Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control.DOI: http://dx.doi.org/10.7554/eLife.00400.001

Gene activation by steroid hormone receptors involves the recruitment of the steroid receptor coactivator (SRC)/p160 coactivator LXXLL motifs to activation function 2 (AF2) in the ligand binding domain. For the androgen receptor (AR), AF2 also serves as the interaction site for the AR NH 2 -terminal FXXLF motif in the androgen-dependent NH 2 -terminal and carboxyl-terminal (N/C) interaction. The relative importance of the AR AF2 site has been unclear, since the AR FXXLF motif interferes with coactivator recruitment by competitive inhibition of LXXLL motif binding. In this report, we identified the X chromosome-linked melanoma antigen gene product MAGE-11 as an AR coregulator that specifically binds the AR NH 2 -terminal FXXLF motif. Binding of MAGE-11 to the AR FXXLF ␣-helical region stabilizes the ligand-free AR and, in the presence of an agonist, increases exposure of AF2 to the recruitment and activation by the SRC/p160 coactivators. Intracellular association between AR and MAGE-11 is supported by their coimmunoprecipitation and colocalization in the absence and presence of hormone and by competitive inhibition of the N/C interaction. AR transactivation increases in response to MAGE-11 and the SRC/p160 coactivators through mechanisms that include but are not limited to the AF2 site. MAGE-11 is expressed in androgen-dependent tissues and in prostate cancer cell lines. The results suggest MAGE-11 is a unique AR coregulator that increases AR activity by modulating the AR interdomain interaction.The androgen receptor (AR) is a member of the steroid receptor subfamily of nuclear receptors. Like other steroid receptors, AR has multiple domains involved in ligand and DNA binding and transcriptional activation. Recently, several unique properties of AR that distinguish it from other steroid receptors have gained attention. High-affinity androgen binding stabilizes AR (26), which is in contrast to most steroid receptors that are down regulated by agonist binding. Agonistinduced AR stabilization results in part from the NH 2 -terminal and carboxyl-terminal (N/C) interdomain interaction mediated by the androgen-dependent interaction between the AR NH 2 -terminal FXXLF motif and activation function 2 (AF2) in the ligand binding domain (16,17). The FXXLF motif 23 FQNLF 27 is part of an amphipathic ␣-helical region that is similar in structure to the LXXLL motifs of the steroid receptor coactivator (SRC)/p160 family of coactivators. The AR FXXLF motif is highly conserved among vertebrates, supporting its functional importance across species (13, 18). Recent cocrystal structures and binding studies have confirmed preferential binding of the FXXLF motif to the AR AF2 site and adaptability of AF2 to coactivator LXXLL motif binding through an induced-fit mechanism (15, 22). FXXLF motif binding to AF2 requires binding of ligands that display agonist activity in vivo (27). In transient transfection reporter gene assays, the N/C interaction is required for the activation of some, but not all, androgen-regulated genes (2, 18)...

Melanoma antigen gene protein-A11 (MAGE-11) of the MAGE family of cancer germ-line antigens increases androgen receptor (AR) transcriptional activity through its interaction with the AR NH 2 -terminal FXXLF motif. The present study investigated the regulatory mechanisms that control MAGE-11 expression during androgen deprivation therapy and prostate cancer progression. Studies include the CWR22 xenograft model of human prostate cancer, clinical specimens of benign and malignant prostate, and prostate cancer cell lines. MAGE-11 mRNA levels increased 100-to 1,500-fold during androgen deprivation therapy and prostate cancer progression, with highest levels in the castration-recurrent CWR22 xenograft and clinical specimens of castration-recurrent prostate cancer. Pyrosequencing of genomic DNA from prostate cancer specimens and cell lines indicated the increase in MAGE-11 resulted from DNA hypomethylation of a CpG island in the 5′ promoter of the MAGE-11 gene. Sodium bisulfite sequencing of genomic DNA from benign and malignant prostate tumors and prostate cancer cell lines revealed DNA hypomethylation at individual CpG sites at the transcription start site were most critical for MAGE-11 expression. Cyclic AMP (cAMP) also increased MAGE-11 expression and AR transcriptional activity in prostate cancer cell lines. However, cAMP did not alter DNA methylation of the promoter and its effects were inhibited by extensive DNA methylation in the MAGE-11 promoter region. Increased expression of the AR coregulator MAGE-11 through promoter DNA hypomethylation and cAMP provides a novel mechanism for increased AR signaling in castration-recurrent prostate cancer. (Mol Cancer Res 2009;7(4):523-35)

The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report, we demonstrate the extent of AR transactivation in the presence of 5a-androstane-3a,17b-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17b-hydroxysteroid dehydrogenase 6 (17b-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17b-HSD6 in prostate-derived cell lines and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor-bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis.

The NH 2 -terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH 2 -terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH 2 -terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH 2 -terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH 2 -terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH 2 -terminal domains and other intrinsically unstructured transcriptional regulatory proteins.Steroid receptors depend on multiple domains for their function as ligand-dependent transcriptional activators. The DNAand ligand-binding domains have been studied extensively and have a high degree of structural and functional conservation. In contrast, the largely unstructured NH 2 -terminal domains (1-3) are highly variable in size and sequence, and the molecular mechanisms that contribute to transactivation are not well understood. The size of the NH 2 -terminal region of steroid receptors increases with evolutionary expansion (4, 5), from estrogen receptor-␣ (185 amino acid residues) to the glucocorticoid receptor (GR 1 ; 420 residues), androgen receptor (AR; 558 residues), progesterone receptor (B form; 566 residues), and mineralocorticoid receptor (602 residues). In contrast, transcription factors such as p53, NF-B, and VP16 typically have transcriptional activation domains of Ͻ100 residues.

Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwiseunidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.

Androgen receptor (AR)-mediated gene regulation involves interactions with coregulatory proteins that include the melanoma antigen gene protein-A11 (MAGE-11). To understand the functional significance of sequence similarity between MAGE-11 and the adenovirus early protein E1A, we determined whether MAGE-11 contributes to AR transcriptional activity through an interaction with p300, a potent and ubiquitous transcriptional regulator. Here, we report that MAGE-11 interacts with the NH 2 -terminal region of p300 through the MAGE-11 MXXIF motif 185 MXXIF 189 , with transcriptional activity depending on the MAGE-11 F-box and MAPK phosphorylation. The MAGE-11-and p300-dependent increase in AR transactivation required the NH 2 -terminal regions of AR and p300, p300 acetyltransferase activity, and the AR FXXLF motif 23 FQNLF 27 interaction with MAGE-11. MAGE-11 linked AR to p300 and the p160 coactivator, transcriptional intermediary protein 2 (TIF2). The p300 NH 2 -terminal FXXLF motif 33 FGSLF 37 was required for transcriptional activation by TIF2. Increased expression of p300 decreased the ubiquitinylation of MAGE-11 and transiently increased endogenous MAGE-11 levels. Autoacetylation of p300 and decreased acetylation of TIF2 were evident in the MAGE-11, p300, and TIF2 complex. The studies suggest that MAGE-11 links NH 2 -terminal domains of AR and p300 to promote transcriptional synergy through a cadre of FXXLF-related interacting motifs.

The androgen receptor (AR) is a ligand-activated transcription factor that interacts with coregulatory proteins during androgen-dependent gene regulation. Melanoma antigen gene protein 11 (MAGE-11) is an AR coregulator that specifically binds the AR NH 2 -terminal FXXLF motif and modulates the AR NH 2 -and carboxyl-terminal N/C interaction to increase AR transcriptional activity. Here we demonstrate that epidermal growth factor (EGF) signaling increases androgen-dependent AR transcriptional activity through the posttranslational modification of MAGE-11. EGF in the presence of dihydrotestosterone stabilizes the AR-MAGE complex through the site-specific phosphorylation of MAGE-11 at Thr-360 and ubiquitinylation at Lys-240 and Lys-245. The time-dependent EGF-induced increase in AR transcriptional activity by MAGE-11 is mediated through AR activation functions 1 and 2 in association with the increased turnover of AR and MAGE-11. The results reveal a dynamic mechanism whereby growth factor signaling increases AR transcriptional activity through the covalent modification of an AR-specific coregulatory protein. Sequence conservation of the MAGE-11 phosphorylation and ubiquitinylation sites throughout the MAGE gene family suggests common regulatory mechanisms for this group of cancer-testis antigens.The androgen receptor (AR) is a ligand-dependent transcription factor that mediates the effects of the biologically active androgens testosterone and dihydrotestosterone (DHT) on gene regulation required for male sex development and function. Androgen-dependent gene regulation involves interactions between AR and androgen response element DNA and coregulatory proteins that link to the transcriptional machinery of chromatin. Like other steroid receptors, AR has a multidomain structure that includes the NH 2 -terminal activation domain 1 (AF1), which has evolved during mammalian evolution (9, 11) and is considered predominant in AR function. Activation function 2 (AF2) in the ligand binding domain functions as a binding site for SRC/p160 coactivator LXXLL motifs and for the FXXLF motif in the AR NH 2 terminus and in several putative AR coregulatory proteins (23,25,27). Cell-free fluorescence binding studies have shown that the AR20-30 FXXLF motif peptide binds to AF2 with 5-to 10-fold-higher affinity than an LXXLL motif containing a peptide from the most active ARSRC/p160 coactivator (23) and is the basis of the androgen-dependent AR NH 2 -terminal and carboxyl-terminal (N/C) interaction important for androgen-induced gene activation (7,25,26,30,40). The AR N/C interaction slows the dissociation rate of bound androgen (62), stabilizes AR binding to androgen response element DNA (60), and appears to be involved in domain swapping between an intramolecular AR monomer in the cytoplasm and an intermolecular antiparallel AR dimer in the nucleus (39, 51). The importance of the N/C interaction for AR function is supported by studies on the human androgen insensitivity syndrome, in which naturally occurring single amino acid mutations...