cc  >> Welcome to Frontiers  of Life Science.  I think it's our eighth session  with two classes left to go.  And today we have Dana Huebert  and she is a third-year graduate  student in cellular and  molecular biology.  And the topic is "Epigenetics:  When Genes Aren't Enough."  So Dana, if you would give us  a little history of who you are  in addition to that topic,  we would appreciate it.  Welcome to Dana Hubert.  (applause)  >> Thank you very much.  Is the microphone on?  Yes? Great, thank you.  As you said, my name is  Dana Huebert, I'm a third-year  graduate student in the lab  of Audrey Gesch at the  University of Wisconsin.  We're in the Genetics  Department.  And affiliated with the  Genome Center of Wisconsin. 

 And what I'm going to be talking  about today, epigenetics, is  actually interesting, he  brought up in the introduction  about the "New York Times"  article about the human genome  and what we know about it.  And what I'm going to be talking  about today is largely  what we don't know about it,  and where active research is  going on to try to understand  what all those genes mean  and how they're regulated,  things of that nature.  So, I have written this talk for  a largely uninformed audience,  so if at any time you feel like  I'm going too slowly  or too quickly, by all means  raise your hand and tell  me to get moving  or get to the interesting stuff.  I'm not even going to talk  about what epigenetics is for  a little bit, I'm actually going  to start out with cells  and with DNA.  So, as you probably know,  cells are made up of, really,  four primary components.  DNA, proteins, lipids which  are fats, and carbohydrates.  And so the bulk of what I'm  going to be talking about today  deals with DNA and also  a little bit with proteins.  So, if your cell is here,  your DNA is in the nucleus.  And the DNA is made up of  several chromosomes, which are  ultimately made up of single  stranded chemical molecules  called DNA.  And my lab mate, Scott, who  some of you might have heard  speak last year,  likes to compare this  to an encyclopedia. 

 So here is a picture of an  encyclopedia pictured in 1751.  And encyclopedia, as you know,  is essentially just the  compendium of all  human knowledge wrapped up  into separate volumes  and separate topics.  In this particular encyclopedia,  there were 35 volumes.  Each volume contains several  different pieces of information  on various things  that the human population  should know how to do.  Here are some simple examples:  chemistry, economics, etc.  Similarly, the human genome  is like an encyclopedia.  It contains all of the  information necessary  for your cells to do  what they need to do.  And the human genome, like your  encyclopedia section is divided  into volumes or chromosomes,  which is one single part  of the genome that contains many  but functionally unrelated  topics.  In your encyclopedia, they're in  alphabetical order, or separated  potentially by topic.  The chromosome is really the  position of genes, and doesn't  necessarily have anything to do  with their function is. 

 Then we have the DNA itself,  which is the physical material  that makes up your genome.  And specific pieces of  information, which are known as  genes are meaningful regions of  information that can be separate  from the rest of the genome.  So in the human genome then, we  have two sets of 23 volumes, or  23 chromosomes that make up the  entire information set.  We have about, well this is a  slight over-estimate.  I think this has been reduced to  about 20,000 protein coding  genes in the human genome.  And the average gene is 3,000  bases long.  Bases is basically a term that  we use for the letters that make  up DNA: A, T, C, and G, if  you've heard about them before.  There are about three billion  total letters in the human  genome.  So the Human Genome project  then, essentially went through  and sequenced all of these  various letters.  And so, one important thing to  note is that the DNA sequence  that came out of that was not of  a single individual, or even of  a certain cell type. 

 It was actually a collection of  many different individuals who  donated blood, essentially.  They knew that they were  donating, but they don't know if  they made it into the final  mishmash of people who got  sequenced.  So what that means is that what  we really have is a summary of  the information from a  collection of people, but not  any one individual.  Since then, several individuals  have been sequenced.  And it's possible for people  with enough money in the right  context to get sequenced.  But the one that we use, really,  for reference is a collection of  people.  So, what we know then is these  three billion base pairs are  letters of sequence.  And what we learned then from  the Human Genome Project is a  prediction of all the genes in  the genome, as well as what  order they're in.  Because as I said before, the  order doesn't necessarily have  anything to do with their  function.  We have predictions of some of  the gene functions, which come  both from studies in the human  cells, and also from comparing  those genes to genes in other  organisms so you have more well  studied, more thoroughly  studied, like yeast or mouse,  or something like that. 

 And we also know in which  regions of the genome provide  information and which are just  "junk," things that we don't  think are providing any  information, but are relics from  evolutionary history.  So there's a fair amount of the  DNA that's informational.  Some of it is informational in  that it makes proteins.  Some of it is informational in  that it regulates which genes  are being active or inactive at  any time.  And then there's a fair amount  of the genome that we have no  idea what it is.  And because we have no idea what  it is, we assume it's junk.  Take that for what it's worth.  So an important question is what  are genes and what aren't genes.  So genes are really information,  DNA that codes for a protein.  Then the example that we give in  the lab is that a recipe is not  a cake. 

 So having the information to  make a cake does not actually  give you a functional output  necessarily, rather, it gives  you the potential.  This says, the genes a cell has  represent all the things that  cell might do.  They don't tell you much about  what the cell is actually doing.  That's an important point,  because the genome alone, then,  can't explain everything.  So in this picture, Gene Kelly  and Frank Sinatra have 99%  identical genomes.  And we could go through and  sequence each of them and get  the whole collection of their  DNA, and then potentially make  comments on what's different  between Gene Kelly and Frank  Sinatra, tap dancing skills not  excepted.  So we could look at the  difference in their unique  codes, and that would tell us  something about the differences  between them.  However, Frank Sinatra's eyes  and his toes have 100% identical  genomes.  But obviously, they serve very  different functions.  And so, what is it about the  genome in his eyes and the  genome in his feet, which at the  DNA sequence level are  identical, but allows them to  perform very different  functions. 

 That's what I'm going to be  talking about today.  So as we talked about, DNA is  really an archive of all of the  information in a cell and what  potential functions the cell  could carry out.  You may have heard of essential  dogma, that DNA makes RNA which  makes protein.  So this DNA sequence here,  with these four genes,  1, 2, 3, and 4, can be copied  then into a disposable  active copy, called RNA.  And this RNA then is information  that we can use to make  functional proteins, but that we  can turn on and off as  necessary.  We can get rid of these copies  if we need to, if those  functions become no longer  necessary.  And of course, RNA can be turned  into the functional tools, which  are the proteins or in the  encyclopedia example, cakes,  shoes and hammers.  So really what this means is  there's no difference between  the DNA in Frank Sinatra's eyes  and his toes, but there is a  difference in the RNA level,  which genes are active or  inactive at any given time.  And that subsequently leads to a  difference in the protein  content of any given cell.  So just to kind of demonstrate  that more explicitly, here we  have four genes that are present  in each cell type. 

 And in this case, potentially  we're going to turn on specific  genes, genes one and three that  may code for, say eye color, or  the ability to dilate your  eyes.  In this case, we're going to  turn on genes three and four,  which have separate functions.  So this is probably sounding a  little repetitive, but what that  means is that we have a genome  and we have a transcriptome.  It's called a transcriptome  because RNA is known as  transcripts.  And so the transcriptome is the  set of all RNA molecules present  in a cell at a particular time,  and reflects the genes that are  active at any given time.  And it's therefore dynamic and  responsive.  That's key, because it allows  cells to react to either  internal signals that are coming  from your body, or external  signals that are coming from the  environment.  So what didn't we learn, then,  from the human genome project?  We still, knowing the sequence  doesn't tell us anything about  which genes are active or  inactive, either in an  individual cell, or even in an  individual person.  So you probably know that you  and the person sitting next to  you have a lot of differences  between you.  And some of that can be at the  DNA level, but some of it is  also at the ability of RNAs to  be expressed or activated. 

 So an important question is what  determines which genes are  active or inactive, and how do  reproducing cells maintain  active and inactive genes.  For example, it is important to  me and some of us in this room  that our hearts continue to make  heart cells.  And so, once a cell has decided  that it is a heart cell, it  needs to pass on that same  information about what RNAs are  necessary to be a heart cell to  every future daughter cell that  comes along.  So, there's an element of  maintenance of RNA expression.  That is really what makes up  epigenetics.  So from the human genome  project, we didn't learn  anything about the epigenetics  of humans.  And you may know that epi means  above.  So anything about the level  above the actual genetic, the  genome level.  We'll talk very explicitly about  what that means.  So the term epigenetics refers  to changes in gene expression  that are caused by mechanisms,  other than changes in the  underlying DNA. 

 So the DNA remains the same, but  there are changes in gene  expression that are caused by  external factors.  These changes can remain through  cell divisions, and sometimes  can remain for multiple  generations.  And we know that, for example  in studies in yeast, that if we  give them an external stimulus,  like a stress, if we heat them  up, that they will remember that  heat and pass that information  on to their daughter cells.  So it's sometimes just a matter  of a few cell divisions.  And it's sometimes multiple  generations of an entire  organism, in the case of yeast.  The key to this is that there's  no change to the underlying DNA  sequence.  So there are a lot of diseases,  for example breast cancer is a  good example where much of what  is known about what causes  breast cancer is it's actually a  mutation on the DNA level.  That's not what we're talking  about here.  We're talking about things above  that level, where the DNA stays  the same, but the activation of  RNA changes.  And a good example of this is  the process of cellular  differentiation. 

 So that's what I'm going to talk  about next.  So you might know that in all--  well, Wisconsin's a great place  to talk about this.  All organisms, especially  multicellular organisms have  what are known as stem cells,  cells that have the potential to  become any other cell type in  the body.  And so, the first stem cell  arises when an egg is  fertilized.  That's the first cell that's  going to make up any new  organism.  And that will ultimately become  all of the cells in the body.  So as this cell, which has four  genes, becomes four different  cell types, one of which remains  a stem cell, and others which  may be say, heart, lung and eye  cells, for example.  Different genes are turned on  and off.  So in a stem cell, we have genes  one and two, and a heart cell  two and three, etc.  So each cell type is really  defined by its gene expression  signature, by the signature of  genes that are on and off in  each case. 

 And then as this particular cell  goes on to become an entire  tissue's worth of cells, that  signature is passed on as the  tissue develops.  So the transcriptome determines  the function of the cell and is  remembered as the tissue  develops, and continuously, the  transcriptome signature is  continuously regenerated as new  cells are created.  A second example of this comes  from plants.  So as you've probably noticed,  it's quite cold now, so these  trees that used to have colored  leaves are now moving toward  a leafless existence.  And as we come around back to  spring, eventually they'll  regenerate those leaves.  An important point for plants  is that they can't get to this  stage too early.  They can't make leaves before  spring, because that would be  detrimental to the life of the  plant.  And so, what they do is they  rely on cues from the  environment, namely cold weather  and long days, to allow them to  know when it's the appropriate  time to start making leaves  again.  And so, in that way, there are  changes that occur that occur at  the transcriptome level, and the  RNA that were being expressed  that remind this plant that  we've been through winter, that  winter was long enough, so it's  not the case last week when it  was 70 degrees, all of our trees  started putting out new buds.  They know based on chemical and  environmental signals that that  doesn't mean spring has arrived  and it's probably going to get  cold again. 

