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Polycomb group (PcG) proteins are part of a conserved cell memory system that conveys epigenetic inheritance of silenced transcriptional states through cell division. Despite the considerable amount of information about PcG mechanisms controlling gene silencing, how PcG proteins maintain repressive chromatin during epigenome duplication is still unclear. Here we identified a specific time window, the early S phase, in which PcG proteins are recruited at BX-C PRE target sites in concomitance with H3K27me3 repressive mark deposition. Notably, these events precede and are uncoupled from PRE replication timing, which occurs in late S phase when most epigenetic signatures are reduced. These findings shed light on one of the key mechanisms for PcG–mediated epigenetic inheritance during S phase, suggesting a conserved model in which the PcG–dependent H3K27me3 mark is inherited by dilution and not by de novo methylation occurring at the time of replication.
Author Summary
During embryonic development, pluripotent cells divide and use their potential to differentiate into a variety of cells with identical genomes but different phenotypes. The emerging concept suggests that the DNA sequence information is not the sole determinant of cell identity. Indeed, epigenetic mechanisms, acting via chromatin organization, control transcriptome complexity and contribute to maintain cell fate. Polycomb-group proteins (PcG) are epigenetic transcriptional regulators that maintaining gene silencing programs through cell division. During S phase, in addition to DNA, the entire epigenome needs to be duplicated. A key question that remains to be addressed is how epigenetic marks are transmitted to subsequent generations. In this study we propose a model for PcG epigenetic inheritance during replication. We found that, during S phase, PcG engagement and characteristic H3K27me3 histone mark deposition on target sites are restricted to a brief interval occurring before DNA replication of the same regions. By increasing the dose of PcG binding the system would prevent potential weakening of silencing control, which is challenged at the time of replication, allowing proper transmission of epigenetic marks to the next generation and preservation of cell identity.
Citation: Lanzuolo C, Lo Sardo F, Diamantini A, Orlando V (2011) PcG Complexes Set the Stage for Epigenetic Inheritance of Gene Silencing in Early S Phase before Replication. PLoS Genet 7(11): e1002370. https://doi.org/10.1371/journal.pgen.1002370
Editor: Asifa Akhtar, Max-Planck-Institute of Immunobiology, Germany
Received: April 20, 2011; Accepted: September 20, 2011; Published: November 3, 2011
Copyright: © 2011 Lanzuolo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from Fondazione Telethon (TCP00094), Associazione Italiana Ricerca sul Cancro AIRC, and EU-FP7 The Epigenome Network of Excellence (LSHG-CT-2004-503433). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The genes of the Polycomb group (PcG) prevent changes in cell lineage identity by maintaining silenced transcription patterns throughout cell division via chromatin structure [1]. To date, four PcG-encoded protein complexes have been isolated from different organisms: Pho Repressive Complex (PhoRC), Polycomb Repressive Deubiquitinase Complex (PR-DUB), Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). Biochemical studies revealed that the zinc finger protein Pleiohometic (PHO) of PhoRC is required for PRC2 targeting [2]–[4] while Enhancer of zeste (E(Z)), the Histone Methyl Transferase (HMTase) subunit of PRC2, marks lysine 27 of histone H3 [5]–[8]. This chromatin mark is specifically recognized by PRC1 complex through the chromo-domain present in the Polycomb protein (PC) [5]. PRC1 complex has several catalytic functions believed to be important for transcriptional repression. By electron microscopy, it has been shown that PRC1 induces compaction of defined nucleosomal arrays in vitro [9]. Components of PRC1 can also function as E3 ligase for H2A ubiquitylation [10]. On other hand, PR-DUB complex is able to deubiquinate H2A [11] and, interestingly, both activities are required for proper gene silencing in vivo. In Drosophila, PcG function is mediated by specialized epigenetic DNA modules called Polycomb Response Elements (PREs), which organize repressed PcG target genes at a distance via chromatin structure and nuclear architecture [12]–[16]. Notably, similar cis-elements were recently reported in mammals [17], [18]. The characteristic feature of the PcG memory system is the mitotic inheritability of gene expression patterns. However, the mechanism by which PcG proteins maintain repressive chromatin during cell division is poorly understood. In mammals, it has been proposed that PRC2 binds to its own methylation mark H3K27me3 to re-establish epigenetic signatures after replication [19], [20]. In Drosophila, by in vitro and partially in vivo assay, it has been observed that PSC, a chromatin compacting subunit of PRC1 complex, remains bound to chromatin during replication [21]. Such an association suggests that, in principle, epigenetic players could be transferred from maternal to daughter strands. To date however, direct evidence for existence of these models in vivo is still lacking. In particular, the time at which the parental marks are imposed and how tightly the process of PcG epigenetic inheritance is coupled to replication have not been determined. To address these questions we used the D. melanogaster embryonic Schneider 2 cell line (S2) to analyse replication timing, PcG proteins binding, , H3K27me3 mark deposition, dynamics of PRE mediated higher order structures and transcriptional repression during S phase. Our data suggest a putative conserved mechanism for epigenetic inheritance, identifying a critical time window before replication, during which the PcG memory system sets the stage for subsequent epigenome duplication.
Repressed PREs replicate during late S phase
We first measured BX-C PRE replication timing in S2 embryonic cell line where the homeotic genes of Bithorax Complex (BX-C) are silenced. The relative abundance of nascent DNA synthesised during different fractions of the S phase was determined by bromodeoxyuridine triphosphate (BrdU) labelling and FACS (fluorescence-activated cell sorting) sorting [22]. DNA was prepared from an equal number of cells representing the first and last stages of the S phase, hereafter referred to as "early" and "late" (Figure 1A). BrdU-labelled DNA was immunoprecipitated from these S-phase specific fractions to enrich for genomic sequences that replicate during the labelling period. We then performed quantitative real-time PCR (qRT-PCR), using primers specific for Fab-7, Mcp, bxd, bx PREs, and control regions, the latters consisting of CG108735 gene locus and dodeca repeats, which are early and late replicating sequences, respectively [22]. Ratios between the amounts of amplified products in early and late S phase showed that repressed PREs replicate late during S phase (Figure 1B), in agreement with the Drosophila genome wide replication timing database [23]. We then repeated the experiment using synchronized S2 cells to confirm that BX-C PRE replication timing was comparable to the FACS-sorted cells. Upon release from hydroxyurea (HU) block (Figure S1A), cells synchronously proceeded through S phase over the next 2 hours (end of S phase). Cells representative of early S phase were pulse-labelled with BrdU at the start of S phase and collected after 1 h from the HU block release, while cells representing late S phase were pulse-labelled after 1 h and collected after 2 h from HU block release. Quantification of the relative amount of PRE sequences after BrdU immunoprecipitation (Figure S1B) confirmed that repressed PREs are late replicating in synchronized S2 cells.
(A) Experimental strategy to collect cells of the early and late S phase using FACS sorting. Cell-cycle profile of D. melanogaster S2 cells after propidium iodide staining. Cells between the G1 and G2 peaks are in S phase. Gates indicate early and late S phase fractions. (B) Replication timing of PREs as measured by quantitative Real Time PCR (qRTPCR). Ratios between the amplified products in early and late S phase are shown. We amplified positive controls for the early and late S phase and gene names correspond to their entries in FlyBase. All data points were generated from an average of five independent experiments. Standard error of the mean is indicated. Two-tailed t-test was applied for statistical analysis. Asterisks indicate statistically relevant differences; α = 0.05. P values: CG108735/dodeca: P = 0.0047; CG108735/Fab-7: P = 0.013; CG108735/mcp: P = 0.002; CG108735/bxd: P = 0.0048; CG108735/bx: P = 0.0053.
Higher order interactions are dynamic during replication
We have previously demonstrated by Chromosome Conformation Capture (3C) and fluorescent in situ hybridization (FISH) that all major elements bound by PcG proteins, including PREs and core promoters, interact at a distance in the repressed state, resulting in a topologically complex structure necessary for the maintenance of BX-C silencing [16]. Similar results were obtained in mammals [24], [25]. In order to investigate whether PRE-mediated BX-C higher order structures are disrupted during replication, we used 3C analysis to monitor DNA/DNA interactions between PcG targets during S phase (Figure 2). We used synchronized cells collected 1 h and 2 h after the release from the HU block as representative of early and late S phase, respectively (Figure S1A). In comparing crosslinking frequencies of different fractions, we found that BX-C promoters were interacting with all PREs during early S phase, while during replication (late S phase) most PRE/promoters interactions were impaired (Figure 2B and 2C). These data may partially explain the previously reported dynamic nature of higher order interactions [13], [16] and suggest that during replication epigenetic higher order structures are altered and need to be reconstituted at each cell cycle. Interestingly, the frequencies of interaction between analysed PRE elements were stable throughtout DNA replication (Figure 2D), suggesting that PRE-PRE clustering may serve as the scaffolding template for PcG inheritance.
(A) The scheme shows the Bithorax Complex (BX-C), including transcription units and genetically characterized regulatory regions. (B–D) Crosslinking frequencies observed in cells collected 1 h and 2 h from HU block release (ES, LS) are in red and in light green respectively. (B) Crosslinking frequencies, normalized on the ES fraction, between the AbdB γ promoter and PREs. (C) Crosslinking frequencies, normalized on the ES fraction, between the abdA promoter and PREs. (D) PRE/PRE crosslinking frequencies, normalized on the ES fraction. All data points were generated from an average of at least four independent experiments. Standard error of the mean is indicated. Two-tailed t-test was applied for statistical analysis. Asterisks indicate statistically relevant differences; α = 0.05. P values: AbdB γ promoter/Fab-7: P = 0.004; AbdB γ promoter/Mcp: P = 0.0001; AbdB γ promoter/bxd: P = 0.008; AbdB γ promoter/bx: P = 0.07; abdA promoter/Fab-7: P = 0.001; abdA promoter/Mcp: P = 0.0001; abdA promoter/bxd: P = 0.026; abdA promoter/bx: P = 0.004; Fab-7/bxd: P = 0.22; Fab-7/bx: P = 0.7.
