The autoimmune regulator (AIRE) is expressed in the thymus, particularly in thymic medullary epithelial cells (mTECs), and is required for the ectopic expression of a diverse range of peripheral tissue antigens by mTECs, facilitating their ability to perform negative selection of auto-reactive immature T-cells. The expression profile of peripheral tissue antigens is affected not only by AIRE deficiency but also with variation of AIRE activity in the thymus.AIRE transcription start site including AIRE minimal promoter for single nucleotide polymorphism (SNPs) and identified two SNPs -655R (rs117557896) and -230Y (rs751032) respectively. To study the effect of these variations on AIRE promoter activity we generated a Flp-In host cell line which was stably transfected with a single copy of the reporter vector. Relative promoter activity was estimated by comparing the luciferase specific activity for lysates of the different reporter AIRE promoter-reporter gene constructs including AIRE-655G AIRE-230C, AIRE-655G AIRE-230T and AIRE-655A AIRE-230C. The analysis showed that the commonest haplotype AIRE-655G AIRE-230C has the highest luciferase specific activity (p<0.001). Whereas AIRE-655G AIRE-230T has a luciferase specific activity value that approaches null. Both AIRE promoter polymorphic sites have one allele that forms a CpG methylation site which we determined can be methylated in methylation assays using the M.SssI CpG methyltransferase.Therefore we screened 591bp upstream of the AIRE-230Y is in a conserved region of the promoter and is adjacent to a predicted WT1 transcription factor binding site, suggesting that AIRE-230Y affects AIRE expression by influencing the binding of biochemical factors to this region. Our findings show that AIRE-655GAIRE-230T haplotype could dramatically alter AIRE transcription and so have an effect on the process of negative selection and affect susceptibility to autoimmune conditions. AIRE) gene plays a key role in this process of negative selection by regulating the promiscuous expression of PTAs in mTECs [Self tolerance processes in the thymus play a key role in thymocyte maturation and in preventing the development of auto-reactive T-cells, and consequential autoimmune reactions. Negative selection of immature thymocytes with auto-reactive potential is an important part of this process and in order to be effective, requires the body’s repertoire of self-antigens to be presented to these immature cells. This task is carried out in the medullar region of the thymus, predominantly by the presentation of peripheral tissue antigens (PTAs) by medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) to immature thymocytes . The Autin mTECs , 2.AIRE gene [AIRE and susceptibility to these secondary autoimmune conditions.Autoimmune Polyglandular syndrome 1 (APS-1) is a monogenic disorder caused by loss-of-function mutations in the IRE gene , 4, charIRE gene . APS-1 pIRE gene , suggestAire knockout mice observed that both T-cell deletion in the thymus reduced and incidence of diabetes increased as the amount of Aire decreased [in vitro and in vivo [AIRE gene, affected its transcriptional activity, and thus could have a functional downstream effect.The level of Aire expression has been shown to have functional effects in several studies using murine models , 8. An eecreased . Other s in vivo . Here, wAIRE promoter was split into two regions for screening and polymerase chain reaction (PCR) employed to amplify each region. The first region  was amplified using primers EIF3 (CTCAGAGAAGGAAAGGACTTGCC) and PF REV (TCGCTCTCGGGAAGTCTC) and the second (Region 2: 478bp) using primers HP FOR (GTCTCGGCTTTGCCCCATTCGA) and HP REV (TGCAGCCTCAGAAGCCGGCGTA). For each region, the PCR product from an unknown sample was mixed 50:50 with the PCR product from a control sample of known sequence and PCR products heteroduplexed by heating to 95°C for 5 minutes then reducing the temperature by 0.1°C every 4 seconds until 25°C was reached. Heteroduplexed samples were analysed using a transgenomic WAVE nucleic acid fragment analysis system. Region 1 samples were analysed at temperatures of 62°C, 64°C and 65.4°C and Region 2 samples at 63.3°C, 64.8°C, 65.9°C and 68.6°C.The first 591bp of the Genomic DNA samples from healthy humans were extracted by standard methods from blood samples obtained from the Trent Blood Transfusion Service (Sheffield). All individuals gave informed consent in accordance with approval by the Trent Multi-Centre Research Ethics Committee, Derby.AIRE-230Y SNP (single nucleotide polymorphism) [Our samples had previously been genotyped for the orphism) . The -65AIRE promoter haplotypes under study were PCR amplified using primers EIF3 and PIR (ACGCCCCTGGCTCCTTGTG), ligated into a pCR2.1 TA vector  and then transformed into TOP10 competent cells (Invitrogen). Each promoter variant was subsequently sub-cloned upstream of the firefly luciferase reporter gene in the pGL3-basic vector  to generate pGL3-P(haplotype) AIRE reporter constructs for transient reporter gene assays. The AIRE promoter and firefly luciferase construct  were then sub-cloned into the pcDNA5/FRT expression vector (Invitrogen), with the AIRE promoter replacing the CMV promoter. These pcDNA5/FRT/P(haplotype)AIRE-firefly luciferase reporter constructs were used for the stable reporter gene assays. Direct sequencing was used to confirm the identity of the promoters after each cloning step.The variant TEC 1A3 cells  were maintained in RPMI 1640 medium (Lonza) supplemented with 10% foetal calf serum (Lonza) and 1% penicillin/streptomycin. Previous research studying the AIRE promoter has been carried out using this cell line .LacZeo vector following the Flp-In system manual . In brief, this process involved the transfection of TEC 1A3 cells with pFRT/LacZeo vector using FuGENE6 reagent (Roche). Cells were grown in media supplemented with 250μg/ml Zeocin to select for those containing the pFRT/LacZeo vector integrated into the genome, with cloning rings used to isolate foci to collect individual cell lines. Southern blotting was used to screen the different cell lines to identify those containing only a single integrant of the pFRT/LacZeo vector. Relative expression efficiency from the FRT region for each TEC 1A3/pFRT/LacZeo cell line generated was estimated by comparing β-galactosidase activity using the β-gal assay kit (Invitrogen). The cell line with the highest β-galactosidase activity was chosen to be the TEC 1A3 host cell line for subsequent reactions.A TEC 1A3 cell line containing a single FRT (Flp recombinase target site) was generated using the pFRT/AIRE-firefly luciferase reporter constructs (20:1 ratio) using FuGENE6 reagent (Roche) per transfection. Cells were maintained in media supplemented with hygromycin B at 150μg/ml to select for those containing integrated pcDNA5/FRT/P(haplotype)AIRE-firefly luciferase reporter constructs.These TEC 1A3 Flp-In host cells were co-transfected with pOG44 with a pcDNA5/FRT/P(haplotype)24 hours after seeding into 6-well plates, cells from each promoter haplotype variant of the reporter gene cell lines were washed with PBS before lysis with Glo lysis buffer (Promega). Lysates were assayed in triplicate for luciferase expression using the Bright-Glo assay kit (Promega), using a known concentration of recombinant luciferase (Promega) as a reference sample to normalise luminescence data. Protein concentration (μg/ml) of lysates was quantified in triplicate using the BCA protein assay kit (Perbio). Promoter efficiency was quantified by comparing specific activity  for the different promoter haplotypes.Difference in relative expression efficiency across the different promoter haplotypes were assessed by one-way analysis of variance (ANOVA) and used Tukey-Kramer multiple comparison test to assess the differences between the individual haplotypes. Analyses were performed using GraphPad PRISM version 5.5’- CGTGTTAGTTTAAGATGGTGAGG-3’ and APLR BSP 5’- CAAATCTCGAACCCGAAACACTA-3’, following the manufacturers recommended thermocycling program with an annealing temperature of 50°C and 35 cycles of amplification. The resultant PCR product was ligated into pCR4-TOPO-TA sequencing vector, cloned into TOP10 chemically competent E.coli (Invitrogen). The cloned vectors extracted and purified from overnight bacterial cultures using QIAprep Spin miniprep kit and finally sequenced to confirm methylation of CpG sites at -655/-654 and at -230/-229.The pcDNA5/FRT/P(GC)AIRE-firefly luciferase construct was first methylated using the M.SssI CpG methyltransferase (N.E.B) and then subjected to the bisulphite conversion process and subsequent DNA clean-up using the EpiMark Bisulpfite conversion kit (N.E.B). This DNA was PCR amplified using Epimark Hotstart Taq polymerase (N.E.B) and the primers APF BSP http://www.ebi.ac.uk/Tools/msa/mafft/ and The sequence corresponding to exon 1 and the first 1kb upstream of exon 1 of AIRE in six different mammalian species  were aligned using the MAFFT alignment program using default parameters (Multiple Alignment using Fast Fourier Transform)  http://wwww.dcode.org.Predicted transcription factor binding sites were identified using rVista 2.0  to analyse the human and chimpanzee sequences which had been aligned using zpicture  www.dcodAIRE transcription start site (TSS) in 32 human control DNA samples using transgenomic WAVE dHPLC (denaturing high performance liquid chromatography). We observed two different novel peaks which were identified by direct sequencing as the AIRE-655R (rs117557896) and AIRE-230Y (rs751032) polymorphisms, both of which have been reported in dbSNP. AIRE-230Y is found within the minimal AIRE promoter region [AIRE-655R SNP, and complement the genotype data we had previously obtained for the AIRE-230Y SNP [We screened the first 591bp upstream of the E-655R rs7557896 ar region  Fig 1)..AIRE tra230Y rs7532 polymoAIRE-230 and AIRE-655 polymorphisms we genotyped 161 control Caucasian samples for the two polymorphisms  whereas AIRE-230 was informative with the frequency of AIRE-230T allele of 0.10. The 1000 genomes project [AIRE-655R, but our data for AIRE-230Y is in keeping with the 1000 genomes project data, with the C allele reported at a frequency of 0.885 and the T allele at 0.115 within the GBR sub-population. These frequencies are almost identical to the overall allele frequencies when accounting for all populations. However, there are differences in allele frequencies between the reported subpopulations, with the African population reported as C:0.995 T:0.006 at one end of the scale and the Asian population as C:0.718 T:0.282 on the other end.To determine the allelic distribution of orphisms . We foun project  does notAIRE-655G AIRE-230C with frequency of 0.89 whereas AIRE-655G AIRE-230T and AIRE-655A AIRE-230C have frequencies of 0.10 and 0.01 