Today's paper is really neat. The group made a mouse mutant that had a craniofacial defect (read: messed-up looking face) and traced it to a molecular/genetic pathway. And all in 4 jam-packed figures and tons of experiments. I've got a soft spot in my heart for the craniofacial researchers, so this paper jumped out at me.
The title is a mouthful: The E3 ubiquitin ligase Wwp2 regulates craniofacial development through mono-ubiquitylation of Goosecoid. The 30 second summary: mice lacking the gene Wwp2 have odd faces and jawsf. Wwp2 gene makes a protein that modifies, and activates another protein, Gsc (Goosecoid) which binds to and activates yet another gene, Sox6, which is a key regulator of normal head and face development.
The Data:
Using a gene trap method of deleting genes in mice, the Wwp2 gene was deleted. The knock-out mice were born, but had defects in their jaws and face: they had stubby noses and domed heads. The way they made the mice they could trace where the Wwp2 gene should be expressed by using a blue dye. They traced where the normal expression of Wwp2 was using the dye. They used a gene already known to be involved in craniofacial development, Sox9 as a control, and showed Wwp2 was necessary for normal development. They looked further into Sox9 and found that when it is overexpressed, Wwp2 expression increases too. Could they be linked? They did direct DNA binding assays to show Sox9 does, indeed, bind to and controls the Wwp2 gene.
Great. Moving on, the Wwp2 gene makes a protein called a ubiquitin ligase. Wwp2 protein then acts on other proteins to decorate them with these teeny itty bitty modifications called ubiquitin. Often, this targets the protein for destruction in the proteosome, but not always. If a long chain of ubiquitin is added, the protein is destroyed; if only one little ubiquitin is added onto the target protein it can change the protein's behavior: increase or decrease it's activity, change where it likes to hang out in the cell, etc. The first question is what protein does Wwp2 modify. Second question is what does that modification mean for a cell, particularly one that is trying to become a jaw or nose.
They had a few hints as to the first question. Based on the protein sequence recognized by Wwp2, they came up with four candidates and quickly showed by direct protein-protein binding assays and ubiquitination detecting assays that Wwp2, indeed, added ubiquitin to the Goosecoid protein (Gsc). The second question, what does Gsc getting a ubiquitin from Wwp2 do to a cell, took a bit more work. They looked at if Gsc got sent to the trash can after Wwp2 ubiquitinated it. Nope, Gsc levels stayed the same. It so happens that Gsc is a transcription factor: a protein that acts at the DNA level to change expression of other genes. They looked at how many ubiquitin molecules Wwp2 added to Gsc, and only one was added, suggesting the ubiquitination of Gsc by Wwp2 changes Gsc's activity on genes at the DNA level. So begins what probably was a stab in the dark that got lucky. They did gene expression analysis to determine if the expression of any genes potentially involved with craniofacial development changed when Gsc was modified by ubiquitination.
And in comes Sox6. Sound familiar? Well, sort of. In the same family as Sox9, Sox6 is yet another transcription factor that is involved in cartilage biology... just the sort of thing that could direct normal growth of bone, and also just what, when disrupted, could lead to abnormalities.
The result:
OK, so I skipped some of the experiments. But really, they were very thorough. And this post is already getting long. A cool little model from this paper is that the first gene they looked at, Wwp2, is turned on by Sox9, goes on to influence another protein, Gsc, which act on another gene, Sox6 to guide normal head and face development in mice. Talk about a cascade effect.
There's always more work to be done. I'd like to see the phenotype of the craniofacial defects be echoed in a Sox6 mutant. This would shed more light if this is the only body region affected by this particular signal cascade. I've no doubt there's lots of literature on Sox9, but I'd like to see what the instigating signal to the little cells are that kicks off this whole signal sequence. I mean, not every cell ends up being a part of your nose or cheek, so what sets these cells off on this fate. And then, linking to humans, are defects in this pathway seen in humans bearing craniofacial anomalies? And don't you love saying cranifacial anomaly? I do.
