text:
Copyright © 2008 Elsevier Inc. All rights reserved.
Molecular Cell, Volume 29, Issue 1, 92-101, 18 January 2008
Add/View Comments (0)A TFTC/STAGA Module Mediates Histone H2A and H2B Deubiquitination, Coactivates Nuclear Receptors, and Counteracts Heterochromatin Silencing
Yue Zhao2, Guillaume Lang1, Saya Ito2, Jacques Bonnet1, Eric Metzger3, Shun Sawatsubashi2, Eriko Suzuki2, Xavier Le Guezennec4, Hendrik G. Stunnenberg4, Aleksey Krasnov5, Sofia G. Georgieva5, Roland Schüle3, Ken-Ichi Takeyama2, Shigeaki Kato2, László Tora1, , and Didier Devys1, ,
1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U 596, Université Louis Pasteur de Strasbourg, BP 10142-67404 ILLKIRCH Cedex, CU de Strasbourg 67404, France
2 Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan
3 Universitäts-Frauenklinik und Zentrum für Klinische Forschung, Klinikum der Universität Freiburg, Breisacherstrasse 66, 79106 Freiburg, Germany
4 Department of Molecular Biology, NCMLS, Radboud University Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands
5 Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia
Corresponding author
- Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo.
Transcription initiation by RNA polymerase II (Pol II) is a highly regulated process that requires the coordinated action of numerous factors. Coactivators are multisubunit complexes that are recruited to promoters by gene-specific activators to facilitate transcription initiation either by direct interaction with general transcription factors (GTFs) or Pol II or indirectly through modification of chromatin structure (reviewed in Li et al., 2007 and Martinez, 2002).
The yeast 2 MDa SAGA coactivator complex and its Drosophila and human homologs, the TBP-free TAF complex (TFTC) and the SPT3/TAF9/GCN5 acetyltransferase complex (STAGA), are multisubunit complexes that facilitate access of GTFs to DNA through histone acetylation mediated by the catalytic activity of the GCN5 subunit (Martinez, 2002). Human homologs of most SAGA subunits have been identified in STAGA and TFTC, and the three-dimensional structure of TFTC can be superimposed to that of SAGA (Wu et al., 2004). In addition, biochemical and functional characterization demonstrated that the described human complexes are almost identical (hereafter called TFTC/STAGA complex; reviewed in Nagy and Tora, 2007). Thus, genetic and structural studies point to an evolutionarily conserved function of these complexes with modular organization that relates to their multiple activities in regulating gene expression.
Gcn5 and other SAGA or TFTC/STAGA subunits have been shown to play a role in ligand-dependent gene activation by nuclear receptors (Wallberg et al., 2000 and Yanagisawa et al., 2002, and references therein). These observations suggested a model in which nuclear receptors recruit, in addition to other coactivator complexes, the SAGA/TFTC/STAGA complex, and that this contributes to chromatin remodeling via a mechanism involving histone acetylation.
Biochemical studies identified new components of the yeast SAGA complex that confer additional functions to the complex (Powell et al., 2004,Rodriguez-Navarro et al., 2004,Sanders et al., 2002). A ubiquitin-specific protease, Ubp8, was shown to be incorporated into SAGA through interaction with Sgf11 and to confer a histone H2B ubiquitin protease activity to the complex (Daniel et al., 2004,Henry et al., 2003,Ingvarsdottir et al., 2005,Lee et al., 2005). H2B has been shown to be monoubiquitinated (H2Bub1) on Lys 123 in S. cerevisiae by the E2-conjugating enzyme Rad6 and the E3-ligase enzyme Bre1 (reviewed in Zhang, 2003). Rad6-mediated H2Bub1 was found to regulate subsequent methylation of histone H3 at Lys 4 and Lys 79 through a "trans tail" mechanism and was shown to be required for subtelomeric gene silencing (Zhang, 2003). In addition to its role in gene silencing, H2B ubiquitination plays an important role in activation of specific genes. H2Bub1 seems to be transient at the promoter of SAGA-dependent genes, and both ubiquitination and deubiquitination of H2B are required for optimal gene activation (Daniel et al., 2004,Henry et al., 2003). Interestingly, Sus1, which was shown to be a component of both SAGA and the Sac3-Thp1 mRNA export complex (Rodriguez-Navarro et al., 2004), interacts with Sgf11 and Ubp8 and regulates the deubiquitination activity of the complex (Kohler et al., 2006). In higher eukaryotes, histone H2A is also monoubiquitinated (H2Aub1), a modification which has not been reported in yeast. H2Aub1 is more prevalent (5%–15%) when compared with that of H2Bub1 (1% to 2%). H2A ubiquitination is mediated by the Polycomb repressive complex 1 (PRC1) and is crucial for Polycomb-mediated gene silencing and X inactivation (de Napoles et al., 2004,Joo et al., 2007,Wang et al., 2004).
In this study we identified ATXN7L3, USP22, and ENY2 as the human orthologs of yeast Sgf11, Ubp8, and Sus1, respectively, and show that they are integral components of TFTC/STAGA complex. These three proteins together form a module of the TFTC/STAGA complex, which specifically removes the ubiquitin moiety from monoubiquitinated histones H2A and H2B. We demonstrate that the deubiquitination module of the Drosophila TFTC/STAGA complex counteracts heterochromatin silencing in vivo and both the Drosophila and the human deubiquitination module are required for full transcriptional activation by the androgen receptor (AR). These findings suggest that the deubiquitinase activity of the TFTC/STAGA complex, in addition to its histone acetyltransferase (HAT) activity, participates in the dynamic turnover of histone modifications during the transcription cycle and functions as a positive cofactor for activation by nuclear receptors.
USP22, ATXN7L3, and ENY2 Are Components of TFTC/STAGA Complex
Homologs of recently identified yeast SAGA subunits (i.e., Ubp8, Sgf11, and Sus1) remained to be identified in the mammalian TFTC/STAGA complex. We analyzed a highly purified TFTC fraction (Wieczorek et al., 1998) by MS-MS mass spectrometry and identified peptides corresponding to USP22, ATXN7L3, and ENY2 (see the Experimental Procedures ). Human USP22 is the closest homolog of yeast Ubp8 having a similar organization to the yeast ubiquitin protease possessing an N-terminal zinc finger domain (ZnF-UBP) and a C-terminal ubiquitin carboxyl-terminal hydrolase motif (UCH-2; Figure 1A). Human ATXN7L3 is the homolog of the yeast Sgf11 protein containing a highly conserved zinc finger domain at its N-terminal end (ZnF-Sgf11; Figure 1B). We have previously described ATXN7L3, an unknown protein at that time, based on its homology to ATXN7, another subunit of the TFTC/STAGA complex (Helmlinger et al., 2004). Indeed, ATXN7 and ATXN7L3 share a second C-terminal domain, the so-called SCA7 domain (InterPro: IPR013243), which is highly conserved through all members of the ATXN7 gene family (Figure 1B). In addition, we identified a peptide corresponding to ENY2, a 101 amino acid protein that is homologous to yeast Sus1.
To test whether the identified polypeptides are indeed TFTC/STAGA subunits, polyclonal antibodies against synthetic peptides corresponding to USP22, ATXN7L3, and ENY2 were raised. These antibodies efficiently recognized the corresponding recombinant proteins overexpressed in Sf9 cells at the expected size, 60 kDa for USP22, 42 kDa for ATXN7L3, and 13 kDa for ENY2 (see Figure S1 available online). We thus analyzed whether USP22 and ATXN7L3 would be present in a highly purified TFTC preparation. Western blot analysis using anti-USP22 and anti-ATXN7L3 detected a specific signal at the expected size in the HeLa cell nuclear extract and showed that USP22 and ATXN7L3 were enriched in the purified TFTC similarly to known components of TFTC/STAGA such as TRRAP, GCN5, and TAF10 (Figure 1C). In order to confirm that USP22, ATXN7L3, and ENY2 are integral components of the TFTC/STAGA complex, we performed a series of coimmunoprecipitation experiments on HeLa cell nuclear extract using antibodies against USP22, ATXN7L3, or ENY2. Following these immunoprecipitations, we analyzed USP22 and ATXN7L3-associated proteins by western blot and identified different TFTC subunits, such as TRRAP, ATXN7, GCN5, SPT3, or TAF10 (Figure 1D, lanes 2 and 3). Similarly, TRRAP, GCN5, and TAF10 were detected in an anti-ENY2 immunoprecipitation (Figure 1E). None of the TFTC subunits were detected in a control immunoprecipitation using preimmune sera (Figure 1D, lane 5 and Figure 1E, lane 2). TBP, a component of TFIID, but not of TFTC/STAGA, was not found associated with USP22, ATXN7L3, or ENY2. We previously showed that ATXN7 is a specific subunit of TFTC/STAGA (Helmlinger et al., 2004). Western blot analysis of complexes obtained after an ATXN7 immunopurification showed that USP22 and ATXN7L3 associate with ATXN7 together with other components of TFTC/STAGA (Figure 1D, lane 4). Taken together, these results indicated that USP22, ATXN7L3, and ENY2 are bona fide subunits of the TFTC/STAGA complex.
Identification of the TFTC/STAGA Deubiquitination Subcomplex
Having established that ATXN7L3, USP22, and ENY2 are components of the TFTC/STAGA complex, we next asked how these subunits interact and incorporate into TFTC/STAGA. In order to identify putative interactions between these proteins, 293T cells were transfected with expression vectors encoding Flag-tagged ATXN7L3 or USP22, or HA-tagged ENY2, and proteins were immunoprecipitated using anti-ATXN7L3 or anti-HA antibodies. Immunoblot analysis of purified complexes revealed an interaction between ATXN7L3 and USP22 (Figure 2A, lane 1) and between ATXN7L3 and ENY2 (Figure 2A, lanes 3 and 7), but not between ENY2 and USP22 (Figure 2A, lanes 8). When the three proteins were coexpressed, they formed a stable complex that could be purified both with anti-ATXN7L3 or anti-HA antibodies (Figure 2A, lanes 2 and 6). Formation of a stable TFTC module composed of ATXN7L3, USP22, and ENY2 was further confirmed by coinfection of Sf9 cells with different combinations of baculoviruses and immunopurification of ATXN7L3-associated proteins (Figure 2B). Note that when USP22 and ENY2 were coexpressed in the absence of recombinant ATXN7L3, no signal corresponding to these proteins could be detected after an ATXN7L3 immunoprecipitation (Figure 2A, lane 4 and Figure 2B, lane 5).
To analyze whether ATXN7L3 is required for USP22 association with TFTC/STAGA, we have downregulated ATXN7L3 expression by an siRNA treatment. HeLa cells were transfected with siRNA against ATXN7L3, cells were grown for 48 hr, TFTC/STAGA was purified by an ATXN7 immunoprecipitation, and the levels of TFTC/STAGA subunits were compared to those of complexes purified from control cells (Figure 2C). While the amount of GCN5 did not change in the purified complexes, USP22 was significantly reduced in the complex prepared from the cells where ATXN7L3 was knocked down (Figure 2C). These results together suggest that ATXN7L3, USP22, and ENY2 form a stable subcomplex and that USP22 and ENY2 are recruited into TFTC/STAGA by ATXN7L3 (Figure 2D).
We further asked which domains of ATXN7L3 and USP22 are involved in the formation of this subcomplex. Cotransfection and coimmunoprecipitation experiments revealed that the ATXN7L3 ZnF-Sgf11 domain alone is able to interact with both ENY2 and USP22 as efficiently as the full-length ATXN7L3 (see Figure S3 B). Similar experiments using USP22 deletion mutants showed that its interaction with ATXN7L3 required the ZnF-UBP domain, but not the catalytic domain of USP22 (data not shown). Thus, the formation of this module would be driven by interactions between ATXN7L3 and USP22 through their respective ZnF-Sgf11 and ZnF-UBP domains, and between ATXN7L3 ZnF-Sgf11 and ENY2 (Figure 2D). Finally, we showed that two different TFTC/STAGA subunits, TAF5L and ATXN7, interacted with this module and may mediate its association with TFTC/STAGA (Figure 2B and Figures S2 and S3 ).
Deubiquitination Activity of TFTC/STAGA
It has been shown that the yeast SAGA specifically removes ubiquitin moiety from H2Bub1 (Henry et al., 2003). We thus tested whether the human TFTC/STAGA complex has a similar activity on human H2Bub1. Although it plays an important role in transcription regulation, H2Bub1 is expected to represent less than 2% of H2B in human cells. To detect H2Bub1, HEK293T cells were cotransfected with expression vectors encoding a Flag-tagged histone H2B (Flag-H2B) and/or an HA-tagged ubiquitin (HA-Ub). Cells were grown for 48 hr, and recombinant histone H2B was immunoprecipitated using an anti-Flag antibody. When testing the purified proteins by immunoblotting, the anti-HA antibody specifically detected H2Bub1 (Figure 3A, lanes 4 and 5), while the anti-Flag antibody revealed the unmodified histone H2B and a faint signal corresponding to H2Bub1. The specific detection of H2Bub1 was also confirmed by the absence of signal when Flag-H2B or HA-Ub was omitted from the transfection reactions (Figure 3A, lanes 2 and 3).
