AsiSI restriction enzyme in normal human fibroblasts. Based on immunofluorescent staining, the co-localization of PRC1 proteins with components of the DNA damage response (DDR) in these primary cells is unconvincing. Moreover, using chromatin immunoprecipitation and deep sequencing (ChIP-seq), which detects PRC1 proteins at common sites throughout the genome, we did not find evidence for recruitment of PRC1 components to AsiSI-induced DSBs. In contrast, the S2056 phosphorylated form of DNA-PKcs and other DDR proteins were detected at a subset of AsiSI sites that are predominantly at the 5′ ends of transcriptionally active genes. Our data question the idea that PcG protein recruitment provides a link between DSB repairs and transcriptional repression.A growing body of evidence suggests that Polycomb group (PcG) proteins, key regulators of lineage specific gene expression, also participate in the repair of DNA double-strand breaks (DSBs) but evidence for direct recruitment of PcG proteins at specific breaks remains limited. Here we explore the association of Polycomb repressive complex 1 (PRC1) components with DSBs generated by inducible expression of the  Drosophila comprises equimolar amounts of the Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce) proteins, is credited with the mono-ubiquitination of histone H2A on lysine 119 (H2AK119ub) The Polycomb group (PcG) proteins are important for establishing the patterns of gene expression in different cell types H2A ubiquitination is also implicated in the repair of DNA double-strand breaks (DSBs) The three PIKK kinases are each capable of phosphorylating the histone 2A variant H2AX on S139 Drosophila embryos There are several lines of evidence suggesting that PRC1 proteins are involved in the repair of DSBs. First, a number of PRC1 proteins have been found to co-purify with γH2AX and other DDR proteins and their association is enhanced by DNA damage Drosophila. Here we investigated whether multiple PRC1 complexes also congregate at specific DSBs generated by conditional expression of the AsiSI restriction enzyme in primary HFs. Contrary to expectations, we found no evidence that PRC1 proteins are stably associated with persistent DSBs.Using a panel of antibodies that support chromatin immunoprecipitation (ChIP) of endogenous PRC1 proteins in normal human fibroblasts (HFs), we previously established that multiple PRC1 components co-localize at common sites in the genome AsiSI:ER and a pBABE-based retroviral vector encoding the HA-tagged AsiSI:ER fusion protein were generously provided by Dr Gaëlle Legube The BF strain of human breast fibroblasts AsiSI:ER fusion protein, cells were grown to near confluence and incubated in medium containing 4-hyrodoxy tamoxifen (OHT) at a final concentration of 300 nM. Control cells received an equivalent amount of methanol, which was used as a solvent for the OHT. For most experiments, cells were fixed after 4 h and analyzed by either immunofluorescence or chromatin immunoprecipitation (ChIP).To activate the The antibodies used for different applications in this study are listed in 3 cells were seeded on coverslips in 12-well culture plates and after 24 h they were washed once in PBSA for 1 min and fixed in 3.7% formaldehyde in PBSA for 15 min at room temperature. Cells were then subjected to 4×20 sec washes in PBSA, permeabilized in 0.1% Triton X-100 in PBSA for 15 min at room temperature and washed again. Four drops of Image-iT FX Signal Enhancer (Molecular Probes) were added to each well and incubated for 30 min at room temperature in a humid atmosphere. After incubation, cells were washed and blocked in 3% BSA in PBSA for 1 h. Cells were then incubated with primary antibody (previously diluted in 3% BSA in PBSA) overnight at 4°C or for 1 h at room temperature. See Approximately 10invert 710 microscope using sequential scanning. Zen 2009 software (Zeiss) was used. DAPI was excited using a 405 nm laser line, the Alexa Fluor 488 with a 488 nm laser line and the Alexa Fluor 555 with a 561 nm laser line. Spatial sampling was 0.04 µm per pixel in the X/Y plane and between 0.2 µm and 0.3 µm per pixel in the Z plane. The pinhole aperture was set to 1 airy unit. For each antibody pair, Z-stacked images of three representative nuclei were collected and the images were deconvoluted using Huygens Essential software (SVI). Co-localization analyses were performed using Imaris 7.6 software (Bitplan) and automatically selected intensity thresholds in the region of interest, (set by the DAPI channel) to generate Pearson’s correlation coefficient (PCCs) values.Images were acquired using a 40x/1.3 DIC Plan Apochromat lens under oil immersion and a Zeiss LSM ChIP assays were performed as described To generate sufficient quantities of DNA for sequence analyses, parallel ChIPs were performed using approximately 5 µg of antibody with 500 µg chromatin. The recovered material was pooled and concentrated to a minimum of 0.2 µg/µl. DNA samples were end-repaired, poly-A tailed and Illumina single-end adapters were ligated following the standard Illumina protocol with minor adjustments. Agencourt AMPure XP beads at 0.8x ratio were used to size select out adapter dimers after adapter ligation. The Illumina kit Phusion enzyme was replaced by Kapa HiFi HotStart ready mix. Post PCR, AMPure XP beads were used at a 1∶1 ratio to maintain size integrity and to allow use of the Invitrogen SizeSelect E-gel system. Samples were finally purified with QAIquick gel extraction kit and quality controlled on the DNA 1000 BioAnalyser 2100 chip before clustering and subsequent 50–51 bp single end sequencing on the Illumina HiSeq 2500.http://novocraft.com) with default parameters and subsequently filtered to allow for a single mismatch per read. Duplicate reads were removed using the Picard MarkDuplicates programme  with default parameters. Peak calling was performed using MACS  and 12.5 µl Express qPCR Supermix with premixed ROX (Invitrogen). The primer sets are listed in All reactions were performed in triplicate in 96-well plates (Applied Biosystems) with the following combination of reagents: 2 µl DNA, 10 µl HAsiSI restriction enzyme and a modified hormone-binding domain from the estrogen receptor AsiSI fusion protein, as detected with an antibody against the HA-tag (AsiSI recognition sites. There are 1219 exact matches of the 8 bp AsiSI recognition site (GCGATCGC) in the human genome but as the sequence is subject to CpG methylation, only a subset of sites are likely to have been cleaved AsiSI:ER fusion protein . As the positive control for recruitment of DDR proteins to AsiSI sites analyzed above, standard peak calling algorithms, such as MACS GATA6, in line with our previous observations  of the AsiSI sites , in the promoter region of the PCGF1 gene, is juxtaposed to a known PRC1 target gene, TLX2. Whereas pDNA-PKcs was detected at PCGF1, specifically in the OHT-treated cells, there was no corresponding OHT-dependent enrichment for MEL18. In contrast, MEL18 showed very robust enrichment at TLX2 in both control and treated cells, with the same characteristic binding pattern as other PRC1 proteins , there was also an H3K4me3 peak  of them were associated with the promoter regions of annotated genes. Again, the vast majority (94%) of these genes were deemed to be active as judged by H3K4me3 profiles. Interestingly, several loci showed distinct pairs of pDNA-PKcs and H3K4me3 peaks associated with different transcription start sites . We did not observe the converse situation, namely the appearance of novel PRC1 peaks whose association with AsiSI sites is OHT-dependent.As PRC1 complexes are generally viewed as transcriptional repressors, it could be argued that they are less likely to be found on transcriptionally active genes. This could lead to an inverse correlation between PRC1 binding and the AsiSI-induced DSBs, even though the enrichment was often an order of magnitude lower than observed for PRC1 proteins at PRC1 target genes. In the few published studies that have reported binding of PcG proteins at a specific DSB, the enrichment was weak and some of the PRC1 components appeared to bind more avidly to the engineered DSBs than to bona fide PcG targets AsiSI sites. In this context, we note that several of the published studies have described partial co-localization, involving only a subset of PcG and a subset of DDR proteins. Taken together, the data suggest that the proposed link between PcG proteins and DNA repair is more tenuous than presently assumed.We have suggested that the binding profiles of PRC1 components in HFs are best explained by the concept of Polycomb bodies in which multiple variants of the canonical PRC1 complex act collectively rather than individually Figure S1AsiSI-induced DNA damage foci in U2OS cells by indirect immunofluorescence. A). AsiSI:ER-transduced U2OS cells were treated for 4 h with or without OHT and co-stained with DAPI and antibodies against either the HA-tag on the fusion protein (green) or γH2AX (red). Right panels show quantification of the HA intensity (voxels) and number of γH2AX foci in 20 representative nuclei, +/− OHT. Error bars represent the standard deviation and *** signifies a P value<0.001 in a student’s t-test. B. Similar analyses comparing the staining for γH2AX (red) and 53BP1, BMI1 or RING2 (green). Images were deconvoluted using Huygens Essential software and the Imaris program was used to generate a co-localization channel (yellow). Middle panel shows Pearson’s correlation coefficient values for the indicated pairs of markers in three representative nuclei. Right panel shows equivalent analyses for RING1/RING2 and BMI1/MEL18 co-localization based on additional data not shown.Visualizing (TIFF)Click here for additional data file.Figure S2AsiSI sites. The panels show DNA sequence tag densities following ChIP-seq with the pDNA-PKcs and MEL18 antibodies in AsiSI:ER-transduced Hs68 cells, before and after addition of OHT, as indicated. Input refers to parallel analyses of the chromatin before immunoprecipitation. The H3K4me3 track refers to genome-wide enrichment of H3K4me3 in the Hs68 strain of HFs from a previously deposited dataset (GEO accession number 40740). A. examples of clustered AsiSI sites on chromosomes 19 and 14. B and C. examples at which pDNA-PKcs binding extends for a considerable distance on either side of the AsiSI site.Examples of ChIP-seq data at representative (TIFF)Click here for additional data file.Figure S3AsiSI:ER-transduced Hs68 cells, before and after addition of OHT, as indicated. The profiles are aligned with equivalent data for CBX6, CBX7, CBX8, RING1 and RING2 in normal cells, from a previously deposited dataset (GEO accession number 40740). At the HOXD cluster (A) and GATA6 (B) there are no predicted AsiSI sites. C. An example where pDNA-PKcs and PRC1 binding occurs discretely on adjacent loci. D. Two examples of pDNA-PKcs peaks that are present in both OHT-treated and untreated cells and at or near a known PRC1 target.Co-localization of MEL18 and other PRC1 components at selected target loci. The panels show DNA sequence tag densities following ChIP-seq with the pDNA-PKcs and MEL18 antibodies in (TIFF)Click here for additional data file.Figure S4Examples of ChIP-seq data where AsiSI sites coincide with PRC1 peaks.The panels show DNA sequence tag densities following ChIP-seq with the pDNA-PKcs and MEL18 antibodies in AsiSI:ER-transduced Hs68 cells, before and after addition of OHT, as indicated. Input refers to parallel analyses of the chromatin before immunoprecipitation. The H3K4me3 track refers to genome-wide enrichment of H3K4me3 in the Hs68 strain of HFs from a previously deposited dataset (GEO accession number 40740). In all three examples, the pDNA-PKcs peak is specifically observed in OHT-treated cells whereas the MEL18 signal is not affected by addition of OHT.(TIFF)Click here for additional data file.Figure S5AsiSI:ER activation. The panels show DNA sequence tag densities following ChIP-seq with the pDNA-PKcs and MEL18 antibodies in AsiSI:ER-transduced Hs68 cells, before and after addition of OHT, as indicated. Input refers to parallel analyses of the chromatin before immunoprecipitation. The H3K4me3 track refers to genome-wide enrichment of H3K4me3 in the Hs68 strain of HFs from a previously deposited dataset (GEO accession number 40740).Examples of pDNA-PKcs peaks that are independent of (TIFF)Click here for additional data file.Table S1Antibodies used in this study and their applications.(DOCX)Click here for additional data file.Table S2Oligonucleotide primers used for analyses of ChIP DNA by qPCR. Genome co-ordinates are based on the Feb 2009, GRCh37/hg19 release of the human genome sequence according to the UCSC genome browser. Primers used in (XLSX)Click here for additional data file.Table S3AsiSI sites at which a pDNA-PKcs peak was detected in OHT-treated cells.List of (XLS)Click here for additional data file.


