Associate Professor of Biomedical Sciences Control of Gene Expression Laboratory 412 Dodge Hall of Engineering (248) 370-2079 mitton@oakland.edu

Biography

Kenneth P. Mitton received his Ph.D. in biochemistry from the University of Western Ontario in 1994. He has completed post-doctoral training Fellowships at Virginia Tech (Biochemistry, 1994-95), the National Institutes of Health, National Eye Institute (Cell Biology, 1995-1997), and Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, the University of Michigan (Molecular Biology, Control of Gene Expression, 1997-2001).

Research

Control of Gene Expression in the developing and mature retina: genes and epigenetic regulation

Early attempts at gene therapy inadvertently introduced new problems despite the good intention of correcting the original defect. Ignorance of the full system that controls gene expression in any tissue increases the chance of such unfortunate results. Our research, and that of other labs, is crucial to understand as much as possible regarding the gene regulatory networks that result in the formation of normal photoreceptor cells. This knowledge, in turn, has set the stage to support the design of gene therapies that will be safe and effective on human patients with retinal degenerations. My lab has focused on a specific window of retinal development when photoreceptors cells have been born to when they are mature and functional in the mammalian neural retina. During this time thousands of genes must become activated to transform photoreceptor progenitor cells into mature light-detecting machines we know as rod and cone photoreceptors. Over the last several years, myself, many undergraduate students (Biological Sciences and Biochemistry), technologists Xiao Zhang (M.Sc.) and Ed Guzman (B.Sc.), and ERI Vision Science Postdoctoral Fellows Raghuveer S. Mali, Ph.D. and Padmaja Tummala Ph.D., have contributed to novel information discovered in my lab.

We use specific photoreceptor genes, such as Rhodopsin, as model genes to uncover what kinds of mechanisms are involved in turning on such genes. Rhodopsin is the light detecting protein of our eyes and the Rhodopsin gene is only expressed in rod photoreceptors. Many retinal diseases in humans are due to mutations in the genetic code of genes like the Rhodopsin gene. Diseases such as Retinitis Pigmentosa (RP). These investigations have helped us understand that many different transcription factors (proteins) cooperate to activate a cell-specific gene like Rhodopsin or Pde6b, often by directly binding with each other as they also bind to the control regions of genes they activate. These proteins include CRX, NRL, NR2E3, FIZ1 and NR1D1 (circadian clock gene). Some of these transcription factor genes cause retinal degenerations in humans when mutations alter their DNA sequence (CRX, NRL, NR2E3).

As the mouse and human genome sequences (3 billion basepairs of DNA) became available, we have broadened our investigations to capture novel gene regulation events across the entire genome using Chromatin Immunoprecipitation and Affymetrix GeneChip Tiling arrays (ChIP-on-Chip). With this technology we have discovered hundreds of gene activation events that were not detected by previous expression microarray (RNA) studies. Using ChIP-on-Chip technology, we produced the first genome-wide maps of RNA-Polymerase-II bound in gene promoters as photoreceptors mature in the mouse neural retina (Tummala et al., 2010, Molecular Vision). We have also made these maps available for the worldwide research community using a genome browser interface that only requires a simple web-browser (Internet Explorer, Firefox, Safari). This enzyme is responsible for producing messenger RNA from active genes, and thus changes in the amount of this polymerase is a novel method to detect gene activation. Our use of this method was one of the first applied it to a native tissue (neural retina), as opposed to cells in culture.

Genetic Diseases of the Retina in Young Children: Mechanisms in Norrie’s Disease and FEVR
(Collaboration with Dr. Kimberly Drenser, MD, PhD)

Norrin is an extra cellular signaling protein that activates some members of the Wnt receptor family such as Fzd4. Mutations in the genes for Norrin (NDP) and FZD4 cause abnormal development of the retinal vasculature in humans, leading to blindness. While there are many different mutations occurring in these genes, we do not know precisely how these mutations disrupt the normal function of Wnt-receptor pathways to cause disease. Can Norrin or other compounds affecting the Wnt signaling pathways be used as potential therapy? To address these questions, we must first understand how these mutations change the normal function of these pathways. Using methods first developed to study the regulation of gene promoters, we have developed a cell-culture based system to compare the function of Fzd4 receptor mutations. The effects of mutations are sometimes surprising, and the results are critical to designing therapeutic strategies that will not make the condition worst.

Epigenetic Regulation in Retinal Development and Ageing

Most recently we have started to explore how chromatin architecture is regulated in the neural retina, as genes are activated or inactivated during retinal development and aging. Epigenetic regulation refers to the control of the packaging of chromatin (DNA and histones) in a cell nucleus. Through epigenetic regulation a gene can be silent or active, without any alterations to the DNA sequence itself. (Cvekl and Mitton, 2010, Heredity) Many natural and man-made chemicals in our food, water, and medicines cause change to DNA methylation and Histone actetylation. These are two chemical changes that oppose each other to affect the availability of a gene for expression. DNA methylation of gene promoters causes chromatin to become tightly packaged away, silencing the gene, while acetylation of specific histones (such as H3 on Lysine-9) promoters unpacking of the gene and gives access to the DNA for transcription factors to bind and recruit RNA-Polymerase-II to the gene. Mammalian DNA is most often methylated at Cytosines immediately preceding a Guanine (CpG) in DNA. (Mitton and Guzman, 2010, Experimental Eye Research) We can look for these chemical motifs using technology such as ChIP-on-Chip and methylation specific PCR to map the locations of 5-Methylcytosine.

