Cells can adapt to environment and development by reconstructing their transcriptional networks to regulate diverse cellular processes without altering the underlying DNA sequences. These alterations, namely epigenetic changes, occur during cell division, differentiation and cell death. Numerous evidences demonstrate that epigenetic changes are governed by various types of determinants, including DNA methylation patterns, histone posttranslational modification signatures, histone variants, chromatin remodeling, and recently discovered chromosome conformation characteristics and non-coding RNAs (ncRNAs). Here, we highlight recent efforts on how the two latter epigenetic factors participate in the sophisticated transcriptional process and describe emerging techniques which permit us to uncover and gain insights into the fascinating genomic regulation. Phenotypic diversity of cells and their response and adaptation to the environment are achieved through the regulation of distinct gene expression in particular temporal-spatial context. In this context, transcription control is a crucial step  to regulate transcription of target gene   to orchestrate long-range/short range of chromatin organization across multiple spatial scales. These interactions and chromatin topology would rearrange according to the external environment changes, and these alternations would directly affect the transcription process. The golden rule in three-dimensional regulation of transcription is that the dynamic balance between effective genome packaging and accessibility within the nuclear space should be established to adapt to environment changes and homeostasis. In this process, several crucial nodes of genome topology can be proposed  of the glucocorticoid receptor (GR) by acting as a decoy RNA version of the “glucocorticoid response element (GRE)”, thus competing with DNA GREs for binding to the GR DBD. Gas5, acting as a “riborepressor” of the GR, thus influences cell survival and metabolic activities during starvation by modulating GR-induced transcriptional activity of endogenous glucocorticoid-responsive genes  not only provides the template for telomeric DNA synthesis, but also serves as a flexible scaffold for tethering TERT/Est1p/Ku/Sm into the complex  gene and upregulated upon DNA damage, where it acts as molecular ‘ligand’ for RNA-binding protein TLS and promotes an allosteric effect to release it from the inactive conformation. This recruitment of TLS by lncRNACCND1 leads to the inhibition of the HAT functions of CBP/p300 and repression of CCND1 transcription  interacts with a range of differentiation mRNAs (e.g. KRT80) to mediate their stabilization. TINCR–mRNA interaction occurs through ‘TINCR box’, a 25-nucleotide motif which is strongly enriched in interacting mRNAs and required for TINCR binding , and brings at least three genes together around their own transcription sites. The co-localization of these different gene loci is lost upon deleting the lncRNA Firre locus or knocking down hnRNPU. This multichromosomal nuclear interaction could be achieved by RNA-protein-DNA loop and bring the specific gene locus into proximity that may be far away in linear sequence  play important roles in chromosomal conformation  could examine subcellular localization of lncRNA Firre in embryonic stem cells (Hacisuleyman et al., Emerging developments in technologies have made it possible to create high resolution genome-wide maps of physical interactions along genomic regions and ncRNAs. First of all, in the aspect of three-dimensional chromatin network research, advanced imaging technologies (for example single-molecule RNA fluorescence in vivo systems will help bridge the gap between live cell imaging and in vitro biochemistry. With research underway in all of these areas, the rapid progress in this field over the past decade should continue unabated.Comprehensive understanding of three hierarchical levels of transcriptional regulator is contingent on the upgrading and innovation of technologies. These will come in several areas ranging from classic molecular biology techniques to bioinformatics tools. Despite that many mysteries about gene transcription are still under investigation, the overall picture is gradually clear: interdisciplinary techniques that enable imaging analysis of complex


LDLR 5′UTR and promoter located at c.-13A>G, c.-101T>C, c.-121T>C and c.-215A>G was investigated using in silico and in vitro assays, and a systemic bioinformatics analysis of all 36 reported promoter variants are presented. Bioinformatic tools predicted that all four variants occurred in sites likely to bind transcription factors and that binding was altered by the variant allele. Luciferase assay was performed for all the variants. Compared with wild type, the c.-101T>C and c.-121T>C variants showed significantly lower mean (±SD) luciferase activity , suggesting that these variants are causal of the FH phenotype. No significant effect on gene expression was seen for the c.-13A>G or c.-215A>G variants (96±15 and 100±12%), suggesting these variants are not FH causing. Similar results were seen for the c.-101T>C and c.-121T>C variants in lipid-depleted serum. However, a significant reduction in luciferase activity was seen in the c.-215A>G variant in lipid-depleted serum. Electrophoretic-mobility shift assays identified allele-specific binding of liver (hepatoma) nuclear proteins to c.-121T>C and suggestive differential binding to c.-101T>C but no binding to c.-215A>G. These data highlight the importance of in vitro testing of reported LDLR promoter variants to establish their role in FH. The functional assays performed suggest that the c.-101T>C and c.-121T>C variants are pathogenic, whereas c.-13A>G variant is benign, and the status of c.-215A>G remains unclear.Familial hypercholesterolaemia (FH) is an autosomal dominant inherited disease characterised by increased low-density lipoprotein cholesterol (LDL-C) levels. The functionality of four novel variants within the  The functionality of four variants identified in the literature6 in patients with a diagnosis of heterozygous FH within the LDLR 5′UTR (c.-13A>G) or promoter  were analysed using in silico and in vitro assays. Previous in vitro luciferase results have shown that the variant c.-120C>T had a significant reduction in luciferase activity compared with the wild type.10 This variant was used as a positive control for these experiments. The location of known transcription factor binding sites in the LDLR promoter and the positions of the tested variants are shown in Familial hypercholesterolaemia (FH) is an autosomal dominant disease characterised by high levels of low-density lipoprotein cholesterol (LDL-C)DLR gene , many of1 The c.-13A>G and c.-121T>C variants were reported in FH patients in the literature.12 Standard nomenclature was used for describing variants (www.hgvs.org), and the locus reference genomic sequence LRG_274 for LDLR was used.The c.-101T>C and c.-215A>G variants were identified by the NE Thames Regional Genetics Service laboratories, Great Ormond Street Hospital (GOSH), in patients with a clinical diagnosis of heterozygous FH according to the UK criteria.www.genomatrix.de) and MATCH (http://www.gene-regulation.com/pub/programs.html#match).14 These tools were used to identify potential transcription factor binding sites in the region where the variants in the LDLR promoter were found. The matrix score calculates a match between the sequence and the matrix. The scores range from 0 to 1, with 1 indicating an exact match.Two transcription factor prediction tools were used: MatInspector . Site-directed mutagenesis was completed to create plasmids containing the c.-13G, c.-101C, c.-121C and c.-215G alleles. All constructs were confirmed by sequencing. The plasmids were transfected into Huh7 cells, a human hepatoma liver cell line ). The cells were grown using Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, or 10% lipid-depleted serum .The c.-600 bp to c.-5 bp region of the 7 Cells were lysed using Passive Lysis Buffer  and luciferase expression was determined using the Dual Luciferase Reporter Assay System (Promega), and measured in the Tropix TR717 Microplate Luminometer . Luciferase activity was determined as the mean of 8–12 transfections with the assay performed in triplicate. EMSAs were carried out, as previously described, to determine transcription factor binding within the variant site.8Transfection was prepared when a confluence of 80% was reached. The pUC18 and pGL3 control were used as negative and positive controls, respectively. The cells were transfected using Lipofectamine 2000  and Opti-MEM serum-free medium according to the manufacturer's instructions and as previously described.In silico analysis indicated that all of the variants were likely to be within transcription factor binding sites , one or both of the programmes used predicted that binding was likely to be significantly modified by the presence of the variant allele, for example, the predicted binding of CREB to c.-101T but not c.-101C, and the binding of HIFIA to c.-101C and not c.-101T. However, although the c.-13G allele showed the abolition of two transcription factors, this binding was not verified in MATCH. Moreover, in MatInspector, the c.-120C>T variant showed an abolition of OCT1 binding site, indicating a biological significance. However, this result was not seen in the MATCH results.ng sites . With thLDLR expression in the promoter sequences containing the variant alleles compared with the wild type. These assays were repeated in multiples of 8–12 for each variant and are presented as the average of three independent runs. Compared with the wild type, the c.