To characterize the deubiquitination activity of TFTC/STAGA, we used the immunopurified fraction of H2B, which contains both unmodified H2B and H2Bub1 as a substrate in the in vitro deubiquitination assay. Two independent TFTC preparations decreased the level of H2Bub1 significantly, when analyzed with an anti-HA antibody, while the level of unmodified H2B was equal in each reaction (Figure 3B, compare lane 2 to lanes 3 and 4). The levels of H2Bub1 were further decreased when the amount of purified TFTC was increased in the deubiquitination assay, suggesting a dose-dependent effect (Figure 3B, lanes 4 and 5). Because in mammalian cells an important fraction of histone H2A is monoubiquitinated on Lys 119, we investigated the deubiquitination activity of TFTC/STAGA on the H2Aub1. Recombinant Flag-H2A-HA-Ub was prepared as described above for H2Bub1 and used as a substrate in an in vitro deubiquitination assay. Two different TFTC preparations could deubiquitinate H2Aub1 with a high efficiency (Figure 3C). From these results we conclude that the human TFTC/STAGA complex deubiquitinates H2Aub1 and H2Bub1.
Both nonstop, the Drosophila Homolog of USP22, and dSgf11, the Drosophila Homolog of ATXN7L3, Counteract Genomic Silencing Mediated by Heterochromatin
To characterize the in vivo function of the TFTC/STAGA deubiquitination activity, we followed a genetic approach in Drosophila. The Drosophila homolog of USP22 is called nonstop (Figure 1A) and was shown to be required for glial cell and neuronal development (Poeck et al., 2001). To determine a possible role of nonstop in the modulation of chromatin structure in vivo, we have used the well-defined In(1)Wm4h system that allows the identification of modifiers of position effect variegation (PEV) (Tartof et al., 1984). The In(1)Wm4h inversion brings the white locus close to a heterochromatic breakpoint leading to variegated expression of the white gene and a mosaic pattern of eye pigmentation. We found that mutation of one nonstop copy (not02069, 75D4) or a deficient line, lacking the chromosomal region containing nonstop (Df[3L]ED225, 75C1-75D4), gave significant decrease in pigment area in this PEV model, resulting in a 2-fold decrease of pigment levels in In(1)Wm4h (Figure 4A). In good agreement, we also showed that overexpression of full-length (FL) Nonstop increased eye pigmentation to wild-type levels in the In(1)Wm4h PEV model (Figure 4B). To further investigate which domain of Nonstop is required for the effect on PEV modification, we constructed Nonstop N- or C-terminal truncation mutations. Transgenic fly lines expressing only the deubiquitinase catalytic domain of Nonstop (UCH-2) had significant increase in pigment area in the eye when tested in the In(1)Wm4h PEV model (although weaker than FL), whereas there was no modification of PEV in transgenic fly lines expressing only the ZnF-UBP domain of Nonstop (Figure 4B). These data strongly suggest that the deubiquitination activity of Nonstop is required for the modification of PEV in the In(1)Wm4h system.
Because both yUbp8/ySgf11 and their human homologs USP22/ATXN7L3 seem to exert their activity in a common subcomplex, next we set out to identify the Drosophila homolog of ySgf11 or ATXN7L3. The CG13379 gene product (hereafter called dSgf11) was identified as a homolog of yeast Sgf11 and human ATXN7L3 (Figure 1B). We then investigated the effect of mutations of the dSgf11 gene on the In(1)Wm4h PEV system. dSgf11, similarly to nonstop, acted as an enhancer of PEV as its mutations led to decreased white expression assessed by eye pigmentation (Figure 4C). In contrast, mutation of dRpd3, a histone deacetylase, had no visible effect on PEV in the In(1)Wm4h system (Figure 4C). These results together suggest that both Nonstop and its partner dSgf11 are required to counteract heterochromatic gene silencing in vivo.
The Subunits of the Drosophila TFTC/STAGA Deubiquitination Module Are Required for Androgen Receptor-Mediated Transactivation
The fact that TFTC/STAGA has been implicated as a chromatin-modifying cofactor for activated transcription and that both Nonstop and dSgf11 counteract heterochromatic gene silencing suggested that the deubiquitination module of dTFTC/STAGA may play a coactivator role for transcriptional activators by modifying chromatin. To test this possibility, we developed an androgen receptor (AR)-associated position effect variegation (AR-PEV) model in Drosophila. In this system, the AR or a truncated AR harboring the ligand-independent AF-1 domain (AR[AF-1]) is expressed in the Drosophila eye using the glass multimer reporter (GMR) gene promoter (Figure 5A). A reporter construct that contains the white gene and a gene encoding the green fluorescent protein (GFP) controlled by eight AR-responsive elements (AREs) was inserted into a heterochromatic region leading to a mosaic red eye phenotype (Figures 5A and 5B). In this system, the AR counteracted silencing by heterochromatin spreading in a ligand-dependent manner (data not shown). The ligand-independent AR(AF-1) led to higher transactivation of the reporter genes and was hereafter used to analyze the AR-dependent alteration of chromatin structure. Two different mutations of nonstop (not02069 and Df[3L]ED225) gave significant decrease (2.2-fold) in the pigment area in this AR-dependent PEV model (Figure 5B). In good agreement, we also show that overexpression of Nonstop increased eye pigmentation to wild-type levels in the AR(AF-1)-PEV model (data not shown).
We further analyzed the effects of Nonstop on AR-dependent gene activation in vivo by testing AR(AF-1)-mediated transactivation on the GFP reporter gene expression system. In order to be able to detect GFP expression, we used additional transgenic lines expressing AR(AF-1) in the Drosophila eye using a GMR GAL4 driver in a GAL4-UAS system. The ARE-GFP reporter was inserted in euchromatic region and further mobilized into pericentric heterochromatin leading to variegated expression of GFP reporter (Y.Z., K.-I.T., and S.K., unpublished data). In agreement with our results obtained with the above described AR-dependent PEV model, this reporter system confirmed again that nonstop loss-of-function mutations, not02069 and Df(3L)ED225, dominantly reduce AR(AF-1) transactivation of the reporter transgene inserted in pericentric heterochromatin (Figure 5C). In contrast, but consistent with the loss-of-function effects, nonstop overexpression (UAS-nonstop) significantly increased GFP transactivation by AR(AF-1) (Figure 5C).
Having established that overexpression of Nonstop-mediated AR-dependent gene activation in pericentric region, we investigated whether this activity requires the association of Nonstop with TFTC/STAGA complex. To this end, we examined how mutations of different TFTC/STAGA components affect Nonstop-mediated AR(AF-1) transcriptional activation in vivo using the second GFP transactivation system. As shown in Figure 5D, Nonstop overexpression enhanced AR(AF-1) transactivation of the ARE-GFP reporter gene expression. In good agreement with our biochemical interaction studies, mutations of dSgf11 (CG13379e01308) and e(y)2 (Df(1)m259-4) significantly reduced GFP expression (Figure 5D). Importantly, the coactivation activity of Nonstop was not modified by mutation of dTAF5 (Taf5EY01764b), which is a specific component of TFIID. Taken together, these results suggest that the deubiquitination activity of Drosophila TFTC/STAGA, mediated by Nonstop, requires the components of the TFTC/STAGA deubiquitination module to play an important role in AR-dependent transactivation in vivo.
The Human Deubiquitination Module and Its Activity Are Positive Modulators of AR-Mediated Transactivation in Human Cells
The fact that Drosophila Nonstop, Sgf11, and E(y)2 increased in vivo transactivation activity of the AR on silenced reporter transgenes suggested that these proteins play a coactivator role in AR activation. To further investigate this hypothesis, we analyzed putative interactions of USP22 or ATXN7L3 with AR in mammalian cells. Cotransfection in HEK293T cells and coimmunoprecipitation experiments revealed that AR interacted with ATXN7L3 in a ligand-dependent manner, similarly to GCN5, while interaction between AR and USP22 was detected also in the absence of ligand (see Figure S6 A). As AR and USP22 interacted in vivo, we next tested whether AR could be a substrate of the TFTC/STAGA deubiquitination activity. Coexpression of increasing amounts of USP22 with AR in 293T cells revealed that the AR steady-state levels were not modified, indicating that AR is not a substrate of USP22 (see Figure S6 B).
In order to determine whether USP22, ATXN7L3, and ENY2 are required for AR-dependent transcriptional activity in human cells, we performed AR-dependent reporter assays. 293T cells were transfected with an AR-dependent reporter gene together with expression vectors encoding AR alone, or together with USP22, ATXN7L3, and ENY2 in the presence or absence of ligand (Figures 6A and 6B). USP22, ATXN7L3, and ENY2 significantly enhanced AR-mediated transactivation in the presence of ligand (2.5- to 3.5-fold compared to AR alone), similarly to the effect of the GCN5 HAT (Figures 6A and 6B). Furthermore, we observed that USP22 dominantly increased also ER- or GR-mediated transactivation in similar reporter assay analyses (data not shown). These results suggested that USP22, ATXN7L3, and ENY2 can function as coactivators for nuclear receptor-mediated transactivation. We next examined whether the deubiquitination activity of USP22 is required for coactivation of AR transcriptional activity. To this end we constructed an expression vector encoding a USP22 mutant in which the two conserved histidine residues in the catalytic site (His box) were mutated to alanines. Such mutations in the active site of ubiquitin-specific proteases were shown to result in undetectable levels of catalytic activity (Hu et al., 2002). Expression of this inactive USP22 catalytic mutant did not change the ligand-induced activation of the AR-dependent luciferase reporter (Figure 6A), further demonstrating that USP22 deubiquitination activity is required for its nuclear receptor coactivation function. In good agreement with our observations in Drosophila, ATXN7L3, ENY2, and USP22, but not the USP22 catalytic mutant, increased transactivation also by the ligand-independent AR(AF-1) in similar reporter assays (Figure 6C).
We then asked whether the TFTC/STAGA deubiquitination activity is required for endogenous AR-dependent gene activation in vivo. To this end, we turned to LNCaP prostate tumor cells in which the AR is constitutively expressed and analyzed the androgen-induced expression of the endogenous AR target gene KLK2 after downregulation of USP22. Transfection of LNCaP cells with siRNA against USP22 led to a 55% reduction of USP22 mRNA and protein levels (Figure 6D and data not shown). Induction of KLK2 expression by the synthetic AR agonist R1881 in LNCaP cells transfected with control siRNA was 3-fold and significantly reduced in cells transfected with siRNA against USP22 (1.6-fold) (Figure 6D). This result together with the observed interaction between the AR and both ATXN7L3 and USP22 suggested that these proteins are recruited together to the promoters of AR-dependent genes upon ligand induction. To test this hypothesis, chromatin was prepared from LNCaP cells treated with or without ligand and subjected to chromatin immunoprecipitation (ChIP) using anti-AR and anti-USP22 antibodies. Recruitment of the AR was significantly increased at the promoter region of KLK2 gene after ligand addition (Figure 6E). Similarly, USP22 associated with the chromatin of KLK2 promoter in a ligand-dependent manner (Figure 6E). No significant association of AR and USP22 with chromatin at an intergenic region could be detected, and the amount of histone H3 at KLK2 promoter remained constant, further demonstrating specificity (Figure 6E). Taken together, these results indicate that upon ligand induction TFTC/STAGA is recruited to the promoter of AR-dependent genes where its deubiquitination activity facilitates gene expression.
In this study, we identified three subunits of the 2 MDa GCN5-containing TFTC/STAGA complex. We show that the ubiquitin protease USP22 forms a subcomplex with ATXN7L3 (ySgf11 homolog) and ENY2 (ySus1 homolog). Importantly, we demonstrate that the catalytic domain of the ubiquitin protease together with dSgf11 is necessary to modify PEV in the Drosophila eye, pointing to the involvement of the deubiquitination module in chromatin remodeling. Furthermore, we show that the subunits of the deubiquitination subcomplex are required for the full activity of the ligand-induced AR and other nuclear receptors in vivo. Thus, to our knowledge we established for the first time the deubiquitination activity of the metazoan TFTC/STAGA HAT complexes as a second positive chromatin-modifying function.
TFTC/STAGA Complex Has a Modular Organization that Links the Deubiquitination Function to HAT, Activator Binding, and Transcription Initiation
An overlay of the yeast SAGA and the human TFTC structures indicated, in good agreement with their similar subunit composition (Nagy and Tora, 2007), a high degree of structural conservation in size and shape (Wu et al., 2004). Several SAGA subunits have been localized in one of the five domains of the complex (Wu et al., 2004). Gcn5 was detected in the central region of SAGA and colocalized with Ada2 and Ada3, indicating that this central domain harbors the HAT activity. We have now identified a module of TFTC/STAGA, which is responsible for the deubiquitination activity of this complex on H2Aub1 and H2Bub1. This module is composed of three subunits, among which ATXN7L3 (hSGF11) interacts with TAF5L and ATXN7 and allows the recruitment of both USP22 and ENY2 to TFTC/STAGA. Because yTAF5 localizes close to the Gcn5-containing HAT module, it is tempting to speculate that a functional connection may exist between the HAT and the deubiquitination modules. Along these lines it is interesting to note that neither free USP22 nor a stable recombinant subcomplex, composed of TAF5L, ATXN7L3, ENY2, and USP22, can deubiquitinate H2Aub1 or H2Bub1 in vitro (see Figure S4 ), suggesting that additional TFTC subunits are required for the regulation of this activity.
