00:08:34.03 as egg size changes, how does the embryo or the species adjust to using 00:12:02.23 where we're activating hunchback gene expression you can measure those concentrations 00:04:20.09 Such that after two and a half hours this single cell has now transformed itself 00:11:16.14 in turn, is that somehow during the course of evolution of these insects 00:06:18.18 What you can't see yet is an obvious difference in the concentration of fluorescence So these proteins put in a melanogaster egg will produce 00:03:08.13 on this earth and how processes evolve and have changed during By using electrophoretic mobility shift assay (EMSA),[88] the activation profile of transcription factors can be detected. The homeodomain that binds DNA 00:10:29.29 then in individual embryos somehow at 30% egg length you've got to have the same bicoid 00:17:42.16 the protein is not anchored, the protein is thought to diffuse from this source of 00:06:36.17 and you can see in those nuclei at the anterior end of the egg, you can see an 00:28:01.14 by its distribution over time, and it follows what would be expected of 00:16:57.22 if you think about it, 600 or 700 molecules in a nucleus if we consider that If you go outside of 00:15:55.14 concentration of bicoid in the nucleus in those nuclei that are actually making 00:19:07.06 and responding to bicoid. 00:03:03.05 of not just fruit fly embryos but more broadly of all organisms 00:10:10.18 the process, we'll watch it one more time, all the cells are the same, 00:27:00.06 measure molecular movement. And what we found is that's not true. If you look at values at the behavior here, Similar mechanisms have been proposed in the context of all alternative phylogenetic hypotheses, and the role of transcription factors in the evolution of all species. And it's after that process that's called 00:06:33.02 there's one other thing that's really interesting about this phase 00:16:11.07 is that if we have a graded distribution for protein, a maternal protein 00:20:45.00 that allow you to manipulate the sequences and structures 00:15:51.06 immediately outside the embryo. 00:04:37.16 Drosophila busckii that make eggs that are even smaller than Drosophila, 00:28:47.11 and ask how fast does it move compared to what he was measuring with EGFP bicoid, 00:25:12.09 and what was unexpected at the time was that in contrast to many genes 00:05:00.01 that they make are substantially bigger. 00:00:55.03 such that different concentrations of the bicoid protein at different points 00:11:52.15 would be to change bicoid as the species evolve they change bicoid, TET enzymes do not specifically bind to methylcytosine except when recruited (see DNA demethylation). 00:28:14.22 that cells can make choices on? 00:31:43.07 the size of the radius of bicoid is independent of its actual size and all molecules Spatial bistability generates hunchback expression sharpness in the Drosophila embryo. The initial changes occur without any cell division. It's basically a 10% difference. Synthetic reconstruction of the hunchback promoter specifies 00:11:23.03 the bicoid gradient has changed. 00:15:45.28 And you can measure that and show that these distributions overlapped 00:07:27.27 this point, it becomes a very interesting point for us in development, And in a model, like the one we are dealing with here, where We engineer light-controlled variants of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. If you actually look though, The cells make choices 00:02:41.06 of the bicoid protein is that it arises from the synthesis of the bicoid protein from What's been conserved in bicoid is 00:04:23.27 that these different fly species make, they are also different sizes. You can plot out that response. 00:19:55.29 Discovered by Christine Nusslein-Volhard and Wolfgang Driever more than Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders. 00:06:46.14 the big green bottle fly, Lucilia versus Drosophila melanogaster There are other things beginning 00:07:42.15 all of them activate transcription at this critical 2 hour period in response to bicoid 00:18:04.16 a hunchback control region and activating transcription by interacting with 00:35:56.24 are the products more of that movement. Other transcription factors differentially regulate the expression of various genes by binding to enhancer regions of DNA adjacent to regulated genes. 00:13:58.03 really want to know is how is it that spatial and temporal patterns of 00:12:49.01 just begun to be transcribed at this stage that we talked about before. So those are 00:13:54.13 products that doesn't really answer the question because if what you 00:22:57.06 simple physical parameters. Additional recognition specificity, however, may be obtained through the use of more than one DNA-binding domain (for example tandem DBDs in the same transcription factor or through dimerization of two transcription factors) that bind to two or more adjacent sequences of DNA. On the genomic level, DNA-sequencing[86] and database research are commonly used. Results: 00:08:43.03 a bicoid gradient to establish pattern. The Hox transcription factor family, for example, is important for proper body pattern formation in organisms as diverse as fruit flies to humans. Can we actually measure degradation? [14] Therefore, approximately 10% of genes in the genome code for transcription factors, which makes this family the single largest family of human proteins. 