PcG proteins and repressive mark H3K27me3 are enriched at PREs before replication
To dissect the dynamics of PcG proteins binding during S phase, we performed Chromatin Immunoprecipitation (ChIP) in synchronized S2 cells. Chromatin collected from G1/S, early and late S phase was immunoprecipitated with antibodies against PHO (Figure 3A), PC (Figure 3B) and E(Z) (Figure 3C), which are members of PhoRC, PRC1 and PRC2 complexes, respectively. Notably, all three PcG proteins were present at PREs during S phase, in agreement with and further corroborating previous reports [19]–[21]. However, we found that the amount of PcG proteins bound to target sites varied over S phase progression. In particular, we observed a striking increase, up to 10 fold, in early S phase (Figure 3A–3C), followed by a dramatic drop in PcG binding in late S phase, returning to G1/S basal levels. Thus PcG complexes engagement is uncoupled from and precedes target sites replication. To analyse PcG dependent HMTase function on chromatin we measured the levels of histone lysine methylation during S phase with antibodies that recognize total H3 and H3K27me3. Although total H3 levels at PREs did not change between G1/S and early S phase fractions (Figure S2A), the ratio between H3K27me3 and H3 peaked in early S phase (up to 10 fold; Figure 3D) following PcG protein loading onto PREs (Figure 3A–3C). Moreover, we observed a consistent drop of H3K27me3 from early to late S-phase while total H3 showed only a mild increase during PRE replication, suggesting that H3K27me3 trend during replication depends on mark deposition and not on replication dependent histone fluctuation. Little is known about in vivo dynamics of chromatin proteins during S-phase. Thus, as a further control, we looked at Topoisomerase II (TOPO II) an enzyme that plays a crucial role in DNA replication and binds PREs in Drosophila [26]. As shown in Figure S2B, we found that the amount of TOPO II at PREs did not change during S-phase, proving that the observed dynamic is specific for PcG complexes. As an additional control we performed ChIP experiments for repressive H3K9me3 mark that is also present on PREs [27], [28]. Interestingly, H3K9me3, also controlled by PcG proteins [28], showed a trend similar to H3K27me3 (Figure S2C) during S phase, suggesting that PcG epigenetic signatures are inherited at the same time during replication. Recently, phosphorylation of Serine 28 on histone H3 (H3S28ph) via mitogen and stress activated kinases, has been proposed as a novel mechanism that induces PcG chromatin displacement, counteracting the H3K27me3 docking site [29], [30]. To explore the contribution of the H3Ser28ph mark in S phase dependent PcG protein binding, additional ChIP experiments were performed. We found a progressive increase in H3Ser28ph mark from G1/S to late S phase (Figure S2D) both on PREs and the bw negative control, likely due to its role in mitosis [31]. Although we cannot completely exclude that H3K27me3 mark recognition by specific antibodies may be partially influenced by histone phosphorylation, the trend of H3Ser28ph mark did not correlate with the observed PcG and H3K27me3. This suggests that, at least during PRE replication, a putative signal dependent mechanism for PcG protein displacement by phosphorylation of Ser28 of histone H3 does not appear to be required.
(A–D) ChIP analysis are presented as percentage of input chromatin precipitated for each region. Mock enrichment is below 0.003% of the input. (A–C) ChIP analysis with antibodies against Pho, Pc and E(z) respectively on synchronized cells. Data obtained in HU treated cells (G1/S) are shown in yellow. Data obtained in cells collected 1 h and 2 h from HU block release (ES and LS) are in red and light green respectively. As negative control we used the promoter region of brown (bw) that is repressed in S2 but it is not under the control of PcG proteins [55]. Each graph shows the result from at least three independent immunoprecipitation reactions done on different chromatin preparations. Standard error of the mean is indicated. (D) ChIP enrichment for H3K27me3 normalized to histone H3 density. The graph shows the result from two independent immunoprecipitation reactions done on different chromatin preparations. Standard deviation is indicated. (E) Western blots of total protein extracts from synchronized S2 cells. Actin was used as a loading control. Quantifications of protein bands normalized on actin and relative to G1/S fraction are shown. (F) Western blots of histone extracts using antibodies that specifically recognize H3K27me3 and H3, as loading control. Quantifications of H3K27me3 and H3 bands relative to G1/S fraction are shown.
Next we examined whether global levels of PcG proteins and H3K27me3 would be S phase regulated. Western blot and RT-PCR analysis revealed that both E(Z) and PC reached their maximum peak of expression during early S phase (Figure 3E and Figure S2E), while PHO showed only a slight increase. These dynamics were not observed for TOPO II, used as control (Figure 3E). The same trend was observed for total H3K27me3 while H3 levels remained constant throughout S phase (Figure 3F). We conclude that PcG proteins quantitatively engage their target sites and enrich for H3K27me3 epigenetic mark in early S phase, preceding PRE replication.
Despite the repression imposed on BX-C by PcG proteins in S2 cells it is possible to detect basal transcription levels of homeotic genes. To measure the correlation between the amount of PcG bound to its repressive function, we performed transcriptional analysis in synchronized S2 cells (Figure S3A). Different primer pairs were used to discriminate the mature and the primary transcripts of two homeotic genes, Ubx and abdA. We found a slight transcriptional increase in late S phase when Polycomb proteins are reduced on their targets (Figure S3A) and the analysed sequences are replicated (Figure S3B). However, we observed the same trend also with the late replicating bw negative control (Figure S3A and S3B), indicating that this effect is not dependent on PcG protein levels. We performed additional transcriptional analysis on synchronized cells treated with dsRNA against Pho, PC and Ez, which give rise to homeotic gene derepression (Figure S3C), and against Gfp as a control. The reproducible transcriptional trend found in PcG depleted cells lends support to the view that it is not dependent of PcG binding. These results suggest that, although continously repressed during S phase, some transcripts escape the restraint at the moment of DNA replication.
Global levels of Polycomb protein and repressive mark H3K27me3 decrease from early to late S phase
As PcG proteins form discrete bodies in the nucleus [32], [33], we followed PC and H3K27me3 localization pattern during replication. To identify S phase, a S2 population of cells was pulse-labelled with BrdU and then analyzed by immunofluorescence (Figure 4A–4D and Figure S4A). As expected, PC does not colocalize with constitutive heterochromatin and it is excluded from replication foci (Figure 4A). This is in agreement with data in mammals showing no colocalization of the PRC1 subunit CBX8 with BrdU foci [19]. In order to perform a deeper analysis of PC dynamics, we then followed the time-lapse of the S phase by measuring nuclei dimensions. First, we used FACS to measure the mean cell size of two fractions representing early and late S phase (Figure 1A). We found that cells belonging to the early S phase were smaller compared with cells of the late S phase (Figure S4B), indicating that as S phase progresses, cell dimension increases. This allowed us to study protein distribution throughout S phase by immunofluorescence. Second, we quantified PC protein levels by measuring the intra-nuclear mean fluorescent intensity in BrdU positive cells. We plotted these values, classifying cells by dimension, and we found that PC amount decreased with S phase progression (Figure 4E). As a control, we repeated the experiment to study TOPO II dynamics and we found a more diffuse distribution of the protein in the cell (Figure 4B) and a slight decrease in overall levels in late S phase compared to early (Figure 4F), in agreement with Western blot results. In parallel, we followed the activity of PRC2 complex by analysing the distribution and the amount of H3K27me3 mark in BrdU positive cells. As observed for PRC1, we found that the repressive mark is excluded from constitutive heterochromatin and replication foci (Figure 4C) and that the relative nuclear intensity of H3K27me3 fluorescence decreased with a similar trend (Figure 4G). In H3 immunostaining, used as control, we found a strong signal in heterochromatin foci (Figure 4D) and relative nuclear fluorescence remained constant throughout S-phase (Figure 4H). Taken together, these data confirm results from ChIP and Western blot analysis, clearly showing specific dynamics of PC and histone H3K27me3 mark during replication.
(A–D) Representative examples of D. melanogaster S2 nuclei with double immunostaining using PC (A), Topoisomerase II (B), H3K27me3 (C) and H3 (D) with anti-BrdU antibodies. Scale bar = 10 µm. (E–H) Quantification of PC (E), Topoisomerase II (F), H3K27me3 (G) and H3 (H) intranuclear mean fluorescence intensity during S phase. Image stacks of 138, 208, 187 and 87 nuclei, respectively, with different expression levels were recorded using constant image acquisition parameters. All values are background corrected. Nuclei were grouped into six categories based on volumes measured in pixel units (1∶6000; 2∶ 7000; 3∶ 8000; 4∶9000; 5∶ 10000; 6∶11000). Mean PC, TOPO II, H3K27me3 or H3 relative fluorescence intensities were then determined for each category by averaging over all nuclei of a class. Standard error of the mean is indicated. One-way ANOVA was applied for statistical analysis. α = 0.05. P values: PC p = 0.001; H3K27me3 p = 0.0001.
