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Molecular Cell Mar 2024RNA polymerase II (RNA Pol II) can backtrack during transcription elongation, exposing the 3' end of nascent RNA. Nascent RNA sequencing can approximate the location of...
RNA polymerase II (RNA Pol II) can backtrack during transcription elongation, exposing the 3' end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome-wide distribution of more persistent backtracking are unknown. Consequently, we developed a method to directly sequence the extruded, "backtracked" 3' RNA. Our data show that RNA Pol II slides backward more than 20 nt in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where RNA Pol II pauses near promoters and intron-exon junctions and is enriched in genes involved in translation, replication, and development, where gene expression is decreased if these events are unresolved. Histone genes are highly prone to persistent backtracking, and the resolution of such events is likely required for timely expression during cell division. These results demonstrate that persistent backtracking can potentially affect diverse gene expression programs.
Topics: Humans; RNA Polymerase II; RNA; Transcription, Genetic; DNA-Directed RNA Polymerases
PubMed: 38340716
DOI: 10.1016/j.molcel.2024.01.019 -
Phase-separation antagonists potently inhibit transcription and broadly increase nucleosome density.The Journal of Biological Chemistry Oct 2022Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene...
Biomolecular condensates are self-organized membraneless bodies involved in many critical cellular activities, including ribosome biogenesis, protein synthesis, and gene transcription. Aliphatic alcohols are commonly used to study biomolecular condensates, but their effects on transcription are unclear. Here, we explore the impact of the aliphatic dialcohol, 1,6-hexanediol (1,6-HD), on Pol II transcription and nucleosome occupancy in budding yeast. As expected, 1,6-HD, a reagent effective in disrupting biomolecular condensates, strongly suppressed the thermal stress-induced transcription of Heat Shock Factor 1-regulated genes that have previously been shown to physically interact and coalesce into intranuclear condensates. Surprisingly, the isomeric dialcohol, 2,5-HD, typically used as a negative control, abrogated Heat Shock Factor 1-target gene transcription under the same conditions. Each reagent also abolished the transcription of genes that do not detectably coalesce, including Msn2/Msn4-regulated heat-inducible genes and constitutively expressed housekeeping genes. Thus, at elevated temperature (39 °C), HDs potently inhibit the transcription of disparate genes and as demonstrated by chromatin immunoprecipitation do so by abolishing occupancy of RNA polymerase in chromatin. Concurrently, histone H3 density increased at least twofold within all gene coding and regulatory regions examined, including quiescent euchromatic loci, silent heterochromatic loci, and Pol III-transcribed loci. Our results offer a caveat for the use of HDs in studying the role of condensates in transcriptional control and provide evidence that exposure to these reagents elicits a widespread increase in nucleosome density and a concomitant loss of both Pol II and Pol III transcription.
Topics: Chromatin; Chromatin Immunoprecipitation; Nucleosomes; RNA Polymerase II; Transcription, Genetic; Glycols
PubMed: 35963432
DOI: 10.1016/j.jbc.2022.102365 -
Oxidative Medicine and Cellular... 2021Biomolecule metabolism produces ROS (reactive oxygen species) under physiological and pathophysiological conditions. Dietary factors (alcohol) and carcinogens (EGF, DEN,... (Review)
Review
Biomolecule metabolism produces ROS (reactive oxygen species) under physiological and pathophysiological conditions. Dietary factors (alcohol) and carcinogens (EGF, DEN, and MNNG) also induce the release of ROS. ROS often causes cell stress and tissue injury, eventually resulting in disorders or diseases of the body through different signaling pathways. Normal metabolism of protein is critically important to maintain cellular function and body health. Brf1 (transcript factor II B-related factor 1) and its target genes, RNA Pol III genes (RNA polymerase III-dependent genes), control the process of protein synthesis. Studies have demonstrated that the deregulation of Brf1 and its target genes is tightly linked to cell proliferation, cell transformation, tumor development, and human cancers, while alcohol, EGF, DEN, and MNNG are able to induce the deregulation of these genes through different signaling pathways. Therefore, it is very important to emphasize the roles of these signaling events mediating the processes of Brf1 and RNA Pol III gene transcription. In the present paper, we mainly summarize our studies on signaling events which mediate the deregulation of these genes in the past dozen years. These studies indicate that Brf1 and RNA Pol III genes are novel biological targets of ROS.
Topics: Cell Proliferation; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Humans; RNA Polymerase III; Reactive Oxygen Species; TATA-Binding Protein Associated Factors
PubMed: 34646425
DOI: 10.1155/2021/5888432 -
Nature Communications Jul 2023Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or...
Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) when transcription termination is disrupted by the viral ICP27 protein. This is accompanied by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperone FACT is not sufficient to induce dOCRs in ΔICP22 infection but increases dOCR induction in wild-type HSV-1 infection. Interestingly, this is accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone repositioning downstream of genes in the wake of Pol II in HSV-1 infection.
Topics: Humans; Histones; Herpesvirus 1, Human; Transcription, Genetic; Viral Proteins; Herpes Simplex; Chromatin; Immediate-Early Proteins
PubMed: 37524699
DOI: 10.1038/s41467-023-40217-w -
Genes Jan 2023DNA polymerase eta (Pol η) is a Y-family polymerase and the product of the gene. Autosomal recessive inheritance of mutations is the cause of the xeroderma... (Review)
Review
DNA polymerase eta (Pol η) is a Y-family polymerase and the product of the gene. Autosomal recessive inheritance of mutations is the cause of the xeroderma pigmentosum variant, a cancer predisposition syndrome. This review summarizes mounting evidence for expanded Pol η cellular functions in addition to DNA lesion bypass that are critical for maintaining genome stability. In vitro, Pol η displays efficient DNA synthesis through difficult-to-replicate sequences, catalyzes D-loop extensions, and utilizes RNA-DNA hybrid templates. Human Pol η is constitutively present at the replication fork. In response to replication stress, Pol η is upregulated at the transcriptional and protein levels, and post-translational modifications regulate its localization to chromatin. Numerous studies show that Pol η is required for efficient common fragile site replication and stability. Additionally, Pol η can be recruited to stalled replication forks through protein-protein interactions, suggesting a broader role in replication fork recovery. During somatic hypermutations, Pol η is recruited by mismatch repair proteins and is essential for V gene A:T basepair mutagenesis. Within the global context of repeat-dense genomes, the recruitment of Pol η to perform specialized functions during replication could promote genome stability by interrupting pure repeat arrays with base substitutions. Alternatively, not engaging Pol η in genome duplication is costly, as the absence of Pol η leads to incomplete replication and increased chromosomal instability.
Topics: Humans; Gene Duplication; DNA-Directed DNA Polymerase; DNA; Genomic Instability
PubMed: 36672916
DOI: 10.3390/genes14010175 -
Acta Virologica 2022Porcine xenograft transplantation raises concerns in humans about the risk of infection with porcine endogenous retroviruses (PERV) as they are an integral part of the...
Porcine xenograft transplantation raises concerns in humans about the risk of infection with porcine endogenous retroviruses (PERV) as they are an integral part of the pig genome and are therefore very difficult to exclude. In this study, for the first time, a relationship between the provirus genes sequences and released virions from pig cell line and the embedded sequence of this retrovirus in infected human cells was analyzed. PERV infection of human cells HEK-293 and HeLa and detection of PERV in pig PK-15 cells and supernatant were assessed by QPCR or RT-QPCR using primers specific for envA, envB, gag, pol genes and LTR region. Sequence analysis was performed at the DNA level and changes in the amino acid sequence were deduced in silico. Fifty nucleotide substitutions (45 in pol, 3 in gag and one each in envA and envB) were detected and most of these were heterozygous (42), which were present mainly in PK-15 cells. Our results show that sequence of the pol gene and the Pol protein is less conserved compared to the other PERV genes and PERV with some polymorphisms were not released from pig cells or/and do not infect human cells. PERV virions with a homozygous allele system were released from PK-15 cells, although their sequence replicated on the basis of the heterozygous PERV provirus sequence in PK-15. The newly discovered selective transduction of human cells with PERV will be helpful in studying the characteristics and genetic variability of the retrovirus genes to ensure safe xenotransplantation. Keywords: PERV; porcine endogenous retroviruses; infection; genetic polymorphism; xenotransplantation.
Topics: Animals; Endogenous Retroviruses; HEK293 Cells; HeLa Cells; Humans; Proviruses; Swine; Transplantation, Heterologous
PubMed: 35766468
DOI: 10.4149/av_2022_201 -
Molecular Cell Jan 2024Cellular homeostasis is constantly challenged by a myriad of extrinsic and intrinsic stressors. To mitigate the stress-induced damage, cells activate transient survival... (Review)
Review
Cellular homeostasis is constantly challenged by a myriad of extrinsic and intrinsic stressors. To mitigate the stress-induced damage, cells activate transient survival programs. The heat shock response (HSR) is an evolutionarily well-conserved survival program that is activated in response to proteotoxic stress. The HSR encompasses a dual regulation of transcription, characterized by rapid activation of genes encoding molecular chaperones and concomitant global attenuation of non-chaperone genes. Recent genome-wide approaches have delineated the molecular depth of stress-induced transcriptional reprogramming. The dramatic rewiring of gene and enhancer networks is driven by key transcription factors, including heat shock factors (HSFs), that together with chromatin-modifying enzymes remodel the 3D chromatin architecture, determining the selection of either gene activation or repression. Here, we highlight the current advancements of molecular mechanisms driving transcriptional reprogramming during acute heat stress. We also discuss the emerging implications of HSF-mediated stress signaling in the context of physiological and pathological conditions.
Topics: Proteostasis; Transcription Factors; Heat-Shock Response; Molecular Chaperones; Chromatin; Heat Shock Transcription Factors
PubMed: 38103561
DOI: 10.1016/j.molcel.2023.11.024 -
Nature Jan 2020Transcription challenges the integrity of replicating chromosomes by generating topological stress and conflicts with forks. The DNA topoisomerases Top1 and Top2 and the...
Transcription challenges the integrity of replicating chromosomes by generating topological stress and conflicts with forks. The DNA topoisomerases Top1 and Top2 and the HMGB family protein Hmo1 assist DNA replication and transcription. Here we describe the topological architecture of genes in Saccharomyces cerevisiae during the G1 and S phases of the cell cycle. We found under-wound DNA at gene boundaries and over-wound DNA within coding regions. This arrangement does not depend on Pol II or S phase. Top2 and Hmo1 preserve negative supercoil at gene boundaries, while Top1 acts at coding regions. Transcription generates RNA-DNA hybrids within coding regions, independently of fork orientation. During S phase, Hmo1 protects under-wound DNA from Top2, while Top2 confines Pol II and Top1 at coding units, counteracting transcription leakage and aberrant hybrids at gene boundaries. Negative supercoil at gene boundaries prevents supercoil diffusion and nucleosome repositioning at coding regions. DNA looping occurs at Top2 clusters. We propose that Hmo1 locks gene boundaries in a cruciform conformation and, with Top2, modulates the architecture of genes that retain the memory of the topological arrangements even when transcription is repressed.
Topics: Chromatin Assembly and Disassembly; DNA Replication; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA, Cruciform; DNA, Fungal; DNA, Superhelical; G1 Phase; Gene Expression Regulation, Fungal; Genes, Fungal; High Mobility Group Proteins; Mutation; Nucleic Acid Hybridization; Nucleosomes; Open Reading Frames; RNA Polymerase II; RNA, Fungal; S Phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription, Genetic
PubMed: 31969709
DOI: 10.1038/s41586-020-1934-4 -
Oncogene Feb 2024Mutations in APC, found in 80% of colon caner, enhance β-catenin stabilization, which is the initial step of colonic tumorigenesis. However, the core transcriptional...
Mutations in APC, found in 80% of colon caner, enhance β-catenin stabilization, which is the initial step of colonic tumorigenesis. However, the core transcriptional mechanism underlying the induction of colon cancer stemness by stable β-catenin remains unclear. Here, we found that inducible inhibition of β-catenin suppressed elongation of Pol II and RNA polymerase-associated factor 1 complex (PAF1C) around the transcription start site (TSS) of LGR5. Moreover, stable β-catenin enhanced the formation of active Pol II complex cooperatively with CDC73 and CDK9 by facilitating the recruitment of DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) complexes to the Pol II complex. Subsequently, stable β-catenin facilitated the formation of the Pol II-DSIF-PAF1C complex, suggesting that stable β-catenin induces cancer stemness by stimulating active Pol II complex through NELF and PAF1C. Furthermore, NELF or PAF1C inhibition recapitulated the changes in cancer stemness-related gene expression induced by the inhibition of stable β-catenin and suppressed colon cancer stemness. Additionally, the chemical inhibition of CDK12 (a downstream transcription CDK of PAF1C) suppressed colon cancer stemness. These results suggest that NELF and PAF1C are the core transcriptional machineries that control expression of colon cancer stemness-inducing genes and may be therapeutic targets for colon cancer.
Topics: Humans; beta Catenin; Colonic Neoplasms; Nuclear Proteins; RNA Polymerase II; Transcription Factors; Transcription, Genetic; Transcriptional Elongation Factors
PubMed: 38182897
DOI: 10.1038/s41388-023-02930-0 -
Journal of Medical Virology Jul 2023The HIV-1 pandemic has persisted for four decades, and poses a major challenge to global public health. Shenzhen, a city with large number of migrant populations in...
The HIV-1 pandemic has persisted for four decades, and poses a major challenge to global public health. Shenzhen, a city with large number of migrant populations in China, is suffering HIV-1 epidemic. It is necessary to continuously conduct the molecular surveillance among newly diagnosed HIV-1 patients in these migrant population. In this study, plasma samples of newly diagnosed and ART-naive HIV-1 infections were collected from Shenzhen city in China. The partial genes of HIV-1 gag and pol were amplified and sequenced for the analysis of genotype, drug resistance, and molecular transmission network. Ninety-one sequences of pol gene were obtained from newly diagnosed HIV-1 infections in Shenzhen, and seven HIV-1 subtypes were revealed in this investigation. Among them, the circulating recombinant form (CRF) 07_BC was the mostly frequent subtype (53.8%, 49/91), followed by CRF01_AE (20.9%, 19/91), CRF55_01B (9.9%, 9/91), unique recombinant forms (URFs) (8.8%, 8/91), B (3.3%, 3/91), CRF59_01B (2.2%, 2/91), and CRF08_BC (1.1%, 1/91). The overall prevalence of pretreatment drug resistance (PDR) was 23.1% (21/91), and 52.38% (11/21) of the PDR was specific for the nonnucleotide reverse transcriptase inhibitors (NNRTIs). Furthermore, a total of 3091 pol gene sequences were used to generate 19 molecular transmission clusters, and then one growing cluster, a new cluster, and a cluster with growth reactivation were identified. The result revealed that more sexual partner, CRF_07BC subtype, and seven amino acid deletions in gag p6 region might be the influencing factors associated with the high risk of transmission behavior. Compared with CRF01_AE subtype, CRF07_BC subtype strains were more likely to form clusters in molecular transmission network. This suggests that long-term surveillance of the HIV-1 molecular transmission should be a critical measure to achieve a precise intervention for controlling the spread of HIV-1 in China.
Topics: Humans; HIV-1; Phylogeny; HIV Infections; Genes, pol; HIV Seropositivity; Genotype; China; Drug Resistance, Viral
PubMed: 37477806
DOI: 10.1002/jmv.28973