Just another pretty face

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!