Early S phase dynamics of H3K27me3 repressive mark and EZH2 are conserved in human cells
Previous reports indicated that PRC1 and 2 complexes are localized at silent INK4/ARF locus in proliferating mouse embryonic fibroblasts (MEF) and that this locus is replicated during late S phase [34]. This evidence was further supported by large scale analysis in Hela cells in which the presence of PcG H3K27me3 repressive mark positively correlated with late-replicating genomic regions, suggesting that PcG targets replicate during late S phase [35], [36]. To check if early S-phase PcG dynamics observed in Drosophila cells would be evolutionary conserved, we analysed the levels of H3K27me3 mark in synchronised mammalian cells. Three different human cell lines (293, Hela S3 and Hela B) were treated with HU and, after block release, several fractions from G1/S (HU block) to late S/G2 phase were collected (Figure 5A). H3K27me3 total amounts were measured by western blot analysis (Figure 5B). Strikingly, all three cellular systems revealed a trend similar to Drosophila S2 cells, showing H3K27me3 enrichment in the first part of the S phase and then a gradual reduction along with S phase progression. Interestingly, it has been previously shown that Hela cells collected 6 hours after G1/S block release can be considered representative of late replication [35], indicating that H3K27me3 deposition precedes duplication of potentially silenced, late replicating sequences. In the same samples, to correlate the H3K27me3 mark deposition with PcG dependent HMTase function on chromatin we analysed the human homologue of Drosophila Ez protein, EZH2 (Figure 5C). As for Drosophila, western blot analysis indicate that increase in H3K27me3 was accompanied by increase in EZH2 levels. These findings suggest that a characteristic timing of PRC2 activity and H3K27me3 deposition preceding PcG target replication may be a key and evolutionary conserved mechanism for epigenetic inheritance of gene silencing. Interestingly, the H3K27me3 drop in late S phase was not followed by a comparable decrease of EZH2 levels, suggesting that other cell-cycle coupled mechanisms could be involved in EZH2 HMT activity regulation.
(A) Cell-cycle profile of human 293, Hela B and Hela S3 cells stained with propidium iodide before and after HU block release. Cells treated with HU are in G1/S phase. After HU block release, cells were collected at each hour, as indicated. (B) Western blots of histone extracts using antibodies that specifically recognize H3K27me3 and H3, as loading control. (C) Western blots of total protein extracts from synchronized cells. Actin was used as a loading control.
At each cell cycle, the integrity of genetic and epigenetic information is challenged during DNA replication, when chromatin undergoes a wave of disruption and subsequent restoration in the wake of the passage of the replication machinery. It is well described that assembly of core histones is coincident with DNA replication and takes place at the replication fork [37]–[41]. However, temporal re-establishment of epigenome structure during cell division remains a key question in epigenetic research.
In mammals, mechanisms of heterochromatin formation involve the sequential recruitment of HMT, deposition of histone mark and binding of HP1 chromodomain protein at the replication fork [42]. In yeast, generation of short interfering RNAs from centromeric repeats in S-phase allows the loading of heterochromatin factors that, in turn, restore the H3K9me2 mark after replication [43], [44]. Despite the considerable amount of information about PcG catalytic and repressive functions, by now mechanisms of PcG mediated epigenetic inheritance at cell division are not fully understood. Increasing evidences suggest that PcG mediated epigenetic signatures are cell cycle regulated, being controlled in S and M phases, when cells are subjected to profound modifications of chromosomal components and nuclear structure [45]–[48].
The key question, still open, is how PcG dependent epigenetic marks are inherited when the genome is replicated. Studies in mammalian cells suggest that all three proteins of PRC2 in the trimeric complex are required to form a combined binding surface that can recognize the H3K27me3 modification, thus generating a positive feedback loop that helps to propagate H3K27me3 mark through DNA replication. Since also PRC1 can recognize H3K27me3, via chromodomain proteins, this could also be a mechanism for recruiting new PRC1 complexes following DNA replication [19], [20]. Notably, it has been shown that in Drosophila chromatin histone proteins turn over faster than cell cycle suggesting that in principle they may be loaded on DNA not only during S phase [49]. Indeed, the ability of PcG proteins to bind their own mark, occurring during all phases of cell cycle and reinforcing the epigenetic repressed status of target genes, could partially explain the stability of epigenetic signatures despite their high turnover in the cell [19], [20]. On other hand, during replication, stability of epigenetic marks is challenged by the replication fork passage. Hence, specific mechanisms of epigenetic inheritance in S-phase must be provided in order to preserve cell identity.
We addressed this issue by analyzing the in vivo, cell cycle dependent dynamics of PcG proteins and their role in maintaining BX-C homeotic gene silencing. By using different experimental approaches, we show that components of the three major PcG complexes follows a characteristc dynamics in S-phase and, notably, it is uncoupled from replication timing. We found that, in early S phase, endogenous PcG protein levels increase and PcG complexes chromatin loading and enrichment for characteristic H3K27me3 mark are strongly enhanced (Figure 3). All these events precede late S-phase when PREs are replicated (Figure 1) and most of epigenetic signatures, such as looping between regulatory sequences (Figure 2), PcG binding and H3K27me3 mark levels (Figure 3 and Figure 4), appear to be challenged. These results are supported by experiments on synchronized mammalian cells showing a conserved dynamics of PcG proteins and H3K27me3 mark through S phase (Figure 5). Of note, our data are in line with previous findings based on mass spectrometry quantification on parental versus newly deposited histones showing that the establishment of H3K27me3 patterns during cell cycle takes place to large extent before replication [47]. Further, the conclusions that can be drawn from these data are strongly reminiscent of centromeric heterochromatin duplication in which epigenetic inheritance of histone variant CENP-A (centromeric protein A) is restricted to a brief interval in G1 and subsequent dilution occurs during S phase [50], [51]. Thus, we propose a mechanism for PcG epigenetic signature inheritance in which H3K27me3 mark is actually inherited by dilution and not by de novo methylation occurring at the time of replication.
Overexpression of PcG proteins and consequent changes in specific chromatin landscapes have been extensively documented in human cancer [52], where control on the cell cycle is lost, and cells constantly enter S phase. We suggest that higher levels of PcG proteins characteristic of cancer cells might be needed to maintain transcriptional repression on differentiation genes and oncosuppressors through S phase. The identification of PcG regulated epigenetic inheritance time window may be relevant for cell reprogramming by allowing the modulation of cell memory function [48], [53].
Materials and Methods
Culture cell growth
Drosophila embryonic S2 cells were grown at 25°C in serum-free insect culture medium (HyQ SFX; Hyclone, Logan, UT). 293, Hela B, and Hela S3 were cultured in Dulbecco Modified Eagel's Medium (DMEM) supplemented with penicillin/streptomycin and 10% fetal bovine serum (Euroclone).
Replication timing analysis
Exponentially growing S2 cells (1×106 cells/ml) were cultured in presence of 50 µM Bromodeoxyuridine (BrdU) for 60 min. For sorting, cells were divided into aliquots containing 5×106 cells per tube, washed with cold PBS, resuspended in 0.5 ml of cold PBS, fixed with drop by drop addition of 5 ml of 70% cold ethanol and incubated for 1 h on ice. Cells were then washed with PBS, resuspended in PBS/RNase A (1 mg/ml) 30 min at 37°C followed by addition of Propidium Iodide (20 µg/ml) and incubated 30 min in the dark at 4°C. On the basis of DNA content, cells were sorted into different S phase fractions using two selective gates representing roughly the first and the last thirds of S phase. Equal numbers of cells from each cell cycle fraction (150,000) were sorted (using a Becton Dickinson or a Moflo, Coulter) into microcentrifuge tubes containing lysis buffer (50 mM TrisHCl pH 8; 10 mM EDTA; 0,8% SDS; supplemented with 0.2 mg of proteinase K per ml). For analysis after HU synchronization, cells representing early S phase were pulse-labelled with BrdU at the beginning of S phase and collected 1 h from HU block release, while cells representing late S phase were pulse labelled with BrdU after 1 h and collected after 2 h from HU block release and resuspended in lysis buffer. The aliquots, collected either by FACS or after synchronization, were incubated at 50°C for 2 h in lysis buffer and then stored at −20°C. Lysates were then extracted once with phenol-chloroform, and phenol was extracted again with an additional volume of TE1X. DNA was precipitated with sodium acetate and ethanol and resuspended in 500 µl of TE. DNA was sonicated to an average size of 0.5 kb, and an aliquot of 100 µl was checked on agarose gel. DNA was heat denatured 10 min at 95°C and cooled on ice. Then 50 µl of 10× phosphate buffer (1 M Sodium phosphate [pH 7.0], 1.4 M NaCl; 0.5% Triton X-100) and 40 µl of mouse anti-BrdU DNA monoclonal antibody (25 mg/ml Becton Dickinson) were added to each tube. After 2 h of constant rocking at room temperature, protein AG plus agarose beads (Santa Cruz Biotechnology) were added and incubation continued for an additional hour at room temperature with rocking. DNA-protein complexes were pelleted by microcentrifuging for 5 min at 4°C. After washing with 750 µl of 1× phosphate buffer, pellets were resuspended in 200 µl of digestion buffer (50 mM Tris HCl pH 8, 10 mM EDTA, 0,5% SDS, 250 µg/ml proteinase K). Digestion was allowed to proceed overnight at 37°C and then for 1 h at 50°C after the addition of 100 µl of fresh digestion buffer. DNA was extracted and precipitated as above, briefly dried and resuspended in 40 µl of TE. RT PCR was performed using 1 µl of each nascent strand sample as template. Primer sequences: CG10873-f 5′agcttgctgcgcagcgag3′, CG10873-r 5′tctccaggcagaagactaagg3′; dodeca-f 5′actggtcgcgtactggtcc3′, dodeca-r 5′gtctcgtactctgtcccgtatt3′; fab-7-f 5′gaaaatgcccaacaaaatgc3′, fab-7-r 5′cgctgtctcgcctcttcttc3′; mcp-f 5′tgcggacgccatttgacac3′, mcp-r 5′gagccacgcagcgagttc3′; bxd-f 5′tcgtcgcttgtttggataattact3′, bxd-r 5′tgcggtgataaggtccataatc3′; bx-f 5′ttattgttgctacaccgctg3′, bx-r 5′agtaggtgccgcgtatgtg3′; CG3436–f 5′atcgctaacagccatgtcgg3′, CG3436-r 5′cttaccgattcaaggagcgc3′; CG4345-f 5′ttcccgagtctctcaccgc3′, CG4345-r 5′acaggaacccacaccactgac3′; Ubxpr-f 5′tcagccctcctccatgatg3′, Ubxpr-r 5′ccaaatcgcagttgccagtg3′; abdApr-f 5′ttgagtcagggagtgagcc3′, abdApr-r 5′cgctttgagtcgttggagac3′; bwpr-f 5′tgatgagcgacaattagctgg3′, bwpr-r 5′tgtccgtctgtctgtctgtc3′.
