New Target Genes Controlled by the Bradyrhizobium japonicum Two-Component Regulatory System RegSR

ABSTRACT RegSR-like proteins, members of the family of two-component regulatory systems, are present in a large number of proteobacteria in which they globally control gene expression mostly in a redox-responsive manner. The controlled target genes feature an enormous functional diversity. In Bradyrhizobium japonicum, the facultative root nodule symbiont of soybean, RegSR activate the transcription of the nitrogen fixation regulatory gene nifA, thus forming a RegSR-NifA cascade which is part of a complex regulatory network for gene regulation in response to changing oxygen concentrations. Whole-genome transcription profiling was performed here in order to assess the full regulatory scope of RegSR. The comparative analysis of wild-type and ΔregR cells grown under oxic and microoxic conditions revealed that expression of almost 250 genes is dependent on RegR, a result that underscores the important contribution of RegR to oxygen- or redox-regulated gene expression in B. japonicum. Furthermore, transcription profiling of ΔregR bacteroids compared with wild-type bacteroids revealed expression changes for about 1,200 genes in young and mature bacteroids. Incidentally, many of these were found to be induced in symbiosis when wild-type bacteroids were compared with free-living, culture-grown wild-type cells, and they appeared to encode diverse functions possibly related to symbiosis and nitrogen fixation. We demonstrated direct RegR-mediated control at promoter regions of several selected target genes by means of DNA binding experiments and in vitro transcription assays, which revealed six novel direct RegR target promoters.

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Thyroid Hormone-Dependent Gene Expression in Differentiated Embryonic Stem Cells and Embryonal Carcinoma Cells: Identification of Novel Thyroid Hormone Target Genes by Deoxyribonucleic Acid Microarray Analysis

Endocrinology ◽

10.1210/en.2004-1177 ◽

2005 ◽

Vol 146 (2) ◽

pp. 776-783 ◽

Cited By ~ 13

Author(s):

Yan-Yun Liu ◽

Gregory A. Brent

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Thyroid Hormone ◽

Early Development ◽

Target Genes ◽

Es Cells ◽

Embryonic Stem ◽

Wild Type ◽

Ec Cells

Abstract T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRα P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.

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238.Determination of differentially displayed oxygen-sensitive genes in bovine blastocysts

Reproduction Fertility and Development ◽

10.1071/srb04abs238 ◽

2004 ◽

Vol 16 (9) ◽

pp. 238

Author(s):

A. J. Harvey ◽

M. Kirstein ◽

A. Navarrete-Santos ◽

K. L. Kind ◽

B. Fischer ◽

...

Keyword(s):

Gene Expression ◽

Differential Display ◽

Target Genes ◽

Hypoxia Inducible Factor ◽

Developmental Competence ◽

Myelogenous Leukemia ◽

Bovine Embryo ◽

Nadh Dehydrogenase Subunit 2 ◽

Regulated Gene Expression

Oxygen-regulated gene expression in the bovine embryo contrasts markedly with that observed in the mouse. Under low (2%) oxygen moderate changes in gene expression are observed in the bovine blastocyst, compared with 3- to 4-fold increases in the mouse. We have determined that these moderate gene expression changes are most likely regulated by Hypoxia-Inducible Factor (HIF)-2 transcription factor activity in the bovine, in the absence of HIF1, although HIF2 target genes are largely unknown. The aim of this study was to screen, by differential display RT-PCR, for putative oxygen-regulated transcripts that might confer developmental competence in blastocysts cultured under varying oxygen atmospheres post compaction. In vitro-produced bovine blastocysts were generated using standard protocols. Compact morulae were randomly allocated to treatments under either 2%, 7% or 20% oxygen for 72 h from Day 5. Blastocyst RNA was isolated using TriReagent and samples were reverse transcribed using Superscript II. cDNA was amplified using 10-mer primers in reactions containing 32Pα-labelled dCTP. Resulting bands were detected by autoradiography, excised, purified and ligated into pGEMT vectors for transformation and sequencing. Seven clones were identified as having high homology with known sequences in GenBank. Real-time PCR was undertaken to confirm oxygen-regulation using Sybr green master mix. Myotrophin mRNA was significantly increased following 2% oxygen culture, compared with 20% cultured blastocysts (P�<�0.01), as was GLUT1 (P�<�0.01). The expression of anaphase-promoting complex showed a significant association with oxygen, being higher in 2% cultured blastocysts (P�<�0.05). Acetyl-coA-acetyltransferase I, chronic myelogenous leukemia tumor antigen (CML66), cyclin I, NADH dehydrogenase subunit 2 and ribonucleotide reductase M1, genes identified using differential display, were not altered by post compaction oxygen concentration. This study has identified potentially HIF2-specific regulated genes, and supports the hypothesis that reduced oxygen concentrations post-compaction may influence bovine embryo development through oxygen-regulated changes in gene expression.