respectively. AIRE-655A AIRE-230T haplotype was not found in our cohort and therefore not included the reporter gene assay.In our samples, the common haplotype was Reporter gene assays are commonly used to assess the effect of polymorphisms on the activity of genetic regulatory regions. Unfortunately, when performing these assays using transient transfection of the reporter vector, there are many variables that can influence the observed activity besides the genetic variation under investigation . TherefoAIRE promoter/firefly luciferase plasmid construct into the host TEC 1A3 Flp-In cell lines, relative promoter activity was estimated by comparing the luciferase specific activity  for 12 lysates of each different reporter cell lines  upstream of AIRE was compared across six different mammalian species by multiple sequence alignment using MFATT . This reAIRE gene, and determined the effect on transcriptional activity for the different alleles of two naturally occurring polymorphisms found within this region.In this study we developed a reporter gene assay system to investigate polymorphisms in the region immediately upstream of the The use of a stably integrated reporter gene assay system has minimised any effect on our final results that might have been caused by experimental variables such as the amount of DNA; the amount of transfection control vector; the method of transfection; and the purity of transfected DNA, all of which have been reported to affect results from transient transfection reporter gene assays . In addiAIRE promoter region, it is not entirely unsurprising that this polymorphism would alter expression the most, we would expect a partial reduction in AIRE expression comparable to those observed in mice as a result of the -230T allele, especially for individuals homozygous for -230T. In fact, phenotypic differences, in terms of incidence of autoimmune activity and changes in PTA expression, have been observed in murine studies where Aire expression has been partially reduced (50–63%) [AIRE -230Y SNP has the potential to affect AIRE regulated promiscuous gene expression, and consequently have an effect of negative selection and autoimmune susceptibility. It is also important to mention that the highest AIRE promoter activities are given by the commonest haplotype AIRE-655G AIRE-230C  and -230T (GT promoter) alleles caused a reduction in luciferase expression compared to the expression observed for the most frequent alleles (GC promoter) for these SNPS, it was the -230T allele that had the greatest effect, with luciferase specific activity reduced by approximately 95%. As the -230Y SNP and not the -655R SNP is found within the minimal (50–63%) –9. We woIRE-230C . HoweverThe -230Y SNP is located in a gap in the promoter sequence that lies between two confirmed transcription factor binding sites (upstream of a NF-Y binding site and downstream of an Ets binding site) , 14. ThiAIRE promoter, especially as this site is adjacent to a CpG site that was found to be differentially methylated in different cell lines. This is of particular note, as demethylation of the AIRE promoter was found to play a major role in up-regulating the expression of AIRE in TEC 1A3 cells [In addition to the change in sequence brought about by the -230Y SNP, there is a CpG methylation site  that is A3 cells , yet in AIRE gene and susceptibility to alopecia areata and vitiligo, another autoimmune disorder associated with APS-1 [AIRE has been found to be a determinant for predisposition to rheumatoid arthritis in the Japanese population [AIRE polymorphisms in large cohorts of patients with autoimmune diseases, in particular those associated with APS-1.These findings are in keeping with our previously identified genetic association between polymorphisms in the th APS-1 , 18, 19.pulation . It is tS1 FigClustalW output displayed, with nucleotide specific colour highlighting added to the output. Above the alignments we have listed the tested SNPs, the CpG sites and confirmed TFBS that have been identified on the human sequence , 14. The(DOC)Click here for additional data file.S1 Table161 control samples were tested with frequencies of haplotypes formed by the two polymorphisms calculated.(DOCX)Click here for additional data file.


SUMOylation, as part of the epigenetic regulation of transcription, has been intensively studied in lower eukaryotes that contain only a single SUMO protein; however, the functions of SUMOylation during mammalian epigenetic transcriptional regulation are largely uncharacterized. Mammals express three major SUMO paralogues: SUMO-1, SUMO-2, and SUMO-3 . Herpesviruses, including Kaposi’s sarcoma associated herpesvirus (KSHV), seem to have evolved mechanisms that directly or indirectly modulate the SUMO machinery in order to evade host immune surveillance, thus advancing their survival. Interestingly, KSHV encodes a SUMO E3 ligase, K-bZIP, with specificity toward SUMO-2/3 and is an excellent model for investigating the global functional differences between SUMO paralogues.We investigated the effect of experimental herpesvirus reactivation in a KSHV infected B lymphoma cell line on genomic SUMO-1 and SUMO-2/3 binding profiles together with the potential role of chromatin SUMOylation in transcription regulation. This was carried out via high-throughput sequencing analysis. Interestingly, chromatin immunoprecipitation sequencing (ChIP-seq) experiments showed that KSHV reactivation is accompanied by a significant increase in SUMO-2/3 modification around promoter regions, but SUMO-1 enrichment was absent. Expression profiling revealed that the SUMO-2/3 targeted genes are primarily highly transcribed genes that show no expression changes during viral reactivation. Gene ontology analysis further showed that these genes are involved in cellular immune responses and cytokine signaling. High-throughput annotation of SUMO occupancy of transcription factor binding sites (TFBS) pinpointed the presence of three master regulators of immune responses, IRF-1, IRF-2, and IRF-7, as potential SUMO-2/3 targeted transcriptional factors after KSHV reactivation.Our study is the first to identify differential genome-wide SUMO modifications between SUMO paralogues during herpesvirus reactivation. Our findings indicate that SUMO-2/3 modification near protein-coding gene promoters occurs in order to maintain host immune-related gene unaltered during viral reactivation. SUMOylation was initially identified as a reversible post-translational modification that controls a variety of cellular processes, including cellular signal transduction, replication, chromosome segregation, and DNA repair –3. The gherpesviridae and have been inevitably linked to counteracting the host’s antiviral properties. SUMOylation has been found to affect most of the immediate-early and early proteins of herpesviruses, which are usually transcriptional factors. BZLF1 and Rta of Epstein-Barr virus (EBV)   and against SUMO-2/3 , as well as rabbit non-immune serum IgG , were used for the ChIP and ChIP-reChIP assays. Rabbit monoclone antibodies specific against IRF-7  was used for the ChIP-reChIP assays.After 12 hours of K-Rta induction to allow KSHV reactivation in the TREx-F3H3-K-Rta BCBL-1 cell line, 1 × 107 cells that had been resuspended in 30 ul of ddH2O and ChIP-seq library construction was then carried by following the sample preparation protocol from Illumina. Short reads (100 bp) from both ends (paired-end sequencing) of size-selected (400 bp) DNA fragments were selected and subjected to high throughput sequencing on an Illumina® Genome AnalyzerII System. The ChIP-Seq data was aligned onto the human genome hg19 build using UCSC. Around 6 × 107 reads were mapped for each sample after filtering and quality control (QC) were carried out. In this study we used the enriched region detection method of Avadis NGS  to localize potential protein binding sites in order to delineated the SUMO-1 and SUMO-2/3 binding patterns.For ChIP-Seq assay, ChIPed DNA was prepared from 5 × 10The binding sites were verified by SYBR® Green Based qPCR using a CFX connect™ real-time PCR detection system . Specific primer sets were designed around the identified binding sites for this purpose.II. Sequencing reads were first trimmed with human ribosomal RNA sequences  by Bowtie (version 1.0.0) with default parameters and then aligned the high quality reads to human reference genome hg19 using TopHat (version 2.0.8b) with Bowtie version 2.1.0 and samtools (version 0.1.9) with transcriptome information obtained from Ensembl Release 70 and NonCode v3.0. The transcript abundances were estimated in fragments per kilobase of transcript per million mapped reads (FPKM) by Cufflinks version 2.1.1. Genes from all three samples with FPKM > 0.05 were considered to be expressed and were used for the remaining analysis. Differential gene expression of the samples (K-Rta induction for 12 and 24 hours vs. control) was analyzed by comparing FPKM. For RT-PCR, 2 μg of total RNA was reverse-transcribed using SuperScript™ III First-strand synthesis system (Invitrogen) and Oligo-dT. qPCR was carried out based on the manufacturer’s protocol .Total RNA was harvested using TRIzol reagent  from TREx-F3H3-K-Rta BCBL-1 at 12 and 24 hours after K-Rta induced viral reactivation according to the manufacturer’s instructions. RNA-seq was conducted at the Sequencing Core of National Research Program for Genomic Medicine at National Yang-Ming University VYM Genome Research Center using an Illumina Genome AnalyzerTransfected 293 T cells were collected in modified radioimmune precipitation assay (RIPA) buffer  supplemented with 1X protease inhibitor cocktail . TCLs were incubated with anti-FLAG M2-agarose  overnight at 4°C. Immune complexes were captured by protein A and protein G Sepharose beads. Beads were washed, and the bound proteins were analyzed by immunoblotting. Antibodies used for immunoblotting were anti-IRF-1 , anti-IRF-2 , anti-IRF-7 (GeneTex), anti-SUMO-2/3 antibodies.SUMO modifications of transcription regulatory proteins and chromatin modifying enzymes are linked to the epigenetic regulation of gene transcription. SUMO-1 and SUMO-2/3 have both common and distinct substrates, but their global functional roles in epigenetic regulation have not as yet been fully investigated. As mentioned earlier, DNA viruses have evolved different strategies that allow them to manipulate the SUMO pathway in a manner that helps their survival. We previously identified a KSHV lytic protein, K-bZIP, as a viral SUMO E3 ligase with specificity toward SUMO-2/3 . Using Kin vivo binding sites of SUMO-1 and SUMO-2/3 were analyzed using massively parallel chromatin immunoprecipitation in combination with high throughput sequencing (ChIP-Seq); these processes were carried out on a K-Rta-inducible KSHV infected primary effusion lymphoma (PEL) cell line, TREx-F3H3-K-Rta BCBL-1. Chromatin samples from TREx-F3H3-K-Rta BCBL-1 cells before and after K-Rta induction to allow KSHV reactivation were isolated and subjected to the ChIP assay using ChIP grade SUMO-1 and SUMO-2/3 antibodies. High-throughput sequencing was then performed to measure the binding of SUMO-1 and SUMO-2/3 from a single run of ChIP assay. Approximately the same number (6 × 107) of reads from the KSHV un-induced and induced samples were mapped to the human reference genome, hg19. Using an enrichment peak calling algorithm, we found a total of 31315 and 45846 high confidence SUMO-1 and SUMO-2/3 enrichment regions, respectively, in the non-reactivated control cells. After K-Rta induction for 12 hours, a total of 39626 SUMO-1 enrichment regions (an increase in 8 K peaks of ~1.3-fold compared to the control cells) and 86479 high confidence SUMO-2/3 enrichment regions (an increase in 40 k peaks of ~1.9-fold compared to the control cells) were identified , whereas the binding to the gene bodies themselves (transcribed regions), the transcription end sites (TESs), the regions upstream of the gene, the regions downstream of the gene and the intergenic regions were low Figure D and 2E.Typical SUMO binding sites are focal and consist of no more than a few hundred base pairs, a pattern reminiscent of the “peaks” associated with transcription factors. A large number of known SUMO conjugates in mammals are transcription factors. To predict the potential chromatin-associated transcription factors (TFs) that are SUMOylated, we annotate SUMO target sites within promoter regions in relation to transcription factor binding sites (TFBS) from the Transfac Matrix Database (v7.0) created by Biobase. This database contains 258 TFBS weight matrices that represent the potential DNA binding sites of 176 TFs across the genome. The SUMO enriched peaks for each TFBS in the promoter region were normalized with their own distribution frequency. Potential SUMO target TFs were ranked by percentage and Hampel Identifier was used to identify the TFBSs that were significantly correlated with SUMO binding . The numThe top twenty potential SUMO-1 and SUMO-2/3 target TFs before and after KSHV reactivation are listed in Tables The presence of highly enriched SUMO-2/3 binding sites around the promoter regions during viral reactivation suggest that SUMO-2/3 might be directly or indirectly targeting a large group of transcription factors during KSHV reactivation. In order to pinpoint the most important gene-regulating TFs that are targeted by SUMO-2/3 during viral reactivation, we collected genes with SUMO-2/3 targeted TFBSs at their promoter before and after viral reactivation and group them into an up-group (SUMO peaks increase >1.5X), a down-group (SUMO peaks decrease >1.5X) and a no-change-group (SUMO peaks variants within 1.5X). When we ranked the TFs by total gene number, the top 10 most important gene-regulating TFs targeted by SUMO-2/3 after KSHV reactivation could be identified Figures  and 5. IThe identification of IRF-1, IRF-2 and IRF-7 as potential SUMO-2/3 targets during KSHV reactivation suggests that the viral SUMO E3 ligase K-bZIP may be involved in this phenomenon. To address this, we first cloned all three IRFs using cDNA of BCBL-1 cells. Then 293 T cells were transiently co-transfected with Flag-tagged IRF-1, IRF-2 or IRF-7, T7-tagged SUMO-2 and SUMO-3, and HA-tagged K-bZIP; this was followed by immunoblotting or immunoprecipitation using Flag antibody. The results showed an increase in SUMO modification of IRF-1 and IRF-2 when there was overexpression of K-bZIP , low expression (FPKM 0.05 ~ <1: 6503), medium expression (FPKM 1 ~ <10: 5597), high expression (FPKM 10 ~ <100: 5390), and very high expression (FPKM >100: 564). We found that between 27% and 37% of the very high and high expression group, about 16% of the medium expression group, about 4% of the low expression group, and about 1% of the no expression group promoters were labeled by SUMO-1 or SUMO-2/3  analysis of genes that are targeted by SUMO-1 and SUMO-2/3 before and after viral reactivation using the IPA software. We found that the genes targeted by SUMO-2/3 after viral reactivation are significantly involved in several pathways related to cellular immune responses, cytokine signaling, cell growth, apoptosis and cancer Table . We furtSUMO is a multifaceted modifier of chromatin structure. SUMO modification of chromatin proteins regulates a range of cellular processes including transcription, replication, DNA repair and chromosome segregation. SUMOylation has long been believe to be associated with gene silencing or repression. However, global mapping of chromatin binding by SUMO in yeast  and DrosHerpesviruses have evolved multiple mechanisms to target SUMOylation pathways, including modulating SUMO conjugation enzymes  and deconjugation enzymes  as well as by directly targeting SUMOylated proteins . InteresThe complete sequence of the human genome was obtained more than a decade ago; nevertheless, our understanding of this genome is far from complete. The emerging concept from Encyclopedia of DNA Elements (ENCODE) is that biochemical functions of a genome can be assigned by systematically identifying the functional elements within the genome . PatternSUMO binding onto chromatin must occur via either the modification of chromatin remodeling proteins or the modification of transcription factors, both of which bind to the genome. SUMO shows focal peaks or areas of high occupancy within the promoter region near TSSs. The focal and gene-selective nature of SUMO occupancy resembles the peaks associated with transcription factors, which suggests that there is SUMO modification of TFs. Motif scanning is a powerful method to facilitate the identification of DNA binding motifs (or transcription factor binding motifs) from peaks defined by ChIP-seq. This method has been widely used to distinguish the transcription regulation of one or a few TFs. SUMO modifications are able to occur in many dozens of known TFs as well as being likely to occur in many currently unknown TFs. Using the current findings, it is probably too complex and too time consuming to carrying full scale motif scanning to identify potential SUMO target TFs. Therefore, as an alternative, we used an annotation method that directly annotates SUMO peaks in the promoter region in relation to transcription factor binding sites (TFBS). The details of this method have been submitted in another article . BrieflyThe growing links between the viral and cellular SUMO systems makes SUMO a potential target for antiviral therapy . IdentifIn summary, we found that SUMO-1 and SUMO-2/3 share a highly similar binding landscape on chromatin. They are preferentially enriched in promoter regions and are associated with highly transcribed genes. Differential chromatin-binding profiles of the SUMO paralogues are able to be observed during herpesvirus reactivation. We found that SUMO-2/3 peaks significantly increased in promoter regions during viral reactivation and this was associated with the genes that do not undergo changes in transcription level. TFs identification and GO analysis suggests that SUMO-2/3 preferentially target immune pathways during viral reactivation.Chia-Yang Cheng is the co-first author.


ChIP-seq provides a wealth of information on the approximate location of DNA-binding proteins genome-wide. It is known that the targeted motifs in most cases can be found at the peak centers. A high resolution mapping of ChIP-seq peaks could in principle allow the fine mapping of the protein constituents within protein complexes, but the current ChIP-seq analysis pipelines do not target the basepair resolution strand specific mapping of peak summits.The approach proposed here is based on i) locating regions that are bound by a sufficient number of proteins constituting a complex; ii) determining the position of the underlying motif using either a direct or a de novo motif search approach; and iii) determining the exact location of the peak summits with respect to the binding motif in a strand specific manner. We applied this method for analyzing the CTCF/cohesin complex, which holds together DNA loops. The relative positions of the constituents of the complex were determined with one-basepair estimated accuracy. Mapping the positions on a 3D model of DNA made it possible to deduce the approximate local topology of the complex that allowed us to predict how the CTCF/cohesin complex locks the DNA loops. As the positioning of the proteins was not compatible with previous models of loop closure, we proposed a plausible “double embrace” model in which the DNA loop is held together by two adjacent cohesin rings in such a way that the ring anchored by CTCF to one DNA duplex encircles the other DNA double helix and vice versa.A motif-centered, strand specific analysis of ChIP-seq data improves the accuracy of determining peak positions. If a genome contains a large number of binding sites for a given protein complex, such as transcription factor heterodimers or transcription factor/cofactor complexes, the relative position of the constituent proteins on the DNA can be established with an accuracy that allow one to deduce the local topology of the protein complex. The proposed high resolution mapping approach of ChIP-seq data is applicable for detecting the contact topology of DNA-binding protein complexes.The online version of this article (doi:10.1186/s12864-016-2940-7) contains supplementary material, which is available to authorized users. In chromatin immunoprecipitation combined with sequencing (ChIP-seq), DNA fragments represented by sequence reads correspond to those regions that can be chemically cross-linked with a protein in question. In case of transcription factors (TFs), the term “peak” is generally used to denote a loosely defined region to which an elevated number of reads can be mapped compared to the background, while the peak summit shows the highest coverage of the region. Peak summits are known to more-or-less coincide with the corresponding DNA elements . It is aThe organization of interphase chromatin is mediated, among other mechanisms, by the dynamic formation of loop structures held together by cohesin, an evolutionarily conserved ring-like protein complex. The tripartite cohesin ring itself consists of RAD21, SMC1 and SMC3 proteins  and is bin situ Hi-C study pointed out that CTSs flanking the loops had a strand specific orientation, where generally the 5’ CTS was on the forward strand while the 3’ CTS was on the reverse strand . CTCF and cohesin  ChIP-seq datasets of each cell or tissue type In the cases of CTCF and cohesin samples with common origin, the measurements were carried out under identical conditions.ii)Sequencing was carried out on Illumina platform.iii)The