The first and arguably most critical level of gene expression is transcription of the genetic information from DNA into RNA. Within this process, transcription initiation stands out as the key step that influences all downstream events. Central to initiation are promoters, DNA sequences that interact with the key enzyme of transcription, RNA polymerase. A single transcription unit may be controlled by one or multiple promoters, a strategy found across all domains of life. This review highlights how combinatorial promoter arrangements control gene expression in microorganisms, with brief comparisons to other organisms. We also explore how these promoter architectures function as sensors for stress-related molecules, such as antibiotics. We examine how these insights can be applied to predict previously unidentified mechanisms of antibiotic resistance. Finally, the use of dual-promoters in synthetic biology is outlined and discussed.

Heterogenous transcription start site (TSS) usage dictates the 5' leader structure and function of unspliced HIV-1 RNAs (usRNA). We and others have previously reported that expression and Rev/CRM1-mediated nuclear export of HIV-1 usRNA in macrophages activates MDA5, MAVS, and innate immune signaling cascades. In this study, we reveal that MDA5 sensing of viral usRNA is largely determined by TSS and cytoplasmic RNA function in macrophages. We show that HIV-1 usRNAs (cap1G) that are preferentially destined for dimerization and viral genome packaging are specifically targeted by MDA5, while efficiently translated (cap3G) usRNAs are immunologically silent. Using mutant viruses which generate usRNA with altered 5' leader structure, or inclusion of a retroviral constitutive transport element which drives mRNA-like NXF1-dependent nuclear export of viral usRNA, we show that transcription initiation site and nuclear export pathway choice are major determinants of both HIV-1 usRNA immunogenicity and cytoplasmic function. In total, we identify innate immune response modulation as a consequence of the well-conserved heterogenous TSS usage among ancestral and extant HIV-1 isolates in humans, and shed light on how MDA5 discriminates between self and non-self RNAs.

Gene transcription is activated through the interaction between cis-regulatory elements (CREs) and transcription factors (TFs). CREs serve as templates to provide binding sites, whereas TFs provide functions to directly initiate transcription. Current research mainly focuses on deciphering cis-regulatory code, while neglecting TF regulatory code. However, CREs alone are not sufficient to determine the binding of TFs, which makes the interpretation of cis-regulatory code ambiguous. In this study, we systematically analyze 13 TF binding profiles associated with transcription initiation and encode them as TF sequence to explore the TF regulatory code. Furthermore, we propose a deep learning model named DeepTF to predict gene expression from TF sequence. Results show that TF binding exhibits conserved positional preferences and combinatorial patterns in promoters and DeepTF is able to predict gene expression with high accuracy (AUROC = 0.97). Meanwhile, cross-cell-line validation (AUROC > 0.90) further confirms the model's transferability. Model interpretation reveals DeepTF successfully captures the TF regulatory grammar associated with gene expression. Compared with the cis-regulatory code, our proposed TF regulatory code is better suited for investigating the relationship between TF binding positions or combinatorial patterns and gene expression. Collectively, DeepTF simplifies gene expression prediction and provides clear biological insights of transcriptional regulation.

Histone H3K9me3 silences repetitive elements and represses non-lineage genes during early development, but its role in organogenesis is understudied. Here, we show that H3K9me3 deposition is dynamic during epidermis morphogenesis and essential for lineage diversification. We ablate Suv39h1, Suv39h2, and Setdb1 histonemethyltransferases, in the embryonic mouse epidermis, to induce H3K9me3 loss. This causes complete failure of keratinocyte differentiation, skin barrier formation, hair follicle development, and Merkel cell specification. Single-cell transcriptomics reveals aberrant cell fates with mixed epidermal subtype identities and dysregulated non-lineage and lineage-specific transcription programs. Affected pathways include differentiation, metabolism, cell cycle, cytoskeletal organization, and extracellular matrix. H3K9me3 primarily restricts RNA Pol II transcription initiation at key developmental promoters and enhancers and has minimal direct effect on promoter-proximal pause release. We uncover a cooperative and indispensable role for Suv39h1, Suv39h2, and Setdb1 in gene expression control of epidermal morphogenesis, establishing H3K9me3 as a critical developmental determinant of skin organogenesis.

RNA polymerase II transcribes all protein-coding genes in eukaryotes, with its C-terminal domain (CTD) acting as a regulatory platform throughout the transcription cycle. Despite its simple sequence, the conserved heptad repeats encode a regulatory grammar critical for transcription initiation, elongation, and termination. Using structural analysis, single-molecule imaging, and synthetic CTD-engineered Pol II constructs, we define the minimal sequence requirements across transcriptional stages. We show that while SP motifs are essential phosphorylation sites, flanking residues can tolerate variability. In contrast, the periodic positioning of tyrosine residues is indispensable for pre-initiation complex (PIC) formation through interactions with the Mediator, which are disrupted upon Ser5 phosphorylation, triggering promoter escape. This supports a model where sequential tyrosine engagement stabilizes PIC assembly, and progressive phosphorylation propels Pol II escape. Site-specific Ser2/5 phosphorylation orchestrates 3'-end processing factor recruitment. These findings define the functional grammar of the Pol II CTD and explain how a low-complexity sequence achieves regulatory specificity.

Spt5 is a universally conserved multidomain transcription elongation factor that acts as a component of all Pol II elongation complexes. Structural studies indicate that several of Spt5's central KOW domains lie adjacent to the Pol II stalk, composed of subunits Rpb4 and Rpb7. However, the in vivo functions of Spt5's central KOW domains are unknown. Here we show that Spt5 and Rpb4/7 jointly modulate 3'-end formation and co-transcriptional chromatin integrity in Saccharomyces cerevisiae. We identify mutations in the SPT5 KOW2-3 domains and RPB7 that cause cryptic initiation of transcription and alter 3'-end formation of RNA transcripts. Molecular readthrough assays reveal allele-specific changes at both GAL10 and SNR13, consistent with impacts on termination mediated by Cleavage and Polyadenylation Factor (CPF)-Cleavage Factor (CF) and the Nrd1-Nab3-Sen1 (NNS) pathway. Proteomic experiments with isolated KOW domains enrich factors from both pathways as well as chromatin regulators, overlapping known Rpb7 interactors. Together, these findings support a model in which the Spt5 KOW2-4/Pol II stalk region acts as a recruitment platform that coordinates pre-mRNA processing and chromatin dynamics during elongation, revealing new roles for the central KOW domains of Spt5.

The human P2X7 purinergic receptor is an ATP-gated ion channel expressed across a wide range of cell types, including immune, epithelial, glial, and various cancer cells. Despite its broad expression and diverse functions, little is known about the transcriptional regulation of the P2RX7 gene. In this study, the transcription initiation site of the P2RX7 gene was mapped 149/148 nucleotides upstream of the ATG start codon by using a sequencing-based primer extension method. To characterise the promoter region of the P2RX7 gene, eight DNA fragments were cloned into a dual-colour luciferase reporter vector system. The promoter activity of each fragment was assessed in transfected HEK-293 and COS-7 cells. Predicted promoter binding sequences were identified, and fluorescence-based super-electrophoretic mobility shift assay confirmed that the transcription factors YY1 and C/EBPβ directly bind to the P2RX7 promoter region.

A key step in gene expression is the opening of DNA at the transcription start site (TSS), allowing RNA polymerase to begin RNA synthesis. In eukaryotes, RNA polymerase II initiates transcription within the pre-initiation complex (PIC), which includes TFIIH. The XPB subunit of TFIIH drives DNA unwinding at the TSS through ATP-dependent activity. However, some promoters enable transcription without XPB, likely due to intrinsically unstable DNA that does not require active unwinding. This mechanism may reflect an evolutionary remnant of archaeal transcription.

