Session 7: Autoimmunity and Allergy
Transcript of Part 2: Transcription Factor Aire Orchestrates T Cell Tolerance
00:00:08.15 So, I'm Diane Mathis. 00:00:10.07 I'm a professor of immunology 00:00:11.29 at Harvard Medical School, 00:00:13.17 and this talk, my second one, 00:00:17.23 is focused on one particular mechanism 00:00:21.29 of enforcing T cell tolerance, 00:00:24.07 which depends on the transcription factor Aire. 00:00:29.11 Now, in my first talk, 00:00:31.09 I explained to you that 00:00:34.13 tolerance mechanisms are needed 00:00:37.25 because of the random manner in which 00:00:40.25 the repertoires of antigen-specific receptors 00:00:44.15 displayed on B cells and T cells 00:00:48.09 are generated during their differentiation 00:00:51.24 in the thymus and bone marrow. 00:00:56.10 And then I went on to 00:00:59.21 define and describe several of the mechanisms, 00:01:03.00 the major mechanisms of imposing tolerance. 00:01:07.13 And then, finally, 00:01:10.10 I explained that these tolerance mechanisms 00:01:15.01 break down, actually, relatively frequently, 00:01:17.20 and the result is the development of autoimmune disease, 00:01:21.15 diseases like type-1 diabetes, or myasthenia gravis, 00:01:25.26 or multiple sclerosis. 00:01:30.11 Now, this talk is focused on 00:01:36.00 one of the more recently discovered 00:01:40.06 means of enforcing tolerance, 00:01:42.17 that is, it was discovered about 15 years ago, 00:01:45.25 and it depends on a transcription factor called Aire. 00:01:51.25 It's an important mechanism 00:01:54.03 and a fascinating mechanism, 00:01:57.08 at least I think so and I hope you'll agree with me 00:01:59.29 by the time we get to the end of the talk. 00:02:03.25 this mechanism actually makes... 00:02:07.26 links together two of the important mechanisms, 00:02:12.14 one in central tolerance 00:02:14.11 and one in peripheral tolerance. 00:02:16.28 So, the Aire story starts with 00:02:20.17 a human disease called APS-1, 00:02:24.17 for autoimmune polyglandular syndrome type-1. 00:02:29.08 And these individuals have 00:02:31.15 severe Candidiasis infections 00:02:34.13 of mucosal surfaces. 00:02:36.02 They also have autoimmune attack 00:02:38.27 on the parathyroid glands 00:02:40.19 and autoimmune attack on the adrenal glands. 00:02:43.25 Now, these are the three most frequent symptoms 00:02:46.01 and they're the symptoms which are used 00:02:49.22 to diagnose the disease, 00:02:51.19 but all of the individuals with APS-1 00:02:55.10 have multiple other autoimmune manifestations. 00:02:58.17 They might have type-1 diabetes 00:03:01.00 or they have autoimmune attack on the ovaries or the liver, 00:03:03.18 and what these organ targets are 00:03:07.08 varies from individual to individual, 00:03:09.02 even for people that are in the same family 00:03:12.02 and have the same mutation. 00:03:16.10 So, in 1997, 00:03:19.21 two groups independently cloned 00:03:23.09 the gene underlying APS-1 00:03:25.08 and they called the protein that it encodes 00:03:29.01 Aire, for autoimmune regulator. 00:03:30.29 Aire was a quite large protein 00:03:33.21 of more than 500 amino acids, 00:03:35.27 and by now more than 50 mutations, 00:03:38.15 scattered through the Aire gene, 00:03:42.29 have been identified in different individuals with APS-1. 00:03:50.11 So, from the beginning, 00:03:52.07 Aire was thought to be some kind of transcriptional regulator, 00:03:57.00 and I'll show you later on that this, indeed, 00:03:59.16 turns out to be the case. 00:04:02.24 So, one important clue 00:04:05.04 to how Aire is working came 00:04:09.05 just from knowing where it's expressed. 00:04:10.28 So, it's expressed primarily in the thymus, 00:04:14.01 not by the differentiating T cells themselves, 00:04:17.11 but rather by the stromal cells, 00:04:19.16 the epithelial cells, 00:04:23.07 which nurture their differentiation and allows the different processes 00:04:27.24 that need to take place for T cell maturation. 00:04:31.16 They're localized... 00:04:34.13 Aire is localized in the medulla 00:04:36.10 and, within the medulla, 00:04:38.21 specifically in a very small subset of epithelial cells 00:04:42.27 which we call medullary epithelial cells, 00:04:45.14 and they make up only 0.5% 00:04:48.27 of the stromal cells in the thymus. 