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Post-translational modifications of histones play important roles in maintaining normal transcription patterns by directly or indirectly affecting the structural properties of the chromatin. In plants, methylation of histone H3 lysine 4 (H3K4me) is associated with genes and required for normal plant development.
We have characterized the genome-wide distribution patterns of mono-, di- and trimethylation of H3K4 (H3K4me1, H3K4me2 and H3K4me3, respectively) in Arabidopsis thaliana seedlings using chromatin immunoprecipitation and high-resolution whole-genome tiling microarrays (ChIP-chip). All three types of H3K4me are found to be almost exclusively genic, and two-thirds of Arabidopsis genes contain at least one type of H3K4me. H3K4me2 and H3K4me3 accumulate predominantly in promoters and 5' genic regions, whereas H3K4me1 is distributed within transcribed regions. In addition, H3K4me3-containing genes are highly expressed with low levels of tissue specificity, but H3K4me1 or H3K4me2 may not be directly involved in transcriptional activation. Furthermore, the preferential co-localization of H3K4me3 and H3K27me3 found in mammals does not appear to occur in plants at a genome-wide level, but H3K4me2 and H3K27me3 co-localize at a higher-than-expected frequency. Finally, we found that H3K4me2/3 and DNA methylation appear to be mutually exclusive, but surprisingly, H3K4me1 is highly correlated with CG DNA methylation in the transcribed regions of genes.
H3K4me plays widespread roles in regulating gene expression in plants. Although many aspects of the mechanisms and functions of H3K4me appear to be conserved among all three kingdoms, we observed significant differences in the relationship between H3K4me and transcription or other epigenetic pathways in plants and mammals.
Post-translational modifications of histones play important roles in maintaining normal transcription patterns by directly or indirectly affecting the structural properties of the chromatin. Histone modifications are highly complex due to the large number of residues that can be modified as well as the variety of modification types (for example, methylation, acetylation, phosphorylation and ubiquitination, and so on) [1]. In addition, in the case of lysine methylation, a lysine residue can be mono-, di- or trimethylated with potentially different effects on chromatin structure [2-4]. Some histone modifications can directly alter chromatin structure. For example, acetylation of specific residues in the globular core domains of histones weakens the histone-DNA interactions, resulting in a relatively 'open' chromatin structure that facilitates transcription [5,6]. In contrast, other modifications (such as lysine methylation on the amino-terminal tail of H3) do not grossly affect chromatin structure per se, but interact with additional factors. For example, several groups of proteins have been shown to preferentially bind histone H3 methylated at lysine 4 (H3K4me): the human chromatin remodeling and assembly factor hCHD1 (human homolog of Chromodomain helicase DNA binding protein 1) binds H3K4me through its chromodomain [7,8], the chromatin remodeling complex NURF (Nucleosome remodeling factor) binds H3K4me through the PHD (plant homeodomain) domain of its large subunit BPTF (Bromodomain PHD finger transcription factor) [9], the H3K9me3 and H3K36me3 demethylase JMJD2A (Jumonji domain containing 2A) binds H3K4me (and H4K20me3) through its Tudor domain [10,11], and members of the ING (Inhibitor of growth) family of tumor suppressor proteins bind H3K4me through the PHD domain [12,13].
Four lysine residues on histone H3 were found to be methylated in Arabidopsis thaliana by mass spectrometry studies (H3K4, H3K9, H3K27 and H3K36) [14,15]. Di-methylation of histone H3 lysine 9 (H3K9me2) is required for the transcriptional silencing of transposons and other repetitive sequences [16,17], whereas H3K27me3 is primarily involved in the developmental repression of endogenous genes [18-21]. Recent genome-wide profiling studies in Arabidopsis have shown that H3K9me2 is highly enriched in the pericentromeric heterochromatin where transposons and other repeats cluster [22-25], whereas H3K27me3 is mostly distributed in the transcribed regions of a large number of euchromatic genes and bound by the chromodomain-containing protein LIKE HETEROCHROMATIN PROTEIN-1 (LHP1) [23,26,27]. H3K36me is required for normal plant development, but the genome-wide distribution of this modification and its role in transcriptional regulation remain unclear [28-31]. Finally, H3K4me2 is primarily distributed in endogenous genes but not transcriptionally silent transposons, as shown by a previous study of a 1-Mb heterochromatic region in Arabidopsis [22].
Only one H3K4 methyltransferase (SET1; SET domain containing 1) has been identified in yeast (Saccharomyces cerevisiae), and it has been proposed the differential methylation of H3K4 can be attributed to the kinetics of the dissociation of SET1 from the elongating RNA polymerase [32]. Multiple putative H3K4 methyltransferases homologous to SET1 have been identified in Arabidopsis [33-36]. Several lines of evidence suggest that in Arabidopsis distinct H3K4 methyltransferase complexes may also contribute to the differential accumulation of H3K4me1, H3K4me2 and H3K4me3 at specific loci. For example, loss of the H3K4 methyltransferase ATX1 (Arabidopsis homolog of Trithorax 1) leads to a mild reduction in global H3K4me3 level and eliminates H3K4me3 at specific loci, but has no detectable effect on H3K4me2 [37]. In contrast, the loss of a closely related H3K4 methyltransferase, ATX2, results in locus-specific defects in H3K4me2 but does not appear to affect H3K4me3 [38]. Examination of H3K4me levels at several genes revealed that the types of H3K4me present at individual genes may differ significantly [38,39]. Interestingly, the atx1 mutant exhibits several developmental abnormalities, whereas the atx2 mutant is phenotypically normal [38-40]. Furthermore, results from transcriptional profiling studies indicated that ATX1 and ATX2 likely regulate two largely non-overlapping sets of genes [38]. It therefore appears that there may be significant differences in the mechanism, localization and function H3K4me1, H3K4me2 and H3K4me3.
Here we report a genome-wide analysis of H3K4me1, H3K4me2 and H3K4me3 in Arabidopsis using chromatin immunoprecipitation (ChIP) and whole-genome tiling microarrays (ChIP-chip). We found that all three types of H3K4me are distributed exclusively within genes and their promoters, and that approximately two-thirds of genes contain at least one type of H3K4me. In addition, H3K4me3, H3K4me2 and H3K4me1 are distributed with a 5'-to-3' gradient along genes, where H3K4me3 and H3K4me2 are enriched in the promoters and 5' end of transcribed regions with H3K4me3 distributed slightly upstream of H3K4me2, and H3K4me1 is depleted in promoters but enriched in the transcribed regions with an apparent 3' bias. Interestingly, we found that genes associated with different combinations of H3K4me are expressed at different levels and with different degrees of tissue specificity. Furthermore, genome-wide comparisons between H3K4me and other epigenetic marks revealed preferential co-localization between H3K4me2 and H3K27me3, and between H3K4me1 and CG DNA methylation in the transcribed regions of genes. Finally, the relationship between H3K4me and DNA methylation was further examined by genome-wide profiling of H3K4me in a DNA methylation mutant. The results suggested that H3K4me and DNA methylation may not directly interfere with each other in Arabidopsis, and that these two epigenetic pathways interact primarily through transcription.
Results and discussion
Genome-wide profiling of H3K4me1, H3K4me2 and H3K4me3
Arabidopsis chromatin enriched for H3K4me was isolated by ChIP using antibodies that specifically recognize H3K4me1, H3K4me2 and H3K4me3 (Figure S1 in Additional data file 1). As a control, nucleosomal DNA was isolated by ChIP using an antibody against histone H3 regardless of its modifications. H3K4me ChIP samples were compared to the control nucleosomal DNA by hybridization to Affymetrix whole-genome tiling microarrays that represent approximately 97% of the sequenced Arabidopsis genome at 35-bp resolution.
H3K4me1, H3K4me2 and H3K4me3 regions identified here are highly consistent with results from recently published studies [38] (Figure S2 in Additional data file 1). In addition, real-time PCR validations were performed at a number of randomly chosen loci, all of which yielded results consistent with the ChIP-chip data (Figure S3 in Additional data file 1). Finally, only 0.10%, 0.66% and 0.57% of the chloroplast genome was falsely identified as containing H3K4me1, H3K4me2 and H3K4me3, respectively. Taken together, these results indicate that the ChIP-chip data here provide an accurate representation of the genome-wide distribution of H3K4me with a relatively low false positive rate.
H3K4me1, H3K4me2 and H3K4me3 accumulate exclusively in genes
A total of 15,475 (7.77 Mb) H3K4me1, 12,781 (7.17 Mb) H3K4me2 and 15,894 (14.48 Mb) H3K4me3 regions were identified as described above, representing 6.45%, 6.0% and 12.1% of the sequenced nuclear genome, respectively. All three types of H3K4me are highly enriched in the gene-rich euchromatin and absent from pericentromeric heterochromatin regions where transposons and other repetitive sequences cluster (Figure 1a). Such a euchromatic distribution may largely reflect the fact that H3K4me1, H3K4me2 and H3K4me3 localize almost exclusively in genes: 96.7%, 93.3% and 95.7% of all H3K4me1, H3K4me2 and H3K4me3 regions, respectively, are in or overlap with transcribed regions of genes or their promoters (defined as the 200-bp regions upstream of transcription start sites). Only a small fraction of the remaining H3K4me1, H3K4me2 and H3K4me3 regions (0.6%, 1.3% and 1.5% of total, respectively) overlap with intergenic repetitive sequences such as transposons. The distribution of HK4me in a representative eukaryotic region is shown in Figure 1b.
Figure 1. Distribution of H3K4me in the Arabidopsis genome. (a) Chromosomal distribution of H3K4me. Top row: the total length of repetitive sequences (y-axis, left-side scale) and number of genes per 100 kb (y-axis, right-side scale). Bottom panels: chromosomal distribution of H3K4me1, H3K4me2 and H3K4me3. X-axis: chromosomal position; y-axis: the total length of genomic regions containing H3K4me1, H3K4me2 and H3K4me3 per 100 kb, respectively. Arrows indicate the heterochromatic knob on chromosome 4. (b) Local distribution of H3K4me1, H3K4me2, H3K4me3, other epigenetic marks (DNA methylation, H3K9me2, H3K27me3, nucleosome density, small RNAs) and transcription activity in an approximately 40-kb euchromatic region on chromosome 1. Repetitive sequences are shown as filled red boxes on top. Individual genes are shown in open red boxes (arrows indicate direction of transcription; filled light blue boxes, exons; light blue lines, introns). Distribution of H3K4me on the gene labeled by a red asterisk is enlarged and shown in detail at the bottom.
Differential distribution of H3K4me1, H3K4me2 and H3K4me3 within genes
A total of 18,233 genes (approximately 68.0% of all annotated genes) were found to contain H3K4me in their promoters and/or transcribed regions, including 8,571 with H3K4me1, 10,396 with H3K4me2 and 14,712 with H3K4me3. The distribution patterns of H3K4me at the 5' regions of genes were determined by aligning genes by their transcription start sites, and the percentage of genes containing H3K4me in their promoters and the 5' transcribed regions was determined. Similarly, the distribution patterns of H3K4me at the 3' regions of genes were determined by aligning genes by the 3' end of their transcribed regions. These analyses were performed on a set of 5,809 genes that meet the following two criteria. First, they are located 1 kb or more away from the upstream and downstream genes such that ambiguity introduced by neighboring genes can be minimized. Second, they are longer than 1 kb so that there is sufficient gene space to determine the distribution of H3K4me. We further classified the 5,809 genes into four groups according to their length: long genes (>4 kb, 691 genes), intermediate genes (3 to 4 kb, 828 genes; 2 to 3 kb, 1,768 genes) and short genes (1 to 2 kb, 2,522 genes).
The distribution patterns of H3K4me on long genes are shown in Figure 2a. H3K4me1 is present at relatively low level at the 5' and 3' termini of transcribed regions, but is enriched in the internal regions with a slight 3' bias. In contrast, H3K4me2 and H3K4me3 are both enriched in the 5' end with H3K4me3 distributed slightly upstream of H3K4me2. Both H3K4me2 and H3K4me3 are also enriched in the promoters (200 bp upstream of transcription start sites) and 5' flanking regions (200 to approximately 400 bp upstream of transcription start sites), but are absent in the 3' half of the transcribed regions or the 3' flanking regions of the long genes.
Figure 2. Distribution of H3K4me relative to genes. (a) Distribution of H3K4me at the 5' and 3' ends of genes. 'Isolated' genes are divided into four groups according to their length (see text for details). Genes belonging to each length group were aligned at the transcription start sites, and the percentage of genes containing H3K4me in their promoters or 5' ends is determined at 200-bp intervals (left y-axis). Similarly, genes belonging to each length group were aligned at the end of transcribed regions, and the percentage of genes containing H3K4me in their 3' ends or downstream flanking regions is determined at 200-bp intervals (right y-axis). The first and last 500 bp, 1 kb, 1.5 kb and 2 kb are shown for genes that are 1 to 2 kb, 2 to 3 kb, 3 to 4 kb and >4 kb in length, respectively. (b) Distribution of H3K4me across genes. Each gene (thick horizontal bar) was divided into 20 intervals (5% each interval), and the 1-kb regions upstream and downstream of each gene (thin horizontal bars) were divided into 50-bp intervals. The percentage of genes with H3K27me3 in each interval was graphed (y-axis). (c) Relationship between gene length and H3K4me. Genes are divided into eight categories according to the combination of H3K4me (see text for details), and the percentage of genes within each length group that are associated with a particular combination of H3K4me is shown (y-axis). (d) Length distribution of genes associated with different combinations of H3K4me. X-axis: gene length in kb (200 bp per bin); y-axis: the percentage of genes associated with a particular combination of H3K4me that are of the corresponding length. A small number of genes longer than 8 kb are not shown.
A comparison of the distribution patterns of H3K4me on long genes and intermediate or short genes revealed several common features as well as some interesting differences. First, as gene length decreases, significantly smaller fractions of genes were found to contain H3K4me1, but the relative position of H3K4me1 in genes (that is, internal regions with a 3' bias) remains similar. Second, the distribution patterns of both H3K4me2 and H3K4me3 at the 5' ends of short or intermediate genes are largely similar to those on long genes, although the shortest genes seem to contain a lower level of H3K4me3 at the 5' end. Third, as gene length decreases, significantly more genes were found to contain H3K4me2 and H3K4me3 in their 3' regions. For example, in the last 200 bp, 10.8- and 13.3-fold more short genes contain H3K4me2 and H3K4me3 than long genes, respectively.
In order to obtain a more continuous view of the distribution of H3K4me, we analyzed the average distribution levels of H3K4me across entire genes. To do this, we divided the transcribed region of each gene into 20 bins (5% of the gene length per bin), and divided the 1-kb upstream and downstream flanking regions of each gene into 20 bins (50 bp per bin). The percentage of genes containing H3K4me in each bin was then determined (Figure 2b). Consistent with the results described above, H3K4me1 is highly enriched within the transcribed regions, but it is present at very low levels in promoters and 3' flanking regions. In addition, H3K4me1 is present at significantly higher levels and spans broader regions on longer genes. In contrast, H3K4me2 and H3K4me3 are enriched in promoters and the 5' half of transcribed regions, at comparable levels on genes with different lengths. Although H3K4me2 and H3K4me3 extend further towards the 3' end on shorter genes relative to gene length, the absolute positions remain virtually constant: regardless of gene length, the highest levels of H3K4me2 and H3K4me3 were found at approximately 600 to 800 bp and 400 to 600 bp downstream of transcription start sites, respectively (Figure 2a). In addition, for genes in all the length groups, H3K4me2 and H3K4me3 appear to be enriched (that is, present at the same or higher levels as they are at transcription start sites) downstream of transcription start sites for approximately 1.5 kb and 1 kb, respectively (Figure 2a).
The observation that H3K4me2 and H3K4me3 appear to cover the 5' regions of genes for a relatively constant length suggests that the length of a given gene may affect the association of this gene with different types of H3K4me, in particular H3K4me1. For example, while all three types of H3K4me are positively correlated with gene length (Figure 2b), such a relationship is significantly more pronounced for H3K4me1. To further study the relationship between gene length and H3K4me, we classified the 5,809 genes into 8 categories based on the 8 possible combinations of their associated H3K4me: H3K4me1 only (me1+me2-me3-), H3K4me2 only (me1-me2+me3-), H3K4me3 only (me1-me2-me3+), H3K4me1 and H3K4me2 but no H3K4me3 (me1+me2+me3-), H3K4me1 and H3K4me3 but not H3K4me2 (me1+me2-me3+), H3K4me2 and H3K4me3 but not H3K4me2 (me1-me2+me3+), H3K4me1, H3K4me2 and H3K4me3 (me1+me2+me3+), and no H3K4me (me1-me2-me3-). The frequencies of occurrences of these combinations within each length group were then determined. As shown in Figure 2c, all combinations that include H3K4me1 (regardless of H3K4me2 and H3K4me3) showed a strong positive correlation with gene length, and all combinations of H3K4me2 and H3K4me3 (in the absence of H3K4me1) showed a negative correlation with gene length. In addition, genes associated with H3K4me1 (me1+me2-me3-, me1+me2+me3-, me1+me2-me3+, me1+me2+me3+) are generally longer than average, with me1+me2-me3+ and me1+me2+me3+ genes being significantly longer and including very few genes shorter than 2 kb (Figure 2d). In summary, by every measure, longer genes show higher levels of H3K4me1.
