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Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways†
Article first published online: 18 AUG 2011
DOI: 10.1002/jcb.23185
Copyright © 2011 Wiley-Liss, Inc.
Additional Information
How to Cite
Pichler, G., Wolf, P., Schmidt, C. S., Meilinger, D., Schneider, K., Frauer, C., Fellinger, K., Rottach, A. and Leonhardt, H. (2011), Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways. J. Cell. Biochem., 112: 2585–2593. doi: 10.1002/jcb.23185
- †
Publication History
- Issue published online: 18 AUG 2011
- Article first published online: 18 AUG 2011
- Accepted manuscript online: 19 MAY 2011 12:11PM EST
- Manuscript Accepted: 11 MAY 2011
- Manuscript Received: 1 APR 2011
Funded by
- Deutsche Forschungsgemeinschaft (DFG)
- Uhrf1;
- Uhrf2;
- DNA methylation;
- Histone modifications;
- Epigenetics
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Gene expression is regulated by DNA as well as histone modifications but the crosstalk and mechanistic link between these epigenetic signals are still poorly understood. Here we investigate the multi-domain protein Uhrf2 that is similar to Uhrf1, an essential cofactor of maintenance DNA methylation. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA, the substrate of maintenance methylation, and enhances binding to H3K9me3 heterochromatin marks. FRAP analyses revealed that localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor domain and depend on H3K9 trimethylation but not on DNA methylation. Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation. While uhrf1 is mainly expressed in pluripotent stem cells, uhrf2 is upregulated during differentiation and highly expressed in differentiated mouse tissues. Ectopic expression of Uhrf2 in uhrf1−/− embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences. We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells. J. Cell. Biochem. 112: 2585–2593, 2011. © 2011 Wiley-Liss, Inc.
DNA methylation and histone modifications are major epigenetic marks involved in the regulation of gene expression, inheritance of chromatin states, genome stability, and cellular differentiation [Bird, 2002; Kouzarides, 2007; Reik, 2007]. Misregulation of epigenetic pathways, like erroneous DNA methylation, may lead to cancer and other diseases [Jones and Baylin, 2007]. Open questions concern the crosstalk and mechanistic link between different epigenetic signals.
Genome-scale DNA methylation studies revealed a connection between DNA methylation and histone modifications. Specifically, DNA methylation correlates with the absence of H3K4 methylation and presence of H3K9 methylation [Meissner et al., 2008]. This correlation may in part be caused by DNA methyltransferases specifically recognizing histone modifications. For instance, the de novo DNA methyltransferase Dnmt3a and its cofactor Dnmt3L specifically recognize unmethylated H3K4 mediated by the ATRX-Dnmt3-Dnmt3L (ADD) domain [Ooi et al., 2007; Otani et al., 2009]. Dnmt1, which is involved in maintenance methylation during DNA replication and DNA repair [Leonhardt et al., 1992; Mortusewicz et al., 2005], specifically methylates hemimethylated DNA [Bestor and Ingram, 1983; Pradhan et al., 1997] and associates with constitutive heterochromatin via its targeting sequence (TS) domain [Easwaran et al., 2004].
Recently, Uhrf1 (also known as Np95 or ICBP90) has been shown to link DNA and histone modifications and has emerged as an essential cofactor for the maintenance of genomic DNA methylation. Genetic ablation of uhrf1 leads to remarkable genomic hypomethylation, a phenotype similar to dnmt1−/− embryonic stem cells (ESCs) [Bostick et al., 2007; Sharif et al., 2007]. Uhrf1 binds hemimethylated DNA via a SET and RING associated domain (SRA) domain and targets Dnmt1 to its substrate of maintenance DNA methylation [Bostick et al., 2007; Sharif et al., 2007; Arita et al., 2008; Avvakumov et al., 2008; Hashimoto et al., 2008; Qian et al., 2008; Rottach et al., 2010]. This targeting activity of Uhrf1 is based on specific binding to the heterochromatin mark H3K9me3 via a tandem Tudor domain (TTD) [Karagianni et al., 2008; Rottach et al., 2010]. In addition, Uhrf1 interacts with Dnmt3a and Dnmt3b and with histone modifying enzymes like HDAC1, G9a, and Tip60 [Unoki et al., 2004; Achour et al., 2009; Kim et al., 2009; Meilinger et al., 2009]. Finally, Uhrf1 displays E3 ubiquitin ligase activity for histone H3 [Citterio et al., 2004] and is involved in large scale reorganization of chromocenters [Papait et al., 2008].
Interestingly, a second member of the Uhrf family, Uhrf2, harbors similar domains [Bronner et al., 2007]. Until now, the only known function of Uhrf2 is a role in intranuclear degradation of polyglutamine aggregates [Iwata et al., 2009]. In this study, we systematically investigated the function and interplay of distinct Uhrf2 domains in DNA and histone tail substrate recognition and report first hints on cell-type specific functions of Uhrf1 and Uhrf2.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Expression Constructs
Expression constructs for GFP, RFP-PCNA, Uhrf1-GFP, and GFP constructs of Dnmt1 were described previously [Sporbert et al., 2005; Fellinger et al., 2009; Meilinger et al., 2009]. All Uhrf2 expression constructs were derived by PCR from mouse uhrf2-myc cDNA (MR210744, ORIGENE). To obtain GFP fusion constructs, the uhrf1 cDNA [Rottach et al., 2010] was replaced by uhrf2 encoding PCR fragments in the pCAG-uhrf1-GFP vector. The deletion and point mutant expression constructs were derived from the corresponding wild-type constructs by overlap extension PCR [Ho et al., 1989] and PCR-based mutagenesis. The following start and end amino acids were chosen: Uhrf2 tandem Tudor domain, amino acids 118–312; Uhrf2 PHD domain, amino acids 325–395; Uhrf2 tandem Tudor–PHD domain, amino acids 118–395; Uhrf1 tandem Tudor–PHD domain, amino acids 121–370. The linker exchange constructs were derived by PCR using overlapping primers that contained the partial linker sequence. Amino acid sequences of the linkers: Uhrf1: KERRPLIASPSQPPA; Uhrf2: GAHPISFADGKF. All constructs were verified by DNA sequencing. Throughout this study enhanced GFP constructs were used and for simplicity referred to as GFP fusions.
Cell Culture, Transfection, Cell Sorting, and Differentiation
HEK293T cells, MEFs, and ESCs were cultured and transfected as described [Schermelleh et al., 2007; Rottach et al., 2010] with the exception that Lipofectamin (Invitrogen) was used for transfection of MEFs. E14 uhrf1−/− ESCs were transfected with Uhrf1-GFP and Uhrf2-GFP expression constructs using FuGENE HD (Roche) according to the manufacturer's instructions. ESCs were sorted for GFP positive cells 48 h after transfection with a FACS Aria II instrument (Becton Dikinson). ESC strains wt E14, wt J1, and E14 uhrf1−/− were cultured and differentiated to embryoid bodies as described [Szwagierczak et al., 2010]. The ESC strain wt JM8A3.N1 (EUCOMM, Germany) was cultured in Knockout D-MEM (Gibco-BRL, Grand-Island, NY) medium containing 10% fetal bovine serum (PAA Laboratories GmbH, Austria), 0.1 mM β-mercaptoethanol (Gibco-BRL), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (PAA Laboratories GmbH). The medium was supplemented with 1,000 U/ml recombinant mouse LIF (Millipore, Temecula, CA).
