00:31:38.27 about 6 microns squared per second of the movement of a molecule of about 00:08:41.11 of the egg. WebThe first identified morphogen, and perhaps the best studied, is the transcription factor Bicoid 6. 00:16:35.08 we've learned that what's central here is that this protein gradient that we can 00:12:02.23 where we're activating hunchback gene expression you can measure those concentrations 00:02:17.12 they are very sensitive to the size of the egg. Below is a brief synopsis of some of the ways that the activity of transcription factors can be regulated: Transcription factors (like all proteins) are transcribed from a gene on a chromosome into RNA, and then the RNA is translated into protein. It's basically a 10% difference. For the previous two parts of this lecture we've 00:10:53.13 in a bigger egg, is bigger, proportionately bigger, because if we then Although the sharpening phenomenon was observed over a decade ago, it is still poorly understood. 00:14:52.07 that would result in a catastrophe for development 00:09:08.04 would be to keep the bicoid gradient the same shape 00:04:11.15 like to have, as direct as possible, a measure of actual concentration in the given 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. And I think the reason that Development. 00:23:44.12 to detect molecules at different distances from the anterior end of the egg, 00:33:33.06 in embryos that carry a GFP-histone so we'll be able to eventually 00:14:50.16 in terms of number of molecules, in terms of absolute concentration. 3 minutes to precisely measure morphogen concentration Cell. 00:32:19.21 with or being bound to other molecules and therefore ultimately we can explain that 00:16:36.29 of the egg. 00:02:51.27 that I'd like to talk about. 00:24:45.08 if you look at those movements and those measurements that you can measure 00:22:59.23 of the egg, how does that movement of bicoid protein from the site of its synthesis 00:10:21.22 of bicoid really is directly reflecting or is directly controlling those gene expression patterns, DNA within nucleosomes is inaccessible to many transcription factors. 2017. 00:35:48.28 establish and move molecules like bicoid and that the stable gradients that we see 00:08:09.00 Even though the eggs are bigger and even though the cells are bigger Because we know that ultimately all 10 embryos 00:16:17.13 in the egg. 00:25:27.17 in the Drosophila egg, and yet we know that the gradient is already there 00:34:56.04 to the random walks that are produced by diffusion. 00:01:29.26 So one of the goals of our work is not just to understand how embryos develop, So one of the things we'll talk about is 00:35:37.17 like diffusion. 00:09:22.18 during the course of evolution. 00:13:12.21 what these concentrations are. bicoid transcription factor Clipboard, Search History, and several other advanced features are temporarily unavailable. 00:25:49.18 to a problem that must have been old and has always existed for all embryos, There are really two simple ways Morphogen - Wikipedia 00:35:56.24 are the products more of that movement. 00:14:43.08 uniformly distributed throughout the egg, but what we've learned now is that [47] TET enzymes play a central role in demethylation of methylated cytosines. [3][4] Transcription factors are members of the proteome as well as regulome. 00:05:47.08 features of all these eggs though is even though they are different sizes 00:19:15.26 on the one hand and then change the size of the egg 00:18:21.27 end of the egg the concentration will fall, this is a transcription factor Whether it was the hundredth cell at this end or the zero cell or the one cell 00:01:45.05 distributions of bicoid or constant transcription patterns. 00:22:47.05 traces is not that initial distribution, but the final distribution is achieved by And lastly, there is one other interesting point 00:34:07.17 and I'll point this out to you when we watch the move one more time, 00:24:25.15 it was at a time where we were coming to realize as molecular biologists that And so 00:04:29.16 So Drosophila melanogaster for example makes eggs that are about 00:01:08.29 I think of the importance to us as biologists right now, beginning to try to view 00:01:45.03 that are essential for life. Now I'd like to 00:19:25.07 in the embryo and are involved in establishing those spatial patterns. 00:24:48.28 you find that they are very small, that the diffusions constants are very small. They suggest that binding a single factor to both enhancers and promoters 00:09:09.06 head of the embryo that's marked off by this fold, it's called the cephalic or So these proteins put in a melanogaster egg will produce On the genomic level, DNA-sequencing[86] and database research are commonly used. 00:27:00.06 measure molecular movement. 