The distribution patterns of H3K4me2 and H3K4me3 described here are similar to results from analyzing genes on chromosomes 4 and 10 in rice [41]. That is, in both species, H3K4me2 and H3K4me3 are enriched in the promoters and the 5' ends of transcribed regions, with H3K4me3 peaking slightly upstream of H3K4me2 (at approximately 400 to 600 bp and approximately 600 to 800 bp downstream of transcription start sites, respectively; Figure 2a). These results suggest that H3K4me2 and H3K4me3 may be involved in both transcription initiation and the early stages of transcription elongation. In contrast, the internal distribution of H3K4me1 observed here suggests that H3K4me1 might be primarily involved in the elongation step during the transcription of longer genes. Alternatively, the apparent preferential accumulation of H3K4me1 in the transcribed regions may be because this modification is reduced at gene ends (that is, H3K4 is preferentially di- or trimethylated at the 5' ends and unmethylated at the 3' ends).
Association of different combinations of H3K4me1, H3K4me2 and H3K4me3 with differential gene expression patterns
To further test the relationship between H3K4me and transcription, we compared the expression level and tissue specificity of genes associated with different combinations of H3K4me, using a previously published expression profiling dataset [42]. Of the 5,809 genes described above, 5,479 were analyzed here, as expression data were available for these genes. As shown in Figure 3a, me1+me2-me3+, me1+me2+me3+ and me1-me2-me3+ genes are highly expressed, whereas me1+me2-me3-, me1-me2+me3- and me1+me2+me3- genes are expressed at very low levels. The me1-me2+me3+ group includes genes with a wide range of expression levels and seems to be enriched for moderately expressed genes. In addition, me1+me2-me3+, me1+me2+me3+ and me1-me2-me3+ genes exhibit very low levels of tissue specificity, while me1+me2-me3-, me1-me2+me3- and me1+me2+me3- genes are highly tissue specific (Figure 3b). Taken together, these results suggest that H3K4me3 is associated with and likely plays important roles in active transcription. H3K4me1 and H3K4me2, in the absence of H3K4me3, are preferentially associated with tissue-specific genes that are generally not expressed at the developmental stage assayed in this study. These results are consistent with previous reports that although H3K4me2 is generally associated with genes in Arabidopsis, its presence does not always correlate with active transcription [37].
Figure 3. Genes with different expression levels and patterns are associated with different combinations of H3K4me. (a) Distribution of expression levels of genes associated with different combinations of H3K4me. X-axis: gene expression level determined in a previous study (log2 scale) [42]. Y-axis: the percentage of genes with corresponding H3K4me combination and expression level. (b) The degree of tissue-specific expression of genes associated with different combinations of H3K4me, as measured by entropy (x-axis). Y-axis: the percentage of genes with corresponding H3K4me combination and entropy values.
Relationship between H3K4me and H3K27me3
In Drosophila, the Trithorax (TRX) family of H3K4 methyltransferases and the Enhancer of Zeste (E(z)) family of H3K27 methyltransferases function antagonistically to activate or repress a largely overlapping set of genes, respectively [43,44]. Interestingly, many genes are associated with both H3K4me and H3K27me3 in mammalian stem cells, and such a 'bivalent' histone modification has been suggested to play an important role in stem cell renewal and differentiation [45]. Similarly, the co-existence and antagonistic functions of H3K4me3 and H3K27me3 have been described at the FLC and AGAMOUS genes in Arabidopsis [38,39,46-48]. We have indeed detected H3K4me2, H3K4me3 and H3K27me3 at the FLC gene. However, we found that AGAMOUS contains a low level of H3K4me2 but no significant level of H3K4me3. This apparent discrepancy is likely due to the different tissues used in the experiments: young seedlings were used in this studywhereas a previous study used mature rosettes (Z Avramova, personal communication).
We have previously found that H3K27me3 is associated with 4,000 to 5,000 tissue-specific genes in their repressed state in Arabidopsis [26]. In order to test whether a preferential association of H3K4me with H3K27me3 exists that could indicate a functional connection, we first determined the fraction of genes with each combination of H3K4me that are also associated with H3K27me3. As shown in Table 1, we found that me1-me2-me3- and me1-me2+me3- genes are associated with H3K27me3 more frequently than expected. In addition, the association frequencies of me1+me2-me3-, me1+me2+me3- and me1-me2+me3+ genes with H3K27me3 are all lower than expected. Finally, me1-me2-me3+, me1+me2-me3+, and me1+me2+me3+ genes are even more depleted of H3K27me3. It should be noted that the differences in transcription levels cannot fully account for the differential association of H3K4me genes with H3K27me3. For example, the me1-me2-me3- and me1-me2+me3- genes are significantly more frequently associated with H3K27me3 than me1+me2-me3- and me1+me2+me3- genes, but these four categories of genes are expressed at very similar levels (Figure 3). The relationship between H3K4me and H3K27me3 was further examined by directly testing whether they co-localize to the same genomic regions. To do this, we determined the presence of each type of H3K4me in H3K27me3-containing genomic regions. As a control, we also determined the presence of H3K4me in a set of randomly chosen regions with the same length and genomic distributions of H3K27me3-containing regions. As shown in Table 2, whereas H3K4me1 and H3K4me3 are significantly depleted in H3K27me3-containing regions, H3K4me2 was found to overlap with H3K27me3 slightly more frequently than with random control regions.
Table 1. Co-localization of H3K4me and H3K27me3 in genes
Table 2. Co-localization of H3K4me and H3K27me3 in the same genome regions
It should be noted that the starting materials in this study (young seedlings) included many distinct cell types. It is likely that some genes are associated with H3K4me3 when they are expressed in some cell types, but are associated with H3K27m3 elsewhere when they are transcriptionally repressed. It is therefore possible that the low frequency of co-localization between H3K4me3 and H3K27me3 described here may still represent an overestimate. It is also possible, however, that co-localization of H3K4me3 and H3K27me3 at a given gene only occurs in specific cell types or during certain developmental stages. If this is the case, our results generated using mixed cell types from a single development stage could represent a gross underestimate of the prevalence of bivalent chromatin modification in plants. Future studies at cell-specific levels should more directly address the exact extent to which plant genes are bivalently modified. In any event, our results seem to indicate a mutually exclusive relationship between H3K4me3 and H3K27me3 at many genes in Arabidopsis seedlings. In animals, the H3K4 demethylase JARID1A (Jumonji, AT rich interactive domain 1A)/RBP2 (Retinol binding protein 2) is recruited to genomic targets through its interaction with the H3K27me3 methyltransferase complex Polycomb repressive complex (PRC) 2, where RBP2 mediates transcriptional repression by demethylating H3K4me3 to H3K4me2 (and to a lesser extent, H3K4me2 to H3K4me1) [49,50]. In addition, the H3K4me3-specific demethylase JARID1D interacts with Ring6a (Really interesting new gene 6a)/MBLR (Mel18 and Bmi1-like RING finger protein), which is closely related to the PRC1 components Bmi1 (B Lymphoma Mo-MLV insertion region 1) and Mel18 [51]. Interestingly, two Arabidopsis RING finger proteins, AtRING1a and AtRING1b, have been recently found to interact with the H3K27me3 methyltransferase CURLY LEAF and the H3K27me3-binding protein LIKE HETEROCHROMATIN PROTEIN1, and are required for the transcriptional repression of H3K27me3 target genes [52]. The general mutual exclusion between H3K4me3 and H3K27me3 as well as the more frequent overlap of H3K4me2 and H3K27me3 suggest that similar mechanisms might also function in plants. That is, plant H3K4me3 demethylase(s) may function in transcriptional repression by interacting with PRC1 and/or PCR2. If this is the case, a fraction of the H3K4me2 in the Arabidopsis genome could be the demethylation product of H3K4me3.
We also observed that H3K4me1 tended not to co-localize with H3K27me3. One contributing factor could be the differential distribution patterns of these histone modifications along genes: H3K4me1 tends to be present at the 3' half of long genes, whereas H3K27me3 does not exhibit similar preferences for either location within genes or gene length (Figure S4 in Additional data file 1). Furthermore, H3K4me1 was present more frequently on ubiquitously expressed housekeeping genes, while H3K27m3 was more frequently present on tissue-specific genes.
Relationship between H3K4me and DNA methylation
Cytosine DNA methylation is an epigenetic silencing mechanism important for the developmental regulation of endogenous genes and the transcriptional silencing of transposons [53-56]. A mechanistic relationship between DNA methylation and H3K4me has been described in mammals, where the DNA methyltransferase (DNMT) homolog DNMT3L specifically interacts with histone H3 containing unmethylated lysine 4 [57]. That DNMT3L also binds and stimulates the activity of the de novo DNA methyltransferase DNMT3A suggests that H3 with unmethylated K4 may play a role in targeting de novo DNA methylation in mammals [57-59]. However, a distinct small interfering RNA (siRNA)-directed pathway is responsible for de novo DNA methylation in plants [60-62], and an interaction between DNA methyltransferase and histone has not been reported.
Three DNA methylation pathways have been described in plants: METHYLTRANSFERASE 1 (MET1) is a homolog of mammalian DNMT1 and primarily functions in maintaining DNA methylation in the CG sequence context ('CG methylation') [63-66]. The DOMAIN REARRANGED METHYLASE (DRM) (homologous to mammalian DNMT3) interacts with the siRNA pathway and is required for de novo DNA methylation in all sequence contexts as well as the maintenance of DNA methylation in the CHH context (H = A, C or T; 'CHH methylation') [60-62]. The CHROMOMETHYLASE3 is specific to plant genomes and interacts with the H3K9me2 pathway to maintain DNA methylation in the CHG sequence context ('CHG methylation') [67,68].
The genome-wide distribution of DNA methylation in Arabidopsis has been determined by a number of studies using microarray analyses or ultra-high-throughput deep sequencing of bisulfite treated DNA [22,25,69-77]. Results from these studies are largely consistent: CG, CHG and CHH methylation is highly enriched in transposons and other repetitive sequences, suggesting that the RNA interference, H3K9me2 and DNA methylation pathways function together in the transcriptional repression at these loci. DNA methylation is generally depleted in the promoters and 5' ends of endogenous genes. However, over one-third of Arabidopsis genes contain DNA methylation exclusively in the CG sequence context that is enriched in the 3' half of their transcribed regions (termed 'body-methylation'). Most body-methylated genes are expressed at moderate to high levels, and it is therefore unclear whether CG methylation alone in the transcribed regions of genes plays a direct and significant repressive role in transcription.
In order to determine the relationship between DNA methylation and H3K4me in Arabidopsis, we compared DNA methylation levels in genomic regions containing H3K4me to the whole-genome average of DNA methylation. As shown in Table 3, CHG and CHH methylation is significantly depleted in genomic regions containing H3K4me1, H3K4me2 or H3K4me3. CG methylation is also significantly depleted in H3K4me2- and H3K4me3-containing regions. In stark contrast, we found that CG methylation is highly enriched in H3K4me1-containing regions (Table 3). In addition, nearly two-thirds of H3K4me1-containing regions (8,841 of 14,599, approximately 60.6%) with two or more CG dinucleotides are methylated at two or more CG sites, compared to approximately 7.0% (842 of 12,100) and approximately 11.7% (1,750 of 14,918) for H3K4me2- and H3K4me3-containing regions, respectively.
Table 3. The percentage of cytosine residues that are methylated in CG, CHG or CHH sequence contexts in H3K4me-containing genomic regions*
The low level of CHG and CHH methylation in H3K4me-containing regions can be explained by the virtual absence of siRNAs and H3K9me2 within actively transcribed endogenous genes. The lack of CG methylation in H3K4me2- and H3K4me3-containing regions could be due to an active mutual exclusion mechanism (for example, MET1 may be discouraged from chromatin containing H3K4m2 or H3K4me3) similar to what was recently described between DNA methylation and the deposition of the histone variant H2A.Z [78], or simply the differential localization of DNA methylation and H3K4me2/H3K4me3 relative to genes (a 5' bias for H3K4me2/H3K4me3 and a 3' bias for DNA methylation). The high level of CG methylation in H3K4me1-containing regions was unexpected. It is possible that CG methylation and H3K4me1 interact with each other and therefore co-localize at the 3' transcribed regions of genes. It is also possible that the overlap of these two epigenetic marks merely reflects their preferential localization in the similar regions of highly expressed genes. In either case, these results indicate that CG methylation per se and H3K4me1 do not appear to interfere with each other. Finally, genomic regions free of H3K4me frequently lack DNA methylation, suggesting that the absence of H3K4me alone is insufficient to trigger DNA methylation.
Ectopic H3K4me in met1 is associated with transcriptional de-repression
In order to test whether direct mechanistic links exist between DNA methylation and H3K4me (that is, whether DNA methylation per se excludes H3K4me2/H3K4me3 and whether gene body methylation facilitates H3K4me1), we determined the genome-wide distribution of H3K4me in the met1 mutant by ChIP-chip. Previous studies have shown that loss of MET1 eliminates CG methylation as well as substantial fractions of CHG and CHH methylation, resulting in massive transcriptional reactivation of transposons [71,72,74,76,77].
All three types of H3K4me were found to be present at much higher levels in the pericentromeric heterochromatin regions in met1 (Figure 4). A closer examination revealed that hyper-H3K4me in met1 is almost always associated with ectopic over-expression of transposons or pseudogenes (Figure 4). However, the loss of DNA methylation does not appear to directly trigger hyper-H3K4me. In contrast to the transcription-independent ectopic accumulation of H2A.Z in DNA hypomethylated regions in the met1 mutant [78], no major change in H3K4me was observed in genomic regions that are DNA-hypomethylated but not transcribed. This suggests that the ectopic transcriptional activity resulting from the loss of DNA methylation, but not the loss of DNA methylation per se, is associated with hyper-H3K4me. In addition, at nearly all genes, a complete loss of gene body methylation in met1 had no significant effect on H3K4me1, H3K4me2 or H3K4me3 (Figure 4), suggesting that CG methylation in genes is dispensable for the normal accumulation of H3K4me1.
Figure 4. Comparisons of H3K4me accumulated in wild-type Arabidopsis (Wt, green) and the met1 mutant (light brown). Left: chromosome-level changes in H3K4me, showing the ectopic accumulation of H3K4me in the pericentromeric heterochromatin. Chromosome 5 is shown as an example (Wt, green; met1, light brown). X-axis: chromosome position; y-axis: the percentage of H3K4me on chromosome 5 in the corresponding region (in 100 kb bins). Right: local changes in DNA methylation, H3K4me and transcription in a euchromatic region (top right) and a heterochromatic region (bottom right) on chromosome 5. The five genes shown in the euchromatic region likely encode cellular proteins and their expression patterns are unaffected in the met1 mutant. These are (from left to right): At5g56210, WPP-DOMAIN INTERACTING PROTEIN 2; At5g56220, nucleoside-triphosphatase; At5g56230, prenylated rab acceptor (PRA1) family protein; At5g56240, unknown protein. The six genes shown in the heterochromatic region are all transposon-encoded genes. These are (from left to right): At5g32925, CACTA-like transposase; At5g32950, CACTA-like transposase, At5g32975, similar to En/Spm-like transposon protein; At5g33000, Transposable element gene; At5g33025, gypsy-like retrotransposon; At5g33050, gypsy-like retrotransposon. Note that the overexpression of At5g32950 and At5g33050 is associated with ectopic accumulation of H3K4me.
