Search

The precise and accurate identification and quantification of transcriptional start sites (TSSs) is key to understanding the control of transcription. The core promoter consists of the TSS and proximal non-coding sequences, which are critical in transcriptional regulation. Therefore, the accurate identification of TSSs is important for understanding the molecular regulation of transcription. Existing protocols for TSS identification are challenging and expensive, leaving high-quality data available for a small subset of organisms. This sparsity of data impairs study of TSS usage across tissues or in an evolutionary context. To address these shortcomings, we developed Smart-Seq2 Rolling Circle to Concatemeric Consensus (Smar2C2), which identifies and quantifies TSSs and transcription termination sites. Smar2C2 incorporates unique molecular identifiers that allowed for the identification of as many as 70 million sites, with no known upper limit. We have also generated TSS data sets from as little as 40 pg of total RNA, which was the smallest input tested. In this study, we used Smar2C2 to identify TSSs in Glycine max (soybean), Oryza sativa (rice), Sorghum bicolor (sorghum), Triticum aestivum (wheat) and Zea mays (maize) across multiple tissues. This wide panel of plant TSSs facilitated the identification of evolutionarily conserved features, such as novel patterns in the dinucleotides that compose the initiator element (Inr), that correlated with promoter expression levels across all species examined. We also discovered sequence variations in known promoter motifs that are positioned reliably close to the TSS, such as differences in the TATA box and in the Inr that may prove significant to our understanding and control of transcription initiation. Smar2C2 allows for the easy study of these critical sequences, providing a tool to facilitate discovery.

Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5' untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.

Background Promoters, non-coding DNA sequences located at upstream regions of the transcription start site of genes/gene clusters, are essential regulatory elements for the initiation and regulation of transcriptional processes. Furthermore, identifying promoters in DNA sequences and genomes significantly contributes to discovering entire structures of genes of interest. Therefore, exploration of promoter regions is one of the most imperative topics in molecular genetics and biology. Besides experimental techniques, computational methods have been developed to predict promoters. In this study, we propose iPromoter-Seqvec – an efficient computational model to predict TATA and non-TATA promoters in human and mouse genomes using bidirectional long short-term memory neural networks in combination with sequence-embedded features extracted from input sequences. The promoter and non-promoter sequences were retrieved from the Eukaryotic Promoter database and then were refined to create four benchmark datasets. Results The area under the receiver operating characteristic curve (AUCROC) and the area under the precision-recall curve (AUCPR) were used as two key metrics to evaluate model performance. Results on independent test sets showed that iPromoter-Seqvec outperformed other state-of-the-art methods with AUCROC values ranging from 0.85 to 0.99 and AUCPR values ranging from 0.86 to 0.99. Models predicting TATA promoters in both species had slightly higher predictive power compared to those predicting non-TATA promoters. With a novel idea of constructing artificial non-promoter sequences based on promoter sequences, our models were able to learn highly specific characteristics discriminating promoters from non-promoters to improve predictive efficiency. Conclusions iPromoter-Seqvec is a stable and robust model for predicting both TATA and non-TATA promoters in human and mouse genomes. Our proposed method was also deployed as an online web server with a user-friendly interface to support research communities. Links to our source codes and web server are available at https://github.com/mldlproject/2022-iPromoter-Seqvec.

Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements—notably the TATA box—as well as promoter GC content and promoter-proximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detect species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S minimal promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features. A massively parallel reporter assay was used to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum in two assay systems, uncovering the sequence features affecting promoter strength and facilitating promoter strength prediction and synthetic design.

The

The effects of rapid acute depletion of components of RNA polymerase II (Pol II) general transcription factors (GTFs) that are thought to be critical for formation of preinitiation complexes (PICs) and initiation in vitro were quantified in HAP1 cells using precision nuclear run-on sequencing (PRO-Seq). The average dependencies for each factor across >70 000 promoters varied widely even though levels of depletions were similar. Some of the effects could be attributed to the presence or absence of core promoter elements such as the upstream TBP-specificity motif or downstream G-rich sequences, but some dependencies anti-correlated with such sequences. While depletion of TBP had a large effect on most Pol III promoters only a small fraction of Pol II promoters were similarly affected. TFIIB depletion had the largest general effect on Pol II and also correlated with apparent termination defects downstream of genes. Our results demonstrate that promoter activity is combinatorially influenced by recruitment of TFIID and sequence-specific transcription factors. They also suggest that interaction of the preinitiation complex (PIC) with nucleosomes can affect activity and that recruitment of TFIID containing TBP only plays a positive role at a subset of promoters.

The manner in which newborn coding sequences and their transcriptional competency emerge during the process of gene evolution remains unclear. Here, we experimentally simulated eukaryotic gene origination processes by mimicking horizontal gene transfer events in the plant genome. We mapped the precise position of the transcription start sites (TSSs) of hundreds of newly introduced promoterless firefly luciferase (LUC) coding sequences in the genome of Arabidopsis thaliana cultured cells. The systematic characterization of the LUC-TSSs revealed that 80% of them occurred under the influence of endogenous promoters, while the remainder underwent de novo activation in the intergenic regions, starting from pyrimidine-purine dinucleotides. These de novo TSSs obeyed unexpected rules; they predominantly occurred ∼100 bp upstream of the LUC inserts and did not overlap with Kozak-containing putative open reading frames (ORFs). These features were the output of the immediate responses to the sequence insertions, rather than a bias in the screening of the LUC gene function. Regarding the wild-type genic TSSs, they appeared to have evolved to lack any ORFs in their vicinities. Therefore, the repulsion by the de novo TSSs of Kozak-containing ORFs described above might be the first selection gate for the occurrence and evolution of TSSs in the plant genome. Based on these results, we characterized the de novo type of TSS identified in the plant genome and discuss its significance in genome evolution.

Selection of suitable promoters is crucial for the efficient expression of exogenous genes in transgenic animals. Although one of the most effective promoters, the β-actin promoter, has been widely studied in fish species, it still remains unknown in the economical important African catfish (Clarias gariepinus). In this study, the β-actin promoter of African catfish (cgβ-actinP) was cloned and characterized. In addition, recombinant plasmid pcgβ-actinP-EGFP with enhanced green fluorescent protein (GFP) gene as the reporter gene was constructed to verify the transcriptional activity. We obtained a cgβ-actinP fragment length of 1405 bp, consisting 104 bp of the 5′ proximal promoter, 96 bp of the first exon, and 1205 bp of the first intron. Similar to those of other fish species, cgβ-actinP contains three key transcription regulatory elements (CAAT box, CArG motif, and TATA box). GFP-specific fluorescent signals were detected in chicken embryonic fibroblasts cells (DF-1 cells) transfected with pcgβ-actinP-EGFP, which was approximately 1.11 times of the positive control. In addition, GFP was effectively expressed in zebrafish larvae microinjected with linearized cgβ-actinP-EGFP, with expression rate reaching approximately 49.84%. Our data indicate that cgβ-actinP could be a potential candidate promoter in the practice of constructing “all fish” transgenic fish.