RNA Isolation, cDNA Synthesis, and Quantitative Real-Time PCR
RNA isolation and cDNA synthesis were performed as described [Szwagierczak et al., 2010]. Equal amounts of cDNA were used for Real-time PCR with TaqMan Gene Expression Master Mix (Applied Biosystems) on the 7500 Fast Real-time PCR System (Applied Biosystems) according to the manufacturer's instructions. The following TaqMan Gene expression assays were used: Gapdh (Assay ID: Mm99999915_g1), uhrf1 (Assay ID: Mm00477865_m1) and uhrf2 (Assay ID: Mm00520043_m1). Gene expression levels were normalized to Gapdh and calculated using the comparative CT Method (ΔΔCT Method).
In Vitro DNA Binding and Histone-Tail Peptide Binding Assay
The in vitro binding assays were performed as described previously [Frauer and Leonhardt, 2009; Rottach et al., 2010]. NoCpG DNA substrates were produced in a primer extension reaction [Frauer and Leonhardt, 2009] others by hybridization of two DNA oligos (Supplementary Fig. S7B–D). Histone-tail peptides were purchased as TAMRA conjugates (PSL, Germany; Supplementary Fig. S7A). Peptides were added in a molar ratio 1.5:1 (peptide/GFP fusion) and the binding reaction was performed at RT for 15 min with constant mixing. For combined assays, samples were additionally incubated with either H3K9me3 or H3K9ac histone-tail peptides in a molar ratio 1.5:1 (peptide/GFP fusion) or increasing amount of DNA substrate as indicated. The binding reaction was performed at RT for 60 min with constant mixing.
Immunoflourescence Staining and Antibodies
For immunostaining, MEF cells and ESCs were grown on cover slips and transiently transfected with Uhrf2-GFP (MEF cells), or co-transfected with Uhrf2-GFP and RFP-PCNA (ESCs). Cells were fixed with 2.0% or 3.7% formaldehyde in PBS and permeabilized in PBS containing 0.2% Triton X-100. The post-translational histone modification H3K9me3 was detected via a rabbit primary antibody (Active Motif) and a secondary anti-rabbit antibody conjugated to Alexa Fluor 594 (Molecular Probes, Eugene, OR). The antibodies were diluted 1:1,000 or 1:500, respectively, in PBS containing 0.02% Tween-20 and 2% BSA. GFP-Binder (ChromoTek, Germany) was used to boost GFP signals and was labeled with Alexa Fluor 488. Cells were counterstained with DAPI and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images of the cells were obtained using a TCS SP5 AOBS confocal laser scanning microscope (Leica, Wetzlar, Germany) with a 63x/1.4 NA Plan-Apochromat oil immersion objective. GFP, Alexa Fluor 488, RFP, and Alexa Fluor 594 were excited with a 488-nm argon laser and a 561-nm diode laser, respectively. Image series were recorded with a frame size of 512 × 512 pixels, a pixel size of 100 nm and with a detection pinhole size of 1 Airy Unit.
Live Cell Microscopy and Fluorescence Recovery After Photobleaching (FRAP) Analysis
Live cell imaging and FRAP analyses were performed as described [Schermelleh et al., 2007] with the exception that imported images were intensity normalized, converted to 8-bit and Gauss-filtered (2 pixel radius). Data sets showing lateral movement were corrected by image registration using the StackReg plug-in of ImageJ [Abramoff et al., 2004] starting with a frame when approximately half recovery was reached. Within the first 30 s after bleaching, images were taken every 150 ms and then in intervals of 1 s.
DNA Methylation Analysis
Genomic DNA was isolated with the QIAmp DNA Mini Kit (Qiagen) and 1.5 µg were bisulfite converted using the EZ DNA Methylation-Gold Kit (Zymo research) according to the manufacturer's instructions. Primer sequences for major satellites were AAAATGAGAAACATCCACTTG (forward primer) and CCATGATTTTCAGTTTTCTT (reverse primer). For amplification we used Qiagen Hot Start Polymerase in 1× Qiagen Hot Start Polymerase buffer supplemented with 0.2 mM dNTPs, 0.2 µM forward primer, 0.2 µM reverse primer, 1.3 mM betaine (Sigma) and 60 mM tetramethylammonium-chloride (TMAC, Sigma). Major satellites were amplified in a single amplification and pyrosequencing reactions were carried out by Varionostic GmbH (Ulm, Germany).
Statistical Analysis
Results were expressed as means ± SD or means ± SEM. The difference between two mean values was analyzed by Student's t-test and was considered as statistically significant in case of P < 0.05 (*) and highly significant for P < 0.001 (**).
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Opposite Expression Pattern of uhrf1 and uhrf2 During Differentiation
Recently, Uhrf1 has emerged as an essential factor for the maintenance of DNA methylation. Sequence analyses revealed that Uhrf2 harbors five recognizable domains similar to Uhrf1 (Fig. 1A), but its role in the regulation of DNA methylation is still unclear. We compared the expression pattern of uhrf1 and uhrf2 in ESCs and somatic cells, during differentiation and in differentiated mouse tissues (Fig. 1B–D and Supplementary Fig. S1). Interestingly, both genes show opposite expression patterns; while uhrf1 is expressed in ESCs and down regulated during differentiation, which is consistent with previous reports [Muto et al., 1995; Fujimori et al., 1998; Hopfner et al., 2000], uhrf2 is upregulated and highly expressed in differentiated mouse tissues. The switch in the expression pattern argues against a functional redundancy of both genes and is consistent with the drastic loss of DNA methylation in uhrf1−/− ESCs despite the presence of intact uhrf2 alleles. Therefore, the opposite expression pattern of both genes suggests different functional roles of uhrf1 and uhrf2 in development.
Cooperative Binding of Repressive Epigenetic Marks by Uhrf2
To investigate DNA and histone-tail binding preferences of Uhrf2 in vitro, we used a versatile binding assay developed for GFP fusion proteins [Rothbauer et al., 2008; Frauer and Leonhardt, 2009; Rottach et al., 2010]. Similar to Uhrf1, histone-tail peptide binding assays revealed that Uhrf2 preferentially binds to H3(1–20) and H3K9me3 peptides (Fig. 2A). This binding activity of Uhrf2 is mediated by the TTD but not the PHD domain (Fig. 2B). Consistently, acetylation of H3K9, underrepresented in heterochromatin, prevented the binding of Uhrf2 and its TTD. The binding of Uhrf1 to H3K9me3 is mediated by an aromatic cage in the TTD [Rottach et al., 2010]. Site-directed mutagenesis of Uhrf2 changing the two conserved tyrosine residues to alanine (Y214A Y217A) (Supplementary Fig. S2) abolished specific peptide binding (Fig. 2B) and supports a function of the aromatic cage in H3K9me3 recognition.
Whereas Uhrf1 preferentially binds to hemimethylated DNA, Uhrf2 failed to show a preference for hemi-over unmethylated DNA (Fig. 2C). These differences in DNA binding preferences between Uhrf1 and Uhrf2 were confirmed by electrophoretic mobility shifts (Supplementary Fig. S3). To further investigate the functional interplay between DNA and histone binding we performed combined binding assays (Fig. 2D). Interestingly, binding to heterochromatin-specific H3K9me3 peptides induced a significant preference of Uhrf2 for hemi-over unmethylated DNA. Uhrf1 already on its own showed preference for hemimethylated DNA that was further enhanced by binding to H3K9me3 peptides. To test the specificity of this cooperativity we mutated the aromatic cage in Uhrf2 that is necessary for H3K9me3 histone-tail peptide binding. The mutated Uhrf2 (Y214A Y217A) showed comparable DNA binding activity as the wild-type Uhrf2 but addition of heterochromatin-specific H3K9me3 peptides did not induce preference for hemi-over unmethylated DNA (Fig. 2D).
In the reverse experiment, addition of DNA enhanced binding of Uhrf1 and Uhrf2 to the H3K9me3 peptide (Fig. 2E,F). This was not observed for the DNA binding mutant of Uhrf1 (Uhrf1ΔSRA) which showed constant peptide binding with increasing DNA concentrations. These findings suggest that single binding events of distinct Uhrf2 domains lead to multivalent engagement of different repressive epigenetic marks. In fact, multivalent engagement of DNA and histone tail peptides via the SRA domain and the TTD, respectively, results in affinity enhancement and additional specificity for hemimethylated DNA, the substrate of maintenance methylation.
Cellular Localization and Dynamics of Uhrf2 Depend on Histone H3K9 Methylation
To monitor the subcellular localization of Uhrf2, we expressed Uhrf2-GFP constructs in cells with different genetic backgrounds. In wild type (wt) ESCs, Uhrf2 is localized in the nucleus and is enriched at pericentric heterochromatin (PH) (Fig. 3A,B and Supplementary Fig. S4A–C). To investigate which epigenetic marks at PH are recognized by Uhrf2 we determined the localization of Uhrf2 in genetically modified ESCs either lacking all three major DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) [Tsumura et al., 2006] or ESCs lacking the two major H3K9 methyltransferases Suv39H1/H2 (Suv39h dn) [Lehnertz et al., 2003]. TKO cells are practically devoid of genomic DNA methylation and Suv39h dn ESCs show substantially reduced H3K9me3 levels. We found Uhrf2 localized at PH in TKO but not in Suv39h dn ESCs, indicating that localization of Uhrf2 is dependent on H3K9 but not on DNA methylation (Fig. 3A). Consistently, immunostaining of wt mouse embryonic fibroblasts (MEFs) showed co-localization of Uhrf2 and H3K9me3 marks at PH, which was not observed in Suv39h dn MEFs [Peters et al., 2001] (Fig. 3B). Also, mutations in the TTD (Uhrf2 Y214A Y217A) that abolished binding to H3K9me3 peptides in vitro disrupted enrichment at PH in wt MEFs (Fig. 3B). The dependence of Uhrf2 localization on H3K9me3 was also confirmed by quantitative correlation analysis (Supplementary Fig. S4D,E).
To investigate the effect of H3K9me3 on the dynamics of Uhrf2 in living cells we performed quantitative fluorescence recovery after photobleaching (FRAP) analyses in wt and Suv39h dn MEFs. We chose to bleach half nuclei to include a representative number of interactions from different nuclear domains and structures in the bleached area [Rottach et al., 2010]. Recovery of Uhrf2-GFP fluorescence in Suv39h dn MEFs (half-time t1/2 = 5.9 ± 0.6 s) and of the TTD mutant in wt MEFs (t1/2 = 3.2 ± 0.4 s) was considerably faster than the recovery of Uhrf2-GFP in wt MEFs (t1/2 = 11.8 ± 0.6 s) pointing to a crucial role of H3K9me3 in Uhrf2 dynamics in living cells (Fig. 3C). Taken together, these results clearly demonstrate that the interaction of Uhrf2 with the heterochromatin mark H3K9me3 is required for the localization at PH and affects binding dynamics in living cells.
Cooperative Binding of the Combined Uhrf2 TTD–PHD Domain
Recently, several studies showed multivalent binding to histone-tail peptides [Ruthenburg et al., 2007]. In case of Uhrf1 and Uhrf2, the TTD is followed by a second histone-tail binding domain, a PHD domain (Fig. 1A). As the isolated PHD domains of Uhrf1 and Uhrf2 did not show binding to H3 histone-tail peptides (Fig. 2B) [Rottach et al., 2010], we tested whether the combination of the PHD and the TTD results in cooperative histone-tail binding. Surprisingly, the combined TTD–PHD domain of Uhrf2 displayed a fourfold increased binding to H3K9me2/me3 in comparison to the single TTD, which was not observed for the corresponding construct of Uhrf1 (Figs. 2B and 4A).
Sequence alignments of the combined domains revealed two striking differences between Uhrf1 and Uhrf2. Firstly, Uhrf2 harbors an additional stretch of 33 highly conserved amino acids present in the TTD (Supplementary Fig. S5A). Secondly, the linker region between the TTD and PHD domain of Uhrf2 is highly conserved, whereas this region is highly diverse in Uhrf1 (Supplementary Fig. S5A). To test which sequence is responsible for the observed cooperative interplay between PHD and TTD, we generated and tested different hybrid and deletion constructs (Supplementary Fig. S5B). Notably, replacement of the native linker in the Uhrf2 TTD–PHD construct by the Uhrf1 linker caused decreased relative binding ratios to H3K9me2/3 comparable to the single Uhrf2 TTD (Fig. 4B). Transferring the Uhrf2 linker to the Uhrf1 TTD–PHD construct as well as deletion of the Uhrf2 stretch region did not affect the binding to H3K9me3 peptides (Fig. 4B).
These results suggest that the cooperative interplay of different Uhrf2 domains, which is responsible for the increased binding to heterochromatin marks, is dependent on the highly conserved linker region connecting the TTD and PHD domains. A similar functional importance of linker sequences has been described for BPTF and histone lysine demethylases [Li et al., 2006; Horton et al., 2010].
Uhrf1 and Uhrf2 Are Not Functionally Redundant in ESCs
To investigate whether Uhrf1 and Uhrf2 are functionally redundant we performed interaction and rescue assays. Like Uhrf1, also Uhrf2 interacts with Dnmts (Supplementary Fig. S6) suggesting a similar function in DNA methylation. To test for such a functional role, we ectopically expressed Uhrf2-GFP or Uhrf1-GFP in uhrf1−/− ESCs and determined DNA methylation levels at major satellites by pyrosequencing. While ectopic expression of Uhrf1-GFP led to significant increase of DNA methylation levels at CpG sites of major satellite DNA in uhrf1−/− ESCs, Uhrf2-GFP did not restore DNA methylation at these sites (Fig. 5). These results point to functional differences between Uhrf1 and Uhrf2 in vivo.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Over the past decades many different histone modifications were discovered that are involved in epigenetic gene regulation. A key question is how these histone marks are linked to DNA methylation pattern and how this complex epigenetic information is integrated and translated into defined chromatin structures and gene expression levels. Epigenetic regulators that bind DNA and histone marks are ideally suited to link these pathways and intramolecular interactions between different binding domains may contribute to substrate specificity and epigenetic regulation [Hashimoto et al., 2009].
Recently, Uhrf1, an essential factor for the maintenance of DNA methylation, has been shown to bind to repressive DNA and histone modifications via an SRA and a tandem Tudor domain, respectively. Here we provide the first systematic characterization of the second member of the Uhrf family, Uhrf2, and demonstrate that Uhrf2 binds to the H3K9me3 heterochromatin mark via an aromatic cage of a tandem Tudor domain (TTD). Mutations in the aromatic cage abolished binding to H3K9me3 histone-tail peptides in vitro and prevented enrichment of Uhrf2 at pericentric heterochromatin in vivo. Interestingly, similar mutations in the aromatic cage of Uhrf1 prevented repression of p16INK4A [Nady et al., 2011] suggesting a link between H3K9me3 binding and a function of Uhrf proteins in gene repression.
Our results point to a complex regulation of substrate recognition by Uhrf2 involving cooperative binding domains and critical linker sequences. In contrast to Uhrf1, preferential binding of Uhrf2 to hemimethylated DNA, the substrate of DNA maintenance methylation, was only induced upon simultaneous binding to H3K9me3 histone-tail peptides. Binding of Uhrf1 and Uhrf2 to DNA in turn enhanced binding to H3K9me3 histone-tail peptides. Consistently, SILAC-based proteomic analysis identified enrichment of UHRF1 at nucleosomes containing repressive DNA and H3K9 methylation marks [Bartke et al., 2010]. Together, these data demonstrate a cooperative interplay between DNA and histone tail binding domains of Uhrf1 and Uhrf2. A similar effect was reported for MSL3 that specifically binds to H4K20me1 via a chromodomain only in the presence of DNA [Kim et al., 2010].
An additional level of complexity was added by recent studies showing multivalent binding of histone-tail peptides by mixed two-effector modules [Ruthenburg et al., 2007]. Notably, the combined TTD–PHD domain of Uhrf2, but not of Uhrf1, showed enhanced binding to H3K9me3 histone-tail peptides. This cooperativity was dependent on the highly conserved linker region connecting the TTD and PHD domains. Similarly, an important role was attributed to the linker sequence between the histone binding domain (PHD) and the histone modifying domain of jumanji histone lysine demethylases [Horton et al., 2010].
The dramatic loss of DNA methylation in uhrf1−/− ESCs [Bostick et al., 2007; Sharif et al., 2007] is remarkable, especially considering the presence of the uhrf2 gene, which encodes a highly similar protein as demonstrated in this study. As one possible explanation for this lack of functional redundancy we found, in contrast to uhrf1, relatively low uhrf2 mRNA levels in ESCs, which were not affected by genetic uhrf1 ablation. Moreover, both genes also show opposite expression patterns during differentiation. The failure of ectopically expressed Uhrf2 to restore DNA methylation in uhrf1 deficient cells clearly points to functional differences between both proteins in vivo. However, more definitive insights into the specific function(s) of Uhrf2 will require targeted mutations and subsequent analyses of pluripotent as well as differentiated cells. Based on the cooperative binding of Uhrf2 domains to repressive DNA and histone marks we propose that Uhrf2 might contribute to a tighter control of gene repression in differentiated cells as compared to a less stringent control by Uhrf1 in pluripotent ESCs.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
The authors are grateful to En Li (Novartis Institutes for Biomedical Research, Boston, MA) for J1 ESCs, to Masaki Okano (RIKEN Center for Developmental Biology, Kobe, Japan) for TKO ESCs, to Thomas Jenuwein (Max-Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany) for the Suv39h dn MEFs and to Gunnar Schotta (Adolf-Butenandt-Institute, Faculty of Medicine, Ludwig Maximilians University Munich, Germany) for the Suv39h dn ESCs. This work was supported by the Nanosystems Initiative Munich (NIM) and by grants from the Deutsche Forschungsgemeinschaft (DFG) SFB646 and SFB684 to H.L. G.P., C.S.S., K.S., and C.F. were supported by the International Doctorate Program NanoBioTechnology (IDK-NBT) and the International Max Planck Research School for Molecular and Cellular Life Sciences (IMPRS-LS). PW is a fellow of the Graduate School Life Science Munich (LSM).
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
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Supporting Information
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- Abstract
- Acknowledgements
- Supporting Information
Additional Supporting information may be found in the online version of this article.
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jcb_23185_sm_SuppInfo.pdf | 1929K | Supplemental Information |
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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RE: transfer of memorial request
Hi Libby, I've transferred management of the entire Hanrahan clan so that you can create links, etc. Take care, Ann
Added by
on Nov 20, 2014 7:53 PM
Glenn Fleming
O.Frank & Ida Waddell
Thank you so very much for the wonderful pictures of Frank & Ida's names in the Woodland Cemetery Mausoleum.I very much appreciate your hard work.
Best regards,
Glenn Fleming
Added by
Glenn Fleming
on May 27, 2014 2:46 PM
Mark Schornstein
Thank You!
Thanks so much for the 4 photos I requested from the Quincy Woodland Mausoleum (Starmann and Shulian husband and wives). I've been back to Quincy several times, but it seems I never had enough time to make an appointment to get inside the Mausoleum to get my photos. Your help to me is greatly appreciated.
Many Blessings to You!
Mark Schornstein
Added by
Mark Schornstein
on May 25, 2014 8:12 PM
John Gavelek
Xfer Julius Gavelek
Please xfer control to me when you get a chance.
Added by
John Gavelek
on Apr 21, 2014 4:21 PM
Cathy Dohm Young
RE: Flora Cabell
Thank you so much for photographing these headstones! I know the weather we have had this winter in Illinois. You must have taken your very first opportunity to do this and I really appreciate it. Looking up Flora Cabell's obituary was very kind of you also. I will post it and more about this family when I get the opportunity.
Added by
Cathy Dohm Young
on Mar 20, 2014 1:21 PM
Cathy Dohm Young
RE: Flora Cabell
Hi Libby,
Thank you so much for Flora Cabell obituary. I wish ALL obituaries said where the burial was going to be.
Added by
Cathy Dohm Young
on Mar 20, 2014 1:15 PM
Geri Hemp Mercer
RE: Davis family
Thank you.
Added by
Geri Hemp Mercer
on Nov 15, 2013 12:31 PM
Geri Hemp Mercer
Davis, Henry Cornelius
would you be able to add the link to Henry Davis' parents?
Cornelius Davis #120066297 and Catherine Davis 120066503 ? thank you.
