In this episode of the Epigenetics Podcast, we talked with Tanja Vogel from the University Clinics Freiburg about her work on epigenetic modifications in stem cells during central nervous system development.
During our discussion, Dr. Vogel shared that she and her team have investigated H3K79 methylation and its functional significance, which remains a topic of debate in the scientific community. They’ve also investigated the role of DOT1L in neural development and its implications for neuronal networks, as disrupting DOT1L can lead to conditions such as epilepsy and schizophrenia. They explored the function of the SOX2 enhancer in the presence or absence of DOT1L enzymatic inhibition.
The conversation then shifts to FoxG1, a vital player in forebrain development. The team uncovered its role in chromatin accessibility and its connection to microRNA processing. Their study, utilizing ChIP-Seq, reveals FoxG1’s interactions with enhancer regions and other transcription factors, like NeuroD1.
welcome to the epigenetics podcast from active Motif join host Dr Stefan Dillinger for lively discussions with leading epigenetics researchers hear about their past experiments what they’re working on now and what’s coming next you know their papers now get to know them and discover the stories behind the science hello and welcome to this episode of the epigenetics podcast today I’m happy to welcome Tanya Fogle from the University clinics tror on this show please let me briefly introduce you to our audience you got your PhD from the medical school hanova in Germany in 1997 you then moved on to do a post talk at the MC human genetics unit Edinburgh and at the MPI for biophysical chemistry in gingan and started your own group at the University of gutan in 2005 you then became professor in 2010 and joined the University of fryberg in 2011 where you are still today a question I like to ask every guest to start off our little podcast is how did you did you become interested in biology in the first place and then in pursuing a career in science okay that um is a very nice startup and um yeah I’m happy to share that basically due to my my school training I was uh very broadly interested in basically everything so um I um had biology as um sort of one of these major subjects but also history and uh initially I was planning to become a teacher for history and something else but um at the time I was afraid that I would end up as a taxi driver and so I thought okay maybe I take um a subject that is uh more upfront and thought that molecular biology would be would be a nice um way to go for and then I decided to um study biochemistry and plain biochemistry at the time there were only like four um universities offering courses and uh I was lucky to um to get um um admission to the University of Hanover and there I studied then biochemistry and uh yeah retrospectively speaking um I’m really happy that I took this um um chance decision um and that I was then diving into into molecular um biology basically and biochemistry which is my subject of training what about the history of science would that have been something that is a good question retrospectively as well but uh it’s hypothetical so History of Science um is yeah this is this is the past right and now I think I’m more eager to learn about the unsolved problems that are lying there before us and uh this is really driving driving me every day that um I want to uh unpack the boxes sometimes um harboring the the Pandora yeah maybe we can we can look at some of the boxes that you already packed and unpacked um so um your science centers around how epigenetic modifications instruct stem cells during central nervous system development Fox G1 and the ethology of foxg1 syndrome and signaling Pathways and the connection to chromatin obviously so I want to start in 2010 there you highlighted the contribution of piston methylation in the neuronal specification of upper layer neurons in an af9 mutant Mouse model can you talk about this story this must have been one of the first paper out of your own lab right yeah this is basically uh well that is still part of my post-doctoral training I would say because I started the project um when I joined the group of pet Gru and the max plank Institute of biophysical chemistry in gingan um and there basically we were interested to um identify factors that um showed sort of a regional expression um in um the forbrain um because this is a highly organized and aized structure and um we were and are still interested how this develops and um then I um came across this transcription factor af9 or mllt3 it is translocation um hotspot and fuses with um ml which is hisone h3k4 um um methyl trans phrase and at the time basically uh it was that that is has a strong Ling to Le leukemia and at the time it was not clear how these um modifiers and to which um af9 um or ml3 belongs um which under mutation mainly show homeotic trans formations what these um molecules would do in the brain and you know homeotic um transformation during development is also um mediated by polycom um protein complex and this basically attracted my my interest in exploring how basically Hawks Regulators that are not expressed in the brain what they are doing there and that basically drove me um towards exploring ml3 af9 and for this I was happy to to characterize this mouse model and for the first time show that um the um histone H3 k79 methylation basically has an impact of specifying a specific subpopulation of neurons which are these um upper lay nurin and that was also my first attempt to um to do chromatin immuno precipitation um they’re um teaming up with the lab of um Steven Johnson at the time in gutan and that was yeah my first real contact I would say with the chromatin by precipitation and some qpcrs so talks when it comes to chromatin are mostly about modifications of the hisone tail so Li in 79 is this still considered part of the tail or I mean it’s always where does the question where does the tail end is k79 already part of the core is there something special about k79 that makes it maybe difficult to chip um yes multiple questions so first of all I would uh consider that k79 is really in the nucleus on the core and not in the tail and I think that is also um shared by all colleagues working on on on k79 mation um it is very well chable nowadays so there is there’s not a problem um what poses um more a problem at least for my lab is to basically show the enzyme. 