F-type lectins (Fucolectins) are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion. In our previous research, we found that two homologous Fucolectin genes, AjFTL-1 and AjFTL-2, exhibited different expression profiles after lipopolysaccharides (LPS) challenge in Apostichopus japonicus. However, the transcriptional regulation mechanism of these two genes remains largely unknown. In this study, the 5′ flanking regions of AjFTL-1 and AjFTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid (EPC) cell system. First, in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB, CREB, and CREBP1, and both contain a TATA box. Additionally, luciferase assay and progressive 5′ truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells. Moreover, the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan (PGN) and mannan (MAN), while AjFTL-2 promoter was prominently regulated by LPS and MAN, indicating AjFTL-1 and AjFTL-2 genes showed different transcriptional regulation pattern under different immune stimulation. More importantly, analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites (−769 bp to −761 bp, −185 bp to −172bp) in AjFTL-1 and one potential binding site (−530 to −517bp) in AjFTL-2. Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity, but not AjFTL-1 promoter activity. These findings indicated that in marine invertebrates, different Fucolectin members differ in transcription regulations and expression patterns, and might play different roles in immune defenses during pathogen infection.

Gene expression vectors are useful and important tools that are commonly used in a variety of experiments, including expression of foreign genes, functional analysis of genes of interest and complementation experiments. In this study, a hybrid promoter, combining the adh1+ upstream activating sequence (UAS) of fission yeast and the GAL10 core promoter of budding yeast, was constructed to enable high level expression depending on the presence of zinc in culture medium for fission yeast. When the hybrid promoter was cloned on the multicopy plasmid, it was fully induced and repressed within 10 h in the presence and absence of zinc, respectively. The kinetics of induction and reduction were similar to those of the endogenous adh1+ mRNA. In contrast, native adh1+ promoter lost its tight repression in zinc-depleted condition when it was cloned on the plasmid. Because adh1+ UAS-specific transcription factors have not yet been identified, we identified UAS elements involved in zinc sensing by characterizing this hybrid promoter. We also found that the expression level increased by the TATA box mutation, GATAA, in the presence of zinc.

Genes are transcribed from transcription start sites (TSSs), and their position in a genome is strictly controlled to avoid mis-expression of undesired regions. In this study, we designed and developed a methodology for the evaluation of promoter context, which detects proximal promoter regions from − 200 to − 60 bp relative to a TSS, in Arabidopsis and rice genomes. The method positively evaluates spacer sequences and Regulatory Element Groups, but not core promoter elements like TATA boxes, and is able to predict the position of a TSS within a width of 200 bp. An important feature of the evaluation/prediction method is its independence of the core promoter elements, which was demonstrated by successful prediction of all the TATA, GA, and coreless types of promoters without notable differences in the accuracy of prediction. The positive relationship identified between the evaluation scores and gene expression levels suggests that this method is useful for the evaluation of promoter maturity.

RNA polymerase II (Pol II)-mediated transcription in eukaryotic cells starts with assembly of preinitiation complex (PIC) on core promoter, a DNA sequence of ∼100 base pairs. The transcription PIC consists of Pol II and general transcription factors TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH. Previous structural studies focused on PIC assembled on TATA box promoters with TFIID replaced by its subunit, TATA box-binding protein (TBP). However, the megadalton TFIID complex is essential for promoter recognition, TBP loading onto promoter, and PIC assembly for almost all Pol II-mediated transcription, especially on the TATA-less promoters, which account for ∼85% of core promoters of human coding genes. The functions of TFIID could not be replaced by TBP. The recent breakthrough in structure determination of TFIID-based PIC complexes in different assembly stages revealed mechanistic insights into PIC assembly on TATA box and TATA-less promotes and provided a framework for further investigation of transcription initiation.

MicroRNAs (miRNA) are non-coding 20-24 nucleotide long RNAs regulating gene expression. In this study, we have characterized and analysed expression of miRNAs in Pigeonpea by using bioinformatics and experimental tools. We identified 116 miRNAs belonging to 32 phylogenetic families. Further, transcription start sites of miRNA genes revealed abundance and unique arrangement of adenine at +1 and thymine at -1 position. Promoter analysis exhibited presence of 19 most prevalent motifs which comprises majorly of TATA box and MYC domains. In total, 252 miRNA-targets were identified and found to be involved in various developmental processes and stress responses. Moreover, genome-wide localization studies demonstrated clustering of cca-miRNA 395 and 169 genes. The tandem and segmental duplication events were observed suggesting miRNA genes have been originated parallelly with protein coding genes. The expression analysis revealed induction of cca-miR169a, 398a and 408 miRNAs under drought stress highlighting their involvement. Conversely, down-regulation of their putative targets (NFYA, SOD, and UCLA, respectively) confirmed regulatory role of miRNAs in their expression emphasising the negative relationship between these miRNAs and targets in Pigeonpea. This study reports vast repertoire of miRNA genes which further can be experimentally characterized to elucidate their functions in various biological processes and can be recommended for Pigeonpea improvement programs.

In this study, we cloned the ATP-binding cassette (ABC) subfamily G member 2 gene (Abcg2 gene; breast cancer resistance protein) from the liver of the marine pufferfish Takifugu rubripes, and examined its tissue distribution and gene structure. The full-length complementary DNA (cDNA) was 1977 base pairs (bp), comprising 15 exons in chromosome 17, and was assigned to the teleost Abcg2b paralogue. The open reading frame was 1839 bp in length, and encodes 612 amino acids constituting Walker A, Q-loop, C motif, Walker B, D-loop, and H-loop sequences in the first half and six transmembrane domains in the second half. Gene expression of Abcg2 was predominantly found in the intestines, followed by the liver and kidneys. The Kozak motif and the polyadenylation signal sequences were at respective ends of the transcript. The putative core promoter sequences including the transcription factor II B recognition elements, the TATA box, the initiator element, and the downstream core promoter element were found in the transcript and its upstream genomic DNA sequence. The hypoxia response element and the estrogen response element were located in the upstream genomic DNA sequence, suggesting that the pufferfish Abcg2 gene possesses the same regulation mechanism of transcription as the human ABCG2 gene.

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.

