Functional importance of Myc-associated zinc finger protein for the human parathyroid hormone (PTH)/PTH-related peptide receptor-1 P2 promoter constitutive activity

The aim of the present study was to analyze the functional importance for the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR1) gene P2 promoter activity of the putative proximal Myc-associated zinc finger protein (MAZ) site localized at position bp -45 to -39 bp, taking advantage of a G/A mutation identified at position -40 in the human sequence. Wild-type 'full-length' (1285P2) and truncated (760P2) promoter sequences were inserted upstream to the luciferase basic (pLucB) and enhancer (pLucE) reporter gene expression vectors. Transient transfections in osteoblast-like SaOS-2 cells and renal cells (RC.SV3A2) showed that the -40 G/A mutation significantly impaired transcriptional activity of wild-type 1285P2-pLucB and 760P2-pLucE promoter constructs. Further truncation of the P2 sequence demonstrated that the sequence -109/-37 bp was essential for promoter activity. Co-transfection with a MAZ expression vector did not modify the wild-type 1285P2-pLucB construct reporter activity but significantly increased 2-fold the mutated construction activity (P<0.05). Electrophoretic mobility shift assays using SaOS-2 nuclear extracts and a double-stranded DNA fragment encompassing the -45 to -39 putative MAZ site (ds-MAZ-oligo) disclosed two specific DNA-protein complexes. Complex II (fast moving) had a lower affinity for the mutated MAZ motif than for the wild-type MAZ motif while complex I (slow moving) had the same affinity for both wild-type or mutated MAZ sequences. Competition studies with Sp1 consensus oligonucleotide (ds-Sp1-oligo) markedly reduced complex I intensity, with a concomitant increase in that of complex II. Finally, ribonuclease protection assays showed that P2-specific PTHR1 mRNA transcript expression was significantly decreased in SaOS-2 cells transfected with ds-MAZ-oligo as compared with that for control (P<0.001) and ds-Sp1-oligo (P<0.05). Taken together, our studies suggest that the putative -45 to -39 MAZ-binding site regulates the constitutive activity of human PTHR1 P2 promoter.

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Disruption of a Locus Encoding a Nucleolar Zinc Finger Protein Decreases Tachyzoite-to-Bradyzoite Differentiation in Toxoplasma gondii

Infection and Immunity ◽

10.1128/iai.73.10.6680-6688.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6680-6688 ◽

Cited By ~ 26

Author(s):

Padmini Vanchinathan ◽

Jeremy L. Brewer ◽

Omar S. Harb ◽

John C. Boothroyd ◽

Upinder Singh

Keyword(s):

Toxoplasma Gondii ◽

Zinc Finger ◽

Insertional Mutagenesis ◽

Zinc Finger Protein ◽

Chemical Mutagenesis ◽

Selection Strategy ◽

Wild Type ◽

Altered Expression ◽

Finger Protein ◽

Bradyzoite Differentiation

ABSTRACT During its life cycle in intermediate hosts, Toxoplasma gondii exists in two interconverting developmental stages: tachyzoites and bradyzoites. This interconversion is essential for the survival and pathogenicity of the parasite, but little is known about the genetic mechanisms that control this process. We have previously generated tachyzoite-to-bradyzoite differentiation (Tbd−) mutants using chemical mutagenesis and a green fluorescent protein-based selection strategy. The genetic loci responsible for the Tbd− phenotype, however, could not be identified. We have now used an insertional mutagenesis strategy to generate two differentiation mutants: TBD-5 and TBD-6 that switch to bradyzoites at 10 and 50% of wild-type levels, respectively. In TBD-6 there is a single insertion of the mutagenesis vector 164 bp upstream of the transcription start site of a gene encoding a zinc finger protein (ZFP1). Disruption of this locus in wild-type parasites reproduces the decreased stage conversion phenotype. ZFP1 is targeted to the parasite nucleolus by CCHC motifs and significantly altered expression levels are toxic to the parasites. This represents the first identification of a gene necessary for efficient conversion of tachyzoites to bradyzoites.

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A Krüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collagen Gene

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.4256-4267.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 4256-4267 ◽

Cited By ~ 19

Author(s):

Kazuhiro Tanaka ◽

Noriyuki Tsumaki ◽

Christine A. Kozak ◽

Yoshihiro Matsumoto ◽

Fumihiko Nakatani ◽

...

Keyword(s):

Zinc Finger ◽

Promoter Activity ◽

Expression Vector ◽

Zinc Finger Protein ◽

Zinc Finger Proteins ◽

Limb Bud ◽

Collagen Gene ◽

Specific Expression ◽

Finger Protein ◽

Krab Domain

ABSTRACT Type XI collagen is composed of three chains, α1(XI), α2(XI), and α3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the −530-bp promoter segment of the α2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.

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A Krüppel-Associated Box-Zinc Finger Protein, NT2, Represses Cell-Type-Specific Promoter Activity of the α2(XI) Collage Gene

Molecular and Cellular Biology ◽

10.1128/mcb.01723-06 ◽

2006 ◽

Vol 26 (21) ◽

pp. 8215-8216

Author(s):

Kazuhiro Tanaka ◽

Noriyuki Tsumaki ◽

Christine A. Kozak ◽

Yoshihiro Matsumoto ◽

Fumihiko Nakatani ◽

...

Keyword(s):

Zinc Finger ◽

Promoter Activity ◽

Zinc Finger Protein ◽

Cell Type ◽

Finger Protein ◽

Cell Type Specific

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Zinc finger protein 521 is required for anabolic action of parathyroid hormone

Bone ◽

10.1016/j.bone.2009.03.079 ◽

2009 ◽

Vol 44 ◽

pp. S227-S228

Author(s):

K. Yamana⁎ ◽

R. Kiviranta ◽

H. Saito ◽

E. Hesse ◽

D. Correa ◽

...

Keyword(s):

Parathyroid Hormone ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Finger Protein ◽

Anabolic Action

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The CHY-type zinc finger protein FgChy1 regulates polarized growth, pathogenicity, and microtubule assembly in Fusarium graminearum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-20-0206-r ◽

2020 ◽

Author(s):

Shulin Cao ◽

Wei Li ◽

Chaohui Li ◽

Guanghui Wang ◽

Wenqiang Jiang ◽

...

Keyword(s):

Filamentous Fungi ◽

Zinc Finger ◽

Fusarium Graminearum ◽

Zinc Finger Protein ◽

Microtubule Assembly ◽

Polarized Growth ◽

Wild Type ◽

Finger Protein ◽

Associated Proteins ◽

Hyphal Tip

Microtubules, as transport tracks, play important roles in hyphal tip growth in filamentous fungi, but microtubule-associated proteins involved in polarized growth remain unknown. Here, we found that one novel zinc finger protein, FgChy1, is required for microtubule morphology and polarized growth in Fusarium graminearum. The Fgchy1 mutant presented curved and directionless growth of hyphae. Importantly, the conidia and germ tubes of the Fgchy1 mutant exhibited badly damaged and less organized beta-tubulin cytoskeletons. Compared with the wild type, the Fgchy1 mutant lost the ability to maintain polarity and was also more sensitive to the anti-microtubule drugs carbendazim and nocodazole, likely due to the impaired microtubule cytoskeleton. Indeed, the hyphae of the wild type treated with nocodazole exhibited a morphology consistent with that of the Fgchy1 mutant. Interestingly, the disruption of FgChy1 resulted in the off-center localization of actin patches and the polarity-related polarisome protein FgSpa2 from the hyphal tip axis. A similar defect in FgSpa2 localization was also observed in the nocodazole-treated wild-type strain. In addition, FgChy1 is also required for conidiogenesis, septation, sexual reproduction, pathogenicity and deoxynivalenol production. Overall, this study provides the first demonstrations of the functions of the novel zinc finger protein FgChy1 in polarized growth, development and virulence in filamentous fungi.

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