Vicinity Of Transcription Start Site Research Articles

Targeting super-enhancer activity for colorectal cancer therapy.

In addition to genetic variants and copy number alterations, epigenetic deregulation of oncogenes and tumor suppressors is a major contributor in cancer development and propagation. Regulatory elements for gene transcription regulation can be found in promoters which are located in the vicinity of transcription start sites but also at a distance, in enhancer sites, brought to interact with proximal sites when occupied by enhancer protein complexes. These sites provide most of the specific regulatory sequences recognized by transcription factors. A sub-set of enhancers characterized by a longer structure and stronger activity, called super-enhancers, are critical for the expression of specific genes, usually associated with individual cell type identity and function. Super-enhancers show deregulation in cancer, which may have profound repercussions for cancer cell survival and response to therapy. Dysfunction of super-enhancers may result from multiple mechanisms that include changes in their sequence, alterations in the topological neighborhoods where they belong, and alterations in the proteins that mediate their function, such as transcription factors and epigenetic modifiers. These can become potential targets for therapeutic interventions. Genes that are targets of super-enhancers are cell and cancer type specific and could also be of interest for therapeutic targeting. In colorectal cancer, a super-enhancer regulated and over-expressed oncogene is MYC, under the influence of the WNT/β-catenin pathway. Identification and targeting of additional oncogenes regulated by super-enhancers in colorectal cancer may pave the way for combination therapies targeting the super-enhancer machinery and signal transduction pathways that regulate the specific transcription factors operative on them.

The proteasome activator PA200 regulates expression of genes involved in cell survival upon selective mitochondrial inhibition in neuroblastoma cells.

The conserved Blm10/PA200 activators bind to the proteasome core and facilitate peptide and protein turnover. Blm10/PA200 proteins enhance proteasome peptidase activity and accelerate the degradation of unstructured proteasome substrates. Our knowledge about the exact role of PA200 in diseased cells, however, is still limited. Here, we show that stable knockdown of PA200 leads to a significantly elevated number of cells in S phase after treatment with the ATP synthase inhibitor, oligomycin. However, following exposure to the complex I inhibitor rotenone, more PA200‐depleted cells were in sub‐G1 and G2/M phases indicative of apoptosis. Chromatin immunoprecipitation (ChIP) and ChIP‐seq data analysis of collected reads indicate PA200‐enriched regions in the genome of SH‐SY5Y. We found that PA200 protein peaks were in the vicinity of transcription start sites. Gene ontology annotation revealed that genes whose promoters were enriched upon anti‐PA200 ChIP contribute to the regulation of crucial intracellular processes, including proliferation, protein modifications and metabolism. Selective mitochondrial inhibitors induced PA200 redistribution in the genome, leading to protein withdrawal from some gene promoters and binding to others. Collectively, the results support a model in which PA200 potentially regulates cellular homeostasis at the transcriptional level, in addition to its described role as an alternative activator of the proteasome.

Abstract 3332: Genome-wide DNA methylation profiling of peripheral blood leukocytes identifies predictors of aggressive prostate cancer

Abstract Background: Prostate cancer (PCa) is the most commonly diagnosed cancer for men in the United States. Most of the PSA screening-detected PCa are slow-growing and not life-threatening. However, the majority of PSA-detected PCa receive aggressive treatment, which is often associated with significant morbidity. Clinical variables, such as Gleason Score (GS), PSA level, and tumor stage, are not sufficient to distinguish aggressive from indolent diseases at diagnosis. Biomarkers are urgently needed for more accurate prediction of individual tumor behavior and patient prognosis. DNA methylation has shown profound impact on cancer development and progression. In this study, we performed a genome-wide DNA methylation profiling of peripheral blood leukocytes to identify predictors of aggressive PCa. Methods: We performed leukocyte DNA methylation profiling in 140 GS=6 PCa patients and 147 GS ≥ 8 patients using the Illumina's Methylation 450K BeadChip. The differential methylated CpG position (DMP) was identified using the ChAMP analysis package implemented in R. Top differentially methylated CpG sites between less aggressive (GS=6) and more aggressive (GS≥ 8) patients were used to develop a signature to best cluster these two groups using semi-supervised recursively partitioned mixture modeling (SS-RPMM) algorithm. The white blood cell subtype composition was analyzed with RefbaseEWAS method. Ingenuity pathway analysis was used to find the enriched pathways that may determine aggressive prostate cancer. Results: Analyzing all the assayed CpG sites together, the mean methylation level appeared to be lower in GS ≥ 8 patients than in GS=6 patients. This global DNA hypomethylation was only significant at CpG sites located within gene body, shore, shelf, opensea and 3' UTR regions, but not in promoter and regions close to transcription start sites. There were many individual hypermethylated CpG sites in GS ≥ 8 patients that are mostly located in CpG islands within promoter and in the vicinity of transcription start sites. We then split the patients into a training (70 GS=6 and 73 GS ≥ 8 patients) and testing set (70 GS=6 and 74 GS ≥ 8 patients) to identify and validate a CpG methylation signature that best differentiates GS=6 and GS≥ 8 patients. The signature can identify a subgroup of aggressive PCa patients with higher risks of biochemical recurrence (HR=2.57, 95% Cl, 0.99-6.68). We also analyzed the white blood cell subtypes in patients and found that natural killer (NK) cell proportion is significantly decreased in patients who experienced biochemical recurrence (P=0.045), indicating weakened immune response. Conclusion: These data suggest that DNA methylation in peripheral blood leukocytes may serve as valuable biomarkers to supplement clinical variables to predict aggressive PCa at diagnosis and worse prognosis of PCa patients. Citation Format: Yuyan Han, Jia Li, Mutian Zhang, Jeri Kim, Xifeng Wu, Deqiang Sun, Jian Gu. Genome-wide DNA methylation profiling of peripheral blood leukocytes identifies predictors of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3332.