Chromosome conformation capture (3C)
The 3C assay was performed as previously described [16].
Antibodies against PC were kindly provided by R. Paro, antibodies against Topoisomerase II by D. Arndt-Jovin, and antibodies against Pho and E(z) by J. Muller. Commercial rabbit polyclonal antibodies against methylated Lysine 27 of histone H3 (Upstate, 07-449), methylated Lysine 9 of histone H3 (Abcam, ab8898), phosphorilated Serine 28 of histone H3 (Upstate, 07-145), histone H3 (Abcam, ab1791) and EZH2 (Diagenode pAb-039-050) were used.
Chromatin immunoprecipitation
ChIP experiments were performed as previously described [27] with minor modifications. After synchronization, cells of different phases of cell cycle were fixed in 1% formaldehyde for 15 min at room temperature and quenched by addition of glycine at 125 mM final concentration for 5 min at room temperature before being placed on ice. Cells were washed once with ice-cold PBS, resuspended in ice-cold cell lysis buffer (5 mM Pipes pH 8; 85 mM KCl; 0.5% NP40; 1 mM PMSF; 1× Protease Inhibitors) and left on ice for 10 min. After centrifugation at 2000 rpm for 5 min, nuclei were resuspended in ice-cold nuclear lysis buffer (50 mM TrisHCl pH 8.0; 10 mM EDTA; 0.8% SDS; 1 mM PMSF; 1× Protease Inhibitors) and left for 10 min on ice. Chromatin was sonicated in the presence of glass beads (150–200 mm, Sigma), spun for 10 min at maximum speed at 4°C, diluted to 0.2% SDS with dilution buffer (10 mM Tris–HCl pH 8.0, 0.5 mM EGTA, 1% Triton X-100, 140 mM NaCl), then split into aliquots and processed immediately for IP. For pre-clearing and antibody recovery, Protein A/G Plus-agarose beads (Santa Cruz Biotechnology) were used. After washing, samples and control chromatin (input) were incubated in the presence of 2 µl of Rnase cocktail (DNase-free, Ambion) overnight at 65°C. Then, samples were adjusted to 0.5% SDS and 0.5 mg/ml proteinase K and incubated for additional 2 h at 55°C. The DNA was phenol–chloroform extracted and precipitated. The final pellet was resuspended in 30 µl of TE and stored at 4°C for RT-PCR analysis. Primer sequences are indicated above.
Protein and histone extraction
Total proteins were prepared by resuspending 2×106 S2 or 1×106 mammalian cells in extraction buffer (50 mM TrisHCl pH 7.6; 0.15 M NaCl; 5 mM EDTA; 1× Protease Inhibitors; 1% Triton X-100). Three pulses of 10 sec sonication at 30% amplitude were performed to allow dissociation of protein from chromatin and solubilization.
For histone extraction, 8×106 S2 cells were washed in cold 1× PBS and resuspended in 800 µl of extraction buffer (10 mM Hepes pH 8; 0.1 mM MgCl2; 0.1 mM EDTA; 2 mM PMSF; 1× Protease Inhibitors; 1 mM NaF; 1 mM Na3VO4) and passed through a needle on ice. After incubation of 10 min on ice, cells were centrifuged for 10 min at 4°C at 2000 rpm. Pellets were washed with 400 µl of extraction buffer, resuspended in 100 µl of 0.2 N HCl and incubated overnight at 4°C with constant rocking. After 10 min of centrifugation at 13000 rpm 4°C, the supernatant was run on 12% SDS-PAGE. Alternatively, 30 µg of mammalian or Drosophila protein extracts were treated for 1 h with 4 units of DNAse (Turbo DNAse Ambion) at 37°C. For Western blot analysis, the densities of protein bands were measured using Image J software program.
Real-time PCR analysis
Total RNA was isolated with Trizol reagent (Invitrogen). 1 µg of RNA from each sample was subjected to cDNA synthesis using a Quantitect reverse transcription kit (Qiagen). DNA from ChIP, 3C or cDNA preparation was amplified in 20 µl reaction mixtures in the presence of 10 µl 2× QuantiTect SYBR Green master mix (Qiagen) and 0.5 µM of corresponding primers. Real-time PCR was performed with the DNA Engine Opticon 2 (MJ). Copy number was determined using the cross-point (Cp) value, which is automatically calculated using the Opticon Monitor 2 software (MJ). Primer sequences: rtgapdh-f 5′aagggaatcctgggctacac3′, rtgapdh-r 5′accgaactcgttgtcgtacc3′; rtpho-f 5′tcagttggttcacaccggtg3′, rtpho-r 5′gaggtatcttcactctggctg3′; rtpc-f 5′ttcaagactcaagtgctgcc3′, rtpc-r 5′ccatgggaaataagcaggag3′; rtez-f 5′ctgtggctgagatcaactcc3′, rtez-r 5′gacaggtcttggtcagcatg3′; rtbw-f 5′tcgctgtgcctcgagtgg3′, rtbw-r 5′aatcgccgccagcagcg3′; rtUbx-f 5′agtgtcagcggcggcaac3′, rtUbx-r 5′agtctggtagaagtgagcccg3′; rtabdA-f 5′caaatacaacgcaacccgagac3′, rtabdA-r 5′agcgatcgtgttgctgctg3′; utrgapdh-f 5′cgaactgaaactgaacgagag3′, utrgapdh-r 5′ttgacatcgatgaagggatcg3′; utrUbx-f 5′gttcgatggcaacggattgg3′, utrUbx-r 5′tgacggatttcctcgaatctg3′; utrabdA-f 5′aactcactgtgtgcggttcg3′, utrabdA-r 5′tcaagtgcgtgagtgtgtgtg3′; utrbw-f 5′agtcggcacatcacatagcc3′, utrbw-r 5′gttccagaaactgtagttgctc3′.
For BrdU labelling, exponential S2 cells were grown for one hour in the presence of 50 µM BrdU. 106 cells were centrifuged, resuspended in 0.4 ml of medium and placed at room temperature (RT) for 30 min on a Poly-Lysine coated slide (22 mm×22 mm). Fixation was performed in 4% paraformaldehyde 1× PBS for 10 min at RT. Cells were washed 3 times with PBT (PBS 1×, 0.1% Tween 20), incubated for 1 h at RT with RNAseA (100 µg/ml in PBT) and for 10 min at RT with PBS, 0.5% Triton. After washing cells again in PBS, they were incubated for 2 min at RT in 0.07N NaOH, briefly rinsed twice in PBS and blocked in PBS/1%BSA. All antibody hybridizations were carried out in a humid atmosphere at 37°C. Anti-PC and anti-H3K27me3 antibodies were incubated for 12–16 h while anti-BrdU antibody was incubated for 1 h. Washes were done in PBT. DNA was counterstained with DAPI, and glasses were mounted in Vectashield Antifade (Vector Laboratories). Images were taken with a Nikon ECLIPSE 90i microscope (100× objective) that was equipped with a digital camera (Nikon Coolpix 990) and NIS-Element software. Fluorescence quantification was done by determining the intranuclear mean fluorescence intensity using an Image J software program that computes area, mean, and grey values.
Exonic fragments of 600 bp, 1400 bp, 658 bp or 810 bp, respectively, from Gfp, Pc, Pho or E(z) genes, were amplified by PCR, creating T7 polymerase binding sites for the transcription of both strands. RNAi was performed as described previously [54]. Primer sequences: Gfp 5′acgtaaacggccacaagttc3′-5′tgctcaggtagtggttgtcg3′; Pc 5′attggcaagttaagcacgggca3′-5′acatcctggatcgccgcctca3′; Pho 5′acagtacgatgaagatataggc3′-5′tgatctgaactgagcttatagg3′; E(z) 5′tcgaaggcattatgaatagcac3′-5′atccgcatcttcagtctcc3′.
Supporting Information
Figure S1.
Experimental strategy to measure the timing of DNA replication using HU synchronization (A) Cell-cycle profile of D. melanogaster S2 cells stained with propidium iodide before and after HU block release. Cells treated with HU are in G1/S phase. After 1 h from the HU block release, cells are considered in early S phase, while cells collected 2 h from the release are in late S phase. (B) Replication timing of PREs as measured by Real Time PCR (qRTPCR). Ratios between the amplified products in early and late S phase are shown. We amplified positive controls for the early and late S phase and gene names correspond to their entries in FlyBase. All data points were generated from an average of four independent experiments. Standard error of the mean is indicated. Two-tailed t-test was applied for statistical analysis. Asterisks indicate statistically relevant differences; α = 0.05. P values: 108735/dodeca: P = 0.033; 108735/Fab-7: P = 0.013; 108735/mcp: P = 0.011; 108735/bxd: P = 0.006; 108735/bx: P = 0.006.
Figure S2.
PcG dependent repressive mark are enriched at PREs before replication. (A–D) ChIP analysis with antibodies against H3 (A), Topoisomerase II (B), H3K9me3 (C) and H3S28ph (D) on synchronized cells. ChIP analysis are presented as percentage of input chromatin precipitated for each region. Mock enrichment is below 0.003% of the input. ChIP enrichment for H3 modifications are normalized to histone H3 density. Data obtained in HU treated cells (G1/S) are shown in yellow. Data obtained in cells collected 1 h and 2 h from HU block release (ES and LS) are in red and light green respectively. Each graph shows the result from at least three independent immunoprecipitation reactions done on different chromatin preparations. Standard error of the mean is indicated. (E) Quantification of transcription by qRTPCR. The transcription levels of PcG mRNA are shown as percentage of Gapdh expression. All data points were generated from the results of six independent experiments. Standard error of the mean is indicated.