Interestingly, the two different TFTC/STAGA subunits, ATXN7 (ySgf73 homolog) and ATXN7L3 (ySgf11 homolog) share a conserved domain and may have a related function within the complex. A polyglutamine expansion in ATXN7 has been identified as the mutation responsible for an inherited neurodegenerative disorder, Spinocerebellar ataxia type 7 (SCA7). It remains to be investigated whether a polyglutamine expansion in ATXN7 deregulates the TFTC/STAGA deubiquitination activity and whether this would account for chromatin modifications and transcriptional alterations previously identified in SCA7 models (Helmlinger et al., 2006).
The yeast and the Drosophila homolog of hENY2 (Sus1 and E[y]2, respectively) were identified as subunits of SAGA and an anchoring and mRNA export (AMEX) complex, homolog of the yeast Sac3-Thp1-Sus1 complex (Kurshakova et al., 2007,Rodriguez-Navarro et al., 2004). Sus1 or dE(y)2 as subunits of AMEX function together with SAGA/TFTC in the anchoring of a subset of transcription sites to the nuclear pore complexes (NPCs) to achieve efficient transcription and mRNA export (Cabal et al., 2006,Kurshakova et al., 2007). The association of USP22 and ENY2 in the same TFTC/STAGA module would therefore suggest that two activities, histone deubiquitination and mRNA export, may also be coregulated within this complex. Although we showed that TFTC/STAGA deubiquitinates H2Aub1 and H2Bub1, USP22 could act on other nonhistone targets as suggested by the accumulation of several ubiquitinated proteins after inactivation of Drosophila nonstop (Poeck et al., 2001).
Different histone deubiquitinases involved in transcriptional regulation have been identified in metazoan. Drosophila USP7 specifically deubiquitinates H2Bub1 and regulates Polycomb-mediated silencing (van der Knaap et al., 2005). Recently, two ubiquitin proteases, Ubp-M/USP16 and 2A-DUB/KIAA1915/MYSM1, were identified as specific H2A deubiquitinases regulating Hox gene expression and AR-dependent gene activation (Joo et al., 2007,Zhu et al., 2007). In contrast to the specific recognition of either H2Aub1 or H2Bub1 by these enzymes, we showed that TFTC/STAGA is active on both H2Aub1 and H2Bub1 in vitro. Thus, it seems that, in addition to these enzymes that seem to be active only at specific target genes, TFTC/STAGA has a broader specificity to counteract heterochromatin silencing and to regulate gene activation by nuclear receptors. Alternatively, the action of TFTC/STAGA on either H2Aub1 or H2Bub1 could be restricted to specific genes in vivo.
The Functional Link between the HAT and the Deubiquitination Activities of TFTC/STAGA in Gene Activation
Genetic and biochemical analysis in yeast and Drosophila suggested that the 2 MDa GCN5-containing complexes have multiple roles in the cells: global deposition of histone H3 acetylation marks along the entire chromosomes and targeted acetylation of histones at specific loci. Our results obtained with the ubiquitin protease mutants in both the In(1)Wm4h PEV system or the AR-dependent PEV model suggest that, similarly to its HAT activity, the deubiquitination module of SAGA/TFTC is influencing both the global chromatin architecture, as a modifier of PEV, and the gene-specific function of the complex, as a nuclear receptor coactivator. This intriguing parallel raises the following questions: is the TFTC/STAGA HAT activity in vivo functionally connected with deubiquitination activity within the complex, and which activity is needed first? Interestingly, all the subunits of the deubiquitination module of TFTC/STAGA are able to function as coactivators during the activation process of AR, similarly to what has been demonstrated for individual subunits of the HAT module (Meng et al., 2004,vom Baur et al., 1998) or for the entire 2 MDa HAT complexes (Yanagisawa et al., 2002). In this respect the HAT and the deubiquitination modules seem to fulfill similar functions suggesting that the two activities together are necessary to achieve full activation of nuclear receptors on chromatin templates. Histone acetylation is thought to open up the chromatin, which may suggest that the deubiquitination activity of the complex would work on templates, which have been previously acetylated by the complex.
Our study pinpoints the monoubiquitination of histone H2A and H2B as a mark that is timely regulated for appropriate transcription in metazoan organisms and further emphasizes that the precise turnover of chromatin modifications is crucial for the control of gene expression. The association of both HAT and deubiquitination activities in the TFTC/STAGA complex provides an attractive mechanism by which the so called "cross-talk" between given histone marks is coordinated within the same regulatory complex. Further mechanistic studies will be required to address the exact link between these activities and the other chromatin-modifying complexes to understand how these sequential events participate in chromatin remodeling and gene activation.
Experimental Procedures
Plasmids, Fly Stocks, and Genetics
Plasmid construction, generation of transgenic flies, fly stocks, and crossing schemes are presented in the Supplemental Experimental Procedures .
Mass Spectrometry
Mass spectrometry was performed using LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) essentially as described previously (Olsen et al., 2004). Peptides identified were as following: SPHIPYK, DFFLSDR, and ITSNCTIGLR corresponding to USP22; SHLSLVGTASGLGSNKK and SLLTTQCGVISEHTK corresponding to ATXN7L3; and TFLAQHASL corresponding to ENY2.
The anti-USP22 (2391 and 2392) and anti-ENY2 (2584 and 2585) polyclonal antibodies were obtained by immunization of rabbits with peptides corresponding to residues 2–15 (VSRPEPEGEAMDAE) of human USP22 and to residues 50–66 (CKEVIKEKGLEHVTVDD) of human ENY2. Rabbits and mice immunization with ATXN7L3 peptides corresponding to residues 278–296 (LQWDGSSDLSPSDSGSSKT) and to residues 335–353 (KKKPKPPAPPTPSIYDDIN) produced the 2326 polyclonal and the 2ATX-2B1 monoclonal antibodies and the 2325 polyclonal and 1ATX-2D7 monoclonal antibodies, respectively.
In Vitro Deubiquitination Assay
293T cells transfected with Flag-H2A or Flag-H2B (gift form R.G. Roeder) and/or HA-Ub (gift from I. Dikic) plasmids were collected in phosphate buffer saline 1X (PBS1X) containing 20 mM N-ethylmaleimide (NEM) and 1 mM PMSF. Whole-cell extracts prepared in modified RIPA buffer (50 mM Tris-HCl [pH 7.9], 150 mM NaCl, 5 mM EDTA, 15 mM MgCl2, 1% NP40, 0.5% sodium deoxycholate, 1 mM DTT, 1X protease inhibitor, 1 mM PMSF, and 20 mM NEM) were immunoprecipitated using M2 beads (Sigma). After extensive washing, bound proteins were eluted with an excess of free Flag peptide. Eluted proteins were used as a substrate in a deubiquitination reaction with different amounts of highly purified TFTC complex prepared as described (Wieczorek et al., 1998). The reaction was performed in 100 mM Tris-HCl (pH 7.9), 5% glycerol, 1 mM DTT, and 1 mM EDTA for 2 hr at 37°C. Unmodified and monoubiquitinated recombinant H2A or H2B were analyzed by immunoblotting using anti-Flag and anti-HA antibodies.
We thank D. Helmlinger, O. Bombarde, M. Vermeulen, M. Oulad-Abdelghani, and G. Duval for their contribution to this work. We thank Z. Nagy and M.E. Torres Padilla for critically reading the manuscript, and R.G. Roeder, I. Dikic, T. Nagase, and E. Martinez for generous gifts of different reagents. Research in the authors' laboratory is supported by grants from the the Agence Nationale de la Recherche (ANR-05-MRAR-O29-01), from the Fondation de la Recherche Médicale (FRM, DLC20060206408), and from Connaitre les Syndromes Cerebelleux to D.D. and from ANR (05-BLAN-0396-01; Regulome), European Community (HPRN-CT 00504228 and STREP LSHG-CT-2004-502950) to L.T.
Cabal et al., 2006 (2006). SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature 441, 770–773. CrossRef | PubMed
Daniel et al., 2004 (2004). Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription. J. Biol. Chem. 279, 1867–1871. CrossRef | PubMed
de Napoles et al., 2004 (2004). Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev. Cell 7, 663–676. Abstract | Full Text | PDF (623 kb) | CrossRef | PubMed
Helmlinger et al., 2004 (2004). Ataxin-7 is a subunit of GCN5 histone acetyltransferase-containing complexes. Hum. Mol. Genet. 13, 1257–1265. CrossRef | PubMed
Helmlinger et al., 2006 (2006). Glutamine-expanded ataxin-7 alters TFTC/STAGA recruitment and chromatin structure leading to photoreceptor dysfunction. PLoS Biol. 4, e67. doi:10.1371/journal.pbio.0040067 | PubMed
Henry et al., 2003 (2003). Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. Genes Dev. 17, 2648–2663. CrossRef | PubMed
Hu et al., 2002 (2002). Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde. Cell 111, 1041–1054. Abstract | Full Text | PDF (1080 kb) | CrossRef | PubMed
Ingvarsdottir et al., 2005 (2005). H2B ubiquitin protease Ubp8 and Sgf11 constitute a discrete functional module within the Saccharomyces cerevisiae SAGA complex. Mol. Cell. Biol. 25, 1162–1172. CrossRef | PubMed
Joo et al., 2007 (2007). Regulation of cell cycle progression and gene expression by H2A deubiquitination. Nature 449, 1068–1072. CrossRef | PubMed
Kohler et al., 2006 (2006). The mRNA export factor Sus1 is involved in Spt/Ada/Gcn5 acetyltransferase-mediated H2B deubiquitinylation through its interaction with Ubp8 and Sgf11. Mol. Biol. Cell 17, 4228–4236. CrossRef | PubMed
Kurshakova et al., 2007 (2007). SAGA and a novel Drosophila export complex anchor efficient transcription and mRNA export to NPC. EMBO J. 26, 4956–4965. CrossRef | PubMed
Lee et al., 2005 (2005). The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex. Mol. Cell. Biol. 25, 1173–1182. CrossRef | PubMed
Li et al., 2007 (2007). The role of chromatin during transcription. Cell 128, 707–719. Abstract | Full Text | PDF (854 kb) | CrossRef | PubMed
Martinez, 2002 (2002). Multi-protein complexes in eukaryotic gene transcription. Plant Mol. Biol. 50, 925–947. CrossRef | PubMed
Meng et al., 2004 (2004). Human ADA3 binds to estrogen receptor (ER) and functions as a coactivator for ER-mediated transactivation. J. Biol. Chem. 279, 54230–54240. CrossRef | PubMed
Nagy and Tora, 2007 (2007). Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation. Oncogene 26, 5341–5357. CrossRef | PubMed
Olsen et al., 2004 (2004). Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol. Cell. Proteomics 3, 608–614. CrossRef | PubMed
Poeck et al., 2001 (2001). Glial cells mediate target layer selection of retinal axons in the developing visual system of Drosophila. Neuron 29, 99–113. Abstract | Full Text | PDF (2252 kb) | CrossRef | PubMed
Powell et al., 2004 (2004). Cluster analysis of mass spectrometry data reveals a novel component of SAGA. Mol. Cell. Biol. 24, 7249–7259. CrossRef | PubMed
Rodriguez-Navarro et al., 2004 (2004). Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116, 75–86. Abstract | Full Text | PDF (538 kb) | CrossRef | PubMed
Sanders et al., 2002 (2002). Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry. Mol. Cell. Biol. 22, 4723–4738. CrossRef | PubMed
Tartof et al., 1984 (1984). A structural basis for variegating position effects. Cell 37, 869–878. Abstract | | CrossRef | PubMed
van der Knaap et al., 2005 (2005). GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol. Cell 17, 695–707. Abstract | Full Text | PDF (643 kb) | CrossRef | PubMed
vom Baur et al., 1998 (1998). The yeast Ada complex mediates the ligand-dependent activation function AF-2 of retinoid X and estrogen receptors. Genes Dev. 12, 1278–1289. CrossRef | PubMed
Wallberg et al., 2000 (2000). Recruitment of chromatin remodelling factors during gene activation via the glucocorticoid receptor N-terminal domain. Biochem. Soc. Trans. 28, 410–414. CrossRef | PubMed
Wang et al., 2004 (2004). Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873–878. CrossRef | PubMed
Wieczorek et al., 1998 (1998). Function of TAF(II)-containing complex without TBP in transcription by RNA polymerase II. Nature 393, 187–191. CrossRef | PubMed
Wu et al., 2004 (2004). Molecular architecture of the S. cerevisiae SAGA complex. Mol. Cell 15, 199–208. Abstract | Full Text | PDF (607 kb) | CrossRef | PubMed
Yanagisawa et al., 2002 (2002). Nuclear receptor function requires a TFTC-type histone acetyl transferase complex. Mol. Cell 9, 553–562. Abstract | Full Text | PDF (484 kb) | CrossRef | PubMed
Zhang, 2003 (2003). Transcriptional regulation by histone ubiquitination and deubiquitination. Genes Dev. 17, 2733–2740. CrossRef | PubMed
Zhu et al., 2007 (2007). A histone H2A deubiquitinase complex coordinating histone acetylation and H1 dissociation in transcriptional regulation. Mol. Cell 27, 609–621. Abstract | Full Text | PDF (1488 kb) | CrossRef | PubMed
Publication Information
Received: June 19, 2007
Revised: November 2, 2007
Accepted: December 15, 2007
Published: January 17, 2008