-13G and c.-215G alleles showed a (mean±SD) 96±15 and 100%±12% level of luciferase activity, indicating that this variant is unlikely to be FH causing. The c.-101T>C and c.-121T>C variants showed a 64±8 and 72±8% lower luciferase activity compared with the wild type . All transfection assays were repeated using cells grown in 10% lipid-depleted serum in order to induce LDLR promoter expression.8 This resulted in a 2.5-fold higher expression of the wild-type construct, with similar increases seen for all variants (data not shown). However, as shown in P<0.001). Remarkably, the c.-215G allele showed a similar and significant reduction in luciferase activity in the absence of lipid (48±10% P<0.001).Luciferase assays were performed to investigate the EMSAs were used in an attempt to identify nuclear factors binding differentially to the c.-101T>C, c.-121T>C and c.-215A>G variants. Extracts from a hepatocarcinoma cell line Huh7 were used, with a probe for the SREBP as a positive control. As shown in in-vitro assay of the effect on transcriptional strength provides the strongest evidence. Four reported variants identified in FH patients within the LDLR 5′UTR and promoter were investigated.6 The c.-13A>G variant showed no difference in luciferase activity, suggesting it is not FH causing, whereas c.-101C and c.-121C showed a significant lower expression, suggesting that these variants are likely to be pathogenic and causal of the FH phenotype seen in these patients, and the status of c.-215A>G remains unclear.In order to correctly report to a referring clinician whether or not an identified promoter variant is likely to be FH causing, bioinformatics predictions may be helpful, and although evidence that the variant affects binding of a nuclear protein by an EMSA may be suggestive, an 15 Both programmes used also suggested that transcription factors would bind to the DNA sequences and that the variants would destroy binding. This is particularly clear for the c.-101T>C allele that is located in the TATA-box and a previously documented CREB binding site.7 Moreover, there were major differences between the two programmes; for example, although MatInspector showed a high score for the binding of two transcription factors, MATCH did not find any binding sites. Of the identified transcription factor binding sites (LDLR expression. The SIRE (sterol-independent regulatory element), located c.-94 to c.-110 of the promoter, binds CREB in a cholesterol-independent manner.7 CREB was predicted to bind to the wild-type sequence, but was abolished in the variant sequence. Moreover, another predicted transcription factor, OCT1, has been implicated as being involved in lipoprotein lipase (LPL) expression, and is critical for hydrolysis of triglycerides in lipoproteins.17 Although OCT1 was predicted to bind by MatInspector and MATCH, there are no major differences in the scores for the variant and wild-type binding sites. These algorithms lack the complexity to take account of physiological conditions. The binding of transcription factors to a specific DNA sequence is dependent on various intracellular and extracellular stimuli within a cell. Therefore, although some transcription factors are predicted to bind to these sequences (with scores as high as 1.0), this does not take into account in vivo conditions such as chromatin structure, and are hence not ideal representations of what occurs in the cell. Bioinformatics tools should not be used in isolation but as a guide for experimental research.The bioinformatic programmes were not helpful in this instance. Both programmes (MatInspector and MATCH) suggested that the c.-13A>G variant destroys binding for a nuclear transcription factor and yet this variant did not affect luciferase activity. This is in agreement with previous studies that have shown no causal effect of variants within the 5′UTR region.ng sites , CREB isLDLR expression, this factor is involved in expression of PCSK9,18 a key gene involved in cholesterol metabolism, and hence the prediction of its binding here may be correct.The EMSA assay was also inconclusive: one out of the three functional sites determined by luciferase assay showed no evidence of binding of a nuclear protein, and this may be because of the protein requiring a longer binding site than the probe provided or nonphysiological binding conditions. Although we are not aware of any direct evidence for a role of HIF1A in the control of LDLR transcription, with the key nucleotides identified at c.-218, c.-219 and c.-231,19 but no variants at these sites have been reported so far. Of the 23 variants between c.-207 and c.-90, where the binding sites for the key transcription factors SREBP1, SP1 and the TATA box CREBP are located, 14 have been tested and all found to have a profound effect on luciferase levels. Excluding the two examined here, the percentage of wild-type activity reported ranges from 0 to 40% (mean 15%). The modestly lower ~50% luciferase noted for the variants tested here  therefore raises the possibility that these may not be FH causing, although the data obtained are based on 8–12 replicates and three repeat experiments, and the lower transcriptional data are statistically robust. To address this, we have used c.-120C>T as a positive control that has been reported to have only 3% luciferase activity compared with the wild-type promoter sequence.10 In our hands, we observe that the variant has 83±9% activity compared with the wild type, confirming that the variant is FH causing but that the effect is not so drastic as previously observed. This could be because of the differences in cell culture conditions; for example, Francova et al10 used the HepG2 cell line compared with the Huh7 hepatoma cells that we used. However, the reduction in luciferase activity observed with the novel variants tested here are similar to the reported FH-causing c.-120C>T variant, strongly supportive of a causal effect of our variants. Finally, of the eight variants located after the start of transcription (c.-93 to c.-79), two have been tested and, as with the c.-13A>G tested here, none have a significant effect on promoter strength.15 Our data suggest that although there is no effect on luciferase activity for the c.-215G allele, there was a significant reduction in luciferase activity when supplemented in lipid-depleted conditions. In lipid-depleted conditions, SREBPs activate the transcription of LDLR by binding to SRE. Therefore, when lipid levels are low, LDLR expression is increased. However for the c.-215G allele construct, in lipid-depleted conditions, luciferase expression was reduced compared with the wild-type construct. Therefore, because lipid-depleted conditions mimic what occurs in vivo, these data support the view that this variant is possibly causal of the FH phenotype, but the status of this variant is still unclear, and co-segregation analysis in the relatives of this patient may resolve this issue. The data obtained from this study have been submitted to the UCL LDLR variant database (https://grenada.lumc.nl/LOVD2/UCL-Heart/home.php?select_db=LDLR).In order to put the present findings in context, we have carried out a systematic review of the published promoter and 5′UTR variants and examined the functional assay data reported. Including the four variants examined here, there are 36 variants reported, of which 20 have been directly tested using reporter assays, usually luciferase . Of the LDLR promoter region and all the four sites tested here show strong conservation across six primate and six mammalian species (12 or from single samples sent to the diagnostic laboratory. Co-segregation of the variant with elevated lipid levels in relatives would strengthen the inference that they are FH causing. For all patients, DNA was tested for the common FH-causing mutations in the APOB and PCSK9 genes and for the Portuguese patient the entire PCSK9 gene was screened, but it remains a formal possibility that their elevated LDL-C levels may be caused by an unidentified variant in these genes. Finally, for the EMSAs we did not carry out detailed competition or supershift experiments to confirm the specificity or identity of the nuclear proteins binding to the alleles, as in our experience these type of experiments are difficult to quantify with accuracy and often give equivocal results that may not be of direct relevance to the in vivo situation because the examined element is not embedded within the full chromatin context.There is high cross-species conservation across the whole of the  species . It is nIn vitro luciferase reporter assays for identified promoter variants are important in determining the pathogenicity of promoter variants, allowing diagnostic reporting with greater confidence and subsequent cascade testing in the relatives of patients with these variations. This study highlights the difficulty of relying solely on in silico methods for the prediction of the pathogenicity of DNA sequence variants. Ideally, functional assays should be used to determine effects; however, the dilemma faced by many diagnostic service laboratories is that such tests are expensive and time consuming, and the relevant expertise and resources are rarely available in-house. Therefore, close links with research groups are key to the transfer of expert knowledge to diagnostic service laboratories. In conclusion, the functional assays performed indicate that c.-101T>C and c.-121T>C are likely to be pathogenic, whereas the c.-13A>G variant appears to be benign and the status of c.-215A>G remains unclear.


New methods for epigenome editing now make it possible to manipulate the epigenome in living cells with unprecedented specificity and efficiency. These ground-breaking approaches are beginning to yield novel insights into the function of individual chromatin marks in the context of cellular phenotype. Our desire to manipulate genomes is not new; selective breeding, genetic and, more recently, genome engineering have greatly advanced our understanding of how genes shape phenotypes. On the cellular level, however, differences in phenotype are mainly determined by epigenetic processes. Millions of chromatin marks have been profiled across the human genome in different tissues and cell types. Yet, we are just beginning to understand how chromatin-based mechanisms contribute to the establishment, control and inheritance of gene activities. Epigenetic research has long been hindered by the lack of experimental methods that would allow the targeted manipulation of chromatin marks in living cells, since mutational approaches and pharmacological inhibition alter marks globally and nonspecifically. Epigenome engineering now fills this gap, making it experimentally possible to modify individual chromatin marks at specific user-defined sites. Targeted modification is achieved by fusing an effector, usually an endogenous chromatin-modifying enzyme or its minimal catalytic domain, to a programmable DNA-binding domain. The DNA-binding domain may be a synthetic zinc finger (ZF) or a TAL protein (TALE), which can be engineered to bind to a DNA sequence of choice. Alternatively, the chromatin modifier is fused to a catalytically inactive version of the Cas9 protein (dCas9), which is targeted via a separate, synthetic RNA molecule known as the guide RNA (gRNA). The base sequence of the gRNA determines the DNA-binding specificity of the fusion protein. A range of chromatin modifiers have already been engineered in this way, representing the first components of an epigenetic ‘toolbox’ Table . These cIt has long been unclear which of the large number of chromatin marks that have been catalogued over the years possess real gene-regulatory capabilities. Evidence was based mainly on statistical association of chromatin marks with expression levels of associated genes. However, it is worth bearing in mind that correlation does not necessarily imply causation. The observation that loss of chromatin modifiers causes strong phenotypes, which can often be interpreted as a consequence of transcriptional deregulation, provided further support. However, chromatin-modifier proteins also have non-histone targets, and epigenetic marks play roles in chromatin-based processes other than transcription. A causal role for chromatin marks in regulation of transcription has been convincingly demonstrated for some model loci, including the Hox and imprinted gene clusters (for example ), but itBy directing chromatin modifiers to a range of sites at different genomic loci and measuring resulting changes in transcription of associated candidate genes, a number of functional chromatin marks have now been identified. For example, demethylating H3K4me2 at putative enhancers with TALE–LSD1 or dCas9–LSD1 leads to downregulation of proximal genes . RemovalCollectively, these studies show that manipulation of single chromatin marks at relevant sites can significantly alter levels of transcription and that this effect depends on both the enzymatic activity of the chromatin modifier and its binding to the target site. However, some questions remain regarding the efficiency of chromatin modification using synthetic modifiers. While epigenetic editing can clearly influence transcription at a specific site, manipulation in close vicinity often produces no effect. This could reflect inherent differences in gene regulatory potential between different loci. Alternatively, it is possible that some sites cannot be modified efficiently for technical reasons , other layers of gene regulation  or immediate reversal of the newly acquired change . Thus, it remains to be addressed how many marks are functional and where in the genome these must be located in order to influence transcription.OCT4 [In addition to a more detailed investigation into the efficiency of targetable chromatin modifiers, it will be important to consider whether the reported transcriptional changes are not just statistically significant, but also biologically meaningful. To some extent this is a semantic issue: while the term ‘gene activation’ suggests that gene functions have been put into effect, it can also be used to describe a minor increase in RNA levels relative to background. It would be important to compare engineered gene expression with physiological levels of expression where this is possible. In addition, analyses of protein levels , 6 can eOCT4 , throughEffectively, we now have an emerging epigenetic ‘toolbox’ for epigenome engineering, ushering in a new era of functional epigenomics. Based on the astonishing pace with which this field is moving, more chromatin modifiers will without doubt be added to the growing list of available tools. We anticipate that it will soon be possible to dissect the effect of combinations of altered chromatin marks using orthologous targeting platforms.The ease of targeting chromatin modifiers through an RNA-based rather than protein-based DNA-binding mechanism will further enable the unbiased discovery of functional epigenetic marks using screening approaches. Screens will constitute a significant step forward from the analysis of the effect of a single mark on a single locus enabling the interrogation of many potentially functional marks in a single experiment. Libraries of guide RNAs can be constructed that are either specific to a limited range of loci of interest or potentially unbiased and genome-wide. Loss-of-function genetic screens and gain-of-function transcriptional activator screens have already been implemented , but no Eventually, some of the findings may be expected to be translated into therapeutic use by adopting epigenetic engineering technologies for in vivo situations. There are already first indications that this may be feasible based on targeted epigenetic engineering of candidate promoters in the brain of living mice, positively affecting addiction-related and depression-related behavior . However


Plasmodium falciparum, the causative agent of malaria, has been poorly understood. To elucidate stage-specific epigenetic regulation, we performed genome-wide mapping of multiple histone modifications of P. falciparum. Further to understand the differences in transcription regulation in P. falciparum and its host, human, we compared their histone modification profiles.Role of epigenetic mechanisms towards regulation of the complex life cycle/pathogenesis of P. falciparum. Interestingly, we found that H3K36me2 acts as a global repressive mark and gene regulation is fine tuned by the ratio of activation marks to H3K36me2 in P. falciparum. This novel mechanism of gene regulation is supported by the fact that knockout of SET genes (responsible for H3K36 methylation) leads to up-regulation of genes with highest occupancy of H3K36me2 in wild-type P. falciparum. Moreover, virulence (var) genes are mostly poised and marked by a unique set of activation (H4ac) and repression (H3K9me3) marks, which are mutually exclusive to other Plasmodium housekeeping genes.Our comprehensive comparative analysis suggests distinct mode of transcriptional regulation in malaria parasite by virtue of poised genes and differential histone modifications. Furthermore, analysis of histone modification profiles predicted 562 genes producing anti-sense RNAs and 335 genes having bidirectional promoter activity, which raises the intriguing possibility of RNA-mediated regulation of transcription in P. falciparum which can influence parasite virulence and pathogenicity. The observed differences in the histone code and transcriptional regulation in P. falciparum and its host will open new avenues for epigenetic drug development against malaria parasite.Our study reveals unique plasticity in the epigenetic regulation in The online version of this article (doi:10.1186/s13072-015-0029-1) contains supplementary material, which is available to authorized users. Plasmodium falciparum causing most of the malaria-associated mortality. P. falciparum exhibits a complex life cycle progressing through multiple developmental stages in two hosts. The clinical manifestation of malaria is a result of parasite development in red blood cells (RBCs), where it completes its asexual intra-erythrocytic cycle (IEC). During the 48-h IEC, parasite invades RBC and develops into a ring stage, followed by trophozoite and schizont stages. Nuclear division during the schizont stage results in the formation of 16–32 merozoites, which can infect the new RBCs. To sustain chronic infection in human hosts, parasite undergoes rapid transitions between morphological states, a mechanism of immune evasion that contributes to pathogenicity. These rapid transitions between morphological states are orchestrated by multiple types of transcriptional and epigenetic regulations [Malaria is a major public health problem in many developing countries, with the parasite P. falciparum genome encodes the four conserved core histones [P. falciparum [P. falciparum genes are activated only once during the infected RBC cycle attesting the importance of stringent gene regulation in stage-specific manner [Nucleosome is the fundamental unit of chromatin, in which 147 base pairs of DNA are wrapped around histone octamer consisting of two copies each of the four core histone proteins H3, H4, H2A and H2B [histones . The N-thistones . Extensilciparum –10. Impoc manner , 12. Epic manner .Saccharomyces cerevisiae and Tetrahymena thermophila, a large proportion of the P. falciparum genome is constitutively acetylated [P. falciparum [P. falciparum, the extent to which gene-specific combinatorial patterns exist remains to be determined. In this study, by characterizing promoters and analyzing histone modifications at three different IEC developmental stages of P. falciparum; ring, trophozoite and schizont, we have unraveled the epigenetic landscape associated with gene regulation. We show that activation marks H3K4me2, H3K4me3, H3K9ac, H3K14ac and histone H4ac are located at the promoter and body of the genes like other eukaryotes and contrary to the earlier observations in P. falciparum [P. falciparum and gene expression is governed by the ratio of activation marks to H3K36me2. Furthermore, relevance of this epigenomic landscape is highlighted by the integration