In Saccharomyces cerevisiae, class II gene promoters contain two classes of TATA elements: the TATA box and the TATA-like element. Functional loss of TFIID and SAGA transcription complexes selectively impacts steady-state mRNA levels expressed from TATA-like element-containing (i.e., TATA-less) and TATA box-containing promoters, respectively. While nascent RNA analysis has revealed that TFIID and SAGA are required for both types of promoters, the division of their roles remains unclear. We show here that transcription from the PGK1 promoter decreased in some cases by more than half after disruption of the TATA box or SPT3 (spt3Δ), whereas spt3Δ did not affect transcription from the TATA-less promoter, consistent with the prevailing view that Spt3 functions specifically in a TATA box-dependent manner. Transcription from this promoter was abolished in the spt3Δ taf1-N568Δ strain but unaffected in the taf1-N568Δ strain, regardless of TATA box presence, suggesting that TFIID was functionally dispensable for PGK1 transcription at least in the SPT3 strain. Furthermore, transcription from the TATA box-containing PGK1 promoter was slightly reduced in the taf1 strain lacking TAND (taf1-ΔTAND) upon temperature shift from 25 to 37 ℃, with restoration to normal levels within 2 h, in an Spt3-dependent manner. Interestingly, when SPT3 was reintroduced into the spt3Δ TAF1, spt3Δ taf1-N568Δ or spt3Δ taf1-ΔTAND strain, TATA box-dependent transcription from this promoter was largely restored, but TFIID independence in transcription was not restored, especially from the TATA-less promoter, and transient TAND/Spt3-dependent fluctuations of transcription after the temperature shift were also not recapitulated. Collectively, these observations suggest that Spt3 has multiple functions in PGK1 transcription, some of which may be intimately connected to Taf1 function and may therefore become unrestorable when the TFIID and SAGA functions are simultaneously compromised.

A farnesyl diphosphate synthase (FPS) cDNA and promoter region was cloned from Sanghuangporus baumii. The gene contains a 150-bp 5'-untranslated region (UTR), a 154-bp 3'-UTR, and a 1062-bp open reading frame (ORF) encoding a 354 amino acid polypeptide. The FPS-DNA includes three exons (nucleotides 1 -123, 184-321, and 505-1305) and two introns (nucleotides 124-183 and 322-504). The FPS protein has a molecular weight of 40.73 kDa, it is hydrophilic with a theoretical isoelectric point of 5.13, and the secondary and three-dimensional structure were analysed. There is a transcription start site at nucleotides 1318-1368 of the promoter, which includes typical eukaryotic promoter elements (TATA Box, CAAT Box, ARBE, AT-rich element, G-box, MBS, Sp1, LTR). FPS was expressed in Escherichia coli BL21, and the recombinant protein (63.41 kDa) was subjected to dodecyl sulphate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE). FPS transcription was measured during different developmental stages, and expression in 11 and 13 days mycelia was upregulated 49.3-fold and 125.4-fold, respectively, compared with 9 days mycelia controls. Through analysing, S. baumii triterpenoid content was correlated with the transcription level of FPS during different development stages, and the triterpenoid content peaked at day 15 (7.21 mg/g).

Annulohypoxylon stygium is an ascomycete that helps Tremella fuciformis produce the fruiting body in wild state or artificial cultivation. Although the interaction between these two fungi is well known, the underlying molecular mechanism is limited. In this study, the 981 bp and 886 bp promoter sequences of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and α-tubulin gene have been cloned, respectively. Sequence analysis showed that gpd promoter contained nine CAAT boxes, and single TGACG-motif, TATA box, ABRE motif, STRE motif, MYB motif, and W box. The α-tubulin promoter included eight CAAT boxes, three STRE, two TATA boxes and MYB, single Box 4, CAT-box, CCAAT-box, TGA-element, and ABRE. Subsequently, we evaluated the promoters' function by constructing four vectors pGEH, pGRH, pTEH, and pTRH to drive fused enhanced green fluorescent protein and hygromycin B phosphotransferase (egfp-hph) or red fluorescent protein and hygromycin B phosphotransferase (rfp-hph) expression under the control of gpd or α-tubulin promoters in A. stygium. The integration of the transformed DNA into A. stygium genome was verified by PCR, Southern blot, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). All the results indicated that the two promoters could drive egfp-hph and rfp-hph expression. This result could provide help in gene functional studies by using gpd and α-tubulin promoters to direct gene over-expression or build dual promoter silencing systems.

A promoter is a short DNA sequence near the start codon, responsible for initiating the transcription of a specific gene in the genome. The accurate recognition of promoters is important for achieving a better understanding of transcriptional regulation. Because of their importance in the process of biological transcriptional regulation, there is an urgent need to develop in silico tools to identify promoters and their types in a timely and accurate manner. A number of prediction methods have been developed in this regard; however, almost all of them are merely used for identifying promoters and their strength or sigma types. The TATA box region in TATA promoter influences the post-transcriptional processes; therefore, in the current study, we developed a two-layer predictor called "iProm-Zea" using the convolutional neural network (CNN) for identify TATA and TATA less promoters. The first layer can be used to identify a given DNA sequence as a promoter or non-promoter. The second layer can be used to identify whether the recognized promoter is the TATA promoter. To find an optimal feature encoding scheme and model, we employed four feature encoding schemes on different machine learning and CNN algorithms, and based on the evaluation results, we selected a one-hot encoding scheme and a CNN model for iProm-Zea. The 5-fold cross validation testing results demonstrated that the constructed predictor showed great potential for identifying promoters and classifying them as TATA and TATA less promoters. Furthermore, we performed cross-species analysis of iProm-Zea to evaluate its performance in other species. Moreover, to make it easier for other experimental scientists to obtain the results they need, we established a freely accessible and user-friendly web server at http://nsclbio.jbnu.ac.kr/tools/iProm-Zea/.

Different workers have found different bases of the spacer of the sigma 70 promoter of Escherichia coli to be important, depending on the base sequence of the two hexameric boxes of the naturally occurring promoter they were working on. Besides, there was no clue as to why particular bases worked better than others in particular positions. This necessitated a fresh look at the spacer region of a model promoter comprising all the consensus promoter elements. Randomisation of the three bases of the spacer in positions -15 to -13 with respect to the transcription initiation site, has elicited more than 50-fold variation in activity of the promoter, the highest and the lowest activities being 14,391(the three bases being GCA) and 264 Miller units (the three bases being AAA) respectively. Pairs of promoters of very similar activities were observed, even when the bases in these three positions were very different. The promoters with similar activities had similar three dimensional structures of the promoter DNA, as determined by molecular dynamics simulations. Randomisation of the three bases in positions -18 to -16 of the promoter that contained the triplet GCA in positions -15 to -13, resulted in promoters with highest activity of 15,759 (the triplet upstream of GCA being TAT) and lowest activity of 1,882 (the triplet upstream of GCA being AAA). Good correlation between the bending angles of the promoter DNAs and promoter activities could be observed, the R

Noise in expression of individual genes gives rise to variations in activity of cellular pathways and generates heterogeneity in cellular phenotypes. Phenotypic heterogeneity has important implications for antibiotic persistence, mutation penetrance, cancer growth and therapy resistance. Specific molecular features such as the presence of the TATA box sequence and the promoter nucleosome occupancy have been associated with noise. However, the relative importance of these features in noise regulation is unclear and how well these features can predict noise has not yet been assessed. Here through an integrated statistical model of gene expression noise in yeast we found that the number of regulating transcription factors (TFs) of a gene was a key predictor of noise, whereas presence of the TATA box and the promoter nucleosome occupancy had poor predictive power. With an increase in the number of regulatory TFs, there was a rise in the number of cooperatively binding TFs. In addition, an increased number of regulatory TFs meant more overlaps in TF binding sites, resulting in competition between TFs for binding to the same region of the promoter. Through modeling of TF binding to promoter and application of stochastic simulations, we demonstrated that competition and cooperation among TFs could increase noise. Thus, our work uncovers a process of noise regulation that arises out of the dynamics of gene regulation and is not dependent on any specific transcription factor or specific promoter sequence.

Background Cotton is a valuable economic crop and the main significant source of natural fiber for textile industries globally. The effects of drought and salt stress pose a challenge to strong fiber and large-scale production due to the ever-changing climatic conditions. However, plants have evolved a number of survival strategies, among them is the induction of various stress-responsive genes such as the ribosomal protein large (RPL) gene. The RPL gene families encode critical proteins, which alleviate the effects of drought and salt stress in plants. In this study, comprehensive and functional analysis of the cotton RPL genes was carried out under drought and salt stresses. Results Based on the genome-wide evaluation, 26, 8, and 5 proteins containing the RPL14B domain were identified in Gossypium hirsutum, G. raimondii, and G. arboreum, respectively. Furthermore, through bioinformatics analysis, key cis-regulatory elements related to RPL14B genes were discovered. The Myb binding sites (MBS), abscisic acid-responsive element (ABRE), CAAT-box, TATA box, TGACG-motif, and CGTCA-motif responsive to methyl jasmonate, as well as the TCA-motif responsive to salicylic acid, were identified. Expression analysis revealed a key gene, Gh_D01G0234 (RPL14B), with significantly higher induction levels was further evaluated through a reverse genetic approach. The knockdown of Gh_D01G0234 (RPL14B) significantly affected the performance of cotton seedlings under drought/salt stress conditions, as evidenced by a substantial reduction in various morphological and physiological traits. Moreover, the level of the antioxidant enzyme was significantly reduced in VIGS-plants, while oxidant enzyme levels increased significantly, as demonstrated by the higher malondialdehyde concentration level. Conclusion The results revealed the potential role of the RPL14B gene in promoting the induction of antioxidant enzymes, which are key in oxidizing the various oxidants. The key pathways need to be investigated and even as we exploit these genes in the developing of more stress-resilient cotton germplasms.

Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

We aimed to analyze the occurrence and clinical and genetic characteristics of spinocerebellar ataxia type 17 (SCA17) among Russian patients with progressive cerebellar ataxia or Huntington disease-like phenotype.Genetic analysis of CAG/CAA repeats in TBP gene was carried out in 217 patients, including 153 patients with progressive unspecified ataxia and 64 patients with Huntington disease-like phenotype. SCA types 1, 2, 3, 6 and 8, Friedreich's ataxia, CANVAS and Huntington disease were preliminarily excluded.Six unrelated patients with SCA17 (2.8 %) were identified (43-57 CAG/CAA repeats in TBP gene). Two patients had a positive family history. Age at the disease onset ranged from 15 to 47 years. The core clinical syndrome included progressive cerebellar ataxia, dysarthria, movement disorders, cognitive impairment, and psychiatric symptoms. One patient had epilepsy with rare generalized tonic-clonic seizures. Another patient with diffuse muscle atrophy and small expansion size (43 CAG/CAA repeats) had myopathic changes in skeletal muscles on EMG study. We also described a patient with a large expansion size of 57 CAA/CAG repeats with early onset and rapid disease progression.SCA17 is a relatively rare cause of progressive disorders with ataxia and chorea, but it should be considered in the spectrum of differential diagnosis in such patients. Most of our SCA17 cases were sporadic which should be kept in mind when planning genetic testing in patients with spinocerebellar ataxia and chorea.

The transcription machinery of archaea can be roughly classified as a simplified version of eukaryotic organisms. The basal transcription factor machinery binds to the TATA box found around 28 nucleotides upstream of the transcription start site; however, some transcription units lack a clear TATA box and still have TBP/TFB binding over them. This apparent absence of conserved sequences could be a consequence of sequence divergence associated with the upstream region, operon, and gene organization. Furthermore, earlier studies have found that a structural analysis gains more information compared with a simple sequence inspection. In this work, we evaluated and coded 3630 archaeal promoter sequences of three organisms, Haloferax volcanii, Thermococcus kodakarensis, and Sulfolobus solfataricus into DNA duplex stability, enthalpy, curvature, and bendability parameters. We also split our dataset into conserved TATA and degenerated TATA promoters to identify differences among these two classes of promoters. The structural analysis reveals variations in archaeal promoter architecture, that is, a distinctive signal is observed in the TFB, TBP, and TFE binding sites independently of these being TATA‐conserved or TATA‐degenerated. In addition, the promoter encountering method was validated with upstream regions of 13 other archaea, suggesting that there might be promoter sequences among them. Therefore, we suggest a novel method for locating promoters within the genome of archaea based on DNA energetic/structural features., A structural parametrization of genetic information into physical attributes enables the location of transcription factor binding sites in the archaeal genome. This study poses a novel method for promoter sequence location in putative regulators of archaea.

SUMMARYRNA polymerase II (Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.HIGHLIGHTSSingle molecule microscopy reveals unexpected dynamics of RNA Pol II and GTFsMultiple Pol IIs cluster on UAS/enhancer-bound activators before binding the core promoterPol II, TFIIF, and TFIIE, but not TFIIH, can pre-assemble at the UAS/enhancerActivators increase the rates of Pol II and GTF association with DNAeTOC BlurbSingle-molecule microscopy experiments by Baek et al. show that RNA polymerase II and basal transcription factors TFIIF and TFIIE preassemble on UAS/enhancer-bound activators, poised for loading into initiation complexes with TFIIH at the core promoter. Transcription activators kinetically enhance factor recruitment, creating a localized cluster of polymerases at the UAS/enhancer.

Resolving the mechanism of salt stress tolerance in plants becomes a necessity to eventually develop salt-tolerant varieties by determining the key players in tolerance mechanism. Our previous results on transcriptome analysis of common bean under salt stress have revealed an upregulation in a putative chalconeO-methyltransferase (pvChOMT) transcript. In the present study, overexpression of pvChOMT gene in Arabidopsis transgenic lines enhanced salt tolerance. In turn, transgenic plants presented enhanced root growth, better water content, better mass preservation with osmoprotectant accumulation, and higher germination rates in saline conditions. Alignment analysis and predicted structure of pvChOMT have implicated it as the homologue of Medicago sativa chalcone O-methyltransferase (1FP1) with same substrate specificity. Promotor analysis of pvChOMT has shown a TATA box at − 145 position putative cis-acting elements within 1.5-kb upstream of the gene known to be activated through ABA-dependent and ABA-independent manner. Our results suggest that pvChOMT can be a good candidate gene to improve crops for salt stress tolerance.

