Chromatin mishandling in Huntington disease: potential links to pathogenesis and points of therapeutic intervention

Huntington disease (HD) is a hereditary neurodegenerative disorder that affects 3,000 Canadians. Patients with a HD mutation experience adult-onset psychiatric symptoms, cognitive impairment and motor disturbances that progressively worsen over many years and lead to death. Despite a massive world-wide research effort, there is still no means of prevention, no treatment, and no cure for HD. Recent studies confirm that psychiatric and cognitive changes occur in at-risk individuals several years before formal HD diagnosis. The ability to understand and reverse these early cognitive changes may lead to a cure for HD. Chromatin is the genomic DNA-protein complex that specifies how to make proteins and when and where to do so. Recent studies show that the way genetic material is packaged into chromatin in brain cells can be altered by early life experiences (such as learning), and can affect behaviour. Alterations in chromatin regulation have been observed in depression, bipolar disorder and schizophrenia. Many effective psychiatric drugs have been found to influence chromatin regulation. Mendel Grant is examining a possible link between chromatin and the onset and progression of HD. She is testing her hypothesis that environmentally-responsive chromatin changes (such as those induced by learning) do not function normally in a mouse model of HD. This could underlie cognitive dysfunction observed in HD patients and animal models. If chromatin is shown to be misregulated in HD, the use of FDA-approved neuropsychiatric drugs that alter chromatin regulation could be a promising therapy. Information yielded from this study may also be applicable to other neurodegenerative disorders including Alzheimer and Parkinson diseases and psychiatric conditions such as depression.

The demi-globule hypothesis: a thermodynamic model for prion protein template-directed misfolding

Prions are the causative agents for many brain diseases of humans and animals. In animals, the most well-known prion-related disease is bovine spongiform encephalopathy, or mad cow disease. The human prion diseases are Creutzfeldt-Jakob disease, kuru, and fatal familial insomnia. All these diseases cause brain cell death leading to difficulty walking, dementia, muscle spasms, and seizures. They are invariably fatal, and there are currently no treatments available. The incidence of human prion diseases is roughly 1 per million people per year. Unlike most other infectious diseases that are spread by bacteria or viruses, prion diseases appear to be caused by misfolded protein molecules. Protein is made of long chains of amino acids, and how a protein folds determines its function. A misfolded prion protein can interact with a normally-folded prion protein and cause it to also misfold, setting off a chain reaction that results in widespread brain cell death. How the misfolded prion causes the normal prion to misfold remains unclear. However, a new theory called the demiglobule hypothesis proposes that the misfolded prion binds to the normal prion and causes a key part of the normal protein structure, called a beta sheet, to come apart. This ultimately results in a misfolded shape. William Guest is using a variety of theoretical and experimental techniques from physics, chemistry and biology to determine whether the demiglobule hypothesis can account for prion protein misfolding. If the results of this project support the demiglobule hypothesis, researchers will know much more about how prion conversion occurs. This knowledge could ultimately enable the development of methods to block prion conversion, thereby stopping the spread of disease.

Development of natural killer (NK) cells from a novel progenitor

Natural Killer (NK) cells are immune cells with an important role in the first line of defense against cancer formation, metastases (spread) of tumour cells, and infections by viruses and other pathogens. Once known solely for their role in the innate immune system, responding to pathogens in an immediate and non-specific way, research now suggests that certain NK cells might also be involved in regulating the more targeted adaptive immune response. It is believed that a subset of NK cells in the lymph node is largely responsible for this function. With new knowledge about subsets of NK cells that have specialized functions, researchers are now looking at how these NK cells arise. It is possible that in addition to the NK cells that develop in bone marrow, other immature NK cells travel to different parts of the body where they mature in specific microenvironments that affect their function. In the lung this could mean a better NK cell response to cancer metastases or virus infection, whereas lymph node NK cells might be better at interacting with other immune system components. Timotheus Halim’s research seeks to find immature NK cells (NK cell progenitors) in sites other than the bone marrow. NK cell progenitors have already been found in the lymph node and lung. Now, Halim is using different mouse models to evaluate how important these progenitors are in forming mature NK cells. He is also determining if the NK cells that arise from these novel NK cell progenitors have specialized functions. A better understanding of NK cell development and function is critical in understanding the overall management of the immune system. Ultimately, this knowledge could help in the development of immunotherapy and other forms of treatment against cancer and infection.