00:04:52.27 And the reason that this finding elicited interest 00:04:57.29 was that, at that time, 00:05:00.06 there was a body of data that was growing larger and larger 00:05:02.22 that these cells 00:05:06.08 actually express a large repertoire of RNA transcripts 00:05:10.04 encoding what we normally think of as 00:05:14.01 proteins particular for fully differentiated cells. 00:05:18.19 So, for example, one is insulin 00:05:21.09 or there might be myelin basic protein, 00:05:24.10 or a heart protein, or a liver protein. 00:05:28.04 In fact, when it was looked at very carefully, 00:05:32.13 it was found that many tissues in the body, 00:05:36.04 more than 30, 00:05:37.25 are represented by transcripts 00:05:41.13 in this very small population of cells. 00:05:46.17 And so the notion developed that these transcripts 00:05:50.11 would be translated into proteins 00:05:53.02 and, actually, if you have good antibodies 00:05:55.19 you can find these proteins 00:05:57.09 -- you can find insulin in these cells, for example -- 00:06:00.29 and these proteins would be degraded 00:06:04.03 by normal mechanisms of antigen processing 00:06:07.08 and loaded onto MHC molecules 00:06:09.17 and be shuttled to the surface of the cell. 00:06:13.07 And then, as the self-reactive, differentiating T cell 00:06:17.23 is percolating through the thymus, 00:06:20.06 if its T cell receptor recognizes 00:06:22.23 this peptide-MHC complex 00:06:25.28 in a particular window of affinity or avidity, 00:06:30.04 T cell tolerance will take place. 00:06:34.03 And it was our hypothesis that, 00:06:36.12 actually, Aire is controlling the transcription 00:06:39.20 of this repertoire of transcripts 00:06:44.11 encoding peripheral tissue antigen... 00:06:48.20 proteins, or PTAs is what we call them. 00:06:51.23 And that was because of the overlap 00:06:55.25 in where Aire is expressed, 00:06:57.17 as well as the fact that individuals that have an Aire mutation 00:07:01.04 have a multi-organ autoimmune disease. 00:07:04.06 So, to evaluate our hypothesis, we 00:07:09.19 -- and, actually, other investigators, independently -- 00:07:12.29 made mice which were lacking Aire 00:07:16.02 -- Aire knockout mice. 00:07:18.24 And when we looked at these mice, indeed, 00:07:21.00 they had inflammatory infiltrates in several organs. 00:07:27.03 I'm showing the salivary gland, here, and the thyroid gland, 00:07:29.20 where you can see these infiltrating leukocytes, 00:07:33.18 which you don't see in the wild type... 00:07:36.18 corresponding wild type tissues. 00:07:39.24 These mice also have auto-antibodies, 00:07:42.16 circulating auto-antibodies, 00:07:44.13 against many different organs. 00:07:46.18 So, basically, what you do here is 00:07:49.28 you just take serum from a wild type mouse 00:07:52.22 or a knockout mouse and use it... 00:07:56.04 incubate it with normal tissue 00:07:59.03 and then come in with a secondary step, 00:08:01.24 which allows you to light up 00:08:05.23 those regions where the antibody bound. 00:08:09.06 And so, the serum from the Aire knockout mice, 00:08:11.10 but not from the Aire wild type, 00:08:12.29 lit up a lot of different organs, 00:08:15.01 and actually some specific structures in different organs. 00:08:20.15 So, it was the parietal cells of the stomach 00:08:22.07 and the rods and cones region of the retina, for example. 00:08:26.01 Then a very critical experiment 00:08:30.12 was the one that's depicted here, 00:08:32.10 where we isolated RNA 00:08:35.22 from medullary epithelial cells 00:08:38.14 coming from an Aire wild type mouse, 00:08:40.18 on the x axis, 00:08:42.19 or an Aire knockout mouse, on the y axis, 00:08:44.27 and what I'm showing you is 00:08:47.16 whole-genome expression profiling, 00:08:52.13 where each dots represents expression of a particular gene 00:08:57.10 and how it's expressed in the wild type 00:08:59.24 is its point on the y axis... on the x axis, 00:09:03.09 and how it's expressed in the knockout is its point on the [y axis]. 00:09:06.06 And where its induced by Aire 00:09:10.15 falls below the diagonal. 00:09:12.14 And when we looked at this, 00:09:14.28 what we found was that 00:09:17.11 Aire actually induced hundreds of transcripts 00:09:19.25 in medullary epithelial cells, 00:09:21.24 in particular, transcripts that were encoding these PTA proteins, 00:09:27.11 or peripheral tissue antigens. 