Negative Roles of a Novel Nitrogen Metabolite Repression-Related Gene, TAR1, in Laccase Production and Nitrate Utilization by the Basidiomycete Cryptococcus neoformans

ABSTRACT The multicopper oxidase laccase is widespread in fungi and has great industrial importance. One puzzle regarding laccase production in the basidiomycetous yeast Cryptococcus neoformans is that it is inhibited by high temperature (e.g., 37°C). In this paper, we report identification of a nitrogen metabolite repression-related gene, TAR1, which is responsible for laccase repression. Disruption of TAR1 results in a significant increase in the level of LAC1 mRNA at 37°C. The putative protein Tar1 shares a moderate level of similarity with the nitrogen metabolite repressors Nmr1 and NmrA from Neurospora crassa and Aspergillus nidulans, respectively. Likewise, Tar1 has a negative role in the utilization of nitrate. Furthermore, the structure of Tar1 is unique. Tar1 lacks the long C-terminal region of Nmr1 and NmrA. It contains the canonical Rossmann fold motif, GlyXXGlyXXGly, whereas Nmr1 and NmrA have variable residues at the Gly positions. Interestingly, the promoter region of TAR1 contains three TTC/GAA repeats which are likely the heat shock factor (Hsf) binding sites, implying that Hsf has a role in laccase inhibition. TAR1 mediation of temperature-associated repression of LAC1 suggests a novel mechanism of laccase regulation and a new function for Nmr proteins. Our work may be helpful for industry in terms of promotion of laccase activity.

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Transcriptional regulation of the human manganese superoxide dismutase gene: the role of specificity protein 1 (Sp1) and activating protein-2 (AP-2)

Biochemical Journal ◽

10.1042/bj3620401 ◽

2002 ◽

Vol 362 (2) ◽

pp. 401-412 ◽

Cited By ~ 44

Author(s):

Yong XU ◽

Sureerut PORNTADAVITY ◽

Daret K. ST CLAIR

Keyword(s):

Binding Sites ◽

Promoter Region ◽

Manganese Superoxide Dismutase ◽

Expression Vector ◽

Binding Motifs ◽

Specificity Protein 1 ◽

Manganese Superoxide ◽

Negative Role ◽

Specificity Protein

Manganese superoxide dismutase (MnSOD) plays an important role in regulating cellular redox conditions. Expression of MnSOD has been shown to protect against damage by oxidative stress and to suppress the malignant phenotype of human cancer cells. We have previously cloned the human MnSOD (SOD2) gene and analysed its 5′ proximal promoter, which has been characterized by a lack of a TATA or CAAT box and the presence of multiple GC boxes. To define further the molecular mechanisms for the regulation of MnSOD expression, multiple transcription factor-binding motifs containing overlapping specificity protein 1 (Sp1)- and activator protein (AP)-2-binding sites were identified by DNase I footprinting analysis. Functional studies in three cell lines with different levels of Sp1 and AP-2 proteins suggested that the cellular levels of these proteins may differentially regulate transcription via GC-binding motifs in the human SOD2 promoter. Co-transfection of an Sp1 expression vector resulted in an increase in the transcription of the promoter-driven reporter gene. In contrast, co-transfection of the AP-2 expression vector caused a decrease in transcription. Direct mutagenesis analysis of Sp1- and AP-2-binding sites showed that Sp1 is essential for transcription of the human SOD2 gene, whereas AP-2 plays a negative role in the transcription. Immunoprecipitation of Sp1 and AP-2 proteins demonstrated that Sp1 interacts with AP-2 in vivo. Two-hybrid analysis revealed that interaction between Sp1 and AP-2 plays both a positive and negative role in the transcription of the reporter gene in vivo. Taken together, our data indicate that AP-2 down-regulates transcription of the human SOD2 gene via its interaction with Sp1 within the promoter region. These findings, coupled with our previous observation that several cancer cell lines have mutations in the promoter region of the human MnSOD gene, which lead to an increase in an AP-2-binding site and a decrease in the promoter activity, signal the importance of understanding the promoter structure and the regulation of the human SOD2 gene by Sp1 and AP-2.

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Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein

Journal of Virology ◽

10.1128/jvi.74.5.2084-2093.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2084-2093 ◽

Cited By ~ 23

Author(s):

Joel Schaley ◽

Robert J. O'Connor ◽

Laura J. Taylor ◽

Dafna Bar-Sagi ◽

Patrick Hearing

Keyword(s):

Dna Binding ◽

Binding Site ◽

Transient Expression ◽

Binding Sites ◽

Promoter Region ◽

Fluorescent Protein ◽

Protein Accumulation ◽

Promoter Regions ◽

Single Binding Site

ABSTRACT The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

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Structure of the canine pancreatic colipase gene includes two protein-binding sites in the promoter region

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)82126-3 ◽

1993 ◽

Vol 268 (15) ◽

pp. 11312-11320

Author(s):

S. Fukuoka ◽

D.E. Zhang ◽

Y. Taniguchi ◽

G.A. Scheele

Keyword(s):

Protein Binding ◽

Binding Sites ◽

Promoter Region ◽

Protein Binding Sites

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Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

International Journal of Molecular Sciences ◽

10.3390/ijms22031157 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1157

Author(s):

Pablo Aza ◽

Felipe de Salas ◽

Gonzalo Molpeceres ◽

David Rodríguez-Escribano ◽

Iñigo de la Fuente ◽

...

Keyword(s):

Protein Engineering ◽

Directed Evolution ◽

Signal Peptide ◽

Laccase Activity ◽

Laccase Production ◽

Wide Range ◽

Mating Factor ◽

Conserved Amino Acids ◽

Basidiomycete Fungi

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

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Global regulation of mitochondrial biogenesis in Saccharomyces cerevisiae: ABF1 and CPF1 play opposite roles in regulating expression of the QCR8 gene, which encodes subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2872-2883.1992 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2872-2883

Author(s):

J H de Winde ◽

L A Grivell

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome C ◽

Mitochondrial Biogenesis ◽

Binding Sites ◽

Promoter Region ◽

S Phase ◽

Negative Regulator ◽

Global Regulation ◽

Two Factors ◽

Efficient Induction

The multifunctional DNA-binding proteins ABF1 and CPF1 bind in a mutually exclusive manner to the promoter region of the QCR8 gene, which encodes 11-kDa subunit VIII of the Saccharomyces cerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR). We investigated the roles that the two factors play in transcriptional regulation of this gene. To this end, the overlapping binding sites for ABF1 and CPF1 were mutated and placed in the chromosomal context of the QCR8 promoter. The effects on transcription of the QCR8 gene were analyzed both under steady-state conditions and during nutritional shifts. We found that ABF1 is required for repressed and derepressed transcription levels and for efficient induction of transcription upon escape from catabolite repression, independently of DNA replication. CPF1 acts as a negative regulator, modulating the overall induction response. Alleviation of repression through CPF1 requires passage through the S phase. Implications of these findings for the roles played by ABF1 and CPF1 in global regulation of mitochondrial biogenesis are discussed.

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Conserved POU/OCT- and GATA-binding sites in 5'-flanking promoter region of mammalian WNT8B orthologs

International Journal of Oncology ◽

10.3892/ijo.30.5.1273 ◽

2007 ◽

Author(s):

Masuko Katoh ◽

Masaru Katoh

Keyword(s):

Binding Sites ◽

Promoter Region

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Two Mutations in the Promoter Region of the Human Protein C Gene Both Cause Type I Protein C Deficiency by Disruption of Two HNF-3 Binding Sites

Journal of Biological Chemistry ◽

10.1074/jbc.270.41.24216 ◽

1995 ◽

Vol 270 (41) ◽

pp. 24216-24221 ◽

Cited By ~ 30

Author(s):

C. Arnold Spek ◽

Judith S. Greengard ◽

John H. Griffin ◽

Rogier M. Bertina ◽

Pieter H. Reitsma

Keyword(s):

Binding Sites ◽

Promoter Region ◽

Protein C ◽

Human Protein ◽

Type I ◽

Protein C Deficiency

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Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1721 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1721-1732 ◽

Cited By ~ 68

Author(s):

C A Bunker ◽

R E Kingston

Keyword(s):

Binding Sites ◽

Transcriptional Repression ◽

Fusion Proteins ◽

Mammalian Cells ◽

Polycomb Group ◽

Polycomb Group Proteins ◽

Related Gene ◽

Activation Domains ◽

Sex Combs ◽

Significant Homology

The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.

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The snail repressor establishes a muscle/notochord boundary in the Ciona embryo

Development ◽

10.1242/dev.125.13.2511 ◽

1998 ◽

Vol 125 (13) ◽

pp. 2511-2520 ◽

Cited By ~ 4

Author(s):

S. Fujiwara ◽

J.C. Corbo ◽

M. Levine

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Sites ◽

Promoter Region ◽

Muscle Cells ◽

Specific Pattern ◽

Binding Assays ◽

Heterologous Promoter ◽

Present Evidence ◽

Tail Muscle

Previous studies have identified a minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra), which is sufficient to direct a notochord-specific pattern of gene expression. Here we present evidence that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages.

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