HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells

eLife ◽

10.7554/elife.08955 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 24

Author(s):

Jonathan E Reeder ◽

Youn-Tae Kwak ◽

Ryan P McNamara ◽

Christian V Forst ◽

Iván D'Orso

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Immune Cells ◽

Rna Structure ◽

Molecular Mechanisms ◽

Target Genes ◽

Regulatory Mechanisms ◽

Messenger Rnas ◽

Transcriptional Regulatory Mechanisms ◽

Cellular Transcription

HIV encodes Tat, a small protein that facilitates viral transcription by binding an RNA structure (trans-activating RNA [TAR]) formed on nascent viral pre-messenger RNAs. Besides this well-characterized mechanism, Tat appears to modulate cellular transcription, but the target genes and molecular mechanisms remain poorly understood. We report here that Tat uses unexpected regulatory mechanisms to reprogram target immune cells to promote viral replication and rewire pathways beneficial for the virus. Tat functions through master transcriptional regulators bound at promoters and enhancers, rather than through cellular ‘TAR-like’ motifs, to both activate and repress gene sets sharing common functional annotations. Despite the complexity of transcriptional regulatory mechanisms in the cell, Tat precisely controls RNA polymerase II recruitment and pause release to fine-tune the initiation and elongation steps in target genes. We propose that a virus with a limited coding capacity has optimized its genome by evolving a small but ‘multitasking’ protein to simultaneously control viral and cellular transcription.

Download Full-text

Eukaryotic RNA Polymerases: The Many Ways to Transcribe a Gene

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.663209 ◽

2021 ◽

Vol 8 ◽

Author(s):

Marina Barba-Aliaga ◽

Paula Alepuz ◽

José E. Pérez-Ortín

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Environmental Changes ◽

Gene Promoter ◽

Rna Polymerases ◽

Regulatory Mechanisms ◽

Gene Copy ◽

Synthesis Rate ◽

Promoter Strength ◽

Messenger Rnas

In eukaryotic cells, three nuclear RNA polymerases (RNA pols) carry out the transcription from DNA to RNA, and they all seem to have evolved from a single enzyme present in the common ancestor with archaea. The multiplicity of eukaryotic RNA pols allows each one to remain specialized in the synthesis of a subset of transcripts, which are different in the function, length, cell abundance, diversity, and promoter organization of the corresponding genes. We hypothesize that this specialization of RNA pols has conditioned the evolution of the regulatory mechanisms used to transcribe each gene subset to cope with environmental changes. We herein present the example of the homeostatic regulation of transcript levels versus changes in cell volume. We propose that the diversity and instability of messenger RNAs, transcribed by RNA polymerase II, have conditioned the appearance of regulatory mechanisms based on different gene promoter strength and mRNA stability. However, for the regulation of ribosomal RNA levels, which are very stable and transcribed mainly by RNA polymerase I from only one promoter, different mechanisms act based on gene copy variation, and a much simpler regulation of the synthesis rate.

Download Full-text

The Mediator Subunit MED16 Transduces NRF2-Activating Signals into Antioxidant Gene Expression

Molecular and Cellular Biology ◽

10.1128/mcb.00785-15 ◽

2015 ◽

Vol 36 (3) ◽

pp. 407-420 ◽

Cited By ~ 28

Author(s):

Hiroki Sekine ◽

Keito Okazaki ◽

Nao Ota ◽

Hiroki Shima ◽

Yasutake Katoh ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Target Genes ◽

Antioxidant Response ◽

Wild Type ◽

Antioxidant Genes ◽

Antioxidant Gene Expression

The KEAP1-NRF2 system plays a central role in cytoprotection. NRF2 is stabilized in response to electrophiles and activates transcription of antioxidant genes. Although robust induction of NRF2 target genes confers resistance to oxidative insults, how NRF2 triggers transcriptional activation after binding to DNA has not been elucidated. To decipher the molecular mechanisms underlying NRF2-dependent transcriptional activation, we purified the NRF2 nuclear protein complex and identified the Mediator subunits as NRF2 cofactors. Among them, MED16 directly associated with NRF2. Disruption ofMed16significantly attenuated the electrophile-induced expression of NRF2 target genes but did not affect hypoxia-induced gene expression, suggesting a specific requirement for MED16 in NRF2-dependent transcription. Importantly, we found that 75% of NRF2-activated genes exhibited blunted inductions by electrophiles inMed16-deficient cells compared to wild-type cells, which strongly argues that MED16 is a major contributor supporting NRF2-dependent transcriptional activation. NRF2-dependent phosphorylation of the RNA polymerase II C-terminal domain was absent inMed16-deficient cells, suggesting that MED16 serves as a conduit to transmit NRF2-activating signals to RNA polymerase II. MED16 indeed turned out to be essential for cytoprotection against oxidative insults. Thus, the KEAP1-NRF2-MED16 axis has emerged as a new regulatory pathway mediating the antioxidant response through the robust activation of NRF2 target genes.