Gene and genome synthesis through pair-wise ligations of short oligonucleotides

While the fundamental unit of life is the cell, it is the cell’s DNA which instructs how a cell is to function. These microscopic blueprints can be read. Reading DNA sequences has enabled genes to be identified and revealed insights about the causes of many human diseases such as Huntington’s and numerous cancers. Reading and understanding how DNA functions, however, is only half the challenge in genetics research. To correct genetic errors accumulated in diseased genes, it is necessary to also write in DNA. But while DNA is essential in all genetics research, it is difficult to produce. Methods to rewrite or create new DNA sequences from scratch are limited. Regions of DNA can be copied, but there are relatively few methods of generating new fragments. Nucleotides are the characters of the DNA language. A 1,000 nucleotide gene costs approximately $1,000 to construct. Given that the average human gene is more than 3,000 nucleotides long, and that regulatory regions of DNA can be tens of thousands of nucleotides long, the cost of producing DNA becomes daunting. Daniel Horspool is researching a new laboratory technique for DNA construction. While the primary method involves adding one nucleotide at a time chemically to the growing sequence, the new technique relies on stitching multi-nucleotide fragments together in parallel. This process could be much faster and less error prone than the conventional method, and by using microfluidic devices, DNA could be produced at a much lower cost. The research could lead to an approach for constructing complete genes, which would be an important new tool for basic and applied biomedical research. The research could ultimately contribute to efforts to realize the promise of genomic and personalized medicine.

Computational simulation of transcription factor binding for the prediction of regulatory regions in DNA sequences

The regulation of when and where a gene is turned on (gene expression) is a complex process, fundamental to how a cell behaves and interacts with the environment around it. Abnormal changes in gene regulation are associated with many diseases, including cancer, asthma, and obesity. One class of proteins involved in the regulation of genes are transcription factors (TFs). TFs recognize and bind to short sequences of DNA near the genes they regulate and act to increase or decrease the expression of their target gene. The binding interaction between TF proteins and DNA is affected by an array of biophysical factors in the cell nucleus, making this complicated process a good candidate for computational modelling through bioinformatics. Bioinformatics is a relatively new field in which computational approaches are used to study biological problems; a field that unites computer science, statistics and the life sciences. Rebecca Hunt Newbury is developing a software simulator to model the TF binding process in a dynamic, interactive setting, with the intent of predicting the locations in a DNA sequence at which TFs will bind and regulate genes. From there, she will begin to incorporate the spatial relationships and combinatorial interactions between TFs that result in the different expression responses of genes. Hunt Newbury’s research will contribute to clearly defining the regions of DNA that participate in regulating a gene. Her work may ultimately contribute to new approaches for combating diseases caused by abnormal gene regulation.

Global view of pre-mRNA splicing in Saccharomyces cerevisiae

Proteins, the molecules that carry out most cellular functions, are synthesized according to information contained in DNA sequences. Converting information from DNA into a protein requires an intermediate step in which the DNA sequence is copied into a molecule called mRNA. In humans, there is an essential biochemical process called pre-mRNA splicing, in which certain (non-coding) portions of the sequence are removed and the remaining protein-coding portions are joined together to form a template for protein synthesis. This is a complex process with multiple steps, and even small errors can be dangerous. Many diseases, such as some cancers, Alzheimer’s disease, and Parkinson’s syndrome, can be attributed to defects in the pre-mRNA splicing machinery. Perhaps due to the complexity and requirement for absolute precision in splicing, the molecular machine that carries out splicing – termed the spliceosome – is enormous. For both humans and yeast, its components number well over 100. However, several splicing proteins likely remain unidentified. A thorough understanding of splicing will require a complete inventory of its parts. Paul Kahlke is identifying novel splicing factors in yeast, which serves as a laboratory model for human splicing. His objective is to uncover previously unknown splicing factors and to determine whether existing candidate proteins are indeed integral to splicing. His studies take a whole genome approach, testing many genes one by one to see which ones are involved in splicing. By screening a large number of genes, Kahlke hopes to identify several new splicing factors and gain insight into the function of known pre-mRNA splicing factors.

Structure and RNA-cleaving function of apurinic/apyrimidinic endonuclease 1 (APE1)

Cancer occurs when cells won’t stop growing. The source of this malfunction is often an alteration in DNA, the genetic instructions for making different proteins inside a cell. To make proteins, a gene is copied out from the DNA into a molecule called mRNA. The mRNA travels out of the cell nucleus and into the cytoplasm, where its genetic instructions are used as a template for synthesizing proteins. C-myc mRNA serves as a template for synthesizing the c-Myc protein. This protein plays fundamental roles in regulating growth, differentiation, and cell death in virtually all mammalian cells, and it is implicated in diverse human cancers. In fact, it has been estimated that c-Myc dysfunction contributes to one-seventh of all cancer deaths. One way that cells regulate their level of proteins is by destroying mRNA in the cytoplasm by chemically cutting, or cleaving, its molecular structure. Recently, the enzyme APE1 was discovered to cleave c-myc mRNA. This opens the potential for using APE1 to reduce or eliminate levels of c-Myc protein in the cytoplasm as a potential treatment for cancer. Wan Kim is exploring the function of APE1 as an mRNA destroyer. He is identifying the key amino acids and mechanisms APE1 uses to cleave c-myc mRNA, and determining whether the enzyme cleaves any other types of mRNA. Kim hopes to generate valuable insights into how APE1 can degrade c-myc mRNA and influence gene expression. Also, the study will provide useful information on the potential design of novel approaches in cancer treatment.