00:09:31.05 Now, these data which I've shown you 00:09:33.13 were the original data that we got back in 2002, 00:09:37.19 and we could see that Aire was controlling hundreds of transcripts. 00:09:40.12 And more recently, there are more performant methods 00:09:43.24 to look at gene expression, 00:09:47.24 and, for example, what we call RNAseq, 00:09:51.08 which allows us to look at a lot more transcripts 00:09:53.12 and in a more quantitative way, 00:09:55.10 and what we found there surprised us in the fact that 00:09:59.10 Aire actually induces thousands of transcripts, 00:10:02.28 more than a quarter of the genome. 00:10:08.18 And then, the last experiment is 00:10:12.03 what I call closing the circle, 00:10:15.01 and what we did there was to take Aire knockout mice, 00:10:18.02 and in those mice there are circulating auto-antibodies, 00:10:22.15 and some of these are against stomach proteins, 00:10:26.02 which I showed you on the previous slide. 00:10:29.18 We then isolated... 00:10:31.04 we then identified the antigen 00:10:33.09 that these stomach antibodies saw 00:10:37.00 and found out that it's the mucin protein. 00:10:41.02 And then we went back 00:10:43.02 and looked into the medullary epithelial 00:10:47.26 gene expression profile and found that, indeed, 00:10:50.25 Aire was regulating mucin transcripts, 00:10:53.16 because when Aire wasn't there 00:10:56.13 mucin transcript levels in the thymus were reduced. 00:10:59.04 Other investigators did similar experiments, 00:11:02.24 one with an eye antigen 00:11:05.26 and another with salivary gland antigen, 00:11:08.29 and came out with the same conclusion. 00:11:11.21 So, I think that the model which we proposed 00:11:16.17 actually turned out to be correct, 00:11:18.23 according to a number of criteria, 00:11:20.19 and the model was close enough to the human disease 00:11:24.23 that it could be used to dissect specific mechanisms. 00:11:31.15 So, let's look a little bit at the cellular mechanisms involved. 00:11:36.07 First of all, you might have noticed that 00:11:40.04 I was very vague about what happened 00:11:42.18 once the differentiating thymocytes 00:11:44.23 saw the MHC-self-peptide complex. 00:11:46.19 I just said T cell tolerance takes place. 00:11:49.23 Now, there are several mechanisms 00:11:52.28 by which this might happen. 00:11:55.26 It could be that Aire promotes negative selection, 00:11:59.11 or clonal deletion, 00:12:01.14 of the self-reactive effector cells 00:12:03.25 that are going to get out into the periphery 00:12:07.08 and wreak havoc. 00:12:09.05 It could instead, or in addition, 00:12:11.15 be that Aire promotes positive selection 00:12:14.09 of regulatory T cells 00:12:16.25 that control the activities of these effector T cells. 00:12:21.13 Or Aire could do something completely different, 00:12:23.12 maybe something totally unexpected, 00:12:25.16 for example, it could control 00:12:28.15 antigen-presenting cells 00:12:30.20 or some other kind of innate cell 00:12:34.14 that starts off an autoimmune response. 00:12:40.00 Indeed, we found that Aire does promote negative selection, 00:12:43.23 or clonal deletion of self-reactive cells, 00:12:45.22 and I'll show you the original experiment 00:12:51.03 which demonstrated that. 00:12:53.11 So, this takes advantage of a trick 00:12:58.14 that immunologists use, 00:13:00.10 the creation of T cell receptor transgenic mice, 00:13:02.22 and this just tries to get around the fact that 00:13:08.06 any particular antigen specificity 00:13:10.09 is usually at a very low frequency, 00:13:12.17 so only 1 in 10^4 to 1 in 10^6 T cells 00:13:17.09 will be specific for a particular antigen. 00:13:19.19 And so, what one can do is 00:13:22.16 isolate T cell receptor transgenes 00:13:25.08 from a particular T cell clone 00:13:27.07 that are already rearranged, 00:13:29.10 introduce those into mice, 00:13:31.13 and that will shut off endogenous T cell receptor gene rearrangements, 00:13:38.16 and so the mouse will have a repertoire 00:13:40.14 that's highly skewed for that antigen specificity. 00:13:43.24 And in this case we add an additional twist onto that, 00:13:47.04 in that we create one mouse 00:13:50.18 that has a neo-self-antigen 00:13:53.19 expressed somewhere, 00:13:55.06 and then we create a T cell receptor transgenic mouse 00:13:57.20 that's capable of seeing this neo-self antigen. 