 So there are changes that go on  at the RNA level and at the  transcriptome level that allow  plants to know when the  appropriate time to put leaves  forth is.  And I'm going to talk about how  that happens kind of later in  the talk.  So the environment can cause  changes, in which genes are  active without changing the  genome.  Are there any questions so far?  No, okay.  So how do we know what genes are  active, what RNAs are present in  any given cell.  So here's an example of a white  blood cell.  And so what we can do then,  using genomic technologies, is  isolate all of the RNAs from  this cell, label them with a  dye, and put them on a  microarray, and I'm going to go  through the details of this in  just a minute.  Put them on a microarray so  that we can tell which of these  genes were present in the  beginning.  And we can turn that into an  informatics kind of color coded  scene.  And then ingeniously come up  with which RNAs were present in  our original sample. 

 And I'll tell you about how that  works.  So each gene in the genome  has a unique sequence.  In this case, notice the last  six or seven are TATCATC.  And in this case,  they are TGGGTTT.  So these are two unique genes.  And as you probably know, DNA  in the body is double stranded,  which means it has a complement  that is essentially this in  reverse,  A is always bonded to T.  C is always bonded to G.  And that provides a mechanism  for us to know, for example,  for replication to occur.  So you can separate those  strands and put them back  together again by filling in the  other half.  We use that to our advantage  then, by putting together single  strands of DNA, which will bind  to DNA that we extract from the  cells, if they're complementary. 

 So for example, if I have this  tag on a slide for example, I  can take RNAs from the cell and  only if they're complementary,  will they bind.  Is that clear?  So that allows us, then, to use  microarrays, which are small,  little glass slides that are  spotted with thousands of unique  DNA probes.  So it's actually possible now in  some smaller organisms called  yeast, to take the entire genome  in small little fragments and  put them on an array like this.  Then we can interrogate the  whole genome at one time.  So I can isolate RNA from the  cell, put it on this slide.  And if its complementary  sequence is already present on  the slide, it will light up with  a fluorescent color.  So we can say this particular  RNA was present in my original  sample.  So we start out with our slide.  This has DNA, and we know the  sequence of each of these DNAs  that's been spotted on the  slide.  Each of these little squares on  the spotted array is made up of  several different unique  position. 

 And each position here  represents one gene.  So for example, this gene here  is Pkc1 in this example.  Then we can take our, what this  really represents is thousands  of copies of the same exact  strand of DNA.  So that's how we get to, well,  I'll talk about it in a minute.  I'm getting ahead of myself.  >> How long and how short is a  gene?  >> It depends on the organism.  So in yeast, most genes are  about 1,000 base pairs long.  In humans, it's closer to 3,000.  But they can be upwards or  10,000 or 100,000 base pairs.  >> Per gene?  >> Per gene. 

 Now, on our slides here, we  don't need the whole sequence in  order to be able to identify any  individual gene that was present  in our RNA sample.  So each gene is so unique that  we can find 30 base pairs in a  row, for example, that represent  that gene.  And if it hybridizes, then we  know that the whole gene was  present in our original sample.  Does that make sense?  >> It does.  What does the term hybridize  mean?  >> That's a very important  point.  So this ability to bind here to  its complementary sequence which  we put on the array, we call  hybridization.  It forms hybrid of the RNA that  we took out of our cells and the  DNA that we had on the slide to  begin with, that we know the  sequence of.  >> You have some information  that would cause you to make a  30-long, that you're trying to  match up?  >> Right, so in most organisms  now, sometimes it takes more  than one array slide to cover  the whole genome.  But we can find a 30 base pair  unique tag for each gene.  And then each of these little  spots here can represent every  gene in the entire genome. 

 So in yeast, there are 6,000  genes.  And on this slide, all 6,000 of  those genes are represented.  So the spots are tiny.  >> The fabrication of a  complementary segment is a very  rapid process in the lab now?  >> Yes, it is.  There are several ways to do it,  but it's less than a day-long  process.  So this is a spotted array, one  in which we actually take the  DNA out of a tube with a robot  with little pins, and we spot it  on that array.  Then they bind to the glass and  they're on there forever.  So once those DNAs that we're  going to put on the slide as our  template have been generated, we  have those forever.  They don't need to be  continuously re-made.  There are other types of arrays,  though. 

 This one over here is called an  Oligo array, where they actually  synthesize the DNA probes, one  at a time.  So you start with your layer of  glass, you put on what's called  a mask.  This is getting kind of  detailed, but you asked.  So you put on a mask.  Say the first base pair of 30  out of 100 spots that you're  going to put on there was C.  So you put on a mask, masking  everything else.  And you put your base pairs C  over the top and it binds to  those places that are still  open.  Now you put on a new mask for  the next base pair, which is  maybe A, for 20 of those.  And now all of those spaces  that are open have A.  That way, you can build these  little strands on your slide, as  long as you want them. 