Added by
Geri Hemp Mercer
on Nov 09, 2013 5:10 PM
Bonnie Parker
RE: George Hopkins
Thanks for posting the inscription!
Added by
Bonnie Parker
on Aug 12, 2013 6:46 AM
Connie Clark
RE: Alvine & Thomas Reid
You are very welcome Libby, it was our pleasure!! This cemetery was close to us, there were other Reid's there, already have photos :) take care, Connie and Wayne
Added by
Connie Clark
on Jun 08, 2013 1:24 PM
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Follow me... but not that close! By Faye Brozek on 26 January 2015 BMX Mike 'Hucker' Clark in mid-air collision - when riding with your mate gets too close for comfort.
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Raditudes: Season ends at Decoy Jam Season 2014, Episode 10 Decoy Jam in England is the backdrop to the final episode of Raditudes' premiere season.
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Raditudes: Vans Kill the Line Comp Season 2014, Episode 8 Anthony and Hucker are in France for the Vans Kill the Line competition in Peynier.


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Join Steve Corkran as he talks about how potential Raiders draft picks fared at the NFL Combine, and which of the players would fit best in Oakland.
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) Eric Church, Luke Bryan and the new kings of country Jason Aldean, Dierks Bentley also among the top talents in country music. |
(Los Angeles Times/Ricardo DeAratanha.)) Did Tom DeLonge quit Blink-182? Mark Hoppus and Travis Barker say Alkaline Trio guitarist Matt Skiba will play next show with band. |


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Jacob's Well Thrift Store
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Jacob's Well Thrift Store - This companies industries/categories
Primary SIC Code : 59320000
This major group includes retail establishments not elsewhere
classified. These establishments fall into the following
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vehicles, trailers, and boats are classified in major group 55; and
those primarily selling used mobile homes in SIC 5271.
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are classified in SIC 5093. Establishments primarily engaged in
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James H. Plott, 85, of Ooltewah, died on Thursday, January 24, 2013, at his home.
James was a graduate of Ooltewah High School and attended the University of Tennessee in Chattanooga.
He was a staff sergeant in the United States Air Force specializing in propeller instruction; and a member of the American Legion Post 247. James was also a member of Harrison Masonic Lodge 114 where he served as Worshipful Master and attained the level of 32nd Degree Mason. He was the Past Worthy Patron of Harrison Eastern Star #57.
James owned and operated his own business, Plott's Auto Repair Shop for over 50 years. He was a member of Fairview Baptist Church where he served as a deacon and treasurer. For many years he was the Precinct Chairman over the Ooltewah #1 Precinct for elections.
He was preceded in death by his parents, James Lee Plott and Nannie Boyd Plott; and first wife, June Scoggins Plott.
Pallbearers will be James' cousins, Francis Rogers, Charles Rogers, Jim Rogers, Ronnie Rogers, Steve Rogers and David Dill.
Funeral services will be held at 11 a.m. on Saturday, Jan. 26, in the Valley View Chapel of Chattanooga Funeral Home with Reverend Richard Jones and Reverend James Vaughn officiating. Burial will follow in Fairview Baptist Church Cemetery.
The family will receive friends from 2–4 and 6-8 p.m. on Friday, Jan. 25, at the funeral home.
The James Plott family would like to express their gratitude for the excellent and compassionate care the Avalon Hospice gave to their loved one.
Please share your thoughts and memories online at www.ChattanoogaValleyViewChapel.com.
Arrangements are by Chattanooga Funeral Home, Crematory & Florist, Valley View Chapel, 7414 Old Lee Highway.
January 29, 2015
Iva Marie Williams Bickford, born on September 23, 1927 in Dunlap, died on Thursday, January 29, 2015 at her home in Rossville, Georgia. She was a very strong God loving woman and faithful ... (click for more)
Marian Grace Elrod Maxwell, 86, of Chattanooga, died on Wednesday, January 28, 2015. Marian was a lifelong resident of the Chattanooga area and a 1945 graduate of Central High School. She ... (click for more)
Allen "Randy" Rand Niswonger, 57, of Ringgold, Georgia died on Tuesday, January 27, 2015. A resident of the Ringgold area for the past five years, he was formerly of Milan, Ga. Randy was ... (click for more)
Iva Marie Williams Bickford, born on September 23, 1927 in Dunlap, died on Thursday, January 29, 2015 at her home in Rossville, Georgia. She was a very strong God loving woman and faithful to her church and its members at Rossville Church of Christ. She was preceded in death by her husband, Tom Bickford; her brother, Cecil Williams; sister, Velma L. Holland. She is survived ... (click for more)
Marian Grace Elrod Maxwell, 86, of Chattanooga, died on Wednesday, January 28, 2015. Marian was a lifelong resident of the Chattanooga area and a 1945 graduate of Central High School. She was a member of First Baptist Church Golden Gateway and the Mary Sunday School Class. She was preceded in death by her husband, Kermit Y. Maxwell, DDS; step-son, Ron ... (click for more)
William Crandel McNabb, 101, of Dunlap, Tennessee, died Wednesday, January 28, 2015. The son of the late Thomas Crawford and Florence Almeda Knight McNabb, he was a graduate of Grundy County High School where he drove the school bus while in high school. During World War II, he worked for Combustion Engineering in Chattanooga. He lived for many years in Jasper, ... (click for more)
Charles Neal Logan, 80, of Trenton, GA, died Wednesday, January 21, 2015. He was self-employed small engine and chainsaw repairman and operated Charlie's Saw Shop for many years. He was a member of the Poplar Springs Baptist Church. He was preceded in death by his parents, John and Ella Logan; brothers, June, Jack, Bill and Corky Logan; and one sister, Katheryn Shankles. ... (click for more)
A female student stabbed a male student twice at Lookout Valley Middle High School on Thursday morning. Sheriff Jim Hammond the students are seventh graders. He said the boy was taken by ambulance and went into surgery after the mid-morning stabbing. He said the boy received a superficial wound to the neck and a more serious wound to the stomach. However, ... (click for more)
Chattanooga Police Chief Fred Fletcher has given a maximum punishment to an officer who fired four shots toward a vehicle after he said the driver backed into his vehicle with him standing behind it. Chief Fletcher sustained a finding of "improper use of force – discharge of firearm." He suspended Officer Alex Olson for 30 days without pay – the maximum suspension allowed ... (click for more)


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Riedel Communications, pioneer of advanced intercom and radio technology, has added several new products to its Performer digital partyline intercom series. The world's first digital partyline intercom system featuring high-quality digital audio now provides 2 and 4-channel master stations, rack-mount, wall-mount and desktop speaker stations, call light indicators and 2-channel beltpack headset stations.
