text:
Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans
DOI: 10.1186/gb-2014-15-2-r21
© González-Aguilera et al.; licensee BioMed Central Ltd. 2014
Received: 18 September 2013
Accepted: 3 February 2014
Published: 3 February 2014
Laminopathies are diseases characterized by defects in nuclear envelope structure. A well-known example is Emery-Dreifuss muscular dystrophy, which is caused by mutations in the human lamin A/C and emerin genes. While most nuclear envelope proteins are ubiquitously expressed, laminopathies often affect only a subset of tissues. The molecular mechanisms underlying these tissue-specific manifestations remain elusive. We hypothesize that different functional subclasses of genes might be differentially affected by defects in specific nuclear envelope components.
Here we determine genome-wide DNA association profiles of two nuclear envelope components, lamin/LMN-1 and emerin/EMR-1 in adult Caenorhabditis elegans. Although both proteins bind to transcriptionally inactive regions of the genome, EMR-1 is enriched at genes involved in muscle and neuronal function. Deletion of either EMR-1 or LEM-2, another integral envelope protein, causes local changes in nuclear architecture as evidenced by altered association between DNA and LMN-1. Transcriptome analyses reveal that EMR-1 and LEM-2 are associated with gene repression, particularly of genes implicated in muscle and nervous system function. We demonstrate that emr-1, but not lem-2, mutants are sensitive to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions.
We identify a class of elements that bind EMR-1 but do not associate with LMN-1, and these are enriched for muscle and neuronal genes. Our data support a redundant function of EMR-1 and LEM-2 in chromatin anchoring to the nuclear envelope and gene repression. We demonstrate a specific role of EMR-1 in neuromuscular junction activity that may contribute to Emery-Dreifuss muscular dystrophy in humans.
The nuclear envelope (NE) is an essential structure conserved in all eukaryotes that separates the nucleus from the cytoplasm [1]. It is characterized by the presence of two concentric lipid membranes, the outer nuclear membrane (ONM), which is continuous with the endoplasmic reticulum, and the inner nuclear membrane (INM). The two membranes are fused where large nuclear pore complexes create transport channels to control trafficking of molecules between the nucleus and the cytoplasm. Numerous membrane proteins are integrated in the membrane bilayer. In the ONM, membrane proteins interact with microtubules, actin filaments and other cytoplasmic components. In the INM of higher eukaryotes, membrane proteins interact with the nuclear lamina, a protein meshwork of intermediate filaments that serves as a scaffold to maintain the morphology of the nucleus. INM and nuclear lamina proteins also interact with chromatin [2].
The NE is an important player in a number of cellular functions, including maintenance of structural integrity of the nucleus, chromatin organization, transcriptional regulation, DNA replication, cell-cycle control, differentiation, nuclear migration, and apoptosis [1, 3–5]. Reflecting these many functions, mutations in NE components have been associated with numerous human diseases. In particular, a predominant group of diseases linked to alterations in the NE is caused by mutations in genes encoding the nuclear lamins (LMNA, LMNB1 and LMNB2 in humans) and is referred to as laminopathies. Laminopathies include diverse pathologies, such as muscular dystrophies, cardiomyopathies, bone disorders, neurological diseases, lipodystrophies and premature aging [5, 6]. Laminopathy mutations have also been identified in several genes encoding ONM and INM proteins. Emery-Dreifuss muscular dystrophy (EDMD) was initially linked to mutations in the EMD gene, which encodes the INM protein emerin [7], but can also be caused by mutations in LMNA and NE genes SYNE1, SYNE2 and TMEM43[6]. EDMD develops during childhood and manifests as degenerative muscle atrophy with cardiac defects [6]. Although clinical investigations focus on muscle abnormalities in EDMD patients, mice lacking emerin were found to display minimal muscle dysfunction but diminished motor coordination [8], suggesting that emerin could have additional functions apart from regulation of muscle formation and integrity. Interestingly, differences between humans and mice in relative expression levels of emerin and lamina-associated polypeptide 1 (LAP1) were recently proposed to explain the absence of muscle dystrophy in emerin mutant mice [9]. Since most NE genes are expressed ubiquitously, a main challenge is to understand how laminopathy mutations result in tissue-specific phenotypes [5, 6]. One proposed hypothesis argues that an alteration of the integrity of the NE - for example, by affecting the nuclear lamina or the connection of the NE with the cytoskeleton - could lead to a structural weakness and a decrease in the ability of the nucleus to resist mechanical stress [10–12]. This could be of special relevance in muscle and skeletal disorders. However, symptoms affecting others tissues, such as the nervous system, cannot be explained simply by mechanical problems. Instead, several studies indicate that the NE contributes to chromatin organization by direct interaction with chromatin factors. For instance, the INM LEM-domain-containing proteins (LAP2, emerin and MAN1) interact with the chromatin binding protein BAF (also known as BANF1), histone deacetylases as well as with the nuclear lamins [2, 13, 14]. The nuclear lamins also interact with chromatin remodelers involved in heterochromatin formation, such as heterochromatin protein HP1 or the NURD complex [15].
Analyses of different organisms and cell types have shown that the NE is mainly associated with heterochromatin and silenced DNA [16–19]. Consistent with this, alterations in the distribution of heterochromatin marks, such as histone H3 lysine 9 methylation (H3K9me) and H3K27me, have been observed in murine and human myoblast cells harboring mutations in the LMNA gene [20, 21]. Mutations in NE proteins can also lead to a reduction of transcription of muscle differentiation genes [22]. Although this reduction could at least partially be due to the role of NE components in heterochromatin formation, it has also been proposed that INM proteins can sequester transcription factors to the nuclear periphery and impede their binding to target genes. For example, in humans and mice, emerin physically interacts with lmo7 and β-catenin, two transcription factors involved in muscle differentiation [23, 24], whereas in humans LEMD3/MAN1 tethers Smads to the NE, thereby affecting connective tissue differentiation [25–27].
While the importance of the NE as a regulator of nuclear architecture and gene expression is becoming increasingly evident, the molecular mechanisms underlying this remain elusive. In particular, the contributions of individual NE proteins to tissue-specific functions are not well understood. Characterization of the chromatin domains that interact with the NE is required to decipher how the NE contributes to nuclear organization. Until now, most experiments have focused on cultured cells, whereas few studies have been performed on cells within intact organisms. We therefore decided to analyze the DNA associated with two components of the NE, lamin/LMN-1 and emerin/EMR-1, in whole adult Caenorhabditis elegans. Due to its amenability to genetic manipulations, C. elegans is particularly suitable to genomic analyses across several genotypes and developmental stages [28]. Our results show that both LMN-1 and EMR-1 are associated with lowly expressed genes. As expected, similar DNA profiles were observed for the two proteins, but we also identified elements bound by only one of the two. 'EMR-1 only' elements were enriched for muscle and neuronal genes, which became accessible to LMN-1 association when emr-1 was deleted. Furthermore, we observed that EMR-1 acts redundantly with another LEM domain containing protein, LEM-2, to repress transcription, consistent with their functional redundancy during mitosis [29], development and myogenesis [30], and signaling [31]. Finally, we demonstrate that EMR-1, but not LEM-2, is required for proper neuromuscular junction activity. Together, this study demonstrates the importance of EMR-1 in the control of chromatin organization and gene expression of muscle and neuronal genes, thereby providing clues as to how defects in INM protein function can have tissue-specific consequences.
Identification of chromatin anchored to LMN-1 and EMR-1 by DamID
To investigate the specific role that different components of the NE play in the control of chromatin organization and gene expression, we generated genome-wide interaction maps of the sole C. elegans nuclear lamina protein, lamin/LMN-1, and the inner nuclear membrane protein emerin/EMR-1, in adult C. elegans nematodes using DamID [32]. This technique is based on the in vivo expression of chimeras of the Escherichia coli DNA adenine methyltransferase (Dam) and a chromatin-interacting protein. Upon interaction of the Dam-fused protein with chromatin, adenines in the vicinity are methylated. These DNA regions are subsequently identified by microarray analysis or high-throughput sequencing. DamID has been successfully used in many organisms and has been demonstrated to reliably identify NE-associated sequences [17, 19]. To minimize experimental variation, we created C. elegans strains containing single copy insertions of the chimeric transgenes in chromosome II (Figure S1A in Additional file 1; Materials and methods). We used the promoter of the heat shock-inducible gene hsp-16.41, and due to the requirement for only trace expression levels of the chimeric protein, performed the DamID experiments without induction [19]. Without heat shock, transgene expression is undetectable by immunofluorescence or western blot analyses: however, after heat shock, the nuclear peripheral localization of chimeric proteins was readily observed (Figure S1B, C in Additional file 1). We also generated a control strain expressing GFP::Dam (which freely diffuses throughout the nucleus; Figure S1D in Additional file 1) to normalize for local chromatin accessibility [19]. Under normal growth conditions, nematodes containing chimeric proteins were fertile and indistinguishable from wild-type (Bristol N2) animals. We conclude that the Dam-fusion proteins mimic the localization of their endogenous counterparts and their presence did not produce any apparent deleterious effects.
To investigate nuclear organization in differentiated cells we performed DamID on young adults. Thus, the DamID signals we describe in the sections below represent the mixture of cell types of which the nematode is composed. We isolated adenine-methylated DNA from three biological replicates for each strain (Dam::LMN-1, Dam::EMR-1 and GFP::Dam) and analyzed it on C. elegans whole-genome high-density tiling microarrays. We normalized the DamID signals using MA2C [33] (Materials and methods) and then all MA2C data were quantile normalized to facilitate comparison between strains. The replicas were highly correlated (Pearson correlation between replicates ranging from 0.88 to 0.98; Figure S2A in Additional file 1) and genomic profiles of LMN-1 and EMR-1 association were constructed by averaging the three replicates.
Maps of LMN-1 and EMR-1 reveal global similarities and association with silent DNA
To precisely define LMN-1-associated domains (LADs) and EMR-1-associated domains (EADs), we converted positive and negative DamID scores to +1 and -1, respectively, and used a window-based method to identify DNA regions continuously associated with LMN-1 or EMR-1 [18] (Materials and methods). Using a false discovery rate <5%, we identified 1,021 LADs and 1,106 EADs, each of which covers 37.5% and 37.8% of the genome, respectively (Figure 1C; Table S1 in Additional file 2). The size of LADs and EADs ranged from 8 to 300 kb with a median size of 22.7 kb and 23.9 kb for LMN-1 and EMR-1, respectively (Figure 1D). A large proportion of LADs and EADs was overlapped: 89% of DNA in LADs was also associated with EMR-1. LADs and EADs at the chromosome arms were separated by non-associated regions ('gaps'), which have a median size of 13.5 kb and 19.2 kb for LMN-1 and EMR-1, respectively (Figure 1D). It is important to note that genomic features, such as open reading frames, promoters and intergenic regions are found in both domains and gaps (Figure 1C).
Like LEM-2-associated regions [18], the fraction of DNA regions occupied by LMN-1 or EMR-1 in autosomes was positively correlated with the size of the autosomes (Figure 1E). In addition, we observed an equal or higher proportion of each autosome bound by LMN-1 than EMR-1. In contrast, the X chromosome showed a lower association with LMN-1 compared to EMR-1, suggesting that EMR-1 might play a distinct role in anchoring the X chromosome to the NE (Figure 1E; see below).
