Swi/Snf chromatin remodeling regulates transcriptional interference and gene repression.

Alternative transcription start sites can affect transcript isoform diversity and translation levels. In a recently described form of gene regulation, coordinated transcriptional and translational interference results in transcript isoform-dependent changes in protein expression. Specifically, a long undecoded transcript isoform (LUTI) is transcribed from a gene-distal promoter, interfering with expression of the gene-proximal promoter. Although transcriptional and chromatin features associated with LUTI expression have been described, the mechanism underlying LUTI-based transcriptional interference is not well understood. Using an unbiased genetic approach followed by functional genomics, we uncovered that the Swi/Snf chromatin remodeling complex is required for co-transcriptional nucleosome remodeling that leads to LUTI-based repression. We identified genes with tandem promoters that rely on Swi/Snf function for transcriptional interference during protein folding stress, including LUTI-regulated genes. This study provides clear evidence for Swi/Snf playing a direct role in gene repression via a cis transcriptional interference mechanism.

Transcriptional interference by RNA polymerase III affects expression of the Polr3e gene.

Overlapping gene arrangements can potentially contribute to gene expression regulation. A mammalian interspersed repeat (MIR) nested in antisense orientation within the first intron of the Polr3e gene, encoding an RNA polymerase III (Pol III) subunit, is conserved in mammals and highly occupied by Pol III. Using a fluorescence assay, CRISPR/Cas9-mediated deletion of the MIR in mouse embryonic stem cells, and chromatin immunoprecipitation assays, we show that the MIR affects Polr3e expression through transcriptional interference. Our study reveals a mechanism by which a Pol II gene can be regulated at the transcription elongation level by transcription of an embedded antisense Pol III gene.

DNA Processing in the Context of Noncoding Transcription.

RNA polymerase (RNAP)II frequently transcribes non-protein-coding DNA sequences in eukaryotic genomes into long noncoding RNA (lncRNA). Distinct molecular mechanisms linked to the position of lncRNA relative to the coding gene illustrate how noncoding transcription controls gene expression. Here, we focus on the impact of the act of lncRNA transcription on nearby functional DNA units. We review the biological significance of the act of lncRNA transcription on DNA processing, highlighting common themes, such as mediating cellular responses to environmental changes. This review combines the background of chromatin signaling with examples in several organisms to clarify when functions of ncDNA can be interpreted through the act of RNAPII transcription.

Unique features of transcription termination and initiation at closely spaced tandem human genes.

The synthesis of RNA polymerase II (Pol2) products, which include messenger RNAs or long noncoding RNAs, culminates in transcription termination. How the transcriptional termination of a gene impacts the activity of promoters found immediately downstream of it, and which can be subject to potential transcriptional interference, remains largely unknown. We examined in an unbiased manner the features of the intergenic regions between pairs of 'tandem genes'-closely spaced (< 2 kb) human genes found on the same strand. Intergenic regions separating tandem genes are enriched with guanines and are characterized by binding of several proteins, including AGO1 and AGO2 of the RNA interference pathway. Additionally, we found that Pol2 is particularly enriched in this region, and it is lost upon perturbations affecting splicing or transcriptional elongation. Perturbations of genes involved in Pol2 pausing and R loop biology preferentially affect expression of downstream genes in tandem gene pairs. Overall, we find that features associated with Pol2 pausing and accumulation rather than those associated with avoidance of transcriptional interference are the predominant driving force shaping short tandem intergenic regions.

Imprinted Long Non-Coding RNAs in Mammalian Development and Disease.

Imprinted genes play diverse roles in mammalian development, homeostasis, and disease. Most imprinted chromosomal domains express one or more long non-coding RNAs (lncRNAs). Several of these lncRNAs are strictly nuclear and their mono-allelic expression controls in cis the expression of protein-coding genes, often developmentally regulated. Some imprinted lncRNAs act in trans as well, controlling target gene expression elsewhere in the genome. The regulation of imprinted gene expression-including that of imprinted lncRNAs-is susceptible to stochastic and environmentally triggered epigenetic changes in the early embryo. These aberrant changes persist during subsequent development and have long-term phenotypic consequences. This review focuses on the expression and the cis- and trans-regulatory roles of imprinted lncRNAs and describes human disease syndromes associated with their perturbed expression.

Emerging Roles of Non-Coding RNA Transcription.

Metazoan genomes are broadly transcribed by RNA polymerase II (RNAPII), but surprisingly few of these RNAs encode proteins. Accordingly, there is great interest in understanding the origins and potential roles of the vast array of non-coding RNAs (ncRNAs) that are produced. We present here emerging evidence that the act of transcription and the presence of nascent RNA at a locus is often central to function, rather than specific ncRNA sequences or structures. We highlight examples wherein transcription elongation through a regulatory region modulates chromatin structure and/or transcription factor occupancy, and describe how nascent RNA contributes to the local epigenetic landscape through sequence-independent interactions with chromatin regulators. Finally, we discuss current strategies for probing the potential functions of ncRNA transcription.

Analysis of the organization and expression patterns of the convergent Pseudomonas aeruginosa lasR/rsaL gene pair uncovers mutual influence.

The two adjacent genes encoding the major Pseudomonas aeruginosa quorum-sensing regulator, LasR, and its opponent, RsaL, overlap in their coding 3' ends and produce mRNA transcripts with long untranslated 3' ends that overlap with the sense transcripts of the gene on the opposing DNA strand. In this study, we evaluated whether the overlapping genes are involved in mutual regulatory events and studied interference by natural antisense transcripts. We introduced various gene expression constructs into a P. aeruginosa PA14 lasR/rsaL double deletion mutant, and found that although complementary RNA is produced, this does not interfere with the sense gene expression levels of lasR and rsaL and does not have functional consequences on down-stream gene regulation. Nevertheless, expression of lasR, but not of rsaL, was shown to be enhanced if transcription was terminated at the end of the respective gene so that no overlapping transcription was allowed. Our data indicate that the natural organization with a partial overlap at the 3' ends of the lasR/rsaL genes gives rise to a system of checks and balances to prevent dominant and unilateral control by LasR over the RsaL transcriptional regulator of opposing function.

Intragenic transcriptional interference regulates the human immune ligand MICA.

Many human genes have tandem promoters driving overlapping transcription, but the value of this distributed promoter configuration is generally unclear. Here we show that MICA, a gene encoding a ligand for the activating immune receptor NKG2D, contains a conserved upstream promoter that expresses a noncoding transcript. Transcription from the upstream promoter represses the downstream standard promoter activity in cis through transcriptional interference. The effect of transcriptional interference depends on the strength of transcription from the upstream promoter and can be described quantitatively by a simple reciprocal repressor function. Transcriptional interference coincides with recruitment at the standard downstream promoter of the FACT histone chaperone complex, which is involved in nucleosomal remodelling during transcription. The mechanism is invoked in the regulation of MICA expression by the physiological inputs interferon-γ and interleukin-4 that act on the upstream promoter. Genome-wide analysis indicates that transcriptional interference between tandem intragenic promoters may constitute a general mechanism with widespread importance in human transcriptional regulation.

Lost Small Envelope Protein Expression from Naturally Occurring PreS1 Deletion Mutants of Hepatitis B Virus Is Often Accompanied by Increased HBx and Core Protein Expression as Well as Genome Replication.

Hepatitis B virus (HBV) transcribes coterminal mRNAs of 0.7 to 3.5 kb from the 3.2-kb covalently closed circular DNA, with the 2.1-kb RNA being most abundant. The 0.7-kb RNA produces HBx protein, a transcriptional transactivator, while the 3.5-kb pregenomic RNA (pgRNA) drives core and P protein translation as well as genome replication. The large (L) and small (S) envelope proteins are translated from the 2.4-kb and 2.1-kb RNAs, respectively, with the majority of the S protein being secreted as noninfectious subviral particles and detected as hepatitis B surface antigen (HBsAg). pgRNA transcription could inhibit transcription of subgenomic RNAs. The present study characterized naturally occurring in-frame deletions in the 3' preS1 region, which not only codes for L protein but also serves as the promoter for 2.1-kb RNA. The human hepatoma cell line Huh7 was transiently transfected with subgenomic expression constructs for envelope (and HBx) proteins, dimeric constructs, or constructs mimicking covalently closed circular DNA. The results confirmed lost 2.1-kb RNA transcription and HBsAg production from many deletion mutants, accompanied by increases in other (especially 2.4-kb) RNAs, intracellular HBx and core proteins, and replicative DNA but impaired virion and L protein secretion. The highest intracellular L protein levels were achieved by mutants that had residual S protein expression or retained the matrix domain in L protein. Site-directed mutagenesis of a high replicating deletion mutant suggested that increased HBx protein expression and blocked virion secretion both contributed to the high replication phenotype. Our findings could help explain why such deletions are selected at a late stage of chronic HBV infection and how they contribute to viral pathogenesis. IMPORTANCE Expression of hepatitis B e antigen (HBeAg) and overproduction of HBsAg by wild-type HBV are implicated in the induction of immune tolerance to achieve chronic infection. How HBV survives the subsequent immune clearance phase remains incompletely understood. Our previous characterization of core promoter mutations to reduce HBeAg production revealed the ability of the 3.5-kb pgRNA to diminish transcription of coterminal RNAs of 2.4 kb, 2.1 kb, and 0.7 kb. The later stage of chronic HBV infection often selects for in-frame deletions in the preS region. Here, we found that many 3' preS1 deletions prevented transcription of the 2.1-kb RNA for HBsAg production, which was often accompanied by increases in intracellular 3.5-, 0.7-, and especially 2.4-kb RNAs, HBx and core proteins, and replicative DNA but lost virion secretion. These findings established the biological consequences of preS1 deletions, thus shedding light on why they are selected and how they contribute to hepatocarcinogenesis.

Dynamics of DNA replication in a eukaryotic cell.

Each genomic locus in a eukaryotic cell has a distinct average time of replication during S phase that depends on the spatial and temporal pattern of replication initiation events. Replication timing can affect genomic integrity because late replication is associated with an increased mutation rate. For most eukaryotes, the features of the genome that specify the location and timing of initiation events are unknown. To investigate these features for the fission yeast, Schizosaccharomyces pombe, we developed an integrative model to analyze large single-molecule and global genomic datasets. The model provides an accurate description of the complex dynamics of S. pombe DNA replication at high resolution. We present evidence that there are many more potential initiation sites in the S. pombe genome than previously identified and that the distribution of these sites is primarily determined by two factors: the sequence preferences of the origin recognition complex (ORC), and the interference of transcription with the assembly or stability of prereplication complexes (pre-RCs). We suggest that in addition to directly interfering with initiation, transcription has driven the evolution of the binding properties of ORC in S. pombe and other eukaryotic species to target pre-RC assembly to regions of the genome that are less likely to be transcribed.

Close arrangement of CARK3 and PMEIL affects ABA-mediated pollen sterility in Arabidopsis thaliana.

Abscisic acid (ABA) signaling is a vital plant signaling pathway for plant responses to stress conditions. ABA treatment can alter global gene expression patterns and cause significant phenotypic changes. We investigated the responses to ABA treatment during flowering in Arabidopsis thaliana. Dipping the flowers of CARK3 T-DNA mutants in ABA solution, led to less reduction of pollen fertility than in the wild type plants (Col-0). We demonstrated that PMEIL, a gene located downstream of CARK3, directly affects pollen fertility. Due to the close arrangement of CARK3 and PMEIL, CARK3 expression represses transcription of PMEIL in an ABA-dependent manner through transcriptional interference. Our study uncovers a molecular mechanism underlying ABA-mediated pollen sterility and provides an example of how transcriptional interference caused by close arrangement of genes may mediate stress responses during plant reproduction.

Transcription of cis Antisense Small RNA MtlS in Vibrio cholerae Is Regulated by Transcription of Its Target Gene, mtlA.

Vibrio cholerae, the facultative pathogen responsible for cholera disease, continues to pose a global health burden. Its persistence can be attributed to a flexible genetic tool kit that allows for adaptation to different environments with distinct carbon sources, including the six-carbon sugar alcohol mannitol. V. cholerae takes up mannitol through the transporter protein MtlA, whose production is downregulated at the posttranscriptional level by MtlS, a cis antisense small RNA (sRNA) whose promoter lies within the mtlA open reading frame. Though it is known that mtlS expression is robust under growth conditions lacking mannitol, it has remained elusive as to what factors govern the steady-state levels of MtlS. Here, we show that manipulating mtlA transcription is sufficient to drive inverse changes in MtlS levels, likely through transcriptional interference. This work has uncovered a cis-acting sRNA whose expression pattern is predominantly controlled by transcription of the sRNA's target gene.IMPORTANCEVibrio cholerae is a bacterial pathogen that relies on genetic tools, such as regulatory RNAs, to adapt to changing extracellular conditions. While many studies have focused on how these regulatory RNAs function, fewer have focused on how they are themselves modulated. V. cholerae expresses the noncoding RNA MtlS, which can regulate mannitol transport and use, and here we demonstrate that MtlS levels are controlled by the level of transcription occurring in the antisense direction. Our findings provide a model of regulation describing how bacteria like V. cholerae can modulate the levels of an important regulatory RNA. Our work contributes to knowledge of how bacteria deploy regulatory RNAs as an adaptive mechanism to buffer against environmental flux.

Interactions between the transcription and replication machineries regulate the RNA and DNA synthesis in the herpesviruses.

The temporal coordination of viral gene expression is imperative for the regulation of the herpesvirus replication cycle. While the main factors of this transcriptional coordination are known, the subtler control mechanisms of gene expression remain elusive. Recent long read sequencing-based approached have revealed an intricate meshwork of overlaps between the herpesvirus transcripts and the overlap of the replication origins with noncoding RNAs. It has been shown that the transcriptional apparatuses can physically interfere with one another while transcribing overlapping regions. We hypothesize that transcriptional interference regulates the global gene expression across the herpesvirus genome. Additionally, an overall decrease in transcriptional activity in individual viral genes has been observed following the onset of DNA replication. An overlap of the replication origins with specific transcripts has also been described in several herpesviruses. The genome-wide interactions between the transcriptional apparatuses and between the replication and transcriptional machineries suggest the existence of novel layers of genetic regulation.

Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis.

Hyaluronic acid (HA) is a high-value biopolymer that is produced in large scales using attenuated strains ofgroup C streptococci. However, due to the pathogenicity and fastidious nature of these bacteria, the development of bioprocesses for HA production centered on robust 'Generally Recognized as Safe (GRAS)' organisms, such as Bacillus subtilis, is of increased interest. Here, we report metabolic engineering of novel B. subtilis strains in which the carbon flux has been partially diverted from central metabolism, i.e. the pentose phosphate pathway (PPP) and glycolysis, into HA biosynthesis. First, an improved base strain of B. subtilis was engineered for more effective HA production with less susceptibility to catabolite repression when expressing genes from a xylose-inducible promoter. Subsequently, Clustered Regularly Interspaced Palindromic Repeats interference (CRISPRi) was applied to reduce the expression of individual pfkA or zwf in the base strain, leading to substantial improvements to the HA titer with a concomitant decrease in the molecular weight (MW). On the other hand, multiplexed repression of both pfkA and zwf expression resulted in increases to the HA titer of up to 108% and enhancements to the MW, compared to the base strain. Moreover, the addition of exogenous HA monomers, i.e. glucuronic acid (GlcUA) and N-acetyl-glucosamine (GlcNAc), to B. subtilis cultures markedly improved the HA MW but decreased the HA titer, providing insights into the mechanism of HA biosynthesis by streptococcal hyaluronan synthase (SeHAS) in B. subtilis. Our study demonstrates the successful application of metabolic engineering strategies to establish B. subtilis as an effective platform for high-level HA production.

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis.

Hyaluronic acid (HA) is a high-value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust "Generally Recognized as Safe (GRAS)" organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase (HAS) of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production. Elevation of membrane CL levels via overexpressing components involved in the CL biosynthesis pathway, and redistribution of CL along the lateral membrane via repression of the cell division initiator protein FtsZ resulted in increases to the HA titer of up to 204% and peak molecular weight of up to 2.2 MDa. Moreover, removal of phosphatidylethanolamine and neutral glycolipids from the membrane of HA-producing B. subtilis via inactivation of pssA and ugtP, respectively, has suggested the lipid dependence for functional expression of SeHAS. Our study demonstrates successful application of membrane engineering strategies to develop an effective platform for biomanufacturing of HA with B. subtilis strains expressing Class I streptococcal HAS.

The Molecular Biology of HIV Latency.

HIV remains incurable due to the existence of a reservoir of cells that harbor intact integrated genomes of the virus in the absence of viral replication. This population of infected cells remains invisible to the immune system and is not targeted by the drugs used in the current antiretroviral therapies (cART). Reversal of latency by the use of inhibitors of chromatin-remodeling enzymes has been studied extensively in an attempt to purge this reservoir of latent HIV but has thus far not shown any success in clinical trials. The full complexity of latent HIV infection has still not been appreciated, and the gaps in knowledge prevent development of adequate small-molecule compounds that can effectively perturb this reservoir. In this review, we will examine the role of epigenetic silencing of HIV transcription, posttranscriptional regulation, and mRNA processing in promoting HIV-1 latency.

Methylation of an intragenic alternative promoter regulates transcription of GARP.

Alternative promoter usage has been proposed as a mechanism regulating transcriptional and translational diversity in highly elaborated systems like the immune system in humans. Here, we report that transcription of human glycoprotein A repetitions predominant (GARP) in regulatory CD4 T cells (Tregs) is tightly regulated by two alternative promoters. An intragenic promoter contains several CpGs and acts as a weak promoter that is demethylated and initiates transcription Treg-specifically. The strong up-stream promoter containing a CpG-island is, in contrast, fully demethylated throughout tissues. Transcriptional activity of the strong promoter was surprisingly down-regulated upon demethylation of the weak promoter. This demethylation-induced transcriptional attenuation regulated the magnitude of GARP expression and correlated with disease activity in rheumatoid arthritis. Treg-specific GARP transcription was initiated by synergistic interaction of forkhead box protein 3 (Foxp3) with nuclear factor of activated T cells (NFAT) and was underpinned by permissive chromatin remodeling caused by release of the H3K4 demethylase, PLU-1. Our findings describe a novel function of alternative promoters in regulating the extent of transcription. Moreover, since GARP functions as a transporter of transforming growth factor ß (TGFß), a cytokine with broad pleiotropic traits, GARP transcriptional attenuation by alternative promoters might provide a mechanism regulating peripheral TGFß to avoid unwanted harmful effects.

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents.

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.

Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure.

The mouse is extensively used to model human folate metabolism and therapeutic outcomes with antifolates. However, the folylpoly-γ-glutamate synthetase (fpgs) gene, whose product determines folate/antifolate intracellular retention and antifolate antitumor activity, displays a pronounced species difference. The human gene uses only a single promoter, whereas the mouse uses two: P2, akin to the human promoter, at low levels in most tissues; and P1, an upstream promoter used extensively in liver and kidney. We deleted the mouse P1 promoter through homologous recombination to study the dual-promoter mouse system and to create a mouse with a humanized fpgs gene structure. Despite the loss of the predominant fpgs mRNA species in liver and kidney (representing 95 and 75% of fpgs transcripts in these tissues, respectively), P1-knockout mice developed and reproduced normally. The survival of these mice was explained by increased P2 transcription due to relief of transcriptional interference, by a 3-fold more efficient translation of P2-derived than P1-derived transcripts, and by 2-fold higher stability of P2-derived FPGS. In combination, all 3 effects reinstated FPGS function, even in liver. By eliminating mouse P1, we created a mouse model that mimicked the human housekeeping pattern of fpgs gene expression.

MetJ-Based Mutually Interfering SAM-ON/SAM-OFF Biosensors.

SAM (S-adenosylmethionine) is an important metabolite that operates as a major donor of methyl groups and is a controller of various physiological processes. Its availability is also believed to be a major bottleneck in the biological production of numerous high-value metabolites. Here, we constructed SAM-sensing systems using MetJ, an SAM-dependent transcriptional regulator, as a core component. SAM is a corepressor of MetJ, which suppresses the MetJ promoter with an increasing cellular concentration of SAM (SAM-OFF sensor). The application of transcriptional interference and evolutionary tuning effectively inverted its response, yielding a SAM-ON sensor (signal increases with increasing SAM concentration). By linking two genes encoding fluorescent protein reporters in such a way that their transcription events interfere with each other's and by placing one of them under the control of MetJ, we could increase the effective signal-to-noise ratio of the SAM sensor while decreasing the batch-to-batch deviation in signal output, likely by canceling out the growth-associated fluctuation in translational resources. By taking the ratio of SAM-ON/SAM-OFF signals and by resetting the default pool size of SAM, we could rapidly identify SAM synthetase (MetK) mutants with increased cellular activity from a random library. The strategy described herein should be widely applicable for identifying activity mutants, which would be otherwise overlooked because of the strong homeostasis of metabolic networks.