text:
X inactivation in mammals results in the transcriptional silencing of an X chromosome in females, and this inactive X acquires many of the epigenetic features of silent chromatin. However, not all genes on the inactive X are silenced, and we have examined the TIMP1 gene, which has variable inactivation amongst females. This has allowed us to examine the features permitting expression from the otherwise silent X by comparing inactive X chromosomes with and without TIMP1 expression.
Expression was generally correlated with euchromatic chromatin features, including DNA hypomethylation, nuclease sensitivity, acetylation of histone H3 and H4 and hypomethylation of H3 at lysines 9 and 27. Demethylation of the TIMP1 gene by 5-azacytidine was able to induce expression from the inactive X chromosome in somatic cell hybrids, and this expression was also accompanied by features of active chromatin. Acetylated histone H3 continued to be observed even when expression was lost in cells that naturally expressed TIMP1; while acetylation was lost upon TIMP1 silencing in cells where expression from the inactive X had been induced by demethylation. Thus ongoing acetylation of inactive X chromosomes does not seem to be simply a 'memory' of expression.
We propose that acetylation of H3 is an epigenetic mark that predisposes to TIMP1 expression from the inactive X chromosome in some females.
Studies have shown considerable individual variability in the level of expression of genes (e.g. [1,2]). In general, however, humans cannot tolerate imbalances for expression of substantial numbers of genes, as demonstrated by the lethality of the majority of chromosomal aneuploidies. Aneuploidy for the sex chromosomes is better tolerated, being observed in approximately 1/500 births [3], presumably because all but one X chromosome is inactivated. X chromosome inactivation ensures the dosage equivalence of X-linked genes between females who have two X chromosomes and males who have a single X chromosome and the sex-determining Y chromosome [4]. However, more than 15% of human X-linked genes escape inactivation, being expressed from both the active and inactive X chromosome [5]. While such an escape from inactivation may maintain dosage equivalence for X-linked genes with Y homologs, the majority of human genes that escape inactivation no longer have functional Y equivalents, and thus may show relative overexpression in females (reviewed in [6]). Substantially fewer genes have been shown to escape inactivation in mice. Although this species difference in expression could reflect less extensive murine expression surveys, a reduced number of genes escaping inactivation is supported by the less drastic phenotype caused by monosomy of the X chromosome in mice (reviewed in [7]). In humans, the over or under-expression of genes that escape inactivation is a major contributor to the phenotypes associated with X chromosome aneuploidies, but may also contribute to expression differences between chromosomally normal males and females (e.g. [8]).
The study of genes that escape X inactivation can provide insight into such phenotypes, as well as contributing to our understanding of epigenetic silencing mechanisms. Inactivation occurs early in mammalian development, and the stable silencing of the X chromosome involves the acquisition of many features of heterochromatin. It is not known if escape from inactivation is a resistance to the initial silencing event, or rather reflects a high frequency of reactivation of an initially silenced gene, as data appear to support both possibilities. Many genes that escape inactivation in humans are clustered together, which may be indicative of regions that are resistant to the initial signal (e.g. [9]). However, analysis of Smcx, one of the few mouse genes expressed from the murine inactive X, has shown reactivation of the gene during early development [10]. Surprisingly, recent results have shown that Smcx has a histone modification pattern suggested to demarcate biallelically rather than monoallelically-expressed (imprinted and other X-linked) genes [11], suggesting that the gene is committed to escape inactivation prior to undergoing inactivation.
In addition to the genes that are subject to, or escape from, inactivation, there are some human genes that show heterogeneous X chromosome inactivation, being expressed from the inactive X in some females, but silenced on the inactive X in others [5,12,13]. Such genes provide an opportunity to study the same region when silent or active on an inactive X chromosome; and can thus provide insights into the features allowing expression from the inactive X chromosome. The human inactive X chromosome maintains its silent status when isolated in a mouse/human somatic cell hybrid, providing a model system to study the inactive X chromosome apart from its active counterpart. The largest survey of gene expression from the inactive X chromosome [5] analysed expression in a panel of nine inactive-X containing hybrids. That study defined heterogeneous inactivation as expression in three to six of the nine hybrids, which was observed for 60 of the 624 X-linked genes analysed. This variable expression is not restricted to the hybrid system, but has also been demonstrated in cells from females [5,12,13].
We now report the further characterization of one of these genes, the X-linked tissue inhibitor of metalloproteinases, TIMP1, located in Xp11.23. Our previous studies have demonstrated that TIMP1 is variably expressed from the inactive X in both somatic cell hybrids and human females. When TIMP1 is expressed from the inactive X, flanking genes (including ARAF1 and ELK1 which lie ~20 and 55 kb from TIMP1, respectively) are not expressed, suggesting that expression is being controlled in a gene-specific rather than regional fashion [12]. Quantitative RNase protection assays showed substantial variability in expression levels from the active X, precluding using expression levels to determine inactive X expression in females. Further studies in hybrids demonstrated that TIMP1-expressing clones were unstable and that methylation does not appear to be the principle controlling feature allowing variable expression of TIMP1 from the inactive X chromosome [14]. In this study we report the analysis of other features characteristic of an inactive X to determine which features might predispose TIMP1 to expression from the inactive X chromosome in a subset of females.
The inactive X acquires many of the general features of heterochromatin (reviewed in [15]), and we have now examined replication timing, nuclease sensitivity, and histone modifications for TIMP1. Late replication of the inactive X at the chromosome level is observed after Giemsa staining following bromodeoxyuridine incorporation [16]. Regions such as the distal and proximal short arm that contain a large proportion of genes that escape inactivation are not delayed in their replication, supporting a regional basis to escape from inactivation. Replication of individual X-linked genes has been analysed by fluorescent in situ hybridization (FISH) or amplification of BuDR incorporated DNA after flow cytometry. Although correspondence is not complete between different techniques [17], such methods have generally shown that genes that escape inactivation are early replicating (reviewed in [18]). DNase sensitivity is a general feature of active chromatin, and promoters of genes subject to inactivation have been seen to be less available for digestion by nucleases (e.g. [19]). Modifications to the histones associated with the inactive X are reflective of both general heterochromatic changes and ones specific to the facultative heterochromatin of the inactive X. Using antibodies to acetylated histones the inactive X chromosome stains very palely [20], while antibodies to histone H3 methylated at lysine (K) 9 (H3mK9) are generally associated with heterochromatin and those to methylated lysine 27 (H3mK27) specifically mark the inactive X chromosome [21,22]. Chromatin immunoprecipitation (ChIP) has revealed that genes subject to inactivation show limited acetylation and elevated histone methylation (H3mK9/27) at their promoters relative to genes escaping inactivation [23,24].
Replication timing of TIMP1 in human cells
To evaluate the influence of replication timing upon expression of TIMP1, cell lines from a female with and a female without TIMP1 expression from the inactive X [12] were analysed by DNA FISH using probes for the TIMP1 and HPRT loci. As shown in the schematic of Figure 1, cells that have not replicated either X chromosome will show two single signals, and almost one half of the cells were of this type. Of the cells with at least one double signal, indicating replication of the locus, considerable asynchrony was observed for the HPRT locus, as 37% of all cells analysed showed single-double (SD) signals and 17% showed the double-double (DD) signals. The TIMP1 locus generally showed lower asynchrony of replication than HPRT (28% of cells were S/D vs 37% for HPRT, P < 0.0001). The cell line with TIMP1 expression from the inactive X (cell line 2 – HSC593) showed a small trend towards having a lower percentage of cells that replicated asynchronously than the GM07059 cell line which does not have TIMP1 expression from the inactive X (25% vs 29%); however this difference was not significant (P = 0.17). The observed shift in replication timing, while not statistically significant, may be a contributing factor in the expression of TIMP1 from the inactive X. However, it is likely that additional changes are also involved, as TIMP1 is within 20 kb of the ARAF1 gene and 55 kb from ELK1 (see Figure 2A), and thus at least one of these genes is likely to share a replication domain with TIMP1 despite remaining silent when TIMP1 is expressed [12].
Figure 1. Replication asynchrony for the TIMP1 (T – white bars) and HPRT (H – grey bars) genes assessed by FISH. The bars show the frequency of nuclei exhibiting unreplicated (single/single); asynchronously replicated (single/double) and completely replicated (double/double) signals as shown in the schematic below. The TIMP1 and HPRT probes were individually hybridized to interphase nuclei of the same preparations. Two human female lymphoblast cell lines were examined for the approximate degree of replication asynchrony, one that inactivated TIMP1 (1 – GM07059) and one that expressed TIMP1 from the Xi (2 – HSC593). For TIMP1, 313 cells were counted from 3 separate cell harvests for cell line 1 (T1) and 217 cells for cell line 2 (T2) from two separate harvests. 144 cells were examined for HPRT from 2 slides of one harvest for cell line 1 (H1) and 97 cells from a single harvest for cell line 2 (H2). Between different cell harvests of the same cell line the maximal difference in frequencies of nuclei in each of the three replication classes was 3%.
Figure 2. Methylation of TIMP1. A. The genes in the region surrounding TIMP1 are shown with the gene name written above the line diagram for the gene (with vertical lines representing exons and small arrows showing transcriptional orientation). The number listed on the line below at the 5' end of the genes indicates the CpG island density. The TIMP1 and ZNF147 genes do not have enough CpG sites to qualify as a CpG island so there is no number listed. The SYN1, and PFC genes are listed in grey because these genes (which are expressed in a tissue-specific manner) were not examined in this study. The presence of repeat elements (SINE, LINE and LTR) is indicated by the vertical lines below the genes. This figure is based on data generated by the UCSC browser http://www.genome.ucsc.edu/ webcite hg17 of NCBI Build 35. B. Somatic cell hybrids analysed for TIMP1 activity. In addition to the stable active X chromosomes that express TIMP1 and are unmethylated at the 5' end of the gene (Xa/+/U) there are several categories of inactive X chromosomes. Most inactive X chromosomes previously analysed do not express TIMP1 and are methylated (Xi/-/M). Other inactive X chromosomes express TIMP1 and are unmethylated (Xi/+/U), while an intermediary class of hybrids showed both DNA methylation and lower expression levels (Xi/+/M). Subcloning of these latter cells showed that they were unstable, giving rise to additional methylated expressing clones as well as methylated silent clones and expressing unmethylated clones. The arrows show approximate proportion of cells of each class derived from subcloning. Subclones further characterized are listed below, as are clones derived by 5-azacytidine treatment and their subclones (see Tables 1 and 2). C. DNA methylation of clones from four of the demethylated clones listed in Table 1. DNA from each clone was digested with EcoRI alone (U), EcoRI plus HpaII (II) or EcoRI plus HhaI (I). Primers for the 5' end of TIMP1 and ARAF1 that flank HpaII or HhaI methylation-sensitive restriction enzyme sites were used to assess methylation, while amplification of MIC2, which is unmethylated on both active and inactive X chromosomes, served as a control for complete digestion with the methylation-sensitive enzymes. D. Comparison of expression levels in subclones of two 'sibling' subclones of t11-az-10 differing in methylation states. The t11-az-10-7 and t11-az-10-10 clones are striped, with their subclones shown to their right. Methylation (dark fill) was observed for t11-az-10-7 and its subclones while t11-az-10-10 and its subclones were unmethylated (unfilled). All subclones continued to express both TIMP1 and ARAF1 as assayed by RT-PCR. Despite the relative stability of the methylated TIMP1+ culture, the TIMP1 expression level was significantly lower in the methylated cultures (p < 0.01). E. Methylation analysis by bisulphite treatment. The 5' end of the TIMP1 gene was sequenced after bisulfite conversion, which changes unmethylated Cs to Us but leaves methylated Cs unchanged. Therefore, the presence of a C indicates that the CpG was methylated. The following CpG sites were analyzed: HhaI sites (stars) at -3 and +31; and three other sites not analyzed by methylation-sensitive enzymes (circles) at +11, +17, +20. The HpaII sites (triangles) at +61 and +81 were used in methylation-sensitive assays but were not reliably analysed by bisulphite sequencing. The open circles indicate unmethylated CpGs whereas the filled circles represent methylated CpGs. The shaded circles designate that both converted and unconverted bases were seen after sequencing, indicating that both methylated and unmethylated CpGs were present. Cell lines are listed, the male cells were GM7057 and the female cells were GM7059. t11-az-9-3 is a TIMP1- subclone of t11-az-9.
DNA methylation of TIMP1 in somatic cell hybrids
Analysis of alterations to the inactive X chromosome is complicated by the presence of the active X chromosome in female cells, so we have analysed features of inactive X chromosomes isolated in mouse/human somatic cell hybrids. We have previously described the characterization of DNA methylation status and expression levels in a number of these hybrids [14] and Figure 2B presents an outline of the hybrids analysed in this study. In addition to hybrids retaining the active X chromosome (t60-12 (t60) and AHA11aB1 (AHA)), in which TIMP1 was unmethylated and expressed, three classes of inactive X-containing hybrids were previously described. Those that were methylated and did not have TIMP1 expression (Xi/-/M – t11-4Aaz-5 (t11) and t48-1a-1Daz4a (t48)) or expressed TIMP1 and were unmethylated (Xi/+/U – t75-2maz34-1a (t75)) were stable, with no gain or loss of expression. In contrast, the two hybrids that expressed TIMP1 but were methylated at the TIMP1 promoter (Xi/+/M – t86-B1maz1b-3a (t86) and t81-az1D (t81)) demonstrated instability as subclones could be either methylated and expressing (Xi/+/M) or methylated and silent (Xi/-/M) as well as occasionally unmethylated and expressing (Xi/+/U). Consistently the Xi/-/M and Xi/+/U subclones tended to be stable while Xi/+/M subclones were unstable. As the methylated-expressing clones (Xi/+/M) are unstable and give rise to a mixed population of methylated silent and unmethylated expressing clones, these clones do not indicate if loss of methylation follows, or predisposes to, expression.
To further test the role of methylation in regulating expression in the TIMP1 region, an inactive X-containing hybrid that did not express TIMP1 (t11) was treated with 5-azacytidine, which is known to induce reactivation of X-linked genes in somatic cell hybrids. As there is not a selectable marker for TIMP1 reactivation, clones were initially selected for HPRT reactivation to ensure that the clones had been demethylated and potentially increase the frequency of reactivation, as co-reactivation of different X-linked genes has been observed after 5-azacytidine treatment [25]. 15 HPRT+ clones were selected in HAT media, and examined by RT-PCR for TIMP1 as well as flanking gene expression (Table 1). After three passages, six clones showed expression of TIMP1. A number of flanking genes were also observed to reactivate with 5-azacytidine treatment; with one clone expressing ARAF1, five expressing ELK1, three expressing ZNF41 and eight expressing ZNF157. The single ARAF1-expressing clone, and all five ELK1-expressing clones, were observed in TIMP1+ clones, and all three ZNF41 positive clones were ZNF157 positive. The TIMP1 and ZNF157 genes lack a CpG island (see Figure 2A), and TIMP1 and ZNF157 showed the highest (6/15 and 8/15) reactivation frequencies after 5-azacytidine treatment. However, ARAF1 showed the lowest reactivation frequency (1/15) despite having a smaller CpG island than either ELK1 or ZNF41, so reactivation frequency was not simply a reflection of CpG density.
Table 1. Expression of TIMP1 and surrounding genes following 5-azacytidine induced reactivation of HPRT
Despite 5-azacytidine treatment and subsequent expression in some clones, DNA methylation continued to be observed at 5' end of ARAF1 and TIMP1 in these clones, as shown in Figure 2C. Given our previous results demonstrating instability of expression in the presence of methylation, four clones were subcloned after 7 weeks of culture. Analysis of expression and DNA methylation for TIMP1 (Table 2) showed that, as expected, all subclones of a non-expressing clone (t11-az-4) remained silent. For both t11-az-8 and 9, the majority of clones lacked TIMP1 expression. Only two weakly positive clones were identified for each line, and even these subclones lost expression by 12 weeks in culture. Three subclasses of hybrids were observed for the t11-az-10 subclones, reminiscent of the situation seen in hybrids from females who spontaneously express TIMP1 from the inactive X. t11-az-10 was the only reactivated clone that expressed ARAF1, and nine of the 14 TIMP1-expressing subclones expressed ARAF1. The single subclone that was unmethylated at TIMP1 (t11-az-10-10) expressed TIMP1, and also expressed ARAF1, which was also unmethylated. t11-az-10-7 was methylated yet expressing for both TIMP1 and ARAF1. These clones (t11-az-10-10 and t11-az10-7) were further subcloned. The TIMP1 expression level of the subclones was determined by RNase protection. The t11-az-10-7 subclones retained DNA methylation, and expressed a lower level of TIMP1 than the unmethylated t11-az-10-10 subclones (Figure 2D).
Table 2. Stability of methylation and expression of TIMP1 in subclones of four 5-azacytidine induced HPRT reactivants
DNA methylation analysis was routinely based on methylation-sensitive restriction enzyme digestion followed by PCR that examined 4 sites, 3 of which were over 50 bp downstream of the transcription start site (Figure 2E). To examine methylation of additional CpG dinucleotides closer to the promoter, methylation analysis by bisulfite modification followed by sequencing of PCR products was performed. Three additional CpGs near the TIMP1 transcription start yielded the same methylation pattern as had been observed at the restriction enzyme sites. Direct sequencing of the PCR product was performed to allow the identification of heterogeneous populations. This approach is complementary to the methylation-sensitive restriction enzyme approach, which gave a positive signal (amplification) for low levels of methylated DNA. The high background corresponding to the unconverted (methylated) Cs, presumably due to their under-representation in the sequence, prohibits identification of low levels of methylated bases by direct sequencing. However, no background of the converted base (A) was present and thus this technique is sensitive to the presence of low levels of unmethylated DNA. If the peak corresponding to the converted base (A) was demonstrably higher than the unconverted (G), the site was called unmethylated (white circles in Figure 2E). Completely methylated sites (as seen for the inactive X hybrid) are indicated by filled in black circles. Sequencing also yielded a mix of unmethylated (converted) and methylated (unconverted) sites. This was observed in a female cell line, where the methylated (unconverted – G) peak height was greater than or equal to the unmethylated (converted – A) peak height. Such a mix, demarcated by the grey circles, was also observed for the t11-az-10-7 cells, although in this case the unmethylated peak was consistently lower than the methylated peak.
DNase sensitivity of TIMP1 in somatic cell hybrids
Nuclease sensitivity of the TIMP1 promoter, the promoter of the nearby ARAF1 gene, as well as the TIMP1 gene body and the anonymous DNA marker DXS8037 was assessed in a series of these clones (Figure 3). The promoters of TIMP1 and ARAF1 were sensitive to nuclease on the active, but not the inactive X chromosome, while the gene body or intergenic regions were generally resistant on both active and inactive X chromosomes. The TIMP1 promoter was also sensitive to digestion in the unmethylated expressing (Xi/+/U) TIMP1 clones, while it was insensitive in both the TIMP1-expressing, and silent methylated clones (Xi/+/M and Xi/-/M). A similar effect was observed for the demethylated clones, with both TIMP1 and ARAF1, being expressed in both the t11-az-10-10 and t11-az-10-7 clones, but only sensitive to nuclease digestion in the t11-az-10-10 clone, where they were unmethylated.
Figure 3. Nuclease sensitivity of TIMP1. Nuclei from various cell lines (as indicated on the left) were treated with increasing amounts of DNase I (0, 0.1, 0.25, 0.5, 1.0 units) as represented by the increasing breadth of the triangle. The DXS8037 primers flank a non-coding region and were used to check that equal digestion and PCR amplification occurred across all cell lines. The cell line designations indicate X activity, TIMP1 expression status, and methylation status as shown in Figure 2B.
Histone modifications
A growing number of specific modifications to the histone tails have been associated with both constitutive and facultative heterochromatin. To assess the potential role of such modifications in the escape from inactivation of TIMP1, we analysed the hybrids by ChIP with antibodies to acetylated histone H3 (antibody recognizes acetylated K9 and 14), acetylated histone H4 (antibody recognizes acetylated K 5, 8, 12, and 16) and methylated histone H3 (antibody to di-methyl K9, although the antibody will cross-react with tri-methyl K27 see: http://www.upstate.com/browse/productdetail.asp?ProductId=07-212 webcite). The promoter regions of TIMP1, ARAF1, ELK1 and the XIST gene (the latter is expressed solely from the inactive X chromosome) were analysed (shown in Figure 4). Additional analyses of the promoters of the PGK1 gene that is expressed only from the active X chromosome, and the ZFX gene that is expressed from both active and inactive X chromosomes, gave the anticipated results for active and inactive X chromosomes (data not shown). Antibody to acetylated H3 immunoprecipitated the TIMP1, ARAF1 and ELK1 promoters for the active, but not inactive X-containing hybrids, while the reverse was observed for the XIST promoter. ELK1 was not expressed in the inactive X hybrids examined, and was not immunoprecipitated. However for TIMP1 and ARAF1 association with acetylated histone H3 was observed for all expressing clones whether on the active or inactive X chromosome. In addition, immunoprecipitation of the TIMP1 promoter was observed for Xi/-/M clones (t86-6P/t86-1U and t81-4). Immunoprecipitation was completely concordant with expression for the acetylated histone H4 antibody at all of the genes examined. Methylation of H3 at K9 corresponds to silent chromatin, and thus, as anticipated, results were generally opposite to those seen for acetylation. Immunoprecipitation was observed when the gene was silent – i.e. XIST on the active X and TIMP1, ARAF1 and ELK1 on the inactive X. However, in all cases where there was expression in the presence of ongoing promoter methylation (for both ARAF1 and TIMP1) the promoter was immunoprecipitated by the methylated H3 antibody. Thus we detect a distinctive pattern of histone acetylation and methylation that does not correspond simply with the expression or DNA methylation status of the TIMP1 gene.