Previous studies in C. elegans embryos and other organisms have shown that the chromatin regions associated with the NE are marked predominantly by histone modifications characteristic of heterochromatin and are transcriptionally silent [17–19]. To investigate if this distribution is maintained in adult nematodes, we analyzed the levels of H3K4me3 and H3K36me3, both associated with transcriptional activity [35], in LADs and EADs. Sliding window analyses across EMR-1 domain boundaries showed that both LADs and EADs have lower H3K4me3 and H3K36me3 levels, and instead, gaps have higher levels of these two marks, suggesting that transcriptionally active chromatin is mainly localized in the nuclear interior in C. elegans adults (Figure 1F; Figure S3B in Additional file 1), as it is in embryos [18]. However, low H3K4me3 and H3K36me3 levels were not exclusively observed in LADs and EADs but also inside gaps, suggesting that not all chromatin away from the NE is transcriptionally active (Figure S3B in Additional file 1).
Chromosome architecture is maintained during development
Interestingly, we found a clear difference in the behavior of the central part of the chromosomes. In early embryos, LMN-1 MA2C scores for the central part of the chromosomes were close to zero, while in adult nematodes LMN-1 association with chromosome centers was significantly lower (Figure 2C, D). Taken together, these data suggest that during adulthood segregation of chromatin into NE-associated and NE-excluded domains is more pronounced, especially at the central region of the chromosomes, which seems to be excluded from the nuclear periphery in most cells.
Local differences between LMN-1 and EMR-1 DNA association reveal a specific role of EMR-1 in the regulation of muscle and neuronal genes
Although LMN-1 is ubiquitously expressed and physically interacts with EMR-1 [36], we also found elements associated exclusively with EMR-1 (Figure 3B, C). Contrary to LMN-1-enriched regions described above, these elements were more abundant close to the central part of the autosomes and on the X chromosome (Figure 3B; Figure S4 in Additional file 1). A systematic search identified 340 'EMR-1 only' elements (Materials and methods) ranging from 339 bp to 3,328 bp and with a median size of 763 bp (Table S1 in Additional file 2). These elements were included in EADs and surrounded by LADs (Figure 3C; Figure S4B in Additional file 1). 'EMR-1 only' elements occurred significantly more often in promoters than expected, and occurred less often than expected in exons and introns (Figure 3C, D). Interestingly, Gene Ontology (GO) analyses of genes placed in or immediately downstream of EADs identified 29 GO categories, most of which were related to muscle and neuronal physiology, including signaling transduction at synapses (G-protein coupled receptor protein signaling pathway, P = 2.41e-4; synaptic transmission, P = 0.01; acetylcholine catabolic process, P = 0.04), behavior (behavior, P = 0.04), reproduction (reproductive process, P = 0.01) or locomotion (regulation of locomotion, P = 0.006) (Figure 3E; Table S5 in Additional file 3). Employing the same criteria used to defined 'EMR-1 only' elements, we identified 97 'LMN-1 only' elements ranging from 406 bp to 2,097 bp and with a median size of 661 bp (Table S1 in Additional file 2). These 'LMN-1 only' elements were located almost exclusively in the large regions with high LMN-1/EMR-1 DamID ratios previously described (Figure 3B; Figure S4C in Additional file 1) and they were not enriched for genome features or GO term categories.
The identification of 'EMR-1 only' elements indicates that EMR-1 could have a predominant function in certain tissues. Indeed, we have observed that EMR-1 expression varies significantly between tissues (Figure S5 in Additional file 1). To further investigate whether LMN-1 and EMR-1 could play tissue-specific roles in chromatin organization, we analyzed LMN-1 and EMR-1 DamID scores of 6,954 genes that are expressed in a tissue-specific manner, as previously defined by others (Table S2 in Additional file 2; see Materials and methods for references). Of 6,954 genes, 4,909 (70.6%) were not anchored to the NE as reflected by the low MA2C values derived from DamID experiments performed in the whole animal (Figure S6A in Additional file 1). Importantly, in the 2,045 genes with a positive MA2C score (that is, anchored to the NE in most cells of the nematode), we observed significantly more association of intestinal, germline, and ubiquitous genes with LMN-1 than with EMR-1 (Figure 3F). In contrast, muscle and neuronal genes were associated with EMR-1 to a higher degree than they were with LMN-1 (Figure 3F; Figure S6B in Additional file 1), which is consistent with the GO analysis of 'EMR-1 only' elements. Our data thus suggest that EMR-1 is specifically involved in gene regulation in muscle and neurons.
Lack of EMR-1 or LEM-2 produces local changes in nuclear organization
The unexpected finding of 'EMR-1 only' elements led us to speculate how they can be established. We imagine three scenarios for their formation: (1) EMR-1 is not only restricted to the NE but can diffuse into the nuclear interior; (2) EMR-1 localizes to patches of the NE devoid of LMN-1; or (3) EMR-1 is recruiting proteins to these DNA elements that inhibit access of LMN-1.
Although lack of EMR-1 expression did not produce massive reorganization of the genome, we identified regions that were detached from the nuclear periphery upon EMR-1 deletion (Figure 4C; Table S1 in Additional file 2). These regions were specifically enriched in introns (Figure 4D) and GO analyses revealed that they were associated with genes involved in development of reproductive organs and muscles (Table S6 in Additional file 3).
Depletion of EMR-1 and LEM-2 increases gene expression globally but reduces transcription of chromatin modifying enzymes
Since LMN-1 DamID profiles of emr-1 or lem-2 single mutants did not reveal dramatic changes in LMN-1 domain organization, we analyzed gene expression in animals lacking expression of both EMR-1 and LEM-2. To bypass the embryonic lethality produced by the simultaneous deletion of EMR-1 and LEM-2, we knocked down emr-1 expression by RNA interference (RNAi) in lem-2 mutants. L1 larvae homozygous for the lem-2(tm1582) allele were fed with bacteria expressing emr-1 double-stranded RNA. These nematodes developed to adulthood at the normal rate but produced a large number of dead embryos, which verified the effectiveness of the RNAi treatment [29]. As illustrated in Figure 5B, a general increase in RNA levels for all genes was observed in both singly and doubly depleted animals: in lem-2(tm1582); emr-1(RNAi) doubly depleted animals, the RNA levels of more genes were significantly altered relative to wild type (9.7% of protein-coding genes compared with 2.2% and 0.48% in emr-1(gk119) and lem-2(tm1582) single mutants, respectively; Figure 5B; Figure S8C in Additional file 1), most likely reflecting the redundant functions of EMR-1 and LEM-2.
Analysis of the genes that lose their NE interaction in emr-1 mutants revealed a negative correlation between NE association and gene expression (Figure S8E in Additional file 1). Combined with the decreased association of chromatin to the nuclear periphery observed by LMN-1 DamID in the single mutants (Figure 4B), this result clearly indicates a role for EMR-1 and LEM-2 in gene repression. However, consistent with previous studies [34, 40], we observed that not all the genes that were up-regulated in the emr-1 mutant changed their position relative to the NE (Figure S8D in Additional file 1). This indicates that multiple processes influence the relationship between NE association and gene expression (see Discussion).
When we analyzed genes expressed in specific tissues, we also found that effects were maximal in the doubly depleted animals. Interestingly, an increase in expression of intestinal, muscle and serpentine genes was observed whereas germ line and ubiquitous genes were repressed (Figure 5C). Because the number of neuronal genes in this data set was very low (n = 14) we expanded our analysis to include 338 genes identified as pan-neuronal genes in larvae [41] (Table S2 in Additional file 2). Of these, 141 genes are associated with the nuclear periphery as judged from positive EMR-1 DamID MA2C scores. Interestingly, the pan-neuronal genes were significantly up-regulated in doubly depleted animals (Figure S6C in Additional file 1). These results were confirmed by GO analyses since GO categories related to the nervous system and locomotion were enriched among the significantly up-regulated genes (Figure 5D; Table S8 in Additional file 3). In contrast, down-regulated genes were associated with GO terms related to reproduction and basic cellular processes like cell cycle or transcription (Figure 5D). Intriguingly, genes related to chromatin organization were over-represented among the transcripts with reduced expression (P = 1.17e-6). In fact, the expression of 18 of the 38 chromatin modifying enzymes described in C. elegans[42] was significantly affected in the doubly depleted animals (47%, compared to 9.7% of all genes; Figure 5E; Table S8 in Additional file 3). We cannot rule out the possibility that some of the changes in expression levels of chromatin modifier enzymes could be secondary effects of chromatin reorganization upon EMR-1 and LEM-2 depletion. Nevertheless, these data support the idea that EMR-1 and LEM-2 could be especially important for regulating the activity of genes encoding chromatin-remodeling activities.
EMR-1 is required for proper neuromuscular junction activity
We determined the time-course for aldicarb-induced paralysis in wild type, emr-1 and lem-2 single mutants and in lem-2 nematodes heterozygous for the emr-1 mutation (emr-1(gk119)/+; lem-2(tm1582)). We did not test for EMR-1/LEM-2 double depletion because RNAi is less effective in C. elegans neurons and the homozygous double mutants proceeding from heterozygous animals arrest during larval development [30]. Compared with wild-type animals, emr-1 mutants were hypersensitive to aldicarb (50% of animals were paralyzed after approximately 35 minutes for emr-1 mutants versus approximately 55 minutes for wild-type animals), whereas lem-2 mutants retain normal aldicarb sensitivity (Figure 6B). Heterozygous emr-1(gk119)/+ mutants were equally sensitive to aldicarb as wild-type animals, indicating that a single copy of emr-1 is sufficient to maintain proper neuromuscular junction activity.
At this moment, we cannot distinguish whether the paralysis observed in emr-1 mutants is due to changes in neurotransmitter release or muscle perception, or a combination of the two. Interestingly, lem-2 mutants present alterations in muscle structure and function [30] and, unlike emr-1 mutants, do not show altered aldicarb sensitivity. This indicates that EMR-1 and LEM-2 are not redundant in all their functions, making C. elegans a good model to study the tissue-specific roles of these two proteins.
A comparison of the genome-wide DNA association profiles of LMN-1, the only lamin protein in C. elegans, and of the INM protein emerin/EMR-1 indicates that EMR-1 defines two types of chromatin domains: a major type consisting of LADs and a minor type devoid of LMN-1 and enriched for genes expressed in specific tissues. LADs are conserved from embryos to adults and epigenetic marks suggest they are transcriptionally silent at both developmental stages (this study and [34]). In contrast, genes associated with EMR-1 only show higher expression and belong to functional categories that respond to stimuli. The difference between these two classes of chromatin domains might be mediated by posttranslational modifications of EMR-1 as well as other NE associated proteins. For instance, phosphorylation of human emerin has been shown to be important for the association with BAF [45, 46].
