Podcasts about bhlh

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.19.533192v1?rss=1 Authors: Sajjad, M., Zahoor, I., Rashid, F., Rattan, R., Giri, s. Abstract: The metabolic need of the premature oligodendrocytes (Pre-OLs) and mature oligodendrocytes (OLs) are distinct. The metabolic control of oligodendrocyte maturation is not fully understood. Here we show that the terminal maturation and higher mitochondrial respiration in the oligodendrocyte is an integrated process controlled through pyruvate dehydrogenase (Pdh). Combined bioenergetics and metabolic studies show that mature oligodendrocytes show elevated TCA cycle activity than the premature oligodendrocytes. Our signaling studies show that the increased TCA cycle activity is mediated by the activation of Pdh due to inhibition of pyruvate dehydrogenases isoform-1 (Pdhk1) that phosphorylates and inhibits Pdh. Accordingly, when Pdhk1 is directly expressed in the premature oligodendrocytes, they fail to mature. While Pdh converts pyruvate into the acetyl-CoA by its oxidative decarboxylation, our study shows that Pdh also activates a unique molecular switch required for oligodendrocyte maturation by acetylating the bHLH family transcription factor Olig1. Pdh inhibition via Pdhk1 blocks the Olig1-acetylation and hence, oligodendrocyte maturation. Using the cuprizone model of demyelination, we show that Pdh is deactivated during the demyelination phase, which is reversed in the remyelination phase upon cuprizone withdrawal. In addition, Pdh activity status correlates with the Olig1-acetylation status. Hence, the Pdh metabolic node activation allows a robust mitochondrial respiration and activation of a molecular program necessary for the terminal maturation of oligodendrocytes. Our findings open a new dialogue in the developmental biology that links cellular development and metabolism. These findings have far-reaching implications for the development of therapies for a variety of demyelinating disorders including multiple sclerosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.23.509112v1?rss=1 Authors: Masuda, A., Ajima, R., Saga, Y., Hirata, T., Zhu, Y. Abstract: Commissural neurons play the crucial role to connect neuronal information from both sides of the nervous system in bilaterians by projecting their axons contralaterally across the midline. These neurons are highly heterogenous in their developmental origins, neurotransmitters and neurophysiology, as many disparate neuron classes contain commissural neurons. In mammals, most commissural axons from the spinal cord to the midbrain, cross the midline via the floor plate, guided predominantly by a conserved molecular mechanism, i.e., Robo3 and DCC expressing commissural axons are guided by Netrin-1 secreted from the floor plate and ventral neural tube. So far, no common transcriptional program has been uncovered for specifying the axon laterality across the highly heterogenous commissural neurons. In this study, we identified a pair of highly-related basic helix-loop-helix (bHLH) transcription factors, Nhlh1 and Nhlh2, as such a transcriptional program. We found that Robo3 promoter contains multiple copies of Nhlh1/2 binding sites and forced expression of Nhlh1/2 can induce ectopic Robo3 expression and contralateral axon projection in the hindbrain and the midbrain. We then generated mutant mice deficient in both genes and found a marked reduction of Robo3 and a total lack of ventral commissures from the spinal cord to the midbrain. This is the first report of a global transcriptional mechanism that controls the laterality of all floor plate-crossing commissural axons via activating Robo3 expression. Nhlh1 and Nhlh2 should provide the key to deciphering the principle underlying the specific and balanced production of contralateral- versus ipsilateral-projection neurons from the spinal cord to the midbrain. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Point mutations can have a huge impact on the genome depending on where in the genetic code they occur. To illustrate this, we're going to be looking at a case study in the form of almonds- formerly poisonous nuts which had their metaphorical fangs taken out by a single base change... Sources for this episode: 1) Hardy, E. R., Encyclopaedia Britannica (2021), Saint Basil the Great (online) [Accessed 22/05/2021]. 2) Herron, J. C. and Freeman, S. (2015), Evolutionary Analysis (Fifth Edition, Global Edition). Harlow: Pearson Education Limited. 3) Leman, J., Scientific American (2019), The Bitter Truth: Scientists Sequence the Almond Genome (online) [Accessed 18/05/2021]. 4) Petruzzello, M., Encyclopaedia Britannica (2021), almond (online) [Accessed 18/05/2021]. 5) Sánchez-Pérez, R., Pavan, S., Mazzeo, R., Molodovan, C., Cigliano, R. A., Del Cueto, J., Ricciardi, F., Lotti, C., Ricciardi, L., Dicenta, F., López-Marquéz, R. L. and Møller, B. L. (2019), Mutation of a bHLH transcription factor allowed almond domestication, Science 364(6445): 1095-1098. 6) Author unknown, Wikipedia (date unknown), Almond (online) [Accessed 18/05/2021].

