Richard Day, Ph.D.
Professor, Full Member of
Indiana University Graduate Faculty
Department of Cellular & Integrative Physiology
Indiana University School of Medicine
635 Barnhill Drive, Room 333
Indianapolis, Indiana 46202-5120
E-mail: rnday @ iupui.edu
Education / Training
- 1978, B.S., University of Colorado, Boulder, CO
- 1980, M.S., State University of NY, Fredonia, NY
- 1987, Ph.D., University of Rochester, Rochester, NY
- 1990, Postdoctoral Fellowship, University of Iowa, Iowa City, IA
Research Biography Summary
Since 1996, my funded research has applied quantitative fluorescence microscopy techniques to visualize the intra-nuclear distribution and behaviors of proteins that are labeled with different color variants of the fluorescent proteins. These studies use the combination of molecular biology, biochemistry, and live-cell imaging approaches to determine how specific gene regulatory complexes are assembled in the intact cell nucleus. We are using the time-resolved fluorescence lifetime imaging microscopy (FLIM) to quantify Förster resonance energy transfer (FRET) to determine how certain disease-causing point mutations can affect specific nuclear protein interactions. I have over one hundred publications, with extensive experience in live- cell imaging. In addition, I have been teaching in the annual Cold Spring Harbor Laboratory or Marine Biological Laboratory live-cell imaging courses yearly since 1998, and I am currently the co-organizer for the Workshop on FRET microscopy, held annually at the University of Virginia since 2001.
Contribution to Science
1. My early research focused on how the interactions between cell-specific transcription factors, their co- regulatory protein partners and the nuclear receptors function in concert to regulate gene expression. We used biochemical, genetic, and molecular approaches to determine how the
rotein complexes that are necessary for pituitary specific gene transcription are assembled. Our studies demonstrated that Pit-1, a pituitary-specific homeodomain transcription factor, orchestrates the activities of a network of transcription factors and co-regulatory proteins that function to control pituitary gene transcription. Together, these studies showed how Pit-1 interacted cooperatively with nuclear receptors and other transcription factors and coregulatory proteins to regulate prolactin (PRL) and growth hormone gene expression.
a)Day, R.N., Koike, S., Sakai, M., Muramatsu, M., Maurer, R.A. 1990. Both Pit-1 and theestrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Molecular Endocrinology 4:1964-1971. PMID: 2082192.
b)Day, R.N. Day, K.H. 1994. An alternatively spliced form of Pit-1 represses prolactin gene expression. Molecular Endocrinology 8:374-381. PMID: 8152427.
c)Day, R.N., Liu, J., Sundmark, V., Kawecki, M., Berry, D., Elsholtz, H.P. 1998. Selective inhibition of PRL gene transcription by the ets domain factor, ERF. Journal Biological Chemistry 273:31909-31915. PMID: 9822660.
d)Schaufele F, Enwright JF III, Wang X, Teoh C, Srihari R, Erickson R, MacDougald OA, Day, R.N. 2001.
CCAAT/enhancer binding protein alpha assembles essential cooperating factors in common subnuclear domains. Molecular Endocrinology 15:1665-1676. PMID: 11579200.
2. Importantly, we complemented these structural and functional studies with live cell imaging studies. This integrative approach allowed us to determine how the subnuclear positioning of protein complexes contributes to the selective expression of tissue-specific genes. We used fluorescence microscopy to visualize the intranuclear distribution and interactions of proteins labeled with a variety of different color fluorescent proteins. These studies in living cells showed how the assembly of cooperating factors at particular intranuclear sites is critical for the regulation of cell-specific gene expression. We used this approach to demonstrate that Pit-1 specifically recruited nuclear receptors and other transcription factors and coregulatory proteins to the nuclear sites it occupied, providing evidence that Pit-1 can direct cooperating factors to particular sites in the nucleus.
a) Day, R.N. 1998. Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy. Molecular Endocrinology 12:1410-1419. (Cover Article). PMID: 9731708.
