Molecular Characterization Molecular Characterization of the Canine HMGB1of the Canine HMGB1

Presented by Melissa Metcalfe and Shauna Presented by Melissa Metcalfe and Shauna MaguireMaguire

Canine GenomeCanine Genome

An overviewAn overview

First Sequenced by Celera with the First Sequenced by Celera with the DNA from Shadow, a male Standard DNA from Shadow, a male Standard Poodle, in 2002. (WGS 80% Poodle, in 2002. (WGS 80% coverage rough draft)coverage rough draft)

Next major sequencing, done by the Next major sequencing, done by the Broad Institute, was of Tasha, a 7 Broad Institute, was of Tasha, a 7 year old female boxer. (95% year old female boxer. (95% coverage)coverage)

Genome InformationGenome Information

Genome Size 2.8x10Genome Size 2.8x109 9 bpbp

Gene sequences + EST’s 900Gene sequences + EST’s 900 BAC end sequences 668BAC end sequences 668 Sequence Tag sites (STS) 106Sequence Tag sites (STS) 106 2n Chromosome number 782n Chromosome number 78 38 autosomes + sex 38 autosomes + sex

chromosomeschromosomes

Why sequence the canine Why sequence the canine genome?genome? Human genome and dog genome are Human genome and dog genome are

evolutionarily and physiologically similar.evolutionarily and physiologically similar. The canine genome could help us understand The canine genome could help us understand

analogous human hereditary diseases. Half of analogous human hereditary diseases. Half of dog hereditary diseases have a human analog.dog hereditary diseases have a human analog.

Canines have been selectively bred for many Canines have been selectively bred for many years and their pedigrees have been kept. years and their pedigrees have been kept. They are also have high levels of inbreeding. They are also have high levels of inbreeding. These factors make them ideal for genetic These factors make them ideal for genetic studies.studies.

Improvement of canine therapeutic treatments Improvement of canine therapeutic treatments and purebred bloodline maintenance.and purebred bloodline maintenance.

HMGB1HMGB1 High mobility group box protein 1High mobility group box protein 1

Originally identified as a transcriptional regulatory Originally identified as a transcriptional regulatory molecule that can modify chromatin structure by molecule that can modify chromatin structure by bending DNA.bending DNA.

Recently, it has been attracting considerable interest Recently, it has been attracting considerable interest by oncologists because it has been said to have a by oncologists because it has been said to have a “double life”.“double life”.

What does it mean a “double life”?What does it mean a “double life”?

HMGB1 HMGB1

Besides its function as a transcriptional activator, HMGB1 Besides its function as a transcriptional activator, HMGB1 has been found outside of the nucleus by secretion from has been found outside of the nucleus by secretion from macrophages.macrophages.

As a extracellular protein, HMGB1 participates in As a extracellular protein, HMGB1 participates in developmental and differentiation processes, triggers and developmental and differentiation processes, triggers and modulates many of the inflammatory cascades in the body, modulates many of the inflammatory cascades in the body, and may be involved in the metastic invasion programme and may be involved in the metastic invasion programme of cancer cells.of cancer cells.

Specifically, HMGB1 is a ligand for the receptor for Specifically, HMGB1 is a ligand for the receptor for advanced glycation end products (RAGE) thus activating advanced glycation end products (RAGE) thus activating p38p38mapkmapk ,JNK, and p42/p44 ,JNK, and p42/p44mapkmapk which are key signaling which are key signaling pathways. pathways.

This signal via a receptor induces inflammatory responses.This signal via a receptor induces inflammatory responses.

SepsisSepsis

Is the body’s systemic inflammatory Is the body’s systemic inflammatory response to infection or trauma.response to infection or trauma.

Can also be a caused by an infecting agent Can also be a caused by an infecting agent (such as bacteria).(such as bacteria).

The body can mount a widespread The body can mount a widespread inflammatory response to an infection that inflammatory response to an infection that can quickly become out of control and cause can quickly become out of control and cause even more harm.even more harm.

Can cause shock, organ damage, permanent Can cause shock, organ damage, permanent disability, or death.disability, or death.

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

HMGB1HMGB1..

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

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

Northern Blot AnalysisNorthern Blot Analysis

Performed to define a basic expression pattern of the protein in canineheart, lung, muscle, kidney, and spleen tissue.

Lane 1 – KidneyLane 2 – SpleenLane 3 – SpleenLane 4 – HeartLane 5 – HeartLane 6 – lungLane 7 – MuscleLane 8 - Fibroblasts

FFluorescentluorescent I Inn S Situitu HHybridizationybridization

Makes it possible to map any Makes it possible to map any cloned locus to its position on the cloned locus to its position on the metaphase chromosome.metaphase chromosome.

Good for quickly obtaining the Good for quickly obtaining the chromosomal position of a newly chromosomal position of a newly cloned locus. Does not require cloned locus. Does not require mapping to be “in relation” to mapping to be “in relation” to another.another.

FISH protocolFISH protocol

Drop cells onto glass slideDrop cells onto glass slide Gently denature DNA with DNaseGently denature DNA with DNase Hybridize with fluorescent probe and Hybridize with fluorescent probe and

wash away unhybridized probewash away unhybridized probe Expose to UV light and take a picture Expose to UV light and take a picture

of the fluorescent chromosome with of the fluorescent chromosome with a Fluorescence microscope.a Fluorescence microscope.

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

ConclusionConclusion

““The molecular characterization of the canine HMGB1 The molecular characterization of the canine HMGB1 gene and protein showed that humans and dogs share gene and protein showed that humans and dogs share an identical HMGB1 protein.”an identical HMGB1 protein.”

Molecular targeting of HMGB1 in dogs is very Molecular targeting of HMGB1 in dogs is very significant for therapeutic approaches in humans due significant for therapeutic approaches in humans due to the similarity of genesis and development of to the similarity of genesis and development of diseases in both species.diseases in both species.

Thus, the canine shows great potential as a model Thus, the canine shows great potential as a model organism in the study of human diseases.organism in the study of human diseases.