My laboratory's chief interest revolves around the structure and control of a human gene called SRC (pronounced SARK). This gene codes for an enzyme which regulates the rate at which a cell divides as well as its ability to move and migrate. If there is too much of the enzyme or it is in an activated state this can contribute to the development of cancer, particularly tumors of the large intestine and breast. We have isolated the complete human gene and have spent the last several years learning what makes SRC tick. To do this we have been studying the genes promoters. Promoters are discrete regions of a gene which act like the volume control on a Hi-Fi regulating the level of SRC produced. Our hope has been to find ways of turning down the volume on the SRC gene. Most lately we have found that a chemical called butyrate (which is a simple natural by-product of bacterial fermentation in the colon) can do exactly this! This provides an exciting link between a colon cancer gene and the food we eat and may help explain how high fiber diets are thought to inhibit the development of bowel cancer. Currently, we are working hard to understand how butyrate and related chemicals are able to control the SRC promoters. Ultimately, this may help us develop new drugs to control SRC levels in tumors such as breast cancer where SRC levels are high. This research is supported by the Canadian Institutes of Health Research and the Saskatchewan Cancer Agency.
B.Sc. (Hons) University of Salford (UK),
Ph.D. University of Calgary
Several projects are currently underway in our laboratory, all concerned in one manner or another with regulation of the human SRC gene. The SRC gene encodes a non-receptor tyrosine kinase, pp60c-src, and is the human homologue of the transforming gene of Rous Sarcoma Virus. c-Src has been linked to a bewildering array of signaling pathways impacting cellular proliferation, transformation, differentiation, survival, adhesion and migration.
Our work stems from the numerous observation over the years that link overexpression and activation of c-Src to the development of various human cancers, especially those of the colon and breast. We recently identified a subset of tumor cell lines that constitutively overexpress c-Src mRNA and protein. Scott Dehm has in turn shown that this results from SRC transcriptional activation. Therefore, unlike most groups working on c-Src, we have concentrated our efforts on understanding the structure and regulation of the SRC gene itself, rather than the gene product. We have isolated the SRC gene and shown that it is regulated by at least two promoters each associated with its own distinct exon. Differential promoter usage and subsequent splicing to a common downstream exon results in SRC transcripts with identical coding capacity but with different 5' noncoding regions. The proximal SRC1A promoter bears many hallmarks of a housekeeping gene including high GC content and multiple start sites. Mark Boyd originally demonstrated that SRC1A is regulated by the Sp1 family of transcription factors while Shawn Ritchie has been studying several long Polypurine:Polypyrimidine tracts (TC1, TC2 and TC3) present in the promoter. Shawn's studies revealed that these tracts contribute significantly to the transcriptional activity of the SRC1A promoter. He also showed that binding of a factor we called SPy (SRC Pyrimidine binding factor) was responsible for this activity. Recently, Shawn purified this SPy factor and identified it as hnRNP-K.
The distal SRC1alpha promoter is located just 1.0kb upstream of the SRC1A promoter. While the SRC1A promoter appears to regulate expression in many tissues, the SRC1alpha promoter is expressed in a much more restricted fashion and is controlled by Hepatic Nuclear Factor (HNF-1). HNF-1 is a homeodomain containing transcription factor regulating various genes in tissues such as intestine and liver. HNF-1 may thus be an important factor in regulating overexpression of SRC in tumors originating in these tissues. A major focus of the lab is now to understand how these two closely linked promoters are differentially regulated in a variety of normal and malignant cells.
Project 1: Regulation of c-Src gene expression by Histone Deacetylase (HDAC) inhibitors.
HDAC inhibitor have generated much interest of late because of their potential chemotherapeutic and cancer chemopreventative properties. For example, butyrate is a highly abundant colonic component generated by the bacterial fermentation of dietary fiber and is thought to exert a protective effect against colon cancer. Very recent work by Calley Kostyniuk, Scott Dehm and Danielle Batten has shown that Butyrate and Trichostatin A (a highly specific HDAC inhibitor) are efficient inhibitors of c-Src gene expression.
This appears to be a direct effect on the SRC promoters. We are now very actively pursuing the mechanism behind this inhibition and expanding our observations to other genes which are either induced or repressed by these interesting agents.