Enhancement of transcription by a splicing-competent intron is an evolutionarily conserved feature among eukaryotes. The molecular mechanism underlying the phenomenon, however, is not entirely clear. Here we show that the intron is an important regulator of promoter directionality. Employing strand-specific transcription run-on (TRO) analysis, we show that the transcription of mRNA is favored over the upstream anti-sense transcripts (uaRNA) initiating from the promoter in the presence of an intron. Mutation of either the 5′ or 3′ splice site resulted in the reversal of promoter directionality, thereby suggesting that it is not merely the 5′ splice site but the entire splicing-competent intron that regulates transcription directionality. ChIP analysis revealed the recruitment of termination factors near the promoter region in the presence of an intron. Removal of intron or the mutation of splice sites adversely affected the promoter localization of termination factors. We have earlier demonstrated that the intron-mediated enhancement of transcription is dependent on gene looping. Here we show that gene looping is crucial for the recruitment of termination factors in the promoter-proximal region of an intron-containing gene. In a looping-defective mutant, despite normal splicing, the promoter occupancy of factors required for poly(A)-dependent termination of transcription was compromised. This was accompanied by a concomitant loss of transcription directionality. On the basis of these results, we propose that the intron-dependent gene looping places the terminator-bound factors in the vicinity of the promoter region for termination of the promoter-initiated upstream antisense transcription, thereby conferring promoter directionality. Eukaryotic genes differ from their prokaryotic counterparts in having intervening non-coding sequences called introns. The precise biological role of introns in eukaryotic systems remains unclear even more than forty years after their initial discovery. One function of intron that has been remarkably conserved during evolution is their ability to enhance the transcription of genes that harbor them. How does the intron regulate transcription, however, is not known. Here we show that the intron enhances gene expression by affecting direction of the promoter-initiated transcription. In the presence of an intron, polymerase tends to transcribe the downstream coding region producing mRNA, while in the absence of a splicing-competent intron polymerase starts transcribing promoter upstream region producing upstream antisense RNA (uaRNA). Intron-mediated promoter directionality was dependent on gene looping, which is the interaction off the promoter and terminator region of a gene in a transcription-dependent manner. We show that the intron-dependent gene looping facilitates the recruitment of termination factors in the promoter-proximal region. The recruited termination factors stop uaRNA synthesis thereby conferring directionality to the promoter-bound polymerase. Although introns were discovered more than four decades ago, their precise physiological role in biological systems still remains an enigma , 2. One Although less than 5% of genes in budding yeast contain introns, the intron-containing genes contribute nearly 28% of mRNA produced in yeast cells , 16. We Here we show that the transcription directionality of a subset of genes in yeast is dependent on the presence of a splicing-competent intron. The intron facilitates the recruitment of CF1, CPF and Rat1 termination complexes in the vicinity of the promoter region. We provide evidence that the intron-dependent gene looping facilitates the recruitment of termination factors near the promoter region. The recruited termination factors selectively terminate uaRNA synthesis, thereby conferring directionality to the promoter-initiated transcription.Research conducted during last eight years has confirmed that the nucleosome free region located at the 5′ end of most RNAPII-transcribed genes contains two unidirectional promoters , 26. EacIMD4, ASC1 and APE2, in the promoter-proximal upstream antisense and downstream sense direction in the presence and absence of an intron. We constructed strains with the intron-less version of these three genes following the strategy described in Moabbi et al., (2012) [IMD4, ASC1 and APE2 deceased by 2.5 to 10 fold upon deletion of the intron  . The mRNe intron . In contf intron . These rIMD4, ASC1 and APE2 in the sense direction (mRNA synthesis) upon deletion of intron  analysis as described in Medler and Ansari (2015) . Brieflyf intron . Simultaf intron . These rf intron , and corTo gain an insight into the role of the intron in promoter directionality, we calculated the directionality index by dividing nascent mRNA level with nascent uaRNA level for each tested gene in the presence and absence of an intron. The directionality indices in the presence of intron for these three genes ranged from 4 to 25 . Upon deACT1 intron into an intron-less IMD4 gene as previously described  [IMD4 in the presence of a wild type native intron, in the presence of a wild type ACT1 intron, in the absence of an intron, in the presence of 5′ splice site mutated and a 3′ splice site mutated ACT1 intron. The insertion of a wild type ACT1 intron brought about a 10-fold increase in transcription of mRNA  . The 5′ ., 2012) . Both mu of mRNA . The enh of IMD4 . Simulta1 intron . A 6–8 f1 intron . The mut1 intron . The dro1 intron . A logicIMD4 and ASC1 genes by ChIP as described in Al Husini et al., (2013) [In mammalian cells, uaRNA synthesis is terminated in a poly(A)-dependent manner by the cleavage and polyadenylation machinery , 21. In , (2013) . The terIMD4 and ASC1 in the presence and absence of an intron. Nab3 was recruited at both the 5′ and 3′ ends of IMD4 with almost equal intensity in the presence of an intron  and PTA1 (pta1-td) by strand-specific TRO approach. The results show that uaRNA transcription increased by about 5-fold upon shifting of rna15-2 cells to elevated temperature . No such increase in uaRNA transcription was observed in the isogenic wild type cells at elevated temperature  (The experiments described above clearly demonstrated an increase in the promoter recruitment of factors required for poly(A)-dependent termination of transcription in the presence of an intron. It was, however, not clear if the recruited termination factors were enhancing transcription directionality by affecting uaRNA transcription. We therefore examined nascent uaRNA and mRNA levels in the promoter-proximal region of perature . A similperature . The incperature . In contperature . The enh10 fold) . These eINO1 and ASC1 [IMD4 and APE2 also exhibit a similar intron-dependent change in gene conformation. We therefore performed ‘Chromosome Conformation Capture’ (CCC) analysis of IMD4, ASC1 and APE2 in the presence and absence of their native wild type intron in the same batch of cells that were used for measuring transcription directionality in IMD4, ASC1 and APE2 in the presence of the native wild type intron  . A gene and ASC1 , but it e intron . The looe intron . Thus, tIMD4 gene in the presence of a wild type, a 5′ splice site mutated as well as a 3′ splice site mutated ACT1 intron. A robust P1T1 looping signal was observed for IMD4 in the presence of the wild type ACT1 intron, which was almost to the same extent as in the presence of the native intron  [sua7-1 strain  . We havee (E62K) . This ste (E62K) , 39–41. 1 strain . To rulenscripts . If it w1 strain . These fsua7-1 strain  sites and U1-binding sites has been shown to influence the recruitment of termination factors in the promoter-upstream region, which in turn terminates uaRNA synthesis , 21. TheIf gene looping enhances transcription directionality by facilitating the recruitment of termination factors in the promoter-proximal region, the next logical question is why the promoter-recruited termination factors selectively terminate uaRNA synthesis, while mRNA transcription continues unabated. We have already shown that the activator-dependent gene looping facilitates recycling of polymerase from the terminator to the promoter for transcription in sense direction . The intThe emerging view is that a vast majority of RNAPII-transcribed genes in yeast and mammalian systems have bidirectional promoters. The regulation of promoter directionality is critical for optimal transcription of these gene. We show a novel role of introns in yeast in regulating promoter directionality through looping-mediated recruitment of termination factors at the promoter. Less than 5% of yeast genes contain introns. In contrast, a vast majority of genes in higher eukaryotes contain introns. It is, therefore, tempting to speculate that introns might have a similar mechanistic impact on transcription directionality in higher eukaryotes as well.Yeast strains used in this study are listed in 600 ~0.6. Equal number of cells were used for strand-specific RT-PCR, CCC, ChIP or strand-specific TRO assays. The rna15-2 and pta1-td mutants were grown at permissive temperature (25°C) till A600 reached 0.5. Cells were then shifted to non-permissive temperature (37°C) for 90 minutes and processed for strand-specific TRO analysis.Cultures were started by inoculating 5 ml of YP-dextrose medium with colonies from a freshly streaked plate and grown at 30°C with gentle shaking. Next morning, overnight grown cultures were diluted (1:100) to appropriate volume and grown to AIMD4 gene were Alu1 and Dra1, were obtained from New England Biolabs. Each experiment was performed with at least four independently grown cultures. The P1T1 PCR signals were normalized with respect to F1-R1 PCR signals.CCC experiments were performed as described previously . The priChIP experiments  were performed as described previously . DiffereTranscription analysis was performed by RT-PCR approach as described previously . The priThe strand-specific ‘Transcription Run-On’ (TRO) assay was performed as described previously in . The priThe data shown in Figures is the result of at least four biological replicates. The quantification and statistical analysis was performed as described in . Error bS1 FigIMD4, ASC1 and APE2 in the presence (black bars) and absence of intron (white bars) to detect the expression of mRNA or uaRNA. The transcript level of 18S was used as a control for normalization.