1. Discovery of the first example of protein interactions between two important DNA-binding domains.
The bZIP (basic leucine zipper) and Homeobox Domains (triple helix) were already well known DNA-binding domains and mostly regarded as single-function domains when I accepted the challenge to work with Anand Swaroop at the Kellogg Eye Center, in 1997, to find proteins that physically interact with NRL (Neural Retina Leucine Zipper). I was interested in the opportunity to take my new signal transduction experience at the NEI/NIH (Russell lab) and learn about where those signals end up and how they work in the nucleus at genes. Anand Swaroop was interested in finding a PhD student or post-doc who wanted to take a chance on applying yeast two-hybrid protein interaction trapping to look for unknown protein interactions with NRL (Neural Retina Leucine Zipper). While scientists generally assumed that transcription factors could not be used as baits in yeast two-hybrid surveys, because they are auto-activating of the yeast’s reporter genes, we felt it was worth trying to make separate baits of the NRL DNA-binding domain and the NRL transactivation domain. I created these Bait-hybrid protein constructs by DNA engineering. Our strategy paid off, in that I found the first evidence of a direct physical interaction of CRX (Cone Rod Homeobox) with the NRL b-ZIP domain. While surprising that the DNA-binding domain of NRL was also important for a protein-protein interaction, we completed other experiments to show that CRX was also using its DNA-binding domain, called a homeobox domain, for this protein-protein interaction too. This not only explains physically why NRL and CRX activate the Rhodopsin promoter with positive synergy, but it was also the first discovery that these important DNA-binding domain families (bZIP and Homeobox) can have protein-interaction functions in addition to their known DNA-binding activity.
- Mitton KP, Swain PK, Chen S, Xu S, Zack DJ, Swaroop A (2000) The leucine zipper of NRL interacts with the CRX-homeodomain: A possible mechanism of transcriptional synergy in rhodopsin regulation. Journal of Biological Chemistry 275, 29794-29799
2. Proving that the first know mutation of NRL (S50T) could alter NRL function sufficiently to be responsible for Retinitis Pigmentosa in two UK families.
I carried out the site-directed mutagenesis to construct and express human NRL(S50T) for mammalian cell-transfection assays, to discover that this mutation of NRL does not cause a loss of NRL function. Surprisingly, it creates a super-activating NRL that activates the expression of rod-specific gene promoters (like the Rhodopsin gene) at ten times normal NRL. This finding was important as one fo the first examples of a super-functional transcription factor mutation causing a human disease.
- Bessant DAR*, Payne AM*, Mitton KP*, Wang Q-L, Swain PK, Plant C,Bird AC, Zack DJ, Swaroop A, Bhattacharya SS (1999). A Mutation in NRL is Associated with Autosomal Dominant Retinitis Pigmentosa. *equivalent contributions. Nature Genetics 21(4):355-6
3. Producing the first genome-wide maps of RNA-Polymerase-II binding around transcription start sites of maturing photoreceptors. The quest to find which genes are activated in rod-photoreceptor cells as they mature from photoreceptor progenitors to mature rods in vivo had been approached by several technologies including expression microarrays and RNA-sequencing. Additionally, cell sorting was used on retinas after proteolytic digestion. Results from those studies revealed several-thousand gene activation events, but were still limited to detect changes in the expression of genes of mid to high expression.
While exploring the recruitment of transcription co-factors to gene promoters with NRL in my first NIH-grant, using Chromatin Immunoprecipitation (ChIP), we reasoned that activated genes should have have increases in the amount of RNA-Polymerase-II around their transcription start sites regardless of the transcript abundance. Furthermore, due to the fact that a good ChIP experiment only captures a protein-bound DNA target at a frequency of 1 in 1,000 – 10,000 cells, RNA-Polymerase-II ChIP with the intact neural retina would essentially represent rod-specific chromatin, even without using enzymatic isolation of photoreceptors. That is, at least 70% of the mouse retina’s chromatin comes from rod photoreceptor cells. Our Pol-II mapping was the first of its kind done with the neural retina and we were able to reveal an additional 800 gene activation events to the several thousand previously reported using RNA expression analysis. We are also one of the first laboratories to make this kind of genome-wide data truly available to all vision science researchers by not only depositing the data in the GEO database, but also by providing easy and fast visualization of the data for anyone to load into the free Genome Browser interfaces at UCSC and the NEI. Two outside laboratories have now used our data to support their own gene specific studies, which I was happy to provide to them upon request.
- 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
4. Discovery of the rod-specific, alternative, promoter responsible for activation of the Mef2c gene during the late phase of rod-maturation.
If one looked carefully at several of the first published expression microarray studies of the developing mouse neural retina, you could see that some Mef2c gene probes suggested increased Mef2c gene expression in the maturing retina. Furthermore, the dramatic loss of Mef2c expression in the NRL-knockout mouse indicated a substantial production of some Mef2c transcripts must rod-derived. However, real time PCR analysis with available probe-sets would indicate that Mef2c expression was low and unchanged. Our RNA-Polymerase-II mapping revealed a new transcription start site appearing in mature rod photoreceptors downstream of the known transcription start sites for brain, muscle and cardiac muscle. By designing splice specific Taqman PCR assays we confirmed that retinal Mef2c gene expression was dramatically increased after age 15 days and was rod-specific. Our additional experiments confirmed that this rod-specific promoter was completely dependent on transactivation by NRL itself. It is also an unusual example of a gene activation event in rod-cells that occurs in the final and late stage of rod-maturation, after age P15.
- Hao H, Tummala P, Guzman E, Mali RS, Gregorski J, Swaroop A, Mitton KP* (2011) The transcription factor Neural Retina Leucine zipper (NRL) controls photoreceptor-specific expression of myocyte enhancer factor Mef2c from an alternative promoter. J Biol Chem 286, 34893-34902 https://doi.org/10.1074/jbc.M111.271072
5. Different effects of the histone deacetylase inhibitor (& anticonvulsant), Valproic Acid, on photoreceptor loss in different models of retinal degeneration.
My research into the mechanisms regulating photoreceptor gene expression involved changes to histone acetylation associated with developmental activation of photoreceptor genes. An FDA approved anticonvusant medication, VPA, was found to be a potent HDAC inhibitor decades after its entry into the clinic, and the medical community was rushing to test VPA off-label for almost any kind of neurological disorder. The hypothesis is that VPA would promote the expression of “neuroprotective genes”. However, I thought that this assumption, while possible, was far from proven and that VPA also has the potential to alter the expression of hundreds or thousands of genes and this might accelerate photoreceptor degeneration or slow degeneration depending on the underlying diseases etiology. The enrolment of patients in North America and Korea for testing VPA treatment of Retintis Pigmentosa was also underway with no requirement to identify the specific gene mutation causing their disease. My lab used two different mouse genetic models, Rd1 and Rd10, to demonstrate that VPA slowed photoreceptor loss in the Rd1 model but it also accelerates degeneration in the Rd10 model. Furthermore, we produced the first data to show that systemic VPA treatment can alter the expression of numerous photoreceptor genes in vivo. This research indicates that any clinical trials in humans need to compare placebo and VPA treatment only between patients that have the same molecular etiology (same mutation) for their retinitis pigmentosa. If patients of different disease etiology are combined in analysis there is a real risk of missing detection of a benefit for families with a specific mutation and missing detection of potential harm to families with a different mutation.
- Mitton KP*, Guzman AE, Deshpande M, Byrd D, DeLooff C, Mkoyan K, Zlojutro P, Wallace A, Metcalf B, Laux K, Sotzen J, Tran T (2014). Different effects of valproic acid on photoreceptor loss in Rd1 and Rd10 retinal degeneration mice. Mol Vis 20, 1527-1544 http://www.molvis.org/molvis/v20/1527
6. Norrin treatment improves ganglion cell survival in an oxygen-induced retinopathy model of retinal ischemia.
Highlights
• Norrin treatment accelerates recovery of the mouse OIR model from ischemic insult.
• SD-OCT can compare NFL/GCL (nerve fiber layer/ganglion cell layer) thickness in vivo.
• Norrin treatment counters thinning of the NFL/GCL in the mouse OIR model.
• Norrin treatment increases the surviving population density of RGCs in OIR retinas.
This paper is one of the first to use the in vivo imaging methods of intrinsic fluorescence with a transgenic mouse strain to see individual ganglion cells in the living mouse eye, and to even follow their morphology over a period of many days in the mouse model of oxygen-induced retinopathy. This was done with a Phoenix Research Labs’ system, in this case the Micron-III version of their imaging system. We used a light filter set recommended by Phoenix to image yellow-fluorescent protein (YFP). Axons and dendrites could be seen on single cells in anesthetized mice. Amazing!
We also employed SD-OCT (Spectral Domain – Optical Coherence Tomography) to capture 3D structural records of the mouse retina and then to measure the changes in thickness of the very thin Nerve Fiber Layer / Ganglion Cell Layer (NFL/GCL).
The ability to use these imaging systems in vivo, which are also used in clinical analysis of the Human retina, enables us to see disease processes as they progress and to use far fewer mice to get the answers to research questions. In this case we were testing the ability of Norrin (Norrie’s Disease Protein) to be used to help avascular regions of retina recover their vasculature more quickly and improve the survival of RGCs (retinal ganglion cells) from the stress of low oxygen. RGCs are the cells that form our optic nerves. Millions of RGCs per eye have axons that extend all the way into connections with our brain. This bundle of a million “wires”, or axons, is the optic nerve.
Our research here and that of other laboratories suggest that Norrin and other agents might have use to maintain a better vasculature in diseases where the blood vessels and capillaries are damaged, such as ROP, Diabetic Retinopathy and AMD.
- Wendy A. Dailey, Kimberly A. Drenser, Sui Chien Wong, Mei Cheng, Joseph Vercellone, Kevin K. Roumayah, Erin V. Feeney, Mrinalini Deshpande, Alvaro E. Guzman, Michael Trese, Kenneth P. Mitton (2017) Norrin treatment improves ganglion cell survival in an oxygen-induced retinopathy model of retinal ischemia. Experimental Eye Research, Vol 164, November 2017, Pages 129-138. https://doi.org/10.1016/j.exer.2017.08.012
- Wendy A. Dailey, Kimberly A. Drenser, Sui Chien Wong, Mei Cheng, Joseph Vercellone, Kevin K. Roumayah, Erin V. Feeney, Mrinalini Deshpande, Alvaro E. Guzman, Michael Trese, Kenneth P. Mitton (2017) Ocular coherence tomography image data of the retinal laminar structure in a mouse model of oxygen-induced retinopathy. Data in Brief, Vol 15, December 2017, Pages 491-495. https://doi.org/10.1016/j.dib.2017.09.075
List of More Published Works in MyBiography at the National Library of Medicine:
What goes around, comes around: connections and fate.
(How I got to where I am now.)
The ERI was founded in 1968 by V. Everett Kinsey, PhD and Venkat Reddy, PhD, just as Oakland University became an independent State University in 1969. Dr Kinsey was awarded the prestigious Lasker Clinical Medical Research Award Prize in 1956 for coordinating clinical trials that established the effect of incubator oxygen on retinopathy of prematurity (ROP). This was also a pioneering example of the multi-center clinical trial. Dr Kinsey is also recognized in NIH’s history for proposing the creation of the National Eye Institute within the National Institutes of Health (Bethesda MD). He personally convinced several renowned clinician scientists to support this idea, which resulted in the creation of the NEI by an Act of Congress in 1968. One of those doctors, Karl Kupfer, became the NEI’s first Director.
So Dr Kinsey was laying the groundwork to make my career as a vision scientist possible long before I was in high school. I first found myself in eye research as I began my PhD training in the lab of John R. Trevithick, PhD, in the Department of Biochemistry at the University of Western Ontario (Recently renamed Western University). John is one of the pioneer researchers who delved into the human lens as a model to study aging and lens development. There I worked on a diabetic rat model to uncover how a cascade of metabolic effects caused the loss of key antioxidation cycles and eventual damage to cellular ATP production and ion-transport. The external examiner for my PhD thesis was Mike Riley, PhD, (ERI Emeritus Professor). My first post-doctoral training at Virginia Tech resulted when an NEI scientist (Donna Garland PhD) passed my CV to John Hess and Edwin Bunce at Virginia-Tech, who were seeking a PhD to complete some lens research. My second post-doctoral fellowship was in the NEI, an excellent education in signal transduction at the center of the biomedical research universe. A posting on Dr Wistow’s bulletin board (NEI) led to my third post-doctoral training in the regulation of gene expression at the Kellogg Eye Center (U.Mich) with Anand Swaroop’s Lab group.
Now, I find myself a Faculty member at Oakland University, in the Eye Research Institute founded by Dr Kinsey. I literally owe my career to Dr Kinsey, who I never had a chance to meet in person. As a Canadian, it is also interesting that another Canadian made Oakland University possible, through her generosity and vision for the people of Michigan and Oakland County: Matilda Dodge Wilson!
Circles and connections continue, as I find myself helping to direct the ERI’s Pediatric Retinal Research Laboratory, and with a support from the VRRF and NIH, we are exploring Vascular Endothelial Growth Factor mechanisms in the human retina and developing novel recombinant protein therapeutics that may be used in the future for ROP and Diabetic Retinopathy.
The hand of Dr Kinsey seems to still be influencing my path. My personal story illustrates that while we are doing science we have a pay-forward obligation to train students who will become the next generation of scientists, engineers and medical doctors. It is impossible to pay back all of my past teachers and mentors in the complex web of fate that brought me to this point. So in science we pay-forward, by introducing undergraduate science students to the real world of bioscience research and training graduate level and medical researchers too.
Ken Mitton, PhD 