00:04:34.04 500 microns long. 00:16:37.27 because you can measure that optically from the images that you have, 00:23:59.09 the biophysical parameters associated with the generation 00:13:31.10 before the cells have begun to change shape that direct the cells And if you think in terms 00:31:26.22 it doesn't go down to zero. [62], Many transcription factors are either tumor suppressors or oncogenes, and, thus, mutations or aberrant regulation of them is associated with cancer. 00:11:02.15 of bicoid can provide stable sufficient information about position to activate precise Not only of the embryo and for embryonic 00:01:40.22 and cell biological parameters that might give rise to these constant 00:05:05.06 that expresses this EGFP bicoid transgene that Thomas Gregor made. 00:10:40.09 the ventral side, and we can see that other things are going on. 00:13:52.05 and now I tell you well that's because their expressing different gene 00:20:56.12 So I'll stop there and thank you for your attendance. 00:15:21.07 vitelline membrane and so what Thomas was able to do was to take these EGFP-labeled 00:20:50.25 is quantitative. The action of Bicoid the transcription factor is relatively well understood, although the finer points keep emerging. 00:15:01.14 in part, or at least it was easier to do in flies, is that we're using EGFP and many argue 00:18:14.00 about bicoid and hunchback is the extent of this binding and occupancy One possibility would be that the embryo 00:12:10.04 measure the concentration of bicoid in a given nucleus and the output You localize an RNA and then when that RNA 00:06:06.02 So what we're going to see is the bicoid gradient in this living embryo and in the embryo 00:34:15.11 is that associated with these nuclear replications are massive movements 00:11:38.10 you can see is that it's as expected, highest at the anterior end here and falls 00:02:55.21 in the laboratory, in the way that we measure them, in the stocks 00:20:16.22 and it's my own belief that the future of developmental biology 00:33:44.12 syncytial embryos. 00:02:55.07 was interested in was in this simple cartoon sense, of how an information gradient 00:29:25.06 at least some molecules moving from the anterior end of the egg [30], Many transcription factors, especially some that are proto-oncogenes or tumor suppressors, help regulate the cell cycle and as such determine how large a cell will get and when it can divide into two daughter cells. 00:10:10.16 at the bicoid distribution in these other insect species, what we observed 00:28:08.00 simple diffusion and allowed Thomas to calculate for dextrans of different sizes 00:09:09.24 at the daughter nucleus that comes to lie closest to the position of the mother nucleus 00:07:42.25 products and then the sense is that's when it begins to transcribe its own 00:22:24.04 which is very finely localized to the anterior end. 00:16:30.13 of bicoid, this 8 nM value, and you know the actual volume of the nucleus 00:03:48.17 that binds to something that binds to something that binds 00:30:14.10 diffusion or to Brownian motion if you will, is its size dependence in that the 00:18:43.15 because actually hunchback is not the only gene in the Drosophila genome that can In my second lecture I describe experiments using EGFP tagged Bicoid to follow Bcd gradient establishment in living embryos, and to test various aspects of the simple model. 00:00:11.22 professor at Princeton University. 00:18:50.28 of the bicoid protein that are essential for it to make The Origins of Anterior-Posterior Polarity - Developmental WebIn Drosophila, a maternally supplied transcription factor called Bicoid plays a particularly important role. 00:34:34.27 are not going to move molecules in any particular direction, but 00:02:06.01 patterns constant, but the actual sizes of the eggs are constant. National Library of Medicine 00:33:14.28 of this molecule through what you can think of as a stable cytoplasm Many large TF families form complex homotypic or heterotypic interactions through dimerization. 00:01:36.10 one of the things that we really have to be able to do is to measure and supply numbers. 00:10:48.11 is this movement of cells, and watch right here you'll see a fold forming. 00:04:20.09 Such that after two and a half hours this single cell has now transformed itself 00:08:26.20 begins to reflect the active contribution of genes in the embryo itself. 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. 00:16:05.06 something like 8 nM. The https:// ensures that you are connecting to the 00:24:01.03 suggests that you need diffusion constants of the order of The Bicoid protein establishes the dorsal-ventral body 00:19:39.23 over and over again. 00:23:07.11 how is that able to establish a stable gradient. 00:06:52.14 the unfertilized egg before fertilization and what the embryo 00:20:56.05 put gene products in the egg to establish pattern. 00:04:37.20 a living probe, a probe that could allow us to follow the bicoid protein in living embryos Due to their important roles in development, intercellular signaling, and cell cycle, some human diseases have been associated with mutations in transcription factors. 00:06:25.11 at the anterior end and the posterior end. 2020;137:79-117. doi: 10.1016/bs.ctdb.2019.11.016. 00:06:26.11 is also observed in all of these other insects and it also is observed 00:00:26.24 and maternal proteins that are localized to particular regions of the egg, 00:27:33.28 provides an interesting opportunity to study how evolutionary pathways 00:09:03.06 concentration. 00:06:36.17 and you can see in those nuclei at the anterior end of the egg, you can see an [44] For gene transcription to occur, a number of transcription factors must bind to DNA regulatory sequences. 00:04:23.27 that these different fly species make, they are also different sizes. The anterior determinant bicoid of Drosophila is a derived Hox 00:12:26.16 with patterns of gene expression. 00:14:54.02 that are supplied and necessary during these early stages are mostly Bicoid functions without its TATA-binding protein-associated 00:16:50.08 there were about 697 molecules per nucleus. 00:18:36.05 major transcriptional activation occurs in the embryo but In Drosophila during the early stages 00:22:44.05 distribution that the cells are going to use to make their developmental 00:05:51.12 if you actually look at the development of these embryos 00:06:41.04 say this is an embryo, at the syncytial Drosophila embryo from 00:08:13.13 the patterns per cell are exactly the same. 00:03:56.05 and there are also disgusting flies like house flies and blow flies that 00:23:04.03 begins to make a protein, the protein diffuses. Can we 00:17:45.21 about how bicoid or how any transcription factor controls or activates transcription 00:20:28.23 in development will depend heavily on our ability to combine 00:19:00.24 And then the other question is what are the features that change This is also true with transcription factors: Not only do transcription factors control the rates of transcription to regulate the amounts of gene products (RNA and protein) available to the cell but transcription factors themselves are regulated (often by other transcription factors). 00:22:05.01 that I've listed here, this orthodenticle, giant, or Krppel, obviously are also responding 00:12:30.16 is being expressed right here, and you ask how much of a difference is there And in particular, in that simple 00:11:31.05 no growth. 00:08:14.20 fertilization, protein is being made, translation is continuous if the embryo is making more So you could 00:01:41.02 And the experiments that I'm going to present, although you'll see their not fully complete 00:29:40.09 Now, strictly speaking, what Thomas's measurements argue for dextran is that 00:01:20.02 and study in embryos are the same kinds of processes that involve 00:36:23.29 that if biological parameters like cytoplasmic flows control the ultimate shape 00:19:12.14 Krppel gene then and the hunchback gene define domains D research in Basel, Swizterland. Many proteins that are active in the nucleus contain nuclear localization signals that direct them to the nucleus. 00:08:59.08 the time when the nuclei first migrate out to the surface, and you can measure the bicoid 2022;2383:33-44. doi: 10.1007/978-1-0716-1752-6_2. And then in the very last part, 00:13:40.10 So to test that possibility, you need to begin to hope to identify 00:09:03.04 And as you change the length of the egg, one strategy 00:15:03.12 that are established in these eggs, actually using the Calliphora protein 00:25:08.20 less than what you'd actually need to visibly establish the gradient. 00:29:59.18 being transported or binding to something or being moved by other mechanisms. 00:08:23.09 for you to keep in mind though that now what we're going to be looking at 00:17:08.10 any GFP-tagged protein put into a Drosophila egg make a gradient 00:20:56.06 and trying to figure out whether it is in a room with 690 or 630 because it's activation 00:13:24.08 that I've indicated here or changes in other regions of the protein 00:08:36.09 Stabilization would actually be really important because if the nucleus wants to know 00:02:55.15 processes, and that's my goal. And what Thomas found, And that becomes important 00:17:51.00 the property of the protein that allows it to build gradients of a 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. 00:18:09.18 other proteins that are the actual transcriptional activators. 00:27:38.29 you can see this biologically inert but fluorescent molecule moving through 00:24:19.29 and those remarkable initial characterizations, the expectation was, 00:02:35.24 diffusive constant and establishes a particular constant shape 00:13:35.14 and control their cell behaviors. 00:15:08.27 for the remainder of my talk is a protein called bicoid. 00:16:11.16 predicted from in vitro binding studies for what would be required 00:33:18.17 such that diffusion could establish a stable gradient. 