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The polyglutamine-expanded androgen receptor has increased DNA binding and reduced transcriptional activity.
Belikov S
,
Bott LC
,
Fischbeck KH
,
Wrange Ö
.
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Expansion of a polyglutamine-encoding trinucleotide CAG repeat in the androgen receptor (AR) to more than 37 repeats is responsible for the X-linked neuromuscular disease spinal and bulbar muscular atrophy (SBMA). Here we evaluated the effect of polyglutamine length on AR function in Xenopus oocytes. This allowed us to correlate the nuclear AR concentration to its capacity for specific DNA binding and transcription activation in vivo. AR variants with polyglutamine tracts containing either 25 or 64 residues were expressed in Xenopus oocytes by cytoplasmic injection of the corresponding mRNAs. The intranuclear AR concentration was monitored in isolated nuclei and related to specific DNA binding as well as transcriptional induction from the hormone response element in the mouse mammary tumor virus (MMTV) promoter. The expanded AR with 64 glutamines had increased capacity for specific DNA binding and a reduced capacity for transcriptional induction as related to its DNA binding activity. The possible mechanism behind these polyglutamine-induced alterations in AR function is discussed.
Fig. 1. Experimental design and quantification of AR in oocyte nuclei. (A) Xenopus oocytes were injected with mRNA into the cytoplasm and with ssDNA into the nucleus and harvested for analysis at indicated time (h). (B) Increasing concentrations of [3H]-R1881 were incubated with oocytes either injected or not injected with mRNA coding for ARQ25 and next day taken for analysis of [3H]-R1881 in manually isolated nuclei. (C) Oocytes were injected with the indicated amounts of mRNA coding for ARQ25 or ARQ64 and were then analyzed either by quantification of nuclear [3H]-R1881 or (D) by Western blot (WB), A.U. indicates arbitrary units.
Fig. 2. Comparison of ARQ25 and ARQ64. (A) Quantification of MMTV transcription by S1 nuclease protection analysis of oocytes injected with either 2.1; 3.1; 4.6 or 6.9 ng ARQ25 mRNA 2.7; 4.1; 6.1 or 9.2 ng of ARQ64 mRNA and then 3 ng ssDNA pMMTV:M13 and exposed to 100 nM R1881. A.U. indicates arbitrary units. The diagram below shows MMTV transcription as a function of nuclear [AR](µM) based on [3H]-R1881 analysis. Gray shadow indicates the curve fitting of all data by software Curve Expert Pro v.2.2.0. (B) Autoradiogram of primer extension from DMS in vivo footprinting of aliquots of the same oocytes as in Fig. 2A. Specific DNA sites for AR (ARE) are indicated on the left side together with binding sites for other proteins (not expressed here), radioactive bands protected in presence of AR, i.e. DMS methylation protected, are marked to the right with empty circles and reference bands for loading control as filled circles. Quantification of the average value of the protected bands, two lanes per oocyte pool, is shown as columns below with the average deviation of double samples as error bars. The last lane was lost in ARQ25. (C) AR-DNA binding, based on DMS methylation protection, plotted as a function of intranuclear [AR], based on quantification of nuclear [3H]-R1881. The curves are calculated based on the Curve Expert Pro v 2.2.0 software. (D) Transcription of MMTV RNA analyzed by S1 nuclease was plotted as a function of AR-DNA binding activity from DMS in vivo footprinting.
Fig. 3. Comparison of nuclear and cytosolic distribution of ARQ25 and ARQ64 (A) in presence of androgen agonist R1881 or (B) androgen antagonist enzalutamide. Oocytes were injected with 3.5 ng ARQ25 mRNA or 6.9 ng of ARQ64 mRNA followed by 3 ng ssDNA pMMTV:M13 as in Fig. 1A. 28 h later oocytes were harvested and processed for SDS PAGE and WB (see Section 2). 0.75 of oocyte equivalent of cytosol or nuclear extract was applied on each lane. The smaller ARQ64 sub-band of MW ∼120 kDa was also present in this experiment since the re-amplification of the pβARQ64 described above was done later. The ratio of the main band and the smaller band of ARQ64 remained constant when comparing cytosolic and nuclear ARQ64 (data not shown).
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