of RNA sequencing, anti-sense transcripts [P. falciparum [P. falciparum.Post-translational modifications of histones influence gene expression which can be decoded to decipher the function of underlying DNA sequence. Unlike higher eukaryotes, but similar to etylated , 15. Surlciparum . In contlciparum . Howeverlciparum . We alsonscripts  and genelciparum . Thus, oP. falciparum, we performed chromatin immunoprecipitation (ChIP)-coupled high-throughput sequencing (ChIP-seq) of active , inactive (H3K9me3 and H3K27me3), elongation (H3K79me3) and regulatory element (H3K4me1) histone modifications at three different stages of intra-erythrocytic cycle  are grouped together indicating a cumulative action presumably for efficient gene expression  in ntil now . To undeP. falciparum and its host Homo sapiens, we first interrogated how the histone modifications are represented between them. Towards this, first we characterized promoters in P. falciparum using the RNA sequencing data from different stages [P. falciparum IEC and compared it with profiles of histone modifications in human . Another major repression mark in human, H3K9me3, exhibits weak global profile in P. falciparum as it is present only on a subset of genes corroborating the earlier findings [Plasmodium, H2A.z was present neither at the promoters nor in the gene body. H2A.z occupancy indeed was found flanking the gene unit suggesting that it acts differently in Plasmodium presumably as a guard or marker of transcription unit, as suggested earlier [P. falciparum. Thus, the absence of repressive mark H3K27me3 and presence of H3K9me3 only on a subset of genes in P. falciparum suggest differential mode of gene regulation in the malaria parasite as compared to its host, human.To understand the mechanism(s) of differential transcriptional regulation in t stages . Next, wfindings , 20. Incfindings . However earlier , 22. H3KP. falciparum. We systematically calculated the enrichment levels of histone modifications in the gene body of all the P. falciparum genes at trophozoite stage and compared them with relative gene expression categorized based on RNA sequencing  to activation marks , which might play an important role towards gene regulation in P. falciparum. To test this hypothesis, we divided P. falciparum genes at trophozoite stage into nine categories ranked on the basis of their expression level and then systematically calculated the ratio of enrichment levels of putative global repressive to activation marks . Genes in cluster 3 are associated with stimulus-dependent functions  Cluster 1 (411 genes), peaks at the 5′ end of the gene (red squared and labeled 1), Cluster 2 (562 genes), peaks at the 3′ end of the genes (green squared and labeled 2), Cluster 3 (335 genes), peaks observed at the center of the gene body (blue squared and labeled 3) and Cluster 4 2442 genes), with very low levels of histone modifications (black squared and labeled 4)  gene families such as var, rifin and stevor, which are shown to play a central role in enabling host immune evasion and promoting pathogenesis. These multicopy gene families exhibit monoallelic expression, which is shown to be regulated by epigenetic modifications [var and rifin genes. We calculated the enrichment of multiple histone modifications  and histone variant H2A.z on CVM genes and compared them with 500 ring-expressed genes in stage-specific manner  of transcription regulation between them. Our comprehensive histone modification analysis shows that most of the chromatin in P. falciparum is poised, indicative of the fact that parasites undergo closed mitosis and their chromosomes do not condense during nuclear division [P. falciparum [P. falciparum as reported earlier [P. falciparum. Thus, the observed discrepancy in distribution of histone modifications between this study and earlier study [P. falciparum. Thus, our analysis suggests that all these activation marks work together to establish more open chromatin required for the regulation of transcription. However, the elongation marks H3K36me3 and H3K79me3, which are associated with active transcription and resetting of the initial chromatin in human cells [P. falciparum suggesting a new mode of communication between chromatin and transcription [Plasmodium suggesting its function in boundary maintenance. Our analyses thus corroborate other studies which suggest that P. falciparum has expanded its repertoire of gene regulation by anti-sense RNA [P. falciparum. Thus, these findings can be used to explore the differences in transcriptional regulation between the P. falciparum and its host to develop inhibitors against the malaria parasite.In this study, by contrasting the histone modifications from division . In humadivision . A mechadivision . This crdivision . Thus, Hdivision . In conclciparum . We wond earlier , 18. To er study  may havean cells , show mocription . Surprisense RNA –37 and bense RNA  but we iP. falciparum lacks H3K27me3 histone modification  or the interplay between these. tudy and ). Hence,genicity . This inrganisms , 40. Furvar genes. Many mechanisms have been suggested for the regulation of var genes, including involvement of non-coding RNAs [var genes [Most of the knowledge of clonally variant multicopy (CVM) genes has been acquired through the study of ing RNAs , bidirecing RNAs , introniing RNAs , insulating RNAs , transcring RNAs  and uniqing RNAs , H3K36meing RNAs  and H2A.ing RNAs . In thisar genes . Interesar genes . Thus, oP. falciparum. The comprehensive epigenomic map generated in this study is in concordance with earlier expression analyses suggests that these data can be used to overcome many conceptual gaps in our understanding of P. falciparum transcription. We also found that transcriptional regulation by the virtue of poised chromatin and differential histone modifications is unique to P. falciparum, which could have evolved for higher rate of transcription. Furthermore, for the first instance, we identified H3K36me2 as a global repressive mark in P. falciparum and revealed a unique mode of regulation of gene expression by altering the ratio of activation and repression marks. Importantly, the observed differences in the epigenetic code and transcriptional regulation in P. falciparum and its host will open new avenues for targeting the epigenetic machinery for drug development in malaria as well as other related parasites in future.In this study, we have generated high-resolution genome-wide profiles of several histone modifications and integrated them with publicly available data for histone modifications in P. falciparum strain 3D7 was cultured as previously described [−1 gentamicin sulfate at 37 °C. RBCs were prepared from fresh whole blood obtained from a healthy donor and stored at 4 °C for at least 1 day. Increased yields were obtained by subculturing the cells every 2 days for 6–8 h before invasion. This was achieved by equally dividing the contents of each flask into two or more flasks and quickly restoring the hematocrit between 1 and 1.5 % in the required volume of culture medium. The culture was synchronized with 5 % sorbitol and the parasites were collected at 18, 30 and 40 hpi for chromatin immunoprecipitation.escribed . BrieflyP. falciparum [P. falciparum lysate , H3K4me3 (Upstate 07-443), H3K9ac (Upstate 06-942), H3K9me3 (Upstate 07-442) antibodies were validated earlier in lciparum , 49. H3KInfected RBCs were crosslinked with 1 % formaldehyde  for 10 min, lysed and sonicated in sonication buffer  to obtain an average chromatin size of 200–400 bp. Chromatin was pre-cleared using 50 µl of a 50 % protein A Sepharose  slurry for 1 h at 4 °C with gentle inverting. Immunoprecipitations were carried out in 1 ml of IP buffer . Three µg antibody was used per 20 µg purified chromatin. Input chromatin was obtained after preclearing by de-crosslinking and purified using the Qiaquick column (Qiagen) according to manufacturer’s instructions. Immunoprecipitations were carried out with inverting at 4 °C for 14–16 h. The samples were then incubated with 50 µL of a 50 % Protein A Sepharose slurry for 3 h at 4 °C with gentle inverting. IP samples were reverse-crosslinked and the DNA was purified using a Qiaquick column (Qiagen). Target sites obtained from ChIP-seq analysis were further validated by quantitative PCR using Power SYBR Green Master Mix (Applied Biosystems). For sequential ChIP, at least four ChIP assays (4 × 20 µg purified chromatin) were used for the first IP (H3K9ac). Following standard washing, elution was performed with 10 mM DTT . The eluates from four ChIPs were combined, diluted at least 30 times with ChIP dilution buffer and secondary antibody (H3K4me3) was incubated overnight. The subsequent steps were performed as for regular ChIP.ChIP-seq libraries for all the samples were prepared from around 5 ng of DNA using fragment library construction kit from Life technologies for SOLiD sequencing. Briefly ChIPed DNA samples were end repaired and adapters were ligated using T4 DNA ligase. Ligated DNAs were subsequently amplified using adapter specific bar-coded primers for 15 cycles. DNA purification at every step was performed using Agencourt XP beads (Beckman Coulter). Library profiles were assessed using Agilent bioanalyzer 2100 high-sensitivity DNA kit. Equimolar amount of libraries were pooled and 50 bp reads were sequenced in-house using SOLiD 4.0 sequencer (Applied Biosystems).Reference genome  was converted to color space using bowtie-build. ChIP-seq data were mapped using the Bowtie allowing two mismatches. Bed files were generated using Samtools . ChIP-seRaw BED files are used as input for ad hoc (WIG) density file creation script as described in . Reads aPlasmodium falciparum, a reference-based transcriptome was assembled using previously reported RNA-seq data covering 8 samples collected at different time points of erythrocytic cycle stage [To determine the Transcription Start Site of coding gene in le stage . Here, Ple stage . In the le stage  and thisle stage . From thhttp://fullmal.hgc.jp/ [Plasmodium falciparum genome  using BLAT tool [Validation of the identified TSS from the current analysis was carried out by comparing with 5 prime EST sequence data available at .hgc.jp/ . A list LAT tool  and EST LAT tool . ResultiWe extracted the tag density in a 1.5 kb window surrounding the TSSs and gene body using the program seqMINER which generates heatmap as well as the profiles . The seqhttp://www.ncbi.nlm.nih.gov/gds) under the accession number: GSE23867  and GSM840279 (Human); . UCSC genome browser was used for data visualization [ChIP-seq datasets were downloaded from the public data bank Gene Expression Omnibus (lization . Number Genes were categorized based on increasing expression levels (based on reads assigned per kilobase of target per million mapped reads (RPKM)) using the RNA-seq data . Datasehttp://madiba.bi.up.ac.za/). REVIGO (revigo.irb.hr) was used to summarize and visualize gene ontology terms.Gene ontology analysis was performed using MADIBA ; an onliTotal tag density of histone modifications was calculated as described above in average profile calculation over the CVM genes and 500 ring expressed genes in a stage-specific manner. The data were visualized using the matrix2png .P. falciparum growth. RNA was treated with DNaseI (Ambion) as described in the manufacturer’s protocol followed by phenol–chloroform extraction. RT-PCR was performed using 500 ng total RNA. For each gene, reverse transcription (RT) was performed using a forward and reverse primer. After first strand cDNA synthesis PCR was performed using sense and anti-sense primers. Sequences of primers used for strand-specific RT-PCR are provided in the Additional file Total RNA was isolated at three different stages of E. coli (DH5α) cells. The resulting colonies were screened via restriction digestion for presence of EGFP insert. A number of positive clones were selected and subjected for large-scale DNA purification via CsCl density gradient method. All positive clones were confirmed by sequencing. Primers used for EGFP amplification were designed such that EGFP insertion occurred in an orientation opposite to that of the DsRed monomer protein. DNA elements (from cluster 3) with predicated bidirectional activity from P. falciparum were cloned in sense and anti-sense to M-cherry and EGFP, respectively. Positive clones for each element along with the pDR vector were transfected in P. falciparum by electroporation. Cells were fixed and analyzed by confocal microscope to score for EGFP and M-cherry expression 48 h post electroporation. The primers used in construction of bidirectional promoters are listed in the Additional file To score for bidirectional transcription of putative DNA elements from Cluster 3, we designed a dual reporter vector (pDR) using pmCherry-N1 as a backbone in which, EGFP and M-cherry were cloned in sense and anti-sense orientation to each other. Briefly, pmCherry-N1 vector was digested with Ase1 and Nhe1 to remove the CMV promoter. EGFP sequence was PCR amplified using pEGFP-N1 vector as a template, along with SV40 polyadenylation signal to make sure that there is no difference in RNA stability of Cherry and EGFP. Amplified EGFP product was purified by extraction using phenol:chloroform and subsequently digested with Ase1 and Nhe1. The restriction digestion products were purified by running on gel followed by purification by gel extraction column (Qiagen). Vector (pmCherry-N1) and insert (EGFP) were ligated and transformed into All the stage-specific ChIP-sequencing data for histone modifications are submitted to Gene Expression Omnibus (GEO) database under the accession number GSE63369 (Additional file


ATRX and MeCP2 belong to an expanding group of chromatin-associated proteins implicated in human neurodevelopmental disorders, although their gene-regulatory activities are not fully resolved. Loss of ATRX prevents full repression of an imprinted gene network in the postnatal brain and in this study we address the mechanistic aspects of this regulation. We show that ATRX binds many imprinted domains individually but that transient co-localization between imprinted domains in the nuclei of neurons does not require ATRX. We demonstrate that MeCP2 is required for ATRX recruitment and that deficiency of either ATRX or MeCP2 causes decreased frequency of long-range chromatin interactions associated with altered nucleosome density at CTCF-binding sites and reduced CTCF occupancy. These findings indicate that MeCP2 and ATRX regulate gene expression at a subset of imprinted domains by maintaining a nucleosome configuration conducive to CTCF binding and to the maintenance of higher order chromatin structure. ATRX gene cause ATR-X syndrome, a severe intellectual disability disorder , and that loss of ATRX prevented the repression of maternal H19 transcription, as well as other co-regulated imprinted genes in the postnatal brain  including imprinting control regions (ICRs) at many imprinted domains. MeCP2 is required for ATRX binding, and loss of ATRX or MeCP2 results in increased nucleosome occupancy over CCCTC-binding factor (CTCF) binding sites. This failure to maintain an extended linker region necessary for stable CTCF binding explains reduced CTCF binding and the altered long-range chromatin interactions and gene expression at two imprinted regions, Atrx gene was conditionally deleted in the mouse forebrain as previously described . For allele-specific expression studies, pups were obtained by mating 129Sv female mice with Mus musculus castaneus males . nullMeCP2 mice were generated by crossing loxPMeCP2 females (Jackson Laboratories Stock #007177) with a ubiquitous Cre line driven by the EIIa promoter (Jackson Laboratories Stock #003724). Animal studies were conducted in compliance with the regulations of The Animals for Research Act of the province of Ontario, the guidelines of the Canadian Council on Animal Care, and the policies and procedures approved by the University of Western Ontario Council on Animal Care.The escribed . The Atret al.  using the Biotin and DIG-Nick Translation Kits (Roche Diagnostics) and purified using the High Pure PCR Product Purification Kit (Roche Diagnostics) as per manufacturer's instructions. Slides were washed in 50% formamide/2× SSC for 2× 5 min and 2× SSC for 2× 5 min. Sections were incubated with the primary antibody for 1 h at room temperature, washed for 15 min in PBS and incubated with the secondary antibody for 1 h. Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI)  and mounted in Slowfade Gold Antifade Reagent (Invitrogen). The primary antibodies used were as follows: anti-ATRX H-300 , anti-DIG  and anti-Biotin . Secondary antibodies used were as follows: goat anti-rabbit Alexa 594  and goat anti-mouse Alexa 488 . Images were taken at 0.3 μm intervals across the 8 μm section using the Olympus FV1000 confocal microscope and FV10-ASW 2.1 image acquisition software (Olympus). Volocity software (PerkinElmer) was used to compile 3D images and distances were measured using Volocity 3D measurement tools. For co-localization analysis, FISH signals with a center-to-center distance of <1 μm were considered to be interacting , equilibrated in 30% sucrose–phosphate buffered saline (PBS), frozen in O.C.T. (Tissue Tek) and sectioned at 8 μm. Antigen retrieval was performed using 0.3% sodium citrate (Sigma-Aldrich) for 1 h. Slides were dehydrated in an ethanol series of 70% for 2 min, 90% for 2 min, and 100% for 5 min, followed by denaturation in 70% formamide/2× SSC for 5 min at 65°C. Slides were again dehydrated as described above and then incubated with 0.05 μg of DIG and/or biotin-labeled probe/hybridization buffer  overnight at 37°C in a humidified chamber. Probes were prepared by nick translation of BAC DNA (eracting .Chromatin immunoprecipitation (ChIP) was conducted as previously described  with antn alignment mode. During alignment duplicate sequences were removed, up to three mismatches were allowed, and reads that aligned to more than one location were discarded. Genome-wide data tracks were generated using custom Perl scripts to extend reads to their fragment lengths and normalized to 20 million reads. Data was visualized in the UCSC Genome Browser.Raw sequencing data for ATRX embryonic stem cell ChIP-sequencing was downloaded from the NCBI Sequence Read Archive (accession number: GSE22162), and aligned to the mouse genome using Bowtie version 0.12.8 in the −® Green master mix (BioRad) on a Chromo-4 thermocycler (MJ Research) using the following conditions: 35 cycles of 95°C for 30 s, 57.5°C for 30 s, and 72°C for 1 min. Quantification was achieved using the Ct method of quantification and normalized to amplification of Gapdh and Beta-actin.Neonatal mouse forebrain was dissected, rinsed in 37°C Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) and passed through a 70-μm cell strainer  to ensure single cell suspension. The cell suspension was incubated at 37°C for 30 min to equilibrate. Cells were fixed in 1% formaldehyde (Sigma-Aldrich) for 5 min and rinsed three times with cold PBS containing protease inhibitors (Roche Diagnostics). Cells were resuspended in lysis buffer  and flash-frozen and thawed three times, nuclei were centrifuged and resuspended in micrococcal nuclease digestion buffer (NEB). Micrococcal nuclease  was added and incubated at 37°C for 5 min, then quenched with EDTA. Cells were lysed with 1% SDS and cross-links reversed by incubation at 65°C for 5 h, followed by RNAse and PK digestion and phenol/chloroform extraction. DNA was subsequently digested with McrBC enzyme (NEB) for 2 h at 37°C. DNA was amplified in duplicate with iQ™ SYBRH19 ICR that regulates imprinting at the H19/Igf2 domain was previously reported to interact with other imprinted domains located on other chromosomes , a chromosome conformation capture (3C)-based technique that identifies genome-wide interactions in vivo from a single bait sequence  and 29 intrachromosomal (cis) regions that were common between the two replicates , with 11 interactions occurring within 50 kb of the bait. Local contacts include the Ins2, Igf2os and Igf2 genes, Igf2 differentially methylated region 1 (DMR1), matrix-attachment region 3 (MAR3), centrally conserved domain (CCD) and the H19 promoter  showed interactions within 1 or 2 Mb of imprinted genes including Grb10, Dlk1, Gtl2, Zac1 and Mest. Genomic intervals that include other imprinted gene network members also showed some enrichment, but failed to reach our threshold. The observation that interactions occur across a broad region surrounding imprinted genes, as opposed to specific restriction fragments, might reflect the dynamic nature of these interactions and transient co-localization of this network of imprinted genes.The omosomes ,35 in a omosomes ,35. Give. 3C libr Figure  and d. Torebrain . The impH19/Igf2 and other imprinted domains in single brain cells in vivo. 3D-DNA fluorescent in-situ hybridization (FISH) of neonatal forebrain cryosections showed that imprinted genes exhibiting increased expression in the ATRX-null forebrain  co-localize with H19/Igf2 in neocortical cells with the exception of Peg10/Sgce . The co-localization frequencies are characteristic of transient events and are consistent with previously reported co-localization data  