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.04.283036v1?rss=1 Authors: Jimeno-Martin, A., Sousa, E., Daroqui, N., Brocal-Ruiz, R., Maicas, M., Flames, N. Abstract: To search for general principles underlying neuronal regulatory programs we built an RNA interference library against all transcription factors (TFs) encoded in C. elegans genome and systematically screened for specification defects in ten different neuron types of the monoaminergic (MA) superclass. We identified over 90 TFs involved in MA specification, with at least ten different TFs controlling differentiation of each individual neuron type. These TFs belong predominantly to five TF families (HD, bHLH, ZF, bZIP and NHR). Next, focusing on the complexity of terminal differentiation, we identified and functionally characterized the dopaminergic terminal regulatory program. We found that seven TFs from four different families act in a TF collective to provide genetic robustness and to impose a specific gene regulatory signature enriched in the regulatory regions of dopamine effector genes. Our results provide new insights on neuron-type regulatory programs that could help better understand specification and evolution of neuron types. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.24.262626v1?rss=1 Authors: Klann, M., Issa, A. R., Alonso, C. R. Abstract: All what we see, touch, hear, taste or smell must first be detected by the sensory elements in our nervous system. Sensory neurons, therefore, represent a critical component in all neural circuits and their correct function is essential for the generation of behaviour and adaptation to the environment. Here we report that a gene encoding the evolutionarily conserved microRNA (miRNA) miR-263b, plays a key behavioural role in Drosophila through effects on the function of larval sensory neurons. Several independent experiments support this finding. First, miRNA expression analysis by means of a miR-263b reporter line, and fluorescent-activated cell sorting coupled to quantitative PCR, both demonstrate expression of miR-263b in Drosophila larval sensory neurons. Second, behavioural tests in miR-263b null mutants show defects in self-righting: an innate and evolutionarily conserved posture control behaviour that allows the larva to return to its normal position if turned upside-down. Third, competitive inhibition of miR-263b in sensory neurons using a miR-263b sponge leads to self-righting defects. Fourth, systematic analysis of sensory neurons in miR-263b mutants shows no detectable morphological defects in their stereotypic pattern. Fifth, genetically-encoded calcium sensors expressed in the sensory domain reveal a reduction in neural activity in miR-263b null mutants. Sixth, miR-263b null mutants show a reduced touch-response behaviour and a compromised response to sound, both characteristic of larval sensory deficits. Furthermore, bioinformatic miRNA target analysis, gene expression assays, and behavioural phenocopy experiments suggest that miR-263b might exert its effects, at least in part, through repression of the bHLH transcription factor atonal. Altogether, our study suggests a model in which miRNA-dependent control of transcription factor expression affects sensory function and behaviour. Building on the evolutionary conservation of miR-263b, we propose that similar processes may modulate sensory function in other animals, including mammals. Copy rights belong to original authors. Visit the link for more info

This month on Episode 9 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the January 31 and February 14, 2020 issues of Circulation Research and talks with Dr Joe Miano and DrThomas Quertermous about their article Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway.   Article highlights:   Wang, et al. Multi-Omics Integration Study of AF   Heianza, et al. Antibiotics and Risk of Mortality   Dikalova, et al. Sirt3 Reduces Hypertension and Vascular Dysfunction   Hu, et al. Lipid Overload Acetylates Drp1 in the Heart Transcript Dr St. Hilaire: Hi, welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm from the Vascular Medicine Institute at the University of Pittsburgh. Today I'm going to share with you four articles that we selected from the January 31st and February 14th issues of Circulation Research. I'm also going to have a discussion with corresponding authors, Drs. Joe Miano and Thomas Quertermous about their study on the role of TCF21 and smooth muscle cell lineage specificity in coronary artery disease. So first, the highlights. The first article I'm sharing with you is titled, Integrative Omics Approach to Identifying Genes Associated with Atrial Fibrillation. First author is Biqi Wang, and the corresponding author is Honghuang Lin from Boston University School of Medicine in Boston, Massachusetts. Atrial fibrillation, or Afib, is the most common form of heart arrhythmia and in the US alone there's somewhere between three and six million individuals with this condition. AFib can be either idiopathic or inherited, and genome-wide association studies, or GWAS studies, have identified hundreds of genetic loci that are linked to AFib. However, these loci explained only a small percentage of inherited cases. This suggests that there are many more AF related genes yet to be discovered. To try and identify these elusive a-fibrillated loci, this study integrated data from previously performed transcriptome, epigenome and GWAS studies. The TWAS and EWAS, as the transcriptome and epigenome-wide studies are short-handedly called, was collected from more than 150 Afib patients, and over 2,000 control individuals. While existing GWAS data, that's genomic data, was collected from tens of thousands of AFib and control participants. By combining and analyzing the data from these