Valproic acid, an antiseizure drug, is one example of prescribed medicine that inhibits histone-deacetylases, leading to increased histone acetylation and abnormal increases in gene expression. This effect was not know when the drug first entered use, and almost $1-billion of this drug are prescribed in the US each year. Valproic acid is now used in some specific leukemia chemotherapies to restore the expression of tumor suppressor genes in cancer cells. There is fast growing interest among clinicians to test this drug in many age related diseases, including retinal degenerations, to increase the expression of genes such as heat-shock proteins might slow damage and cell death in affected tissues. Unfortunately, we currently do not have genome-wide data to understand epigenetic regulation in normal retinal development and ageing, nor how they are affected by Valproic acid or inhibitors of DNA-methyl-transferases. Using ChIP we can address this lack of knowledge using the mouse retina model and also donor human retinal tissue.

Biomarkers in Human Age-related Disease

Stroke, leads to the death of critical brain functions including vision and may also directly affect the eye locally. Thus, some patients experiencing stroke first alerted by their eye doctor. Stroke and other stresses that affect our fixed tissues, even migraine attacks, have secondary effects on circulating white blood cells, altering their gene expression patterns. We have started pilot projects in collaboration with Beaumont Hospital’s Surgery Department (Dr. Charles Shanley, Dr Graham Long) and the Beaumont Biobank to use RNA-Polymerase-II mapping (ChIP-on-Chip) to detect active genes in blood cells from patients with carotid atherosclerotic disease. This pilot project is almost complete, supported in part by a recent 2009 Beaumont – Oakland University Multidisciplinary Research Award (Mitton, Shanley, Long). One goal is to find a panel of novel gene-expression biomarkers that will help clinicians monitor when patients arterial plaques are becoming dangerously unstable and more inflammatory. Another goal is discover changes in leukocyte gene expression that are involved in driving plaque formation.
 

With Dr. Kimberly Drenser (Ophthalmology WBH, and ERI), we are also interested in expanding into similar biomarker studies to monitor the progress and treatment effectiveness in retinal conditions such as AMD and diabetic retinopathy. Blood based biomarkers are ideal, since blood collection is minimally invasive and is already part of normal clinical practice. Only a few milliliters of blood are required for genome-wide analysis of blood cell gene expression. The process we have piloted with Surgery and the Biobank, is the same process ready for use on patients with any condition.

Dr Mitton’s Website: http://web.mac.com/eyeresearch

Recent Publications:

Mitton KP*, Guzman AE (2010) Focus on Molecules: 5-Methylcytosine, a possible epigenetic link between ageing and ocular disease. Exp Eye Research, In Press, Available online 8 July 2010, DOI: 10.1016/j.exer.2010.06.026.

Tummala P,  Mali RS, Guzman E, Zhang X, Mitton KP*. (2010) Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation. Mol Vision 16, 252-271 (http://www.molvis.org/molvis/v16/a32)

Cvekl A*, Mitton KP*. (2010) Epigenetic regulatory mechanisms in vertebrate development and disease. Heredity (doi:10.1038/hdy.2010.16)

Simpanya MF, Wistow G, David LL, Giblin FJ, Mitton KP*. (2008) Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank  Mol Vision; 14:2413-2427 (http://www.molvis.org/molvis/v14/a278)

Mali RS, Peng G-H, Zhang X, Dang L, Chen S, Mitton KP*. (2008) FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo. BMC Mol Biol 9, 87 (17 pages) http://www.biomedcentral.com/1471-2199/9/87

Mali RS, Zhang X, Hoerauf W, Doyle S, Devitt J, Loffreda-Wren J, and Mitton KP*. (2007) FIZ1 is Expressed During Photoreceptor Maturation, and Synergizes with NRL and CRX at Rod-Specific Promoters, in vitro. Exp Eye Res 84, 349-360

Friedman JS, Khanna H, Swain PK, DeNicola R, Cheng H, Mitton KP, Weber CH, Hicks D, and Swaroop A. (2004) The minimal transactivation domain of the basic motif-leucine zipper transcription factor NRL interacts with TATA-binding protein. J Biol Chem 279, 47233-41.

Cheng, H., H. Khanna, E.C. Oh, D. Hicks, K.P. Mitton and A. Swaroop. (2004) Photoreceptor specific nuclear receptor NR2E3 functions as a transcriptional activator in rod photoreceptors. Hum Mol Genet 13, 1563-1575.

Mitton KP, Swain PK, Khanna H, Dowd M, Apel IJ, and Swaroop A. (2003) Interaction of retinal bZIP transcription factor NRL with Flt3-interacting zinc-finger protein Fiz1: Possible role of Fiz1 as a transcriptional repressor. Hum Mol Genet 12, 1-10.