Pituitary cell specific gene regulation

My research seeks to determine 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 are using biochemical, genetic, and molecular approaches to determine how the protein complexes that are necessary for pituitary specific gene transcription are assembled. For example, our studies show 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. Using biochemical and molecular genetic approaches, we demonstrated that Pit-1 and the basic-leucine zipper transcription factor C/EBPa cooperate in the activation of prolactin and growth hormone gene expression. Interestingly, we also found that C/EBPa preferentially localized to regions of centromeric heterochromatin in mouse pituitary cells - regions of the genome typically associated with gene silencing (see Enwright et al., 2003).
We are complementing these structural and functional studies with live cell imaging studies. This integrative approach allows us to define how the subnuclear positioning of protein complexes contributes to the selective expression of tissue-specific genes. We are using fluorescence microscopy to visualize the intranuclear distribution and interactions of proteins labeled with a variety of different color fluorescent proteins (FPs; see Fig. 1). The fluorescence images shown here illustrate the ability of Pit-1 to reorganize corepressor protein complexes in the nucleus of living pituitary cells. The top image demonstrates that the corepressor protein complexes are localized in subnuclear focal bodies (green, top image). The corepressor focal bodies are distinct from those formed by PML (red, top image), and localize to nuclear regions outside centromeric heterochromatin (blue, top image). Significantly, the co-expression of Pit-1 reorganized the corepressor protein complexes into a distribution that precisely overlapped the web-like distribution of Pit-1 (cyan, bottom image). In contrast, the PML-bodies were not reorganized by Pit-1 (red, bottom image). These studies in living cells indicate that the assembly of cooperating factors at particular intranuclear sites is critical for the regulation of cell-specific gene expression (see Voss et al., 2005).
Fig. 1. Pit-1 organizes corepressor protein complexes. Data first published in Voss et al., 2005.
We also used this approach to show that Pit-1 recruited C/EBPa from the regions of heterochromatin to the nuclear sites it occupied, providing evidence that Pit-1 can direct cooperating factors to particular sites in the nucleus. We then used the technique of Förster resonance energy transfer (FRET; see Lab Resources) microscopy to define the mechanisms that control the assembly of these protein complexes. This approach allowed us to demonstrate how certain disease-causing point mutations in Pit-1 disrupted its network of protein interactions (see Demarco et al., 2006a). These results have broad implications for many human diseases that have been linked to mutations in the homeodomain proteins.

These live-cell imaging techniques provide unique insights into the assembly of the transcription factor complexes that control specific gene transcription. What was missing, however, were methods that directly measure both the dynamics and interactions of proteins simultaneously. We developed a novel technique that fills this void, allowing us to quantify the dynamics of protein interactions in living cells. The method we developed uses the photoactivated green fluorescent protein (PA-GFP) as an acceptor fluorophore for FRET. The mobility of PA-GFP labeled proteins can be directly measured by activating PA-GFP in a discrete region of the cell and then monitoring the distribution of the newly fluororescent protein. If the PA-GFP labeled proteins interact with other proteins that are labeled with the cyan FP (CFP), and provided the average distance between the fluorophores is less than about 80Å, then the donor fluorescence will be quenched. Therefore, by monitoring changes in the CFP signal following the activation of PA-GFP, we can quantify the dynamics of the interactions between the labeled proteins. This technique, called photo-quenching FRET (PQ-FRET), provides direct measurements of protein mobility, protein exchange with macromolecular complexes and protein-protein interactions in living cells. We have used this technique to define the dynamic interactions between the heterochromatin protein-1 alpha (HP1a) and C/EBPa in regions of centromeric heterochromatin in mouse pituitary cells (see Demarco et al., 2006b).

We plan to use this approach to continue to define the interactions of transcription factors with other nuclear proteins, including proteins that function to modify chromatin structure. We are particularly interested in mechanisms of transcriptional repression. We have begun studies to determine how the dynamic interactions between HP1a, a protein involved in the maintenance of repressive heterochromatin domains, and transcription factors can function to regulate the expression of specific genes (see Demarco et al., 2006b). The organization of heterochromatin domains depends on the methylation of underacetylated histone tails by the Su(var)3-9 histone methyltransferase, which modifies histone H3 on lysine 9 (MeK9H3). HP1a is recruited to this epigenetic mark through the direct binding of the chromodomain to the MeK9H3. Several groups have proposed that the binding of HP1a to MeK9H3 and the recruitment of Su(var)3-9 may allow HP1a to move along the chromatin fiber, spreading the regions of repressive chromatin. Our hypothesis is that the dynamic interactions of HP1a with tissue-specific transcription factors may function to control the spread of silencing, allowing the selective expression of genes. We are currently applying our integrative approach to address these important questions.

The research in the Day laboratory is funded by the National Institutes of Health, and we gratefully acknowledge the past support of the NSF, the Center for Biological Timing, and the American Cancer Society.



Postdoctoral position available; follow this link for more information
Department of Medicine, PO Box 800578, University of Virginia Health System, Charlottesville, VA 22908-0578
Email: Phone: 434 982 3623