Alisha Clinger and Dr. Laura Clarke Bridgewater, Microbiology and Molecular Biology

Improper development of collagen is the cause for many cartilage-based diseases, including osteoarthritis as wells as various skeletal disorders. The Col11a2 (type XI collagen á2 subunit) gene is critical to cartilage development and is necessary for cartilage cells to be able to perform their specific functions (1,5). The development of collagen is controlled by the regulation of various collagen genes by specific proteins. Certain proteins bind to regions in the DNA called enhancers and promoters and this activates the gene. Dr. Laura Bridgewater’s laboratory at Brigham Young University has analyzed three enhancer regions for the Col11a2 gene. Each of these enhancer regions have sites that are homologous to the high mobility group (HMG) protein-binding consensus sequence. These HMG sites are specific sequences of DNA that are found in many different sites that bind proteins. The purpose of my experiment is to determine whether the protein SOX9 binds to the three enhancer regions of the Col11a2 gene in vivo (under conditions found in the living cell).

SOX9 was chosen to test because it has been shown to bind to the HMG sites of the enhancer regions of the Col11a2 gene in vitro (under laboratory conditions, not the same as the living cell). (1) Researchers tested the binding of SOX9 in vitro by performing Electrophoretic Mobility Shift Assays (EMSAs) and transient transfections. However, both of these methods create situations where the binding of the protein SOX9 to DNA could be more favorable than it would actually be in the cell. I am trying to solidify the assumptions of the in vitro experiments by actually testing the binding of SOX9 to Col11a2 enhancer regions by using in vivo formaldehyde cross-linking and chromatin immunoprecipitation (ChIP).

During this past year, I have created a protocol to determine whether or not SOX9 bound in vivo to the three enhancer regions (B/C, D/E, and F/G) in the Col11a2 gene. To create my protocol I adapted the procedure outlined by Orlando, Strutt, and Paro (3). I used rat chondrosarcoma (RCS) cells because they are easily cultured and closely resemble cartilage cells, which are very difficult to obtain and culture.

Fixation and Sonication

First, I add a formaldehyde solution to the cells and this strongly binds any proteins that are closely associated with the DNA to the DNA. The cell and nuclear membranes are then broken up in a buffer solution. Next, the protein-DNA complexes are sonicated, subjected to high frequency sound waves that shear DNA.

Immunoprecipitation

I split the sonicated solution into four different tubes and then add SOX9 antibody to one tube, control antibodies to two other tubes (negative control), and no antibody to the last tube (negative control). Immunoprecipitation uses antibodies to separate certain proteins from a solution. In my experiment, the SOX9 proteins should be cross-linked to the DNA HMG sites, so when I separated out the SOX9 proteins with the SOX9 antibody the DNA bound to SOX9 should also be separated out.

DNA purification and PCR

After immunoprecipitation, I used proteases to destroy the proteins and isolate the DNA. I then used a process called Polymerase Chain Reaction (PCR) to amplify the DNA fragments so I could compare them on an agarose gel to positive controls, which consisted of mouse genomic DNA.

This past year has been spent fine tuning my protocol and learning the experimental procedures necessary to perform this experiment. I was able to complete five trials of the experiment, however, in each one I had positive results in my negative controls. I tried to decrease the amount of possible DNA contamination, yet I still found positive results in my negative controls. Recently, while reading a paper about formaldehyde cross-linking and chromatin immunoprecipition I found that when using mammalian cells an extra purification procedure is needed to reduce the amount of background DNA (6). This purification procedure requires the use of radioactivity, a cesium chloride gradient, and centrifugation at very high speeds for an extended period of time. Right now I am in the process of working with my faculty advisor and the undergraduate student I am training to figure out if it is absolutely necessary to do this step and if we can obtain the materials necessary to perform this extra purification step.

In the future we can use this procedure to confirm that a protein binds to a specific DNA sequence in vivo. Determining which proteins bind to the enhancers of cartilage-specific genes, such as Col11a2, can ultimately increase our understanding of the regulatory processes that are involved in the expression of these genes, which are essential to proper collagen development.

1. Brigdewater, L., Lefebvre, V., and de Crombrugghe, B. (1998) “Chondrocyte-specific Enhancer Elements in the Col11a2 Gene Resemble the Col2a1 Tissue-specific Enhancer”. The Journal of Biological Chemistry vol.273 no.24, 14998-15006.
2. Lefebvre, V., Huang, W., Harley, V., Goodfellow, P., and de Crombrugghe, B. (1997) “SOX9 Is a Potent Activator of the Chondrocyte-Specific Enhancer of the Proá1(II) Collagen Gene”. Molecular and Cellular Biology vol.17, no.4, 2336-2346.
3. Orlando, V., Strutt, H., and Paro, R., (1997) “Analysis of Chromatin Structure by in Vivo Formaldehyde Cross-Linking”. Methods vol.11, 205-214.
4. Wathelet, M., Lin, H., Parekh, B., Ronco, L., Howley, P., and Maniatis, T., (1998) “Virus Infection Induces the Assembly of Coordinately Activated Transcription Factors on the IFN-â Enhancer in Vivo”. Molecular Cell vol.1, 507-518.
5. Bridgewater, L., Walker, M., Miller, G., Ellison, T., Holsinger, D., Chen, R., Potter, J., Winkel, V., Zhang, Z., McKinney, S., and de Crombrugghe, B., “Mutational Analysis of Three Chondrocyte-specific Enhancer Elements from the Mouse Col11a2 Gene”.
6. Orlando, V. (2000) “Mapping chromosomal proteins in vivo by formaldehydecrosslinked- chromatin immunoprecipitation”. Trends in Biochemical Science 25(3): 99- 104.