Analyzing the conservation of DNA and protein sequences has proven to be an incredibly valuable approach in structural and molecular biology. The fundamental principle underlying the use of conservation data is the idea that conserved sequences are invariant for functional reasons. Changing the amino acid or base at these conserved sites, it is assumed, would lead to a catastophic structural disruption of a protein, eliminate an important functional group, or (in the case of DNA elements) substantially distort expression levels or splicing. If this is so, genetic elements that are ultraconserved (>200 bp segments that are 100% conserved between human, mouse, and rat genomes) should be positively essential to the survival of an organism. However, a recent study published in PLoS Biology (open access) indicates that removing at least some of these ultraconserved elements (UCEs) has little or no effect on development or longevity in mice.
Ahituv et al. knocked out four ultraconserved elements in C57BL/6J mice, none of which are known to code for protein. This is normal; more than half of these ultraconserved regions (481 have been identified) are not transcribed into RNA. It's thought that these non-coding regions are important for gene regulation, and some preliminary experiments suggested that this would be the case for these four. But homozygous or hemizygous (some of these genes were on the X chromosome) knock-out mice developed normally, for the most part, and were born in roughly the ratio one would expect from a Punnett square. Gender ratios and litter sizes were also normal -- this is particularly compelling in the case of the 2 UCEs located on the X chromosome. If losing these UCEs were fatal or even mildly problematic, we would expect to see some evidence of this in the gender ratio of the pups.
Element deletion was not entirely without effect. Knocking out one of them caused a very low incidence of male mice born with only one kidney. Another had a slight effect on the expression of a gene called Sox3 in the developing brain. However, the genes flanking these deletion sites have much more serious effects when they are deleted or otherwise disrupted -- several of these cause embryonic lethality. So the UCEs have enormously reduced effects compared to the flanking genes.
If they're not important, why are these regions conserved? That's a tough question, and in a way hinges on a false impression. This particular disruption of these regions did not adversely affect an extremely inbred pool of mice that were well-fed and kept in controlled climates. That is, these organisms are genetically abnormal and face little stress. Their responses to these mutations may not be indicative of mice in general, or even of other strains of laboratory mice. Moreover, these regions may only be important in stress conditions that are nonetheless ordinary -- development under conditions where some nutrient is absent or rare, for instance. Or the effect may only be felt after many cumulative generations.
Another possibility is that of redundancy or subtlety of phenotype. Some other element may be acting to do the job once performed by these UCEs. There is a similar story with growth factors, expressed proteins that are essential but nonetheless seemed not to produce a phenotype in mice unless several were knocked out of the genome simultaneously.
And finally, there is a possibility that the reason UCEs do not mutate is not that they are essential but that they can easily gain function that is fatal. That is, mutations turn the UCE into a toxic piece of DNA, but some minor function keeps them in the genome. This interpretation is not as well supported by existing data, however, which suggest that it is specifically the loss of these elements that reduces fitness.
As usual, this interesting work raises more questions than it answers. Understanding the reasons why UCEs are preserved with such high fidelity through various species will likely contribute a great deal to our understanding of expression dynamics and evolution.
Ahituv N, Zhu Y, Visel A, Holt A, Afzal V, Pennacchio, L. A., Rubin, E. M. "Deletion of Ultraconserved Elements Yields Viable Mice" PLoS Biology Vol. 5, No. 9, e234
Ahituv et al. knocked out four ultraconserved elements in C57BL/6J mice, none of which are known to code for protein. This is normal; more than half of these ultraconserved regions (481 have been identified) are not transcribed into RNA. It's thought that these non-coding regions are important for gene regulation, and some preliminary experiments suggested that this would be the case for these four. But homozygous or hemizygous (some of these genes were on the X chromosome) knock-out mice developed normally, for the most part, and were born in roughly the ratio one would expect from a Punnett square. Gender ratios and litter sizes were also normal -- this is particularly compelling in the case of the 2 UCEs located on the X chromosome. If losing these UCEs were fatal or even mildly problematic, we would expect to see some evidence of this in the gender ratio of the pups.
Element deletion was not entirely without effect. Knocking out one of them caused a very low incidence of male mice born with only one kidney. Another had a slight effect on the expression of a gene called Sox3 in the developing brain. However, the genes flanking these deletion sites have much more serious effects when they are deleted or otherwise disrupted -- several of these cause embryonic lethality. So the UCEs have enormously reduced effects compared to the flanking genes.
If they're not important, why are these regions conserved? That's a tough question, and in a way hinges on a false impression. This particular disruption of these regions did not adversely affect an extremely inbred pool of mice that were well-fed and kept in controlled climates. That is, these organisms are genetically abnormal and face little stress. Their responses to these mutations may not be indicative of mice in general, or even of other strains of laboratory mice. Moreover, these regions may only be important in stress conditions that are nonetheless ordinary -- development under conditions where some nutrient is absent or rare, for instance. Or the effect may only be felt after many cumulative generations.
Another possibility is that of redundancy or subtlety of phenotype. Some other element may be acting to do the job once performed by these UCEs. There is a similar story with growth factors, expressed proteins that are essential but nonetheless seemed not to produce a phenotype in mice unless several were knocked out of the genome simultaneously.
And finally, there is a possibility that the reason UCEs do not mutate is not that they are essential but that they can easily gain function that is fatal. That is, mutations turn the UCE into a toxic piece of DNA, but some minor function keeps them in the genome. This interpretation is not as well supported by existing data, however, which suggest that it is specifically the loss of these elements that reduces fitness.
As usual, this interesting work raises more questions than it answers. Understanding the reasons why UCEs are preserved with such high fidelity through various species will likely contribute a great deal to our understanding of expression dynamics and evolution.
Ahituv N, Zhu Y, Visel A, Holt A, Afzal V, Pennacchio, L. A., Rubin, E. M. "Deletion of Ultraconserved Elements Yields Viable Mice" PLoS Biology Vol. 5, No. 9, e234
1 comment:
Dude, what if they are all hinges....
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