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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 ๐›พ:ฮด 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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. 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.

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