Trimethylation of histone 3 lysine 4 (H3K4me3) is a mark of active transcription, and its regulatory role in RNA polymerase II-mediated transcription has been well-studied. However, if and how this mark regulates RNA polymerase I (RNA Pol I) is not known. Here, we used customized genome assemblies for rDNA to demonstrate that KMT2A and KMT2F bind to entire rDNA loci. The binding of these enzymes was mirrored by the binding of H3K4me2 and H3K4me3 marks. Using biochemical assays, we demonstrate the interaction of KMT2-specific subunits with RNA Pol I transcriptional machinery. Our findings reveal KMT2F as the primary KMT depositing the H3K4me3 on rDNA. Loss of H3K4me3 adversely affects the epigenetic landscape and leads to repression of the rDNA locus. Mechanistically, using mammalian cells as a model system, we demonstrate that KMT2F promotes the formation of the pre-initiation complex by RNA Pol I. Our findings highlight the thus far undiscovered role of H3K4me3 in the transcriptional initiation of rDNA genes.

Aberrant activation of TAL1 , a key oncogenic driver, defines a major subgroup comprising ∼30% of childhood T-lineage acute lymphoblastic leukemias (T-ALLs). We and others have shown that somatic non-coding mutations within upstream and intronic cis -regulatory regions of TAL1 contribute to transformation by creating binding sites for MYB and other transcription factors. Here we investigated cis -regulatory mechanisms mediated by somatic mutations occurring in an intergenic region located 29 kilobase pairs downstream of the canonical TAL1 transcription initiation site, implicated in 6% of TAL1 -expressing T-ALLs. These somatic variants include i) complex indels resulting in de novo MYB transcription factor binding sites (TFBSs) and ii) internal tandem duplications (ITDs) encompassing canonical MYB TFBSs. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed binding of the TAL1 core regulatory circuit (CRC) transcription factors MYB, GATA3, and RUNX1, resulting in enhancer activity mediated by sequences with the mutant allele. Strikingly, ChIP-seq peaks for the repressive H3K27me3 mark and the active H3K27ac mark co-existed across TAL1 regulatory sequences but enriched for different haplotypes. TAL1 transcription from the mutant haplotype initiated from a promoter located within exon 4 of the canonical TAL1 transcript, resulting in a short isoform normally expressed by hematopoietic stem cells (HSC). Interestingly, neither the isoform expression nor the enhancer activity could be predicted by the sequence-to-function deep learning artificial intelligence (AI) model AlphaGenome, emphasizing the importance of experimental validation. Our findings indicate that selection for cis -regulatory, non-coding variants leads to reactivation of enhancers normally active in HSC but silenced in differentiated lineages during normal hematopoietic cell development.

The transition of RNA polymerase II from initiation to elongation requires the timely exchange of many factors. Some initiation factors and elongation factors contact the same RNA polymerase II surfaces in an apparently mutually exclusive manner. For example, space occupied by the initiation factor TFIIH and elongation factor Spt4/5 (also known as DSIF) are predicted to overlap. To determine whether Spt4/5 actively displaces TFIIH, or binds only after TFIIH dissociates, labeled yeast nuclear extracts were combined with single-molecule fluorescence microscopy to observe their dynamics in real time. TFIIH categorically dissociates before Spt4/5 arrives, with a mean interval of ~35 seconds. Therefore, Spt4/5 binding is not required to dissociate TFIIH from RNA polymerase II.

Astrocytes are central regulators of neuroinflammation, yet the mechanisms by which they convert common inflammatory signals into cell type-specific transcriptional responses remain poorly understood. Here we mapped transcription initiation genome-wide in primary mouse astrocytes stimulated with interleukin-1B (IL-1B) and defined the active regulatory elements that drive astrocyte reactivity. We find that inducible enhancer transcription in astrocytes is encoded by a transcription-initiation grammar in which lineage-restricted transcription factors, particularly NFIA and TEAD4, cooperate with inflammatory transcription factors such as NF-κB, AP-1 and IRF to drive stimulus-dependent transcription activation. The motifs of these inflammatory transcription factors show a strong positional bias upstream of induced transcription start sites, supporting their direct role in controlling initiation upon stimulation. Moreover, NF-κB and TEAD4 motifs are preferentially associated with sites showing altered patterns of transcription initiation in response to inflammatory stimulus. Comparison with stimulated macrophages revealed that, despite substantial overlap in induced genes, astrocytes exhibit a largely distinct enhancer repertoire, indicating that shared inflammatory signals are interpreted through cell type-specific regulatory landscapes. Finally, transcribed astrocyte regulatory elements are functionally conserved in human astrocytes and are enriched for genetic risk variants associated with neurological disorders. Together, these findings define a cell type-specific regulatory logic for astrocyte inflammatory responses and link astrocyte enhancer regulation to human disease susceptibility.