See you next week!
Showing posts with label genes. Show all posts
Showing posts with label genes. Show all posts
Thursday, January 13, 2011
Wednesday, July 7, 2010
Egad! Why will the girl not sleep?
I'm not a very good sleeper. Ask my parents. They'll tell you stories of me at age 3 or so wandering the house in the wee hours of the morning, because I was just awake. And I recall many nights, even now, where it will take me 2 hours to fall asleep. And naps? Unless I'm so sleepy I'm stumbling, forget about it. In college they called me the "sleep camel" because I would sleep 5-6 hours per night for a few weeks then spend one entire weekend in bed, catching up. We all have our own sleep patterns that are unique to us, and very few of us fit the average 8-9 hours per night, 10 pm to 6 am kind of pattern. So it didn't surprise me when my first daughter exhibited similar sleep patterns to me. After all, she has my nose, my eyebrows, my smile, why not my sleep habits too? It got me thinking if there was a genetic basis to sleep behaviors. And guess what, the answer is absolutely!
Sleep patterns, or more broadly, circadian rhythms, can be thought of as how we live around that 24 hour day/night cycle. It is well appreciated that circadian rhythms influence nearly every biological process in our bodies: metabolism, cell cycle, tissue repair, digestion, gene transcription, you name it. So it should come as no surprise that a complex network of genes and proteins dedicated to keeping us in-sync with the sun. A quick search of the Gene Ontology database for "Circadian Rhythm" produced 487 distinct protein products associated with the day/night cycle.
The genetic basis of sleep patterns are most easily thought of as complex traits: many genes are involved, and the equation is muddled by environment. For example, our sleep patterns as a society changed drastically with the advent of electricity. So how can we separate out what component of sleep behavior is genetic and what is environment, or learned behavior? One easy way is to look at extreme examples where researchers have identified unusual patterns running in families and performed exhaustive searches to identify single genes associated with those behaviors. While mutations in these genes are exceedingly rare, one can imagine how subtle differences in these genes, or others yet to be discovered, can contribute to a sort of "genetic sleep fingerprint" that is common in a family.
"Short Sleeper Gene"
Another rare reported sleep phenotype reported in families is that of short sleep duration. These individuals typically sleep only 6.5 hours or so per night, rather than the 8-9 hours more typical of humans. The trait was mapped to the gene DEC2 (He et al, 2009). The gene DEC2 codes for a transcription factor, a protein that controls the expression of other genes. DEC2, and a related gene DEC1, are found to regulate expression of other circadian rhythm genes such as Clock/BMAL1 and PER1 by hooking onto their promoters, little DNA bits upstream of the gene, and turning on expression (Honama et al, 2002). And, at least in the case of DEC1, gene expression is regulated by light exposure.
"Early Bird Gene".
The disorder known as familial advanced sleep phase syndrome (FASPS) is a rare, inherited disorder characterized with early rising. Individuals typically wake at 4 AM, and go to sleep at an early time as well, about 7 PM. While this disorder is considered non pathogenic, individuals are normal and healthy, it does cause some psychological difficulties as patients feel "out of step" with the rest of society. In 2005, researchers at UCSF studied a family with three generations of FASPS sufferers, and identified a gene, CKI delta, or hPeriod2, associated with the early sleep, early rise phenotype (Xu et al, 2005). They reported in 2007 that mice carrying two mutated copies of the mutated Period2 gene exhibit similar shifts in sleep patterns (Cy et al, 2007 and Zheng et al, 1999). The mice had changes to their metabolism, their hormones (estrogen levels were altered), neurotransmitter levels, and alcohol consumption. Talk about broad effects.
