Monday, February 20, 2012

Transgenic mice to study FGFR3 signaling in vivo – Part 1



FGFR3 is part of a complicated network that is required for normal bone growth and development. Achondroplasia-associated mutation increases the negative role of FGFR3 (anti-proliferative), causing growth inhibition. Although FGFR3 appears to have an anti-proliferative role in bones, it has been shown to promote proliferation in other tissues. So, what makes FGFR3 function differently in different tissues, and why is it important for us to understand this? To be able to identify and test molecular targets for drug, we have to find out how does Ach-associated FGFR3 mutation cause increased growth inhibition in bones. We need the sequence of events downstream of FGFR3 signaling that are negatively affected by this mutation. By comparing proliferative vs inhibitory role of FGFR3, we might get some indication of what is happening in bones due to the mutations. All of this requires dissecting out the molecular details of FGFR3 signaling and regulation. Although the initial experiments need to be carried out in cultured cells, they will need to be eventually tested in live animals - genetically engineered mice in our case. Our plan is to use such mice (transgenic mice) models to understand the role of FGFR3 signaling in bone growth. First, it will be useful to understand what are transgenic mice and how are they generated.


Transgenic mice models are frequently used in the study of human diseases due to their genetic similarities with humans. They are easy to manipulate and work with. Most importantly, they provide a method to study the effect of a single gene or protein without disturbing any other factors in the animal. In simple terms, transgenic mice expressing the mutant form of a disease-related gene would mimic the conditions associated with the human disease, and thus allow us to observe and study the effect of that mutation in the system.


Generation of transgenic mice involves several steps. Foreign genetic material - the transgene - is incorporated into the genome of a mouse to generate transgenic mice. There are several methods available for the method of delivery of the transgene and efficient incorporation of the transgene into the genome. Following link (2 part video) provides an overview of how transgenic system works. http://www.youtube.com/watch?v=ujZHrR1mro8


Also, for further reading:
1. Van Keuren ML et al. “Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes.” Transgenic Res. 2009 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3016422/?tool=pubmed)
2. Connelly CS et al. “The role of transgenic animals in the analysis of various biological aspects of normal and pathologic states.” Exp Cell Res. 1989. (http://www.ncbi.nlm.nih.gov/pubmed/2670592)

More on ChIP to identify DNA-protein interactions



Progress:

Our current focus is to identify a role for sICD (the small intracellular protein fragment formed by the cleavage of FGFR3 receptor) using chromatin immunoprecipitation (ChIP). We would like to find out whether sICD protein could interact with DNA to change the genetic environment of the cell and alter the outcome of FGFR3 signaling. We have conducted preliminary experiments to establish the ChIP method to look at DNA-protein interactions in cartilage cells. These ChIP experiments will be combined with ChIP-sequencing analysis to recognize a functional role for sICD protein.



ChIP-sequencing is a powerful tool that gives information on DNA-protein interaction sites on a genome-wide (in the whole organism) level. Briefly, ChIP isolates pieces of DNA that are occupied (or bound) by a transcription factor (typically a protein) within the whole genome. The isolated pieces of DNA represent various sites within the whole genome that the protein binds. Loosely speaking, if a protein occupies or binds to a certain region (or site) of a DNA, it is probable that it may participate in regulating expression of that gene. The information encoded by these pieces of DNA is determined by sequencing simply by determining the arrangement of the nucleotide bases adenine (A), thymine (T), guanine (G), and cytosine(C) within that DNA. ChIP-sequencing will provide valuable information and lead to better understanding of the system. More on ChIP-sequencing can be found here:
In our next blog update, we will present a second research that is being conducted in the lab using transgenic mice to help characterize the role of FGFR3 signaling in achondroplasia

The progress on ChIP

PROGRESS: We have adapted the ChIP assay to cartilage cells, which will help us to identify "target genes" that are regulated by nuclear FGFR3 and potentially involved in achondroplasia. 
TECHNICAL NOTE: The genetic material of a cell is found inside the nucleus and is stored in the form of DNA, which is organized into linear segments called genes.  Proteins referred to as transcription factors bind to DNA and turn the genes on and off.  We think the sICD portion of FGFR3 (see earlier blog) acts as a transcription factor for genes involved in achondroplasia and the ChIP assay will be used as a tool to identify them. ChIP stands for Chromatin Immunoprecipitation.  The ChIP technique allows us to isolate pieces of DNA that are bound by the protein of interest - the FGFR3-sICD in this case.  For our experiments, we will analyze cultured chondrocyte cells using ChIP to first show that the sICD fragment binds to DNA. Once the region of the DNA to which sICD binds to is identified, we will then determine which specific genes bind to and are presumably regulated by the sICD. 

Achondroplasia - an introduction to our approach

As discussed under Research, the root cause of achondroplasia is too much “anti-growth” activity of FGFR3 in the cells of growing bones.  That is, increase in FGFR3 activity within the cells of growing bones results in specific signals that reduce or block growth. Several therapeutic strategies have been proposed to reduce this activity, and most are targeted to the cellular networks that carry instructions from FGFR3 to nucleus within the cells. These networks are complex and dispersed throughout the cell. We have discovered a new pathway that bypasses these established networks and potentially allows instructions to be transferred directly from FGFR3 residing on the surface of a cell to its nucleus, where the regulation of genes involved in controlling growth occurs.  We believe this novel pathway mediates many of the anti-growth signals of FGFR3, including those responsible for achondroplasia.  We also believe that defining this new pathway will lead to the identification of new and potentially better targets for therapeutic intervention for achondroplasia.


Indeed, we have already identified one potential target.  A key feature of this new pathway involves cutting of the intact FGFR3 molecule present at the cell surface to produce a small fragment that moves to the nucleus to deliver its anti-growth message, and promotes nuclear signaling of FGFR3.  To our surprise, the machinery that cuts FGFR3 also cleaves a protein implicated in Alzheimer’s disease.  Other than sharing this common protein cutting machinery, the two proteins and also the two diseases are completely unrelated.  In fact, abnormal cutting of this protein in many patients with Alzheimer’s disease has identified this cutting machinery as a therapeutic target for Alzheimer’s disease, which in turn has led to considerable investment by the pharmaceutical industry in drugs to correct the abnormality.


So how is this relevant to achondroplasia?  As we investigated the cleavage of FGFR3, we discovered that achondroplastic FGFR3 is cut abnormally creating a scenario similar to that for Alzheimer’s disease in which abnormal cleavage contributes to disease.  If so, the distinct possibility arises that drugs designed to favorably alter the cleavage of the Alzheimer protein might also normalize the cleavage of FGFR3 and at least partially alleviate the manifestations of achondroplasia.  In economic terms, our findings raise the possibility of leveraging the enormous investment in Alzheimer’s drugs for a novel treatment for achondroplasia. 

Our goal is to move our project from its current stage of initial discovery to the point where it merits further development by industry.  For this we need to define the new pathway in more depth.  We need to demonstrate that this pathway is involved and necessary for normal FGFR3 effects on growing bone, that it is altered in achondroplasia, and that restoration of normal cleavage corrects the disturbance caused by the achondroplasia mutation.  In short, we need to show that the newly discovered pathway influences bone growth, plays a role in achondroplasia and is amenable to therapeutic manipulation. We anticipate reaching these goals over the next 2-3 years.


We will discuss how we are addressing these points in future blogs.  However, for this blog suffice it to say that many of our experiments are being performed in cultured cells.  We have developed and continue to refine assays that measure precisely how much of the cleaved FGFR3 fragment reaches the cell nucleus.  By comparing normal FGFR3 to FGFR3 bearing the achondroplasia and other relevant mutations, we can begin to understand the impact of the mutations on the fate of the cleaved fragments.  We are also setting up assays to determine the precise location of cleavage within the FGFR3 protein and how this is affected by the achondroplasia mutation.  Once this information is known, we will determine if Alzheimer drugs can normalize cleavage and transfer of the fragment to the nucleus. 


We expect there to be direct target genes of FGFR3 in the nucleus.  To identify them, we are using a technique called Chromatin Immunoprecipitation (ChIP).  It will be explained in a future blog.  Identifying even a few FGFR3 target genes will provide valuable insights into how FGFR3 regulates bone growth.  Measuring the expression of these genes will further validate the effects of the achondroplasia mutation and of candidate drugs on FGFR3 functions. 


To complement our cell culture studies, we are also genetically engineering mice to validate the importance of the FGFR3 nuclear signaling pathway in live animals.  FGFR3 in these mice will be tagged to allow microscopic and biochemical detection.  The mice will have normal FGFR3, FGFR3 bearing the achondroplasia mutation, FGFR3 bearing a mutation that blocks cleavage and prevents the fragment from being produced and FGFR3 comprised of only the cleavage fragment destined for the nucleus.  By analyzing and comparing these mice in detail, we will determine the biologic significance of the direct nuclear signaling by FGFR3.  These mice will also be used to test the Alzheimer’s drugs.
As noted above, future blogs will focus on progress within the different aspects of this overall investigation.