Intergenic Elements Research Articles

Documentation of virulence and races of Xanthomonas citri pv. malvacearum in India and its correlation with genetic diversity revealed by repetitive elements (REP, ERIC, and BOX) and ISSR markers.

Thirty-four Xanthomonas citri pv. malvacearum (Xcm) isolates collected from three cotton-growing zones of India were subjected for virulence and race documentation and further correlated with genetic diversity as revealed by repetitive elements [repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC) and BOX elements] and intersimple sequence repeat (ISSR)-PCR analyses. Among the 34 isolates tested for virulence on susceptible cultivar LRA 5166, 7 were recorded as highly virulent (HV), 16 were moderately virulent (MV) and 11 were less virulent (LV). Eight different races were recorded by using ten cotton host differentials. Twenty-two isolates (65%) belonged to race 18. Twelve isolates (35%) pertained to races 3, 5, 6, 7, 8, 11 and 13. REP, ERIC, BOX, combined repetitive elements, and ISSR analyses revealed the presence of 7, 10, 9, 11, and 8 clusters, respectively, at similarity coefficient of 0.70 in dendrograms. Principal coordinate analysis (PCoA) exhibited 76.4% and 77.5% cumulative variability for combined repetitive elements and ISSR analyses. ERIC produced the highest polymorphic information content (PIC) value (0.928). A lot of intra-pathovar variability was observed in virulence and genomic fingerprinting among Xcm isolates. Many of the isolates grouped based on geographical origin irrespective of virulence or race. The spread of the pathogen races in India might be due to the transport of germplasm lines and seed materials from one place to others.

Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk.

Osteoarthritis (OA) is a common, multifactorial and polygenic skeletal disease that, in its severest form, requires joint replacement surgery to restore mobility and to relieve chronic pain. Using tissues from the articulating joints of 260 patients with OA and a range of in vitro experiments, including CRISPR-Cas9, we have characterized an intergenic regulatory element. Here, genotype at an OA risk locus correlates with differential DNA methylation, with altered gene expression of both a transcriptional regulator (RUNX2), and a chromatin remodelling protein (SUPT3H). RUNX2 is a strong candidate for OA susceptibility, with its encoded protein being essential for skeletogenesis and healthy joint function. The OA risk locus includes single nucleotide polymorphisms (SNPs) located within and flanking the differentially methylated region (DMR). The OA association SNP, rs10948172, demonstrates particularly strong correlation with methylation, and two intergenic SNPs falling within the DMR (rs62435998 and rs62435999) demonstrate genetic and epigenetic effects on the regulatory activity of this region. We therefore posit that the OA signal mediates its effect by modulating the methylation of the regulatory element, which then impacts on gene expression, with RUNX2 being the principal target. Our study highlights the interplay between DNA methylation, OA genetic risk and the downstream regulation of genes critical to normal joint function.

Marked Global DNA Hypomethylation Is Associated with Constitutive PD-L1 Expression in Melanoma

SummaryConstitutive expression of the immune checkpoint, PD-L1, inhibits anti-tumor immune responses in cancer, although the factors involved in PD-L1 regulation are poorly understood. Here we show that loss of global DNA methylation, particularly in intergenic regions and repeat elements, is associated with constitutive (PD-L1CON), versus inducible (PD-L1IND), PD-L1 expression in melanoma cell lines. We further show this is accompanied by transcriptomic up-regulation. De novo epigenetic regulators (e.g., DNMT3A) are strongly correlated with PD-L1 expression and methylome status. Accordingly, decitabine-mediated inhibition of global methylation in melanoma cells leads to increased PD-L1 expression. Moreover, viral mimicry and immune response genes are highly expressed in lymphocyte-negative plus PD-L1-positive melanomas, versus PD-L1-negative melanomas in The Cancer Genome Atlas (TCGA). In summary, using integrated genomic analysis we identified that global DNA methylation influences PD-L1 expression in melanoma, and hence melanoma's ability to evade anti-tumor immune responses. These results have implications for combining epigenetic therapy with immunotherapy.