"Short Sleeper Gene"
Another rare reported sleep phenotype reported in families is that of short sleep duration. These individuals typically sleep only 6.5 hours or so per night, rather than the 8-9 hours more typical of humans. The trait was mapped to the gene DEC2 (He et al, 2009). The gene DEC2 codes for a transcription factor, a protein that controls the expression of other genes. DEC2, and a related gene DEC1, are found to regulate expression of other circadian rhythm genes such as Clock/BMAL1 and PER1 by hooking onto their promoters, little DNA bits upstream of the gene, and turning on expression (Honama et al, 2002). And, at least in the case of DEC1, gene expression is regulated by light exposure.
Transgenic mice that have both copies of their mouse-version of DEC2 gene mutated are more active and sleep less than mice with the normal, functional gene. But mice that have both copies deleted do not exhibit this phenotype, suggesting the mutation seen in these mice (and by extension in humans) is a functional variant, retaining some activity but altered in either specificity or regulation.
"Night Owl Gene"
"Night Owl Gene"
And rounding out our sleep gene sampling is a gene that when mutated results in people staying up late. Well, it's not really been shown in people yet, but in mice, mutations in the gene Fbxl3 resulted in the mice having an "overtime" phenotype, where their circadian rhythm was pushed from 24 to about 27 hours (Siepka et al, 2007). This gene encodes an f-box protein - a protein involved in the ubquitin ligase/proteasomal degradation pathway. So, Fbxl3 tags proteins to be degraded and recycled. Work coming from the Pagano lab showed that the circadian rhythm protein Cry2 was ubiquitinated by Fbxl3, and this results in the protein Clock to be reactivated (Busino et al, 2007). So, Fbxl3 plays a key role in the timekeeping mechanism in a cell, and without it, well, cells, and mice, don't follow the sun.
This is just a teeny, isty-bitsy sampling of some interesting genes involved in keeping us awake in the day and sleeping at night. I keep these genes, and the 484 more identified in the circadian rhythm pathway, in mind when my 2 1/2 year old decides that 10.5 hours of sleep per day is sufficient for her, and that 5:30 AM is a perfectly reasonable time to wake. She, like me and her dad and everyone else, was born with her own sleep patterns. I do try to coax her into that extra half-hour in the morning, but I know that as long as I provide the right opportunity and conditions for her, she will get the sleep that she needs. Maybe she's a sleep camel too.
REFERENCES:
SCF-Fbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins. Science 316: 900-904, 2007
The transcriptional repressor DEC2 regulates sleep length in mammals. Science 325: 866-870, 2009.
Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature 419: 841-844, 2002.
Circadian mutant Overtime reveals F-box protein FBXL3 regulation of Cryptochrome and Period gene expression. Cell129: 1011-1023, 2007.
Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, Saigoh N, Saigoh K, Ptácek LJ, Fu YH .Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome. Nature. 2005 Mar 31;434(7033):640-4.Xu Y, Toh KL, Jones CR, Shin JY, Fu YH, Ptácek LJ. Modeling of a human circadian mutation yields insights into clock regulation by PER2.
Cell. 2007 Jan 12;128(1):59-70.
The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400: 169-173, 1999.
Wednesday, June 16, 2010
Gene Wednesday: LALBA
Righto! Here's post number six, and guess what.... six people signed up to follow this little blog. Talk about motivating factors.
Today is the inaugural Gene Wednesday. I love genes. I love genetics. So here's where I'll spotlight one gene. I promise, all these posts this week, though they seem disjointed, are leading to something.
Today's gene is LALBA. It codes for the protein human alpha-lactalbumin. It's located on human chromosome 12q13 (the long arm of chromosome 12). As you can probably guess by the name, it's the principle protein in milk. But it's oh so much more. It binds to to another protein, lactose synthase, and makes lactose, the principle sugar found in milk. LALBA is only expressed in mammary tissue, and is hormonally regulated. It is highly conserved among mammals, and mice that have both copies of LALBA deleted make a thick milk lacking lactose that does not sustain the pups, showing that LALBA is involved in lactose synthesis and that lactose is necessary for sustaining infants.
