The androgen receptor (AR) is involved in the development and maintenance of the male phenotype, and plays a role in the growth of metastasized prostate cancer. To perform its function, the AR binds to promoter/enhancer regions of androgen-regulated genes. Although its biochemical and structural properties are relatively well established, little is known about AR function in its most relative context, the living cell. Research questions we typically attempt to answer are: how do receptors find their target sites in DNA?, where and when are interactions with coregulators required?, by which molecular mechanisms do receptors and coregulators contribute to activation of the genes they regulate?, in what way is specificity of gene activation achieved?, and finally: how are these mechanisms and pathways affected in prostate cancer cells, and by which molecular mechanisms do therapeutic antagonists interfere with this aberrant behavior?

To answer the questions formulated in 1. we develop and apply green fluorescent protein (GFP) technology and advanced quantitative live cell imaging methods (Farla et al., 2005; van Royen et al., 2007). In pioneering research, we found that many nuclear factors involved in transcription and/or DNA-repair interact with target sites in a much more dynamic way than previously anticipated (Houtsmuller et al., Science, 1999). In addition, we showed that transcription/repair factor TFIIH is able to rapidly switch its activity between transcription and DNA-repair (Hoogstraten et al., Mol Cell, 2002). In subsequent research we found that ARs are freely mobile in the nucleus and very dynamically interact with target sites, residence times on promoters being in the order of only tens of seconds (Farla et al., 2004). In spite of this dynamic behavior, ARs quickly after activation by testosterone, form tiny stable structures in the nucleus (speckles) which most likely are the sites of active transcription. From this research a picture arises where stable ‘transcription factories’ exist, but their components continually exchange between structure and nucleoplasm (Dinant et al., 2009). Such a scenario may be important to enable the cell to quickly respond to internal signals or changing environmental conditions. In more in-depth research using a novel quantitative technique especially developed for this purpose (van Royen et al., 2009), we revealed a conformational change of the AR after DNA-binding, allowing coregulators to bind only when the AR is associated with promoter/enhancers. In this way the AR itself regulates the timing of coregulator interactions (van Royen et al., 2007). In parallel to the above we investigated the behavior of ARs bound to therapeutic antagonists to find that, in contrast to what was previously thought, antagonists prevent stable AR DNA-binding (Farla et al., 2005).

We are currently developing several novel types of methodology to further investigate AR function and to unravel the structure and function (and dysfunction in prostate cancer) of the hallmarks of AR activity: the speckles in which they accumulate and which most likely represent the site of active transcription. Our working hypothesis is that these represent specific types of previously postulated transcription factories (recent review in: Carter, Eskiw, Cook, Biochem Soc Trans. 36:585-9, 2008). The two most important approaches we are currently developing are single molecule tracking and super resolution microscopy. Using single molecule tracking we aim to study the behavior of individual receptors and receptor mutants involved in prostate cancer, their interaction with transcription sites, and their response to AR-antagonists. More importantly, single molecule tracking may allow us to study interaction of coregulators and general transcription factors with transcription sites, which is not possible in more conventional microscopy since they accumulate in very small amounts or interact only very briefly with transcription sites. We have recently received grants from NWO and Nefkens foundation and already implemented the required instruments for this research (spinning disk and TIRF microscopes equipped with highly sensitive cameras). Using super-resolution microscopy we aim to study the ultrastructure of alleged transcription factories. In this line of research we also aim to include other steroid receptors, starting with estrogen and progesterone receptors. We have implemented a 4Pi confocal microscope for this purpose.

Next to this fundamental research, which lays the basis for interpretation of aberrant AR behavior in prostate cancer, we intend to study patient cells, freshly derived from human prostate carcinomas (collaboration with van Leenders and Trapman). To enable quantitative microscopic studies, we will fuse cells stably expressing AR-GFP with living patient cells. The fusion technique was successfully applied in previous research, where we studied histone dynamics in AR-speckles (ms. In preparation). After fusion, AR-activation by testosterone will lead to entry of the tagged ARs in patient cancer nuclei, allowing application of the full set of fluorescence assays, which are either already routinely used or under development and to be implemented in the near future.

Transcription (AR)

1.      Van Royen ME, Cunha SM, Brink MC, Mattern KA, Nigg AL, Dubbink HJ, Verschure PJ, Trapman J, Houtsmuller AB. Compartmentalization of androgen receptor protein-protein interactions in living cells. J Cell Biol 177: 63-72 (2007)

2.      Farla P, Hersmus R, Trapman J, Houtsmuller AB. Antiandrogens prevent stable DNA-binding of the androgen receptor. J Cell Sci 118:4187-98, (2005)

3.      Farla P, Hersmus R, Geverts B, Mari PO, Nigg AL, Dubbink HJ, Trapman J, Houtsmuller AB The androgen receptor ligand-binding domain stabilizes DNA binding in living cells J Struct Biol 147:50-61 (2004)

4.      Dinant C, Luijsterburg MS, Höfer T, von Bornstaedt G, Vermeulen W, Houtsmuller AB, van Driel R. Assembly of multiprotein complexes that control genome function. J Cell Biol. 185:21-6 (2009)

5.      van Royen ME, Dinant C, Farla P, Trapman J Houtsmuller AB FRAP and FRET methods to study nuclear receptors in living cells. Meth Mol Biol 464:363-85 (2009)

DNA-repair

6.      Essers J, Vermeulen W, Houtsmuller AB. DNA damage repair: anytime, anywhere? Curr Opin Cell Biol. 18:240-6. (2006)

7.      Hoogstraten D, Bergink S, Ng JM, Verbiest VH, Luijsterburg MS, Geverts B, Raams A, Dinant C, Hoeijmakers JH, Vermeulen W, Houtsmuller AB. Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC J Cell Sci 121:2850-9 (2008)

8.      Dinant C, de Jager M, Essers J, van Cappellen WA, Kanaar R, Houtsmuller AB, Vermeulen W. Activation of multiple DNA repair pathways by sub-nuclear damage induction methods. J Cell Sci. 120:2731-40 (2007)

9.      Mari PO, Florea BI, Persengiev SP, Verkaik NS, Bruggenwirth HT, Modesti M, Giglia-Mari G, Bezstarosti K, Demmers JA, Luider TM, Houtsmuller AB, van Gent DC. Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4. Proc Natl Acad Sci U S A. 103:18597-602. (2006)

10.  Van Den Boom V, Citterio E, Hoogstraten D, Zotter A, Egly JM, Van Cappellen WA, Hoeijmakers JH, Houtsmuller AB, Vermeulen W DNA damage stabilizes interaction of CSB with the transcription elongation machinery. J Cell Biol 166:27-36 (2004)