(A) Schematic depiction of a gene with and without intron indicating the sense (mRNA) or the promoter-initiated upstream anti-sense (uaRNA) transcripts. TSS is transcription start site, and TTS indicates transcription termination site. (B) RT-PCR analysis of (PDF)Click here for additional data file.S2 FigIMD4 gene containing wild type ACT1 intron, 5ꞌ splice site mutated ACT1 intron mutated, 3ꞌ splice site mutated ACT1 intron. A1 and A2 indicate the position of primer pairs used in RT-PCR analysis to monitor splicing. TSS is transcription start site, and TTS indicates transcription termination site. (B) Pre-mRNA of IMD4 is efficiently spliced in the wild type (WT), but not in splicing defective mutants. Lanes 1 and 3 show RT-PCR results of mRNA of IMD4 with wild type ACT1 intron. Lanes 2 and 4 show RT-PCR results of mRNA of IMD4 with 5ꞌ splice-site (5ꞌ SS) and 3ꞌ splice-site (3ꞌSS) mutations in ACT1 intron.(A) Schematic depiction of (PDF)Click here for additional data file.S3 FiguaRNA and mRNA transcription as well as transcription directionality remains unaffected in a wild type strain at elevated temperature (A) Schematic depiction of a gene with intron. TSS represents transcription start site, and TTS represents transcription termination site. (B) Quantification of TRO analysis of IMD4, ASC1 and APE2 in wild type (FY23) at 25°C (black bars) and 37°C (grey bars) to detect the expression of mRNA or uaRNA. The transcript level of 18S was used as a control for normalization. (C) Directionality indices of IMD4, ASC1 and APE2 in wild type cells at 25°C (black bars) and 37°C (grey bars).(PDF)Click here for additional data file.S4 FigASC1 and IMD4 is efficiently spliced in the wild type (WT) and the looping defective sua7-1 cells. Lane 1 shows PCR of genomic DNA using A1-A2 primers to indicate the size of unspliced RNA. Lanes 2 and 3 show RT-PCR results of mRNA of indicated genes in the wild type and sua7-1 strains respectively.(A) Schematic depiction of an intron-containing gene indicating the position of primers used in RT-PCR analysis to monitor splicing. TSS is transcription start site, and TTS indicates transcription termination site. (B) Pre-mRNA of (PDF)Click here for additional data file.S5 FigIMD4, ASC1 and APE2 in wild type (black bars), and sua7-1 strain (grey bars) to detect the expression of mRNA or uaRNA. The transcript level of 18S was used as a control for normalization. P values were calculated by two-tailed student t-test. (C) Directionality indices of IMD4, ASC1 and APE2 in wild type (black bars), and sua7-1 strain (grey bars).(A) Schematic depiction of a gene with intron indicating the sense (mRNA) or the promoter-initiated upstream anti-sense (uaRNA) transcripts. TSS is transcription start site, and TTS indicates transcription termination site. (B) Strand-specific TRO analysis of (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Arabidopsis thaliana chromosomes dissected into subkilobase segments, which is required for gene-level resolution in this species with a gene-dense genome. We found that the repressive H3K27me3 histone mark is overrepresented in the promoter regions of genes that are in conformational linkage over long distances. In line with the globally dispersed distribution of RNA polymerase II in A. thaliana nuclear space, actively transcribed genes do not show a strong tendency to associate with each other. In general, there are often contacts between 5′ and 3′ ends of genes, forming local chromatin loops. Such self-loop structures of genes are more likely to occur in more highly expressed genes, although they can also be found in silent genes. Silent genes with local chromatin loops are highly enriched for the histone variant H3.3 at their 5′ and 3′ ends but depleted of repressive marks such as heterochromatic histone modifications and DNA methylation in flanking regions. Our results suggest that, different from animals, a major theme of genome folding in A. thaliana is the formation of structural units that correspond to gene bodies.The three-dimensional packing of the genome plays an important role in regulating gene expression. We have used Hi-C, a genome-wide chromatin conformation capture (3C) method, to analyze  The spatial organization of the genome in the nucleus is critical for many cellular processes . It has Microscopy-based methods have limited power to reveal fine-grained chromatin structures at the kilobase level and therefore have recently been complemented by PCR and sequencing-based methods. The chromosome conformation capture (3C) approach, which targets specific loci, has been widely used to examine juxtaposition between specific transcription units and remote enhancer elements . A more Arabidopsis thaliana, a possible explanation being the absence of canonical insulator proteins in this species and in other plants . Such power-law behavior, which reflects the physical property and packing patterns of chromatin in nuclear space, has been typically found for chromatin interactions over genomic distances at the Mb scale, suggesting that it is feasible to adapt previously established methods , which we used to determine the statistical significance of each pair of chromatin interactions. Biases that affected the count number of each interaction that appeared in our sequencing reads, such as fragment length, PCR efficiency, mappability, and restriction site density of the corresponding chromatin segments, were taken into account . Because sequencing depth is a major limiting factor for the number of chromatin loops that can be confidently identified . With stringent read mapping and filtering, we retained over 162 million Hi-C reads from our combined data set, of which around 125 million reads were from intra-chromosomal interactions . In total, we identified over 20,000 chromatin loops from the euchromatic chromosome arms (q < 0.01) .We developed a pipeline to identify statistically significant patterns indicative of small chromatin loops (see details in Methods). For every chromatin interaction observed in our data set, we estimated its expected sequencing counts based on a spline-fitting approach . All chrentified , we adde-C reads . For eacFLOWERING LOCUS C (FLC) locus , we re-identified another known loop, between the AT2G34655  promoter and the PINOID (PID) coding region , a chemical that blocks auxin transport  locus . A 3C exthe gene . Our Hi-rst exon , which irst exon . With a ransport . PID RNApression . The detTFL1 and FT, both of which have rather restricted spatial and/or circadian expression patterns  . We also examined whether chromatin loops were enriched for specific chromatin states that are reflected in correlated epigenetic marks. From a nine-chromatin-state annotation, we found that chromatin loops larger than 6 kb tended to avoid state 4, which corresponded to intergenic or distal promoter regions with substantial H3K27me3 levels  that are not expressed in leaves   B.Supplemental Table 3). The control sets had substantially fewer pairs  having H3K27me3 marks on both promoters  and condensin subunit CAP-D3 (AT4G15890) has revealed that these proteins can form nonoverlapping speckles throughout the euchromatic nucleoplasm of interphase nuclei, suggesting their involvement in organizing interphase chromatin  complexes, which include cohesin and condensin, play critical roles in organizing chromatin structure . Althoughromatin . It willSupplemental Table 4). This observation suggests that in A. thaliana, transcribed genes have only a weak tendency to cluster in three-dimensional space. Chromosomal-scale compartmentalization of chromatin has been shown in human cells, where active and inactive chromatin reside in different domains (“AB” compartment)  . A simili-C maps . Highly A. thaliana genes do not predominately couple with each other over long distances. At short distances, interactions between chromatin strongly bound by Pol II mainly occur within genes.On the other hand, when we examined chromatin loops smaller than 6 kb, we found that a subset of them had strong Pol II binding on both interacting partners . To addrSupplemental Fig. 7). This pattern was not specific to the boundaries of transcribed sequences, as we found similar patterns when analyzing gene body regions a few hundred bp inward from TSSs or TTSs , suggesting that at a local level, many A. thaliana genes adopted self-loops.Because our Pol II analysis revealed that many highly expressed genes formed chromatin loops E, we exaSupplemental Table 5), corresponding to 12% of the 14,672 candidate genes, for which our algorithms could potentially identify gene loops . In parallel, on a genome-wide scale, we further examined the interaction directionality bias of regions close to gene bodies. The results suggest an “insulating effect” within a few kb upstream of highly expressed genes: Chromatin located within this range preferentially showed interaction with regions further upstream, but not downstream . This insulating effect was in line with our previous finding that highly expressed genes are enriched at insulator-like regions . The causality between the level of gene expression and the structure of its flanking chromatin regions is unclear, and some gene loop structures in A. thaliana may simply be the consequence of adjacent opened chromatin due to highly transcribed genes. If this is the case, for transgenes having strong promoters such as the cauliflower mosaic virus 35S promoter, the regions flanking insertion sites might acquire a new chromatin conformation that ultimately influences the expression of host genes adjacent to the transgene. Likewise, the presence of a highly expressed host gene next to a transgene insertion might contribute to variation in transgene activity.We found potential links between gene loops and transcriptional activity of neighboring chromatin regions. Compared to the background, genes neighboring regions with loops did not differ in terms of transcriptional direction or gene length but were expressed at higher levels . These results suggest that gene loops in plants are positively associated with transcriptional activity.We assessed the connection between gene loops and expression levels. As mentioned above, because of possible influences from nearby highly expressed genes, we compared genes with self-loops that had at least one highly expressed gene within 5 kb and genes without such neighbors. In both groups, self-looping genes tended to have higher expression levels than non-self-looping genes G. Upon gSupplemental Fig. 11). Neither did classification by aspects of gene structure, such as exon/intron size, exon density, first exon length, and GC content, reveal differences (data not shown). Because looped genes tended to be expressed at higher levels . Similar to animals, the A. thaliana H3 variant H3.3 is generally associated with transcribed genes, with H3.3 being preferentially enriched around TTSs (For genes which were silent or expressed at low levels (level 1–3), we found that gene loops were associated with enrichment for histone variant H3.3 in the gene body near TSSs and TTSs, and at the same time with depletion for DNA methylation and several heterochromatic marks in flanking regions . The difund TTSs . Indepenund TTSs . In animund TTSs , being rund TTSs . BecauseA. thaliana Hi-C data has revealed important details of chromatin structure at very high resolution. Similar to the enrichment of interactions between H3K27me3-marked promoters, and interactions between H3K27me3-marked enhancers and their target sites that have been discovered in animals from high-resolution Hi-C data  was grown at 23°C in long days (16 h light/8 h dark) on half-strength Murashige & Skoog (MS) medium supplemented with 1% sucrose and 0.3% Phytagel. The aerial portions of 10-d-old seedlings were harvested at Zeitgeber time (ZT) 6 h for Hi-C and ChIP-seq library preparation.Hi-C libraries were constructed as described  using DpSupplemental Table 1.Read mapping, removal of PCR duplicates, and read filtering were performed as described  with a mSupplemental Fig. 3). During chromatin loop calling, the distance between two segments was approximated as the genomic distance between their centers. Similar to observations by For chromatin digestion, we used a four-cutter restriction enzyme, DpnII, which produces mostly chromatin fragments below 500 bp . To defiSupplemental Fig. 1).In Hi-C experiments, in addition to chimeric reads coming from statistically significant chromatin loops, the following background reads are also expected: (1) reads from self-ligated fragments; (2) reads from random collisions of chromatin fragments during ligation; and (3) reads from stochastically formed chromatin loops, with interaction frequency largely dependent on physical distance. We focused on intra-chromosomal interactions between 2 and 25 kb. Among all filtered intra-chromosomal Hi-C reads, ∼25% fell in this interval, indicating that distance-dependent stochastic looping was a dominant factor. In this interval, we found that a log-linear relationship held between interaction frequency and intra-chromosomal distance , … } were fitted with the “smooth.spline” function in the R “stats” package (with “spar” set as 0.2). We ensured that at the end of fitting, the C values were monotonically decreasing with respect to the D values. Based on the regression model f(D), the contact probabilities with neighbors were calculated for each anchor point (segment) in the genome asj = 1, 2, … n) are all upstream and downstream segments within the distance range of 2–25 kb, li is the length (bp) of segment i, and Di is the distance (bp) between the anchor point and segment i.All reads corresponding to contacts between segments having 2–25 kb distance were extracted from filtered Hi-C reads, except for reads that mapped to segments located in centromeric regions . A table describing these Hi-C reads counts is available in strategy  was emplp) with neighboring segments were adjusted to account for technical biases. First, we considered biases due to uneven PCR amplification and different mapping ability, which directly affected the likelihood that a Hi-C read finally appeared in our filtered reads list. For example, considering an extreme situation, in which all upstream segments of segment X are either nonmappable or are very inefficiently amplified during PCR, after filtering, segment X might appear to only interact with its downstream regions. Thus, the contact probabilities p of segment X with its upstream regions are actually 0, since they are not detectable under our protocol. Accordingly, the probabilities p with downstream regions should be doubled. Therefore, the p1, p2, …pn values of interacting partners of an anchor segment were adjusted based on their “visibility,” which is in turn associated with PCR (mainly due to GC content biases) and mappability biases. We assayed these biases by analyzing genomic resequencing data of A. thaliana, in which the genomic DNA are fragmented randomly. Thus, prior to library amplification, all chromatin segments that we defined in this study should be equally represented in genomic resequencing data sets. After mapping, the differences of sequencing depth among chromatin segments were largely due to biases in PCR and the mapping process. We analyzed 10 genomic resequencing data sets from A. thaliana Col-0, with each of them having about 20 million reads . Thus, we further revised pAB asf(EB) is the regression model describing the Hi-C sequencing depth of a chromatin segment B as a function of the number of fragment ends in segment B.Another technical bias was restriction cutting site density, which we reported previously on a Hi-C map normalized with 2-kb windows . When usAB = X + Y, where X and Y had negative binomial distributions as X ∼ NB and Y ∼ NB, describing a combination event of taking segment A and segment B as anchor points. Therefore, the probability of observing k reads wasA and B are background reads on segment A and B excluding AB reads, pAB and pBA are adjusted contact probability described above, andp value of observing k reads asCAi is the read count between segment A and its interacting partner i, and Atotal and itotal the sequencing depth of segment A and segment i, respectively. By scanning through all interacting partners of segment A, we used the estimated Â as the total number of failed trials (considering segment A as the anchor point) in our negative binomial distribution model. At the end of the p value calculation, multiple testing correction was performed with the Benjamini-Hochberg method to obtain q values. Among all contacts observed, we selected those with q values less than 0.01 for downstream association analysis. Association analysis pipelines regarding promoter-promoter interactions, gene body to gene body interactions, and gene loops are illustrated in Supplemental Figure 14.For each pair of chromatin segments (segment A and segment B), we observed its contact AB and considered it as q values below 0.01. We only retained interacting partners within the search radius, which was the distance from the focal point to the TTS. In this way, a gene was identified as having gene loop conformation if its focal point(s) had at least one interacting partner located at the 3′ downstream end with respect to the direction of transcription  as focal points. After each focal point was assigned to the corresponding chromatin segment, we looked for all interacting partners of this segment in our data set with cription C. Note t2, 10 µM ZnSO4, 1% Triton X-100, 0.05% SDS) and incubated with anti-Pol II antibody (Abcam ab5408), or equally divided and incubated with anti-H3 (Abcam ab1791) and anti-H3K27me3 antibodies . After overnight incubation at 4°C, 10 µL Protein A/G magnetic beads (Pierce) were added and incubated at 4°C for 2 h. The beads were washed at 4°C as follows: 3× with IP buffer, 1× with IP buffer having 500 mM NaCl, and 1× with LiCl buffer  for 5 min each. Chromatin retained on beads were incubated in 200 µL elution buffer  at 65°C for 6 h, followed by Proteinase K treatment at 37°C for 1 h. DNA was extracted with a standard phenol-chloroform method, and all subsequent end repairing, A-tailing, adaptor ligation, and library amplification steps were done following a standard protocol (Illumina). The final library was sequenced on an Illumina HiSeq 2000 instrument with 1×101-bp reads.Seedlings were fixed under vacuum for 30 min with 1% formaldehyde in MC buffer  at room temperature. After fixation, seedlings were incubated at room temperature for 5 min under vacuum in MC buffer with 0.15 M glycine. Nuclei were isolated as described , and nucA. thaliana reference genome (TAIR10) using Bowtie 2 v2.2.4  under accession number SRP064711.Hi-C and ChIP-seq short read data from this study have been submitted to the NCBI Sequence Read Archive (SRA;


LTBP1 and LTBP4 had two regions initiating different transcripts. Most of the family members were expressed in a range of mesenchymal and other cell types, often associated with use of alternative promoters or transcription start sites within a promoter in different cell types. FBN3 was the lowest expressed gene, and was found only in embryonic and fetal tissues. The different promoters for one gene were more similar to each other in expression than to promoters of the other family members. Notably expression of all 22 LTBP2 promoters was tightly correlated and quite distinct from all other family members. We located candidate enhancer regions likely to be involved in expression of the genes. Each gene was associated with a unique subset of transcription factors across multiple promoters although several motifs including MAZ, SP1, GTF2I and KLF4 showed overrepresentation across the gene family. FBN1 and FBN2, which had similar expression patterns, were regulated by different transcription factors. This study highlights the role of alternative transcription start sites in regulating the tissue specificity of closely related genes and suggests that this important class of extracellular matrix proteins is subject to subtle regulatory variations that explain the differential roles of members of this gene family.The fibrillins and latent transforming growth factor binding proteins (LTBPs) form a superfamily of extracellular matrix (ECM) proteins characterized by the presence of a unique domain, the 8-cysteine transforming growth factor beta (TGFβ) binding domain. These proteins are involved in the structure of the extracellular matrix and controlling the bioavailability of TGFβ family members. Genes encoding these proteins show differential expression in mesenchymal cell types which synthesize the extracellular matrix. We have investigated the promoter regions of the seven gene family members using the FANTOM5 CAGE database for human. While the protein and nucleotide sequences show considerable sequence similarity, the promoter regions were quite diverse. Most genes had a single predominant transcription start site region but  •We examine expression, promoter use and enhancers for the fibrillin/LTBP gene family.•Promoter switching was observed for most family members.•Multiple enhancers were identified for all family members.•Family members overlapped in tissue specificity with some unique expression patterns.•A degree of redundancy among family members is possible. In rats and mice the FBN3 gene appears to have degenerated and does not produce a functional mRNA FBN3 is likely to be active since transcripts have been detected (data from http://www.ensembl.org). There are a variable number of annotated LTBP genes across species, from two in fish to four in mammals: LTBP1, LTBP2, LTBP3 and LTBP4. It is possible that one or more of the gene family members take over the role of FBN3 in rats and mice.In vertebrates, including eutherian, marsupial and monotreme mammals, birds, reptiles and fish, fibrillins are encoded by three genes, Fbn1 mRNA is ubiquitous in mesenchymal cell types Fbn2 appears more restricted in expression . A similar pattern was reported for human FBN2FBN3 expression is restricted to embryonic/fetal tissues http://biogps.org; Expression of fibrillin/LTBP family members is principally found in cells and tissues of mesenchymal origin. In mouse, Consistent with their function in mesenchymal cell types, mutations in members of this gene superfamily result in phenotypes that primarily affect connective tissue types (reviewed in FBN1FBN2 (unpublished). However those data included few tissues showing expression of FBN3 in humans. The current FANTOM5 project involves many more tissue and cell types likely to express members of the fibrillin and LTBP families 154700; FBN1 OMIM: 134797), congenital contractural arachnodactyly , primary congenital glaucoma  and cutis laxa with severe systemic abnormalities  The FANTOM  projects co-ordinated by the RIKEN Institute in Japan have provided extensive information on gene expression in human and mouse and allowed the identification and characterization of a large number of gene promoter regions http://fantom.gsc.riken.jp/5/.This work is part of the FANTOM5 project. Data downloads, genomic tools and co-published manuscripts have been summarized at 22.1Five human cell lines and a primary adult derived mesenchymal stem cell culture were used to analyze expression patterns of fibrillin/LTBP gene family members. SAOS-2 (sarcoma osteogenic 2) is a hypotriploid line derived from the osteosarcoma of an 11-year-old Caucasian female and has been in culture since 1973 2.2http://www.amsbio.com/rna-bee.aspx). RNA was quantified using a Nanodrop spectrophotometer and cDNA was synthesized using MMLV reverse transcriptase  and an annealing temperature of 60 °C http://www.roche-applied-science.com, GAPDH was used as the reference gene . Quantitative PCR was performed in triplicate using LightCycler 480 SYBR Green 1 Master Mix (Roche) on the LightCycler 480 machine (Roche). Products were quantified using software supplied by the manufacturer.Expression of fibrillin/LTBP family members was initially examined in cell lines using quantitative reverse transcriptase PCR from RNA extracted from the cell lines described above. In addition RNA was extracted from cell pellets from Day 2 of embryoid body formation from two human ES cell lines, H1 http://www.amsbio.com/rna-bee.aspx). RNA was quantified using a Nanodrop spectrophotometer and cDNA was synthesized using MMLV reverse transcriptase  and an annealing temperature of 60 °C http://www.roche-applied-science.com, Supplementary Table 1). Human GAPDH was used as the reference gene . Quantitative PCR was performed in triplicate using LightCycler 480 SYBR Green 1 Master Mix (Roche) on the LightCycler 480 machine (Roche). Products were quantified using software supplied by the manufacturer.Expression of fibrillin/LTBP family members was initially examined in cell lines using quantitative reverse transcriptase PCR from RNA extracted from the cell lines described above. In addition RNA was extracted from cell pellets from Day 2 of embryoid body formation from two human ES cell lines, H1 The presence of the encoded proteins was ascertained using immunofluorescence with a range of antibodies. 20,000 NHDF cells per well were seeded into an eight well chamber slide (NUNC). After 7 days, the cells were fixed with 95% ethanol and 5% acetic acid for 20 min and washed with PBS. The samples were blocked with 1% BSA in PBS for 1.5 h prior to addition of a 1:100 dilution of the primary antibody and incubated over night at 4 °C. Following a wash with PBS, the secondary antibody was added at 1:1000 dilution, for 1 h at room temperature. The cells were then washed with PBS, and mounted with ProGold + DAPI (Invitrogen). Samples were viewed using Zeiss LSM 710 confocal microscope and analyzed with ZEN software at standard settings (Zeiss). Details of antibodies used are supplied in The presence of the encoded proteins was ascertained using immunofluorescence with a range of antibodies. 20,000 NHDF cells per well were seeded into an eight well chamber slide (NUNC). After 7 days, the cells were fixed with 95% ethanol and 5% acetic acid for 20 min and washed with PBS. The samples were blocked with 1% BSA in PBS for 1.5 h prior to addition of a 1:100 dilution of the primary antibody and incubated over night at 4 °C. Following a wash with PBS, the secondary antibody was added at 1:1000 dilution, for 1 h at room temperature. The cells were then washed with PBS, and mounted with ProGold + DAPI (Invitrogen). Samples were viewed using Zeiss LSM 710 confocal microscope and analyzed with ZEN software at standard settings (Zeiss). Details of antibodies used are supplied in Supplementary Table 1.2.3http://fantom.gsc.riken.jp/zenbu/). Composite promoters are those where two or more robust peaks occur within 100 bp of each other − 5The tissues and cells used for the FANTOM5 analysis of transcription initiation sites have been described in a parallel publication 2.4Express3DAll 81 DPI-detected robust promoters for the seven genes were clustered using Biolayout The parallel paper 2.5We downloaded the whole-genome alignment of the human genome with 45 other vertebrate genomes from the UCSC Genome Browser database 33.1We used quantitative reverse transcriptase PCR (qRT-PCR) to examine expression of gene family members in cell lines including H1 and RH1 embryonic stem cells, ADMSC mesenchymal stem cells from adipose tissue, SAOS2 and MG63 osteosarcoma cell lines and NHDF fibroblasts, . The resFBN1 expression was consistently high in ADMSC, NHDF and MG63 cell types but minimal in early ES cells (FBN2 was highly expressed early in embryoid body formation from H1 and RH1 embryonic stem cells and in SAOS2 and NHDF cells (FBN3 was present in both ES cell lines (H1 and RH1), with no expression in the remaining cell types  and 21 (LTBP2) robust promoters peaks associated with members of the gene family. FBN1 along with regulatory elements detected by the ENCODE project To assess further the tissue distribution and regulation of gene family members, we used FANTOM5 CAGE-based data for promoter expression. There were between 1 (FBN3) and 21 (LTBP2) robust promoters peaks associated with members of the gene family. FBN1 along with regulatory elements detected by the ENCODE project To assess further the tissue distribution and regulation of gene family members, we used FANTOM5 CAGE-based data for promoter expression. There were between 1  and this was attributable to LTBP1 , LTBP2  and FBN2 .Almost all the promoters were broad promoters in CpG islands and there were no TATA box promoters . The maj21 = 13.0; p = 0.0003) and this was attributable to LTBP1 , LTBP2  and FBN2 .Almost all the promoters were broad promoters in CpG islands and there were no TATA box promoters . The majority of robust peaks were within 100 bp of another peak, forming 18 composite promoters FBN1FBN1 CAGE tags localized to the main composite promoter region containing p1@FBN1 (Exon A), but p11@FBN1 is probably associated with the rarely used Exon B of previous reports FBN2 , were associated with the short transcript ENST00000507835.1. Two promoters were identified in the 3′ noncoding region of the LTBP2 gene . They may be associated with a short overlapping transcript from LOC730019.There were known transcripts  starting in the region of the majority of the detected promoters, and most known alternative transcripts were supported by this analysis . NotablyFBN1FBN1 CAGE tags localized to the main composite promoter region containing p1@FBN1 (Exon A), but p11@FBN1 is probably associated with the rarely used Exon B of previous reports FBN2 , were associated with the short transcript ENST00000507835.1. Two promoters were identified in the 3′ noncoding region of the LTBP2 gene . They may be associated with a short overlapping transcript from LOC730019.There were known transcripts  starting in the region of the majority of the detected promoters, and most known alternative transcripts were supported by this analysis (Supplementary Table 2). Notably, promoters for the three alternative first exons of FBN2 transcript ENST00000508053.1 which has an additional six 5′ non-coding exons , nor were there promoters for two long transcripts (AY203940 and AK022050AY203940AK022050) which overlap FBN3. Similarly there was no evidence for a promoter for the longest reported LTBP4 transcript, NM_003573 (LTBP4 A. —ENST00000545697 and ENST00000204005).Some published transcripts were not supported by CAGE tags. For example, there was no support at the robust level for a promoter for the long FBN2 transcript ENST00000508053.1 which has an additional six 5′ non-coding exons , nor were there promoters for two long transcripts (AY203940 and AK022050AY203940AK022050) which overlap FBN3. Similarly there was no evidence for a promoter for the longest reported LTBP4 transcript, NM_003573 .Some published transcripts were not supported by CAGE tags. For example, there was no support at the robust level for a promoter for the long LTBP1, alternate transcripts with a long or short 5′ UTR have been reported (LTBP1 D.). However, there was no CAGE support for the transcript with the shorter UTR (ENST00000354476.3). Instead, a small TSS cluster was detected midway between the main (long) start site about 200 bp upstream of the annotated beginning of the putative shorter transcript, which may indicate that the shorter transcript is incomplete. Similarly a number of identified TSS in LTBP3 did not correlate with known transcripts .Transcription start sites were also detected where there was no identified transcript. For instance, at the 5′ end of LTBP1, alternate transcripts with a long or short 5′ UTR have been reported . However, there was no CAGE support