Figure 4. ChIP analysis of TIMP1 expressing or non-expressing hybrid clones. PCR products were amplified from DNA at various X-linked gene promoters after ChIP using antibodies to the modifications listed below the panels for the cell lines listed across the top of each panel (see Figure 2B for derivation of lines). t11-az-10-7a is a subclone of t11-az-10-7 (see Figure 2D). The DNA template for 'no Ab' lanes was prepared following the ChIP procedure without an antibody. A. PCR amplification products after ChIP with acetylated histone H3. Similar analysis to A, using antibody to acetylated histone H4 (panel B), or methylated histone H3 (C).
The facultative heterochromatin of the inactive X chromosome is a fascinating system to study the epigenetic modifications associated with silent chromatin as both an active and inactive version of most X-linked genes exist in female cells. However, not all genes on the 'inactive' X chromosome are subject to silencing [26], and these genes that escape inactivation must somehow maintain an active state on an otherwise silent chromosome. TIMP1 is subject to inactivation in many females, and we now show that when subject to inactivation the anticipated features of inactive chromatin are assembled – promoter DNA hypermethylation, nuclease insensitivity and histone methylation and hypoacetylation. In other females, however, we have previously demonstrated that TIMP1 continues to be expressed from the inactive X chromosome, and in this study we have exploited such chromosomes to analyze the epigenetic features that are associated with expression from the inactive X. By comparing expression of TIMP1 that occurs naturally with that induced by the demethylating agent 5-azacytidine we are also able to propose which feature may predispose to expression of TIMP1 in otherwise silent chromatin. We observed that expression of TIMP1 was associated with an active chromatin structure, despite the presence of the gene on the inactive X chromosome, except in three situations.
First, in female cell lines with or without TIMP1 expression from the inactive X there was a very similar extent of replication asynchrony, suggesting that the expression of TIMP1 was ocurring from a late-replicating region of the X chromosome. This is not surprising, as the genes flanking TIMP1, which are not variable in their inactivation, are located within 55 kb, so it is likely that at least one of ARAF1 or ELK1 shares a replication origin with TIMP1. We assessed replication timing by FISH, and detected a frequency of replication asynchrony for the HPRT locus in close agreement with the frequencies previously reported for this gene [27]. It has been suggested that the 'double dot' versus 'single dot' pattern may reflect chromatid association in addition or instead of replication asynchrony [17]. Regardless of the underlying cause, the results were highly reproducible and consistently showed less asynchrony for the TIMP1 locus relative to the HPRT locus, although still in the range that is generally considered asynchronous [28]. This reduced asynchrony at TIMP1 could reflect, or contribute to, weaker epigenetic silencing being established in this region of the X chromosome. There was a slight, but not statistically significant, trend for the female showing expression of TIMP1 from the inactive X chromosome to show even less replication asynchrony for TIMP1, however it seems unlikely that such a small difference could contribute substantially to TIMP1 expression from the inactive X, and thus other factors must be involved in permitting the expression of TIMP1 from the inactive X.
The other two situations in which expression and active chromatin features were not concordant were found in the somatic cell hybrids, and these results are summarized in Figure 5. As shown in the grey box, DNA methylation and additional features of silent chromatin were detected along with expression in the Xi/+/M (t86, t81) cells. We attribute this to heterogeneity in the population of cells, as subcloning yielded both methylated/silent and unmethylated/expressing clones. In addition to DNA methylation, these clones were observed to be insensitive to DNase and have methylated H3K9 residues near the TIMP1 promoter. As the assays for these features relied on PCR, a positive signal could be obtained from a subpopulation of cells with a silent chromatin structure, while another population of cells could be positive for expression and active chromatin modifications (histone acetylation). We also suggest that heterogeneity accounts for expression in the presence of DNA methylation in several of the demethylated clones, including the t11-az-10-7 clone. However, unlike the mixed population of subclones obtained with all other Xi/+/M cells, eight of eight subclones of t11-az-10-7 were methylated and expressing, and six of these subclones examined by RPA showed a consistently reduced level of expression relative to their unmethylated counterparts. While it was surprising that subcloning did not isolate distinct subpopulations, the presence of an unmethylated subpopulation was detectable by bisulphite sequencing. Thus heterogeneity again seems the most likely explanation for DNA methylation, DNase insensitivity and histone H3K9 methylation in these expressing cells. However, without single cell assays it is not possible to rule out that there are methylated cells that express TIMP1 at reduced levels and show additional features of silent chromatin. These two classes of clones were the only exceptions to concordance between nuclease sensitivity and gene expression at the promoters of the TIMP1 and ARAF1 genes; and previous studies of the nucleosomal organization of the HPRT1 gene promoter have shown that methylation does not directly affect the differential positioning of nucleosomes on active and inactive X-linked promoters [19]. Thus we believe that the methylated/expressing/nuclease insensitive clones reflect the presence of a subpopulation of silent cells. This heterogeneity demonstrates the unstable nature of silencing for TIMP1 in these cells.
Figure 5. Summary of chromatin features observed in somatic cell hybrid clones for the TIMP1 gene. Most subclones and demethylated clones follow the patterns seen for the active and inactive X hybrids that are outlined in bold. A positive (+) designates the presence of the feature, while a negative (-) depicts the absence of the feature, while ND means that the feature has not been examined in that class of clones. The assays used are PCR-based and would detect a small population of cells. DNase sensitivity is listed as the inverse – DNase resistance – as it is the presence of undigested (resistant) DNA that will yield a 'positive' PCR signal. In each category of clones not all clones have been examined for all features. For the clones shaded in grey the results are an amalgamation of results anticipated from an active and inactive X, and we suggest these clones represent a mixed population. This suggestion is generally supported by sub-cloning experiments (see text for discussion). The ongoing H3 acetylation of the Xi/-/M hybrids (highlited in darker grey) cannot be attributed to heterogeneous cell populations, and since it is not seen for demethylated hybrids that have lost TIMP1 expression (t11-az/-/M) we suggest that this modification reflects a predisposing feature of inactive X chromosomes that express TIMP1.
The third exception is the most interesting, and is highlighted in Figure 5 with a dark grey fill. Acetylation of histones is generally seen for active genes, and acetylated histone H4 showed complete concordance with expression for all genes. However, the Xi/-/M clones (t86-6P and 1 U as well as t81-4) showed ongoing acetylation at H3 despite having lost TIMP1 expression from the inactive X chromosome. The Xi/-/M subclones were derived from two different unstable, naturally expressing inactive X chromosomes (in t86 and t81), however expression of TIMP1 in these hybrids was now stably silenced, and thus this result was not a reflection of a mixed population of cells. Furthermore, acetylation cannot simply reflect a failure of this region to reset chromatin structure, as the demethylated t11-az-9-10 hybrid which had lost expression had also lost acetylation. Methylation of H3 K9 was observed in these silent clones, so the Xi/-/M cells, despite being a homogeneous population, appear to show both methylation and acetylation of histone H3. This may reflect the lack of specificity of the antibodies used for these experiments, as the acetylated H3 antibody recognizes acetylation at residues 9 and 14, while the antibody to methylated K9 can cross-react with methylation of K27. Thus immunoprecipitation by both antibodies may reflect modification of specific lysines. It is also possible that only a particular set of nucleosomes show the acetylation mark, and as the sonicated fragments immunoprecipitated in the assay averaged ~600 bp, modifications on several different nucleosomes flanking the primers used could result in immunoprecipitation. Regardless of the specific site of modification, acetylation of histone H3 was the one feature that was consistently associated with X chromosomes that were naturally predisposed to expression from the inactive X chromosome, regardless of expression status. Thus we propose that histone H3 acetylation differs at the TIMP1 genes in females, predisposing some females to expression from the inactive X. Unfortunately this hypothesis is difficult to test, as females generally show acetylation at TIMP1, due to the presence of the active X chromosome. It would be necessary to study clonal populations of cells from a female with a polymorphism close enough to the promoter to be analysed by ChIP, and currently no such polymorphisms are known. Our previous work did not show an association between expression of TIMP1 and a downstream polymorphism in exon 5 [12].
A different predisposing epigenetic mark for expression from the inactive X has previously been reported in mice. Methylation of H3 at K4 was restricted to the promoter in undifferentiated embryonic stem cells for genes that would subsequently be expressed monoallelically (i.e. genes subject to X inactivation or imprinting), while autosomal genes or genes that escape inactivation had H3 K4 methylation in the gene body as well as the promoter [11]. Such methylation was observed in the body of the Smcx gene which is initially silenced, and then reactivates, reminiscent of the H3 acetylation mark and instability of silencing that we observe for TIMP1. While many genes escape inactivation in humans, TIMP1 provides us with a unique opportunity to examine the gene in its expressed and silent state in hybrid somatic cells. Thus, it will be interesting to study additional chromatin modifications for TIMP1, including H3 K4 methylation, as well as additional modifications recently associated with the inactive X chromosome such as H4 K20 methylation [29] or H2A ubiquitinylation [30,31]. Even more interesting would be to determine if the changes seen are observed for additional X-linked genes that are variable in their inactivation status, if suitable model systems could be developed.
