Functional Domains of c-myc Promoter Binding Protein 1 Involved in Transcriptional Repression and Cell Growth Regulation

ABSTRACT We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library. Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells. To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed. This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites. Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244). The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region. When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited. Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common. Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression. Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-mycpromoter. In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1. Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation.

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Ubc9 interacts with chicken ovalbumin upstream promoter-transcription factor I and represses receptor-dependent transcription

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0320069 ◽

2004 ◽

Vol 32 (1) ◽

pp. 69-86 ◽

Cited By ~ 29

Author(s):

S Kobayashi ◽

H Shibata ◽

I Kurihara ◽

K Yokota ◽

N Suda ◽

...

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Transcriptional Repression ◽

Binding Domain ◽

Adrenocortical Adenoma ◽

Dna Binding Domain ◽

Upstream Promoter ◽

Chicken Ovalbumin ◽

Repression Domain ◽

Conjugating Enzyme

Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors involved in regulation of neurogenesis and organogenesis. COUP-TF family members are generally considered to be transcriptional repressors and several mechanisms have been proposed to underlie this activity. To explore novel transcriptional coregulators for COUP-TFs, we used the COUP-TFI as bait in a yeast two-hybrid screen of an adrenocortical adenoma cDNA library. We have identified Ubc9, a class E2 conjugating enzyme of small ubiquitin-related modifier (SUMO)-1 as a COUP-TFI corepressor. Ubc9 interacts with COUP-TFI in yeast and in glutathione S-transferase pulldown and coimmunoprecipitation assays. Fluorescence imaging studies show that both Ubc9 and COUP-TFI are colocalized in the nuclei of transfected COS-1 cells. The C-terminal region of Ubc9 encoding amino acids 59-158 interacts with the C-terminus of COUP-TFI encoding amino acids 383-403, in which transcriptional repression domains are located. Mammalian one-hybrid assays utilizing a variety of Ubc9 fragments fused to Gal4 DNA-binding domain show that a Ubc9 fragment encoding amino acids 1-89 contains autonomous transferrable repression domain. Transfection of Ubc9 into COS-1 cells markedly enhances transcriptional repression by Gal4 DNA-binding domain-fused to COUP-TFI(155-423), but not by Gal4-COUP-TFI(155-388) which lacks a repressor domain. Coexpression of a C-terminal deletion mutant of Ubc9(1-58), which fails to interact with COUP-TFI, but retains a transcriptional repression domain, has no effect on Gal4-COUP-TFI-mediated repression activity. These findings indicate that interaction of Ubc9 with COUP-TFI is crucial for the corepressor function of Ubc9. Overexpression of Ubc9 similarly enhances COUP-TFI-dependent repression of the promoter activity of the bovine CYP17 gene encoding steroid 17alpha-hydroxylase. In addition, the C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, continues to function as a COUP-TFI corepressor. Our studies indicate that Ubc9 functions as a novel COUP-TFI corepressor, the function of which is distinct from its SUMO-1 conjugating enzyme activity.

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The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5128 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5128-5137 ◽

Cited By ~ 10

Author(s):

M M Witte ◽

R C Dickson

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Zinc Finger ◽

Binding Specificity ◽

Binding Domain ◽

Dna Binding Domain ◽

Transcription Activator ◽

Activator Proteins ◽

Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

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The DNA-binding domain of HPV-16 E2 protein interaction with the viral enhancer: Protein-induced DNA bending and role of the nonconserved core sequence in binding site affinity

Virology ◽

10.1016/0042-6822(90)90109-5 ◽

1990 ◽

Vol 174 (2) ◽

pp. 557-575 ◽

Cited By ~ 26

Author(s):

Camille L. Bedrosian ◽

Deepak Bastiav

Keyword(s):

Dna Binding ◽

Binding Site ◽

Protein Interaction ◽

Dna Bending ◽

Binding Domain ◽

Dna Binding Domain ◽

Hpv 16 ◽

E2 Protein ◽

Core Sequence

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Role of GCR2 in transcriptional activation of yeast glycolytic genes.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3834 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3834-3842 ◽

Cited By ~ 45

Author(s):

H Uemura ◽

Y Jigami

Keyword(s):

Dna Binding ◽

Fusion Protein ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain ◽

Gene Products ◽

Lacz Expression ◽

Genetic Method ◽

Potential Interactions

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

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XBF-2 is a transcriptional repressor that converts ectoderm into neural tissue

Development ◽

10.1242/dev.125.24.5019 ◽

1998 ◽

Vol 125 (24) ◽

pp. 5019-5031 ◽

Cited By ~ 6

Author(s):

F.V. Mariani ◽

R.M. Harland

Keyword(s):

Dna Binding ◽

Neural Development ◽

Ectopic Expression ◽

Transcriptional Repressor ◽

Neural Plate ◽

Binding Domain ◽

Expression Cloning ◽

Dna Binding Domain ◽

Neural Fate ◽

Striking Contrast

We have identified Xenopus Brain Factor 2 (XBF-2) as a potent neuralizing activity in an expression cloning screen. In ectodermal explants, XBF-2 converts cells from an epidermal to a neural fate. Such explants contain neurons with distinct axonal profiles and express both anterior and posterior central nervous system (CNS) markers. In striking contrast to X-ngnR-1a or X-NeuroD, ectopic expression of XBF-2 in Xenopus embryos results in an expansion of the neural plate to the ventral midline. The enlarged neural plate consists predominantly of undifferentiated neurons. XBF-2 lies downstream of the BMP antagonists noggin, cerberus, and gremlin since ectodermal explants expressing these molecules exhibit strong expression of XBF-2. While XBF-2 does not upregulate the expression of secreted neural inducers, it downregulates the transcription of BMP-4, an epidermal inducer. We show that XBF-2 acts as a transcriptional repressor and that its effects can be phenocopied with either the engrailed or hairy repressor domain fused to the XBF-2 DNA-binding domain. A fusion of the DNA-binding domain to the activator domain of VP16 blocks the effects of XBF-2 and prevents neural plate development in the embryo. This provides evidence that a transcriptional repressor can affect both regional neural development and neurogenesis in vertebrates.

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A position-dependent transcription-activating domain in TFIIIA

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7496-7506.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7496-7506

Author(s):

X Mao ◽

M K Darby

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Zinc Finger ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Rna Polymerase Iii ◽

Binding Domain ◽

Dna Binding Domain ◽

5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

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482. A Mutational Analysis Screen of the phiC31 Integrase DNA Binding Domain Identifies Critical Amino Acids Defining Activity and Binding to Attachment Sites

Molecular Therapy ◽

10.1016/s1525-0016(16)39885-9 ◽

2008 ◽

Vol 16 ◽

pp. S181-S182

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Mutational Analysis ◽

Binding Domain ◽

Dna Binding Domain ◽

Phic31 Integrase ◽

Attachment Sites

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Mad proteins contain a dominant transcription repression domain.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5772 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5772-5781 ◽

Cited By ~ 116

Author(s):

D E Ayer ◽

C D Laherty ◽

Q A Lawrence ◽

A P Armstrong ◽

R N Eisenman

Keyword(s):

Dna Binding ◽

Transcriptional Repression ◽

Binding Domain ◽

Full Length ◽

Dna Binding Domain ◽

Interaction Domain ◽

Transcription Repression ◽

Helix Loop Helix ◽

Repression Domain

Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.

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