The viral responsive protein 15 from the black tiger shrimp Penaeus monodon (PmVRP15) is a highly responsive gene upon white spot syndrome virus (WSSV) challenge. It is identified from hemocyte and important for WSSV trafficking and assembly. However, the knowledge of PmVRP15 gene regulation is limited. In the present study, the genome organization and 5'upstream promoter sequences of PmVRP15 gene were investigated. The PmVRP15 gene was found to contain 4 exons interrupted by 3 introns and the start codon was located in the exon 2. The transcription start site and TATA box were also determined from the 5' upstream sequence. By using the narrow down experiment, the 5' upstream promoter active region was determined to be at the nucleotide positions -525 to +612. Mutagenesis of the putative transcription factor (TF) binding sites revealed that the binding site of interferon regulatory factor (IRF) (-495/-479) was a repressor-binding site whereas those of the octamer transcription factor 1 (Oct-1) (-275/-268) and the nuclear factor of activated T-cells transcription factor (NFAT) (-228/-223) were activator-binding sites. This is the first report on the transcription factors that might play essential roles in modulating the PmVRP15 gene expression. Nevertheless, the underlying regulation mechanism of PmVRP15 gene expression needs further investigation.

The protozoan parasite Trichomonas vaginalis is a common human pathogen from one of the earliest-diverging eukaryotic lineages. At the transcriptional level, the highly conserved Inr element of RNA pol II-transcribed genes surrounds the transcription start site and is recognised by IBP39, a protein exclusive of T. vaginalis. Typical TATA boxes have not been identified in this organism but, in contrast, BLAST analyses of the T. vaginalis genome identified two genes encoding putative TATA-binding proteins (herein referred to as TvTBP1 and TvTBP2). The goal of this work was to characterise these two proteins at the molecular level. Our results show that both TvTBPs theoretically adopt the saddle-shaped structure distinctive to TBPs and both Tvtbp genes are expressed in T. vaginalis. TvTBP1 did not complement a Saccharomyces cerevisiae mutant lacking TBP; however, TvTBP1 and TvTBP2 proteins bound T. vaginalis DNA promoter sequences in EMSA assays. We propose that TvTBP1 may be part of the preinitiation transcription complex in T. vaginalis since TvTBP1 recombinant protein was able to bind IBP39 in vitro. This work represents the first approach towards the characterisation of general transcription factors in this early divergent organism.

Avian leukosis virus (ALV) caused tremendous economic losses to poultry industry all over the world, especially in China. One natural recombinant ALV strain, designated as HB2015032, was isolated from indigenous chickens with neoplastic diseases in Hubei, China. The complete proviral genome of HB2015032 is 7703 bp in length. Sequence analysis showed that the Env of HB2015032 exhibited 99.3% similarity with that of a ALV subgroup K (ALV-K) isolate JS11C1 at amino acid level. Phylogenetic analysis revealed that both gp85 and gp37 of HB2015032 were clustered in the same branch with JS11C1 and other ALV-K strains isolated from Chinese indigenous chickens in recent years. However, the pol gene, the 3' untranslated region (3' UTR), and the 3' long terminal repeat (3' LTR) of HB2015032 were more closely related to ALV-J prototype HPRS-103, and clustered in the same branch with ALV-J strains. Furthermore, the pol gene of HB2015032 contained a premature stop codon that resulted in a truncated Pol protein with 22 amino acid residues missing, which was a unique feature of the pol gene of ALV-J. 3'UTR of HB2015032 containing entire DR1, E element and U3. E element of HB2015032 contained one base deletion, which resulted in a c-Ets-1 binding site. In addition, U3 region of HB2015032 contains most of the transcription regulatory elements of ALV-J, including two CAAT boxes, Y boxes, CArG boxes, PRE boxes, NFAP-1 boxes, and one TATA box. These results suggest that isolate HB2015032 was a novel recombinant ALV-K containing the ALV-K env gene and the ALV-J backbone and exhibiting high pathogenicity.

Saccharopine dehydrogenase (EC 1.5.1.7) regulates the last step of fungal lysine biosynthesis. The gene (Fvsdh) encoding saccharopine dehydrogenase was identified and cloned from the whole genome of Flammulina velutipes. The genomic DNA of Fvsdh is 1257 bp, comprising three introns and four exons. The full-length complementary DNA of Fvsdh comprises 1107 bp with a deduced amino acid sequence of 368 residues. A 1,000-bp promoter sequence containing the TATA box, CAAT box, and several putative cis-acting elements was also identified. The results of tissue expression analysis showed that the expression level of the Fvsdh gene was higher in the pileus than in the stipe whether in the elongation or maturation stage. Further research showed that the lysine contents were 3.03 and 2.95 mg/g in maturation-pileus and elongation-pileus, respectively. In contrast, the lysine contents were 2.49 and 2.07 mg/g in elongation-stipe and maturation-stipe, respectively. To study the function of Fvsdh, we overexpressed Fvsdh in F. velutipes and found that Fvsdh gene expression was increased from 1.1- to 3-fold in randomly selected transgenic strains. The lysine contents were also increased from 1.12- to 1.3-fold in these five transformants, except for strain T3, in which the lysine contents were the same as the control. These results indicate that the expression of the Fvsdh gene can affect the lysine content of F. velutipes.

Transcriptional cofactors (COFs) communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression1. Although some COFs have been shown to prefer certain promoter types2–5 over others (for example, see refs 6,7), the extent to which different COFs display intrinsic specificities for distinct promoters is unclear. Here we use a high-throughput promoter-activity assay in Drosophila melanogaster S2 cells to screen 23 COFs for their ability to activate 72,000 candidate core promoters (CPs). We observe differential activation of CPs, indicating distinct regulatory preferences or ‘compatibilities’8,9 between COFs and specific types of CPs. These functionally distinct CP types are differentially enriched for known sequence elements2,4, such as the TATA box, downstream promoter element (DPE) or TCT motif, and display distinct chromatin properties at endogenous loci. Notably, the CP types differ in their relative abundance of H3K4me3 and H3K4me1 marks (see also refs 10–12), suggesting that these histone modifications might distinguish trans-regulatory factors rather than promoter- versus enhancer-type cis-regulatory elements. We confirm the existence of distinct COF–CP compatibilities in two additional Drosophila cell lines and in human cells, for which we find COFs that prefer TATA-box or CpG-island promoters, respectively. Distinct compatibilities between COFs and promoters can explain how different enhancers specifically activate distinct sets of genes9, alternative promoters within the same genes, and distinct transcription start sites within the same promoter13. Thus, COF–promoter compatibilities may underlie distinct transcriptional programs in species as divergent as flies and humans. A screen of 23 transcriptional cofactors for their ability to activate 72,000 candidate core promoters in Drosophila melanogaster identified distinct compatibility groups, providing insight into mechanisms that underlie the selective activation of transcriptional programs.

Transcription by RNA polymerase II initiates at the core promoter, which is sometimes referred to as the “gateway to transcription.” Here, we describe the properties of the RNA polymerase II core promoter in Drosophila. The core promoter is at a strategic position in the expression of genes, as it is the site of convergence of the signals that lead to transcriptional activation. Importantly, core promoters are diverse in terms of their structure and function. They are composed of various combinations of sequence motifs such as the TATA box, initiator (Inr), and downstream core promoter element (DPE). Different types of core promoters are transcribed via distinct mechanisms. Moreover, some transcriptional enhancers exhibit specificity for particular types of core promoters. These findings indicate that the core promoter is a central component of the transcriptional apparatus that regulates gene expression.

Transcription factor II D (TFIID), a multiprotein complex consisting of TATA-binding protein (TBP) and 13-14 TBP-associated factors (Tafs), plays a central role in transcription and regulates nearly all class II genes. The N-terminal domain of Taf1p (TAND) can be divided into two subdomains, TAND1 and TAND2, which bind to the concave and convex surfaces of TBP, respectively. The interaction between TAND and TBP is thought to be regulated by TFIIA, activators and/or DNA during transcriptional activation, as the TAND1-bound form of TBP cannot bind to the TATA box. We previously demonstrated that Drosophila TAND1 binds to TBP with a much stronger affinity than yeast TAND1 and that the expression levels of full-length chimeric Taf1p, whose TAND1 is replaced with the Drosophila counterpart, can be varied in vivo by substituting several methionine residues downstream of TAND2 with alanine residues in various combinations. In this study, we examined the transcriptional activation of the GAL1-lacZ reporter or endogenous genes such as RNR3 or GAL1 in yeast cells expressing various levels of full-length chimeric Taf1p. The results showed that the substitution of TAND1 with the Drosophila counterpart in yeast TFIID weakened the transcriptional activation of GAL1-lacZ and RNR3 but not that of GAL1. These findings strongly support a model in which TBP must be released efficiently from TAND1 within TFIID upon transcriptional activation.

Synthetic targeted optimization of plant promoters is becoming a part of progress in mainstream postgenomic agriculture along with hybridization of cultivated plants with wild congeners, as well as marker-assisted breeding. Therefore, here, for the first time, we compiled all the experimental data—on mutational effects in plant proximal promoters on gene expression—that we could find in PubMed. Some of these datasets cast doubt on both the existence and the uniqueness of the sought solution, which could unequivocally estimate effects of proximal promoter mutation on gene expression when plants are grown under various environmental conditions during their development. This means that the inverse problem under study is ill-posed. Furthermore, we found experimental data on in vitro interchangeability of plant and human TATA-binding proteins allowing the application of Tikhonov’s regularization, making this problem well-posed. Within these frameworks, we created our Web service Plant_SNP_TATA_Z-tester and then determined the limits of its applicability using those data that cast doubt on both the existence and the uniqueness of the sought solution. We confirmed that the effects (of proximal promoter mutations on gene expression) predicted by Plant_SNP_TATA_Z-tester correlate statistically significantly with all the experimental data under study. Lastly, we exemplified an application of Plant_SNP_TATA_Z-tester to agriculturally valuable mutations in plant promoters.

The assembly of transcription complexes on eukaryotic promoters involves a series of steps, including chromatin remodeling, recruitment of TATA-binding protein (TBP)-containing complexes, the RNA polymerase II holoenzyme, and additional basal transcription factors. This review describes the transcriptional regulation by TBP and its corresponding homologs that constitute the TBP family and their interactions with promoter DNA. The C-terminal core domain of TBP is highly conserved and contains two structural repeats that fold into a saddle-like structure, essential for the interaction with the TATA-box on DNA. Based on the TBP C-terminal core domain similarity, three TBP-related factors (TRFs) or TBP-like factors (TBPLs) have been discovered in metazoans, TRF1, TBPL1, and TBPL2. TBP is autoregulated, and once bound to DNA, repressors such as Mot1 induce TBP to dissociate, while other factors such as NC2 and the NOT complex convert the active TBP/DNA complex into inactive, negatively regulating TBP. TFIIA antagonizes the TBP repressors but may be effective only in conjunction with the RNA polymerase II holoenzyme recruitment to the promoter by promoter-bound activators. TRF1 has been discovered inDrosophila melanogasterandAnophelesbut found absent in vertebrates and yeast. TBPL1 cannot bind to the TATA-box; instead, TBPL1 prefers binding to TATA-less promoters. However, TBPL1 shows a stronger association with TFIIA than TBP. The TCT core promoter element is present in most ribosomal protein genes inDrosophilaand humans, and TBPL1 is required for the transcription of these genes. TBP directly participates in the DNA repair mechanism, and TBPL1 mediates cell cycle arrest and apoptosis. TBPL2 is closely related to its TBP paralog, showing 95% sequence similarity with the TBP core domain. Like TBP, TBPL2 also binds to the TATA-box and shows interactions with TFIIA, TFIIB, and other basal transcription factors. Despite these advances, much remains to be explored in this family of transcription factors.

Regulatory interactions between enhancers and core promoters are fundamental for the temporal and spatial specificity of gene expression in development. The central role of core promoters is to initiate productive transcription in response to enhancer’s activation cues. However, it has not been systematically assessed how individual core promoter elements affect the induction of transcriptional bursting by enhancers. Here, we provide evidence that each core promoter element dynamically alters “enhancer responsiveness” by changing functional parameters of transcriptional bursting in developing Drosophila embryos. Quantitative live imaging analysis revealed that the timing and the continuity of burst induction are common regulatory steps on which core promoter elements impact. We also show that core promoters use the upstream TATA box as an amplifier of transcriptional bursting upon interaction with enhancers. Genome editing analysis of the pair-rule gene fushi tarazu further revealed that the endogenous TATA box and the downstream core promoter element are both essential for the correct expression and the function of key developmental genes in early embryos. We suggest that core promoter elements serve as a key regulatory module in converting enhancer activity into transcription dynamics during animal development.

Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.

IFN-β is a unique member of type I IFN in humans and contains four positive regulatory domains (PRDs), I-II-III-IV, in its promoter, which are docking sites for transcription factors IFN regulatory factor (IRF) 3/7, NF-κB, IRF3/7, and activating transcription factor 2/Jun proto-oncogene, respectively. In chicken IFN-β and zebrafish IFNφ1 promoters, a conserved PRD or PRD-like sequences have been reported. In this study, a type I IFN gene, named as xl-IFN1 in the amphibian model Xenopus laevis, was found to contain similar PRD-like sites, IV-III/I-II, in its promoter, and these PRD-like sites were proved to be functionally responsive to activating transcription factor 2/Jun proto-oncogene, IRF3/IRF7, and p65, respectively. The xl-IFN1, as IFNφ1 in zebrafish, was transcribed into a long and a short transcript, with the long transcript containing all of the transcriptional elements, including PRD-like sites and TATA box in its proximal promoter. A retroposition model was then proposed to explain the transcriptional conservation of IFNφ1, xl-IFN1, and IFN-β in chicken and humans.