number of mapped reads was above 10 million. NCBI Build 37/hg19 and NCBI Build 38/mm10 were used as human and mouse reference genomes, respectively.Human and mouse ChIP-seq, ChIA-PET, ChIP-exo and DNase-seq data were downloaded from the NCBI Sequence Read Archive [, http://ENCODE) [, http://Processing of raw data  was carried out with an in-house developed ChIP-seq analysis pipeline  using thAs we expected a single shift or no shift between the CTCF and the cohesin complex, we differentiated these two groups of proteins. As the average resolution of the ChIP-seq coverage is about a few tens of base pairs, we calculated the average position of the sites bound by CTCF (consensus peak summits) based on the raw peak summits if these were present in at least two samples and were closer than 51 bps. Consensus peak summits for cohesin were determined in the same way. Finally, we collected those consensus CTCF peak summits that were closer to a consensus cohesin summit than 51 bps. Direction of the shift between the consensus peak summits of CTCF and cohesin was determined and showed that the cohesin is almost always downstream compared to the CTCF protein on the DNA.Motif enrichments were determined by findMotifsGenome.pl  from theChIP-fragment coverage of CTCF/cohesin samples were plotted on their own CTS set  by annotatePeaks.pl using -hist 1 parameter . The maxTo further investigate the strand specific shift between CTCF and cohesin peak summits, we compared sample duos and trios that were derived from the same cell or tissue type with identical condition. In these cases, for each comparison, peaks were selected that overlapped with the consensus CTS set. Instead of the summit predictions of MACS2, we used the ones located with PeakSplitter, which was developed to discriminate subpeaks (in case of overlapping peaks) and thus gives more accurate local maxima. The distance of the summits relative to the reference points was established by closestBed (BEDtools) . FirstlyTo identify the genomic location and coverage of CTCF/cohesin proteins with near-single-nucleotide accuracy, we used publicly available HeLa DNase-seq and ChIP-exo data .The single-nucleotide resolution border peak detection was executed with “model based analysis of ChIP-exo” (MACE) .The DNase-seq bam files were downloaded directly from the ENCODE database . The rawThe identified ChIP-exo and DNase-seq borders were then compared with the processed ChIP-seq data and are shown in Additional file ChIA-PET  data was used to collect CTSs that are involved in CTCF mediated chromatin looping. The CTCF ChIA-PET data were downloaded from the public database of ENCODE as a processed interaction set in “junction BED” format . We usedWe identified CTSs under RAD21 and CTCF co-occupied region  with theThe two sides of one interaction are actually two sections of the DNA (ChIA-PET DNA section with variable size). The ChIA-PET DNA sections contain CTSs, which are involved in CTCF-mediated DNA looping. To identify these we searched for the closest CTS to the midpoint of the ChIA-PET DNA sections that showed convergent motif orientation with the CTS of the other side if there was interaction .We constructed a consensus interaction set from the replicas using intersectBed . 8482 in3C, Chromosome conformation capture; AR, androgen receptor; Bp, base pair; ChIA-PET, Chromatin Interaction Analysis with Paired-End-Tag sequencing; ChIP-exo, chomatin immunoprecipitation with exonuclease digestion and high-throughput sequencing; ChIP-seq, chromatin immunoprecipitation followed by sequencing; CTCF, CCCTC-binding factor; CTS, CTCF specific recognition sites; DHS, DNase I hypersensitivity sites; DNase-seq, deoxyribonuclease I digestion with high-throughput sequencing; ENCODE, ENCyclopedia Of DNA Elements; kb, kilobase; FOXA1, Forkhead box protein A1; MACE, model based analysis of ChIP-exo; MACS, Model-based Analysis of ChIP-Seq data; PET, paired-end tag; RAD21, Double-strand-break repair protein rad21 homolog; SMC, Structural Maintenance of Chromosomes; SMC1/3, constitutes the core subunits of the cohesin complexes involved in sister chromatid cohesion; STAG2, Cohesin subunit SA-2; TF, transcription factor; TFBS, transcription factor binding siteAdditional file 1:Figure S1. The reproducibility of ChIP-seq peak shifts in a HeLa cell experiment. Figure S2. Shift between CTCF and cohesin bound sites in mouse cells. Figure S3. The boundaries of genomic regions covered by CTCF/cohesin. Figure S4. Shift between CTCF/cohesin proteins in human cell lines. Figure S5. Box plot representation of the strand specific shift between CTCF and cohesin proteins in human cell lines. Figure S6. Shift between CTCF/cohesin proteins in mouse cell and tissue types. Figure S7. Box plot representation of the strand specific shift between CTCF and cohesin proteins in mouse cell and tissue types. Figure S8. Distance distribution of cohesin proteins relative to the CTCF in human cell lines. Figure S9. Distance distribution of cohesin proteins relative to the CTCF in mouse cell and tissue types. Figure S10. DNA modeling. The model of the CTCF binding site (CTS) and a consensus prediction of 16964 aligned binding sites shows that the DNA double helix is not inherently curved in this region (inset), and that it is slightly less curved and more flexible than the surrounding regions. Figure S11. Mapping the shift values onto the B-DNA. Figure S12. Shift between interacting transcription factors (positive control). Figure S13. Lack of shift between interacting transcriptional regulator proteins (negative control). Table S3. Steps of ChIP-seq analysis pipeline. Table S4. Results of statistic analysis in case of two coherent samples. Table S5. Results of statistic analysis in case of more then two coherent samples. Table S6. Summary table of CTCF-cohesin samples. Table S7. Average values of CTCF/cohesin proteins related to CTS. Table S8. Median and Mean distance from CTCF summits. Table S9. Standard deviation of protein distances near CTSs. Table S10. Relative positions of the co-occupied transcription regulators used as controls. (DOCX 1860 kb)Supplementary Materials. Additional file 2:Table S1 and S2. Basic statistics  of human and mouse datasets, respectively. (XLSX 58 kb)Additional file 3:Table S11. Investigation of curvature/bendability on CTS-centered sequences. The table shows the sequence of 400 bp frame of 16964 CTSs, which were identified with ChIA-PET analysis sequences. (XLSX 1305 kb)


Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. In the present paper, we analyze 32 cytology-mapped polytene chromosome interbands. We estimated molecular locations of these interbands, described their molecular and genetic organization and demonstrate that polytene chromosome interbands contain the 5′ ends of housekeeping genes. As a rule, interbands display preferential “head-to-head” orientation of genes. They are enriched for “broad” class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC) components. In two regions, 10A and 100B, coding sequences of genes whose 5′-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called “grey” bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. We developed a special algorithm to computationally process protein localization data generated by the modENCODE project and show that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date. Drosophila polytene chromosomes have served as the best available model of eukaryotic interphase chromosome. They are prominent for their banding pattern formed by dark transverse stripes , which encompass large chunks of chromatin material. These bands alternate with fine, lighter-colored stripes that have less material and are more loosely packed. Such light-transparent structures between bands are known as interbands.Drosophila genome, methods to even approximately map band/interband borders on a physical map are still lacking.Genetic organization of bands and interbands defined as the pattern that sets positioning of genes and genetic features relatively to the structural elements of a chromosome, is still largely elusive. This is due to the fact that despite the availability of the Yet, many interesting hypotheses regarding the genetic organization of bands and interbands in polytene chromosomes have been put forth. Some of the points of these hypotheses were experimentally validated, so we believe it is important to consider them below.Genes were proposed to reside in interbands Very interesting conclusions were made regarding the general meaning of banding pattern: bands were regarded as hosting inactivated genes, interbands were represented by the genes in a steady state of activity Several recent technological advances have dramatically moved forward our understanding of polytene chromosome organization. First, efforts of the modENCODE project have produced genome-wide profiling data for many proteins that specifically localized to bands or interbands in interphase chromosomes  and “BLACK” (repression mechanism not defined) chromatin types associated with repressed chromatin, “YELLOW” chromatin contained ubiquitously expressed genes, whereas “RED” chromatin harbored active genes with more complex expression patterns. “GREEN” chromatin type was defined by enrichment of heterochromatin-specific proteins HP1 and Su(var)3–9 , proteins of origin recognition complexes (ORC). Moreover, they show clustering of DNaseI hypersensitive sites (DHS) . They differ in many aspects of their protein and genetic make-up, as well as in DNA compactization Previously, we showed that polytene chromosomes and interphase chromosomes from dividing cells display identical organization. Namely, interbands from polytene chromosomes and the corresponding DNA sequences from cell line chromosomes share similar features in terms of localization of open chromatin-type proteins. Consequently, banding pattern appears as a fundamental organization principle of interphase chromosomes. In both types of chromosomes, homologous interbands and bands have identical physical borders and length; importantly, they also associate with identical sets of proteins In the present work, using various cytological approaches, we first characterized a new set of precisely mapped interbands, and then processed the modENCODE data on localization of active chromatin proteins using a custom-designed computation model. This analysis suggests that interphase chromosome interbands contain constantly active promoter regions of ubiquitously active genes. Coding sequences of these genes, at least in two regions studied, map to adjacent loosely compacted early-replicating “grey” bands. In contrast, densely packed, late-replicating bands of polytene chromosomes appear to preferentially harbor tissue-specific genes.To analyze the interbands' protein make-up and to explore their molecular organization, these structures must be accurately mapped on both cytological, electron microscopy (EM) and physical maps. In this study, we present the molecular-genetic analysis of a set of interbands , which we believe were unambiguously identified at the cytology level; 21 of these interbands were mapped earlier A group of 11 interbands was characterized in detail here, by comparing Bridges' polytene maps, EM data, modENCODE protein localization data and mapping of IH regions For illustrative purposes, below we provide detailed description of mapping data for interband regions found in 7F1-2 and 100B.Nrg gene 03670 , blue – 16.8% (4006 fragments), green – 22.5% (8903 fragments) and magenta – 48.0% (5148 fragments). The sizes of the cyan chromatin fragments range between 0.2 and 39.6 kb (2.7 kb average), blue - 0.2-46.8 (4.9 kb average), green - 0.2-46.8 kb (3.1 kb), magenta - 0.2-82.8 kb (11.2 kb average).The fraction of the Genome browser-compatible tracks showing the positions of all four chromatin states can be found in the In our downstream analysis, we used a set of 32 interbands described above and whose positions on the cytology map were established with maximum accuracy. This set of interbands is also molecularly well-characterized. Therefore, it can be used for fine analysis of interbands, i.e. for mapping of proteins and functional chromatin elements enriched in these interbands. For each of these interbands, detailed maps of associated proteins and other open chromatin features were constructed; Nrg probe, cyan state, various promoter types identified by Hoskins et al. Interband 7F1-2/7F3-4 shows nearly perfect overlap between the FISH signal from the 5′-Very similar trends are clearly observed for all other interbands as well –S5: althAll 32 interbands studied here display similar organization. First of all, 100% of interbands harbor cyan chromatin fragments, 5′-UTRs of genes, they show low nucleosome density and overlap with the positions of state 1 chromatin  shows that for w* P{EP}G400 the FISH signal maps immediately proximal to 10A1-2, i.e. it is located in the adjacent interband  across the genome. Results of this genome-wide analysis are presented in As was mentioned above, in polytene chromosomes there are three basic structural types: interbands and two types of bands – large IH bands composed of tightly compacted late-replicating material, and loosely compacted small bands replicating early. High-resolution mapping of chromosomal structures presented in this study makes it possible to match the positions of genes and chromosomal structures on a scale of the physical map. One such comparison for the region 10A is illustrated in In polytene chromosomes, two prominent late-replicating IH bands 10A1-2 and 10B1-2 flank a group of six faint loosely compacted bands that are barely detectable under the light microscope. They are depicted grey on Bridges' map dco gene of about 3 kb maps between these two interbands, hence it likely corresponds to the miniature band 100B3. Thus, in the regions where interbands alternate with grey bands, the interbands generally tend to comprise 5′-ends of the genes and correspond to cyan chromatin state, whereas grey bands harbor gene coding sequences. This observation forms the basis of a hypothesis that in the context of interphase chromosome, many other regions may share the same pattern of genetic organization .Yet another region, 100B, may serve to further illustrate of this trend. As was mentioned above, this region harbors a group of two interbands and a loosely compacted very faint band 100B3  barely vSo, the genes mapping to grey bands/interbands, in fact, may occupy two polytene chromosome structures: interband hosts the 5′-end of a gene encompassing its regulatory part, the first exon and intron, whereas the structural part of the gene is found in the neighboring loosely compacted grey band. In this respect, localization of cyan fragments serves as a marker of 5′-ends of genes and interbands, while blue chromatin may correspond to loosely compacted grey bands and coding parts of the genes. Thus, loosely compacted grey bands and the adjoining interbands can be viewed as linked structures: gene promoter locates to the interband, gene coding region resides in the adjacent grey band.In the set of 32 interband/grey bands studied here, we identified 65 genes . Late-reDrosophila genome, P-elements preferentially integrate into interbands. Genome-wide analysis was indicative of predominant integration of P-element transposons into replication origins (ORC-positive regions) Our earlier analysis of 12 interband regions from polytene chromosomes We further confirm this observation, as we compare the insertions of transposons and chromosomal structures. Clearly, the interbands serve as the hotspots of transposon insertions, which is evident on the molecular and genetic maps of all interbands studied to this end .When this analysis is performed genome-wide, and localization data for all the P-element insertions referenced in the FlyBase  are used, 78.3% of all insertions map to the cyan chromatin states, which constitutes only 12.7% of the genome sequence (see above). This translates into 6.2-fold higher frequency compared to the random distribution of insertions. Importantly, we observe a pronounced decrease in insertion frequencies in other chromatin types. P-element based transposons are 9.1-fold less likely to land in magenta chromatin  is suggestive of their specific organization and function. We compared expression patterns of genes found in the late-replicating IH bands vs those located in 32 interbands/grey bands (see the list in −16 Mann-Whitney test).In the region 9F13 – 10B3, interband/grey band genes  are active across almost all tissues analyzed. At the same time, expression pattern of genes found in large IH bands 10A1-2 and 10B1-2 is much more restricted . SimilarTo compare the magnitude of transcription for the genes found in IH bands and 32 interbands  .Drosophila transcriptome have been published, utilizing high-throughput RNA-seq technique . For each gene, abundance of RNA transcripts was measured throughout development  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032933/).Recently, many interesting datasets characterizing Using these data, we confirmed that expression of 238 genes mapping to IH bands  in comparison with magenta fragments  .Taken together, these data are indicative of the constantly high transcription level of genes whose 5′-ends locate to the interbands or to the cyan chromatin state in the genome. These genes are active across the majority of cell types (i.e. they may be referred to as ubiquitously active or multiply active genes).D. melanogaster embryos identified 12454 promoters in 8037 genes. It was shown that distribution of transcription start sites (TSS) within these promoters forms a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% cases. This led to the conclusion that these distributions are generally determined by static elements such as local DNA sequence, rather than by dynamic signals such as histone modifications. As it turned out, Drosophila promoters are characterized either by broad region of distributed TSSs, or by single TSS defining a discrete promoter. These patterns are consistent with a definition of “broad” and “peaked” promoter classes in the human and mouse genomes Recent high-resolution mapping of promoters active in the On a genome-wide scale, 83.6% (6436) of broad-class promoters map to cyan (interband-enriched) chromatin fragments . This arDrosophila genome showed high frequency of bi-directional promoters activating expression of genes arranged in a “head-to-head” configuration with less than 2000 base pairs of intervening sequence Interestingly, analysis of promoter architecture in the human and Drosophila genome, there are 3357 head-to-head oriented genes with less than 2000 base pairs between them (FlyBase). Of these, 5′-ends of 2027 genes (60.4%) map to cyan chromatin, which constitutes only 12.7% of the genome. In stark contrast, only 46 “bi-directional” genes (1.4%) are found within blue chromatin (16.8% genome). Green (22.5%) and magenta chromatin types (48.0%) encompass 448 (13.4%) and 760 “bi-directional” genes (22.6%), accordingly  and those of Filion et al. As compared to IH bands, it is far less trivial to provide accurate mapping for interbands and grey bands, because these regions are fully replicated and are much smaller. Yet, using a combination of EM, P-element tagging and FISH, we were able to unambiguously map the positions of 32 interbands. Using the data on the features of interband chromatin, we developed a mathematical model that defines four basic chromatin states in the drosophila genome. This model allows identification of interband regions chromosome-wide. Accordingly, the limits of the DNA sequences corresponding to interbands were defined as borders of cyan fragments.With these data in hands, we proceeded to analyze the molecular and epigenetic organization of interbands. Interbands clearly displayed features of transcriptionally active regions: H3K4me3 histone modification, lower nucleosome density and histone H1 dips, presence of DHS, localization of RNA polymerase II and components of nucleosome remodeling complexes such as NURF, ISWI, WDS. One characteristic feature of interbands is that they are specifically bound by the chromodomain-containing CHRIZ protein Drosophila melanogaster polytene chromosomes According to different estimates, there are 3500–5000 bands and interbands in The major finding of our analysis of functional organization of interbands is that they typically encompass 5′-regions of multiply active genes (constitutively and actively transcribed). In a number of instances, we observe that short genes can be entirely engulfed by interbands see , howeverOur findings, therefore, resonate well with several early ideas regarding the interplay of structural and functional organization of banding pattern in polytene chromosomes. These include interband localization of multiply active genes, and the hypothesis of a single functional unit composed of band+interband (see Introduction).Recently, there has been an avalanche of publications describing various types of domain organization in the genomes of eukaryotes Kharchenko et al. Using the modified Hi-C approach based on the ligation of chromatin fragments that are found in close proximity in cross-linked chromatin, high-resolution chromosomal contact maps were generated  on polytene chromosomes was performed as described Immunostaining was performed as described To analyze the distribution of P-element insertions within the euchromatic part of the genome , we used insertion coordinates tagged as “transposable_element_insertion_site” from FlyBase release 5.50. We had to