The discovery of regions associated with EMR-1 but not with LMN-1 was surprising, since the two proteins physically interact at the NE and LMN-1 is required to localize EMR-1 properly [36]. Interestingly, although we could find 'EMR-1 only' elements in all chromosomes, they were especially abundant in the X chromosome. We do not know why the X chromosome has fewer interactions with LMN-1 compared to the autosomes, nor can we explain the particular enrichment of 'EMR-1 only' elements on the X chromosome. Our experiments were performed with C. elegans hermaphrodites, in which both X chromosomes undergoes approximately two-fold chromosome-wide transcriptional repression to achieve dosage compensation [47]. It is possible that the interaction with EMR-1 or other INM proteins could be important for this transcriptional repression. Our data suggest that the anchoring of the X chromosome to the NE could be more pronounced than previously inferred from LEM-2 ChIP data [18] or by LMN-1 DamID ([34] and this study).
We found that 'EMR-1 only' elements were surrounded by chromatin interacting with both LMN-1 and EMR-1. This indicates that 'EMR-1 only' elements are located at or very close to the NE, but raises the question as to why LMN-1 is not able to bind to these chromatin regions. Given that the absence of EMR-1 and/or LEM-2 allows the 'EMR-1 only' elements to interact more frequently with LMN-1, we hypothesize that EMR-1 and LEM-2 physically prevent LMN-1 from binding to these regions. It is known that human and mouse emerin interacts physically with transcription factors involved in muscle development, including β-catenin [23] and Lmo7 [24], and with chromatin associated factors, such as BAF and HDAC3 [13, 48]. Similarly, the mammalian LEM-2-like protein MAN1 interacts with Smads at the NE to regulate transforming growth factor-β signaling [49]. C. elegans EMR-1 and LEM-2 may also associate with DNA-binding proteins and such protein complexes may hinder LMN-1 from binding to 'EMR-1 only' elements.
The NE is generally associated with repressed DNA and several of our observations suggest that EMR-1 and LEM-2 may also be involved in anchoring of silent heterochromatin to the nuclear periphery in specific tissues. First, genes within EADs are less expressed than genes in gaps. Second, we have demonstrated that, during adulthood, depletion of EMR-1 and/or LEM-2 caused a partial release of LADs from the NE and increased transcription of several hundred genes. These results are consistent with the delocalization and overexpression of repetitive heterochromatic transgene arrays observed upon simultaneous depletion of EMR-1 and LEM-2 in adult nematodes [50]. Interestingly, although chromatin associated with EMR-1 is less transcribed than chromatin found in gaps, genes in 'EMR-1 only' elements are significantly more expressed than genes in other EMR-1 domains. It is possible that the accumulation of transcription factors around 'EMR-1 only' elements might poise these regions for a rapid response to environmental stimuli. Supporting this idea, 'EMR-1 only' elements are enriched for GO categories related to signaling pathways and transcription. However, we have also noticed that gain or loss of NE anchoring do not always correlate with changes in gene expression. This has also been observed in other organisms. For instance, approximately one-third of genes that move towards or away from the NE during mouse embryonic stem cell (ESC) differentiation do not change transcriptional activity [40]. Similarly, changes in expression of genes that gain or lose lamin interaction after ecdysone treatment is marginal in Drosophila Kc cells [19]. Finally, mouse ESCs lacking B type lamins show altered transcription profiles but the alterations do not correlate with lamin B1 association in wild-type ESCs [51]. The reason for this lack of concordance between NE anchoring and silencing is not clear. Peric-Hupkes and colleagues [40] proposed that genes that move away from the NE during ESC differentiation are 'unlocked' to become activated at later steps of development. Likewise, results from a recent study in human adipose stem cells, indicate that dissociation of promoters from the nuclear lamins produces chromatin changes that enhance transcriptional permissiveness but do not necessarily elicit gene activation [52]. The molecular details of these intermediate states are unknown. The observation in C. elegans that the sequential action of two H3K9 methyltransferases promotes first anchoring of DNA to the NE and afterwards transcriptional repression has provided mechanistic insight into the uncoupling of the two processes [34].
Our data also indicate that genes specifically expressed in muscles and neurons are enriched in EADs. Furthermore, expression levels of genes related to locomotion and behavior were affected in emr-1 and lem-2 mutants. Several studies on Emery-Dreifuss muscular dystrophy patients and model organisms support the role of emerin and LEMD2 in muscle integrity, but their potential role in neurons had not been explored. Here we show that in C. elegans, localization and expression of genes related to neurotransmitter recycling, acetylcholine receptor network and ion-channels are affected in emr-1 and lem-2 mutants. Interestingly, neuromuscular junction activity is strongly affected in emr-1 but not in lem-2 mutants, indicating that the two genes are not redundant in all their functions. The role of emerin in neuronal activity may not be restricted to C. elegans since emerin knockout mice suffer relatively mild muscle defects and altered motor coordination [8]. In rodents, expression of emerin but not LEMD2 has been detected in neuroretinal cells, while both proteins are equally present in muscles [53]. Moreover, mutations in several other NE components, such as lamin A, BAF, lamin B1, and the LINC complex (which connects the NE to the cytoskeleton), are associated with neuropathies affecting both the peripheral and central nervous system [54–58]. Future analyses are needed to investigate whether emerin also contributes to proper neuronal physiology in humans.
Finally, we have demonstrated that, in adult nematodes, the NE-associated heterochromatin domains are highly enriched at the ends of autosome arms and at the left arm of chromosome X. This configuration is very similar to the one observed during embryogenesis [18, 34]. However, we also found differences in NE-genome associations during development. These include few local changes in LADs placed in the chromosome arms and a general decrease in association of LMN-1 with the central part of the chromosomes in adults. Developmental changes in chromatin distribution have been observed in several organisms. The location of integrated small transgene arrays has been reported to be developmentally regulated, being randomly positioned in embryonic nuclei and accumulated in the nuclear interior in larvae [59]. The integration sites of these transgene arrays are unknown but their repositioning away from the NE coincides with transcriptional activation [59]. In murine embryonic stem cells, approximately 10% of genes change their interaction with the nuclear lamina during in vitro differentiation [40]. Together, these results suggest that, across evolution, global nuclear organization is established early in development and only a few specific regions change their localization in response to developmental requirements or changes.
The comparison of chromatin associated with the conserved NE proteins LMN-1/lamin and EMR-1/emerin presented here reveals a large overlap between LMN-1- and EMR-1-associated domains but also the existence of numerous elements that interact with EMR-1 only. Muscle and neuronal genes are enriched in these 'EMR-1 only' elements and are deregulated in animals depleted for EMR-1 and LEM-2. Moreover, differential expression of EMR-1 and LEM-2 was observed across tissues.
The requirement for EMR-1 for the proper spatial and transcriptional control of muscle and neuronal genes demonstrated here prompts the idea that variation in NE composition among tissues may contribute to the correct adaptation of the organism to the environment. Combined with the defects in neuromuscular junction activity in emr-1 mutants reported here, we propose that development of Emery-Dreifuss muscular dystrophy may involve altered nuclear architecture and gene expression, especially in muscles and neurons.
Materials and methods
Strains and plasmids
Strains carrying a single-copy insertion of Dam-fusion constructs were made using the MosSCI technique [60]. Strain and plasmid construction are described in Additional file 4.
Immunofluorescence and western blot
Nematodes grown at 20°C were heat-shocked 1 h at 33°C and left to recover for 2 h at 20°C. Embryos were obtained and processed as described [61]. Anti-MYC primary antibody (Sigma-Aldrich, catalog number C3956, St. Louis, MO, USA) was diluted 1:100 for immunofluorescence and 1:400 for western blotting. Secondary goat anti-rabbit Alexa Fluor 633-conjugated antibody (Invitrogen, catalog number A21071, Carlsbad, CA, USA) diluted 1:1,000 and peroxidase-conjugated antibody (Sigma-Aldrich, catalog number A0545) diluted 1:5,000 were used for immunofluorescence and western blotting, respectively.
DamID experiments
DamID experiments were performed using three biological replicates and all cultures were grown in parallel. DamID strains were synchronized from eggs prepared by hypochlorite treatment of gravid adults fed with OP50 E. coli and left to hatch overnight at 20°C in S-medium. Then, from each strain, approximately 35,000 L1s were grown in 50 ml S-medium containing GM119, a Dam-E. coli strain (F-dam-3 dcm-6 metB1 galk2 galT22 lacY1 tonA31 tsx-78 supE44 mtl-1). This food source was used to avoid contamination by methylated E. coli DNA. Cultures were grown with continuous agitation (180 rpm) at 20°C for 53 h. At this time point the cultures were highly enriched for non-gravid young adults. Nematodes were frozen at -80°C until further processing.
Methylated genomic DNA (gDNA) was purified and amplified from 30 mg nematodes using a protocol based on [62] with some modifications. Briefly, 2.5 μg of gDNA isolated with DNAeasy kit (QIAGEN, Venlo, Limburg, Netherlands), was digested with 10 units DpnI (New England Biolabs, Ipswich, MA, USA) overnight in 10 μl to cut at GmATC sites. DpnI was inactivated (80°C, 20 minutes) and then gDNA was ligated to double-stranded adaptors with 5 units T4 DNA ligase (5 U/μl; Roche, Basel, Switzerland) in a volume of 20 μl. After inactivation of the ligase (65°C, 10 minutes), DNA fragments were digested with 5 units DpnII (New England Biolabs) in a final volume of 80 μl to cut non-methylated GATC sites, thereby preventing PCR amplification of non-methylated gDNA. Methylated DNA was amplified using adaptor-specific primers. PCR reactions were performed using 1 μl PCR Advantage enzyme mix (50×; Clontech, Otsu, Shiga, Japan), 10 μl of DpnII digestion and the following program: (1) 68°C for 10 minutes, (2) 94°C for 1 minute, (3) 65°C for 5 minutes, (4) 68°C for 15 minutes, (5) 94°C for 1 minute, (6) 65°C for 1 minute, (7) 68°C for 10 minutes, (8) go to step 5 3×, (9) 94°C for 1 minute, (10) 65°C for 1 minute, (11) 68°C for 2 minutes, (12) go to step 9 20×. To achieve sufficient material, two PCR reactions from the same DpnII digestion were pooled and purified with Qiaquick PCR purification kit (QIAGEN). Amplified Dam-methylated DNA was labeled and hybridized by the Roche Nimblegen Service Laboratory. Dye orientation of experiments is indicated in Table S4 in Additional file 2.
DamID data processing
Nimblegen 2.1 M whole genome tiling arrays, with 50 bp probes, designed against WS180 (ce5) genome assembly, were used for all experiments. Data were analyzed with MA2C [33], which was used to normalize the log2 ratio (log2 [Dam::fusion/GFP::Dam control]) of each probe based on the probe behavior estimated by its GC content, and then to smooth the value by assigning the median across sliding windows of 300 bp. The resultant values are MA2C scores. Subsequently, data were subjected to quantile normalization over all available replicates and backgrounds to facilitate comparison between strains. Finally, the mean of normalized MA2C scores from the three independent biological replicates was calculated. We then converted the chromosome coordinates to WS190 (ce6) genome assembly using the Lift-Over tool from the UCSC genome browser [63] and used these values for the subsequent domain calling and sliding window analyses. In some cases, to facilitate chromosome-scale data visualization by reducing the number of data points, MA2C scores were averaged within non-overlapping 100 kb windows across the genome as indicated in the figure legends.
