p54(nrb) acts as a transcriptional coactivator for activation function 1 of the human androgen, receptor
ABSTRACT The androgen receptor (AR) has two transactivation functions that have been mapped to the N- and C-terminal domains and designated as activation function-1 (AF-1) and AF-2, respectively. While the molecular basis for AF-2 function has been well studied, little is known about AF-1 coregulators. Therefore, we attempted to identify AF-1-interacting proteins from HEK293 cells by biochemical purification followed by mass fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Purified AF-1 region-interacting proteins were found to contain nuclear RNA-binding protein p54(nrb), polypyrimidine tract-binding protein-associated splicing factor (PSF), paraspeckle protein 1 (PSP1), and PSP2, which are assumed to be involved in pre-mRNA processing. p54(nrb) interacted with AR via the A/B domain in a ligand-dependent manner. Reflecting the physical interaction between p54(nrb) and the AR A/B domain, AR AF-1 function was potentiated by p54(nrb). Our results suggest that p54(nrb) functions as a coactivator of AR that potentiates transcription, and presumably splicing as well.
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ABSTRACT: We investigated the relationship between oct4 gene expression patterns and CpG sites methylation profiles during ES cell differentiation into neurons, and identified relevant binding factor. The oct4 gene expression level gradually declined as ES cell differentiation progressed, and the CpG sites in the oct4 proximal enhancer (PE) and promoter regions were methylated in concert with ES cell differentiation. An electro-mobility shift assay (EMSA) showed that putative proteins bind to CpG sites in the oct4 PE/promoter. We purified CpG binding proteins with DNAbinding purification method, and NonO was identified by liquid chromatography-mass spectrometry. EMSA with specific competitors revealed that NonO specifically binds to the conserved CCGGTGAC sequence in the oct4 promoter. Methylation at a specific cytosine residue (CC* GGTGAC) reduced the binding affinity of NonO for the recognition sequence. Chromatin immunoprecipitation analysis confirmed that NonO binds to the unmethylated oct4 promoter. There were no changes in the NonO mRNA and protein levels between ES cells and differentiated cells. The transcriptional role of NonO in oct4 gene expression was evaluated by luciferase assays and knockdown experiments. The luciferase activity significantly increased threefold when the NonO expression vector was cotransfected with the NonO recognition sequence, indicating that NonO has a transcription activator effect on oct4 gene expression. In accordance with this effect, when NonO expression was inhibited by siRNA treatment, oct4 expression was also significantly reduced. In summary, we purified NonO, a novel protein that binds to the CpG island of oct4 promoter, and positively regulates oct4 gene expression in ES cells.Moleculer Cells 11/2012; 35(1). DOI:10.1007/s10059-013-2273-1 · 2.24 Impact Factor
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ABSTRACT: To identify genes linked to early stages of disruption of brain sexual differentiation, hypothalamic region-specific microarray analyses were performed using a microdissection technique with neonatal rats exposed to endocrine-acting drugs. To validate the methodology, the expression fidelity of microarrays was first examined with two-round amplified antisense RNAs (aRNAs) from methacarn-fixed paraffin-embedded tissue (PET) in comparison with expression in unfixed frozen tissue (UFT). Decline of expression fidelity when compared with the 1x-amplified aRNAs from UFTs was found as a result of the preferential amplification of the 3' side of mRNAs in the second round in vitro transcription. However, expression patterns for the 2x-amplified aRNAs were mostly identical between methacarn-fixed PET and UFT, suggesting no obvious influence of methacarn fixation and subsequent paraffin embedding on expression levels. Next, in the main experiment, neonatal rats at birth were treated subcutaneously either with estradiol benzoate (EB; 10 microg/pup) or flutamide (FA; 250 microg/pup), and medial preoptic area (MPOA)-specific microarray analysis was performed 24 h later using 2x-amplified aRNAs from methacarn-fixed PET. Numbers of genes showing constitutively high expression in the MPOA predominated in males, implying a link with male-type growth supported by perinatal testosterone. Around 60% of genes showing sex differences in expression demonstrated altered levels after EB treatment in females, suggesting an involvement of genes necessary for brain sexual differentiation. When compared with EB, FA affected a rather small number of genes, but fluctuation was mostly observed in females, as with EB. Moreover, many selected genes common to EB and FA showed down-regulation in females with both drugs, suggesting a common mechanism for endocrine center disruption in females, at least at early stages of post-natal development.Developmental Neurobiology 02/2007; 67(3):253-69. DOI:10.1002/dneu.20349 · 4.19 Impact Factor
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ABSTRACT: P54nrb is a protein implicated in multiple nuclear processes whose specific functions may correlate with its presence at different nuclear locations. Here we characterize paraspeckles, a subnuclear domain containing p54nrb and other RNA-binding proteins including PSP1, a protein with sequence similarity to p54nrb that acts as a marker for paraspeckles. We show that PSP1 interacts in vivo with a subset of the total cellular pool of p54nrb. We map the domain within PSP1 that is mediating this interaction and show it is required for the correct localization of PSP1 to paraspeckles. This interaction is necessary but not sufficient for paraspeckle targeting by PSP1, which also requires an RRM capable of RNA binding. Blocking the reinitiation of RNA Pol II transcription at the end of mitosis with DRB prevents paraspeckle formation, which recommences after removal of DRB, indicating that paraspeckle formation is dependent on RNA Polymerase II transcription. Thus paraspeckles are the sites where a subset of the total cellular pool of p54nrb is targeted in a RNA Polymerase II-dependent manner.Molecular Biology of the Cell 12/2005; 16(11):5304-15. DOI:10.1091/mbc.E05-06-0587 · 4.55 Impact Factor