Sunday, September 9, 2012

A little discussion about the ribosomal RNA genes
I mentioned in the last post about the rRNA genes (also called rDNA loci).  The reason why these are important starts with what is called the central dogma of molecular biology, which is that DNA -> RNA -> Protein.  That means that the information for everything in your cell is contained within your DNA.  That information is transcribed into RNA, which is chemically very similar to DNA.  It is also mobile.  It moves from the nucleus of your cells out into the cytoplasm where its information is translated into protein.  The picture below, found at, illustrates this graphically.  The way that the RNA is translated into protein is by something called the ribosome, .  This ribosome is actually an RNA based machine, that is 3 RNA molecules do the actually synthesizing of the protein. These are called ribosomal RNAs, or rRNAs.  Because every protein in the cell needs the ribosome and these rRNAs to be made, they are obviously found in very high abundance. The way this is possible is because they are in a very high copy number.  Typically you have 2 copies of each gene, one from mom and one from dad.  But you have 200 copies of these genes (100 from mom and 100 from dad).  They are found in what we call head to tail repeats.  This means they come right in a row with the end of one going right up against the start of the next, sorta like floats in a parade.  The interesting thing about these genes is that in actively dividing cells, all 200 are turned "on".  But as cells develop into more mature types, some are turned "off".  Most commonly, half are turned off but I've also seen many cases where most are turned off.
Essentially, one can think of these as the engine that allows a cell to grow and divide.  This has made the regulation of these genes very important to the study of severe metastatic tumors and cancer treatment.  Because it seems that when a cell becomes cancerous, its less likely to become a life threatening and severely aggressive tumor unless its able to turn all these genes "on".
Its an interesting field of study to those of us, like myself, that are interested in basic molecular biological questions because of the problem that differentially regulated repeats pose to the study of the cell.  Sequence is very important to biology. The sequence of DNA is what differentiates one gene from another.  And from these sequences, we can understand how a gene is turned on and off.  Most commonly, whenever you have identical DNA sequences, they behave identically.  However, this is occasionally not true. In this instance, it is the crux of biologists like myself to determine how the cell can tell the difference between two identical sequences. This is a common theme in a study that has recently gained notoriety, called epigenetics. 

A chance to play catch-up

A little summary of the Giles Lab, and how it came to be. 
(also, a summary of some science sprinkled throughout and again at the bottom)
Its been a long time since the last entry into this blog. So I’ll start with happened since the last time. I was a postdoc at the NIH/NIDDK in Gary Felsenfeld’s lab for six years. It seemed like forever. I started there to work on RNAi and its effect on chromatin structure, and that was what I worked on. For anyone whoever reads this I'd like to not sacrifice the science, but I'd love for non-scientists to also have a chance to know what I'm talking about. So, I'll give a sentence or two whenever possible to help clarify things. I can also throw in a link to sites like wikipedia that can explain things too. So that end, RNAi is a process that earned its discover's the nobel prize in 2005(?) or near there. They showed that very small RNAs, things that were previously believed to be nothing more than degraded and useless, had a huge role in things as varied as prevent viral infection to regulating cancer. They had been implicated in effecting genome structure and function in a yeast, but nothing yet in humans. This is what I wanted to work on.  

