Transcriptional induction of heat shock protein (HSP) genes is accompanied by dynamic changes in their 3D structure and spatial organization, yet the molecular basis for these phenomena remains unknown. Using chromosome conformation capture and single-cell imaging, we show that genes transcriptionally activated by Hsf1 specifically interact across chromosomes and coalesce into diffraction-limited intranuclear foci. Genes activated by the alternative stress regulators Msn2/Msn4, in contrast, do not interact among themselves nor with Hsf1 targets. Likewise, constitutively expressed genes, even those interposed between HSP genes, show no detectable interaction. Hsf1 forms discrete subnuclear puncta when stress activated, and these puncta dissolve in concert with transcriptional attenuation, paralleling the kinetics of HSP gene coalescence and dissolution. Nuclear Hsf1 and RNA Pol II are both necessary for intergenic HSP gene interactions, while DNA-bound Hsf1 is necessary and sufficient to drive heterologous gene coalescence. Our findings demonstrate that Hsf1 can dynamically restructure the yeast genome.

The advent of large-scale gene expression technologies has helped to reveal in eukaryotic cells, the existence of thousands of non-coding transcripts, whose function and significance remain mostly poorly understood. Among these non-coding transcripts, long non-coding RNAs (lncRNAs) are the least well-studied but are emerging as key regulators of diverse cellular processes. In the present study, we performed a survey in bovine Longissimus thoraci of lincRNAs (long intergenic non-coding RNAs not overlapping protein-coding transcripts). To our knowledge, this represents the first such study in bovine muscle.

Functionally altered biological mechanisms arising from disease-associated polymorphisms, remain difficult to characterize when those variants are intergenic, or, fall between genes. We sought to identify shared downstream mechanisms by which inter- and intragenic single nucleotide polymorphisms (SNPs) contribute to a specific physiopathology. Using computational modeling of 2 million pairs of disease-associated SNPs drawn from genome wide association studies (GWAS), integrated with expression Quantitative Trait Loci (eQTL) and Gene Ontology functional annotations, we predicted 3,870 inter-intra and inter-intra SNP pairs with convergent biological mechanisms (FDR<0.05). These prioritized SNP pairs with overlapping mRNA targets or similar functional annotations were more likely to be associated with the same disease than unrelated pathologies (OR>12). We additionally confirmed synergistic and antagonistic genetic interactions for a subset of prioritized SNP pairs in independent studies of Alzheimer's disease (entropy p=0.046), bladder cancer (entropy p=0.039), and rheumatoid arthritis (PheWAS case-control p<10-4). Using ENCODE datasets, we further statistically validated that the biological mechanisms shared within prioritized SNP pairs are frequently governed by matching transcription factor binding sites and long-range chromatin interactions. These results provide a "roadmap" of disease mechanisms emerging from GWAS and further identify candidate therapeutic targets among downstream effectors of intergenic SNPs.

In recent years, considerable advances have been made in the characterization of protein-coding alterations involved in the pathogenesis of melanoma. However, despite their growing implication in cancer, little is known about the role of long noncoding RNAs in melanoma progression. We hypothesized that copy number alterations (CNAs) of intergenic nonprotein-coding domains could help identify long intergenic noncoding RNAs (lincRNAs) associated with metastatic cutaneous melanoma. Among several candidates, our approach uncovered the chromosome 6p22.3 CASC15 (cancer susceptibility candidate 15) lincRNA locus as a frequently gained genomic segment in metastatic melanoma tumors and cell lines. The locus was actively transcribed in metastatic melanoma cells, and upregulation of CASC15 expression was associated with metastatic progression to brain metastasis in a mouse xenograft model. In clinical specimens, CASC15 levels increased during melanoma progression and were independent predictors of disease recurrence in a cohort of 141 patients with AJCC (American Joint Committee on Cancer) stage III lymph node metastasis. Moreover, small interfering RNA (siRNA) knockdown experiments revealed that CASC15 regulates melanoma cell phenotype switching between proliferative and invasive states. Accordingly, CASC15 levels correlated with known gene signatures corresponding to melanoma proliferative and invasive phenotypes. These findings support a key role for CASC15 in metastatic melanoma.

A great mass of long noncoding RNAs (lncRNAs) have been identified in mouse genome and increasing evidences in the last decades have revealed their crucial roles in diverse biological processes. Nevertheless, the biological roles of lncRNAs in the mouse retina remains largely unknown due to the lack of a comprehensive annotation of lncRNAs expressed in the retina.