 Not as long as you want them to  be, there are some technological  limits as to how long they can  get.  But 60 to 100 base pairs is well  within the range.  Yeah?  >> Those arrays, are those  commercially available or do you  build those for each test?  >> It's cheaper to build them  yourself if you have the  technology, but it's also kind  of the old-school way of doing  it.  Most people buy them now,  because having the appropriate  robots is a significant  investment.  So it used to be that everybody  did it this way.  You made them in-house.  And so they're really only  usable one time, but you could  make hundreds of them at a time,  and they last for a while.  It used to be that everybody did  it this way.  Now though, NimbleGen, which is  a company based here in Madison,  and others from other states and  other places in the country,  actually will make them for you  if you have an organism in mind.  Some of the major lab organisms,  like mouse, yeasts, worms, rats,  for example, all have entire  arrays that you can order  online. 

 If you wanted to do an  experiment in your basement, it  would totally be possible.  Other questions?  All right, so as you were  noting, the key to this whole  process is the hybridization.  So once we've made our array  with these strands of DNA  attached to a glass slide, then  we can take our sample that we  isolated from cells, and if a  complementary DNA exists in our  RNA collection, it will bind.  And if we label these with  fluorescent dyes, if we label  our sample with fluorescent  dyes, then we get a colored spot  where that RNA has bound.  Since we know the identity of  this particular spot, then we  can say, okay, Pkc1 was present  in my original RNA sample.  Typically, we do this in two  colors.  So we take a healthy cell and a  cancer cell.  We label one of them red and one  of them green.  Then we put them together and  put them on the same array so  that we can say, okay, if a spot  is red, that means that RNA was  more present in a cancer cell  than it was in my healthy cell.  Or conversely, that means it was  more present in my healthy cell  than in my cancer cell. 

 That's how a lot of the studies  that inform us about what RNAs  are necessary for making a  cancer cell, so we can  potentially go in and get rid of  them, and make those cells  healthy again, is done with this  type of genome-wide  interrogation.  So we can say, of all of the  genes that are possible in the  genome, which ones are active  right now in this particular  cell.  So with this information then,  we've moved one step up from the  Human Genome Project.  Now, in addition to knowing the  DNA sequence, we also know which  RNAs are active in any  particular cell type.  And we can take Frank Sinatra's  eyes, not anymore, of course,  but take his eyes and take his  toes, and say what's different  at the RNA level about these two  different tissues.  But that's not really the  information we want, right?  What we really want to know is  what determines which genes are  active in either of these cases,  because that's the level where  we could actually go in and make  changes if we wanted to.  For example, in the example  about healthy cells versus  cancer cells, or any other  disease.  So how is the genome regulated?  There are too many steps to even  really list, that are required  to activate a gene.  But the first step is to  regulate competence for  transcription.  I'll talk about what that means. 

 But really what it means by  undoing the packaging of the  genome.  So DNA in any given human cell  is about two meters long, or  about six feet.  People are always shocked to  hear that.  But cells are about ten  micrometers long.  So 20,000 times smaller in  length than the length of the  DNA that has to fit in there.  So if this mouse was any given  human cell, his DNA stretching  from his tail would reach  Oregon, Wisconsin, about 20  kilometers from here.  So obviously, that takes an  enormous amount of packaging to  be able to fill the DNA into  every single cell.  The counter to that, then, is if  your DNA is so tightly packaged,  how do you even access it to do  the things you need to do, like  replicate your DNA or transcribe  particular genes that you care  about.  So the key is you have to be  able to open the encyclopedia in  order to read the words.  You have to open your genome in  order to get access to the parts  that you care about. 

 The key is, you don't have to  see every page to read one page.  So you can open directly to the  page that you care about that  has the information that you  need for a specific process.  Then you can use that in every  subsequent daughter cell, if you  can remember which page you were  on at the beginning.  So packaging then, is a method  of regulating which parts of the  genome are active and inactive.  I'm just going to go through  this in some detail, because it  will be important for the  downstream floods.  But if we start out with our  double stranded DNA, our two  meters of DNA, the way the  packaging occurs is by wrapping  them around proteins, called  histones, which I hear you've  heard about before.  And these histones plus DNA  complex is known as a  nucleosome.  These nucleosomes then can come  together, in sequence,  stretching across and form  together higher order of  structures, like chromosomes,  which allow the DNA then to be  packaged inside the cell.  Two other features of the  nucleosome, one is that if this  is a binding site, for example,  it's necessary for certain  enzymes to recognize a gene, and  transcribe it.  You may have heard of an enzyme  called polymerase, which goes  through on the DNA, and actually  makes the RNA copies. 

 That's one step that's required  for regulating the genome.  If this is a binding site for  that, when we wrap this up in  our histone proteins, our  nucleosomes, the binding site is  no longer accessible.  So that's what I mean when I say  that this part of our  encyclopedia has been closed off  from anything that we might want  to do to that DNA, like  transcribe it into RNAs.  A second feature is that the  proteins that make up this  nucleosome have little tails on  the end of them that can be  chemically modified.  So some of those chemical  modifications might include  acetylation or methylation.  In this case, "Me" stands for  methylation.  But these marks can be put on  specific nucleosomes, on  specific genes.  And so this provides us a method  to mark specific genes for  activation or repression.  I'll go through that in one  second.  So I show this only because I  think it's a cool picture. 