TWAS, EWAS, AND GWAS studies, the team was able to identify an additional 1700 genes that were associated with AFib. Now this is compared to the original 206 loci that were identified by the GWAS studies alone. Many of these new genes are involved in cardiac development as well as the regulation of the heart and the muscle cells. The additional gene hunting power afforded by co-analyzing multiple Omics data is not only helpful for approaching AFib but is really setting a platform upon which future studies might be done to provide novel insights for numerous other diseases of complex ideology. The second article I will highlight is titled, Duration and Life-Stage of Antibiotic Use and Risks of All-Cause and Cause-Specific Mortality, a Prospective Cohort Study. The first author is Yoriko Heianza, and the corresponding author is Lu Qi from Tulane University in New Orleans, Louisiana. So microbiome is a word that is used to describe all of the microbes; the bacteria, the fungi, the protozoa, and the viruses that live on and also live inside the human body. And how our microbiome influences human health as well as disease state is a new and hot research topic. Alterations to the gut microbiome have been suggested to influence the risk of developing certain chronic diseases, including cancer and cardiovascular disease. There are many factors that influence the constituents of the gut microbiome; things such as your diet, your environment, your stress level, but another factor that can significantly alter the gut microbiome is the use of antibiotics. So there's preliminary evidence that suggests long-term antibiotic use may be linked to increased mortality in adult women, and now this study defined that link in more detail. The authors performed a large-scale population study of antibiotic use in middle aged and older women with a follow up period of 10 years. Over 37,000 women who were in middle age or in late age at the start of the study show that long durations of antibiotic use, which was defined as using antibiotics over two or more months, was associated with increased risk of all-cause mortality and of cardiovascular disease-related mortality in late adulthood, even after adjusting for risk factors such as age, lifestyle, diet and obesity. While no such association was apparent in middle-aged women, the risk for older women was more pronounced if they had also used antibiotics during middle life. And middle life is defined as between the age of 40 and 59 years of age. This suggests that risk of mortality due to antibiotic use may be cumulative. While antibiotics are unquestionably beneficial for saving lives, the link is not necessary causative, and the results indicate a potential risk may exist that could be factored into prescription decisions. Obviously, there's much more details that need to be worked out, but this is quite a provocative study. While antibiotics unquestionably saved lives and the link is not necessarily causative, the results indicate a potential risk may exist that could be factored into prescription decisions. Moving to a metabolism theme, the next article I want to share with you is titled, Mitochondrial Deacetylase Sirt3 Reduces Vascular Dysfunction and Hypertension While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation and Oxidative Stress. The first author is Anna Dikalova and the corresponding author is Sergey Dikalov, and the work was completed at Vanderbilt University. Hypertension affects about a third of the global adult population. That's a huge number of individuals. It's a risk factor for stroke, myocardial infarction and heart failure. Although blood pressure lowering treatments are widely available, hypertension remains uncontrolled in about 30% of patients who are on those treatments. A thorough understanding of the complex pathophysiology of the condition would facilitate the search for much needed alternate treatments for this third of patients with hypertension. To that end, these investigators studied the role of Sirt3, which is an enzyme that tends to be at the lower than usual levels in blood vessels of patients with hypertension. Sirt3 regulates metabolic and antioxidant functions, and alterations in either of these functions can contribute to cardiovascular disease and vascular dysfunction. The team showed that mice genetically engineered to over express Sirt3 had healthier blood vessels and lower blood pressure than control animals who were subjected to experimentally induced hypertension. By contrast, Sirt3 depletion was shown to cause vascular inflammation and increased signs of vascular aging in mice. The team also confirmed that humans with hypertension exhibit low levels of Sirt3; however, the mechanism causing Sirt3 to be low in certain people is not clear. These data suggest that boosting Sirt3 may be potential therapy for hypertension; however, of course, more studies must be conducted to thoroughly investigate this. The last article I want to share with you before we switch to our interview is titled, Increased Drp1 Acetylation by Lipid Overload Induces Cardiomyocyte Death and Heart Dysfunction. The first author is Qingxun Hu and the corresponding author is Wang from the University of Washington School of Medicine in Seattle, Washington. In the heart, fat molecules are the main energy source. However, excessive lipids caused from diet induced dyslipidemia, AKA eating too much fat, can lead to cardiomyocyte dysfunction. It's known that lipid overload in the heart can cause increased activity of dynamin-related protein one, or Drp1. Drp1 is an enzyme that regulates mitochondrial fission, but exactly how Drp1 becomes activated due to lipid overload is entirely unclear. The authors of this paper confirmed that Drp1 activity and mitochondrial fission are abnormally