Immune responses triggered by pathogen-associated molecular patterns (PAMPs) are key to pathogen defense, but drivers and stabilizers of the growth-to-defense genetic reprogramming remain incompletely understood in plants. Here, we report a time-course study of the establishment of PAMP-triggered immunity (PTI) using cap analysis of gene expression. We show that around 15% of all transcription start sites (TSSs) rapidly induced during PTI define alternative transcription initiation events. From these, we identify clear examples of regulatory TSS change via alternative inclusion of target peptides or domains in encoded proteins, or of upstream open reading frames in mRNA leader sequences. We also find that 60% of PAMP response genes respond earlier than previously thought. In particular, a cluster of rapidly and transiently PAMP-induced genes is enriched in transcription factors (TFs) whose functions, previously associated with biological processes as diverse as abiotic stress adaptation and stem cell activity, appear to converge on growth restriction. Furthermore, examples of known potentiators of PTI, in one case under direct mitogen-activated protein kinase control, support the notion that the rapidly induced TFs could constitute direct links to PTI signaling pathways and drive gene expression changes underlying establishment of the immune state.

In vitro reconstitution of a biological complex or process normally involves assembly of multiple individually purified protein components. Here we present a strategy that couples expression and assembly of multiple gene products with functional detection in an in vitro reconstituted protein synthesis system. The strategy potentially allows experimental reconstruction of a multi-component biological complex or process using only DNA templates instead of purified proteins. We applied this strategy to bacterial transcription initiation by co-expressing genes encoding Escherichia coli RNA polymerase subunits and sigma factors in the reconstituted protein synthesis system and by coupling the synthesis and assembly of a functional RNA polymerase holoenzyme with the expression of a reporter gene. Using such a system, we demonstrated sigma-factor-dependent, promoter-specific transcription initiation. Since protein synthesis, complex formation and enzyme catalysis occur in the same in vitro reaction mixture, this reconstruction process resembles natural biosynthetic pathways and avoids time-consuming expression and purification of individual proteins. The strategy can significantly reduce the time normally required by conventional reconstitution methods, allow rapid generation and detection of genetic mutations, and provide an open and designable platform for in vitro study and intervention of complex biological processes.

RNA polymerase II (Pol II)-mediated transcription in metazoans requires precise regulation. RNA Pol II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). Here, we show that RPAP2 binds hypo-/hyper-phosphorylated Pol II with undetectable phosphatase activity. The structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents and disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in inhibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating a critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to inhibit PIC assembly and transcription initiation and suggests a transcription checkpoint.

Transcription initiation is finely regulated to ensure proper expression and function of genes. The regulated transcription initiation in response to various environmental stimuli in a classic model organism Saccharomyces cerevisiae has not been systematically investigated. In this study, we generated quantitative maps of transcription start sites (TSSs) at a single-nucleotide resolution for S. cerevisiae grown in nine different conditions using no-amplification nontagging Cap analysis of gene expression (nAnT-iCAGE) sequencing. We mapped ∼1 million well-supported TSSs, suggesting highly pervasive transcription initiation in the compact genome of the budding yeast. The comprehensive TSS maps allowed us to identify core promoters for ∼96% verified protein-coding genes. We corrected misannotation of translation start codon for 122 genes and suggested an alternative start codon for 57 genes. We found that 56% of yeast genes are controlled by multiple core promoters, and alternative core promoter usage by a gene is widespread in response to changing environments. Most core promoter shifts are coupled with altered gene expression, indicating that alternative core promoter usage might play an important role in controlling gene transcriptional activities. Based on their activities in responding to environmental cues, we divided core promoters into constitutive class (55%) and inducible class (45%). The two classes of core promoters display distinctive patterns in transcriptional abundance, chromatin structure, promoter shape, and sequence context. In summary, our study improved the annotation of the yeast genome and demonstrated a much more pervasive and dynamic nature of transcription initiation in yeast than previously recognized.

In plants, epigenetic regulation is critical for silencing transposons and maintaining proper gene expression. However, its impact on the genome-wide transcription initiation landscape remains elusive. By conducting a genome-wide analysis of transcription start sites (TSSs) using cap analysis of gene expression (CAGE) sequencing, we show that thousands of TSSs are exclusively activated in various epigenetic mutants of Arabidopsis thaliana and referred to as cryptic TSSs. Many have not been identified in previous studies, of which up to 65% are contributed by transposons. They possess similar genetic features to regular TSSs and their activation is strongly associated with the ectopic recruitment of RNAPII machinery. The activation of cryptic TSSs significantly alters transcription of nearby TSSs, including those of genes important for development and stress responses. Our study, therefore, sheds light on the role of epigenetic regulation in maintaining proper gene functions in plants by suppressing transcription from cryptic TSSs.

The promoter landscape of several non-human model organisms is far from complete. As a part of FANTOM5 data collection, we generated 13 profiles of transcription initiation activities in dog and rat aortic smooth muscle cells, mesenchymal stem cells and hepatocytes by employing CAGE (Cap Analysis of Gene Expression) technology combined with single molecule sequencing. Our analyses show that the CAGE profiles recapitulate known transcription start sites (TSSs) consistently, in addition to uncover novel TSSs. Our dataset can be thus used with high confidence to support gene annotation in dog and rat species. We identified 28,497 and 23,147 CAGE peaks, or promoter regions, for rat and dog respectively, and associated them to known genes. This approach could be seen as a standard method for improvement of existing gene models, as well as discovery of novel genes. Given that the FANTOM5 data collection includes dog and rat matched cell types in human and mouse as well, this data would also be useful for cross-species studies.

Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA and a divergent non-coding (DNC) transcript of unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. Using high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements, we identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC transcription. Nascent transcription profiling showed a genome-wide role of Hda1C in repression of DNC transcription. Live-cell imaging of transcription revealed that mutations in the Hda3 subunit increased the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC transcription regions, supporting DNC transcription repression by histone deacetylation. Our data support the interpretation that DNC transcription results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

In transcription initiation, all RNA polymerase molecules bound to a promoter have been conventionally supposed to proceed into elongation of transcript. However, for Escherichia coli RNA polymerase, evidence has been accumulated for a view that only its fraction can proceed into elongation and the rest is retained at a promoter in non-productive form: a pathway branching in transcription initiation. Proteins such as GreA and GreB affect these fractions at several promoters in vitro. To reveal the ubiquitous existence of the branched mechanism in E. coli, we searched for candidate genes whose transcription decreased by disruption of greA and greB using a DNA array. Among the arbitrarily selected 11 genes from over 100, the atpC, cspA and rpsA passed the test by Northern blotting. The Gre factors activated transcription initiation from their promoters in vitro, and the results demonstrated that the branched mechanism is exploited in vivo regulation. Consistently, decrease in the level of the GreA in an anaerobic stationary condition accompanied a decrease in the levels of transcripts of these genes.

Transcriptional activation of enhancer and sigma(54)-dependent promoters requires efficient interactions between enhancer-binding proteins (EBP) and promoter bound sigma(54)-RNA polymerase (E sigma(54)) achieved by DNA looping, which is usually facilitated by the integration host factor (IHF). Since the lengths of the intervening region supporting DNA-loop formation are similar among IHF-dependent and IHF-independent promoters, the precise reason(s) why IHF is selectively important for the frequency of transcription initiation remain unclear. Here, using kinetic cyclization and in vitro transcription assays we show that, in the absence of IHF protein, the DNA fragments containing an IHF-binding site have much less looping-formation ability than those that lack an IHF-binding site. Furthermore, when an IHF consensus-binding site was introduced into the intervening region between promoter and enhancer of the target DNA fragments, loop formation and DNA-loop-dependent transcriptional activation are significantly reduced in a position-independent manner. DNA-looping-independent transcriptional activation was unaffected. The binding of IHF to its consensus site in the target promoters clearly restored efficient DNA looping formation and looping-dependent transcriptional activation. Our data provide evidence that one function for the IHF protein is to release a communication block set by intrinsic properties of the IHF DNA-binding site.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.

A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs and recruits general transcription factors to initiate transcription. The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle. Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition. This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression, and determined the positions of H3K4me3-marked promoter-associated nucleosomes. Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (+1) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts.

Sporulation of budding yeast is a developmental process in which cells undergo meiosis to generate stress-resistant progeny. The dynamic nature of the budding yeast meiotic transcriptome has been well established by a number of genome-wide studies. Here we develop an analysis pipeline to systematically identify novel transcription start sites that reside internal to a gene. Application of this pipeline to data from a synchronized meiotic time course reveals over 40 genes that display specific internal initiations in mid-sporulation. Consistent with the time of induction, motif analysis on upstream sequences of these internal transcription start sites reveals a significant enrichment for the binding site of Ndt80, the transcriptional activator of middle sporulation genes. Further examination of one gene, MRK1, demonstrates the Ndt80 binding site is necessary for internal initiation and results in the expression of an N-terminally truncated protein isoform. When the MRK1 paralog RIM11 is downregulated, the MRK1 internal transcript promotes efficient sporulation, indicating functional significance of the internal initiation. Our findings suggest internal transcriptional initiation to be a dynamic, regulated process with potential functional impacts on development.

Transcription initiation and RNA processing govern gene expression and enable bacterial adaptation by reshaping the RNA landscape. The aim of this study was to simultaneously observe these two fundamental processes in a transcriptome responding to an environmental signal. A controlled σE system in E. coli was coupled to our previously described tagRNA-seq method to yield process kinetics information. Changes in transcription initiation frequencies (TIF) and RNA processing frequencies (PF) were followed using 5' RNA tags. Changes in TIF showed a binary increased/decreased pattern that alternated between transcriptionally activated and repressed promoters, providing the bacterial population with transcriptional oscillation. PF variation fell into three categories of cleavage activity: (i) constant and independent of RNA levels, (ii) increased once RNA has accumulated, and (iii) positively correlated to changes in TIF. This work provides a comprehensive and dynamic view of major events leading to transcriptomic reshaping during bacterial adaptation. It unveils an interplay between transcription initiation and the activity of specific RNA cleavage sites. This study utilized a well-known genetic system to analyze fundamental processes and can serve as a blueprint for comprehensive studies that exploit the RNA metabolism to decipher and understand bacterial gene expression control.

Enhancers are modular regulatory elements that are central to the spatial and temporal regulation of gene expression. Bidirectional transcription initiating at enhancers has been proposed to mark active enhancers and as such has been utilized to experimentally identify active enhancers de novo.

Marburg virus (MARV) is an enveloped, negative-sense RNA virus from the filovirus family that causes outbreaks of severe, frequently fatal illness in humans. Of the seven MARV proteins, the VP30 protein stands out because it is essential for viral growth but lacks a definitive function. Here, we used model MARV genome RNAs for one or two reporter genes and the MARV VP40, glycoprotein (GP), and VP24 genes to demonstrate that VP30 is dispensable for the transcription of some genes but critical for transcription reinitiation at the GP gene. This results in the loss of the expression of GP and downstream genes and the impaired production of infectious particles when VP30 is absent. Bicistronic minigenome assays demonstrate that the VP40 gene end/GP gene start junction specifically confers VP30 dependence. A region at the GP gene start site predicted to form a stem-loop contributes to VP30 dependence because the replacement of the GP stem-loop with corresponding sequences from the MARV VP35 gene relieves VP30 dependence. Finally, a Cys3-His zinc binding motif characteristic of filovirus VP30 proteins was demonstrated to be critical for reinitiation at GP. These findings address a long-standing gap in our understanding of MARV biology by defining a critical role for VP30 in MARV transcription. IMPORTANCE Marburg virus and Ebola virus encode VP30 proteins. While the role of VP30 in Ebola virus transcription has been well studied, the role of VP30 in the Marburg virus life cycle is not well understood. The work here demonstrates that different gene start sites within the Marburg viral genome have variable levels of dependence on Marburg virus VP30, with its expression being critical for transcription reinitiation at the GP gene start site. These findings address a long-standing question regarding Marburg virus VP30 function and further our understanding of how Marburg virus gene expression is regulated.

Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5'-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5'-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5'-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5'-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5'-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription.

The application of deep sequencing to map 5' capped transcripts has confirmed the existence of at least two distinct promoter classes in metazoans: "focused" promoters with transcription start sites (TSSs) that occur in a narrowly defined genomic span and "dispersed" promoters with TSSs that are spread over a larger window. Previous studies have explored the presence of genomic features, such as CpG islands and sequence motifs, in these promoter classes, but virtually no studies have directly investigated the relationship with chromatin features. Here, we show that promoter classes are significantly differentiated by nucleosome organization and chromatin structure. Dispersed promoters display higher associations with well-positioned nucleosomes downstream of the TSS and a more clearly defined nucleosome free region upstream, while focused promoters have a less organized nucleosome structure, yet higher presence of RNA polymerase II. These differences extend to histone variants (H2A.Z) and marks (H3K4 methylation), as well as insulator binding (such as CTCF), independent of the expression levels of affected genes. Notably, differences are conserved across mammals and flies, and they provide for a clearer separation of promoter architectures than the presence and absence of CpG islands or the occurrence of stalled RNA polymerase. Computational models support the stronger contribution of chromatin features to the definition of dispersed promoters compared to focused start sites. Our results show that promoter classes defined from 5' capped transcripts not only reflect differences in the initiation process at the core promoter but also are indicative of divergent transcriptional programs established within gene-proximal nucleosome organization.

Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5' terminal oligopyrimidine (5'TOP) sequence, dramatically increasing the range of genes potentially subjected to 5'TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K-Akt-mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.

The molecular process of transcription by RNA Polymerase II is highly conserved among eukaryotes ("classic model"). A distinct way of locating transcription start sites (TSSs) has been identified in a budding yeast Saccharomyces cerevisiae ("scanning model"). Herein, we applied genomic approaches to elucidate the origin of the scanning model and its underlying genetic mechanisms. We first identified TSSs at single-nucleotide resolution for 12 yeast species using the nAnT-iCAGE technique, which significantly improved the annotations of these genomes by providing accurate 5' boundaries for protein-coding genes. We then inferred the initiation mechanism of each species based on its TSS maps and genome sequences. We discovered that the scanning model likely originated after the split of Yarrowia lipolytica and the other budding yeasts. Species that use the scanning model showed an adenine-rich region immediately upstream of the TSS that might facilitate TSS selection. Both initiation mechanisms share a strong preference for pyrimidine-purine dinucleotides surrounding the TSS. Our results suggest that the purine is required to accurately recruit the first nucleotide, thereby increasing the chances of a messenger RNA of being capped during mRNA maturation, which is critical for efficient translation initiation during protein biosynthesis. Based on our findings, we propose a model for TSS selection in the scanning-model species, as well as a model for the stepwise process responsible for the origin and evolution of the scanning model.

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity.