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Bending and adaptability to proteins of the cAMP DNA-responsive element: molecular dynamics contrasted with NMR.

ABSTRACT: DNA bending is assumed to play a crucial role during recognition of the cAMP-responsive element (CRE) by transcription factors. However, diverging results have been obtained for the bending direction of the unbound double helix. The refined NMR structures present a bend directed toward the minor groove, while biochemical methods conclude that there is a bend toward the major groove. The present 10-ns molecular dynamics (MD) simulation of d(GAGATGACGTCATCTC)(2), which contains the octamer CRE in its center, was carried out with AMBER in explicit water and counterions. It shows that CRE is a flexible segment, although it is bent slightly toward the major groove (7 degrees -8 degrees ) on the average. The MD structure agrees with both the biochemical results and unrefined NMR data. The divergence with the NMR refined structures suggests an improper electrostatic parameterization in the refinement software. The malleability of the central CpG is certainly the major contribution to the curving of the whole CRE segment in both the unbound and bound states. Comparison with the crystal structure of CRE bound to GCN4 shows that the deformation induced by the protein is concentrated mainly on the CpG step, rendering the bound structure of CRE closer to the structure of the 12-0 tetradecanoylphorbol-beta-acetate-responsive element.

SUBMITTER: Derreumaux S

PROVIDER: S-EPMC1300966 | biostudies-other | 2000 Aug

REPOSITORIES: biostudies-other

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Bending and adaptability to proteins of the cAMP DNA-responsive element: molecular dynamics contrasted with NMR.

Derreumaux S S Fermandjian S S

Biophysical journal 20000801 2

DNA bending is assumed to play a crucial role during recognition of the cAMP-responsive element (CRE) by transcription factors. However, diverging results have been obtained for the bending direction of the unbound double helix. The refined NMR structures present a bend directed toward the minor groove, while biochemical methods conclude that there is a bend toward the major groove. The present 10-ns molecular dynamics (MD) simulation of d(GAGATGACGTCATCTC)(2), which contains the octamer CRE in ...[more]

PMID: 10920000

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Project description:Generally, knowledge of the mechanism regulating gene expression in primary spermatocytes is incomplete. We have used the lactate dehydrogenase gene (Ldhc) as a model to explore these mechanisms during spermatogenesis. Its 100-bp core promoter contained two essential elements common to many genes, a GC box and a CRE site. Here we report results that support a model in which transcription factor MYBL1 acts as a coactivator directing tissue-specific expression via the CRE cis element. We hypothesize that this is a common mechanism involving activation of multiple genes in the primary spermatocyte. MYBL1 is expressed predominantly as a tissue-specific transcription factor in spermatocytes and breast epithelial cells. Our finding that LDHC expression is lost in 21-day testes of MYBL1 mutant mice supports our hypothesis. In the GC1-spg germ cell line exogenous MYBL1 induces activity 4- to 8-fold, although extracts from these cells do not show MYBL1 binding activity for the Myb consensus sequences in the Ldhc promoter by EMSA. Rather, MYBL1 stimulates expression from a synthetic promoter containing only CRE elements, suggesting MYBL1 activates the promoter by interacting with protein that binds to a CRE element. Mutation of three Myb sites does not affect Ldhc promoter activity significantly (P > 0.05). CREB-binding protein (CBP) is a coactivator that interacts with CRE-binding protein CREB. We show that the transactivation domain (TAD) in MYBL1 interacts with the KIX domain in CBP, and the TAD domain and DNA binding domain in MYBL1 each interact with the CREB N-terminal domain. MYBL1 also stimulated expression from testis-specific genes Pgk2 (phosphoglycerate kinase 2) and Pdha2 (pyruvate dehydrogenase alpha 2) promoters, each of which contains CRE promoter elements and is expressed in primary spermatocytes. We propose that MYBL1 directs germ cell-specific activation via the CRE site of certain genes that are activated specifically in the primary spermatocyte, although other, more indirect effects of MYBL1 remain a possible explanation for our results.

Dataset Information

Bending and adaptability to proteins of the cAMP DNA-responsive element: molecular dynamics contrasted with NMR.

Publications

Bending and adaptability to proteins of the cAMP DNA-responsive element: molecular dynamics contrasted with NMR.

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