Figure S3.
Transcriptional profile of homeotic genes during S phase progression is not affected by PcG proteins depletions. (A) Quantification of transcription levels of mature and primary transcripts of indicated genes by Real Time PCR in untreated S2 and Gfp-dsRNA, Pc-dsRNA, Pho-dsRNA or Ez-dsRNA treated S2 cells. Data obtained in HU blocked cells (G1/S) are shown in yellow. Data obtained in cells collected 1 h and 2 h from HU block release (ES and LS) are in red and light green respectively. Transcription levels are shown as percentage of Gapdh expression. No amplification was detected in the absence of RT. All data points were generated from the results of at least four independent experiments. Standard error of the mean is indicated. (B) Replication timing of analysed promoters as measured by qRTPCR. Ratios between the amplified products in early and late S phase are shown. We amplified positive controls for the early and late S phase and gene names correspond to their entries in FlyBase. All data points were generated from an average of at least three independent experiments. Standard error of the mean is indicated. Two-tailed t-test was applied for statistical analysis. Asterisks indicate statistically relevant differences; α = 0.05. P values: CG3436/CG4345: P = 0.0002; CG3436/Ubx promoter: P = 0.003; CG3436/abdA promoter: P = 0.004; CG3436/bw promoter: P = 0.01. (C) Quantification of transcripts by qRTPCR. Expression level of homeotic genes in GFP-RNAi S2 cells (blue), in PHO-dsRNA treated cells relative to GFP-RNAi S2 (violet), in PC-dsRNA treated cells relative to GFP-RNAi S2 (brown), in Ez-dsRNA treated cells relative to GFP-RNAi S2 (green). Transcriptional levels are shown as percentage of Gadph expression. All data points were generated from an average of four different experiments. Standard error of the mean is indicated.
Figure S4.
(A) Negative control of immunofluorescence experiment. Representative examples of S2 nuclei with double immunostaining using only secondary antibodies. (B) Cell dimensions in S phase measured by FACS. Dot plot indicating the mean FSC (Forward Scatter) of early (ES, red) and late S (LS, green) phase in 5 independent S2 populations. Student t test was applied for statistical analysis; α = 0.05. P = 0.0009.
We thank Luca Battistini for providing lab space and funding to perform the work done by AD, Laura Pisapia and Maria Teresa Cencioni for technical support at FACS. We thank Donna Arndt-Jovin, J. Muller, and R. Paro for kindly providing antibodies that have been essential for this study. We are grateful to Gennaro Oliva for help in graphic design. We thank Genevieve Almouzni, Marco Foiani, Maria Vivo, Filippo Cernilogar, Holly Bream, and all members of the lab for stimulating discussions and constructive criticisms.
Author Contributions
Conceived and designed the experiments: CL VO. Performed the experiments: CL FLS. Analyzed the data: CL FLS VO. Contributed reagents/materials/analysis tools: AD VO. Wrote the paper: CL VO.
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text:
It's bedtime at my house.
My youngest always begs for a story. It's Wednesday night well past her bedtime, but I send her off with good news, "Climb into bed with a good book."
She chose "The Fantastic Flying Books of Mr. Morris Lessmore."
This book surprised me with encouragement for my writer's heart.
In the middle of the story, I found a breath of life for my often-doubting, inner writer. The words sank in.
"Everyone's story matters," said Morris.
And all the books agreed.
I wanted to stop reading and linger on the words, but I pressed forward and finished. We closed the book and prayed. I kissed my girl goodnight.
On my way out, I nipped the book and took it to my office. I needed to see those words again, to breathe in the fresh words of encouragement.
Discouragement comes easily for me as a writer. I've heard all writers wonder if they're good enough and, more importantly, if their story matters.
This book says so, but that's not what makes it true. Everyone's story matters because each person uniquely reflects God's image.
Imperfect as we are, God wants to mold us into his image so we might beautifully display his image to the world.
He uses our life story—with all the hardships and the victories—to transform us. As we tell our story, and especially as writers writing the story he's placed on our hearts, we bring hope, life, and color to this world https://viagraforgreece.com/.
Lexington Word Weavers member Cheryl Simpson writes real stories from her real life — a life made whole through the gospel.
Check out her blog @ Realing In Life
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subset: commoncrawl


text:
(An Incredible Revelation of God's Love)
Jesus Did It!
By: Norm Rasmussen
To view this full-length testimony on video,
click here:
Have you ever heard someone say: "God is love"? I never used to know what that really meant, until something happened one day that opened my understanding of God's unconditional love for each of us - for me personally - and I want to share with you how that happened.
Before I do that, however, I'm going to share a Truth with you that every one of us need to get hold of, if we want the very best possible relationship with God that we can have. God loves you and I not for what we do ... but for who He IS, and for what He has already DONE. Once you get hold of this mind-blowing truth, your relationship with God will soar unlike ever before!
Now the testimony:
By 1984, I had been a born again Christian for four years. God had done so much in my life over those four years, but God knew I needed a revelation of His unconditional love (God accepting us with all our character flaws, among other things) before I could grow in my relationship with Him.
I couldn't get my mind off God - which is how it all started. I found myself unable to concentrate at my job. I was physically at work, but mentally constantly thinking about God. At home with my wife, I didn't want to be distracted by having to fulfill my obligations as a spouse. I just wanted to be alone and be with God. It was not like me, I assure you, to be like this.
Yet - supernaturally, something was happening. Something inside me was growing in intensity, and I was so caught up in it at the time that I didn't fully realize what was happening. All I knew was that I had an incredibly powerful desire to commune with God and never quit communing.
After three days of being this way, I told my wife, Kathleen, that I was going to take the day off and just go spend it with God. I told her I had to. Her blessings were upon my request. She seemed to understand.
I drove my car to a deserted road for privacy and parked. I couldn't wait to put all distractions aside so I could concentrate 100% of my attention and affections on God. It was the burning desire of my whole being.
I spent maybe an hour - possibly more - in my car just communing with God, telling Him I loved Him. Thanking Him for all He had done for me. Thanking Him and verbally expressing to Him all of my appreciation for all He had done for me . . . when I had never deserved it.
Oh yes - that's right. You see, for over 20 years, I wasn't convinced there was a God! How could there be? All the suffering and pain and heartache in this world? Evil and greedy people getting their way, while good and unselfish people getting very little? No way- there couldn't be a God . . . at least a loving God. On top of that, I had prayed many times through my life, and my prayers never seemed to get answered the way I wanted them to. No - there was no God, which I was quite certain of.
But one fateful Saturday morning, after crying out to God all night, my life changed forever. I met God on His terms - the Holy Bible's terms - not mine. Overnight, my skepticism was gone. God had suddenly become as real as the sky is blue.
Years later - this was partly why I now had to be alone with God. I had been one of the world's greatest skeptics, and God had grabbed me by the shoelaces and pulled me out of the pit of blindness and despair that I had dug for myself.
No doubt it was a work of the Holy Spirit - I realized later. A burning desire to praise God had begun to build up in me several days prior to the ultimate climax. As each day had passed, all I wanted to do was sing praises to Him. At work, it became such a distraction that I decided to take a few spontaneous vacation days off, just so I could put my total focus on praising the Lord.
Never had I experienced such a thing. This was not a voluntary act on my part - something I had just decided to do. Something inside me to praise my Creator burned deeply - day and night, and it kept intensifying. Trying to sleep ... I was unable to do that, because it was like a broken record of praise kept going over and over in my mind and spirit. I had to get alone with God and praise Him forever. Nothing could stand in the way of that.
I drove my car to an old deserted two-track rode where I felt I would not be bothered by anyone.
My muscles were sore so I stepped out of my car. I started walking slowly down the dirt road to loosen out the kinks, but soon found that even walking distracted me. So I just stood there in the dusty road, with arms raised, loving on God; that is, telling Him over and over how much I loved and appreciated Him and praising and thanking Him every way I could think of.
Silly to some I'm sure - I was getting somewhat frustrated for not having a better way to tell God just how much love I felt for Him at the moment. With my hands still in the air, I began to praise Him more loudly. That was so wonderful. A new avenue to express my heart of love toward Him that felt like my heart was going to burst from it!
I must have stood there praising and worshiping Him for over half an hour, engulfed and enraptured by a closeness of what I had grown to know as intimacy with the Holy Spirit, when all of a sudden a distinct calm swept over me. Then God spoke something to me in my spirit that made me drop to my knees in the dust with tears streaming down my face. This is the "meat" of what He said:
"My child, I love you so much. Do you know that I love you as much as any great person you could ever imagine? As much as I loved the Apostle Paul. As much as I love the Pope or Billy Graham. As much as I love anyone else that you might think I would love more than you. I love you equally as much as I love them.
"But do you know what? I love the worst of sinners as much as I love you. No matter who you might think is the worst of sinners, I love that person as much as I love you, or Billy Graham, or anyone else you can think of. That's the nature of my love. It is not partial, nor is it conditional.
"Listen carefully however: No - I do not love sin. I hate sin. But I love the sinner".
"Yes - the favor of my peace rests more and more on those who put away sin in their life, and they will be rewarded for doing so in eternity, but do not confuse my love with my favor.
"You are trying to earn my love, son. There is nothing you can do to earn my love, so stop trying. There is nothing anyone can do to earn my love - from the worst of sinners to the most obedient Christians - so quit trying to earn my love. It's a free gift ... just as Salvation is a free gift.
"It's important, my child, that you know WHY I love impartially like this. The reason is because of WHO I AM. It is my nature and the essence of My being. I AM love. I AM unconditional love. I AM impartial love.
"I proved my love at the cross. At the cross, my Son, Jesus Christ, died for the sins of everyone ever born on earth, or whoever will be born. I sent Him to earth for this primary purpose.