text:
Now this is what we like to see – children getting involved in animation from a young age. This week we heard that Tate is teaming up with Aardman Animations, the team behind Wallace and Gromit, to create a new film. And, they want children to help them brainstorm. Check out the video on our YouTube channel.
Back to the future of 3D cinema
I've mentioned 3D movies before on the blog, but this week another article on the topic caught my eye. Pixar has said that it's hoping 3D films will encourage people to watch movies at the cinema – rather than pirate copies or illegal downloads at home.
Traditional Animation makes a comeback
The first trailer for the upcoming Disney Feature The Princess and the Frog has just been released. It heralds a return to the hand drawn traditional animation that Disney is famous for. I for one am excited to see it and hope that it heralds the rebirth of traditional animation as a whole.
Monsters vs Aliens: mixed bag
I watched Dreamworks newest animation feature, "Monsters vs Aliens" recently and found it to be both truly amazing and really bad. Some bits of animation were brilliant, funny and a joy to watch (such as the cracking the computer code dance scene), while others were disappointing (like the General's entrance and a lot of his dialogue). I felt that the story was just above average. Not bad and not brilliant.

subset: commoncrawl


text:
Connect with Us
Podcasts & RSS Feeds
All Content |
View all podcasts & RSS feeds |
Now Playing
Loading streams...
Now Playing
Loading stream...
KUER News Staff
News Director
Reporter / Morning Edition Host
Reporter and Host for All Things Considered
Online Editor
The Two-Way
7:21 am
Mon July 16, 2012
Top Stories: Romney, Obama Spar; Fierce Fighting In Damascus
By Mark Memmott
Good morning.
Our early headlines:
-- New Week, Same Argument: Romney, Obama & Who's Being Dishonest.
-- Military Chief's Removal, Rise In Hemlines: Hopeful Signs In North Korea?
-- Retail Sales Dip For Third Straight Month, But Are Still Up From Year Earlier.
-- Here's A Scoop: When News Breaks, People Check YouTube For Videos.
Some other stories making news:
-- "Fierce Fighting In Damascus — Live Updates." (The Guardian)
-- Russian Foreign Minister Accuses West Of "Blackmail" Over Syrian Sanctions. (BBC News)
-- "Microsoft, NBC Call It Quits; Dissolve MSNBC.com." (The Associated Press)
-- Egyptian Protesters Chant "Monica, Monica, Monica," Throw Tomatoes And Shoes As Secretary Of State Clinton's Motorcade Passes. (CNN.com)
Copyright 2012 National Public Radio. To see more, visit http://www.npr.org/.


text:
Skip to this page's content
With more than 60 undergraduate majors and five graduate schools, Simmons offers too many programs to list here. Check them all out »
300 The Fenway Boston, MA02115 617-521-2000
Search Simmons Search
Home » Undergraduate College » Site Tools
Below you will find some useful tools for the Simmons Undergraduate Site:
Got questions about Simmons? Email or call your Simmons admission counselor »
Come visit! Want to find out what Simmons is like? Check out our upcoming admission events.
Apply to Simmons Start your application today!