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The new Performer master stations CR-4 (4-channel) and CR-2 (2-channel) are the ideal choice for setting up a stand-alone digital partyline system. Depending on the setup, the integrated power supply of the 19"/1RU device can power up to 32 Performer devices – such as beltpacks, split-boxes or desktop speaker stations – per line. Additional power-supplies easily expand the possibilities. The clear user interface ensures ultimate performance and flexibility. The color-illuminated buttons are ideal for applications in operational environments. The remote mic-kill function allows the user to silence any open microphone on the intercom channels. The CR-4/CR-2 features an additional program input that can be mixed individually to each of the intercom channels. Other features include individual listen volume controls for all partylines, Call and S-Call, IFB, and an on-stage function for using the intercom microphone with a PA system. The CR-4/CR-2 can be operated using a headset or an integrated powerful loudspeaker with a gooseneck microphone. The new Performer CR-4 / CR-2 Master Station are scheduled to start shipping in October.
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The CD-2 Desktop Speaker/Headset Station provides the same feature set as the CR-2 Master Station with the exception of the internal power-supply. This makes the CD-2 ideal for operation as a desktop speaker station or – in combination with an external power-supply – as a 2-channel master station for setting up a stand-alone digital partyline system.
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Riders Roads and retrace steps
... Got the rolls I wanted, only to find they were like rocks so binned them down the street. Next the supermarket for said supplies. Language choices are Spanish, Catalan, French or Vietnamese. So I wander around a huge supermarket looking for products that only a lady will use. Not something I am unfamiliar with, just the location is new. So I found the Tena pads for men, but no help. Now its 9am, the place has just opened and I am one of about 4 ...
... if you wanted. There were so many religious shops selling everything religious you can think of right next to each other. There would have had to be about 100 of them in Lourdes. We also saw so many priests and nuns in full outfits strolling around. Prayers, masses, rosary and confession go all day every day. You can buy candles that are 2m high to put at the alters. Back on the train to Paris and our ...
... looking down on the town and the basilica is built on top of the grotto. We met a lovely lady named Mary Irwin from Brisbane. She also works for a school looking after after-school care at St Bernadette's at Redcliffe. We spent the entire time with Mary and found we had a lot in ...
Haute Route Day Six
... Lanterne Rouge' appeared and they told us they would be shutting down in 20 mins. Hustle boys, hustle.
Let me explain. The 'Lanterne Rouge' is one of the traditions of the Haute Route and is a rider that comes at the back and ushers the slow riders along and marks what will likely be the cut off time, beyond which, riders will not get an official standing in the race but may continue. One of our fears before the start was that we would not ...
Haute Route Pyrenees Day 5: Tormallet Time Trial
Day 5: Tormallet time trial. 18.5km and 1500m of ascent.
The day was overcast and damp - it had rained overnight but the forecast was good. Everyone was feeling reasonable apart from Bevan who was still hurting. In spite of a later start time of 10.00 for the first riders, we still had to get up at very early to ferry us and our bikes up the 32km from Lourdes to Luz St Saveur. So much for the anticipated and hoped for sleep in.
Let me set the ...


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Hi, I'm Nathalie, born and raised in France and currently living in the South Bay. My daughter Maya Rose is 19 months old. So excited about this group! Looking forward to meeting you all soon!
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Ocean Cruises Inc. released a statement on an attempted act of piracy against one of its cruise ships. On November 30, 2008, at approximately 0928 local time, 0528 GMT, M/S Nautica was transiting through the Gulf of Aden within the prescribed Maritime Safety Protection Area which is patrolled by international anti-piracy task forces. As the vessel sailed past several groups of non-hostile fishing vessels, two small skiffs were sighted by the Officer on Duty and deemed potentially hostile. The skiffs, approaching from a range of approximately 3,300 ft, attempted to intercept the vessel's course. Captain Jurica Brajcic and his officers immediately began evasive maneuvers and took all prescribed precautions. Nautica was immediately brought to flank speed and was able to out run the two skiffs. One of the skiffs did manage to close the range to approximately 9 yards and fired eight rifle shots in the direction of the vessel before trailing off. No one aboard Nautica was harmed and no damage was sustained. All guests and crew onboard are safe and there were no injuries. All requisite international authorities have been notified and all anti-piracy precautions were in place prior to the event and all necessary measures were taken during the event.
For the first time ever, the general public will be admitted free to the Naval Future Force Science and Technology EXPO general exhibit hall Feb. 4-5 where they
Shipbuilder General Dynamics National Steel & Shipbuilding Co. – NASSCO – will christen the USNS Lewis B. Puller (MLP 3 AFSB) Saturday, February 7, 2015 in San Diego.
UK-listed energy giant BP has decided to relinquish two exploration blocks to Indonesia, after a survey of the blocks found them to be high risk, the company said on Friday.


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Bali Hai resort looks like it hasn't changed in three decades. Sadly, its cottages and amenities look run-down and dirty. The restaurant has improved a bit on its cuisine, and it still has a good view of the West Philippine Sea, though.
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Video: Ohio cop risks own life to save suicidal teen
The girl was found crying on a dangerously narrow ledge over a highway
By PoliceOne Staff
ROCKY RIVER, Ohio —An officer put her own life on the line when she realized the only way to bring a teen to safety was to join her on a narrow ledge above a highway.
Officer Kim Forkins, an 18-year veteran with Rocky River police, found the girl standing on the ledge after receiving several 911 calls from passersby.
"She was very despondent," Forkins told Cleveland.com. "She was standing on the ledge and crying. I tried to talk to her, but she didn't want to talk at all. She wasn't responding to anything I or anyone else was saying to her."
After securing the girl with a rope with a second off-duty officer holding her by the arm, Forkins feared the girl still couldn't safely be helped off the ledge. Forkins ventured onto the narrow ledge, risking her own life to save the girl.
"I got the feeling she really didn't want to jump," Forkins said, "but I was very worried she might fall. We didn't want to give up securing her, and that's why I wanted that rope around her and Nate reached over to grab her arm."
The teen and Forkins were taken down using a bucket from a fire truck.
Forkins was honored in Nov. for the Aug. 10 incident by the FBI Citizens Academy of Cleveland, according to the article.
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Aloha there at this site, where you may aquire hot ringtones for your Android smartphone. Today we have a ringtone of "派大星" by 蔡依林 as well as 1993 other ringtones by 蔡依林. "派大星" is a real hit from the "Myself" album that was created by 蔡依林 at their label together with other 34 tracks. You can listen to all 6 蔡依林 – "派大星" ringtone variations available here and download it for free right now. You can also select other 9 albums produced by 蔡依林 for all time. "派大星" is referred to Chinese, and Audiko offer a huge amount of Chinese melodies absolutely for free. And finally, we have collected lyrics for this track and you can view it here as well. So feel free to browse, listen, download ringtones at Audiko.net. Make your phone sound cool!
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Count all occurrences of characters from a given set in a string.
