Towards a rational approach to promoter engineering: understanding the complexity of transcription initiation in prokaryotes.

Promoter sequences are important genetic control elements. Through their interaction with RNA polymerase they determine transcription strength and specificity, thereby regulating the first step in gene expression. Consequently, they can be targeted as elements to control predictability and tuneability of a genetic circuit, which is essential in applications such as the development of robust microbial cell factories. This review considers the promoter elements implicated in the three stages of transcription initiation, detailing the complex interplay of sequence-specific interactions that are involved, and highlighting that DNA sequence features beyond the core promoter elements work in a combinatorial manner to determine transcriptional strength. In particular, we emphasize that, aside from promoter recognition, transcription initiation is also defined by the kinetics of open complex formation and promoter escape, which are also known to be highly sequence specific. Significantly, we focus on how insights into these interactions can be manipulated to lay the foundation for a more rational approach to promoter engineering.

Transcription-replication interactions reveal bacterial genome regulation.

Organisms determine the transcription rates of thousands of genes through a few modes of regulation that recur across the genome1. In bacteria, the relationship between the regulatory architecture of a gene and its expression is well understood for individual model gene circuits2,3. However, a broader perspective of these dynamics at the genome scale is lacking, in part because bacterial transcriptomics has hitherto captured only a static snapshot of expression averaged across millions of cells4. As a result, the full diversity of gene expression dynamics and their relation to regulatory architecture remains unknown. Here we present a novel genome-wide classification of regulatory modes based on the transcriptional response of each gene to its own replication, which we term the transcription-replication interaction profile (TRIP). Analysing single-bacterium RNA-sequencing data, we found that the response to the universal perturbation of chromosomal replication integrates biological regulatory factors with biophysical molecular events on the chromosome to reveal the local regulatory context of a gene. Whereas the TRIPs of many genes conform to a gene dosage-dependent pattern, others diverge in distinct ways, and this is shaped by factors such as intra-operon position and repression state. By revealing the underlying mechanistic drivers of gene expression heterogeneity, this work provides a quantitative, biophysical framework for modelling replication-dependent expression dynamics.

A mutation rate model at the basepair resolution identifies the mutagenic effect of polymerase III transcription.

De novo mutations occur at substantially different rates depending on genomic location, sequence context and DNA strand. The success of methods to estimate selection intensity, infer demographic history and map rare disease genes, depends strongly on assumptions about the local mutation rate. Here we present Roulette, a genome-wide mutation rate model at basepair resolution that incorporates known determinants of local mutation rate. Roulette is shown to be more accurate than existing models. We use Roulette to refine the estimates of population growth within Europe by incorporating the full range of human mutation rates. The analysis of significant deviations from the model predictions revealed a tenfold increase in mutation rate in nearly all genes transcribed by polymerase III (Pol III), suggesting a new mutagenic mechanism. We also detected an elevated mutation rate within transcription factor binding sites restricted to sites actively used in testis and residing in promoters.

Transcriptional linkage analysis with in vivo AAV-Perturb-seq.

The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a high-throughput manner1,2. Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome3,4 genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and previously undescribed pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2-deletion mouse model. Our findings suggest that the 22q11.2-deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of genes associated with disease susceptibility that are important for dysfunctional RNA processing and synaptic function. Our study establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.

Elongation rate of RNA polymerase II affects pausing patterns across 3' UTRs.

Yeast mRNAs are polyadenylated at multiple sites in their 3' untranslated regions (3' UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Slow Pol II derivatives favor upstream poly(A) sites, and fast Pol II derivatives favor downstream poly(A) sites. Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation. Here, we investigate the effect of Pol II elongation rate on pausing patterns and the relationship between Pol II pause sites and poly(A) sites within 3' UTRs. Mutations that affect Pol II elongation rate alter sequence preferences at pause sites within 3' UTRs, and pausing preferences differ between 3' UTRs and coding regions. In addition, sequences immediately flanking the pause sites show preferences that are largely independent of Pol II speed. In wild-type cells, poly(A) sites are preferentially located < 50 nucleotides upstream from Pol II pause sites, but this spatial relationship is diminished in cells harboring Pol II speed mutants. Based on a random forest classifier, Pol II pause sites are modestly predicted by the distance to poly(A) sites but are better predicted by the chromatin landscape in Pol II speed derivatives. Transcriptional regulatory proteins can influence the relationship between Pol II pausing and polyadenylation but in a manner distinct from Pol II elongation rate derivatives. These results indicate a complex relationship between Pol II pausing and polyadenylation.