for the transcript with the shorter UTR (ENST00000354476.3). Instead, a small TSS cluster was detected midway between the main (long) start site about 200 bp upstream of the annotated beginning of the putative shorter transcript, which may indicate that the shorter transcript is incomplete. Similarly a number of identified TSS in LTBP3 did not correlate with known transcripts .Transcription start sites were also detected where there was no identified transcript. For instance, at the 5′ end of FBN1 promoter region contained a characteristic pyrimidine (T-rich) stretch, about 70 nucleotides upstream of the transcription start site associated with p1@FBN1 FBN2 promoter was in a C-rich region but the promoter region of the alternative transcript (p14@FBN2 and p18@FBN2) was not in a CpG island and was AT rich. The FBN3 promoter was also pyrimidine rich but shared no sequence homology with the other genes. The region around p1@LTBP1 contained alternating strings of purines and of pyrimidines.Although most promoters were located in CpG islands, there was little conservation of sequence around the promoters. Many of the promoter regions themselves were pyrimidine-rich (on the coding strand). The coding strand of the main FBN2 A. —red box compared with B.), which is almost exclusively expressed in testis, a tissue type not represented in the cell lines examined in the ENCODE Project.The major promoters were all supported by regulatory elements detected in the ENCODE project FBN2 A. —red box compared with B.), which is almost exclusively expressed in testis, a tissue type not represented in the cell lines examined in the ENCODE Project.The major promoters were all supported by regulatory elements detected in the ENCODE project 3.3LTBP1 . Promoters within a compound promoter were associated with the same predicted enhancer(s)  . All proions see , while aLTBP1 . Promoters within a compound promoter were associated with the same predicted enhancer(s) . This enhancer was not correlated with the other FBN2 promoters. p12@LTBP2 was uniquely associated with a bidirectional expression peak 50 kb upstream (Supplementary Table 3). In contrast, p10@LTBP3, p12@LTBP3 and p21@LTBP3 located within the second and third exons of the major LTBP3 transcript were correlated with one of the same predicted enhancers as the major promoter region containing p1@LTBP3. For LTBP4, each promoter tended to be associated with a different enhancer.p7@FBN1 and p25@FBN1, initiating the alternative first exon, Exon C 3.4The promoters all showed the characteristics of tissue restricted expression, as defined in the parallel paper The promoters all showed the characteristics of tissue restricted expression, as defined in the parallel paper http://fantom.gsc.riken.jp/5/sstar/ and To investigate this tissue restricted expression further, we used a number of ontologies http://fantom.gsc.riken.jp/5/sstar/ and Supplementary Table 4). Promoters in one composite cluster within a gene tended to be expressed in tissues associated with the same ontology terms    was highly expressed in adult heart samples and also expressed in tissue and cells types of mesenchymal origin. This composite promoter was associated with mesenchyme ontology terms including fibroblast but also with many muscle related terms , the high expression levels in cardiovascular cell types also led to a preponderance of terms associated with the vasculature   . Similaral cells . p2@LTBPyoblast) . The maied terms . Althougar cord) .FBN3 were strongly expressed in cells associated with ontology terms indicating mesenchymal origin  (Supplementary Table 4). Similar ontology terms were found for the main promoters of FBN2, which was expressed in a range of cells from fetal and embryonic tissues, fibroblasts, osteoblasts, placenta, hair follicle and lens epithelial cells  (Supplementary Table 4). The main composite promoter region of LTBP1  was highly expressed in adult heart samples and also expressed in tissue and cells types of mesenchymal origin. This composite promoter was associated with mesenchyme ontology terms including fibroblast but also with many muscle related terms (Supplementary Table 4). Although the promoter regions identified for LTBP2 were associated with some generic mesenchymal ontology terms (such as fibroblast and myoblast), the high expression levels in cardiovascular cell types also led to a preponderance of terms associated with the vasculature  (Supplementary Table 4).In general, the major promoters of all gene family members other than al cells . p2@LTBPpresumptive neural plate, neural tube, neural rod, structure with developmental contribution from neural crest, future spinal cord)  but also with terms associated with other organs . p7@LTBP2 demonstrated significant expression levels in both hematopoietic and epithelial samples and was enriched for terms reflection this expression pattern   . p3@FBN2pression . p2@LTBPal cell) . The higtron 25,  displayeve organ .presumptive neural plate, neural tube, neural rod, structure with developmental contribution from neural crest, future spinal cord) (Supplementary Table 4). p3@FBN2 peaked in hematopoietic cell types, and was associated with ontology terms such as classical monocyte as well as terms reflecting mesenchyme/mesoderm expression (Supplementary Table 4). p2@LTBP2 was also expressed in some hematopoietic cells while p3@LTBP4 was associated with lymphocytes of the immune system  but also with terms associated with other organs . p7@LTBP2 demonstrated significant expression levels in both hematopoietic and epithelial samples and was enriched for terms reflecting this expression pattern  (Supplementary Table 4). The high expression promoter of LTBP4, p1@LTBP4, was enriched in ontology terms reflecting an endodermal origin. p14 @FBN2 and p18@FBN2, located in intron 25,  displayed highest expression in testis and both promoters were associated with ontology terms such as testis, male reproductive organ, gonad and external genitalia/reproductive organ (Supplementary Table 4).A number of promoters were expressed in tissues of other origins. Thus p7@FBN1 and p1@LTBP3 were enriched in ontology terms reflecting development of the nervous system   l. Promotanscript  were higus layer .FBN3, p2@FBN3, was almost exclusively expressed in cells of fetal and embryonic origin. This expression pattern was associated with ontology terms such as embryonic stem cell, neuronal stem cell, neuroectodermal cell as well as some terms relating to pigmentation  (Supplementary Table 4l). Promoters of LTBP1 leading to the longer transcript  were highly expressed in embryonic and extraembryonic cell/tissue types including chorionic and amniotic membrane cells, fetal heart, epithelial and endothelial tissue samples. Within this region, p3@LTBP1 had the highest activity, and transcription from this region was found primarily in fetal heart. It was most strongly associated with ontology terms such as extraembryonic cell/structure, mesenchyme, compound organ and membranous layer (Supplementary Table 4).Some promoters were associated with early development. The sole robust promoter for LTBP1 promoter containing p1@LTBP1 showed strong trimethylation of H3K4 in human lung fibroblasts (NHLF), while the composite promoter containing p3@LTBP1 was strongly trimethylated in most of the cell lines, except embryonic stem cells and a human lymphoblastoid cell line (Gm12878) (LTBP1 B.).Variability of promoter use in different cell types was supported by the H3K4Me3 track from the ENCODE data , where dLTBP1 promoter containing p1@LTBP1 showed strong trimethylation of H3K4 in human lung fibroblasts (NHLF), while the composite promoter containing p3@LTBP1 was strongly trimethylated in most of the cell lines, except embryonic stem cells and a human lymphoblastoid cell line (Gm12878) .Variability of promoter use in different cell types was supported by the H3K4Me3 track from the ENCODE data , where different colored peaks (representing trimethylation levels in different cell types) can be seen for different promoters. For example, the composite 3.5Express3D. Biolayout Express3D employs a statistical approach to look at transcript-to-transcript similarities in expression pattern across the samples analyzed, by calculation of a Pearson pairwise correlation matrix LTBP2 promoters formed a separate cluster (Cluster 1) with no edges to promoters for any of the other genes, even at this low correlation level. This indicates that the LTBP2 promoters are more similar in expression pattern to each other than to any other promoters. FBN2 promoters formed two separate clusters (Clusters 3 and 11) and LTBP1 promoters were also in two clusters (Clusters 4 and 8) suggesting two patterns of expression for these genes. In contrast, at this low level of correlation LTBP3 and LTBP4 promoters were frequently in the same cluster (Clusters 5 and 7), or associated with the single FBN3 promoter (Cluster 6), showing that these promoters share some similarity of expression pattern. Some LTBP3 and LTBP4 promoters formed separate distinct clusters (Cluster 12 and Clusters 9 and 10 respectively). One LTBP1 and six LTBP3 promoters also clustered with the FBN1 promoters (Cluster 2).To explore further the similarities and differences in promoter expression across the gene family, we used RLE-normalized tag counts Express3D. Biolayout Express3D employs a statistical approach to look at transcript-to-transcript similarities in expression pattern across the samples analyzed, by calculation of a Pearson pairwise correlation matrix LTBP2 promoters formed a separate cluster (Cluster 1) with no edges to promoters for any of the other genes, even at this low correlation level. This indicates that the LTBP2 promoters are more similar in expression pattern to each other than to any other promoters. FBN2 promoters formed two separate clusters (Clusters 3 and 11) and LTBP1 promoters were also in two clusters (Clusters 4 and 8) suggesting two patterns of expression for these genes. In contrast, at this low level of correlation LTBP3 and LTBP4 promoters were frequently in the same cluster (Clusters 5 and 7), or associated with the single FBN3 promoter (Cluster 6), showing that these promoters share some similarity of expression pattern. Some LTBP3 and LTBP4 promoters formed separate distinct clusters (Cluster 12 and Clusters 9 and 10 respectively). One LTBP1 and six LTBP3 promoters also clustered with the FBN1 promoters (Cluster 2).To explore further the similarities and differences in promoter expression