Molecular Characterization of the Canine HMGB1

1. Molecular Characterization of the Canine HMGB1 Presented by Melissa Metcalfe and Shauna Maguire

2. Canine Genome An overview

3. First Sequenced by Celera with the DNA from Shadow, a male Standard Poodle, in 2002. (WGS 80% coverage rough draft) Next major sequencing, done by the Broad Institute, was of Tasha, a 7 year old female boxer. (95% coverage)

4. Genome Information • Genome Size 2.8x109 bp • Gene sequences + EST’s 900 • BAC end sequences 668 • Sequence Tag sites (STS) 106 • 2n Chromosome number 78 • 38 autosomes + sex chromosomes

5. Why sequence the canine genome? • Human genome and dog genome are evolutionarily and physiologically similar. • The canine genome could help us understand analogous human hereditary diseases. Half of dog hereditary diseases have a human analog. • Canines have been selectively bred for many years and their pedigrees have been kept. They are also have high levels of inbreeding. These factors make them ideal for genetic studies. • Improvement of canine therapeutic treatments and purebred bloodline maintenance.

6. HMGB1 • High mobility group box protein 1 • Originally identified as a transcriptional regulatory molecule that can modify chromatin structure by bending DNA. • Recently, it has been attracting considerable interest by oncologists because it has been said to have a “double life”. • What does it mean a “double life”?

7. HMGB1 • Besides its function as a transcriptional activator, HMGB1 has been found outside of the nucleus by secretion from macrophages. • As a extracellular protein, HMGB1 participates in developmental and differentiation processes, triggers and modulates many of the inflammatory cascades in the body, and may be involved in the metastic invasion programme of cancer cells. • Specifically, HMGB1 is a ligand for the receptor for advanced glycation end products (RAGE) thus activating p38mapk ,JNK, and p42/p44mapk which are key signaling pathways. • This signal via a receptor induces inflammatory responses.

8. Sepsis • Is the body’s systemic inflammatory response to infection or trauma. • Can also be a caused by an infecting agent (such as bacteria). • The body can mount a widespread inflammatory response to an infection that can quickly become out of control and cause even more harm. • Can cause shock, organ damage, permanent disability, or death.

9. Figure 1. Structure of the genomic elements and cDNA of the canine HMGB1.

10. Table 1. Detailed analysis of the canine HMGB1 cDNA and genomic elements.

11. Fig. 3 Comparison of Canine, Human, Mouse, and Bovine HMGB1 Protein

12. Northern Blot Analysis Performed to define a basic expression pattern of the protein in canine heart, lung, muscle, kidney, and spleen tissue. Lane 1 – Kidney Lane 2 – Spleen Lane 3 – Spleen Lane 4 – Heart Lane 5 – Heart Lane 6 – lung Lane 7 – Muscle Lane 8 - Fibroblasts

13. Fluorescent In Situ Hybridization Makes it possible to map any cloned locus to its position on the metaphase chromosome. Good for quickly obtaining the chromosomal position of a newly cloned locus. Does not require mapping to be “in relation” to another.

14. FISH protocol • Drop cells onto glass slide • Gently denature DNA with DNase • Hybridize with fluorescent probe and wash away unhybridized probe • Expose to UV light and take a picture of the fluorescent chromosome with a Fluorescence microscope.

15. Metaphase spread after FISH with signals on both chromosomes (a) and after GTG-banding (b).

16. Conclusion • “The molecular characterization of the canine HMGB1 gene and protein showed that humans and dogs share an identical HMGB1 protein.” • Molecular targeting of HMGB1 in dogs is very significant for therapeutic approaches in humans due to the similarity of genesis and development of diseases in both species. • Thus, the canine shows great potential as a model organism in the study of human diseases.