text:
Ranked among the nation's most integrated healthcare systems, Community Health Network is Central Indiana's leader in providing convenient access to exceptional healthcare services, where and when patients need them—in hospitals, health pavilions, workplaces, schools and homes.
Rapid stabilization of symptoms through evidence-based treatment is a key goal for the stabilization treatment model at Community's behavioral health pavilion.
"We want to bring patients back to a higher level of functioning than when they entered the hospital and engage them in ongoing outpatient care," says Eric Crouse, Ph.D., vice president for behavioral health services.
The stabilization model involves teaching patients life skills in a group setting to help them better cope with their condition and use interpersonal support systems to live happier, more symptom-free lives. It is an evidence-based best practice that is used in some of the best inpatient psychiatric hospitals across the country.
The program puts the patient at the center of the treatment and is tailored based on his or her individual needs. Group sessions offer a range of topics, including preserving normalcy, decreasing the impact of crisis, problem solving, anger management, communication/assertiveness and managing relationships. Patients and family members also receive guidance about how medications help treat a condition, and how to work with a psychiatrist on an ongoing basis to make best use of a medication.
"Patients need to understand the notion that they will need to stay in some form of therapy," notes Crouse. "It is highly unlikely that everything will be perfect when they leave the hospital."
As part of the stabilization model, offerings at all levels of Community's behavioral health services have been enhanced to increase the likelihood that patients will follow through with ongoing care. This includes improved interdisciplinary work and communication between psychiatric nurses, social workers and psychiatrists. "We have brought the structure of our program to a higher level," says Crouse," in terms of the impact we can make on people who need our assistance."


text:
Hey, thanks you guys.
I will try to update my iphone. Although still a bit scary haha.
One question, do u recommend restore from my backup or set up a new phone?
That doesnt says anywhere, in the guide.
Hey, thanks you guys.
I will try to update my iphone. Although still a bit scary haha.
One question, do u recommend restore from my backup or set up a new phone?
That doesnt says anywhere, in the guide.
I restored from my backup and it worked perfectly. You can try it too.
And BTW, the thrill is part of the iPhone's fun
** If you just want to support hackint0sh.org with a donation, please click here **.
Hahaha i know that the thrill is part of it. Its just that i dont want to bricked it, i use it a lot, so thats the main reason.
But i think i have it pretty clear now.
I will try to follow step by step, but im at work now. So i will check it out when im home. By the way, all the winpwn process from start i got to have my iphone connected to the pc, right??
thanks got that part figured out....thought all was good but now instead of error 1604 its just a error 6. I did everything step by step.
Ok, 3 things make sure you are doing right.
1. Check if you got the right firmware?
2. Make sure you put it correctly in DFU mode
3. Make sure you put the root partition above 512 mb
Hope that helps!
too bad you cant use the resizer on 1.1.4
has anyone made a complete error list for the iphone cuz id love to kno what error(6) means cuz i get that no matter what i do
MSN/AIM? PM me
If you want to become a Hackint0sh supporter click here.
I DO READ PM's
"Just because I'm losing
Doesn't mean I'm lost
Doesn't mean I'll stop
Doesn't mean I will cross
Just because I'm hurting
Doesn't mean I'm hurt
Doesn't mean I didn't get what I deserve
No better and no worse "
Had issues but did exactly what you said and flawless jailbreak....You are the Man Law! again. appreciate the info. I was on 1.1.4 and upgraded to 2.0.1 and champ! it worked
Tho I dont like 2.0 at all.. all the free stuff now costs and up to 9.99 on some that were once free!! I reverted back to 1.1.4 for now... 2.0 just not worth it to me,, the iphone seemed slower, graphics better but the free apps are junk and barely used what is available for data.. so Meh! 1.1.4 it is
i have the phone in dfu mode...im pretty sure. when i do the process of getting into dfu itunes detects the phone, while the phone screen shows no logo. I then shift restore and it gets to the point of actually restoring the software and than bam error 6.
DFU mode to be exact.... open itunes, plug in iphone, look at a clock.. exactly 10 secs hold home and power at the 10 sec mark release power and keep on home until itunes detects it.... after this do a regular restore, let itunes do the work. then do this a second time and do the winpwn and you should be all set... Be warned it will sit there like it isnt doing anything for a while depending on your pc speed.. it took me about 20-30min to do the restore in DFU...
I think the confusion to alot of this lies in the DFU mode.. DFU is a BLACK screen, not the secondary screen you pick.


text:
Harry & Draco Dolls
Unbleached muslinFelt
Embroidery thread
Pencil or fabric marker
Using the pattern below, cut out the pattern and then trace around it directly onto the wrong side of the fabric with pencil or disappearing fabric marker. Don't cut the fabric yet!Pin the traced fabric to another piece of fabric, right sides facing each other.
Sew around your tracing, leaving an opening in the top of the head for stuffing.
Cut around the sewn doll, leaving just a little bit of fabric as a seam allowance.
Very carefully clip around the curves right up to the seam, without cutting the seam.
Turn the doll right side out so the seams are now on the inside.
Stuff the doll with fiberfill, then sew up the top of its head by hand.
Sewing hair
Make the yarn longer rather than shorter, though, because you can always cut it after it's attached.
Some doll hair tutorials:
How to knot on yarn doll wigs.
Make a little wig. This one is pretty easy to adapt to all doll head sizes.
Local public libraries have plenty of books on cloth doll making. There are a lot of tips in these.a
The author suggests embroidering the face before sewing, because, if a mistake is made, it would be better to do it on fabric that can be replaced, rather than having to replace an entire doll.
Here is an online dictionary of embroidery stitches.
Final Product
Aliciajoy's tutorial was taken with permission from Sock Monkey Rescue.Please note that the patterns and tutorials you find here have been designed by Harry Potter fans all over the Internet. The authors alone hold the copyrights and licences to these patterns and tutorials, which means you CANNOT use their patterns to make something that you will sell to others afterwards. You can use them to make things for yourself. You can make some for your friends and ask them to pay for supplies. You CANNOT, however, ask them to pay you to do it as though you had created this pattern by yourself, or try to sell you crafts to a local store.
Think about it. Would you take a Prisoner of Azkaban book, photocopy it, put your name in big red letters on the front cover and try to sell it in your local library? The answer is, obviously, no. Well, selling crafts you have made but not designed would be just as bad!
Also note that the tutorials, recipes and patterns found here have not been tested and that The Leaky Cauldron's Harry Potter Crafts section is not responsible for any mistakes they may contain. If you do find something wrong in one of them, however, please e-mail us to let us know.
On that note, Harry crafting to all!


text:
Open Daily
Extended Hours
Alexandria King Sleigh BedSKU: 200040820
Compare $1,339.99
Art Van Price $699.99
Availability: Available in mid March
Bed features a sleigh style headboard. Stocked in queen and king size. Bed includes: Headboard, Footboard and side rails.
Dimensions:Height - 60.00" Width - 82.00" Depth - 91.00"
Add To Wish List
Art Van Price $499.99
Art Van Price $249.99
Finish: Autumn brown finish.
Construction: Hardwood solids and Cathedral cherry veneers.
Care Instructions: Use a soft, clean polish moistened cloth to dust. Never use soap and water. Always use a fine furniture polish.


text:
Ads by Google AdSense
Be the first to rate this station!
1 Tune InsMadera County Fire Department of Madera, CA, U.S.A., provides fire protection, rescue capabilities, and emergency medical service to its residents.
Playlist, tracklist, top songs played coming soon...
PC requires Windows Media Player - Mac additionally requires Real Player - See FAQs for more info on how to listen to live streaming radio.


text:
Chairperson John Hunt * Vice Chairperson Thomas McNamara *
Student Member Jeff Tomasich NOTICE OF MEETING CANCELLATION PARKS & COMMUNITY SERVICES COMMISSION Wednesday, September 10, 2008 Please be advised that the Parks & Community Services Commission meeting scheduled for Wednesday, September 10, 2008 has been canceled. The next scheduled meeting will be held on Wednesday, October 8, 2008, in the Council Chambers, San Ramon City Hall, 2222 Camino Ramon. Please refer any questions to the Parks & Community Services Department, (925) 973-3369. I hereby certify the attached Parks and Community Services agenda was posted 72 hours prior to the noted meeting: Dated: August 19, 2008 |
Parks & Community Services Dept | P&CS Commission | P&CS Commission Agenda History


text:
Carissa Zerga, Lea Tate, Amber Gilbert, Donna DeMarchi, Christina Kaiser , Rebecca Beckett, Michelle Cavil, Penny Morin and Jessica VanValkenburgh, committee members for United for Families' Le Bal Masque, which raised $57,000 raised for the nonprofit.
Buy this photoBe the first to post a comment!
Want to participate in the conversation? Become a subscriber today. Subscribers can read and comment on any story, anytime. Non-subscribers will only be able to view comments on select stories.


text:
Casual (3)
Dress (5)
Sport (12)
Quartz (17)
Stainless Steel (15)
Sterling Silver (1)
Titanium (4)
Compare up to 5 Items
Receive a $50 Offer When You Sign Up for Email*
No Thank You
*Valid for new registrations only. A welcome email will be sent with your offer information. Privacy Policy


text:
KPMG provides Audit, Tax, and Advisory services. KPMG LLP is the Canadian member firm of KPMG International.
Working in partnership with Free The Children, KPMG is helping our people make a meaningful contribution to development by putting our skills to work - internationally, in our communities, and with our families.
Information is becoming the "power resource" and organizations are looking at how to leverage it. How can your company derive value from data?
The Canadian FATCA Intergovernmental Agreement (IGA) was announced on February 5, 2014. FATCA will require all non-US entities worldwide to undertake additional reporting. Find out how the IGA may affect your business.
KPMG's Energy professionals can help clarify the issues and enhance your competitive advantage. We serve organizations operating in three sectors: Energy Services, Oil & Gas, and Power & Utilities.
Our Financial Services teams have the experience, technical skills, industry focus, and global resources to help clients address and overcome their business challenges.
This annual tax guide will give you quick answers to many practical questions that come up when doing tax and financial planning.
Answer today's questions, and ask tomorrow's with KPMG's revamped Thought Leadership App.
KPMG's commitment to provide a supportive and innovative workplace for our people has been recognized year after year.
Our FIT Magazine will give you an inside look at a career with KPMG, by reading about personal experiences, our culture and the limitless opportunities available with the firm.
This section is dedicated to special topics and interests. Each of these discrete sections present research, opinion and KPMG thought leadership.
Subscribe to receive email alerts when new content is available.
Subscribe now.


text:
Results 6 to 10 of 11
July 1st, 2013 09:40 AM #6
I'm with renrose on this one. Calista is great!
"Everybody is a genius. But if you judge a fish by it's ability to climb a tree, it will spend it's whole life believing that it is stupid." - Albert Einstein.
14, writer (http://www.wattpad.com/user/arboretum), and obsessed with names since forever.
Plucky Heroines - Lyla, Waverly, Caroline, Lucy, Linnea, Early, Arielle, Ariadne, Felicity 'Fliss', Livienne, Ketura 'Kit', Laura
Dashing Heroes - Waylon, Luca, Wilder,Stephen, Daniel, Lysander, Levi, Nathaniel, Adam
July 1st, 2013 06:25 PM #8
Callista is an awesome name, that's true, but here's the thing: what sort of parents would name one daughter Callista and the other one Ava? I know, I know, they're only half-sisters, but still...Callista is most-people-haven't-heard-it-before weird and Ava is an everyone-knows-it kinda name. Just saying. Also, I don't know if Ethan and Callista work together...hard to say. But that's just my opinion, which most people aren't fond of listening to, mostly because I have very odd opinions, in most cases, at least.
"I read books when I was a kid, lots of books. Books always seemed like magic to me. They took you to the most amazing places. When I got older, I realized I couldn't find books that took me to all the places I wanted to go. To go to those places, I had to write some books myself."
― Pat Murphy, The Wild Girls
"Like everyone else, I am going to die. But the words – the words live on..."
― J. Michael Straczynski
July 1st, 2013 10:27 PM #10
I think Kelsey works with all your standards.
courage is what it takes
to stand up and speak
courage is also what it takes
to sit down and listen
July 2nd, 2013 12:02 AM #12
Well, based on how she described the plot, the mother was the only one naming the main character, whereas the father/his wife named her half sister. Two completely different naming parties that weren't necessarily aware of each other.
However, I still like Kelsey.I'm just trying to behave as I think a friend should behave. Granted, I haven't had much practice.
~Elphaba Thropp, Wicked, by Gregory Maguire
July 2nd, 2013 08:30 AM #14Senior Member
- Join Date
- Apr 2013
- Posts
- 308
I like Kelsey.