Our genome-wide analysis of H3K4me1, H3K4me2 and H3K4me3 led to several interesting results. First, a large number of genes were found to contain H3K4me: at a single developmental stage, approximately two-thirds of all Arabidopsis genes contain at least one type of H3K4me. This suggests that H3K4me may be required for the normal expression or a large number of genes in plants. Second, H3K4me1, H3K4me2 and H3K4me3 are enriched in different regions in their target genes. H3K4me2 and H3K4me3 are distributed in the promoters and 5' regions with H3K4me3 slightly more upstream, whereas H3K4me1 is mostly located within the transcribed regions. Our H3K4me3 results are highly consistent with those recently published by van Nocker and colleagues [47]. Importantly, very similar distribution patterns of H3K4me1, H3K4me2 and H3K4me3 were also found in yeast, human and other plants (for example, H3K4me2 and H3K4me3 in rice) [32,41,79-81], which suggests that many aspects of the mechanisms and functions of H3K4me may be highly conserved during evolution. Third, we found that genes with different expression levels and tissue specificity are associated with different assortments of H3K4me1, H3K4me2 and H3K4me3, suggesting that the three types of H3K4me may have different effects on chromatin structure and transcription. In particular, whereas H3K4me3 appears to be generally associated with actively transcribed genes, our results do not support a direct role of H3K4me1 and H3K4me2 in transcriptional activation: H3K4me1 and H3K4me2 do not appear to have an additive effect on H3K4me3 with regard to transcription levels and, in the absence of H3K4me3, they are not preferentially associated with active transcription. Interestingly, our observation that H3K4me2 (but not H3K4me1 or H3K4me3) often overlaps with H3K27me3 raises the possibility that the accumulation of H3K4me2 at some loci in the Arabidopsis genome might result from demethylation of H3K4me3 by histone demethylases associated with PcG complexes. Fourth, unlike in mammalian stem cells, H3K4me3 and H3K27me3 do not appear to preferentially co-localize on a genome-wide level in Arabidopsis. A second significant difference between plants and mammals is that, in mammals, H3K4me3 is present at active promoters as well as a large number of 'poised' promoters [82], whereas in plants, the presence of H3K4me3 is usually correlated with active transcription. Finally, we observed strong negative correlations between H3K4me2/H3K4me3 and all three types of DNA methylation, and between H3K4me1 and CHG and CHH DNA methylation. However, the loss of DNA methylation does not generally trigger hyper-H3K4me in the corresponding genomic region, indicating that DNA methylation per se may not inhibit H3K4me. Our results do suggest that DNA methylation may interfere with H3K4me indirectly through transcriptional repression, as ectopic transcription was observed in the vast majority of the cases where DNA hypomethylation and hyper-H3K4me occur at the same genes. Interestingly, H3K4me1 is highly correlated with the CG methylation that exists within the transcribed regions of genes. Although the retention of H3K4m1 in the met1 mutant indicates that CG DNA methylation is not required for the accumulation of H3K4me1, it is possible that H3K4me1 might play a role in the colonization of CG DNA methylation within the transcribed regions of genes.
Materials and methods
Arabidopsis thaliana plants (accession Col) were grown on soil under continuous light for 3 weeks, and the aerial part of the seedlings was harvested. The met1-3 mutant plants were grown under the same conditions and harvested at a similar developmental stage. Chromatin was fragmented to 300 to 1,200 bp (mostly 600 to 800 bp) by sonication, and ChIP was performed as previously described using antibodies purchased from Abcam (anti-H3K4me1, ab8895; anti-H3K4me2, ab7766; anti-H3K4me3, ab8580; anti-H3, ab1791) (Cambridge, MA, USA) [26]. The specificities of anti-H3K4me antibodies were validated by dot blot analysis (Figure S1 in Additional data file 1). ChIP samples were amplified, labeled, and hybridized to microarrays as previously described [26,72]. Four biological replicates were performed for H3K4me1 and H3K4me3, and eight biological replicates were performed for H3K4me2. For each H3K4me ChIP, an H3 ChIP was performed to isolate nucleosomal control DNA. Microarray hybridization intensities from probes that match a unique genomic region were analyzed using Tilemap with the Hidden Markov model option, as previously described [83]. All raw microarray data (CEL files) have been deposited in Gene Expression Omnibus [GEO:GSE13613]. Processed data showing the enrichment of H3K4me can be viewed online [84]. The gene expression data used here were from a previous comprehensive transcriptional profiling study (data for 7- to 14-day-old seedlings were used here for analysis of gene expression levels, and data for all tissue types and developmental stages were used here to analyze tissue specificity) [42]. The gene annotations used here are according to TAIR7. Real-time PCR validation of ChIP-chip results was performed using the SYBR Green I Master kit (Roche; Indianapolis, IN, USA) on a Roche Light Cycler 480. The PCR parameters are: 1 cycle of 10 minutes at 95°C, 40 cycles of 10 s at 95°C, 10 s at 60°C, and 20 s at 72°C. PCR primer sequences are listed in Table S1 in Additional data file 1.
ATX: Arabidopsis homolog of Trithorax; ChIP: chromatin immunoprecipitation; DNMT: DNA methyltransferase; H3K4me: H3 methylated at lysine 4; JARID: Jumonji, AT rich interactive domain; MET1: METHYLTRANSFERASE 1; PHD: plant homeodomain; PRC: Polycomb repressive complex; RBP: Retinol binding protein; SET1: SET domain containing 1; siRNA: small interfering RNA.
Authors' contributions
XZ, YVB and SEJ designed the experiments. YVB and XZ performed the experiments. XZ, MP and SEJ analyzed the data. SC contributed reagents/materials/analysis tools. XZ wrote the paper.
Additional data files
The following additional data are available with the online version of this paper: Figures S1 to S4 and Table S1 (included in Additional data file 1).
Additional data file 1. Figure S1: dot blot analysis showing the specificity of antibodies used here. Figure S2: comparison of the H3K4me distribution patterns determined here and those reported in a recent locus-specific study [38]. Figure S3: real-time PCR validation of H3K4me ChIP-chip results. Figure S4: length distribution of H3K27me3 target genes. Table S1: real-time PCR primer sequences.
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XZ was supported by a Faculty Research Grant (JR-040) from the University of Georgia. YVB was supported by USPHS National Research Service Award GM07104. Jacobsen lab research was supported by NIH grant GM60398. SEJ is an investigator of the Howard Hughes Medical Institute.
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Zilberman D, Gehring M, Tran RK, Ballinger T, Henikoff S: Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription.
Nat Genet 2007, 39:61-69. PubMed Abstract | Publisher Full Text
Zhang X, Yazaki J, Sundaresan A, Cokus S, Chan SW, Chen H, Henderson IR, Shinn P, Pellegrini M, Jacobsen SE, Ecker JR: Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis.
Cell 2006, 126:1189-1201. PubMed Abstract | Publisher Full Text
Reinders J, Delucinge Vivier C, Theiler G, Chollet D, Descombes P, Paszkowski J: Genome-wide, high-resolution DNA methylation profiling using bisulfite-mediated cytosine conversion.
Genome Res 2008, 18:469-476. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mathieu O, Reinders J, Caikovski M, Smathajitt C, Paszkowski J: Transgenerational stability of the Arabidopsis epigenome is coordinated by CG methylation.
Cell 2007, 130:851-862. PubMed Abstract | Publisher Full Text
Zilberman D, Henikoff S: Genome-wide analysis of DNA methylation patterns.
Development 2007, 134:3959-3965. PubMed Abstract | Publisher Full Text
Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, Jacobsen SE: Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.
Nature 2008, 452:215-219. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lister R, O'Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, Ecker JR: Highly integrated single-base resolution maps of the epigenome in Arabidopsis.
Cell 2008, 133:523-536. PubMed Abstract | Publisher Full Text
Zilberman D, Coleman-Derr D, Ballinger T, Henikoff S: Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks.
Nature 2008, 456:125-129. PubMed Abstract | Publisher Full Text
Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, Lee W, Mendenhall E, O'Donovan A, Presser A, Russ C, Xie X, Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander ES, Bernstein BE: Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.
Nature 2007, 448:553-560. PubMed Abstract | Publisher Full Text
Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K: High-resolution profiling of histone methylations in the human genome.
Cell 2007, 129:823-837. PubMed Abstract | Publisher Full Text
Pokholok DK, Harbison CT, Levine S, Cole M, Hannett NM, Lee TI, Bell GW, Walker K, Rolfe PA, Herbolsheimer E, Zeitlinger J, Lewitter F, Gifford DK, Young RA: Genome-wide map of nucleosome acetylation and methylation in yeast.
Cell 2005, 122:517-527. PubMed Abstract | Publisher Full Text
Guenther MG, Levine SS, Boyer LA, Jaenisch R, Young RA: A chromatin landmark and transcription initiation at most promoters in human cells.
Cell 2007, 130:77-88. PubMed Abstract | Publisher Full Text
Ji H, Wong WH: TileMap: create chromosomal map of tiling array hybridizations.