Integrated microfluidic technologies for optimization of hematopoietic stem cell expansion

Blood contains different types of specialized cells. Red cells are responsible for oxygen transport, white cells ensure body’s defense against infections, and platelets initiate clotting to limit the loss of blood after an injury. These cells are constantly renewed, and are manufactured in the centre of the bones in a sponge-like tissue called bone marrow. Hematopoietic stem cells are a small subset of cells found in bone marrow that have the astounding ability to self-renew and divide, and to differentiate into a variety of mature blood cells. They are often used to treat blood-related diseases or given after cancer treatment. Although stem cells have great potential for regenerative medicine, they are extremely rare and they are difficult to expand in the lab, because they very readily differentiate into other cell types. The multiple factors that influence their self-renewal are poorly understood. Véronique Lecault is exploiting the potential of microfluidic technology, an engineering advance that allows thousands of different experiments to be performed in tandem upon a device the size of a microscope slide. Across rows and rows of miniature cell culture chambers, individual hematopoietic stem cells can each be exposed to different chemical conditions and tracked over time. This makes determining the specific environments that will allow the cells to be expanded much more efficient. This technology could lead to the ability to produce more hematopoietic stem cells for use in disease therapies. It could also help researchers gain a better understanding of stem cell biology, perhaps leading to the discovery of new ways to identify and purify these rare cells.

Vascular Endothelial Growth Factor Signalling in Cardiac Allograft Vasculopathy

Atherosclerosis, also known as hardening of the arteries, is a common vascular disease caused by the buildup of a waxy plaque on the inside of blood vessels. This narrowing of blood vessels can cause blood clots, leading to heart attack or stroke. In almost half of all heart transplant patients, an accelerated form of hardening of the arteries, known as Transplant Vascular Disease (TVD), occurs in the transplanted heart. In fact, TVD is a leading cause of death one year after transplantation. The exact mechanisms behind this process remain unclear. Blood vessels are lined with endothelial cells, specific cells that create a barrier between blood and the artery. An important factor in TVD is damage to endothelial cells. This damage increases the size of gaps between cells, allowing fats to accumulate in artery walls. One protein that causes endothelial “”leakiness”” is called Vascular Endothelial Growth Factor (VEGF). VEGF is also important in many other serious diseases, such as cancer and degenerative eye diseases. David Lin is expanding on previous research that showed that VEGF is increased in the muscle cells in arteries of transplanted hearts. He is studying in detail the mechanisms by which VEGF alters the function and structure of endothelial cells. By learning how VEGF works in transplanted hearts, Lin hopes his research will lead to the development of new ways to maintain the health of heart blood vessels following transplantation.

Investigation of genetic networks involving genes that confer chromosome stability in S. cerevisiae and C. elegans

Chromosomes are found in all organisms that have a cell nucleus, and carry the organism’s hereditary material. During cell division, chromosomes divide and distribute equally to the daughter cells. Errors in the division process can result in daughter cells that contain an incorrect number of chromosomes. Known as aneuploidy, this state is responsible for many genetic diseases and is characterized by a specific type of genome instability known as Chromosome Instability (CIN). Because chromosome stability is a fundamental requirement across all organisms, it can be studied using simpler organisms, such as baker’s yeast. Genome wide screens on yeast have identified approximately 300 genes important for maintaining chromosome stability (CIN genes). A subset of these genes has also been found to be mutated at an elevated rate in some human cancers, which suggests that these genes contribute to tumour progression and development. Mutations in these key genome stability genes may also represent an Achilles’ heel for tumours. Enhancing chromosome instability to a point where tumour cells can no longer function and reproduce could halt their division or lead to cell death. Jessica McLellan is studying the subset of CIN genes mutated in colon cancer. She specifically aims to identify genes that, when mutated in combination with a CIN gene mutation, lead to cell death. By exploiting the mutations seen in many types of cancers, this project could lead to the development of novel cancer therapeutics that are less harmful to non-cancer cells than current treatments. McLellan’s research will increase our understanding of the complex biological processes that ensure genome stability and the mechanisms by which these processes can become deregulated.