text:
Biography Speech Titles Scheduling Honorarium
Tracy is the widow of Christopher Klicka, who was Senior Counsel of Home School Legal Defense Association. Married 21 years, they were blessed with seven children, whom they homeschooled since their birth. Through many trials over the years (including the near—death of their twins during her pregnancy, and Chris' struggles with multiple sclerosis), Tracy has learned firsthand what walking by faith is all about. She has seen over and over again the outpouring of God's grace in her life, and the precious importance of the Scriptures in sustaining and empowering believers. Her message for moms is one of hope and joy in God, who is able to make all grace abound to us (II Corinthians 9:8). She has written dozens of articles, has been interviewed on both national and international media, and has spoken at numerous state homeschool conventions to encourage women to see God's faithfulness as they take him at His Word.
Biography | Scheduling
If you would like to schedule Tracy Klicka as a speaker at an upcoming event where her husband, Chris, is scheduled to speak, please email her at email@example.com.
Tracy's requested fee is $500 for up to three workshops. There is no fee for her vocal performance at an event where she is scheduled to speak.


text:
Blue Book® Suggested Retail Value Fuel Economy (city/hwy) General PicturesAdditional Photo Galleries2014 Chevrolet SS GM returns to its roots with this big, powerful rear-wheel-drive car. Note: The 2013 Chevrolet Sonic LT Manual Sedan pictured below is representative of the 2013 Chevrolet Sonic LS Manual Sedan.
Video ReviewsThere are no 2013 Chevrolet Sonic video reviews. However, the following might be helpful: 2012 Chevrolet Sonic "The 2012 Chevrolet Sonic features an attractive design, impressive fuel economy, and selection in… Additional VideosChevrolet Cruze Advanced peek into Chevy's sharp-looking new compact 4-door sedan. advertisement |


text:
The Job You Selected Has Expired...
Our patented CareerBuilder.com matching technology takes the information in the job you selected and uses it to find other similar jobs for you.
Jobs like High School Math and Sceince Teacher at Sacramento Waldorf School (Expired)
We're sorry, but we cannot recommend any similar jobs. Please select a different job or try again later.


text:
Join Date: Sep 2005
Posts: 5
Duplicates in Calendar permalink
The calendar on my iPad2 is synced via iCloud to both of my Macs. For some reason, all of the events on the iPad2 calendar are duplicated. However, that is not the case on either the calendar on iCloud or my Macs.
Unfortunately, if I delete any of the dupes on the iPad2, that event will be completely deleted from both iCloud & my Macs (since they are all synced).
So, how can I delete the duplicated events on the iPad2 without affecting those same events on iCloud or my Macs,
Thank you kindly for any help.


text:
Share on: | |||
Other Media Options: | |||
User Services - Frequently Asked QuestionsMany of the common problems and questions that our User Services staff handle are included on these web pages and documents. Please check through the topics for a solution to your personal computing issue. If a solution is not found on these web pages our User Services group will be happy to help you. Please call the User Services Helpdesk at extension 1208, (518) 320-1208 or send your question via the Helpdesk Request Form.


text:
Loading... Market Forces (edition 2004)by Richard K. Morgan
Work detailsMarket Forces by Richard K. Morgan
Sign up for LibraryThing to find out whether you'll like this book. No current Talk conversations about this book. Market Forces offers a near future ruled de facto by corporations, where international geopolitics are no longer the arena of national governments as a means for dominance in long term, but instead a tool for corporate profits - cold, efficient and brutal battleground for money and nothing else. This has given a rise to a deadly competition between mega-companies, often expressed by permitted firefight/races to beat another to a deal. Interesting ideas, reflecting some of the fears of our times. I did find the world-building, set-up and description a bit lacking, and the action and fights are not on par with other works in the Altered Carbon series. Otherwise a decent book with enough action but most importantly, with key intriguing ideas that do strike some raw nerve, especially for those interested in current worldwide geopolitics and economy. For the latter alone - highly recommend it. P.S. I only gave a relatively low rating due to somewhat awkward execution, since the author did change gears and style from his established skill in high-tech mid-far future action. Would be cool to see him give something like this another go - I would read that. like reading brin's "kiln people" only with less connection with reality. despite the fact that the whole premise of the book is untenable, i found myself finishing the book. this is a different morgan, less profane, less angry, ultimately more charming. Morgan came so close to pulling this off, but fell at the final hurdle. Set in the 2050s this book tells of the rise of globalisation to such an extent that individual companies pout in rival bids to control politically or militarily sensitive areas in the world. The senior executives of these companies gain advancement by duelling with their colleagues, usually through the medium of car-borne dogfights. However, the characterisation was very weak - in fact, there wasn't one character for whom I felt any empathy - and stretched credibility beyond breaking point. I'd say a practicing free-market economist has blood on his hands, or he isn't doing his job properly. I did not enjoy this book. I read it all the way through, mostly because I was on vacation and had limited reading materials with me, but also because for some reason, I kept expecting it to get better. I guess after reading the Takeshi Kovacs books, I just expected more from Richard Morgan than this overblown frat boy's playground. Chris Faulkner is a rapidly advancing investment banker. But in Faulkner's line of work, investments aren't made in stocks or bonds, but rather in warring factions in third-world wars. Chris and his buddies make their money by betting on who's going to win, and then helping that side to do so. In an all-too-macho twist, the investment guys at Faulkner's firm don't get promotions based on their performance. Rather, they engage in "road rage duels" - duking it out on the streets of London in hyper-decked-out cars until someone dies. I get that this was supposed to represent a negative commentary on capitalism, but the premise of this novel was a total joke. Road rage duels? Are you kidding me? And I was so surprised by the macho attitude the entire book was predicated on - surprised, and offended. This was a huge departure from the seemingly progressive Richard Morgan of the Kovacs series, who seemed to lean towards not only feminism, but feminism of a radical bent. Perhaps in this novel, he is making fun of the corporate world's emphasis on masculinity, but even so, it was unpalatable to the extreme. There is not a single believable female character in the entire novel. The women that do exist are one-dimensional and fit snuggly into cliched stereotypes. I had rather enjoyed the other Richard Morgan novels I read. This was a piece of garbage. Hopefully whatever else he comes out with will more resemble the earlier stuff than this. Don't waste your time. Enjoying this! - But not as much as Altered Carbon. Strange violent future where contract negotiation is played out in a "Death Race 2000" kind of scenario on the roads. If you don't get killed you win the contract to "manage conflict" overseas (Like Afghanistan) but it needs to be profitable! :-) no reviews | add a review
References to this work on external resources. Wikipedia in English (2)Amazon.com Amazon.com Review (ISBN 0345457749, Paperback)Richard Morgan, the award-winning author of Altered Carbon and Broken Angels, strikes out into new territory with Market Forces, leaving behind the farflung battlegrounds of Takeshi Kovacs for the not-so-distant future of corporate Earth. Here, Morgan extrapolates a world where commodities trading reaches a brutal pitch and the outcomes of banana republic uprisings are the new market. Now, on the road to success, the brokers of the new economy compete for status and promotions via road rage on the freeways of new London.Morgan's conflicted protagonist, Chris Faulkner, is a comer known for one spectacular kill that shot him to the top of mid-range global capital firm. He parlays his reputation and skills as a driver into a job in the emerging field of "Conflict Investment" at the world's hottest and hardest firm. Soon he finds himself running with the big dogs and rises to the top of a brutal realm, but his ascent is quickly threatened by vicious senior partners, gold-digging suitors, fame, fair-weather friends, and his own nagging conscience. Market Forces is at once an anti-globalization treatise and anime fantasy meets The Road Warrior. Morgan employs the graphic-novel imagery of his two previous novels to create a disturbingly brutal picture of slash-and-burn capitalism run amok. There are times when Faulker's moral quandries seem hollow in the face of his actions but this isn't Crime and Punishment. Enjoy the ride and "come back with blood on your wheels or don't come back at all." --Jeremy Pugh Amazon.com Exclusive Content A Winning Translation: An Exclusive Essay by Richard Morgan His novels may paint a bleak picture of the future, but Richard Morgan has a great attitude toward language, and one word in particular. Read his Amazon.com exclusive essay and find out why he'll never consider himself, or anyone else, anything worse than an occasional non-winner. (retrieved from Amazon Mon, 30 Sep 2013 13:22:40 -0400) "Chris Faulkner has just landed the job of his life. But Shorn Associates are market leaders in Conflict Investment. They expect results, they expect the best. Chris has one very high-profile kill to his credit already, but he will have to drive hard and go for kill after kill if he's to keep his bosses happy. All he has to do in the meantime is keep his marriage alive. And himself..." "Thirty years from now the big movers in global capitalism have moved on from commodities. The big money is in Conflict Investment. And that's where Shorn Associates comes in. They keep a careful watch on the wars of liberation and revolution that burn constantly around the world. They gauge who the winners will be and sell them arms, intelligence and power. In return for a slice of the action when the war is won. The reward for the investors? A stake in the new nation." "It's cynical, brutal and it has nothing to do with democracy and the rule of law. So what else is new?" "The executives in this lethal game bid for contracts, fight for promotion, secure their lives on the roads. Fighting lethal duels in souped-up, heavily armoured cars on the empty motorways of the future." "Has Chris Faulkner got what it takes to make a real killing?"--BOOK JACKET.… (more) (summary from another edition) |
Google Books — Loading... Popular coversRatingAverage:
Is this you?Become a LibraryThing Author. |


text:
Search 0 items in your bag |
Women's TankinisIf bikinis and full swimsuits aren't your style, but you still want to wear something fashionable, then look no further than our range of stunning, plus size tankinis. With supportive, figure-flattering tops and control bottoms, it's easy to find a piece to make you feel beautiful. |


text:
The North Face Boys' Komit Short-Sleeve Performance Tee
Item # 702893
Key Features
Lightweight 100% polyester jersey VaporWick fabric
Designed to dry fast and wick away moisture
UPF 50 sun protection shields against harmful rays
Self-fabric color blocking creates visual interest
Flatlock stitching ensures chafe-free comfort
Our Price $25.00
Sale Price $17.50
Availability: Sold Out
Prices, promotions, styles and availability may vary by store and online.
back to topProduct Description
The North Face Komit Short-Sleeve Performance Tee is built to withstand whatever rugged outdoor adventures your little one puts it through. The t-shirt is made from lightweight 100% polyester jersey fabric that's bolstered with The North Face's exclusive VaporWick technology, ensuring fast drying and excellent moisture wicking. Built-in UPF 50 sun protection helps shields your child's skin against harmful UV rays. Self-fabric color blocking creates visual interest, while contrast flatlock stitching ensures chafe-free comfort all day long. A reflective logo at the left chest lends distinctive The North Face styling. Imported.


