Although genetic or epigenetic alterations have been shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed.Here, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data.Our study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes. The organization of the human genome in the nucleus is non-random and important for proper gene expression -4. For iGiven the relationship between genome architecture and activity, it is not surprising that human disease can sometimes be attributed to defects in genome organization. Compelling evidence for the role of three-dimensional (3D) chromatin organization in human disease comes from studies on laminopathies like the Emery-Dreifuss muscular dystrophy. It was shown that this disease could originate from mutations in a lamin protein that specifically causes abnormal retention and silencing of muscle-specific genes at the nuclear envelope . The impHOX genes.Overall, genome architecture is guided by chromatin interactions with nuclear landmarks like the lamina, and by intra- and inter-chromosomal contacts mediated by chromatin-binding proteins. In addition to transcription factors that form DNA loops between enhancers and promoters, proteins like CTCF, SATB1, and the Cohesin complex are thought to be master regulators of genome organization. Protein complexes such as those containing Polycomb group proteins or the mixed lineage leukemia (MLL) protein might be equally important in shaping the human genome -23. MLL HOX family members [HOX genes was reported to be a dominant mechanism of leukemic transformation by MLL fusions [The MLL gene is a common target in non-random chromosomal translocations associated with both acute lymphoblastic (ALL) and acute myeloid (AML) leukemia, with over 50 different translocation partners identified so far -27. Thes members . In fact fusions .The mechanisms by which different MLL fusions activate transcription or lead to either AML or ALL are poorly understood. However, the observation that many translocation partners are elongation factors that co-exist in a super elongation complex (SEC) with the fusion proteins suggests that they can activate transcription at the elongation step . InteresHOXA gene cluster [HOXA region is considered, chromatin architecture has the information value to distinguish between leukemia types and subtypes.In infants, MLL is translocated in over 50% of acute leukemias ,35, wher cluster . From thHOX clusters encode transcription factors that are important for embryonic development and hematopoietic lineage regulation [HOX expression is found in various types of human cancers including lung cancer [HOXA9 and 10 for instance are oncogenes overexpressed in various leukemia types and are direct targets of MLL fusion oncoproteins [HOX genes organized into 13 paralogue groups and divided into four clusters named A, B, C, and D located on different chromosomes [HOXA cluster spans over 100 kbp on chromosome 7 and encodes 11 transcription factors  technology  protein . We also included three embryonic carcinomas (EC) samples in this training set to increase diversity. These are known to encode only the wt MLL protein and express no HOXA genes ([wt protein are AML or ALL. When comparing the average HOXA interaction frequencies (IFs) in leukemia samples expressing MLL fusions to those encoding only wt MLL, we could find marked differences in contact frequencies between neighbors  and between distal fragments interacting long-range  classifS of 30 samples shown in Figure s in S, a classifier was trained on the 29 remaining samples (S - {s}) and then used to predict the class of s. The result of this cross-validation procedure is then reported as one entry in the confusion matrix shown in Figure wt protein could be classified with 83% accuracy by 3D-SP . The information gain estimates the reduction of entropy in the classification achieved by each contact, and can therefore be used to identify discriminatory interactions. As expected, there were much fewer contacts than those displaying large averaged IFs differences  and the cluster, suggesting that these two regions are differentially regulated in leukemias expressing MLL fusions. This result was interesting in light of our previous report that differentiation of THP-1 promyelomonocytic leukemia cells into macrophages is accompanied by transcription repression of 5’ end genes and the formation of long-range contacts between the ends of the cluster [HOXA11 and HOXA13 regions are important for the observed conformational changes is unclear but mapping of CTCF and cohesin by ChIP-seq shows that the two proteins bind to these regions   Figure A. In con) Figure B,C and dwt and fusions with the training set  expressed significant levels of HOXA genes, while one of the MLL fusion leukemia cell lines (Karpas-45) did not express the genes at all and yet, all were correctly classified , all HOXA gene expression (62%) or on the 5C data  but remained slightly lower than 5C classification with an SVM  as defined by growth curves. All cells were grown at 37°C in 5% COHOXA gene expression was measured by quantitative real-time polymerase chain reaction and is shown relative to actin. The RNeasy© Mini kit (Qiagen) was used to extract total RNA from the samples as described by the manufacturer. The samples were then treated with DNAseI (NEB), and re-purified on Qiagen columns. Two micrograms of the resulting total RNA was used in each reverse transcription reaction with the Superscript® III reverse transcriptase kit (Invitrogen™) and oligo (dT)20. The SsoFast™ EvaGreen© Supermix (Bio-Rad) was used to quantify the samples by RT-qPCR with a Bio-Rad CFX96™ (C1000™ series) real-time system. Quantification was performed by two-fold serial dilutions of total cDNA. The size and specificity of amplified products was verified on agarose gels containing 0.5 μg/mL ethidium bromide and/or by verifying the melting temperatures of PCR amplicons. An AlphaImager© HP coupled to a 12-bit digital camera and equipped with the AlphaView® image acquisition and analysis software  was used to document and analyze the gels. All RT-qPCR primer sequences used in this study were previously described [escribed , and theescribed .HOXA gene cluster [The chromosome conformation capture (3C) and 3C-carbon copy (5C) techniques were used as previously described to characterize the chromatin organization of a region containing the  cluster ,51. The We modified the previously published approach to normalize interaction frequency data  to accoup) and was trained using sequential optimization minimization, with 1-vs-1 pairwise classification for multi-class problems [in vivo. The SVM was trained and evaluated using a leave-one-out cross-validation approach to obtain a classifier with a maximum-margin hyperplane in the transformed feature space. The predictive power of individual features was evaluated using the information gain score , which measures the reduction of the entropy of the class distribution when the feature is considered. Contacts with large information gains confer the highest amount of predictive power. Informative contacts were selected by determining if the IF values were statistically different between sample types. This was done by calculating P values using a two-sided Student’s t-Test in R [vs. wt, or MLL-AF9 vs. MLL-ENL) t-test was performed for each feature independently, and features that were retained had reported P values below a threshold of 0.01 (P <0.01).A support vector machine (SVM) classifier  was implproblems ,64. Eachest in R . The twoThe Matthews Correlation Coefficient (MCC) was calculated as a measure of the quality of the classifications performed by the SVM, with values close to +1 indicating perfect prediction, values close to 0 indicating random prediction, and values close to -1 indicating complete disagreement between the prediction and the observation. The MCC statistic was calculated from the confusion matrix as:Chromatin immunoprecipitation was conducted as described previously  with 5 μFor BARCODE ChIP-seq libraries (Rad21), FASTX-Toolkit (version 0.0.13)  was usedThe data and source code of our software are available through our website with instructions at the following address: . The sou3C: Chromosome conformation capture; 5C: 3C-carbon-copy; SVM: Support vector machine.The authors declare that they have no competing interests.MR designed, developed, and carried out the computational analysis, and helped to draft the manuscript. MAF participated in designing the 5C experiments, produced part of the 5C datasets, and participated in the mRNA quantification by real-time quantitative PCR. JC carried out part of the 5C experiments, participated in the quantification of mRNAs by real-time quantitative PCR, and helped to draft the manuscript. XQDW participated in generating part of the 5C experiments, and in the quantification of mRNAs by real-time quantitative PCR. HM performed the ChIP-seq experiments and their analysis. MB participated in the design and coordination of the study, and helped to draft the manuscript. JD conceived the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.Supplementary Materials and methods, Supplementary Figure legends, and Supplementary References. The Supplementary Materials and methods contain a detailed description of the cell lines analyzed in this study. It also describes the 3C and 5C protocols used to capture chromatin organization, and the computational approach developed to calculate normalized interaction frequencies (IFs). This file also contains the figure legends for  [ome.php) .Click here for fileThis file contains the source code for all the software used in this study.Click here for file


The neonatal intestine is a very complex and dynamic organ that must rapidly adapt and remodel in response to a barrage of environmental stimuli during the first few postnatal weeks. Recent studies demonstrate that the zinc finger transcriptional repressor Blimp1/Prdm1 plays an essential role governing postnatal reprogramming of intestinal enterocytes during this period. Functional loss results in global changes in gene expression patterns, particularly in genes associated with metabolic function. Here we engineered a knock-in allele expressing an eGFP-tagged fusion protein under control of the endogenous regulatory elements and performed genome wide ChIP-seq analysis to identify direct Blimp1 targets and further elucidate the function of Blimp1 in intestinal development. Comparison with published human and mouse datasets revealed a highly conserved core set of genes including interferon-inducible promoters. Here we show that the interferon-inducible transcriptional activator Irf1 is constitutively expressed throughout fetal and postnatal intestinal epithelium development. ChIP-seq demonstrates closely overlapping Blimp1 and Irf1 peaks at key components of the MHC class I pathway in fetal enterocytes. The onset of MHC class I expression coincides with down-regulated Blimp1 expression during the suckling to weaning transition. Collectively, these experiments strongly suggest that in addition to regulating the enterocyte metabolic switch, Blimp1 functions as a gatekeeper in opposition to Irf1 to prevent premature activation of the MHC class I pathway in villus epithelium to maintain tolerance in the neonatal intestine. The transcriptional repressor Blimp1/Prdm1 plays a pivotal role in the metabolic switch that occurs in the small intestine during the suckling to weaning transition. Notably, expression profiling of perinatal Blimp1-deficient small intestine revealed premature activation of metabolic genes normally restricted to post-weaning enterocytes. To further elucidate the function of Blimp1 in intestinal development, we engineered a novel Blimp1-eGFP-fusion knock-in mouse strain to perform ChIP-seq analysis. In addition to identifying which metabolic genes are direct Blimp1 targets, ChIP-seq analysis revealed a highly conserved Blimp1/Irf-1 overlapping sites that function to control MHC class I antigen processing during acquisition of neonatal tolerance in the first weeks after birth during early colonization of the intestinal tract by commensal microorganisms. Moreover, immunohistochemical analysis of human fetal intestine suggests that a BLIMP1/IRF-1 axis may also function in human intestinal epithelium development. Blimp1 feration . In the feration . At lateferation . Blimp1 feration .c-Myc expression to block proliferation in B cells, macrophages, and sebaceous gland progenitors [c-Myc is not a key target in activated effector T cells. Rather Blimp1 blocks IL-2 production required for T cell proliferation [+ T-cell lineage Blimp1 selectively attenuates Th1 subset development by extinguishing expression of IFNγ, Tbx21 and Bcl6 [Nfat5, Fos, Dusp16 and Prdm1 itself are direct targets in the skin epidermis [Considerable data strongly suggests that Blimp1 transcriptional targets are cell type-specific. For example Blimp1 directly represses genitors ,9,10. Inferation ,12. Withand Bcl6 . Nfat5, pidermis . Recent pidermis . Cooperapidermis .Blimp1 is strongly expressed in the intestinal epithelium throughout fetal development but beginning at birth becomes dramatically down-regulated in the crypt progenitors, corresponding to the adult intestinal stem cell compartment ,16,17. CCyp4v3, Slc16a5 and Myo18b. Interestingly, comparisons of our ChIP-seq peaks with those reported for P19 EC cell and human HeLa cell datasets revealed several highly conserved genomic targets, including key components of the MHC class I peptide-loading pathway [To further investigate Blimp1 functional contributions during this developmental transition and identify its direct targets in immature intestinal enterocytes, we created a knock-in allele engineered to express an enhanced green fluorescent protein (eGFP)-tagged fusion protein reactive with the well-characterized ChIP quality anti-GFP monoclonal antibody. Here we demonstrate that the fusion protein faithfully reconstitutes Blimp1-dependent functional activities. Thus homozygous embryos exclusively expressing the eGFP-tagged Blimp1-fusion protein develop normally, and healthy homozygous adults recovered at the predicted Mendelian ratios were indistinguishable from wild type littermates. Moreover, the knock-in allele efficiently rescues Blimp1-dependent plasma cell differentiation. EGFP-tagged Blimp1 was strongly expressed in the developing intestine allowing us to undertake unbiased ChIP-seq analysis. Several candidate target genes strongly up-regulated in conditional loss mutants were identified as direct targets including in vivo under physiological conditions in the intestinal epithelium. We found overlapping Blimp1/Irf1 binding sites proximal to the promoters of 24% (24 of 99) of human/mouse conserved Blimp1 target genes including Psmb8, Psmb10, Psme1, Tapbp and Erap1. These findings strengthen the idea that Blimp1 occupancy directly antagonizes Irf1-dependent activation of MHC class I antigen presentation. Consistent with this suggestion we demonstrate that Irf1 is constitutively expressed in the fetal intestine and throughout postnatal stages. The onset of MHC class I expression in the developing intestine precisely coincides with down-regulated Blimp1 expression and the appearance of crypt-derived Blimp1-negative adult enterocytes during the suckling to weaning transition. Besides its role in governing the switch to adult metabolic pathways, Blimp1 co-occupancy at these Irf1-target genes promotes neonatal tolerance in the first weeks after birth during early colonization of the intestinal tract by commensal microorganisms.SELEX experiments revealed that the Blimp1 consensus-binding motif closely resembles the IRF-E sequence  recognizin vivo under physiological conditions, we engineered a novel eGFP-tagged Blimp1 knock-in allele Prdm1BEG by introducing the cDNA expression cassette into the first coding exon  with the present ChIP-seq Blimp1 binding events. We observed significant enrichment of Blimp1 binding sites proximal to up-regulated genes  (365.2 fold increased in mutants) predicted in silico to contain multiple Blimp1 binding sites proximal to its promoter [Arg2) (174.5 fold increased in mutants) gave no detectable ChIP-seq peak. In vivo in the context of the E18.5 embryonic intestine, compacted chromatin near genomic regions surrounding these potential target sites appears to be inaccessible. We observed robust Blimp1 binding proximal to the c-Myc P1 promoter  after Blimp1 to the de novo identified consensus DNA binding motif underlying all Blimp1 ChIP-seq peaks (in situ hybridization (ISH) database [Irf1 transcripts are present in the developing gut tube as early as E14.5 prior to villus formation  [Irf1 and Prdm1, but importantly the closely related family member Irf2, known to bind the same IRF-E consensus sequence as Irf1, is only minimally co-expressed in the intestine   demonstrntestine [20,36,40de novo Irf1 DNA binding motif identified here and that described by Garber et al. [P = 3.3 x 10−11) [P = 3.4 x 10−6) as well as IRF2 (P = 5.1 x 10−6). Roughly 33% IRF1/BLIMP1 overlap was predicted in human myeloma cells [Next to directly identify Irf1 targets in the E18.5 small intestine we performed ChIP-seq analysis. We identified 1996 binding events  broadly r et al.  were indx 10−11) , Blimp1 ma cells , whereasFunctional annotation of the 331 overlapping Irf1/Blimp1 ChIP-seq peaks using GREAT revealed significant enrichment of GO terms associated with MHC class I antigen processing . To evalin utero and early postnatal stages.To directly examine MHC class I expression in the developing small intestine, we performed Western blot experiments. Irf1 is strongly expressed at E16.5 and throughout postnatal intestinal development . In contThe ability of Blimp1 to mediate gene silencing and re-organize the chromatin landscape at its target genes depends on recruitment of histone-modifying enzymes . Recent in utero at approximately 12 weeks [ARG2 expression [PRDM1 and very low levels of ARG2 and SIS [Human placenta architecture, the duration of pregnancy, suckling behaviors and time frames of intestinal maturation have been adapted to fit the lifestyles of different mammalian species. In mice, crypt progenitors emerge from the inter-villous epithelium only after birth. Migration into the underlying mesenchyme leads to the formation of mature crypts and crypt-derived Blimp1-negative adult enterocytes gradually repopulate the intestinal epithelium during the suckling to weaning transition . By cont12 weeks . Considepression . Additio and SIS .As shown in + T lymphocyte subsets [+ T cell homeostasis [Target gene expression controlled by the zinc finger transcriptional repressor Blimp1/Prdm1 has been extensively characterized in the context of B-cell terminal differentiation to become antibody secreting plasma cells, the development of diverse CD4 subsets ,49 as weeostasis ,50–53. IIt has been known for many years that the Blimp1 consensus motif closely overlaps with the IRF-E sequence recognized by IRF1/IRF2 ,29. The Psmb10 promoter contained 8 motif matches. The Tap1 and Psmb8 bi-directional promoter also displays overlapping occupancy [The present experiments reveal considerable overlap between Blimp1 and Irf1 ChIP-Seq peaks. When further analyzed using Weeder, we identified an IRF-E DNA binding motif GAAAGTGAAA  underlyiccupancy ,57. ClosIrf1 constitutively expressed in the developing thymus plays an essential role in positive and negative selection of the CD8+ T cell repertoire [+ T lymphocytes are directed against peptides derived from cytosolic proteins degraded by the proteasome that are translocated across the ER membrane by the TAP1/TAP2 transporter, stabilized by interactions with the dedicated class I chaperone Tapasin, and edited by ER aminopeptidases [Previous experiments demonstrate that MHC class I surface expression, absent at early embryonic stages of development is interferon-inducible ,59. Simipertoire ,61. MHC ptidases . T cell ptidases . During ptidases . Interesptidases . We demoin utero but maturation of the mucosal immune system is incomplete at birth. Neonatal immune tolerance during postnatal colonization by commensal bacteria is essential for the establishment of a well-balanced host-commensal relationship. It is widely accepted that maternal tolerance of the allogenic fetus is largely dependent on the absence of embryonic MHC class I expression [Besides its essential role in absorption of nutrients, the intestinal epithelium also function as a protective barrier to prevent infection. The formation of gut-associated lymphoid tissues including the Peyer’s patches is initiated pression ,59. The Prdm1, Il33, Psmb9, Psmb10, Psmg4, Psme2, Tap1, Tap2, Tapbp, and Erap1.Note added in proof. While this manuscript was under review, another genome wide Blimp-1 ChIP data set was published by Saitou and colleagues  Quantitative dynamics of chromatin remodelling during germ cell specification from mouse embryonic stem cells. Cell Stem Cell). The autPrdm1-KI-F: AGAAACCAGCGCTTCTGTTTTAGTACGCGGAGC and Prdm1-KI-R: GAGAGGCGCGCCGAGAACTAGTCTCTGCCAGTCCTTGAAACTTCACGGAGCC with the bacterial artificial chromosome bMQ-375h16 as template. The PCR product was digested  and cloned to introduce SpeI and AscI (underlined) cloning sites into Prdm1 exon 3. The Blimp1-eGFP fusion construct plus SV40 polyadenylation signal [Prdm1 exon 3. Finally, a fragment containing a Frt flanked Neomycin resistance cassette [For the 5’ homology region, a StuI-XbaI fragment was excised from a genomic subclone  and intrn signal  excised cassette , the MfeSouthern blot screening was performed as described using the restriction enzyme and probe combinations shown in Prdm1CA/CA animals to the Villin cre transgenic strain were genotyped as described [Genotyping of mice carrying the novel eGFP knock-in (BEG) allele was performed with Forward primers BEG Mut (GTTATTGGCGTGGTAAGTAAGG) and WT (AGGCATCCTTACCAAGGAAC) and Reverse primers BEG Mut (ATTTATCACTGTGAGCTCTCCAG) and WT (GCTGAAGGGAGGAAGAAATG). The cycling conditions were 94°C for 20 s, 58°C for 30 s, and 72°C for 45 s for 35 cycles. Conditional mutants selectively lacking Blimp1 function in the developing intestine, generated by crossing escribed . The tarescribed ,27. All Cell cultures to induce plasma cell differentiation and Western blot analysis of LPS-stimulated splenocytes were performed as described previously . To insuCell staining experiments were performed as described  using a Placental and intestinal tissue samples were fixed overnight in 4% paraformaldehyde (PFA) in PBS, dehydrated in ethanol, embedded in paraffin and sectioned (6 μm) Immunohistochemistry was performed as described . PrimaryPrdm1BEG/BEG, or Irf1 ChIP of WT and Villin-cre conditional Blimp1 mutants, [Small intestines were dissected, flushed with PBS, cut into small pieces and cross-linked with 1% formaldehyde in PBS for 15 min at 4°C followed by 35 min at 25°C  and subsmutants, ) samplesDe novo identification of motifs within ChIP-seq peaks was performed using MEME suite tools [Prdm1-/- small intestine [www.ncbi.nlm.nih.gov/geo, accession no. GSE29658) was calculated by chi-square test.Sequence reads were mapped to the mm9 mouse genome release with Stampy using default parameters . Peak cate tools . Functiote tools . Regionsntestine   on a Rotor-Gene Q (Qiagen). Primers were designed to amplify 100–200 bp regions central to ChIP-seq peak genomic coordinates. Selected genes included c-Myc, Psmb8, Psmb9, Tap1, Tap2 and Tapbp [Prdm1 (45.3 fold), Slc16a5 (16.1 fold), Gsta1 (16.8 fold), 2210407C18Rik (15.7 fold), Trib3 (2.9 fold) and Cyp4v3 (68.9 fold) [Prdm1 gene was used as a negative control. Primer sequences are shown in QPCR analysis of triplicate GFP or control IgG ChIP, and input samples of nd Tapbp  and sevePrdm1 exon 1A and Prdm1 exon 1B transcripts whereas total Prdm1 transcripts were detected with primers Ex4For and Ex5Rev [Total RNA from yolk sac and embryonic small intestinal tissues was isolated using an RNeasy Mini kit (Qiagen), and reverse transcription-PCR (RT-PCR) was performed using the OneStep RT-PCR kit (Qiagen) as previously described . Primersd Ex5Rev .The ChIP-seq data have been deposited in NCBI GEO with the accession number GSE66069.S1 FigPrdm1BEG/BEG small intestine GFP ChIP samples relative to input DNA. GFP ChIP of wild type (WT) and normal mouse IgG ChIP of Prdm1BEG/BEG and wild type were included as negative controls. A region within the 3’UTR of Prdm1 was used as an internal control. Significant enrichment of all genomic regions corresponding to Blimp1 ChIP-seq peaks was observed selectively in GFP-ChIP of Prdm1BEG/BEG samples. Data represent mean % of input +/- SEM of triplicate samples per group. *P<0.05 relative to WT GFP ChIP samples.Real-time PCR quantification of triplicate E18.5 (PDF)Click here for additional data file.S2 FigPrdm1 exon 1A and Prdm1 exon 1B transcripts whereas total Prdm1 transcripts were detected with Primers Ex4For and Ex5Rev. A STO fibroblast sample is included as a negative control.Primers Ex1AFor and Ex1BFor in combination with Ex3Rev1 distinguish (PDF)Click here for additional data file.S3 FigDe novo analysis (MEME) of all Blimp1 ChIP-seq peaks (n = 2689) identified a binding motif with significant similarity to both BLIMP1 and IRF1 motifs (JASPAR_ CORE_2014_Vertebrates database).(PDF)Click here for additional data file.S4 FigP = 1.8 x 10−119 in comparison to whole genome for the same region. (B) The distance of each Irf1 ChIP-seq peak from the nearest TSS binned at 5kb intervals.(A) Distribution of all Irf1 peak locations in comparison to whole genome at defined genomic regions. * (PDF)Click here for additional data file.S5 FigLsd1, CoREST, Hdac1/2 and G9a expression levels in embryonic and postnatal small intestine. We observed roughly similar Lsd1, CoREST, Hdac1/2 and G9a expression levels at embryonic (E16.5) through to post weaning stages (P28). A STO fibroblast sample is included as a negative control for Blimp1 expression.(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.S5 Dataset(XLSX)Click here for additional data file.S6 Dataset(XLSX)Click here for additional data file.


