Pinctada fucata. We isolated a genomic clone containing the nacrein gene and cloned the 5′-flanking region. Within the 1336 bp 5′ flanking region, we identified putative cis-acting elements, including the TATA box (TATAAAA) at −82 bp, and AP1 (−819 bp) and Oct-1 (−1244 bp) binding sites. In addition to the mantle, the nacrein gene is also expressed in the adductor muscle, liver, and foot. These results showed that nacrein not only takes part in the formation of the hard tissue but also might be involved in acid–base balance, ion transport, and maintenance of ionic concentration. In vitro transcription experiments showed that the addition of human c-jun activates transcription from the nacrein promoter. This is the first report of a promoter from a gene that controls the formation of the hard tissue of mollusk shells.The carbonic anhydrase nacrein participates in the formation of the nacreous or prismatic layer of  Carbonic anhydrases (CA)(EC 4.2.1.1) are zinc-containing enzymes that catalyze the hydration of carbon dioxide and the dehydration of bicarbonate:CAs are widely distributed in all living things, from eukaryotes to prokaryotes, and play an important role in acid–base balance, ion transport, and maintenance of ionic concentration Cameron , 1996. THaliotis tuberculata, Linnaeus  among them had carbonic anhydrase activity and calcium binding capability  specimens were obtained from Gokasho Bay  in August 2006.Recombinant human c-jun was purchased from Promega. Pearl oyster  using the QuickPrep Total RNA Extraction Kit  according to the manufacturer’s instructions.RNA was isolated from various tissues of RNA samples (20 μg per lane) were separated by electrophoresis through a 15 % agarose gel containing 0.66 M formaldehyde and were then transferred to positively charged nylon membranes . The membrane was hybridized with a nacrein cDNA of approximately 2.3 kbp or human β-actin cDNA and labeled with digoxigenin-dUTP by the random primer method (Boehringer Mannheim). Hybridization and detection were performed as described previously  vector, which was designated pBDEV4. The plasmid pBDEV4 has two HincII sites. To remove the putative AP1 binding site, it was necessary to remove the HincII site between the EcoRV and XhoI sites. Therefore, plasmid pBDEV4 was digested with SalI and XhoI followed by self-ligation. The putative AP1 binding site is located between the SmaI and HincII sites. The SmaI site is in the multiple cloning site, and the HincII site is just to the 3′ side of the putative AP1 binding site. The plasmid pBDEV4 was digested with SmaI and HincII followed by self-ligation. The plasmid with the putative AP1 binding site deleted was designated pBDDS1.The pBDEN78 has two 2, 2.25 μl H2O, and 1.0 μl linearized nacrein promoter/5′-flanking region (50 ng/ml) as a template. The reactions were initiated by the addition of 4.0 μl HeLa extract and incubated at 30 °C for 1 h. Reactions were terminated by adding 87.5 μl Stop solution. After phenol/chloroform treatment, RNA transcripts were collected by ethanol precipitation. The precipitate was dissolved in water.The in vitro transcriptions were performed by the following modified protocol of HeLaScribe nuclear extract in vitro transcription system, provided by the manufacturer (Promega). DIG RNA Labeling mix (rNTP: Boehringer Mannheim) was used to label run-off transcripts. Reaction mixtures of 12.5 μl were prepared by combining 1.5 μl HeLaScribe nuclear extract (58.2 μg) with 1.0 μl DIG RNA labeling mix (10×), 0.75 μl 50 mM MgClTranscripts were separated by 1.2 % agarose gel electrophoresis and then transferred to a nylon membrane (Boehringer Mannheim) by Northern blotting. After baking at 80 °C for 2 h, the filters were visualized with BCIP/NBT reagent, according to the manufacturer’s instructions (Boehringer Mannheim).5 lambda FIX II phages was screened by plaque hybridization using the nacrein cDNA as the probe. Five positive plaques were isolated and designated clones A, B, C, D, and E. Restriction analysis showed that clones B, C, and D were identical (data not shown). The same analysis showed that clones A and E were different (data not shown). The length of the genomic fragment of clone D was longer than that of clone A. Moreover, although the 5′-flanking region was the same length in clones A and D, the breeding ratio of clone D was higher than that of clone A. Therefore, clone D was used for analysis.A library of approximately 5 × 10MboI restriction site within the fifth intron of the nacrein gene . The DNA was completely digested with EcoRI and NotI. After agarose gel electrophoresis, Southern blotting was performed, followed by hybridization with the 82 bp NotI-DraI fragment of the 5′ end of the nacrein cDNA  is functional in the in vitro transcription system using HeLa nuclear extracts  increased dependent on the amount of c-jun added. Deletion analysis showed that c-jun functions through the putative AP1 binding site located at −830 bp. These results suggest that the AP1 binding site and the TATA box of the nacrein gene are functional in an in vitro transcription system using Hela nuclear extracts.We examined the promoter activity of the nacrein gene in an in vitro transcription system using HeLa nuclear extracts. The addition of human c-jun protein to the in vitro transcription system increased the amount of transcript from the nacrein promoter. The amount of the 500-nt transcript (corresponding to the region between a putative transcription start site to an C-jun functions as a transcription factor and forms a homo dimer or a hetero dimer with c-Fos, to form the transcription factor AP1 (Kerppola and Curran In the mantle epithelium, nacrein mRNA is actively transcribed and translated. After translation, the signal peptide causes the secretion of nacrein from the mantle epithelium. The synergistic interaction between the promoter and a transcriptional regulatory element, such as an enhancer, regulates this transcription. The nacrein promoter is the first promoter from a gene that controls hard tissue formation of mollusk shells to be reported.


Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci  in the mouse mammary gland before and after functional differentiation.Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization.A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the full functional capacity of the mammary gland. Profound morphological and functional changes take place in the mammary gland during key reproductive stages, and the gland undergoes repeated cycles of functional differentiation with reproduction during the life of a mammal. In the mouse, as in most other mammals, the first postnatal-phase of development occurs at puberty under the influence of puberty-associated hormones and local growth factors Mammary glands are unique to mammals and are the basis of the survival and existence of these species. Unlike most organs and tissues, which undergo most of their development Mammary epithelial cell differentiation during puberty and pregnancy is mediated through a succession of cell fate decisions that include changes in expression patterns, increases of cell type-specific expression, and silencing of genes not required for cell identity or cell proliferation Chromatin organization plays a key role in transcriptional regulation during development and differentiation Most evolutionary conserved regions (ECRs) outside of coding regions correspond to Distal Regulatory Elements (DREs) . DREs seLittle is known about the chromatin changes that take place during mammary gland development and functional differentiation Since milk protein genes such as the casein genes and Wap encode the major milk proteins, which are secreted by the functionally differentiated epithelial cells in high amounts during lactation, they serve as markers for functionally differentiated cells. It has been suggested that the coordinated regulation of the casein genes is due to distal regulatory elements located within the casein gene cluster We hypothesized that chromatin organization is part of the regulation of gene expression in the functionally differentiating mammary gland. Open chromatin structure is expected on regulatory elements of genes expressed during lactation in the lactating mammary gland, while a closed chromatin structure is expected on regulatory elements of non-expressed genes. Additionally, lactation-specific genes are expected to be associated with closed chromatin in non-expressing and non-mammary tissues. Accordingly, we investigated several markers of chromatin structure in milk protein gene genomic regions in lactating or late-pregnant mammary gland tissue and compared this to non-mammary tissue (liver tissue).To determine the presence of histone modifications associated with open chromatin in milk protein gene loci in mammary and non-mammary tissues, we performed ChIP-seq analysis using an antibody against H3K4me2. H3K4me2 is associated with open chromatin and marks active and poised gene promoters as well as active distal regulatory elements To further establish the presence of open chromatin on the promoters of milk protein genes and evolutionary conserved regions (ECRs), we used conventional ChIP for Histone H3–acetylation (H3Ac)–associated with open chromatin. H3Ac was enriched on the promoters of the casein genes and Wap, while no enrichment was detected in liver ,3B, S3B.We analyzed the Csn1s1, Csn2 and Csn3 gene promoters for DHS in late pregnant (day 16) and lactating mammary gland tissue. We found DHS overlapping the proximal promoter regions of these genes . The BetDNA methylation is thought to represent a closed chromatin structure and has been associated with repression of gene expression Taken together, the above data establish that milk protein genes reside in genomic regions with an open chromatin organization, in the lactating mammary gland but not in non-mammary tissues