Sliding window analysis was performed using the left boundaries corresponding to the left edges of all EMR-1 domains (Table S1 in Additional file 2). Both sides of boundaries and sliding window analyses with LMN-1 boundaries provided the same results. Boxplots for gene classes were generated by averaging MA2C scores of probes included in regions between the transcription start site and end site. Coordinates for these sites were obtained from the UCSC genome browser (WS190 genome assembly). Analyses for enrichment at promoters (up to 3 kb before the transcription start site), exons and introns were performed using the CEAS program [64]. ChIP-chip data of young adult H3K4me3 and H3K36me3 marks [35], and the embryonic LEM-2 mark [18] are from modENCODE [65] (accession IDs modENCODE_3552, modENCODE_3559, modENCODE_2729, respectively). Embryonic LMN-1 DamID data [34] were obtained from the Gene Expression Omnibus (GEO; accession GSE37226). Gene sets used in tissue-specific analyses were defined previously and are listed in Table S2 in Additional file 2: muscle [66], intestine [67], ubiquitous, serpentine, germ line [68], and neuron [69].
Definition of LMN-1- and EMR-1-associated domains
LMN-1- and EMR-1-associated domains were defined as previously described [18]. Briefly, we transformed positive and negative MA2C scores to +1 and -1, respectively. We averaged the binary values in 200-probe windows (approximately 10 kb), sliding one probe (50 bp offset) across the genome to identify windows with high average binary values. To have an estimation of the false positive windows, we performed the same procedure on a GFP::Dam/GFP::Dam MA2C normalized dataset. From this, we defined positive windows when a binary window value is ≥0.8, at which the false discovery rate is <5% (Figure S2 in Additional file 1). We then joined any overlapping (≥1 bp) windows to generate the LMN-1- and EMR-1-associated domains (Table S1 in Additional file 2).
The definition of chromosomal arms and chromosomal centers for comparison of embryonic and adult LMN-1 association profiles (Figure 2D) was based on the boundaries of LADs in adult nematodes. Coordinates of chromosome arms: chromosome I, 1 to 3,745,632 and 10,809,938 to 15,072,421; chromosome II, 1 to 4,708,341 and 11,877,168 to 15,279,323; chromosome III, 1 to 3,508,994 and 9,947,268 to 13,783,681; chromosome IV, 1 to 7,317,812 and 12176625 to 17,493,785; chromosome X, 1 to 4,191,936.
To define 'EMR-1 only' elements, we selected probes with a MA2C score ≥1 in EMR-1 DamID experiments and <0 in LMN-1 DamID experiments. We defined 'EMR-1 only' elements as the regions that contain at least 10 positive probes, each separated ≤500 bp from the neighboring positive probe (Table S1 in Additional file 2). We performed the same procedure to identify 'LMN-1 only' elements (Table S1 in Additional file 2).
RNAi and RNA extraction
To simultaneously deplete EMR-1 and LEM-2, BN19 animals homozygous for the lem-2(tm1582) allele were subjected to emr-1 RNAi-mediated knockdown. RNAi feeding bacteria were grown in LB at 37°C with agitation (180 rpm) until OD600 = 0.8 and then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) during 19 h at 20°C. The RNAi bacteria expressed double-stranded RNA corresponding to either full-length emr-1 cDNA or, as negative control, 185 bp of unrelated sequence from the L4440 empty plasmid. The next day, the bacteria were collected and resuspended in 1/20 initial volume of S-medium with 1 mM IPTG. Approximately 40,000 synchronized L1 nematodes obtained by hypochlorite treatment and hatching overnight at 20°C without food were grown in 50 ml of S-medium containing 5 ml of resuspended RNAi bacteria, 100 μg/ml ampicillin and 1 mM IPTG at 20°C during 53 h with continuous agitation (180 rpm). At this point the cultures accumulated non-gravid young adults. Nematodes were recovered, frozen immediately in liquid N2 and kept at -80°C until further processing. Part of the culture was grown for an additional day, which confirmed the expected embryonic lethal phenotype in the EMR-1 LEM-2 double depletion. RNA extraction was performed using 20 to 30 mg of nematodes. The nematode cuticle was ruptured by three rounds of freezing in liquid N2 and thawing at 30ºC and RNA was purified using the RNeasy Mini kit (QIAGEN).
Expression profiling
Sequencing libraries were prepared using the TruSeq RNA sample preparation kit according to the manufacturer's instructions (Illumina, San Diego, CA, USA). Briefly, polyA-containing mRNA was isolated from 1 μg of total RNA using poly-T oligo-attached magnetic beads and processed as described [70]. Pools of three indexed libraries were mixed (multiplexed) at equimolar ratios to yield a total oligonucleotide mix of 10 nM. Finally, the resulting libraries were sequenced on the Illumina Genome Analyzer IIx platform to generate 150 bp single-end reads. Three pooled indexed libraries were sequenced in each flow cell lane.
Raw RNA sequence data were processed [70] and aligned with the WS231 genome version using TopHat. Next, results were sorted and indexed using Samtools. Coverage vectors for each sample were extracted under R statistical environment using Rsamtools library. The C. elegans GTF annotation from Ensembl was applied as reference for the exon genomic ranges, parsed into R data structures. Exon expression was calculated as the median coverage for every exon genomic range as stated by annotation. Exon expression data from our nine different samples (three N2, two BN19, two BN20, and two BN24) were normalized by quantile normalization and locus expression was defined as the median of all the expression hits from distinct exons annotated to a single locus. Library siggenes in R was applied to select the differentially expressed loci using the false discovery rate-based SAM method [71]. Genes presenting a Δ value >4 were considered to be significantly altered (Table S3 in Additional file 2). Resulting data are available with accession numbers summarized in Table S4 in Additional file 2. For the analysis of emr-1(gk119) and lem-2 (tm1582) mutants, the sets of genes expressed in different tissues were the same as in the DamID analysis (Table S2 in Additional file 2).
Gene Ontology analysis
GO analyses were performed using DAVID [72] and only the 'Biological Processes' tree was used in our study. For the study of DamID 'EMR-1 only' elements, as they were enriched in promoters and regulatory regions, the closest but not overlapping genes were selected. In the other cases, we selected genes with ≥50% of the open reading frame included within the domains.
Aldicarb sensitivity experiments
Sensitivity to aldicarb was determined by assaying the time course of the onset of paralysis following acute exposure of a population of animals to this drug. In each experiment 20 to 25 young adults were placed on freshly made drug plates (1 mM aldicarb; Fluka Sigma-Aldrich ref. 33386) and prodded every 10 minutes over a 2 h period to determine if they retained the ability to move. Nematodes that failed to respond to touch were classified as paralyzed. Each experiment was repeated four times by two independent experimenters.
Data access
DamID and RNA sequencing data performed in this study are available from GEO [73] accession IDs GSE44188 and GSE44682, respectively (described in Table S4 in Additional file 2).
- bp:
base pair
- ChIP:
chromatin immunoprecipitation
- DamID:
DNA adenine methyltransferase identification
EMR-1-associated domain
Emery-Dreifuss muscular dystrophy
embryonic stem cell
- gDNA:
genomic DNA
Gene Expression Omnibus
Gene Ontology
inner nuclear membrane
isopropyl β-D-1-thiogalactopyranoside
LMN-1-associated domain
nuclear envelope
outer nuclear membrane
polymerase chain reaction
- RNAi:
RNA interference.
We are grateful to Bas van Steensel for reagents and advice as well as to Ozren Bogdanovic for bioinformatics assistance. Peter Meister and two anonymous reviewers are acknowledged for insightful comments on the manuscript. This work was funded by the Spanish Ministry of Science and Innovation (BFU-2010-15478 to PA), and the European Regional Development Fund. Some nematode strains used in this work were provided by the National Bioresource Project for the Nematode C. elegans (directed by Shohei Mitani), the International C. elegans Gene Knockout Consortium, and the Caenorhabditis Genetic Center (University of Minnesota, Minneapolis, MN), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
Authors' Affiliations
- Mekhail K, Moazed D: The nuclear envelope in genome organization, expression and stability. Nat Rev Mol Cell Biol. 2010, 11: 317-328. 10.1038/nrm2894.PubMedPubMed CentralView ArticleGoogle Scholar
- Wilson KL, Foisner R: Lamin-binding proteins. Cold Spring Harb Perspect Biol. 2010, 2: a000554-PubMedPubMed CentralView ArticleGoogle Scholar
- Bermejo R, Kumar A, Foiani M: Preserving the genome by regulating chromatin association with the nuclear envelope. Trends Cell Biol. 2012, 22: 465-473. 10.1016/j.tcb.2012.05.007.PubMedView ArticleGoogle Scholar
- Gomez-Cavazos JS, Hetzer MW: Outfits for different occasions: tissue-specific roles of Nuclear Envelope proteins. Curr Opin Cell Biol. 2012, 24: 775-783. 10.1016/j.ceb.2012.08.008.PubMedPubMed CentralView ArticleGoogle Scholar
- Zuela N, Bar DZ, Gruenbaum Y: Lamins in development, tissue maintenance and stress. EMBO Rep. 2012, 13: 1070-1078. 10.1038/embor.2012.167.PubMedPubMed CentralView ArticleGoogle Scholar
- Mendez-Lopez I, Worman HJ: Inner nuclear membrane proteins: impact on human disease. Chromosoma. 2012, 121: 153-167. 10.1007/s00412-012-0360-2.PubMedView ArticleGoogle Scholar
- Bione S, Maestrini E, Rivella S, Mancini M, Regis S, Romeo G, Toniolo D: Identification of a novel X-linked gene responsible for Emery-Dreifuss muscular dystrophy. Nat Genet. 1994, 8: 323-327. 10.1038/ng1294-323.PubMedView ArticleGoogle Scholar
- Ozawa R, Hayashi YK, Ogawa M, Kurokawa R, Matsumoto H, Noguchi S, Nonaka I, Nishino I: Emerin-lacking mice show minimal motor and cardiac dysfunctions with nuclear-associated vacuoles. Am J Pathol. 2006, 168: 907-917. 10.2353/ajpath.2006.050564.PubMedPubMed CentralView ArticleGoogle Scholar
