cdx2 is expressed exclusively in the trophectoderm, which is the outer lining tissue of the embryo. What we started doing is the signing method that could allow us to study the nuclear organization of the embryo in 3D. What you are seeing here is an example of an 8-cell stage embryo that has been processed with a chromosome paint for the chromosome 17, and has a RNA-FISH signal this time for Oct 4, and the nuclei are depicted in blue.

As I said to you, we set up conditions to understand these in 3D and to combine DNA-FISH and RNA-FISH. What you are seeing on the right this time is a represented 16-cell stage embryo, and what you can see in the small right dot is a RNA-FISH signal for Oct 4. What this basically means is that you can pinpoint the cells that are, for example, not expressing Oct 4 that would mean that there is no spot in that particular nucleus, like the one on the right at this moment, but also the ones that have one allele active or two allele active. In other words, the RNA-FISH can give you an idea of the ongoing transcription in individual cells. When we applied this to the embryo what we noticed is that in the case of Nanog, which is on the red, so with a red spot on the bottom picture, is expressed primarily from only one allele in the embryo. This was different to other genes like Oct 4, which is in yellow at the bottom, where you can see the two alleles lighting up. This is what's happening only at the very early stages of development 4-cell stage and 8-cell stage.

When we examined embryos a little later when the pluripotencies are formed, which are the inner cells of the late blastocyst, what we noticed is that Nanog was expressed from 2 alleles. So there seems to be a predominant monoallelic expression at the earlier stages of development, following the pluripotent cells form, Nanog could be mainly expressed from two different alleles. We then went on and analyzed the expression of Nanog also in ES cells, because Nanog is also important for maintenance of stem cells in vitro, and what we developed is an approach where we targeted a GFP and a cherry in the endogenous alleles of Nanog. The alleles allowed us to see the transcription and activity, because we introduced a best signal at the three-pronged region of the GFP and the cherry. That basically means that the GFP and the cherry are going to be very unstable, and they are not going to interfere with the protein function because of the presence of a 2A peptide between the coding region of Nanog and the GFP.

So we used these GFP and cherry reporters as an indication of the transcription and activity of the Nanog loci. What you see on the right is a time lapse analysis of these ES cells where the red reflects, of course, expression of one allele and the green of the other allele, and the yellow of two alleles. What we found in analyzing these movies is that, actually, the cells switched color and that basically means that one cell is expressing a green allele, it can also change to the red allele, or it can also change to the yellow color, which means that they will be expressed through alleles. This basically allowed us to conclude that Nanog is subject to what we called 'Allele Switching', and it also relates in gene expression. The summary of this analysis is present here where you see that there is up to 14 per cent of changes in allelic expression of Nanog. The model that we proposed is following from what we think is that it is important that early during development there is primarily one allele of Nanog being fired, and that is probably because we need to keep relatively low levels of Nanog proteins at the earlier stages of development.

However, when the epiblast is formed, which is the pluripotent compartment of the embryo, perhaps we need more protein of Nanog and, therefore, the second allele is switched on. The next question that we asked is, okay, so these are the transcription factors, but could there be a chromatin environment permissive for the action of such transcription factors in the cells in the embryo? What we asked more specifically was, do highly plastic cells need a particular chromatin environment? Does the chromatin define gradually different cellular states, for example, the very plastic cells are the left, the zygotes on the 2-cell stage compared to the later stages of development where we already see differentiated cells? One can think about different hypothesis, for example, that some cells start being different perhaps at the first stage, or on the eighth stage. These differences might be setting up slowly some excuse for making the cells into a differentiation pathway, or not, or we can think that perhaps what happens is that the cells just lose gradually their potency and at some point they just differentiate.

These hypotheses are not mutually exclusive and there could be a contribution of mechanisms that will be important to set the first lineage allocation, which is visual at the blastocyst, as I mentioned at the beginning. As I mentioned to you, we wanted to understand whether there could be a permissive chromatin environment for the action of transcription. So what we aimed to do, was to generate single cell epigenetic landscape in the embryo, and by epigenetic landscape I meant expression of chromatin modifiers. So we used Fluidigm, which basically is a microfluidics-based approach that allows to work with a very small amount of regions and liquids, and that is very well amenable to understand single cell profiling. As you see on this diagram, we generated single cells from all the different stages of development, and making also sure that in the blastocyst their outer parts, or the trophectoderm which is depicted in yellow, was distinguishable from the inner cell mass which is depicted in grey.