00:08:41.11 of the egg. What have we been able 00:01:11.18 Now, we've seen that if you examine this gradient in Drosophila embryos 2020 Jul 29;9:e49758. 00:34:39.22 will we believe contribute, if bicoid is being associated with the cytoplasm, 00:03:30.08 maybe half a millimeter or 500 microns long. 00:16:24.23 the course of evolution it's not bicoid that has changed to allow for the 00:20:17.26 And so I think there are a couple of interesting things that happen. 00:04:31.26 along the anterior with the future head and future tail anterior/posterior axis Optogenetic control of the Bicoid morphogen reveals fast and 00:09:30.05 you could adjust with the size of the egg not by changing 00:02:52.19 And if you look at Drosophila eggs in the particular way you raise them 00:07:12.00 and look at the nuclear distributions you can see that all these eggs 00:01:41.01 how they control their cell shape, how they control all these processes 00:20:56.05 put gene products in the egg to establish pattern. 00:04:34.04 500 microns long. The problem is actually, to make the problem graphic just The other interesting thing, the question that then though 00:18:09.18 other proteins that are the actual transcriptional activators. 00:02:25.02 hunchback expression up to 48% egg length, and you are doing that 00:04:59.14 and these are actually living photographs at different levels of a single Drosophila embryo 00:16:51.02 using other tagged proteins, EGFP, we've altered the bicoid area, 00:11:57.01 Because the concentrations are so reproducible, particularly in the region right here Epub 2019 Dec 27. The cells that are As such, they are vital for many important cellular processes. Having it both ways: transcription factors that bind DNA and RNA And if you think in terms Once we had an EGFP-bicoid molecule Transcription Factor - an overview | ScienceDirect Topics Many studies have provided a number of alternative combinations to the non-Yamanaka factors. 00:29:29.09 can produce gradients of the kind that we see with bicoid. 00:18:50.28 of the bicoid protein that are essential for it to make 00:05:16.10 again without cell division so that you have from two nuclei to four nuclei, eight nuclei. We know that those cell shape changes correlate 00:10:36.07 of the embryo. 00:12:15.23 and thus establish gradients of comparable shape when function There are really two simple ways So the interesting conclusion then from these Our results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Kruppel . 00:16:30.13 of bicoid, this 8 nM value, and you know the actual volume of the nucleus 00:11:58.19 And it's that kind of cell shape change that produces ultimately 00:11:02.15 of bicoid can provide stable sufficient information about position to activate precise 00:09:26.24 that is at cycle 10, just after 10 cycles to just when the nuclei made it out to the surface Gregor T, Wieschaus EF, McGregor AP, Bialek W, Tank DW. 00:08:44.24 with 6,000 cells, 100 cells all arranged along the anterior/posterior axis Unauthorized use of these marks is strictly prohibited. 00:04:00.28 concentrations. 00:18:32.13 for that activation remain permanently bound or whether there's a dynamic So one of the things that Thomas did by following individual embryos from 00:21:39.15 to those measured concentrations. You can see that it's highly [34] TAL effectors contain a central repeat region in which there is a simple relationship between the identity of two critical residues in sequential repeats and sequential DNA bases in the TAL effector's target site. Shuttling Homeoproteins and Their Biological Significance. 00:30:32.08 that you measure will depend on the size of the molecules. [5][6][7], A defining feature of TFs is that they contain at least one DNA-binding domain (DBD), which attaches to a specific sequence of DNA adjacent to the genes that they regulate. 00:10:57.13 of the embryo, because obviously that's where the embryo wants to have muscles. 00:28:08.00 simple diffusion and allowed Thomas to calculate for dextrans of different sizes 00:01:02.07 I wanted to talk about these experiments is that they provided a wonderful example 00:02:37.24 through a basic description of the process and then work our way through 00:20:14.10 why was that result so exciting, what was so important about it. All those things 00:02:35.24 diffusive constant and establishes a particular constant shape Now the transgene Exemplified by the prototypic Xenopus TFIIIA protein, and more recently by mammalian p53, this list of transcription factors includes WT1, TRA1, bicoid, the It has an essential role in brain neuron epigenetic reprogramming. How does bicoid protein move. Why do they do that, and what's the nature of those 00:07:21.00 going from the anterior to the posterior end of the egg. 00:17:10.21 it to the cytoplasm in the anterior end of the egg. 00:14:08.00 which is a very high degree of cooperativity, which means that very little change [39] Important classes of transcription factors such as some nuclear receptors must first bind a ligand while in the cytoplasm before they can relocate to the nucleus.