increased in the hearts of mice fed a high-fat diet, and these mice also exhibit signs of heart dysfunction. They show similar effects in monkeys who were fed a high-fat diet. Interestingly, Drp1 mRNA was not altered in the hearts of mice. However, Drp1 protein acetylation was increased. So this suggests post-translational modifications are regulating its activity in dyslipidemia. The team went on to perform experiments on cultured rat cardiomyocytes, and they found that incubation with saturated fatty acid palmitate led to the acetylation of Drp1, and thus its activation. And this activation resulted in an excess of mitochondrial fission, which reduced cell viability. By contrast, mutation of Drp1 to prevent its acetylation protected the cells. Together, the results reveal the mechanism of how dyslipidemia can contribute to heart cell dysfunction. Further, this data suggests that Drp1 activity or acetylation state could be novel targets for treating obesity-related heart disease. Okay, we're now going to switch over to the interview portion of the podcast. I have with me Dr Thomas Quertermous, the William G Erwin Professor of Medicine and the Director of Research in the division of cardiovascular medicine at Stanford University. And Dr Joe Miano, Professor and Jay Harold Harrison Distinguished University Chair in vascular biology at the Medical College of Georgia at Augusta University. And today we're going to be discussing their manuscript titled Coronary Disease Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway. So welcome to both of you. Thank you for joining me. Dr Miano: Thank you. Dr Quertermous: Thank you. Dr St. Hilaire: So I'm going to start with you, Dr Quertermous. You've been taking a genomics approach to identify factors that contribute to coronary artery disease. And I'm wondering if you could just give us a brief summary of your work thus far and how it brought you to this current study? Dr Quertermous: Well, as you know, the classical risk factors for coronary artery disease and vascular disease in general really only contribute about 30% of the total risk and the remainder has not been studied, and not been investigated, and can't currently be targeted by therapeutics. So the goal is to try and better understand what are the molecular mechanisms in the blood vessel wall that must contribute the remainder of the risk. And so with the advent of genome-wide association studies and the identification of genes and loci, we've been able to begin to uncover the signaling pathways and mechanisms of disease risk. Dr St. Hilaire: And so the one we're most interested in today, this TCF21, you pulled that up out of one of your GWA studies, or how did we get to this? Dr Quertermous: Well, it's an interesting story. I first cloned that gene about 15 years ago when I was trying to understand vascular development, and it's a basic helix loop helix factor, and I was, well, a number of labs at that point in time were cloning this class of transcription factor to try and better understand developmental processes, and so it was one of a number of bHLH proteins that we cloned at the time. I did some work on it and then named it Pod-1 at that point in time, and then lost interest, and went away from it. And then I was involved in the cardiogram genome-wide association study for coronary artery disease, and I was sitting at my desk one night, and I was watching the hits coming in, you know, as we were doing the association, as we were doing the analysis, and I saw this gene, TCF21, and I thought, "Well, I don't really know what the heck that gene is." And so I was going back and forth between our data and a spreadsheet on the web, and I saw that I had published on this gene, and I was like, "Wow, I didn't even know that I had written a paper about this gene." And then it became clear that it was the bHLH factor I'd cloned some time ago. And then knowing what I knew about the development, that this gene is involved in early processes that lead to the formation of the coronary artery, and in particular the development of smooth muscle cells, then I became super interested, and I said, "Okay, my gene, I'm coming back to you. You and me are going to have a great future together." And that was really how I got started. Dr St. Hilaire: It re-found you. Dr Quertermous: It found me, I guess in this case, yes, and so I began then to work very seriously, because it's hard to try and understand mechanisms. And so we had a good starting point. We had a transcription factor so I could quickly identify the targets downstream of that, and I can link it into some cell biology that I already had some insights into. Dr St. Hilaire: That's a really neat story. I like that. It's kind of penicillin-esque. Dr Quertermous: Thank you. Dr St. Hilaire: Dr Miano, those of us familiar with smooth muscle cells appreciate that they are plastic, that they have this ability to kind of switch their phenotypes per se, and those of us familiar with that also then know about the myocardin and serum response factor pathway. But for our listeners who are less familiar with that, could you maybe give a brief background about what myocardin SRF pathway is and what smooth muscle cell phenotype modulation is? Dr Miano: Sure. I wish I could say, as my colleague said, that I cloned one of those factors, but I didn't. I've been interested in SRF since I was a graduate student actually. Actually went to Eric Olson's lab to look for what we affectionately called back then SmyoD, which stands for smooth muscle myo D. So at that time, we didn't understand what the factors were, even the signaling, that directed cells to become differentiated smooth muscle cells. So I went to Eric's lab looking for SmyoD. Of course I didn't find it. I found some other things. Worked