What we wanted to obtain is quantitative expression, information and developmental time to understand the dynamics of the expression of these chromatin modifiers, also spatial information, and what we wanted is to obtain combinatorial data on the expression part. So we analyzed a number of chromatin modifiers on the list as depicted for you, but you see that we included histone lysine methyltransferases, polycomb PRC1, zinc finger set proteins, arginine methyltransferases. Also the transcription factors as controls, internal controls such as RPLP0 and Actin B, PRC2, DNA methyltransferases and also histone lysine demethylases. Then we asked were there, for example, in the pluripotencies of the embryo which are depicted in orange, the ICM, could be different to differentiated cells in the embryo which are depicted in purple, the trophectoderm or TE. What you are seeing here is a principal component analysis of the expression pattern of 36 chromatin modifiers of these two lineages of the blastocyst, and each dot represents a single cell. What you can see is that the two groups of cells can segregate quite clearly.

The advantage of this method is that you can also identify genes that are contributing to the identity of each of these two groups*. In this case, for example, we found the Dnmt3b and Dnmt3L are enriched in the trophectoderm and this is why they are on the left side of the panel. Whereas Prdm14 is highly enriched in the inner cell mass. The advantage of having the single cell data is that you can also identify intraembryonic differences. For example, one of the things that we found is that in analyzing 4-cell stage embryos, individual cells displayed different levels of Prdm14, which disappears at zinc finger containing protein. What you are seeing here is the protein levels in red for immunostaining of a 4-cell stage embryo. You can see that the two nuclei at the top are almost devoid of Prdm14 signal, whereas the two blastocysts* at the bottom have very strong levels of Prdm14. Remember that I told you that Prdm14 is also highly enriched in the inner cell mass, so one could ask whether Prdm14 containing cells early on, could be more prone to be part of the inner cell mast in the blastocyst.

The way we address this is depicted at the bottom, is by expressing certain amounts of Prdm14 with a HA tag, coupled with GFP RNA. You can then trace the cells by the protein from the injected cells by looking at the GFP signal in the embryo, and asking in the blastocyst stage after 3D reconstitution, whether the green cells are located in the inner cell mass compartment, or in the trophectoderm compartment. The results of those analysis are in the graph on the top, that show that a proportion of the progeny of the Prdm14 expressing cells that is located to the inner cell mass is higher when you express Prdm14, compared to the GFP control. If you delete the zinc finger of Prdm14, which is the last bar on the right, you lose that effect, which suggests that the zinc fingers are made of Prdm14, is essential for promoting the inner cell mass allocation. In conclusion of what I have been showing you, we think that the embryonic chromatin is unique and we would like to envisage this as the totipotent cell has a very specific chromatin configuration that is also visible just by globally organizing.

The take-home messages are the following, so the embryo has a particularly dynamic chromatin, there are many changes in short periods of time and terms of histone change in terms of histone modifications, and also the expression of some chromatin modifiers, but also the embryos are very sensitive to changes in chromatin modifications. I would say that perhaps one of the most important features of these embryonic chromatin is the lack of conventional heterochromatins, and this is because the typical specifics of heterochromatic marks like K9 methylation that I mentioned, are either not present or remodeled very rapidly, but also heterochromatic regions, such as retrotransposons are activated. In conclusion, we think that our work will be essential to understand the general mechanisms that are behind cell fate allocation, cell plasticity and reprogramming. I could say that from a more general perspective, we think that it will have major implications for our understanding of stem cell biology development, chromatin biology, epigenetics and also human reproduction.

This is my acknowledgements slide where I would like to acknowledge all the members in my lab that have performed the work that I mentioned, together with the former lab members, but also our collaborations and our funding. This is the end of my presentation and I hope that you can meet us in Strasbourg for the Abcam meeting that we are organizing in October, and I will now hand it over to Sonja who will talk to you for some minutes. I will be back to answer some of your questions. Thank you.

SF:    Thank you Maria-Elena for such an interesting talk. I'm sure you will have plenty of exciting questions from our listeners. Hello, I would like to take this opportunity to tell you a bit more about some of the resources and products that Abcam has available for epigenetics research. We have recently launched a new epigenetics microsite at www.abcam.com/Epigenetics. Here we have grouped all our epigenetics resources in one place to make it quicker and easier for you to find our latest tools covering the key mechanisms of DNA methylation, histone modifications and non-coding RNAs. This includes articles, selected products, protocol hints and tips, as well as relevant webinars and events. If you want to get hold of a hardcopy of a specific poster, you can request download copies from our website. Also, please feel free to contact us if you have any suggestions or comments for new posters, or protocol ideas.