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EPCR-dependent PAR2 activation by the blood coagulation initiation complex regulates LPS-triggered interferon responses in mice

Blood ◽

10.1182/blood-2014-11-610717 ◽

2015 ◽

Vol 125 (18) ◽

pp. 2845-2854 ◽

Cited By ~ 35

Author(s):

Hai Po H. Liang ◽

Edward J. Kerschen ◽

Irene Hernandez ◽

Sreemanti Basu ◽

Mark Zogg ◽

...

Keyword(s):

Gene Expression ◽

Blood Coagulation ◽

Target Genes ◽

Adaptor Protein ◽

Messenger Rnas ◽

Initiation Complex ◽

Induced Expression ◽

Regulated Gene Expression

Abstract Infection and inflammation are invariably associated with activation of the blood coagulation mechanism, secondary to the inflammation-induced expression of the coagulation initiator tissue factor (TF) on innate immune cells. By investigating the role of cell-surface receptors for coagulation factors in mouse endotoxemia, we found that the protein C receptor (ProcR; EPCR) was required for the normal in vivo and in vitro induction of lipopolysaccharide (LPS)-regulated gene expression. In cultured bone marrow–derived myeloid cells and in monocytic RAW264.7 cells, the LPS-induced expression of functionally active TF, assembly of the ternary TF-VIIa-Xa initiation complex of blood coagulation, and the EPCR-dependent activation of protease-activated receptor 2 (PAR2) by the ternary TF-VIIa-Xa complex were required for the normal LPS induction of messenger RNAs encoding the TLR3/4 signaling adaptor protein Pellino-1 and the transcription factor interferon regulatory factor 8. In response to in vivo challenge with LPS, mice lacking EPCR or PAR2 failed to fully initiate an interferon-regulated gene expression program that included the Irf8 target genes Lif, Iigp1, Gbp2, Gbp3, and Gbp6. The inflammation-induced expression of TF and crosstalk with EPCR, PAR2, and TLR4 therefore appear necessary for the normal evolution of interferon-regulated host responses.

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120. OXYGEN REGULATED GENE EXPRESSION IN MOUSE CUMULUS CELLS

Reproduction Fertility and Development ◽

10.1071/srb09abs120 ◽

2009 ◽

Vol 21 (9) ◽

pp. 39

Author(s):

K. Tam ◽

K. Banwell ◽

D. Froiland ◽

D. Russell ◽

K. Kind ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Oxygen Concentration ◽

In Vitro Maturation ◽

Target Genes ◽

Cumulus Cells ◽

Differentially Expressed ◽

Oxygen Levels ◽

Regulated Gene Expression

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that mediate the expression of a range of genes in response to low oxygen. Previously we showed that subsequent developmental outcomes were influenced by oxygen levels during in vitro maturation. The aim of the current study was to examine the effects of varying oxygen concentration during in vitro maturation of mouse COCs on expression of HIF target genes in the cumulus cells. I mmature COCs were collected from the ovaries of eCG-stimulated CBAB6F1 females (21 d) and cultured for 17-18 h under 2, 5 or 20% O2. Hyaluronidase-treated and recovered cumulus cells were collected and mRNA extracted for analysis. A microarray approach (Affymetrix 430_2) was used to identify genes in cumulus cells that were differentially expressed under varying oxygen concentrations (2, 5, 10 and 20%). This revealed 218 differentially expressed probes, of which 34 were up-regulated with decreasing oxygen levels. The great majority of these were classified as HIF-regulated genes. Specific analysis from real time RT-PCR of HIF regulated target genes Slc2a1, Ldha, Pgk1, Eno1, Ndrg1, Bnip3 were all significantly up-regulated (by at least 5–fold) when cells were cultured at 2% or 5% oxygen, when compared to 20% oxygen. Hif-1a mRNA decreased when cumulus cells were cultured in 2%, compared to 20% oxygen. This study demonstrates that cumulus cell gene expression is influenced by oxygen concentration, and suggests that these effects are mediated by the HIF transcription factors.

