Formin-mediated nuclear actin at androgen receptors promotes transcription.

Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology. The androgen receptor (AR) binds androgens mediating gene expression for sexual, somatic and behavioural functions, and is involved in various conditions including androgen insensitivity syndrome and prostate cancer1. Here we identified functional mutations in the formin and actin nucleator DAAM2 in patients with androgen insensitivity syndrome. DAAM2 was enriched in the nucleus, where its localization correlated with that of the AR to form actin-dependent transcriptional droplets in response to dihydrotestosterone. DAAM2 AR droplets ranged from 0.02 to 0.06 µm3 in size and associated with active RNA polymerase II. DAAM2 polymerized actin directly at the AR to promote droplet coalescence in a highly dynamic manner, and nuclear actin polymerization is required for prostate-specific antigen expression in cancer cells. Our data uncover signal-regulated nuclear actin assembly at a steroid hormone receptor necessary for transcription.

Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals.

RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7-3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals.

Cryo-EM structures of human RNA polymerase I.

RNA polymerase I (Pol I) specifically synthesizes ribosomal RNA. Pol I upregulation is linked to cancer, while mutations in the Pol I machinery lead to developmental disorders. Here we report the cryo-EM structure of elongating human Pol I at 2.7 Å resolution. In the exit tunnel, we observe a double-stranded RNA helix that may support Pol I processivity. Our structure confirms that human Pol I consists of 13 subunits with only one subunit forming the Pol I stalk. Additionally, the structure of human Pol I in complex with the initiation factor RRN3 at 3.1 Å resolution reveals stalk flipping upon RRN3 binding. We also observe an inactivated state of human Pol I bound to an open DNA scaffold at 3.3 Å resolution. Lastly, the high-resolution structure of human Pol I allows mapping of disease-related mutations that can aid understanding of disease etiology.

Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states.

RNA polymerase III (Pol III) synthesizes transfer RNAs and other short, essential RNAs. Human Pol III misregulation is linked to tumor transformation, neurodegenerative and developmental disorders, and increased sensitivity to viral infections. Here, we present cryo-electron microscopy structures at 2.8 to 3.3 Å resolution of transcribing and unbound human Pol III. We observe insertion of the TFIIS-like subunit RPC10 into the polymerase funnel, providing insights into how RPC10 triggers transcription termination. Our structures resolve elements absent from Saccharomyces cerevisiae Pol III such as the winged-helix domains of RPC5 and an iron-sulfur cluster, which tethers the heterotrimer subcomplex to the core. The cancer-associated RPC7α isoform binds the polymerase clamp, potentially interfering with Pol III inhibition by tumor suppressor MAF1, which may explain why overexpressed RPC7α enhances tumor transformation. Finally, the human Pol III structure allows mapping of disease-related mutations and may contribute to the development of inhibitors that selectively target Pol III for therapeutic interventions.

Structure of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation.

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

Structural Basis for the Activation of the Deubiquitinase Calypso by the Polycomb Protein ASX.

Ubiquitin C-terminal hydrolase deubiquitinase BAP1 is an essential tumor suppressor involved in cell growth control, DNA damage response, and transcriptional regulation. As part of the Polycomb repression machinery, BAP1 is activated by the deubiquitinase adaptor domain of ASXL1 mediating gene repression by cleaving ubiquitin (Ub) from histone H2A in nucleosomes. The molecular mechanism of BAP1 activation by ASXL1 remains elusive, as no structures are available for either BAP1 or ASXL1. Here, we present the crystal structure of the BAP1 ortholog from Drosophila melanogaster, named Calypso, bound to its activator, ASX, homolog of ASXL1. Based on comparative structural and functional analysis, we propose a model for Ub binding by Calypso/ASX, uncover decisive structural elements responsible for ASX-mediated Calypso activation, and characterize the interaction with ubiquitinated nucleosomes. Our results give molecular insight into Calypso function and its regulation by ASX and provide the opportunity for the rational design of mechanism-based therapeutics to treat human BAP1/ASXL1-related tumors.

ORP-Mediated ER Contact with Endocytic Sites Facilitates Actin Polymerization.

Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enriched at ER contacts sites. ORPs promote non-vesicular lipid transport and work as lipid sensors in the context of many cellular tasks, but the determinants of their distinct localization and function are not understood. Here, we demonstrate that the yeast endocytic invaginations associate with the ER and that this association specifically requires the ORPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Disruption of the ER contact with endocytic sites using ORP, VAP, myosin-I, or reticulon mutants delays and weakens actin polymerization and interferes with vesicle scission. Finally, we provide evidence suggesting that ORP-dependent sterol transfer facilitates actin polymerization at endocytic sites.

A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT.

Bromodomains are critical components of many chromatin modifying/remodelling proteins and are emerging therapeutic targets, yet how they interact with nucleosomes, rather than acetylated peptides, remains unclear. Using BRDT as a model, we characterized how the BET family of bromodomains interacts with site-specifically acetylated nucleosomes. Here we report that BRDT interacts with nucleosomes through its first (BD1), but not second (BD2) bromodomain, and that acetylated histone recognition by BD1 is complemented by a bromodomain-DNA interaction. Simultaneous DNA and histone recognition enhances BRDT's nucleosome binding affinity and specificity, and its ability to localize to acetylated chromatin in cells. Conservation of DNA binding in bromodomains of BRD2, BRD3 and BRD4, indicates that bivalent nucleosome recognition is a key feature of these bromodomains and possibly others. Our results elucidate the molecular mechanism of BRDT association with nucleosomes and identify structural features of the BET bromodomains that may be targeted for therapeutic inhibition.