[39]. 00:32:40.14 but one model, all that we know is that it can't be explained by Brownian diffusion. 00:20:21.16 in general and the refined understanding of problems 00:22:09.05 the bicoid gradient is itself established. There are two mechanistic classes of transcription factors: Transcription factors have been classified according to their regulatory function:[12], Transcription factors are often classified based on the sequence similarity and hence the tertiary structure of their DNA-binding domains:[93][11][94][10]. Multiple transcription factors important in cell differentiation and lineage specification, including NANOG, SALL4A, WT1, EBF1, PU.1, and E2A, have been shown to recruit TET enzymes to specific genomic loci (primarily enhancers) to act on methylcytosine (mC) and convert it to hydroxymethylcytosine hmC (and in most cases marking them for subsequent complete demethylation to cytosine). A protein that the mother has directly put into the egg. WebWe engineer light-controlled versions of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. 00:04:07.06 The bigger flies are the uglier ones, the smaller flies are nicer and sweeter, generally. Bicoid evolved at a point in the history 00:24:48.28 you find that they are very small, that the diffusions constants are very small. 00:17:45.21 about how bicoid or how any transcription factor controls or activates transcription So we can see here, that other genes that can be activated by 00:02:01.23 which is the embryo that we work on in the lab develops, and point out to you 00:17:13.00 or choosing to activate hunchback only varies by 10%, and so what it requires 00:24:02.27 of biological information? 00:07:37.19 doing something repetitive over and over using only maternally supplied gene WebChristiane Nsslein-Volhard was the first to identify a morphogen, Bicoid, one of the transcription factors present in a gradient in the Drosophila syncitial embryo. 00:23:08.18 of what kinds of things can impact on that, things like how fast 00:19:43.24 bicoid was the first such maternal RNA which was functionally demonstrated The site is secure. 00:12:30.16 is being expressed right here, and you ask how much of a difference is there Transcription factors interact with their binding sites using a combination of electrostatic (of which hydrogen bonds are a special case) and Van der Waals forces. In this network, the expression of four So what we're 00:23:37.14 the establishment of the gradient over time. 00:16:00.28 the choice whether to activate hunchback expression or not, was of the range 00:01:20.02 and study in embryos are the same kinds of processes that involve 00:08:13.13 the patterns per cell are exactly the same. 00:07:38.25 are all so closely related even though their sizes are different, is that 00:21:04.17 is going to depend on that. Hox gene - Wikipedia Our results recapitulate known 00:22:24.16 they are as accurate, whether part of that accuracy depends on interactions or People had even speculated that mothers may 00:19:11.26 How is it that you are able to maintain the property of the protein 00:10:53.11 These are future muscle cells that are going to be brought into the interior 2022 Apr 1;11:e74509. 00:35:20.07 in the cytoplasm. Many proteins that are active in the nucleus contain nuclear localization signals that direct them to the nucleus. 00:07:16.09 at 2.5 hours have the same number of nuclei. 00:01:24.18 phenomenon that we've come to understand in a vague-ish kind of cartoon way, but that Many large TF families form complex homotypic or heterotypic interactions through dimerization. 00:07:53.18 things, to go on to the next step in development. Abstract. 00:12:40.06 that of Drosophila melanogaster bicoid, and if you look at that 00:29:21.08 more than enough of what you need to make a gradient. 00:09:35.15 the control responses of individual genes, but by somehow 00:03:57.03 or the intensity of the staining that you see and how that relates to absolute 00:26:56.22 and knowing at what scale and over what time frames we have to 00:34:26.13 kind of turbulent patterns that appear to have no overall directionality 00:17:28.23 there's 690 molecules or 630 or 770. Because transcription factors can bind a set of related sequences and these sequences tend to be short, potential transcription factor binding sites can occur by chance if the DNA sequence is long enough. 00:00:59.12 But over the past 20 years we have also learned that all these process 00:29:40.09 Now, strictly speaking, what Thomas's measurements argue for dextran is that The sample is detected on a western blot. 00:36:33.27 and distribution of bicoid, then those biological parameters 00:01:57.08 first describe to you a little bit about how the fruit fly embryo, 00:05:30.03 the EGFP-bicoid is expressed as RNA in the mother, localized appropriately, and in fact if you 00:06:03.20 or partition them into individual cells. 