analysis, with primer design guided by the results of the 4C experiment. The approach included an EcoRI primary digestion, which divides the 140 kb H19/Igf2 region into 45 fragments stretching from the Ins gene to the H19 enhancers . We designed a forward primer and Taqman probe to the H19 ICR (EcoRI restriction fragment used as bait), and 32 reverse primers in other EcoRI fragments covering negative intergenic regions as well as key genomic elements identified by 4C. Confirmation of interaction frequencies at a subset of sites was obtained by designing a second primer at the other end of the restriction fragment. To verify that our 3C experiments yield reliable data, we first tested the approach using neonatal liver. The results were comparable to those obtained by Qiu et al. . We observed minimal or no interaction with a number of other sites , substantiating the specificity of the identified interactions. We then repeated the 3C analysis in control and ATRX null neonatal forebrain tissue isolated from littermate-matched pups. In forebrains lacking ATRX protein, we detected fewer interactions across the H19/Igf2 domain, with significant reductions specifically at the Igf2 DMR1 (region E/F: P = 0.001 and 0.0003), MAR3 (region I: P = 0.0052 and 0.0038), CCD (region L: P = 0.0001 and 0.0001) and the endodermal enhancer (region Q: P = 0.0004 and 0.0105) , the intergenic site between MAR3 and the CCD (region K) and the region downstream of the H19 enhancers (region T) were not affected   and the Igf2 DMR1, MAR3, CCD, as well as with the endodermal enhancer, similar to the results obtained in the ATRX-deficient brain  Figure . We also) Figure  at the Hn Figure . DecreasN Figure , showingH19 ICR or occurs at other imprinted genes. Given that ATRX localizes with MeCP2 at the Gtl2 DMR within the Gtl2/Dlk1 domain, and controls CTCF binding at this site  imprinted domain, including the Dlk1 gene (regions F and G), intergenic DMR (region N), and many intergenic sites  , and intergenic sites . Interactions with the intergenic DMR were not significantly affected by the loss of ATRX or MeCP2 (region N). Furthermore, allele-specific nucleosome analysis revealed that loss of MeCP2 or ATRX caused a significant increase in nucleosome occupancy at the Gtl2 DMR CTCF biding site  Figure  and e. Te Figure , suggesttranschromosomal interactions occur independently of ATRX. Rather, we show that ATRX binds directly to each imprinted domain and mediates intrachromosomal interactions and controls gene expression at each domain in parallel. Interestingly, we find that interchromosomal interactions do not occur with specific genomic elements, but are enriched in areas surrounding imprinted genes. This result suggests that these interactions are not functional, but rather consequences of co-localization within the nucleus, perhaps at transcription factories. A recent study reported that Hi-C analysis in single cells shows consistent local interactions between cells, but that interchromosomal interactions are highly variable  parallel the maternal-specific effects following loss of CTCF , epigenetic modifications and changes in long-range chromatin interactions.ATRX is thought to bind G-quadruplex DNA and was proposed to resolve these structures to facilitate DNA replication and transcription ,15,43. T binding . Thus, c binding ,12. In tme locus , and mayDlx5/Dlx6 imprinted domain was proposed to control chromatin looping and gene expression  to regulate chromatin structure at these imprinted domains. Genome-wide ChIP studies have shown that MeCP2 binds across the genome in a histone-like manner and affects the expression of multiple genes . Moreovsability . Continu


Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.The online version of this article (doi:10.1007/s00018-015-1850-1) contains supplementary material, which is available to authorized users. Inflammation is a part of the immune system’s response to infection and injury. The usual outcome of an inflammatory cellular response is its successful resolution and repair of tissue damage, whereas persistence and dysfunction of the inflammatory response has been implicated in the pathogenesis of diseases such as arthritis, cancer, neurodegenerative and cardiovascular diseases .Key players in the generation of the inflammatory response are the cyclooxygenase enzymes COX1 and COX2, which catalyze the conversion of arachidonic acid into pro-inflammatory prostaglandins and trigger the production of other pro-inflammatory chemokines and cytokines . COX1 isOur work has focused on understanding the mechanisms regulating induced COX2 expression at the level of gene transcription. COX2 is regulated by a diverse group of transcription factors such as AP1, CRE, HIF, SP1 and STAT –11, but In this study, we used a combination of quantitative kinetic modeling and experimental validation to describe a novel mechanism for controlling the expression of human cyclooxygenase 2 (hCOX2): a molecular logic AND gate composed of two NREs acting in tandem to give a switch-like property to hCOX2 expression. We also report that this logic gate is absent in the mouse COX2 promoter, which instead produced an analog response to NFκB. Thus, our results demonstrate an additional level of complexity in the specific gene activation by NFκB in different species.2 humidified incubator.Human HEK293 and HT29 and mouse MEF cells were cultured in DMEM high-glucose medium supplemented with 10 % FCS and 100 U/ml penicillin–streptomycin in a 5 % CO2, 10 mM KCl, 200 mM sucrose, 0.5 mM DTT, 0.25 % NP-40 and 1× PIC), centrifuged , the supernatant containing the cytosolic extract was stored at −20 °C. The nuclear pellet was washed again in buffer A, suspended in buffer C , incubated for 30 min on ice and pelleted again, the supernatant containing the soluble nuclear proteins was stored at −80 °C. Protein concentrations were quantified using a Lowry assay and normalized accordingly. Samples were separated by SDS-PAGE and immunoblotted using the following antibodies: goat COX2 , mouse β-actin , rabbit p65 , rabbit lamin A/C . Secondary antibodies, anti-mouse IgG HRP conjugate  and anti-rabbit IgG HRP conjugate  were used. Development was performed with the Detection kit Pierce® ECL Western Blotting Substrate (Thermo Scientific). Stripping buffer was used to allow for the detection of all the above-mentioned antigens on the same membrane.To prepare whole cell extracts, cells were lysed with RIPA buffer, centrifuged  and the supernatant stored at −20 °C. For the preparation of nuclear and cytoplasmic extracts, cells were lysed in buffer A  to the centrifuged beads and heated with shaking at 65 °C. 240 µL of the supernatant was recovered and this step was repeated. 470 µL of elution buffer was now added to the input samples, and treated as the ChIP samples from now on. Samples were treated with RNAse A for 30 min at 37 °C. Crosslinks were reversed by incubating the samples overnight at 65 °C. Samples were than treated with proteinase K . The DNA was than isolated by phenol/chloroform precipitation, and resuspended in 60 µL nuclease free water. Purified DNA (3 µL) was amplified using human COX-2 promoter primers  using a thermocycler program . Samples were run on a 2 % agarose gel using ethidium bromide to visualize a 150-bp product for NRE1 and a 165-bp product for NRE2. For the mouse NRE site, we had technical difficulties in amplifying a single PCR product (data not shown), thus we used Taqman chemistry, which combines specific primers and probes to prevent the formation of unspecific products and primer dimers. Thus, chromatin precipitation analysis of the mouse NRE site was performed using quantitative-reverse-transcription PCR (qPCR), and the results are shown as fold change to the unstimulated p65 precipitated chromatin, after normalization to the input samples. The sequences for the Taqman reagents are: forward 5′-AGACTGCGCCCCAGT-3′, reverse 5′-CCGGGATCTAAGGTCCTAACTAAGG-3′, probe 5′-GGGAGAGGTGAGGGGAT-3′). The following program was used 50 °C for 2 min, followed by 95 °C for 10 min; then 39 cycles of 95 °C for 15 s followed by 60 °C for 60 s, in the 7900HT Fast Real-Time PCR System (Life Technologies).Chromatin immunoprecipitation (ChIP) assays were performed as previously described with some modifications . HEK293 All transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The empty vector used for cloning is the pGluc vector (NEB) driven by a minimal promoter . The pGlr as a function of the TF concentration can be derived using a thermostatistical approach. It is useful to rescale the concentration of the relevant TF with its dissociation constant. Thus, a dimensionless, relative concentration variable [TF]rel is obtained rel has to be modeled separately , the fact that in the thermostatistical model the exponent n is higher for the AND gate than for the 1-site model suggests that the AND gate exhibits more switch-like behavior relative to the promoter with a single binding site. In doing so, the analytical thermostatistical approach is consistent and complementary to the numerical kinetic modeling approach described below.We see that To quantitatively analyze and predict steady-state dynamics of the COX2 gene expression under different modes of regulation by a transcriptional factor , we developed three general kinetic models that describe: (1) a promoter that is regulated by a transcriptional factor through a single TF-Promoter binding site; (2) a promoter regulated by a transcriptional factor through two TF-Promoter binding sites following a OR gate and (3) a promoter regulated by a transcriptional factor through two TF-Promoter binding sites following an AND gate regulation. For convenience, we will refer to these models as the “1-site”, “2-site OR-gate” and “2-site AND-gate” models, respectively. These models are schematically illustrated in Fig. An optimal modeling strategy keeps the model simple, yet biologically relevant and importantly capable of generating meaningful predictions. To this end, the models formulated are kept to minimal details, containing several biologically reasonable assumptions and simplifications: (1) The 1-site model is straightforward. Binding of the TF to its promoter follows mass-action kinetic laws of simple association/dissociation. We assume that the promoter becomes active and transcription is ON as soon as the binding site is occupied, and turned OFF when the TF dissociates from the promoter. (2) In the 2-site models, we assumed that binding between the TF and two sites occurs independently: the association/dissociate rates between the TF and either of the binding sites are the same when both sites are initially unoccupied, or when one of them is already occupied by the TF. In the OR gate model, transcription is ON as long as at least one site is occupied whereas in the AND gate model, transcription is ON only if both sites are occupied.