In the code below $CharSet is the set of characters to count. $SearchStr is the string to count them in.
my $Set = qr/[^$CharSet]/; length join "", split $Set, $SearchStr;
Wrapped up in a rather more general sub:
sub CountSetChars { my $Count; my $CharSet = shift; my $Set = qr/[^$CharSet]/; my $Param; while ($Param = shift) { if (defined @$Param) {map {$Count += CountSetChars ($CharSet, $_)} @$Param;} elsif (ref $Param) {$Count += length join "", split $Set, @$Param;} else {$Count += length join "", split $Set, $Param;} } return $Count; }
There's a very pretty way to count them with the tr// operator, my $vowel_count = tr/AEIOUYaeiouy//; If you want the character list in a variable, you need to string-eval: my $foo_count = eval "tr/$foo//";
After Compline,Zaxo
Use subroutines for code reuse Master regular expressions Write my GUIs with Perl Abandon CGI in favor of some modern web framework Put my code in modules Create CPAN distributions test and document my code as I write it Write OOP with or without animal help Finally switch to a modern Perl style Create a killer application with Perl Detoxify from Perl and PerlMonks Choose another language because Perl is dead Learn to program for mobile devices Complete my secret world domination project Something else None, because the road to hell is paved with good intentions
Results (237 votes), past polls


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The fluffy Keeshond is an attention-craving, family-friendly breed that looks like a smaller version of a Samoyed or Husky. Keeshonds quickly become a part of the family and are generally great playmates for kids.
Average weight: 35 to 65 pounds
Pro: moderate activity level
Con: requires regular brushing and grooming
One parent says: "Keeshonds are extremely loving. They live to please their human family. This breed requires more attention than most and will do anything to get that attention. They refuse to be ignored."
Next: Labrador Retriever
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Just days after learning about the dangers of being trapped inside a hot car, 3-year-old Keith Williams' quick thinking saved the life of a 68-year-old man stuck inside his sweltering vehicle.
Bob King was waiting for his wife outside Vestal Baptist Church in Knoxville, Tennessee, on Saturday. The car doors automatically locked, and he didn't have the key. As the temperature inside the car hit 120 degrees, King — who over the past six months has had two strokes and gone through cancer treatment — became weak. He saw Keith walk by, and began to knock at the window to get his attention.
"I hollered at him and he just looked at me kind of funny and I said, 'Get help, get help,'" King told ABC News. Keith had just learned about car safety from his mother, and tried to open the door himself. When he couldn't, he ran to Pastor Jack Greene, and began to say, "Locked, locked, locked" and "hot, hot, hot." He pulled Greene to King's car, where the good reverend opened the door after several tries. King fell out and almost hit his head on the pavement, but was quickly brought into the air-conditioned church and given water.
King was grateful for his tiny helper, and Keith knew that what he did was special. "He said, 'I saved life' after I brought Bob inside," Greene said. "He is such a good kid. He is an inspiration and blessing to us." --Catherine Garcia
Japanese Prime Minister Shinzo Abe on Sunday condemned the Islamic State's apparent killing of journalist Kenji Goto, calling it a "despicable and horrendous act of terrorism."
ISIS on Saturday released a video purporting to show Goto's decapitated body after its demand of a prisoner exchange went unmet. Though the video has yet to be authenticated, both Japan and the U.S. have released statements tacitly confirming it is real.
"To the terrorists, we will never, never forgive them for this act," Abe said.
As you may have heard, the Super Bowl is finally here. So ahead of the big game, Saturday Night Live showed what it would be like if the Seattle Seahawks' Marshawn Lynch and Richard Sherman hosted their own talk show. Incredibly, a special guest even got he media-averse Lynch (played by Kenan Thompson) to crack a smile and open up a little bit. — Jon Terbush
Egypt on Sunday said it freed Al Jazeera journalist Peter Greste and would soon deport him to his native Australia.
Greste was arrested in December 2013 and accused of publishing false news, sparking an international outcry from free press advocates who considered the charges bogus. Egypt has not said what it plans to do with two other Al Jazeera reporters, Mohamed Fahmy and Baher Mohamed, who were imprisoned along with Greste.
Novak Djokovic on Sunday defeated Andy Murray in four sets — 7-6, 6-7, 6-3, 6-0 — to claim a record fifth Australian Open title.
The top-ranked player in the world, Djokovic remained perfect in Australian Open finals while claiming his eighth Grand Slam. Murray, who has never won the event, settled for his fourth second place finish in Melbourne.
Japanese officials said they are attempting to authenticate a video released on Saturday which shows the apparent beheading of Kenji Goto, a hostage of Islamic State militants, The Associated Press reports.
The video, titled "A Message to the Government of Japan," shows an apparent ISIS militant with a British accent, along with Goto, who kneels in an orange jumpsuit and does not speak in the minute-long video.
The militant speaks directly to Japanese Prime Minister Shinzo Abe, saying that Abe's "decision to take part in an unwinnable war," is to blame for Goto's beheading.
"Let the nightmare for Japan begin," he adds.
Japanese and Jordanian officials had been attempting to negotiate through indirect channels for the release of Goto and Jordanian pilot Muath al-Kasaesbeh.
After nearly 7,000 miles in the air, Troy Bradley of Albuquerque and Leonid Tiukhtyaev of Russia came back to earth on Saturday, landing their helium-filled balloon four miles offshore of Baja California, The Associated Press reports.
Bradley (left) and Tiukhtyaev | (AP Photo/Tami Bradley-Two Eagles Balloon Team)
The Two Eagles Balloon team took off from Japan early on Sunday morning; a little less than a week later, they had broken at least two long-standing records for ballooning; the pilots easily exceeded the previous distance record of 5,209 miles, and also set a new duration record of six days, 16 hours, and 38 minutes in the air.
The official distance and time of the flight still has to be confirmed by the Federation Aeronautique Internationale, so in the meantime, feast your eyes on the views from above. —Sarah Eberspacher
Crossing the Pacific Ocean | (AP Photo/Two Eagles Balloon Team)
Passing over Mount Fuji | (AP Photo/Two Eagles Balloon Team)
Jurors in the murder trial of former New England Patriots player Aaron Hernandez will be allowed to watch the Super Bowl, so long as they remain vigilant, Bristol County Superior Court Judge Susan Garsh warned them on Friday.
"I am not going to forbid you from watching the Super Bowl if that's something that's really important to you," CNN reports that Garsh told the jurors. "(But), you hear that word, you've got to walk out of the room."
"That word" is of course "Hernandez." As a former star for the Patriots who helped carry the team to a Super Bowl victory three years ago, Aaron Hernandez's name could very well come up during Sunday's broadcast featuring New England and the Seattle Seahawks.
The jurors had heard two days of testimony as of Friday; Hernandez, 25, has pleaded not guilty to the 2013 killing of Odin Lloyd, who was dating the sister of Hernandez's fiancee.
While saying that "we're not there yet," outgoing Defense Secretary Chuck Hagel told CNN on Friday that the United States may eventually have to send ground troops back into Iraq.
"I think (turning back Islamic State militants) may require a forward deployment of some of our troops," Hagel said. "Whether we get there or not, I don't know."
Hagel noted that the ground troops could remain in non-combat roles, such as intelligence gathering on ISIS targets. There are already 4,500 U.S. troops in Iraq, which are operating in training and advising capacities with Iraqi forces.
The National Highway Traffic Safety Administration has recalled more than 2 million vehicles from Toyota, Chrysler, and Honda due to possible air bag problems, Reuters reports.