Crosstalk between PI(4,5)P2and CK2 modulates actin polymerization during endocytic uptake.

A transient burst of actin polymerization assists endocytic budding. How actin polymerization is controlled in this context is not understood. Here, we show that crosstalk between PI(4,5)P2and the CK2 catalytic subunit Cka2 controls actin polymerization at endocytic sites. We find that phosphorylation of the myosin-I Myo5 by Cka2 downregulates Myo5-induced Arp2/3-dependent actin polymerization, whereas PI(4,5)P2cooperatively relieves Myo5 autoinhibition and inhibits the catalytic activity of Cka2. Cka2 and the PI(4,5)P2-5-phosphatases Sjl1 and Sjl2, the yeast synaptojanins, exhibit genetic interactions indicating functional redundancy. The ultrastructural analysis of plasma membrane invaginations in CK2 and synaptojanin mutants demonstrates that both cooperate to initiate constriction of the invagination neck, a process coupled to the remodeling of the endocytic actin network. Our data demonstrate a holoenzyme-independent function of CK2 in endocytic budding and establish a robust genetic, functional, and molecular link between PI(4,5)P2and CK2, two masters of intracellular signaling.

Structural and functional characterization of a phosphatase domain within yeast general transcription factor IIIC.

Saccharomyces cerevisiae τ55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (τ55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of τ55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phosphoserine- and phosphotyrosine-containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phosphoproteomic study identified additional phosphopeptides as possible targets that show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify τ55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities, and provide a small set of regulated phosphosite targets in vivo.

RWDD1 interacts with the ligand binding domain of the androgen receptor and acts as a coactivator of androgen-dependent transactivation.

During embryogenesis, the development of the male genital is dependent on androgens. Their actions are mediated by the androgen receptor (AR), which functions as a transcription factor. To identify AR coregulators that support AR action during the critical time window of androgen-dependent development in the genital tubercle of male mice, we performed yeast two-hybrid screenings with cDNA libraries of genital tubercles from male mouse embryos using human AR as bait. RWD domain containing 1 (RWDD1) was identified as an AR-interacting protein from three independent libraries of the embryonic days E15, E16 and E17. The interaction between the AR and RWDD1 was confirmed in vitro and in vivo and the ligand binding domain of the AR was shown to be sufficient to mediate the interaction. RWDD1 enhanced AR-dependent transactivation in reporter assays with promoters of different complexity and in different cell lines. These results suggest that RWDD1 functions as a coactivator of androgen-dependent transcription.

Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites.

Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C(ext)) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C(ext). The TH1 domain prevented binding of the Myo5 C(ext) to the yeast WIP homologue Vrp1, Myo5 C(ext)-induced actin polymerization and recruitment of the Myo5 C(ext) to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C(ext). Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.

46,XY disorders of sex development--the undermasculinised male with disorders of androgen action.

Insensitivity to the action of androgens is a common cause of undermasculinisation in 46,XY individuals. These disorders are a result of the failure of major androgens to act via the intracellular androgen receptor and, thus, the genomic effects of androgen signalling are disrupted. The phenotype of affected individuals can vary considerably, depending on the dysfunction of the receptor. In childhood, the diagnosis is often complicated due to the lack of sensitive biochemical determinants, whilst during adolescence and in adults, the diagnosis can be readily made because of the striking clinical feminisation and a conclusive laboratory analysis. A variety of mutations in the androgen receptor have been analysed, providing insight into the complex pathways of intracellular processing and signal transduction via the androgen receptor. Endocrine therapy in androgen-insensitivity syndrome is controversial, because till date the special hormonal profiles in androgen insensitivity have not been acknowledged in replacement strategies.

Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane.

Endocytosis in yeast requires actin and clathrin. Live cell imaging has previously shown that massive actin polymerization occurs concomitant with a slow 200-nm inward movement of the endocytic coat (Kaksonen, M., Y. Sun, and D.G. Drubin. 2003. Cell. 115:475-487). However, the nature of the primary endocytic profile in yeast and how clathrin and actin cooperate to generate an endocytic vesicle is unknown. In this study, we analyze the distribution of nine different proteins involved in endocytic uptake along plasma membrane invaginations using immunoelectron microscopy. We find that the primary endocytic profiles are tubular invaginations of up to 50 nm in diameter and 180 nm in length, which accumulate the endocytic coat components at the tip. Interestingly, significant actin labeling is only observed on invaginations longer than 50 nm, suggesting that initial membrane bending occurs before initiation of the slow inward movement. We also find that in the longest profiles, actin and the myosin-I Myo5p form two distinct structures that might be implicated in vesicle fission.

TEDS site phosphorylation of the yeast myosins I is required for ligand-induced but not for constitutive endocytosis of the G protein-coupled receptor Ste2p.

The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.

Real-time detection of hyperosmotic stress response in optically trapped single yeast cells using Raman microspectroscopy.

Living cells survive environmentally stressful conditions by initiating a stress response. We monitored changes in the Raman spectra of optically trapped Saccharomyces cerevisiae yeast cell under normal, heat-treated, and hyperosmotic stress conditions. It is shown that when glucose was used to exert hyperosmotic stress, two chemical substances-glycerol and ethanol-can be monitored in real time in a single cell.