such as the liver. GAS-(Stat), C/ebp sites and other transcription-factor-binding-sites have been extensively mapped in casein gene promoters and BCE  cells DNA methylation levels at ECR3 are similar in MEC and non-MEC from pre-pubertal animals, 2.5–3 weeks of age , while tAs the BCE sequence does not contain CpGs and the proximal promoter sequences (<500 bp from TSS) of Csn1s1 and Csn1s2a did not allow design of working DNA methylation assays for these genes, these regions were not analyzed. However, for all measurable regions, the results are consistent with a loss of DNA methylation near milk protein genes as the mammary gland prepares for lactation.ChIP-seq analysis showed that, in the casein gene region, levels of H3K4me2 are low in mature virgin MECs compared to lactating mammary gland tissue . WhereasLike the casein region, the Wap gene showed similarly low levels of H3K4me2 in virgin MEC, except for a peak that coincides with HSS0–a DHS identified in mouse, rat (rHSS1) and rabbit lactating mammary gland ELF5 is an important regulator of functional differentiation during pregnancy, regulating the establishment of the secretory alveolar lineage and expression of some milk protein genes Consistent with the fact that the amphiregulin (Areg) and progesterone receptor (Pgr) genes are mainly expressed in the virgin MEC, H3K4me2 was markedly enriched on these genes in the virgin MEC . In the Together, these results indicate that chromatin organization gradually changes with the development and functional differentiation of the mammary gland and is associated with changes in gene expression.Cessation of lactation results in involution of the mammary gland, which is accompanied by a profound reorganization of the mammary gland. Many of the epithelial cells that produced milk undergo apoptosis, while the adipocytes reconstitute their lipid content DNAseI hypersensitivity (DHS), histone H3 acetylation (H3Ac) enrichment, and H3K4-di-methylation (H3K4me2) enrichment are each indicative of open chromatin In contrast, in liver tissue the casein gene cluster region lacks H3K4me2 and has higher levels of DNA methylation relative to mammary gland tissue, both of which are indicative of closed chromatin structure. Furthermore, our preliminary data show enrichment for H3K27me3–another histone modification associated with developmentally inactivated chromatin–in the casein locus in liver tissue (data not shown). These findings are consistent with data from Wen et al A lack of H3K4me2 enrichment at the promoters and throughout most of the casein gene region in the virgin MEC, together with a trend towards higher levels of DNA methylation in virgin MEC, suggest a more closed chromatin organization relative to lactation in the mature virgin gland. However some ECRs (ECR3 and ECR19) show a reduction of DNA methylation from pre- to post-puberty and an open chromatin organization marked by H3K4Me2 enrichment in the mature virgin, suggesting an early role for these ECR in gene regulation in the casein region. Similar changes in chromatin organization between the virgin and lactating mammary gland were seen in the WAP gene region–Hss0 is enriched for H3K4me3 in the virgin gland. These epigenetic changes are consistent with the high level of expression of these genes during lactation.In contrast to the casein and Wap gene regions, H3K4me2 enrichment of the Lalba and Expi gene regions in the mature virgin gland indicates that these regions already have an open chromatin organization at this stage of development. These seemingly contradictory results highlight the fact that gene expression is the summation of different mechanisms of regulation. This suggests that these genes are regulated differently and that an open chromatin organization at these genes is not enough for gene expression. Interestingly in a study examining the role of Sim2s in mammary gland function Wellberg et al After involution of the mammary gland, a time when casein genes are not expressed at high levels, DNA methylation levels at the Csn1s2b casein gene promoters are higher than during lactation and pregnancy and a similar trend was present for the Csn2 and Csn3 promoters, whereas the DNA methylation levels at ECR3 and 19 remain low. This suggests a more closed chromatin state at the promoters in the involuted gland while the chromatin organization at potential DRE (ECR3 and 19) appears to remains open.Together, these data indicate that epigenetic modifications influence temporal regulation of gene expression in the mammary gland. Moreover, our results suggest that there are distinct groups of genes, each with their own dynamic pattern of epigenetic regulation during mammary gland development.Upon involution, milk protein gene expression is down-regulated, tissue remodeling takes place and morphologically the gland resembles the pre-pregnant state. The transcriptional state of the mammary gland is at least partially reset to the level prior to the onset of lactation Higher DNA methylation levels at the casein gene promoters after involution than during lactation and pregnancy  could beStudies of DREs in different tissues and in other gene regions such as beta-globin http://insulatordb.uthsc.edu/http://genome.ucsc.edu/). ECR19 is located between the region harboring four of the casein genes, which are expressed in the mammary gland, and the Odam gene, which is expressed in tooth-associated and other epithelia Transcription factor binding site predictions as well as genomic location suggest that ECR19 may serve as a chromatin-organizing element. ECR19 and a 1 kb region immediately flanking have predicted binding sites for the chromatin organizer CTCF Our findings should be interpreted with the following caveats. First, the virgin MEC preparations are not 100% pure mammary epithelial cells, and likewise neither does lactating mammary tissue consist of 100% epithelial cells, but rather both are (significantly) enriched for MECs. Second, two recent studies demonstrated lineage specific  epigenetic marks in mammary epithelial cells We have intensely studied a small subset of genes, particularly the casein genes, so there are limits to which these findings can apply to the whole genome. However, similar findings for other spatially, temporally and lineages regulated genes in different tissues 2 exposure and tissues were isolated from animals at different ages and times of mammary gland development.Balb/c and ICR mice were obtained from Harlan laboratories and housed in an American Association of Laboratory Animal Care-accredited facility at Baylor College of Medicine following guidelines outlined by the institutional Animal Care and Use Committee. The protocol was approved by institutional Animal Care and Use Committee of Baylor College of Medicine (AN-3455). Animals were euthanized by COMammary Epithelial Cell (MEC) enriched organoid preparations were obtained by enzymatic digestion of mammary gland tissue and differential sedimentation as described by Genomic DNA was isolated from MEC and whole tissues using standard proteinase K and phenol/chloroform methods −ΔΔCt method Total RNA was isolated from whole tissue  or MEC pellets  using Trizol (Invitrogen) and treated with DNaseI followed by clean-up using RNAeasy kit (Qiagen). RNA concentration was measured with Ribogreen (Invitrogen) measured on Q-bit and quality was determined using Agilent Bioanalyzer. mRNA was converted into cDNA using SuperScript III reverse transcriptase according to manufacturer’s protocol (Invitrogen). Relative expression was determined using CyberGreen . Next, cells were incubated in Nuclear Isolation Buffer ) on ice for 20 min. Nuclei were released from cells by Dounce homogenization (loose pestle) with 10–15 strokes. Nuclei were collected by centrifugation at 500 g at 4°C for 5 min. Nuclei were lysed in nuclear lysis buffer ) and chromatin was sheered to a size of 200–1000 bp by sonication (Branson D-450) using 15 sec bursts , 30 sec rests, at 90 to 100% Amplitude. 50–150 µg of chromatin was used per Immunoprecipitation (IP) in 1× dilution of ChIP Dilution Buffer (IPDB (10×): 16.7 mM Tris-Cl pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Trition X100, 0.01% SDS, plus 10 mM Na Butyrate, 10 mM PMSF and protease inhibitors ). Chromatin was precleared with protein-A Sepharose beads  and normal rabbit serum (Sigma Cat# 15006) for 1 hr at 4°C. Chromatin was incubated with 5 µg antibody specific for H3Ac (06–599. Upstate-Millipore); H3K4me2 ; Pol II   overnighReal-Time-PCR, values were calculated relative to a standard curve of genomic input DNA and were normalized to GAPDH values in mammary gland or liver.For ChIP-seq, chromatin was prepared as described by Wagschal et al Raw reads generated from Illumina/Solexa GAII were mapped to mouse reference genome (NCBI37/mm9) using Eland (Illumina) with maximally 2 mismatches tolerated.ChIP-seq data  have bee2, 0.1 mM PMSF, 0.5 mM DTT) with MgCl2 and CaCl2 added to 0.4 mM and 2 mM respectively at a standard DNA: DNaseI  concentration for increasing time. The reaction was stopped with 20 mM EDTA, 1% SDS. DNA was isolated with proteinase K treatment and purified by phenol/chloroform extraction. DHS were detected by Southern blotting using probes designed to the ends of informative restriction fragments, or by Real-Time-PCR as described by McArthur et al DHS mapping was performed as described by Millot et al Up to 1 µg genomic DNA was treated with Bisulfite (BS) using the Epitech Bisulfite kit according to manufacturer’s instructions (Qiagen). PCR primers specific for Bisulfite treated DNA were designed with methyl-primer express software (ABI). PCR was performed using Bisulfite DNA specific primers .Sequence was retrieved from available genome assembly. Sequence was analyzed for potential CTCF binding sites at the CTCF binding site data base , signaling pathways, gene expression and structural variation of the genome  will lead to a better understanding of the functioning of the mammary gland in health and disease, and may contribute to new strategies for enhancement of lactation performance and breast cancer treatment.Broadly, our data demonstrate chromatin remodeling in both a tissue-specific and developmental stage-specific manner. These data also suggest that the evolutionary conservation of certain sequences in promoters and distal regions is due to their role in regional gene expression through control of chromatin state. Employing the mammary gland as a model for developmental biology, we expect these findings to be widely applicable to the development of other tissues and organs in mammals and in other organisms.Figure S1Gene expression of genes in genomic region as shown in – and S2. Expression of genes in the casein gene region (A), Albumin (ALB) and tyrosine aminotransferase (Tat) gene (B), and other milk protein gens: genes in the Whey Acidic Protein gene region, alpha-lactalbumin , and extracellular proteinase inhibitor (Expi) (C), in liver (red) and lactating mammary gland (blue). Expression array data (GDS1805) for liver  and lactating mammary gland  (PDF)Click here for additional data file.Figure S2H3K4me2ChIP-seq results for Albumin (Alb) and tyrosine aminotransferase (Tat) gene expressed in liver. ChIP-seq H3K4me2 of liver (red) lactating mammary gland (blue) and virgin MEC (black) tissue. Black arrow indicates location and transcriptional direction of Alb and Tat, Green box indicates Albumin enhancer location.(PDF)Click here for additional data file.Figure S3ChIP on select region in the casein gene cluster, Wap Alb and Tat genes. ChIP assays on mouse lactating mammary gland (white bars) and liver (Black bars) tissue. MG and liver chromatin was immuno-precipitated using antibodies against-acetylated-Histone-H3 and di-methylated-Lysine4 of H3 (H3K4me2), DNA was isolated, and DNA samples were analyzed using real-time PCR. (A) enrichment in samples immuno-precipitated with antibodies to (A) H3K4me2 ). (B) H3Ac , compared to input (un-precipitated sample) and normalized to enrichment in the housekeeping gene GAPDH, based on real-time PCR analysis of the indicated amplicons. (C), representative PCR results (n = 3) of ChIP analysis of ECRs . Alb: albumin gene & Tat; tyrosine aminotransferase (both liver specific); Csn1s1: alpha-s1casein; Csn2: beta casein: Csn1s2a: alpha-s2a casein; Csn1s2b: alpha-s2b casein; Csn3: kappa casein; Wap-p: Whey Acid Protein promoter; Wap-H2: Wap-Hypersensitive site 2; ECR1: evolutionary conserved region 1; ECR3; ECR6; beta-casein upstream enhancer (BCE); ECR16, ECR19, ECR21 (evolutionary conserved regions).(PDF)Click here for additional data file.Figure S4Tissue-specific DHS on casein promoters and ECRs .(A) Csn1s1 (Alpha casein) promoter: Nuclei were isolated from 16day pregnant and liver tissue, exposed to increasing amounts of DNase1, genomic DNA isolated and digested with EcoRI. Regions in the chromatin hypersensitive to DNase1 are detected as sub-bands of the EcoRI band (red line and red arrow), size of the sub-bands indicates the location of a DNase1 hypersensitive site . Red blocks indicate location of probe used. (B) Nuclei were isolated from 16day pregnant and8 day lactating mammary gland tissue, exposed to increasing amounts of DNase1, genomic DNA isolated and digested with EcoRI. Analyzed with a probe identifying DHS at the CSn2 (beta casein) promoter (left panel) or DHS around the BCE (right panel). (C) Csn3 (kappa-casein) Nuclei were isolated from 16day pregnant and liver tissue, exposed to increasing amounts of DNase1, genomic DNA isolated and digested with EcoRI. (D) ECR19: Nuclei were isolated from 16day pregnant and liver tissue, exposed to increasing amounts of DNase1, genomic DNA isolated and digested with EcoRI. Similarly Nuclei were isolated from MEC and non-MEC cell preparations isolated from mammary glands of 8 week old virgin animals, exposed to increasing amounts of DNase1, genomic DNA isolated, PCR performed with primers flanking the HS in ECR19. Values were normalized to fragment that is not DHS in these tissues; DHS is expressed as fraction of fragments left relative to non-MEC and zero point.(PDF)Click here for additional data file.Figure S5DNA methylation around ECR6 during mammary gland development and in Liver. Genomic DNA isolated from 3 week virgin (3V) mammary gland, 12 week virgin MEC (12Ve) or non-MEC (12Vs) cells, 8day lactating mammary gland and Liver was digested with EcoRI alone (lanes 3) or in combination with either MspI (lanes 1) or methylation sensitive HpaII (lane2). ECR6 sequence was used as probe in southern blot analysis. Complete digestion with MspI or HpaII results in the detection of a ∼1.3 Kb band indicated with red arrows. Repeat sequences (LINE and LTR) are indicated by light gray blocks. .(PDF)Click here for additional data file.Figure S6Real-Time-RT-PCR expression analysis of lactating mammary gland expressed genes. Q-RT-PCR at different developmental stages in MEC isolated from mammary gland tissue of 13 week virgin (13wV), 3 days pregnant (3dP) and whole 0and 8dL MG) as well as liver from a 8d lactating animal (8dL Lvr). Real-time-RT-PCR data are normalized to K18 and expressed relative to 13wV MEC.(PDF)Click here for additional data file.Figure S7ChIP-seq information.(A) TableS4 H3K4me2 ChIP-seq reads used for analysis. H3K4me2 peak frequency 5 kb (B) and 20 kb (C) around TSS, in liver, Mammary epithelial cells isolated from 12 week virgin (MEC), and Lactating mammary gland tissue (Mg) based on MACs peak calling.(PDF)Click here for additional data file.Table S1Primers used for ChIP q-PCR, regular ChIP-PCR, DnaseI-PCR, Bisulfite sequencing, and MassArray analysis.(XLSX)Click here for additional data file.Table S2DNA methylation levels from MassArray analysis.(XLSX)Click here for additional data file.Table S3Antibodies used for ChIP.(DOCX)Click here for additional data file.


AUTS2, mutations of which are associated with autism and intellectual disabilities. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We also pinpointed concomitant changes in histone modifications between samples.Copy number variants (CNVs) influence the expression of genes that map not only within the rearrangement, but also to its flanks. To assess the possible mechanism(s) underlying this “neighboring effect”, we compared intrachromosomal interactions and histone modifications in cell lines of patients affected by genomic disorders and control individuals. Using chromosome conformation capture (4C-seq), we observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together. The newly identified interacting genes include We conclude that large genomic rearrangements can lead to chromatin conformation changes that extend far away from the structural variant, thereby possibly modulating expression globally and modifying the phenotype.GEO Series accession number: GSE33784, GSE33867. They significantly contribute to genetic variation, covering more nucleotide content per genome than single nucleotide polymorphisms  of genomic segments is frequent in human per se, i.e. without changes in gene dosage were shown to profoundly impact the phenotypic outcome, as some phenotypic traits present in Smith-Magenis (deletion) and Potocki-Lupski syndromes  mouse models were not rescued by restoration of the copy number in a strain carrying both the deletion and duplication on different alleles cis-acting regulators CNVs impact tissue transcriptomes on a global scale by modifying the level and timing of expression of genes that localize within the CNV As chromatin structure plays an important role in gene regulation, we anticipate that CNVs will affect the chromatin structure on a large scale, and hence possibly modify the clinical phenotype. However, studies investigating the impact of a structural aberration on long-range chromatin structure have been lacking. Here, we explored the chromosome-wide effect of a structural rearrangement on chromatin structure. First, we studied, by chromosome conformation capture, whether non-hemizygous genes neighboring a rearrangement and known to be affected in their expression levels also show changes in chromatin structure. Second, we monitored local chromatin changes as determined by histone modifications in the same cell lines with a structural rearrangement.GBAS, ASL, KCTD7, HIP1, POR and MDH2  are modified in their relative expression levels in lymphoblastoid and/or skin fibroblast cell lines of WBS patients ZNF107, a gene located between the GBAS and ASL that did not show any significant change in expression in WBS versus Control cell lines . After removal of the strong local background signal, we used a statistical segmentation algorithm to detect significantly interacting regions without imposing a fixed window size (see methods) Supplementary Table S2).We have previously shown that ll lines Table 1 P = 0.09 for GBAS, P<0.05 for all other 6 viewpoints, permutation test with number of permutations N = 10000). We also found a positive correlation between the number of viewpoints with which a region interacts and the gene density of that particular region: regions interacting with all, five (excluding GBAS), two or only a single viewpoint(s) have a gene density of 4.8×10−2, 4.1×10−2, 1.7×10−2, 0.3×10−2 RefSeq genes/kilobase, respectively. We then compared chromosome-wide interactions with the ENCODE data set of expressed genes from the GM12878 lymphoblastoid cell line P = 0.25 for ZNF107, and P<0.05 for all other viewpoints, permutation test with number of permutations N = 1000). We also investigated whether regulatory elements were enriched in the interacting regions. Towards this goal we used the ENCODE datasets of different regulatory marks from the same GM12878 cell line including H3K4me1, H3K27ac, p300, CTCF, DNaseI hypersensitive sites (DHSs) and FAIRE sites Supplementary