OsDREB1b, is transcriptionally induced by cold stress and over-expression of OsDREB1b results in increase tolerance towards high salt and freezing stress. This spatio-temporal expression of OsDREB1b is preceded by the change in chromatin structure at the promoter and the upstream region for gene activation. The promoter and the upstream region of OsDREB1b genes appear to be arranged into a nucleosome array. Nucleosome mapping of ∼700bp upstream region of OsDREB1b shows two positioned nucleosomes between −610 to −258 and a weakly positioned nucleosome at the core promoter and the TSS. Upon cold stress, there is a significant change in the nucleosome arrangement at the upstream region with increase in DNaseI hypersensitivity or MNase digestion in the vicinity of cis elements and TATA box at the core promoter. ChIP assays shows hyper-acetylation of histone H3K9 throughout the locus whereas region specific increase was observed in H3K14ac and H3K27ac. Moreover, there is an enrichment of RNA PolII occupancy at the promoter region during transcription activation. There is no significant change in the H3 occupancy in OsDREB1b locus negating the possibility of nucleosome loss during cold stress. Interestingly, cold induced enhanced transcript level of OsDREB1b as well as histone H3 acetylation at the upstream region was found to diminish when stressed plants were returned to normal temperature. The result indicates absolute necessity of changes in chromatin conformation for the transcription up-regulation of OsDREB1b gene in response to cold stress. The combined results show the existence of closed chromatin conformation at the upstream and promoter region of OsDREB1b in the transcription “off” state. During cold stress, changes in region specific histone modification marks promote the alteration of chromatin structure to facilitate the binding of transcription machinery for proper gene expression.The rice ortholog of DREB1,  The highly complex structure of chromatin imparts resistance to several nuclear processes including transcription RD29A, RD29B, RD20 and At2g20880 ADH1 and PDC1COR15a and AtGLOS3 that there was a decrease in H3K27me3 mark (transcription repression) for the transcription, in response to cold stress COR15a and AtGLOS3 decreased but the low H3K27me3 status was maintained in these plants for 3days at normal growth temperature. These findings suggest that rapid alteration of epigenetic marks and there reversibility with a potential to keep a memory to give plants the flexibility to respond to environmental stress.Plant being sessile organism has the ability to sense and respond quickly in response to environmental cues for its adaptation and survival. Abiotic stress such as drought, high salt content and temperature changes negatively influence both the growth and survival of plants RD29A and RD29B genes which contain DRE/CRT or ABRE elements COR15a and AtGOL3 where again the H3 density decrease initially at the promoter region during transcription but the occupancy increases to normal within 1d after returning to normal temperature cis-regulatory element during transcription activation showed enhanced DNaseI hypersensitive sites (DHS) in many plant genes such as Arabidopsis ADHChlamydomonas HSP70A and RbcS2The presence of a positioned nucleosome at the promoters prevents the binding of transcription factors to the promoter sites and hence inhibits transcription DREB1a, DREB1b, DREB2a and DREB2b are the major transcription factors that regulate cold, high salt or dehydration inducible gene expression in plants DREB1/CBF genes has been shown to be induced by cold stress but not by dehydration or salt stress OsDREB1 genes not only show heightened tolerance to freezing temperature but also to drought or high-salt condition OsDREB1b genes in rice during cold stress. Our results indicate that hyperacetylation at the promoter and upstream region of DREB1b gene promotes the alteration in the chromatin structure during transcription. These changes in the chromatin structure are not random but occur in a region specific manner to increase the accessibility of stress responsive TF binding sites for transcription.The Dehydration responsive element binding proteins (DREBs) especially Oryza sativa L. cv. IR-64 seeds were germinated according to protocol described elsewhere Oryza sativa L. cv. IR-64 were surface sterilized with 0.1% (w/v) HgCl2 for 15 min, washed and allowed to germinate over water-soaked sterile gauge in trays at 37°C in dark for 3 days. The germinated seedlings were grown in water-soaked sterile gauge in trays in presence of 0.25X Murashige and Skoog (MS) complete media at 30°C under 16 h light and 8 h dark photoperiodic cycle with 50% relative humidity and 700 lmol photons m−2s−1 for the desired period in a plant growth chamber. For cold-stress treatment, the 17 day-old seedlings were transferred to 4°C for varying time periods ranging from 2 hrs to 16 hrs; while the control plants were maintained at 30°C. For recovery experiments, 17-cold seedlings were first incubated at 4°C for 2 hrs and then transferred to growth chamber at 30°C for indicated time period.OsDREB1b (+648 to +843) and OsDREB2a (+3411 to +3600). Rice actin (OSJNBa0005K07) gene was used as internal control. The primers used to amplify the 3′region of OsDREB1b, OsDREB2a and actin were listed in RNA samples were isolated 17 days old rice seedlings using Trizol reagent (invitrogen) as described by manufacturer protocol. 12 µg of total RNA from control and stress treated samples were used for RNA blot and hybridised with probes specific for the 3′ region of 2; 5 mM β-mercaptoethanol; 10 mM spermidine; 1 mM PMSF and Protease cocktail inhibitors). The extract was filtered twice through two layers of Miracloth and the filtrate was centrifuged at 4000 rpm for 30 minutes at 4°C. The pellet was resuspended gently with 2 ml of extraction buffer 2  and centrifuged at 13000 rpm at 4°C for 10 minutes. The pellet was re-suspended again in 0.5 ml of extraction buffer 2 and the solution was layered slowly on top of 0.5 ml of extraction buffer 3 and centrifuged at 13000 rpm at 4°C for one hour. The nuclear pellet was washed consecutively in washing buffer  and storage buffer  and was finally resuspended in storage buffer for subsequent experiments.Rice seedlings (10–12 grams) were homogenized using liquid Nitrogen in 200 ml ice cold extraction buffer1  of MNase (Worthington). The reaction mixture was incubated at 37°C for 20 minutes and was then terminated with 1%SDS and 50 mM EDTA. The nucleosomal DNA was extracted with equal volume phenol:chloroform (v/v). For DNaseI digestion, the nuclei were resuspended in DNaseI buffer  and digested as indicated in figure legend. For Indirect end-labelling experiments, the MNase digested chromatin was further digested with restricted endonuclease as indicated and the purified DNA was separated in a 1% agarose gel, transferred on nylon N+ membrane and Southern hybridized by standard protocols OsDREB1b locus.Nuclei resuspended in storage buffer were resuspended in Storage Buffer supplemented with 1.5 mM CaCl3 at 65°C for 15 min, mixed with 20 µl of 5M NaCl and incubated overnight at 65°C to reverse the formaldehyde cross-linkages. The next day, 10 µl of 0.5M EDTA; 20 µl of Tris-HCl, pH 6.5 and 1 µl of Proteinase K (20 mg/ml) was added to each elute and incubated at 45°C for 1 hr and extracted with phenol: chloroform: isoamyl alcohol (25∶24:1) and chloroform. After precipitation with ethanol in the presence of 50 µg of glycogen, the purified DNA pellets were suspended in 50 µL of TE and analyzed by Q-PCR.ChIP assay was adapted from protocols described previously For Western blot, nuclear protein was isolated by lysing the nuclei isolated from control and stress treated plants. The nuclear extract was run on 15% SDS-PAGE, blotted on PVDF membrane and probed with antibodies against histone H3, H3K9ac, H3K14ac and H3K27ac.OsDREB1b and OsDREB2a. The % input values thus obtained for these regions were calculated from the standard curve. To normalise the ChIP data, the rice actin promoter region was used. The % input values for DREB locus were normalised with the values obtained for actin promoter and represented as “normalised % input” as done by Pavangadkar et al OsDREB1b ChIP results were analysed by semi-quantitative PCR. The PCR was done with 1 µl diluted ChIP DNA (1∶2.5 times) in a 30 µl reaction volume containing 0.15 mM primers, 1.5 mM MgCl2, 0.15 mM dNTPs and 5% DMSO. The data was also normalised with values obtained for actin promoter obtained under similar PCR condition. To determine the significance of the results, the values obtained for stress treated samples were compared with control and analysed by student’s t test. The significant changes (P≤0.05) were marked in the figures. The primers used for ChIP analysis were listed in Real time PCR was carried out in 20 µl reaction volume containing 1X buffer with Syber Green and 0.25 µM primers . For each set, input samples were diluted and used for generating a standard curve. The amount of DNA pulled down for individual region was calculated using input standard curve. PCR was carried out with primers specific for the promoter and upstream region of OsDREB2a was used as control locus in this case. The amount of PCR product obtained for different digestion time point was normalised with the amount present at 0 time point. The normalised signal was plotted against time to compare the degradation rate for each condition.Enzyme (MNase and DNase I) accessibility assay to monitor chromatin remodelling in control and stressed nuclei was done as described by Chua et al. (2001). Nuclei from control and stress treated seedlings were digested with MNase (30 U/ml) and DNase I (5 U/ml) for different time period (as indicated in the figure legend). The digested mixture was extracted with equal volume of Phenol: Chloroform (1∶1 v/v) and precipitated with ethanol. Three replicates of each nuclease treatment were performed and analysed by quantitative PCR. The enzyme accessibility at the actin promoter and OsDREB1b genes during cold stress was analysed using northern blot hybridization. For this, RNA was isolated from 17 days old rice seedlings  treated at cold temperature (4°C) for different time periods and the blot was hybridised with probes generated from the 3′ terminal of DREB genes  and a basal state (Control).The expression of EB genes . The resOsDREB1b gene, we initially