exclude 718 insertions  from further analysis, as they mapped to multiple chromatin types or to the regions where the model failed to return a specific value.Using R-language, we quantitatively described the distribution of promoter shapes To analyze promoter orientation in pairs of adjacent genes, we used gene models from FlyBase release 5.50. Gene pairs were chosen so that no annotated gene bodies mapped between these genes. A custom R script (available upon request) was used to analyze the overlap of gene pairs with chromatin states in our model. We assumed that the chances of two genes being oriented “head-to-head”, “tail-to-tail” and “head-to-tail” under random distribution would be 25%, 25% and 50%, respectively.http://flybase.org/static_pages/feature/previous/articles/2013_05/rna-seq_bulk.html, description: http://flybase.org/reports/FBrf0221009.html). Genomic coordinates of both expression datasets were converted so as to match the FlyBase release 5.50.Two data sources were used for gene expression analysis. For processing the data from the FlyAtlas project prcomp function in R. Since we analyzed pre-normalized log2-converted ChIP-chip data, no scaling was applied. Data clustering was done using RHmm library package in R Data from the fly modENCODE project were extensively used in our analyses and appropriate datasets are listed in the Figure S1Cytological identification of the interbands in the 100B region of the Drosophila melanogaster 3R chromosome. A, D – Comparison between Bridges revised map A) and Electron Microscopic map of the region 100A (D) . Increased part of the interbands proximally and distally to the 100B3 small grey band is shown in the rectangle. B, C - Immunofluorescent localization of CHRIZ in the region. The interbands 100B1-2/100B3 and 100B3/100B4-5 are marked by asterisks. E, F - FISH localization of the DNA containing a fragment of the dco gene (arrow in F), phase contrast as a control (C).(TIF)Click here for additional data file.Figure S2Heatmap showing correlation between protein distributions along the X chromosome. Spearman correlation matrix between protein binding data on the X chromosome . Pairwise correlation values are presented and color-coded according to the color map shown on the bottom left. Spearman correlation distances are illustrated by the dendrogram on the left of the graph. The cluster of proteins to be analyzed in more detail was identified using the appropriate stopping rule (TIFF)Click here for additional data file.Figure S3Localization of proteins and genomic features (fly modENCODE) around the interbands 100B1-2 - 100B4-5. Dashed red vertical lines show the edges of cyan state chromatin in the region which conditionally reflect the location of the interbands. A – gene map (RefSeq Genes). B – Localization of promoter broad, unknown and peacked types according to Hoskins et al. (2011) C – Localization of 4-state chromatin types according to the algorithm developed in this paper. Only cyan and green chromatin types map to this genome region. D – Five-state chromatin types in Kc cells by Filion et al. E - 9-chromatin states in S2 cells by Kharchenko et al. F - 9-chromatin states in BG3 cells by Kharchenko et al. G – Nucleosome density according to Henikoff et al. H – Localization of histone H1 dips in Kc cells by Braunschweig et al. I - DNAse I hypersensitivity sites  in S2, BG3, and Kc cells by Kharchenko et al. J - ORC2-binding sites in S2, BG3, Kc cells and salivary glands by Eaton et al. K – Enrichment profiles of NSL complex components: NSL1 binding profile from salivary glands by Raja et al. L – Enrichment regions of various proteins specific for interbands and active chromatin (fly modENCODE). The list of interband-specific proteins is taken from Demakov et al. (TIF)Click here for additional data file.Figure S4Localization of proteins and genomic features (modENCODE) around the interband 1A8/1B1-2. Red dotted vertical lines are according to edges of cyan state chromatin in the region which conditionally reflect the location of the interbands. A – gene map (RefSeq Genes). B – Localization of pICon3C(1A) reference transposon, of promoter broad and unknown types according to Hoskins et al. (2011) C – Localization of 4-state chromatin types according to the algorithm developed in this paper. Only cyan and green chromatin types map to this genome region. D – Five-state chromatin types in Kc cells by Filion et al. E - 9-chromatin states in S2 cells by Kharchenko et al. F - 9-chromatin states in BG3 cells by Kharchenko et al. G – Nucleosome density according to Henikoff et al. H – Localization of histone H1 dips in Kc cells by Braunschweig et al. I - DNAse I hypersensitivity sites  in S2, BG3, and Kc cells by Kharchenko et al. J - ORC2-binding sites in S2, BG3, Kc cells and salivary glands by Eaton et al. K – Enrichment profiles of NSL complex components: NSL1 binding profile from salivary glands by Raja et al. L – Enrichment regions of various proteins specific for interbands and active chromatin (fly modENCODE). The list of interband-specific proteins is taken from Demakov et al. (TIF)Click here for additional data file.Figure S5Localization of proteins and genomic features (modENCODE) around the interband 10A7/10A8-9. Red dashed vertical lines are according to edges of cyan state chromatin in the region which conditionally reflect the location of the interbands. A – gene map (RefSeq Genes) and position of the reference transposon insertion P{EPgy2}EY09320 (arrow). B – Localization of promoter broad, peacked and unknown types according to Hoskins et al. (2011) C – Localization of 4-state chromatin types according to the algorithm developed in this paper. Only cyan and green chromatin types map to this genome region. D – Five-state chromatin types in Kc cells by Filion et al. E - 9-chromatin states in S2 cells by Kharchenko et al. F - 9-chromatin states in BG3 cells by Kharchenko et al. G – Nucleosome density according to Henikoff et al. H – Localization of histone H1 dips in Kc cells by Braunschweig et al. I - DNAse I hypersensitivity sites  in S2, BG3, and Kc cells by Kharchenko et al. J - ORC2-binding sites in S2, BG3, Kc cells and salivary glands by Eaton et al. K – Enrichment profiles of NSL complex components: NSL1 binding profile from salivary glands by Raja et al. L – Enrichment regions of various proteins specific for interbands and active chromatin (fly modENCODE). The list of interband-specific proteins is taken from Demakov et al. (TIF)Click here for additional data file.Figure S6FISH localization of transposon insertions in the polytene chromosome interband regions 10A1-2/10A3 , 10A3/10A4-5  and 10A7/10A8-9 .A - C – overlay of FISH signal (green) and phase contrast. D – F – overlay of FISH signal (green), phase contrast and DAPI (blue). G – I – overlay of FISH signal (green) and DAPI (blue). Arrows point to the FISH signals in polytene chromosome regions.(TIF)Click here for additional data file.Figure S7Box-and-whiskers diagram reflecting activity of genes located in the set of select 32 interbands (A) and intercalary heterochromatin bands (B). To plot this diagram, data from Chintapalli et al. X axis. Mean mRNA expression  is shown on the Y axis. Median value is shown as thick black line; open boxes represent the data between 25 and 75 percentile (50% of data points). Whiskers extend to the most extreme data points which are no more than 1.5 times the length of the box away from the box. Separate circles represent outliers.(TIFF)Click here for additional data file.Figure S8Box-and whiskers diagram reflecting activity of genes located in the cyan (A) and magenta (B) chromatin types in the whole genome. Labeling is the same as in the (TIFF)Click here for additional data file.Figure S9Box-and-whiskers diagram showing mean expression of genes located in the 32 interband chromatin according to modENCODE RNA-seq data. The list of datasets on temporal, tissue, treatment and cell line expression is given below the x axis. Y axis shows gene expression levels  (according to (TIFF)Click here for additional data file.Figure S10Box-and-whiskers diagram reflecting mean activity of genes located in IH bands according to modENCODE RNA-seq data. Labeling is the same as in the Figure S9.(TIFF)Click here for additional data file.Figure S11Box-and-whiskers diagram reflecting mean activity of genes located in the cyan type of chromatin in the whole genome according to modENCODE RNA-seq data. Explanations as in the Fig. S9.(TIFF)Click here for additional data file.Figure S12Box-and-whiskers diagram reflecting mean activity of genes located in magenta type of chromatin in whole genome according to modENCODE RNA-Seq data. Explanations as in the Fig. S9.(TIFF)Click here for additional data file.Figure S13Different types of genome organization domains described for the region 9F13 – 10B3.A - Genomic coordinates around the 10A1-2 and 10B1-2 bands (red dashed lines). B - Track showing our four-state chromatin types. The span of 10A1-2 the 10A1-2 and 10B1-2 bands is indicated above the track. C - Map of five-state chromatin types in Kc cells by Filion et al. D - Position of 9 chromatin states in S2 cells by Kharchenko et al. E - Position of 9 chromatin states in BG3 cells by Kharchenko et a. F - Physical domains by Sexton et al. G - Physical domains by Hou et al. H - Select genes from FlyBase which according to Weber, Hurst I - Select genes from FlyBase referenced as housekeeping in Feller et al. J - FlyBase genes. Genes that we classify as housekeeping are shown in red. K - NSL1 enrichment profile in S2 cells according to Feller et al. L - NSL1 enrichment in salivary glands according to Raja et al. M - NSL3 enrichment in S2 cells according to Lam et al. (TIF)Click here for additional data file.Figure S14Values of Calinski-Harabasz criterion at different numbers of states used for HMM clustering of D. melanogaster X chromosome sequences.(TIF)Click here for additional data file.Text S1Identification of a new set of interbands in Drosophila melanogaster polytene chromosomes.(DOC)Click here for additional data file.Table S1Lists of genes, whose 5′-ends map to the cytologically defined interbands and genes located completely in the intercalary heterochromatin bands.(XLS)Click here for additional data file.Table S2Coordinates and descriptions of probes selected for FISH mapping on polytene chromosomes (release 5.12).(DOC)Click here for additional data file.Table S3The list of ChIP-chip protein binding data sets used for clusterization .(XLS)Click here for additional data file.File S1Track of 4-state chromatin model developed in this study.(GZ)Click here for additional data file.File S2Track of head-to-head oriented genes.(GZ)Click here for additional data file.File S3Scripts in R language for building 4-state chromatin model.(GZ)Click here for additional data file.