"There is no greater act of love than someone dying on behalf of another ... especially when the one giving their life is totally innocent - totally undeserving - taking full punishment upon themself that rightly should be administered to the one who is guilty.
"My Son ... fully one with Me in the Godhead ... died on behalf of others; He died on behalf of sinners. He took their punishment. He took their condemnation, so they might receive life eternal. He became their pardon from eternal death. If you had been the only sinner on earth, He would have died just for you. He died for every person who has lived on planet Earth, or ever shall live.
"That's how much I love you. That's how much He loves them. That's how much we love every person ever created."
[Note: Some additional dialogue that transpired on that day can be accessed on the following link: Understanding God's Unconditional Love]
As the weeks and months went by following that fateful day on the dusty road, I began to see the great significance of what God had spoken to me. I had been giving money for the furtherance of the gospel to earn God's love, and didn't even realize my motive behind it. I had been sharing the Good News of the gospel everyway I felt led of the Holy Spirit to do so to earn God's love. I prayed, fasted, studied my Bible, went to church -- everything I was doing that pertained to the things of God -- I was "doing" to try to earn His love, and didn't even realize my unconscious motive behind it.
Since then, things have changed. Now I give money for the furtherance of the Kingdom of God to be built in the lives of people because of love, not because of fear that He won't love me if I don't. I share what God has done in my life every way I know how as I believe the Holy Spirit is leading me to do so for love, not for fear that He won't love me if I don't. Now I'm finally able to understand that when everything I do for God is done out of the motive of love . . . giving with no expectation of getting back . . . Christianity is so much more rewarding, for truly indeed as the scripture says:
Perfect love does cast out fear. (See: I John 4:18)
God gave His life for you and me. It was a sacrifice of love. In appreciation, we give ourselves fully to God as the natural expression of our sacrifice of love back to Him. It's not always easy ... but it should be our highest daily priority.
It is only wise to share another revelation the Holy Spirit imparted to me shortly after the above happened on the dusty road. God's unconditional love for each of us, from the worst of sinners to the most godliest of saints, will not keep a person from going to hell in and of itself. God has declared in His Word that every person has God's wrath upon them until they turn their lives over to Jesus Christ. Once they do that, and mean business, God's wrath is instantly removed, for all time and eternity, if that person continues to walk in intimate relationship with Jesus Christ until their time is done here on earth. That doesn't mean a Christian won't stumble and fall. Sometimes we fail to realize Christians are not perfect on this side of eternity, and thank God ... there is always forgiveness. Yet it does mean that we are to emulate what David did when He sinned against God: He eventually repented. Repentance is what keeps the grace of God overshadowing us, when we sin.
"He who believes in the Son has everlasting life; and he who does not believe the Son shall not see life, but the wrath of God abides on him." (John 3:36)
But God demonstrates His own love toward us, in that while we were still sinners, Christ died for us. Much more then, having now been justified by His blood, we shall be saved from wrath through Him. (Romans 5:8-9)
IMPORTANT NOTE: In the video series (see at top) I did on this subject, I spoke briefly of God's unconditional love toward Hitler, and Jeffery Dalhmers, as examples. Please don't anyone assume, if you watched this video series, that I implying in any way that Hitler and/or Jeffrey Dalmers are not in hell, if they didn't ask for God's forgiveness and accept Jesus Christ prior to their dying. God's unconditional love in and of itself does NOT exempt anyone from ending up in hell. What keeps a person from hell is their love for Truth, and their obedience to it. Please read the following writing for greater understanding on the topic: Why Would A Loving God Send Someone To Hell?
Here is another writing that I recommend everyone read to gain greater insight into the love of God. Brother John Piper has revelation of God's love that can be fully trusted in my opinion, and in fact he has been given revelation of from the Holy Spirit that the entire Body of Christ needs to be made aware of, if they have yet to receive it fully. Here is John's precious writing: For WHOM Did Jesus Taste Death?
JESUS DID IT! - or - JESUS DID IT!
(Please paste one of the above links onto your Facebook page - website - blog - video; etc.)
What if a born again Christian sins? Will that change God's unconditional love for that person? Absolutely not! Sin does not, and cannot, stop the flow of God's unconditional love toward a person, whether they are spiritually born again or not. It can stop and/or hinder God's favor toward that person until that person acknowledges their sin to God, asks for forgiveness, (which 1 John 1:9 promises they will receive), and gets that sin out of their life ... but it cannot stop, alter, or hinder in any way ... God's unconditional love for every person born into this world. Here is the assurance we have from God's Word of this incredible promise:
Who shall separate us from the love of Christ? Shall tribulation, or distress, or persecution, or famine, or nakedness, or peril, or sword? (Romans 8:35)
For I am persuaded that neither death nor life, nor angels nor principalities nor powers, nor things present nor things to come, nor height nor depth, nor any other created thing, shall be able to separate us from the love of God which is in Christ Jesus our Lord. (Romans 8:38-39)
If you should want, you can stop loving God, but that does not, and cannot stop God's unconditional love for you. It's simply impossible to stop! The death of Jesus Christ established the law of unconditional love for sinners that forever will never be able to be reversed, changed, or altered (even if God should want to, though it's impossible now for Him to even want to ... that's how cemented in stone His unconditional love for sinners is).
Now here is something that will blow the minds of most of us earthlings, it is so profound. If you could afford to put a million dollars in the offering plate in church every time it goes by, that will not gain you more of God's unconditional love. On the other hand, if you never place one penny in that offering plate as it goes by the rest of your life, it will not stop God's unconditional love for you. We can pray 70 times a day ... we can faithfully attend Church every time it's open ... we can read our Bible hours on end ... we can give all we have to the poor until we're poor just like them -- and that will not earn us God's unconditional love, or more of God's unconditional love. None of us can earn God's unconditional love by anything we do or not do. Nor can any of us stop God's unconditional love by what we do or don't do, no matter how pleased God might be with our doing. [Our Spirit-led "doing" certainly can and will lay up for ourselves treasure in heaven, and it can and usually does release God's favor toward us ... but don't confuse "doing" and "obedience" with God's unconditional love. (See Matthew 6:19-21 and 2 Corinthians 9:6)].
Let's address this issue: "Well, if God is a God of love, why does He allow all the suffering and hardship that people experience here in this life? Why does he allow war and killing and inhumane suffering of every kind? "
One of the attributes or expressions of God's love for every human being is His expression of free will. God loves every person so much that He gives that person choice. Choice to read and obey God's Word, or the ability to disbelieve it and disobey it. God gives us the choice to kill others, hurt others, steal from others, abuse others ... whatever we want to do to others. God allows leaders and rulers to wage war against others, if that is what they choose to do. He has given each of us the gift of free choice, to do as we please. What we do with that free choice will ultimately determine our destinies -- our future reward in heaven, or punishment in hell.
If God did not give us this gift of free choice, then the only alternative we would have would be robots. We would only be able to do what our Creator said we could and couldn't do, and that is not love at all, but dictatorship and control against our free will.
We give God a "bad rap" when we say that God cannot be a God of love, simply because if He was, He wouldn't be allowing all the hurt and the pain that is going on around this planet. God is a God of free choice -- we must always remember that. Furthermore, we must never forget that God has allowed Satan and his demon helpers some free will as well to have influence over the affairs of mankind ... yet for a season. There is more pain and suffering caused by the devil's doings than what most people realize.
If you would like to read Norm's born again testimony, click on the following link: http://precious-testimonies.com/BornAgain/L-N/Norm.htm
If you would like to read Norm's wife's born again testimony, click on the following link: http://precious-testimonies.com/BornAgain/h-k/kathleen.htm
Once a person can begin to grasp just how much the sacrifice of Jesus Christ on the Cross released the flow of God's unconditional love toward sinners, which every person born into this world is ... that person will never be able to turn their back on Jesus Christ again. The devil may be able to put the right kind of temptation to sin in front of you, and you might bite. You may in fact bite over and over. But the more you keep biting, the worse you'll begin to hate that sin you keep repeating, simply because the love of God simply won't let you stay content with your sin indefinitely.
Jesus Christ stands at the door of each of our hearts and knocks. If we'll open that door for Him, the first thing He wants to say to us is the most profound expression of God's love that God could express: "My Father's love for you is so deep that I died for you to prove it."
Does that sound too good to be true? Not once you grasp the nature of God's love for you!
I said this at the very beginning, and I'll end it by saying: God loves you and I not for what we do ... but for who He IS, and for what He has already DONE.
In truth, God's love for us is in fact unconditional ... yet not completely. There is a crucially important condition to it. It must be mixed with Truth. The truth, the whole truth, and nothing but the truth. That truth is recorded in the New Testament Bible.
To help us share the Best News every person needs to hear on this planet, randomly click FOR JESUS on just three (3) of the JESUS DID IT! links below. It will take just a few moments of your time. Please - that's all you are asked to do. God will reward you! (Of course, be highly encouraged to forward one or more of these video clips to those who may have never heard what Jesus did for them on the cross ... especially young people). Thank you so much!
For anyone living in the continental U.S. who would like 50 FREE copies [In English] of JESUS DID IT! to be made available to others, simply email us requesting them and provide us your mailing address.
Our email: email@example.com
A Special Message: http://www.precious-testimonies.com/Exhortations/f-j/HelpingShareTheMessageOfTheCross.htm
If this testimony/writing has blessed you, would you please take a few moments and share with us HOW it has blessed you? Your feedback is very important. Please mention the author of this testimony/writing and the testimony/writing title when you email your comments. Thank you so very, very much! Email: firstname.lastname@example.com
Dear Reader - are you at peace with God? If not, you can be. Do you know what awaits you when you die? You can have the assurance from God that heaven will be your home, if you would like to be certain. Either Jesus Christ died for your sins, or He didn't (He did!). Are you prepared to stand before God on the Judgment Day and tell Him that you didn't need the shed blood of Jesus Christ on the Cross to have your sins forgiven and get in right-standing with God? We plead with you ... please don't make such a tragic mistake.