Table S3). A large fraction of the interacting regions are shared between multiple viewpoints on the long arm of HSA7 . For example, 23% (28/121) of the regions found to interact with POR also interact with the ASL, KCTD7, HIP1 and MDH2 viewpoints. They cluster however less with the GBAS viewpoint, which maps to the short arm of HSA7. An exception is ZNF107, which maps close to the centromere, and interacts frequently with the other side of the centromere. The robustness of the 4C assays is finally further exemplified by the fact that many of the reported interactions are identified reciprocally (see below).We assessed whether our data are consistent with known features of chromosome conformation. As previously published we observed the strongest interactions close to the viewpoints, a clustering of gene-dense regions and possible regulatory regions and that loci interact more frequently with regions along the same chromosomal arm ). For the three genes immediately downstream of the WBS deletion , we observed higher interactions with the entire WBS deletion region when compared to the region telomeric to these viewpoints. This could in part be due to spatial clustering of active gene-dense regions ELN), LIMK1, EIF4H and CLIP2 genes . We also found interactions with the centromeric low-copy repeat (LCR) region, but we cannot exclude that this merely reflects its high similarity with the nearby telomeric LCR. Alternatively, as the HIP1, POR and MDH2 viewpoints are immediately adjacent to the telomeric LCR, this interaction loop might be a chromatin loop caused by the mispairing of these two repetitive and highly homologous sequences. Existence of such loop was postulated to facilitate excision and thus deletion of the intervening sequence causing WBS ZNF107, ASL and KCTD7 that map at a greater distance of the WBSCR than the telomeric viewpoints, also loop with that genomic interval albeit not as strongly  . Overall there is no drastic reorganization of the chromatin. In most cases, interactions are not abrogated but only modified in their intensity in cells with the 7q11.23 microdeletion consistent with the maintenance of one normal allele. From 58% (GBAS) to 89% (MDH2 viewpoint), of the interacting regions are shared between the Ctrl and WBS cell lines. We next calculated changes in interaction frequency in both cell lines and determined positive and negative ratio Bricks, corresponding to interactions that are significantly increased or decreased in WBS cells, respectively . We found that interactions within the WBSCR are on average decreased approximately two-fold in the WBS cells for the viewpoints mapping close to the WBSCR , consistent with normal looping intensity in the remaining allele and absence of interaction in the deleted allele (KCTD7 and a region around the CLIP2 and GTF2IRD1 genes within the WBSCR were more than two fold diminished in WBS cells. We used the ENCODE datasets from the GM12878 lymphoblastoid cell line to search for regulatory elements within this region HIP1 the closest viewpoint maps more than 1 Mb away). We hypothesized that only specific DNA/gene loops between regions on opposite sides of the WBSCR might be changed with the deletion, complicating the chromatin landscape. Corroboratively, in WBS cells the GBAS viewpoint is closer in space to the HIP1, POR and MDH2 genes, while the POR viewpoint and the AUTS2 gene interact less (Supplementary Table S3), both at positive and negative ratio Bricks. In some instances, we identified interesting patterns of changes: around genes particularly, an increased interaction in WBS cells was concomitant with flanking reduction of looping intensity . This observation suggests that chromatin reorganization is not dramatic, but rather that the intensity of long-range interactions is modified locally around certain loci. This is consistent with other work that showed that chromatin reorganization is mirrored in local changes in interactions , consistent with the clustering of open, actively transcribed regions , suggesting that chromatin clustering might be determined more by the presence of genes than accessibility of the chromatin  show a statistically significant change in chromatin opening between Ctrl and WBS cells . HIP1 shows an increase in H4K20me1 that does not parallel its diminished expression in WBS cells. However, it also presents a significant increase in H3K27me3 marks, which parallels its change in expression  and H3K27me3 (trimethylation of Lysine 27 of histone H3), as proxies for open and condensed chromatin, respectively pression Table 1.Structural variants have been shown to capture 10% to 25% of the expression variance KCTD7 viewpoint and the region between CLIP2/GTF2IRD1 is abolished in WBS cells rather than diminished by 50%, suggesting allele-specific chromatin interaction, which was recently postulated by studying the inactive X chromosome First, deletion of specific long-range regulatory elements within the rearrangement, such as enhancers and/or boundary elements, can cause changes in their finely tuned regulatory function and thus in the expression of their target genes. Concordantly, we detect alterations of intrachromosomal interactions  between expression-affected gene loci and the rearranged interval in WBS cells using chromosome conformation capture. Some of these alterations go beyond the expected two-fold decrease. Specifically, we observe that the interaction between the cis-acting DNA regulatory elements, a structural rearrangement could also exert its effect on gene expression by changing the overall chromatin topology and DNA accessibility. Genes might be co-regulated by clustering into “chromatin globules” independently of functional relationship ZNF107 locus hinting that other mechanisms must also be at play.Second, in addition to modifying specific AUTS2 gene. Its looping architecture, chromatin structure changes and expression modification in WBS cells designate this gene as a potential candidate in some of the phenotypes shown by WBS or WBRdupS patients. AUTS2 is mutated or translocated in autistic patients and individuals with intellectual disabilities AUTS2 expression is modified in other tissues.Some of these modifications may be associated with specific phenotypic features observed in genomic disorders patients. A tantalizing example from our study is the http://www.unil.ch/Jahia/site/fbm/op/edit/pid/36053), made exception of the WBS (GM13472) and Control  lines that were obtained from the Coriell Institute for Medical Research Biobank (http://www.coriell.org/).All lymphoblastoid cell lines used in this study were collected with written appropriate informed consent and approval of the local ethics committee  with addition of 10% fetal calf serum and 1% penicillin-streptomycin. The rearrangement was examined by array CGH using Human CGH 3x720K whole-genome tiling array (Nimblegen) following the manufacturer's protocol. Known changes in the expression levels of 7 exponentially growing cells were harvested and crosslinked with 1% formaldehyde, lysed and cut with the restriction enzyme BglII. After ligation and reversal of the crosslinks, the DNA was purified to obtain the 3C library. This 3C library was further digested with NlaIII and circularized to obtain a 4C library. The inverse PCR primers to make the 4C-seq templates were designed to contain the Illumina adaptor tails, as well as the bait-specific sequences for each of the seven loci we interrogated. The list of primers is described in Supplementary Table S1. The seven viewpoints were selected at the BglII fragment containing the transcriptional start sites of four genes located upstream of the WBSCR , and three other genes located immediately downstream of the WBSCR . For the three nearby downstream viewpoints, we amplified at least 0.6 µg of 4C template, whereas for the further away upstream viewpoints, we amplified at least 1 µg of 4C template (using about 100 ng per inverse PCR reaction). We multiplexed the 4C-seq templates by pooling the samples in equimolar ratios in two sets, representing 3 viewpoints each . Replicate 4C libraries were prepared for both the Ctrl and the WBS cell lines. We randomly selected three viewpoints  and replicated the experiments. All 4C-seq multiplexed samples were analyzed on a Illumina GAIIx flow-cell using a 76-bp single-end sequencing run. These studies were completed with a 4C assay with viewpoint mapping at the transcriptional start site of ZNF107. This gene did not show any significant change in expression in WBS versus Control cell lines The 4C-seq assay was performed as described in BgIII fragments. Reads were then normalized to the total number of reads. A running mean algorithm was applied to smooth the data (19 fragments per window). As the data from the three replicated viewpoints were strongly correlated , we used the average of each data point for these experiments. To remove the strongly interacting local “background” region, we modeled the data to apply a profile correction similar to the one described in GBAS and MDH2, the first and last viewpoint, respectively . As long-range interactions are more prone to random ligation events, we used a more stringent FDR threshold of 0.001 for the genomic space outside of these borders . Interacting regions were then defined by merging consecutive Bricks. To determine differentially interacting regions between the WBS and Ctrl cells, we first computed the log2 ratio of WBS over Ctrl of the smoothed profile corrected data and selected ratio Bricks that were specific to either WBS or Ctrl . To assess the significance of those regions we quantified the number of reads inside each Brick, and averaged over consecutive Bricks within each region. We then compared the distribution of the (Ctrl+WBS) log counts in these regions versus Bricks outside by a Wilcoxon rank-sum test. We obtained very significant p-values , indicating that selected ratio Bricks contained a significantly higher number of reads than all Bricks. 