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The development of multicellular organisms and the uniqueness of each cell are achieved by distinct transcriptional programs. Multiple processes that regulate gene expression converge at the core promoter region, an 80 bp region that directs accurate transcription initiation by RNA polymerase II (Pol II). In recent years, it has become apparent that the core promoter region is not a passive DNA component, but rather an active regulatory module of transcriptional programs. Distinct core promoter compositions were demonstrated to result in different transcriptional outputs. In this mini-review, we focus on the role of the core promoter, particularly its downstream region, as the regulatory hub for developmental genes. The downstream core promoter element (DPE) was implicated in the control of evolutionarily conserved developmental gene regulatory networks (GRNs) governing body plan in both the anterior-posterior and dorsal-ventral axes. Notably, the composition of the basal transcription machinery is not universal, but rather promoter-dependent, highlighting the importance of specialized transcription complexes and their core promoter target sequences as key hubs that drive embryonic development, differentiation and morphogenesis across metazoan species. The extent of transcriptional activation by a specific enhancer is dependent on its compatibility with the relevant core promoter. The core promoter content also regulates transcription burst size. Overall, while for many years it was thought that the specificity of gene expression is primarily determined by enhancers, it is now clear that the core promoter region comprises an important regulatory module in the intricate networks of developmental gene expression.

Contents Egg production is an important economic trait in poultry, and it is of great significance to study the key genes and functional SNPs that affect egg laying performance. Follicle‐stimulating hormone (FSH) plays an important physiological role in the reproductive performance of humans and animals by binding to its receptor (FSHR). Studies have shown that there are many transcriptional regulatory elements in the 5′ flanking region of the FSHR gene that interact with transcription factors to regulate FSHR transcription. In this study, DNA sequencing was used to identify SNPs in the FSHR promoter sequence in both Dongxiang and Suken chickens. To detect the activity of the chicken FSHR gene promoter, we analysed the characteristics of the sequence and constructed three deletion vectors. We confirmed that the region (−18/−544) was the core promoter. Furthermore, five polymorphisms, including a 200‐bp indel at −869, C−1684T, C−1608T, G−368A and T−238A, were detected in both the Dongxiang and Suken chickens. The age at first egg (AFE) for different genotype of −869 indel in Suken chicken was significantly different (p

BACKGROUND & AIMS: Our understanding about the interaction of the Hepatitis B virus (HBV) with its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict Hepatitis C Virus. We aim to dissect its role in HBV infection that remains a big threat to global health. METHODS: SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV infected cell cultures and in vivo in HBV replicating mice evaluating HBV replication markers. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) to investigate protein-DNA interactions. RESULTS: In HBV infected cell cultures, downregulation of SART1 did not affect HBV cccDNA but resulted in markedly enhancing HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV infected mice persistently replicating HBV. Inhibition of Janus kinases showed no effect SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with HNF4α proximal P1 promoter element. CONCLUSIONS: We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression that is essential for transcription of HBV cccDNA. LAY SUMMARY: Hepatitis B virus (HBV) infects hepatocytes and establishes a covalently closed circular DNA (cccDNA), which remains a major obstacle for antiviral treatments. Spliceosome associated factor 1 (SART1) has been described to inhibit hepatitis C virus infection through direct regulation of interferon stimulated genes. In this study, by using various HBV models, we demonstrate that SART1 restrains HBV cccDNA transcription through suppression of HBV key transcription factor HNF4α.

Background Core promoters have a substantial influence on various steps of transcription, including initiation, elongation, termination, polyadenylation, and finally, translation. The characterization of core promoters is crucial for exploring the regulatory code of transcription initiation. However, the current understanding of insect core promoters is focused on those of Diptera (especially Drosophila) species with small genome sizes. Results Here, we present an analysis of the transcription start sites (TSSs) in the migratory locust, Locusta migratoria, which has a genome size of 6.5 Gb. The genomic differences, including lower precision of transcription initiation and fewer constraints on the distance from transcription factor binding sites or regulatory elements to TSSs, were revealed in locusts compared with Drosophila insects. Furthermore, we found a distinct bimodal log distribution of the distances from the start codons to the core promoters of locust genes. We found stricter constraints on the exon length of mRNA leaders and widespread expression activity of the distant core promoters in locusts compared with fruit flies. We further compared core promoters in seven arthropod species across a broad range of genome sizes to reinforce our results on the emergence of distant core promoters in large-sized genomes. Conclusions In summary, our results provide novel insights into the effects of genome size expansion on distant transcription initiation.

Background Core promoter controls transcription initiation. However, little is known for core promoter diversity in the human genome and its relationship with diseases. We hypothesized that as a functional important component in the genome, the core promoter in the human genome could be under evolutionary selection, as reflected by its highly diversification in order to adjust gene expression for better adaptation to the different environment. Results Applying the “Exome-based Variant Detection in Core-promoters” method, we analyzed human core-promoter diversity by using the 2682 exome data sets of 25 worldwide human populations sequenced by the 1000 Genome Project. Collectively, we identified 31,996 variants in the core promoter region (− 100 to + 100) of 12,509 human genes (https://dbhcpd.fhs.um.edu.mo). Analyzing the rich variation data identified highly ethnic-specific patterns of core promoter variation between different ethnic populations, the genes with highly variable core promoters, the motifs affected by the variants, and their involved functional pathways. eQTL test revealed that 12% of core promoter variants can significantly alter gene expression level. Comparison with GWAS data we located 163 variants as the GWAS identified traits associated with multiple diseases, half of these variants can alter gene expression. Conclusion Data from our study reals the highly diversified nature of core promoter in the human genome, and highlights that core promoter variation could play important roles not only in gene expression regulation but also in disease predisposition.

VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs&rsquo, presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon&ndash, intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1&ndash, 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.

Phenylalanine (Phe) is an essential aromatic amino acid synthesized by plants and the final reaction in its biosynthesis is catalyzed by AROGENATE DEHYDRATASEs (ADTs). In Arabidopsis thaliana, six isoforms (AtADT1-ADT6) are capable of performing this reaction. To analyze the expression patterns of each AtADT, in silico data were used to describe AtADT mRNA expression during development in A. thaliana and Solanum lycopersicum (tomato). Eight stable A. thaliana transgenic lines with ADT promoter-eGFP/GUS reporter constructs were also tested and analyzed under standard conditions during development. This showed that AtADTs are differentially regulated through their promoters, which results in the variation of their expression. ADT expression patterns were described and putative roles in channeling carbon to different Phe-derived metabolites were proposed for each ADT, associating expression data in plant organs with different Phe-derived metabolites. This thesis is a valuable information resource and foundation for future high-resolution studies of ADT expression.

Background While there is an ongoing trend to identify single nucleotide substitutions (SNSs) that are linked to inter/intra-species differences and disease phenotypes, short tandem repeats (STRs)/microsatellites may be of equal (if not more) importance in the above processes. Genes that contain STRs in their promoters have higher expression divergence compared to genes with fixed or no STRs in the gene promoters. In line with the above, recent reports indicate a role of repetitive sequences in the rise of young transcription start sites (TSSs) in human evolution. Results Following a comparative genomics study of all human protein-coding genes annotated in the GeneCards database, here we provide a genome-scale portrait of human-specific short- and medium-size (≥ 3-repeats) tri- and tetranucleotide STRs and STR motifs in the critical core promoter region between − 120 and + 1 to the TSS and evidence of skewing of this compartment in reference to the STRs that are not human-specific (Levene’s test p

Genome‐wide transcription studies are revealing an increasing number of “dispersed promoters” that, unlike “focused promoters”, lack well‐conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well‐defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters. Here, we have analyzed regions of transcription initiation in the eukaryotic parasite Trypanosoma brucei, in which RNA polymerase II transcription initiation occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site‐specific and genome‐wide assays, we identified GT‐rich promoters that can drive transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context‐dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with RNA pol II enrichment and gene expression, pointing to a role in RNA maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes. Our findings show that even highly dispersed, unregulated promoters contain specific DNA elements that are able to induce transcription and changes in chromatin structure.

Nuclear receptor subfamily 5 group A member 2 (NR5A2), also referred to as LRH-1 or FTF, is an orphan nuclear hormone receptor that is involved in regulating embryonic development, ovarian granulosa cell differentiation, gonadal sex differentiation, and steroidogenesis in mammals. However, little is known about how NR5A2 regulates reproduction in sheep. In this study, we amplified the promoter sequence of NR5A2 and determined that its core promoter region ranged from -721 nt to -281 nt. A T > G polymorphism at -700 nt was detected in the core promoter region. Association analysis found that the litter sizes of Hu ewes at their second and average parities with genotype GG (2.20 ± 0.20 and 1.97 ± 0.06, respectively) were significantly higher than those of ewes with genotype TG (1.68 ± 0.10 and 1.74 ± 0.05, respectively) (p < 0.05) and TT (1.67 ± 0.10 and 1.62 ± 0.06, respectively) (p < 0.05). The litter size of Hu ewes at their third parity with genotype GG (2.10 ± 0.10) was significantly higher than that of ewes with genotype TT (1.56 ± 0.12) (p < 0.05). A luciferase assay showed that the -700G allele increased the luciferase activity relative to the -700T allele. Furthermore, the -700T > G polymorphism created a novel binding site for metal-regulatory transcription factor 1 (MTF-1). A competitive electrophoretic mobility shift assay confirmed that MTF-1 specifically bound with the G-type promoter of NR5A2. An overexpression experiment demonstrated that MTF-1 was involved in the alteration of NR5A2 transcription activity and further increased NR5A2 gene mRNA expression. Our findings revealed that the -700T > G polymorphism promoted NR5A2 expression due to the positive effects on NR5A2 gene transcription activity by MTF-1 and thereby increased fecundity in Hu sheep.

Background In mammals, fine-tuned regulation of gene expression leads to transcription initiation from diverse transcription start sites (TSSs) and multiple core promoters. Although polysome association is a critical step in translation, whether polysome selectively uses TSSs and core promoters and how this could impact translation remains elusive. Results In this study, we used CAGE followed by deep sequencing to globally profile the transcript 5′ isoforms in the translatome and transcriptome of human HEK293 cells at single-nucleotide resolution. By comparing the two profiles, we identified the 5′ isoforms preferentially used in translatome and revealed a widespread selective usage of TSSs (32.0%) and core promoters (48.7%) by polysome. We discovered the transcription initiation patterns and the sequence characteristics that were highly correlated with polysome selection. We further identified 5804 genes significantly enriched or depleted in translatome and showed that polysome selection was an important contributing factor to the abundance of related gene products. Moreover, after comparison with public transcriptome CAGE data from 180 human tissues and primary cells, we raised a question on whether it is a widely adopted mechanism to regulate translation efficiency by changing the transcription initiation sites on the transcription level in cells of different conditions. Conclusions Using HEK293 cells as a model, we delineated an indirect selection toward TSSs and core promoters by the translation machinery. Our findings lend additional evidence for a much closer coordination between transcription and translation, warranting future translatome studies in more cell types and conditions to develop a more intricate regulatory model for gene expression. Electronic supplementary material The online version of this article (10.1186/s12864-019-5650-0) contains supplementary material, which is available to authorized users.

Animal promoters initiate transcription either at precise positions (narrow promoters) or dispersed regions (broad promoters), a distinction referred to as promoter shape. Although highly conserved, the functional properties of promoters with different shapes and the genetic basis of their evolution remain unclear. Here we used natural genetic variation across a panel of 81 Drosophila lines to measure changes in transcriptional start site (TSS) usage, identifying thousands of genetic variants affecting transcript levels (strength) or the distribution of TSSs within a promoter (shape). Our results identify promoter shape as a molecular trait that can evolve independently of promoter strength. Broad promoters typically harbor shape-associated variants, with signatures of adaptive selection. Single-cell measurements demonstrate that variants modulating promoter shape often increase expression noise, whereas heteroallelic interactions with other promoter variants alleviate these effects. These results uncover new functional properties of natural promoters and suggest the minimization of expression noise as an important factor in promoter evolution. Fil: Schor, Ignacio Esteban. European Molecular Biology Laboratory Heidelberg; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Degner, Jacob F.. European Molecular Biology Laboratory; Alemania Fil: Harnett, Dermot. European Molecular Biology Laboratory; Alemania Fil: Cannavò, Enrico. European Molecular Biology Laboratory; Alemania Fil: Casale, Francesco P.. European Bioinformatics Institute; Reino Unido Fil: Shim, Heejung. Purdue University; Estados Unidos Fil: Garfield, David A.. European Molecular Biology Laboratory; Alemania Fil: Birney, Ewan. European Bioinformatics Institute; Reino Unido Fil: Stephens, Matthew. University of Chicago; Estados Unidos Fil: Stegle, Oliver. European Bioinformatics Institute; Reino Unido Fil: Furlong, Eileen E. M.. European Molecular Biology Laboratory; Alemania

Although the importance of cis-acting mutations on detoxification enzyme genes for insecticide resistance is widely accepted, only a few of them have been determined as concrete mutations present in genomic DNA till date. The overexpression of a cytochrome P450 gene, CYP9M10, is associated with pyrethroid resistance in the southern house mosquito Culex quinquefasciatus. The haplotypes of CYP9M10 exhibiting overexpression (resistant haplotypes) belong to one specific phylogenetic lineage that shares high nucleotide sequence homology and the same insertion of a transposable element. Among the resistant haplotypes, allelic progression involving an additional cis-acting mutation and gene duplication evolved a CYP9M10 haplotype associated with extremely high transcription and strong pyrethroid resistance. Here we show that a single nucleotide substitution G−27A, which is located near the transcription start site of CYP9M10, is involved in the progression of the duplicated haplotype lineage. The deletion of a 7-bp AT-rich sequence that includes nucleotide −27 inhibited the initiation of transcription from the original transcriptional initiation site. The mutation was suspected to reside within a core promoter, TATA-box, of CYP9M10.

This study was aimed at identifying the active control area of chicken DAZL gene core promoter, to screen optimum inducers of the DAZL gene, thus to enhance the differentiation of embryonic stem cells into spermatogonial stem cells. Fragments of chicken DAZL gene promoter were cloned into fluorescent reporter plasmids and transfected into DF-1 cells. Then Dual-Luciferase® Reporter Assay System was used to identify the activity of the DAZL gene under different inducers. Our studies showed that the DAZL core promoter region for the Suqin yellow chicken was −383 to −39 bp. The dual-luciferase® reporter showed that all-trans retinoic acid (ATRA), a retinoic acid receptor alpha agonist (tamibarotene/Am80), or estradiol (E2) could significantly enhance DAZL transcription. The in vitro inductive culture of chicken ESCs demonstrated that, with ATRA treatment, DAZL transcription peaked at 6 days and then decreased slowly, whereas, DAZL transcription was continuous and peaked at 10 days with Am80 treatment. E2 treatment significantly increased DAZL expression after 8 days. All three treatments were associated with the appearance of male germ cell (MGC)-like cells on day 10. These results provide the optimum inducer screening of the DAZL gene and lay the foundation for further screening of compounds that can induce the differentiation of ESCs into MGCs in vitro.

Background Development is largely driven by transitions between transcriptional programs. The initiation of transcription at appropriate sites in the genome is a key component of this and yet few rules governing selection are known. Here, we used cap analysis of gene expression (CAGE) to generate bp-resolution maps of transcription start sites (TSSs) across the genome of Oikopleura dioica, a member of the closest living relatives to vertebrates. Results Our TSS maps revealed promoter features in common with vertebrates, as well as striking differences, and uncovered key roles for core promoter elements in the regulation of development. During spermatogenesis there is a genome-wide shift in mode of transcription initiation characterized by a novel core promoter element. This element was associated with > 70% of male-specific transcription, including the use of cryptic internal promoters within operons. In many cases this led to the exclusion of trans-splice sites, revealing a novel mechanism for regulating which mRNAs receive the spliced leader. Binding of the cell cycle regulator, E2F1, is enriched at the TSS of maternal genes in endocycling nurse nuclei. In addition, maternal promoters lack the TATA-like element found in zebrafish and have broad, rather than sharp, architectures with ordered nucleosomes. Promoters of ribosomal protein genes lack the highly conserved TCT initiator. We also report an association between DNA methylation on transcribed gene bodies and the TATA-box. Conclusions Our results reveal that distinct functional promoter classes and overlapping promoter codes are present in protochordates like in vertebrates, but show extraordinary lineage-specific innovations. Furthermore, we uncover a genome-wide, developmental stage-specific shift in the mode of TSS selection. Our results provide a rich resource for the study of promoter structure and evolution in Metazoa. Electronic supplementary material The online version of this article (10.1186/s12864-018-4504-5) contains supplementary material, which is available to authorized users.

The signals that direct the initiation of transcription ultimately converge at the core promoter, which is the gateway to transcription. Here we provide an overview of the RNA polymerase II core promoter in bilateria (bilaterally symmetric animals). The core promoter is diverse in terms of its composition and function yet is also punctilious, as it acts with strict rules and precision. We additionally describe an expanded view of the core promoter that comprises the classical DNA sequence motifs, sequence-specific DNA-binding transcription factors, chromatin signals, and DNA structure. This model may eventually lead to a more unified conceptual understanding of the core promoter.

The TCT core promoter element is present in most ribosomal protein (RP) genes in Drosophila and humans. Here we show that TBP (TATA box-binding protein)-related factor TRF2, but not TBP, is required for transcription of the TCT-dependent RP genes. In cells, TCT-dependent transcription, but not TATA-dependent transcription, increases or decreases upon overexpression or depletion of TRF2. In vitro, purified TRF2 activates TCT but not TATA promoters. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) experiments revealed the preferential localization of TRF2 at TCT versus TATA promoters. Hence, a specialized TRF2-based RNA polymerase II system functions in the synthesis of RPs and complements the RNA polymerase I and III systems.

Transmembrane protein 174 (TMEM174) is a type III transmembrane protein with no clear signal peptide. The N and C terminals are located inside the cell. Our previous study demonstrated high expression of TMEM174 in the kidney and its potential involvement in renal cancer based on its capacity to stimulate cell proliferation. However, the mechanism by which TMEM174 promotes proliferation at the transcriptional level remains to be elucidated. In the present study, the TMEM174 promoter region was amplified from whole blood DNA. Six different regions of the regulatory sequences of the TMEM174 promoter region including ~2.5 kb of the upstream region were cloned into the dual luciferase expression vector pGL3-basic. Comparison of the activity of these fragments in dual luciferase reporter assays revealed higher levels of activity for the fragments spanning -186 to +674, -700 to +674, -1,000 to +674 and -2,500 to +1 bp. Lower levels of activity were detected for the fragments spanning -466 to +674 and -890 to +674 bp. The highest activity was detected for the fragment spanning -186 to +674 bp. Electrophoretic mobility shift assay (EMSA) was performed to determine effective transcription factor binding sites. Specific binding of the cyclic-AMP response element binding (CREB) within the TMEM174 gene promoter region was demonstrated, although binding of the activator protein-1 (AP-1) was also detected in this region. In conclusion, these results suggest that the core promoter region of the human TMEM174 gene is located within the region spanning -186 to +674 bp and that the transcription factors CREB and AP-1 are involved in the transcriptional regulation of this gene.

DNA sequence signals in the core promoter, such as the initiator (Inr), direct transcription initiation by RNA polymerase II. Here we show that the human Inr has the consensus of BBCA+1BW at focused promoters in which transcription initiates at a single site or a narrow cluster of sites. The analysis of 7678 focused transcription start sites revealed 40% with a perfect match to the Inr and 16% with a single mismatch outside of the CA+1 core. TATA-like sequences are underrepresented in Inr promoters. This consensus is a key component of the DNA sequence rules that specify transcription initiation in humans.

Identification of promoter region is an important part of gene annotation. Identification of promoters in eukaryotes is important as promoters modulate various metabolic functions and cellular stress responses. In this work, a novel approach utilizing intensity values of tilling microarray data for a model eukaryotic plant Arabidopsis thaliana, was used to specify promoter region from non-promoter region. A feed-forward back propagation neural network model supported by genetic algorithm was employed to predict the class of data with a window size of 41. A dataset comprising of 2992 data vectors representing both promoter and non-promoter regions, chosen randomly from probe intensity vectors for whole genome of Arabidopsis thaliana generated through tilling microarray technique was used. The classifier model shows prediction accuracy of 69.73% and 65.36% on training and validation sets, respectively. Further, a concept of distance based class membership was used to validate reliability of classifier, which showed promising results. The study shows the usability of micro-array probe intensities to predict the promoter regions in eukaryotic genomes.

The tumor suppressor protein p53 plays a role in cellular responses to cancer-initiating events by regulating progress through the cell cycle. Several recent studies have shown that p53 transactivates expression of the members of the proapoptotic microRNA-34 family, which are underexpressed in several cancers. We demonstrate here that the latency-associated cluster of microRNAs (miRNA) encoded by an oncogenic herpesvirus, gallid herpesvirus 2 (GaHV-2), is a direct target of p53. Robust transcriptional activity was induced in three avian cell lines by a sequence mapping 600 base pairs (bp) upstream of the cluster of miRNAs. We found transcription start sites for the pri-miRNA transcript at the 3′ end of this transcription-inducing sequence. The promoter has no consensus core promoter element, but is organized into a variable number of tandem repeats of 60-bp harboring p53-responsive elements (RE). The minimal functional construct consists of two tandem repeats. Mutagenesis to change the sequence of the p53 RE abolished transcriptional activity, whereas p53 induction enhanced mature miRNA expression. The identification of a viral miRNA promoter regulated by p53 is biologically significant, because all avirulent GaHV-2 strains described to date lack the corresponding regulatory sequence, whereas all virulent, very virulent, and hypervirulent strains possess at least two tandem repeats harboring the p53 RE.

For the last couple of years, a method that permits the collection of precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high-throughput manner was established. We applied this novel method, 'tss-seq', to elucidate the transcriptome of tachyzoites of the Toxoplasma gondii, which resulted in the identification of 124,000 TSSs, and they were clustered into 10,000 transcription regions (TRs) with a statistics-based analysis. The TRs and annotated ORFs were paired, resulting in the identification of 30% of the TRs and 40% of the ORFs without their counterparts, which predicted undiscovered genes and stage-specific transcriptions, respectively. The massive data for TSSs make it possible to execute the first systematic analysis of the T. gondii core promoter structure, and the information showed that T. gondii utilized an initiator-like motif for their transcription in the major and novel motif, the downstream thymidine cluster, which was similar to the Y patch observed in plants. This encyclopaedic analysis also suggested that the TATA box, and the other well-known core promoter elements were hardly utilized.

A plasmid carrying 1.3-fold HBV genome was constructed from a HBV strain that caused five consecutive cases of fulminant hepatitis (pBFH2), and HepG2 cells were transfected with pBFH2 or its variants. The pBFH2 construct with A1762T/G1764A, G1862T, and G1896A showed the largest amount of core particle-associated intracellular HBV DNA, but no significant increase of extracellular HBV DNA in comparison with the wild construct, suggesting that these mutations might work together for retention of the replicative intermediates in the cells. The retention might relate to the localization of hepatitis B core antigen (HBcAg) in the nucleus of HepG2, which was observed by confocal fluorescence microscopy. HBcAg immunohistochemical examination of liver tissue samples obtained from the consecutive fulminant hepatitis patients showed stronger staining in the nucleus than acute hepatitis patients. In conclusion, the fulminant HBV strain caused retention of the core particles and the core particle-associated HBV DNA in the cells.

Hepatitis B virus (HBV) clone 4B replicated much more efficiently than clone 2A of the same genotype. Introduction of its T1753C, A1762T, G1764A, and C1766T core promoter mutations into the 2A genome greatly enhanced genome replication and suppressed HBeAg expression. Here we show that these effects are mediated by transcriptional up regulation of pregenomic RNA and suppression of precore RNA. Analysis of chimeric constructs suggested that the 5′ end of the 2A core gene conferred higher level of pregenomic RNA, but less core protein and genome replication relative to the 4B sequence. Genome maturity of secreted virions was reduced by mutations present in the core protein of the 2A genome but enhanced by mutations found in the 4B core protein. The 4B core protein migrated faster than that of clone 2A. The possible links among the various phenotypes and the responsible mutations remain to be established.

Core promoters are minimal regions sufficient to direct accurate initiation of transcription and are crucial for regulation of gene expression. They are highly diverse in terms of associated core promoter motifs, underlying sequence composition and patterns of transcription initiation. Distinctive features of promoters are also seen at the chromatin level, including nucleosome positioning patterns and presence of specific histone modifications. Recent advances in identifying and characterizing promoters using next-generation sequencing-based technologies have provided the basis for their classification into functional groups and have shed light on their modes of regulation, with important implications for transcriptional regulation in development. This review discusses the methodology and the results of genome-wide studies that provided insight into the diversity of RNA polymerase II promoter architectures in vertebrates and other Metazoa, and the association of these architectures with distinct modes of regulation in embryonic development and differentiation.

The core promoter (CP) of the viral genome plays an important role for hepatitis B virus (HBV) replication as it directs initiation of transcription for the synthesis of both the precore and pregenomic (pg) RNAs. The CP consists of the upper regulatory region and the basal core promoter (BCP). The CP overlaps with the 3’-end of the X open reading frames and the 5’-end of the precore region, and contains cis-acting elements that can independently direct transcription of the precore mRNA and pgRNA. Its transcription regulation is under strict control of viral and cellular factors. Even though this regulatory region exhibits high sequence conservation, when variations appear, they may contribute to the persistence of HBV within the host, leading to chronic infection and cirrhosis, and eventually, hepatocellular carcinoma. Among CP sequence variations, those occurring at BCP may dysregulate viral gene expression with emphasis in the hepatitis B e antigen, and contribute to disease progression. In this review these molecular aspects and pathologic topics of core promoter are deeply evaluated. Fil: Quarleri, Jorge Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina

The objective of this study was to examine hepatitis B virus (HBV) subgenotypes and mutations in enhancer II, basal core promoter, and precore regions of HBV in relation to risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Southeast China. A case-control study was performed, including chronic hepatitis B (CHB; n=125), LC (n=120), and HCC (n=136). HBV was genotyped by multiplex polymerase chain reaction and subgenotyped by restriction fragment length polymorphism. HBV mutations were measured by DNA sequencing. HBV genotype C (68.2%) predominated and genotype B (30.2%) was the second most common. Of these, C2 (67.5%) was the most prevalent subgenotype, and B2 (30.2%) ranked second. Thirteen mutations with a frequency >5% were detected. Seven mutation patterns (C1653T, G1719T, G1730C, T1753C, A1762T, G1764A, and G1799C) were associated with C2, and four patterns (C1810T, A1846T, G1862T, and G1896A) were associated with B2. Six patterns (C1653T, G1730C, T1753C, A1762T, G1764A, and G1799C) were obviously associated with LC, and 10 patterns (C1653T, G1730C, T1753C, A1762T, G1764A, G1799C, C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with CHB. Four patterns (C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with LC. Multivariate regression analyses showed that HBV subgenotype C2 and C2-associated mutation patterns (C1653T, T1753C, A1762T, and G1764A) were independent risk factors for LC when CHB was the control, and that B2-associated mutation patterns (C1810T, A1846T, G1862T, and G1896A) were independent risk factors for HCC when LC was the control.

Recent structural and biochemical studies of human TFIID have significantly increased our understanding of the mechanisms underlying the recruitment of TFIID to promoter DNA and its role in transcription initiation. Structural studies using cryo-EM revealed that modular interactions underlie TFIID's ability to bind simultaneously multiple promoter motifs and to define a DNA state that will facilitate transcription initiation. Here we propose a general model of promoter binding by TFIID, where co-activators, activators, and histone modifications promote and/or stabilize a conformational state of TFIID that results in core promoter engagement. Within this high affinity conformation, we propose that TFIID's extensive interaction with promoter DNA leads to topological changes in the DNA that facilitate the eventual loading of RNAP II. While more work is required to dissect the individual contributions of activators and repressors to TFIID's DNA binding, the recent cryo-EM studies provide a physical framework to guide future structural, biophysical, and biochemical experiments.

Background The core promoter is the region flanking the transcription start site (TSS) that directs formation of the pre-initiation complex. Core promoters have been studied intensively in mammals and yeast, but not in more diverse eukaryotes. Here we investigate core promoters in oomycetes, a group within the Stramenopile kingdom that includes important plant and animal pathogens. Prior studies of a small collection of genes proposed that oomycete core promoters contain a 16 to 19 nt motif bearing an Initiator-like sequence (INR) flanked by a novel sequence named FPR, but this has not been extended to whole-genome analysis. Results We used expectation maximization to find over-represented motifs near TSSs of Phytophthora infestans, the potato blight pathogen. The motifs corresponded to INR, FPR, and a new element found about 25 nt downstream of the TSS called DPEP. TATA boxes were not detected. Assays of DPEP function by mutagenesis were consistent with its role as a core motif. Genome-wide searches found a well-conserved combined INR+FPR in only about 13% of genes after correcting for false discovery, which contradicted prior reports that INR and FPR are found together in most genes. INR or FPR were found alone near TSSs in 18% and 7% of genes, respectively. Promoters lacking the motifs had pyrimidine-rich regions near the TSS. The combined INR+FPR motif was linked to higher than average mRNA levels, developmentally-regulated transcription, and functions related to plant infection, while DPEP and FPR were over-represented in constitutively-expressed genes. The INR, FPR, and combined INR+FPR motifs were detected in other oomycetes including Hyaloperonospora arabidopsidis, Phytophthora sojae, Pythium ultimum, and Saprolegnia parasitica, while DPEP was found in all but S. parasitica. Only INR seemed present in a non-oomycete stramenopile. Conclusions The absence of a TATA box and presence of novel motifs show that the oomycete core promoter is diverged from that of model systems, and likely explains the lack of activity of non-oomycete promoters in Phytophthora transformants. The association of the INR+FPR motif with developmentally-regulated genes shows that oomycete core elements influence stage-specific transcription in addition to regulating formation of the pre-initiation complex.

Background The transformation of a developing epithelium into an adult structure is a complex process, which often involves coordinated changes in cell proliferation, metabolism, adhesion, and shape. To identify genetic mechanisms that control epithelial differentiation, we analyzed the temporal patterns of gene expression during metamorphosis of the Drosophila wing. Results We found that a striking number of genes, approximately 50% of the Drosophila transcriptome, exhibited changes in expression during a time course of wing development. While cis-acting enhancer sequences clearly correlated with these changes, a stronger correlation was discovered between core-promoter types and the dynamic patterns of gene expression within this differentiating tissue. In support of the hypothesis that core-promoter type influences the dynamics of expression, expression levels of several TATA-box binding protein associated factors (TAFs) and other core promoter-associated components changed during this developmental time course, and a testes-specific TAF (tTAF) played a critical role in timing cellular differentiation within the wing. Conclusions Our results suggest that the combinatorial control of gene expression via cis-acting enhancer sequences and core-promoter types, determine the complex changes in gene expression that drive morphogenesis and terminal differentiation of the Drosophila wing epithelium.

Hepatitis B virus (HBV) genomes are small, semi-double-stranded DNA circular genomes that contain alternating overlapping reading frames and replicate through an RNA intermediary phase. This complex biology has presented a challenge to estimating an evolutionary rate for HBV, leading to difficulties resolving the evolutionary and epidemiological history of the virus. Here, we re-examine rates of HBV evolution using a novel data set of 112 within-host, transmission history (pedigree) and among-host genomes isolated over 20 years from the indigenous peoples of the South Pacific, combined with 313 previously published HBV genomes. We employ Bayesian phylogenetic approaches to examine several potential causes and consequences of evolutionary rate variation in HBV. Our results reveal rate variation both between genotypes and across the genome, as well as strikingly slower rates when genomes are sampled in the Hepatitis B e antigen positive state, compared to the e antigen negative state. This Hepatitis B e antigen rate variation was found to be largely attributable to changes during the course of infection in the preCore and Core genes and their regulatory elements.

It is now well known that vertebrates use multiple types of core promoter to accomplish differentiated tasks in Pol II-dependent transcription. Several transcriptional characteristics are known to be associated with core types, including distribution patterns of transcription start sites (TSSs) and selection between tissue-specific and constitutive expression profiles. However, their relationship to gene structure is poorly understood. In this report, we carried a comparative analysis of three Arabidopsis core types, TATA, GA, and Coreless, with regard to gene structure. Our genome-wide investigation was based on the peak TSS positions in promoters that had been identified in a large-scale experimental analysis. This analysis revealed that the types of core promoter are related with the room for promoters that is measured as the distance from the TSS to the end of the upstream gene, the distance from the TSS to the start position of the coding sequence (CDS), and the number and species of the cis-regulatory elements. Of these, it was found that the distance from the TSS to the CDS has a tight, inverse correlation to the expression level, and thus the observed relationship to the core type appears to be indirect. However, promoter length and preference of cis-elements are thought to be a direct reflection of core type-specific transcriptional initiation mechanisms.

Background Microsporidia are obligate intracellular eukaryotic parasites with genomes ranging in size from 2.3 Mbp to more than 20 Mbp. The extremely small (2.9 Mbp) and highly compact (~1 gene/kb) genome of the human parasite Encephalitozoon cuniculi has been fully sequenced. The aim of this study was to characterize noncoding motifs that could be involved in regulation of gene expression in E. cuniculi and to show whether these motifs are conserved among the phylum Microsporidia. Results To identify such signals, 5' and 3'RACE-PCR experiments were performed on different E. cuniculi mRNAs. This analysis confirmed that transcription overrun occurs in E. cuniculi and may result from stochastic recognition of the AAUAAA polyadenylation signal. Such experiments also showed highly reduced 5'UTR's (E. cuniculi genes presented a CCC-like motif immediately upstream from the coding start. To characterize other signals involved in differential transcriptional regulation, we then focused our attention on the gene family coding for ribosomal proteins. An AAATTT-like signal was identified upstream from the CCC-like motif. In rare cases the cytosine triplet was shown to be substituted by a GGG-like motif. Comparative genomic studies confirmed that these different signals are also located upstream from genes encoding ribosomal proteins in other microsporidian species including Antonospora locustae, Enterocytozoon bieneusi, Anncaliia algerae (syn. Brachiola algerae) and Nosema ceranae. Based on these results a systematic analysis of the ~2000 E. cuniculi coding DNA sequences was then performed and brings to highlight that 364 translation initiation codons (18.29% of total CDSs) had been badly predicted. Conclusion We identified various signals involved in the maturation of E. cuniculi mRNAs. Presence of such signals, in phylogenetically distant microsporidian species, suggests that a common regulatory mechanism exists among the microsporidia. Furthermore, 5'UTRs being strongly reduced, these signals can be used to ensure the accurate prediction of translation initiation codons for microsporidian genes and to improve microsporidian genome annotation.

Fulminant and severe viral hepatitis are frequently associated with mutant hepatitis B virus (HBV) strains. In this study, the genetic background of a viral strain causing severe subfulminant outcome in heart-transplanted patients was studied and compared with viral hepatitis B strains that were not linked to severe liver disease in the same setting. A total of 46 patients infected nosocomially with HBV genotype A were studied. Five different viral strains were detected, infecting 3, 9, 5, 24, and 5 patients, respectively. Only one viral strain was found to be associated with the subfulminant outcome and 3 patient deaths as a consequence of severe liver disease. The remaining 43 patients with posttransplantation HBV infection did not show this fatal outcome. Instead, symptoms of hepatitis were generally mild or clinically undiagnosed. Comparison of this virus genome with the four other strains showed an accumulation of mutations in the basic core promoter, a region that influences viral replication, but also in hepatitis B X protein (HBX) (7 mutant motifs), core (10 mutant motifs), the preS1 region (5 mutant motifs), and the HBpolymerase open reading frame (17 motifs). Some of these variations, such as those in the core region, were located on the tip of the protruding spike of the viral capsid (codons 60 to 90), also known in part as an important HLA class II-restricted epitope region. These mutations might therefore influence the immune-mediated response. The viral strain causing subfulminant hepatitis was, in addition, the only strain with a preCore stop codon mutation and, thus, hepatitis B e antigen (HBeAg) expression was never observed. The combination of these specific viral factors is thought to be responsible for the fatal outcome in these immune-suppressed heart-transplant recipients.

The FE65 gene encodes a nuclear protein of unknown function that is expressed in several areas of the rat nervous system during development and in the adult animal, particularly in somatic and visceral ganglia. FE65 mRNA is abundant in neuronal cell lines, whereas it is barely detectable in non-neuronal cells. We identified the two transcription start sites of theFE65 gene and we isolated the rat genomic fragment containing one of these two transcriptional start sites. We demonstrate that this fragment contains a promoter able to direct an efficient transcription of a reporter gene in PC12 cells and in NTERA2 cells upon their differentiation with retinoic acid, whereas it functions poorly in non-neuronal cells, such as Rat2 fibroblasts and BRL hepatocytes. This promoter is composed of two regions. The first includes a c/s-element whose removal greatly decreases the transcriptional efficiency in all cells examined and which forms similar complexes with proteins from PC12 and Rat2 cells. This cis-element binds Sp1 or another GC-binding factor. The second cis-element encompasses the transcription start site and is still able to direct transcription only in neuronal cells. The DNA-protein complexes formed by this cis-element in neuronal cells differ from those formed in non-neuronal cells. The analysis of point mutations in this region indicates that the proteins that bind to this cis-element interact with both overlapping and distinct nucleotide sequences. © 1994 Oxford University Press.

The RNA polymerase II core promoter is a structurally and functionally diverse transcriptional regulatory element. There are two main strategies for transcription initiation – focused and dispersed initiation. In focused initiation, transcription starts from a single nucleotide or within a cluster of several nucleotides, whereas in dispersed initiation, there are several weak transcription start sites over a broad region of about 50 to 100 nucleotides. Focused initiation is the predominant means of transcription in simpler organisms, whereas dispersed initiation is observed in approximately two-thirds of vertebrate genes. Regulated genes tend to have focused promoters, and constitutive genes typically have dispersed promoters. Hence, in vertebrates, focused promoters are used in a small but biologically important fraction of genes. The properties of focused core promoters are dependent upon the presence or absence of sequence motifs such as the TATA box and DPE. For example, Caudal, a key regulator of the homeotic gene network, preferentially activates transcription from DPE- versus TATA-dependent promoters. The basal transcription factors, which act in conjunction with the core promoter, are another important component in the regulation of gene expression. For instance, upon differentiation of myoblasts to myotubes, the cells undergo a switch from a TFIID-based transcription system to a TRF3–TAF3-based system. These findings suggest that the core promoter and basal transcription factors are important yet mostly unexplored components in the regulation of gene expression.

In trypanosomatids, the RNA polymerase I (RNAPI)-dependent promoters controlling the ribosomal RNA (rRNA) genes have been well identified. Although the RNAPI transcription machinery recognizes the DNA conformation instead of the DNA sequence of promoters, no conformational study has been reported for these promoters. Here we present the in silico analysis of the intrinsic DNA curvature of the rRNA gene core promoters in Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. We found that, in spite of the absence of sequence conservation, these promoters hold conformational properties similar to other eukaryotic rRNA promoters. Our results also indicated that the intrinsic DNA curvature pattern is conserved within the Leishmania genus and also among strains of T. cruzi and T. brucei. Furthermore, we analyzed the impact of point mutations on the intrinsic curvature and their impact on the promoter activity. Furthermore, we found that the core promoters of protein-coding genes transcribed by RNAPI in T. brucei show the same conserved conformational characteristics. Overall, our results indicate that DNA intrinsic curvature of the rRNA gene core promoters is conserved in these ancient eukaryotes and such conserved curvature might be a requirement of RNAPI machinery for transcription of not only rRNA genes but also protein-coding genes.