To get to know God; to be at peace with God; to have your sins forgiven; to make certain heaven will be your home for eternity; to make certain that you are in right-standing with God right now ... please click here to help understand the importance of being reconciled to God. What you do about being reconciled to God will determine where you will spend eternity, precious one. Your decision to be reconciled to God is the most important decision you'll ever make in this life.
The staff and our ministry supporters so greatly appreciate hearing how God is touching lives for His glory through this outreach. If this ministry has blessed you in some special way, would you please consider taking a brief moment and share your blessing with us? Simply email us at: email@example.com
We truly thank each of you who allow us to publish your testimony, for those who faithfully pray (and fast) for this outreach, for those of you who help support the ministry financially, and for those of you who pass along these testimonies and other ministry writings to others. Especially to prisoners! The part the Holy Spirit has you play is vital in helping win lost souls and being engaged in discipleship, and we can never thank you enough for the labor of love and support you provide on behalf of our Lord Jesus Christ.
Precious Testimoniesis help supported financially by those God directs to sow into this ministry. We ask each person reading this to please ask God on an on-going basis if He would have you sow a financial gift to this evangelistic outreach of His - trust that He will clearly communicate His will to you in the matter - then simply be obedient. Please feel free to contact us if you have any questions about the current financial needs of this outreach, or any other questions you may have.
No salaries are paid to anyone from donations. Our staff consists strictly of volunteers. All love gifts are used to help pay for the expenses of this outreach (Mostly for providing FREE Jesus Did It! God's Best News Flyers around the globe).
For convenience, you can simply click on the secure Pay Pal donate button below if you want to donate by credit card. Otherwise, you can send your precious gift to:Precious Testimonies, P.O. Box 516, Jenison, MI 49429.
Precious Testimoniesis a non-denominational 501-C-3 evangelistic ministry, and financial love offerings to this ministry are tax-deductible for those who qualify. A financial summary can be viewed by clicking on the following link: http://www.precious-testimonies.com/General/u-z/YearlyContributions.htm
Inquiries or comments are welcome at our E-mail address:
Thank You, and God bless you!


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NOS is a popular energy drink that is part of the ever-growing energy drink industry that has already exceed $4.8 billion, according to Health Services at the University of New Hampshire 1. The NOS energy drink is very recognizable and is usually sold in a blue bottle that resembles the nitrous oxide cartridge used in racing cars. True to design, NOS's slogan is, "Human horsepower."
What Is In The NOS Energy Drink?
The regular NOS Energy Drink is 16 oz. and 220 calories in total. According to the manufacturer's website, the drink contains 28 g of carbohydrates per serving, with 27 g coming from sugar from high fructose corn syrup. There are are two servings per NOS energy drink, so you will consume 54 g of sugar per can or bottle. NOS also contains vitamin B-12, L-carinitine, taurine and ginseng as part of its energy complex. The University of New Hampshire notes that an entire 16 oz can or bottle of NOS contains 260 mg of caffeine 1. The smaller 11 oz bottle contains 172 mg of caffeine, the bigger 22 oz bottle contains 343 mg of caffeine.
What Are The Benefits Of The NOS Energy Drink?
The primary benefit of energy drinks such as NOS is a short-term energy boost. The large amounts of caffeine and sugar in NOS may give you an immediate energy boost. Caffeine is a central nervous system stimulant that will increase your alertness and may reduce fatigue in the short-term. According to the Virtual Mass Spectrometry Laboratory at Carnegie Mellon University, caffeine is rapidly absorbed by your body and reaches your bloodstream in about 30 minutes. The panax ginseng in NOS may also also improve short-term alertness, mental and physical endurance, according to the University of Maryland Medical Center.
What Are The Potential Dangers Of The NOS Energy Drink?
The stimulant properties of the NOS Energy Drink present several potential dangers and side effects. Boise State University explains that high doses of caffeine from energy drinks can dramatically increase your heart rate and blood pressure 12. This can lead to heart palpitations or an irregular heart beat, especially if you are sensitive to caffeine. This is why Boise State University discourages you from using energy drinks during exercise 2. The high fructose corn syrup in the NOS Energy Drink can also increase your body fat, especially if you consume the drink on a daily basis. Princeton University psychology department explains that high fructose corn syrup encourages fat storage at a far greater rate than regular sugar 3. This fat storage is particularly in the form of belly fat. There is a sugar-free version of the NOS energy drink that does not carry this side effect.
The NOS Energy Drink and Alcohol
When energy drinks are combined with alcohol, the combination can be fatal, according to Boise State University 2. Since alcohol is a depressant and NOS is a stimulant, the combination can reduce fatigue and mask how intoxicated you really are. Mixing caffeine and alcohol can cause you to become dehydrated very quickly if you are not drinking water. If you are dehydrated, your body will metabolize alcohol at a much slower rate. You should never combine high powered stimulants with alcohol, especially if you are going to be driving within a few hours.
The high fructose corn syrup in the NOS Energy Drink can also increase your body fat, especially if you consume the drink on a daily basis. Boise State University explains that high doses of caffeine from energy drinks can dramatically increase your heart rate and blood pressure. The University of New Hampshire notes that an entire 16 oz can or bottle of NOS contains 260 mg of caffeine.
- Amit Somvanshi/IndiaPicture/Getty Images


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PVC ball valves are installed in water supply lines for household water use and sometimes in garden watering systems. The valve has an inlet in each end for the supply pipe to enter, and depending on the valve can be cemented to the pipe or tightened in place with nuts. The ball itself has a hole drilled through the center, which when aligned with the supply pipe (by turning the valve handle), allows water to rush through the hole and serve the attached appliances. Loosening the valve is a quick process, but replacement may also be necessary.
Step 1
Turn the water off at the home's main shut-off valve. Attempt to open and close the valve by turning its handle several times by hand.
Step 2
Squirt lubricating spray where the valve handle enters into the valve, and wait 20 minutes. Attempt to turn the valve handle again, by hand. If it is still hard to turn, tap the valve slightly with a hammer, and place a pipe wrench around the handle to turn it (position a rag between the handle and wrench to keep the wrench from chewing up the handle). Keep attempting to turn the handle on and off for a few minutes.
Step 3
Turn the water back on if the valve loosens and keep turning the valve until its looseness reaches an acceptable level. However, if the valve handle refuses to budge, it needs to be replaced.


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Atlanta Fixture has been servicing Atlanta and the Southeastern United States for over 70 years. An industry leader in the distribution of foodservice supply products in Georgia, Alabama, Mississippi, South Carolina, North Carolina and Florida.
Atlanta Refrigeration Service works closely with Atlanta Fixture to provide customers with Turn-key installation services on equipment purchases. Atlanta Refrigeration can also provide factory authorized start-ups on most equipment. To find out more about Atlanta Fixtures, please visit their web site at http://www.atlantafixture.com/
Links > Strategic Partners >


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First of all, try everything that you can to get employed. Tell all of your friends, relatives and acquaintances that you are out of work and need a job.
Also, go to all the Job Fairs that you can. There is a Job Fair & Career Expo at Reliant Center in Houston on Jan. 29, 2014, at 3pm. Go and talk to employers, and either find a job or find out what employers are looking for in the way of skills or experience.
Once you have employment, come in and see us, and you may qualify for chapter 13 bankruptcy. We can file such a case quickly, and as soon as we do, your car or truck can no longer be repossessed, so long as you comply with your chapter 13 plan.
Chapter 13 can stop a repossession or foreclosure, so long as you will have regular income and it is sufficient to fund a plan. Call us at 713-772-8037 and set up your first consultation with us, to see what can be done in your situation. And good luck on the job hunt!


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Livas crafts
To discover crafting possibilities in Latvia and whole world
Storage Solutions
Monday, March 10, 2014
Ont The First Day Of School
I don't know how about you, but in my family, the first day of every schoolyear must be celebrated by going to some eating establishement. Well I would have written "restaurant", but since my kids rarelly get to fast food establishments, this is what they chose for their special meal least year :)
Once agaqin I played along with the
CSI challenge
- this time with their special additional challenge of this month.
Looks like I just can't stop making layouts using watercolor wash and stenciling. Well, at least this time I also added some modeling paste to give extra dimension to my page.
I rummaged through mu stash and found some old metallic accents to add as well as small bits of lace, fabric roses and that little fabric heart.
Journalling was done on a simple piece of notebook paper and I also added some border stickers. I had to go over them with ProMarkers to brighten up their colors a bit - to match the overall colors.
Even thou these photos were taken using my cellphone, this layout is very lovely and it sure is a moment to remember.
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Sunday, March 9, 2014
Waiting For The New Year
This week I also played along with the
CSI challenges
and made this New Years eve's layout of my girls. I just loved this color palette and it resonated with that photo so well.
I started with a really light watercolor wash as a background and then went over with teal acrylic paint and a damask stencil. Then I also added some stenciled splatters with golden glitter. I also added the same stencilling directly onto my photo.
I made several clusters from my handmade paper flowers, some store bought flowers and handmade glittered leaves.
As the challenge asked for journalling to be included in an envelope - I made one from light blue vellum as well as the note where the journalling is written on. I used my white Gelly Roll pen to write.
I just love this page and I hope that you do too.
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Monday, March 3, 2014
In The Kindergarten
It has been a really long time since I last created a scrapbook layout - way too long for my liking. So I am now working quite hard to remedy that.
I've always liked
CSI challenges
and so I decided to play along with their challenge 111. I must apologize for the quality of my pictures - they are taken using my cell phone, as my camera at the moment is traveling along with my sister somewhere in South Americas.
