A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding

Krylov, Dmitry; Kasai, Kenji; Echlin, Deborah R.; Taparowsky, Elizabeth J.; Arnheiter, Heinz; Vinson, Charles

doi:10.1073/pnas.94.23.12274

Cited by 60 publications

(75 citation statements)

References 28 publications

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“…In our analysis, expression of the A-Mitf dominantnegative mutant, which should have prevented DNA binding by TFE3-related proteins (Krylov et al, 1997), had little effect on CDK4 expression in normal or breast cancer cell lines (data not shown). This result would exclude a major involvement of the TFE3 family in these cells.…”

Section: Discussionmentioning

confidence: 98%

“…The A-USF and A-Max constructs were chosen for this analysis because of their specificity and efficiency in inhibiting the DNA-binding activity of proteins of the USF or Myc family, respectively (Krylov et al, 1997;Qyang et al, 1999). A nearly fivefold decrease in the activity of the CDK4 promoter was observed in the presence of cotransfected A-USF, indicating an essential role of endogenous USF…”

Section: Functional Importance Of the Cdk4 E Boxes In Nontumorigenic mentioning

confidence: 99%

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Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc

et al. 2004

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USF and c-Myc are basic helix-loop-helix transcription factors with similar DNA-binding specificities, but antagonistic effects on cellular transformation. In order to determine how these opposite functions correlate with the transcriptional activities of the two factors on particular downstream targets, we investigated the roles of USF and c-Myc in expression of CDK4, a known direct target of cMyc. Overexpression of either c-Myc or USF2, but not USF1, stimulated the expression of CDK4 promoterdriven reporter genes in the non-tumorigenic mammary epithelial MCF-10A cells. Dominant-negative mutants specific to either Myc or USF family proteins inhibited reporter gene activity as well as endogenous CDK4 expression, demonstrating involvement of both USF and Myc in CDK4 transcriptional control. In contrast, in two different breast cancer cell lines where USF is transcriptionally inactive and c-Myc is overexpressed, CDK4 promoter activity was no longer responsive to either transcription factor. Accordingly, chromatin immunoprecipitation revealed significantly lower levels of both USF and c-Myc bound to the endogenous CDK4 promoter in breast cancer cells than in MCF-10A cells, with a concomitant decrease in associated histone H3 acetylation. These results suggest that a major switch in the transcriptional control of CDK4 occurs during breast carcinogenesis, with likely alteration of cell cycle regulation.

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Section: Discussionmentioning

confidence: 98%

Section: Functional Importance Of the Cdk4 E Boxes In Nontumorigenic mentioning

confidence: 99%

Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc

et al. 2004

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“…Therefore we have decided to follow a dierent strategy, that consists in the design, by molecular modelling, of a new protein domain which acts from within, by altering the balance among dierent factors of one of these networks, the Max network. Naturally occurring dominant negative mutations in bHLHZip proteins, like those in the Mi gene (Moore, 1995), as well as previously designed dominant mutations (Krylov et al, 1997) aect the function of the basic region, thus abolishing DNA binding. Our target was instead protein dimerization.…”

Section: Discussionmentioning

confidence: 99%

Design and properties of a Myc derivative that efficiently homodimerizes

et al. 1998

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bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and speci®c proteinprotein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and o cell proliferation, dierentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization speci®city. We report that Myc recognition speci®city is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes eciently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding eciency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.

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“…To determine whether these endogenous proteins were USF itself, we cotransfected expression vectors encoding mutants with dominant negative e ect on the activity of the USF or, as a control, the Myc family of transcription factors. A-USF and A-Max are constructs in which the basic region of USF1 or Max, respectively, was replaced by an acidic sequence (Qyang et al, 1999;Krylov et al, 1997). This substitution greatly stabilizes heterodimer formation between the wild-type and mutant proteins.…”

Section: Transcriptional Activity Of Usf In a Non-tumorigenic Breast mentioning

confidence: 99%

Loss of USF transcriptional activity in breast cancer cell lines

1999

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USF is a family of transcription factors that are structurally related to the Myc oncoproteins and also share with Myc a common DNA-binding speci®city. USF overexpression can prevent c-Myc-dependent cellular transformation and also inhibit the proliferation of certain transformed cells. These antiproliferative activities suggest that USF inactivation could be implicated in carcinogenesis. To explore this possibility, we compared the activities of the ubiquitous USF1 and USF2 proteins in several cell lines derived from either normal breast epithelium or breast tumors. The DNAbinding activities of USF1 and USF2 were present at similar levels in all cell lines. In the non-tumorigenic MCF-10A cells, USF in general, and USF2 in particular, exhibited strong transcriptional activities. In contrast, USF1 and USF2 were completely inactive in three out of six transformed breast cell lines investigated, while the other three transformed cell lines exhibited loss of USF2 activity. Analyses in cells cultured from healthy tissue con®rmed the transcriptional activity of USF in normal human mammary epithelial cells. These results demonstrate that a partial or complete loss of USF function is a common event in breast cancer cell lines, perhaps because, like Myc overexpression, it favors rapid proliferation.

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A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding

Abstract: We describe a method to design dominantnegative proteins (

Cited by 60 publications

References 28 publications

Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc

Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc

Design and properties of a Myc derivative that efficiently homodimerizes

Loss of USF transcriptional activity in breast cancer cell lines

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