mapped the nucleosome arrangement of promoter proximal region of OsDREB1b locus by MNase digestion combined with indirect end-labelling technique NcoI restriction enzyme digestion and hybridised with DNA corresponding to the promoter region (−74 to −232) as shown in In order to understand how the nucleosomes alter during the cold induced transcription of OsDREB1b using PCR based approach (see method). In this approach, the region of interest was divided by a series of primers that were15–25 bp apart from each other so that a combination of each primer set (forward and reverse primer) can amplify 150 to 200 bp DNA fragment that may correspond to at least one nucleosome (OsDREB1b: nucleosome −I(−40 to −232), nucleosome −II(−258 to −415) and nucleosome −III (−440 to −610) (We next determined the position of these nucleosomes at 30–50 bp resolution at the upstream regulatory region of cleosome . Nuclei to −610) . NucleosOsDREB1b locus during transcription activation in response to cold stress, we monitored the acetylation status of H3K9, H3K14 and H3K27 residues respectively, using ChIP assay. The promoter proximal region and TSS of OsDREB1b locus (−800 to +350 bp) was divided into 4 different regions: region Ia (−232 to −40); region Ib: (−415 to −258); region II (−610 to −440) and region III (+157 to 307). The primers were designed against all the four regions and used for amplifying the corresponding fragment from immunoprecipitated chromatin. The immuprecipitated DNA for region Ib and II of OsDREB1b was analysed by real time PCR (Syber green based). For the region Ia (−232 to −40) and region III (+157 to 307) we were unable to quantitate ChIP data using Real time PCR. The exact reason for this failure is not known. The only possibility is that the promoter and TSS region from −230 to +250 of OsDREB1b has high GC content (>75%) and previous reports have suggested that Syber green dye prefer AT rich dsDNA compared to GC rich OsDREB1b gene we proceeded our analysis with our semi-quant data. We have used actin promoter region as internal control to normalise the ChIP data for real time as well as semi-quantitative data.Higher order chromatin structure imparts structural constraint for the transcriptional machinery to move. Changes in the post-translational modifications of histone N-terminal tail residues particularly acetylation of histone residue, play a pivotal role in determining the chromatin state of a gene and controlling transcription. In order to understand how histone acetylation changes in OsDREB1b gene during cold stress. Histone H3K9 acetylation shows 2 to 4 fold (p<0.05) increases throughout the 800 bp upstream region of OsDREB1b. Along with H3K9 acetylation there are other H3 residues which get differentially enriched in a region specific manner in response to cold stress. Region Ia which covers the predicted TATA box and TFIIb binding site shows an significant enrichment in H3K14 acetylation mark  upon transcription activation  increase in H3K14 acetylation marks during cold stress along with gradual increase in H3K9 acetylation in cold treated nuclei (4 hr stress time point) compared to control (OsDREB1b (data not shown). To further investigate whether these changes in histone marks observed in the OsDREB1b locus is because of a global change of post translational modifications of histone H3 in response to cold, we performed western blot with total protein extract isolated from control and cold stress treated plants. Our results indicate that there is no significant change in H3K9 and H3K27 acetylation at the global level in response to cold stress whereas H3K14ac gets reduced after cold treatment , was carried out. The ChIP results clearly indicate enrichment of initiating RNA Pol II predominantly in region Ia of OsDREB1b promoter in response to 2 hr and 4 hr of cold stress   in increase in RNA Pol II occupancy around TSS during cold stress. In case of transcriptionally repressed OsDREB2a locus, no significant change in RNA Pol II occupancy was observed during cold stress (OsDREB2a in response to cold stress.To determine whether the above changes in the histone H3 marks at the  p≤0.03) . As mentd stress . The resOsDREB1b locus during transcriptional activation was examined by measuring the changes in nuclease accessibility (MNase and DNaseI). Both MNase and DNaseI have different DNA digestion characteristics and exhibit different sequence specificities. In chromatin context the digestion profile of these enzymes is influenced by not only the presence of nucleosomes but also the conformation assumed by nucleosomes at different transcriptional states. Hence, the changes in the accessibility of these two endonucleases at the OsDREB1b locus at different stages of cold stress should reflect the conformational changes in the chromatin structure in response to the environmental cue.The positioning of nucleosomes on promoters and upstream regulatory elements is inhibitory to transcription as it occludes the access of both transcription factors and the transcriptional machinery to their cognate sites. Transcriptional activation therefore requires active repositioning of nucleosomes which can in part be accomplished by changes in the epigenetic marks that facilitates “opening” of the chromatin structure at a locus OsDREB1b locus and normalised to the amount of DNA present at “0” digestion time point. The normalised values where then plotted against time to compare degradation rates between three transcription states.Nuclei representative of all three transcription states  were isolated and digested with MNase and DNase I for increasing time points. DNA was isolated; subjected to quantitative PCR reaction using primers for different region of OsDREB1b locus in cold treated samples were enhanced compared to control nuclei. Based on three independent experiment result it was found that in cold treated nuclei, 50% decrease in MNase digestion was observed within 10 min of incubation in both region I (p<0.02)  nfc 102 gene of maize during UV-B treatment OsDREB1b gene in our case.Nuclease accessibility experiment shows, the rate of degradation of the amplification at region I  and II (Upstream:−794 to −440) of (p<0.02)  and regi(p<0.02)  and withm region . IncreasOsDREB1b transcription when cold-exposed plants were returned to a normal growth temperature. 17 days old rice seedlings were exposed to cold temperature (4°C) for 2 hrs and then allowed to grow at normal temperature for 6 hrs and 24 hrs. Northern analysis indicates that the level of OsDREB1b which was highly elevated during cold stress, reaches basal level within 6 hrs of incubation at normal growth temperature  and studies have shown that high GC content sequence does not favour nucleosome occupancy and promote nucleosome depleted region OsDREB1b nucleosome arrangement indicates the presence of a nucleosome within +157 to +307 leaving a 200 bp weakly positioned sequence around TSS for nucleosome occupancy. Furthermore, our nucleosome mapping analysis was found to be in consonance with the Kaplan distribution model OsDREB1b are: (i) weakly positioned −I and +I nucleosome providing heterogeneous population of nucleosome occupancy at the core promoter and TSS during transcriptionally repressed state. The nature of this nucleosome occupancy introduces competition for forming a stable pre-initiating complex for initiating transcription (ii) the two positioned nucleosomes flanking the TATA box and TSS provides partial blockage for the progression of RNA polymerase during transcriptionally repressed state.In order to better understand the transcription regulation of rice s region . PCR bass region . Interescis-element for transcription factor binding. Yeast PHO5 transcription is a well studied model showing increase in DNase I HSs at the upstream region of the locus for the binding of transcription factor Pho4 Adh and HSP18, tomato proteinase inhibitor-1, Chlamydomonas HSP70A and RbcS2 gene during transcription activation. In each case, the DNase HSs were found to be extended in the regulatory region like promoter, enhancer and other cis elements during transcription “on” state.Various studies in yeast and animal system have shown that the disruption of nucleosome structure at the regulatory regions is pre-requisite for the onset of transcription OsDREB1b gene in control plants as evident by transcription result . Many stress responsive TF binding sites with high score; mainly ABRE elements, bZIP binding sites, Myc like HLH binding sites are found to be masked within nucleosome −II and nucleosome −III of OsDREB1b gene. Hence, in order to obtain proper threshold of transcript of OsDREB1b during cold stress, nucleosomes remodeling seems to be a pre-requisite at promoter proximal region to uncover these cis elements.Sequence analysis and sequence prediction software shows putative TATA box and TFIIB binding sites reside within the first 100 bp sequence upstream from TSS of OsDREB1b transcription there is an increase in the acetylation of histone H3 residues within promoter and upstream region. Histone H3K9 acetylation was found to be increased throughout the 800 bp region, whereas H3K14 and K27 acetylation is more biased towards core promoter and upstream region respectively .PCR based Strategy to map nucleosomes at the promoter proximal and upstream region of (TIF)Click here for additional data file.Figure S2OsDREB1b locus. The ChIP DNA was used to amplify for different regions of OsDREB1b locus from control and cold treated samples. These PCR products were separated on 2% agarose gel.Gel documentation of the ChIP PCR products for (TIF)Click here for additional data file.Figure S3OsDREB2a during cold stress. (A and B) Relative MNase accessibility in control and cold stress treated nuclei (2 Hr and 4 Hr) was detected with PCR based method. (C and D) Relative DNase I accessibility in control and cold stress treated nuclei (2 Hr and 4 Hr) was detected with PCR based method. The amount of DNA amplified at each time point was normalised to that at time 0 and plotted against time to compare the rate of degradation. (E and F) PCR products showing the amplification of upstream and promoter proximal region of OsDREB2a in MNase and DNase I treated nuclei isolated from control and cold treated plants.Alteration of chromatin structure at the upstream region of (TIF)Click here for additional data file.Table S1List of primers used for this study.(DOC)Click here for additional data file.


Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, two largest subunits of RNAP II, at the clamp, protrusion and wall domains. These interactions may lock the clamp to the closed conformation and enclose the DNA being transcribed in the central cleft of RNAP II. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR.Spt5, a transcription elongation factor, and Rpb4, a subunit of RNA polymerase II (RNAP II) that forms a subcomplex with Rpb7, play important roles in transcription elongation and repression of transcription coupled DNA repair (TCR) in eukaryotic cells. How Spt5 physically interacts with RNAP II, and if and/or how Spt5 and Rpb4/7 coordinate to achieve the distinctive functions have been enigmatic. By site-specific incorporation of the unnatural amino acid  RNA polymerases (RNAPs), which carry out transcription in all living organisms, are highly conserved at the level of sequence, structure, function and molecular mechanisms . The mosSaccharomycescerevisiae and humans, respectively) and more complex, consisting of an N-terminal acidic domain, an NGN domain, multiple KOW domains and a C-terminal repeat (CTR) domain , to form a heterodimeric Spt4/5 complex.NusG/Spt5 family proteins are universally conserved transcription elongation factors that play pivotal roles in transcription and transcription related processes by binding to RNAP and interacting with other transcription-associated factors . Bacteri) domain . The NGNS. cerevisiae and human Spt5 bound to Spt4 have been solved  is a DNA repair pathway that removes a wide variety of bulky and/or helix-distorting lesions that generally obstruct transcription, such as UV-induced cyclobutane pyrimidine dimers (CPDs) ,13. Tran forward ,17–19. C forward . The bioe in TCR . Howevere in TCR  or Spt4 e in TCR . Rad26 ie in TCR  or any se in TCR . TherefoS. cerevisiae. We found that Spt5 interacts with the clamp, protrusion, wall and Rpb4/7 stalk domains of RNAP II. The binding sites of Spt5 on RNAP II partially overlap with those of the transcription initiating factor TFIIE. Disruption of the interactions between Spt5 and Rpb4/7 by deleting the Spt5 domains that extensively interact with Rpb4/7 decreases transcription elongation and derepresses TCR. Our results suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR.To gain insights into the mechanisms that underlie the functions of Spt5 in eukaryotic cells, we mapped site-specific interactions between Spt5 and RNAP II in E. coli tRNACUA and tyrosyl-tRNA synthetase genes  was obtained from Dr Steven Hahn. Multi-copy LEU2 plasmids bearing genes of interest (GOI)  with a TAG codon replacing a desired amino acid codon were created using plasmid pESC-LEU (Stratagene) as vector . The LEU2 gene on the original vector contains the leucine tRNA gene tRNA3Leu, which starts −463 nucleotides upstream of the start codon of the LEU2 gene  into the protein of interest. The tRNA3Leu gene was therefore inactivated by removing the sequence between the HpaI and SfoI sites in the gene. To increase detection sensitivity, the Flag tag contained in the original vector was converted to 3×Flag tag by inserting two Flag sequences between the SacI and BglII sites. The GAL1–10 promoter sequence on the vector was removed and replaced with the GOI encompassing their native promoters and coding sequences with a TAG codon replacing an amino acid codon of interest. All mutations were confirmed by DNA sequencing.Plasmid pLH157 bearing the genetically engineered EU2 gene . We founURA3 plasmid pRS416 . Deletion of genes was performed using procedures previously described , which is toxic to cells with a functional URA3 gene, to select for cells that had lost the URA3 (pRS416) plasmids bearing the respective wild type GOI. The loss of the URA3 plasmids and the gain of the LEU2 plasmids in the respective transformed yeast cells were confirmed by PCR.The pGOI-TAG plasmids encoding Rpb1, Rpb2, Rpb4, Rpb7 and Spt5 with a TAG codon replacing a desired amino acid codon were transformed into respective yeast strains (Supplementary Table S1). Plasmids pRS415-SPT5, pRS415-SPT5ΔKOW4 and pRS415-SPT5ΔKOW4-5 were transformed into yeast strains whose genomic A600 ≈ 1.0) and harvested. The harvested cells from 15 ml of culture were washed twice with ice-cold H2O, resuspended in 20 ml ice-cold 2% glucose, and split into two aliquots. One aliquot was kept on ice and the other was transferred into a glass petri dish (10 cm in diameter) and irradiated with 365 nm UVA for 15 min  on ice. The UVA source was an array of 12 fluorescent black light tubes  mounted on a home-made wooden structure (the light tubes were 5 cm apart on the structure). The UVA source gave a dose rate of 60 J/m2/s at a distance of 13 cm. The cells were harvested, resuspended in 400 μl of 20% trichloroacetic acid and broken by vortexing with 400 μl of glass beads for 30 min. The proteins were pelleted by centrifugation at 16 000 × g for 15 min at 4°C, washed with ice-cold 80% acetone, resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and subjected to Western blot. Specific SDS-PAGE conditions for Western blot detection of cross-linkings between different proteins are presented in Supplementary Table S2. Rpb1 on the blots was detected with 8WG16 (Neoclone), which recognizes the CTRs of Rpb1. 3×Flag and 3×Myc-tagged proteins were detected with anti-Flag M2 (Sigma) and anti-c-Myc (Genscript) antibodies, respectively. Blots were incubated with SuperSignal® West Femto maximum-sensitivity substrate (Pierce) and scanned with the VersaDoc Imaging System (BioRad).Yeast cells having plasmid pLH157 and the pGOI-TAG plasmids encoding Bpa-substituted proteins of interest were grown at 30°C in synthetic dextrose (SD) medium containing 0.5 mM Bpa (Bachem) to late log phase  by sonication with a Bioruptor (Diagenode) for 15 min (30 s on and 30 s off). The sample was clarified by centrifugation and the supernatant was transferred to a fresh tube. The sample was added with 1 ml of immunoprecipitation buffer and SDS to a final concentration of 0.1%. Fifty microliters of the sample was saved as ‘input’ and the remaining was mock-immunoprecipitated (without addition of antibody) or immunoprecipitated with 4 μg of 8WG16. The levels of Rpb1, Rpb4 and 3×Myc-tagged Spt5 were detected with 8WG16, 2Y14 (Neoclone) and anti-Myc antibodies, respectively, on western blots.For detection of co-immunoprecipitation of Spt5 and Rpb4 with core RNAP II, 90 ml of log phase yeast cells were harvested and resuspended in 0.6 ml of chromatin preparation buffer . The cells were broken by vortexing with acid-washed glass beads, and the chromatin fractions were collected by centrifugation at 20 000 × For detection of the cellular levels of Rad26, log phase yeast cells were harvested and the whole cell extracts were prepared using the glass beads and trichloroacetic acid method (see above). The levels of 3×Flag-tagged Rad26 and Rpb1 were detected using anti-Flag and 8WG16 antibodies, respectively, on a western blot.Yeast cells were grown at 30°C in SD medium to saturation, and sequential 10-fold serial dilutions were made. For temperature sensitivity test, the diluted samples were spotted onto YPD  plates and incubated at 25, 30 and 37°C. For UVC sensitivity assay, the diluted samples were spotted onto YPD plates, irradiated with different doses of 254 nm UV light  and incubated at 30°C in the dark. For mycophenolate sensitivity assay, the diluted samples were spotted onto SD plates containing different concentrations of MPA and incubated at 30°C. After 3–8 days of incubation the plates were photographed.A600 ≈ 1.0), cross-linked with 1% formaldehyde and lysed by vortexing with glass beads. The cell lysates were sonicated by using a Bioruptor (Diagenode) to shear the chromatin DNA to an average size of 200 bp and clarified by centrifugation at 4°C. An aliquot from each of the clarified lysates was saved as an input. The remaining lysates were immunoprecipitated with anti-Rpb1 antibody 8WG16 or mock-immunoprecipitated. DNA fragments corresponding to different regions of the RPB2 gene in the input, immunoprecipitated and mock-immunoprecipitated samples were quantified in triplicates by using real-time PCR. Primers used for amplifying the different regions of the RPB2 gene are shown in Supplementary Table S3. The number of molecules in each immunoprecipitated sample was subtracted by that in the corresponding mock-immunoprecipitated sample  and then normalized to that in the corresponding input. Each ChIP assay was repeated three times. The levels of RNAP II association with the different regions of RPB2 gene in cells expressing a truncated Spt5 were normalized to those in cells expressing the full-length wild type Spt5. The Student's t-test was used for statistical analysis.ChIP assays were performed as described previously . BrieflyA600 ≈ 1.0), irradiated with 120 J/m2 of 254 nm UV  and incubated in YPD medium in the dark at 30°C. At different times of the repair incubation, aliquots were removed and the genomic DNA was isolated using a hot SDS procedure as described previously .The TS of eviously ,30. Briein vivo site-specific cross-linking technique . The yeast cells were cultured in a medium containing Bpa. Whole cell extracts were prepared directly from the cells or following irradiation of the cells with UVA. The proteins were resolved on SDS-PAGE, which disrupts non-covalent protein–protein interactions and separates proteins primarily based on protein sizes, and subjected to Western blot. Bpa substitutions at Rpb1 H281 and E291, located at the coiled coil of the RNAP II clamp, caused slower migrating bands of Spt5 upon UVA irradiation . This indicates that Rpb4/7 directly interacts with other proteins in addition to Spt5. This is consistent with the fact that Rpb4/7 also interacts with various transcription factors during transcription initiation and executes some non-transcriptional activities, including mRNA transport .To date, only the crystal structure of the yeast Spt5 NGN domain bound to Spt4 has been reported . To gainLike Rpb4, the CTR domain of Spt5, which can be phosphorylated by the Bur1/2 kinase complex, plays an important role in repression of TCR . We wond23In view of the observations that the archaeal Spt5 NGN interacts with the RNAP clamp and has close proximity to the protrusion , it is h23We attempted to dock model structures of different Spt5 KOW domains [obtained by using the I-TASSER server ] onto thin vitro .,38.in viin vitro . Howeverin vitro , indicatspt5ΔKOW4) cells grew almost normally, Spt5 KOW4-5 deleted (spt5ΔKOW4-5) cells had growth defects especially at an elevated temperature (37°), suggesting a defect in transcription elongation . The RNAP II densities in the transcription start site (TSS) in spt5ΔKOW4 and spt5ΔKOW4-5 cells were not significantly different from that in wild type cells  were immunoprecipitated with antibody 8WG16, which recognizes the CTRs of Rpb1, the largest subunit of RNAP II. The immunoprecipitated fragments located at different regions of the s Figure  and D. Ts Figure , indicatA Figure  and B. Ce Figure . It is qrad7Δ or rad16Δ cells. A nucleotide resolution method that uses streptavidin magnetic beads and biotinylated oligonucleotides to facilitate isolation and strand-specific end-labeling of DNA fragments of interest was used for the analysis  decreased TCR in the transcribed region of the