The path to having my own lab
We managed to get one manuscript out on the subject, in Nature Cell Biology. It wasn’t accepted until the fourth try. Its acceptance was greatly helped by a talk that I gave at a Keystone Symposia on epigenetics and chromatin. An editor from the journal was there and approached Gary to say that they would be interested in the work. Its no guarantee, of course, when they approach you like this. But its just a slight wink/nod in the right direction towards publication. We submitted it twice and finally got it in. I needed to follow that work up and I tried to do so by pursuing the genome-wide localization pattern of human Argonaute 2. This technique required the new process known as ChIP-seq, which required the ability to work with linux –based systems. Many of the bench molecular biologists at that time were “doing” chip-seq, but this occurred by virtue of a tight working relationship with a full-time, dedicated biostats colleague. An example of this was the lab next to ours, Dan Camerini’s group. His lab had 3 bioinformatics experts as postdocs. The rest of the lab merely had to do the ChIP and then hand it off to the bioinformatics folks and then the got their beautiful genome-wide binding data back. I had no such luxury and if I was going to stay in this game I knew I had to figure out a way to do these anlyses myself. With the great help of some of Dan Camerini’s postdocs, mainly Kevin Brick and Ivan Gregoritti, I was able to do many of the computational analysis needed from genome-wide, high-throughput sequencing analysis. I was able to put together a manuscript that consisted of the Argonaute 2 genome wide binding and a series of hypothesis driven analysis and comparisons with other publicly available databases. I was able to conclude that the Ago2 binding sites were enriched for repeat regions, and regions enriched for short RNA production and H3K9me3 levels. We sent this manuscript to Molecular Cell and it was reviewed but rejected. They wanted more experimental evidence and clearly thought a bioinformatics journal was more appropriate. This was annoying because less than a year previous, many labs were publishing entire manuscripts that were nothing more than an analysis of the genome wide distributions of a single histone modification. The main lab to do this was Keji Zhao, also at the NIH. But in science, the burden of proof and the bar for what is a good manuscript changes constantly. And although these types of papers were great in 2008, by mid 2009, they were no longer acceptable. I kept up working on this manuscript by re-doing the Ago2 ChIP-seq, including some immunoflourescne done in a collaboration by Gaelle Lefevre and myself, and also doing some small RNA-IP seq. We resubmitted but the reviewers always wanted more controls, and more information. However, this was data was good enough to impress two universities into giving me a job interview, University of Alabama in Huntsville, and University of Alabama at Birmingham. I was given an offer by both places and had no choice but to accept the job at UAB. I was also given a offer to visit the University of North Dakota, but I didn’t actually go visit b/c I accepted the offer at UAB first.  