1l itself and where it is present right so we are mainly working in the mouse and we think that antibodies recognizing do one L in the mouse are not really convincing in in all circumstances but the mark itself does not make um huge problems so we have um and colleagues have generated multiple data sets right showing um really nice chips SEC tracks and also doing um M chip and qpcr um usually gives um really um faithful results with the respective controls so I would consider that that this is not a problem um the meaning of k79 methylation that is um I think a difficult um part right so um literature is full of um report reports claiming it’s an activating Mark and uh it is of course Associated also with um active transcription um we usually pick uh and also report on um some Contra contrasting results which um show for example that k79 dimethylation specifically is also found at um loai that um are not active but are more more silenced or now days I would say that they are sort of poised because we see um um a nice cross talk also to k27 um trimethylation and this is also not um a single observation by my lab but also other um um colleagues report on on on um this m this correlation between the two marks and that might be an interesting um way basically to follow up research or Al on k79 methylation right so um I I don’t know basically the meaning right I mean you find the different methylations mono and trimethylation at different um sites um I said we are we are mainly focusing on the DI methylation and um we or I don’t know what it is what what the meaning of this of this meth relation really is now because I think that we sometimes have like conflicting results between genetic knockdown and or removal um of the dot1l protein and then its enzymatic inhibition so that does not necessarily follow a straight um um interpretation and the black and white pattern but it is sort of difficult to to untangle there is no reader for this Mark as of yet and or I mean only few reports claiming there would be um and of course there’s also no way to um um actively get rid of the mark As we um think um as of yet so it is then the only option right to to dilute um um this modification through um um cell cycle regulation or to going through multiple rounds of of of cell division which then does not play any role the nervous system if we look at neurons because neurons are terminally differentiated cells which um do not divide right and so um that would basically mean once you have k79 methylation at One S should stick there right yeah so you you touched already uh on on many points of my next questions so one of of those would be1l right it’s an interaction part of the af9 protein and it’s I think you mentioned it just now that the reader no not the reader because there is none the rer of this modification um you started to investigate 1l um so what did you find about the function of1 l in stem cells I think you you looked at it in neuro stem cells um and also in the process of neuronal development MH so um the function of 1l is is we um well looking looking at all um data that we have acquired from different um um neuroanatomical locations including the forbrain and there the dorsal and the vental tanyon for example and the hippocampus we looked we looked in the cerebellum and also in the spinal cord um I have the feeling that dot1l is um really a gatekeeper or a state preserver um um of a given State either being a stem cell so you have to overwrite um um the information um um that1 L is is basically um um referring to uh to trans transfer to the next stage right for example you have a stem cell and the stem cell wants to become a neuron at some point so you have to actively overwrite the information that oneel um um is is given to activate programs um that allow a cell to express um genes that are necessary for neuronal differentiation and this basically is an observation that really holds true for all of the um and systems that we have looked at um but also um if you if you look at um at the next stage if a stem cell has um acquired neuronal fate already And1 is um regulating transcriptional programs that are implicated in pathf finding and to to Really place then the cell um um into the network that it belongs to right that is a um peculiarity I think of the nervous system development that um after a stem cell um um has left the the active um stem cell um proliferating um State and becomes differentiated that it also has to migrate um um to the to the site where it then um finds it um well it its last um um location for example um in the cortex that means that you um um migrate as um as a young neuron from um a part where you are near The ventricle um towards the outer surface right and that can be quite a substantial way that you have to to um overcome or if if you are a specialized uh neuron and you’re born in the ventral tanon you migrate um um tangentially um even longer um to find um um your place within um the cortex and within cortical networ and um here we have found that1 L also is implicated in um um finding the terminal place where um a young neuron basically then integrates into into the system and that um is of of relevance right that because you could um um imagine that you have diseases where um you have a disbalance between um or within a neuronal network where you have over excitation or under excitation right and this is something um that you don’t want to happen because it can manifest in in diseases like for example epilepsy or schiz Fria or I don’t know any or there’s a whole bunch of of more um um neurodevelopmental diseases that impact the balances um of activity within a neuronal network which would be a very important role right I mean every protein or every factor that is involved in neural development neuronal development this is very important and probably