Bacillus subtilis Zur transcription factor recognizes a specific DNA motif, the Zur box, to repress expression of genes in response to zinc availability. Although several Zur-regulated genes are well characterized, a genome-wide mapping of Zur-binding sites is needed to define further the set of genes directly regulated by this protein.The cis-repressive effect (4- to 9-fold) but the function of Zur at these sites remains unclear. Zur binding to intragenic Zur boxes could prime an intricate mechanisms of regulation of the transcription elongation, possibly with other transcriptional factors. However, the disruption of zinc homeostasis in Δzur cells likely affects many cellular processes masking direct Zur-dependent effects. Finally, most Zur-binding sites were located near or within genes responsive to disulfide stress. These findings expand the potential Zur regulon and reveal unknown interconnections between zinc and redox homeostasis regulatory networks.Using chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip), we reported the identification of 80 inter- and intragenic chromosomal sites bound by Zur. Seven Zur-binding regions constitute the Zur primary regulon while 35 newly identified targets were associated with a predicted Zur box. Using transcriptional fusions an intragenic Zur box was showed to promote a full Zur-mediated repression when placed within a promoter region. In addition, intragenic Zur boxes appeared to mediate a transcriptional Our findings considerably expand the potential Zur regulon, and reveal a new level of complexity in Zur binding to its targets via a Zur box motif and via a yet unknown mechanism that remains to be characterized.The online version of this article (doi:10.1186/s12866-015-0345-4) contains supplementary material, which is available to authorized users. Zinc is an essential trace element for all forms of life. It serves as structural scaffold for protein folding and as cofactor for many enzymes and DNA-binding proteins ,2. HowevBacillus subtilis, transcription of genes involved in zinc homeostasis is regulated by Zur, a metalloprotein that binds Zn(II) as corepressor  in bothhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64671).The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE64671 (


Multiple sirtuins are encoded by each eukaryotic genome, raising the possibility of cooperativity or functional overlap. The scope and variety of chromatin binding sites of the sirtuins in any specific organism remain unclear.The sirtuins are a conserved family of NADSaccharomyces cerevisiae. Unexpectedly, Sir2, Hst1 and Sum1, but not the other sirtuins, exhibit co-enrichment at several classes of chromatin targets. These include telomeric repeat clusters, tRNA genes, and surprisingly, the open reading frames (ORFs) of multiple highly expressed RNA polymerase II-transcribed genes that function in processes such as fermentation, glycolysis, and translation. Repression of these target genes during the diauxic shift is specifically dependent on Sir2/Hst1/Sum1 binding to the ORF and sufficiently high intracellular NAD+ concentrations. Sir2 recruitment to the ORFs is independent of the canonical SIR complex and surprisingly requires Sum1. The shared Sir2/Hst1/Sum1 targets also significantly overlap with condensin and cohesin binding sites, where Sir2, Hst1, and Sum1 were found to be important for condensin and cohesin deposition, suggesting a possible mechanistic link between metabolism and chromatin architecture during the diauxic shift.Here we utilize the ChIP-seq technique to identify and functionally characterize the genome-wide targets of the sirtuins, Sir2, Hst1 to Hst4, and the DNA binding partner of Hst1, Sum 1, in This study demonstrates the existence of overlap in sirtuin function, and advances our understanding of conserved sirtuin-regulated functions, including the regulation of glycolytic gene expression and condensin loading. They uetylated . As a reticipate ,5. This Saccharomyces cerevisiae encodes five, known as Sir2, and Hst1 through Hst4 (homologs of Sir two)  dCTP  by random priming. The labeled probe was mixed with 100 µl of sonicated salmon sperm DNA (10 mg/ml), boiled to denature, and then hybridized to the membrane for 1 hour at 68°C in QuickHyb solution. The membrane was washed two times at room temperature in 2×-SSC + 0.1% SDS and once at 60°C in 0.1×-SSC + 0.1% SDS. The washed membrane was exposed to X-ray film for autoradiography.Genomic DNA (10 µg) was digested with om pYPLV , and labACT1 or ALD2. As indicated in some figures, to determine the fold induction, gene transcript levels in the mutant strains were also normalized to the levels in the wild-type strain. Results reflect the average fold induction (relative to the induction in the wild-type strain) from three biological replicates. Where indicated in the figures, the standard deviation was calculated.Synthesis of cDNA from total RNA and PCR reactions with SYBR green PCR master mix was performed as previously described . The oli2SO4, followed by measuring the absorbance at 540 nM.The amount of glucose in the growth medium was assayed with a glucose assay kit (Sigma) using the glucose oxidase system. One milliliter of culture was removed at each time point from the indicated cultures and centrifuged at 10,000x g for 5 minutes to clarify the supernatant. The supernatant was further diluted 250-fold with deionized water and 250 µl of the diluted sample was subjected to the assay. Briefly, the reaction was started by adding 500 µl of assay reagent that contains o-dianisidine and glucose oxidase/peroxidase. After reacting for exactly 30 minutes at 37°C, the reaction was stopped by adding 500 µl of 12 N H+/NADH ratio, we utilized a fluorescent NAD/NADH detection kit . Yeast cells were inoculated into 100 ml YPD medium and grown at 30°C in a shaker. A quantity of 2 × 106 cells was collected for each time point and then washed twice with 2 ml of phosphate-buffered saline. After removal of the final supernatant, the cell pellet was resuspended in 200 µl of the NAD or NADH extraction buffer supplied in the kit, and the rest of the protocol performed according to the manufacturer's instructions.To determine the NADChIP: chromatin immunoprecipitation; IGS: intergenic spacer; IGV: Integrative Genomics Viewer; IP: immunoprecipitation; ORF: open reading frame; rDNA: ribosomal DNA; TGM: tRNA gene-mediated; WT: wild type.The authors declare that they have no competing interests.ML contributed to conceptualization of the experiments, carried out most of the molecular biological experiments, and drafting of the manuscript. VV performed bioinformatic and statistical analysis with the ChIP-seq data and contributed to the writing. KP contributed to statistical analysis of the ChIP-seq data and diauxic shift gene expression analysis. SB contributed to conceptualization of the experiments, drafting the manuscript, and data analysis. JS conceived of the study, contributed to the experimental design, data analysis, and drafting of the manuscript. All authors read and approved the final manuscript.Supplementary online material. This file contains Supplemental materials and methods, Figures S1 to S7, and Tables S1 to S7.Click here for file


FMR1) gene and subsequent DNA methylation of the promoter region, accompanied by additional epigenetic histone modifications that result in a block of transcription and absence of the fragile X mental retardation protein (FMRP). The lack of FMRP, involved in multiple aspects of mRNA metabolism in the brain, is thought to be the direct cause of the FXS phenotype. Restoration of FMR1 transcription and FMRP production can be obtained in vitro by treating FXS lymphoblastoid cell lines with the demethylating agent 5-azadeoxycytidine, demonstrating that DNA methylation is key to FMR1 inactivation. This concept is strengthened by the existence of rare male carriers of a FM, who are unable to methylate the FMR1 promoter. These individuals produce limited amounts of FMRP and are of normal intelligence. Their inability to methylate the FMR1 promoter, whose cause is not yet fully elucidated, rescues them from manifesting the FXS. These observations demonstrate that a therapeutic approach to FXS based on the pharmacological reactivation of the FMR1 gene is conceptually tenable and worthy of being further pursued.Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability, caused by CGG expansion over 200 repeats  at the 5′ untranslated region (UTR) of the fragile X mental retardation 1 ( A paradigmatic condition belonging to this latter group is the Fragile X syndrome , an X-linked condition with hemizygous males more severely affected than females, characterized by cognitive impairment, behavioral abnormalities , muscular hypotonia and some physical signs  [FMR1, which contains in its 5′ untranslated region (UTR) a polymorphic CGG triplet repeat [Hereditary disorders of the epigenetic machinery are a newly delineated group of multiple congenital anomalies and intellectual disability (ID) syndromes resulting from mutations in genes encoding components of the epigenetic machinery (acting chidism) . Up to 6chidism) , correspt repeat ), is theFMR1 gene but are characterized by opposed epigenetic changes, with decreased transcription in FXS patients (loss-of-function) and increased transcription in PM carriers, resulting in a toxic gain-of-function of the excess mRNA [A number of CGG between 55 and 200 defines another class of alleles, the premutated alleles (PM). The PM alleles are at high risk of expanding into FM alleles through maternal meiosis and confer a risk to develop fragile X-associated tremor/ataxia syndrome  and fragile X-associated primary ovarian insufficiency . Overall, these disorders, known as FRAXopathies  or fragiess mRNA .FMR1 silencing in human embryonic stem cells (hESCs)-derived neurons is mediated by its own mRNA which hybridizes to the complementary CGG repeat on the DNA sequence to form an RNA:DNA duplex. Disrupting the interaction of the mRNA with the CGG repeat seems to prevent silencing of the promoter, supporting a mechanism of RNA-directed gene silencing. However, it should be noted that the hESCs employed in this work were already partially methylated, casting some doubts on the conclusions reached by the authors [FMR1 transcription in presence of CGG expansion. It should be noted that FMR1 epigenetic gene silencing takes place already in FXS-hESCs. Using 12 FXS-hESCs it was observed that FMR1 hypermethylation occurs during the undifferentiated state and thus earlier than 11 weeks and it is tightly linked to FMR1 transcriptional inactivation [This scenario is complicated by the description of rare individuals with apparent normal phenotype, carriers of an unmethylated full mutation (UFM) with a CGG triplet over 200 repeats, which is completely unmethylated and retains an euchromatic configuration, compatible with both transcription and translation of the gene, even though at higher and lower levels respectively, compared to normal ,12,13. I authors . In any tivation .FMR1 gene was identified by positional cloning by Verkerk et al. [The k et al. . It contk et al.  and prodk et al. , with a FMR1 locus includes (from 5′ to 3′) an upstream methylated region, the promoter region, the CGG stretch and the coding sequence. The boundary of the methylated region is located approximately 650–800 nucleotides upstream from the CGG sequence. In FXS alleles this boundary is no longer visible, given that promoter and CGG stretch are also methylated, while it is preserved in UFM alleles [FMR1 promoter region. CTCF protein was recently considered as a possible regulator of FMR1 expression, probably modulating its transcription through chromatin loop formation [The  alleles ,20. The The promoter contains approximately 56 CpG sites (CpG island) and includes three initiator-like (InR) sequences localized about 130 nucleotides (nt) upstream the CGG sequence. The transcription starts from one of these three transcription start sites within a region of approximately 50 nt, and the size of the CGG repeat may act as a downstream enhancer/modulator of transcription. Initiation shifts to the upstream sites when the size of the CGGs expands .FMR1 gene structure with the main classes of alleles and their transcriptional activity is depicted in The polymorphic CGG repeat is located in the 5′ UTR of exon 1. Based on the CGG expansion range, three