2011 Apr 22;6(4):e19122. Unable to load your collection due to an error, Unable to load your delegates due to an error. 00:09:03.04 And as you change the length of the egg, one strategy Due to the nature of these chemical interactions, most transcription factors bind DNA in a sequence specific manner. If that is true then what it will argue is that the establishment Chapter 21 Flashcards | Quizlet 00:29:59.18 being transported or binding to something or being moved by other mechanisms. And so 00:18:57.03 of bicoid protein and so show expression only in the anterior most Bicoid If it's being made at the anterior end 00:22:54.06 in a stable form. 00:09:22.04 and this was quite remarkable, is that at the earliest points when we could measure the RNA, 00:09:43.07 that establish the bicoid gradient itself such that in bigger eggs 00:08:36.09 Stabilization would actually be really important because if the nucleus wants to know 00:15:28.24 of the egg, and then grades off in cells as we move more and more posterior 00:18:45.09 that we're in the process of doing where you can identify the regions 00:08:02.23 paired or evenskipped, they show exactly comparable scaled patterns. WebThe first identified morphogen, and perhaps the best studied, is the transcription factor Bicoid 6. 00:09:39.03 changing the manner or changing the physical properties One simple strategy 00:30:36.29 as you see here, you see the behavior of the diffusion constants that you measure 00:03:12.09 consistent that such a mechanism could function to pattern Below is a partial list of some of the major families of DNA-binding domains/transcription factors: The DNA sequence that a transcription factor binds to is called a transcription factor-binding site or response element.[61]. 00:28:34.06 of approximately the size of bicoid. In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. 00:18:04.06 but other proteins do not have that property. This website uses cookies to remember you and improve your experience. 00:20:11.16 is again the kind of quantitation that we talked about in the second lecture 00:08:24.05 directly or indirectly in response to the bicoid gradient. The way that the bicoid gene activates transcription 00:09:57.15 This seemed to us initially a less likely alternative, partially because 00:34:21.02 of the cytoplasm. [83][84], The transcription factors have a role in resistance activity which is important for successful biocontrol activity. 00:07:35.13 the other remarkable similar thing between all these insects that 00:12:15.29 of that concentration in terms of hunchback protein expression. 00:12:03.12 the changes that we see here that will say separate the future head Cofactors are interchangeable between specific gene promoters; the protein complex that occupies the promoter DNA and the amino acid sequence of the cofactor determine its spatial conformation. When injected into plants, these proteins can enter the nucleus of the plant cell, bind plant promoter sequences, and activate transcription of plant genes that aid in bacterial infection. Bethesda, MD 20894, Web Policies Background Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). So what I'm going National Library of Medicine [23][24] Another example is the transcription factor encoded by the sex-determining region Y (SRY) gene, which plays a major role in determining sex in humans. 00:31:53.09 are going to move in the egg at about 6 microns squared per second And the way that its able to do this transformation so rapidly 00:08:04.26 not only where something new begins to happen, you stop mitosis, So what we'd like to know is what are the 00:17:08.10 any GFP-tagged protein put into a Drosophila egg make a gradient 2017. 00:16:08.20 So we know that what that's telling us is that the fact that Dipteran bicoids 00:04:29.16 So Drosophila melanogaster for example makes eggs that are about 00:06:26.11 is also observed in all of these other insects and it also is observed 00:01:18.00 problems more quantitatively, to try to establish actual numbers for the biological 00:16:59.18 with an NLS, a nuclear localization sequence, and localizing the RNA 00:22:05.01 that I've listed here, this orthodenticle, giant, or Krppel, obviously are also responding 00:05:05.06 that expresses this EGFP bicoid transgene that Thomas Gregor made. Does it continue to rise? It's still a single cell. 00:12:26.16 with patterns of gene expression. 00:07:22.29 So it begins to make what we call zygotic RNAs and zygotic proteins. 00:03:24.02 with that model arises if you go outside of Drosophila. Examples include the following: Language links are at the top of the page across from the title. 00:21:26.00 on concentration levels, but on the ability of nuclei, or cells, What these experiments are telling us is that 00:01:36.10 one of the things that we really have to be able to do is to measure and supply numbers. Disclaimer. 00:02:30.14 different patterns of gene activity. 