If you have any questions regarding Abcam products, please contact our scientific support team who will be very happy to help you with any query you might have. We have scientific support teams in the US, in Hong Kong, in Europe and in Japan. I would like to highlight that we have multiple language support in German, French and Spanish, as well as in Portuguese, Chinese and Japanese, so please do not hesitate to contact us. If you use large quantities of one product, you might want to look into buying in bulk. Bulk-buying will help you to save money and minimize the variability in your experiment. For more information about our bulk options, please contact our sales team at sales@abcam.com. I would now like to point out Abcam's range of epigenetic products. In addition to an increasing range of ChIP grade antibodies, we offer relevant biochemicals, for example, HDAC inhibitors. We also offer a range of HDAC and SIRT activity kits, as well as ChIP kits that have been designed so that you can spend less time doing the experiment, and more time thinking about the design and outcome.

Today, I will just mention our range of EpiSeeker ChIP kits of cross-link ChIP. Our one step and plant ChIP kits have been optimized for mammalian and plant DNA, respectively. These kits do not contain a preselected antibody and are, therefore, adaptable to any target of choice. The EpiSeeker range also includes kits optimized for methylated or acetylated histone modifications, and can be used either for cells or tissue starting material. We also have kits for immunoprecipitation of methylated DNA. These kits contain specific antibodies to enrich methylated or hydroxymethylated DNA, respectively. These kits are also useful to complement other DNA methylation experiments, such as bisulfite modifications where it is not possible to differentiate between methylated and hydroxymethylated cytosines. More details on these kits and other EpiSeeker products can be found at abcam.com/EpiSeeker. You may ask what are the advantages of using the EpiSeeker ChIP kits instead of following the conventional ChIP method? Well, the reaction takes place on a 96 well plate so they are easy to standardize. It only takes five hours. The kits contain all main reagents, except the cross-linking, and except for the general kits, the kits all contain a preselected ChIP grade antibody which has been optimized. The precipitator DNA can be used straightaway for downstream processes, such as ChIP on ChIP or ChIP-seq.

Lastly, I would like to highlight two upcoming Abcam conferences as these are probably of interest to you. First, the Crossing Boundaries: Linking Metabolism to Epigenetics meeting and that is taking place in Boston in May. Second, the Chromatin and Epigenetics: From Omics to Single Cells meeting that is taking place in Strasbourg in October, as Maria-Elena just mentioned. If you would like more information about this meeting, please visit the meeting website at www.abcam.com/Strasbourg2014. Without further delay, I will pass you over to Maria-Elena who is ready to answer the questions we've received during the webinar. Thank you very much for your attention.

METP:    Thank you, Sonja. I'm going to now answer a couple of questions that were arranged during the webinar. So the first question that I got is I mentioned that the heterochromatin is transcribed, and whether it is also translated? The answer is it depends the regions that have been analyzed so far, for example, the major satellites do not close to any protein as far as I know, but the line does close for a couple of proteins, the ORF1 and ORF2, and at least ORF1 has been shown to be present in the embryo. So, in that case, yes, it is transcribed and it is also translated. The second question that I've received is the following: Why do I think that some retrotransposons become activated after fertilization? What we think is that, as I mentioned, the K9 methylation is not present in the paternal chromatin, the lack of K20 trimethylation, also of K64 trimethylation, so there is a lack of heterochromatic marks. So what we think is that this provides, we know of an opportunity that is permitted for the activation of these regions. I guess that the following question would be relevant, whether the activation of the retrotransposons could be actually important for development at all, or it is just a side-effect of this remodeling of heterochromatic marks.

Now, the following is going to be the last question, which is following the experiments about reorganization of major satellites in most cells it is known that the nuclear membrane is a silent environment. Why? The embryo does not seem to be a silent environment, and so what I would probably argue is that perhaps the nuclear organization in the embryo is somehow reversed, at least in terms of heterochromatin seems to be localized in the center of the nucleus at the beginning, and not in the periphery. But we believe that at some point this is reversed perhaps around the 8-cell stage, and heterochromatin starts being accumulated in the membrane. So we think that, again, this is a nuclear organization feature of the embryo. So with that question, I thank you for your attendance and I will pass it over to Abcam again. Thank you.

Thank you Maria-Elena. That is the end of our webinar and we hope that you have found it useful to your work. If you do have any questions about what has been discussed in this webinar, or have any technical query, please do not hesitate to contact our scientific support team who will be very happy to help you. They can be contacted at technical@abcam.com. Well, I'd like to thank you again for attending, and I'd like to say good luck with your research.