|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
I. Analyses of intracellular dynamics of mutant androgen receptors (AR) derived from two androgen insensitivity syndrome (AIS) patients.
Intracellular dynamics of two different AR mutants (AR-C579F, AR-F582Y) derived from AIS patients were analysed using GFP-fused mutant ARs. The wild-type AR was ligand-dependently translocated into the nucleus where it formed fine subnuclear foci. Surprisingly, after the addition of dihydrotestosterone (DHT), the two mutant ARs initially formed large cytoplasmic dots, many of which were found to be close to mitochondria by electron microscopy. Subsequently, a proportion of the ligand-bound mutant ARs gradually entered the nucleus to form a smaller number of larger dots compared with the wild-type AR. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the intranuclear mobilities of the mutant ARs were decreased compared with that of the wild-type AR. These results suggest that the abnormal translocation, localization and mobility o
f the mutant ARs may be the cause of AIS in these subjects.
II. Analyses of transcriptional repression mechanisms by novel corepressors, TZF and Tob
We showed that testicular zinc finger protein, TZF, was a novel corepressor for AR. TZF was ligand-dependently colocalized with AR and interacted with the N-terminus of AR. A central portion (amino acids 512-663) of TZF, TZF(512-663), is responsible for both binding to AR and repressing the transactivation. TZF recruited endogenous histone deacetylase 2 (HDAC2) and formed a complex with agonist-bound AR. Imaging analyses showed that TZF and TZF(512-663) were recruited by AR and simultaneously impaired distinct AR foci formation. Quantification of the foci number using a three-dimensional imaging method revealed that the number of intranuclear AR foci was related to its transactivation activity. Moreover, increased levels of TZF dissociated a coactivator, TIF2, from the AR foci and vice versa. These results indicate that the ligand-dependent transactivation function of AR is quantitatively related to its intranuclear foci formation, and suggest that corepressors, such as TZF, act on these intranuclear events competitively with coactivators.
We also found that Tob, which was regarded as a negative regulator of osteoblast function, repressed transactivation function of AR, ER and GR. Less