Alu exaptation enriches the human transcriptome by introducing new gene ends

ABSTRACTIn mammals, transposable elements are largely silenced, but under fortuitous circumstances may be co-opted to play a functional role. Here, we show that when Alu elements are inserted within or nearby genes in sense orientation, they may contribute to the transcriptome diversity by forming new cleavage and polyadenylation sites. We mapped these new gene ends in human onto the Alu sequence and identified three hotspots of cleavage and polyadenylation site formation. Interestingly, the native Alu sequence does not contain any canonical polyadenylation signal. We therefore studied what evolutionary processes might explain the formation of these specific hotspots of novel gene ends. We show that two of the three hotspots might have emerged from mutational processes that turned sequences that resemble polyadenylation signals into full-blown canonical signals, whereas one hotspot is tightly linked to the process of Alu insertion into the genome. Overall, Alu elements may lie behind the formation of 302 new gene end variants, affecting a total of 243 genes. Intergenic Alu elements may elongate genes by creating a downstream cleavage site, intronic Alu elements may lead to gene variants which code for truncated proteins, and 3′UTR Alu elements may result in gene variants with alternative 3′UTR.

Smek1/2 is a nuclear chaperone and cofactor for cleaved Wnt receptor Ryk, regulating cortical neurogenesis

The receptor-like tyrosine kinase (Ryk), a Wnt receptor, is important for cell fate determination during corticogenesis. During neuronal differentiation, the Ryk intracellular domain (ICD) is cleaved. Cleavage of Ryk and nuclear translocation of Ryk-ICD are required for neuronal differentiation. However, the mechanism of translocation and how it regulates neuronal differentiation remain unclear. Here, we identified Smek1 and Smek2 as Ryk-ICD partners that regulate its nuclear localization and function together with Ryk-ICD in the nucleus through chromatin recruitment and gene transcription regulation. Smek1/2 double knockout mice displayed pronounced defects in the production of cortical neurons, especially interneurons, while the neural stem cell population increased. In addition, both Smek and Ryk-ICD bound to the Dlx1/2 intergenic regulator element and were involved in its transcriptional regulation. These findings demonstrate a mechanism of the Ryk signaling pathway in which Smek1/2 and Ryk-ICD work together to mediate neural cell fate during corticogenesis.

Genome-wide identification of novel intergenic enhancer-like elements: implications in the regulation of transcription in Plasmodium falciparum

BackgroundThe molecular mechanisms of transcriptional regulation are poorly understood in Plasmodium falciparum. In addition, most of the genes in Plasmodium falciparum are transcriptionally poised and only a handful of cis-regulatory elements are known to operate in transcriptional regulation. Here, we employed an epigenetic signature based approach to identify significance of previously uncharacterised intergenic regions enriched with histone modification marks leading to discovery of enhancer-like elements.ResultsWe found that enhancer-like elements are significantly enriched with H3K4me1, generate unique non-coding bi-directional RNAs and majority of them can function as cis-regulators. Furthermore, functional enhancer reporter assay demonstrates that the enhancer-like elements regulate transcription of target genes in Plasmodium falciparum. Our study also suggests that the Plasmodium genome segregates functionally related genes into discrete housekeeping and pathogenicity/virulence clusters, presumably for robust transcriptional control of virulence/pathogenicity genes.ConclusionsThis report contributes to the understanding of parasite regulatory genomics by identification of enhancer-like elements, defining their epigenetic and transcriptional features and provides a resource of functional cis-regulatory elements that may give insights into the virulence/pathogenicity of Plasmodium falciparum.

Investigation of the human H3.3B (H3F3B) gene expression as a novel marker in patients with colorectal cancer.