Alpha-lactalbumin appears to do much more than make lactose. It can bind together into a multimeric protein that has apoptotic properties - it can help kill tumor-like cells but not normal cells in culture. Some data suggest it also has microbicidal activity And recently, a protein dubbed HAMLET (human alpha-lactalbumin made lethal to tumor cells) is alpha-lactalbumin folded in a different way when exposed to acidic environments that, as the somewhat awkward name suggests, appears to specifically kill tumor cells. So one gene, one protein, lots of different functions. Neat!
Today is the inaugural Gene Wednesday. I love genes. I love genetics. So here's where I'll spotlight one gene. I promise, all these posts this week, though they seem disjointed, are leading to something.
Today's gene is LALBA. It codes for the protein human alpha-lactalbumin. It's located on human chromosome 12q13 (the long arm of chromosome 12). As you can probably guess by the name, it's the principle protein in milk. But it's oh so much more. It binds to to another protein, lactose synthase, and makes lactose, the principle sugar found in milk. LALBA is only expressed in mammary tissue, and is hormonally regulated. It is highly conserved among mammals, and mice that have both copies of LALBA deleted make a thick milk lacking lactose that does not sustain the pups, showing that LALBA is involved in lactose synthesis and that lactose is necessary for sustaining infants.
Alpha-lactalbumin appears to do much more than make lactose. It can bind together into a multimeric protein that has apoptotic properties - it can help kill tumor-like cells but not normal cells in culture. Some data suggest it also has microbicidal activity And recently, a protein dubbed HAMLET (human alpha-lactalbumin made lethal to tumor cells) is alpha-lactalbumin folded in a different way when exposed to acidic environments that, as the somewhat awkward name suggests, appears to specifically kill tumor cells. So one gene, one protein, lots of different functions. Neat!
Wednesday, February 17, 2010
Shhhhh - quieting the troublesome genes
Welcome to the inaugural post of Why You Will Love Science. Today we discuss a paper published in the Proceedings of the National Academy of Sciences discussing some really cool emerging technology. The title, "Lipid-like materials for low-dose, in vivo gene silencing" is a great summary. This seemed like an excellent paper to start with because it delves into some relatively recently understood fundamental basics of biology, the authors work diligently to show it works across different species, including nonhuman primates, and the work shows some exciting progress towards new types of medicines - specifically in the hot arena of gene therapy. So let's begin, shall we?
So you're not left hanging, here's the punchline. The authors have developed a way to get small nucleic acid particles targeting specific genes to turn down the expression of those genes into liver cells of animals. Not only did they turn down the expression - or silence - one gene, but up to five genes simultaneously in the same animal. And they were able to do this at very low levels of "drug" with no obvious side effects. Pretty cool? Definitely.
First a bit of background.
siRNA are little pieces of genetic material that takes the product from a gene, the messenger RNA, and diverts it from making a protein, and generally targets it for destruction. It's a way your cells have of regulating genes, amazingly only discovered about 20 years ago. This paper looks at using siRNA to do gene therapy - modifying a gene to treat a disease. This is a tricky thing to do because not only is it really hard to get genetic material into a cell - there's a little something called an immune system in the way - and once genetic material gets to the target cell, it's difficult to generate a sustained response in the cell.
So what did they do?
They first created a library of fat-like particles called lipidoids of varying sizes in a way that was fast (3 days) and could be completely automated. This is important for future applications. Next, they tested how effective each little fat bubble was at getting the siRNA into a cell. Most were not effective but a few looked really promising. They chose a few really potent ones and tested in mice.
The first test in mice was to target a liver-specific gene, Factor VII. They chose one particle type that was effective at a very low dose, and added a few modifications to make it more invisible to the immune system and less sticky with other blood proteins, giving the particles an extra chance to get into cells. The most potent one was several hundred fold more potent than the current method. They dubbed the particle C12-200. With a very low dose they observed lowered levels of Factor VII for up to 25 days, with little side effects in the mice.