4C data are deposited under accession number GSE33867.4C-seq data were analyzed as described in −4. To permute the interacting regions we used shuffleBed from BEDtools version 2.10.1 To estimate if the long-range interactions of the seven viewpoints were significantly enriched in genes or histone modifications we performed permutation tests (n = 10000) with all RefSeq genes or histone modified regions identified by SICER with a FDR = 1×106 cells were centrifuged and used directly in the ChIP assay. Cells were lysed by addition of cell lysis buffer  and 10- minute incubation on ice. Next, chromatin was sheared using a Bioruptor sonicator (Diagenode) at medium power settings (30 seconds on – 30 seconds off cycles for 45 minutes). Sonication efficiency was tested by reversing cross-links of a chromatin sample and running the obtained DNA on a 1.5% agarose gel. Fragmented chromatin was used directly in the ChIP assay or frozen at −80°C for latter usage.Crosslinking was performed by adding formaldehyde solution (Sigma Aldrich) to the cells in growth medium to a final concentration of 0.5%. After 5-minute incubation at room temperature, cross-linking agent was quenched with glycine. 1×10GAPDH and intron 5 of the GRM8 gene were used for the H4K20me1 and H3K27me3 ChIP, respectively. The same primer pairs were used reciprocally as negative controls.ChIP was performed as suggested in 6 and 3.3 and 15.3×106 for H4K20me1 and H3K27me3, respectively.Sequencing libraries of immunoprecipitated DNA samples were prepared as described by the manufacturer (Illumina) and then sequenced on two lanes of an Illumina GAIIx flow-cell each . Sequencing reads were mapped to the human reference genome  using Bowtie algorithm allowing 2 mismatches and no seed −4 defined by SICER for the Ctrl and the rearranged sample and further used these islands to assess statistical significance of differential modification of a given region using the DEseq package The identification of ChIP-enriched regions was performed assuming a Poisson distribution of the tag counts by using SICER Figure S1Reproducibility of 4C-seq experiments. (A) Mirror plot of the windowed 4C scores of two biologically independent replicates using MDH2 as viewpoint (Pearson correlation  = 0.97). (B) Overview of the number of mappable reads per viewpoint and per cell line, as well as Pearson correlation coefficient between bioreplicates.(PDF)Click here for additional data file.Figure S2Steps followed to generate the ratios BRICKS. To allow the identification of BRICKS with a negative log2 ratios, we run the domainogram algorithm by sorting the data on an ascending order, which puts the high negative ratios on top position of the initial ranking. The two sets of BRICKS have been treated independently, in the following way: 1) selecting and grouping consecutive BRICKS as described in the material & methods, 2) removing BRICKS found in both datasets (for overlaps greater than 5%), 3) removing genomics gaps  from BRICKS and 4) excluding BRICKS that were not part of a selected BRICKS in either Ctrl or WBS BRICKS. Finally both sets were grouped together to form a unique set of BRICKS.(PDF)Click here for additional data file.Figure S3Heatmap showing the percent coverage of HSA7 by Bricks of each viewpoint, as well as the percent of HSA7 that overlaps between Bricks of the different viewpoints, indicating that the viewpoint interactions cluster by their linear chromosomal position.(PDF)Click here for additional data file.Figure S4Close-up of the interactions of the seven viewpoints with the WBSCR in cells from a healthy control individual. The two areas highlighted in grey show the strongly interacting regions at the LCRcen (centromeric LCR) and the region within WBSCR. Pink boxes indicate the mapping of genes within the WBSCR.(PDF)Click here for additional data file.Figure S5Interactions of seven genes on HSA7 in cells from a WBS patient. Windowed 4C signal of each of the seven viewpoints along the entire chromosome. The black ticks below each graph show the location of the Bricks. The density of genes is shown at the bottom. Areas highlighted in blue pinpoint some examples of strong correlation of gene-dense regions with H4K20me1 marks and highly interacting regions. The mapping of the viewpoints and the WBSCR is indicated at the bottom.(PDF)Click here for additional data file.Figure S6Close-up of the interactions of the seven viewpoints with the WBSCR in cells from a WBS patient. The two areas highlighted in grey show the strongly interacting regions at the LCRcen (centromeric LCR) and the region within WBSCR. Pink boxes indicate the mapping of genes within the WBSCR.(PDF)Click here for additional data file.Figure S7Examples of regions with modified interactions with the POR viewpoint. The y-axis represents postprocessed normalized counts. The log2-fold change of the windowed 4C data in WBS over Ctrl cells is plotted. Positive or negative Bricks are indicated below each viewpoint graph, by blue or red bars, respectively. In WBS cells, the region around the CDK6 gene (A) or sonic hedgehog (SHH gene) (B) interacts with the POR gene, whereas in Ctrl cells, the flanking regions interact more frequently, indicating local changes in interactions.(PDF)Click here for additional data file.Table S14C-seq primer sequences.(PDF)Click here for additional data file.Table S2Overview of the location of Bricks per viewpoint for control and WBS cells, as well as the ratio.(PDF)Click here for additional data file.Table S3Correlation analyses between six different marks of regulatory elements and interacting regions in Ctrl cells (Ctrl Bricks), in all differential interacting regions significantly decreased (negative ratio Bricks) or increased (positive ratio Bricks) in WBS versus Ctrl cells. Permutation test with number of permutation  = 1000. Significant p-values are highlighted in grey.(PDF)Click here for additional data file.


MEN1) is mutated in the inherited disease multiple endocrine neoplasia type I, and has several documented cellular roles, including the activation and repression of transcription effected by several transcription factors. As an activator, MEN1 is a component of the Set1-like mixed lineage leukemia (MLL) MLL1/MLL2 methyltransferase complex that methylates histone H3 lysine 4 (H3K4). MEN1 is localized to the signal transducer and activator of transcription 1 (STAT1)-dependent gene, interferon regulatory factor 1 (IRF1), and is further recruited when IRF1 transcription is triggered by interferon-γ signaling.The tumor suppressor menin (IRF1. While MEN1 knockdown does not impact the rate of transcription, IRF1 heteronuclear transcripts become enriched in MEN1-depleted cells. The processed mRNA and translated protein product are concomitantly reduced, and the antiviral state is attenuated. Additionally, the transcription start site at the IRF1 promoter is disrupted in the MEN1-depleted cells. The H3K4 demethylase, lysine specific demethylase 1, is also associated with IRF1, and its inhibition alters H3K4 methylation and disrupts the transcription start site as well.RNAi-mediated knockdown of MEN1 alters the H3K4 dimethylation and H3 acetylation profiles, and the localization of histone deacetylase 3, at Taken together, the data indicate that MEN1 contributes to STAT1-activated gene expression in a novel manner that includes defining the transcription start site and RNA processing. Eukaryotic gene expression is regulated by dynamic nuclear signaling events that occur at the chromatin template and include post-translational modification of the histone proteins, via methylation, phosphorylation, acetylation and ubiquitination. Depending upon the position of a lysine residue in the histone amino acid sequence, histone lysine methylation is associated with either an activated or a repressed transcriptional state ,2. For eSaccharomyces cerevisiae, a single complex containing the histone methyltransferase (HMT) Set1 is responsible for the methylation of histone H3K4. In mammalian systems, six homologs of Set1 contribute to different Set1-like HMT complexes to accomplish this modification -labeled IRF1 (Open Biosystems IHS1380-97433442) and GAPDH (Open Biosystems IHS1380-97434647) cDNAs prepared using the DECAprime II kit . Phosphor screens were exposed to probed blots, and the band intensity was quantified with Storm 840 Imager and ImageQuant TL software .Total RNA was extracted from shRNAmir-MEN1 cells were grown in HyClone DMEM/high glucose medium supplemented with 10% tetracycline-screened fetal bovine serum and 10% antibiotic/antimycotic (Fisher). Cells were cultured with or without 2 μg/mL of doxycycline (Fisher Scientific). Approximately 8000 cells per well were plated in 96-well dishes and allowed to grow overnight. Cells were untreated, treated with IFN-γ  or IFN-α  for 12 hours and then infected with various dilutions of VSV for approximately 15 hours. The viral medium was removed, cells were fixed with 5% formaldehyde and stained with 0.05% crystal violet. shRNAmir-NS cells did not phenocopy 2fTGH cells in this assay, perhaps due to an off-target effect that becomes relevant in this biological response assay. Therefore shRNAmir-MEN1 (-) doxycycline cells were used as the control.CPE assays (n = 2) were performed as described in . The titIRF1 specific primers designed to exon 3 (forward: 5'-CCCAGCTCCGGAACAAACAGGCATCC-3') for 3'RACE, or exon 3 (reverse: 5'-GGATGCCTGTTTGTTCCGGAGCTGGGC-3') for 5' RACE or exon 10 (forward: 5'-CAGTCACAGACAGAACAGTCAGCAGCCC-3') for 3' RACE. Products were gel purified and cloned into the TOPO vector (pCR4-TOPO). TOP10 competent cells were transformed and plasmid DNA was isolated from multiple clones using the Qiagen Spin Miniprep Kit . DNA was then sequenced with M13F and M13R primers provided in the TOPO cloning