H3.3 histone is a replacement histone subtype that is express in entire cell cycle phases and overexpress in transcriptionally active regions, promoter regions, and intergenic or intragenic regulatory elements. This histone encoded by two genes termed H3.3A (H3F3A) and H3.3B (H3F3B). Mutations of these two genes lead to some human cancers such as chondroblastoma, osteosarcoma, and epithelial ovarian cancer. The aims of this study were to quantitatively examine the expression of H3.3B gene in colorectal cancer (CRC) and to correlate their expression level with demographics and clinicopathological characteristics. We investigated H3.3B gene expression in CRC by relative quantitative real-time polymerase chain reaction (real-time PCR) technique for the first time. For this purpose, total RNA extracted, then cDNA synthesized and H3.3B gene expression was evaluated with specific primers by real-time PCR in tumoral tissues and adjacent normal tissues of 36 patients with CRC, then statistical analysis was performed using SPSS software. The results of this study indicated that H3.3B gene significantly overexpressed in tumoral tissue than adjacent normal tissue. Furthermore, statistical analysis represented the significant correlation between the H3.3B gene expression and some of the clinicopathological characteristics. Our study showed that H3.3B gene expression changes can be useful as a probable prognosis biomarker in the early stages of CRC before it metastasized.

Single-cell profiling reveals that eRNA accumulation at enhancer-promoter loops is not required to sustain transcription.

Enhancers are intergenic DNA elements that regulate the transcription of target genes in response to signaling pathways by interacting with promoters over large genomic distances. Recent studies have revealed that enhancers are bi-directionally transcribed into enhancer RNAs (eRNAs). Using single-molecule fluorescence in situ hybridization (smFISH), we investigated the eRNA-mediated regulation of transcription during estrogen induction in MCF-7 cells. We demonstrate that eRNAs are localized exclusively in the nucleus and are induced with similar kinetics as target mRNAs. However, eRNAs are mostly nascent at enhancers and their steady-state levels remain lower than those of their cognate mRNAs. Surprisingly, at the single-allele level, eRNAs are rarely co-expressed with their target loci, demonstrating that active gene transcription does not require the continuous transcription of eRNAs or their accumulation at enhancers. When co-expressed, sub-diffraction distance measurements between nascent mRNA and eRNA signals reveal that co-transcription of eRNAs and mRNAs rarely occurs within closed enhancer–promoter loops. Lastly, basal eRNA transcription at enhancers, but not E2-induced transcription, is maintained upon depletion of MLL1 and ERα, suggesting some degree of chromatin accessibility prior to signal-dependent activation of transcription. Together, our findings suggest that eRNA accumulation at enhancer–promoter loops is not required to sustain target gene transcription.

Transcriptional control of transglutaminase 2 expression in mouse apoptotic thymocytes

Transglutaminase 2 (TGM2) is a ubiquitously expressed multifunctional protein, which participates in various biological processes including thymocyte apoptosis. As a result, the transcriptional regulation of the gene is complex and must depend on the cell type. Previous studies from our laboratory have shown that in dying thymocytes the expression of Tgm2 is induced by external signals derived from engulfing macrophages, such as retinoids, transforming growth factor (TGF)-β and adenosine, the latter triggering the adenylate cyclase signaling pathway. The existence of TGF-β and retinoid responsive elements in the promoter region of Tgm2 has already been reported, but the intergenic regulatory elements participating in the regulation of Tgm2 have not yet been identified. Here we used publicly available results from DNase I hypersensitivity analysis followed by deep sequencing and chromatin immunoprecipitation followed by deep sequencing against CCCTC-binding factor (CTCF), H3K4me3, H3K4me1 and H3K27ac to map a putative regulatory element set for Tgm2 in thymocytes. By measuring eRNA expressions of these putative enhancers in retinoid, rTGF-β or dibutiryl cAMP-exposed thymocytes we determined which of them are functional. By applying ChIP-qPCR against SMAD4, retinoic acid receptor, retinoid X receptor, cAMP response element binding protein, P300 and H3K27ac under the same conditions, we identified two enhancers of Tgm2, which seem to act as integrators of the TGF-β, retinoid and adenylate cyclase signaling pathways in dying thymocytes. Our study describes a novel strategy to identify and characterize the signal-specific functional enhancer set of a gene by integrating genome-wide datasets and measuring the production of enhancer specific RNA molecules.

Intergenic Alu exonisation facilitates the evolution of tissue-specific transcript ends.