Long intergenic noncoding RNAs (lincRNAs) play a crucial role in many biological processes. The rat is an important model organism in biomedical research. Recent studies have detected rat lincRNA genes from several samples. However, identification of rat lincRNAs using large-scale RNA-seq datasets remains unreported. Herein, using more than 100 billion RNA-seq reads from 59 publications together with RefSeq and UniGene annotated RNAs, we report 39,154 lincRNA transcripts encoded by 19,162 lincRNA genes in the rat. We reveal sequence and expression similarities in lincRNAs of rat, mouse and human. DNA methylation level of lincRNAs is higher than that of protein-coding genes across the transcription start sites (TSSs). And, three lincRNA genes overlap with differential methylation regions (DMRs) which associate with spontaneously hypertensive disease. In addition, there are similar binding trends for three transcription factors (HNF4A, CEBPA and FOXA1) between lincRNA genes and protein-coding genes, indicating that they harbour similar transcription regulatory mechanisms. To date, this is the most comprehensive assessment of lincRNAs in the rat genome. We provide valuable data that will advance lincRNA research using rat as a model.

The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

Analyses of genome reduction in obligate bacterial symbionts typically focus on the removal and retention of protein-coding regions, which are subject to ongoing inactivation and deletion. However, these same forces operate on intergenic spacers (IGSs) and affect their contents, maintenance, and rates of evolution. IGSs comprise both non-coding, non-functional regions, including decaying pseudogenes at varying stages of recognizability, as well as functional elements, such as genes for sRNAs and regulatory control elements. The genomes of Buchnera and other small genome symbionts display biased nucleotide compositions and high rates of sequence evolution and contain few recognizable regulatory elements. However, IGS lengths are highly correlated across divergent Buchnera genomes, suggesting the presence of functional elements. To identify functional regions within the IGSs, we sequenced two Buchnera genomes (from aphid species Uroleucon ambrosiae and Acyrthosiphon kondoi) and applied a phylogenetic footprinting approach to alignments of orthologous IGSs from a total of eight Buchnera genomes corresponding to six aphid species. Inclusion of these new genomes allowed comparative analyses at intermediate levels of divergence, enabling the detection of both conserved elements and previously unrecognized pseudogenes. Analyses of these genomes revealed that 232 of 336 IGS alignments over 50 nucleotides in length displayed substantial sequence conservation. Conserved alignment blocks within these IGSs encompassed 88 Shine-Dalgarno sequences, 55 transcriptional terminators, 5 Sigma-32 binding sites, and 12 novel small RNAs. Although pseudogene formation, and thus IGS formation, are ongoing processes in these genomes, a large proportion of intergenic spacers contain functional sequences.

The applicability of a newly-designed PCR primer pair in examination of methanogenic Archaea in a digester treating plant biomass was evaluated by Ribosmal Intergenic Spacer Analysis (RISA). To find a suitable approach, three variants of RISA were tested: (1) standard, polyacrylamide gel-based, (2) automated, utilized capillary electrophoresis (GA-ARISA), and (3) automated microfluidics-based (MF-ARISA). All three techniques yielded a consistent picture of archaeal community structure changes during anaerobic digestion monitored for more than 6 weeks. While automated variants were more practical for handling and rapid analysis of methanogenic Archaea, the gel-based technique was advantageous when micro-organism identification was required. A DNA-sequence analysis of dominant bands extracted from the gel revealed that the main role in methane synthesis was played by micro-organisms affiliated with Methanosarcina barkeri. The obtained results revealed that RISA is a robust method allowing for detailed analysis of archaeal community structure during organic biomass conversion into biogas. In addition, our results showed that GA-ARISA has a higher resolution and reproducibility than other variants of RISA and could be used as a technique for tracking changes in methanogenic Archaea in an anaerobic digester.

Patients with advanced ovarian cancer usually exhibit high mortality rates, thus more efficient therapeutic strategies are expected to be developed. Recent transcriptomic studies revealed that long intergenic noncoding RNAs (lincRNAs) can be a new class of molecular targets for cancer management, because lincRNAs likely exert tissue-specific activities compared with protein-coding genes or other noncoding RNAs. We here show that an unannotated lincRNA originated from chromosome 10q21 and designated as ovarian cancer long intergenic noncoding RNA 1 (OIN1), is often overexpressed in ovarian cancer tissues compared with normal ovaries as analyzed by RNA sequencing. OIN1 silencing by specific siRNAs significantly exerted proliferation inhibition and enhanced apoptosis in ovarian cancer cells. Notably, RNA sequencing showed that OIN1 expression was negatively correlated with the expression of apoptosis-related genes ras association domain family member 5 (RASSF5) and adenosine A1 receptor (ADORA1), which were upregulated by OIN1 knockdown in ovarian cancer cells. OIN1-specifc siRNA injection was effective to suppress in vivo tumor growth of ovarian cancer cells inoculated in immunodeficient mice. Taken together, OIN1 could function as a tumor-promoting lincRNA in ovarian cancer through modulating apoptosis and will be a potential molecular target for ovarian cancer management.

The Escherichia coli RutR protein is the master regulator of genes involved in pyrimidine catabolism. Here we have used chromatin immunoprecipitation in combination with DNA microarrays to measure the binding of RutR across the chromosome of exponentially growing E. coli cells. Twenty RutR-binding targets were identified and analysis of these targets generated a DNA consensus logo for RutR binding. Complementary in vitro binding assays showed high-affinity RutR binding to 16 of the 20 targets, with the four low-affinity RutR targets lacking predicted key binding determinants. Surprisingly, most of the DNA targets for RutR are located within coding segments of the genome and appear to have little or no effect on transcript levels in the conditions tested. This contrasts sharply with other E. coli transcription factors whose binding sites are primarily located in intergenic regions. We suggest that either RutR has yet undiscovered function or that evolution has been slow to eliminate non-functional DNA sites for RutR because they do not have an adverse effect on cell fitness.

Identification of diffuse signals from the chromatin immunoprecipitation and high-throughput massively parallel sequencing (ChIP-Seq) technology poses significant computational challenges, and there are few methods currently available. We present a novel global clustering approach to enrich diffuse CHIP-Seq signals of RNA polymerase II and histone 3 lysine 4 trimethylation (H3K4Me3) and apply it to identify putative long intergenic non-coding RNAs (lincRNAs) in macrophage cells. Our global clustering method compares favorably to the local clustering method SICER that was also designed to identify diffuse CHIP-Seq signals. The validity of the algorithm is confirmed at several levels. First, 8 out of a total of 11 selected putative lincRNA regions in primary macrophages respond to lipopolysaccharides (LPS) treatment as predicted by our computational method. Second, the genes nearest to lincRNAs are enriched with biological functions related to metabolic processes under resting conditions but with developmental and immune-related functions under LPS treatment. Third, the putative lincRNAs have conserved promoters, modestly conserved exons, and expected secondary structures by prediction. Last, they are enriched with motifs of transcription factors such as PU.1 and AP.1, previously shown to be important lineage determining factors in macrophages, and 83% of them overlap with distal enhancers markers. In summary, GCLS based on RNA polymerase II and H3K4Me3 CHIP-Seq method can effectively detect putative lincRNAs that exhibit expected characteristics, as exemplified by macrophages in the study.

Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN-, MERVL-, and ERVK-associated and essential for mouse embryonic development beyond the two-cell stage. LincGET is expressed in late two- to four-cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two-cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA-protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis-regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two-cell stage. Thus, as the first functional ERV-associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.

Mineral dust-induced gene (mdig) had been linked to the development of human lung cancers associated with environmental exposure to mineral dust, tobacco smoke or other carcinogens. In the present studies, we demonstrated that the overexpression of mdig in A549 adenocarcinomic human alveolar type II epithelial cells decreases the heterochromatin conformation of the cells and de-represses the transcription of genes in the tandemly repeated DNA regions. Although mdig can only cause a marginal decrease of the total histone H3 lysine 9 trimethylation (H3K9me3), a significant reduction of H3K9me3 in the promoter region of H19, the paternally imprinted but maternally expressed gene transcribing a large intergenic non-coding RNA (lincRNA), was observed in the cells with mdig overexpression. Silencing mdig by either shRNA or siRNA not only increased the level of H3K9me3 in the promoter region of H19 but also attenuated the transcription of H19 long non-coding RNA. Demethylation assays using immunoprecipitated mdig and histone H3 peptide substrate suggested that mdig is able to remove the methyl groups from H3K9me3. Clinically, we found that higher levels of mdig and H19 expression correlate with poorer survival of the lung cancer patients. Taken together, our results imply that mdig is involved in the regulation of H3K9me3 to influence the heterochromatin structure of the genome and the expression of genes important for cell growth or transformation.

Transcription initiation by RNA polymerase II is unidirectional from most genes. In plants, divergent genes, defined as non-overlapping genes organized head-to-head, are highly represented in the Arabidopsis genome. Nevertheless, there is scarce evidence on functional analyses of these intergenic regions. The At5g06290 and At5g06280 loci are head-to-head oriented and encode a chloroplast-located 2-Cys peroxiredoxin B (2CPB) and a protein of unknown function (PUF), respectively. The 2-Cys peroxiredoxins are proteins involved in redox processes, they are part of the plant antioxidant defence and also act as chaperons. In this study, the transcriptional activity of a small intergenic region (351 bp) shared by At5g06290 and At5g06280 in Arabidopsis thaliana was characterized.