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UCSC Genome Bioinformatics [http://epigenomics.mcdb.ucla.edu/H3K4m1m2m3/] webcite


text:
Details of further Java exploits are coming to light, while Oracle has still yet to release a fix for its customers.
Since Computing reported on Michael Schierl's research into the Java 7 exploit, Immunity Products' Esteban Guillardoy has submitted findings that suggest two separate security holes are driving the exploit.
"The first bug was used to get a reference to the sun.awt.SunToolkit class, that is restricted to applets, while the second bug invokes the getField public static method on SunToolkit using reflection with a trusted immediate caller bypassing a security check," Guillardoy wrote on Immunity's blog.
As knowledge of the exploit, which affects all versions of Java 7, becomes greater and more widespread, the eyes of Java users will look to Oracle for a patch or fix before its next scheduled update on 16 October 2012.
An Oracle spokesperson today told Computing that there is no comment available at the moment on the security exploit. The spokesperson supplied a link to the Oracle Software Security Assurance Blog, which has not been updated since 10 August 2012, in response to the Security Alert CVE-2012-3132 Oracle Database Server exploit.
Oracle's post reminds users that "it is unfortunate when the technical details of a security vulnerability are disclosed before a fix could be made available, especially when the disruption resulting from having to deal with an unplanned patch, and the amount of time required by customers to apply the patch, may yield less of a security posture improvement than other security effort."
Security companies such as DeepEnd, however, argue that as Oracle continues to do nothing, the revelation of technical details – which allow action to be taken by security groups as well as hackers – are all that stand between the community and total vulnerability.

subset: commoncrawl


text:
This is a program that can print your karaoke song books. This program connects to the developer´s servers and obtains the list of themes and lyrics for the songs included on your karaoke CDs. This way, you can create and print song books for each of the CDs you own, and you can also export that information to use it in other programs.
This is a free converter. Free PCM to Zen Audio Converter is professionally designed to help you convert PCM to Zen Audio files easily and fast. With his user-friendly interface, it is easy to use for all its users. With this program you can complete your conversion as easy as possible. This program runs on Windows XP, 2003, Vista (32bit, 64bit).
Free PCM 2 AAC Convert is a professional conversion program which allows you to convert your PCM to AAC format with fast speed and great output quality. The program is freeware and does not contain any form of malware, including spyware, virus, spam or trojans, so it is safe to install.
APR is a call recorder software for desktop computers.
1) Records ALL incoming phone calls.
2) Logs date, time, duration, caller ID.
3) Compressed wav files.
4) Post process your wav files.
5) Archiving.
C.A.T.S. FLASH Utility Program is developed to read and program the FLASH memory in the '94/'95 LT1 PCMs. The program uses your PC's serial port to communicate with the PCM over the vehicles ALDL port.
With this program you can read the original calibration stored in your car's PCM and save it to your hard drive so you make changes to the calibration using the C.A.T.S. Tuner program.
CDA Converter ActiveX is an ActiveX component for software developers. With this component you can convert CDA tracks from an audio CD to many types of audio files, such as MP3, WMA, PCM WAV, ACM WAV, OGG and APE.
This component is actually the light version of the Audio CD Ripper AX and it function the same, without the FreeDB component.
This ActiveX component can join many types of audio files, such as: MP3, WMA, WAV, ACM, OGG, APE, AIF, VOX, SND and CDA tracks from an Audio CD.
The destination can be any one of MP3, WMA, WAV, ACM, OGG and APE audio files.
This ActiveX component can handle with the ID3 tags, can get full information about the audio files, supports many events, runs fast and easy to use.
Registry Shower 2012 scans your system registry and detects both mild and critical errors. They are the usually the cause of common system problems like slowness and instability. The program is capable of repairing those errors, thus increasing your system's speed and helping you keep an error-free system. Registry Shower 2012 has been designed to be usable by both novice and expert users.
Kaspersky Anti-Virus 2012 is the latest software from the Kaspersky Lab designed to make your computer immune to almost any threats.
Kaspersky Anti-Virus uses the latest technologies in antivirus development to offer means of protection against multiple types of virus, trojan, worm threats, spam, hackers for your computer or notebook.
Serial Key Generator is a program to help developers generate serial numbers for applications. You can generate serial keys using a custom number of columns and characters per column. The sequence of numbers/digits can be defined in the application. The output can be saved as CSV or TXT documents.
Guidelines for applications using Serial/IP to access serial devices
using public/private networks and wireless data services.
Use remote serial devices on TCP/IP networks through
Serial/IP virtual COM ports that work like local COM ports.
Serial/IP virtual COM ports appear in the list. Selecting a port shows its current settings.
The UI is hopefully self explanatory. If you want to disable some of the UI goodies (like the list editor) you can do that in the Settings menu by choosing "Use Lists In Grid"
This software allows you to create customized content with a few mouse clicks and then view it on your monitor. You can supply up to 5 players on an associated editor with content.
PDF-XChange 'Lite' 2012 is the popular solution for the creation of Adobe compatible PDF files from virtually any Windows application, the list is endless. PDF-XChange 'Lite' 2012 is FREE for non commercial use and is a simplified version of our PDF-XChange Standard 2012 print driver - retaining the 'Industrial' strength PDF creation engine shared by all of our PDF creation tools !.
With the List & Label Report Designer you can quickly and easily enhance your application with powerful features. You can enhance your applications by adding only a few lines of code to include all types of evaluations: reports, subreports, multi-tables, crosstabs, charts, Gantt charts, gauges, forms, labels, serial letters, more than 50 barcode formats, web reporting.
This software offers a solution for users who need to keep an organized inventory of multiple units of equipment. After entering vital data such as serial numbers and item descriptions a print-ready Excel spreadsheet is automatically generated. Keep inventory lists with maintenance logs and capital expense records. This software will save you time by quickly generating organized templates.
WinCatalog 2012 helps you to keep your music and film collections neatly organized, which will allow you to find a specific title or track in just seconds. This versatile application can also scan all your computer drives, helping you to locate and organize all those loose audio and video files that are gradually becoming harder and harder to find.
PDFCreator 2012 allows you to create PDF files from any printable file or document. The program installs a virtual printer on your system, which you can then select just as you do with any other printer. Thus, all you need to do is to open the file you want to convert, and "print" it selecting "PDFCreator 2012" as the printer. This way you can get professional PDF files in just a few seconds.
Autodesk®Design Review is a free program used for creating and reviewing DWF files. An open, published, and secure file format developed by Autodesk, DWF enables you to combine and publish rich 2D- and 3D-design data and share it with others. Design Review enables your entire project or product team to view, print, measure, and markup DWF, DWG, DXF, PDF.


text:
Lotus Saudi Arabia / Bahrain
The introduction of the Lotus brand to Saudi Arabia and Bahrain has been met with a very high level of regional interest. The brand has a widely appreciated heritage of sporting success and its high profile participation in Formula One racing is still remembered.
Today Lotus is an industry leader in automotive design and engineering specializing in chassis and suspension and consulting with most of the world's leading brand manufacturing.
The founder, Mr Colin Chapman, was a very talented engineer and keen racing driver. It was his passion for success both on and off the racing track that shaped his philosophies of "Performance Through Lightweight", "Great Ride & Handling", "Innovation" and "Fun To Drive", philosophies that are still true to Lotus today, 27 years after his death and that can be seen and felt by looking at and driving every Lotus car that comes off our production line at Hethel in England. Every Lotus driver can experience our passion for "Pure Sports Car Performance" and every Lotus owner is welcomed into our family to share this passion.
At Al Ghassan Motors, we have the passion, determination and a real understanding of Lotus values and just like Lotus, Al Ghassan Motors is unconventional, inventive, vibrant and straight talking. We feel sure that all Saudi and Bahraini sports car enthusiasts are delighted by our commitment to Lotus and that we are making these extraordinary products available to them. We would like to extend an open invitation to all sports car enthusiasts to come and visit the Al Ghassan Lotus showrooms in Jeddah and also Riyadh, Al Khobar and Bahrain to share our passion and to become part of our family.


text:
- Filed Under
About 40 people gathered late Monday afternoon in the old Stone Chapel at Drury University for a memorial service for the 20 children and six school employees killed Friday in Newtown, Conn.
Adam Lanza, 20, the gunman, took his own life.
Those in attendance at Drury were offered words of assurance from the Bible as well as words of advice from a grief counselor. ...


text:
The Logitech C920 is the first webcam we've tested that delivers solid 1080p video chats in Skype, and it's pretty great otherwise, too.Read the full review →
As low as
conclusion:We like it
If you spend a lot of time doing Skype video chats, here's your chance to do them in full 1080p HD. As long as you have good lighting and a relatively new PC running Windows 7 (the c920 isn't Mac compatible), your chat partners will see you in all your HD glory (it's up to you whether or not that's a good thing). If you're not a Skype user, the c920 will work with other video chat services, including Google and Yahoo, at 720p.
90-Day Price History
Critic reviews
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Video quality | 2 comments
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Logitech's C920 is a new high-end webcam that's even better than the excellent C910 it's due to replace. Picture quality and sound quality are top-notch. It's a cut above all the other webcams we've reviewed.Read the full review →
Video-call enthusiasts should take a good look at the Logitech HD Pro Webcam C920 as it offers noticeably better image quality, and Logitech did not crank up the "sharpening" filter on that one, so the picture looks more natural.Read the full review →
The Logitech HD Pro Webcam C920 lives up to the "pro" in its name, taking care of any needs an amateur vlogger or parent away on a business trip might have. With virtually no setup besides plug in and go, this is a webcam that's guaranteed to last for any practical purposes.Read the full review →
I'm returning this webcam after testing it on skype. I was on a 7 person Skype call which was recorded. I saw the video recording and was astounded at how terribly my video looked. There was a lot of obvious compression, and my video looked worse than every other person's (mostly people with...Read the full review →
This is the webcam I currently use, and I have to say it is amazing and worth every penny. It has great quality and a great microphone as well as a amazing sleek design. It's perfect for Skype and amazing for recording things like YouTube videos. The included software is also pretty cool. Overall...Read the full review →
A very good webcam. I buyed it and was impressed by the image quality. Defineatly something that I will not have to upgrade for many years. But it was also very expensive. I'm not skyping that often with video chat but when i tried it out i couldn't get it run in HD. It was more like 360p. I...Read the full review →
Fantastic video quality and design. Average sound.Read the full review →
I am using mine connected to my MacMini to watch over the main entryway into my house. For that, it is great. If I need video conf, I will use the one built-into my MacBook Pro.Read the full review →
How it stacks up
Logitech HD Pro C920 87 from $49
Nest Cam 76 from $169
Sony PlayStation Camera for…
Logitech HD Webcam C270 from $19
Sony PlayStation Eye 83 from $7


text:
James V. Brownson is James I. and Jean Cook Professor of New Testament at Western Theological Seminary, Holland, Michigan, and author of the new book Bible, Gender, Sexuality: Reframing the Church's Debate on Same-Sex Relationships.
* * *
You all know the story of the blind men and the elephant. One thinks it's a tree; another a snake. It all depends on what part of the elephant they are touching.
One of the things I try to argue in my book, Bible, Gender, Sexuality, is that something like this happens in a lot of the discussion about "nature" and same-sex intimate relationships as depicted in Romans 1:26-27. One of the things Paul says about these relationships is that they are "contrary to" or "outside of" nature. But different people have very different ways to interpret what Paul means by "nature."
On the more progressive side of the broader debate over the interpretation of Romans 1, we see a couple of different perspectives. For some, Paul is speaking here about heterosexual men who act against their "natural" heterosexual orientation by engaging in sex with other men. By this view, Paul says nothing about gay men who are acting in accordance with what is "natural" for them in terms of sexual orientation. Others on this side of the larger debate focus not so much on "nature" as one's individual disposition, and more on "nature" as social consensus, or "what everybody knows." Often, these folks cite texts like 1 Cor 11:14, "Does not nature itself teach you that if a man wears long hair, it is degrading to him?" In this view, "nature" refers to little more than commonly-held social assumptions, which can change dramatically in different times and cultures.
Those on the more conservative side of the broader debate strongly disagree with both of these readings. They insist that what Paul means by "nature" in this text is all about human anatomy — the "fittedness" of male and female sex organs. By this argument, "nature" has nothing to do with one's individual "orientation," or even social consensus. Instead, it has everything to do with the way that human bodies are designed. Same-sex intimate relationships are wrong, by this view, because they involve a misplacement of body parts.
In Bible, Gender, Sexuality, I explore all these arguments. I'm not convinced that "nature" in the ancient world refers primarily to the shape of body parts, though I think the ancient view of nature included an awareness of the link between sex and procreation. But at a deeper level, I also argue that for the ancients, "nature" was an all-encompassing vision, including individual disposition and social consensus, as well as the physical and biological world. The various dimensions emphasized by different sides of the contemporary debate were all part of the ancient discussion. All the blindfolded folks are holding on to part of the elephant. But what the ancients were looking for at the deepest level, when they spoke of "nature," was a convergence of individual disposition, social consensus, and the biological world. They wanted to take off the blindfold, to see the whole, and to determine how it all fit together.
What might it mean to embrace such an integrative vision today, when we have a different social consensus, and when many people are recognizing that one's individual sexual orientation, regardless of its origin, is often deeply persistent and resistant to change? Human biology hasn't changed, but many other aspects of the ancient vision for "nature" have changed in the modern world. Pursuing an integrated and converging vision for "nature" will be considerably more complicated in our contemporary context. Where should we put the emphasis today, and how do we integrate these various dimensions of what is "natural" or "contrary to nature?" One my book's aims is to explore the various aspects of that more complicated vision.
Click to order James Brownson's Bible, Gender, Sexuality: Reframing the Church's Debate on Same-Sex Relationships.


text:
Netscape has been building up its Internet Keywords system since it was implemented last July as part of its Smart Browsing enhancements, and it has turned into an impressive resource that can make locating web sites much easier.
The idea of Internet Keywords is simple. Users should be able to enter a word or words into the web location box of their browser and be taken to a relevant site, regardless of whether they entered a proper web address. So under the system, a user can enter "united airlines" where they'd ordinarily enter a web address and be taken straight to the United Airlines web site, rather than having to enter http://www.ual.com.
When Netscape launched its system, users were either taken directly to a relevant web site or given matching results from the Netscape Search search engine. Occasionally, they might also have been dumped into one of the Netscape site's channels, such as "autos" linking to a page within Netcenter.
Now, the keywords system is quite likely to land you on an intermediary page, where Netscape displays a variety of options that seem suited to your query.
"We quickly learned that there isn't any one thing that you can send people to, especially in terms of the generic categories," Ken Hickman, the senior product manager at Netscape who oversees Smart Browsing. "So, we introduced this intermediate page, that allows us to point at multiple answers in respect to what you type in."
The results are easy to understand and helpful -- sometimes even better than what the major search engines dish up. For instance, imagine a search for "united." It could mean United Airlines -- but it could also mean United Artists. The intermediary page solves the problem. It lists several major companies that all include the word United in their names. Likewise, the page lists common domains that have the word in them.
Another good example is a search for "airlines." Here, you're shown a link to a matching category from the Netscape site relating to airlines. Netscape also offers to take you to its online booking area -- as an option, it can be appreciated. If it had been forced upon you, it might have been resented.
Using Internet Keywords is easy. Just enter a word or words into the location box and hit Enter. Netscape will then try to resolve your term in one of several ways.
First, it will check to see if the word or words have been linked directly to a web site. This is commonly the case for trademarks, brand names, company names or other common words where Netscape feels comfortable that it knows what most users want. Entering "white house" is a perfect example. You're taken directly to the US White House site.
If there are no defined keywords, then you may be taken to an intermediary page. This will happen if you search for a term that might match several trademarks, such as the "united" example above. This will also happen in response to many generic terms, such as "music" and "movies."
If the term matches no intermediary pages, then Netscape will scan to see if there are any domain names that seem to match your words. If so, you'd be taken directly to a web site.
Finally, if all the above attempts fail, Netscape will forward your request to its Excite-powered Netscape Search service, where you'll be shown web pages that match your topic.
Netscape says it intends to further add to the system. Editors are constantly watching query logs and creating pages in response to popular searches. For instance, they have already made up a page in response to a search for "melissa virus," Hickman said.
In the next few months, more content on these pages will begin to come from the Netscape Open Directory. Currently, most of the directory categories listed come from the Excite directory, which has been rebranded for Netscape's use.
That also makes being listed in the Open Directory much more important. As the system grows in popularity, it can become an important source of new traffic. If you still haven't submitted to the Open Directory, get started soon.
Netscape also says it may either enhance the Open Directory submission process to help further develop the Internet Keywords system, or it may launch a submission mechanism just for the system's needs. This is because it would like to solicit greater guidance from site owners on the best place to link within their sites, in relation to trademarked terms.
One welcome twist to the Internet Keywords system is that you will get different results if you have set your language to French, German or Japanese. For instance, searching for "yahoo" with a browser set to French would take you to Yahoo France, not Yahoo in the US. More of this will be a well received by those outside the US who are looking for local content.
Anyone can try this. Just go to the Preferences menu option, then open Navigator and click on Languages. Click on Add to select an additional language, then use the arrow keys to move your choice to the top, so it's the default language.
Netscape also has a number of "keyverbs" that work within the Internet Keywords system. For instance, use the word "quote" followed by a company symbol, and you'll get a current stock quote. For instance, "quote aol" would bring back the AOL stock price. You can also use the word "help" followed by a topic to get help about Netscape's products and services.
Ordinarily, there's no need to use the "go" keyverb, which is an explicit command for the browser to do an Internet Keywords search. But if you go through a proxy server, a single word query will probably fail, because the browser will try to find a local server. In these cases, start your query with "go."
Netscape has also augmented the Alexa-provided information that's available when clicking on the What's Related button, which is next to the Location box. What's Related displays links that seem similar to the page you are viewing, along with helpful information about the site. Additionally, when at some major company sites, you will also see relatively new options allow you to retrieve a stock quote or news items.
A link at the bottom of the What's Related list will also take you to a web page version of the What's Related information presented, which is useful in case you want to print or save the information for later reference.
Internet Keywords and What's Related are built into Netscape Communicator 4 and 4.5. Those running Internet Explorer 4 can also download a "tune-up" to add Netscape's Internet Keywords to the Microsoft browser.
IE4 users might also find they've been accidentally converted to using Internet Keywords without realizing it. When you install Netscape Communicator, it will ask if you want to "Use Netscape Netcenter to search the Web." If you say yes, then any Internet Explorer installation will also be changed to use Netscape's Internet Keywords. The install program does warn that this will happen, but this may be overlooked by some.
Don't choose this option if you want Explorer to stay as it is. Netscape will still configure itself to use Internet Keywords (and on the off chance it doesn't, then go to Preferences, choose Navigator, then Smart Browsing, then click on Enable Internet Keywords).
If Explorer has already been changed on your system, you'll need to install the Tweak UI utility to get back to normal. Click on the General tab, then change the Internet Explorer search engine option from Custom to the search engine of your choice.
Netscape Communicator 4.5 Home Page
Download the latest version, which has the Smart Browsing features described above.
About Internet Keywords
More information about Internet Keywords, including the ability to use an online form to search for terms using the system. There's a helpful FAQ page, plus links to the IE4 tune-up mentioned above.
Netscape Integrates Directory
The Search Engine Report, Feb. 2, 1999
More about the Netscape Open Directory.
NewHoo: Yahoo Built By The Masses
The Search Engine Update, June 22, 1998
Includes tips on submitting to the Netscape Open Directory.
Smart Browsers Ease Searching
The Search Engine Report, July 1, 1998
Learn more about how Autosearch and other search assistance features work in older versions of Internet Explorer.
Alexa: Searching Serendipity And More
The Search Engine Report, Jan. 9, 1998
More about information provided by the Alexa service.


text:
The Problem with Programming
Page 2 of 3
TR: The idea behind C++ was that programmers would work harder in return for more-efficient code. Bell Labs wanted a language that a few really smart people would use to write code that would run on computers like Electronic Switching Systems (ESS) that weren't very fast. Today, there are a lot of software developers and computers are very fast. Does that vitiate the point of C++?
BS: C++ wasn't designed specifically for the large switching machines, but for a huge range of applications. Bell Labs was the home of an incredible range of interesting projects spanning every scale and using essentially every kind of computer and operating system. But yes, the average Bell Labs programmer was significantly more able than most people's notion of an "average programmer," and reliability and performance (in that order) were considered significantly more important than in most other places.
Performance is still an issue in many of the applications that I'm interested in: responsiveness of interfaces, start-up and close-down time of applications. Software developers have neutralized the astounding performance of modern computer hardware by adding layer upon layer of overelaborate [software] abstractions. We seem to have hit the limits of linear speedup for hardware, but in many cases, we could win a couple of orders of magnitude back from the software.
That said, C++ has indeed become too "expert friendly" at a time where the degree of effective formal education of the average software developer has declined. However, the solution is not to dumb down the programming languages but to use a variety of programming languages and educate more experts. There has to be languages for those experts to use–and C++ is one of those languages.
TR: In retrospect, in designing C++, wasn't your decision to trade off programmer efficiency, security, and software reliability for run time performance a fundamental mistake?
BS: Well, I don't think I made such a trade-off. I want elegant and efficient code. Sometimes I get it. These dichotomies (between efficiency versus correctness, efficiency versus programmer time, efficiency versus high-level, et cetera.) are bogus.
What I did do was to design C++ as first of all a systems programming language: I wanted to be able to write device drivers, embedded systems, and other code that needed to use hardware directly. Next, I wanted C++ to be a good language for designing tools. That required flexibility and performance, but also the ability to express elegant interfaces. My view was that to do higher-level stuff, to build complete applications, you first needed to buy, build, or borrow libraries providing appropriate abstractions. Often, when people have trouble with C++, the real problem is that they don't have appropriate libraries–or that they can't find the libraries that are available.
Other languages have tried to more directly support high-level applications.
That works, but often that support comes at the cost of specialization. Personally, I wouldn't design a tool that could do only what I wanted–I aim for generality.


text:
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EFR spendt $250k + shares on a flier with VUI (which may come to fruition eventually)...so it is a NON-EVENT/IRRELEVANT.....down the road, who knows???
They may end up taking over VUI for virtually nothing and waiting for the tide to turn down the road...who knows?
The bottom line is that our maximum exposure was the $250k in cash.....meaningless....


text:
Everything At Once chords by Lenka, added: July 21st, 2011
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Intro: Dm
As sly as a fox as strong as an ox
As fast as a hare as brave as a bear
As free as a bird as neat as a word
As quiet as a mouse as big as a house
Dm C
All I wanna be,
Dm C
All I wanna be, oh oh oh
Dm C A
All I wanna be is everything
As mean wolf as sharp as a tooth
As deep as a bite as dark as the night
As sweet as a song as right as a wrong
As long as a road as ugly as a toad
As pretty as a picture hanging from a fixture
As strong as a family, strong as I wanna be
Bright as day as light as play
As hard as nails as grand as a whale
Dm C
All I wanna be, oh oh oh
Dm C
All I wanna be, oh oh oh
Dm C A
All I wanna be is everything
Everything at once
Dm Bb
Everything at once, ohh ohh ohh
Everything at once
As warm as the sun as silly as fun
As cool as a tree as scary as the sea
As hot as fire as cold as ice
Sweet as sugar and everything nice
As old as time as straight as a line
As royal as a queen as buzzed as a bee
Stealth as a tiger, smooth as a glide
Pure as a melody pure as I wanna be
Dm C
All I wanna be, oh oh oh
Dm C
All I wanna be, oh oh oh
Dm C A
All I wanna be is everything
Everything at once


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Fuel moves from the gas tank to the engine with the assistance of a fuel pump. For a powerful and stable fuel flow to the motor of your Suzuki Sj413, the pump must work properly. To steer clear of reduced engine performance or squandering gasoline, the fuel pump should be working properly to deliver gas to the engine. Your Suzuki Sj413 fuel pump needs to be changed immediately if it starts to diminish in effectiveness or displays indications of problems to ensure your vehicle's smooth operation.
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A family of brands printed on quality products, HFW is printed by Cumpy's T-Shirt Shop in Claycomo, Missouri. All of our brands can be custom printed. Find Hillbilly Fight Wear here or at an arena of civil discourse near you. Yes, You Can.
Thump A Stranger
Published journal of Brian Imes, named Top Fighter Blog by USA TODAY 3 times in 2011. Also published by NY Daily News on election day 2010 and by Pro MMA Now for 3 years straight.
Hillbilly Fight Wear is The Brand of professional mixed martial artist, Brian Imes. Inspired to compete in the sport by his brother, Brad Imes, Brian has developed a family of brands that represent his philosophy, Thumpology. Brad, The Hillbilly Heart Throb, competed in 21 professional bouts, including appearances in the UFC, The Ultimate Fighter Season 2, and the International Fight League.
Brian has carried HFW all across the country; competing, promoting, sponsoring and advocating for civil discourse.
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