The 3′ untranslated regions (3′ UTRs) of transcripts serve as important hubs for posttranscriptional gene expression regulation. Here, we find that the exonisation of intergenic Alu elements introduced new terminal exons and polyadenylation sites during human genome evolution. While Alu exonisation from introns has been described previously, we shed light on a novel mechanism to create alternative 3′ UTRs, thereby opening opportunities for differential posttranscriptional regulation. On the mechanistic level, we show that intergenic Alu exonisation can compete both with alternative splicing and polyadenylation in the upstream gene. Notably, the Alu-derived isoforms are often expressed in a tissue-specific manner, and the Alu-derived 3′ UTRs can alter mRNA stability. In summary, we demonstrate that intergenic elements can affect processing of preceding genes, and elucidate how intergenic Alu exonisation can contribute to tissue-specific posttranscriptional regulation by expanding the repertoire of 3′ UTRs.

An oncogenic enhancer enemy (N-Me) in T-ALL

Constitutive activation of NOTCH signaling plays a central role in the pathogenesis of over 60% of T-cell acute lymphoblastic leukemias (T-ALL) harboring activating mutations in NOTCH1 or disruption of FBXW7, a critical negative regulator of NOTCH signaling.1 Expression of oncogenic NOTCH1 drives T-cell transformation activating a transcriptional program that includes multiple genes promoting leukemia cell growth and proliferation. Among these, the MYC oncogene has been highlighted as an important mediator of NOTCH1 induced transformation as NOTCH1 signaling induces transcriptional upregulation of MYC expression and NOTCH1 and MYC share multiple common target genes involved in leukemia cell growth.2 Still, demonstrating direct regulation of MYC by NOTCH1 has remained elusive as NOTCH1 fails to activate MYC promoter activity in reporter assays. This enigmatic link between NOTCH1 and MYC in T-ALL has been now clarified with the identification of N-Me (for NOTCH MYC enhancer), a long-range distal enhancer controlling MYC expression and T-cell transformation downstream of NOTCH1 (Fig. 1).3 The N-Me enhancer is located +1,427 kb from MYC in a large gene desert region extending telomeric from the MYC locus and represents the strongest peak of NOTCH1 binding in ChIP-seq analysis of T-ALL cells. Notably, and despite its distal genomic location, this regulatory element directly interacts with and controls MYC promoter activity as demonstrated by chromatin configuration 3C analysis and reporter assays. The functional importance of N-Me in the control of MYC expression is further supported by multispecies DNA sequence homology analyses which revealed remarkable conservation of this region in mammals, birds and reptiles. In addition, analysis of functional chromatin marks showed bona fide active enhancer features including P300 occupancy, high levels of H3K4me1 and low levels of H3K4me3 associated with N-Me. Moreover, detailed epigenetic profiling across 64 haematopoietic and non-haematopoietic cell lines and tissues revealed that this regulatory element is located within a major super-enhancer specifically active in T-cells. Consistently with these results, analysis of N-Me knockout and conditional knockout mice demonstrated a critical role for this long-range enhancer in T-cell development and NOTCH1-induced T-ALL.3 Thus, N-Me deficient mice showed marked thymic atrophy with depletion of CD4 CD8 double positive thymocytes. This phenotype was cell autonomous and was associated with decreased Myc expression in DN3, DN4 and ISP T-cell progenitors. Importantly, these T-cell developmental defects recapitulate those observed in T-cell specific Myc knockout mice,4 and were rescued by retroviral expression of MYC, formally demonstrating a strict requirement for N-Me mediated Myc expression in T-cell development. In addition, the NOTCH-N-Me-Myc regulatory axis showed to be essential for NOTCH1 induced T-ALL. Thus, N-Me knockout bone marrow progenitor cells failed to generate leukemia upon retroviral expression of oncogenic NOTCH1, and secondary deletion of N-Me in fully established NOTCH1-induced T-ALL resulted in profound antileukemic effects and almost complete suppression of leukemia initiating cell activity. Strikingly, loss of one copy of N-Me showed intermediate phenotypes including partial reduction of thymocyte numbers; reduced penetrance of T-ALL upon oncogenic NOTCH1 activation; and delayed tumor progression of fully established NOTCH1-driven tumors. These results highlight the exact dose-dependent function of the NOTCH1-NMe-Myc regulatory circuitry. Finally, genomic profiling of human leukemias identified recurrent focal duplications encompassing the N-Me enhancer in T-ALL supporting that increased N-Me dosage may contribute to T-cell transformation by strengthening NOTCH1 driven MYC expression. Figure 1. Schematic representation of N-Me activity during thymocyte development and in NOTCH1-induced T-ALL. Overall our results highlight the importance of long-range enhancers involved in the control of MYC expression in the pathogenesis of human cancer and are in line with previous results in myeloid leukemia and colon cancer.5,6 Still, the detailed characterization of the N-Me enhancer opens new outstanding questions that remain to be addressed. Most intriguingly, NOTCH1 and MYC play important developmental roles in multiple tissues other than T-cells and NOTCH signaling regulation of MYC expression has been implicated in the pathogenesis of breast cancer and other solid tumors;7,8 however N-Me seems to be strictly T-cell specific, suggesting that other as yet unidentified NOTCH-controlled MYC enhancers may be responsible for MYC expression outside T-cells. In addition, it is unclear what factors drive the T-cell specific activity of the N-Me enhancer. Finally, it has not escaped our attention that our identification of N-Me duplications in T-ALL support a potentially important and broader role for chromosomal copy number alterations and mutations occurring in intergenic regulatory elements in the pathogenesis of human cancer.