For the year of 2013, I have created an album that I will show you in some other post. The size of this album is an A4, so all my further layouts will also be the size of A4.
I started out by misting page with various shades Color Wash and then stenciled over with gesso.
Next I made a panel that I dryembossed using TH Woodgrain embossing folder. I also added some splatters and starburst rays using Th stencils and Walnut Stain Distress ink.
Then I made a cluster of various patterned papers with torn edges and put the picture of Emily right on top of it.
I did some fussy cutting from a patterned paper of roses and added clusters of handmade and store bought paper flowers. there is also a piece of painted chipboard flourish and lots of brown liquid pearls.
Journalling for this page was printed in several blocks of couple words and then scattered all over the page - one of the demands from CSI challenge.
I really like this layour and I'm sorry that you can't see it properly.
Hope you will enjoy,
Best regards,
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This year has been a whirlwind of change, the least of which is not the leaps and bounds my child is growing in. I wanted to write a few memories down, just so I won't forget them.
- A few nights ago we sang our bathtime song, which goes "Bath time, here we go! / Bath times a good time, you know! / Bath time, everybody LOVES bath time it's bath time, you know!" and repeats ad infinitum. We sang it before bath time like we do almost every night. After bath, we were downstairs snuggling and playing and I told Justin that we needed to put Benjamin to bed, soon, so we could take a shower. Benjamin proceeded to look at me and say, "No Mommy, I need shower too, it's shower time. Shower time, here we go! Shower times a good time you know!" Sorry, Benjamin, but it didn't get you out of bedtime.
- I made whoopie pies for coworkers and as the cakes were cooling I gave Benjamin one. He enjoyed it a lot – "Yum, delicioso!" After a few minutes, he started whining, and I asked him what was wrong. His response? "I don't have a cookie in my mouth!!"
- Did Benjamin get another cookie? Yes, yes he did!
- I feel bad sometimes, because I work so much and everyone else gets to be home with Benjamin more than I do. To combat that, I try to take Benjamin with me to social events that would drive me husband crazy. So the other day we went to a concert in a local park where they were playing 80s music. Benjamin and I danced and danced and danced. We had SO much fun!
- During the concert, Benjamin got antsy and wanted to get away from the loud music, so we took a walk. I followed him all the way to the edge of the park, where we played around. At the end of the walk I told him it was time to head back, and we could be like Dora and go "through the woods, across the field, to the concert!" So we did that, and midway through I made the mistake of saying "We need to keep an eye out for Swiper! That tricky fox is always trying to get out stuff!" So… we get ALMOST to the concert, and he turns to a random stranger and starts holding out his arm yelling, "SWIPER NO SWIPING! SWIPER NO SWIPING! SWIPER NO SWIPING!!" I was so embarrassed. Apologized. Moved on. Poor lady was freaked out…
- Last night Benjamin was cranky and overtired. It was a rough hour before bedtime, but we survived, and I was snuggling with him in our rocking chair before bed. I asked him if he wanted to pray, or sing, and he wanted to pray so we did. I said, after our initial prayers, "Benjamin, who do you want to bless?" He said Frankie (our dog). So we prayed, "Dear God, please bless Frankie." Then I asked him, "who else do you want to bless?" He thinks for a minute. Lucky, he says, the other dog. "Dear God, please bless Lucky." I ask him who else. He says, in the cutest voice, "Me." Oh, Benjamin, you want God to bless you? "Yes," he says to me, and my heart swells and gets all sentimental, and we pray a blessing over him too. I thought how brave of him, to be willing to ask God, but then I realized he doesn't know any better. He is at the age where he can boldly go to the throne without baggage. What a lesson for me!


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Blog | Media | Shop
Increase membership, make your Aktion Club more visible and share with others what you are doing by creating your own website. Here are some basics about websites.
Once your club website is live on the Internet, please submit the URL (Web site address) via e-mail. Include your club key number, club name, city/state and contact info in case we have a question. Upon review and approval, a link will appear on the Aktion Club Website Links page. This is a great way to gain exposure for your club and attract future Aktion Clubbers.
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For the past 18 consecutive months, the U.S. has led the world's crude oil production. It surpassed Russia and Saudi Arabia in June and August of 2018. One year later, the U.S. surpassed its own production record by 14.6 percent.Read More
Tag: Saudi Arabia
US to Send Troops, Military Equipment to Saudi Arabia, UAE
WASHINGTON – The U.S. is sending additional military forces and air defense equipment to Saudi Arabia and the United Arab Emirates, the Pentagon said Friday night.Read More
Commentary: The World Needs More Oil, but the Green New Deal Democrats Say No
In the flash of an eye, the world's oil supply was reduced by 5 percent after the Sept. 14 attacks on Saudi Arabian oil facilities including the Abqaiq oil processing facility, which sent oil prices soaring in trading on Sept. 16 and then a subsequent pullback today upon Saudi announcements that production will be restored within weeks.Read More
Department of Education Going After Elite Colleges for Allegedly Taking and Hiding Foreign Cash
by Luke Rosiak The Department of Education is going after U.S universities over supposed ties to foreign governments, after some allegedly took huge quantities of foreign cash and hid it from regulators. At the top of the list are Georgetown University and Texas A&M, which have taken hundreds of…Read More
US Reliance on OPEC Oil Hits 30-Year Low
by Michael Bastasch U.S. crude oil imports from the Saudi Arabian-led OPEC fell to a 30-year low, according to the latest federal figures. OPEC imports fell to 1.5 million barrels per day in March, which is the lowest level since March 1986, the U.S. Energy Information Administration reported Thursday.…Read More
Gavin Newsom Under Pressure to End Drilling as California's Reliance on Saudi Oil Soars
by Michael Bastasch Environmentalists are optimistic Democratic California Gov. Gavin Newsom will further restrict, or even end, oil and gas drilling, despite his state's increasing reliance on oil shipped from abroad. So-called "keep it in the ground" activists want to see Newsom ban new oil and gas leases, but…Read More
OPEC Scraps April Meeting but Keeps Oil Cuts in Place
Reuters Oil producer group OPEC on Monday scrapped its planned meeting in April and will decide instead whether to extend output cuts in June, once the market has assessed the impact of U.S. sanctions on Iran and the crisis in Venezuela. A ministerial panel of OPEC and its allies…Read More
IHOP Boycotts Tucker Carlson But Not Saudi Arabia
by Peter Hasson Breakfast chain IHOP cited its company "values" to justify boycotting Fox News host Tucker Carlson's show, even as the company profits from its businesses in Saudi Arabia, a notorious violator of human rights. Saudi Arabia's repeated human rights violations include "unlawful killings," "official gender discrimination against…Read More
Elite Universities Hide Information on Funding From Ultraconservative Nation of Qatar
by Luke Rosiak The nation of Qatar, a Sharia-law monarchy that has been accused of trying to influence other countries' governments, gave $1 billion to elite American universities since 2011, according to Department of Education data. Some universities have refused to discuss where strings are attached to that money.…Read More
Commentary: Big Media's Power Games and the Khashoggi Affair
by Joseph Duggan Jamal Khashoggi was a thoroughly charming and charismatic person. In March 2012, I took the last available seat at a luncheon table at the 20th Public Relations World Congress in Dubai. By sheer accident I found myself sitting next to Khashoggi and conversing with him for…Read More
Mnuchin Pulls US Out of Saudi Investment Conference
U.S. Treasury Secretary Steve Mnuchin has pulled out of an investment conference next week in Saudi Arabia, as Riyadh continues to face questions about its involvement in the disappearance and alleged killing of a U.S.-based Saudi journalist in Turkey. Mnuchin made the announcement Thursday on Twitter, following numerous Western corporate…Read More


text: Contents IT Business Management Previous Topic Next Topic Revert bucket assignments Subscribe Log in to subscribe to topics and get notified when content changes. ... SAVE AS PDF Selected Topic Topic & Subtopics All Topics in Contents Share Revert bucket assignments Reverting bucket assignments means taking the expenses out of the segments and accounts and leaving it in the bucket. Reverting buckets also deletes existing allocation lines for the working fiscal period that are associated with this bucket. Before you beginRole required: cost_transparency_admin or cost_transparency_analyst About this taskYou can revert bucket assignments for a single bucket or all buckets. Note: You can also revert expenses outside the workbench. Procedure To revert bucket assignments for a single bucket, perform the following actions: Click the information icon on a bucket to open the account summary. Click Unallocate Bucket. To revert bucket assignments for a single bucket and delete existing allocation lines for that working fiscal period, perform the following actions: click the unassign bucket icon () on the bucket. Click Yes on the confirmation window. You can also delete existing allocation lines for all fiscal periods by clicking click here on the confirmation window, and then clicking Delete. Figure 1. The Unallocate Bucket window To revert all bucket assignments, perform the following actions: Click the Gear icon () next to Allocation Setup. Click Unassign All Buckets. Confirm the action. Related tasksView bucket contentsSplit bucketsReview assigned expensesVerify the total amount allocatedPreview an allocation from a bucketRelated conceptsRollups for allocationsBucket assignments On this page Send Feedback Previous Topic Next Topic


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The Coffee House blog is covering tomorrows front page coverage of Budget 09. And it won't make pleasant reading in Numbers 10 & 11 Downing Street.
The Daily Mail's headline is the rather droll 'Alistair in wonderland'.
The Telegraph blasts 'The return of class war'.
The Guardian's has 'Darling's great squeeze'.
The FT's is 'Darling gambles on growth'.
The Sun lists the bad economic news and has a headline about how 'At least it's sunny'; appropriately it is meant to rain this Sunday.
The Times has the wonderful double—or is it triple—entendre, 'Red all over'.
The Independent highlights the breach of Labour's manifesto promise not to change income tax rates with the line 'That's rich!'.
The Daily Express's front page is simply, 'They've ruined Britain'.
Who do you think is going to win the battle for the budget narrative? Hmm...