kit. Sixteen or seventeen clones were sequenced for each of the different conditions. A Fisher's exact test determined significance.RNA was isolated from 2fTGH cell lines under various conditions. RACE-PCR was performed using the Gene Racer Kit (Invitrogen). PCR products were generated using a 5' primer or 3' primer provided in the kit, as well as 2-PCPA: trans-2-phenylcyclopropylamine; bp: base pairs; CAGE: cap analysis of gene expression; ChIP: chromatin immunoprecipitation; CoREST: RE1-silencing transcription factor co-repressor; CPE: cytopathic effect; CTD: C-terminal domain; CTSS: CAGE tag starting site; DMEM: Dulbecco's modified Eagle's medium; HDAC: histone deacetylase; HMT: histone methyltransferase; hnRNA: heterogeneous nuclear mRNA; IFN: interferon; IgG: immunoglobulin G; kb: kilobases; LSD1: lysine specific histone demethylase 1 isoform a; me2: dimethylation; me3: trimethylation; MEN1: multiple endocrine neoplasia type 1; MLL: mixed lineage leukemia; NCoR: nuclear receptor corepressor; NR: nuclear receptor; PCR: polymerase chain reaction; qRT-PCR: quantitative real time PCR; RACE: rapid amplification of cDNA ends; Set1: Set domain containing 1A; SMRT: silencing mediator of retinoid and thyroid receptors; SnRNP: small nuclear ribonucleoprotein; STAT: signal transducers and activators of transcription; TSS: transcription start site; VSV: vesicular stomatitis virus.The authors declare that they have no competing interests.MAH conceived of and directed the study and wrote the manuscript. LBA designed the experimental strategy, performed the experiments and contributed to the manuscript preparation. SS performed the aberrant TSS RT-PCR assays and HDAC3 complex component ChIP experiments. LML designed and performed the rescue assays. All authors read and approved the final manuscript.IRF1 hnRNA transcripts are enriched in the MEN1-depleted cell line. (a-e) qRT-PCR to quantitate IRF1 total and pre-mRNA expression in shRNA-MEN1 and shRNA-NS cell lines that were uninduced (un) or treated with IFN-γ. IRF1 expression was normalized to β-actin and presented as fold change relative to the uninduced, shRNA-NS condition. Numbers are the base pair location of the primers used on the IRF1 gene; see Figure P ≤ 0.01, *P ≤ 0.05.Click here for fileshRNAmir-MEN1 cell line. (a-b) Increased rate of transcription does not account for elevated hnRNA pool in the qRT-PCR to quantitate IRF1 pre-mRNA and total RNA expression in shRNA-MEN1 and shRNA-NS cell lines that were uninduced or treated with IFN-γ and collected at indicated time points. IRF1 expression was normalized to β-actin and presented as fold change relative to the uninduced, non-silencing shRNAmir condition. Error bars are standard error (n = 4). **P ≤ 0.01.Click here for fileCPE assay MEN1 protein levels. (a) MEN1 western blot of whole cell extracts prepared from shRNA-MEN1 cells treated with doxycycline for 24 hours or not. GAPDH served as the loading control.Click here for filePhosphoserine 2 Polymerase II levels are not altered in the MEN1-depleted cell line.  Chromatin immunoprecipitation of shRNA-MEN1 and shRNA-NS cell lines treated with IFN-γ for 90 minutes or uninduced using the antibody specific for the phosphoserine 2 form of Pol II.Click here for fileAberrant TSS transcripts at Pol I, Pol II and Pol II genes. (a) qRT-PCR to quantitate transcripts initiated from a TSS upstream of the canonical TSS for IRF1, GAPDH and RN7SK (a Pol III gene) in shRNA-MEN1 and shRNA-NS cell lines. IRF1, GAPDH and RN7SK mRNA transcripts and aberrant TSS transcripts were normalized to β-actin; the aberrant TSS transcripts were calculated as a fraction of total mRNA, and presented as fold enrichment relative to the non-silencing shRNAmir condition. Error bars are standard error (n = 2). A student's one-tailed t-test determined significance. *P ≤ 0.05. (b) Effective qPCR primers could not be designed at the TSS of the 45S rRNA that is processed to 18S rRNA  and so endpoint PCR was used to attempt to detect aberrant TSS transcripts. 25 cycles of PCR did not generate an amplicon, nor did a second round of 25 cycles using an aliquot (10%) of the first reaction as template (arrow), but when genomic DNA was used a template an amplicon of the correct size was produced (positive control). This experiment was done for two biological replicate cDNA preparations, though only one is shown. 18S ORF primers detect the RN18S1 rRNA. RT+ indicates reverse transcriptase was included in the cDNA reaction while RT- indicates it was omitted.Click here for file


Mot1p-NC2 regulators of TATA-binding protein (TBP), we detected synthetic genetic interactions indicative of suppression of intragenic transcription. Conditional depletion of Mot1p or NC2 in absence of the ISW1 remodeler, but not in the absence of other chromatin remodelers, activated the cryptic FLO8 promoter. Likewise, conditional depletion of Mot1p or NC2 in deletion backgrounds of the H3K36 methyltransferase Set2p or the Asf1p-Rtt106p histone H3-H4 chaperones, important factors involved in maintaining a repressive chromatin environment, resulted in increased intragenic FLO8 transcripts. Activity of the cryptic FLO8 promoter is associated with reduced H3 levels, increased TBP binding and tri-methylation of H3K4 and is independent of Spt-Ada-Gcn5-acetyltransferase function. These data reveal cooperation of negative regulation of TBP with specific chromatin regulators to inhibit intragenic transcription.Chromatin structure in transcribed regions poses a barrier for intragenic transcription. In a comprehensive study of the yeast chromatin remodelers and the  Saccharomyces cerevisiae, these complexes are divided over four families based on their ATPase subunit: ISWI , INO80 , CHD and SWI/SNF (SWI/SNF and RSC) (The repeating unit of chromatin is the nucleosome particle consisting of ∼147 bp of DNA wrapped around an octamer of histones . Chromatand RSC) .FLO8 gene  complex  and transcription factor IIB (TFIIB) for TBP binding, thereby inhibiting PIC formation . In vivo, Mot1p and NC2 are concomitantly recruited to active promoters, where they form a complex with TATA-bound TBP to evict TBP from the promoter on ATP hydrolysis by Mot1p  formation starts with recruitment of the TATA-binding protein (TBP) . The ass complex ,20. In vdrolysis ,22. NC2 ormation ,24. In cn  to allow binding of TBP to low-affinity binding sites (TATA-less)  . InteresTA-less) . SAGA is)  technique developed by Laemmli and colleagues  transcription at specific loci. Further, chromatin immunoprecipitation (ChIP) analysis revealed a cooperative control mechanism of the Isw1p chromatin remodeler and Asf1p histone chaperone with the negative TBP regulators in maintaining a repressive barrier for intragenic transcription by restricting TBP binding to the cryptic FLO8 promoter.Here, we performed a comprehensive genetic analysis to investigate interplay of the TBP regulators, lleagues , allowinS. cerevisiae strains used in this study are listed in Supplementary Table S1. They were derived from HHY168 (Euroscarf #Y40343). Cells were grown in yeast extract peptone dextrose (YPD) or synthetic complete medium supplemented with 2% glucose at the indicated temperature. To create strains with a gene deletion or to C-teriminally fuse FKBP12-rapamycin-binding (FRB) domain of human mTOR to a protein of interest homologous recombination using polymerase chain reaction (PCR) generated DNA fragments was performed and verified by PCR. Details of primers and plasmids used are listed in Supplementary Tables S2 and S3, respectively. To generate the catalytic dead mutant isw1K227R, a linearized pRS406 plasmid containing a fragment of an ISW1 catalytically inactive mutation (a gift from Toshio Tsukiyama) was integrated using the standard pop-in/pop-out method  and harvested. For liquid growth curves, cells were diluted to OD600 of 0.15 in YPD in 24 - or 48-well plates at 30°C in a Tecan Infinite F200 instrument under continuous shaking. OD600 was recorded every 10 min. Rapamycin (1 μg/ml) was added at an OD600 of 0.15  where indicated.For spot assays, overnight cultures from single colonies in YPD at 30°C were diluted to an OD32P-labeled strand-specific or double-stranded DNA probes were used. For strand-specific probes, a cold PCR template was made by amplification. For detection of the sense transcripts, a labeled single-stranded DNA probe was generated from the template by using the reverse primer in a linear PCR reaction. Double-stranded probes were generated using the RediPrime II kit . The membrane was washed twice with 2× SSC  at room temperature, once with 2× SSC + 1% SDS at 65°C, once with 1× SSC + 1% SDS at 65°C and once with 0.5× SSC + 1% SDS at 65°C. Analysis was carried out using a Storm 820 phosphorimager .RNA isolation and northern blotting was carried out as described previously . RNA wasChIP was carried out as described previously , with miMot1p and NC2β .In our previous studies of and NC2β , we usedand NC2β , which rcultures . In contcreening . To alloMot1p and NC2 with chromatin structure regulators, the MOT1-FRB, NC2α-FRB and NC2β-FRB alleles were combined with deletions of the catalytic subunits of all non-essential chromatin remodelers , the H3/H4 chaperones (Δasf1 and Δrtt106) and the H3K36 methyltransferase (Δset2). We exploited the AA technique further by creating strains that could be depleted for two essential nuclear proteins at once, by creating FRB-tagged alleles of INO80, SNF2 or STH1. Genetic interactions were tested by analyzing growth on plates . This revealed interactions with the chromatin remodelers ISW1  and CHD1  and a strong interaction with INO80 . No genetic interactions were observed with ISW2 , FUN30 , SWR1  or SNF2 . As expected , no synthetic growth effects of STH1 with MOT1, NC2α or NC2β were apparent. In conclusion, depletion of Mot1p, NC2α or NC2β results in synthetic growth phenotypes with depletion of Isw1p, Chd1p