Setd1a regulates progenitor B‐cell‐to‐precursor B‐cell development through histone H3 lysine 4 trimethylation and Ig heavy‐chain rearrangement

SETD1A is a member of trithorax-related histone methyltransferases that methylate lysine 4 at histone H3 (H3K4). We showed previously that Setd1a is required for mesoderm specification and hematopoietic lineage differentiation in vitro. However, it remains unknown whether or not Setd1a controls specific hematopoietic lineage commitment and differentiation during animal development. Here, we reported that homozygous Setd1a knockout (KO) mice are embryonic lethal. Loss of the Setd1a gene in the hematopoietic compartment resulted in a blockage of the progenitor B-cell-to-precursor B-cell development in bone marrow (BM) and B-cell maturation in spleen. The Setd1a-cKO (conditional knockout) mice exhibited an enlarged spleen with disrupted spleen architecture and leukocytopenia. Mechanistically, Setd1a deficiency in BM reduced the levels of H3K4me3 at critical B-cell gene loci, including Pax5 and Rag1/2, which are critical for the IgH (Ig heavy-chain) locus contractions and rearrangement. Subsequently, the differential long-range looped interactions of the enhancer Eμ with proximal 5' DH region and 3' regulatory regions as well as with Pax5-activated intergenic repeat elements and 5' distal VH genes were compromised by the Setd1a-cKO. Together, our findings revealed a critical role of Setd1a and its mediated epigenetic modifications in regulating the IgH rearrangement and B-cell development.

The Role of Transcription Factor Tfii-I/GTF2I in the Response to Cellular Stress in Hematopoietic Cells

The transcription factor ATF3 and the heme regulated eIF2α kinase (HRI) have previously been shown to be involved in the stress response in hematopoietic cells. Many stress-responsive genes are regulated at the level of transcription elongation and are characterized by paused RNA polymerase II (Pol II) at the promoter region. Indeed, analyzing data from the public ENCODE project revealed that the HRI and the ATF3 genes contained paused Pol II at the promoter and at upstream intergenic regulatory elements. In this study we performed a genomic and proteomic analysis of transcription factor TFII-I (also known as GTF2I) in human erythroleukemia K562 cells. TFII-I is a ubiquitously expressed transcription factor previously implicated in regulating genes involved in stress response. TFII-I exhibits both positive and negative effects on transcription. TFII-I is a relatively large protein (about 120 Kda) and contains DNA binding, nuclear localization, and multiple protein/protein interaction domains. Using biotinylation tagging technology and high-throughput sequencing, we determined sites of chromatin interactions for TFII-I. This study revealed that TFII-I binds downstream of Pol II peaks at the ATF3 and HRI gene loci (Fig 1). A proteomic analysis revealed that TFII-I interacts with the elongation factor elongin A, which has been shown to help Pol II overcome the paused state. We demonstrate that Elongin A only associates with the ATF3 gene in response to cellular stress. The stress induction of ATF3 gene transcription requires the function of TFII-I. Furthermore, in the absence of induction, transcription factors E2F6, and E2F4 as well as the histone deacetylase I (HDAC1) bind to the HRI and ATF3 genes in close proximity to the Pol II and TFII-I peaks (Fig. 1). E2F4/6 and HDAC1 have been implicated in the repression of gene expression. The DNA elements that recruit TFII-I, Pol II and the repressor proteins interact over long distances in K562 cells as shown by ChIA-Pet data available from the ENCODE project (Fig.1). Moreover, we demonstrate that TFII-I directly interacts with E2F6 using co-immunoprecipitation assays (Fig. 2). Partial depletion of TFII-I by shRNA led to a decrease in stress mediated transcription without impairing basal transcription levels. The data demonstrate that TFII-I regulates stress-responsive transcription of the HRI and ATF3 genes in hematopoietic cells. In the absence of stress, TFII-I occupies promoter and long distance regulatory elements together with Pol II, E2F6/E2F4, and HDAC1. These proteins may be part of a chromatin loop that keeps Pol II in a repressed configuration. We suggest that upon stress induction, TFII-I recruits Elongin A, which converts Pol II into a transcriptionally active configuration. Transcription elongation disrupts formation of a repressive chromatin loop and leads to transcription activation of the HRI and ATF3 genes. Figure 1 Diagram of the HRI and ATF3 gene loci showing interactions of TFII-I, Pol II, E2F4, E2F6, and HDAC1 as well as RNA seq, and Pol II ChIA-PET data. The ChIA-PET data show interactions of chromatin fragments that have Pol II bound. The arrows indicate the direction of transcription of the HRI gene and from the two promoters of the ATF3 gene (P1 and P2). Figure 1. Diagram of the HRI and ATF3 gene loci showing interactions of TFII-I, Pol II, E2F4, E2F6, and HDAC1 as well as RNA seq, and Pol II ChIA-PET data. The ChIA-PET data show interactions of chromatin fragments that have Pol II bound. The arrows indicate the direction of transcription of the HRI gene and from the two promoters of the ATF3 gene (P1 and P2). Figure 2 Input; TFII-I Figure 2. Input; TFII-I Figure 3 Co-Immunoprecipitation experiment showing the interaction between TFII-I and E2F6. K562 cell nuclear extracts were subjected to precipitation with IgG control or TFII-I specific antibodies. The material was fractionated by SDS-PAGE, transferred to a nylon membrane and probed with an antibody specific for E2F6. Figure 3. Co-Immunoprecipitation experiment showing the interaction between TFII-I and E2F6. K562 cell nuclear extracts were subjected to precipitation with IgG control or TFII-I specific antibodies. The material was fractionated by SDS-PAGE, transferred to a nylon membrane and probed with an antibody specific for E2F6. Disclosures No relevant conflicts of interest to declare.

Oncogene regulation. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element.

In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, that recruit much of the cell's transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits its H3K27 acetylase-binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, which suggests a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells.