−12 L [E. coli these have been measured for the lac promoter and its TF regulators (repressor and activator) which is about 0.0027 nM−1 s−1 for association rate [−1 for dissociation rate [−1 s−1 and dissociation rate = 0.002 s−1 in the 2-site models, and 0.02 s−1 for dissociation rates in the 1-site model as reasonable values.NFκB p65 copies number has been measured in TNF-stimulated T-leukemia cell lines to be 120,000 . Assumin−12 L , the copion rate  and abouion rate . For ourThe same parameter values as given in Table β is the offset level, Rmax is the maximal response, x50 is the half-maximal threshold and H is the Hill coefficient which indicates strength of the switch. Fitting was implemented with the NonlinearModelFit function in Mathematica 8.The steepness of a dose–response curve is often indicated by the Hill coefficient value resulting from fitting the curve with a Hill function . The folSequences and species alignment for the COX2 promoters were obtained from the University of California Santa Cruz Genome Browser .Tumor necrosis factor α (TNFα) was obtained from Sigma (Ireland).n = 3 independent times unless otherwise indicated and data are expressed as the mean ± SEM. Statistical significance was calculated by Student’s t test for the comparison of two datasets or one-way ANOVA followed by Bonferroni’s multiple comparison test  for more than two datasets. P < 0.05 was considered statistically significant.All experiments were carried out a minimum of cis-regulatory response elements.The inflammatory stimulus TNFα induces nuclear localization of p65, one of the main subunits of NFκB , 18). WeIt has been shown that a single nucleotide can determine which co-factors are recruited to the NFκB response element (NRE) , and speWe next compare the transcriptional activities of the human COX2 promoter to the mouse equivalent . These experiments are performed in HEK293 cells, so that the responses of the human and mouse promoters to the same inflammatory stimulus can be compared. Our data indicate that the response from the mouse COX2 promoter is linear  to a promoter containing either one binding site . On the contrary, the same analysis for the 2-site AND gate model consistently displayed the bell-shaped activity dependence across the sets despite having variability in the activity amplitude and optimal peak . Calculating the mean curve and curves within one standard deviation further confirmed the robustness of the predicted bell-shape response . Taken together, these ensemble simulations demonstrate that the prozone effect is an exclusive and robust feature of the 2-site AND gate model.To experimentally test the predicted prozone effect for various expression systems, we use a sequence from the interleukin-6 promoter which was previously shown to contain only one functional NRE  as exampOur mathematical model predicts that a promoter with a single TFBS is likely to produce a linear increase in promoter activity Fig. a, whereaWhen there is a graded signal output to an input, the cell requires a large change in the inflammatory stimulus (input) to achieve a maximum expression of COX2  can also contribute to species-specific expression patterns of the same gene [Species-specific differential regulatory mechanisms of gene expression, as demonstrated here for COX2, may contribute to the recently reported discordance observed between human and mouse models of inflammatory diseases . The stuame gene . Speciesame gene . Our worCandidate drugs are usually developed and tested in mouse models, but have very poor success rates when moved to clinical trials . COX2 inIn conclusion, the logic AND gate comprised of the two NREs characterized in this study represents a novel regulatory mechanism for the multitude regulation modes exhibited by NFκB , 48. OurSupplementary material 1 (PDF 2368 kb)Below is the link to the electronic supplementary material.


Plant Epigenetics: From Genotype to Phenotype Keystone Symposium held in Taos, New Mexico, USA, 15–19 February, 2016.A report on the  Plant Epigenetics: From Genotype to Phenotype Keystone Symposium. This meeting marked the latest contribution to the plant epigenetics field by the Epigenomics of Plants International Consortium, a US National Science Foundation-funded group that aims to disseminate plant epigenetics information to support and elevate this community of researchers. The conference began with a keynote address from Steven Jacobsen , who provided an introduction and framework for the rest of the presenters to build from. Oral presentations ranged from established leaders in the field, junior faculty, and postdoctoral researchers, and the meeting included lively poster sessions filled with a wealth of unpublished data and associated models. Presentations ranged in organism, approach, and specific topic; however, seven strong themes stood out.In February 2016, the plant epigenetics community assembled in Taos, New Mexico, for the Arabidopsis plants. She produced vigor mimics that are not hybrids, but have many of the same benefits. In addition to investigating the underlying mechanisms, several presentations focused on using epigenomic markers for agricultural improvement. Felix Seifert  discussed his research on maize heterosis, using small RNA distribution patterns to predict vigor relationships. Jon Reinders (DuPont Pioneer) used DNA methylation patterns as markers to predict key phenotypes in maize breeding programs.The most notable trend at this meeting was how, on many fronts, this field has recently deciphered complex epigenetic inheritance patterns at the molecular chromatin level, particularly for agricultural traits. For example, Robert Martienssen (Cold Spring Harbor Laboratory) identified the epiallele responsible for poor fruit production in the oil palm. His group generated an early assay to genotype palms before transplantation, saving valuable land resources for those trees that will produce high-quality fruit. Elizabeth Dennis  demonstrated that hybrid vigor acts on key metabolic pathways early in development to produce increased energy for trans-generational effect and toward epigenetic regulation within a single generation. Stress responses are being investigated at the single gene level and at the whole chromosome level with the formation and disassociation of heterochromatic chromocenters. Ortrun Mittelsten Scheid  demonstrated chromocenter dissociation during high temperature stress, and the number of days required to reset the normal organization of heterochromatin. Similarly, Fredy Barneche  demonstrated light-induced chromocenter formation on de-etiolation, and the role of H1 linker histone variation in this process. Switching to plant defense (perhaps plant offense would be a more appropriate term), Claude Becker  showed how certain plants produce and exude chemical compounds that are converted in the soil into histone deacetylase inhibitors. These compounds inhibit the growth of nearby competitor plants—a process called allelopathy. Claude determined which genes and pathways are perturbed in the affected plants.A second notable trend was the focus on the epigenetic regulation of stress responses, shifting away from a One stress-related topic that received considerable attention at the meeting was priming, whereby a plant is more resistant to a stressor if it has previously encountered that same stress. Priming occurs within a single generation and constitutes an epigenetic memory that is being leveraged for crop improvement. Zoya Avramova (University of Nebraska) discussed the transcriptional memory of drought on particular stress response genes, while Anna Amtmann  investigated osmotic and salt stress. She showed that the initial priming stress must occur at a specific young developmental stage, and discussed the histone deacetylation necessary for the priming response.FLOWERING LOCUS C (FLC) gene, and a long non-coding RNA produced from this same locus responsible for the regulation of FLC. They performed cutting-edge single-molecule fluorescence in situ hybridization that followed the pairing and expression dynamics of the key regulatory FLC locus through the prolonged response to cold required for vernalization. Ido Keren (SUNY-Stony Brook) researched a histone deubiquitinase that acts on FLC when the gene is activated, while the memory of cold treatment at FLC is governed by histone H3 trimethylation of lysine 27(H3K27me3). Iva Mozgová, a postdoctoral fellow with Lars Henning  researched the role of the polycomb repressive complex proteins that initiate the H3K27me3 mark in regulating somatic embryogenesis. Toshiro Ito  investigated the timed induction of H3K27me3 in floral stem cells and in flower development. Both Toshiro and Doris Wagner (University of Pennsylvania) discussed the mechanism by which the chromatin modifiers that direct H3K27me3 are recruited or evicted from their target loci. They elegantly demonstrated that developmentally induced transcription factors control this recruitment and eviction.The epigenetic regulation of genes underlies the more complex inheritance patterns of traits involved in crop improvement and stress tolerance. Several presentations focused on the epigenetic regulation of single genes, which drive cellular memory of the environment or developmental timing. Caroline Dean’s lab  discussed the memory of winter held at the chromatin level that is responsible for vernalization, the process by which prolonged exposure to cold temperatures promotes flowering. Her lab investigated the timing of flowering controlled by the b1 locus, which is dependent on a series of seven tandem repeats far upstream of the b1 coding region. Maike investigated the chromatin structure of the repeats and their regulation by proteins involved in RNA-directed DNA methylation (RdDM). Karen McGinnis (Florida State University) is studying the same RdDM mutants in maize; these produce striking phenotypes that she has connected to regulation by the phytohormone abscisic acid and altered nucleosome positioning. Mario Arteaga Vázquez  also spoke about paramutation at the b1 locus, and then transitioned his research to examples of paramutation in Marchantia, a liverwort and rudimentary land plant that is an up-and-coming system for plant molecular biology.The most perplexing epigenetic gene regulation is paramutation, whereby the repressed state of an allele can be transferred to an active allele. Maike