The CUP2 gene product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein

1989 ◽  
Vol 9 (9) ◽  
pp. 4091-4095 ◽  
Author(s):  
C Buchman ◽  
P Skroch ◽  
J Welch ◽  
S Fogel ◽  
M Karin
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Regulatory Gene ◽  
Point Mutations ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Dna Binding Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Binding Activity ◽  
Yeast Strains

CUP2 is a regulatory gene controlling expression of CUP1, which encodes the Cu-binding yeast metallothionein. CUP2, which is identical to the ACE1 gene, encodes a Cu-regulated DNA-binding protein. The CUP2 protein contains a cysteine-rich DNA-binding domain dependent on Cu+ and Ag+ ions which bind the cysteine residues and direct the refolding of the metal-free apoprotein. CUP2 mutant alleles from Cu-sensitive yeast strains have point mutations affecting the DNA-binding activity. These results establish CUP2 as the primary sensor of intracellular Cu+ in the yeast Saccharomyces cerevisiae, functioning as a Cu+-regulated transcriptional activator.

scholarly journals The CUP2 gene product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein.

1989 ◽  
Vol 9 (9) ◽  
pp. 4091-4095 ◽  
Author(s):  
C Buchman ◽  
P Skroch ◽  
J Welch ◽  
S Fogel ◽  
M Karin
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Regulatory Gene ◽  
Point Mutations ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Dna Binding Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Binding Activity ◽  
Yeast Strains

CUP2 is a regulatory gene controlling expression of CUP1, which encodes the Cu-binding yeast metallothionein. CUP2, which is identical to the ACE1 gene, encodes a Cu-regulated DNA-binding protein. The CUP2 protein contains a cysteine-rich DNA-binding domain dependent on Cu+ and Ag+ ions which bind the cysteine residues and direct the refolding of the metal-free apoprotein. CUP2 mutant alleles from Cu-sensitive yeast strains have point mutations affecting the DNA-binding activity. These results establish CUP2 as the primary sensor of intracellular Cu+ in the yeast Saccharomyces cerevisiae, functioning as a Cu+-regulated transcriptional activator.

scholarly journals A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1

1994 ◽  
Vol 14 (3) ◽  
pp. 1852-1860
Author(s):  
K Nakagomi ◽  
Y Kohwi ◽  
L A Dickinson ◽  
T Kohwi-Shigematsu
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Contact ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Sequence Context ◽  
Dna Binding Activity

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.

scholarly journals A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1.

1994 ◽  
Vol 14 (3) ◽  
pp. 1852-1860 ◽  
Author(s):  
K Nakagomi ◽  
Y Kohwi ◽  
L A Dickinson ◽  
T Kohwi-Shigematsu
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Contact ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Sequence Context ◽  
Dna Binding Activity

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.

scholarly journals Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

1991 ◽  
Vol 11 (9) ◽  
pp. 4356-4362 ◽  
Author(s):  
M N Kanaan ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

scholarly journals Silencing of the inhibitor of DNA binding protein 4 (ID4) contributes to the pathogenesis of mouse and human CLL

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 862-871 ◽  
Author(s):  
Shih-Shih Chen ◽  
Rainer Claus ◽  
David M. Lucas ◽  
Lianbo Yu ◽  
Jiang Qian ◽  
...  
Keyword(s):  
Dna Binding ◽  
Promoter Methylation ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Lymphocytic Leukemia ◽  
Dominant Negative ◽  
Helix Loop Helix ◽  
Dna Binding Activity ◽  
Inhibitor Of Dna Binding

Abstract Inhibitor of DNA binding protein 4 (ID4) is a member of the dominant-negative basic helix-loop-helix transcription factor family that lacks DNA binding activity and has tumor suppressor function. ID4 promoter methylation has been reported in acute myeloid leukemia and chronic lymphocytic leukemia (CLL), although the expression, function, and clinical relevance of this gene have not been characterized in either disease. We demonstrate that the promoter of ID4 is consistently methylated to various degrees in CLL cells, and increased promoter methylation in a univariable analysis correlates with shortened patient survival. However, ID4 mRNA and protein expression is uniformly silenced in CLL cells irrespective of the degree of promoter methylation. The crossing of ID4+/− mice with Eμ-TCL1 mice triggers a more aggressive murine CLL as measured by lymphocyte count and inferior survival. Hemizygous loss of ID4 in nontransformed TCL1-positive B cells enhances cell proliferation triggered by CpG oligonucleotides and decreases sensitivity to dexamethasone-mediated apoptosis. Collectively, this study confirms the importance of the silencing of ID4 in murine and human CLL pathogenesis.

scholarly journals Characterization of the Pathway-Specific Positive Transcriptional Regulator for Actinorhodin Biosynthesis inStreptomyces coelicolor A3(2) as a DNA-Binding Protein

1999 ◽  
Vol 181 (22) ◽  
pp. 6958-6968 ◽  
Author(s):  
Paloma Arias ◽  
Miguel A. Fernández-Moreno ◽  
Francisco Malpartida
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Wild Type ◽  
Biosynthetic Genes ◽  
Dna Binding Activity ◽  
Orf4 Protein

ABSTRACT The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the act cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type actII-ORF4 gene but not with theactII-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified fromEscherichia coli cultures; both showed specific DNA-binding activity for the actVI-ORF1–ORFA andactIII-actI intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the −35 regions of the corresponding promoters, showing the consensus sequence 5′-TCGAG-3′. Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the actgenes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the act biosynthetic genes.

Regulation of Ldb1 Stability and Transcriptional Complex Assembly by Single-Stranded DNA-Binding Protein SSDP2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1739-1739
Author(s):  
Zhixiong Xu ◽  
Xianzhang Meng ◽  
Ying Cai ◽  
Lalitha Nagarajan ◽  
Stephen J. Brandt
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Lim Domain ◽  
Complex Assembly ◽  
Single Stranded Dna ◽  
Dna Binding Activity ◽  
E Box ◽  
Transcriptional Complex

Abstract The LIM domain-binding protein Ldb1 is known to form higher order complexes with LIM-homeodomain and LIM-only (LMO) proteins to regulate diverse developmental programs, including hematopoiesis. The level of Ldb1 is critical for its cellular roles, and its turnover is regulated by the E3 ubiquitin ligase RLIM. Single-stranded DNA-binding protein (SSDP), an Ldb1-interacting partner, is an essential gene for embryonic development and has been shown to regulate axis formation in Xenopus and wing development in Drosophila through Ldb1; however, the mechanisms by which SSDPs regulate these and other developmental programs are still obscure. We previously reported that a DNA-binding complex containing the basic helix-loop-helix protein TAL1/SCL, its DNA-binding partner E47, zinc finger protein GATA-1, LIM domain protein LMO2, and Ldb1 stimulates Protein 4.2 (P4.2) transcription in erythroid progenitors through tandem E box-GATA elements in the gene’s proximal promoter. We have now established that SSDP2 is associated with this complex (by supershift analysis) and occupies the promoter of this gene (by chromatin immunoprecipitation analysis) in murine erythroleukemia (MEL) cells. Further, enforced expression of SSDP2 in these cells stimulated P4.2 reporter activity and accumulation of P4.2 and beta-globin mRNAs, and cotransfection of SSDP2 with the five originally identified components of this complex further increased promoter activity in reporter analysis. Importantly, overexpression of SSDP2 in MEL cells significantly increased Ldb1 protein half-life and steady-state levels of Ldb1 and LMO2 protein. This effect on Ldb1 stability required the Ldb1-interacting domain of SSDP2, consisting of its first 94 amino acids (SSDP2(1–94)), and was also observed in Cos7L and CHO cells. We showed, in addition, that SSDP2 or SSDP2(1–94), but not an Ldb1 interaction-defective mutant, prevented RLIM-mediated degradation of both Ldb1 and LMO2 in transfected cells, that SSDP2 protection of LMO2 degradation required Ldb1, and that SSDP2 directly inhibited RLIM-mediated ubiquitination of Ldb1. Immunoprecipitation analysis revealed that overexpression of SSDP2 or SSDP2(1–94) significantly decreased interaction between Ldb1 and RLIM. Finally, SSDP2 protein expression in differentiating MEL cells paralleled this multi-protein DNA-binding activity and overexpression of SSDP2 in these cells dramatically increased E box-GATA DNA-binding activity, with maximal formation of the ternary complex requiring coexpression of SSDP2, Ldb1, and LMO2. Together, these studies reveal a positive role for SSDP2 in erythroid gene expression and identify a biochemical function for SSDP2 in regulating Ldb1 stability and transcriptional complex assembly. The mechanism mediating Ldb1 stabilization appears to involve competitive inhibition of RLIM interaction with Ldb1.

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