Epigenetics maintains cell-identity specific gene-expression patterns. However, within a population of isogenic cells of the same identity, a substantial variability in gene expression and responsiveness is still observed. Transcription bursting is a substantial source of this gene-expression variability or ‘noise’, contributing to phenotypic heterogeneity and potentially driving both physiological and pathological processes such as differentiation or tumorigenesis and drug resistance. Identification of transcription-bursting dynamics at a genome-wide scale has been restricted to inferring bursts in mRNA production computationally from the heterogeneity of mRNA levels in single cell transcriptomic data. Systematic characterisation of the genomic and epigenetic chromatin context of genes with defined transcription bursting behaviour has been incomplete. Here, we measured the bursting of transcription itself by genome-wide nascent RNA sequencing of breast cancer MCF-7 cells upon synchronisation of transcription with a transcription elongation inhibitor and by calibration using live cell imaging of nascent PP7-tagged GREB1 transcription. Comparing across the entire genome, we find transcription bursting to be ubiquitous, with burst sizes of up to 160 transcripts. Transcription bursting is strongly correlated with steady state gene expression between genes, whereas both burst frequency and nascent transcript degradation only correlate weakly. Individual genes deviate strongly from this trend and engage both in anomalous burst size and frequency. We find that the presence of the TATA box or Inr sequence within gene promoters are significantly associated with a larger burst size, as are promoter-associated YY1 and E2F1 transcription-factor binding motifs. Enrichment of the transcription start site with epigenetic marks such as H3K79me2 and H3K27ac is also strongly associated with the transcription burst size. Finally, we show that in these MCF-7 breast-cancer cells, genes with a larger transcription burst size exhibit a larger immediate transcriptional response following endocrine drug treatment. Our genome-wide transcription-bursting analysis method paves the way to elucidate the dynamic role of epigenetic regulation on dynamic transcription in pathophysiology.

During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code., The vertebrate TATA-binding protein (TBP) paralogue (TBPL2) is only expressed in growing oocytes, where TBP is absent. Here the authors highlight a unique role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.

Fabry disease (FD) is a rare hereditary disorder characterized by a wide range of symptoms caused by a variety of mutations in the galactosidase α (GLA) gene. The heterogeneous nuclear ribonucleoprotein (HNRNPH2) gene is divergently paired with GLA on chromosome X and is thought to be implicated in FD. However, insufficient information is available on the regulatory mechanisms associated with the expression of HNRNPH2 and the GLA loci. Therefore, the current study performed bioinformatics analyses to assess the GLA and HNRNPH2 loci and investigate the regulatory mechanisms involved in the expression of each gene. The regulatory mechanisms underlying GLA and HNRNPH2 were revealed. The expression of each gene was associated with a bidirectional promoter (BDP) characterized by the absence of TATA box motifs and the presence of specific transcription factor binding sites (TFBSs) and a CpG Island (CGI). The nuclear run-on transcription assay confirmed the activity of BDP GLA and HNRNPH2 transcription in 293T. Methylation-specific PCR analysis demonstrated a statistically significant variation in the DNA methylation pattern of BDP in several cell lines, including human adult epidermal keratinocytes (AEKs), human renal glomerular endothelial cells, human renal epithelial cells and 293T cells. The highest observed significance was demonstrated in AEKs (P

SOST encodes the sclerostin protein, which acts as a key extracellular inhibitor of the canonical Wnt pathway in bone, playing a crucial role in skeletal development and bone homeostasis. The objective of this work was to assess the functionality of two variants previously identified (the rare variant rs570754792 and the missense variant p.Val10Ile) and to investigate the physical interactors of the SOST proximal promoter region in bone cells. Through a promoter luciferase reporter assay we show that the minor allele of rs570754792, a variant located in the extended TATA box motif, displays a significant decrease in promoter activity. Likewise, through western blot studies of extracellular and intracellular sclerostin, we observe a reduced expression of the p.Val10Ile mutant protein. Finally, using a circular chromosome conformation capture assay (4C-seq) in 3 bone cell types (MSC, hFOB, Saos-2), we have detected physical interactions between the SOST proximal promoter and the ECR5 enhancer, several additional enhancers located between EVT4 and MEOX1 and a distant region containing exon 18 of DHX8. In conclusion, SOST presents functional regulatory and missense variants that affect its expression and displays physical contacts with far reaching genomic sequences, which may play a role in its regulation within bone cells.

Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

SummaryThe critical process in development, bifurcation of cellular fates, requires epigenetic H3K27me3 marks propagated by PRC2 complex. However, the precise chromatin loci of functional H3K27me3 marks are not yet known. Here we identify critical PRC2 functional sites at a single nucleosome resolution. We fused a computationally designed protein, EED binder (EB) that competes with EZH2 and thereby disrupts PRC2 function, to dCas9 (EBdCas9) to direct PRC2 inhibition at a precise locus using gRNA. We targeted EBdCas9 to 4 different genes (TBX18, p16, CDX2 and GATA3) and observed epigenetic remodeling at a single nucleosome resolution resulting in gene activation. Remarkably, while traditional TATA box is located 30bp upstream of TSS, we identified a functional TATA box, >500bp of TSS, normally repressed by PRC2 complex. Deletion of this TATA box eliminates EBdCas9 dependent TBP recruitment and transcriptional activation. Targeting EBdCas9 to CDX2 and GATA3 results in trophoblast trans-differentiation. EBdCas9 technology is broadly applicable for epigenetic regulation at a single locus to control gene expression.Graphical Abstract

Gene expression is controlled by regulatory elements with accessible chromatin. Although the majority of regulatory elements are cell type-specific, being in the open chromatin state in only one or a few cell types, approximately 16,000 regions in the human genome and 13,000 regions in the mouse genome are in the open chromatin state in nearly all of the 517 human and 94 mouse cell and tissue types assayed by the ENCODE consortium, respectively. We performed a systematic analysis on the subset of 9,000 human and 8,000 mouse ubiquitously (ubi) open chromatin regions that were also classified as candidate cis-regulatory elements (cCREs) with promoter-like signatures (PLSs) by the ENCODE consortium, which we refer to as ubi-PLSs. We found that these ubi-PLSs had higher levels of CG dinucleotides and corresponded to the genes with ubiquitously high levels of transcriptional activities. Furthermore, the transcription start sites of a vast majority of cell-essential genes are located in ubi-PLSs. ubi-PLSs are enriched in the motifs of ubiquitously expressed transcription factors and preferentially bound by transcriptional cofactors that regulate ubiquitously expressed genes. Finally, ubi-PLSs are highly conserved between human and mouse at the synteny level, but not as conserved at the sequence level, with a high turnover of transcription factor motif sites. Thus, there is a distinct set of roughly 9,000 promoters in the mammalian genome that are actively maintained in the open chromatin state in nearly all cell types to ensure the transcriptional program of cell-essential genes.

Chinese hamster ovary (CHO) cells, with their human-compatible glycosylation and high protein titers, are the most widely used cells for producing biopharmaceuticals. Engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the Chinese hamster genome were computationally predicted and are frequently inaccurate. Here, we revised TSS annotations for 15,308 Chinese hamster genes and 4,478 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. The experimental realignment and discovery of TSSs now expose previously hidden motifs, such as the TATA box. We further demonstrate, by targeting the glycosyltransferase geneMgat3, how accurate annotations readily facilitate activating silent genes by CRISPRa to obtain more human-like glycosylation. Together, we envision our annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

Background In population ecology, the concept of reproductive potential denotes the most vital indicator of chances to produce and sustain a healthy descendant until his/her reproductive maturity under the best conditions. This concept links quality of life and longevity of an individual with disease susceptibilities encoded by his/her genome. Female reproductive potential has been investigated deeply, widely, and comprehensively in the past, but the male one has not received an equal amount of attention. Therefore, here we focused on the human Y chromosome and found candidate single-nucleotide polymorphism (SNP) markers of male reproductive potential. Results Examining in silico (i.e., using our earlier created Web-service SNP_TATA_Z-tester) all 1206 unannotated SNPs within 70 bp proximal promoters of all 63 Y-linked genes, we found 261 possible male-reproductive-potential SNP markers that can significantly alter the binding affinity of TATA-binding protein (TBP) for these promoters. Among them, there are candidate SNP markers of spermatogenesis disorders (e.g., rs1402972626), pediatric cancer (e.g., rs1483581212) as well as male anxiety damaging family relationships and mother’s and children’s health (e.g., rs187456378). First of all, we selectively verified in vitro both absolute and relative values of the analyzed TBP–promoter affinity, whose Pearson’s coefficients of correlation between predicted and measured values were r = 0.84 (significance p p p Conclusions Overall, our findings seem to support a self-domestication syndrome with disruptive natural selection by male reproductive potential preventing Y-linked underexpression of a protein.

Background Hemoglobin is a tetramer consisting of two α-chains and two β-chains of globin. Hereditary aberrations in the synthesis of one of the globin chains are at the root of thalassemia, one of the most prevalent monogenic diseases worldwide. In humans, in addition to α- and β-globins, embryonic zeta-globin and fetal γ-globin are expressed. Immediately after birth, the expression of fetal Aγ- and Gγ-globin ceases, and then adult β-globin is mostly expressed. It has been shown that in addition to erythroid cells, hemoglobin is widely expressed in nonerythroid cells including neurons of the cortex, hippocampus, and cerebellum in rodents; embryonic and adult brain neurons in mice; and mesencephalic dopaminergic brain cells in humans, mice, and rats. Lately, there is growing evidence that different forms of anemia (changes in the number and quality of blood cells) may be involved in (or may accompany) the pathogenesis of various cognitive and mental disorders, such as Alzheimer’s and Parkinson’s diseases, depression of various severity levels, bipolar disorders, and schizophrenia. Higher hemoglobin concentrations in the blood may lead to hyperviscosity, hypovolemia, and lung diseases, which may cause brain hypoxia and anomalies of brain function, which may also result in cognitive deficits. Methods In this study, a search for unannotated single-nucleotide polymorphisms (SNPs) of erythroid genes was initially performed using our previously created and published SNP-TATA_Z-tester, which is a Web service for computational analysis of a given SNP for in silico estimation of its influence on the affinity of TATA-binding protein (TBP) for TATA and TATA-like sequences. The obtained predictions were finally verified in vitro by an electrophoretic mobility shift assay (EMSA). Results On the basis of these experimental in vitro results and literature data, we studied TATA box SNPs influencing both human erythropoiesis and cognitive abilities. For instance, TBP–TATA affinity in the HbZ promoter decreases 6.6-fold as a result of a substitution in the TATA box (rs113180943), thereby possibly disrupting stage-dependent events of “switching” of hemoglobin genes and thus causing erythroblastosis. Therefore, rs113180943 may be a candidate marker of severe hemoglobinopathies with comorbid cognitive and mental disorders associated with cerebral blood flow disturbances. Conclusions The literature data and experimental and computations results suggest that the uncovered candidate SNP markers of erythropoiesis anomalies may also be studied in cohorts of patients with cognitive and/or mental disorders with comorbid erythropoiesis diseases in comparison to conventionally healthy volunteers. Research into the regulatory mechanisms by which the identified SNP markers contribute to the development of hemoglobinopathies and of the associated cognitive deficits will allow physicians not only to take timely and adequate measures against hemoglobinopathies but also to implement strategies preventing cognitive and mental disorders.

Using our previously published Web service SNP_TATA_Comparator, we conducted a genome-wide study of single-nucleotide polymorphisms (SNPs) within core promoters of 68 human rheumatoid arthritis (RA)-related genes. Using 603 SNPs within 25 genes clinically associated with RA-comorbid disorders, we predicted 84 and 70 candidate SNP markers for overexpression and underexpression of these genes, respectively, among which 58 and 96 candidate SNP markers, respectively, can relieve and worsen RA as if there is a neutral drift toward susceptibility to RA. Similarly, we predicted natural selection toward susceptibility to RA for 8 immunostimulatory genes (e.g., IL9R) and 10 genes most often associated with RA (e.g., NPY). On the contrary, using 25 immunosuppressive genes, we predicted 70 and 109 candidate SNP markers aggravating and relieving RA, respectively (e.g., IL1R2 and TGFB2), suggesting that natural selection can simultaneously additionally yield resistance to RA. We concluded that disruptive natural selection of human immunostimulatory and immunosuppressive genes is concurrently elevating and reducing the risk of RA, respectively. So, we hypothesize that RA in human could be a self-domestication syndrome referring to evolution patterns in domestic animals. We tested this hypothesis by means of public RNA-Seq data on 1740 differentially expressed genes (DEGs) of pets vs. wild animals (e.g., dogs vs. wolves). The number of DEGs in the domestic animals corresponding to worsened RA condition in humans was significantly larger than that in the related wild animals (10 vs. 3). Moreover, much less DEGs in the domestic animals were accordant to relieved RA condition in humans than those in the wild animals (1 vs. 8 genes). This indicates that the anthropogenic environment, in contrast to a natural one, affects gene expression across the whole genome (e.g., immunostimulatory and immunosuppressive genes) in a manner that likely contributes to RA. The difference in gene numbers is statistically significant as confirmed by binomial distribution (p < 0.01), Pearson’s χ2 (p < 0.01), and Fisher’s exact test (p < 0.05). This allows us to propose RA as a candidate symptom within a self-domestication syndrome. Such syndrome might be considered as a human’s payment with health for the benefits received during evolution.