b)Enwright III, J.F., Kawecki-Crook, M.A., Voss, T.C., Schaufele, F., Day, R.N. 2003. A Pit-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/Enhancer binding protein alpha required for prolactin gene transcription. Molecular Endocrinology 17(2):209-222. PMID: 12554749.
c) Voss, T.C., Demarco, I.A., Booker, C.F., Day, R.N. 2005. Functional Interactions with Pit-1 Reorganize Corepressor Complexes within the Nucleus. Journal of Cell Science 118: 3277-3288. PMID: 16030140.
d)Demarco, I.A., Voss, T.C, Booker, C.F., Day, R.N. 2006. Dynamic Interactions between Pit-1 and C/EBP alpha in the Pituitary Cell Nucleus. Molecular and Cellular Biology 26:8087-8098. PMID:16908544.
3. These studies also demonstrated that the p42 CCAAT/enhancer-binding protein alpha (C/EBPα) interacted cooperatively with Pit-1 to activate PRL transcription. This was of particular interest because C/EBPα is a basic region-leucine zipper (BZip) transcription factor that functions to direct programs of cellular differentiation. C/EBPα is unusual among transcription factors in that it binds preferentially to α-satellite DNA elements located in regions of centromeric heterochromatin. Furthermore, C/EBPα requires DNA methylation to optimally bind DNA elements in gene promoters. Our published studies showed that the BZip domain of C/EBPα is necessary and sufficient for targeting to the centromeric heterochromatin in mouse cells, and critically demonstrated that the C/EBPα BZip domain directly interacts with heterochromatin protein 1 α (HP1α).
a)Demarco, I.A., Periasamy, A., Booker, C.F., Day, R.N. 2006. Monitoring Dynamic Protein Interactions with Photo-quenching FRET. Nature Methods 3:519-524. PMID: 16791209.
b)Siegel, A.P., Hays, N.M., Day, R.N. 2013. Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. J Biomed Optics 18:25002. PMID: 23392382.
c)Tsekouras, K., Siegel, A.P., Day, R.N., Presse, S. 2015. Inferring Diffusion Dynamics from FCS inHeterogeneous Nuclear Environments. Biophysical journal 109 (1):7-17. PMID: 26153697
4. Over the past decade we have pioneered the use of fluorescence lifetime imaging microscopy (FLIM) to measure Förster resonance energy transfer (FRET) between proteins labeled with FPs inside living cells. FLIM quantifies FRET by the direct measurement of the donor fluorescence lifetime alone. This eliminates problems associated with spectral bleedthrough, making FLIM among the most accurate method for measuring FRET. We have also developed the complementary approach of fluorescence correlation spectroscopy (FCS) to monitor protein dynamics. We are currently applying these live-cell imaging methods to establish how the HP1α-C/EBPα network controls epigenetic processes in living cells.
a)Sun, Y., Hays, N.M., Periasamy, A., Davidson, M.W., Day, R.N. 2012. Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy. Methods in Enzymology: Live Cell Imaging 504:371-91. PMID: 22264545.
b)Day, R.N. 2014. Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy. Methods 66(2):200-207. PMID: 23806643.
c)Siegel, A.P., Baird, M.E., Davidson, M.W., Day, R.N. 2013. Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy. Int. J. Mol. Sci. 14(10):20340-20358. PMID: 24129172.
d)Day, R.N. 2015. Measuring Förster Resonance Energy Transfer Using Fluorescence Lifetime ImagingMicroscopy. Microscopy Today 23(3):44-50.
My laboratory web page is: http://mypage.iu.edu/~rnday/
Cell-Type Specific Regulation of Pituitary Gene Expression
The initiation of programs of gene expression in differentiated cells requires the interactions between cell-type specific transcription factors, nuclear receptors, and coregulatory proteins that occur in the cell nucleus. The focus of our research is to determine how tissue-specific transcription factors in cells of the anterior pituitary regulate the assembly of the protein complexes that control pituitary gene expression. Our studies are showing how Pit-1, a pituitary specific homeodomain transcription factor, orchestrates the activities of a network of transcription factors and co-regulatory proteins that function to control prolactin and growth hormone gene expression.