Gene expression begins with transcription initiation. However, the control of gene activation encompasses events that precede transcription and extend beyond it, and that are prominently regulated by the noncoding genome. Here we discuss the pivotal roles of long noncoding RNAs (lncRNAs) and the related enhancer RNAs (eRNAs) as genomic rheostats of the magnitude and duration of activation of transcription by RNA polymerase II, acting in trans by influencing general transcription factors and co-transcriptional and post-transcriptional processes, and in cis through their interactions with enhancers, promoters and other noncoding RNA genes. We further discuss how these lncRNAs are often produced at active enhancers or boundaries of topologically associating domains and can accumulate in biomolecular condensates. We propose models of lncRNA action that account for pre-existing chromatin states, and for the interactions between DNA, RNA and proteins. Understanding how lncRNAs fit within the dynamic process of transcription activation is essential for obtaining a comprehensive view of the regulation of gene expression.

BTF3, also known as BETA-NAC, BTF3b, BTF3a, and NACB, is a transcription factor originally isolated from HeLa cell extracts. It forms stable complexes with RNA polymerase II and plays a critical role in transcription initiation. Although aberrant BTF3 expression has been reported in certain malignancies, its overall role across diverse tumor types remains poorly defined. This study aimed to elucidate the functional implications of BTF3 across various cancers. BTF3 expression was analyzed using datasets from The Cancer Genome Atlas (TCGA), including TCGA_GTEx, TCGA unpaired, and TCGA paired samples. The prognostic significance of BTF3 across 33 tumor types was evaluated using Kaplan-Meier survival and univariate Cox regression analyses. In cancers where BTF3 expression showed prognostic value, additional clinical correlation analyses were performed. Clear cell renal carcinoma (sample size >500) was selected for nomogram construction to illustrate BTF3's prognostic relevance. The association between BTF3 expression and immune cell infiltration was also examined, alongside functional enrichment analysis to explore involved signaling pathways. BTF3 displayed variable expression across tumor types and was significantly correlated with several clinical parameters. Survival and regression analyses identified BTF3 as a prognostic marker in specific cancers. The nomogram model for clear cell renal carcinoma further supported its predictive value. BTF3 expression was also associated with features of the tumor immune microenvironment. Functional enrichment analysis implicated signaling pathways, including the tuberous sclerosis complex/mechanistic target of rapamycin (TSC/mTOR), phosphoinositide 3-kinase/AKT (PI3K/AKT), and rat sarcoma/mitogen-activated protein kinase (RAS/MAPK) in BTF3-associated tumorigenesis. These findings underscore the heterogeneous expression of BTF3 across malignancies and highlight its potential involvement in modulating the tumor immune landscape and oncogenic signaling pathways. The results expand our understanding of BTF3's role in cancer biology and suggest mechanistic links to well-characterized tumorigenic processes. However, further experimental validation is necessary to substantiate these associations. BTF3 may serve as a promising prognostic biomarker and a potential target for immunotherapeutic intervention across multiple cancer types.

Triple-negative breast cancer (TNBC) is frequently characterized by notably elevated Ki-67 expression, a hallmark of uncontrolled rapid cell-cycle progression. However, the underlying mechanisms remain unclear, leading to limited therapeutic options. In this study, hub gene was identified through integrated bioinformatic analysis of public datasets (TCGA-BRCA and METABRIC). Subsequent functional validation was performed both in vitro and in vivo using siRNA-mediated knockdown and small-molecule inhibitors. Phenotypic effects-including cell viability, cell cycle distribution, DNA synthesis, and clonogenic survival-were comprehensively assessed using MTT assays, flow cytometry, EdU, and colony formation assays. Protein-level changes were confirmed by Western blotting and immunohistochemistry (IHC). To dissect the transcriptional regulation of the key hub gene TTK, we first predicted potential upstream transcription factors using the JASPAR database; binding specificity was then validated through in silico motif analysis, luciferase reporter assays, and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). The mitotic kinase TTK is significantly overexpressed in TNBC compared with non-TNBC breast cancers. Notably, TTK overexpression exhibited a strong positive correlation with elevated Ki-67 indices and reduced overall survival in TNBC patients. Functional validation demonstrated that pharmacological or genetic inhibition of TTK effectively induced G2/M cell-cycle arrest and potently suppressed TNBC proliferation in both in vitro cell cultures and in vivo xenograft models. Mechanistically, TTK overexpression stems from enhanced transcriptional initiation driven by the transcription factor NFYA binding to the CCAAT box in the TTK promoter-an interaction newly identified here. Concurrently, TTK blockade disrupted spindle assembly checkpoint (SAC) signaling via BUB1B/MAD1L1 downregulation, triggering mitotic arrest and catastrophe. Collectively, these findings establish TTK as a key cell-cycle regulator driving TNBC proliferation. More importantly, targeting mitotic control through TTK inhibition represents an efficient strategy to impede the aberrantly fast cell cycle progression in TNBC.

Post-transcriptional regulation is an adaptive response used by living systems to alter protein levels independently of transcription initiation. In bacteria, one mechanism of post-transcriptional regulation involves the binding of small, non-coding RNAs (sRNAs) to target mRNAs to regulate their translation into protein. This regulation generally includes an RNA-binding protein that facilitates the sRNA::mRNA interaction. In the etiological agent of Lyme disease, Borreliella burgdorferi, over 400 temperature-dependent sRNAs have been identified. Here, we characterized the sRNA SR0735, which is upregulated at 37°C (characteristic of mammalian infection). Transcriptomic and proteomic analyses revealed that deleting SR0735 in B. burgdorferi resulted in reduced production of virulence-associated, RpoS-dependent proteins due to a reduction of RpoS and its upstream regulator BosR. We thus renamed SR0735 as BasA for BosR-associated sRNA locus A. Consistent with previous studies that identified BosR as an RNA-binding protein, we demonstrate here that BosR can bind BasA in vitro. Importantly, the loss of BasA significantly attenuated B. burgdorferi infectivity during murine infection, which is consistent with the reduced levels of BosR and RpoS observed. The emerging model suggests that BasA is required for optimal levels of BosR protein through the post-transcriptional regulation of bosR mRNA, which, in turn, promotes the production of RpoS and its downstream genes required for mammalian infectivity and pathogenesis.

Biofilm-associated infections caused by Staphylococcus aureus (S. aureus) remain notoriously difficult to treat due to their pronounced tolerance to most antibiotics. Here, we evaluated the antibiofilm efficacy of the natural product antibiotic corallopyronin A (CorA) across a panel of strains, including clinically relevant strains differing in their biofilm-forming capacities and antibiotic resistance profiles. CorA is an alpha-pyrone antibiotic produced by Corallococcus coralloides. It targets the switch region of the bacterial DNA-dependent RNA polymerase, thereby blocking transcription initiation at a site distinct from the rifampicin-binding pocket, and displays potent activity against staphylococci, including MRSA and rifampicin-resistant S. aureus. In vitro, CorA eradicated and inhibited biofilm formation, outperforming the biofilm-active antibiotics dalbavancin and rifampicin both in optical density measurements and in microscopic analyses. Importantly, CorA had activity against rifampicin-resistant strains in these assays. In a murine foreign body infection model with S. aureus SA113, CorA treatment resulted in a > 4-log10 reduction in bacterial loads on implanted devices and surrounding tissues, comparable with high-dose rifampicin, and significantly reduced local inflammation. These findings position CorA as a promising candidate for preventing and managing staphylococcal biofilm-associated infections, warranting further investigation into its clinical potential.

The 5' untranslated region (5' UTR) of an mRNA is classically viewed as a regulatory region that controls the amount of protein production, but not the resulting protein sequence. Here, we demonstrate that 5' UTR length plays a direct role in alternative N-terminal protein isoform production by controlling start codon selection. We find that very short 5' UTRs enhance leaky ribosome scanning, thereby promoting the production of truncated alternative N-terminal protein isoforms. We also show that endogenous changes in 5' UTR length due to alternative transcription initiation can tune the relative abundance of alternative N-terminal isoforms from the same gene. In addition, we identify mutations in rare genetic diseases that alter 5' UTR length, including a deletion in the VHL 5' UTR in von Hippel-Lindau disease that shifts translation toward the shorter VHLp19 isoform. Together, our results implicate 5' UTR length as a determinant of alternative N-terminal isoform production and reveal an underappreciated mechanism by which noncoding changes can reshape the proteome.

Life has the property to produce from a single genome, the collection of DNA molecules, different cell types, as well as mechanisms for bacteria to adapt to environmental changes. Although regulation can happen at different levels, regulation of transcription initiation, the start of copying DNA into RNA, is the most studied level in bacteria. The collection of regulators and their regulated elements defines transcriptional regulatory networks (TRNs), whose study has driven relevant areas, such as antimicrobial resistance. Their analyses and understanding depend on some few highly manually curated databases. The traditional way to reconstruct these networks is by manual curation of the literature, which is accurate, but also demanding and time-consuming. These limitations have resulted in the shortage and incompleteness of bacterial TRNs. Here, we present a novel ensemble model approach using two GPT-based foundation models (LLaMA-3 and GPT-4o mini) to effectively reconstruct TRNs from the literature. We applied a supervised fine-tuning strategy with sentences from Escherichia coli literature to train models to predict the type of regulatory effect between a transcription factor and a regulated element (gene/operon). To evaluate the performance of reconstructing a curated TRN, we used 264 full-text articles of Salmonella Typhimurium, a pathogen of clinical interest. With the test data, both models obtained significant performance (F1-Score > 0.87, Matthews correlation coefficient > 0.82). For the curated TRN reconstruction, the ensemble approach using the agreement of models correctly reconstructed 80% of the TRN (Recall: 0.80, F1-score: 0.64). We applied the approach to reconstruct a large Salmonella TRN using the literature available at the time on transcriptional regulation of this bacterium (2,278 articles). This network was described with network metrics, over-representation analyses, and compared to existing biological knowledge. Our approach overtook the performance of prior works predicting the effect of the interaction. The analysis of the TRN of the 2,278 articles showed the effectiveness of our approach to reconstruct TRNs of diverse bacteria, as the network aligns with biological knowledge. Thus, our work may support the study of bacteria of biological and clinical interest, especially those without a reconstructed TRN.

Mitochondrial DNA (mtDNA) transcription is essential for cellular energy production and is carried out by a streamlined transcription system in which transcription factor A (TFAM), transcription factor B2 (TFB2M), and the mitochondrial RNA polymerase (PolRMT) assemble at defined promoters to initiate transcription. Previous structural studies elucidated the core initiation mechanism but relied on truncated promoter templates that excluded upstream regulatory DNA interactions. Here, we present two conformations of mitochondrial transcription initiation complexes assembled on the heavy-strand promoter (HSP): a TFAM-bound complex with extended upstream DNA and a TFAM-free complex containing short linear DNA. The TFAM-bound structure reveals a transcription-stimulatory interface between PolRMT and the upstream promoter region (UPR) enabled by TFAM-induced promoter bending. Consistent with this structural observation, UPR truncation reduces transcription from all mtDNA promoters, an effect abolished by mutation of the PolRMT interface. In contrast, the TFAM-free structure reveals a transcription-inhibitory interaction of linear upstream DNA with the PolRMT tether helix, which would sterically clash with TFAM binding. Deletion of the tether helix increases off-target transcription, supporting an autoinhibitory role that enhances promoter specificity. Together, these findings reveal how TFAM-shaped promoter architecture and PolRMT regulatory elements coordinate mitochondrial transcription initiation and regulation.