gene  cells, especially in the region ∼50 nucleotides downstream of the TSS  but play no role in TCR . Therefoanalysis ,30. TCR,s Figure . As expee Figure ,50 or CSe Figure ,52 cellss Figure  and B. NS Figure  and B. TS Figure  but moreS Figure , the SptS Figure  or subunS Figure . Also, i6 Figure . Rather,6 Figure , which mrad7Δ or rad7Δ rad26Δ cells enhanced survival of yeast cells upon UVC (254 nm) irradiation, with the KOW4-5 deletion being more striking  cells , and Spt4/5 compete for binding to the RNAP clamp during the transition from transcription initiation to elongation . Studiesin vitro, Rpb4 and Rpb7 play important roles throughout the transcription cycle in vivo  opposite to the damaged template . Our finrad26Δ and +RAD26 cells. Similarly, deletion of Rpb4 also enhances TCR in rad26Δ and +RAD26 cells (rad26Δ but not RAD26+ cells (We found that deletion of Spt5 KOW4-5 domains, which extensively interact with Rpb4/7, enhances TCR in 6+ cells . In cont6+ cells ,24. ThesE. coli UvrD induces backtracking of RNAP, and thereby exposing DNA lesions shielded by RNAP and allowing NER enzymes to gain access to lesion sites (E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNAP (E. coli and UvrD may antagonize the repression by counteracting NusG. However, in view of the fact that the E. coli RNAP has no Rpb4/7 counterparts and the single KOW domain of E. coli NusG does not directly interact with RNAP, the underlying mechanisms of TCR repression in E. coli and eukaryotes may be somewhat different. By binding to an upstream activating sequence, the transcription activator Fis stimulates transcription of a tRNA gene to an extremely high level and at the same time represses TCR in the gene, except for a short region immediately downstream of the TSS (E. coli is apparently caused by extremely high level of loading of RNAP rather than by stabilization of the transcription elongation complex. During very high level transcription in a tRNA gene in E. coli, an RNAP may arrive at the site of a downstream RNAP stalled at a lesion before the downstream RNAP can initiate or finish the TCR process, resulting in repression of TCR (E. coli. In eukaryotic cells, however, the loading of RNAP II to a gene being transcribed does not appear to be able to reach a level that can repress TCR. The galactose-induced GAL1–10 genes are among the most highly transcribed genes by RNAP II in yeast. However, TCR occurs very rapidly in the GAL1–10 genes in RAD26+ or rad26Δ cells (RPB2 and URA3 genes in rad26Δ cells (rad26Δ cells restores TCR in the RPB2 and URA3 genes (rad26Δ cells, is not caused by extremely high level of loading of RNAP II but may be due to Spt5-coordinated stabilization of the transcription elongation complex. The reason why TCR is less repressed in the highly transcribed GAL1–10 genes in rad26Δ cell is unknown, but may be due to a lower content of a TCR repressor in the RNAP complex engaged in highly transcribed genes.It has been shown recently that the on sites . In cont of RNAP . Therefo the TSS . Howevern of TCR . It is a6Δ cells ,70, indi6Δ cells ,50. HoweA3 genes ,22. ThesSupplementary Data are available at NAR Online.


Escherichia coli (ETEC) cause severe diarrhoea in humans and neonatal farm animals. Annually, 380,000 human deaths, and multi-million dollar losses in the farming industry, can be attributed to ETEC infections. Illness results from the action of enterotoxins, which disrupt signalling pathways that manage water and electrolyte homeostasis in the mammalian gut. The resulting fluid loss is treated by oral rehydration. Hence, aqueous solutions of glucose and salt are ingested by the patient. Given the central role of enterotoxins in disease, we have characterised the regulatory trigger that controls toxin production. We show that, at the molecular level, the trigger is comprised of two gene regulatory proteins, CRP and H-NS. Strikingly, this renders toxin expression sensitive to both conditions encountered on host cell attachment and the components of oral rehydration therapy. For example, enterotoxin expression is induced by salt in an H-NS dependent manner. Furthermore, depending on the toxin gene, expression is activated or repressed by glucose. The precise sensitivity of the regulatory trigger to glucose differs because of variations in the regulatory setup for each toxin encoding gene.Enterotoxigenic  Escherichia coli (ETEC) are the leading bacterial cause of such disease; hundreds of millions of cases occur every year. The severe watery diarrhoea associated with ETEC infections results from the action of enterotoxins. The toxins target human gut epithelial cells and trigger the loss of water and electrolytes into the gut lumen. Oral rehydration therapy can counteract this process. Hence, glucose and salt solutions promote rehydration of the patient. In this work we show that the gene regulatory mechanisms controlling toxin expression respond directly to sugar and salt. Furthermore, we describe a molecular mechanism to explain these effects. Hence, we provide a starting point for the optimisation of oral rehydration solutions to reduce toxin expression over the course of an ETEC infection.Diarrheagenic illness remains a major disease burden in the developing world. Enterotoxigenic  Consequently, H-NS also influences gene expression on a global scale In this work we define the molecular trigger that controls toxin expression in ETEC. We show that CRP and H-NS are key regulatory factors. Strikingly, this allows ETEC to integrate extracellular signals of osmolarity and metabolism to control toxin production. Hence, we propose that ETEC toxicity responds directly to osmo-metabolic flux. Interestingly, the precise regulatory settings are different for each toxin encoding gene. The differences result from i) varying promoter configurations and ii) competition between CRP and H-NS for overlapping DNA targets. This is significant since fluctuations in osmolarity, and changes in the availability of metabolites, are central to ETEC infection and its treatment.E. coli K-12 estA gene are found on plasmids p666 (estA1) and p948 (estA2). A single copy of the eltAB operon is encoded by plasmid p666. We used Chromatin Immunoprecipitation (ChIP) coupled with next-generation DNA sequencing (ChIP-seq) to map CRP and H-NS targets across the ETEC H10407 genome. The binding profiles are shown in E. coli K-12. The most common location for CRP sites was in intergenic regions (66% of targets) whilst a smaller number of targets were found within genes (34%). Consistent with expectations, CRP sites were most frequently located ∼40.5 bp, or ∼92.5 bp, upstream of experimentally determined transcription start sites (TSSs). Surprisingly, CRP binding was restricted to the ETEC chromosome . The PWM was then used to search p948 and p666 for CRP sites. A continuum of over 100 potential CRP targets was identified. However, we recognise that the vast majority of these are likely to be false positives. Hence, we next sought to differentiate between genuine CRP sites and spurious predictions. To do this, predicted sites were scored, grouped, and ranked on the basis of their match to the PWM . Electro targets . Remarka in vivo .estA2. This derivative of the toxin is more commonly associated with human disease and ETEC H10407 is somewhat unusual in also encoding estA1estA2 regulatory region is shown in lacZ reporter plasmid pRW50 to generate a lacZ fusion  in length (5′-TTAAAT-3′) and -35 (5′-TTGCGC-3′) elements were observed at the expected positions upstream of the TSS. Throughout this work we refer to this promoter, highlighted purple in estA2. To confirm CRP binding at the predicted site we used DNase I footprinting . To test this expectation we created a 92 bp PestA1::lacZ fusion, equivalent to the 93 bp PestA2::lacZ fusion described above, and mapped the 5′ end of the resulting mRNA. As expected, the primer extension product was 109 nt in length  eltABeltAB was identified by our ChIP-seq analysis . However, non-specific CRP binding was again detectable at high protein concentrations (lane 14). Hence, CRP does not bind specifically to PeltAB. We hypothesised that previously observed changes in PeltAB activity, in cells lacking CRP, may occur indirectly. To test this, we cloned a 359 bp DNA fragment, containing PeltAB, into our pRW50 lacZ expression system. We also made a truncated 118 bp derivative of this construct where two of the three putative CRP targets were removed. A derivative of the truncated 118 bp construct, where the remaining CRP site was completely ablated by point mutations, was also made. The DNA sequences of the different constructs are shown in eltAB increased 2.5 fold in the absence of CRP. However, the response of PeltAB was identical when the CRP binding sites were removed  and enhanced by salt (green line). As expected, PeltAB activity increased in the presence of both salt and glucose, but induction by salt was more prominent . Thus, we next sought to understand if our model for regulation of LT and ST expression was broadly applicable. We focused our efforts on ETEC E24377A since i) the genome has been sequenced and ii) a vast array of independently generated transcriptomic data are available for this organism estA2, and 1126 bp PeltAB DNA fragment. The sequences are shown in lacZ expression in response to CRP and H-NS was measured. As expected, transcription from PestA2 was repressed by H-NS and activated by CRP whilst PeltAB was repressed by H-NS . However, levels of hns transcription change dramatically (green line) 60 minutes after host cell attachment. As predicted by our model, levels of estA2 and eltA transcription (dashed lines) inversely track changes hns transcript levels. When undertaking this analysis we noticed that, although there was little change in the relative level of crp mRNA between attached and unattached ETEC cells, the absolute level of crp mRNA did fluctuate across the time course of the experiment and between biological replicates. Strikingly, when these absolute mRNA levels are compared there is a clear linear relationship between crp and estA2 expression . H-NS can interfere with this process by competing with CRP for binding at PestA2 (pathway “d”). Finally, CRP can indirectly repress expression of eltAB via an unknown pathway that is influenced by H-NS (“e”). Both pathways “c” and “e” are sensitive to glucose availability because of their dependence on CRP. We speculate that pathway “e” may include H-NS since the effects of salt and sugar on eltAB expression were epistatic  disease symptoms (the extrusion of cations and cAMP into the gut lumen) and treatment  pistatic . Our mod2O, cations, and cAMP (the cofactor for CRP) from the small intestine into the gut lumen estA2 expression. Hence, even if 90% of glucose present in ORT solutions is absorbed before reaching the site of infection, sufficient glucose should be present to down regulate toxin expression. Furthermore, even though salt is able to induce expression of estA2 and eltAB, the effect is only observed at concentrations far higher than those found in ORT solutions.Our model for regulation of ST and LT expression is pertinent to both ETEC mediated disease and its treatment. ST and LT trigger the extrusion of HestA1 and estA2 are oppositely regulated by CRP is intriguing given the similarities between the promoter sequences of these