text:
Skip to Content
If you are seeking a world-class educational experience within a vibrant, Christ-centered learning community, Cedarville is the place for you. Step up to the challenge of our arts, sciences, professional and graduate programs, and achieve more than you ever imagined. Be challenged and transformed by professors who are experts in their fields and invest their lives at Cedarville to mentor and guide students like you.
Cedarville University is an accredited institutional member of the National Association of Schools of Music.
All | 2014 | 2013 | 2012 | 2011 | 2010 | 2009 | 2008 | 2007 | 2006
Found 194 matching news items.
Feb 21 2012Music Therapist, Dr. Deforia Lane, to Speak in ChapelDeforia Lane, Ph.D., believes in the healing power of music. As the director of music therapy at University Hospitals in... continue »
Feb 15 2012Annual Masterworks Concert Conducted by Peter Stafford WilsonMarch 23 marks the date for the annual Masterworks concert at Cedarville University. The Masterworks concert was founded in 2007... continue »
Feb 13 2012Over 650 Attend Worship Leader ConferenceOn January 27–28, 2012, worship leaders from across the region gathered at Cedarville University for a time of training and... continue »
Feb 9 2012Babbie Mason, Award-Winning Singer and Songwriter, Visits CedarvilleCedarville University is excited to welcome Babbie Mason to campus on Monday, February 20. Babbie is a nationally known,... continue »
Feb 6 2012Equipping Students with Excellence in Music and WorshipServing as assistant professor of music at Cedarville University, Patrick Anderson brings a unique set of talent, skill... continue »
Page 10 11 12 13 14 15 16 17 18 19


text:
Reds and 26.2's
Join Date: Jun 2003
Location: Gastonia, NC
Posts: 649
Is it ok to do this?
It's not really baseball related, but for the past year, I've really enjoyed reading what ya'll have to say here in Redszone. Reds fans have to be some of the most knowledgeable baseball fans anywhere, and it shows tremendously here. I don't post a lot, but I read nearly every thread and enjoy the banter.
Would it be ok to ask everyone to tell a little about themselves? Where you're from, what you do for a living, how many are old enough to remember the BRM, family? hobbies? stuff like that.. I know a lot of you live in the Cincy area and know each other personally, but some of us only know you from what you type on here. Hope this isn't too intrusive. As for me, I live in the Charlotte NC suburb of Gastonia, but I lived the first 28 years of my life in West Virginia. I work for a plant that manufactures Freightliner truck parts. I was a teen through the glorious '70's and I get to Cincinnati every year to watch a series. This year. it's the Pirates at the end of June. My other passion is running. I was in Cincinnati 3 weeks ago to run the Flying Pig Marathon in the cold rain! Let me know who you are! please! And Go REDS! |
Join Date: Apr 2000
Location: San Marcos, CA
Posts: 13,977
Re: Is it ok to do this?
Of course it's okay. I made it a sticky.
I'll be back. |
Join Date: Jan 2004
Location: Hamilton, Ohio
Posts: 266
Re: Is it ok to do this?
I don't mind answering you...In fact, I love to talk about myself.
Born and raised in Hamilton (home of Joe Nuxhall), Ohio. I'm 19 and I am a full time student at Miami University but to pay for that I work part time at Angilo's Pizza (a local pizza chain). I'm obviously not old enough to remember the machine but I love Reds history and know quite a bit about that era. I still live at home w/ my parents b/c it's cheaper than living on campus. I've never met anybody from Redszone personally (I'm fairly new to the site). Hobbies I guess I'd say are all sports (but baseball is especially my favorite). I guess my other hobby is music. I play guitar and piano. That's me in a nutshell. |
Go Reds Go!
Join Date: Nov 2002
Location: Northern KY
Posts: 1,856
Re: Is it ok to do this?
I'm 30 years old, for a few more days anyway, so I don't remember the glory days, though I am old enough to remember Johnny Bench as my favorite baseball player ever by far. I still remember being on a family camping trip at Natural Bridge when one of my great-uncles told me that # 5 was going to retire at the end of the season, and then I was at Johnny Bench Night, where I got to see him his his last home run. That's still the greatest single moment I've witnessed at a ballpark, and I'll never forget wanting to go to the 580 gift shop, where they sold home run balls, because I thought they would actually sell that ball there, LOL.
I live in Bellevue Kentucky, a short drive from GAB & work in downtown Cincy. So far, Opening Day has been the only game I've attended this year, but I'm sure I'll get to at least a couple more. I've not met anybody from this board, as I mostly lurk (only about 300-350 posts in a year and a half), but I really do enjoy the site. The "UK" in my name is for the University of Kentucky (congrats to UK alumnus Steve Flesch for winning on the PGA tour today), which is where I went to school and I, of course, love their basketball & football teams (though I wish the best for all the sports.) I also enjoy reading about the Civil War, which has always been a fascinating subject to me, and I enjoy working on genealogy as well.
http://www.civilwarobsession.com |
Smells Like Teen Spirit
Join Date: Apr 2004
Location: Phoenix
Posts: 6,490
Re: Is it ok to do this?
Hey this is a cool idea.
I'll keep it going. I'm 24 - I work as a design engineer for a defense contractor that makes commerical and military rockets and satellites. I've followed the Reds my entire life. I have family in the Cincy area, and every summer I'd fly back and catch a few games in Riverfront. My baseball highlights in life have been going to Riverfront each summer, as well as going to Dodger Stadium each year when the Reds came to town. I also have had the chance to watch Randy Johnson throw 20k's against the Reds, and the thrill of sitting in nosebleed watching the Dbacks win game 7 against the Yankees. Last summer I was privledged to travel to Cincy for a three game set against the Cubs, watching Paul Wilson go mano a mano w/ Kurt Farnsworth. I went to the Univ. of Arizona, so Wildcat Football is the only other sport that can get my blood boiling. I'm currently going to school for a masters in Mech Engineering. When I'm not studying, working, watching baseball or Coll. Football, I'm helping my gf run her small business as well as taking care of the small zoo she's accumulated over the last few years. |
Join Date: Apr 2000
Location: Nashville
Posts: 1,117
Re: Is it ok to do this?
I grew up in Cincinnati - went to the 61 world series w the Yankees as a nine yr old. played drums all my life too and still do, now professionally. I live in NYC and work in various studios / do touring / or do teaching- drum clinics or master classes.
my website is www.billyward.com I write articles for Modern Drumer magazine and I've just released an instructional drum DVD. The Reds aren't in it except for a few shots of me wearing a 1990 Champs shirt and another shot where i'm playing the drums with a Reds beach towel over the toms...oh ... there's a few times I'm wearing a Reds hat. Like many here, I read the posts almost each and everyday - but I post very little. You knuckleheads are far more clever and "on it" than I am! Someday, i hope to be able to meet more of you. Though you might not feel the same as I am what some of you might call a "dreaded liberal". :mhcky21: |
Join Date: Dec 2002
Location: Columbus, Ohio
Posts: 7,120
Re: Is it ok to do this?
I'll be thirty in less than a week, and I'm not happy about it.
I live in Columbus Ohio with my dog Casey, and my soon to be ex roomate. I'm an owner of a stupid little restaurant, and I work too much. When I'm not working, I buy lots and lots of records, go to see bands, and try to make out with my girlfriend as much as possible. For anyone living in the Columbus area, you can see my picture in next week's Columbus Alive. I wrote a short article about the best local records of all time. It was an excellent opportunity to plug my birthday show at the Ravari Room on May 29.
"....then Carps replacement the following year will win ROY and MVP and might get a CY young vote for pitching and playing 2nd base at the same time. I have come to expect those types of scenarios with the Cardinals and it makes me sick." - Lidspinner |
Join Date: Oct 2000
Location: PDX
Posts: 41,448
Re: Is it ok to do this?
Ronald Koal and the Trillionaires. |
Join Date: Apr 2003
Location: Ohio
Posts: 1,633
Re: Is it ok to do this?
I'm 18, and am graduating high school in a few days! I am attending the University of Dayton in the fall. My free time consists of a multitude of things. I play both ice and inline hockey, have been able to skate it seems like my whole life. I've been in various short lived bands with friends that never seem to last although I love my guitar and its a great therapeutic way for me to relax. I am also a big OOTP nut, been in as many as 8 leagues at once and loved every minute despite it killing all free time of mine. When I am not doing that, I am probably working (disinterested movie store kid ) or hanging out with my girlfriend of about two years. I used to pitch and IMO was well above average but I decided to stick with the hockey. I love playing hockey so much yet baseball will always be my true passion. I am not old enough to remember the BRM, heck I wasnt even alive, but I sure remember the 1990 Reds, even got to see us dominate Oakland. If that doesnt make you a baseball fan for life I dont know what will.
And yes! It is defiently ok to do this. |
You know his story
Join Date: Nov 2000
Location: Norfolk, VA
Posts: 7,713
Re: Is it ok to do this?
I moved to Cincy in 1978 at the tender age of 8. The BRM was the toast of major league baseball, but #14 had just moved on to Philly. Arriving here, I was disappointed that we'd missed each other. No doubt he felt the same.
Coming from Toledo, I'd never seen a major league game in person until the day I walked into Riverfront. It was indescribable. Unless you'd been to Riverfront, of course, in which case you know exactly what it was like. As an adult, you could see Riverfront's faults easily, but as a child, all you saw was the baseball. Green below, blue above, bright scoreboard beyond, and the occasional bugle call urging someone to charge. I was hooked. These days I make my home in Montgomery, which is a suburb of Cincinnati. Paul O'Neill lives about a mile north of me. Sean Casey lives about a mile west. So I get to see them from time to time at restaurants and such. (For fun, they pretend not to know me.) By day I work in advertising. By night I either pray for the homeless or drink wine and call talk radio shows, depending on my mood. And I've been pretty moody lately.
Makes all the routine posts. |
Join Date: Apr 2000
Location: Bright, Indiana USA
Posts: 5,573
Re: Is it ok to do this?
Born and raised in Cincinnnati. I was a kid when the BRM was around. I started collecting baseball cards as a kid in 1969 and that's when I began following the Reds. I remember being crushed when Lee May and Tommy Helms trade was announced. That trade became a very important lesson to me about baseball. Joe Morgan was my favorite player for a long time. I remember Dad waking us up to take us to the airport because the Reds were landing soon from the road trip portion of the World Series. My grandma loved the Reds. She would go to every double header she could, and she always stopped at the store before hand to buy the biggest bag of peanuts she could fit in her suitcase of a purse. She was, as I realized later, as infatuated with the Big Klu as creek is with Dunn. I can still hear her talk about how big his arms were. I learned about Klu from her, as he obviously out of baseball by my time. Marty and Joe being on the radio no matter what we are doing outside or in a vehicle is just a part of life. The Reds are my get-away from my everyday stressors. Oh and my baseball cards that I collected as a kid, I left most of them at home when I moved out, thinking they would be safe, Mom gave them to a neighbor kid. I still have a black and white 3 x 5 glossy photo of Pete Rose that is signed from then. The dozen or so cards that I took with me, my kids have.
Artificial intelligence is no match for natural stupidity. |
Join Date: Apr 2004
Location: Trenton, Ohio
Posts: 141
Re: Is it ok to do this?
I'm 38, from Trenton, I deliver pizza, and I'm old enough to remember the Reds playing in Crosley Field.
:al_cohol: |
Join Date: Mar 2002
Posts: 863
Re: Is it ok to do this?
My name is Brian, I am 19 years old and am a student at Mount Union College in Alliance, Ohio where I am trying to decide whether I want to be a history teacher and coach baseball or go into the sports marketing world, either way I want to be involved in the great game of baseball for life. I now live in Canton, Ohio and work at Mulligan's Irish Pub in Canton. I was raised in Mason, Ohio and just moved from there last summer. While living in Mason I went to countless games with my friends, buying top 6 and sneaking down into the better seats usually. My screename is pretty simple really, the summer ball team that I played for was the Mason Buzz and I was #3. Now that I am in college my baseball playing days are probaby behind me, but I'll be dominating on the softball fields in NE Ohio. I am not old enough to have witnessed the BRM first hand but have heard the stories from my father. I do remember the 1990 World Series though. Thats basically me, Reds fan for life.
Join Date: Apr 2004
Posts: 169
Re: Is it ok to do this?
I wear a red suit and fly a lot....seriously, I live in Falls City Nebraska (just about where Missouri, Iowa and Nebraska meet) but I was born in 1954 in Cincy and moved to Dayton as a youngster. I saw my first game in 1961 as a 7 year old and remember mostly my dad. About 1965 I really got hooked because I began playing myself, collecting cards and Derron "Iron Glove" Johnson was my favorite then. He was so bad he was a danger to himself in the field but I still loved him - mostly because he could mash the ball. I went to Stebbins HS, and for a year Dayton Chaminade. Ran away in 71 and joined the Marine Corps and wound up in Vietnam as soon as I was 18. Got home in time to see the Reds-Redsox in 75 getting tickets from a friend of friend who wanted to thank me for my service. Only thank you for Nam for about 20 years - somehow we were the disgraceful ones for serving as honorably as we could even if we didn't agree with much of it. Anyway, now I own and run 3 convenient stores and I'm about the only place between KC and Omaha that has Reds caps and Jerseys for sale in his store! You gotta go to a big city store like Champs to get em but I carry them (yeah, Royals, too, but that's a requisite out here). Still love the Reds - tried to get as interested in KC but it just doesn;t work. No team ever replaces the one of your youth, I think, no matter where you end up.
Join Date: Jun 2000
Location: TeamBoone's Attic
Posts: 12,317
Re: Is it ok to do this?
I'm 29 .... er .... um ..... 38. Raised in upstate NY. Grew up a Yankee fan. Became a Reds fan after a job transfer brought me to the Queen city about 10 years ago. TeamBoone brought me to my first game. I had to love the Reds or I couldn't be her friend anymore.
I work in cell and molecular biology research. I enjoy camping and fishing and love to get back to NY to taunt the bass population. Best moment is waking up on a lake before dawn. Hot coffee in hand. Dangling your feet into the chilly water. Mist rising. The only sounds are jumping fish and an occasional heron or loon. I envy old Katherine Hepburn on Golden Pond.
"A great democracy does not make it harder to vote than to buy an assault weapon." |
Thread Tools | |
Display Modes | |
Board Moderators may, at their discretion and judgment, delete and/or edit any messages that violate any of the following guidelines: 1. Explicit references to alleged illegal or unlawful acts. 2. Graphic sexual descriptions. 3. Racial or ethnic slurs. 4. Use of edgy language (including masked profanity). 5. Direct personal attacks, flames, fights, trolling, baiting, name-calling, general nuisance, excessive player criticism or anything along those lines. 6. Posting spam. 7. Each person may have only one user account. It is fine to be critical here - that's what this board is for. But let's not beat a subject or a player to death, please. |