and Ino80p.To investigate interplay of n plates  or in suexpected , nuclearexpected F. By comMot1p interacts physically with the INO80 remodeler as well as with the ISW1 complex . We detected intragenic transcripts from the well-characterized cryptic FLO8 promoter in Δisw1, Δasf1, Δrtt106 and Δset2 strains  of structural integrity of chromatin in gene bodies. The suppressive effects of Mot1p and NC2 on intragenic transcription are gene-specific, as no effect on STE11 transcription was observed.In short, FLO8. In the presence of Mot1p and NC2, cryptic promoter activity is repressed by clearance of TBP. Analysis of the ATPase-deficient isw1K227R mutant allele confirmed that the enzymatic activity of the ISW1 remodelers is critical for the cooperative effects of Mot1p and NC2 on intragenic FLO8 transcripts , no elevated intragenic transcript levels were observed on deletion of CHD1  at the cryptic promoter of FLO8 . This is expected, as Mot1p does not have chromatin as a substrate, but rather acts on TATA-bound TBP. TBP binding to the cryptic FLO8 promoter is increased about 2-fold on nuclear depletion of Mot1p or NC2α , suggesting that both ISW1 complexes are involved.Our study provides novel insight into control mechanisms that ensure transcription fidelity. Previous analyses stressed the importance of histone chaperones and chromatin regulators in maintenance of a repressive chromatin conformation to prevent spurious intragenic transcription. In a genetic screen for synthetic growth phenotypes of proteins . Our rescription B and C, cription . Possibl complex . Isw1p i complex . DeletioINO80 and MOT1/NC2α/NC2β be explained (A/T)A(A/T)(A/G)] in the coding region are three times more likely to give rise to intragenic transcripts compared with genes lacking such a TATA sequence  sites (FLO8 promoter (+1626)  sites . In supps) sites B and C aegulated . The smapromoter  and is eMot1p, NC2α and NC2β behave similar in our present assays. This agrees well with previous genome-wide mapping data indicating that Mot1p and NC2 bind together to promoter-bound TBP, which is further substantiated by biochemical assays  through ALW [820.02.013 to H.T.M.T.]; CW-TOP [700.57.302 to H.T.M.T.]; National Institutes of Health [GM52461 to P.A.W.]. Funding for open access charge: Own institution and NWO.The Conflict of interest statement. None declared.


Neurospora crassa grown under different light and temperature conditions. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3′ end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs.The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus  High-throughput sequencing has revealed that the overwhelming majority of the eukaryotic genome is transcribed. For example, the ENCODE project has annotated transcription originating from around three quarters of the human genome cis- and trans-NATs. Cis-NATs arise from the same genomic region as their complementary sense transcript, whereas trans-NATs are complementary to transcripts from remote loci. Specific antisense transcripts have been shown to regulate the expression of their sense transcripts via a range of mechanisms including: inhibition of transcription due to steric clashes of the transcriptional machinery; repression of expression due to competition for transcription factors; silencing the expression of the sense protein by RNAi; disruption of post-transcriptional modification and translation of the sense transcript by forming RNA/RNA duplexes; and masking of specific signals on the sense RNA necessary for splicing, stability or degradation , containing transcripts with sequence complementarity to other RNAs. NATs can be divided into cis-NATs are expressed from the opposite genomic strand of 15-50% of protein-coding loci. In Saccharomyces cerevisiae, antisense transcripts associated with more than a thousand genes are expressed Schizosaccharomyces pombe, 2409 protein-coding genes were found to have cis-NAT expression. Again, genes involved in meiosis were more likely to be associated with cis-NAT transcription Aspergillus flavus, differentially regulated NATs are found antisense to genes involved in temperature-sensitive morphogenesis and aflatoxin biosynthesis Fungi provide a simple eukaryotic model in which to understand important and widespread mechanisms of gene regulation. The few genome-wide searches for antisense transcription in fungi have indicated that Neurospora crassa. Although N. crassa is non-pathogenic, it is closely related to a number of important animal and plant pathogens. The N. crassa genome has been fully sequenced and assembled Neurospora displays complex cellular and morphological organisation Neurosporaqrf is a lncRNA transcribed antisense to the circadian clock gene, frequency (frq) qrf expression affects the clock's response to light via chromatin modification at the frq promoter N. crassa predicted the presence of 87 pairs of sense/antisense ORFs using computational methods Of the filamentous fungi, resources for investigating gene function are most advanced in N. crassa using RNAseq from cultures grown in the dark and under conditions of light and temperature stimulation. 477 of our lncRNAs are antisense to annotated protein-coding genes, and we find 38 novel pairs of sense/antisense lncRNAs. We have also characterised protein-coding transcripts that are associated with NATs and determined their expression, NAT overlap, number and position of introns. We report the differential expression of lncRNAs in response to light and temperature using RNAseq data and confirm the expression of several sense/antisense transcript pairs by qPCR. The expression of these candidate sense/antisense pairs was observed in a dicerlike-1, dicerlike-2 double mutant as well as in upf1 mutant strains to determine if they were substrates for RNAi or nonsense mediated decay (NMD) pathways.Here, we annotate a total of 939 novel lncRNAs in 54–3, bd a) and wc-2▵, vvd▵, frq▵, wc-1▵, ▵bd (quad) strains of N. crassa under three conditions . The number of reads obtained for each dataset varied between 15 and 52 million reads. Using the splice-aware mapping tool, Tophat ▵quad datasets mapped to unique locations in the NC10 version of the genome  and that do not overlap an annotated gene. However, 2,652 of these transfrags are located within 500 nts of the ends of annotated protein-coding genes on the same genomic strand, and may therefore represent unannotated terminal exons. Since 92% of annotated protein-coding genes are separated by more than 500 nts  etes see . Unsurpra genome .N. crassa genome database In the BROAD qrf transcript antisense to frequency (frq), for which the transcript boundaries are well-determined . To eliminate the possibility that the antisense lncRNAs could be annotated based on reads that are mapped to the incorrect genomic strand , we examined the mapped reads for splice junctions. 315 of our antisense lncRNAs (66%) have consensus splice junctions  supported by reads spanning the intron. We identified 5 occurrences of the nonconsensus splice junction CT-AC, which could represent incorrectly orientated reads. However, the same predicted lncRNA transcript also contained other consensus splice sites supported by read data, indicating the correct orientation of the antisense transcript. The remaining 162 antisense transcripts have no evidence of introns.In our dataset, 513 annotated protein-coding transcripts, representing over 5% of all annotated genes, display evidence of antisense lncRNA transcription (> = 50 antisense reads). Eleven of these protein-coding transcripts have two antisense lncRNAs separated by more than 200 nts. One such pair represents the wn genes . The lncThe majority (258) of antisense lncRNAs are transcribed convergently with their protein-coding sense partner, such that their 3′ ends overlap. 63 sense/antisense transcripts are divergently transcribed and overlap at their 5′ ends. There are 43 cases of antisense lncRNAs nested within the bounds of the sense protein-coding gene, and 149 annotated sense genes nested within antisense lncRNAs. Almost 97% of our antisense lncRNAs (461) overlap the ORF of the annotated protein-coding transcripts. We also identify 38 pairs of sense/antisense lncRNAs originating from previously unannotated loci . 36 of tp-value 0.001); transcripts with antisense expression have fewer introns at the 5′ and 3′ ends  exhibit any particular characteristics. The lengths of transcripts with associated antisense RNAs are present at a greater proportion in the 1–2 kb size range, however there was no significant difference between the distributions of lengths between transcripts with and without antisense RNA . Similar 3′ ends . The traDivergent transcripts may arise from bidirectional promoters. In our dataset, 355 lncRNAs, including 233 antisense lncRNAs, are located upstream (within 1 kb) of annotated genes on the opposite strand. Without detailed experimental characterisation of a promoter region, it is difficult to determine whether or not a bidirectional promoter is responsible for the expression of divergently transcribed transcripts. However, we find 2 examples, NCU07267/NCU07268_AS and NCU01107/NCU01106_AS, where the divergently transcribed gene and lncRNA are both significantly up-regulated in response to light (see below).et al. It has previously been suggested that the terminators of protein-coding genes could act as promoters for antisense transcripts Our datasets further allowed us to identify lncRNAs under the direct or indirect control of light- or temperature. We used DESeq to determine the differential expression of transcripts between the control dark-grown culture at 25 °C and cultures exposed to light or 30 °C. Eleven lncRNA, were found to be up-regulated in response to light . This antisense transcript is located close to and is expressed divergently from the blue light-induced-3 gene . We therefore suggest that bli-3 and the transcript antisense to NCU07268 may be expressed from a bidirectional promoter. Other predicted sense and antisense transcripts whose expression were verified include the divergently transcribed NCU02607, predicted to code for a hypothetical protein, and its partially complementary antisense transcript. In addition, we confirm the expression of an antisense transcript within which NCU07915  is nested. The antisense transcript overlaps all the introns of the sense transcript and interestingly reads antisense to the third intron of the sense transcript are most abundant. We note that exons 3 and 4 of NCU07915 encode the Mpv17/PMP22 family domain, which is predicted to have pore-forming activity. We also confirmed expression of a known snoRNA that is complementary to the 3′ end of NCU09135 (predicted to code for phosphatidylinositol phospholipase C) and the 5′ end of NCU09136 , and a second antisense lncRNA that is nested within NCU09136  knockout in which expression of dicer-like-1 and dicer-like-2 is abolished. If sense and antisense transcripts form double-stranded RNA recognized by DICER, we might expect to see higher levels of transcripts in the ddicer strain. Another possible consequence of co-expression of sense and antisense transcripts is mis-splicing leading to degradation by the nonsense-mediated decay pathway. NCU04242 is a homolog of upf1ddicer strain. The expression of both the sense and antisense transcripts of NCU09135 and NCU09136 was significantly up-regulated in the ddicer mutant . In some culture conditions we also see up-regulation of the sense transcripts for NCU04182, NCU02607 and NCU07915 and antisense transcripts NCU07268 and NCU02607 in the ddicer mutant. With the exception of NCU02607, significant changes in sense and antisense transcript levels are seen for all sense/antisense pairs in the nmd mutant.Given that the stability of sense/antisense transcript pairs could be controlled by components of the RNA silencing machinery, we assessed the expression of the sense and antisense transcripts by qPCR in a et al. The presence and functional relevance of widespread transcription in eukaryotic genomes is a subject of extensive debate in the literature. Genome-wide studies have shown that a large portion of the mammalian genome is transcribed while only a small fraction codes for proteins et al.The possible modes of action of antisense transcripts are many and varied, and include inhibiting synthesis of their sense transcript, regulating splicing, and controlling the levels of sense RNA via RNAi pathways. It seems likely that features of antisense transcripts, such as the extent and nature of the overlap between sense and antisense transcripts and their expression, may indicate their function. Osato N. crassa sense/antisense transcript pairs (97% of those in the BROAD database) also overlap each other at their 3′ ends. A number of signals required for post-transcriptional modifications are located in 3′ UTRs and we might therefore expect that antisense transcripts play roles in regulating modification of their sense counterpart Neurospora genes, the locations of introns are skewed towards the 5′ end , have been found associated with regions of convergent transcription and linked to dynamic DNA methylation especially prevalent around promoters of the disi-loci ddicer strains should throw light on the presence or absence of disiRNAs mapping to the location of sense/antisense transcript pairs.Some of the NATs that we report here may be required for the production of regulatory siRNAs. Small RNA species, including siRNA, QDE-2-interacting RNA (qiRNA), microRNA-like RNAs (milRNA) and dicer-independent siRNAs, have previously been identified in NCU04242Δ strain, suggesting that they could be potential substrates for the NMD pathway.Recently, two novel classes of non-coding RNA transcript were annotated in yeast: CUTs (cryptic unstable transcripts) and SUTs (stable unannotated transcripts). CUTs are short (<800 nts) and have a very short half-life in the cell, suggesting that their role is achieved via transcription itself N. crassa appear to be transcribed independently of neighbouring genes, we identify examples of potential bidirectional promoters. Seila et al. showed that the transcripts formed from bidirectional promoters in mouse are shorter and are less abundant than the sense RNA While the majority of lncRNAs in N. crassa. The expression profiles of the lncRNAs and antisense transcripts indicate that a variety of mechanisms regulate their expression. However, few examples of antisense transcription have been functionally characterised in any organism. The following questions therefore remain: (1) Which common themes underlie the control of expression of sense and antisense transcripts? (2) Which characteristics of NAT form and expression can be used to predict their mode of action? We suggest that Neurospora can serve as an informative model for studying the function of eukaryotic NATs.Several studies have highlighted the expression of NATs in fungal genomes  and wc-2▵, vvd▵, frq▵, wc-1▵, bd (quad▵) strains were grown in liquid medium  on a rotary shaker (225 rpm) at 25°C. After 24 hours growth in light and 24 hours in darkness, cultures were either harvested, exposed to a pulse of light (580 μW/cm2) for one hour at 25°C, or transferred to 30°C in the dark for one hour (temperature pulse). Cultures  were then harvested, ground under liquid nitrogen and RNA extracted (Qiagen). Two rounds of ribosomal RNA depletion were carried out using the RiboMinus kit (Life Technologies). Sequencing libraries were prepared using the Applied Biosystems SOLiD Total RNA-Seq Kit and sequenced using its SOLiD chemistry at the Genomic Technologies Core Facility . The 50 nt reads were filtered for quality N. crassa OR74A (NC10) genome (http://www.broadinstitute.org/annotation/genome/neurospora) Cultures of wildtype N. crassa revealed a median intron length of 76 nts and 88% of annotated introns are less than 200 nts (▵ RNAseq datasets were classified as novel transcripts. The genomic locations of transfrags were cross-referenced with the locations of annotated transcripts from the BROAD N. crassa genome annotation (NC10) in a strand-dependent fashion. Transcript sequences were searched for coding potential using Coding Potential Calculator Neurospora tetrasperma, Magnaporthe grisea, Chaetomium globosum, Myceliophthora thermophile, Trichoderma reesei, Fusarium graminearum, Aspergillus nidulans, Aspergillus oryzae, Aspergillus fumigatus, Neosartorya fischeri, Coccidioides posadasii, Myceliophthora thermophila, Thielavia terrestris, Yarrowia lipolytica, Candida albicans, Candida glabrata, Saccharomyces castellii, Saccharomyces cerevisiae and Schizosaccharomyces pombe, using WU-BLASTN −5 and requiring a match covering at least 40% of the length of the query and at least 50 bases.To annotate novel transcribed regions, all 12 sequencing datasets were merged, and reads that overlap on the same genomic strand were clustered together. Novel transcripts could be made up of several such clusters that do not overlap due to low expression levels, low read coverage  200 nts . Therefopadj-value reported by DESeq). The SAM files were then used to determine alternative splicing in response to light or temperature using Cufflinks Read counts for every gene were calculated from the SAM file dicerlike-1Δ, RIPdicerlike-2 , and NCU04242Δ (deletion of a homologue of upf1 from Arabidopsis) strains by qPCR. Neurospora was grown under the conditions described above. After 48 hours of light-dark cycle, cultures were either kept at 25°C in the dark, exposed to a pulse of light for one hour at 25°C, or transferred to 30°C in the dark for one hour (temperature pulse). RNA was extracted from the tissues using TRizol. The RNA (100 μg) obtained from the tissues was DNase-treated (New England Biolabs) for 2 hours at 37°C and then the enzyme was heat-inactivated at 75°C for 10 minutes. The RNA was further purified using the RNeasy Plant mini kit (Qiagen). 1 μg of RNA was used to perform strand-specific reverse transcription using the primers shown in We validated the expression of 6 sense and antisense transcripts in wildtype, Figure S1A. Distribution of distance between neighbouring annotated transcripts on the same strand. B. Size distribution of introns in annotated transcripts.(TIF)Click here for additional data file.Figure S2Expression of both the sense and antisense transcript for NCU04182, NCU07268, NCU02607, NCU07915, NCU09135 and NCU09136 in the WT, ddicer and NCU04242Δ (nmd) strains are shown, after growth in the dark, and exposure to light and temperature pulses. Black bars indicate the protein-coding sense transcript and white bars indicate its antisense transcript. Each experiment was repeated with 3 biological replicates. Error bars represent standard deviation. Statistical significance between WT and mutants was determined using Student t test, * indicates p-value <0.05 and ** indicates p-value < = 0.005 . Only significant differences between the mutant and WT strains are shown here.(TIFF)Click here for additional data file.Table S1The number of reads obtained from RNA sequencing and mapped to unique locations in the NC10 version of the genome for each sample in the WT and ▵quad dataset.(XLSX)Click here for additional data file.Table S2List of putative lncRNAs and novel putative protein-coding transcripts.(XLSX)Click here for additional data file.Table S3List of previously annotated sense/antisense pairs.(XLSX)Click here for additional data file.Table S4List of antisense lncRNAs.(XLSX)Click here for additional data file.Table S5FunCat analysis.(XLSX)Click here for additional data file.Table S6List of lncRNA/lncRNA antisense overlaps.(XLSX)Click here for additional data file.Table S7Primers used for strand-specific RT and qPCR.(XLSX)Click here for additional data file.