genes. Differential regulation is dependent on the promoter -35 element . Plasmids pRW50 and pSR are described by Lodge et al.et al.ETEC strain H10407 is described by Crossman estA2 or PeltAB fragments cloned in pRW50 carried in strain M182. The ChIP-seq was done as described extensively by Singh et al.2O. After quantification by PCR each library was amplified, purified and resuspended in 20 µL H2O. Libraries were the sequenced using a HiSeq 2000 sequencer . Sequence reads were aligned to non-repetitive sequences in the E. coli H10407 genome using CLC Genomics Workbench and overall coverage was determined using custom Python scripts. Sequence reads have been submitted to the EBI ArrayExpress database and can be accessed using accession number E-MTAB-2917.Cultures were grown to mid-log phase in M9 minimal medium with 1% (w/v) fructose at 37°C. Targeted ChIP experiments  and 6 weE. coli K-12 MG1655 genome. All but 11 CRP sites in ETEC had a single perfect match in the E. coli K-12 chromosome. For each perfect match the distance from the centre of the CRP site to all transcription start sites was calculated. Transcription start site coordinates are from Kim et al.et al.E. coli K-12 genome, the distance between CRP sites and TSSsChIP-seq peaks were identified as described previously E. coli K-12 genome The PWM describing CRP binding sites was generated using the PREDetector software package and our previous list of 68 CRP binding sites in the et al.crp), UTI189_C1433 (hns), D4754 (eltA) and D4048 (estA). The average signal intensity was calculated and the fold change in transcription in attached compared to planctonic ETEC cells was determined for each time point. The data in crp) and D4048 (estA) compared for each of the two replicates obtained at 30, 60 or 120 minutes after attachment to host cells. Signal intensities obtained after 30 minutes growth in LB medium (three replicates) are also included in this analysis.Data shown in 70 purification was done exactly as described previously 70 at room temperature for 20 minutes prior to use. H-NS was overexpressed in T7 express cells from plasmid pJ414hns. After overexpressing H-NS, cells were collected from the culture by centrifugation and resuspended in buffer A  glycerol) containing 100 mg/ml PMSF. Cells were lysed by sonication and the sample was cleared by centrifugation. The supernatent was loaded directly onto a Heparin column (Amersham) pre-equilibrated with buffer A. A linear NaCl gradient was applied and H-NS was found to elute at approximately 500 mM NaCl. The peak fractions were pooled and diluted 3-fold with buffer A. The sample was then loaded onto an S-FF column (Amersham) pre-equilibrated with Buffer A. A NaCl gradient was applied and H-NS eluted at approximately 550 mM NaCl. The H-NS containing fractions were then dialysed against a buffer containing 20 mM Tris HCl (pH 7.2), 300 mM KCl and 10% Glycerol (v/v)for storage at −80°C.The CRP and σHindIII and then AatII. After digestion, fragments were labelled at the HindIII end using [γ-32P]-ATP and T4 polynucleotide kinase. DNAse I footprints and EMSA experiments were then done as described by Grainger et al.in vitro DNA binding reactions contained a vast excess (12.5 µg ml−1) of Herring sperm DNA as a non-specific competitor. Footprints were analysed on a 6% DNA sequencing gel (molecular dynamics). The results of all footprints and EMSA experiments were visualized using a Fuji phosphor screen and Bio-Rad Molecular Imager FX.DNA fragments for DNAse I footprinting or EMSA assays were excised from pSR by sequential digestion with et al.estA1 or 93 bp PestA2 fragment cloned in pRW50. The 5′ end-labelled primer D49724, which anneals downstream of the HindIII site in pRW50, was used in all experiments. Primer extension products were analysed on denaturing 6% polyacrylamide gels, calibrated with size standards, and visualized using a Fuji phosphor screen and Bio-Rad Molecular Imager FX.Transcript start sites were mapped by primer extension, as described in Lloyd in vitro transcription experiments were performed as described previously Savery et al.et al.2, 500 µM DTT, 50 mM KCl, 100 µg ml−1 BSA, 200 µM ATP, 200 µM GTP, 200 µM CTP, 10 µM UTP with 5 µCi [α-32P]-UTP. The reaction was started by adding purified E. coli RNA polymerase. Labelled RNA products were analysed on a denaturing polyacrylamide gel.The w/v. The addition of “salt” refers to a 3∶1 molar ration of NaCl to KCl. We have arbitrarily described 30 mM NaCl and 10 mM KCl as being a “1%” salt solution.β-Galactosidase assays were done using the protocol of Miller 600 of 1.0. Groups of 10 mice (8–10 week old BALB/c) were infected intranasally with approximately 1×109 colony forming units of bacteria in 100 µl of inoculums according to Byrd et al.Strains of ETEC were grown in Luria Broth (LB) to an ODE coli (ETEC)” has been approved by the Noble Life Sciences IACUC committee. All animal care and use procedures adhere to the guidelines set by the Public Health Service Policy, U.S. Dept. of Agriculture (USDA) and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.The protocol 12-02-015IBT “Oral Immunization of Mice with Enterotoxigenic: S1 FigBinding of CRP to predicted targets in vitro. A) The data show binding of CRP to a target from each of the bins shown in (PDF)Click here for additional data file.S2 FigPromoter DNA fragments used in this work. A) ETEC H10407 PestA2 containing DNA fragments. B) ETEC H10407 derived DNA fragments containing PestA1. C) ETEC H10407 PeltAB containing sequences. D) DNA fragments containing sequences upstream of the estA2 and eltAB genes of ETEC E24377A.(PDF)Click here for additional data file.S3 FigA) Mutations in PestA2 UP-element alter the migration of the promoter DNA on an agarose gel. The DNA sequences used are shown in part (i) and the mobility of the fragments, on an agarose gel, are shown in part (ii). Note that each sample has been loaded in duplicate. B) Mutating the UP-element renders PestA2 uninducible by CRP. Part (i) shows LacZ activity data for the different promoter fragments cloned in pRW50. The pRW50 derivatives were used to transform M182 or the Δcrp derivative. Part (ii) shows the result of in vitro transcription assays using the different promoter fragments, cloned in pSR, as a template.(PDF)Click here for additional data file.S4 FigActivity of different promoter::lacZ fusions in the presence of increasing glucose and salt concentrations. The figure shows β-galactosidase activity measurements for lysates obtained from cultures of M182 carrying the A) estA1 B) estA2 or C) eltAB promoters cloned in pRW50. Panel D) shows β-galactosidase activity values for lysates of M182 and M182Δhns cells, carrying the estA2 promoter, or a derivative lacking the CRP site, cloned in pRW50. Cells were grown in the presence or absence of 2% glucose. Assays were done in M9 minimal medium so that the glucose and salt concentrations could be more accurately controlled.(PDF)Click here for additional data file.S1 TablePutative CRP binding targets on ETEC plasmids p948 and p666 identified by PREDetector.(DOCX)Click here for additional data file.S2 TableStrains, plasmids and oligonucleotides.(DOCX)Click here for additional data file.S3 TableAll CRP binding sites on the ETEC H10407 chromosome identified by ChIP-seq.(DOCX)Click here for additional data file.


CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3′ UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3′ UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3′ UTR isoforms was altered following CELF1 induction, with 3′ UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes.RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their genome-wide functions, we analyzed the transcriptome dynamics following induction of  CELF1-2, which are expressed most highly in heart, skeletal muscle, and brain, and CELF3-6, which exhibit more restricted expression (CELF RNA binding proteins (RBPs) play roles in early embryonic development, heart, and skeletal muscle functions. They are also thought to contribute to DM pathogenesis  and havepression . The CELpression .During normal development, CELF1 and CELF2 proteins are highly expressed in early embryonic stages and are then down-regulated more than 10-fold in skeletal muscle  and the CELF1 in adult mice recapitulates a subset of the misregulated splicing events observed in DM1 skeletal muscle and heart  and can functionally replace it, binding to the EDEN element to control poly(A) tail length and aiding in rapid deadenylation of maternal mRNAs after fertilization  and GU-repeats . The level of CELF1 mRNA increased strongly before declining at the end of the time course, likely as a secondary effect of heart pathology on the Myh1 promoter that drives rtTA . RNA was isolated from three Tet-inducible mice each at 12, 24, and 72 h and 7 d following induction and from three control mice lacking Tet-inducible CELF1 or CELF2 transgenes, treated with doxycycline for 72 h. The MISO software for statistical inference of splicing changes using RNA-seq data (We performed strand-specific paired-end RNA sequencing (RNA-seq) of poly(A)eviously . The CELves rtTA . Howeverves rtTA  and apprves rtTA . Heart pseq data  was usedTmed2 had a Ψ of 79% in the control heart but decreased to 13% after CELF1 induction for 7 d (Z-score” (MZ) defined as MZ = (δ − μ)/σ, where μ and σ are the mean and standard deviation of the null distribution of δ values, respectively. Large positive or negative MZ values indicate consistent increase or decrease, respectively, in the inclusion of an alternative exon over the time course.  We observed 627 and 825 exons responsive to CELF1 induction at a MZ score of 1.8 in heart and skeletal muscle, respectively.We identified thousands of exons whose Ψ values changed between time points at a BF cutoff of five, a value supported by previous studies of RBPs . For exa for 7 d A. To ideCELF1-induced heart and CELF1-induced skeletal muscle, we observed a high correlation (RSpearman = 0.64), suggesting that the functions and regulatory targets of CELF1 in these tissues are quite similar (CELF1 or CELF2. This comparison yielded a moderate positive correlation (RSp = 0.47), suggesting similar but not necessarily identical splicing functions  skipped exons between  similar B. To askunctions C.CELF1 heart to changes that occur during normal heart development. Developmental changes were analyzed by use of an RNA-seq time series (CELF1 induction. Splicing of 198 (85%) of these exons changed in the opposite direction following CELF1 induction from that during heart development, with a fairly strong negative correlation —of the splicing changes that occur during normal heart development   E. In skeegulated G. FurtheCELF1 induction in heart and muscle yielded only modest changes in the mRNA levels of Mbnl1  and Mbnl2  by RNA-seq RPKM and did not change inclusion of Mbnl1 exon 5, which can regulate nuclear-cytoplasmic distribution of MBNL1  or change the localization of MBNL1 by Western blot (data not shown). Finally, inclusion levels of exons bound and regulated by MBNL1, but not bound by CELF1, showed no tendency to change in a direction consistent with an overall decrease in MBNL1 activity (data not shown). These observations indicate that antagonistic regulation of many MBNL-dependent splicing events by CELF proteins results predominantly from direct effects on splicing