Previous examinations of genes that escape inactivation have shown an active chromatin structure (reviewed in [7]), and recent results further demonstrate that epigenetic modifications seem to be heterogeneous in their distribution along the X [32]. These 'flavours' of inactive X chromatin may correspond to clusters of genes that escape inactivation. While genes that escape inactivation tend to be clustered in blocks and enriched on the short arm of the X chromosome, genes such as TIMP1 that are variable in their inactivation are more evenly distributed along the X chromosome ([5], reviewed in [6]), and thus may reflect different mechanisms leading to expression from the inactive X. The CTCF boundary factor has recently been shown to be found between the domain of escape and that of inactivation [33]. It is not known whether such boundaries flank the variably inactivated genes. The five clones that showed reactivation of ELK1 were all TIMP1+, suggesting a co-ordinate reactivation between the two genes. Only one ARAF1 reactivant was identified, and this clone was also TIMP1+. However, this co-ordinate reactivation of TIMP1 with flanking gene(s) is different from the TIMP1-specific expression seen for the inactive X naturally. The ZNF157/41 genes did not seem to show co-ordinate reactivation with the TIMP1 region, however all three ZNF41 positive clones were ZNF157 positive, which suggests they may be in a separately controlled domain.
While association with a CpG island does not differ between genes subject to or escaping from inactivation, genes heterogenous in their inactivation are more likely to lack an island [5], consistent with the TIMP1 promoter being the least CpG dense of the region. Although TIMP1 lacks a CpG island, our analysis suggests that methylation of the few CpGs present near the promoter generally correlates well with silencing. Demethylation resulted in at least transient expression of all genes examined, confirming the importance of DNA methylation in stable maintenance of X chromosome silencing. Examination of the ELK1, TIMP1 and ARAF1 promoters after demethylation showed the presence of DNA methylation, even when the clones were expressing the genes. The majority of these clones subsequently resilenced these genes, as has been observed for other genes following demethylation [34], perhaps reflecting only partial initial demethylation or the spread of silencing from adjacent silent regions that retained DNA methylation or other epigenetic marks of silencing.
It has been proposed that the evolutionarily recent addition of the X short arm may predispose genes located there to escape from silencing [35]. Interestingly, TIMP1 is very close to the evolutionary breakpoint between the region added to the X after marsupial divergence. ARAF1 is found on the marsupial X while TIMP1 and its surrounding gene SYN1 are autosomal in marsupials, suggesting that they are part of the eutherian addition to the human X [36]. Since genes closely flanking TIMP1 are normally subject to X inactivation, even when TIMP1 is expressed, evolutionary history alone cannot explain the escape from inactivation, although it may result in a different genomic context that contributes to expression from the inactive X. However, inspection of genomic features in the region surrounding TIMP1 does not show a substantial difference in the frequency of repetitive elements near TIMP1 relative to the flanking ARAF1 and ELK1 genes (Figure 2A).
Several factors, including reduced replication asynchrony, lower CpG density, and more recent evolutionary addition to the X, may contribute to less stringently controlled inactivation for TIMP1. However, we propose that there is a difference between females for a feature unique to TIMP1 that predisposes some females to expression of the gene. This mark appears to be at least reflected in a difference in acetylation of histone H3 on the inactive X in females predisposed to expression of TIMP1 from the inactive X chromosome. While histone acetylation appears to be a predisposing mark for expression of TIMP1, there may be a different hierarchy of epigenetic modifications permitting expression of other genes from the inactive X. Elucidation of these mechanisms is important not only as a model for epigenetic gene regulation but because genes that escape inactivation contribute to the phenotype of X chromosome anueploidies, and may also result in differential male/female expression levels and disease susceptibilities.
Cell culture
Lymphoblast cell lines were grown in RPMI 1640 media (Stem Cell Technologies) supplemented with 15% fetal calf serum (Cansera), L-glutamine (Invitrogen) and penicillin/streptomycin (Invitrogen). Cells were harvested 12–26 hours after addition of fresh media by centrifugation. The human/rodent somatic cell hybrids were grown in alpha minimal essential media (Invitrogen) supplemented with 7.5% fetal calf serum, L-glutamine, penicillin/streptomycin, and non-essential amino acids (Invitrogen) to sub-confluence before harvesting with trypsin-EDTA (0.25%). Cell lines have been previously described [14]. To generate single cell clones, the hybrid cultures were plated to a final concentration of 3 to 17 cells/60 mm plate. After 5 to 10 days in culture, well-separated colonies were isolated by trypsinization in cloning cylinders and transferred to new 60 mm plates. To induce demethylation, an inactive X-containing hybrid that had never expressed TIMP1 (t11-4Aaz-5) was treated with 5-azacytidine as previously described [37]. The cells were grown in media supplemented with HAT (Invitrogen) to select for HPRT reactivants and then single cell cloned to test for TIMP1-positive cultures. To remove any confounding effects of HPRT selection, the cells were transferred back to alpha minimal essential media after 2 weeks of selection. The expression of genes in the TIMP1 region was determined by RT-PCR as described previously [12].
Replication timing
Approximately 4 × 106 lymphoblast cells were harvested one day after subculture to ensure that they were actively growing. The cell pellet was resuspended in 8 ml of prewarmed hypotonic (0.75 M KCl) and then incubated at 37C for 10 minutes. 2 to 3 ml of fixative (3:1 methanol:glacial acetic acid) was slowly added before spinning at 200 g for 10 minutes. The cell pellet was washed three more times with 5 ml fixative and then resuspended in 10 ml fixative for storage at -20C for up to a week. The nuclei preparations were dropped onto slides and left at room temperature overnight. The slides were then incubated in 2X SSC at 37C for 30 minutes followed by 2 minute room temperature incubations in each of: 70%, 85%, and 95% ethanol, and then allowed to air dry. To denature, the slides were incubated in fresh 70% formamide/2 × SSC at 74C for 2 minutes followed by an ice cold ethanol series of rinses (70%, 85%, 95% for 2 minutes each) and air drying. Probes for both the TIMP1 locus (lambda phage TIMP-3.9X) and the HPRT locus (Hulambda4x-8, ATCC 57236) were labelled with dUTP-digoxigenin (Roche 1745816) by nick translation (Roche 0976776). Unincorporated nucleotides were removed with a PCR clean-up kit (Qiagen), and 1 ul (approximately 100 ng) of labelled probe was mixed with 10 ul of 70% formamide hybridization buffer and 40 ng of human Cot-1 DNA (Invitrogen 15279011). The probes were denatured at 74C for 10 minutes, pre-annealed at 37C for one hour and then added to the prepared slide to hybridize overnight in a humidified chamber at 37C. The slides were washed in 50% formamide/2 × SSC for 15 minutes at 43C, followed by two washes in 2 × SSC for 4 minutes at 37C and then 3 washes at room temperature for 2 minutes each in 1 × PBD (0.1 M NaH2PO4, 0.1 M Na2HPO4, 0.1% Triton X). To visualize the probe, slides were incubated with 500 ng of anti-DIG (sheep – Roche 1207741) conjugated to fluorescein, for 5 minutes at 37C, followed by three two-minute washes in 1 × PBD. To amplify the signal, FITC-anti-sheep (IgG FI-6000 (Vector Laboratories)) was incubated and washed as above. The slides were then counterstained with DAPI mixed with antifade (VectorLabs). Cells were scored for nuclei with single-single, single-double, or double-double TIMP1 or HPRT signals on a Ziess Axioplan II microscope by a single individual who was blinded as to the cell line being analysed.
Methylation analyses
For methylation-sensitive restriction enzyme analysis, genomic DNA was pre-digested with EcoRI at 37C overnight, followed by incubation with 2 ul of RNase at 37C for 15 minutes. After phenol extraction and ethanol precipitation, the DNA was quantified by spectrophotometry. Pre-digested DNA (2 ug) was then incubated overnight at 37C in a total volume of 20 ul with 20 units of one of the following: mock enzyme (uncut), HpaII, or HhaI. An aliquot of 1 ul (100 ng) was then used as a template in the PCR reactions as described previously [14]. All primers flanked a region of genomic DNA that did not contain EcoRI restriction enzyme sites and contained 1–2 HpaII or HhaI sites. For bisulfite analysis, 500 ng of genomic DNA was first denatured with 3 ul of 3 M NaOH at 37C for 10 minutes. After 15 ul of 20 mM hydroquinone and 255 ul of 3.9 M sodium bisulfite were added and mixed well, reactions were left at 50C for 16 hours to allow the unmethylated cytosines to convert to uracil. DNA was purified using the DNA Wizard Clean-Up Kit (Promega). After amplification with the TIMP-S primers (see Table 3), the 3' reverse oligonucleotide was used as the primer for sequencing of the PCR product.
Table 3. Primers for PCR analyses
RNase Protection quantitation of TIMP1 levels
RNase protection analysis was performed with Ambion's RPA II kit, following the manufacturer's directions. RNA probes were isolated after in vitro transcription with 32P-UTP. After solution hybridization overnight of excess antisense radiolabelled probe to 10 ug of total RNA, any unhybridized probe and sample RNA was removed by RNase digestion. The hybridized product was then separated on a native 5% polyacrylamide gel, visualized by autoradiography, and bands quantified by phosphoimager (BioRad FX). The intensity of the TIMP1 fragment was compared to the intensity of the band detected for MIC2 used to control for the amount of input RNA. All RPA results were normalized to one stock RNA to decrease variability between gels (see [14]).
DNaseI sensitivity
The protocol for nuclease sensitivity was adapted primarily from [38]. The somatic cell hybrids were grown to 75% confluence on 60 mm plates before harvesting with 0.25% trypsin-EDTA. The cell pellets were resuspended in 300 ul ice-cold DNase buffer (0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT, and 15 mM Tris-HCL pH7.5). The suspensions were then split into 6 tubes of 50 ul each and another 50 ul of ice-cold DNase buffer with 0.4% Nonidet P40 was added. The tubes were mixed gently and placed on ice for four to five minutes before 100 ul of freshly diluted DNaseI (Invitrogen 18047-019) was added, such that 2.5 U, 1 U, 0.5 U, 0.25 U, 0.1 U, and no enzyme were used. The reaction was then incubated at 25C for 5 minutes, followed by 95C for 15 minutes. The DNA was isolated with a standard phenol/chloroform extraction protocol, quantified with spectrophotometry, and diluted to 60 ng/ul, 12 ng/ul, and 4 ng/ul. 1 ul was used as a PCR template with primers listed in Table 3. The diluted DNAs showed product intensity inversely proportional to concentration, indicating that the PCRs at 25 cycles of amplification were in the linear range of amplification, and the results shown in figure 3 are for the 12 ng/ul template.