a little bit on SRF, but it was actually Daiju Wong in 2000 or 2001 who in a Cell paper described an elegant a way of finding myocardin, what he called myocardin. So SRF myocardin is a transcriptional switch that is necessary and sufficient to make just about any cell a smooth muscle cell. So when myocardin is not present, then smooth muscle cells lose their differentiated state and they become another cell type, depending on who SRF talks to. And so how does a factor that binds a very discrete element like the CArG box, how does it confer cell identity or specific cell states? And it does so through its interaction with these cofactors, one of which is myocardin. And as this paper describes so elegantly, what Tom did in his lab, is that this TCF1 transcription factor, which is DNA binding, unlike myocardin, it does a similar thing in that it competes for SRF binding with myocardin, so it binds myocardin, prevents myocardin's ability to bind SRF, and thereby directs a new program of gene expression. Dr St. Hilaire: Interesting. So it's kind of helping to fine tune that transcriptional regulation. So I always think of smooth muscle cells, they're kind of always in a contractile state when they're healthy, and it's when they're in either unhealthy, or diseased, or a stress state that they're in that more proliferative-like state. And Dr Quertermous, your previous studies have shown that TCF21 is required for the De-differentiation, and proliferation, and migration of smooth muscle cells. However, there was one sentence in the paper that I was slightly confused on and I'm hoping that you can expand about the bigger role of TCF21. And what it said was that TCF21 expression is protective towards human coronary artery disease. And so the data in the paper show that TCF21 inhibits smooth muscle cell contractility. So can you maybe reconcile the bigger mutations or things you identified in the GWAS with the functional activities you're seeing that you presented in the paper, and maybe talk about the timeline in the continuum of atherosclerosis where TCF is maybe good or maybe bad? Dr Quertermous: So this paper is one of a duo of papers, honestly, that the other paper being published in Nature Medicine almost exactly the same time, and so that paper sort of described some of the aspects of TCF21 at a population level and shows that if you look at all of the single base pairs in the genome that regulate disease risk at 6q23.2 and also regulate expression, you can gain an idea of what's the directionality of the expression of TCF21. And those data suggests that the more TCF21 you have, the less your risk of developing coronary artery disease. And Joe and other scientists have worked for a long, long time to characterize this process and characterize the plasticity of this cell type. And note that one can switch the cell back and forth between being a contractile0differentiated cell to a de-differentiated cell, and elegant work by Gary Owens and a number of investigators have profiled the phenotype of the cells that the smooth muscle cell can become if it undergoes this differentiation process. It's not been able to know though up until this point in time whether that's a good process or a bad process. I mean, 15, 10 years ago we thought smooth muscle cells are proliferating, they're creating a space-occupying lesion, they're decreasing the lumen of the blood vessel, and that's got to be a bad thing. And in honesty, I think over the past three or four years, it's been increasingly clear that perhaps the smooth muscle cells are actually doing a good thing. They are stabilizing the lesion, they're creating the fibrous cap, and there's been some nice work correlating the number of smooth muscle cells in the plaque to the risk that that plaque is going to rupture. Dr St. Hilaire: Yeah, that was kind of my next question. Do you think there's more nuance to it's not just contractile, and synthetic? There's much more broader scope and it's not so much a good or a bad smooth muscle cell. Dr Quertermous: I think it depends on the circumstances I guess, but it's important that the smooth muscle cell be able to migrate into the plaque, and begin to produce matrix components which stabilize the plaque, and to form the fibrous cap, and I think if the smooth muscle cell remains in the media as a contractile cell, it's really not able to do those things, right? And so the human genetics data, looking at the directionality, the expression, the different alleles and their expression patterns, and what is the risk allele at? In this region of the genome, it's pretty clear that more TCF21 is good, and what TCF21 does is to promote this phenotypic modulation. And so that suggests that the process as a whole is good. Not just the gene, but what it does. It's really not possible that TCF21 does anything else in the blood vessel wall. It's primarily restricted to the smooth muscle cell. It's not expressed in macrophages, or endothelial cells, or the other cell types that we think are important in the pathophysiology of the disease process. So putting everything together, it looks for the most part, like this is a positive force in the blood vessel wall, this gene and this process. Dr St. Hilaire: Interesting. And speaking to the vessel wall, I thought one of the very cool and really key experiments in the paper was taking your mechanistic in vitro studies into the mouse, and Dr Miano, maybe you could tell us a little bit about how you were able to do that and mutate these smooth muscle specific CArG boxes in a mouse model. Dr Miano: Well, that's a really good question. Again, it's a history. We've wanted to edit CArG boxes, well, mutate back then, for a long, long time, but it wasn't until the CRISPR craze took a foothold that we really recognize now