text:
Download Your 2013 Summer Law Institute Application Here!
Applications for the 2013 Summer Law Institute should be completed and returned by March 25, 2013.
Applications are now being accepted for the LBA's annual Summer Law Institute, a law camp for high school students interested in the law or legal careers. The 2013 camp, scheduled June 9-15, is a partnership between the LBA, University of Louisville Brandeis School of Law and Bellarmine University. It provides a well-rounded intensive program, aimed at providing students insight into the profession.
For those who are accepted, tuition is $150 per student and some scholarships are available to those who qualify.
Questions about the program should be directed to Kate Lindsay at firstname.lastname@example.com or by calling (502)583-5314.
Copyright Louisville Bar Association. All rights reserved. Use of this Web site assumes acceptance of the Terms of Use. |


text:
Morris Whitehouse (1878-1944)
Over the course of his career, Morris Whitehouse designed many important buildings in Portland, the Willamette Valley, and the Columbia Gorge. Born in Portland on March 21, 1878, Whitehouse studied architecture at the Massachusetts Intstitute of Technology, completing his studies in 1905. After a year's study at the American Academy in Rome, he returned to Portland and established a practice. His parents had moved to Portland in 1859, and his father worked first for the Portland Water Company and then for the Portland Gas and Coke Company.
Between 1908 and 1944, Whitehouse entered partnerships with such well-known architects as Edgar Lazarus, Jacques Fouilhoux, Genn Stanton, and Walter Church. He had a reputation for assembling and effectively managing strong design teams. While working with Fouilhoux, the firm designed Portland's Jefferson (1910) and Lincoln (1911) High Schools, Multnomah Athletic Club (1911), University Club (1913), Waverly (1913) and Eastmorland (1918) Country Clubs, the Platt Building (1913), and 705 Davis Street Apartments (1913). With Stanton and Church, Whitehouse designed the Columbia River Gorge Hotel (1921), such notable Portland buildings as the U.S. Courthouse (1932), and Sixth Church of Christ, Scientist (1932). They also did the construction drawings for Temple Beth Israel (1927).
Whitehouse designed the Lake Oswego Country Club in 1923 and collaborated with A.E. Doyle on the Multnomah Stadium (1926). His firm also designed the First Presbyterian Church in Salem (1929). During the Great Depression, Whitehouse and Church worked on the new State Capitol Building in collaboration with Trowbride and Livingston (1936-1938); they also designed the Oregon State Library (1938-1939).
Whitehouse—trained in Beaux Arts historicism and influenced by an Italianate aesthetic from his study in Italy—expressed his stylistic preferences in his designs for the white stucco and red tile Columbia Gorge Hotel and his many clubhouse buildings. His design for the U.S. Courthouse can be seen as a refined or stripped version of Classical Renaissance Revival style.
Whitehouse also worked in more modern styles, such as Art Deco and Moderne. The lobby of the U.S. Courthouse is an impressive mix of classical and Art Deco elements. Cast bronze friezes and grilles in geometric and floral designs are set in marble and cherry and mahogany wood to great effect. The state capitol buildings reflect a similar blend of modern and stripped classical idioms.
The architectural firm Whitehouse established in 1907 continued under his successors for eighty years, making it the longest operating architectural office in Oregon. Whitehouse was active in the Portland Architectural Club and the Oregon chapter of the American Institute of Architects. He also served many years as a member of the Oregon State Board of Architectural Examiners. Whitehouse died in 1944.
Written by:William F. WillinghamFurther Reading:
Bosker, Gideon, and Lena Lencek. Frozen Music: A History of Portland Architecture. Portland: Oregon Historical Society Press, 1985.
King, Bart. An Architecutral Guidebook to Portland. 2d ed. Corvallis: Oregon State University Press, 2007.
Ritz, Richard Ellison, Architects of Oregon. Portland, Ore.: Lair Hill Publishing, 2003.


text:
Cover Story
A New Game Plan for Psychological Science
In the spirit of the "March Madness" college basketball tournament in the US, the Observer showcases the latest methodological innovations in the psychological research playbook.
Presidential Column
Igniting the BRAIN Initiative
The Obama administration's BRAIN initiative is driving remarkable collaboration among scientists from diverse fields. Guest columnist Miyoung Chun reflects on the project's origin, in which she played a seminal role.… More>
Giving Psychological Science Away Online
Sharing scientific findings on social media is more worthwhile — and less time-consuming — than many researchers think, says psychological scientist and science writer Jason Goldman. More>
Remembering Nalini Ambady
A world-renowned social psychologist is remembered for her inspiring mentorship and her groundbreaking research on nonverbal behavior and interpersonal judgments. More>
Stanford SPARQ Sparks Change
A new center honors Nalini Ambady's legacy by sharing social psychological innovations with people working to fight racism, poverty, and other societal problems. More>
Panel Calls for Explicit Rules on Human Subjects Research
Psychological and other social scientists recommend new regulatory definitions for human subjects research. More>
Achieving 'Good Article' Status in Wikipedia
Psychology professor Paula Marentette helped her students achieve a coveted designation on the collaborative online encyclopedia. More>
Money and Morality: Lack of Resources May Lead to Harsher Moral Judgments
Material resources, specifically income, have a sustaining impact on our lives. But new research reveals that our resources, or lack thereof, can also influence how we judge other people.
More>In Diversifying Neighborhoods, How Do Attitudes Shift?
Changes to demographics, housing policies, lending practices, and real estate markets are increasing the sociocultural diversity of many neighborhoods. How will neighborhood residents react to the changes?
More>APS Announces Second Replication Project: Proposals Due April 10, 2014
APS is pleased to announce a new Registered Replication Report project is under way.
More>Got a Dollar? You May Be Happier if You Spend it on Someone Else
A boost to income can increase happiness to a certain degree, but accumulating research suggests how you spend your money may be equally important as the amount you have.
More>Feats of Memory at the 2014 APS Annual Convention
At the 2014 APS Annual Convention, May 22–25 in San Francisco, attendees will have the chance to see the 2012 US Memory Champion in action.
More>Spatial Imagery Improves With Sight
Research with children who have treatable blindness shows that spatial imagery abilities don't depend on sight, but they vastly improve once sight is restored.
More>Taking a Transdiagnostic Approach to Understanding Self-Injury
Millions of people are affected by self-injury, especially adolescents and young adults. Researchers propose a four-function model to guide future research investigating the causes and consequences of self-injury.