Transcriptional Activation by Bacillus subtilis ResD: Tandem Binding to Target Elements and Phosphorylation-Dependent and -Independent Transcriptional Activation

ABSTRACT The expression of genes involved in nitrate respiration in Bacillus subtilis is regulated by the ResD-ResE two-component signal transduction system. The membrane-bound ResE sensor kinase perceives a redox-related signal(s) and phosphorylates the cognate response regulator ResD, which enables interaction of ResD with ResD-dependent promoters to activate transcription. Hydroxyl radical footprinting analysis revealed that ResD tandemly binds to the −41 to −83 region of hmp and the −46 to −92 region of nasD. In vitro runoff transcription experiments showed that ResD is necessary and sufficient to activate transcription of the ResDE regulon. Although phosphorylation of ResD by ResE kinase greatly stimulated transcription, unphosphorylated ResD, as well as ResD with a phosphorylation site (Asp57) mutation, was able to activate transcription at a low level. The D57A mutant was shown to retain the activity in vivo to induce transcription of the ResDE regulon in response to oxygen limitation, suggesting that ResD itself, in addition to its activation through phosphorylation-mediated conformation change, senses oxygen limitation via an unknown mechanism leading to anaerobic gene activation.

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Mutational Analysis of the Signal-Sensing Domain of ResE Histidine Kinase from Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.186.6.1694-1704.2004 ◽

2004 ◽

Vol 186 (6) ◽

pp. 1694-1704 ◽

Cited By ~ 17

Author(s):

Avanti Baruah ◽

Brett Lindsey ◽

Yi Zhu ◽

Michiko M. Nakano

Keyword(s):

Bacillus Subtilis ◽

Transcriptional Activation ◽

Catalytic Domain ◽

Mutational Analysis ◽

Response Regulator ◽

Regulatory Region ◽

Regulatory System ◽

Oxygen Limitation ◽

Nitrate Respiration ◽

Terminal Domain

ABSTRACT The Bacillus subtilis ResD-ResE two-component regulatory system activates genes involved in nitrate respiration in response to oxygen limitation or nitric oxide (NO). The sensor kinase ResE activates the response regulator ResD through phosphorylation, which then binds to the regulatory region of genes involved in anaerobiosis to activate their transcription. ResE is composed of an N-terminal signal input domain and a C-terminal catalytic domain. The N-terminal domain contains two transmembrane subdomains and a large extracytoplasmic loop. It also has a cytoplasmic PAS subdomain between the HAMP linker and C-terminal kinase domain. In an attempt to identify the signal-sensing subdomain of ResE, a series of deletions and amino acid substitutions were generated in the N-terminal domain. The results indicated that cytoplasmic ResE lacking the transmembrane segments and the extracytoplasmic loop retains the ability to sense oxygen limitation and NO, which leads to transcriptional activation of ResDE-dependent genes. This activity was eliminated by the deletion of the PAS subdomain, demonstrating that the PAS subdomain participates in signal reception. The study also raised the possibility that the extracytoplasmic region may serve as a second signal-sensing subdomain. This suggests that the extracytoplasmic region could contribute to amplification of ResE activity leading to the robust activation of genes required for anaerobic metabolism in B. subtilis.

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Histone deacetylase–mediated transcriptional activation reduces proviral loads in HTLV-1–associated myelopathy/tropical spastic paraparesis patients

Blood ◽

10.1182/blood-2007-04-085076 ◽

2007 ◽

Vol 110 (10) ◽

pp. 3722-3728 ◽

Cited By ~ 50

Author(s):

Agnès Lezin ◽

Nicolas Gillet ◽

Stéphane Olindo ◽

Aïssatou Signaté ◽

Nathalie Grandvaux ◽

...

Keyword(s):

Histone Deacetylase ◽

Transcriptional Activation ◽

Gene Activation ◽

Spastic Paraparesis ◽

Viral Gene Expression ◽

Tropical Spastic Paraparesis ◽

Viral Reservoir ◽

Viral Gene

AbstractEpigenetic modifications of chromatin may play a role in maintaining viral latency and thus persistence of the human T-lymphotropic virus type 1 (HTLV-1), which is responsible for HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A major determinant of disease progression is increased peripheral blood proviral load (PVL), possibly via the accumulation of infected cells in the central nervous system (CNS) creating a damaging inflammatory response. Current therapeutic approaches that focus on reducing either cell proliferation, viral replication, or tissue invasion are still unsatisfactory. Contrasting with these inhibitory strategies, we evaluated the efficacy of a novel approach aimed, paradoxically, at activating viral gene expression to expose virus-positive cells to the host immune response. We used valproate (VPA), a histone deacetylase inhibitor that has been used for decades as a chronic, safe treatment for epileptic disorders. Based on in vitro and in vivo data, we provide evidence that transient activation of the latent viral reservoir causes its collapse, a process that may alleviate the condition of HAM/TSP. This represents the first such approach to treating HAM/TSP, using gene activation therapy to tilt the host-pathogen balance in favor of an existing antiviral response. This trial is registered at http://clinicaltrials.gov/as no. NCT00519181.