Non-coding RNAs and mRNAs have been implicated in replication, pathogenesis and host response in HIV infection. However, the impact of long intergenic non-coding RNAs (lincRNAs) on HIV-1 and HIV-2 infection is not known. In this study, we have analyzed expression profiles of lincRNAs and mRNAs in monocyte derived macrophages (MDMs) infected with HIV-1/HIV-2 using microarrays. Our study identified many differentially expressed lincRNAs and mRNAs in MDMs infected with HIV-1/HIV-2 compared to uninfected MDMs. Genes involved in glutathione metabolism and lysine degradation were differentially regulated only in HIV-1 infected MDMs. In HIV-2 infected MDMs, CUL 2, SFRS9, and RBBP4 genes were differentially expressed. Furthermore, we found that plasma levels of lincRNA: chr2: 165509129-165519404 and lincRNA: chr12: 57761837-57762303 were better indicators of HIV-1 infection while lincRNA: chr10:128586385-128592960, XLOC_001148 and lincRNA: chr5:87580664-87583451, were better indicators of HIV-2 infection. In summary, our study has demonstrated that there is substantial alteration in lincRNA and mRNA expression in response to HIV-1/HIV-2 infection. These differentially expressed lincRNAs and mRNAs could serve as prognostic and diagnostic biomarkers of HIV infection and help in the identification of new targets for therapy.

Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/H19 allelic expression. The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O'Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves.

Intramuscular fat (IMF) content is an important trait that can affect pork quality. Previous studies have identified many genes that can regulate IMF. Long intergenic non-coding RNAs (lincRNAs) are emerging as key regulators in various biological processes. However, lincRNAs related to IMF in pig are largely unknown, and the mechanisms by which they regulate IMF are yet to be elucidated. Here we reconstructed 105,687 transcripts and identified 1,032 lincRNAs in pig longissimus dorsi muscle (LDM) of four stages with different IMF contents based on published RNA-seq. These lincRNAs show typical characteristics such as shorter length and lower expression compared with protein-coding genes. Combined with methylation data, we found that both the promoter and genebody methylation of lincRNAs can negatively regulate lincRNA expression. We found that lincRNAs exhibit high correlation with their protein-coding neighbors in expression. Co-expression network analysis resulted in eight stage-specific modules, gene ontology and pathway analysis of them suggested that some lincRNAs were involved in IMF-related processes, such as fatty acid metabolism and peroxisome proliferator-activated receptor signaling pathway. Furthermore, we identified hub lincRNAs and found six of them may play important roles in IMF development. This work detailed some lincRNAs which may affect of IMF development in pig, and facilitated future research on these lincRNAs and molecular assisted breeding for pig.

Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression in both mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of long intergenic nocoding RNAs (lincRNAs) in plant defence responses are yet to be fully explored.

The genomic shock of whole-genome duplication (WGD) and hybridization introduces great variation into transcriptomes, for both coding and noncoding genes. An altered transcriptome provides a molecular basis for improving adaptation during the evolution of new species. The allotetraploid cotton, together with the putative diploid ancestor species compose a fine model for study the rapid gene neofunctionalization over the genome shock. Here we report on Drought-Associated Non-coding gene 1 (DAN1), a long intergenic noncoding RNA (lincRNA) that arose from the cotton progenitor A-diploid genome after hybridization and WGD events during cotton evolution. DAN1 in allotetraploid upland cotton (Gossypium hirsutum) is a drought-responsive lincRNA predominantly expressed in the nucleoplasm. Chromatin isolation by RNA purification profiling and electrophoretic mobility shift assay analysis demonstrated that GhDAN1 RNA can bind with DNA fragments containing AAAG motifs, similar to DNA binding with one zinc finger transcription factor binding sequences. The suppression of GhDAN1 mainly regulates genes with AAAG motifs in auxin-response pathways, which are associated with drought stress regulation. As a result, GhDAN1-silenced plants exhibit improved tolerance to drought stress. This phenotype resembles the drought-tolerant phenotype of the A-diploid cotton ancestor species, which has an undetectable expression of DAN1. The role of DAN1 in cotton evolution and drought tolerance regulation suggests that the genomic shock of interspecific hybridization and WGD stimulated neofunctionalization of non-coding genes during the natural evolutionary process.