Transcription Unit Research Articles

Rational design of hybrid DNA-RNA triplex structures as modulators of transcriptional activity in vitro.

Triplex nanostructures can be formed in vitro in the promoter region of DNA templates, and it is commonly accepted that these assemblies inhibit the transcription of the downstream genes. Herein, a proof of concept highlighting the possibility of the up- or downregulation of RNA transcription is presented. Hybrid DNA-RNA triplex nanostructures were rationally designed to produce bacterial transcription units with switchable promoters. The rate of RNA production was measured using the signal of a transcribed fluorescent RNA aptamer (i.e. Broccoli). Indeed, several designed bacterial promoters showed the ability of induced transcriptional inhibition, while other properly tailored sequences demonstrated switchable enhancement of transcriptional activity, representing an unprecedented feature to date. The use of RNA-regulated transcription units and fluorescent RNA aptamers as readouts will allow the realization of biocomputation circuits characterized by a strongly reduced set of components. Triplex forming RNA oligonucleotides are proposed as smart tools for transcriptional modulation and represent an alternative to current methods for producing logic gates using protein-based components.

Transcriptional analysis of the mfa-cluster genes in Porphyromonas gingivalis strains with one and two mfa5 genes.

The Porphyromonas gingivalis Mfa1 fimbria is composed of the Mfa1 to Mfa5 proteins, encoded by the mfa1 to mfa5 genes, respectively, which are tandemly arranged on chromosomes. A recent study discovered that many P. gingivalis strains possess two mfa5 genes (called herein mfa5-1 and mfa5-2), which are also in tandem. This study examined the transcriptional unit and activity of mfa-cluster genes in strains with one (the ATCC 33277 and TDC60 strains) and two (the HG66 and A7436 strains) mfa5 genes. Complementary DNA was prepared from the total RNA extracted from the bacterial cells in the logarithmic growth phase using a random primer. PCR analysis for the intergenic regions from mfa1 to mfa5 or mfa5-2 showed that mfa1 to mfa5 or mfa5-2 formed a polycistronic gene cluster. Quantitative real-time PCR showed that the mfa1 transcription was 5-10 times higher than that of mfa2 in all the strains. However, mfa2 to mfa5 mostly showed a comparable expression. Both mfa5 genes were comparably transcribed in HG66 and A7436 strains. The transcriptional levels were almost consistent with the respective protein expression levels. In silico analysis identified a transcriptional terminator structure in the intergenic region between mfa1 and mfa2 that was probably responsible for the decreased transcription rate of mfa2 and the downstream genes.

Multiplex CRISPR/Cas9-mediated raffinose synthase gene editing reduces raffinose family oligosaccharides in soybean.

Soybean [Glycine max (L.) Merr.] is an important world economic crop. It is rich in oil, protein, and starch, and soluble carbohydrates in soybean seeds are also important for human and livestock consumption. The predominant soluble carbohydrate in soybean seed is composed of sucrose and raffinose family oligosaccharides (RFOs). Among these carbohydrates, only sucrose can be digested by humans and monogastric animals and is beneficial for metabolizable energy, while RFOs are anti-nutritional factors in diets, usually leading to flatulence and indigestion, ultimately reducing energy efficiency. Hence, breeding efforts to remove RFOs from soybean seeds can increase metabolizable energy and improve nutritional quality. The objective of this research is to use the multiplex Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing system to induce the knockout of soybean raffinose synthase (RS) genes RS2 and RS3 simultaneously to reduce RFOs in mature seeds. First, we constructed five types of multiplex gene editing systems and compared their editing efficiency in soybean hairy roots. We confirmed that the two-component transcriptional unit (TCTU) and single transcriptional unit (STU) systems with transfer RNA (tRNA) as the cleavage site performed better than other systems. The average editing efficiency at the four targets with TCTU-tRNA and STU-tRNA was 50.5% and 46.7%, respectively. Then, we designed four single-guide RNA (sgRNA) targets to induce mutations at RS2 and RS3 by using the TCTU-tRNA system. After the soybean transformation, we obtained several RS2 and RS3 mutation plants, and a subset of alleles was successfully transferred to the progeny. We identified null single and double mutants at the T2 generation and analyzed the seed carbohydrate content of their progeny. The RS2 and RS3 double mutants and the RS2 single mutant exhibited dramatically reduced levels of raffinose and stachyose in mature seeds. Further analysis of the growth and development of these mutants showed that there were no penalties on these phenotypes. Our results indicate that knocking out RS genes by multiplex CRISPR/Cas9-mediated gene editing is an efficient way to reduce RFOs in soybean. This research demonstrates the potential of using elite soybean cultivars to improve the soybean meal trait by multiplex CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-mediated gene editing.

Tandemly repeated genes promote RNAi-mediated heterochromatin formation via an antisilencing factor, Epe1, in fission yeast.

In most eukaryotes, constitutive heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), is enriched on repetitive DNA, such as pericentromeric repeats and transposons. Furthermore, repetitive transgenes also induce heterochromatin formation in diverse model organisms. However, the mechanisms that promote heterochromatin formation at repetitive DNA elements are still not clear. Here, using fission yeast, we show that tandemly repeated mRNA genes promote RNA interference (RNAi)-mediated heterochromatin formation in cooperation with an antisilencing factor, Epe1. Although the presence of tandemly repeated genes itself does not cause heterochromatin formation, once complementary small RNAs are artificially supplied in trans, the RNAi machinery assembled on the repeated genes starts producing cognate small RNAs in cis to autonomously maintain heterochromatin at these sites. This "repeat-induced RNAi" depends on the copy number of repeated genes and Epe1, which is known to remove H3K9me and derepress the transcription of genes underlying heterochromatin. Analogous to repeated genes, the DNA sequence underlying constitutive heterochromatin encodes widespread transcription start sites (TSSs), from which Epe1 activates ncRNA transcription to promote RNAi-mediated heterochromatin formation. Our results suggest that when repetitive transcription units underlie heterochromatin, Epe1 generates sufficient transcripts for the activation of RNAi without disruption of heterochromatin.

Comprehensive Characterization of the Regulatory Landscape of Adrenocortical Carcinoma: Novel Transcription Factors and Targets Associated with Prognosis

We reconstructed a transcriptional regulatory network for adrenocortical carcinoma (ACC) using transcriptomic and clinical data from The Cancer Genome Atlas (TCGA)-ACC cohort. We investigated the association of transcriptional regulatory units (regulons) with overall survival, molecular phenotypes, and immune signatures. We annotated the ACC regulons with cancer hallmarks and assessed single sample regulon activities in the European Network for the Study of Adrenal Tumors (ENSAT) cohort. We found 369 regulons associated with overall survival and subdivided them into four clusters: RC1 and RC2, associated with good prognosis, and RC3 and RC4, associated with worse outcomes. The RC1 and RC3 regulons were highly correlated with the 'Steroid Phenotype,' while the RC2 and RC4 regulons were highly correlated with a molecular proliferation signature. We selected two regulons, NR5A1 (steroidogenic factor 1, SF-1) and CENPA (Centromeric Protein A), that were consistently associated with overall survival for further downstream analyses. The CENPA regulon was the primary regulator of MKI-67 (a marker of proliferation KI-67), while the NR5A1 regulon is a well-described transcription factor (TF) in ACC tumorigenesis. We also found that the ZBTB4 (Zinc finger and BTB domain-containing protein 4) regulon, which is negatively associated with CENPA in our transcriptional regulatory network, is also a druggable anti-tumorigenic TF. We anticipate that the ACC regulons may be used as a reference for further investigations concerning the complex molecular interactions in ACC tumors.

Arginine 58 is indispensable for proper function of the Francisella tularensis subsp. holarctica FSC200 HU protein, and its substitution alters virulence and mediates immunity against wild-type strain

ABSTRACT HU protein, a member of the nucleoid-associated group of proteins, is an important transcription factor in bacteria, including in the dangerous human pathogen Francisella tularensis. Generally, HU protein acts as a DNA sequence non-specific binding protein and it is responsible for winding of the DNA chain that leads to the separation of transcription units. Here, we identified potential HU protein binding sites using the ChIP-seq method and two possible binding motifs in F. tularensis subsp. holarctica FSC200 depending upon growth conditions. We also confirmed that FSC200 HU protein is able to introduce negative supercoiling of DNA in the presence of topoisomerase I. Next, we showed interaction of the HU protein with a DNA region upstream of the pigR gene and inside the clpB gene, suggesting possible regulation of PigR and ClpB expression. Moreover, we showed that arginine 58 and partially arginine 61 are important for HU protein’s DNA binding capacity, negative supercoiling induction by HU, and the length and winding of FSC200 chromosomal DNA. Finally, in order to verify biological function of the HU protein, we demonstrated that mutations in arginine 58, arginine 61, and serine 74 of the HU protein decrease virulence of FSC200 both in vitro and in vivo and that immunization using these mutant strains is able to protect as many as 100% of mice against wild-type challenge. Taken together, our findings deepen knowledge about the role of the HU protein in tularaemia pathogenesis and suggest that HU protein should be addressed in the context of tularaemia vaccine development.

FeMV is a cathepsin-dependent unique morbillivirus infecting the kidneys of domestic cats

Feline morbillivirus (FeMV) is a recently discovered pathogen of domestic cats and has been classified as a morbillivirus in the Paramyxovirus family. We determined the complete sequence of FeMVUS5 directly from an FeMV-positive urine sample without virus isolation or cell passage. Sequence analysis of the viral genome revealed potential divergence from characteristics of archetypal morbilliviruses. First, the virus lacks the canonical polybasic furin cleavage signal in the fusion (F) glycoprotein. Second, conserved amino acids in the hemagglutinin (H) glycoprotein used by all other morbilliviruses for binding and/or fusion activation with the cellular receptor CD150 (signaling lymphocyte activation molecule [SLAM]/F1) are absent. We show that, despite this sequence divergence, FeMV H glycoprotein uses feline CD150 as a receptor and cannot use human CD150. We demonstrate that the protease responsible for cleaving the FeMV F glycoprotein is a cathepsin, making FeMV a unique morbillivirus and more similar to the closely related zoonotic Nipah and Hendra viruses. We developed a reverse genetics system for FeMVUS5 and generated recombinant viruses expressing Venus fluorescent protein from an additional transcription unit located either between the phospho-protein (P) and matrix (M) genes or the H and large (L) genes of the genome. We used these recombinant FeMVs to establish a natural infection and demonstrate that FeMV causes an acute morbillivirus-like disease in the cat. Virus was shed in the urine and detectable in the kidneys at later time points. This opens the door for long-term studies to address the postulated role of this morbillivirus in the development of chronic kidney disease.

Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts.

Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and RNA cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts that meet stringent criteria for expression. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORFs), six novel ORF-containing transcripts, and 15 transcripts encoding for messages that could alter protein functions through truncation or fusion of canonical ORFs. In addition, we detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking distinct gene transcription units. Among these chimeric proteins we detected an evolutionarily conserved protein containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies combined with mass spectrometry can reveal further complexity within viral transcriptomes and resulting proteomes.

A quantitative modelling approach for DNA repair on a population scale.

The great advances of sequencing technologies allow the in vivo measurement of nuclear processes-such as DNA repair after UV exposure-over entire cell populations. However, data sets usually contain only a few samples over several hours, missing possibly important information in between time points. We developed a data-driven approach to analyse CPD repair kinetics over time in Saccharomyces cerevisiae. In contrast to other studies that consider sequencing signals as an average behaviour, we understand them as the superposition of signals from independent cells. By motivating repair as a stochastic process, we derive a minimal model for which the parameters can be conveniently estimated. We correlate repair parameters to a variety of genomic features that are assumed to influence repair, including transcription rate and nucleosome density. The clearest link was found for the transcription unit length, which has been unreported for budding yeast to our knowledge. The framework hence allows a comprehensive analysis of nuclear processes on a population scale.

Ebola virus infection induces a delayed type I IFN response in bystander cells and the shutdown of key liver genes in human iPSC-derived hepatocytes.

SummaryLiver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

Comparative studies of genomic and epigenetic factors influencing transcriptional variation in two insect species.

Different genes show different levels of expression variability. For example, highly expressed genes tend to exhibit less expression variability. Genes whose promoters have TATA box and initiator motifs tend to have increased expression variability. On the other hand, DNA methylation of transcriptional units, or gene body DNA methylation, is associated with reduced gene expression variability in many species. Interestingly, some insect lineages, most notably Diptera including the canonical model insect Drosophila melanogaster, have lost DNA methylation. Therefore, it is of interest to determine whether genomic features similarly influence gene expression variability in lineages with and without DNA methylation. We analyzed recently generated large-scale data sets in D. melanogaster and honey bee (Apis mellifera) to investigate these questions. Our analysis shows that increased gene expression levels are consistently associated with reduced expression variability in both species, while the presence of TATA box is consistently associated with increased gene expression variability. In contrast, initiator motifs and gene lengths have weak effects limited to some data sets. Importantly, we show that a sequence characteristics indicative of gene body DNA methylation is strongly and negatively associate with gene expression variability in honey bees, while it shows no such association in D. melanogaster. These results suggest the evolutionary loss of DNA methylation in some insect lineages has reshaped the molecular mechanisms concerning the regulation of gene expression variability.

Agrobacterium- and a single Cas9-sgRNA transcript system-mediated high efficiency gene editing in perennial ryegrass.

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.

Accurate characterization of dynamic microbial gene expression and growth rate profiles.

Genetic circuits are subject to variability due to cellular and compositional contexts. Cells face changing internal states and environments, the cellular context, to which they sense and respond by changing their gene expression and growth rates. Furthermore, each gene in a genetic circuit operates in a compositional context of genes which may interact with each other and the host cell in complex ways. The context of genetic circuits can, therefore, change gene expression and growth rates, and measuring their dynamics is essential to understanding natural and synthetic regulatory networks that give rise to functional phenotypes. However, reconstruction of microbial gene expression and growth rate profiles from typical noisy measurements of cell populations is difficult due to the effects of noise at low cell densities among other factors. We present here a method for the estimation of dynamic microbial gene expression rates and growth rates from noisy measurement data. Compared to the current state-of-the-art, our method significantly reduced the mean squared error of reconstructions from simulated data of growth and gene expression rates, improving the estimation of timing and magnitude of relevant shapes of profiles. We applied our method to characterize a triple-reporter plasmid library combining multiple transcription units in different compositional and cellular contexts in Escherichia coli. Our analysis reveals cellular and compositional context effects on microbial growth and gene expression rate dynamics and suggests a method for the dynamic ratiometric characterization of constitutive promoters relative to an in vivo reference.

Localization of Epigenetic Markers in Leishmania Chromatin.

Eukaryotes use histone variants and post-translation modifications (PTMs), as well as DNA base modifications, to regulate DNA replication/repair, chromosome condensation, and gene expression. Despite the unusual organization of their protein-coding genes into large polycistronic transcription units (PTUs), trypanosomatid parasites also employ a “histone code” to control these processes, but the details of this epigenetic code are poorly understood. Here, we present the results of experiments designed to elucidate the distribution of histone variants and PTMs over the chromatin landscape of Leishmania tarentolae. These experiments show that two histone variants (H2A.Z and H2B.V) and three histone H3 PTMs (H3K4me3, H3K16ac, and H3K76me3) are enriched at transcription start sites (TSSs); while a histone variant (H3.V) and the trypanosomatid-specific hyper-modified DNA base J are located at transcription termination sites (TTSs). Reduced nucleosome density was observed at all TTSs and TSSs for RNA genes transcribed by RNA polymerases I (RNAPI) or RNAPIII; as well as (to a lesser extent) at TSSs for the PTUs transcribed by RNAPII. Several PTMs (H3K4me3, H3K16ac H3K20me2 and H3K36me3) and base J were enriched at centromeres, while H3K50ac was specifically associated with the periphery of these centromeric sequences. These findings significantly expand our knowledge of the epigenetic markers associated with transcription, DNA replication and/or chromosome segregation in these early diverging eukaryotes and will hopefully lay the groundwork for future studies to elucidate how they control these fundamental processes.

Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is colocalized, genome-wide, with transcribing RNA polymerase (RNAP). Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, colocalization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched upstream (within up to 12-15 kb) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP:EcTopoI interaction by either overexpression of EcTopoI competitor (CTD or inactive EcTopoI Y319F mutant) or deletion of EcTopoI domains involved in the interaction is toxic for cells and leads to excessive negative plasmid supercoiling. Moreover, uncoupling of the RNAP:EcTopoI interaction leads to R-loops accumulation genome-wide, indicating that this interaction is required for prevention of R-loops formation.

Long-distance association of topological boundaries through nuclear condensates

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.

Homology-based reconstruction of regulatory networks for bacterial and archaeal genomes

Gene regulation is a key process for all microorganisms, as it allows them to adapt to different environmental stimuli. However, despite the relevance of gene expression control, for only a handful of organisms is there related information about genome regulation. In this work, we inferred the gene regulatory networks (GRNs) of bacterial and archaeal genomes by comparisons with six organisms with well-known regulatory interactions. The references we used are: Escherichia coli K-12 MG1655, Bacillus subtilis 168, Mycobacterium tuberculosis, Pseudomonas aeruginosa PAO1, Salmonella enterica subsp. enterica serovar typhimurium LT2, and Staphylococcus aureus N315. To this end, the inferences were achieved in two steps. First, the six model organisms were contrasted in an all-vs-all comparison of known interactions based on Transcription Factor (TF)-Target Gene (TG) orthology relationships and Transcription Unit (TU) assignments. In the second step, we used a guilt-by-association approach to infer the GRNs for 12,230 bacterial and 649 archaeal genomes based on TF-TG orthology relationships of the six bacterial models determined in the first step. Finally, we discuss examples to show the most relevant results obtained from these inferences. A web server with all the predicted GRNs is available at https://regulatorynetworks.unam.mx/ or http://132.247.46.6/.

Transcriptional Activation of Biosynthetic Gene Clusters in Filamentous Fungi.

Filamentous fungi are highly productive cell factories, many of which are industrial producers of enzymes, organic acids, and secondary metabolites. The increasing number of sequenced fungal genomes revealed a vast and unexplored biosynthetic potential in the form of transcriptionally silent secondary metabolite biosynthetic gene clusters (BGCs). Various strategies have been carried out to explore and mine this untapped source of bioactive molecules, and with the advent of synthetic biology, novel applications, and tools have been developed for filamentous fungi. Here we summarize approaches aiming for the expression of endogenous or exogenous natural product BGCs, including synthetic transcription factors, assembly of artificial transcription units, gene cluster refactoring, fungal shuttle vectors, and platform strains.

Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a

BackgroundFunctionally related genes in bacteria are often organized and transcribed as polycistronic transcriptional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci.ResultsCas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator’s sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non-polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase.ConclusionsWe conclude that Cas12a-mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intracellular ATP in the markerless ΔatpIBEFHAGDC mutant.

Shared Transcriptional Machinery at Homologous Alleles Leads to Reduced Transcription in Early Drosophila Embryos.

The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. We explored the possibility that interallelic interactions between two homologous alleles might affect gene regulation. Using an MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcripts of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the corresponding hemizygous allele, suggesting the possibility of allelic competition in homozygotes. However, the competition was not observed when the enhancer-promoter interaction was weakened by placing the reporter construct in a different chromosome location or by moving the enhancer further away from the promoter. Moreover, the reporter gene showed reduced transcriptional activity when a partial transcription unit (either an enhancer or reporter gene only) was in the homologous position. We propose that the transcriptional machinery that binds both the enhancer and promoter regions, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. We showed that the degree of allelic interference increased over developmental time as more Pol II was needed to activate zygotic genes. Such allelic competition was observed for an endogenous gene as well. Our study provides new insights into the role of 3D interallelic interactions in gene regulation.