00:21:53.27 uses certain genes like hunchback as guideposts, establishes them with great accuracy 00:05:25.15 that is involved in localizing that RNA to the anterior end of the egg. TFs work alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes. 00:21:43.02 how is it? These transcription factors are critical to making sure that genes are expressed in the right cell at the right time and in the right amount, depending on the changing requirements of the organism. 00:10:41.11 of bicoid protein extends much longer, much farther 00:15:39.20 look at the concentration or measure the intensity of the bicoid EGFP signal 00:05:18.29 oogenesis. 00:26:15.26 and new solutions. 00:18:40.09 those genes are activated by bicoid as a transcription factor PLoS Comput Biol. [13], There are approximately 2800 proteins in the human genome that contain DNA-binding domains, and 1600 of these are presumed to function as transcription factors,[3] though other studies indicate it to be a smaller number. 00:01:48.29 development but for everything that we see today. 00:20:50.03 of those proteins and also the other features of the egg. 00:07:52.23 if you look at say hunchback or giant, the two different gap genes Transcription factors use a variety of mechanisms for the regulation of gene expression. 00:03:13.20 that you see back there were made in fixed embryos where you fix an embryo And this is probably Mutations affecting segment number and polarity in Drosophila. 00:03:29.12 fruit flies and extend your observations to other kinds of embryos identify a two-motif sequence code that is required and sufficient for activation of a specific promoter by the transcription factor Bicoid. 00:11:07.01 the bicoid gradients in the big eggs and the small eggs are [14] Therefore, approximately 10% of genes in the genome code for transcription factors, which makes this family the single largest family of human proteins. And there are other theoretical considerations 00:35:06.20 does not arise necessarily directly from diffusion or from the diffusive movements But what's interesting 00:05:59.16 what we need to do is to go back one time and watch this movie one more time. And one of the things we've learned and that I'll tell you more It's still a single cell. A matter of time: Formation and interpretation of the Bicoid morphogen gradient. 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]. Following fertilization, the single celled embryo undergoes a number of mitotic divisions to produce a ball of cells called a blastula or blastoderm. 00:02:43.02 gene activities and basic cellular functions but what we'd really like 00:34:39.22 will we believe contribute, if bicoid is being associated with the cytoplasm, 00:23:12.18 molecules move, how fast are they degraded, all those things will ultimately 00:13:42.13 the question back. 00:06:47.06 and all the RNAs are supplied by the embryo's mother, put into Once we had an EGFP-bicoid molecule Thus, contrary to recent reports proposing that the Bcd gradient is not sufficient to establish precise positional information, we show that Bcd drives precise and sharp expression of its target genes through a process that depends exclusively on its ability to activate transcription. Furthermore, several artificial Gal4-derived transcription factors expressed as gradients in the embryo share Bcd's ability to drive precise target gene expression with sharp boundaries. 00:09:26.24 that is at cycle 10, just after 10 cycles to just when the nuclei made it out to the surface But to measure concentration in those molecules 00:13:19.16 the orange gene which is a gene called runt. 00:14:47.10 sized gradient in a Drosophila egg and you can imagine 00:29:21.08 more than enough of what you need to make a gradient. So we can see here, that other genes that can be activated by And other pieces of DNA, if we accept 00:12:16.20 and others not? 00:15:28.25 extending out in a visible sense to about 48% where we'd be activating No one knew what the nature of DamID profiling of protein-DNA interactions", "The general transcription factors of RNA polymerase II", "Systematic DNA-binding domain classification of transcription factors", "PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants", "TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions", transcription factor/coregulator deficiencies, https://en.wikipedia.org/w/index.php?title=Transcription_factor&oldid=1163318984, Short description is different from Wikidata, Creative Commons Attribution-ShareAlike License 4.0, C-terminal effector domain of the bipartite response regulators, Caused by mutations in the tumor suppressor, stabilize or block the binding of RNA polymerase to DNA, interaction with other transcription factors (, 1.1.5 Family: Plant G-box binding factors, 1.2.1 Family: Ubiquitous (class A) factors, 1.2.2 Family: Myogenic transcription factors (, 1.3 Class: Helix-loop-helix / leucine zipper factors (, 1.3.1 Family: Ubiquitous bHLH-ZIP factors; includes USF (, 1.3.2 Family: Cell-cycle controlling factors; includes, 2 Superclass: Zinc-coordinating DNA-binding domains, 2.3.1 Family: Ubiquitous factors, includes, 2.3.2 Family: Developmental / cell cycle regulators; includes, 2.3.4 Family: Large factors with NF-6B-like binding properties, 2.5 Class: Zinc fingers of alternating composition, 3.1.1 Family: Homeo domain only; includes, 3.1.3 Family: Homeo domain with LIM region, 3.1.4 Family: homeo domain plus zinc finger motifs, 3.3.1 Family: Developmental regulators; includes, 3.3.3 Family: Cell-cycle controlling factors, 3.6 Class: TEA ( transcriptional enhancer factor) domain, 4 Superclass: beta-Scaffold Factors with Minor Groove Contacts, 4.4.1 Family: Regulators of differentiation; includes (, 4.4.2 Family: Responders to external signals, SRF (, 4.4.3 Family: Metabolic regulators (ARG80), 4.5 Class: beta-Barrel alpha-helix transcription factors, 0 Superclass: Other Transcription Factors. 00:12:56.11 as being involved in protein stability. 00:07:02.28 in the Lucilia and the nuclei in Drosophila melanogaster or busckii [21] The preinitiation complex binds to promoter regions of DNA upstream to the gene that they regulate. 00:07:07.00 these gene products that are supplied by the mother. Background Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). 00:17:21.21 size and a particular distribution. 00:14:04.07 what type of gradients that they make. 00:07:02.16 doing the same thing over and over again using the same gene products, 00:20:11.16 is again the kind of quantitation that we talked about in the second lecture 00:09:45.12 And when you watch this movie again what you can see, this is again 00:19:57.21 interact with other genes in the genome. 00:10:45.08 because of the variabilities of fixation and staining was there's very very little Nat Commun. 00:06:24.23 can begin to become different from each other and show distinct behaviors 00:03:51.22 to the molecule, and you are never really sure then about the levels of staining 00:00:04.17 My name is Eric Wieschaus and I'm a HHMI investigator and professor Our results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Krppel. When Thomas did that, he found that 00:04:17.27 region of the embryo. 00:19:18.14 and change the movement of the proteins. 00:14:43.00 that it moves faster or that it degrades less, would make a Calliphora [87] The protein version of the transcription factor is detectable by using specific antibodies. WebWe engineer light-controlled versions of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. 00:28:53.11 what Thomas saw was that in contrast to the 0.3 microns squared per second If you look at fly eggs, not only are the expression 00:12:52.23 or other regions that have been implicated at least suggestively 00:22:45.19 Another really interesting problem is how it is that the gradient is actually established 00:11:48.09 that we saw in the movie are all happening because individual cells in specific 00:20:22.13 If you look at the model that you have a graded maternal protein that 00:10:20.12 reproducible pattern, so embryos always make a head and they always The function of TFs is to regulateturn on and offgenes in order to make sure that they are expressed in the desired cells at the right time and in the right amo In terms of microns, that is the bicoid gradient 00:04:52.01 that's followed by cell division. 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. 00:13:24.04 of gene expression, of transcription, that have arisen at this stage, 20 minutes 00:36:06.25 of gradients in unfertilized eggs where we see different patterns of movements And all the bicoid molecules, For example, although the consensus binding site for the TATA-binding protein (TBP) is TATAAAA, the TBP transcription factor can also bind similar sequences such as TATATAT or TATATAA. The problem is actually, to make the problem graphic just 00:16:56.00 just put straight, if you can imagine just taking EGFP or EGFP 00:02:01.23 which is the embryo that we work on in the lab develops, and point out to you 00:08:14.01 but it's also associated with adding new gene products. 00:02:10.16 along the anterior to posterior, that is head to tail axis of the embryo depends 00:14:48.13 we have to put the emphasis on mostly these maternal RNAs and proteins Shuttling Homeoproteins and Their Biological Significance. 00:29:54.17 It doesn't actually tell you that molecules are moving by diffusion rather than 00:19:22.15 us to models where what's actually required is, for cells to make choices,. And it's that process 00:27:39.17 are modified. Protein that regulates the rate of DNA transcription, Srpskohrvatski / , Differential enhancement of transcription, Availability of other cofactors/transcription factors, How Genes are Regulated: Transcription Factors, Sun Z, Xu X, He J, Murray A, Sun MA, Wei X, Wang X, McCoig E, Xie E, Jiang X, Li L, Zhu J, Chen J, Morozov A, Pickrell AM, Theus MH, Xie H. EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity.