Cap Analysis Of Gene Expression Research Articles

Effect of Butyric Acid in the Proliferation and Migration of Junctional Epithelium in the Progression of Periodontitis: An In Vitro Study.

Purpose: To elucidate the effects of butyric acid (BA), a metabolite of bacteria involved in periodontitis, and a possible enhancer of the junctional epithelial cells. Methods: A murine junctional epithelial cell line, JE-1, was used to assess the effects of sodium butyrate (NaB) as BA. Cell proliferation, migration and attachment were analyzed. Additionally, gene and promoter expression analysis was performed, i.e., cap analysis of gene expression (CAGE) and gene ontology (GO) term enrichment analysis. Results: NaB affected junctional epithelial cell proliferation, migration and attachment. A high concentration of NaB caused cell death and a low concentration tended to promote migration and adhesion. CAGE analysis revealed 75 upregulated and 96 downregulated genes in the cells after 0.2 mM NaB stimulation for 3 h. Regarding GO term enrichment, the genes upregulated >4-fold participated predominantly in cell migration and proliferation. The results of this study suggest that BA produced from periodontopathic bacteria is involved in periodontal tissue destruction at high concentrations. Furthermore, at low concentrations, BA potentially participates in periodontal disease progression by increasing proliferation, migration and attachment of the junctional epithelium and thereby increasing epithelial down-growth.

A Transcription Start Site Map in Human Pancreatic Islets Reveals Functional Regulatory Signatures.

Identifying the tissue-specific molecular signatures of active regulatory elements is critical to understand gene regulatory mechanisms. Here, we identify transcription start sites (TSS) using cap analysis of gene expression (CAGE) across 57 human pancreatic islet samples. We identify 9,954 reproducible CAGE tag clusters (TCs), ∼20% of which are islet specific and occur mostly distal to known gene TSS. We integrated islet CAGE data with histone modification and chromatin accessibility profiles to identify epigenomic signatures of transcription initiation. Using a massively parallel reporter assay, we validated the transcriptional enhancer activity for 2,279 of 3,378 (∼68%) tested islet CAGE elements (5% false discovery rate). TCs within accessible enhancers show higher enrichment to overlap type 2 diabetes genome-wide association study (GWAS) signals than existing islet annotations, which emphasizes the utility of mapping CAGE profiles in disease-relevant tissue. This work provides a high-resolution map of transcriptional initiation in human pancreatic islets with utility for dissecting active enhancers at GWAS loci.

Flexible analysis of TSS mapping data and detection of TSS shifts with TSRexploreR.

Heterogeneity in transcription initiation has important consequences for transcript stability and translation, and shifts in transcription start site (TSS) usage are prevalent in various developmental, metabolic, and disease contexts. Accordingly, numerous methods for global TSS profiling have been developed, including most recently Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a method to profile transcription start sites (TSSs) on a genome-wide scale with significant cost and time savings compared to previous methods. In anticipation of more widespread adoption of STRIPE-seq and related methods for construction of promoter atlases and studies of differential gene expression, we built TSRexploreR, an R package for end-to-end analysis of TSS mapping data. TSRexploreR provides functions for TSS and transcription start region (TSR) detection, normalization, correlation, visualization, and differential TSS/TSR analyses. TSRexploreR is highly interoperable, accepting the data structures of TSS and TSR sets generated by several existing tools for processing and alignment of TSS mapping data, such as CAGEr for Cap Analysis of Gene Expression (CAGE) data. Lastly, TSRexploreR implements a novel approach for the detection of shifts in TSS distribution.

CAGE-seq reveals that HIV-1 latent infection does not trigger unique cellular responses in a Jurkat T cell model.

The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a GFP reporter under the HIV-1 promoter and an mKO2 reporter under the internal EF1α promoter. This viral construct enables direct identification of HIV-1 both productively and latently infected cells. In this study we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using Cap Analysis of Gene Expression (CAGE).We deep sequenced CAGE tags in uninfected, latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus producing cells had differentially expressed TSSs related to T-cell activation and apoptosis when compared to uninfected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to uninfected cells. Among these, SPP1 and APOE were down-regulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have anti-viral properties. Components of the PI3K/mTOR pathway, MLST8, 4EBP and RPS6, were significant TSSs in productively infected cells, and S6K phosphorylation was increased compared to latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent.Importance: Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used CAGE to compare the transcriptional landscape of latently infected cells with that of non-infected or productively infected cells. We found that latently infected cells and non-infected cells show quite similar transcriptional profiles. Our data suggest that T-cells cannot recognize incoming viral components nor the integrated HIV-1 genome when infection remains latent. These findings should guide future research into widening our approaches to identify and target latent HIV-1 infected cells.

Low Quantity Single Strand CAGE (LQ-ssCAGE) Maps Regulatory Enhancers and Promoters.

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.

Analysis of Enhancer-Promoter Interactions using CAGE and RADICL-Seq Technologies.

Regulation of gene expression is a key feature for higher eukaryotes and how chromatin topology relates to gene activation is an intense area of research. Enhancer-promoter interactions are believed to mediate activation of target genes. Bidirectional transcription represents one hallmark of active enhancers that can be measured using transcriptome technologies such as Cap analysis of gene expression (CAGE). Recently, we have developed RNA and DNA interacting complexes ligated and sequenced (RADICL-Seq) a novel methodology to map genome-wide RNA-chromatin interactions in intact nuclei. Here, we describe how CAGE and RADICL-Seq data can be used to characterize enhancer elements and identify their target genes.

DIANA-miRGen v4: indexing promoters and regulators for more than 1500 microRNAs.

Deregulation of microRNA (miRNA) expression plays a critical role in the transition from a physiological to a pathological state. The accurate miRNA promoter identification in multiple cell types is a fundamental endeavor towards understanding and characterizing the underlying mechanisms of both physiological as well as pathological conditions. DIANA-miRGen v4 (www.microrna.gr/mirgenv4) provides cell type specific miRNA transcription start sites (TSSs) for over 1500 miRNAs retrieved from the analysis of >1000 cap analysis of gene expression (CAGE) samples corresponding to 133 tissues, cell lines and primary cells available in FANTOM repository. MiRNA TSS locations were associated with transcription factor binding site (TFBSs) annotation, for >280 TFs, derived from analyzing the majority of ENCODE ChIP-Seq datasets. For the first time, clusters of cell types having common miRNA TSSs are characterized and provided through a user friendly interface with multiple layers of customization. DIANA-miRGen v4 significantly improves our understanding of miRNA biogenesis regulation at the transcriptional level by providing a unique integration of high-quality annotations for hundreds of cell specific miRNA promoters with experimentally derived TFBSs.

Full-length annotation with multistrategy RNA-seq uncovers transcriptional regulation of lncRNAs in cotton.

Long noncoding RNAs (lncRNAs) are crucial factors during plant development and environmental responses. To build an accurate atlas of lncRNAs in the diploid cotton Gossypium arboreum, we combined Isoform-sequencing, strand-specific RNA-seq (ssRNA-seq), and cap analysis gene expression (CAGE-seq) with PolyA-seq and compiled a pipeline named plant full-length lncRNA to integrate multi-strategy RNA-seq data. In total, 9,240 lncRNAs from 21 tissue samples were identified. 4,405 and 4,805 lncRNA transcripts were supported by CAGE-seq and PolyA-seq, respectively, among which 6.7% and 7.2% had multiple transcription start sites (TSSs) and transcription termination sites (TTSs). We revealed that alternative usage of TSS and TTS of lncRNAs occurs pervasively during plant growth. Besides, we uncovered that many lncRNAs act in cis to regulate adjacent protein-coding genes (PCGs). It was especially interesting to observe 64 cases wherein the lncRNAs were involved in the TSS alternative usage of PCGs. We identified lncRNAs that are coexpressed with ovule- and fiber development-associated PCGs, or linked to GWAS single-nucleotide polymorphisms. We mapped the genome-wide binding sites of two lncRNAs with chromatin isolation by RNA purification sequencing. We also validated the transcriptional regulatory role of lnc-Ga13g0352 via virus-induced gene suppression assay, indicating that this lncRNA might act as a dual-functional regulator that either activates or inhibits the transcription of target genes.

The Direct Interactions with Bone Marrow Microenvironment Confer Resistance to the Inhibition of Oxidative Phosphorylation in AML

Acute myeloid leukemia (AML) cells are highly dependent on oxidative phosphorylation (OxPhos) for survival and continually adapt to the bone marrow (BM) microenvironment. We investigated how the BM microenvironment impacts the response to energy-depriving OxPhos inhibition in AML using a novel complex I OxPhos inhibitor (OxPhosi), IACS-010759. We have reported that OxPhosi-resistant primary AML samples demonstrated higher baseline transcription of genes related to cell adhesion, integrin and/or Rho GTPase family genes that modulate intracellular actin dynamics. (Yang et al. ASH 2019) In this study, we performed Cap Analysis of Gene Expression (CAGE) transcriptome analyses using IACS-010759-sensitive and -resistant AML PDXs. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts (= transcription start sites) and allows investigating promoter structures necessary for gene expression. Primary AML cells from 9 AML PDXs were injected into irradiated NSG mice, which were randomized upon documented engraftment to receive IACS-010759 or vehicle (n = 3/group). The antileukemia efficacy of the treatment was monitored by serial measurements of circulating AML cells. Of the 9 models tested, we defined 4 PDXs as sensitive and 5 as resistant to OxPhos inhibitor therapy. In the resistant models, CAGE analysis of OxPhosi-induced changes (comparing pretreatment with posttreatment) identified upregulation of 77 promoters and downregulation of 207 promoters (log 2-fold change > 3.0, FDR < 0.05, EdgeR), including increased promoter expression (>3.0 fold) of genes associated with adhesion (CCR8,ADGRB2, LAG3, BMF, ATN1, PLXDC1), migration (CCR8, NKX3-2, TMEM123, IGLV7-43, FAM171A1, LBX2, TRAV21, PPP2R5C, BMF, PLXDC1), and actin cytoskeleton dynamics (FAM171A1, BMF, BEST1, PLXDC1). Of note, the 6 adhesion-associated promoters that were upregulated by OxPhosi in 5 of the OxPhosi-resistant mouse models were unchanged or downregulated in the 4 OxPhosi-sensitive models. We then used DEGseq, an R package for identifying differentially expressed genes, to identify promoters whose expression was different between OxPhosi-treated and vehicle-treated groups in the OxPhosi-resistant mouse models. DEGseq detected consistent changes of 214 upregulated and 626 downregulated promoters with OxPhosi treatment in all 5 mouse models. KEGG pathway enrichment analysis was performed with these consistently changed genes and revealed that OxPhos inhibitor treatment significantly upregulated the transcripts of cell adhesion pathway. We then confirmed that BM derived mesenchymal stem cells (MSC) protected OxPhosi-sensitive OCI-AML3 cells; the IC50 of IACS-010759 under MSC coculture was 80-fold higher than in monoculture conditions (IC50; 0.04 nM in monoculture vs. 3.25 nM in coculture), and IACS-010759 (10nM) induced 55% reduction of viable cells in coculture condition as compared to 70% reduction in monoculture. We further observed that OCI-AML3 cells adhered to MSCs were more profoundly protected from OxPhosi induced apoptosis than nonadherent cells. These results indicate that BM stromal cells, in particular those in direct contact with leukemia cells, play a key role in the microenvironment-mediated protection of AML cells from metabolic stress caused by OxPhos blockade. We further observed promoter upregulation of ASS1, coding Argininosuccinate Synthase 1 and of LRP1, coding LDL Receptor Related Protein 1. Argininosuccinate Synthase 1 is an epigenetically regulated key enzyme in the biosynthesis of arginine and energy starvation that induces adaptive transcriptional upregulation of ASS1. LDL Receptor Related Protein 1 plays a major role in lipid metabolism and has been reported to be responsible for hemin-induced autophagy in leukemia cells. These might contribute to intrinsic AML resistance to OxPhosi via activation of compensatory metabolic pathways, amino acid metabolism and lipid metabolism. Taken together, our data highlight the importance of direct interaction with BM stromal cells as well as complementally modification of amino acid- and lipid metabolism for the resistance of AML cells to OxPhos inhibition. While the mechanisms of stroma-leukemia interactions are likely complex, reducing the adhesion of AML cells to nurturing stromal cells ameliorates the resistance to the metabolic and energetic consequences of OxPhos inhibition. Disclosures Andreeff: Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding. Konopleva:Rafael Pharmaceutical: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Sanofi: Research Funding; AstraZeneca: Research Funding; Cellectis: Research Funding; AbbVie: Consultancy, Research Funding; Ablynx: Research Funding; Agios: Research Funding; Ascentage: Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Kisoji: Consultancy; Calithera: Research Funding.

Global Analysis of Transcription Start Sites in the New Ovine Reference Genome (Oar rambouillet v1.0).

The overall aim of the Ovine FAANG project is to provide a comprehensive annotation of the new highly contiguous sheep reference genome sequence (Oar rambouillet v1.0). Mapping of transcription start sites (TSS) is a key first step in understanding transcript regulation and diversity. Using 56 tissue samples collected from the reference ewe Benz2616, we have performed a global analysis of TSS and TSS-Enhancer clusters using Cap Analysis Gene Expression (CAGE) sequencing. CAGE measures RNA expression by 5′ cap-trapping and has been specifically designed to allow the characterization of TSS within promoters to single-nucleotide resolution. We have adapted an analysis pipeline that uses TagDust2 for clean-up and trimming, Bowtie2 for mapping, CAGEfightR for clustering, and the Integrative Genomics Viewer (IGV) for visualization. Mapping of CAGE tags indicated that the expression levels of CAGE tag clusters varied across tissues. Expression profiles across tissues were validated using corresponding polyA+ mRNA-Seq data from the same samples. After removal of CAGE tags with <10 read counts, 39.3% of TSS overlapped with 5′ ends of 31,113 transcripts that had been previously annotated by NCBI (out of a total of 56,308 from the NCBI annotation). For 25,195 of the transcripts, previously annotated by NCBI, no TSS meeting stringent criteria were identified. A further 14.7% of TSS mapped to within 50 bp of annotated promoter regions. Intersecting these predicted TSS regions with annotated promoter regions (±50 bp) revealed 46% of the predicted TSS were “novel” and previously un-annotated. Using whole-genome bisulfite sequencing data from the same tissues, we were able to determine that a proportion of these “novel” TSS were hypo-methylated (32.2%) indicating that they are likely to be reproducible rather than “noise”. This global analysis of TSS in sheep will significantly enhance the annotation of gene models in the new ovine reference assembly. Our analyses provide one of the highest resolution annotations of transcript regulation and diversity in a livestock species to date.

Use of Cap Analysis Gene Expression to detect human papillomavirus promoter activity patterns at different disease stages

Transcription of human papillomavirus (HPV) genes proceeds unidirectionally from multiple promoters. Direct profiling of transcription start sites (TSSs) by Cap Analysis Gene Expression (CAGE) is a powerful strategy for examining individual HPV promoter activity. The objective of this study was to evaluate alterations of viral promoter activity during infection using CAGE technology. We used CAGE-based sequencing of 46 primary cervical samples, and quantitatively evaluated TSS patterns in the HPV transcriptome at a single-nucleotide resolution. TSS patterns were classified into two types: early promoter-dominant type (Type A) and late promoter-dominant type (Type B). The Type B pattern was more frequently found in CIN1 and CIN2 lesions than in CIN3 and cancer samples. We detected transcriptomes from multiple HPV types in five samples. Interestingly, in each sample, the TSS patterns of both HPV types were the same. The viral gene expression pattern was determined by the differentiation status of the epithelial cells, regardless of HPV type. We performed unbiased analyses of TSSs across the HPV genome in clinical samples. Visualising TSS pattern dynamics, including TSS shifts, provides new insights into how HPV infection status relates to disease state.

Expression Patterns of Claudin Family Members During Tooth Development and the Role of Claudin-10 (Cldn10) in Cytodifferentiation of Stratum Intermedium.

There is growing evidence showing that tight junctions play an important role in developing enamel. Claudins are one of the main components of tight junctions and may have pivotal functions in modulating various cellular events, such as regulating cell differentiation and proliferation. Mutations in CLDN10 of humans are associated with HELIX syndrome and cause enamel defects. However, current knowledge regarding the expression patterns of claudins and the function of Cldn10 during tooth development remains fragmented. In this study, we aimed to analyze the expression patterns of claudin family members during tooth development and to investigate the role of Cldn10 in amelogenesis. Using cap analysis gene expression of developing mouse tooth germs compared with that of the whole body, we found that Cldn1 and Cldn10 were highly expressed in the tooth. Furthermore, single-cell RNA-sequence analysis using 7-day postnatal Krt14-RFP mouse incisors revealed Cldn1 and Cldn10 exhibited distinct expression patterns. Cldn10 has two isoforms, Cldn10a and Cldn10b, but only Cldn10b was expressed in the tooth. Immunostaining of developing tooth germs revealed claudin-10 was highly expressed in the inner enamel epithelium and stratum intermedium. We also found that overexpression of Cldn10 in the dental epithelial cell line, SF2, induced alkaline phosphatase (Alpl) expression, a marker of maturated stratum intermedium. Our findings suggest that Cldn10 may be a novel stratum intermedium marker and might play a role in cytodifferentiation of stratum intermedium.

Is the Secret of VDAC Isoforms in Their Gene Regulation? Characterization of Human VDAC Genes Expression Profile, Promoter Activity, and Transcriptional Regulators

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs’ presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon–intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1–3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.

CAGE-seq analysis of osteoblast derived from cleidocranial dysplasia human induced pluripotent stem cells.

Non-coding RNAs (ncRNAs) comprise a major portion of transcripts and serve an essential role in biological processes. Although the importance of major transcriptomes in osteogenesis has been extensively studied, the function of ncRNAs in human osteogenesis remains unclear. Previously, we developed hiPSCs from patients with cleidocranial dysplasia (CCD) caused by runt-related transcription factor 2 (RUNX2) haploinsufficiency. To gain insight into ncRNAs in osteogenesis, we surveyed differential ncRNA expression profiling and promoter differences of RUNX2 using patient-specific iPSCs and cap analysis gene expression (CAGE) technology to define the promoter landscape. Revertant iPSCs (Rev1 iPSCs) edited by CRISPR/Cas9 system to harbor mutation-corrected RUNX2 exhibited increased proximal promoter expression of RUNX2, while CCD iPSCs did not. We identified 2271 ncRNA genes with altered expression levels before and after differentiation, 31 of which showed at least 20-fold higher expression in Rev1 iPSCs. Bioinformatic analysis also categorized AC007392.3, LINC00379, RP11-122D10.1, and RP11-90J7.2 as enhancer regulatory regions, and HOXA-AS2, MIR219-2, and RP11-834C11.3 as dyadic regulatory regions of these ncRNAs. In addition, two miRNAs, termed MIR199A2 and MIR152, were found to have high enrichment of osteogenic-related terms. Upon further examination of the role of MIR152 on osteoblast differentiation, we found that MIR152 knockdown induced upregulation of ALP and COL1A1 in Saos-2 cells. Thus, ncRNAs were found to regulate the osteogenic differentiation potentials of hiPSCs that are used for bone regeneration and repair owing to their differentiation potentials. These data allow understanding ncRNA profiles of hiPSCs during osteogenesis.

The Transcriptional Network That Controls Growth Arrest and Macrophage Differentiation in the Human Myeloid Leukemia Cell Line THP-1.

The response of the human acute myeloid leukemia cell line THP-1 to phorbol esters has been widely studied to test candidate leukemia therapies and as a model of cell cycle arrest and monocyte-macrophage differentiation. Here we have employed Cap Analysis of Gene Expression (CAGE) to analyze a dense time course of transcriptional regulation in THP-1 cells treated with phorbol myristate acetate (PMA) over 96 h. PMA treatment greatly reduced the numbers of cells entering S phase and also blocked cells exiting G2/M. The PMA-treated cells became adherent and expression of mature macrophage-specific genes increased progressively over the duration of the time course. Within 1–2 h PMA induced known targets of tumor protein p53 (TP53), notably CDKN1A, followed by gradual down-regulation of cell-cycle associated genes. Also within the first 2 h, PMA induced immediate early genes including transcription factor genes encoding proteins implicated in macrophage differentiation (EGR2, JUN, MAFB) and down-regulated genes for transcription factors involved in immature myeloid cell proliferation (MYB, IRF8, GFI1). The dense time course revealed that the response to PMA was not linear and progressive. Rather, network-based clustering of the time course data highlighted a sequential cascade of transient up- and down-regulated expression of genes encoding feedback regulators, as well as transcription factors associated with macrophage differentiation and their inferred target genes. CAGE also identified known and candidate novel enhancers expressed in THP-1 cells and many novel inducible genes that currently lack functional annotation and/or had no previously known function in macrophages. The time course is available on the ZENBU platform allowing comparison to FANTOM4 and FANTOM5 data.

Embryonic tissue differentiation is characterized by transitions in cell cycle dynamic-associated core promoter regulation.

The core-promoter, a stretch of DNA surrounding the transcription start site (TSS), is a major integration-point for regulatory-signals controlling gene-transcription. Cellular differentiation is marked by divergence in transcriptional repertoire and cell-cycling behaviour between cells of different fates. The role promoter-associated gene-regulatory-networks play in development-associated transitions in cell-cycle-dynamics is poorly understood. This study demonstrates in a vertebrate embryo, how core-promoter variations define transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. Assessment of cell proliferation across zebrafish embryo segmentation, using the FUCCI transgenic cell-cycle-phase marker, revealed a spatial and lineage-specific separation in cell-cycling behaviour. To investigate the role differential promoter usage plays in this process, cap-analysis-of-gene-expression (CAGE) was performed on cells segregated by cycling dynamics. This analysis revealed a dramatic increase in tissue-specific gene expression, concurrent with slowed cycling behaviour. We revealed a distinct sharpening in TSS utilization in genes upregulated in slowly cycling, differentiating tissues, associated with enhanced utilization of the TATA-box, in addition to Sp1 binding-sites. In contrast, genes upregulated in rapidly cycling cells carry broad distribution of TSS utilization, coupled with enrichment for the CCAAT-box. These promoter features appear to correspond to cell-cycle-dynamic rather than tissue/cell-lineage origin. Moreover, we observed genes with cell-cycle-dynamic-associated transitioning in TSS distribution and differential utilization of alternative promoters. These results demonstrate the regulatory role of core-promoters in cell-cycle-dependent transcription regulation, during embryo-development.

Functional annotation of human long noncoding RNAs via molecular phenotyping.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Epigenetic regulation of spurious transcription initiation in Arabidopsis

In plants, epigenetic regulation is critical for silencing transposons and maintaining proper gene expression. However, its impact on the genome-wide transcription initiation landscape remains elusive. By conducting a genome-wide analysis of transcription start sites (TSSs) using cap analysis of gene expression (CAGE) sequencing, we show that thousands of TSSs are exclusively activated in various epigenetic mutants of Arabidopsis thaliana and referred to as cryptic TSSs. Many have not been identified in previous studies, of which up to 65% are contributed by transposons. They possess similar genetic features to regular TSSs and their activation is strongly associated with the ectopic recruitment of RNAPII machinery. The activation of cryptic TSSs significantly alters transcription of nearby TSSs, including those of genes important for development and stress responses. Our study, therefore, sheds light on the role of epigenetic regulation in maintaining proper gene functions in plants by suppressing transcription from cryptic TSSs.

CDK1 dependent phosphorylation of hTERT contributes to cancer progression

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner.

Promoter-Level Transcriptome Identifies Stemness Associated With Relatively High Proliferation in Pancreatic Cancer Cells.

Both pancreatic intraepithelial neoplasia (PanIN), a frequent precursor of pancreatic cancer, and intraductal papillary mucinous neoplasm (IPMN), a less common precursor, undergo several phases of molecular conversions and finally develop into highly malignant solid tumors with negative effects on the quality of life. We approached this long-standing issue by examining the following PanIN/IPMN cell lines derived from mouse models of pancreatic cancer: Ptf1a-Cre; KrasG12D; p53f/+ and Ptf1a-Cre; KrasG12D; and Brg1f/f pancreatic ductal adenocarcinomas (PDAs). The mRNA from these cells was subjected to a cap analysis of gene expression (CAGE) to map the transcription starting sites and quantify the expression of promoters across the genome. Two RNA samples extracted from three individual subcutaneous tumors generated by the transplantation of PanIN or IPMN cancer cell lines were used to generate libraries and Illumina Seq, with four RNA samples in total, to depict discrete transcriptional network between IPMN and PanIN. Moreover, in IPMN cells, the transcriptome tended to be enriched for suppressive and inhibitory biological processes. In contrast, the transcriptome of PanIN cells exhibited properties of stemness. Notably, the proliferation capacity of the latter cells in culture was only minimally constrained by well-known chemotherapy drugs such as GSK690693 and gemcitabine. The various transcriptional factor network systems detected in PanIN and IPMN cells reflect the distinct molecular profiles of these cell types. Further, we hope that these findings will enhance our mechanistic understanding of the characteristic molecular alterations underlying pancreatic cancer precursors. These data may provide a promising direction for therapeutic research.