Insertion sequence distribution bias in Archaea

Insertion sequences (IS) are common transposable elements in Archaea. Intergenic IS elements are usually less harmful than intragenic ISs, simply because they are less likely to disrupt host gene function. However, because regulatory sequences are intergenic and upstream of genes, we hypothesized that not all intergenic regions are selectively equivalent for IS insertion. We tested this hypothesis by analyzing the distributions of intergenic IS elements within 155 fully sequenced archaeal genomes. Of the 22 genomes with enough IS elements for statistical analysis, five have significantly fewer ISs between divergently oriented neighboring genes than expected by chance, and seven have significantly more ISs between convergently oriented genes. Furthermore, of the 85 genomes with at least one expected IS within each of the three possible neighboring gene orientations (i.e., divergent, convergent, and tandem), 73 genomes have fewer ISs between divergently oriented genes than expected, and 60 have more ISs between convergently oriented genes than expected (both values deviate significantly from binomial probabilities of random distribution). We suspect that these non-random IS distributions are molded by natural selection resulting from differential disruption of neighboring gene regulation, and that this selective pressure has affected transposable element distributions in prokaryotes for billions of years.

Pervasive Transcription of the Human Genome Produces Thousands of Previously Unidentified Long Intergenic Noncoding RNAs

Known protein coding gene exons compose less than 3% of the human genome. The remaining 97% is largely uncharted territory, with only a small fraction characterized. The recent observation of transcription in this intergenic territory has stimulated debate about the extent of intergenic transcription and whether these intergenic RNAs are functional. Here we directly observed with a large set of RNA-seq data covering a wide array of human tissue types that the majority of the genome is indeed transcribed, corroborating recent observations by the ENCODE project. Furthermore, using de novo transcriptome assembly of this RNA-seq data, we found that intergenic regions encode far more long intergenic noncoding RNAs (lincRNAs) than previously described, helping to resolve the discrepancy between the vast amount of observed intergenic transcription and the limited number of previously known lincRNAs. In total, we identified tens of thousands of putative lincRNAs expressed at a minimum of one copy per cell, significantly expanding upon prior lincRNA annotation sets. These lincRNAs are specifically regulated and conserved rather than being the product of transcriptional noise. In addition, lincRNAs are strongly enriched for trait-associated SNPs suggesting a new mechanism by which intergenic trait-associated regions may function. These findings will enable the discovery and interrogation of novel intergenic functional elements.

Expression of the repeat genome and aberrant epigenetic factors in cancer

Nearly half of the human genome consists of noncoding repetitive DNA elements, including tandem satellite repeats in large blocks at the pericentric regions of chromosomes and intergenic repetitive elements. While both repeat types were long thought to remain mostly silent, recent evidence indicates that repeats can be expressed, but the extent and regulation of their expression or their potential function(s) remain to be elucidated. Due to their critical location within regions vital for cell division, it is expected that tight regulation of pericentric satellite sequences is essential for both epigenetic and genetic stability. Our data suggests aberrant expression of pericentric satellite RNA is tightly linked to epigenetic misregulation in cancer. It is well known that epigenetic changes can be important in cancer initiation and progression, but studies have focused primarily on the inappropriate silencing and methylation of tumor suppressor genes. While pathologists have long noted the loss of heterochromatic organization in cancer nuclei, and hypomethylation of satellite DNA has been observed, the misregulation of repeat RNAs has only recently been described. Our results provide a link between overexpression of repeat RNAs and aberrant distribution of epigenetic factors in cancer. Our data suggests regulation of the repeat genome has potentially important roles in both normal and neoplastic cells in their ability to affect distribution and recruitment of epigenetic factors.

The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila.

BackgroundThe question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription.Results and discussionHere we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters.ConclusionsOur data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes.

Global Assessment of Genomic Regions Required for Growth in Mycobacterium tuberculosis

Identifying genomic elements required for viability is central to our understanding of the basic physiology of bacterial pathogens. Recently, the combination of high-density mutagenesis and deep sequencing has allowed for the identification of required and conditionally required genes in many bacteria. Genes, however, make up only a part of the complex genomes of important bacterial pathogens. Here, we use an unbiased analysis to comprehensively identify genomic regions, including genes, domains, and intergenic elements, required for the optimal growth of Mycobacterium tuberculosis, a major global health pathogen. We found that several proteins jointly contain both domains required for optimal growth and domains that are dispensable. In addition, many non-coding regions, including regulatory elements and non-coding RNAs, are critical for mycobacterial growth. Our analysis shows that the genetic requirements for growth are more complex than can be appreciated using gene-centric analysis.

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.