Todd Lincoln Jackson and Dr. Laura Bridgewater, Zoology

Purpose

My purpose is to explore activator protein 4’s (AP-4) role in the expression of the Col11a2 gene. My hypothesis is that AP-4 will bind to the Col11a2 enhancer elements and increase activity of that gene. AP-4 is a ubiquitous transcription factor. Transcription factors are proteins that help regulate cell differentiation and proliferation by binding to enhancer sites to promote or inhibit gene expression. Previous work has shown that the mouse Col11a2 gene contains at least three enhancer sites namely; ABC, DE, and FG [1,2]. Each sequence of the enhancer sites was put through two computer databases of known transcription factors in order to identify possible candidates that could bind to the enhancer elements. AP-4 was shown to have a range of 75%- 89% probability of binding to the enhancer regions [3]. Hence, my research will determine if AP-4 binds to the aforementioned enhancer sites.

Background

Activator proteins are a family of proteins that are transcription factors. For example, AP-1, a ubiquitous transcriptional activator, mediates responses to external signals by regulating the expression of genes involved in growth, differentiation, and stress responses [4,5]. My proposed work deals primarily with AP-4.

Cartilage, which ossifies to form bones, makes up our skeletal structure. Therefore, if cartilage formation is aberrant then the bones will be affected. The extracellular matrix of cartilage consists of three types of collagen, type II, type IX, and type XI. Together these form the cartilage collagen fibrils that give cartilage its unique tensile strength. I am interested in studying the transcriptional activators that regulate the Col11a2 gene, which codes for the alpha 2 subunit of the heterotrimeric collagen XI fibril. This work is important because some serious conditions such as cleft palate, hearing loss, and osteoarthritis may be directly linked to the mutation or abnormal expression of the Col11a2 gene [7]. I will focus my investigation on the effects of the AP-4 protein transcriptional activator on the expression of Col11a2 enhancer elements.

A study done at M.D. Anderson Cancer Center on the Col11a2 gene showed that there were at least three important enhancer regions: ABC, DE, and FG [1]. These enhancer elements were only active in chondrocytes, and therefore chondrocytes must have contained the important transcriptional activators and environment that worked together with the enhancer regions to express the Col11a2 gene.

Systematic mutations were subsequently done on the ABC, DE, and FG enhancer elements to further identify the important segments of the enhancer sequences [2]. The mutational analysis revealed a dramatic decrease in activity when specific regions of the enhancer element were mutated. This was an indication that one or more transcriptional activators may have normally bound to those regions. The sequences of the suspected binding sites were analyzed using a Transfac [10] and Tess [11] computer databases to determine the known transcriptional factors that could have bound to the sequence [3]. AP-4 came out as one of the most probable candidates in both of the searches with between 70-80% probability of binding to a portion each of the enhancer element sequences.

AP-4 is a known transcription factor that has a helix-loop-helix conformation as shown in Figure 1[9]. The helix-loop-helix conformation allows AP-4 to work together with other proteins to bind to the enhancer element. My objective is to systematically test AP-4 to see if it can activate the enhancer elements that control the Col11a2 gene. I will also test AP-4 in conjunction with transcription activator Sox9 to detect changes, if any, in the activity level of gene expression [8]. Sox9 transcriptional activator is expressed primarily in cartilage and is known to play a critical role in cartilage formation. In addition, Sox9 binds to the each of the enhancer elements. The effects of Sox9 on the expression of the Col11a2 have already been studied individually, but not together with AP-4. I will see if AP-4 works together with this transcriptional activator to activate the expression of the Col11a2.

Results and Discussion

Results showed that the enhancer regions were activated in ABC, DE, and FG with the addition of AP-4 above the normal levels by the enzymatic assays (Figure 1). However, it can also be concluded that AP-4 is a minor activator of the enhancer regions when compared with enzymatic activity increase that occurs when Sox9 was added. When Sox9 and AP-4 were transfected together it was hypothesized that is the transcription factors worked together that there would be an additive effect happen in enzyme activity. My results suggest that AP-4 and Sox9 do not directly work together. AP-4 may even compete with Sox9 for binding to the DNA.

Further research will focus on AP-4 and its role in the activation of the Col11a2 gene.