The First Year of the Giles Lab
The job offer at UAB came around April of 2011 and I moved there around August. I did a couple extra experiments before anyone was in the lab and we resubmitted the manuscript with Gaelle and Gary but it was reviewed and rejected. I think brought in a graduate student, Blake Atwood, who developed a QPCR assay system to use for RT-QPCR and ChIP-QPCR analysis of the rDNA locus. I have gotten a little bit ahead of myself here. Our analysis had always suggested that Argonaute 2 was specifically localized to the repetitive regions of the human genome. However, these analyses leave out the ribosomal RNA genes, of which there are ~200 per human cell. This highly repetitive nature of these genes makes it difficult to analyze them. However, we were able to use a single consensus repeat unit of the locus and observe that the tags from the ChIP-seq experiment were highly enriched for the coding region of this gene. So, Blake enabled us to do ChIP-QPCR and RT-QPCR throughout the rDNA locus and we analyzed the effect of knocking down Ago2 on RNA levels throughout the locus. I also brought on a postdoc , named Mariana Saint Just Ribeiro. She was previously in Suming Huang’s lab at Florida. Suming was a postdoc along with me in Gary’s lab so I knew I could trust his recommendation. He made it clear that Mariana was perfectly capable of doing the work but that he didn’t have sufficient time to mentor her. So I took the leap and brought her into the lab. She quickly made an impact by demonstrating a strong effect on histone modifications when Ago2 is reduced. She joined the lab around the start of January 2012. Blake joined a couple months before that. With these new additions in personnel and data, we resubmitted the manuscript to Nature Genetics, and it was triaged. We immediately resubmitted it to Nature Cell Biology, which also triaged it. We immediately resubmitted it to Genes and Development and they sent it out. However, the reviews were mostly negative. Although the reviewers like the area of research and would like to see more work, they weren’t convinced. So now its September of 2012 and I have a new manuscript on this project. We have mostly just rearranged how the data is to be presented. We added some ChIP-QPCR of both endogenous and tagged Ago2 to the rDNA locus to confirm that it is actually bound there as suggested by CHIP-seq. We have also added a new analysis of the sRIP-seq, which demonstrates some information of the biogenesis of the small RNAs to which Ago2 are bound. We utilized a metabolic labeling technique to demonstrated the a loss of Ago2 actually does have an effect on the synthesis rate of the rRNA genes. It also illustrates a change in the processing rate when Ago2 is knocked down. We have added some very interesting data demonstrating that Ago2 binds to SETDB1, a histone lysine methyltransferases. We can show that this is found at the rDNA locus and that its localization depends on Ago2. So now the manuscript is in the hands of Gary and we hope to resubmit it soon, for the umpteenth time. Luckily, this isn’t the only thing that lab has going on. I have also been lucky to be in a collaboration with Hengbin Wang, also at UAB in the Dept. of Biochemistry &Molecular Genetics. His lab initiated a very large scale protein purification project to screen HeLa nuclear extract for its histone deubiquitination activity. Histone ubiquitiination is modification that we don’t know much about, so being able to find the enzymes that regulate it is a huge step. He ended the project with ChIP-seq and RNA-seq data that was done by his Chinese collaborators. However, their analysis didn’t make much sense, so he needed me to take a look at it. I did, and it was quickly apparent that it was going to be a ton of work. Hengbin quickly offered me the chance to be a coauthor. I jumped at the chance. My analysis showed that the loss of this new protein Usp49, caused ~10,000 introns to be retained in the mature mRNA. Furthermore, these introns have a uH2B containing nucleosome positioned at the 5’ splice site. These introns are also highly enriched for uH2B. We submitted this manuscript to cell, where it was triaged. Then we sent it to Science, where it was reviewed but rejected. The reviewers loved the biochemistry but thought that we hadn’t proved the splicing connection. We addressed their concerns to prove that a direct effect on splicing was occurring and then resubmitted it to Nature. They rejected it with the reviews being of the exact opposite nature. They seemed to love the splicing connection but hated the biochemistry. This only goes to show how broken our system of peer-review is! I’ll say that again, its broken! How could their be any truth to a process that could be so varied between 2-5 experts ranging between 2 of the premier journals in science? We are currently hoping to resubmit this to science. My lab currently has 3 people in it, after another very bright graduate student named Jessica Makofske joined the group. Her project is to purify specific chromatin fragments from the rDNA gene. This is no easy feat and is something that would revolutionize chromatin biology. To date, if you want to know which proteins are bound at a given site at a given time you have to have a-priori knowledge of the proteins existence and then check for it. This would most likely be done by ChIP-QPCR or a gel shift analysis. However, if one were able to biochemically purify certain chromatin fragments, mass-spec could be done on the entire locus and we would be able to know all the proteins that are present, as well as all the histone modifications. Our approach is the utlize a combination of nuclease accessibility, velocity sedimentation rates and differential restriction digestion to purify these fragments. This project has already yielded some interesting data.
A summary of some science:
ChIP.  Chromatin immunoprecipitation. First of all, what is chromatin. Well, most people know what DNA is.  But DNA doesn't look like that inside your cells.  Its actually wrapped up like a string wrapped around beads.  The beads are proteins called histones.  And when you have a group of histones combined with the DNA thats wrapped around it, its called a nucleosome.  When you string together a bunch of nucleosomes together and add in all the other non-histone proteins that bind to your DNA, you have chromatin. 
immunoprecipitation is a fancy way for doing the following, taking an antibody that recognizes a protein and attaching it to a bead.  The bead is the size of very very small pebble.  The antibodies are the same kind of antibodies that your cell makes to fend off infections. When this antibody is attached to the very small pebble, you can incubate the antibody:bead complex with your cells and then give it a spin.  The proteins recognized by the antibody:bead will pellet (or precipitate) to the bottom of the tube.  This technique is called immunoprecipitation  If the antibody is desinged to pull down chromatin, its called chromatin immunoprecipitation. 
ChIP-QPCR.  Is doing ChIP and then measuring how much of a given DNA sequence is in the tube using quantitative PCR.  ChIP-seq is a ChIP experiment where instead of doing the PCR step to quantify how much of a given DNA sequence is associated with a given protein, you sequence all the DNA that comes down in the tube.  This will typically yield between 10 and 150 million small sequences and requires quite a large computer to analyze. 

Friday, October 7, 2011

Cosequences of Wealth Disparities part 3 ... i told ya so

I have posted numerous times before about wealth disparity in this nation (and the globe too). I don't mean to blow things out of proportion but it seems that western civilization can be leading to a collapse. The youth have revolted in much of the Arab world, Greece, Spain, Paris, London, New York and many other large cities in the USA. Is this just a blip or a sign of things to come? Well, the old saying is that "its all about the economy stupid" is important here. But its not the only thing. The scariest and most realistic reason why this unrest and these protests are only going to get worse is that the youth have lost their faith in the democratic process to effect change. Good times come and go. If a democracy (or a republic) is functioning as it should, peoples angst will take them to the ballot box and they will create change through voting. But as long as the Gen Xers have been alive they have seen donkeys and elephants parade in and out of the white house and congress; and things have gotten steadily worse for them. And, there is not a "we're in this together" mentality, which could buffer the problem during genuine economic tough times, because people suffer while the wealthiest 1% of the country continue to prosper.