00:14:02.08 So, an example is, for the neo-self antigen, 00:14:06.07 we make a mouse which is expressed ovalbumin, 00:14:10.12 membrane-bound ovalbumin, 00:14:13.04 under the dictates of the rat insulin promoter, 00:14:16.05 so it should be expressed in the pancreas 00:14:19.15 and, as I now should have convinced you, 00:14:21.24 is also expressed in the thymus. 00:14:25.26 And the second mouse, the reporter mouse, 00:14:28.05 is a T cell receptor transgenic mouse 00:14:33.17 which is capable of seeing a peptide 00:14:37.11 for membrane-ovalbumin. 00:14:39.25 So, you can see that when we cross these two mice together, 00:14:42.14 we create a potentially explosive situation 00:14:47.12 where there are many, many T cells 00:14:50.05 expressing this self-reactive specificity. 00:14:53.11 So, this slide shows you what happens 00:14:56.29 in the thymus of these mice. 00:14:59.17 We're looking at thymocytes 00:15:03.09 by staining for the CD4 and CD8 co-receptors, 00:15:07.21 and what you see is that, 00:15:09.15 in the absence of the neo-self antigen, 00:15:13.23 just the plain T cell receptor transgenic, 00:15:15.26 we find a lot of CD4... 00:15:18.15 fully mature CD4 single-positive T cells maturing. 00:15:24.15 And that's because the original clone 00:15:27.04 that we started with was a CD4+ T cell clone, 00:15:30.04 so that's expected. 00:15:31.28 And this is what happens 00:15:34.20 when you now cross the mice to the transgenic line 00:15:38.01 which is expressing the neo-self antigen. 00:15:41.03 In the presence of Aire, these CD45... 00:15:44.08 these CD4+ T cells are absent 00:15:48.24 -- they disappear, they're clonally deleted. 00:15:50.24 However, in the absence of Aire, 00:15:54.14 those T cells come back. 00:15:58.20 So, that clonal deletion does not take place 00:16:01.22 when Aire's not there. 00:16:03.16 And, actually, the animals develop 00:16:06.28 autoreactivity of the pancreas. 00:16:13.12 So, other labs did similar experiments 00:16:17.01 using other TCR transgenic 00:16:20.27 neo-self antigen models 00:16:23.01 and, in addition, 00:16:25.01 non-transgenic systems 00:16:28.04 could demonstrate that Aire was required 00:16:31.12 for clonal deletion. 00:16:33.04 So, I think we can be more specific here 00:16:35.06 and say that Aire is 00:16:39.05 controlling negative selection of self-reactive effector T cells. 00:16:43.16 Now, it turns out that Aire also controls 00:16:46.09 positive selection of regulatory T cells. 00:16:49.29 When we first started studying this, 00:16:51.29 we didn't think that that was the case 00:16:54.06 because we looked at a lot of mice 00:16:56.01 and we didn't see much difference 00:16:58.18 in the frequency or the type of Treg cell 00:17:03.17 that these mice had 00:17:06.13 -- so, CD4+/Foxp3+ regulatory T cells -- 00:17:10.28 and that's because we were always looking at adult mice. 00:17:14.05 Later, we looked earlier 00:17:17.14 and did find that there was a deficit 00:17:19.26 in this regulatory T cell population 00:17:22.29 before 10 days of age. 00:17:25.14 Now, we wondered if this was very meaningful, 00:17:30.01 because it's not a huge difference 00:17:32.06 -- the cells aren't totally gone -- 00:17:34.09 and it's only during this quite narrow time window 00:17:38.01 where one sees the difference. 00:17:40.00 So, we designed an experiment 00:17:42.05 which allowed us to show 00:17:45.03 whether regulatory T cells, during that time period, 00:17:47.20 are important. 00:17:49.07 So, we could do both a loss-of-function experiment 00:17:52.01 and a gain-of-function experiment. 00:17:54.22 So, in the loss-of-function experiment, 00:17:56.24 we took a mouse 00:18:00.19 where we could turn Foxp3 off 00:18:03.20 whenever we wanted to. 00:18:05.28 And so if we turn off Foxp3 00:18:08.14 then we won't get Tregs made during that time window. 00:18:12.14 So, we used that mouse to 00:18:15.03 deplete regulatory T cells 00:18:20.14 that are being generated during the first ten days of life, 00:18:23.16 and when we did that the mice developed multi-organ autoimmunity, 00:18:25.18 as indicated by the shaded blocks 00:18:29.28 that I show you, here. 00:18:32.03 If we did the same experiment, 00:18:34.10 however we took an adult mouse 00:18:37.16 where we depleted Tregs for 10 days, 00:18:39.06 we saw only rare and quite sporadic autoimmunity. 