 This is a micrograph of a cell.  Here's the outside of the cell,  kind of the double white layer.  Here's a pore, sorry, this is  the nuclear membrane, not the  cellular membrane.  Here's a pore in the nucleus  that allows things to go in and  out.  And here is DNA that is very  tightly packaged in these  nucleosomes.  And here is DNA that's open and  available.  So what that means is that some  of the space of the nucleus,  some of the space of the cell,  is being taken up by genes that  are active.  So the packaging is actually  tighter than you would think it  would have to be.  I mean, if everything was  tightly packaged, it would look  like this and take up about this  much space.  But actually, most of the  nucleus is taken up by genes  that are currently active and  able to be transcribed. 

 So how do we mark our genes for  activation and repression?  Here's our same four genes.  In this case, gene 1 and gene 3  have been activated.  And what that really looks like,  if we blow up just this gene 1  region, here's the black regions  to the left, which is usually  used for regulation of a  particular gene.  Here's our actual gene  information coding region.  And here it is wrapped up in  nucleosomes.  So here are some binding sites,  like I said, for the activation  machinery that would actually  come in if possible and  transcribe this gene.  Here are the protein structures  that actually package the gene,  and here are the tails that  stick up, that we can modify to  remind us whether or not these  genes are active or repressed.  So just an example, genes in  this cell really have three  possible states.  They can be active or on.  They can be temporarily off,  which means I'm not using it  right now, but I might use it  later. 

 Or they can be silenced, which  means forever off.  And I'll talk about some cases  where this might be, some  specific examples that might  make this a little clearer.  But to start out with, let's  just say we have an active gene,  and we're going to mark the  histones of that gene in a  particular way, in this case, a  methylation group on a  particular amino acid of our  protein K4, that will always  remind us that this gene is  supposed to be on.  Likewise, we can put a different  mark on.  In this case, methylation on a  different amino acid, K27, that  reminds us that, while I don't  want this gene off forever, I  want to be able to access it  later, I do need it off right  now.  Then the third state, the silent  state, which is marked by a  methylation of a separate amino  acid, K9.  And this serves a purpose of  silencing this region so that  it's never going to be on in  that cell again.  And you can see, I just  demonstrated here one  consequence of turning these off  forever is that the nucleosomes  become much more compact.  And so in that case, this is our  silenced chromatin, our silenced  nucleosomes I mean, and this is  our either active or repressed,  temporarily off.  So these we could still access  if we needed to. 

 But we might not be using them  at this particular moment.  >> From a percentage basis of  those three types, I don't know  how to ask the question, how  much of it is silenced, how much  of it is repressed, how much of  it is active?  >> That is a good question, that  I don't know the answer to for  any particular cell type, but my  guess is that it varies.  It depends on what type of cell  you are.  In yeast, which is  the organism I study,  and why I keep bringing it up--  You know, I wouldn't want to  speculate for fear of being  wrong.  So, I don't know.  The short answer.  I can say, with some certainty,  that a lot of the yeast genome  is active, whereas a lot of the  human genome is actually  repressed or silenced.  So the human genome has a lot  more functionality in any given  cell than it needs, a lot more  potential, because we're so much  more complex.  So the human genome has the  ability to make hearts and lungs  and eyes.  And so, in your liver, all of  those things have been turned  off. 

 In contrast, in a yeast, pretty  much the only functions it has  are the functions it needs to  survive.  And so most of the genome is  actually and in an open,  accessible state.  >> The K9 methylation, what is  that, a molecule?  >> Methylation is a chemical  molecule, yes.  So it's a carbon hydrogen  compound that actually gets put  on the protein.  >> Forever shuts it off, you  can't get it back on?  >> Yep, and so how that works is  kind of a complicated story.  But really what it does is bring  in other proteins that kind of  coat the region and keep it from  being opened later.  Other questions?  Yeah?  >> What you're doing with the  methylation doesn't change the  functioning of the cell, it's  only a tag for you to study it?  >> Right, it's a tag put on by  the cell itself, to remember  which of these genes is which.  But we can use it to our  advantage to figure out where  those regions are.  Does that make sense?  >> Yes.  >> So the cell itself is putting  these marks on them. 

 All right, this actually kind of  gets to your question.  It's possible to pull out all of  the pieces of DNA marked with a  specific modification on the  histone protein.  Then we can take those DNAs, in  much the same way we did to find  out what RNAs were active in the  cell, we can color them with a  dye, put them on a microarray  and say, okay, this region of  the DNA was marked with a K4  methylation, this region of the  DNA was marked with a K27, and  this region was marked with a  K9.  We can actually go through  different cell types, or  different parts of the cell  cycle and say which genes are  marked with active marks at any  given time.  And in some ways, they're so  tightly correlated between  having the mark and being active  or being repressed, that we can  actually use it as a substitute,  in some cases, for measuring the  actual RNA levels in the cell.  So if I know that this DNA  region is marked with an active  mark, I more or less know that  there's RNA being made from it  without actually measuring it.  So what that tells us is where  the active and repressing marks  are throughout the whole genome.  And this is a tool, then, that  we can use to study the  differences between various cell  types.  So if we go back to our two  examples, we'll start with  the animal example.  Remember that in each of these  different cell types, the  transcriptome is a signature  that tells each of these cells  what functions it needs that can  be passed on to every daughter  cell, as the cells are becoming  tissues. 

 And so at the histone level, at  the nucleosome level, what that  means is if we start out with a  stem cell, now I've upgraded our  genome a little, so now we have  12 genes in this particular stem  cell and only gene 6 is on.  At the chromosome level, gene 6,  for example, might be a gene  that's required for replication,  which is a function that all  cells, except potentially  neurons, need to undergo in  order to produce daughter cells.  I should clarify that.  Neurons don't usually make  daughter cells, which is why  they don't need to replicate.  Yes?  >> Could you go back two slides?  >> Two slides.  This one.  >> How do you go from this on  our left to the colored one?  >> That's a whole separate talk.  Well, actually, really, it's a  matter of turning on some genes  and turning off others.  At the most basic level, it's  selectively turning on genes for  a particular function in each  cell.  And as our stem cell is  dividing, it goes through a  process in which it makes more  of itself, so it regenerates  itself, more stem cells, and  sometimes, it goes through a  process where it divides and  makes a new cell type. 

 And that's based on a lot of  signals that are not well  understood.  The genes that are required to  keep this thing a stem cell are  more or less known.  That's Jamie Thomson's lab's  work, among others.  And the genes that are necessary  at various downstream levels are  known.  But how we get from one to the  other is really an area of  active research.  >> So it's not quite known yet.  >> Yes, frontiers of life  science, right there.  Yeah, I mean, we're really at  the level where it's easy to  know what genes are active in  any given cell type, and it's  difficult to know how they got  there, what regulatory steps  needed to happen to turn them on  and others off.  >> When you get to step two.  >> From here to here?  >> No. 

 >> From here to here?  >> Back one.  >> Oh.  >> There.  You obviously learned how to  make all reds from that point  on.  >> Well, the body knows how to  make all reds from one point on.  >> When then you know, or your  group knows.  >> I don't know if that's true  or not.  >> You can take stem cells and  make them myocardial cells.  >> Right, you can in a dish.  I mean, your body obviously can. 

 And trying to understand that is  a point of active research.  Although, recently papers have  come out that can push a stem  cell in a dish into particular  directions.  We can't push them into every  direction.  But you're right, we can make  certain other cell types out of  stem cells, because we know the  signals that are required.  I don't know them, because it's  not really my focus.  But it is known, yes.  >> Thank you.  >> You're welcome.  I feel like that was kind of an  unsatisfactory answer, but it's  the best I can do.  All right, so if we get back to  here, gene 6 is on our stem  cells because it's required for  a function that all downstream  cells are going to require. 

 And so we've marked it with our  active histone mark, our K4  methylation.  And this gene will be on in  every cell that is a daughter of  our stem cells.  Stem cells are tricky, though,  in that they have a method to  mark the rest of these genes as  unknown.  So it's not, I made a false  statement in saying that there  are three options, active,  repressed, temporarily off or  forever off.  There's a specific state in stem  cells that is temporarily off,  but not because I might need  this function later in my own  life, because I might need this  function later in my future  cells, as they become specific  cell types.  Does that make sense?  So for example, there's a gene  here, number 9, that for example  is required for eye color.  If this stem cell becomes a  liver, I want this gene off.  But if it becomes an eye, I want  to keep the potential for this  gene to be activated.  So stem cells have a mechanism  of marking these genes that are  unknown with both activating and  repressing marks at the same  time.  So the cell is smart enough to  know that this gene might become  necessary only in some cell  types, so it keeps these two  marks around so that it can  quickly say, okay, we're active,  or okay, we're repressed,  depending on what cell type it  becomes. 

 So they carry the marks of both  activation and repressing,  simply because they're still  deciding, as they differentiate,  whether that gene will be  necessary.  >> And is the repression mark  the unique molecule that you  understand?  >> As opposed to the activation  mark?  >> A variety of molecules.  >> There are several types of  modifications that can occur,  and I'm really only talking  about one of them.  But these methylation marks, for  example, in the active state we  said was lysine 4 that was  methylated.  In these cells, both lysine 4  and 27 are methylated at the  same time, so that one of them  could be removed in order for  this gene to be marked as on or  off.  Does that answer your question?  No other questions?  So what that means is that as  this cell differentiates, here  was our original state, now as  we go through differentiation,  more genes are becoming turned  on.  And if this is an intermediate  state between stem cell and  finally terminated finished  cell, some genes are still in  the I'm not sure state.  Or some genes, I should say are  still in the I'm not sure state.  So, our gene 6, the one for  replication is still on.  Our gene for eye color is still  marked with both, because we  don't know yet what cell type  we're going to be at the end. 

 But the gene for stomach acid  has been turned off, because we  know that this gene now, as it's  moving through various stages of  differentiation, is not going to  become a stomach cell.  We know in this example.  If this stem cell was going in a  different direction, we may have  turned off eye color and turned  on stomach acid.  But for the sake of example, we  don't want stomach acid in  anything that's going to be near  our eyes.  >> Would a gene 3, for example,  be strictly for stomach acid and  nothing else?  >> Probably.  So you're right in suggesting  that it's more complicated than  I'm making it.  But usually, there are multiple  genes that are required for  multiple functions.  Sometimes they need to come  together in a complementorial  way in order to do a particular  function.  Sometimes they're redundant, so  there's more than one protein  that could do the same function.  And it's an extremely  complicated process that pretty  much all of biology is trying to  reconcile, what's necessary when  and how do proteins come  together. 

 Yeah, you're really asking what  is the active research of  molecular biology doing.  It's that type of research.  So in this intermediate stage,  we have genes that are on, genes  that are off, and genes that are  still in an unsure state.  And as we move forward, then, to  a terminally differentiated  cell, like the eye, we've  reached kind of a final state.  So now, the gene for replication  and the gene for eye color are  on.  And they will be on in  subsequent daughter cells.  The gene for pupil dilation,  though, is unsure, because you  don't need it all the time.  But when you need it, it needs  to be in a state where you can  access it quickly.  So this gene has gone from being  in an unsure state to a  temporarily off state.  And our gene for stomach acid is  still forever off. 

 So in this way, when cells are  undifferentiated, they can use  different marks to remember  which genes to leave competent  for future cell types.  Then as the cells differentiate,  some genes are marked on, and  others are not sure, and others  are marked off.  And once they have been  terminally differentiated, the  cell can keep these marks as  reminders of which genes should  be on and off.  This is really an area of very  active research.  And mostly, right now, we're  trying to identify in different  cell types where these marks  occur, and can we use that  information to understand  something about what makes a  heart a heart and a liver a  liver.  And so, in combination with the  human genome project, a new,  kind of similar large  collaborative effort has been  started called the Human  Epigenome Project, which instead  of mapping the DNA in various  cells, we'll actually try to map  these epigenetics marks that  allow cells to be different cell  types.  So I'll give just one more  example in the ten minutes that  are left.  So in the plant example--  In the stem cell example,  it's really responding to  internal signals from the cells  themselves to allow them to  differentiate.  But humans, like plants, also  have to be able to respond  rapidly to environmental  signals.  So we don't want these  environmental signals to change  our genome, although that's  possible in the case of UV rays,  or something like that. 

 What we want is to be able to  change our epigenome, our  transcriptome, without changing  the genome.  And so, using plants as an  example, here's just one case.  This is a genetic pathway that  leads to flowering.  We have a gene here that's  required for flowering.  Then we have a gene up here  called FLC, Flowering Locust C  that prevents flowering.  So if this gene is on, this gene  is off.  Then we have a gene up here  called Fri or Frigida, which  actually turns on FLC.  So if Fri is on, FLC is on, and  flowering is off.  And so, it requires two signals,  then, in order to turn flowering  on at the appropriate time.  One is cold weather, and that's  an epigenetic response. 

 And one is long days, and that's  actually simply a genetic  response.  Let me go through this in a  little bit more detail, just to  clarify what I'm talking about.  In the fall, then, we're not  making any new leaves.  And in this case, I'm using  flowers as a stand-in for  leaves.  Although the same is true in  perennial plants.  So in the fall, our FLC gene is  on, which means our flowering  gene is off.  So the histones at each of these  various states, FLC is marked  with our active marks.  And the flowering gene is marked  with repressed marks.  Note that it's not off forever,  because when spring comes, we're  going to want to turn this on.  As we move through to winter,  then, cold weather comes in and  stops the activation of this  gene by changing the epigenetic  marks. 

 So where this used to be active,  we've just turned it off.  So you'll note that I've colored  this red, because it really is  off in winter and through the  summer when leaves are being  made.  And there's a totally separate  process that allows this to be  re-set to active.  But in the meantime, it doesn't  go through the repressed state,  it goes directly to off.  And that means that now this  flowering gene, we've repressed  the repressor, so we've made it  so that this thing is no longer  stopping flowering, and we've  made this competent to flower.  Now, it's winter.  We don't actually want to  produce leaves yet.  So there's a second signal  that's required to actually  activate this gene.  And that comes from the addition  of long days.  So in the spring then, the  weather is warm, but because we  have these epigenetic marks  which can be remembered, our  chromatin state actually doesn't  change. 

 So we've removed cold weather,  but because we've turned this  off, it stays off.  And because this guy is off,  this guy can remain competent  for transcription.  The addition of long days, then,  starts off a genetic pathway  that produces a protein that's  required to activate this gene.  So this pentagonal protein can  then come in, bind to a site  that is regulating the  activation of this gene, and now  flowering can occur.  So it's kind of a complex  genetic mechanism, and I've  obviously simplified it down to  four key components.  But the key is that by having  this epigenetic ability to  remember which genes are on and  off, despite the fact that warm  weather occurs, this guy is  still in an off state.  And so, we're ready to flower  after a certain amount of winter  has passed.  And in the presence of long days  in the spring, then we can fully  activate this gene and get  flowers and leaves.  So with that, I'll conclude, and  then I'll be happy to take any  of your questions.  So changes in gene expression  can give different functional  outcomes to cells with identical  genomes. 

 Epigenetic marks to nucleosomes  and histones allow cells to  remember which genes are active  and repressed, even when those  cells divide to the next  generation.  And this memory has been shown  to be necessary for cellular  differentiation in animal cells  and for response to  environmental cells in plants.  And I should say, it's also been  shown to be necessary for  cellular differentiation in  plant cells and response to  environmental signals in  animals, but I didn't talk about  that.  With that, I would be happy to  take any questions that you  have.  >> What we know about triggering  the long-day gene, I don't  understand how that might, how  any scenario whereby that might  work.  >> How do you mean?  >> Well, in other words, it's  got to know.  Something in the plant.  >> Right.  >> Something in the plant has to  know when the days are over a  certain period, because if it  turns on too soon.  And I guess I'm trying to figure  out the light waves, the photons  hitting the cells. 

 >> So it actually has to do with  the angle of the light.  So you can imagine that late in  the day, or as the sun goes  across, it's hitting the earth  at various angles.  And so, plant cells, especially,  because they photosynthesize,  are constantly receiving light  from the environment.  They have the ability then, to  recognize what angles of light  are being given to them.  So in the winter, the angle is  different.  And the length of time that  various angles are present.  >> Have you done some  experiments whereby instead of  changing the length, we change  the angle, and we've proven that  it's the angle that counts?  >> Right, so it actually has to  do also with parts of the  spectrum.  So they can recognize the  difference between red light and  far red light, if you're  familiar.  I know very little about  physics.  I think it's the case that  there's more far red light in  long days than in short days. 

 I hope I'm not screwing that up.  Yes?  Thank you.  In that case, cells can  recognize how much far light  they're receiving relative to  how much red light they're  receiving, and can use that as a  genetic signal to produce  appropriate proteins.  >> One question that I don't  understand.  It appears like there's no  systematic structure to control  the epigenetics system.  If there are events like the  angle of the sun that controls  that specifically.  If they're all like that, it  would seem like there's no real  systematic structure to that.  >> Right, I think it's true that  we don't know what that  structure is yet.  I think that there must be a  structure, because.  >> That's if there is one. 

 >> Yeah, well, because the case  is that all liver cells, for  example, from you and from me,  have very similar epigenetic  marks.  But how that system is set up is  still really an active place of  research.  So I don't doubt that there is  some way for the cell to be able  to interpret a signal that tells  it that it's going to become a  liver cell, turn on all of these  genes, turn off all of those  genes.  But there's a big gap there that  we don't understand yet.  >> If I'm understanding things,  it could be environmental  effects that could cause  differences in epigenes that  could cause differences in  identical twins?  >> Yes, and actually, I was  going to give that example, but  the talk got to be too long.  So it is definitely true that  identical twins raised apart  have different susceptibility to  disease.  So when they go in and look,  that could be for two reasons.  It could be that one of them  lived in Arizona and had more  exposure to sun than the one who  lived in Maine, so it was  actually a UV damage DNA  mutation.  Or, it could be that it's  epigenetic, and it turns out  that cases of both have been  seen.  If you go in and look at the  histone marks in two twins that  were raised separately, they're  different. 

 And they're more different as  people age, so it's actually  accumulating changes in the  epigenome over time.  >> The part about the cell  responding to a gene expression,  I kind of think of the eye  dilating as an example, or the  adrenaline that has to be at the  muscle site instantly, and the  sunlight instantly.  I'm wondering how the gene can  get turned on that fast, that  the light can trigger it and  your eye dilates, or I can see  something and I can be really  scared and run.  >> Really quickly.  You're right, so there's  multiple levels at which the  cell prepares itself for a  future response.  And actually, I study stress in  yeast cells for my research.  And you might say, what stresses  out a yeast, but that's a  separate issue.  (laughter)  Turns out, a lot of things.  They're unicellular,  they don't have a lot of  protection from the environment.  But you're question is about how  can cells rapidly respond to  signals. 

 So there are lots of ways.  One is that they build up, keep  proteins around, like  adrenaline, for example.  You have low levels of  adrenaline all the time.  And so, you have some of what  you need to be able to quickly  respond.  And the transcription process,  and the translation process, is  very rapid, but not  instantaneous.  So you're right, there's a  separate, there are other  pathways that your body has set  up to be able to respond  quickly.  I'm not sure, actually, I mean,  I picked this eye example  because I wanted to talk about  Frank Sinatra, honestly.  And so I'm not sure that eye  dilation is necessarily the best  example.  Because like you said, it   happens so rapidly.  I'm trying to think of a  similar, of something that you  would need, like, hmm,  I can't think of any that  have to do with the eye,  off the top of my head. 

 Well, anyway, the short story is  there are multiple pathways.  And this epigenetic pathway is  slower than a lot of the things  you would need for the fight or  flight response, for example,  which is regulated differently.  So in that case, genes are on.  There are two ways to regulate  that.  One is to keep those genes on,  either at low levels or at high  levels, in the event that you  need those proteins very  rapidly.  The other is to keep them in a  repressed state so that you can  turn them on later if you needed  them.  >> I want to go back to your  twin example and the ability to  inherit and pass that on, once  they have acquired this Arizona  versus Maine resistance.  >> Yes, so that is an important  question.  And a place that's being  actively researched.  But actually, similarly to the  plant example, I think it's more  or less thought that when you  have children, for example, a  lot of those marks are re-set. 

 So in the stem cells that your  offspring have, they've been  re-set to that, we can make any  cell in the body mode.  So the epigenetic marks are not  actually, might not be passed on  from parent to offspring in  humans.  They are passed on from parent  to offspring in yeast, though.  So it's interesting to think  about whether that's true in  some cases, or no cases, or in  all cases.  >> In the case of flowers,  that's been known for a long  time, like in commercial  producers controlling light,  especially the poinsettia,  timing it for Christmas.  But they're also finding that in  milk cows and laying chickens,  they're also affected or can  modify the production level by  artificial light.  >> Oh, really?  So is that related to circadian  rhythms, or like how much light  versus dark that they have?  >> I think that's the  relationship.  Anyway, in both of them, it's  increased the amount, especially  on the shorter day, that they  try to set timers to control  during the dark period,  artificial light.  That's why a lot of times you'll  see, in large dairy herds, that  they've got lights on at night  when you'd think that they'd be  off.  >> That's funny, I've wondered  about that. 

 Thanks, I've been educated.  Additional questions?  I'm happy to stick around, too,  if you have individual  questions.  >> Thank you very much.  >> Thank you.  (applause)