the power of harnessing that and doing the experiments we wanted to do for so long. And so we've previously published on a CArG box in the first intron of the calponin locus, and found that, to our surprise, that a subtle mutation in that element completely abolished expression of calponin into the smooth muscle. So Tom and I were working in parallel and unbeknownst to me, Tom was working on this SRF enhancer in the second intron, and we've known for quite a while that SRF is auto regulated by itself, and there's CArG boxes in it 5-prime promoter, and there's CArG boxes in the interior of the locus as well, including the one in the second intron that Tom describes in the paper here. And so what we've been doing is using CRISPR in the mouse to make these subtle edits in these CArG boxes around the SRF locus. And unlike the affirmation calponin model I just described, if we mutate the two proximal CArG boxes of SRF, we don't see a lot of change in SRF expression. That was really surprising to us, because studies from Bob Schwartz' lab in Houston two decades ago showed those were important, at least in an artificial reporter assay for the autoregulatory loop that he first described. So we moved interior to this CArG box that's really the focus of this paper, highly conserved, much more so than other CArG boxes, and we first deleted the region, which we often do with CRISPR, and found there was a decrease. But we'd like to do more subtle things with CRISPR, which is really the power of this new editing technology. And so we went in and made just, I think it was like four or five base substitutions to create a novel restriction cipher ease in genotyping. And we reported in the paper, you can see that, to compliment Tom's group's data, that in vivo, indeed, that CArG box by itself, nothing else altered within the locus, did cause a, I would call, a substantial decrease in expression of this important regulator. And so that was really our main contribution to this paper. Dr St. Hilaire: Yeah. I know the opportunities are endless, but also complicated and expensive, and I thought this was a beautiful addition to really confirming those mechanistic studies. So I think my next big question is, if TCF21 is,  so important and protective, and perhaps it's upregulation is beneficial, what is regulating it, and do we know how we can potentially modulate this? Dr Quertermous: That's a great question. That's a great question. And so we know a few things; we certainly know that platelet-derived growth factor stimulation of smooth muscle cells will upregulate TCF21, which is sort of surprising. I mean, it's not so surprising, I guess. So we've spent a little time working on that, and there's a micro RNA which regulates the expression level of TCF21, but we haven't spent a lot more time than that, honestly. We spent a lot more time downstream trying to figure out what's the mechanism by which TCF21 works to suppress the smooth muscle contractile phenotype and activate this more de-differentiated migratory phenotype that the smooth muscle cell adopts. So we've not gone upstream, but your question's a really, really good one. We certainly mapped where TCF21 binds across the genome and we've mapped the variation that regulates its expression, and so we've made progress in that direction, and as I said, identified things which are downstream. But we definitely need to spend more time upstream, and I think that's the area of this intersection of molecular science and genomic science, that there are not many groups that really spend much time up above the gene trying to understand. And so we've not spent enough time doing that, and I think that as a community we've not spent enough time doing that, because I think that's where the big payoff can come in terms of therapeutics. Dr St. Hilaire: To that end, I think I'll end with that question. What do you think is the best way that we could leverage your findings in the clinic? Would it be to focus more on the downstream or to try to identify these more upstream factors in TCF21? Dr Quertermous: Well, I think both open up opportunities, right? If we can understand how TCF21 works and what's downstream, and we can activate those processes and activities, then that's good. If we can figure out what's above TCF21, that would be good as well. The danger there is that TCF21 does a lot of things in a lot of different cells in the body. Dr St. Hilaire: So it'd be a little bit harder to focus onto a smooth muscle cell in a plaque than perhaps some of the downstream effects of TCF21? Dr Quertermous: Correct. Right. That's my worry. It's sort of like thinking about TGF beta and you wouldn't really want to try and manipulate TGF beta. Dr St. Hilaire: That's a whole another can of worms. Dr Quertermous: Yeah, it gets you into a lot of difficulties, I think. So we're really pretty focused downstream now and thinking that we can find specific opportunities there that are resident in that smooth muscle cell in the blood vessel that may not be active in other cell types. So that's really our thinking and that's the way we're going. Dr St. Hilaire: Wonderful. Well, thank you so much to both of you for joining me today. I learned a lot and I really thought this was a beautiful, complex, but well-done study, so thank you very much. Dr Miano: Thank you, Cindy. Dr Quertermous: Thank you so much for calming us down, I guess. Dr St. Hilaire: Well, that's it for our highlights from the January 31st and February 14th issues of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the Editorial team of Circulation Research. Some of the copy texts for the highlighted articles was provided by Ruth Williams. Thank you to our guests, Drs Thomas Quertermous and Joseph Miano. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.    