The Organization of Nuclear Function
Using biochemical and molecular genetic approaches, we are determining how Pit-1 coordinates this network of protein interactions. For example, we showed that Pit-1 and the transcription factor C/EBPα cooperate in the activation of pituitary cell-specific gene transcription. Interestingly, our studies also revealed that C/EBPα was preferentially localized to regions of centromeric heterochromatin in mouse pituitary cells - regions of the genome typically associated with gene silencing. Our current studies are focused on determining how the protein interactions that are coordinated by Pit-1 function to remodel densely packaged chromatin, allowing the access of the pituitary-specific transcription factors to target genes. These biochemical and molecular genetic studies, however, are not enough to provide a complete picture of the mechanisms that control transcription.
Imaging Transcription Factor Interactions in Living Cells?
Proteins are in dynamic equilibrium inside the cell, and it is necessary to understand the mechanisms that control the assembly of regulatory protein complexes in the context of their natural environment. To address this, we are using advanced live-cell imaging methods to track the behavior of proteins within the organized microenvironment of the living cell. For example, we used the direct visualization of fluorescent protein-labeled Pit-1 and C/EBPα in living pituitary cells to demonstrate that Pit-1 recruited C/EBPα from the regions of centromeric heterochromatin to the intranuclear sites occupied by Pit-1. These studies are showing how the network interactions of nuclear proteins lead to the formation of dynamic complexes, which in turn are stabilized by interactions with chromatin. What is more, this approach has allowed us to demonstrate how disease-causing mutations in Pit-1 disrupt the network of protein interactions. These results have broad implications for many human diseases that have been linked to mutations in the homeodomain proteins. This analysis of protein interaction networks in their natural environment will be the key to understanding the control of gene expression at the structural level.
Grant Funding: NIH-NIDDK
- Tao, W., M. Rubart, J. Ryan, X. Xiao, C. Qiao, T. Hato, M. W. Davidson, K. W. Dunn, and R. N. Day. 2015. A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy. American journal of physiology. Cell physiology 309:C724-735.
- Day, R.N. 2015. Measuring Förster Resonance Energy Transfer Using Fluorescence Lifetime Imaging Microscopy. Microscopy Today 23(3):44-50.
- Hum, J. M., Day, R.N., Bidwell, J.P., Wang, Y., Pavalko, F.M. 2014. Mechanical loading in osteocytes induces formation of a Src/Pyk2/MBD2 complex that suppresses anabolic gene expression. PLoS One 9(5): e97942.
- Day, R.N. 2014. Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy. Methods 66(2):200-207
- Shaner, N.C., Lambert, G.G., Chammas, A., Ni, Y., Cranfill, P.J., Baird, M.A., Sell, B.R., Allen, J.R., Day, R.N., Israelsson, M., Davidson, M.W., Wang, J. 2013. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods.2413.
- Siegel, A.P., Hays, N.M., Day, R.N. 2013. Unraveling transcription factor interactions with heterochromatin protein 1 using fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. J Biomed Opt 18:25002.
- Hum, J.M., Siegel, A.P., Pavalko, F.M., Day, R.N. 2012. Monitoring biosensor activity in living cells with fluorescence lifetime imaging microscopy. Int J Mol Sci. 13(11):14385-400.
- Day, R.N., Davidson, M.W. 2012. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells. BioEssays 34(5):341-50.
- Sun, Y., Hays, N.M., Periasamy, A., Davidson, M.W., Day, R.N. 2012. Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy. Methods in Enzymology: Live Cell Imaging 504:371-91.
- Sun, Y., Day, R.N., Periasamy, A. 2011. Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. Nature Protocols 6(9):1324-40.
Day Laboratory Home Page
Last update: 3/3/2016