00:18:45.09 Now, for the gain-of-function experiment, 00:18:48.01 what we did was take Aire knockout mice, 00:18:50.15 which, as you saw before, 00:18:52.11 will develop multi-organ autoimmunity, 00:18:55.09 and then we took Tregs that were generated specifically 00:18:58.27 during that 10-day age window just after birth, 00:19:02.14 and we added those in, 00:19:05.20 and those mice were highly protected from autoimmunity. 00:19:08.27 However, if we took regulatory T cells 00:19:12.12 that were made during a 10-day window in an adult, 00:19:16.01 they were not protected. 00:19:17.18 So, by both the loss-of-function and gain-of-function experiments, 00:19:22.14 it was clear that Aire 00:19:27.16 was controlling an important population of regulatory T cells, 00:19:31.16 and this was specifically 00:19:34.29 during a very early time window. 00:19:38.05 Now, that made sense to us, 00:19:40.03 because we had an earlier finding 00:19:42.20 which we were not able to understand 00:19:45.11 until we got these more recent results, 00:19:47.21 and that is that we made a mouse 00:19:49.27 where we could turn Aire on and off at will. 00:19:53.28 So, if we had Aire on during the whole life of the mouse, 00:19:59.11 there was tolerance and the mouse didn't develop any autoimmunity. 00:20:03.10 And if we had Aire off during the whole life of the mouse, 00:20:06.24 it did develop autoimmunity. 00:20:09.23 That wasn't very surprising; 00:20:11.10 that's what we expected at that point. 00:20:13.14 However, what was surprising was that 00:20:17.00 if we had Aire on only for the first 7 or 10 days of life, 00:20:22.15 and then turned it off, 00:20:25.21 the mice were tolerant -- they didn't develop any signs of autoimmunity. 00:20:28.10 And then, conversely, 00:20:31.18 if we had Aire off during the first 7-10 days of life 00:20:35.10 and then turned it on for the rest of the life of the animal, 00:20:38.15 they did develop severe autoimmunity. 00:20:41.15 So, the expression of Aire during this very early, 00:20:47.06 this perinatal time window, 00:20:49.18 seemed to be necessary and sufficient to predict... 00:20:53.13 to protect from the autoimmune disease 00:20:56.02 that develops in the absence of Aire. 00:20:59.23 And so, we can also add that 00:21:03.07 Aire is also functioning 00:21:07.02 to promote positive selection of regulatory T cells. 00:21:10.05 I would be remiss if I didn't add that 00:21:13.13 other experiments in other laboratories 00:21:15.21 have suggested that Aire might have additional functions. 00:21:19.12 It might be involved in 00:21:22.01 differentiation of medullary epithelial cells, 00:21:24.11 differentiation of 𝛾:δ cells, 00:21:27.00 the turnover of medullary epithelial cells, 00:21:31.00 or peripheral tolerance. 00:21:35.15 Okay, ummm... 00:21:37.23 so, I think those are the major points 00:21:40.07 I'd like to make about the cellular mechanisms of Aire. 00:21:42.17 Now, I'd like to turn to the molecular mechanisms. 00:21:45.20 And I'll start out by saying this is an area 00:21:47.28 where it's been very dark for some time, 00:21:52.24 and light is just beginning to be shed. 00:21:57.29 People have been fascinated 00:22:00.04 by the molecular mechanism of Aire 00:22:02.02 since the beginning, 00:22:03.14 and the reason for that is that 00:22:05.22 here is a transcription factor 00:22:08.06 that's controlling thousands of genes 00:22:10.26 in a very small population of cells in the thymus. 00:22:16.11 And these genes, in the periphery, 00:22:20.00 are expressed very differently. 00:22:23.08 They're expressed in different cells, 00:22:25.11 they're expressed at different levels, 00:22:27.07 and they're expressed at different times 00:22:30.10 during the ontogeny of the individual, 00:22:33.22 so how can this one transcription factor do that? 00:22:38.22 So, from the beginning, 00:22:40.08 it was thought that Aire 00:22:43.19 was a transcriptional regulator, 00:22:45.09 and that's because it has 00:22:49.08 structural and functional features 00:22:52.05 of a transcriptional regulator. 