Endurance training remodels sperm-borne small RNA expression and methylation at neurological gene hotspots

Remodeling of the sperm epigenome by lifestyle factors before conception could account for altered metabolism in the next generation offspring. Here, we hypothesized that endurance training changes the epigenome of human spermatozoa. Using small RNA (sRNA) sequencing and reduced representation bisulfite sequencing (RRBS), we, respectively, investigated sRNA expression and DNA methylation in pure fractions of motile spermatozoa collected from young healthy individuals before, after 6 weeks of endurance training and after 3 months without exercise. Expression of 8 PIWI interacting RNA were changed by exercise training. RRBS analysis revealed 330 differentially methylated regions (DMRs) after training and 303 DMRs after the detraining period, which were, in both conditions, enriched at close vicinity of transcription start sites. Ontology analysis of genes located at proximity of DMRs returned terms related to neurological function at the trained state and, to a much lesser extent, at the detrained state. Our study reveal that short-term endurance training induces marked remodeling of the sperm epigenome, and identify genes related to the development of the central nervous system as potential hot spots for epigenetic variation upon environmental stress.

Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection.

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were “DNA methylation-sensitive” genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A. The other half were “DNA methylation-resistant” genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

Genome-Wide Targets Regulated by the OsMADS1 Transcription Factor Reveals Its DNA Recognition Properties.

OsMADS1 controls rice (Oryza sativa) floral fate and organ development. Yet, its genome-wide targets and the mechanisms underlying its role as a transcription regulator controlling developmental gene expression are unknown. We identify 3112 gene-associated OsMADS1-bound sites in the floret genome. These occur in the vicinity of transcription start sites, within gene bodies, and in intergenic regions. Majority of the bound DNA contained CArG motif variants or, in several cases, only A-tracts. Sequences flanking the binding peak had a higher AT nucleotide content, implying that broader DNA structural features may define in planta binding. Sequences for binding by other transcription factor families like MYC, AP2/ERF, bZIP, etc. are enriched in OsMADS1-bound DNAs. Target genes implicated in transcription, chromatin remodeling, cellular processes, and hormone metabolism were enriched. Combining expression data from OsMADS1 knockdown florets with these DNA binding data, a snapshot of a gene regulatory network was deduced where targets, such as AP2/ERF and bHLH transcription factors and chromatin remodelers form nodes. We show that the expression status of these nodal factors can be altered by inducing the OsMADS1-GR fusion protein and present a model for a regulatory cascade where the direct targets of OsMADS1, OsbHLH108/SPT, OsERF034, and OsHSF24, in turn control genes such as OsMADS32 and OsYABBY5 This cascade, with other similar relationships, cumulatively contributes to floral organ development. Overall, OsMADS1 binds to several regulatory genes and, probably in combination with other factors, controls a gene regulatory network that ensures rice floret development.

Comparative Analysis of MicroRNA Promoters in Arabidopsis and Rice

Endogenously-encoded microRNAs (miRNAs) are a class of small regulatory RNAs that modulate gene expression at the post-transcriptional level. In plants, miRNAs have increasingly been identified by experiments based on next-generation sequencing (NGS). However, promoter organization is currently unknown for most plant miRNAs, which are transcribed by RNA polymerase II. This deficiency prevents a comprehensive understanding of miRNA-mediated gene networks. In this study, by analyzing full-length cDNA sequences related to miRNAs, we mapped transcription start sites (TSSs) for 62 and 55 miRNAs in Arabidopsis and rice, respectively. The average free energy (AFE) profiles in the vicinity of TSSs were studied for both species. By employing position weight matrices (PWM) for 99 plant cis-elements, we discovered that three cis-elements were over-represented in the miRNA promoters of both species, while four and ten cis-elements were over-represented in Arabidopsis only and in rice only. Thus, comparison of miRNA promoters between Arabidopsis and rice provides a new perspective for studying miRNA regulation in plants.

Nonspecific Protein-DNA Binding Is Widespread in the Yeast Genome

Recent genome-wide measurements of binding preferences of ∼200 transcription regulators in the vicinity of transcription start sites in yeast, have provided a unique insight into the cis-regulatory code of a eukaryotic genome. Here, we show that nonspecific transcription factor (TF)-DNA binding significantly influences binding preferences of the majority of transcription regulators in promoter regions of the yeast genome. We show that promoters of SAGA-dominated and TFIID-dominated genes can be statistically distinguished based on the landscape of nonspecific protein-DNA binding free energy. In particular, we predict that promoters of SAGA-dominated genes possess wider regions of reduced free energy compared to promoters of TFIID-dominated genes. We also show that specific and nonspecific TF-DNA binding are functionally linked and cooperatively influence gene expression in yeast. Our results suggest that nonspecific TF-DNA binding is intrinsically encoded into the yeast genome, and it may play a more important role in transcriptional regulation than previously thought.

Genome-wide analysis of epigenetic changes in mouse sperm by maternal exposure to endocrine disruptor vinclozolin

The genome-wide epigenetic alterations (DNA methylation) in germ cells have not been extensively studied in mice by exposure to environmental hazards. We first report here that a genome-wide analysis of CpG methylation in F1 mice sperm by maternal exposure to the anti-androgenic compound vinclozolin (VCZ). The gestated (GD) female mice (n=8) were injected intraperitoneally with VCZ (100 mg/kg/day), from GD 8 to GD 15 to induce reproductive toxicity and epigenetic alterations in mice. Our observations showed a loss of normal spermatogenesis and abnormal seminiferous tubule morphology in VCZ-treated F1 males. We also confirmed reduced fertility of the VCZ-exposed F1 male mice and increased abnormality in F2 fetuses. We compared the methylation patterns of five untreated with 13 VCZ-treated F1 mice male via high-resolution CpG microarray analysis to determine CpG methylation alterations in F1 male sperm DNA. The overall methylation between control and treated F1 sperm showed normal distribution, and differentially methylated CpG islands were frequently mapped between the −0.5 kb and +1.0 kb regions of the transcription start site (TSS). We identified more than several hundred epigenetically altered genes by statistical analysis (P<0.05). More than 50% of the statistically significantly methylated loci were also mapped at the vicinity of TSS. We further examined the effects of VCZ on the mouse repeated elements and paternally imprinted genes. VCZ induced no epigenetic alterations in repeated mouse elements (LINE and B1) and the paternally imprinted Dlk1 gene, whereas it did affect the paternally imprinted H19 gene. In conclusion, we successfully explored VCZ-induced genome-wide epigenetic changes on F1 mice sperm DNA by global methylation profiling, and our data will provide important clues for studying the epigenetic changes of germ cells due to exposure to environmental hazards.

Promoter Analysis with Wavelets and Support Vector Machines

Promoters are key control regions for the transcription regulation of genes, usually lying upstream of the genes they control. Promoter prediction is worthwhile not only for the detection of orphan genes but also for understanding the mechanisms that regulate gene expression. Promoter prediction therefore remains one of the primary challenges subjects in bioinformatics in the post-genome era. Many methods are used for promoter prediction, such as the presence of the CpG islands, sequence motifs of transcription factor binding sites, and the statistical and chemo- physical properties in the vicinity of transcription start sites. Among these strategies, we have focused on a method which employs wavelet analysis and support vector machine for promoter prediction. The wavelet analysis is based on localized wave packets characterized by both a range of frequency and a location. In our scheme, information from promoter and non-promoter regions is converted to wavelet space as a positive and a negative set, respectively, and the 2 sets are subsequently used to train a support vector machine. Finally, the support vector machine is utilized for promoter prediction. In this study, we improved the coding method of our prediction strategy and analysed a new set of test data.

Genome-wide in Silico Identification of New Conserved and Functional Retinoic Acid Receptor Response Elements (Direct Repeats Separated by 5 bp)

The nuclear retinoic acid receptors interact with specific retinoic acid (RA) response elements (RAREs) located in the promoters of target genes to orchestrate transcriptional networks involved in cell growth and differentiation. Here we describe a genome-wide in silico analysis of consensus DR5 RAREs based on the recurrent RGKTSA motifs. More than 15,000 DR5 RAREs were identified and analyzed for their localization and conservation in vertebrates. We selected 138 elements located ±10 kb from transcription start sites and gene ends and conserved across more than 6 species. We also validated the functionality of these RAREs by analyzing their ability to bind retinoic acid receptors (ChIP sequencing experiments) as well as the RA regulation of the corresponding genes (RNA sequencing and quantitative real time PCR experiments). Such a strategy provided a global set of high confidence RAREs expanding the known experimentally validated RAREs repertoire associated to a series of new genes involved in cell signaling, development, and tumor suppression. Finally, the present work provides a valuable knowledge base for the analysis of a wider range of RA-target genes in different species.

The long reach of noncoding RNAs

Transcription of genomic loci containing protein-coding genes often yields not only cognate mRNAs but also assorted noncoding RNAs (ncRNAs), which typically map in the vicinity of transcription start sites. A new study shows that far from being random byproducts of gene expression, many long ncRNAs (lncRNAs) are synthesized in a coordinate fashion and control important cellular processes, such as survival in the face of DNA damage.

Unfolding of Non-Telomeric G-Quadruplexes of Varying Stabilities by Replication Protein A

We studied the stability of various G-quadruplex (GQ) structures formed by sub-sections of a highly heterogeneous and guanine rich 45 nucleotide long DNA sequence, called TH-12 and located in the promoter region of tyrosine hydroxylase (TH) gene, using single-molecule FRET and conventional biochemistry methods. TH-12 has seven regions that contain two or more consecutive guanines capable of forming various GQs of potentially different stabilities. TH-12 is within a conserved region of TH promoter, is in the immediate vicinity of transcription start site, and is invariably included for maintaining the promoter activity. This work serves as the first step in identifying possible roles that these GQs, with potentially different stabilities, play in regulating TH expression. Abnormality in TH gene expression is associated with a variety of psychiatric problems, including schizophrenia, depression and bipolar disorder and is also linked to Parkinson's disease. We measured the UV-melting temperatures of these GQ structures formed by sub-sections of TH12 and established that some of these GQs are stable against Watson-Crick base pairing, in the presence of a complementary strand, at physiological potassium concentrations. Finally, we studied the viability of these structures against the DNA binding activity of human Replication Protein A (RPA), the most abundant single strand DNA binding protein in eukaryotes. Most GQs were unfolded at RPA concentrations that correlated with their melting temperatures, while a GQ formed by a certain sub-section of TH-12 remained folded even at micromolar RPA concentrations. We propose that this subsection of the DNA sequence is the most likely candidate for forming a physiologically relevant GQ that can be involved in TH regulation. These studies also demonstrate that it is possible to have stable enough GQs that can not be unfolded by RPA activity at physiological ionic strength.

Analysis of nucleosome positioning in promoters of miRNA genes and protein-coding genes

Nucleosome positioning in promoters is important for gene transcription regulation. In this paper, with a nucleosome prediction model, curvature profile, the characteristics of nucleosome positioning in promoters are analyzed for miRNA genes and protein-coding genes. In the vicinity of transcription start site (TSS), there is a nucleosome-free region (NFR) followed by a positioned nucleosome at ∼200 bp downstream of TSS. A similar characteristic is observed in independent intronic promoters and intergenic promoters, namely, both types of promoters have a longer NFR in 0–−400 bp upstream of TSS. Moreover, transcription factor binding sites (TFBSs) locate in the NFR with a high concentration. However, nucleosome pattern in dependent intronic promoters are like that in protein-coding promoters, with two nucleosomes positioned at −200–−400 bp and −400–−600 bp upstream of TSS. The results indicate nucleosome positioning is probably different in independent miRNA promoters and protein-coding promoters; and positioning seems to be an important factor not only in regulation of protein-coding gene, but also in that of miRNA gene.

Nucleosome positioning—what do we really know?

The positioning of nucleosomes on the DNA of eukaryotic genomes is a major determinant of gene expression. In particular nucleosomes in close proximity to regulatory regions are often more precisely positioned in vivo than nucleosomes located elsewhere. In this article we compare data obtained from the most recent studies by a variety of techniques. We argue that the disparate conclusions in the literature could be a consequence of procedural differences sampling alternative arrays of nucleosomes on the same DNA sequence. Importantly, the ostensibly least invasive techniques identify differences between nucleosomes in the vicinity of transcription start sites in budding yeast and those positioned distally within the transcribed region.

V1R promoters are well conserved and exhibit common putative regulatory motifs

BackgroundThe mouse vomeronasal organ (VNO) processes chemosensory information, including pheromone signals that influence reproductive behaviors. The sensory neurons of the VNO express two types of chemosensory receptors, V1R and V2R. There are ~165 V1R genes in the mouse genome that have been classified into ~12 divergent subfamilies. Each sensory neuron of the apical compartment of the VNO transcribes only one of the repertoire of V1R genes. A model for mutually exclusive V1R transcription in these cells has been proposed in which each V1R gene might compete stochastically for a single transcriptional complex. This model predicts that the large repertoire of divergent V1R genes in the mouse genome contains common regulatory elements. In this study, we have characterized V1R promoter regions by comparative genomics and by mapping transcription start sites.ResultsWe find that transcription is initiated from ~1 kb promoter regions that are well conserved within V1R subfamilies. While cross-subfamily homology is not evident by traditional methods, we developed a heuristic motif-searching tool, LogoAlign, and applied this tool to identify motifs shared within the promoters of all V1R genes. Our motif-searching tool exhibits rapid convergence to a relatively small number of non-redundant solutions (97% convergence). We also find that the best motifs contain significantly more information than those identified in controls, and that these motifs are more likely to be found in the immediate vicinity of transcription start sites than elsewhere in gene blocks. The best motifs occur near transcription start sites of ~90% of all V1R genes and across all of the divergent subfamilies. Therefore, these motifs are candidate binding sites for transcription factors involved in V1R co-regulation.ConclusionOur analyses show that V1R subfamilies have broad and well conserved promoter regions from which transcription is initiated. Results from a new motif-finding algorithm, LogoAlign, designed for this context and more generally for searching large, hierarchical datasets, suggest the existence of common information-rich regulatory motifs that are shared across otherwise divergent V1R subfamilies.

Histone H3 acetylated at lysine 9 in promoter is associated with low nucleosome density in the vicinity of transcription start site in human cell

Nucleosome depletion in the promoters has been indicated in yeasts, suggesting that nucleosome depletion in promoter might be a fundamental feature of eukaryotic transcriptional regulation. We compared the relationship between histone H3 acetylation at lysine 9 (K9) in promoter, gene expression level, and nucleosome density in the vicinity of the transcription start site (TSS), in HepG2 cells (human hepatocellular liver carcinoma cells). We found that the density of nucleosome is relatively low in the close vicinity of TSS flanked by H3 K9 significantly acetylated promoter, compared with that for genes without marked H3 K9 acetylation in promoter, regardless of their transcriptional activation status. Our results imply that the relative nucleosome depletion in the vicinity of TSS is not necessarily associated with active transcription, but with histone H3 K9 acetylation in promoter.

Characterization of transcription initiation, translation initiation, and poly(A) addition sites in the gene-sized macronuclear DNA molecules of Euplotes.

The DNA in the transcriptionally active macronucleus of the hypotrichous ciliate Euplotes crassus exists as short, linear molecules with each molecule encoding a single genetic function. Previous work has indicated that coding regions occupy the majority of macronuclear DNA molecules. In the present study we have defined the transcription initiation sites and poly(A) addition sites for a number of different macronuclear genes in Euplotes crassus. Our results indicate that mature transcripts represent all but approximately 100-200 bases of the non-telomeric sequences in macronuclear DNA molecules. We have also examined the sequences in the vicinity of transcription start sites, poly(A) addition sites, and translation initiation sites for Euplotes species genes in an attempt to define the cis-acting elements that control these processes. Our results indicate that some of the common sequence elements known to control these processes in higher eukaryotes are likely not utilized by Euplotes genes. The data do indicate the presence of other conserved sequences both preceding and at the site of poly(A) addition, as well as at the site of translation initiation. These conserved sequences may serve an analogous role in these organisms. Finally, we have found that most macronuclear DNA molecules have transcription initiation sites within 30 bp of the telomere, suggesting that the telomere may play a role in promoting transcription.