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 @
Phone: 317-274-2166
Fax: 317-274-3318

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

Personal Statement

 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:


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

Selected Publications

  1. 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.
  2. Day, R.N. 2015.  Measuring Förster Resonance Energy Transfer Using Fluorescence Lifetime Imaging Microscopy.  Microscopy Today 23(3):44-50.
  3. 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.
  4. Day, R.N. 2014. Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy. Methods 66(2):200-207
  5. 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.
  6. 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.
  7. 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.
  8. Day, R.N., Davidson, M.W. 2012. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells.  BioEssays 34(5):341-50.
  9. 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.
  10. 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.




PubMed Listings for this Faculty Member 

Day Laboratory Home Page

Last update: 3/3/2016

635 Barnhill Drive, Medical Science Bldg. Room 385 | Indianapolis, IN 46202-5120 | 317-274-7772