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Bmi1 Is Essential for Leukemic Reprogramming of Myeloid Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.1423.1423 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1423-1423

Author(s):

Jin Yuan ◽

Masahiro Takeuchi ◽

Hideyuki Oguro ◽

Masamitsu Negishi ◽

Hitoshi Ichikawa ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Target Genes ◽

Expression Profiles ◽

Myelogenous Leukemia ◽

Wild Type ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Immortalized Cells

Abstract Abstract 1423 Poster Board I-446 The polycomb group (PcG) protein Bmi1 plays an essential role in the maintenance of self-renewing hematopoietic stem cells (HSCs). Derepressed p16Ink4a and p19Arf are tightly associated with a loss of self-renewing capacity of HSCs in Bmi1-deficient mice. Deletion of both Ink4a and Arf genes substantially restores the self-renewal capacity of Bmi1−/− HSCs. Thus, Bmi1 maintains HSCs by acting as a critical failsafe against the p16Ink4a- and p19Arf-dependent senescence pathway. Meanwhile, Bmi1 was originally identified as a collaborating oncogene in the induction of lymphoma and was subsequently reported to be overexpressed in various human cancers including leukemia. Recent studies have demonstrated that PcG proteins bind to multiple regions of the genome and regulate a bunch of target genes. Therefore, we asked whether Bmi1 is essential for leukemic stem cells (LSCs) and tried to identify critical target genes for Bmi1 other than Ink4a and Arf in leukemia. We expressed the MLL-AF9 leukemic fusion gene in purified Lin−Sca-1−c-Kit+CD34+FcγRII/ IIIhi granulocyte/macrophage progenitors (GMPs) from wild-type, Bmi1−/−, Ink4a-Arf−/−, and Bmi1−/−Ink4a-Arf−/− mice and performed in vitro myeloid progenitor replating assay. GMPs from 4 different genetic backgrounds were all immortalized in vitro, although Bmi1-deficient cells showed a slightly decreased replating efficiency. We then infused the immortalized cells into lethally irradiated recipient mice. Mice infused with wild-type and Ink4a-Arf−/− cells developed acute myelogenous leukemia (AML) at 30 to 60 days after infusion. Mice infused with Bmi1−/− cells did not develop leukemia at all. While a significant portion of mice infused with Bmi1−/−Ink4a-Arf−/− cells developed AML, although they took much longer time compared to those mice infused with wild-type and Ink4a-Arf−/− cells. These results indicate that as in HSCs, the Ink4a /Arf locus is one of the major targets for Bmi1 in leukemogenesis. In order to find unknown targets of Bmi1 in LSCs, we compared gene expression profiles of purified c-KithiFcRγII/IIIhiCD34+ cells from Ink4a-Arf−/− and Bmi1−/−Ink4a-Arf−/− immortalized cells. We found that the loss of Bmi1 did not affect the induction of MLL-AF9 target gene expression. By contrast, a number of genes were derepressed in the absence of Bmi1. Among these, Tbx15, a transcriptional co-repressor gene, appeared to be regulated by Bmi1 and a potential tumor suppressor gene in the development of leukemia. Of interest, the majority of derepressed target genes in transformed Bmi1−/−Ink4a-Arf−/− cells, including Tbx15, remained unchanged by re-expression of Bmi1. Correspondingly, re-introduction of Bmi1 to transformed Bmi1−/−Ink4a-Arf−/− cells failed to rescue their compromised leukemogenic activity in vivo. Our findings suggest that Bmi1 is required for faithful epigenetic reprogramming of myeloid progenitors into LSCs by leukemic fusions and contributes to establish LSC-specific transcriptional profiles to confer full leukemogenic activity on LSCs. Disclosures: No relevant conflicts of interest to declare.

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Three New NifA-Regulated Genes in the Bradyrhizobium japonicum Symbiotic Gene Region Discovered by Competitive DNA-RNA Hybridization

Journal of Bacteriology ◽

10.1128/jb.182.6.1472-1480.2000 ◽

2000 ◽

Vol 182 (6) ◽

pp. 1472-1480 ◽

Cited By ~ 29

Author(s):

Andrea Nienaber ◽

Alexander Huber ◽

Michael Göttfert ◽

Hauke Hennecke ◽

Hans-Martin Fischer

Keyword(s):

Nitrogen Fixation ◽

Bradyrhizobium Japonicum ◽

Sequence Similarity ◽

Regulatory Protein ◽

Gene Clusters ◽

Amino Acid Sequence Similarity ◽

Wild Type ◽

Significant Similarity ◽

Symbiotic Gene

ABSTRACT The so-called symbiotic region of the Bradyrhizobium japonicum chromosome (C. Kündig, H. Hennecke, and M. Göttfert, J. Bacteriol. 175:613–622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA. Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown nifA and rpoNmutant cells or from aerobically grown wild-type cells. In addition to the previously characterized nif and fixgene clusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC(nrg stands for NifA-regulated gene). The latter two probably form an operon, nrgBC. The proteins encoded bynrgC and nrgA exhibited amino acid sequence similarity to bacterial hydroxylases andN-acetyltransferases, respectively. The product ofnrgB showed no significant similarity to any protein with a database entry. Primer extension experiments and expression studies with translational lacZ fusions revealed the presence of a functional −24/−12-type promoter upstream ofnrgA and nrgBC and proved the NifA- and RpoN (ς54)-dependent transcription of the respective genes. Null mutations introduced into nrgA and nrgBCresulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants. Thus, the discovery ofnrgA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.

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Targeting gene expression to the head: the Drosophila orthodenticle gene is a direct target of the Bicoid morphogen

Development ◽

10.1242/dev.125.21.4185 ◽

1998 ◽

Vol 125 (21) ◽

pp. 4185-4193 ◽

Cited By ~ 4

Author(s):

Q. Gao ◽

R. Finkelstein

Keyword(s):

Gene Expression ◽

Target Genes ◽

Regulatory Element ◽

Regulatory Region ◽

Repeated Sequence ◽

Drosophila Embryo ◽

Sequence Motif ◽

Specific Expression ◽

Gap Gene

The Bicoid (Bcd) morphogen establishes the head and thorax of the Drosophila embryo. Bcd activates the transcription of identified target genes in the thoracic segments, but its mechanism of action in the head remains poorly understood. It has been proposed that Bcd directly activates the cephalic gap genes, which are the first zygotic genes to be expressed in the head primordium. It has also been suggested that the affinity of Bcd-binding sites in the promoters of Bcd target genes determines the posterior extent of their expression (the Gene X model). However, both these hypotheses remain untested. Here, we show that a small regulatory region upstream of the cephalic gap gene orthodenticle (otd) is sufficient to recapitulate early otd expression in the head primordium. This region contains two control elements, each capable of driving otd-like expression. The first element has consensus Bcd target sites that bind Bcd in vitro and are necessary for head-specific expression. As predicted by the Gene X model, this element has a relatively low affinity for Bcd. Surprisingly, the second regulatory element has no Bcd sites. Instead, it contains a repeated sequence motif similar to a regulatory element found in the promoters of otd-related genes in vertebrates. Our study is the first demonstration that a cephalic gap gene is directly regulated by Bcd. However, it also shows that zygotic gene expression can be targeted to the head primordium without direct Bcd regulation.

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Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽

10.1128/msystems.00980-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Kumari Sonal Choudhary ◽

Julia A. Kleinmanns ◽

Katherine Decker ◽

Anand V. Sastry ◽

Ye Gao ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Target Gene ◽

Target Genes ◽

Rna Seq ◽

Content Type ◽

E Coli ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

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In vivo and in vitro oncogenic effects of HIF2A mutations in pheochromocytomas and paragangliomas

Endocrine Related Cancer ◽

10.1530/erc-13-0101 ◽

2013 ◽

Vol 20 (3) ◽

pp. 349-359 ◽

Cited By ~ 76

Author(s):

Rodrigo A Toledo ◽

Yuejuan Qin ◽

Subramanya Srikantan ◽

Nicole Paes Morales ◽

Qun Li ◽

...

Keyword(s):

Somatic Mutations ◽

Target Genes ◽

Germline Mutations ◽

Vascular Tumors ◽

Wild Type ◽

Pheochromocytoma Pc12 ◽

Hereditary Tumors ◽

Tumor Dna

Pheochromocytomas and paragangliomas are highly vascular tumors of the autonomic nervous system. Germline mutations, including those in hypoxia-related genes, occur in one third of the cases, but somatic mutations are infrequent in these tumors. Using exome sequencing of six paired constitutive and tumor DNA from sporadic pheochromocytomas and paragangliomas, we identified a somatic mutation in the HIF2A (EPAS1) gene. Screening of an additional 239 pheochromocytomas/paragangliomas uncovered three other HIF2A variants in sporadic (4/167, 2.3%) but not in hereditary tumors or controls. Three of the mutations involved proline 531, one of the two residues that controls HIF2α stability by hydroxylation. The fourth mutation, on Ser71, was adjacent to the DNA binding domain. No mutations were detected in the homologous regions of the HIF1A gene in 132 tumors. Mutant HIF2A tumors had increased expression of HIF2α target genes, suggesting an activating effect of the mutations. Ectopically expressed HIF2α mutants in HEK293, renal cell carcinoma 786-0, or rat pheochromocytoma PC12 cell lines showed increased stability, resistance to VHL-mediated degradation, target induction, and reduced chromaffin cell differentiation. Furthermore, mice injected with cells expressing mutant HIF2A developed tumors, and those with Pro531Thr and Pro531Ser mutations had shorter latency than tumors from mice with wild-type HIF2A. Our results support a direct oncogenic role for HIF2A in human neoplasia and strengthen the link between hypoxic pathways and pheochromocytomas and paragangliomas.

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Abstract P244: Transcriptional Regulation of Cardiac Gene Expression by Med13 Alters Global Energy Balance

Circulation Research ◽

10.1161/res.109.suppl_1.ap244 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Chad E Grueter ◽

Brett A Johnson ◽

Xiaoxia Qi ◽

John McAnally ◽

Rhonda Bassel-Duby ◽

...

Keyword(s):

Gene Expression ◽

Energy Balance ◽

Hormone Receptor ◽

Hormone Receptors ◽

Target Genes ◽

Thyroid Hormone Receptor ◽

Cardiac Contractility ◽

Nuclear Hormone Receptor ◽

Mediator Complex

Aberrant cardiac metabolism is associated with obesity, type 2 diabetes and heart failure. The heart requires highly efficient metabolism to maintain the levels of ATP needed for contractility and pump function, however little is known about the role of the heart as a metabolic organ. Nuclear hormone receptors, such as thyroid hormone receptor play an important role in cardiovascular disease by significantly altering expression of genes involved in maintaining metabolic activity. The Mediator, a large multiprotein complex functions as a hub to control gene expression through association with transcriptional activators and repressors. We tested the hypothesis that Med13, a component of the Mediator complex, regulates cardiac function in a gain-of-function mouse model. Trangsenic mice overexpressing Med13 in the heart are lean, have increased energy expenditure, are resistant to high fat diet-induced obesity and have enhanced cardiac contractility. Microarray analysis and biochemical assays show that in vivo and in vitro Med13 selectively inhibits nuclear hormone receptor target genes of energy metabolism. These results implicate the Mediator complex regulates energy balance and cardiac contractility and suggests that the heart may function as a key component of mammalian energy homeostasis.

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