Background: Deregulation of Wnt/beta-catenin signaling is a hallmark of the majority of sporadic forms of colorectal cancer and results in increased stability of the protein beta-catenin. beta-catenin is then shuttled into the nucleus where it activates the transcription of its target genes, including the proto-oncogenes MYC and CCND1 as well as the genes encoding the basic helix-loop-helix (bHLH) proteins ASCL2 and ITF-2B. To identify genes commonly regulated by beta-catenin in colorectal cancer cell lines, we analyzed beta-catenin target gene expression in two non-isogenic cell lines, DLD1 and SW480, using DNA microarrays and compared these genes to beta-catenin target genes published in the PubMed database and DNA microarray data presented in the Gene Expression Omnibus (GEO) database. Results: Treatment of DLD1 and SW480 cells with beta-catenin siRNA resulted in differential expression of 1501 and 2389 genes, respectively. 335 of these genes were regulated in the same direction in both cell lines. Comparison of these data with published beta-catenin target genes for the colon carcinoma cell line LS174T revealed 193 genes that are regulated similarly in all three cell lines. The overlapping gene set includes confirmed beta-catenin target genes like AXIN2, MYC, and ASCL2. We also identified 11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are regulated similarly in DLD1 and SW480 cells and one pathway - the steroid biosynthesis pathway - was regulated in all three cell lines. Conclusions: Based on the large number of potential beta-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T cells as well as the large overlap with confirmed beta-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study Wnt/beta-catenin signaling and associated colorectal carcinogenesis. Furthermore, the confirmed and the newly identified potential beta-catenin target genes are useful starting points for further studies.

Reprogramming of somatic cells into neurons provides a new approach toward cell-based therapy of neurodegenerative diseases. Conversion of postnatal astroglia from the cerebral cortex of mice into functional neurons in vitro can be achieved by forced expression of a single transcription factor. Also skin fibroblasts have been successfully reprogrammed into functional neurons yet through the synergistic action of several transcription factors. A major challenge for the translation of neuronal reprogramming into therapy concerns the feasibility of this approach in adult human tissues. This work demonstrates the potential of perivascular cells isolated from the adult human brain to serve as a substrate prompted to neuronal reprogramming by forced co-expression of neurogenic transcription factors, namely the SRY-related HMG box protein Sox2 and the basic helix loop helix (bHLH) mammalian homologue of achaete-schute-1 Mash1 (also known as Ascl1). The cells used in this study display characteristics of pericytes assessed by immunocytochemistry, fluorescence-activated cell sorting (FACS) and real time RT-PCR. The presence of neural progenitor cells was excluded by real time RT-PCR analysis of mRNAs typically expressed by these cell lineages. Upon expression of Sox2 and Mash1, these cells adopt a neuronal phenotype characterized by the expression of neuronal markers such us ßIII-Tubulin, MAP2, NeuN, GABA and calretinin. Electrophysiological recordings reveal the ability of these cells to fire repetitive action potentials and to integrate into neuronal networks when co-cultured with mouse embryonic neurons. The pericytic nature of the reprogrammed cells was further demonstrated by isolation of PDGFRß-positive cells from adult human brain cultures by FACS and monitoring the Mash1/Sox2-induced neuronal conversion by time-lapse video microscopy. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue non-specific alkaline phosphatase promoter corroborated that pericytes from the adult cerebral cortex can be expanded and reprogrammed in vitro into neurons by co-expression of Sox2 and Mash1. These results demonstrate the feasibility of an in vitro neuronal reprogramming approach on somatic cells isolated from the adult human cerebral cortex which could have important implications in the development of in vivo direct repair strategies in neurodegenerative diseases and brain injury.