- Shin JY, Mendez-Lopez I, Wang Y, Hays AP, Tanji K, Lefkowitch JH, Schulze PC, Worman HJ, Dauer WT: Lamina-associated polypeptide-1 interacts with the muscular dystrophy protein emerin and is essential for skeletal muscle maintenance. Dev Cell. 2013, 26: 591-603. 10.1016/j.devcel.2013.08.012.PubMedPubMed CentralView ArticleGoogle Scholar
- Broers JL, Peeters EA, Kuijpers HJ, Endert J, Bouten CV, Oomens CW, Baaijens FP, Ramaekers FC: Decreased mechanical stiffness in LMNA-/-cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies. Hum Mol Genet. 2004, 13: 2567-2580. 10.1093/hmg/ddh295.PubMedView ArticleGoogle Scholar
- Lammerding J, Schulze PC, Takahashi T, Kozlov S, Sullivan T, Kamm RD, Stewart CL, Lee RT: Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction. J Clin Invest. 2004, 113: 370-378.PubMedPubMed CentralView ArticleGoogle Scholar
- Lombardi ML, Jaalouk DE, Shanahan CM, Burke B, Roux KJ, Lammerding J: The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton. J Biol Chem. 2011, 286: 26743-26753. 10.1074/jbc.M111.233700.PubMedPubMed CentralView ArticleGoogle Scholar
- Demmerle J, Koch AJ, Holaska JM: The nuclear envelope protein emerin binds directly to histone deacetylase 3 (HDAC3) and activates HDAC3 activity. J Biol Chem. 2012, 287: 22080-22088. 10.1074/jbc.M111.325308.PubMedPubMed CentralView ArticleGoogle Scholar
- Somech R, Shaklai S, Geller O, Amariglio N, Simon AJ, Rechavi G, Gal-Yam EN: The nuclear-envelope protein and transcriptional repressor LAP2beta interacts with HDAC3 at the nuclear periphery, and induces histone H4 deacetylation. J Cell Sci. 2005, 118: 4017-4025. 10.1242/jcs.02521.PubMedView ArticleGoogle Scholar
- Pegoraro G, Kubben N, Wickert U, Gohler H, Hoffmann K, Misteli T: Ageing-related chromatin defects through loss of the NURD complex. Nat Cell Biol. 2009, 11: 1261-1267. 10.1038/ncb1971.PubMedPubMed CentralView ArticleGoogle Scholar
- Fedorova E, Zink D: Nuclear genome organization: common themes and individual patterns. Curr Opin Genet Dev. 2009, 19: 166-171. 10.1016/j.gde.2009.02.003.PubMedView ArticleGoogle Scholar
- Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W, Eussen BH, de Klein A, Wessels L, de Laat W, van Steensel B: Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature. 2008, 453: 948-951. 10.1038/nature06947.PubMedView ArticleGoogle Scholar
- Ikegami K, Egelhofer TA, Strome S, Lieb JD: Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2. Genome Biol. 2010, 11: R120-10.1186/gb-2010-11-12-r120.PubMedPubMed CentralView ArticleGoogle Scholar
- Pickersgill H, Kalverda B, de Wit E, Talhout W, Fornerod M, van Steensel B: Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet. 2006, 38: 1005-1014. 10.1038/ng1852.PubMedView ArticleGoogle Scholar
- Scaffidi P, Misteli T: Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome. Nat Med. 2005, 11: 440-445. 10.1038/nm1204.PubMedPubMed CentralView ArticleGoogle Scholar
- Kandert S, Wehnert M, Muller CR, Buendia B, Dabauvalle MC: Impaired nuclear functions lead to increased senescence and inefficient differentiation in human myoblasts with a dominant p.R545C mutation in the LMNA gene. Eur J Cell Biol. 2009, 88: 593-608. 10.1016/j.ejcb.2009.06.002.PubMedView ArticleGoogle Scholar
- Frock RL, Kudlow BA, Evans AM, Jameson SA, Hauschka SD, Kennedy BK: Lamin A/C and emerin are critical for skeletal muscle satellite cell differentiation. Genes Dev. 2006, 20: 486-500. 10.1101/gad.1364906.PubMedPubMed CentralView ArticleGoogle Scholar
- Markiewicz E, Tilgner K, Barker N, van de Wetering M, Clevers H, Dorobek M, Hausmanowa-Petrusewicz I, Ramaekers FC, Broers JL, Blankesteijn WM, Salpingidou G, Wilson RG, Ellis JA, Hutchison CJ: The inner nuclear membrane protein emerin regulates beta-catenin activity by restricting its accumulation in the nucleus. EMBO J. 2006, 25: 3275-3285. 10.1038/sj.emboj.7601230.PubMedPubMed CentralView ArticleGoogle Scholar
- Dedeic Z, Cetera M, Cohen TV, Holaska JM: Emerin inhibits Lmo7 binding to the Pax3 and MyoD promoters and expression of myoblast proliferation genes. J Cell Sci. 2011, 124: 1691-1702. 10.1242/jcs.080259.PubMedView ArticleGoogle Scholar
- Hellemans J, Preobrazhenska O, Willaert A, Debeer P, Verdonk PC, Costa T, Janssens K, Menten B, Van Roy N, Vermeulen SJ, Savarirayan R, Van Hul W, Vanhoenacker F, Huylebroeck D, De Paepe A, Naeyaert JM, Vandesompele J, Speleman F, Verschueren K, Coucke PJ, Mortier GR: Loss-of-function mutations in LEMD3 result in osteopoikilosis, Buschke-Ollendorff syndrome and melorheostosis. Nat Genet. 2004, 36: 1213-1218. 10.1038/ng1453.PubMedView ArticleGoogle Scholar
- Lin F, Morrison JM, Wu W, Worman HJ: MAN1, an integral protein of the inner nuclear membrane, binds Smad2 and Smad3 and antagonizes transforming growth factor-beta signaling. Hum Mol Genet. 2005, 14: 437-445.PubMedView ArticleGoogle Scholar
- Pan D, Estevez-Salmeron LD, Stroschein SL, Zhu X, He J, Zhou S, Luo K: The integral inner nuclear membrane protein MAN1 physically interacts with the R-Smad proteins to repress signaling by the transforming growth factor-beta superfamily of cytokines. J Biol Chem. 2005, 280: 15992-16001. 10.1074/jbc.M411234200.PubMedView ArticleGoogle Scholar
- Gonzalez-Aguilera C, Palladino F, Askjaer P: C. elegans epigenetic regulation in development and aging. Brief Funct Genomics. 2013, doi:10.1093/bfgp/elt048Google Scholar
- Liu J, Lee KK, Segura-Totten M, Neufeld E, Wilson KL, Gruenbaum Y: MAN1 and emerin have overlapping function(s) essential for chromosome segregation and cell division in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2003, 100: 4598-4603. 10.1073/pnas.0730821100.PubMedPubMed CentralView ArticleGoogle Scholar
- Barkan R, Zahand AJ, Sharabi K, Lamm AT, Feinstein N, Haithcock E, Wilson KL, Liu J, Gruenbaum Y: Ce-emerin and LEM-2: essential roles in Caenorhabditis elegans development, muscle function, and mitosis. Mol Biol Cell. 2012, 23: 543-552. 10.1091/mbc.E11-06-0505.PubMedPubMed CentralView ArticleGoogle Scholar
- Huber MD, Guan T, Gerace L: Overlapping functions of nuclear envelope proteins NET25 (Lem2) and emerin in regulation of extracellular signal-regulated kinase signaling in myoblast differentiation. Mol Cell Biol. 2009, 29: 5718-5728. 10.1128/MCB.00270-09.PubMedPubMed CentralView ArticleGoogle Scholar
- Askjaer P, Ercan S, Meister P: Modern techniques for the analysis of chromatin and nuclear organization in C. elegans. WormBook. in press [http://www.wormbook.org/toc_wormmethods.html]
- Song JS, Johnson WE, Zhu X, Zhang X, Li W, Manrai AK, Liu JS, Chen R, Liu XS: Model-based analysis of two-color arrays (MA2C). Genome Biol. 2007, 8: R178-10.1186/gb-2007-8-8-r178.PubMedPubMed CentralView ArticleGoogle Scholar
- Towbin BD, Gonzalez-Aguilera C, Sack R, Gaidatzis D, Kalck V, Meister P, Askjaer P, Gasser SM: Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery. Cell. 2012, 150: 934-947. 10.1016/j.cell.2012.06.051.PubMedView ArticleGoogle Scholar
- Gerstein MB, Lu ZJ, Van Nostrand EL, Cheng C, Arshinoff BI, Liu T, Yip KY, Robilotto R, Rechtsteiner A, Ikegami K, Alves P, Chateigner A, Perry M, Morris M, Auerbach RK, Feng X, Leng J, Vielle A, Niu W, Rhrissorrakrai K, Agarwal A, Alexander RP, Barber G, Brdlik CM, Brennan J, Brouillet JJ, Carr A, Cheung MS, Clawson H, Contrino S, et al: Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project. Science. 2010, 330: 1775-1787. 10.1126/science.1196914.PubMedPubMed CentralView ArticleGoogle Scholar
- Gruenbaum Y, Lee KK, Liu J, Cohen M, Wilson KL: The expression, lamin-dependent localization and RNAi depletion phenotype for emerin in C. elegans. J Cell Sci. 2002, 115: 923-929.PubMedGoogle Scholar
- Cohen M, Tzur YB, Neufeld E, Feinstein N, Delannoy MR, Wilson KL, Gruenbaum Y: Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos. J Struct Biol. 2002, 140: 232-240. 10.1016/S1047-8477(02)00516-6.PubMedView ArticleGoogle Scholar
- Askjaer P, Galy V, Hannak E, Mattaj IW: Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos. Mol Biol Cell. 2002, 13: 4355-4370. 10.1091/mbc.E02-06-0346.PubMedPubMed CentralView ArticleGoogle Scholar
- Holaska JM, Wilson KL: An emerin 'proteome': purification of distinct emerin-containing complexes from HeLa cells suggests molecular basis for diverse roles including gene regulation, mRNA splicing, signaling, mechanosensing, and nuclear architecture. Biochemistry. 2007, 46: 8897-8908. 10.1021/bi602636m.PubMedPubMed CentralView ArticleGoogle Scholar
- Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SW, Solovei I, Brugman W, Graf S, Flicek P, Kerkhoven RM, van Lohuizen M, Reinders M, Wessels L, van Steensel B: Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation. Mol Cell. 2010, 38: 603-613. 10.1016/j.molcel.2010.03.016.PubMedView ArticleGoogle Scholar
- Von Stetina SE, Watson JD, Fox RM, Olszewski KL, Spencer WC, Roy PJ, Miller DM: Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C. elegans nervous system. Genome Biol. 2007, 8: R135-10.1186/gb-2007-8-7-r135.PubMedPubMed CentralView ArticleGoogle Scholar
- Wenzel D, Palladino F, Jedrusik-Bode M: Epigenetics in C. elegans: facts and challenges. Genesis. 2011, 49: 647-661. 10.1002/dvg.20762.PubMedView ArticleGoogle Scholar
- Melcon G, Kozlov S, Cutler DA, Sullivan T, Hernandez L, Zhao P, Mitchell S, Nader G, Bakay M, Rottman JN, Hoffman EP, Stewart CL: Loss of emerin at the nuclear envelope disrupts the Rb1/E2F and MyoD pathways during muscle regeneration. Hum Mol Genet. 2006, 15: 637-651. 10.1093/hmg/ddi479.PubMedView ArticleGoogle Scholar
- Mahoney TR, Luo S, Nonet ML: Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay. Nat Protoc. 2006, 1: 1772-1777. 10.1038/nprot.2006.281.PubMedView ArticleGoogle Scholar
- Tifft KE, Bradbury KA, Wilson KL: Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases. J Cell Sci. 2009, 122: 3780-3790. 10.1242/jcs.048397.PubMedPubMed CentralView ArticleGoogle Scholar
- Hirano Y, Segawa M, Ouchi FS, Yamakawa Y, Furukawa K, Takeyasu K, Horigome T: Dissociation of emerin from barrier-to-autointegration factor is regulated through mitotic phosphorylation of emerin in a xenopus egg cell-free system. J Biol Chem. 2005, 280: 39925-39933. 10.1074/jbc.M503214200.PubMedView ArticleGoogle Scholar
- Ercan S, Dick LL, Lieb JD: The C. elegans dosage compensation complex propagates dynamically and independently of X chromosome sequence. Curr Biol. 2009, 19: 1777-1787. 10.1016/j.cub.2009.09.047.PubMedPubMed CentralView ArticleGoogle Scholar