About 400 cases of inadvertent air bag deployments have been reported in the recalled vehicle models, although the incidents have not resulted in any known deaths, an official for the NHTSA said. The three automakers will have to fix a defective chip in the air bag systems, which will require the entire air bag module and its circuits to be replaced.
Ah, Super Bowl weekend.
We could talk more about Deflategate, but why do that when Key & Peele released this perfectly delightful sketch spoofing Seattle Seahawks players Richard Sherman and Marshawn Lynch?
"Richard Sherman" spends his time critiquing the Academy Award nominations, questioning why Selma and Foxcatcher were overlooked. And "Marshawn Lynch" trots out a new catchphrase, before throwing us a surprise around the 2:20 mark. Check out the fun in the video, below. —Sarah Eberspacher


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A Diary of Dreams by Kevin Pollock Publisher Comments Dreams are fascinating. Reading about other people's is even more fascinating, especially when you can relate some of the dreams to your own. Murder, mystery, suspense, love, hate, happiness; these are but a few of the subjects you will find packed into... (read more) Your price: $10.25 New - Trade Paper add to wish list |
Serotonia by Bret Harvey Publisher Comments In the dreams of Bret Harvey, a huge orange fireball has destroyed much of the landscape. It is a desert now, lifeless and filled with broken houses. Food is scarce. To see a human is rare. The Dark Ones, with their childlike voices and fingers like long... (read more) Your price: $25.50 New - Hardcover add to wish list |
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Out of Body Experience and Mafetic Energy by John V. Goff Publisher Comments I know most people at least know someone or have had Out of Body Experiences in their Life. Most people just consider them to be Dreams but some of us don't. I can say in my personal experience they seem real, as real as walking down the road on a sunny... (read more) Your price: $20.50 New - Trade Paper add to wish list |
The Hidden Power of Dreams: The Mysterious World of Dreams Revealed by Denise Linn Publisher Comments Dreams are secret messages from your soul, as well as from the realm of spirit. They can be your greatest tool for profoundly understanding your life, yet few people recognize how to access this tremendous source of guidance and wisdom. Best-selling... (read more) Your price: $14.95 New - Trade Paper add to wish list |
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The Mind at Night: The New Science of How and Why We Dream by Andrea Rock Publisher Comments Psychologists and philosophers have long grappled with the mysteries of dreaming, and now-thanks largely to recent innovations in brain imaging -neuroscientists are starting to join the conversation. In this groundbreaking book, award-winning journalist... (read more) List Price $15.95 Your price: $8.50 Used - Trade Paper add to wish list |
The Dreamer's Book of the Dead: A Soul Traveler' S Guide to Death, Dying, and the Other Side by Robert Moss Publisher Comments In "The Dreamer's Book of the Dead," Robert Moss explains that we have entirely natural contact with the departed in our dreams, when they come visiting and we may travel into their realms. As we become "active "dreamers, we can heal our relationship... (read more) Your price: $18.95 New - Trade Paper add to wish list |
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Field Guide to Dreams: How to Identify and Interpret the Symbols in Your Dreams by Kelly Regan Publisher Comments At last, a field guide to identifying and interpreting dream symbols, from falling to flying! Dreams can be mysterious, exhilarating, and terrifying—but they don't have to be confusing. With Field Guide to Dreams, you'll unravel... (read more) List Price $15.95 Your price: $5.50 Used - Trade Paper add to wish list |
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Toltec Dreaming: Don Juan's Teachings on the Energy Body by Ken Eagle Feather Publisher Comments Toltec Dreaming explores the many aspects and levels of the dream-state, distinguishing ordinary dreaming from "dreaming awake," a condition of heightened awareness through which the active dreamer ascends to the Dream of Transcendence. Filled with... (read more) Your price: $18.00 New - Trade Paper add to wish list |
Lucid Dreaming: A Concise Guide to Awakening in Your Dreams and in Your Life [With CD] by Stephen Laberge Synopsis What if right now, reading these words, you suddenly realized that you were actually dreaming and that in this domain you could do anything imaginable? That is what it's like to dream lucidly, teaches Stephen LaBerge. With Lucid Dreaming, the author of... (read more) Your price: $14.95 New - Trade Paper add to wish list |


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Four Views on Salvation in a Pluralistic World - eBook
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Format: DRM Protected ePub Vendor: Zondervan Publication Date: 2010 ISBN: 9780310872382 | ISBN-13: 9780310872382 UPC: 025986872380 Availability: In Stock |
Publisher's Description
To read the New Testament is to meet the Old Testament at every turn. But exactly how do Old Testament texts relate to their New Testament references and allusions? Moreover, what fruitful interpretive methods do New Testament texts demonstrate? Leading biblical scholars Walter Kaiser, Darrel Bock and Peter Enns each present their answers to questions surrounding the use of the Old Testament in the New Testament. Contributors address elements such as Divine and human authorial intent, the context of Old Testament references, and theological grounds for an interpretive method. Every author applies his framework to the same three texts so that readers see each methods practical use. Each contributor also receives a thorough critique from the other two authors. A one-stop reference for setting the scene and presenting approaches to the topic that respect the biblical text, Three Views on the New Testament Use of Old Testament gives readers the tools they need to develop their own views on this important subject. The Counterpoints series provides a forum for comparison and critique of different views on issues important to Christians. Counterpoints books address two categories: Church Life and Bible and Theology. Complete your library with other books in the Counterpoints series.
Author Bio
Stanley N. Gundry is executive vice president and editor-in-chief for the Zondervan Corporation. He has been an influential figure in the Evangelical Theological Society, serving as president of ETS and on its executive committee, and is adjunct professor of Historical Theology at Grand Rapids Theological Seminary. He is the author of seven books and has written many articles appearing in popular and academic periodicals.
Dennis L. Okholm (PhD, Princeton) is professor of theology at Azusa Pacific University. Until his death in 2000, Phillips (Ph.D., Vanderbilt University) was associate professor of historical and systematic theology at Wheaton College.Product Reviews
David R. Bess3 Stars Out Of 5March 9, 2002David R. BessThis review was written for Four Views of Salvation in a Pluralistic World.Having studied these four views previously, I was disappointed after finishing this particular title. All of these authors could have made their arguments much more concise and readable, but none of them chose to do so.Hick basically states that many different religions lead to the same God. Pinnock holds that salvation in Christ may be found through other religions. McGrath asserts salvation is only found in a relationship with Christ, and persons who have had no chance to enter that relationship might receive God's mercy in some unknown way. Geivett and Phillips maintain salvation is only found in a relationship with Christ, and persons who have had no chance to enter that relationship will spend eternity separated from God.This book could have and should have been written in such a manner to appeal to the average layperson. These individuals are the ones who struggle with these issues and want to be better informed. Instead, all of these essays are directed towards other scholars. The overly-technical manner in which they are written will not appeal to most persons without professional theological training.For pastors and well-versed teachers, this book provides a helpful summary of four current views on salvation. For everyone else, this volume will be confusing and hard to follow.
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