Chromatin Immunopreciptation Assay (ChIP Assay)
The hybrid cells were grown in a t25 flask and were harvested after treatment with 5 drops of 0.25% trypsin-EDTA for 2 minutes at room temperature, and then washed in 1 × PBS. The cells were placed in a 1.5 ml tube with one ml of 0.37% formaldehyde in minimal essential media (Invitrogen) and incubated at 37C for ten minutes. From this point, the cells were kept on ice. The cells were washed twice with 1/100 proteinase inhibitor cocktail (Sigma) in 1 × PBS and then centrifuged at 2500 rpm for 4 minutes at 4C. The pellet was resuspended in 200 ul SDS lysis buffer (Upstate) with 1/100 proteinase inhibitor cocktail and placed on ice for 10 minutes. The suspension was drawn up with a 25 gauge needle and then sonicated. Immunoprecipitation was performed (Upstate Biochemicals catalogue number 17-295) with the following antibodies: Anti-acetyl H3 against lysine 9 and 14 (catalogue number 06-599); Anti-acetyl H4 against lysine 5, 8, 12, and 16 (catalogue number 06-866); and Anti-dimethyl-H3 against lysine 9 (catalogue number 07-212).
H3 – histone H3; H4 – histone H4; K – lysine; ChIP – chromatin immunoprecipitation; X – X chromosome; Y – Y chromosome; Xi – inactive X; Xa – active X; M – methylated; U – unmethylated; + expressing; - not expressing; S – single signal; D – double signal.
Authors' contributions
CA carried out the molecular genetic studies and data analysis and drafted the manuscript. CB conceived of the study, participated in the experimental design and data analysis and wrote the final manuscript. Both authors read and approved the final manuscript.
This study was supported by a Canadian Institutes of Health Research operating grant (MOP13690).
Cheung VG, Conlin LK, Weber TM, Arcaro M, Jen KY, Morley M, Spielman RS: Natural variation in human gene expression assessed in lymphoblastoid cells.
Nat Genet 2003, 33:422-425. PubMed Abstract | Publisher Full Text
Whitney AR, Diehn M, Popper SJ, Alizadeh AA, Boldrick JC, Relman DA, Brown PO: Individuality and variation in gene expression patterns in human blood.
Proc Natl Acad Sci U S A 2003, 100:1896-1901. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Thomson MW, McInnes RR, Willard HF: Genetics in Medicine. 5th edition. Philadelphia, W.B. Saunders Co.; 1991.
Lyon MF: Gene action in the X-chromosome of the mouse (Mus musculus L.).
Nature 1961, 190:372-373. PubMed Abstract
Carrel L, Willard HF: X-inactivation profile reveals extensive variability in X-linked gene expression in females.
Nature 2005, 434:400-404. PubMed Abstract | Publisher Full Text
Brown CJ, Greally JM: A stain upon the silence: genes escaping X inactivation.
Trends Genet 2003, 19:432-438. PubMed Abstract | Publisher Full Text
Disteche CM, Filippova GN, Tsuchiya K: Escape from X inactivation.
Cytogenet Genome Res 2002, 99:36-43. PubMed Abstract | Publisher Full Text
Craig IW, Harper E, Loat CS: The genetic basis for sex differences in human behaviour: role of the sex chromosomes.
Ann Hum Genet 2004, 68:269-284. PubMed Abstract | Publisher Full Text
Tsuchiya KD, Greally JM, Yi Y, Noel KP, Truong JP, Disteche CM: Comparative sequence and X-inactivation analyses of a domain of escape in human Xp11.2 and the conserved segment in mouse.
Genome Res 2004, 14:1275-1284. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lingenfelter PA, Adler DA, Poslinski D, Thomas S, Elliot RW, Chapman VM, Disteche CM: Escape from X inactivation of Smcx is preceded by silencing during mouse development.
Nat Genet 1998, 18:212-213. PubMed Abstract | Publisher Full Text
Rougeulle C, Navarro P, Avner P: Promoter-restricted H3 Lys 4 di-methylation is an epigenetic mark for monoallelic expression.
Hum Mol Genet 2003, 12:3343-3348. PubMed Abstract | Publisher Full Text
Anderson C, Brown CJ: Polymorphic X-chromosome inactivation of the human TIMP1 gene.
Am J Hum Genet 1999, 65:699-708. PubMed Abstract | Publisher Full Text
Carrel L, Willard HF: Heterogeneous gene expression from the inactive X chromosome: an X-linked gene that escapes X inactivation in some human cell lines but is inactivated in others.
Proc Natl Acad Sci USA 1999, 96:7364-7369. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Anderson CL, Brown CJ: Variability of X chromosome inactivation: effect on levels of TIMP1 RNA and role of DNA methylation.
Hum Genet 2002, 110:271-278. PubMed Abstract | Publisher Full Text
Plath K, Mlynarczyk-Evans S, Nusinow DA, Panning B: Xist RNA and the mechanism of X chromosome inactivation.
Annu Rev Genet 2002, 36:233-278. PubMed Abstract | Publisher Full Text
Willard HF, Latt SA: Analysis of deoxyribonucleic acid replication in human X chromosomes by fluorescence microscopy.
Am J Hum Genet 1976, 28:213-227. PubMed Abstract
Azuara V, Brown KE, Williams RR, Webb N, Dillon N, Festenstein R, Buckle V, Merkenschlager M, Fisher AG: Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication.
Nat Cell Biol 2003, 5:668-674. PubMed Abstract | Publisher Full Text
Chow JC, Brown CJ: Forming facultative heterochromatin: silencing of an X chromosome in mammalian females.
Cell Mol Life Sci 2003, 60:2586-2603. PubMed Abstract | Publisher Full Text
Chen C, Yang TP: Nucleosomes are translationally positioned on the active allele and rotationally positioned on the inactive allele of the HPRT promoter.
Mol Cell Biol 2001, 21:7682-7695. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Jeppesen P, Turner B: The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression.
Cell 1993, 74:281-289. PubMed Abstract | Publisher Full Text
Heard E, Rougelle C, Arnaud D, Avner P, Allis CD, Spector DL: Methylation of histone H3 at Lys-9 is an early mark on the X chromosome during X inactivation.
Cell 2001, 107:727-738. PubMed Abstract | Publisher Full Text
Plath K, Fang J, Mlynarczyk-Evans SK, Cao R, Worringer KA, Wang H, de la Cruz CC, Otte AP, Panning B, Zhang Y: Role of histone H3 lysine 27 methylation in X inactivation.
Science 2003, 300:131-135. PubMed Abstract | Publisher Full Text
Gilbert SL, Sharp PA: Promoter-specific hypoacetylation of X-inactivated genes.
Proc Natl Acad Sci U S A 1999, 96(24):13825-13830. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Rougeulle C, Chaumeil J, Sarma K, Allis CD, Reinberg D, Avner P, Heard E: Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome.
Mol Cell Biol 2004, 24:5475-5484. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hors-Cayla MC, Heuertz S, Frezal J: Coreactivation of four inactive X genes in a hamster x human hybrid and persistence of late replication of reactivated X chromosome.
Somat Cell Genet 1983, 9:645-657. PubMed Abstract | Publisher Full Text
Carrel L, Cottle AA, Goglin KC, Willard HF: A first-generation X-inactivation profile of the human X chromosome.
Proc Natl Acad Sci U S A 1999, 96:14440-14444. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Boggs BA, Chinault AC: Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization.
Proc Natl Acad Sci 1994, 91:6083-6087. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gribnau J, Luikenhuis S, Hochedlinger K, Monkhorst K, Jaenisch R: X chromosome choice occurs independently of asynchronous replication timing.
J Cell Biol 2005, 168:365-373. PubMed Abstract | Publisher Full Text
Kohlmaier A, Savarese F, Lachner M, Martens J, Jenuwein T, Wutz A: A chromosomal memory triggered by Xist regulates histone methylation in X inactivation.
PLoS Biol 2004, 2:E171. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
de Napoles M, Mermoud JE, Wakao R, Tang YA, Endoh M, Appanah R, Nesterova TB, Silva J, Otte AP, Vidal M, Koseki H, Brockdorff N: Polycomb Group Proteins Ring1A/B Link Ubiquitylation of Histone H2A to Heritable Gene Silencing and X Inactivation.
Dev Cell 2004, 7:663-676. PubMed Abstract | Publisher Full Text
Fang J, Chen T, Chadwick B, Li E, Zhang Y: Ring1b-mediated H2A ubiquitination associates with inactive X chromosomes and is involved in Initiation of X-inactivation.
J Biol Chem 2004, 279:52812-5. PubMed Abstract | Publisher Full Text
Chadwick BP, Willard HF: Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome.
Proc Natl Acad Sci U S A 2004, 101:17450-17455. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Filippova GN, Cheng MK, Moore JM, Truong JP, Hu YJ, Nguyen DK, Tsuchiya KD, Disteche CM: Boundaries between chromosomal domains of X inactivation and escape bind CTCF and lack CpG methylation during early development.
Dev Cell 2005, 8:31-42. PubMed Abstract | Publisher Full Text
Hansen RS, Gartler SM: 5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.
Proc Natl Acad Sci U S A 1990, 87:4174-4178. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lahn BT, Page DC: Four evolutionary strata on the human X chromosome.
Science 1999, 286:964-967. PubMed Abstract | Publisher Full Text
Wilcox SA, Watson JM, Spencer JA, Graves JA: Comparative mapping identifies the fusion point of an ancient mammalian X-autosomal rearrangement.
Genomics 1996, 35:66-70. PubMed Abstract | Publisher Full Text
Tinker AV, Brown CJ: Induction of XIST expression from the human active X chromosome in mouse/human somatic cell hybrids by DNA demethylation.
Nucl Acids Res 1998, 26:2935-2940. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gregory RI, Feil R: Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.
Nucleic Acids Res 1999, 27:e32. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Brown CJ, Ballabio A, Rupert JL, Lafreniere RG, Grompe M, Tonlorenzi R, Willard HF: A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome.
Nature 1991, 349:38-44. PubMed Abstract | Publisher Full Text


text:
Helpful (3)
"Awesome startup company!! "
- Comp & Benefits
- Work/Life Balance
- Senior Management
- Culture & Values
- Career Opportunities
I have been working at Signpost full-time (Less than a year)
Everyone in the company including all executives are extremely accessible and open to ideas. New and young reps are able to jump right in and make a difference in a startup environment. Some of the more sr reps are frequently brought in on ideas and planning meetings to give their feedback and help shape the direction of the company.
The culture is awesome. Team happy hours pretty frequently, president's club trips, contests for reps to work out of different offices (Denver, Austin, NYC), etc.
Some people are not a fit for the startup environment. The people that are successful here are fast paced, fun loving people who believe in the company, very passionate and are here to make ridiculous amounts of money. Some of the people who were not a fit are more interested in just coasting by.
Advice to Management
Stay passionate, continue to be accessible to everyone and keep doing a great job.
- Any Location
- Any Job Title
Helpful (12)
"Not bad if you like sales. "
- Comp & Benefits
- Work/Life Balance
- Senior Management
- Culture & Values
- Career Opportunities
I worked at Signpost full-time (Less than a year)
Doesn't RecommendPositive OutlookNo opinion of CEOPros
- casual environment
- happy hours
- good training
- opportunities to win prizes (amazon gift cards, bottles of liquor, etc) through competitions
This is a good job for someone who is interested in sales. Unfortunately, I found a lot of the people hired either haven't done sales before so don't know exactly what to expect coming in or didn't want to do sales but ended up accepting the job because they "had to" and realize this is not a good job for them.Cons
SALARY: base salary is really low. You could be hitting your minimum quota every month and only make a little over $30,000 per year. Few reps (maybe 15-20% out of the entire company) are making $40-45,000+ per year. They'll say in the interview that top performers are making up to $80-90,000 but keep in mind these are only a handful of people and given the stressful and repetitive nature of cold calling it's not common people are consistently performing at these levels.
- sales is hard and cold calling sucks unless it's what you're really passionate about. you should think hard about whether you want to be making 100+ outbound calls a day trying to persuade people who don't want to buy your product to buy. I had never done sales previous to this job and overestimated my ability or desire to cold call.
- can get stressful having to constantly worry about hitting quota. you may be above quota one week or even one month but then it resets and you start from scratch again. this might be a stressful work environment for some
- a lot of times you can only control your success to a certain degree. you can't control how high quality the leads you pull on a certain day are or how receptive the people you talk to will be. you could be doing the exact same thing one week to another but not see the type of performance (sales) you saw previously.
- the job gets very repetitive and mundane. you're cranking out calls everyday giving mostly the same pitch to merchants.
- they'll show you a career path with quick opportunities for promotions but all these promotions mean is a little bump on your base salary (5k each promotion). the job you're doing stays the same regardless of whether you get promoted from rep to exec etc.
- no good career advancement after you hit senior executive status. In the Austin office at least, a lot of management hires are happening from the outside. and after hitting the senior exec status you can either move to the account management side (which is generally a pay cut since there isn't much of a bonus structure involved) or move to national sales which is just more of the same cold calling.
- after you've been doing the job for a couple of months you don't learn any new skills.
- micro manage
- not very good at empathizing-- they act like cold calling is rewarding and fun and never address the common pitfalls it can have
- turn around is REALLY high. (within 9 months 99% of the people that were in the office when I started were gone)
- employee loyalty is low. even if you've been there for a long time and have been successful the company doesn't do anything to reward your loyalty (like being lenient when having an off week or month) and generally act like you are disposable since they are hiring extremely rapidly.Advice to Management
- micro manage less. be less strict about being a couple of minutes late and don't constantly breathe down on reps for things like talk time etc unless they are constantly behind. trust that you've hired good employees and that we're all adults. it's not fun for anyone if a rep doesn't hit their quota.
- show more loyalty towards long-term employees if retention/ high turnover is a concern you have
- be more clear about whether the next few management hires are coming from within or externally so people can have realistic expectations for their career path at signpost -
Helpful (3)
"Terrible MGMT, awful product, great culture and co-workers. JOIN Account Management NOT Sales "
- Comp & Benefits
- Work/Life Balance
- Senior Management
- Culture & Values
- Career Opportunities
I worked at Signpost full-time (Less than a year)
RecommendsPositive OutlookApproves of CEOPros
Amazing culture. There are tons of things to do. There is a keg in office, pingpong table, darts, and small kitchen filled with snacks and drinks. They make the atmosphere fun and inviting. They had plenty of company happy hours where co-workers made me feel comfortable and part of the team. You can also make pretty good cash if you do well with sales.
Management is very unprofessional and rude. 1:1 can sometimes be awful. It's more about lecturing you than giving you advice or plans on how to succeed. Beware of mgmt. they will make it seem like they are nice, but once you are having a bad month, forget about it. On top of that, the Director was dating a Sales rep, who suddenly became a Team Lead? Isn't that against some company policies? Not at Signpost. Also, we feed a bunch of lies to most of our merchants. Sad to say that a lot of our merchants don't receive what we actually pitch.
Advice to Management
Management has got to comfortable. They lack management skills for the most part. Base pay also should not be so low, a bad month doesn't mean you haven't worked hard.

subset: commoncrawl


text: Drag Racing Events 03/20 - 03/21 NHRDA Spring Diesel Nationals NHRDA Presents the 2015 Season Opener at Historic Famoso Raceway Venue: Famoso Raceway Location: Bakersfield, CA 04/11 NHRDA Texas Diesel Nationals NHRDA Presents the Texas Diesel Nationals Venue: Texas Motorplex Location: Ennis, TX 05/29 - 05/30 NHRDA Diesels On The Mountain NHRDA Presents the Diesels On The Mountain Date: May 29th - Sled Pull Venue: Jefferson County Fairgrounds Location: Denver, CO Date: May 30th - Drag Racing Venue: Bandimere Speedway Location: Denver, CO Purchase Tickets! 06/27 NHRDA Midwest Truckin Nationals NHRDA Presents the Mid West Truckin Nationals Venue: Gateway Raceway Location: St. Louis, MO Purchase Tickets! 07/11 NHRDA Pacific Coast Diesel Nationals NHRDA Presents the Pacific Coast Diesel Nationals Venue: Woodburn Dragstrip Location: Woodburn, OR Purchase Tickets! 08/08 NHRDA Canadian Diesel Shootout NHRDA Presents the Canadian Diesel Shootout Venue: Castrol Dragway Location: Edmonton, AB Purchase Tickets! 08/22 NHRDA Big Sky Truck Fest NHRDA Presents the Big Sky Truck Fest Venue: Yellowstone Dragstrip Location: Billings, MT Purchase Tickets! 09/12 NHRDA - Oklahoma Diesel Nationals NHRDA Presents the TBA In Tulsa Oklahoma Venue: Tulsa Raceway Park Location: Tulsa, OK Purchase Tickets! 10/0210/03 NHRDA World Finals - Sled PullNHRDA World Finals - Drag Racing NHRDA Presents the NHRDA World Finals Date: Oct 2nd – Sled Pull Venue: Texas Motorplex Location: Ennis, TX Date: Oct 3rd – Drag Racing Venue: Texas Motorplex Location: Ennis, TX Purchase Tickets! Prior Years Events: 2014


text:
use the following search parameters to narrow your results:
e.g. subreddit:aww site:imgur.com dog
subreddit:aww site:imgur.com dog
see the search faq for details.
advanced search: by author, subreddit...
~25 users here now
Merhaba and welcome to r/Turkey
This subreddit is for news and discussion about Turkey. Posts both in English and Turkish are welcome.
Bu subreddit Türkiye ile ilgili haber ve sohbet ortamıdır. Türkçe veya Ingilizce yazabilirsiniz.
'Ask-I-Memnu' a Turkish drama becomes the most watched drama in the history of Pakistan. (self.Turkey)
submitted 2 years ago by Nishta
The finale of Ask-I-Memnu was aired here not more than 2-3 weeks ago and everyone is pretty much missing it already.
My wife was literally in love with the drama and would love for me to take her to Istanbul in the early 2013. I was wondering if its possible to actually see the places where the drama was shot, Adnan Ziyagil's house to be in specific. She would be very excited to see all of this in person.
Let me know if there is a service or anything that can help us arrange the trip.
[–]gokayn 0 points1 point2 points 2 years ago (2 children)
i knew some people who went to the locations. but it's been a couple of years since the show was on air in Turkey. i'm not sure if they are still available to visit.
but i don't think there is something special about the shooting locations of the show. one sunny day at the Bosphorus would be more than enough to experience the Istanbul atmosphere
[–]Nishta[S] 1 point2 points3 points 2 years ago (1 child)
I get that man. My wife would just simply love to visit this specific house where all these people lived. It would be dream come true experience for her. You know women are very emotional type here in Pakistan and to them these small things mean the world.
I would love it if somehow I can get in touch with someone who can arrange a tour to this house.
[–]foucoult 1 point2 points3 points 2 years ago (0 children)
It's at the Piyasa street of the Sarıyer district of İstanbul. There isn't any organized tours but you can see the house from the outside.
check the google map :
[–]crenaaniglue-sniffing atheist 0 points1 point2 points 2 years ago (1 child)
You can see it from the outside, but I don't think you'd be able to get in. The owner of the house strictly forbids that. Some caretakers managed to sneak some tourists in, in exchange for money, but they either got fired or got sent off to somewhere else to work for doing that.
I'm not sure if they're filming something else over there right now, but even if they aren't, the insides of the house won't be what you saw on the tv. The owner of the house asked the production company to undo all the changes they did there. So it's back to its old, boring self. Just so you know.
[–]Nishta[S] 0 points1 point2 points 2 years ago (0 children)
That is sad. As popular as it had become I thought it'd become more of a tourist spot xD
My luck I guess :) I still might visit Istanbul though.
[–]sinobey 0 points1 point2 points 2 years ago (1 child)
It is located in the main street (the coastal road near the Bosphorus) in Sarıyer; you can see it from the outside. If a show is filmed on a huge house in Turkey, it is most probably a home rented from the owner for the duration of the show. So the owner most probably lives there at the moment, and might not let anyone in.
It makes sense too. I just had this wild idea that they might let tourist in for a set price. I appreciate quick response from all of you though.
π Rendered by PID 606 on app-89 at 2015-04-19 08:13:25.940442+00:00 running 1eeacda country code: US.


text:
PreK-12 Market Headlines RSS Feed
Pinellas County Pioneers "Revolutionary" Evaluation System
Learning Sciences International — Saturday, June 21, 2014
A multi-year research pilot project in Pinellas County, Florida has begun to address many of the challenges inherent in teacher evaluation, particularly the heavy reliance on end-of-year VAM (value added measures) scores. The research pilot, a partnership between Pinellas County Schools and Learning Sciences International Read More »
Smithsonian Education Offers Summer Learning
Smithsonian Center for Learning and Digital Access — Saturday, June 21, 2014
The Smithsonian Center for Learning and Digital Access is helping students avoid the summer learning slide with a collection of self-directed, project-based learning activities through its Smithsonian Quests program. Smithsonian Quests motivate young people with personal coaching from the Smithsonian and reward their achievements with digital badges. Read More »
Hooda Math Releases Free Online Math Manipulatives
Hooda Math — Thursday, June 19, 2014
Hooda Math, K-12's leading resource for free online math games, has released five math manipulatives designed to be completely device agnostic. ... Hooda Math's free math manipulatives are built using HTML5 code, so whether students are on a smartphone, tablet or desktop computer, they can take advantage of these free math manipulatives. Read More »
digedu and Wilson Education Center Announce Strategic Partnership
digedu — Wednesday, June 18, 2014
digedu today announced a partnership with one of Indiana's most prominent Educational Service Centers, Wilson Education Center. The alliance will allow for members of IAESC Procurement, the cooperative purchasing program sponsored by Wilson Education Center, to easily access digedu's products and services at a competitive rate. Read More »
Rourke Educational Media's New Spanish Library-Bound Collections
Rourke Educational Media — Wednesday, June 18, 2014
Filling a void in the K-12 informational text marketplace,Rourke Educational Media announced it is introducing library-bound Spanish collections with science, history and social skills titles targeted for students in kindergarten and elementary and middle schools. Read More »
Scientific Learning Corp. Announces a New Three-Day Protocol
Scientific Learning Corp. — Tuesday, June 17, 2014
Scientific Le arning Corp.today announces a new, shorter three-day protocol. The new three-day protocol will allow preK-12 schools to bring the benefits of the Fast ForWord language, literacy and reading intervention to more students fewer days per week, making it easier to integrate the program into their daily class schedules. Read More »
Curriculum Advantage and Scantron Partenr for Personalized Learning
Curriculum Advantage® Scantron® — Monday, June 16, 2014
The alliance integrates Classworks with Scantron Performance Series® and Scantron Achievement Series® assessment solutions, both recognized for delivering reliable and comprehensive data. Performance Series provides ongoing, relevant information in reading, language arts, mathematics, and life sciences. Classworks integrates with both of the Scantron solutions, taking the precise assessment data provided on individual students to create customized assignments. Read More »
The InvestiGator Club Recommended for MD Early Childhood Curriculum
Robert-Leslie Publishing — Monday, June 16, 2014
The Early Learning Branch of the Maryland State Department of Education has recommended The InvestiGator Club® Prekindergarten Learning System with STEAM for state endorsement as a comprehensive curriculum for three-, four-, and five-year-olds. The program is the only recommended curriculum fully aligned in every category of the Maryland Framework for 3–5 year-old preschool students. Read More »
National PTA and By Kids For Kids Partner to Inspire Student Innovation
National PTA, By Kids For Kids — Monday, June 16, 2014
National PTA and By Kids For Kids (BKFK) announced today a partnership to bring resources to students, families and schools across the country to inspire innovation and entrepreneurship. Through the partnership, National PTA and BKFK will bring the Secret Millionaires Club "Grow Your Own Business Challenge" to PTAs nationwide to empower them to help children develop their own business ideas and establish healthy financial habits. Read More »
Professional Learning Management System Enhancements
Truenorthlogic — Friday, June 13, 2014
Truenorthlogic has released a newly redesigned Professional Learning Management System (PLMS) and a powerful set of search tools in support of the ongoing professional growth and development of teachers, leaders and staff. The PLMS is part of the Truenorthlogic K-12 Talent Management Platform, which is used by more than 500,000 educators in over 1,500 school districts including eight of the largest 20 districts. Read More »


text:
(MICR Toner) LD Remanufactured Replacement for Hewlett Packard CE505X (HP 05X) High-Yield Black Laser Toner Cartridge
The LD remanufactured CE505X (HP 05X) high-yield black laser toner cartridge is professionally remanufactured by LD Products. Let LD help you print your checks for less. Our MICR toners contain the magnetic properties necessary for passing professional checks through a bank's scanning system. LD remanufactured cartridges may contain new internal parts including OPC drums and wiper blades. Purchasing LD remanufactured cartridges is an excellent way to save money without sacrificing quality. The use of the LD remanufactured CE505X / 05X black laser toner does not void your printer's warranty. LD remanufactured laser cartridges are not remanufactured by HP. All of our remanufactured products are offered with a 'One Year 100% Quality Satisfaction Guarantee'.
This remanufactured replacement for HP CE505XMICR with MICR toner works with these printer models:LaserJet P2055dn, P2055X, P2055d
The non-MICR remanufactured replacement for HP CE505X can be found by clicking here
Compatible Printers:
SKU | OEM Number | Cost Per Page | Capacity (ml) | Shelf Life | |
CE505XMICR | CE505X | 6,500* | 1.69 cents | N/A | 24-36 Months |


text:
BakerHostetler Partner Alan Friel Talks Big Data and Data Collection
The TCPA: Deep Dive: Details, Class Actions, Regulations, and Defense Strategies
The TCPA: Basics, Targeted Industries, and Trends
Advertising Law
Polsinelli Podcast - Telephone Consumer Protection Act and Consequences for Corporations
FCPA Compliance and Ethics Report-Episode 137, interview with Loren Steffy
FCPA Compliance and Ethics Report-Episode 130, The Oscars and Compliance, Part IV with Jay Rosen
FCPA Compliance and Ethics Report-Episode 124, The Oscars and Compliance, Part I with Jay Rosen
fcpa Compliance and Ethics Report-Episode 106-interview with Gini Deitrich
A Moment of Simple Justice - Snitching Ain't Easy
Polsinelli Podcasts - Be Careful What you Put in Writing - What Companies Need to Know About Document Retention
Jail Time for Revenge Porn Offenses?
FCPA Compliance and Ethics Report-Episode 3-Interview with Marie Patterson, Vice President of Marketing for Hiperos
What you need to know about Canada's new Anti-Spam Law (CASL)
Why Ending FCC Blackout Rules May Be Broadcast TV's Demise
FCC Proposes New Rules On Local Wireless Siting
Data Center Networks – Interview with Jeff Moerdler, Member, Mintz Levin
Employer Social Media Policies – Interview with Mitch Danzig, Member, Member, Mintz Levin
JOBS Act Implementation Regulations
Polsinelli Podcast - Public Policy Issues in 2013
Chief Judge Loretta A. Preska of the U.S. District Court for the Southern District of New York affirmed Magistrate Judge James C. Francis IV's opinion and ordered that the U.S. Department of Justice (DOJ) could compel...more
..A relatively new site called Wanelo combines the features of a social media site and an e-commerce site by permitting users to "save" products that they are interested in, and showing them to their online friends. The site...more
A perspective on the recent Fourth Amendment and privacy considerations raised by Judge James C. Francis' recent Memorandum and Order: 'In The Matter Of A Warrant To Search A Certain E-Mail Account Controlled And Maintained...more
In many criminal cases, cellular telephone records are an invaluable resource. These records may shed light on whether an alleged victim or witness communicated with someone of importance in a case, and when. There are...more
In April 2013, Rehtaeh Parsons died after her parents took her off life support following a suicide attempt after months of enduring cyber bullying. The harassment was the result of a photo depicting Parsons allegedly being...more
Can the United States search your laptop, camera, iPhone/iPad, or other devices at the border? Read this article on the Fourth Amendment rights and exceptions....more
Last month, we summarized the N.Y. Supreme Court case, Fawcett v. Altieri, which involved the production of electronically stored social media content. What the post did not address, however, was the simple two prong analysis...more
The People of the State of New York, v. Malcolm HARRIS, –N.Y.S.2d–, 2012 WL 1644956 (N.Y. City Crim.Ct. May 7, 2012) On May 15, 2012, we discussed a New York criminal court's decision to deny Defendant Malcolm Harris'...more
The incidents of war happened in the real world is mirrored in cyber space, given the recent incidents of hacking of government websites by state or non state group of hackers for political, military, espionage purposes. As...more
cyber attacks on corporates have become common either by competitors or state sponsored actors of foriegn state, Security to organisations and legal compliance has become important...more
The Electronic Frontier Foundation (EFF) filed a class-action lawsuit against AT&T on January 31, 2006, accusing the telecom giant of violating the law and the privacy of its customers by collaborating with the National...more
Find a Communications & Media Author »
Sign up for your custom alerts now, using LinkedIn ›


text:
1) No short notice booking
2) Please read through description
3) This is Not service apartment. If you break anything, I shall ask for compensation
4) Shoes off inside the premises PLEASE
5) Non smoker ONLY
6) Female or couples; no kid
Thank you Angie for your honest review. I am sorry about the unpleasant check in as I was out of town and could not have arranged it better with time difference and everything. I shall learn from the experience and manage better next time I am not around.
Despite I did not clearly state the kitchen item because new items will be added from time to time, I have specifically mentioned 'Do not break anything' in my house rules. Nevertheless, Airbnb is about another style of travel, to stay in others' home while you are away from home. I believe it is common sense to protect any properties inside others' home if others are kind enough to allow you in. I have entrusted a lot in you to accept you while I was not around. To me, even without prior warning of expensive items, it is not right to damage then ignore the responsibility to do something about it when you can. It is a tragedy that you cracked the lunch plate and do not want to replace it. Honestly, you did not just break my lunch plate. You have broken my confidence and good faith in people which I believe value much, much, much more than USD100. After this very unpleasant incident, I do not want to host any travelers again unless they can earn my trust in prior communication.
Despite all this unhappy encounters, I wish you and your family well.


text:
82 posts found | |
In an effort to avoid a wall of text I'll try to keep this short and sweet.
PvP has destroyed the MMORPG gaming experience for all of us. The idea that a "Wizard" with huge fireballs and freezing ice spells should be a balanced fight vs a dude with a sword and shield is stupid. The idea that a class with a unique spell, making them fun and enjoyable to play in PvE, yet extremely overpowered in PvP, should lose that ability completely is stupid!
Look back at every game that has added PvP and what has become of it.
I'm not saying PvP is bad or should be removed from games. I fully enjoy PvP… In counter strike, in league of legends, in Heroes of Newerth…
MMORPG should never have balanced PvP. MASSIVE does not mean 5v5 or even less. Game balance should be on battlegrounds and huge fights. Never 1v1 or 2v2 encounters.
If a player has a huge fiery death spell that explodes another player in 1 shot, who cares? If they happen to one-shot 1 player in a 100v100 battle once every 10 min that means NOTHING in the grand scheme of things.
We can't have interesting PvE content (or even PvP content) if every class is balanced to be 1v1 capable. At that point everyone ends up being the same with tools to combat everyone else.
cry me a river....there is no reason why pvp should NOT be part of an MMO. It's fun, you even know that. The same can be said about RPing and PvE.....its a matter of taste but to generalize like you are doing is just wrong. |
Novice Member
Joined: 11/24/09 The more you hype an upcoming game in your mind, the more it will fail to meet your expectations. |
It's more than just a class balancing issue. PvP in general is contrary to the entire point of RPGs. More specifically rather, balanced PvP conflicts with progression. Balanced PvP means everyone has to be equal or otherwise very close to it through a rock-paper-scissors setup. RPGs however, are based around character progressio. You level up, obtain gear, learn new abilities, etc. Well, progression runs contrary to PvP balance because it inherently unbalances the scales when you can have people at different levels of progression compete against each other. That said, I do enjoy PvP, but like the OP, I go to games like TeamFortress 2 and the like where they are build from the ground up and balanced specifically for PvP, and not progression. |
Sooner we get rid of all these stupid Wizards in games the better. |
No, pvpers have not destroyed MMORPG gaming. People who think that every single mmorpg should be about balanced "e-sports" all the time though have had an impact. But then I would argue that the whole 1 v 1 thing is driven by the solo player aspect of the market, more so than the pvpers per say. |
I have to agree somewhat with OP, that PVP has ruined a lot of games for me, maybe not the rest of the populace. I'm a casual MMO'er and when I slowl work a toon up to mid level, then come back to the game and find him utterly unplayable due to changes to balance PVP from latest patch, I tend to get PO'd. I'm not a PVP'er but I understand there is an audience for it. This suggestion has been brought up before, but I really wish they would implement a set of 'rules', 'powers' , 'whatever' that are set for PVE and then for PVP have different set/effects. OP does have a good point that a fire-wielding mage vs an armored knight really isn't a contest, in the realm of an MMO. For a PnP game, the knight may win, because of player ingenuity and discussion with a real life thinking GM. There may be environmental factors, quick thinking on a person's part, doing something unconvential or 'tricky' to outwit the wizard, etc. etc. Won't happen in MMO -- you have set parameters that you must follow. |
PVP only really exists in MMO's because it's easy to develop content. Make an arena and let players fight, done! I also tend to agree that it has no place at all in MMO's. These are supposed to be roleplaying games with story and character progression, not an epeen waving contest. I've enjoyed PVP in some games but it causes class balance to shift so drastically and causes so many other problems that in most cases it's not worth it. But again, it's easy content so it goes in and probably isn't going away. The main reason i quit wow was due to the constant class balance changes that were prompted by PVP. It got so tiring not knowing one week if your class was going to be nerfed into oblivion or the FOTM. Just gets old after awhile riding that see saw. I wouldn't have a problem with it if there was a method for them that once you entered a battleground it adjusted your spells on the fly to work differently against players than AI. if a spell was overpowered in PVP but ok in PVE have it automatically toned down just in BG's to avoid balance issues but instead it just gets nerfed or removed all around and your left with nothing but a bunch of generic abilities that dont due anything fun due to possible griefing or overpoweredness in pvp. |
{mod edit} pvp has its place in MMOs, and it is one of the reasons most games retain subscribers or we could say I think the fact that "epic" bosses respawn and drop all kinds of random items that have nothing to do with said boss destroyed mmos, or that thousands upon thousands of people run the same quest doing the same thing for the life of the game has ruined mmos... such a silly comment to throw out there because you wanna frolick with a virtual pet through a wonderland of war as long as the war is only brought to you by stupid AI |
I agree that it is not the PvPer's fault that stupid game companies have not separated PvE and PvP. In an effort to "balance" everything the classes have become quite similiar and boring. Any interesting PvE abilities are destroyed in the name of PvP balance. Another item I feel is hurting the PvE MMO experience is the PvP rush to hit the lvl cap so they can be competitive in PvP. All of the PvP'ers cry if they don't reach lvl cap in a week, and for good reason, as they want to participate in the part of the game that matters to them. Luckly some of the upcoming games are fixing this lvling and PvP issue by making evenyone the same lvl when they enter some instanced PvP. |
PvP should always be a part of MMORPG, but things like instanced battlegrounds, arenas, PvP gear, etc.. should go. Lets keep the focus on adventuring and exploring the world. I want my +5 sword of asskicking from a pile of treasure guarded by a dragon we just managed to kill rather than a goblin in a tuxedo who's obsessed with badges and trinkets and gives stuff away for a handful of them. |
Apprentice Member
Joined: 6/11/08 I think with my heart and move with my head.-Kongos |
I guess I'm going to voice the dissenting opinion here. I don't think MMOs have been 'destroyed' anymore than I think the PvP in the games has contributed to any sort of decline in the games. Kind of the opposite actually. I can not remember winning or losing a single debate on the internet. |
The OP has a great point. Just a shame that as soon as a developer as naive as him puts this into practice, everyone will play a wizard. Or you will have groups of wizards going around 1 shooting content and/or players. Human nature.
Still waiting for your Holy Grail MMORPG? Interesting... |
It's possible for wizards to be powerful without huge fireballs too. WoW mages for example have an incredible arsenal of spells ranging from invulnerability to crowd control.
Still waiting for your Holy Grail MMORPG? Interesting... |
Balancing in PvP is what causes most fun stuff to be toned down to cater to another class. It is the truth but what to do if you do not tone the spell down the people roasted by it will whine and leave and then where would the game be. |
I never PvP because I always PvE in mmorpgs, its too hard to maintain aggro in PvP |
Elite Member
Joined: 1/13/10 Currently playing EVE, SMITE, ArcheAge, and Combat Arms |
Originally posted by tormunda QFT. I remember hanging out at the point south of Trinsic back in the 90's, merrily smacking away at orcs and lizardmen when Crush of Red Army (RA) showed up and fireballed everyone to death. While he was emoting various forms of sodomy over our corpses one of the other ghosts turned to me and said, "Mages... *shrug* there goes the neighborhood." I couldn't help but agree. Cool Crowfall info and speculation - http://www.thewulfden.com/search/label/Crowfall |
Cry babies, battlegrounds and balancing have destoyed mmo's..pvp has been in mmo's since the begining so you must be new to the genre to make a statement like that. |
No problem with pvp, nor with wizards but.. wizards shouldn't be a dime a dozen.. The kind of fireball wielding demigod the OP refers to should be rare, everyone else should at most fart smoke when they try. Unless the game is specifically wizard vs wizard based, I guess, but in most lores, there's usually one powerful wizard and a bunch of ineffective underlings with little or no magic heh ---- |
"The idea that a "Wizard" with huge fireballs and freezing ice spells should be a balanced fight vs a dude with a sword and shield is stupid." Not entirely true. In pnp d&d I could create a sword and board character that specializes in killing mages, but that same character would be much less effective fighitng against brute strength type mobs. I agree with your core idea though. I think if classes weren't so black and white a rock, sissors, paper design would work much better. Right now a rogue is a clothy killer, but if that clothy could build there class so that it was a rogue killer pvp wouldn't be so black and white. With more options per class having imbalance wouldn't be the big of a deal. |
Perhaps the problem isn't a PvP game--but instead a game that tries to do both (PvP and PvE), despite very different objectives, squeezed together into the same "balance". PvE players hate getting nerfed because of some perceived PvP imbalance. PvP players hate being nerfed because of some perceived e-sport imbalance. Yet developers (oddly) try to create one set of rules that encompasses all things and all people. And to a large extent, at the best of times, it doesn't work very well for anyone. Self-pity imprisons us in the walls of our own self-absorption. The whole world shrinks down to the size of our problem, and the more we dwell on it, the smaller we are and the larger the problem seems to grow. |


text:
see more
More like this
University of Phoenix-Nashville Campus
616 Marriott Drive, Suite 150 Nashville, TN 37214
(615) 872-0188
| www.phoenix.edu
University of Phoenix-Nashville Campus is a university in Nashville, TN.
The school is a school of business and management.
There are 1,827 undergraduate students and 178 graduate students at University of Phoenix-Nashville Campus.
The school has its own library and participates in Title IV federal financial aid programs for those who qualify.more
Fields of study offered at University of Phoenix-Nashville Campus
- Business, Management, Marketing, and Support Services (11 majors)
- Communication, Journalism and Public Relations (1 major)
- Computer and Information Sciences and Support Services (7 majors)
- Criminal Justice and Security Services (1 major)
- Education (2 majors)
- Health Professions and Clinical Sciences (4 majors)
- Psychology (1 major)


text:
United Rentals has been punished by concerns about low oil prices, but energy is a tiny slice of its business. Why the shares could get a 50% lift.
Money flow gives a snapshot of relative buying and selling pressure in a stock.
A ratio above one suggests greater buying pressure, below one suggests greater selling.
URI has not issued dividends in more than 2 years


text:
News Archive
January 30, 2006
William Kirby will return to teaching amid grumbling over curricular review and reported split with Summers.
January 30, 2006
Presidents, provosts, deans and professors consider principles and data they need to promote their curricular ideals.
January 27, 2006
AAUP's "Boston experiment" garners another success, say organizers.
January 27, 2006
Scholars issue draft "declaration" calling for expanded role of religion in curriculum and student life -- at religious and secular colleges.
January 27, 2006
Ohio State and UT-Austin -- 2 of the largest universities in U.S. -- may revamp requirements with theme-based sequences.
Search for Jobs
Most Popular
- Viewed
- Commented
- Past:
- Day
- Week
- Month
- Year
Loading results...


text:
Perl Monk, Perl Meditation | |
PerlMonks |
Re: File Manipulationby 2teez (Priest) |
on Aug 23, 2013 at 12:46 UTC ( #1050667=note: print w/ replies, xml ) | Need Help?? |
Hi, NOTE: Since, the key only changes, when the name of server is seen, and that until the next one. It works perfectly well. You could also see perldsc
If you tell me, I'll forget. If you show me, I'll remember. if you involve me, I'll understand. --- Author unknown to me
In Section
Seekers of Perl Wisdom


text:
The rumors have swirled: Jon Lester's on the block. Jacoby Ellsbury's on the block. But are they? And should they be?
Jon Lester's season has been a bitter disappointment thus far. But will it continue. Our Uno's Sports Tonight Question of the Day:
Over the winter, the Los Angeles Angels signed Albert Pujols to the second largest contract in Major League history.
Jon Lester's September struggles -- 1-4, 5.61 in his last five starts, including three losses to the two teams (Yankees and Rays) closest to the Red Sox in the standings -- have raised questio
Jacoby Ellsbury's three-run home run, and Jon Lester's dominating performance helped the Red Sox defeat the Rays at Fenway Park, 3-1.