00:22:53.27 So, structurally, it has a SAND domain 00:22:58.09 and, in other transcription factors, 00:23:00.28 the SAND domain is a DNA-binding domain. 00:23:03.12 However, I should mention that the important 00:23:09.24 amino acids in Aire... 00:23:11.26 the important amino acids for DNA binding in other proteins 00:23:15.25 are mutated in Aire 00:23:18.03 -- they're not the same as they are in these other proteins. 00:23:20.19 Aire has a nuclear localization signal. 00:23:23.10 It has a CARD domain, 00:23:25.11 which is important for homo-oligomerization. 00:23:29.10 It also has two PHD domains, 00:23:31.23 which are used in various types of protein-protein interactions. 00:23:41.02 As far as functional features, 00:23:42.27 Aire is localized in the nucleus, 00:23:45.01 as one would expect for a transcription factor. 00:23:47.06 It can induce transcription 00:23:50.02 -- so, if you make an expression vector where... 00:23:56.25 which will allow Aire expression if you transduce or transfect 00:24:01.17 the plasmid into a cultured cell, 00:24:08.19 and you also transfect a reporter gene 00:24:12.27 driven by the interferon promoter, 00:24:16.20 Aire will induce expression of that reporter. 00:24:20.06 So, it also binds to known transcription factors. 00:24:24.00 The first one identified was CBP, 00:24:27.10 or CREB-binding protein. 00:24:29.09 Now, Aire only binds very weakly 00:24:32.07 and non-specifically to DNA, 00:24:34.22 even though it has the SAND domain, 00:24:38.18 which I mentioned. 00:24:40.04 It seems to be more involved in binding 00:24:43.07 to different chromatin proteins 00:24:45.05 than directly to DNA itself. 00:24:50.17 So, even though it looked and smelled like a transcription factor, 00:24:52.29 there were always some odd things about Aire 00:24:55.18 which made one question 00:24:58.26 whether it was a classical transcription factor 00:25:01.29 that would bind to a promoter and induce or repress transcription 00:25:06.14 of a particular locus. 00:25:09.17 So, first of all, it's regulating thousands of genes, 00:25:14.01 so it seemed unlikely that they would 00:25:16.26 all have a specific binding site for Aire. 00:25:20.06 Secondly, it induces genes 00:25:25.26 which encode proteins that are found in many different cell types. 00:25:29.23 And, thirdly, it's possible to 00:25:34.04 introduce Aire artificially into different cell types, 00:25:38.04 either in culture or making transgenic mice, 00:25:41.21 for example, putting Aire behind the rat insulin promoter, 00:25:44.06 and in all these different cell types 00:25:46.27 Aire will induce batteries of transcripts. 00:25:49.14 However, the particular transcripts 00:25:52.17 that it does induce 00:25:54.27 differ from cell type to cell type, 00:25:56.23 and usually there's only about a 10-20% overlap 00:26:00.01 when you're comparing different cell types. 00:26:04.14 So, people have been working quite hard on this puzzle, 00:26:09.13 and I have to say that 00:26:13.16 we don't have a complete answer yet, 00:26:15.19 but we have learned some very important things 00:26:18.25 over the past couple of years. 00:26:20.21 So, one of them is that Aire 00:26:24.08 partners with many different proteins. 00:26:29.20 Now, the way that this experiment was done 00:26:33.23 was to take chromatin 00:26:37.09 from an Aire-expressing cell, 00:26:39.23 immunoprecipitate Aire 00:26:42.22 and then use mass spectromety... 00:26:47.05 spectrometry... 00:26:49.17 to identify the proteins that are binding to Aire. 00:26:52.05 And of course you have to do 00:26:54.23 many different types of controls 00:26:57.09 to prove the validity of this assay, 00:27:01.06 which were done. 00:27:03.00 And, even after these controls, 00:27:05.19 it became clear that Aire binds to... 00:27:08.20 interacts with scores of proteins, 00:27:10.22 either directly or within the same complex. 00:27:15.10 Up here, I'm showing about 40, 00:27:17.13 but even since this figure was made 00:27:20.07 there are another 5-10 which have been identified. 00:27:23.21 Now, some of these... and these proteins fall into four different classes: 00:27:27.24 nuclear transport, 00:27:30.12 pre-messenger RNA processing, 00:27:32.11 chromatin function, 00:27:34.10 and the DMA-damage response 00:27:38.24 and some transcriptional activation function. 