Physical Peculiarity of Two Sites in Human Promoters: Universality and Diverse Usage in Gene Function.

Since the discovery of physical peculiarities around transcription start sites (TSSs) and a site corresponding to the TATA box, research has revealed only the average features of these sites. Unsettled enigmas include the individual genes with these features and whether they relate to gene function. Herein, using 10 physical properties of DNA, including duplex DNA free energy, base stacking energy, protein-induced deformability, and stabilizing energy of Z-DNA, we clarified for the first time that approximately 97% of the promoters of 21,056 human protein-coding genes have distinctive physical properties around the TSS and/or position -27; of these, nearly 65% exhibited such properties at both sites. Furthermore, about 55% of the 21,056 genes had a minimum value of regional duplex DNA free energy within TSS-centered ±300 bp regions. Notably, distinctive physical properties within the promoters and free energies of the surrounding regions separated human protein-coding genes into five groups; each contained specific gene ontology (GO) terms. The group represented by immune response genes differed distinctly from the other four regarding the parameter of the free energies of the surrounding regions. A vital suggestion from this study is that physical-feature-based analyses of genomes may reveal new aspects of the organization and regulation of genes.

promSEMBLE: Hard Pattern Mining and Ensemble Learning for Detecting DNA Promoter Sequences.

Accurate identification of DNA promoter sequences is of crucial importance in unraveling the underlying mechanisms that regulate gene transcription. Initiation of transcription is controlled through regulatory transcription factors binding to promoter core regions in the DNA sequence. Detection of promoter regions is necessary if we are to build genetic regulatory networks for biomedical and clinical applications, and for identification of rarely expressed genes. We propose a novel ensemble learning technique using deep recurrent neural networks with convolutional feature extraction and hard negative pattern mining to detect several types of promoter sequences, including promoter sequences with the TATA-box and without the TATA-box, within DNA sequences of four different species. Using extensive independent tests and previously published results, we demonstrate that our method sets a new state-of-the-art of over 98% Matthews correlation coefficient in all eight organism categories for recognizing the stretch of base pairs that code for the promoter region within DNA sequences.

Taf1 N-terminal domain 2 (TAND2) of TFIID promotes formation of stable and mobile unstable TBP-TATA complexes.

In eukaryotes, TATA-binding protein (TBP) occupancy of the core promoter globally correlates with transcriptional activity of class II genes. Elucidating how TBP is delivered to the TATA box or TATA-like element is crucial to understand the mechanisms of transcriptional regulation. A previous study demonstrated that the inhibitory DNA binding (IDB) surface of human TBP plays an indispensable role during the two-step formation of the TBP-TATA complex, first assuming an unstable and unbent intermediate conformation, and subsequently converting slowly to a stable and bent conformation. The DNA binding property of TBP is altered by physical contact of this surface with TBP regulators. In the present study, we examined whether the interaction between Taf1 N-terminal domain 2 (TAND2) and the IDB surface affected DNA binding property of yeast TBP by exploiting TAND2-fused TBP derivatives. TAND2 promoted formation of two distinct types of TBP-TATA complexes, which we arbitrarily designated as complex I and II. While complex I was stable and similar to the well-characterized original TBP-TATA complex, complex II was unstable and moved along DNA. Removal of TAND2 from TBP after complex formation revealed that continuous contact of TAND2 with the IDB surface was required for formation of complex II but not complex I. Further, TFIIA could be incorporated into the complex of TAND2-fused TBP and the TATA box, which was dependent on the amino-terminal non-conserved region of TBP, implying that this region could facilitate the exchange between TAND2 and TFIIA on the IDB surface. Collectively, these findings provide novel insights into the mechanism by which TBP is relieved from the interaction with TAND to bind the TATA box or TATA-like element within promoter-bound TFIID.

A comparative analysis of stably expressed genes across diverse angiosperms exposes flexibility in underlying promoter architecture.