Thu, 26 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14293/ https://edoc.ub.uni-muenchen.de/14293/1/Csanadi_Endy.pdf Csanadi, Endy ddc:610, ddc:600, Medizinische Fakultät

Thu, 10 Jun 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11696/ https://edoc.ub.uni-muenchen.de/11696/1/Helferich_Simone.pdf Helferich, Simone ddc:610, ddc:600, Medizi

During the development of a C. elegans hermaphrodite, 131 of the 1090 cells generated die due to programmed cell death, an important process conserved throughout the animal kingdom. Although a genetic pathway for programmed cell death has been established in C. elegans, not much is known about the signals that trigger cell death in cells destined to die. One particular cell-death event, the death of the NSM sister cell, occurs about 430 min after the first division of the zygote, just 20 min after its progenitor cell has undergone an asymmetric cell division. The sister of the NSM sister cell, the NSM, however, survives and differentiates into a serotonergic neuron located in the pharynx. Here, I show that the cell-death activator egl-1 is expressed in the NSM sister cell, which is destined to die, but not in the surviving NSM. In addition, using a candidate gene approach, I found that in hlh-2(bx108lf); hlh-3(bc248lf) animals, 30% of the NSM sister cells survive. This observation suggests that the NSM sister cell death is at least partially dependent on the activity of hlh-2 and hlh-3, which code for bHLH transcription factors. These and additional results suggest that egl-1 expression is directly activated in the NSM sister cell by a heterodimer composed of HLH-2 and HLH 3, which binds to a specific cis-regulatory region of the egl-1 locus. In order to identify additional factors that contribute to the NSM sister cell death, I performed a forward genetic screen. In particular, I screened for mutations that enhance the NSM sister cell survival caused by hlh-2(bx108). This screen resulted in the identification of mutations in at least six genes not previously implicated in this cell-death event. One of these mutations, bc212, is a loss-of-function mutation in the gene dnj-11. dnj-11 codes for a protein with a J domain, which is found in chaperones, as well as two SANT domains, which are implicated in transcriptional regulation. dnj-11 is an essential gene expressed in most if not all cells. Furthermore, it acts in the NSM sister cell death pathway by negatively regulating the activity of the snail-like gene ces-1. dnj-11 is required for the ability of the NSM mother cell to divide asymmetrically. I propose that dnj-11 promotes the death of the NSM sister cell by establishing polarity in the NSM mother cell. Moreover, I present evidence that the snail-like ces-1 gene is involved in establishing polarity in the NSM mother cell as well, revealing a new function of ces-1 in C. elegans.

The cells of the mammalian central nervous system (CNS) arise from multipotential precursor cells. The mechanisms that drive precursor cells toward a distinct cell fate are not well understood. Since transcription factors are known to control fate decisions, I attempted to determine the role of transcription factors Emx1, Emx2 and Pax6 that are particularly interesting since they specify area identities in the mouse telencephalon. To analyze their roles in precursor cells I chose gain-of-function experiments. Overexpression of these transcription factors showed that Emx2, Emx1 and Pax6 affect precursor cells in a region-specific manner. Emx2 transduction increases proliferation by promoting symmetric cell divisions, whereas blockade of endogenous Emx2 by antisense Emx2 mRNA limits the number and fate of progenitors generated by an individual cortical precursor cell. In the Emx2-/- asymmetrical cell divisions are increased in the cerebral cortex in vivo. In contrast to Emx2 Pax6 decreases proliferation. Pax6 deficient cells show more symmetrical cell divisions while Pax6 promotes asymmetric cell divisions in vitro. Emx2 endows in vitro cortical precursor cells with the capacity to generate multiple cell types, including neurons, astrocytes and oligodendrocytes. Emx1 keeps cells in an undifferentiated cell type, while Pax6 increases the proportion of neurons and can also convert astrocytes to neurons. The bHLH transcription factors Olig2 and Mash1 are up-regulated upon Emx2-transduction whereas Pax6 negatively influences those transcription factors and specifically up-regulates Ngn2. Thus, Emx2 is the first cell-intrinsic determinant able to instruct CNS precursors towards a multipotential fate. These results demonstrated an important role of Pax6 as intrinsic fate determinant of the neurogenic potential of glial cells. Taken together, Emx2 and Pax6 have opposing roles in cell proliferation, mode of cell division and cell fate.