00:10:29.07 here, these cells here that are moving into this invagination 00:14:23.05 millimeter through the entire Calliphora egg which is 00:32:03.05 because it suggests that a molecule, even a molecule like bicoid 00:25:33.28 you don't find the bicoid protein. 00:10:53.13 in a bigger egg, is bigger, proportionately bigger, because if we then So one of the things we'll talk about is Fernandes G, Tran H, Andrieu M, Diaw Y, Perez Romero C, Fradin C, Coppey M, Walczak AM, Dostatni N. Elife. Below are some of the important functions and biological roles transcription factors are involved in: In eukaryotes, an important class of transcription factors called general transcription factors (GTFs) are necessary for transcription to occur. 00:07:50.19 and if you look at the patterns of gene expression 00:10:41.11 of bicoid protein extends much longer, much farther 00:01:35.14 but to understand how cells and living organisms control their gene activity, 00:19:39.23 over and over again. Translational Repression by Bicoid : Competition for the Cap [38], In eukaryotes, transcription factors (like most proteins) are transcribed in the nucleus but are then translated in the cell's cytoplasm. 00:19:22.15 us to models where what's actually required is, for cells to make choices,. Part of the question is that we don't really know, 00:23:59.09 the biophysical parameters associated with the generation 00:14:34.06 of the signal and trying to extrapolate concentrations of functional protein 00:16:28.19 How is it formed? 00:09:32.12 the concentration in a given nucleus was fixed for that position and remained constant 00:27:47.15 like to do is focus on those questions. 00:15:34.03 defined molarity of bicoid, in fact 36 nM bicoid and so what this meant is that he could Nsslein-Volhard, C. and E. Wieschaus (1980). The nucleic acid-binding homeodomain of Bicoid has been solved by NMR. The gap gene network of the early Drosophila embryo is a canonical example of such a sophisticated pattern-forming system. 00:09:08.04 would be to keep the bicoid gradient the same shape 00:14:17.14 that will make a large gradient that extends more than a The DNA sequences can then be identified by microarray or high-throughput sequencing (ChIP-seq) to determine transcription factor binding sites. I grew up in Birmingham, Alabama and did my undergraduate work at University of Notre Dame. Bicoid determines sharp and precise target gene It's a transcription factor And what Thomas was able to show In Drosophila during the early stages 00:11:09.09 and the overall changes in morphology in the behaviors of individual cells. 00:11:48.09 that we saw in the movie are all happening because individual cells in specific 00:18:24.10 and then that raises the kinds of questions that you'd like to answer now. 00:21:28.00 So one of the other things that we don't know, and I think it's really important to know One possibility would be that the embryo 00:08:20.11 though just continue on with the description of development, But to measure concentration in those molecules 00:21:17.18 pattern along the entire access of the embryo, other genes responding and ", "Direct inhibition of the NOTCH transcription factor complex", "A promiscuous intermediate underlies the evolution of LEAFY DNA binding specificity", "An Arabidopsis Transcriptional Regulatory Map Reveals Distinct Functional and Evolutionary Features of Novel Transcription Factors", "Molecular Tools for the Yeast Papiliotrema terrestris LS28 and Identification of Yap1 as a Transcription Factor Involved in Biocontrol Activity", "VOMBAT: prediction of transcription factor binding sites using variable order Bayesian trees", "Tyrosine phosphorylation regulates the partitioning of STAT1 between different dimer conformations", "ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions", "Dam it's good! 2022;2383:33-44. doi: 10.1007/978-1-0716-1752-6_2. 00:09:15.00 head fold from the rest of the embryo. You localize an RNA and then when that RNA 00:28:01.12 to fly embryos to figure how molecules move and how stable are the actual patterns The initial localization, the initial pattern is a localized RNA They suggest that binding a single factor to both enhancers and promoters 00:37:25.21 bicoid distributions have changed during evolution. 00:21:31.16 about the whole response is to get a more global sense of what genes respond to bicoid, 00:04:42.17 is that what it's done is that unlike most other organisms where when [43] Pairs of transcription factors and other proteins can play antagonistic roles (activator versus repressor) in the regulation of the same gene. 00:01:30.00 and controlling the behaviors of individual cells in the embryo. 00:14:13.29 if you take a bicoid protein from Calliphora for example 00:17:59.12 in these first two hours, is a gradient of this transcription factor bicoid. So in the last Transcription factors may be activated (or deactivated) through their signal-sensing domain by a number of mechanisms including: In eukaryotes, DNA is organized with the help of histones into compact particles called nucleosomes, where sequences of about 147 DNA base pairs make ~1.65 turns around histone protein octamers.
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