They next wanted to see if they could target multiple genes at the same time. They added in siRNAs that targeted four other liver-specific genes At low doses that were well tolerated by mice they could successfully knock down all five of the genes simultaneously. Very cool.
Next they looked to see if the best lipidoid would work in non-human primates. Again they chose a liver-specific gene. And it worked. At very low doses. With no apparent side-effects.
So why are these little fat bubbles so interesting? Some diseases, such as hepatitis C, have no effective treatments but has unique genes that would be great siRNA targets. We might be able to use these little fat bubbles to target the genes in HepC, shutting them off, and providing a cure. Or, if you had a disease with multiple genes involved, such as cancer or metabolic syndrome, you could target those genes, altering their expression and stopping the disease in their tracks.
What is still missing? Well, they only looked at genes active in one organ, the liver. This made for clean experiments but for other applications, it will be important to know how wide-spread the siRNAs have their effect. For example, in a cancer cell, turning off a gene might be a good thing, but in a normal cell it might have dire consequences. This still needs to be tested. Also, in mice the length of time of gene silencing was quite a long time, but it would be nice to know if they can give a second dose and quiet those genes again.
So you're not left hanging, here's the punchline. The authors have developed a way to get small nucleic acid particles targeting specific genes to turn down the expression of those genes into liver cells of animals. Not only did they turn down the expression - or silence - one gene, but up to five genes simultaneously in the same animal. And they were able to do this at very low levels of "drug" with no obvious side effects. Pretty cool? Definitely.
First a bit of background.
siRNA are little pieces of genetic material that takes the product from a gene, the messenger RNA, and diverts it from making a protein, and generally targets it for destruction. It's a way your cells have of regulating genes, amazingly only discovered about 20 years ago. This paper looks at using siRNA to do gene therapy - modifying a gene to treat a disease. This is a tricky thing to do because not only is it really hard to get genetic material into a cell - there's a little something called an immune system in the way - and once genetic material gets to the target cell, it's difficult to generate a sustained response in the cell.
So what did they do?
They first created a library of fat-like particles called lipidoids of varying sizes in a way that was fast (3 days) and could be completely automated. This is important for future applications. Next, they tested how effective each little fat bubble was at getting the siRNA into a cell. Most were not effective but a few looked really promising. They chose a few really potent ones and tested in mice.
The first test in mice was to target a liver-specific gene, Factor VII. They chose one particle type that was effective at a very low dose, and added a few modifications to make it more invisible to the immune system and less sticky with other blood proteins, giving the particles an extra chance to get into cells. The most potent one was several hundred fold more potent than the current method. They dubbed the particle C12-200. With a very low dose they observed lowered levels of Factor VII for up to 25 days, with little side effects in the mice.
They next wanted to see if they could target multiple genes at the same time. They added in siRNAs that targeted four other liver-specific genes At low doses that were well tolerated by mice they could successfully knock down all five of the genes simultaneously. Very cool.
Next they looked to see if the best lipidoid would work in non-human primates. Again they chose a liver-specific gene. And it worked. At very low doses. With no apparent side-effects.
So why are these little fat bubbles so interesting? Some diseases, such as hepatitis C, have no effective treatments but has unique genes that would be great siRNA targets. We might be able to use these little fat bubbles to target the genes in HepC, shutting them off, and providing a cure. Or, if you had a disease with multiple genes involved, such as cancer or metabolic syndrome, you could target those genes, altering their expression and stopping the disease in their tracks.
What is still missing? Well, they only looked at genes active in one organ, the liver. This made for clean experiments but for other applications, it will be important to know how wide-spread the siRNAs have their effect. For example, in a cancer cell, turning off a gene might be a good thing, but in a normal cell it might have dire consequences. This still needs to be tested. Also, in mice the length of time of gene silencing was quite a long time, but it would be nice to know if they can give a second dose and quiet those genes again.
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