across the gene family, we used RLE-normalized tag counts FBN3 promoter, p2@FBN3, was in this excluded group as were a promoter for FBN2 and LTBP1, seven for LTBP3 and two for LTBP4. Nine of these 11 were singleton promoters while two were within a composite promoter. The network of associations at P ≥ 0.5 is shown in LTBP2 promoters continued to cluster together (Cluster 1) while FBN1 (Clusters 2 and 12), FBN2 (Clusters 3 and 11) and LTBP1 (Clusters 4 and 6) formed two clusters each. p6@LTBP1 clustered with the majority of the FBN1 promoters, indicating an expression pattern more similar to FBN1 than to the other LTBP1 promoters. p7@FBN1 and p25@FBN1, initiating the alternative first exon, Exon C FBN1 promoters. LTBP3  and LTBP4  formed four clusters each. These results suggest that the most diverse expression patterns were found for promoters of LTBP3 and LTBP4, while LTBP2 had the most similar expression pattern across all promoters.At a Pearson correlation coefficient threshold of 0.5, 11 promoters were excluded from the analysis indicating that their expression pattern was sufficiently different from any other promoter that they did not correlate at this level. The sole LTBP2 promoters and single clusters for FBN1, LTPB1 and LTBP4 .Promoters for LTBP genes tended to fail to cluster or formed clusters with other promoters for the same gene, particularly in the primary cell analysis. p1@LTBP1, p2@LTBP1, p4@LTBP1 and p5@LTBP1 formed a small cluster when all samples were analyzed and p3@LTBP1, p7@LTBP1 and p8@LTBP1 formed a cluster when primary cells were analyzed (Supplementary Table 2). However some LTBP promoters clustered with promoters for mesenchymal genes and occasionally were found with promoters for other members of the fibrillin/LTBP gene family. 3.7FBN1 , the motifs for GTF2I, NKX3-1, TBX4-5 and TFAP4 regulated five promoters, while for FBN2 (6 promoters) HIF1A was found to regulate four promoters and TFDP1, KLF4, TFAP2B and TOPORS regulated three promoters. There were no significant regulatory edges for FBN3  for FBN3 l. HoweveFBN1 , the motifs for GTF2I, NKX3-1, TBX4-5 and TFAP4 regulated five promoters, while for FBN2 (6 promoters) HIF1A was found to regulate four promoters and TFDP1, KLF4, TFAP2B and TOPORS regulated three promoters. There were no significant regulatory edges for FBN3 (Supplementary Table 2). However, binding motifs for the transcription factors MAZ, PATZ1, RREB1, SP1 and TFAP4 were detected in the promoter region of FBN3.We used Motif Activity Response Analysis (MARA) LTBP1  and LTBP2 (regulated at 16 promoters) showed significant regulation by the MAZ motif in seven and eight promoters respectively. Five promoters of LTBP1 were also regulated by HIC1 and NFY while four promoters were regulated by PRDM1, RXR and SOX 17. LTBP2 was predicted to be regulated by KLF4 (at 10 promoters), SP1 (at eight promoters), GATA4 and TEAD (at five promoters) and XCPE1 (at four promoters) was associated with LTBP2. Regulation at seven LTBP3 promoters was detected, through motifs for GTF2I and SP1 (at six promoters), MED-1 (at five promoters) and TFAP2 and ZFP161 (at four promoters). Only two LTBP4 promoters were significantly associated with motifs, with TRAF2B found for both  and LTBP2 (regulated at 16 promoters) showed significant regulation by the MAZ motif in seven and eight promoters respectively. Five promoters of LTBP1 were also regulated by HIC1 and NFY while four promoters were regulated by PRDM1, RXR and SOX 17. LTBP2 was predicted to be regulated by KLF4 (at 10 promoters), SP1 (at eight promoters), GATA4 and TEAD (at five promoters) and XCPE1 (at four promoters) was associated with LTBP2. Regulation at seven LTBP3 promoters was detected, through motifs for GTF2I and SP1 (at six promoters), MED-1 (at five promoters) and TFAP2 and ZFP161 (at four promoters). Only two LTBP4 promoters were significantly associated with motifs, with TRAF2B found for both (Supplementary Table 2).4Members of the fibrillin/LTBP gene family appear to play two roles in the extracellular matrix. Firstly, they contribute structurally to the formation of the matrix. In particular, fibrillin-1 and fibrillin-2 are integral components of the 10 nm microfibrils which provide strength and elasticity to the matrix In this study we have examined gene expression and regulation of the seven fibrillin/LTBP family members using results from the FANTOM5 CAGE analysis, a genome wide analysis of transcription start sites and bidirectional putative enhancers FBN1 was highly expressed in a range of connective tissue cells, consistent with previously published transcription factor activity There were multiple examples of differential use of promoters in different tissue types (promoter switching) and this was frequently associated with differences in ENDOCE regulatory elements and distinctive predicted enhancer activity. The main promoter region of FBN1 was highly expressed in a range of connective tissue cells, consistent with previously published transcription factor activity There were multiple examples of differential use of promoters in different tissue types (promoter switching) and this was frequently associated with differences in ENDOCE regulatory elements and distinctive predicted enhancer activity. The main promoter region of FBN2, where p14@FBN2 and p18@FBN2 were expressed in reproductive cell types. The transcript associated with these promoters, ENST00000507835.1, covers coding exons 26 to 34 of the full length FBN2 and the polypeptide would consist of eight cbEGF domains with short N- and C-terminal flanking sequences (http://www.ensembl.org). Preliminary examination of the mouse CAGE data from FANTOM5 showed an equivalent promoter exclusively expressed in mouse testis, suggesting that this transcript has a real function, perhaps involved with sequestration of calcium. Further investigation of this novel transcript should reveal more about its role.Promoter switching with a change in tissue specificity was also seen for LTBP1 is documented to have at least two alternative transcription start regions (http//:www.ensembl.org) and this switch in promoters was confirmed in our analysis. One promoter region demonstrated high expression in early development and low expression in mesenchymal cells  while the alternate promoter region  was associated with mesenchyme-related ontology terms and expressed in heart. The major promoters p1@LTBP1 and p2@LTBP1 were associated with the same transcription factor motifs , while the alternative promoter region was associated with a distinct set of transcription factors motifs .Putative enhancers within 500 kb of the promoter regions were significantly associated with many of the promoters examined. The majority were downstream of the promoter region and some were as close 2 kb from the promoter. In general many promoters in one gene were correlated with the same predicted enhancers although there were a few examples of promoter-specific enhancers, associated with promoters of unique expression pattern.FBN2 showed similar expression patterns and ontology term enrichment to FBN1. However, FBN1 and FBN2 promoters did not cluster together in the co-expression analyses and there was little overlap of transcription factor motif activity between FBN1 and FBN2 promoters, suggesting that there were subtle differences in their expression patterns and regulation. In particular, FBN2 promoters tended to be associated with the epithelial and pluripotency transcription factor KLF4 FBN1 promoters were associated with mesenchymal transcription factors Cluster analysis of expression from the different promoters highlighted the differences between the gene family members. Members of a composite promoter often clustered together, but few promoters for different genes were found in the same cluster. For example, most promoters for LTBP2 A —blue box and C.), for example, showed significant potential to bind GATA4 , for example, showed significant potential to bind GATA4  or Ltbp4 (through p5@LTBP4). Preliminary analysis of FANTOM5 data for mouse indicates that the equivalent of p3@LTBP1 is strongly expressed in embryonic tissue, especially neuron derived neurospheres, while a region equivalent to p5@LTBP4 is expressed in embryonic stem cells , supporting this possibility.Because its expression is restricted to fetal and embryonic tissue types FBN1, FBN2, LTBP2, LTBP3 and LTBP4 have been associated with connective tissue disease in humans. Transgenic and natural mutation mouse models, available for Fbn1, Fbn2, Ltbp1, Ltbp2, Ltbp3 and Ltbp4 also show phenotypic abnormalities of connective tissue (reviewed in Mutations in human 5This study supports the differential roles of members of the fibrillin/LTBP gene family. We have shown the strong mesenchymal expression of most family members and most promoters and highlighted promoter and enhancer switching associated with changes in tissue specificity. We were unable to find evidence for a single “master mesenchyme regulator” but there was considerable overlap in the transcription factor activity associated with these genes. These data aid in better understanding the overall function and activity of the members of the fibrillin/LTBP family.Promoters of the fibrillin/LTBP family members.qPCR primers and antibodies.Promoter characteristics, expression levels, transcription factor associations and expression clusters.Enhancers of fibrillin/LTBP family members.Ontology terms associated with cell types expressing fibrillin/LTBP family member promoters.The following are the supplementary data related to this articlehttp://dx.doi.org/10.1016/j.ymgme.2013.12.006.Supplementary data to this article can be found online at The data in the paper will be freely available.http://fantom.gsc.riken.jp/5/. FANTOM5 enhancers can also be accessed from. http://enhancer.binf.ku.dk.The FANTOM5 atlas is accessible from MRD carried out bioinformatic and molecular genetic analysis, participated in the design of the study and helped to draft the manuscript. RA provided the enhancer analysis. JS and NB supported the analysis and visualization. JKB performed the initial clustering of all CAGE detected promoters. MdH carried out the MARA analysis. HK managed the data handling. ARRF was responsible for FANTOM5 management and concept. KMS conceived this study, participated in its design and coordination, carried out much of the analysis and interpretation, performed the clustering analysis of the promoters and drafted the manuscript. All authors read and approved the final manuscript.


Heterocephalus glaber telomerase RNA(hgTER) were identified: A→G in the first loop of pseudoknot P2b-p3  and G→A in the scaRNA domain CR7-p8b . Analysis of TER promoter regions allowed us toidentify two new transcription factor binding sites. The first one is the ETSfamily site, which was found to be a conserved element for all the analyzed TERpromoters. The second site is unique for the promoter region of TER of thenaked mole rat and is a binding site for the SOX17 transcription factor. Theabsence of one Sp1 site in the TER promoter region of the naked small rat is anadditional specific feature of the promoter area of hgTER. Such variation inthe hgTER transcription regulation region and hgTER itself could provideincreased telomerase activity in stem cells and an extended lifespan toH. glaber.Telomere length, an important feature of life span control, is dependent on theactivity of telomerase (a key enzyme of the telomere-length-maintainingsystem). Telomerase RNA is a component of telomerase and, thus, is crucial forits activity. The structures of telomerase RNA genes and their promoter regionswere compared for the long-living naked mole rat and different organisms. Tworare polymorphisms in  Several studies have shown that stem cell function is impaired during aging-3. A decin vitro: a reverse transcriptase catalytic subunit(TERT ) and telomerase RN A (TER ) that contains a template for telomeresynthesis [Telomerase synthesizes new telomere repeats at the G-strand and thusparticipates in the compensation for telomere loss during replication . Two comynthesis . TERT waynthesis ) outsideynthesis . In contynthesis . In someynthesis  showed tynthesis , providiHeterocephalus glaber, the naked molerat (H. glaber), has a very high life expectancy among rodents( > 28 years vs 1.5–7 years in other rodents), high resistance tocarcinogenesis and retarded aging [The genome and transcriptome of the naked mole rat have recently been sequenced, 17. Heted aging .H. glaber genome can help revealthe reasons for the surprisingly long lifespan of this animal. A number ofgenetic alterations have already been found, which could explain the increasein the DNA repair level, as well as the reduced oxidative damage or reducedreplicative senescence [H. glaber stemcells or telomerase reactivation under a certain type of stimulus. In thisstudy, we have compared the structure of telomerase RN A genes and theirpromoter regions for the longliving naked mole rat and different organisms withan aim to identify the features that may increase hgTER expression andtelomerase activity in stem cells.A comparative study of the nescence , 19. AnoComparison of TER sequenceshgTER gene of H. glaber, weused the whole genome shotgun project entries with IDs: AFSB (GenBank:AFSB00000000.1) and AHKG (Gen- Bank: AHKG00000000.1). We used ClustalW to buildan alignment of the sequences of interest with the already well-describedtelomerase RN A genes. The following reference sequences were used:Cavia porcellus TER (GenBank: AF221929.1), Caviaporcellus WGS assembly (GenBank: AAKN00000000.2), Chinchillachinchilla TER (GenBank: AF221937.1), Chinchillachinchilla WGS assembly (GenBank: AGCD00000000.1), Musmusculus TER (GenBank: NR _001579.1), Mus musculuschromosome 3 (GenBank: NC _000069.6), Rattus norvegicus TER(GenBank: NR _001567.1), Rattus norvegicus chromosome 2(GenBank: NC _005101.3), human TER (GenBank: NR _001566.1), human chromosome 3(GenBank: NC _000003.11), Danio rerio TER (GenBank:EF569636.1), and chromosome 25 (GenBank: CU 651628.3).To search for the Suncusmurinus (GenBank: AF221921), Geomys breviceps(GenBank: AF221930), Microtus ochrogaster (GenBank:AF221909), Mus spretus (GenBank: AY058901), Musmusculus (GenBank: AY058900), Dasyurus hallucatus(GenBank: AF221919), Bufo japonicus (GenBank:AF221913) and Typhlonectes natans (GenBank: AF221910) wereused to perform a secondary structure analysis.The alignment analysis and the influence of polymorphisms on the secondarystructure were performed manually. The sequences of TER s from Comparison of TER promoter areasWe searched for promoter regions using the Jaspar database , restricIdentification of H. glaber TERhgTERC gene  was identified by local BLAST on the basis of the H.glaber genome assembly (WGS record AHKG) and a comparison withmultiple alignment data for mammalian TER sequences [The full equences . The finH. glaberare Hydrochoerus hydrochaeris, Caviaporcellus, Chinchilla chinchilla, andMyocastor coypus [Chinchilla chinchilla(GenBank: AF221937.1) and Cavia porcellus (Gen- Bank:AF221929.1); those were used for further structure comparison. Furthermore, thedata for human TER were used for the analysis due to the availability ofdetailed information about the promoter region and secondary structure of TER .According to phylogenetic data, the closest relatives of r coypus .TER seqComparison of TER promoter areasH. glaber. Thesetools provide the possibility to analyze any sequence data; they are relativelysimple in operation and allow one to deal with large-position weight matriceswith optimal results due to the effective job filtering. We used strictfiltering of the results for both tools to find the most reliable sites.A 500 nt (from the expected transcription start site) promoter region was usedfor the analysis, since the major regulatory elements were found in this areafor human TER (hTER ) . The webThis approach reduced the number of predicted transcription factor bindingsites in the hTER promoter area compared to the number of sites determinedearlier . The elep values were calculated by MAST. The ELK4 scorefor the H. glaber was 14.059 ; 11.056  forCavia porcellus; 12.053  forChinchilla chinchilla; and 10.398  for humans,thus meaning a very high probability (> 90%) of ELK4 transcription factorbinding site occurrence in the TER promoter area, at least from thebioinformatic point of view.The ELK4 transcription factor binding site had never been identified for anyTER before; it is located approximately 170 nt upstream from the start of theTER coding region. We have found this promoter element for all testedsequences; When performing the search, we found that the ELK4 binding site  matrix iCavia porcellus, H. glaber,and Chinchilla chinchilla. The binding sites found havevariations in positions 1, 8, and 9 of PWM, which is consistent with the knownET S binding sites [Multiple alignments showed that a ET S binding site was present in all fourspecies: human, ng sites , 27, 29.H. glaberTER promoter region; it is located ~ 430 nt upstream from thetranscription start site. The fact that the SOX17 site is present in H.glaber but not in other evolutionary related animals may be anindication of an important difference between these species.The SOX17 binding site was detected solely in the The SOX17 protein contains an HMG_box (Pfam domain high-mobility group box)responsible for the high-affinity binding to non-B-type DNA conformations(kinked or unwound) . ThechaBased on the common architecture for most TER s, we assumed that hgTER alsocontains the known secondary structure elements . We mainH. glaber’sclosest relatives (guinea pig and chinchilla) and the TER of model organisms revealed a number of differences. Although mostchanges in H. glaber TER were not unique  and did not affect the functional elements of TER , we managed tofind two rare polymorphisms in the functional region of hgTER . The mappedpolymorphisms are shown in Fig. 1aand Fig. 1b.The comparison of hgTER with the TER of Geomys breviceps andMicrotus ochrogaster) [Dasyurus hallucatus and Suncus murinus[The first polymorphism was a A→G replacement in the first loop ofpseudoknot P2b-p3 at position 111 (according to the hTER nomenclature). Thissubstitution gives rise to the non-canonical “G–U” pair inthe pseudoknot region. Most other TER s have a canonical pair at this position.Moreover, for TER s that have polymorphism A→G111 additional replacementU→C179 takes place that restored the canonical WC pair between bases in111 and 179 positions   The only murinus.H.glaber.The second polymorphism is the replacement G→A in the stem of the scaRN Adomain CR 7-p8b at position 421 (according to the hTER nomenclature). Thissubstitution gives rise to the non-canonical “C–A” pair inthe p8b stem terminus element at position 421. Most TER s have a canonical basepair at this position. Amphibians (toads and typhlonectes) have G421→Areplacement accompanied by C→U transition at position 408, which restoresthe WC pair . Close rH. glaber with available data on other species allowed us toreveal variations both in the promoter region and telomerase RN A structure.Telomerase RN A is a crucial telomerase component, and increased expression oftelomerase RN A and te lomerase activity in stem cells or other tissues atdifferent stages of an animal’s development could be an essential reasonfor its long lifespan. Comparison of the TER gene promoter region and TER inH.glaber .All common elements are located within the ~ 270 nt area, in agreement with the DNase 1protection data for the human regulatory region [H. glaber and itsclose relatives revealed that one or more Sp1 sites can be missing in aparticular organism. For example, the Sp1.2 site is missing in all rodentsand the Sp1.3 site is also absent in Caviaporcellus. In case of humans, the two transcription factors Sp1 andSp3 can bind to the Sp-sites within the promoter. Sp1 stimulates expression,while Sp3 induces dosedependent repression [H. glaber) are thought to cooperate with NF-Yto mediate positive or negative regulatory effects in humans[H. glaber, this may have a positive effect on the TERtranscription efficiency.An analysis of the region 500 nt upstream of hgTER transcription start sideallowed us to identify the regulatory elements known for other organisms and two y region .This repression .The sitn humans. The Sp1n humans. The conn humans. Thus, tH.glaber, the sequence of this site is identical to that of ELK4, but itcan also be used by the other members of the ET S family [The newly identified ELK4 site is located within the 272 nt area furtherdownstream from the transcription start site. It was found in all the studiedspecies, including humans. ELK4 is a member of the ET S family of transcriptionfactors . For H.S family . This faS family .H. glaber. It islocated approximately 430 nt upstream of the gene region, and thus outside ofthe 272 nt promoter area, which was previously shown to be important for hTERtranscription. SOX17 belongs to the family of HMGlike SOX proteins. The SOX17binding site (ACAAT) is identical for the other members of the SOX proteins,and binding of a particular factor depends on the broader context around theconventional site. SOX17 (SRY-box 17) is a transcription factor involved in theregulation of several developmental processes [A SOX17 binding site was found only in rocesses , 40, incH. glaber to regulate the expression oftelomerase RN A and to increase the level of telomerase activity, especially infetal stem cells. This correlates with earlier studies, where long-livingrodent species (such as the H. glaber and Sciuruscarolinensis ) have a higher telomerase activity than mice [Sox17 is highly restricted in its expression within the hematopoietic system tofetal hematopoietic stem cells (HSCs) . It has han mice .Telomerase activity depends not only on the transcriptional level of telomerasecomponents; many other processes are involved, including TER maturation,transport, telomerase assembly, interaction between TER and TERT , etc.Mutations in telomerase RN A can influence these processes.Fig. 1a) is highlyconserved [H. glaber, a non-canonical G–Upair is formed. In contrast to other non-canonical pairs, G–U causes verylittle distortion to the RN A helix structure [We found two rare polymorphisms in hgTER : A111→G and G421→A. TheA111→G transition in hgTER is located in the stem loop of P2b-p3pseudoknot. The P2b-P3 pseudoknot domain. CR 7is required for 3’-end processing, localization, and the stability ofhTER [H. glaber, the non-canonical pair “C–A” inthe p8b stem loop can improve the interaction between hgTC AB1 and hgTER , maketelomerase traffic more effective and thus result in a more efficient telomereelongation.The polymorphism G→A at position 421 leads to the formation of aC–A non-canonical pair in the p8b stem. Most mammals have the canonicalbase pair at this position. It should be mentioned that disruption (C→Gin C–G) of 408–421 base pairs in humans leads to dyskeratosiscongenita . Rodents ofhTER . CR 7 co ofhTER . The tel ofhTER  and driv ofhTER . ForH. Comparison of hgTER and other telomerase RN Agenes suggests that both the unique structure of thepromoter region and the specific polymorphisms inthe functional domains can cause increased expressionof the telomerase RN A gene in stem cells, thusreducing replicative senescence and increasing thelifespan. We hope that our finding of a difference inthe promoter region of telomerase RN A will inspireother researchers to study these processes using invivo mouse models.