I have no political point to make here. There are people who subscribe to a libertarian perspective and think that if you're poor, its your fault (this position could become increasingly difficult to justify because there simply are not enough jobs to go around). And there are more liberal-minded types who believe government has a role to play in helping the poor. The problem with the first perspective is that if you create a society that leaves too many people behind, those people are not going to just go quietly. They exist. They are real. And they are going be angry. The notion of letting capitalism run wild and not caring about the poor can work, so long as the poor a) aren't really too poor and b) the masses of poor people are content with their lots. Neither of these things are the case. I have news for anyone who thinks this is just a nuisance; the only constant in world history is change, the rise and fall of nations and great empires.
This situation must be dealt with.

I told ya so. (obviously so did many others...:))

Tuesday, June 7, 2011

What they should've said..

I'd like to reinvigorate this blog. But I don't think my invective-laden tirades do anybody any good. I believe this to be the case in the main because the topics are too general and vague. Everyone has their "pie-in-the-sky" ideas about how to make the world better, or more commonly, what is wrong with the world. Thus, I'm launching a new feature that I call "what they should've said".
And for this weeks installment I'm going to give my responses to two people who have been in the news lately.
The first is Anthony Weiner. At his press conference what he should've said is this:

"Yes. I took a lewd picture of myself and sent it to some women. I have cheated on my wife. So what. It has nothing to do with my abilities as a legislator, or my ability to improve the lives of my constituents in my district in New York. I realize that what I have done will hurt my family and my wife, but that is my business and I will have to deal with it. I won't say another word about it. As for my future in politics, that is up to the people of (insert name of his district). They put me here, they know what I have done, and they alone will decide my future, as is the mechanism by which this republic functions. I won't waste anyone's time with false tears and manufactured contrition, written by an overly paid publicist, as have so many of my fellow colleagues. I'm not perfect. I've never claimed to be. And I'm no hypocrite. I have not campaigned for, or championed anything that could resemble social conservatism or religious hegemony that so many on the right of the aisle routinely do. I don't want your vote because you think you can have a beer with me. I don't want your vote because you think I have the same morals and ethics as you. I don't want your vote because we worship the same God. I want your vote because you think I'm good at my job, and that I make the lives of the people I'm sent to Washington to represent, better.
I am sorry if I have disappointed anyone. But my personal life is my own business. I have broken no laws; and i will not step down. I'm a good legislator; and I will continue to be. That is all".

If he would've said that..he'd be talked about forever. He'd look strong as an ox! And he might actually help us escape this awful pattern of getting distracted by the personal lives or our elected officials and pay more attention to the routine shredding of the constitution of which they try with all their might to prevent us from noticing.