Download Full-text

The Integrator complex regulates microRNA abundance through RISC loading

10.1101/2021.09.21.461113 ◽

2021 ◽

Author(s):

Nina Kirstein ◽

Sadat Dokaneheifard ◽

Pradeep Reddy Cingaram ◽

Monica Guiselle Valencia ◽

Felipe Beckedorff ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Molecular Mechanisms ◽

Target Genes ◽

Mature Mirnas ◽

Mirna Biogenesis ◽

Important Regulator ◽

Rna Maturation ◽

Nascent Transcripts ◽

Post Transcriptional Regulation ◽

Mirna Duplex

MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The molecular mechanisms underlying miRNA biogenesis from processing of primary miRNA transcripts to formation of mature miRNA duplex are well understood. Loading of miRNA duplex into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing. The Integrator complex has been previously shown to be an important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination. Here, we report that loss of Integrator results in global diminution of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Enhanced UV crosslinking and immunoprecipitation (eCLIP) of Integrator confirms a robust association with mature miRNAs. Indeed, Integrator potentiates Ago2-mediated cleavage of target RNAs. These findings highlight an essential role for Integrator in miRNA abundance and RISC function.

Download Full-text

Elucidating the Mechanisms of Hugan Buzure Granule in the Treatment of Liver Fibrosis via Network Pharmacology

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/8385706 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Yi Zhu ◽

Ming Qiao ◽

Jianhua Yang ◽

Junping Hu

Keyword(s):

Liver Fibrosis ◽

Rna Polymerase Ii ◽

Molecular Mechanisms ◽

Target Genes ◽

Docking Simulation ◽

Interaction Network ◽

Network Pharmacology ◽

Transcription Activator ◽

Active Ingredients ◽

Oxidoreductase Activity

Objective. To holistically explore the latent active ingredients, targets, and related mechanisms of Hugan buzure granule (HBG) in the treatment of liver fibrosis (LF) via network pharmacology. Methods. First, we collected the ingredients of HBG by referring the TCMSP server and literature and filtered the active ingredients though the criteria of oral bioavailability ≥30% and drug-likeness index ≥0.18. Second, herb-associated targets were predicted and screened based on the BATMAN-TCM and SwissTargetPrediction platforms. Candidate targets related to LF were collected from the GeneCards and OMIM databases. Furthermore, the overlapping target genes were used to construct the protein-protein interaction network and “drug-compound-target-disease” network. Third, GO and KEGG pathway analyses were carried out to illustrate the latent mechanisms of HBG in the treatment of LF. Finally, the combining activities of hub targets with active ingredients were further verified based on software AutoDock Vina. Results. A total of 25 active ingredients and 115 overlapping target genes of HBG and LF were collected. Besides, GO enrichment analysis exhibited that the overlapping target genes were involved in DNA-binding transcription activator activity, RNA polymerase II-specific, and oxidoreductase activity. Simultaneously, the key molecular mechanisms of HBG against LF were mainly involved in PI3K-AKT, MAPK, HIF-1, and NF-κB signaling pathways. Also, molecular docking simulation demonstrated that the key targets of HBG for antiliver fibrosis were IL6, CASP3, EGFR, VEGF, and MAPK. Conclusion. This work validated and predicted the underlying mechanisms of multicomponent and multitarget about HBG in treating LF and provided a scientific foundation for further research.

Download Full-text

mRNA adenosine methylase (MTA) deposits m6A on pri-miRNAs to modulate miRNA biogenesis inArabidopsis thaliana

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2003733117 ◽

2020 ◽

Vol 117 (35) ◽

pp. 21785-21795 ◽

Cited By ~ 2

Author(s):

Susheel Sagar Bhat ◽

Dawid Bielewicz ◽

Tomasz Gulanicz ◽

Zsuzsanna Bodi ◽

Xiang Yu ◽

...

Keyword(s):

Secondary Structure ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Structure ◽

Methylation Status ◽

Mirna Biogenesis ◽

Stem Loop ◽

Structure Probing ◽

Plant Transcriptome ◽

Loop Regions

InArabidopsis thaliana, the METTL3 homolog, mRNA adenosine methylase (MTA) introducesN6-methyladenosine (m6A) into various coding and noncoding RNAs of the plant transcriptome. Here, we show that an MTA-deficient mutant (mta) has decreased levels of microRNAs (miRNAs) but accumulates primary miRNA transcripts (pri-miRNAs). Moreover, pri-miRNAs are methylated by MTA, and RNA structure probing analysis reveals a decrease in secondary structure within stem–loop regions of these transcripts inmtamutant plants. We demonstrate interaction between MTA and both RNA Polymerase II and TOUGH (TGH), a plant protein needed for early steps of miRNA biogenesis. Both MTA and TGH are necessary for efficient colocalization of the Microprocessor components Dicer-like 1 (DCL1) and Hyponastic Leaves 1 (HYL1) with RNA Polymerase II. We propose that secondary structure of miRNA precursors induced by their MTA-dependent m6A methylation status, together with direct interactions between MTA and TGH, influence the recruitment of Microprocessor to plant pri-miRNAs. Therefore, the lack of MTA inmtamutant plants disturbs pri-miRNA processing and leads to the decrease in miRNA accumulation. Furthermore, our findings reveal that reduced miR393b levels likely contributes to the impaired auxin response phenotypes ofmtamutant plants.

Download Full-text

PPARβ/δ recruits NCOR and regulates transcription reinitiation of ANGPTL4

Nucleic Acids Research ◽

10.1093/nar/gkz685 ◽

2019 ◽

Vol 47 (18) ◽

pp. 9573-9591 ◽

Cited By ~ 1

Author(s):

Nathalie Legrand ◽

Clemens L Bretscher ◽

Svenja Zielke ◽

Bernhard Wilke ◽

Michael Daude ◽

...

Keyword(s):

Mass Spectrometry ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Receptor ◽

Transcription Initiation ◽

Target Gene ◽

Target Genes ◽

Hdac Inhibition ◽

Inverse Agonists ◽

Transcription Reinitiation

Abstract In the absence of ligands, the nuclear receptor PPARβ/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question how PPARβ/δ represses transcription mechanistically. We show that the PPARβ/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARβ/δ. Reconstitution of knockout cells with PPARβ/δ mutants deficient in basal repression results in diminished recruitment of NCOR, SMRT, and HDAC3 to PPAR target genes, while occupancy by RNA polymerase II is increased. PT-S264 restores binding of NCOR, SMRT, and HDAC3 to the mutants, resulting in reduced polymerase II occupancy. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes, which act in parallel to downregulate transcription.

Download Full-text

Postrecruitment Regulation of RNA Polymerase II Directs Rapid Signaling Responses at the Promoters of Estrogen Target Genes

Molecular and Cellular Biology ◽

10.1128/mcb.00841-08 ◽

2008 ◽

Vol 29 (5) ◽

pp. 1123-1133 ◽

Cited By ~ 63

Author(s):

Miltiadis Kininis ◽

Gary D. Isaacs ◽

Leighton J. Core ◽

Nasun Hah ◽

W. Lee Kraus

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Target Genes ◽

Elongation Factor ◽

Estrogen Signaling ◽

Gene Promoters ◽

Pol Ii ◽

Activator Proteins ◽

Classical Models ◽

Target Promoters

ABSTRACT Under classical models for signal-dependent transcription in eukaryotes, DNA-binding activator proteins regulate the recruitment of RNA polymerase II (Pol II) to a set of target promoters. However, recent studies, as well as our results herein, show that Pol II is widely distributed (i.e., “preloaded”) at the promoters of many genes prior to specific signaling events. How Pol II recruitment and Pol II preloading fit within a unified model of gene regulation is unclear. In addition, the mechanisms through which cellular signals activate preloaded Pol II across mammalian genomes remain largely unknown. We show here that the predominant genomic outcome of estrogen signaling is the postrecruitment regulation of Pol II activity at target gene promoters, likely through specific changes in Pol II phosphorylation rather than through recruitment of Pol II to the promoters. Furthermore, we show that negative elongation factor binds to estrogen target promoters in conjunction with preloaded Pol II and represses gene expression until the appropriate signal is received. Finally, our studies reveal that the estrogen-dependent activation of preloaded Pol II facilitates rapid gene regulatory responses which play important physiological roles in regulating estrogen signaling itself. Our results reveal a broad use of postrecruitment Pol II regulation by the estrogen signaling pathway, a mode of regulation that is likely to apply to a wide variety of signal-regulated pathways.

Download Full-text

Splicing-independent recruitment of spliceosomal small nuclear RNPs to nascent RNA polymerase II transcripts

The Journal of Cell Biology ◽

10.1083/jcb.200706134 ◽

2007 ◽

Vol 178 (6) ◽

pp. 937-949 ◽

Cited By ~ 12

Author(s):

Snehal Bhikhu Patel ◽

Natalya Novikova ◽

Michel Bellini

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Messenger Rnas ◽

Stem Loop ◽

Small Nuclear Ribonucleoprotein ◽

Small Nuclear Rnas ◽

Transcriptional Units ◽

Necessary And Sufficient ◽

Nuclear Ribonucleoprotein ◽

Nascent Transcripts

In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre–messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.

Download Full-text

Flavopiridol Down-Regulates Genes Involved in Cell Cycle Regulation and Tumor Progression in Adults with Refractory or Poor-Risk Acute Leukemia.

Blood ◽

10.1182/blood.v112.11.953.953 ◽

2008 ◽

Vol 112 (11) ◽

pp. 953-953 ◽

Cited By ~ 1

Author(s):

Linda Resar ◽

Joelle Hillion ◽

Katrina Alino ◽

Michelle Rudek ◽

Judith Karp

Keyword(s):

Cell Cycle ◽

Acute Leukemia ◽

Rna Polymerase ◽

Cyclin D1 ◽

Rna Polymerase Ii ◽

Target Genes ◽

Mrna Levels ◽

Cyclin Dependent Kinase ◽

Poor Risk ◽

Before And After

Abstract Acute leukemia in adults continues to be a formidable clinical challenge that demands further investigation to identify more rational therapies. To optimize anti-leukemia therapy, we are investigating the prototypical cyclin dependent kinase (cdk) inhibitor, flavopiridol, in refractory or poor-risk disease. Flavopiridol is a cytotoxic molecule that is thought to induce cell cycle arrest by blocking cyclin-dependent kinase (cdk) function, thereby interfering with RNA Polymerase II activity and globally down-regulating gene expression. In the setting of pan-cdk inhibition, E2F1 is released and appears to drive apoptosis in transformed cells. Consistent with these proposed mechanisms of action, a previous study from our group showed that flavopiridol induces apoptosis in vitro in leukemic blasts from patients with refractory leukemia. Administration of flavopiridol was associated with a decrease in one or more of the following proteins in the leukemic blasts: RNA Polymerase II, STAT3, cyclin D1, Bcl-2, and Mcl-1. Serum VEGF levels also decreased in most patients. We are now investigating mRNA levels of the genes encoding these proteins by quantitative, RT-PCR in leukemic blasts from adult patients with refractory or poor-risk leukemia before and after flavopiridol therapy. We have treated 26 patients with flavopiridol at an escalating, hybrid dose followed by ara-c and mitxantrone. Adequate RNA from leukemic blasts before and after flavopiridol administration was available from 8 of 11 patients studied thus far. All cases (8/8) exhibit a marked decrease in mRNA for VEGF following flavopiridol. mRNA levels for other putative flavopiridol target genes is also decreased in a subset of leukemic blast samples after therapy, as follows: E2F1 (6/8), STAT3 (6/8), Mcl-1 (6/8), RNA Polymerase subunit 2a (3/3), and cyclin D1 (2/3). In contrast, bcl-2 mRNA levels increased after flavopiridol in most cases (7/8), which could represent a compensatory mechanism of leukemic blasts to avoid apoptotic cell death. Our preliminary studies indicate that flavopiridol is cytotoxic in poor-risk and refractory acute leukemia. Studies are underway to determine if down-regulation of any putative target genes correlates with pharmacologic data or clinical responses.

Download Full-text