High-throughput functional characterization of combinations of transcriptional activators and repressors.

Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small-molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools. A record of this paper's transparent peer review process is included in the supplemental information.

Butt-seq: a new method for facile profiling of transcription.

A wide range of sequencing methods has been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed Butt-seq (bulk analysis of nascent transcript termini sequencing), which can produce libraries from purified nascent RNA in 6 h and from as few as 10,000 cells-an improvement of at least 10-fold over existing techniques. Butt-seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcriptional regulation in a tissue, Butt-seq was used to measure the circadian regulation of transcription from fly heads. All the results indicate that Butt-seq is a simple and powerful technique to analyze transcription at a high level of resolution.

What is an enhancer?

Tight control of the transcription process is essential for the correct spatial and temporal gene expression pattern during development and in homeostasis. Enhancers are at the core of correct transcriptional activation. The original definition of an enhancer is straightforward: a DNA sequence that activates transcription independent of orientation and direction. Dissection of numerous enhancer loci has shown that many enhancer-like elements might not conform to the original definition, suggesting that enhancers and enhancer-like elements might use multiple different mechanisms to contribute to transcriptional activation. Here, we review methodologies to identify enhancers and enhancer-like elements and discuss pitfalls and consequences for our understanding of transcriptional regulation.

An unexpected abundance of bidirectional promoters within Salmonella Typhimurium plasmids.

Transcription of the DNA template, to generate an RNA message, is the first step in gene expression. The process initiates at DNA sequences called promoters. Conventionally, promoters have been considered to drive transcription in a specific direction. However, in recent work, we showed that many prokaryotic promoters can drive divergent transcription. This is a consequence of key DNA sequences for transcription initiation being inherently symmetrical. Here, we used global transcription start site mapping to determine the prevalence of such bidirectional promoters in Salmonella Typhimurium. Surprisingly, bidirectional promoters occur three times more frequently in plasmid components of the genome compared to chromosomal DNA. Implications for the evolution of promoter sequences are discussed.

Mirusviruses link herpesviruses to giant viruses.

DNA viruses have a major influence on the ecology and evolution of cellular organisms1-4, but their overall diversity and evolutionary trajectories remain elusive5. Here we carried out a phylogeny-guided genome-resolved metagenomic survey of the sunlit oceans and discovered plankton-infecting relatives of herpesviruses that form a putative new phylum dubbed Mirusviricota. The virion morphogenesis module of this large monophyletic clade is typical of viruses from the realm Duplodnaviria6, with multiple components strongly indicating a common ancestry with animal-infecting Herpesvirales. Yet, a substantial fraction of mirusvirus genes, including hallmark transcription machinery genes missing in herpesviruses, are closely related homologues of giant eukaryotic DNA viruses from another viral realm, Varidnaviria. These remarkable chimaeric attributes connecting Mirusviricota to herpesviruses and giant eukaryotic viruses are supported by more than 100 environmental mirusvirus genomes, including a near-complete contiguous genome of 432 kilobases. Moreover, mirusviruses are among the most abundant and active eukaryotic viruses characterized in the sunlit oceans, encoding a diverse array of functions used during the infection of microbial eukaryotes from pole to pole. The prevalence, functional activity, diversification and atypical chimaeric attributes of mirusviruses point to a lasting role of Mirusviricota in the ecology of marine ecosystems and in the evolution of eukaryotic DNA viruses.

Excessive transcription-replication conflicts are a vulnerability of BRCA1-mutant cancers.

BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations.

UBE2T resolves transcription-replication conflicts and protects common fragile sites in primordial germ cells.

The proper development of primordial germ cells (PGCs) is an essential prerequisite for gametogenesis and mammalian fertility. The Fanconi anemia (FA) pathway functions in maintaining the development of PGCs. FANCT/UBE2T serves as an E2 ubiquitin-conjugating enzyme that ubiquitylates the FANCD2-FANCI complex to activate the FA pathway, but its role in the development of PGCs is not clear. In this study, we found that Ube2t knockout mice showed defects in PGC proliferation, leading to severe loss of germ cells after birth. Deletion of UBE2T exacerbated DNA damage and triggered the activation of the p53 pathway. We further demonstrated that UBE2T counteracted transcription-replication conflicts by resolving R-loops and stabilizing replication forks, and also protected common fragile sites by resolving R-loops in large genes and promoting mitotic DNA synthesis to maintain the genome stability of PGCs. Overall, these results provide new insights into the function and regulatory mechanisms of the FA pathway ensuring normal development of PGCs.

Better late than never: A unique strategy for late gene transcription in the beta- and gammaherpesviruses.

During lytic replication, herpesviruses express their genes in a temporal cascade culminating in expression of "late" genes. Two subfamilies of herpesviruses, the beta- and gammaherpesviruses (including human herpesviruses cytomegalovirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus), use a unique strategy to facilitate transcription of late genes. They encode six essential viral transcriptional activators (vTAs) that form a complex at a subset of late gene promoters. One of these vTAs is a viral mimic of host TATA-binding protein (vTBP) that recognizes a strikingly minimal cis-acting element consisting of a modified TATA box with a TATTWAA consensus sequence. vTBP is also responsible for recruitment of cellular RNA polymerase II (Pol II). Despite extensive work in the beta/gammaherpesviruses, the function of the other five vTAs remains largely unknown. The vTA complex and Pol II assemble on the promoter into a viral preinitiation complex (vPIC) to facilitate late gene transcription. Here, we review the properties of the vTAs and the promoters on which they act.

Prediction of the cell-type-specific transcription of non-coding RNAs from genome sequences via machine learning.

Gene transcription is regulated through complex mechanisms involving non-coding RNAs (ncRNAs). As the transcription of ncRNAs, especially of enhancer RNAs, is often low and cell type specific, how the levels of RNA transcription depend on genotype remains largely unexplored. Here we report the development and utility of a machine-learning model (MENTR) that reliably links genome sequence and ncRNA expression at the cell type level. Effects on ncRNA transcription predicted by the model were concordant with estimates from published studies in a cell-type-dependent manner, regardless of allele frequency and genetic linkage. Among 41,223 variants from genome-wide association studies, the model identified 7,775 enhancer RNAs and 3,548 long ncRNAs causally associated with complex traits across 348 major human primary cells and tissues, such as rare variants plausibly altering the transcription of enhancer RNAs to influence the risks of Crohn's disease and asthma. The model may aid the discovery of causal variants and the generation of testable hypotheses for biological mechanisms driving complex traits.

Enhanced myogenesis through lncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter.

The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.

Identification of an E3 ligase that targets the catalytic subunit of RNA Polymerase I upon transcription stress.

RNA Polymerase I (Pol I) synthesizes rRNA, which is the first and rate-limiting step in ribosome biogenesis. Factors governing the stability of the polymerase complex are not known. Previous studies characterizing Pol I inhibitor BMH-21 revealed a transcriptional stress-dependent pathway for degradation of the largest subunit of Pol I, RPA194. To identify the E3 ligase(s) involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes. We establish Skp-Cullin-F-box protein complex F-box protein FBXL14 as an E3 ligase for RPA194. We show that FBXL14 binds to RPA194 and mediates RPA194 ubiquitination and degradation in cancer cells treated with BMH-21. Mutation analysis in yeast identified lysines 1150, 1153, and 1156 on Rpa190 relevant for the protein degradation. These results reveal the regulated turnover of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14 in response to transcription stress.

The HASTER lncRNA promoter is a cis-acting transcriptional stabilizer of HNF1A.

The biological purpose of long non-coding RNAs (lncRNAs) is poorly understood. Haploinsufficient mutations in HNF1A homeobox A (HNF1A), encoding a homeodomain transcription factor, cause diabetes mellitus. Here, we examine HASTER, the promoter of an lncRNA antisense to HNF1A. Using mouse and human models, we show that HASTER maintains cell-specific physiological HNF1A concentrations through positive and negative feedback loops. Pancreatic ß cells from Haster mutant mice consequently showed variegated HNF1A silencing or overexpression, resulting in hyperglycaemia. HASTER-dependent negative feedback was essential to prevent HNF1A binding to inappropriate genomic regions. We demonstrate that the HASTER promoter DNA, rather than the lncRNA, modulates HNF1A promoter-enhancer interactions in cis and thereby regulates HNF1A transcription. Our studies expose a cis-regulatory element that is unlike classic enhancers or silencers, it stabilizes the transcription of its target gene and ensures the fidelity of a cell-specific transcription factor program. They also show that disruption of a mammalian lncRNA promoter can cause diabetes mellitus.

A spatially resolved stochastic model reveals the role of supercoiling in transcription regulation.

In Escherichia coli, translocation of RNA polymerase (RNAP) during transcription introduces supercoiling to DNA, which influences the initiation and elongation behaviors of RNAP. To quantify the role of supercoiling in transcription regulation, we developed a spatially resolved supercoiling model of transcription. The integrated model describes how RNAP activity feeds back with the local DNA supercoiling and how this mechanochemical feedback controls transcription, subject to topoisomerase activities and stochastic topological domain formation. This model establishes that transcription-induced supercoiling mediates the cooperation of co-transcribing RNAP molecules in highly expressed genes, and this cooperation is achieved under moderate supercoiling diffusion and high topoisomerase unbinding rates. It predicts that a topological domain could serve as a transcription regulator, generating substantial transcriptional noise. It also shows the relative orientation of two closely arranged genes plays an important role in regulating their transcription. The model provides a quantitative platform for investigating how genome organization impacts transcription.

A transcriptional cycling model recapitulates chromatin-dependent features of noisy inducible transcription.

Activation of gene expression in response to environmental cues results in substantial phenotypic heterogeneity between cells that can impact a wide range of outcomes including differentiation, viral activation, and drug resistance. An important source of gene expression noise is transcriptional bursting, or the process by which transcripts are produced during infrequent bursts of promoter activity. Chromatin accessibility impacts transcriptional bursting by regulating the assembly of transcription factor and polymerase complexes on promoters, suggesting that the effect of an activating signal on transcriptional noise will depend on the initial chromatin state at the promoter. To explore this possibility, we simulated transcriptional activation using a transcriptional cycling model with three promoter states that represent chromatin remodeling, polymerase binding and pause release. We initiated this model over a large parameter range representing target genes with different chromatin environments, and found that, upon increasing the polymerase pause release rate to activate transcription, changes in gene expression noise varied significantly across initial promoter states. This model captured phenotypic differences in activation of latent HIV viruses integrated at different chromatin locations and mediated by the transcription factor NF-κB. Activating transcription in the model via increasing one or more of the transcript production rates, as occurs following NF-κB activation, reproduced experimentally measured transcript distributions for four different latent HIV viruses, as well as the bimodal pattern of HIV protein expression that leads to a subset of reactivated virus. Importantly, the parameter 'activation path' differentially affected gene expression noise, and ultimately viral activation, in line with experimental observations. This work demonstrates how upstream signaling pathways can be connected to biological processes that underlie transcriptional bursting, resulting in target gene-specific noise profiles following stimulation of a single upstream pathway.

RSC and GRFs confer promoter directionality by restricting divergent noncoding transcription.

The directionality of gene promoters-the ratio of protein-coding over divergent noncoding transcription-is highly variable. How promoter directionality is controlled remains poorly understood. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription relative to protein-coding transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus to a decrease in promoter directionality. We find that RSC has a modest effect on nucleosome positioning upstream in promoters at the sites of divergent transcription. These promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 DNA-binding protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription relative to coding direction transcription. We propose that any DNA-binding factor, when stably associated with cryptic transcription start sites, forms a barrier which represses divergent transcription, thereby promoting promoter directionality.