Wednesday, January 19, 2011

A commentary on friendship

I've always been fascinated with friendships. I have been blessed with many throughout my life. I can't remember a time when I didn't always have a dozen or so people that I could call to do anything at any given time. I don't think that is anything odd when I was in high school or college. But as I got into my mid-20's I think that the number of people that most people routinely spend time with that are not family or colleagues (the definition of "friends" from here on it)plummets. But my friendship levels did not. They are probably as high as they ever were, or perhaps even higher. I can honestly say that four or five of my closest high school friends, guys who were always hanging out in large groups, have now lost anything resembling that. Their time is spent with family and work first, and maybe with one or two close friends in the little bit of free time that they can round up.
I am inclined to say that I find this sad, but i don't. They are both happy men, with wives and families that they adore. I suppose that it is the natural order of things. Although I don't think that this is particularly sad in general, I think losing all my friendships would suck. I just can't imagine a life without dozens of close friends. And so I prioritize friends as high as I can.
But that leads me to my next question. How high should you prioritize friends? The benefits are obvious; you have fun with your friends. However, when you have a child there can only be so many hours in the day and time spent doing one thing typically takes away from time spent doing something else. It also makes matters more difficult when you have a large group of equal friends. If you had a single friend; you could stay equally close with that person because all your "friend" time could go that direction. But when you have multiple groups; something has got to give.
There is also an emotional cost to trying maintain friends. One thing I've learned being part of a large network of friends is that no matter how one tries to be inclusive, people always get left out. And when this happens, either feelings get hurt or people re-evaluate their friendships. If you only have a small amount of time to hang out with some friends you aren't likely to invest that time with someone who routinely leaves you out. The reasons are not just petty, time spent with friends leads to stories and memories, and these memories compound over time. If you miss out on too many events, you are out of the loop, and hanging out just isn't the same anymore. Everyone has been there before, like say, if a friend of yours from high school has a group of college friends coming over. They will just talk and talk about the good ole times and you are just a fly on the wall. This never works out.
Although this inevitable fall is somewhat petty as well. Your time may not mean jack shit to anyone else, but its infinitely precious to you. And its hard to justify spending it with someone who doesn't seem to want to spend time equally with you. These things happen all the time as friends "fall off". What exactly is that? It doesn't mean that there is a big fight or a change in one's life, but they start making plans less frequently, that frequency hits a tipping point, and then viola! you reach a point of "I used to hang out with them" from "my good friend who I see all the time". These events happen all the time and they fell like real losses. And I suppose the last thing I can say about it is that perhaps this is why most families fall out with their large networks; they seem to be less and less involved in things and then one day , presto, chango, 10 years goes by and nobody has thought to call. when you put it that way , it is definitely sad.

Sunday, November 7, 2010

Consequences of Wealth Dispariites part 2

I've been spouting off about wealth disparity on, and off now for years. You can go back in this blog and see that I've touched on it numerous times. It seems to be coming more and more to the forefront. Nicholas Kristoff has written an excellent piece on the matter in his latest NY Times column: "Our Banana Republic" .
He mentions how our wealth disparity is much worse than some of the countries we loved to call Banana Republics. He then goes on to claim that the real divide in this country is the split between corporate interests and average citizens. The Supreme Court has ruled this year that a corporation is extended the same first amendment rights as actual people when it comes to donating money. This is the prime example illustrating how if there is a war of capital vs. labor in this country (to borrow an old concept that should be brought back), the capital is crushing the labor.
You almost can not get ahead on your own merit, if you live in a major metropolitan area and are a salaried worker. The jobs that used to support that possibility have all been off-shored. And whats worse, is that the liberals in this country continue to support a democratic party that they believe supports their ideals for a more balanced economy, but it was the Clinton Administration that actually got the ball rolling in the free trade arena. For some reason Americans love stuff that has the word "free" in it (free trade, free market, free-dom), but in this case, free trade doesn't really mean trade in the way you think. For some reason stupid middle americans love how this sounds because it appeals to their sense of fairness. But it will not only never benefit anyone who doesn't own a large company that sells or busy things from China, it will most definitely hurt you. It will most likey involve the loss of a job, or the necesarry reduction in wages to keep your job here. Not enough people seem to get this. I didn't. Free trade actually appealed to my sense of fairness. But I realize now that it doesn't work for us. It may work great for a C-level executive of a fortune 500 company; or for anyone who works fairly high up the food chain in such a place. But it will only make everyone elses life worse. Its great that shit at walmart costs 10% less than it would w/o globalization, but that doesn't do you any good if you don't have a job. So the first problem is that not enough people "get it". The second problem is that even if they do, they don't realize that neither party is putting forth ideas that help.
There are millions of unemployed/underemployed people, many living in the upper midwest "rust belt". They have lost all their manufacturing jobs, which are not coming back. What do you propose that these people do? Where are the jobs that are going to allow them to make enough money to buy a house, save money for a nice retirement and be able to put their kids through college. Because without the opportunity to do that, nobody is going to be happy. That is really all the american dream boils down to. It used to be that if the majority of the people had a chance at the very modest aforementioned life, we were happy. But it seems now that we'd rather craft a society where 1 in 1000 people are going to "make it" (however possible) and have millions and millions; and everyone else will struggle. Nobody on capital hill wants to help you if your poor. They don't care about you. The wealth disparity will keep growing, and this is definitely not sustainable.

Obviously, politicians only want to get elected. So they push issues that are easy to understand and get people riled up: immigration, abortion, government spending (without actually outlining percentages of GDP, and putting the spending in perspective), and gun rights, just to name a few. When there are so many more important issues that just don't get discussed. This is mainly because they are complicated issues that are impossible to be on one side or the other. They are not two sided. There is no for or against when the question is "how should government spend its money?", or "how do we get more well paying jobs for people with bachelors degrees or less?", "how do we balance off our trade deficit?", and even, "how far is too far when police put GPS units on peoples cars?". A discussion about any of those issues will not increase ratings at msnbc or fox. Thus, politicians waste little time talking them up. "If you always do what you always did, you'll always get what you always got", may be a stupid corporate slogan to make you work harder, but it carries an important message. Dems and Repubs are just pepsi and coke. You may prefer one flavor slightly to the other, but so long as you are drinking either you are still rotting your teeth.

Friday, October 1, 2010

I hate the Tea Party. I welcome the Tea Party

There is no way for a self-respecting scientist to consider any of the Tea Party candidates for even a second. Since I'm not self-respecting, I'll give it a second. They are the most ignorant, awful, loathsome group of closet racists and sheep that I've ever seen. And they might just do some good.
The problem isn't with liberal or progressive ideals. The problem isn't even with conservative ideals. All of these political ideologies have their proper times and places, and I believe the best governments can use bits and pieces of each to govern most effectively. The problem is corruption and systemic failure. Let me ask any Barrack Obama supporters in what ways is he really that different than any of his predecessors. He may be winding down the war in Iraq, but he is amping it up in Afghanistan. So the peace-niks shouldn't be happy with him. He took on healthcare, but did any liberals really get what they wanted? The only real thing to be happy about is that insurance companies can't drop children. Thats pretty good; but nowhere near what he set out to do. For all the political capital he spent on that fight, we now have a system that is going to require you to pay for insurance. Wow! Do you really think people weren't getting health insurance because they didn't want it? Maybe I'm missing some wonkish details of the policy, but it seems that the only big winners are the insurance companies. The Tea Party wants to undo this on the grounds of it being unconstitutional; and I think they may have a point. This is crap. Believe me, if a republican president passed this bill; the democratic bloggers, main stream media, and just about every American that lives inside a major city or suburb, would be screaming their heads off. He would be called a fascist pig and blah blah blah. But a Dem can get away with it. People should be outraged at this legislation.
What else is there? Well, Obama is OK with the federal government increasing their abilities to invade privacy on the internet,news-8163.html . Obama has not closed gitmo . So on four really big issues :
1) WAR. no significant difference in terms of financial or human output.
2) Health Care. A whole bunch of fuss over a bill that requires you to pay for it, or else suffer a fine.
3) Privacy. He is basically extending things started with Bush's PATRIOT Act.
4) Human Rights. Gitmo is still open. The talk about torture has just stopped. Its been swept under the rug.

This article sums up what I'm trying to say: .

So, back to the Tea Party. What the Obama administration proves is that the only real difference between an elephant and a donkey is the sounds they make. The policies keep promoting a large military; they keep the divide between the wealthiest and most poor growing, they keep decreasing our privacy, and reducing our rights. (how about this one for horrific.. Where is Obama on illegal search and seizure. Apparently its ok for the cops to come into your house and put a GPS device on your car, so long as its not in a garage. Basically, if you can afford a garage, the bill of rights applies to you. If not, well, don't expect any privacy.
The tea party wants to tear everything down. If I didn't depend on a healthy government funded dedication to scientific research for my livliehood, I'd be in favor of this too. There isn't much in Washington that isn't tainted with a corrupt big-budget military industrial complex; hypocritical financial industry or greedy corporate lobbyists. This sounds trite, but they don't give a crap about you. Don't believe me, how about this memo from citibank that states just that. Their consultants did an analysis to show that all but the top ~3% of Americans are heading for ruin, but thats OK, because the wealthiest will have enough money to support their industry..( This came from the Michael Moore movie, "Capitalism. A Love Story"
So the Tea Baggers may be dumb as a box of rocks, but they aren't corrupt yet. If they could stay more libertarian and not so much evangelicals; they might be electable. And who knows, could they be much worse than what we got? Maybe highly educated, super-motivated, Rovian-types in the office is the problem. Some slow moving, big dumb animals might just be what we need.