main classes of alleles are described: normal, with 5–55 CGGs; PM, with 56–200 CGGs and FM, with over 200 CGGs. These latter alleles are subject to hypermethylation and epigenetic silencing, with consequent loss of FMRP. This loss-of-function mutation is the cause of the FXS phenotype. A schematic overview of the FMR1, FMRP can be primarily classified as an RNA-binding protein that regulates translation of several target mRNAs, and particularly those associated with neuronal development. The interaction with target mRNAs is mediated by two K homology (KH) domains and an arginine-glycine-glycine triplet (RGG) box in the central region and C-terminus of the protein, respectively. The protein has a nuclear localization signal (NLS) and a nuclear export signal (NES), used to shuttle from nucleus to cytoplasm and back. The fraction of the protein that stays within the nucleus is only about 4%, the balance being localized in the cytoplasm [The protein product of ytoplasm . FMRP caytoplasm ,28 and Rytoplasm . It alsoytoplasm .Fmr1 knock-out mice synapses and represents the basis for the so-called “mGluR theory” [Fmr1 knockout mice, heterozygous for mGlur5 (50% reduction of expression of this receptor) showed a rescue of the synaptic phenotype [There is evidence that the absence of FMRP results in an increased translation rate of its target mRNAs , which i theory” . The abshenotype .FMR1-mRNA in human breast cancer cells increase the probability for these cells to form lung metastases [In addition to its role in the brain, FMRP plays important functions in other cell types, such as tumor cells. Its mRNA is overexpressed in hepatocarcinoma cells ,35 and Ftastases .Within the nucleus, FMRP acts as a chromatin-binding protein, with a function in the DNA damage response (DDR), playing an important role in gametogenesis. It has been shown that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis .FMR1 gene seems to require several levels of epigenetic regulation , although the critical mechanism involved in the silencing is not fully understood [FMR1 transcription persists despite CGG expansion over 200 repeats.The silencing of the derstood . SilenceIn FM alleles, the cytosines of the CpG island and of the expanded CGGs become methylated, histones 3 and 4 (H3 and H4) are deacetylated, lysine 4 on H3 (H3K4) is demethylated, while lysine 9 on H3 (H3K9) becomes methylated, lysine 27 on H3 is trimethylated (H3K27me3) and lysine 20 on H4 (H4K20) increases its methylation status near the CGG expansion ,42,43. AFMR1 promoter and RNAs transcribed from premutated CGG expansions tend to form hairpins, which may account for the stalling of the 40S ribosomal subunits and consequently for the translation deficit of PM alleles [FMR1-mRNA was detected in the inclusions of both neurons and astrocytes of FXTAS patients [FMR1 is transcriptionally active, despite a CGG expansion above 200 repeats. The DNA is unmethylated and the histone marks are similar to those of WT alleles , with the exception of H3K9 which remains partially methylated like in FM alleles. The major epigenetic modifications in normal, FXS and UFM alleles are reported in FMR1 gene occurs. In FX-ESCs, H3K9 dimethylation of the FMR1 promoter was detected before the occurrence of DNA methylation [FMR1 locus, which remained in a heterochromatic configuration [FMR1 hypermethylation with its consequent transcriptional inactivation occurs during the undifferentiated state and it is associated to loss of H3K4me2, gain of H3K9me3 and is unrelated to CTCF binding [In contrast to the hypoacetylation of FM alleles, PM alleles have 1.5–2 times the normal levels of acetylated H3 and H4 , thought alleles . The inc alleles ,48. The patients . This papatients ,11,12. Thylation  and in iguration . In FXS- binding .FMR1 transcription [FMR1 gene. CTCF depletion with siRNA caused a reduction of both FMR1 and FMR1 antisense RNA 1 (FMR1-AS1) transcription, which however did not appear to be caused by re-methylation of the FMR1 promoter, both in normal and UFM cell lines. Based on these findings, we concluded that CTCF may have a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions. The second protein investigated was the CGG-binding protein (CGGBP1) [FMR1 gene, but not to methylated CGGs. However, CGGBP1 silencing with shRNAs did not affect FMR1 transcription and CGG expansion stability in expanded alleles. Although the strong binding to the CGG tract could suggest a role of CGGBP1 on FMR1 gene expression, these data demonstrate that CGGBP1 has no direct effect on FMR1 transcription and CGG repeat stability.To identify structure-specific proteins that could recruit components of the silencing machinery, we explored the role of two proteins. The first was the DNA binding protein CTCF, which was recently considered as a possible regulator of cription . CTCF bi(CGGBP1) . CGGBP1 FMR1 locus includes several noncoding RNAs (ncRNAs), particularly long ncRNAs (lncRNAs), which can be transcribed from both strands of the gene, sense and antisense. These lncRNAs may act as modulators of transcription or of the epigenetic landscape of the locus of origin. The main ncRNA originated from the FMR1 locus is FMR1-AS1, an antisense transcript absent in FM alleles and upregulated in PM and UFM alleles [FMR1 bidirectional promoter and the second is located in the second intron of the FMR1 gene. In both cases it includes the CGG∙GGC repeat sequence. PM and UFM alleles showed a specific alternative splicing in intron 2 that used a non-consensus CT-AC splice site. Other ncRNAs were further identified, both in the sense and in the antisense direction. FMR4 is transcribed in the antisense orientation, is absent in the FM alleles and slightly overexpressed in PM alleles [FMR5 and FMR6 [FMR1 transcription start site (in the methylated region upstream of the methylation boundary) and is expressed in WT, PM and FM alleles. The latter is an antisense transcript that overlaps exons 15, 16 and 17, as well as the 3′ UTR, splicing out the introns through non-canonical consensus sites, and is silenced in expanded alleles (both PM and FM). Although the exact role of these ncRNAs is not yet elucidated, they could act as regulators of FMR1 transcription, possibly acting as scaffold for the proteins necessary for heterochromatin formation or as a guide for the recruitment of silencing complexes, as described for other loci through RNAi [FMR1 gene silencing in FXS-hESC derived-neurons, ruling out the involvement of RNAi in silencing FM alleles [FMR1 reactivation in a FXS lymphoblastoid cell line [FMR1 transcription while longer FM alleles would rather block transcription and effectively induce local heterochromatin formation [Transcription of the  alleles ,53. It i alleles . Additioand FMR6 . The forugh RNAi . However alleles . These s alleles . It has ell line . The difell line  while thell line . Therefoormation .FMR1 gene silencing: CGG expansion over 200 repeats and epigenetic modifications (mainly DNA methylation), in presence of an intact open reading frame (ORF). These mechanisms lead to the absence of FMRP, whose lack causes the FXS phenotype. Thus, two different approaches could in principle be employed to treat FXS: (a) to normalize the defective functions due to the lack of FMRP, acting on the pathways in which it is involved; and (b) to restore FMR1 expression acting on the epigenetic mechanisms involved in the transcriptional inactivation. Both approaches were tested in vitro (mainly on Fmr1 knock-out mouse brain slices and FXS patient cells) and in vivo . All clinical trials so far were based on evidence obtained on animal models, both mouse and Drosophila. It would be of the utmost importance to test new therapeutic approaches on a human cellular model. FXS-iPS cells were obtained by different groups, and neurons derived from FXS-iPS cells represent a potentially useful cellular model to test new drugs. Two main mechanisms are involved in FMR1 promoter, while in the non-responder subgroup DNA methylation was incomplete. The hypothesis that AFQ056 may affect DNA methylation was not supported by an in vitro study [According to plan (a) above, several clinical trials were conducted, all aimed at correcting the FXS synaptic defect. Most of them stemmed from the discovery of excessive mGLuR signaling at synapses lacking FMRP . Interesro study . Unforturo study ). Considro study .FMR1 gene silent. The hypothesis of reversibility is supported by the existence of UFM individuals who for some yet unknown reasons are unable to methylate their fully expanded CGG tract allowing FMR1 transcription. This approach may be theoretically considered more effective in curing FXS because it goes directly to the cause of the transcriptional silencing.The second approach (plan (b) above) to treat FXS is based on the possibility to revert the epigenetic marks which maintain the mutated FMR1 gene in vitro by inducing DNA demethylation with 5-azadC in lymphoblastoid cell lines from FXS patients. In 1998, we first achieved in vitro reactivation of the FMR1 FM by treating FXS lymphoblastoid cell lines with 5-azadC for 7 consecutive days [FMR1 gene and not amplification of the CGG repeat is the major determinant in abolishing FMRP production [FMR1 promoter region before and after 5-azadC treatment through bisulphite sequencing analysis. We demonstrated that 5-azadC-induced demethylation is partial and transient. After 4 weeks from 5-azadC withdrawal, the FMR1 promoter resumed its methylated status [FMR1 gene reactivation, we undertook a systematic study of its epigenetic status, testing the acetylation and methylation of histones H3 and H4, in three different regions of the gene; promoter, exon 1 and exon 16 before and after treatment of FXS lymphoblastoid cell lines with 5-azadC for 7 consecutive days. The treatment induced histone acetylation, as well as methylation of H3K4, while only partly reducing H3K9 methylation [FMR1 promoter effectively transforming a MFM (inactive) into an UFM (active)  [FMR1 reactivation after treatment. DNA methylation likely represents the main epigenetic mark that switches off the expanded gene. DNA demethylation can be obtained with 5-azacytidine (5-azaC) or, more efficiently, with 5-azadC, that is incorporated as analog of deoxycytidine during cell replication and irreversibly blocks DNA methyltransferases . Severalive days . The reaoduction . Subsequd status . To betthylation . These e(active) . In a mohe drug) . In Figuhe drug)  treated FMR1 reactivating compounds having limited or no toxicity, such as, for instance, histone acetylating drugs. We showed that histone deacetylase inhibitors (butyrate and phenylbutyrate) alone did not reactivate FMR1 in FXS lymphoblastoid cell lines, but were synergistic with 5-azadC in reactivating the silent gene [A limitation to the possible clinical use of 5-azadC is represented by its toxicity. While 5-azaC and 5-azadC are generally well tolerated in the treatment of hematological malignancies , the effent gene .FMR1 in vitro [FMR1 reactivation when used alone in vitro [Valproic acid (VPA), which acts as histone deacetylases inhibitor but not as DNA demethylator, was shown to have a modest effect as transcriptional reactivator of mutant in vitro . VPA is in vitro . In a prin vitro . Similarin vitro , a naturin vitro . Figure FMR1 gene expression was produced in FXS-ESC and iPS derived-neuronal cells. These results provide further insights into the molecular mechanisms of FXS and towards future therapies of trinucleotide repeat disorders [Besides drugs to treat FXS, further perspectives may be taken into account, based on the newly available CRISPR/Cas9 gene editing technique. Recently a targeted deletion of trinucleotide repeats restoring isorders .Among rare genetic disorders, FXS appears to be more amenable than others to an effective pharmacological treatment. FXS is strictly monogenic, practically all patients having the same mutation; the mutation does not affect the coding sequence of the gene but rather its reversible epigenetic status; the pathogenic mechanism is relatively well elucidated; the phenotype  ranges from mild to moderate, and it does not normally include structural defects of tissues or organs. However, as testified by several clinical trials, the effective correction of a genetic defect continues to be a tremendous challenge, still requiring a wider basic knowledge of the pathophysiology underlying the disease.