Stam  discussed her research on paramutation at the maize Arabidopsis reference strain and found that, within this one strain (not a hybrid), one NOR is transcriptionally active while the other is silenced. Fernando Rabanal  came to a similar conclusion with a different quantitative trait locus-based deep sequencing approach. Together, Rabanal and Pikaard demonstrated that the entire NOR operates as a single unit that drives the hierarchical dominance relationships between NORs, a conceptual breakthrough in the nucleolar dominance field.A major breakthrough discussed at the meeting was the distinction between active and inactive nucleolar organizing regions (NORs) within a single ecotype. When hybrids are produced, one set of NORs is dominant to the other set, creating a phenomenon called nucleolar dominance. Craig Pikaard’s laboratory (University of Indiana) mapped the ribosomal RNA variants within the atxr5/6 mutants occurs due to a failure to resolve conflicts when both transcription and replication occur in heterochromatin. Scott Michaels (Indiana University) found nuclear repair pockets in the atxr5/6 mutants responsible for repairing DNA breaks. He speculated that these pockets provide a sequestered chromosome repair microenvironment that provides access for repair proteins and excludes other chromatin modifiers.The epigenetic regulation of crop traits, stress response, and more broadly gene regulation manifest at the molecular level as changes in chromatin structure that can provide a memory of a transcriptional state. A major focal point of the presentations converged on how chromatin structures and domains are established and molecularly delineated, with particular emphasis on the associated histone dynamics that control chromatin structure. Steven Jacobsen discussed the establishment of histone H3 monomethylation of lysine 27 (H3K27me1) chromatin mark by the ATXR5 and ATXR6 proteins. Failure of ATXR5/6 to mark heterochromatin with H3K27me1 results in DNA overreplication. This activates DNA repair pathways that suppress the overreplication-induced DNA damage. He described his model of how overreplication in the Two presentations focused on the idea that certain histones and/or modifications are mutually exclusive (not on the same nucleosome). Fred Berger  focused on the histone variant H2A.W, which is plant-specific and concentrates at the pericentromere. He showed that mononucleosome isolation could identify mutually exclusive histone marks that divide the chromosome into distinct regulatory domains. In parallel, Crisanto Gutiérrez  used live fluorescent reporters to demonstrate that dividing cells have histone H3.1, but this is mutually exclusive with histone H3.3, which replaces H1.1 in the last cell cycle. Two other presentations centered on how histone variants are targeted to specific regions of the chromosome. Inna Lermontova  discussed a factor responsible for directing the centromeric histone H3 variant to the centromere core for assembly, and that this process operates in a cell cycle-specific manner. Paul Fransz  discussed the organization of nuclear chromosomal territories, the association of centromeric DNA with the nuclear envelope, and mutants that perturb this organization.The DNA methylation mark in plants is transmitted from one generation to the next, propagating epigenetic information in a much more direct route compared to animals. Several presentations focused on the mechanism and evolutionary implications of cytosine DNA methylation. Robert Schmitz (University of Georgia) presented an evolutionary history of gene body methylation. Using data from a number of plant species, he focused on the evolutionary function of methylation within genes. Nathan Springer (University of Minnesota) discussed DNA methylation that accumulates in the CHH context (where H is any base but G) in the complex maize genome. Nathan demonstrated that most CHH methylation in maize is at islands that mark the boundary between heterochromatin and a neighboring gene. Along with DNA methylation, Xiaofeng Cao (Chinese Academy of Sciences) researched how the histone demethylase protein IBM1 functions to remove heterochromatic histone marks from genes and restrict this mark to transposable elements, thus providing a distinct boundary force to segregate gene chromatin states in complex genomes such as rice and maize.A key topic addressed in many presentations focused on the evolutionary dynamics of DNA methylation. Nathan Springer presented data demonstrating that methylation within the maize genome is stability-inherited over long periods of time. Magnus Nordborg’s lab  investigated the natural variation of DNA methylation and its correlation with the environment. His research demonstrated that many epigenetic changes between ecotypes have a genetic (sequence) root cause, as single-nucleotide polymorphisms drive gene body methylation from a distance. Frank Johannes  demonstrated that the rate of spontaneous epimutations is highest in the CG sequence context, as these sites are not retargeted by RdDM. He proposed the interesting argument that CG epimutations can be viewed as a molecular clock. After methylation, Jonathan Cahn from Ryan Lister’s lab  identified methylation-binding proteins, with the hope of characterizing how the cell interprets the DNA methylation signal to dictate expression changes, while Jian-Kang Zhu (Purdue University/Shanghai Center for Plant Stress Biology) discussed the specific biochemical steps associated with the removal of DNA methylation via DNA glycosylase enzymes.The function of small RNAs was investigated in several presentations. Jixian Zhai from the Jacobsen laboratory  described his recent sequencing capture of RNA polymerase IV (Pol IV) transcripts, which are cleaved into 24-nucleotide small RNAs that participate in RdDM. Jixian showed that the Pol IV transcripts are very short, and then presented his model proposing that each individual Pol IV transcript is cleaved into one small RNA. Andrzej Wierzbicki (University of Michigan) discussed his lab’s improved methods of identifying RNA polymerase V (Pol V) scaffolding transcripts. He showed that Pol V uses internal promoters and produces bidirectional short transcripts. Lastly, two presentations focused on the mobility of small RNAs. Mathew Lewsey from the Ecker Lab  demonstrated that Pol IV small RNA production does not need to occur in the same cell targeted by RdDM, as endogenous Pol IV small RNAs are mobile across graft junctions and target RdDM at many different transposable element regions in recipient tissues. In addition, R. Keith Slotkin (The Ohio State University) showed transposable element small RNA movement within the pollen grain from terminally differentiated pollen vegetative cells into the sperm cells, suggesting that small RNAs may function between one generation and the next.from phenotype to genotype was also explored, providing breakthroughs in the explanation of complex phenotypic phenomena, such as nucleolar dominance and stress priming. Working both from the bottom (mechanism) up and top (phenotype) down is a major strength of the plant epigenetics community, and this dual approach will enable high-impact science and, more importantly, continued breakthroughs on complex traits.The topics presented at this meeting were highly variable in nature, ranging from the complex regulation of stress and heterosis to the precise mechanisms of RdDM. The subtitle of the meeting “from genotype to phenotype” was investigated at length, but the more complex route


The fine three-dimensional structure of the nucleosomal fiber has remained elusive to genome-wide chromosome conformation capture (3C) approaches. A new study mapping contacts at the single nucleosome level (Micro-C) reveals topological interacting domains along budding yeast chromosomes. These domains encompass one to five consecutive genes and are delimited by highly active promoters. Saccharomyces cerevisiae.Over the past ten years, genome-wide derivatives of the chromosome conformation capture approach  two alternative structures for the nucleosomal fiber have been proposed . These et al. . In addiIn line with the possibility of a polymorphic structure, Hsieh et al. show that several factors can change what they describe as the ‘compaction’ of the chromosomal fiber. Here, the compaction is simply defined as the ratio of long-range over short-range contacts (with short-range being defined as closer than 300 bp). The compaction of each gene was found to be correlated with its transcriptional activity, and the decrease in compaction observed for highly transcribed genes might be attributed to the local disruption of the nucleosomal fiber by active RNA polymerase(s). Consistent with this finding, genes that were upregulated following a diamide treatment were convincingly shown to be less compacted.S. cerevisiae mutants in which chromatin structure is altered. Micro-C confirmed the role of the RSC chromatin remodeling complex and the cohesin complex in chromatin structuring, with defects in these complexes being associated with increased gene compaction. Conversely, other chromatin mutants, such as those with defects in Mediator or the histone deacetylase Rpd3, appeared to induce a significantly reduced level of gene compaction. This change is, however, accompanied by only small changes in transcriptional activity. The effect of these ‘structural mutants’ suggests that changes in compaction can also be modulated by factors other than the progression of DNA polymerase along the genes, either by the potential formation of transient long-range loops or by the modification of inter-nucleosomal interactions. To test for the latter specifically, the authors used variants of histone H4 that were previously characterized in vitro as having a direct effect on nucleosome array folding. Micro-C confirmed the important role of the H4 N-terminal tail in vivo.To investigate the mechanistic basis of gene compaction further, the authors set out to repeat the Micro-C experiment in 23 Finally, the authors took advantage of their high-resolution assay to search for the promoter and terminator gene loops identified previously using a 3C approach [S. cerevisiae at an unprecedented level of resolution, refining our understanding of chromosome organization in this species. Obviously, larger genomes can be investigated next and, if successful, this work will provide answers to hotly debated questions in the field.Micro-C provides an additional tool to investigate the structure of chromatin at fine resolution. In the pioneering study by Hsieh and colleagues, the authors describe gene domains in