00:27:41.19 So, it's not surprising that Aire would bind to proteins 00:27:47.08 involved in nuclear transport, 00:27:48.23 because it is in the nucleus, 00:27:50.27 or chromatin function, 00:27:53.01 because we know it's a transcriptional regulator somehow, 00:27:55.12 but some of the other classes of proteins 00:27:57.26 were somewhat surprising, 00:27:59.21 like pre-mRNA processing. 00:28:01.16 And, after this was found, it... 00:28:05.10 experiments were done which showed that 00:28:08.12 Aire does actually control pre-mRNA processing. 00:28:10.26 And then, lastly, 00:28:14.17 these proteins that are involved in transcription 00:28:19.00 but are also part of the DNA-damage response. 00:28:24.01 So, that's one important thing. 00:28:25.29 The second important thing is that 00:28:28.08 Aire seems to be operating very early 00:28:30.28 in the process of gene transcription 00:28:33.25 by RNA polymerase-II. 00:28:37.05 So, when I was learning about transcription, 00:28:41.25 some years ago, 00:28:44.14 we used to think that there were two types of genes: 00:28:47.24 genes that had RNA polymerase on them, 00:28:50.02 and they were transcribed; 00:28:52.07 and genes that didn't have RNA polymerase on them, 00:28:55.15 and they weren't transcribed. 00:28:56.20 But, over the last, say, 5-8 years, 00:28:59.14 it's become clear that there's a third class of genes 00:29:03.13 which is very important, 00:29:06.09 and these genes are ones which RNA polymerase binds, 00:29:11.01 it moves a little bit into the gene, 00:29:13.09 and then just stops. 00:29:14.29 And these are called paused or stalled polymerase genes, 00:29:20.13 and it's clear that every cell type 00:29:23.11 that's been looked at so far 00:29:25.17 has a group of genes 00:29:27.27 which have paused polymerase on them. 00:29:29.29 So, to explain this a little better, 00:29:34.06 let me give you some detail. 00:29:36.27 So, what happens is that 00:29:40.09 RNA polymerase binds to the transcriptional start site 00:29:43.12 and it has at its C-terminal end 00:29:47.09 a set of repeats of a particular sequence 00:29:50.16 that has serines in it that become phosphorylated. 00:29:54.23 And so it binds at the transcriptional start site 00:29:57.28 and lets its C-terminal domain hang out, 00:30:02.18 and then it clears the promoter, 00:30:06.03 once there has been phosphorylation 00:30:11.06 of the serine residues at position 5 in this repeat 00:30:14.21 by the transcriptional factor TFIIH. 00:30:20.09 It proceeds a little further 00:30:23.22 and then just stops. 00:30:26.07 This is a paused polymerase 00:30:28.21 and it has been thought that one of the reasons that it pauses 00:30:32.11 is to allow time to allow capping of the RNA to take place. 00:30:36.22 Now, pausing is enforced by these two transcription factors, 00:30:40.28 NELF and DSIF, 00:30:44.04 and it's lifted, or released, 00:30:47.16 by the combined action of Brd4 and the heterodimer P-TEFb. 00:30:55.07 So, this set of proteins comes in, 00:30:57.28 causes additional phosphorylation of the C-terminal domain, 00:31:02.09 causes phosphorylation of NELF, 00:31:05.04 which releases it, 00:31:07.13 and turns DSIF into a positive transcriptional regulator. 00:31:13.13 And then elongation can proceed. 00:31:15.29 So, what Aire does is that 00:31:21.26 it releases RNA polymerase pausing 00:31:26.03 on paused genes. 00:31:27.20 And it's now become clear that 00:31:30.24 this site of RNA polymerase pausing 00:31:33.24 is a site of impact for many important types of 00:31:38.15 transcriptional regulation. 00:31:40.02 It's where c-Myc works, 00:31:41.27 it's where much of the regulation of LPS 00:31:44.28 -- lipopolysaccharide -- 00:31:48.22 induced genes in macrophages is controlled, 00:31:53.07 and this is where Aire is operating. 00:31:56.15 So, we have a number of piece of evidence that point to that. 00:32:01.12 First of all, if we go back to 00:32:05.05 our genome-wide gene expression profiles 00:32:07.08 and, instead of looking at the whole gene... 00:32:10.14 the whole... 00:32:13.02 a group of Aire-induced genes and looking at the whole gene, 00:32:15.29 we look at different regions along the gene, 00:32:19.10 what we find is that Aire's impact 00:32:22.11 at the beginning of the gene is relatively low, 00:32:24.29 but it's impact later on is much higher, 00:32:27.26 which is indicative of an effect on elongation. 00:32:33.11 Secondly, Aire interacts directly with 00:32:38.29 Brd4 protein and also P-TEBb proteins. 00:32:43.03 And, thirdly, if we take inhibitors of 00:32:47.01 Brd4 or P-TEFb 00:32:49.28 or things which lift polymerase pausing, 00:32:54.05 those inhibitors also inhibit 00:32:57.15 Aire-induced gene transcription. 00:33:03.10 So, we're quite convinced that this is 00:33:06.27 a key element of Aire's molecular mechanism of action. 00:33:11.12 So, in fact, this mechanism goes quite far 00:33:14.15 in explaining many of these Aire oddities, 00:33:18.26 which I mentioned before. 00:33:21.08 It's clear, then, that it can 00:33:24.01 simultaneously control thousands of genes, 00:33:25.28 because thousands of genes 00:33:27.27 have polymerase paused on them. 00:33:29.15 It can induce 00:33:32.04 peripheral tissue antigen genes 00:33:34.23 associated with many cell types, 00:33:36.15 because, in fact, it's been found that, 00:33:38.22 preferentially, genes that are paused 00:33:42.01 are genes which are going to later be induced 00:33:44.20 or are later important for cell type differentiation. 00:33:49.26 And then, finally, it explains why, 00:33:52.19 if you put Aire into different cell types, 00:33:55.17 different sets of transcripts are induced, 00:33:57.16 and that's because different cell types 00:33:59.28 have different repertoires of paused genes. 00:34:04.18 And it also explains the large number of protein partners 00:34:09.07 that Aire has, 00:34:11.08 and that's because this scaffold, 00:34:14.21 this tail, this C-terminal domain tail, 00:34:18.10 which becomes phosphorylated 00:34:21.04 when polymerase pausing is lifted, 00:34:24.23 is an important scaffold for a number of cellular processes. 00:34:29.06 So, this... these phosphorylation events control RNA capping. 00:34:36.06 They'll also be very important in regulating splicing, 00:34:39.21 elongation, 00:34:42.10 and even nuclear export. 00:34:45.15 And then, lastly, both DSIF 00:34:48.19 and the C-terminal domain 00:34:50.16 have been implicated in controlling different histone modifications. 00:34:56.26 So, that's where we stand today 00:35:01.24 with both the cellular and molecular mechanism of Aire, 00:35:05.12 and of course there are some very interesting questions 00:35:08.00 which remain to be answered. 00:35:10.15 If we look back at the cellular mechanisms, 00:35:13.00 I think the most pressing issue is to determine... 00:35:18.15 is to determine, 00:35:22.16 what's so special about this repertoire of regulatory T cells 00:35:25.04 that develops in the first 10 days of a mouse? 00:35:27.22 Do they have a particular repertoire? 00:35:29.29 What are the antigens that they are seeing? 00:35:32.25 And why are they so important 00:35:35.13 for protecting against autoimmunity, 00:35:37.28 when regulatory T cells are of course generated 00:35:40.17 throughout the life of the animal? 00:35:42.17 If we look at the molecular mechanism, 00:35:45.01 it's clear that we have some important bits and pieces 00:35:51.03 about how Aire operates at the molecular level, 00:35:55.19 but we need to learn, still, some important things, 00:35:58.29 like, how does Aire actually target the genes 00:36:01.22 that it is going to induce? 00:36:04.05 Is it sufficient that the gene just be paused 00:36:06.08 and there's something about that configuration 00:36:08.21 which Aire can recognize, 00:36:10.16 or is there some other element 00:36:14.03 which is used for recognition? 00:36:15.24 And then, finally, what I haven't told you yet 00:36:19.10 is that Aire does control 00:36:22.08 a lot of these peripheral tissue antigen transcripts 00:36:25.11 in medullary epithelial cells, 00:36:27.15 but there are also some that Aire doesn't control. 00:36:30.19 So, is there an Aire-2? 00:36:33.24 And, if that's the cause, what's its identity? 00:36:38.10 there's no homologous protein, 00:36:40.29 or no highly homologous protein, I should say. 00:36:43.16 So, what's its identity, 00:36:46.05 and does it operate in the same way that Aire does. 00:36:49.22 And, lastly, I'd like to thank or acknowledge 00:36:53.26 the people in the lab who have worked on Aire 00:36:56.26 over the past 15 years, 00:37:01.08 and I think found some very interesting things.