Promoters regulate both the amplitude and pattern of gene expression-key factors needed for optimization of many synthetic biology applications. Previous work in Arabidopsis found that promoters that contain a TATA-box element tend to be expressed only under specific conditions or in particular tissues, while promoters that lack any known promoter elements, thus designated as Coreless, tend to be expressed more uniformly. To test whether this trend represents a conserved promoter design rule, we identified stably expressed genes across multiple angiosperm species using publicly available RNA-seq data. Comparisons between core promoter architectures and gene expression stability revealed differences in core promoter usage in monocots and eudicots. Furthermore, when tracing the evolution of a given promoter across species, we found that core promoter type was not a strong predictor of expression pattern. Our analysis suggests that core promoter types are correlative rather than causative in promoter expression patterns and highlights the challenges in finding or building constitutive promoters that will work across diverse plant species.

Transcription factor IID parks and drives preinitiation complexes at sharp or broad promoters.

Core promoters are sites where transcriptional regulatory inputs of a gene are integrated to direct the assembly of the preinitiation complex (PIC) and RNA polymerase II (Pol II) transcription output. Until now, core promoter functions have been investigated by distinct methods, including Pol II transcription initiation site mappings and structural characterization of PICs on distinct promoters. Here, we bring together these previously unconnected observations and hypothesize how, on metazoan TATA promoters, the precisely structured building up of transcription factor (TF) IID-based PICs results in sharp transcription start site (TSS) selection; or, in contrast, how the less strictly controlled positioning of the TATA-less promoter DNA relative to TFIID-core PIC components results in alternative broad TSS selections by Pol II.

Promoter-proximal nucleosomes attenuate RNA polymerase II transcription through TFIID.

A nucleosome is typically positioned with its proximal edge (NPE) ∼50 bp downstream from the transcription start site of metazoan RNA polymerase II promoters. This +1 nucleosome has distinctive characteristics, including the presence of variant histone types and trimethylation of histone H3 at lysine 4. To address the role of these features in transcription complex assembly, we generated templates with four different promoters and nucleosomes located at a variety of downstream positions, which were transcribed in vitro using HeLa nuclear extracts. Two promoters lacked TATA elements, but all supported strong initiation from a single transcription start site. In contrast to results with minimal in vitro systems based on the TATA-binding protein (TBP), TATA promoter templates with a +51 NPE were transcriptionally inhibited in extracts; activity continuously increased as the nucleosome was moved downstream to +100. Inhibition was much more pronounced for the TATA-less promoters: +51 NPE templates were inactive, and substantial activity was only seen with the +100 NPE templates. Substituting the histone variants H2A.Z, H3.3, or both did not eliminate the inhibition. However, addition of excess TBP restored activity on nucleosomal templates with TATA promoters, even with an NPE at +20. Remarkably, nucleosomal templates with histone H3 trimethylated at lysine 4 are active with an NPE at +51 for both TATA and TATA-less promoters. Our results strongly suggest that the +1 nucleosome interferes with promoter recognition by TFIID. This inhibition can be overcome with TBP alone at TATA promoters or through positive interactions with histone modifications and TFIID.

Analysis of the Drosophila and human DPR elements reveals a distinct human variant whose specificity can be enhanced by machine learning.

The RNA polymerase II core promoter is the site of convergence of the signals that lead to the initiation of transcription. Here, we performed a comparative analysis of the downstream core promoter region (DPR) in Drosophila and humans by using machine learning. These studies revealed a distinct human-specific version of the DPR and led to the use of machine learning models for the identification of synthetic extreme DPR motifs with specificity for human transcription factors relative to Drosophila factors and vice versa. More generally, machine learning models could similarly be used to design synthetic DNA elements with customized functional properties.

Core Promoter Regions of Antisense and Long Intergenic Non-Coding RNAs.

RNA polymerase II (POL II) is responsible for the transcription of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs). Previously, we have shown the evolutionary invariance of the structural features of DNA in the POL II core promoters of the precursors of mRNAs. In this work, we have analyzed the POL II core promoters of the precursors of lncRNAs in Homo sapiens and Mus musculus genomes. Structural analysis of nucleotide sequences in positions -50, +30 bp in relation to the TSS have shown the extremely heterogeneous 3D structure that includes two singular regions - hexanucleotide "INR" around the TSS and octanucleotide "TATA-box" at around ~-28 bp upstream. Thus, the 3D structure of core promoters of lncRNA resembles the architecture of the core promoters of mRNAs; however, textual analysis revealed differences between promoters of lncRNAs and promoters of mRNAs, which lies in their textual characteristics; namely, the informational entropy at each position of the nucleotide text of lncRNA core promoters (by the exception of singular regions) is significantly higher than that of the mRNA core promoters. Another distinguishing feature of lncRNA is the extremely rare occurrence in the TATA box of octanucleotides with the consensus sequence. These textual differences can significantly affect the efficiency of the transcription of lncRNAs.

Candidate SNP Markers Significantly Altering the Affinity of TATA-Binding Protein for the Promoters of Human Hub Genes for Atherogenesis, Atherosclerosis and Atheroprotection.

Atherosclerosis is a systemic disease in which focal lesions in arteries promote the build-up of lipoproteins and cholesterol they are transporting. The development of atheroma (atherogenesis) narrows blood vessels, reduces the blood supply and leads to cardiovascular diseases. According to the World Health Organization (WHO), cardiovascular diseases are the leading cause of death, which has been especially boosted since the COVID-19 pandemic. There is a variety of contributors to atherosclerosis, including lifestyle factors and genetic predisposition. Antioxidant diets and recreational exercises act as atheroprotectors and can retard atherogenesis. The search for molecular markers of atherogenesis and atheroprotection for predictive, preventive and personalized medicine appears to be the most promising direction for the study of atherosclerosis. In this work, we have analyzed 1068 human genes associated with atherogenesis, atherosclerosis and atheroprotection. The hub genes regulating these processes have been found to be the most ancient. In silico analysis of all 5112 SNPs in their promoters has revealed 330 candidate SNP markers, which statistically significantly change the affinity of the TATA-binding protein (TBP) for these promoters. These molecular markers have made us confident that natural selection acts against underexpression of the hub genes for atherogenesis, atherosclerosis and atheroprotection. At the same time, upregulation of the one for atheroprotection promotes human health.

Structural basis for TBP displacement from TATA box DNA by the Swi2/Snf2 ATPase Mot1.

The Swi2/Snf2 family transcription regulator Modifier of Transcription 1 (Mot1) uses adenosine triphosphate (ATP) to dissociate and reallocate the TATA box-binding protein (TBP) from and between promoters. To reveal how Mot1 removes TBP from TATA box DNA, we determined cryogenic electron microscopy structures that capture different states of the remodeling reaction. The resulting molecular video reveals how Mot1 dissociates TBP in a process that, intriguingly, does not require DNA groove tracking. Instead, the motor grips DNA in the presence of ATP and swings back after ATP hydrolysis, moving TBP to a thermodynamically less stable position on DNA. Dislodged TBP is trapped by a chaperone element that blocks TBP's DNA binding site. Our results show how Swi2/Snf2 proteins can remodel protein-DNA complexes through DNA bending without processive DNA tracking and reveal mechanistic similarities to RNA gripping DEAD box helicases and RIG-I-like immune sensors.

Broad compatibility between yeast UAS elements and core promoters and identification of promoter elements that determine cofactor specificity.

Three classes of yeast protein-coding genes are distinguished by their dependence on the transcription cofactors TFIID, SAGA, and Mediator (MED) Tail, but whether this dependence is determined by the core promoter, upstream activating sequences (UASs), or other gene features is unclear. Also unclear is whether UASs can broadly activate transcription from the different promoter classes. Here, we measure transcription and cofactor specificity for thousands of UAS-core promoter combinations and find that most UASs broadly activate promoters regardless of regulatory class, while few display strong promoter specificity. However, matching UASs and promoters from the same gene class is generally important for optimal expression. We find that sensitivity to rapid depletion of MED Tail or SAGA is dependent on the identity of both UAS and core promoter, while dependence on TFIID localizes to only the promoter. Finally, our results suggest the role of TATA and TATA-like promoter sequences in MED Tail function.

RNA Polymerase II transcription independent of TBP in murine embryonic stem cells.

Transcription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the pre-initiation complex onto promoter DNA. Decades of research have shown that the TATA-box binding protein (TBP) is essential for Pol II loading and initiation. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells has no global effect on ongoing Pol II transcription. In contrast, acute TBP depletion severely impairs RNA Polymerase III initiation. Furthermore, Pol II transcriptional induction occurs normally upon TBP depletion. This TBP-independent transcription mechanism is not due to a functional redundancy with the TBP paralog TRF2, though TRF2 also binds to promoters of transcribed genes. Rather, we show that the TFIID complex can form and, despite having reduced TAF4 and TFIIA binding when TBP is depleted, the Pol II machinery is sufficiently robust in sustaining TBP-independent transcription.

Genetic Diversity of the LTR Region of Polish SRLVs and Its Impact on the Transcriptional Activity of Viral Promoters.

A long terminal repeat (LTR) plays an indispensable role in small ruminant lentivirus (SRLV) gene expression. In this study, we present the LTR sequence of Polish SRLVs representing different subtypes, and analyzed their impact on SRLV promoter activity, as measured in transient transfection assays. Although certain nucleotide motifs (AML(vis), TATA box and the polyadenylation site (AATAAA)) were conserved across sequences, numerous mutations within the LTR sequences have been identified. Single nucleotide polymorphisms (SNPs) were detected in both regulatory (AP-1, AP-4, Stat and Gas) and non-regulatory sequences, and subtype-specific genetic diversity in the LTR region of Polish SRLVs was observed. In vitro assays demonstrated subtype-specific functional differences between the LTR regions of distinct SRLV subtypes. Our results revealed that the promoter activity of Polish strains was lower (1.64-10.8-fold) than that noted for the K1514 reference strain; however, the differences in most cases were not statistically significant. The lowest promoter activity was observed for strains representing subtype A5 (mean 69.067) while the highest promoter activity was observed for strain K1514 representing subtype A1 (mean 373.48). The mean LTR activities of strains representing subtypes A12, A17, A23, A18 and A24 were 91.22, 137.21, 178.41, 187.05 and 236.836, respectively. The results of the inter-subtype difference analysis showed that the promoter activity of strains belonging to subtype A5 was significantly lower than that for subtype A12 strains (1.32-fold; p < 0.00). The promoter activities of the A5 strain were 1.98-fold and 2.58-fold less active than that of the A17 and A23 strains, and the promoter activities of A12 strains were 1.955 and 1.5 times lower than the promoter activity of A23 and A17 strains, respectively. Furthermore, the promoter activity of A17 strains was 1.3 lower than the promoter activity of A23 strains. Our findings suggest that subtype-specific genetic diversity, mainly in the transcription factor's binding sites, has an impact on their transcriptional activity, producing a distinct activity pattern for the subtypes. This study provides new information that is important for better understanding the function of the SRLV LTR. However, further research including more strains and subtypes as well as other cell lines is needed to confirm these findings.

Bases immediate upstream of the TATAAT box of the sigma 70 promoter of Escherichia coli significantly influence the activity of a model promoter by altering the bending angle of DNA.

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 R2 value being 0.8724. Retardation of electrophoretic mobility of the promoter DNAs correlated well with activity.

Tuning the Transcriptional Activity of the CaMV 35S Promoter in Plants by Single-Nucleotide Changes in the TATA Box.

Synthetic biology uses genetically encoded devices and circuits to implement novel complex functions in living cells and organisms. A hallmark of these genetic circuits is the interaction among their individual parts, according to predefined rules, to process cellular information and produce a circuit output or response. As the number of individual components in a genetic circuit increases, so does the number of interactions needed to achieve the correct behavior, and hence, a greater need to fine-tune the levels of expression of each component. Transcriptional promoters play a key regulatory role in genetic circuits, as they influence the levels of RNA and proteins produced. In multicellular organisms, such as plants, they can also determine developmental, spatial, and tissue-specific patterns of gene expression. The 35S promoter from the Cauliflower Mosaic Virus (CaMV 35S) is widely used in plant biotechnology to direct high levels of gene expression in a variety of plant species. We produced a library of 21 variants of the CaMV 35S promoter by introducing all single nucleotide substitutions to the promoter's TATA box sequence. We then characterized the activity of all variants in homozygous transgenic plants and showed that some of these variants have lower activity than the wild type in plants. These promoter variants could be used to fine-tune the behavior of synthetic genetic circuits in plants.

iPromoter-Seqvec: identifying promoters using bidirectional long short-term memory and sequence-embedded features.

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 .

Mutation analysis of the TATA box-binding protein (TBP) gene in Russian patients with spinocerebellar ataxia and Huntington disease-like phenotype.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

OsTBP2.1, a TATA-Binding Protein, Alters the Ratio of OsNRT2.3b to OsNRT2.3a and Improves Rice Grain Yield.

The OsNRT2.3a and OsNRT2.3b isoforms play important roles in the uptake and transport of nitrate during rice growth. However, it is unclear which cis-acting element controls the transcription of OsNRT2.3 into these specific isoforms. In this study, we used a yeast one-hybrid assay to obtain the TATA-box binding protein OsTBP2.1, which binds to the TATA-box of OsNRT2.3, and verified its important role through transient expression and RNA-seq. We found that the TATA-box of OsNRT2.3 mutants and binding protein OsTBP2.1 together increased the transcription ratio of OsNRT2.3b to OsNRT2.3a. The overexpression of OsTBP2.1 promoted nitrogen uptake and increased rice yield compared with the wild-type; however, the OsTBP2.1 T-DNA mutant lines exhibited the opposite trend. Detailed analyses demonstrated that the TATA-box was the key cis-regulatory element for OsNRT2.3 to be transcribed into OsNRT2.3a and OsNRT2.3b. Additionally, this key cis-regulatory element, together with the binding protein OsTBP2.1, promoted the development of rice and increased grain yield.

Evolutionary Invariant of the Structure of DNA Double Helix in RNAP II Core Promoters.

Eukaryotic and archaeal RNA polymerase II (POL II) machinery is highly conserved, regardless of the extreme changes in promoter sequences in different organisms. The goal of our work is to find the cause of this conservatism. The representative sets of aligned promoter sequences of fifteen organisms belonging to different evolutional stages were studied. Their textual profiles, as well as profiles of the indexes that characterize the secondary structure and the mechanical and physicochemical properties, were analyzed. The evolutionarily stable, extremely heterogeneous special secondary structure of POL II core promoters was revealed, which includes two singular regions-hexanucleotide "INR" around TSS and octanucleotide "TATA element" of about -28 bp upstream. Such structures may have developed at some stage of evolution. It turned out to be so well matched for the pre-initiation complex formation and the subsequent initiation of transcription for POL II machinery that in the course of evolution there were selected only those nucleotide sequences that were able to reproduce these structural properties. The individual features of specific sequences representing the singular region of the promoter of each gene can affect the kinetics of DNA-protein complex formation and facilitate strand separation in double-stranded DNA at the TSS position.

Plant_SNP_TATA_Z-Tester: A Web Service That Unequivocally Estimates the Impact of Proximal Promoter Mutations on Plant Gene Expression.

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.