Project 2: Regulation of the SRC1A promoter (SPy identified!)
Work performed by Shawn Ritchie in the lab has concentrated on an interesting factor called SPy. This factor binds to certain double stranded Pu:Py tracts present in the SRC1A promoter with high sequence specificity. However, the same factor binds to single stranded Py sequences with a higher affinity but relaxed sequence specificity. Deletion or point mutations in these tracts which abolish SPy's ability to bind had dramatic effects on SRC1A promoter activity. Shawn purified SPy and used Mass Spec. analysis to identify it. SPy was found to be identical to heterogeneous Ribonucleoprotein (hnRNP) K. Protein K is a fascinating protein implicated in many cellular processes and binds a variety of biomolecules including DNA, RNA and various proteins (including c-Src itself!). Danielle Batten is now carrying on some of this work as Shawn successfully defended his Ph.D. thesis in May 2002.
In a related project we are studying the induction of c-Src gene expression during monocyte differentiation in the U937 model system. Phorbol ester mediated differentiation of this cell line leads to massive c-Src induction exclusively from the SRC1A promoter.
Project 3: Regulation of the SRC1alpha promoter
Activity of the SRC1alpha promoter is absolutely dependent on the HNF-1 site. However work carried out by Scott Dehm has shown that SRC1alpha promoter activity is actually very weak compared to SRC1A, an observation inconsistent with the high in vivo activity of this promoter in certain cell lines. This has led to our conclusion that undiscovered additional elements, such as enhancers, exist in the SRC gene. We are using a variety of techniques to search for these sites.
Ritchie SA., Pasha MK., Batten DJP., Sharma RK., Olsen DDH., Ross ARS. and Bonham, K. (2003) Identification of the SRC Pyrimidine Binding Protein (SPy) as hnRNP K: Implications in the Regulation of SRC1A Transcription. Nucleic Acids Res. 31: 1502-1513. pdf
Kostyniuk CL., Dehm SM., Batten D. and Bonham K. (2002) The Ubiquitous and Tissue Specific Promoters of the Human SRC Gene are Repressed by Inhibitors of Histone Deacetylases. Oncogene 21: 6340-6347. pdf
Chen Z., Dehm S., Bonham K., Kamencic H., Juurlink B., Zhang, X., Gordon JR. and Xiang J. (2001) DNA Array and Biological Characterization of the Impact of the Maturation Status of Mouse Dendritic Cells on Their Phenotype and Antitumor Vaccination Efficacy. Cellular Immunology 214(1): 60-71. pdf
Dehm S., Senger M. and Bonham K. (2001) SRC transcriptional activation in a subset of human colon cancer cell lines. FEBS Letters 487: 361-371. pdf
Bonham K., Ritchie SA., Dehm SM., Snyder K. and Boyd FM. (2000) An Alternative Human SRC Promoter and its regulation by Hepatic Nuclear Factor-1a. J. Biol. Chem. 275: 37604-37611. pdf
Rajala RVS., Dehm SM., Bi X., Bonham, K. and Sharma RK. (2000) Expression of N-Myristoyltransferase inhibitor protein and its relationship to c-Src levels in human colon cancer cell lines. Biochem. Biophys. Res. Commun. 273: 1116-1120. pdf
Ritchie S., Boyd F.M., Wong, J., and Bonham K. (2000) Transcription of the human c-Src gene is dependent on Sp1, a novel pyrimidine binding factor SPy and can be inhibited by Triplex Forming Oligonucleotides. J. Biol. Chem. 257: 847-854. pdf
Ritchie S. and Bonham K. (1998) The Human c-Src Proto-oncogene Promoter Contains Multiple Targets for Triplex Forming Oligonucleotides. Antisense and Nucleic Acid Drug Dev. 8: 391-400.
Aich P., Ritchie S., Bonham, K., and Lee, JS. (1998) Thermodynamics and Kinetic Studies of the Formation of Triple Helices Between Purine Rich Deoxyribo-nucleotides and the Promoter Region of the c-Src Proto-oncogene. Nucleic Acids Res. 26: 4173-4177. pdf
Bonham, K. and Fujita, D.J. (1993) Organization of the 5' Noncoding Exons of the Human C-SRC Proto-Oncogene and Analysis of its Promoter Region. Oncogene 8: 1973-1981.