- Lee KK, Haraguchi T, Lee RS, Koujin T, Hiraoka Y, Wilson KL: Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF. J Cell Sci. 2001, 114: 4567-4573.PubMedGoogle Scholar
- Bengtsson L: What MAN1 does to the Smads. TGFbeta/BMP signaling and the nuclear envelope. FEBS J. 2007, 274: 1374-1382. 10.1111/j.1742-4658.2007.05696.x.PubMedView ArticleGoogle Scholar
- Mattout A, Pike BL, Towbin BD, Bank EM, Gonzalez-Sandoval A, Stadler MB, Meister P, Gruenbaum Y, Gasser SM: An EDMD mutation in C. elegans lamin blocks muscle-specific gene relocation and compromises muscle integrity. Curr Biol. 2011, 21: 1603-1614. 10.1016/j.cub.2011.08.030.PubMedView ArticleGoogle Scholar
- Kim Y, Sharov AA, McDole K, Cheng M, Hao H, Fan CM, Gaiano N, Ko MS, Zheng Y: Mouse B-type lamins are required for proper organogenesis but not by embryonic stem cells. Science. 2011, 334: 1706-1710. 10.1126/science.1211222.PubMedPubMed CentralView ArticleGoogle Scholar
- Lund E, Oldenburg AR, Delbarre E, Freberg CT, Duband-Goulet I, Eskeland R, Buendia B, Collas P: Lamin A/C-promoter interactions specify chromatin state-dependent transcription outcomes. Genome Res. 2013, 23: 1580-1589. 10.1101/gr.159400.113.PubMedPubMed CentralView ArticleGoogle Scholar
- Solovei I, Wang AS, Thanisch K, Schmidt CS, Krebs S, Zwerger M, Cohen TV, Devys D, Foisner R, Peichl L, Herrmann H, Blum H, Engelkamp D, Stewart CL, Leonhardt H, Joffe B: LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation. Cell. 2013, 152: 584-598. 10.1016/j.cell.2013.01.009.PubMedView ArticleGoogle Scholar
- Mejat A, Decostre V, Li J, Renou L, Kesari A, Hantai D, Stewart CL, Xiao X, Hoffman E, Bonne G, Misteli T: Lamin A/C-mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy. J Cell Biol. 2009, 184: 31-44. 10.1083/jcb.200811035.PubMedPubMed CentralView ArticleGoogle Scholar
- Coffinier C, Chang SY, Nobumori C, Tu Y, Farber EA, Toth JI, Fong LG, Young SG: Abnormal development of the cerebral cortex and cerebellum in the setting of lamin B2 deficiency. Proc Natl Acad Sci U S A. 2010, 107: 5076-5081. 10.1073/pnas.0908790107.PubMedPubMed CentralView ArticleGoogle Scholar
- Mamada H, Takahashi N, Taira M: Involvement of an inner nuclear membrane protein, Nemp1, in Xenopus neural development through an interaction with the chromatin protein BAF. Dev Biol. 2009, 327: 497-507. 10.1016/j.ydbio.2008.12.038.PubMedView ArticleGoogle Scholar
- Wang X, Xu S, Rivolta C, Li LY, Peng GH, Swain PK, Sung CH, Swaroop A, Berson EL, Dryja TP, Chen S: Barrier to autointegration factor interacts with the cone-rod homeobox and represses its transactivation function. J Biol Chem. 2002, 277: 43288-43300. 10.1074/jbc.M207952200.PubMedView ArticleGoogle Scholar
- Gros-Louis F, Dupre N, Dion P, Fox MA, Laurent S, Verreault S, Sanes JR, Bouchard JP, Rouleau GA: Mutations in SYNE1 lead to a newly discovered form of autosomal recessive cerebellar ataxia. Nat Genet. 2007, 39: 80-85. 10.1038/ng1927.PubMedView ArticleGoogle Scholar
- Meister P, Towbin BD, Pike BL, Ponti A, Gasser SM: The spatial dynamics of tissue-specific promoters during C. elegans development. Genes Dev. 2010, 24: 766-782. 10.1101/gad.559610.PubMedPubMed CentralView ArticleGoogle Scholar
- Frøkjær-Jensen C, Davis MW, Hopkins CE, Newman BJ, Thummel JM, Olesen SP, Grunnet M, Jorgensen EM: Single-copy insertion of transgenes in Caenorhabditis elegans. Nat Genet. 2008, 40: 1375-1383. 10.1038/ng.248.PubMedPubMed CentralView ArticleGoogle Scholar
- Rodenas E, Gonzalez-Aguilera C, Ayuso C, Askjaer P: Dissection of the NUP107 nuclear pore subcomplex reveals a novel interaction with spindle assembly checkpoint protein MAD1 in Caenorhabditis elegans. Mol Biol Cell. 2012, 23: 930-944. 10.1091/mbc.E11-11-0927.PubMedPubMed CentralView ArticleGoogle Scholar
- Greil F, Moorman C, van Steensel B: DamID: mapping of in vivo protein-genome interactions using tethered DNA adenine methyltransferase. Methods Enzymol. 2006, 410: 342-359.PubMedView ArticleGoogle Scholar
- UCSC Genome Browser. http://genome.ucsc.edu/,
- Shin H, Liu T, Manrai AK, Liu XS: CEAS: cis-regulatory element annotation system. Bioinformatics. 2009, 25: 2605-2606. 10.1093/bioinformatics/btp479.PubMedView ArticleGoogle Scholar
- modENCODE. http://www.modencode.org,
- Steiner FA, Talbert PB, Kasinathan S, Deal RB, Henikoff S: Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. Genome Res. 2012, 22: 766-777. 10.1101/gr.131748.111.PubMedPubMed CentralView ArticleGoogle Scholar
- Haenni S, Ji Z, Hoque M, Rust N, Sharpe H, Eberhard R, Browne C, Hengartner MO, Mellor J, Tian B, Furger A: Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3′-end-seq. Nucleic Acids Res. 2012, 40: 6304-6318. 10.1093/nar/gks282.PubMedPubMed CentralView ArticleGoogle Scholar
- Gassmann R, Rechtsteiner A, Yuen KW, Muroyama A, Egelhofer T, Gaydos L, Barron F, Maddox P, Essex A, Monen J, Ercan S, Lieb JD, Oegema K, Strome S, Desai A: An inverse relationship to germline transcription defines centromeric chromatin in C. elegans. Nature. 2012, 484: 534-537. 10.1038/nature10973.PubMedPubMed CentralView ArticleGoogle Scholar
- Ruvinsky I, Ohler U, Burge CB, Ruvkun G: Detection of broadly expressed neuronal genes in C. elegans. Dev Biol. 2007, 302: 617-626. 10.1016/j.ydbio.2006.09.014.PubMedView ArticleGoogle Scholar
- Larrayoz IM, de Luis A, Rua O, Velilla S, Cabello J, Martinez A: Molecular effects of doxycycline treatment on pterygium as revealed by massive transcriptome sequencing. PLoS One. 2012, 7: e39359-10.1371/journal.pone.0039359.PubMedPubMed CentralView ArticleGoogle Scholar
- Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A. 2001, 98: 5116-5121. 10.1073/pnas.091062498.PubMedPubMed CentralView ArticleGoogle Scholar
- da Huang W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009, 4: 44-57.PubMedView ArticleGoogle Scholar
- Gene Expression Omnibus. http://www.ncbi.nlm.nih.gov/geo/,
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


text: This was the second time I visited this place (the last time was a year back). My memories from the previous visit were pretty good and I was expecting equally great tasting food this time around. While the food was stiil good and the lunch buffet spread had quite many items, somehow the food tasted a bit bland this time.... More

subset: commoncrawl


text:
Top Claimed Casino Bonuses
What Bonuses are Others Claiming?
Do you ever need help picking the right bonus? With such a huge selection offered on Latest Casino Bonuses, this may often be a problem.
To avoid any confusion and make your choice easier, we have provided for you a special list of bonuses most claimed by our visitors, so far.
How does it all work?
It's rather simple, actually. This is a user-friendly list, which allows LCB's visitors to keep track of all the bonuses they've claimed
You can also keep track of the bonuses you have claimed by letting us know each time you click 'CLAIM' weather you did in fact claim the bonus or not by answering the question that pops up.
In case you want to address our forum in regards to a specific casino bonus, you can do so by clicking the grey icon (i). All other info is right here on this page: the casino name, weather the bonus is an exclusive, amount, wagering requirements and rating.
But if you want to find out more, there's More+ button, which will reveal more details about the bonus, as well as a link to the casino review.
With all the info on this page, you can just stay around and choose the bonus you like best. And enjoy the play, of course!
Exclusive Bonuses
257 Bonuses Found
- Sort:
We are only showing bonuses that accept players from United States.
lcb activities in the last 24 hours
- 33
- new members
- 732
- members online
- 27029
- guests online
- 131
- new posts
- 6198
- free games played


text:
Kids & Family, Religion & Spirituality, ChristianityFollow
|Kids & Family
Need help with raising your kids? Focus on the Family provides tried and true parenting advice to help your children thrive.
March 19th, 2015
Episode 104 of 308 episodes
Dr. Tony Evans shares insights from his own life and book on launching your kids.
You'll need to sign in to write a comment.
Welcome to the daily audio workout for your head.


text:
State Highlights Buttons
This page is archived for historical purposes and is no longer being updated.
Disclaimer: Data and findings provided reflect the content of this particular Tobacco Control State Highlights. Check the STATE System State Highlights reports for more recent information.
- Page last reviewed: February 5, 2016 (archived document)
- Content source:


text:
Algerian novelist Said Khatibi's Kitab al-khataya is not the story of Algeria's black decade, or of Islamist fighters, or of star-crossed love. Instead, it's a book that begins on a crowded bus with a tampon emergency.
Algerian Journalist Jailed for Facebook Poem and Posts Reportedly in a Coma
"Tamalt was taken to intensive care following a hunger strike that lasted more than sixty days. "
Homage to Sadek Aïssat: Music, Dreams and the Cleansing Effect of Urine
"Algerian fiction is better known, in Algeria and beyond, for its narratives of tragedies directly related to war, through which seeps the undiluted memory of ruthless violence. Underground, however, a magnificent layer lies, one that is deeply connected to an ancestral oral literary tradition, to its animals, its magic realism and delicious wit."
An Interview with the Anonymous Author of 'Through Algeria'
In the tradition of Assia Djebar's Fantasia, Algerian author-scholar Nadia Ghanem has a discussion with the anonymous unaccompanied Englishwoman who wrote Through Algeria.
'Lands in Solidarity' and 'Resonances': Producing African Literatures in Algeria
"Our interest for African literature does not boil down to buying book rights so we can reedit them in Algeria. We are trying to contribute to the autonomy of the African literary field."
6 Algerian Women Writers Whose Work Should Be Translated into English
"She is one of the strongest women's voices of Algerian literature on matters of women's rights and who openly discussed issues of rape and violence against of women."
'The Dictator's Last Night': When fact is confused with fiction
"[Y]ou cannot be neutral towards this novel."
'The Bride of Reeds' and Tamazight Literature's from Oral to Written
"[I]n order for Tamazight culture to move forward, we must produce. I hope that our culture will finally take its rightful place in a universal context."
Why Algerian Author Zehira Houfani Quit Writing Detective Novels
"State publishing, as with every other state sector, entered a serious crisis."
A Poem to Mark Tamazight's New Status As an Official Algerian Language
"alas you the clerics who chant
you who are well considered
can't you see that our religion is up for sale"
'How I Smoked All My Books': An Investigation of Smoking, and Fiction, in Algeria
While sex, drugs and pizzas have been stable story elements in Algerian novels over the past decade (for pizzas, read Chawki Amari), a prominent place given to smoking seems relatively new to me in our fiction as far as my… Read More ›
2015 in Algerian Literature: Five to Watch
"To illustrate this, here is a sample of titles released in 2015 from authors we should keep an eye on."


text:
Regional Europe Spain Travel and Tourism Lodging Vacation Rentals
Vacation Rental covers acc. dor dmoz's definition: Generally self-service lodging facilities which: Contain full kitchen facilities, May be rented by the day, week, or month, and Encompass condominiums, cabins, chalets, beach cottages, lodges, and homes. This classification accepts listing for Spain only and only in the English language Esta categoría es para sitios en inglés. Por favor, solicite incluir su sitio en la categoría apropiada en /World/Español. Gracias.
Subcategories 6
Related categories 1
Sites 15
Holiday apartments for rent directly from the owners in Madrid, Barcelona and Malaga.
Offers a selection of private villas in Melorca and Malaga. Includes photos and descriptions.
Casa Mila
4 bedroom vacation villa with pool and views over Javea. Includes description of villa and features.
España Breaks
Villas and apartments for holiday rental throughout mainland Spain, the Canaries and Balearic Islands.
Internet-Cottages Spain
Directory of privately owned villas in Spain, bookable direct with the property owners. Full details, pictures and prices.
Accommodation in villas and apartment to rent in whole Spain. Most listings with photos, price schedules and in Dutch, German and English, some also in French and Spanish.
Offers a holiday villa rental search engine including self-catered villas, houses and apartments to rent.
Rustical Travel
Offers self catering villas in natural settings across rural Spain. Catalogue with descriptions, photos and regional guides.
Solmar Villas
Private villas for rent in mainland Spain, the Canary Islands and the balearic Islands.
Spain Holiday
Offers vacation rentals and accommodations in touristic regions. Apartments, villas and townhouses booked directly with the property owners.
Spain Rentals Direct
Features privately owned villas throughout the country. Photos and descriptions are provided.
Spanish Web
Self catering holiday accommodation listed by the owners together with regional and flight information.
Villa Holidays.org
Three-bedroom villa in L'Escala, Costa Brava, and two bedroom self-catering unit at Villa Lagarto, Costa Blanca. Both have private pools. Includes details, photos, rates, facilities, directions and a location map.
Villa Rental Spain
Find details of villas, apartments, and cortijos for rent direct from the owners.
Villas Spain Rentals
Privately owned property to rent or buy.
Other languages 2
Last update:January 11, 2017 at 8:00:44 UTC
All Languages


text:
read more +
Publisher's Description+
From HU WEN ZENG: #1 Free Game in more than 40 countries#10 Free Game in more than 100 countriesWatch your step, DON'T TOUCH ON ANY WHITE TILES! This is the core rule of this addictive game. Sounds easy? Give it a try, and see how well you stack up against your friends.Features: Charming piano soundtracks Customizable playlist More than 35 awesome game modes Muti-color themes & dozens of color options Smooth gameplay Optimised for high resolution screen Social network sharing We'd love to hear your suggestions and comments! We've got loads of improvements and new features planned, so stay tuned!LIKE PIANO TILES ON FACEBOOK!http://www.facebook.com/PianoTilesFansFOLLOW US ON TWITTER!http://www.twitter.com/PianoTiles
What's new in this version:
Fix Some Bugs
read more +


text:
June 3, 2015 8:20 pm
Download audio
Cybersecurity experts are required to have certain credentials. So how do you acquire them?
ASIS International is one such place as the largest organization for security management professionals worldwide.
Now ASIS's Professional Certification Board has a new president — Christina Duffey. Duffey is also the director for client services for Paragon Protection in Toronto. She joined the DorobekINSIDER to describe professional certification board and the certification process.
This story is part of Federal News Radio's daily Cybersecurity Update brought to you by Tripwire. For more cybersecurity news, click here.
Hiring freeze result of Trump team's review of federal agencies
Hiring freeze result of Trump team's review of federal agencies
Photos of the amazing, moving, important and amusing things happening in the federal community.
A daily update of important moments in the history of the U.S. government.


text:
Contact Us
Credit Reports Guide
Are credit reports really free?
June 15, 2014
No, they are initially free if you go direct, but when you get it from a 3rd party provider, they give you the FREE CREDIT REPORT that you can normally get, but for a fee they charge so that they…Read more →
Why check for my CreditScore?
June 14, 2014
CreditScore web sites like CreditScore.org can help you by providing you with emphasis on checking you credit.Read more →
More on Credit Reports
June 14, 2014
A credit report contains detailed information about your credit history – and bill repayment history – and the status of your credit accounts. This information includes how you often make your payments on time, how much credit you have, how…Read more →
iPhones.com news:
iPhones creating Social Network & Cloud Service in Joint Venture
February 23, 2017
The owners of iPhones/iPhones.com have made an internal announcement engaging with a Social Networking firm to create the next Social Network for all users of iPhones.com. The plan would be to provide Cloud Services, Real Time Streaming, Development of Applications, and Utilities to take some of the hidden features to the next level by empowering […]
February 20, 2017
The diseased knows their time is short.
February 20, 2017
Injured Tiger will attack out of nowhere.
February 20, 2017
The media is accused of selling Fake News for years and years. Do you continue to buy once known?
February 20, 2017
Who will drain the swamp?
February 20, 2017
Who will expose the corruption?
February 20, 2017
Is your nation in a path of destruction?
February 20, 2017
Daniel Scheinman of Cisco Systems – Where did he go after Cisco?
February 11, 2017
Boston Dynamics
February 6, 2017
GasPrices.org NEWS:
Which Cars actually give you better output for your tank?
April 7, 2016
Gas Prices are plan on going down to $2.00/gallon due to Fracking Huge Supplies of Unlimited
April 7, 2016
Gas Prices stay low due to over abundant supply. FRACKING!!!
January 25, 2015
Gas Prices are under to $2.99 for SUPER UNLEADED, right under the mark
December 18, 2014
It is now amazing how the U.S. Dollar is stronger, what the heck, this is amazing.
Can Gas Prices hold the price at around $4/gallon?
September 25, 2014
It really depends on what the global demand is and how crude oil is trading in the overall market place. The understanding is that Gas is unlimited since crude oil is merely a continued regeneration of stick sweet crude that is processed and sucked up to sell to consumers, after going through a processing plant, […]
Gas Prices are going up to $5/gallon thank you Iraq?
June 13, 2014
Are gas prices planning on increasing overnight in the beginning of year 2015? The answer for some states is yes. Particularly, California will be increasing as a tax, which will be an immediate jump overnight. So, keep on the look out if your state is doing it too, chances are, only the high volume states […]
Are Gas Prices really affecting the economy?
March 9, 2014
Or is it a matter of getting used to it? Who really knows!?!?!
Gas Prices debate with Ron Paul
January 11, 2013
What will it take?
July 30, 2012
July 18, 2012
Please Submit your Question or Comments below:
SocialNetworking news:
Lego Life claimed to be a Safe Social Networking for kiddos.
February 17, 2017
FaceBook videos autoplay with sound
February 17, 2017
Twitter rolls back anti-abuse tool
February 17, 2017
Smoking iQuit:
Quit Smoking Cigarettes by throwing them all away
March 26, 2014
Keep no cigarette packs in your sight. Make sure all ash trays, lighters, cigarette packs, or any items related to smoking must be disposed of so there's no sudden reminder that you can easily go back to smoking. There's a guilt situation removed from getting these smoking items out of the way.
Do you know what you can do instead of smoking a cigarette?
March 26, 2014
You can drink coffee or drink a tall glass of water. Coffee is more organic and natural than a processed habit. Water is a great alternative. Doing some walking around is a good idea too.
People knew that e-cigarettes did not help stop smoking. In fact, it encouraged.
March 26, 2014
Smoking using e-cigarettes was a misguiding situation for smokers who were benefitting by taking heavier dosages of nicotine. Since the concentrated mount of nicotine was being inhaled, it was giving more than a buzz, but more of a feeling that gave the smoker a better sensation than the cigarette itself, but took advantage of the […]
March 26, 2014
Breath bettwr, Smell Better and don't feel like lungs are weird.
Ukraine Crisis As It Happened
Is the ukraine crisis over yet???
August 15, 2014
UKRAINE WAR discussions
March 21, 2014
What is CRIMEA?
March 20, 2014
a Ukrainian peninsula between the Black Sea and the Sea of Azov.
A Ukrainian air force officer closes the gate at the Belbek airbase, outside Sevastopol in Crimea
March 20, 2014
Air Pollution in Chinas
Chapter 11 Bankruptcy
Amazon news:
Games iPhones
Briv.com Parent Frip, launches Racex.com
March 1, 2014
This site is all about Racing Flash Games.
Games iPhones
New Games web site Briv.com launches aside from Frip
March 1, 2014
Frip's parent company launches Briv.com, a new flash game web site aimed to provide games for the young and adult generations. As Parent Company Frip diversifies into newer brand sites like Briv.com, Kizi and others, their aim is to scale their flash games delivery to as many players as possible. Keep an eye for their […]
Games iPhones
Software Developers News
Forbes Most Popular Stories
The Nintendo Switch Is A Technological Work Of Art
February 23, 2017
The Nintendo Switch is sleek, impeccably designed video game console that's truly next-gen.
Erik Kain, Contributor
Why I Decided To License Starship's Music To Chick-fil-A
February 23, 2017
"I believe that men should be able to marry men, and women women. I am passionately against anyone who would try to suppress this basic human right. My first thought when Chik-fil-A came to me was, 'F**k no!' But then I decided, 'F**k yes.'"
Forbes Showbiz, Contributor
California: The Physical Collapse Of A Social State
February 23, 2017
Under the one-party Democrat rules, spending on fairness tops $100 billion every year. Meanwhile, the basic infrastructure of the state, so necessary for the economy long and short term, is collapsing.
Thomas Del Beccaro, Contributor
Intuition Is The Highest Form Of Intelligence
February 23, 2017
Albert Einstein said, "The intuitive mind is a sacred gift and the rational mind is a faithful servant. We have created a society that honors the servant and has forgotten the gift."
Bruce Kasanoff, Contributor
Goldberg Vs. Brock Lesnar: Who Has The Edge In WWE Raw Ratings?
February 23, 2017
As Brock Lesnar and Goldberg prepare for a WWE WrestleMania showdown, who has been the bigger ratings draw on Raw?
Alfred Konuwa, Contributor
Re-cap of FaceBook Acquisitions:
Instagram - acquired $1B Waze - acquired $1.1B Attempt for SnapChat (failed) WhatsApp - acquired $16B (google may have offered $10B)
Construction News:
New Homes News:
Buy/Sell Your Website Business:
Interested in buying or selling a web site business.
Contact us
to discuss your proposal.
Content Writers?
If you are a content writer, contact Developers.org to experience a new direction in being a part of a new publishing network.
Solar Power News:
iPhones.com - Smart Phone Guide
Sales / Marketing
Contact E-mail - firstname.lastname@example.com
Your browser does not support the video tag.
Android Developers
If you're an Android Developer, you've come to the right place. Find out how you can join our team. firstname.lastname@example.com
Entrepreneur in the NEWS:
With more and more unnecessary services and products being offered for people to choose, iToxic can be created where people's minds and spirits are pre-occupied with unnecessary toxic clutter. iToxic is a problem in today's society, as the chain reaction of problems can occur, resulting in the necessity of detoxification. Detoxifying the toxicity will result in a more holistic and calmer lifestyle that will eliminate the need and desire to always be affected by the latest and greatest things that appear to be helping people, but in reality, hurting.
Cancel The Account
Why you should "cancel the account" you have that you aren't really using, and especially if the service provider charges you some penalty for termination. You should think twice before signing up since the service provider's methodology is to trap you so you think and delay the process to CANCEL THE ACCOUNT, and while you delay, you just continue to pay and pay and pay away...
Developers Categories
FaceBook Developers
Google Developers
Android Developers
Software Developers
Apple Developers
eBay Developers
Mozilla Developers
NVIDIA Developers
SlashDot Developers
BlackBerry Developers
Twitter Developers
FreeBSD Developers
Intel Developers
FourSquare Developers
Debian Developers
Kongregate Developers
Viddler Developers
Yahoo Developers
Netflix Developers
MassDOT Developers
Janrain Developers
Yammer Developers
Samsung Developers
Recent Posts
Developers Making Progress?
The purpose of creating offspring to keep your legacy alive and knowing how to do it effectively where all the ingedients and characteristics are aligned and right for the hybrid development. Otherwise what is the point? Two wrongs don't make a right.
Sean Penn Interviews El Chapo, the Robinhood of Mexico
Fishy Stories in the media that don't make sense.
More news regarding everything… – What's the news that is relevant to change in the world?
Recent Comments
April 2016
February 2016
January 2016
December 2015
October 2015
September 2015
July 2015
June 2015
May 2015
April 2015
March 2015
February 2015
January 2015
November 2014
October 2014
September 2014
August 2014
July 2014
June 2014
May 2014
April 2014
March 2014
February 2014
January 2014
December 2013
November 2013
October 2013
August 2013
July 2013
June 2013
May 2013
April 2013
March 2013
Developers Making Progress?
No comments
The purpose of creating offspring to keep your legacy alive and knowing how to do it effectively where all the ingedients and characteristics are aligned and right for the hybrid development. Otherwise what is the point? Two wrongs don't make a right.
No comments
Sean Penn Interviews El Chapo, the Robinhood of Mexico
No comments
Fishy Stories in the media that don't make sense.
No comments
« Older Entries
Insurance Dot News:
November 30, 2014
October 21, 2014
Auto Insurance
August 31, 2014
Did you know that every 6 months, you are able to obtain a new quote for your auto insurance premium, which can be adjusted based on the going trends and rate, your own driving history, and such. Premiums change, and if you don't attempt to get a new quote every 6 months, then you miss […]
Vladimir Putin:
Credit Score NEWS
You need to FREEZE your Credit from being checked without your approval – EMERGENCY MEETING TAKING PLACE RIGHT NOW
September 22, 2016
Credit Score
Negative Reports weighing down Credit Score's fullest potential?
September 22, 2016
Credit Score
We need to stop our credit from being checked due to international and domestic criminals.
May 22, 2016
Credit Score
Freeze your Credit Score so it can't be checked.
May 22, 2016
Credit Score
Is the perceived value of a Credit Score being higher than lower really not what it's given the impression to be?
May 11, 2016
In today's world, if you have money, then that's enough to make your purchase. The only time your credit score really matters is when you're going to take a loan from a bank. You don't need to worry about your credit score if you're paying all cash for a purchase. So, in most situations, when […]
Credit Score
Credit Score is failing the people that rely on it?
April 24, 2016
Ask yourself this question.
Credit Score
Credit Score – Is it really helping you or is it just an illusion to make you think it really helps.
March 29, 2016
Perhaps a lower credit score may be in your favor to get you a lower payment.
Credit Score
Can using Cash instead of Credit Card be safer for my identity protection?
December 13, 2015
Whenever you use your credit card, you are basically requesting to borrow money instantly in exchange for disclosing your location, types of purchases you make and promise that you will pay the bill when received in your monthly credit card statement. If you are like Steve Jobs who changes his car every 6 months, then […]
Credit Score
Cleaning Bad Credit is not difficult
June 8, 2015
The ability to clean bad credit is simple and straight forward. It's not rocket science. It's so simple that you will be smiling when you realize how easy it is to correct those mistakes.
Credit Score
My 3Score report is far apart from each of the 3 credit bureau scores – What is the problem?
June 8, 2015
It is highly likely that one or more bureaus have errors or missing information which is providing you a different score than the others, and so what you want to do is check to make sure your report is clear of errors and you may want to submit evidence to the lower score credit bureau […]
Credit Score
Disputes & Conflicts:
Disputes & conflicts We do not control the exact ads served by Google advertisers on the PPC (paid-per-click) ad landing pages. So if you have noticed that some ads served on one of our domains are in conflict with internet advertising laws then please contact us indicating the domain name in question and the nature of the ads you deem are in conflict so we could manually review and take action to correct the situation. Domain owner maintains no relationship with third party advertisers. Reference to any specific service or trademark is not controlled by domain owner and does not constitute or imply its association, endorsement or recommendation. We are doing our best to ensure the use of domains under management is in compliance with domain laws and take the good faith of business conduct seriously. thank you!
iPhones News
iPhones creating Social Network & Cloud Service in Joint Venture
February 23, 2017
The owners of iPhones/iPhones.com have made an internal announcement engaging with a Social Networking firm to create the next Social Network for all users of iPhones.com. The plan would be to provide Cloud Services, Real Time Streaming, Development of Applications, and Utilities to take some of the hidden features to the next level by empowering […]
February 20, 2017
The diseased knows their time is short.
February 20, 2017
Injured Tiger will attack out of nowhere.
February 20, 2017
The media is accused of selling Fake News for years and years. Do you continue to buy once known?
February 20, 2017
Who will drain the swamp?
February 20, 2017
Who will expose the corruption?
February 20, 2017
Is your nation in a path of destruction?
February 20, 2017
Daniel Scheinman of Cisco Systems – Where did he go after Cisco?
February 11, 2017
Boston Dynamics
February 6, 2017
The Inquirer/Submitter claims no legal right to the domain it's making an offer on or any domain that is forwarding to the page that is displayed on this web site and/or url.


text:
GymkidzSports Gymnastics Centres
95 Lennox Street, Newtown, NSW 2042 2-5 km from Ashbury
Best match results for: CATEGORY = Gymnastic Activities for Kids + LOCATION = Ashbury (2193)
95 Lennox Street, Newtown, NSW 2042 2-5 km from Ashbury
18/102-112 Edinburgh Road, Marrickville, NSW 2204 2-5 km from Ashbury
Boundary St, Croydon, NSW 2132 2-5 km from Ashbury
1 Knox St, Belmore, NSW 2192 2-5 km from Ashbury
Cnr Vaucluse and New South Head Rd, Dulwich Hill, NSW 2203
Cnr Queens Rd and William St, Five Dock, NSW 2046 2-5 km from Ashbury
Results include related kids activities and neighbouring locations.
Gymnastic activities for kids - greater than 5km from Ashbury (2193)
215 Forbes St., Darlinghurst, NSW 2010 5-10 km from Ashbury
Club PERÓ has given my daughter loads of confidence and motivation. The teachers are all hand-picked and unique in what they do, they are passionate and love the kids as if they were their own.
Callan Park, Balmain Rd, Rozelle, NSW 5-10 km from Ashbury
This business can travel to you.
80 Waterloo Road, Greenacre, NSW 2190 5-10 km from Ashbury
7-11 Allen Street, Waterloo, NSW 2017 5-10 km from Ashbury
Had my daughter in tears as she is the only one in the class who couldn't do the splits and was made a mockery and example of. Trainers seem young and entire focus of the gym appears to be competition and alienating anyone who doesn't plan to try out for the Olympics. Awful.
What a great place to keep kids fit and healthy. Just what Sydney needed. The service, venue and instructors are all of great quality and dedication. Thanks guys.! We needed you.
I'm so over the moon we finally have an A grad gym in the eastern suburbs! My daughters are extremely excited about starting there.
7 Waratah street, Rushcutters Bay, NSW 2011 5-10 km from Ashbury
Cnr King Georges Rd & Forest Rd, Penshurst, NSW 2222 5-10 km from Ashbury
26a Bunnerong Road, Eastern Suburbs PCYC, Daceyville, NSW 2032 5-10 km from Ashbury
80 Waterloo Road, Greenacre, NSW 2190 5-10 km from Ashbury
100 New South Head Road, Edgecliff, NSW 2027 10-25 km from Ashbury
Olympic Boulevard, Sydney Olympic Park, Newington, NSW 2127 5-10 km from Ashbury
Paine Reserve, Botany Street, Randwick, NSW 2031 5-10 km from Ashbury
Unit 1B, 7-9 Orion Road, Lane Cove West, NSW 2066 5-10 km from Ashbury
Hard to get a place but well worth it when you get it. My kids compete and have done just so well. Coaches great
Fab club. Great coaches very pleasant. Kids love it and so do i. Admin very pleasant too!
Although the center is great and the kids love it there the staff are always friendly however the administration is very poor. There is no consistency between the teachers responses to questions. Children are being told they can move but then can't, the whole re-enrollment is a joke & there is no loyalty given to parents who have been there in the beginning. Parking is a nightmare! Pulled my kids lout because I was fed up with it all
4 College St, Sydney CBD, NSW 2000 5-10 km from Ashbury
Hurstville Aquatic Centre, Cnr King Georges and Forest Road, Hurstville, NSW 2220 5-10 km from Ashbury
Thompson Hall, Young Street, Croydon, NSW 2132 5-10 km from Ashbury
4-6 Linsley St, Gladesville, NSW 2111 5-10 km from Ashbury
525 Crown St, Surry Hills, NSW 2010 5-10 km from Ashbury
North Sydney Pcyc, 224 Falcon St, North Sydney, NSW 2060 5-10 km from Ashbury
Unit 2 / 61 Norman Street, Peakhurst, NSW 2210 5-10 km from Ashbury
Only Available For Ascham Students, 188 New South Head Road, Edgecliff, NSW 2027 5-10 km from Ashbury
Mlc School, Rowley St, Burwood, NSW 2134 5-10 km from Ashbury
Sydney Olympic Park Sports Centre, Grand Parade Sydney Olympic Park, Sydney CBD, NSW 2000 5-10 km from Ashbury
441 Bunnerong Rd, Matraville, NSW 2036 10-25 km from Ashbury
Ryde Rsl Junior Sports Hall Eagle St, Ryde, NSW 2112 10-25 km from Ashbury