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In Vivo Requirement of Activator-Specific Binding Targets of Mediator

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8709-8719.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8709-8719 ◽

Cited By ~ 91

Author(s):

Jin Mo Park ◽

Hye-Suk Kim ◽

Sang Jun Han ◽

Moon-Sun Hwang ◽

Young Chul Lee ◽

...

Keyword(s):

Transcriptional Activation ◽

Specific Binding ◽

Gene Activation ◽

Dna Interaction ◽

Activation Process ◽

Binding Region ◽

Physiological Conditions ◽

Functional Analyses

ABSTRACT There has been no unequivocal demonstration that the activator binding targets identified in vitro play a key role in transcriptional activation in vivo. To examine whether activator-Mediator interactions are required for gene transcription under physiological conditions, we performed functional analyses with Mediator components that interact specifically with natural yeast activators. Different activators interact with Mediator via distinct binding targets. Deletion of a distinct activator binding region of Mediator completely compromised gene activation in vivo by some, but not all, transcriptional activators. These demonstrate that the activator-specific targets in Mediator are essential for transcriptional activation in living cells, but their requirement was affected by the nature of the activator-DNA interaction and the existence of a postrecruitment activation process.

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Transcriptional Activation of the Bacillus subtilis spoIIG Promoter by the Response Regulator Spo0A Is Independent of the C-Terminal Domain of the RNA Polymerase Alpha Subunit

Journal of Bacteriology ◽

10.1128/jb.180.17.4760-4763.1998 ◽

1998 ◽

Vol 180 (17) ◽

pp. 4760-4763 ◽

Cited By ~ 5

Author(s):

Dean A. Rowe-Magnus ◽

Mario Mencía ◽

Fernando Rojo ◽

Margarita Salas ◽

George B. Spiegelman

Keyword(s):

Bacillus Subtilis ◽

Rna Polymerase ◽

Transcriptional Activation ◽

Response Regulator ◽

In Vitro Transcription ◽

Alpha Subunit ◽

Deletion Mutants ◽

Wild Type ◽

Initiation Mechanism

ABSTRACT In vitro transcription from the spoIIG promoter byBacillus subtilis RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A. Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.

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Molecular Analysis of Phr Peptide Processing in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.185.16.4861-4871.2003 ◽

2003 ◽

Vol 185 (16) ◽

pp. 4861-4871 ◽

Cited By ~ 47

Author(s):

Sophie Stephenson ◽

Christian Mueller ◽

Min Jiang ◽

Marta Perego

Keyword(s):

Bacillus Subtilis ◽

Response Regulator ◽

Signal Peptidase ◽

Type I ◽

Peptidase Activity ◽

Amino Terminal ◽

Processing Event ◽

Signal Peptidases

ABSTRACT In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F∼P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.

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Butyrate induces selective transcriptional activation of a hypomethylated embryonic globin gene in adult erythroid cells

Blood ◽

10.1182/blood.v72.5.1536.bloodjournal7251536 ◽

1988 ◽

Vol 72 (5) ◽

pp. 1536-1542

Author(s):

LJ Burns ◽

JG Glauber ◽

GD Ginder

Keyword(s):

Sodium Butyrate ◽

Transcriptional Activation ◽

Globin Gene ◽

Gene Activation ◽

Globin Genes ◽

Erythroid Cells ◽

Hemoglobin Switching ◽

High Level

An animal model of hemoglobin switching has been developed in which anemic adult chickens are treated with 5-azacytidine and sodium butyrate or alpha-aminobutyric acid, thereby resulting in activation of the embryonic rho-globin gene in adult erythroid cells. In vitro nuclear runoff transcription assays using erythroid nuclei from treated birds show that the mechanism of activation of the rho-globin gene is transcriptional whereas no transcriptional activation of the embryonic epsilon-globin gene occurs. The action of 5-azacytidine appears to be as an inhibitor of DNA methylation because other S-phase active cytotoxic drugs, when substituted for 5-azacytidine, do not cause demethylation of the embryonic globin genes, nor do they allow transcriptional activation to occur. Embryonic rho-globin gene activation in this model is not due to selection of primitive erythroid cells since a subpopulation of primitive erythroid cells is not evident either morphologically or when cells are probed for embryonic and adult globin RNA by in situ hybridization. These studies show that demethylation by 5-azacytidine is a prerequisite but not sufficient cis- regulatory event for a high level of transcriptional activation of the embryonic rho-globin gene in adult erythroid cells in vivo. The possible basis for the selective transcriptional activation by sodium butyrate in this system is discussed.

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Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c2 Gene P2 Promoter by the Response Regulator PrrA

Journal of Bacteriology ◽

10.1128/jb.184.2.390-399.2002 ◽

2002 ◽

Vol 184 (2) ◽

pp. 390-399 ◽

Cited By ~ 43

Author(s):

James C. Comolli ◽

Audrey J. Carl ◽

Christine Hall ◽

Timothy Donohue

Keyword(s):

Amino Acid ◽

Rhodobacter Sphaeroides ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Response Regulator ◽

Single Amino Acid ◽

Single Amino Acid Change ◽

Wide Range

ABSTRACT Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the α subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c 2 gene (cycA) promoter, P2, which contained sequences from −73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.

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Regulators of the Bacillus subtilis cydABCD Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD

Journal of Bacteriology ◽

10.1128/jb.00050-07 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3348-3358 ◽

Cited By ~ 14

Author(s):

Ankita Puri-Taneja ◽

Matthew Schau ◽

Yinghua Chen ◽

F. Marion Hulett

Keyword(s):

Bacillus Subtilis ◽

Response Regulator ◽

Regulatory Protein ◽

Negative Regulator ◽

Regulatory Proteins ◽

Regulation Of Respiration ◽

Regulator Protein ◽

Promoter Fusion

ABSTRACT The cydABCD operon of Bacillus subtilis encodes products required for the production of cytochrome bd oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the cydABCD operon. Two additional regulatory proteins for the cydABCD operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the cydA promoter in vivo. ResD binding to the cydA promoter between positions −58 and −107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A ccpA mutant had increased expression from the full-length cydA promoter during stationary growth compared to the wild-type strain. Maximal expression in a ccpA mutant was observed from a 3′-deleted cydA promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the cydA promoter, protecting the region from positions −4 to −33, which contains sequences similar to the CcpA consensus binding sequence, the cre box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the cre box reduced CcpA binding 10-fold in vitro and increased cydA expression in vivo. Thus, CcpA and ResD, along with the previously identified cydA regulator Rex (YdiH), affect the expression of the cydABCD operon. Low-level induction of the cydA promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the cydA promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.

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A Central Role for Canonical PRC1 in Shaping the 3D Nuclear Landscape

10.1101/2019.12.15.876771 ◽

2019 ◽

Cited By ~ 2

Author(s):

Shelagh Boyle ◽

Ilya M. Flyamer ◽

Iain Williamson ◽

Dipta Sengupta ◽

Wendy A. Bickmore ◽

...

Keyword(s):

Transcriptional Activation ◽

Gene Activation ◽

Embryonic Stem ◽

Nuclear Architecture ◽

Regulatory System ◽

Spatial Arrangement ◽

Polycomb Group ◽

List Type

AbstractPolycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus to form observable bodies; a conformation which is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organisation and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb Repressive Complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are seperable events. These findings provide important insights into the function of PRC1, whilst highlighting the complexity of this regulatory system.HighlightsLoss of RING1B substantially disrupts nuclear architecture.PRC1 mediated looping can occur at a Mb scale and is independent of CTCF.Polycomb mediated looping is driven by canonical PRC1 complexes.Multimeric PRC1-mediated interactions occur in vitro and in vivo.Disruption of PRC1-mediated looping is independent of gene activation.

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Bordetella pertussis fim3gene regulation by BvgA: Phosphorylation controls the formation of inactive vs. active transcription complexes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421045112 ◽

2015 ◽

Vol 112 (6) ◽

pp. E526-E535 ◽

Cited By ~ 8

Author(s):

Alice Boulanger ◽

Kyung Moon ◽

Kimberly B. Decker ◽

Qing Chen ◽

Leslie Knipling ◽

...

Keyword(s):

Rna Polymerase ◽

Bordetella Pertussis ◽

Transcription Initiation ◽

Response Regulator ◽

Gene Activation ◽

Virulence Gene ◽

In Vitro Transcription ◽

Transcription Complexes

Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and theBordetella pertussisRR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at Pfim3, the promoter for the virulence genefim3(fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at Pfim3. However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence offim3. We suggest that the presence of nonphosphorylated BvgA down-regulates Pfim3activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating Pfim3activation once the signal for BvgA phosphorylation is removed.

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