very hard to study because if you get rid of it it will be not not be easy to study probably yeah if you’re too harsh in your condition and your factor is too important then of course um the organism is not developing properly and usually um um you have strong phenotypes which lead to um for example perinatal lethality this is also something that um most of our mutants um suffer from there’s a good question why do these do these organisms die which is a question that we cannot answer right um because they have like smaller brains but this is not necessar reason for um not being able to live at all um but um yeah1 L is is is very important for the entire development this is the reason why um the um proper and the full knockout basically does not um reach later developmental stages then in the mouth like seven days right which is very early before you really have um important organ develops the development being initiated so one L um mediates all three degrees of methylation of hdk 79 what we already talked about and it’s animatic activity is critical to modulate Cellular differentiation and reprogramming we already talked about that and you also investigated um the uh accessibility of the so 2 enhancer in this context um what did you learn about the function of this enhancer or about the accessibility yeah this was um also really um um interesting to see that um um HK 79 dimethylation is not restricted to Gene bodies where basically it is it is mainly found but um that within and neural precursor cells um where we were working on but also in in cancer cells um we saw that um k79 dimethylation is also impacting enhancers and through attack sequencing um which we correlated to these chip sequencing um data we saw that um we have the mark specifically at enhancers and they um um impacting upon um being present or being removed the accessibility um toward socks to enhancers funny enough um these socks to enhancers or all accessible sites um um um upon 1l enzymatic inhibition do not necessarily um also have the mark so the enhancer can change its accessibility upon dot1l inhibition without being marked with k79 di methanation which is also um um well another miracle that we that we somehow have to have to solve right and to to understand how how that can be so maybe one L has another function or another Target yeah um I think that one L um has its main function really through its interaction partners and acting in complexes and that maybe the k79 method relation itself is sort of um a flagging of telling the system that1 was there um but I’m not sure whether the mark itself has has a real driving force itself right because that is um yeah it it might be really a bystander and not a a cause really cause um um for the effects that um that we are observing do you know if there is any connection to h3k 37 uh 36 because it’s also found in gene bodies and is an active Mark yeah and there um there are reports um and we also have some um um unpublished um observations that um oneel might um um um Co or co- influence slicing uh which means that it’s also um comigrating with um the the RNA po2 Machinery right we know that1 L um or the complex um that contains 1l is also um interacting with um um the ctd of of RNA Pole to so they travel um along and of course also um um this h3k 36 um trimethylation has similar pattern right so um and and for this I think that at least for some genes where we have this striking um methylation within the gene bodies there is um um a strong correlation to to k36 trith however we never saw massive changes in k36 trith upon entic interference with dot1l itself so I think that they are really um correlating um but not acting causatively together one may say so and not I think we I I heard this like in one of the last episodes um no just because they are at the same place doesn’t mean they are acting together right they must yeah yeah absolutely absolutely this is also what we what we observe now with the k27 triod right where we where we have indication that E2 um is affected um in in some systems by um um changing its expression but sometimes it’s the recruitment um so um there are um yeah ways to cross talk but I think there are um versatile there are there is a plethora of um possibilities how that can be exerted and um it will be really interesting to learn um with with um novel experimentation whether it’s direct or indirect right whether it’s just making space for the other one and uh um not occupying um sufficient um sufficient parts or or places of the chromatin um so that um other factors cannot access for example so this would be a scenario that I could Envision that um explains many of these um cross cross talks which might be indirect and correlative so we are recording this in October 2023 and over the course of the summer like in August September yeah August and September um three papers regarding one I’m just looking at your apartment record here were published um and I think the h3k27 tration um information you just shared was part of one of those um so is there something um you can share about those three studies that that uh yeah you want to share for our latest results yeah this um they basically completely unrelated the the uh yeah the the the bracket or yeah that that is only dot one L so um um first we um um have published um a story of where uh we looked at the enzymatic activity of dot1l and during cortical development um of the glutamatergic that means exit excitatory neuronal lineage um where we used um extensive um single cell um characterization Pati s um initially I was interested of how much of um the 1l information is basically transmitted to a next cell Generation Um however um we faced some some problems in resolving on single cell uh level um the the chip um um results chip sequencing and uh with the Advent now of single cell cut and teag I think that we can um go back to to this initial question but in that story um we were able to basically show that um the inhibition of um1 L um leads to an activation of metabolic programs and that on one side is um um switching to um a state of glycolysis that is a Hallmark of differentiated neurons and on the other hand we saw that um1 L regulates as Asen synthetase um which um as the name says um synthesizes aspirin which is also an important metabolic enzyme for brain development because mutations of that result in smaller brains called micr and there we could show that this inhibition of K of dot1l um leads to the reduction of k79 dimethylation but also to k27 trimethylation um which then basically allows that this Gene um um gets activated prematurely um probably in stem cells which um is followed by differenciation this is also what we have shown then by overx and the aspirin synthetize in the stem cells which is sufficient to drive them um out of their stem cell State and to initiate a differentiation and another paper we basically looked at interneurons um and they um have a different um origin and a different developmental umum logic um but they also depend on um um expression of dot1l there um in that study we also observed that um stem cells exit prematurely the cell cycle they differentiate too early and by this they they are exhausting the pool of of progenitors so that um the last neurons that are basically born um there there are not sufficient um stem cells for them to generate them and this is why numbers drop and um this is uh what we have what we have seen when we do um an interneuron precursor specific knockout of dot1l that we see that the last interneuron population is not made in sufficient numbers and um in the third study that um basically describes the um um the complex or one of the complexes that1 L is part of and there we looked at npm1 nucleo falman one which is also an important um molecule for for cell biology um it is um it has many functions and the function that we were looking on was the organization of the nucleos and um there we basically saw that um the knockdown of npm1 um comes together with um increased k79 diation levels which was also observed by by other colleagues and uh we could show that1 L and npm1 are engaged in a feedback loop of regulating each other’s expression probably to to balance um availability of the respective proteins and uh that this overexpression or the over um the activation of of dot1l expression and k79 diation comes together with um um A disruption um of the nucle architecture probably not because of dot1l but because we reduce npm1 levels but both of them basically impact the expression and um of um a major satellite repeats which which we have observed um um yeah which is which is the main story right you you see it’s it’s it’s not so so much on one L it’s more um characterizing npm1 but um we did not find um really good evidence what both protein could do together in a in a complex I see mhm so Switching gears a little bit another protein next to1l that you are working on is Fox q1 um Fox G1 prevents cell Cy exit by binding to the smut foxal protein complex and you investigated the details of this process um what did you find there MH yeah Fox G1 is one of these transcription factors that are of absolute importance for the developing um um forbrain right is it a Pioneer transcription Factor right it is a transcription Factor yeah a p a Pioneer transcription Factor um Pioneer um I would doubt that it’s a real Pioneer um um and being able to to first open up and then basically allowing um chromatin modifications to to be placed I think it is a modulator um of um of the chromatin oh yeah yes sorry I was I was mixing it up with Fox A1 Fox A1 is is a clear Pioneer right but for Fox 21 this um is has not been shown and um also our um chromatin data do not really propagate this um Pioneer function so it is it’s a modulator of Chromatin um interpretation and accessibility yeah so um yeah Fox J1 is is really important we used it um as a as a um as a cre line for um studying one L and in this context because it’s a Knockin um we also thought okay it might be a good idea to study Fox J1 itself and this is why this is a spin-off and and and being now in on line of of research Fox G1 um has also many many functions um at the chromatin level but also aside from the chromatin level and we um we also studied this umum impact on posttranscriptional regulation for example by um association with um the um micr RNA um processing machineries um and um at the chromatin level we were reporting um earlier um this year um a large study of where we used um also chip sequencing for Fox G1 um also attack sequencing to understand um um what and how um Fox G1 can can influence um gene expression at the chromatin level and fox1 is also mainly reported as a transcriptional repressor as usual we find that it’s also an transcription activator contradicting um um these um earlier findings but I think this is now also accepted in the field that um depending on the context foxg1 can activate or repress and um we found a strong um um correlation to um enhancer regions again and um reduction of fox1 um impacts chromatin accessibility and K 27 AET ination at these sites and uh there we also found that Fox G1 um um is not only acting on its own but that it has important um interacting um um transcription factors such as neurod1 and there we um basically um found which which I find very um attractive also as uh as as way to to regular transcription that uh there is not a clear hierarchy between these um two transcription factors but that they um um influence each other sometimes it’s neurod D1 binding and recruiting Fox G1 sometimes is the other way around and in some occasions they they also um act totally independent from each other so um I think what drives us now with with future questions is really um um how these site directed recruitments and and how these site specific dynamics of uh transcription factors of Chromatin modifiers um Can can be explained right because we know that they are regulated and that they find their place but we don’t understand how that’s that’s going and this is basically where the signaling um comes into play um you have um um mentioned correctly that Fox 21 um acts together with a smart pathway that means it’s under control of the transforming um growth factor beta family TGF beta um but this um is not the only one it’s also um controlled by um fgf signaling that fiber blast growth factor signaling and um it will be really um interesting and also one of our next attempts to understand how signals basically um regulate um these chromatin specific um events that are occuring for transcription factors but also for chromatin modified so you touched many of the questions I wrote down so maybe I can can just pick some um so you mentioned the the the story that you published earlier this year so 2023 and there you used um as you said you used many uh methods together in a multiomic approach so uh why did did you choose this multiomic approach what did it enable you to do what was the the real Advantage here yeah um that was that was um a factor that delayed our our publication quite substantially because uh we did this um um fox21 chip and um I was really surprised to see it at enhancer regions and then uh within we were working in in primary hippocampal neurons and you know data sets are sparse um and not not many um um yeah all make data sets published on this particular um cell types so we were forced to um really show that these are enhancers which then basically um um drove us into performing the k27 aculation um chip sequencing but because we want we did not only want to k27 um acetel we also included um h3k4 trimethyl and um and I think also 27 Tri meth within this data set so that was that was the second multiomic part and then because we were then proving that we are working with enhancers we said okay if foxg1 um um impacts k27 acation it enhances it will probably lead to um differences in chromatin accessibility the way to address that is then to to do attack sequencing which was then the next step that we did and um in the course um um of this experimentation we wanted to explain why k27 acation is changing because Fox G1 is not um a Hy AO transferase or de atilas so it basically um has to interact with something so we did the proteomics analysis we saw the H decks associate with boxg1 and then basically the question is of course if you work on a on a neurod developmental disease or whether you can use this information um to uh Drive therapy and we thought okay there are so many um HTE Inhibitors and if um fox1 um works together with h deex so maybe we can exploit this information for doing therapies and there we then did um um um an inition um um experiment of H deex and this of course meant that we had to um do another um data set and uh look for um transcriptional um alterations which is basically yeah opening one box uh leads to many more boxes that you have to open to understand the biology behind um one point you need to say well now it’s enough right now it’s enough we have done enough experiments let’s let’s write the paper yeah I yeah there was one question after the other and of course now we have um again um um a plate of um questions that we would like to ask um using uh using multiomic approaches which which I find um really really fascinating to work with because we we always have a genome wide view and um there are so many observations that you can draw or that you can that you can um observe um um in the single data sets but if you then combine you have other um views on the regulative regulative mechanisms and um and to untangle these and to understand the the individual molecular Machinery I find absolutely fascinating and driving our research mhm so you already kind of answered the my next question but imagine you are due to submitting a grant proposal tomorrow um what would you have written into that or maybe would have written into that if it was not coming from your lab now so maybe a more imaginative Grant proposal oh well um it’s not that we don’t have many ideas in our head so um obviously for Fox G1 we would like to understand or or to come a little bit closer towards um understanding the human disease because human development is a little bit um um different from what we know from the mouse of course and we would like to um um get funding for um driving our research on human Fox G1 and using brain organoids and now um um putting basically um everything that we have um learned um in in methods but also in in the biological system from the mouse towards the human system so that is what we are doing there and um in regard to to1l research um I mean I would really really love to understand whether K20 k79 methylation has a meaning or not but for this um this question is is is really difficult to untangle and I don’t think that I could write the gr just now I’m always thinking that um larger Labs um equipped with more Technologies they should tackle um these difficult questions but since nothing is basically being published I think that other labs and other pis or other researchers are as clueless as I am understanding this um this this particular Mark um at this very moment so for the last almost 40 minutes now we have taken a journey through your scientific career um did we miss something important or would you like to add something um no I think uh this is this is mainly the the content right of my of my research of course there’s many things that we did not talk about um all um the failed attempts for example ex that um come across or come come alongside our our Scientific careers or these um hypothesis that uh you find absolutely brilliant at the moment that uh you think about them but which um you can never prove um this is of course um also something that um is is part of any any scientific career like probably people do not do not touch on these points too much but um I think we should be and also encouraging um and the younger um and and early research um um careers for not giving up but uh to follow the path and to find um um yeah the right boxes to unpack and it’s clear that sometimes a box is empty um but um yeah nevertheless science is real fun and you can ask so many questions so yeah being curious is uh yeah I think a main driving force so thank you Tanya for your time and for being on the show yeah thanks very much for your time and having me here and uh yeah enjoy Christmas everyone um who might listen 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