rather than indirectly through regulation of MBNL activity.The observed splicing antagonism might result from direct effects of CELF1 on splicing or potentially from indirect effects on MBNL expression by CELF proteins. We found that Mbnl1 depletion and CELF1 induction in muscle and/or heart. These exons were enriched for several Gene Ontology (GO) categories, including “cell differentiation,” “multicellular organismal development,” “microtubule cytoskeleton,” and “cell junction,” suggesting roles in developmental remodeling of the heart . Furthermore, 124 of these exons (60%) preserved the translational reading frame , suggesting that the splicing activities of CELF and MBNL proteins are predominantly involved in shaping the spectrum of protein isoforms expressed in the heart. These observations provide genome-wide evidence for the principle that CELF and MBNL proteins exert opposing effects on the splicing of a large cohort of exons. They also expand the number of developmentally regulated CELF-/MBNL-responsive exons several-fold.In total, 206 alternative exons were responsive to both To identify transcript sites bound by CELF1, we performed CLIP-seq analysis using the 3B1 mouse monoclonal antibody against the endogenous CELF1 protein, yielding 1.6 million, 1.0 million, and 1.6 million reads uniquely mapping to the genome and splice junctions in 16-wk-old C57BL/6 heart, 16-wk-old C57BL/6 muscle, and C2C12 myoblast samples, respectively, after collapsing identical reads. Mapping occurred predominantly to transcribed regions, with enrichment for introns and/or 3′ UTRs , consistent with previous studies .Myadm —positions where CLIP-seq reads differ from the genome —at each CELF1 induction were enriched for CELF1 CLIP clusters . To determine an RNA map describing the splicing regulatory activity of CELF1, we analyzed the location of CELF1 clusters relative to exons responsive to CELF1 overexpression in heart and/or muscle. We found that cassette alternative exons bound by CELF1 were more likely to be repressed following CELF1 induction in heart and muscle  to two different Pumilio (Puf) domains, each recognizing a distinct eight-base Puf motif, as in We observed extensive CELF1 binding to 3′ UTRs, with increased density upstream of the cleavage and polyadenylation site (PAS) A. ConsisCELF1 induction relative to genes not bound by CELF1 . For example, the Clcn1 mRNA, which had prominent CELF1 binding clusters in its 3′ UTR, decreased in expression 50%–70% from its initial level 7 d after CELF1 induction, in both muscle  D. The dincreased D. A simincreased E, with ancreased D and of ncreased B–E assocCELF1 induction in heart, we observed a strong negative correlation (RSp = −0.52)  F. Theref or ALEs G. The huP < 1 × 10−48, Fisher's exact test)  average down-regulation, messages with similar numbers of CELF1 and MBNL1 clusters exhibited little change, and those with greater MBNL1 binding showed increased expression  are bound by both CELF1 and MBNL1; and (3) changed their localization upon Mbnl depletion in the expected direction  .To assess possible functional relationships between the effects of CELF and MBNL proteins on expression, we assessed gene expression changes for sets of genes grouped by extent of CELF1 and MBNL1 binding. Expression changes following pression B. These s study)  includedWithin the set of mRNAs not detectably bound by CELF1, the up-regulation of mRNAs bound more strongly by MBNL1 relative to less-bound messages was unexpected. One possible explanation is that this signal derives from CELF1 CLIP false negatives, i.e., messages that are bound by CELF1 but that failed to crosslink efficiently . Another possibility is that CELF proteins may exert a general nonspecific or low-specificity, mRNA decay-promoting activity at high levels and that MBNL binding protects against this effect as well as the effects of specific CELF1 binding.P < 1.8 × 10−55 by Kolmogorov-Smirnov [KS] test)  C. This pCELF1 induction on messages with proximal (≤50 nt) or distal (>50 nt) pairs of CELF1 and MBNL1 binding sites. The presence of a distal MBNL1 site was sufficient to abrogate the repression normally associated with presence of a CELF1 site, but presence of a proximal MBNL1 site lacked such a protective effect . This phenomenon of “local inhibition” of MBNL1's activity by CELF1 suggests that CELF1 can bind to messages when MBNL1 is bound nearby, and is readily explicable by either of two models: (1) “local inhibition of binding,” in which inhibition results simply from CELF1 inhibiting nearby binding of MBNL1, either directly through steric hindrance or indirectly via effects on local RNA structure; or (2) “local inhibition of function,” in which occupancy of CELF1 binding sites proximal to bound MBNL1 antagonizes the localization/stabilization function conferred by MBNL1 without affecting MBNL1's binding.To ask whether functional antagonism between MBNL1 and CELF1 might depend on binding site proximity, we examined the effects of e effect D. These Under either model, mRNAs containing a proximal pair of MBNL and CELF sites are expected to be more responsive to developmental changes in CELF and MBNL levels than messages containing distal pairs of sites. To explore this idea, we assessed developmental changes in expression of mRNAs grouped by binding site count and presence of proximal or distal CELF/MBNL site pairs E. OveralR-values) in the CELF1 pulldown libraries relative to a control library prepared from input RNA enables assessment of sequence-specific RNA affinity.To help distinguish between the “local inhibition of binding” and “local inhibition of function” models introduced above, we performed a variant of our RNA Bind-n-Seq (RBNS) assay , in whicBy analyzing all RNA 6mers, we observed a strong preference for GU- and UGU-containing motifs such as UGUUGU in all pulldown libraries, consistent with previous studies . By compThe relationship between CELF and MBNL proteins is important both in the development and context of neuromuscular disease, particularly DM. Throughout mouse heart development, CELF protein levels decrease and MBNL levels increase ; in DM, CELF1 induction and specifically enhanced by direct CELF1 binding, but have a lower decay rate when localized to the membrane compartment. Under this model, the localizations and half-lives of more than 1000 messages bound by both factors are determined by the outcome of a competition between the activities of MBNL and CELF proteins.It has recently been established that many genes shift toward greater expression of longer 3′ UTR isoforms during differentiation of muscle cells and likely other cell types . We obseCLCN1 gene, which encodes the major voltage-gated chloride channel that controls the membrane excitability of skeletal muscle, causes the myotonia that is characteristic of DM  included Kcnj2, a cardiac inward rectifier potassium channel whose mutation is associated with a syndrome that involves cardiac arrhythmia  appears to override the protective effect normally conferred by MBNL1 binding D, 6B, eiWe identified several dozen exons exhibiting antagonistic splicing regulation, many of which are developmentally regulated. These exons tend to preserve the reading frame and to reside in genes affecting functions involved in development and cell differentiation (see Results). Human homologs of this set of antagonistically regulated exons may be misregulated in DM to a particularly strong extent, as increased levels of CELF proteins would exacerbate splicing changes that result from MBNL sequestration. The hypothesis that CELF binding to exons tends to repress splicing, suggested by genome-wide analyses, was confirmed in a splicing reporter system, establishing a rule to predict CELF-dependent splicing regulation.CELF1 function intersects with RNA processing in a different way in its impact on the stability of alternative mRNA isoforms that differ in their 3′ UTRs. We observed a large set of genes with developmentally regulated ALEs and tandem UTR isoforms that differentially respond to CELF induction . RegulatTRECUGBP2 transgenic mice were generated by standard techniques and maintained on an FVB background. All reported TRECUGBP2/MHCrtTA bitransgenic mice were the F1 progeny from TRECUGBP2 × MHCrtTA (FVB/N-Tg(Myh6-rtTA)1Jam) matings. TRECUGBP2/MHCrtTA bitransgenic mice induced using doxycycline did not show evidence of ECG, echocardiography abnormalities, or abnormal heart size or morphology after 8 wk of induced expression of CELF2.N-terminal FLAG-tagged human CELF2 was expressed from the transgene previously used for CELF1 . TRECUGBCELF1 and CELF2 were induced in the heart and muscle by feeding mice 2 g/kg doxycycline for 12, 24, 72 h, or 7 d in the relevant transgenic animal. For CELF1 induction in the heart, mice with myosin heavy chain, promoter-driven, reverse tetracycline transactivator (rtTA) and Tet-inducible, N-terminal FLAG-tagged human CELF1 LYLQ isoform were used (data are provided under GEO accession GSE56185) , followed by RNeasy column with DNase treatment (Qiagen). Poly(A)+ RNA was prepared using Oligo dT dynabeads (Invitrogen) and prepared for paired-end RNA-seq (36–40 nt on Illumina GAII).SE56185) . For CEL2 tissue culture dish, sitting on ice, and crosslinked 3 × 400 mJ/cm2. In between each round of cross-linking, the dish was shaken from side to side to redistribute the tissue powder and provide maximum opportunity for all tissue particles to be cross-linked. The tissue was then lysed in RIPA buffer . The lysate was treated with DNase and RNase If (NEB) for 10 min at 37° C, with dilutions of 1:10,000 and 1:50,000 providing optimal RNA fragment lengths for downstream purification. Immunoprecipitation was performed using the 3B1 antibody clone against CELF1 (Millipore) and protein A beads (Invitrogen). The beads were washed twice with RIPA and twice with RIPA containing 1 M NaCl. The 3′ adapter was preadenylated with ImpA /σ), where μ and σ are the mean and standard deviation of the null distribution, respectively.MISO (version 0.4.8)  was usedCELF overexpression, Mbnl1 KO, or heart development) were enumerated, and the number of events regulated among each pair of perturbations was counted (this was the “observed” overlap) (Splicing events regulated in response to each perturbation (overlap) G,H. To coverlap) H was assAnalysis of CLIP data was performed as described in the Supplemental Material  of cassette exons with CLIP clusters within 1 kb of each splice site or within the exon itself was compared to a control set of cassette exons found in similarly expressed genes D, 3B. Co2(fold-change) in expression level in each group was computed, relative to genes with no CLIP clusters or miRNA seeds, respectively  is proportional to the number of core sites × potency of core site + number of extension sites × potency of extension × Ψextension. For ALEs, we considered a model in which log(expression change) is proportional to the number of sites in ALE1 × potency × ΨALE1 + number of sites in ALE2 × potency × ΨALE2. In both cases, coefficients were estimated by least squares regression.The abundance of alternative 3′ UTR isoforms by MISO was performed in “multi-isoform” mode B or in bhttp://www.ncbi.nlm.nih.gov/geo/) under superseries accession number GSE61893.The RNA-seq, CLIP-Seq, and RBNS sequencing data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO;