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Using a structural and logics systems approach to infer bHLH-DNA binding specificity determinants.

De Masi F, Grove CA, Vedenko A, Alibés A, Gisselbrecht SS, Serrano L, Bulyk ML, Walhout AJ - Nucleic Acids Res. (2011)

Bottom Line: These TFs bind E-box (CANNTG) and E-box-like sequences.Validation experiments using mutant bHLH proteins provide support for our inferences.Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Genetics, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Numerous efforts are underway to determine gene regulatory networks that describe physical relationships between transcription factors (TFs) and their target DNA sequences. Members of paralogous TF families typically recognize similar DNA sequences. Knowledge of the molecular determinants of protein-DNA recognition by paralogous TFs is of central importance for understanding how small differences in DNA specificities can dictate target gene selection. Previously, we determined the in vitro DNA binding specificities of 19 Caenorhabditis elegans basic helix-loop-helix (bHLH) dimers using protein binding microarrays. These TFs bind E-box (CANNTG) and E-box-like sequences. Here, we combine these data with logics, bHLH-DNA co-crystal structures and computational modeling to infer which bHLH monomer can interact with which CAN E-box half-site and we identify a critical residue in the protein that dictates this specificity. Validation experiments using mutant bHLH proteins provide support for our inferences. Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.

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Analysis of bHLH residue/DNA-contact frequency. (a) Canonical structure of a bHLH monomer contacting the CANNTG E-box. The E-box nucleotides on each DNA strand are indicated in blue, the bHLH basic region is shown in green and bHLH helices 1 and 2 are indicated in red and cyan, respectively. Residues 1, 15 and 50 indicate the start of the basic region, helices 1 and 2, respectively. (b) Plot indicating the frequency of contacts between bHLH residues and the bases in or flanking the E-box based on seven co-crystal structures. (c) As in (b) but indicating contacts between bHLH residues and the DNA backbone. P, phosphate; dR, deoxyribose. bHLH residue consensus numbering scheme based on Atchley et al. (33).
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Figure 2: Analysis of bHLH residue/DNA-contact frequency. (a) Canonical structure of a bHLH monomer contacting the CANNTG E-box. The E-box nucleotides on each DNA strand are indicated in blue, the bHLH basic region is shown in green and bHLH helices 1 and 2 are indicated in red and cyan, respectively. Residues 1, 15 and 50 indicate the start of the basic region, helices 1 and 2, respectively. (b) Plot indicating the frequency of contacts between bHLH residues and the bases in or flanking the E-box based on seven co-crystal structures. (c) As in (b) but indicating contacts between bHLH residues and the DNA backbone. P, phosphate; dR, deoxyribose. bHLH residue consensus numbering scheme based on Atchley et al. (33).

Mentions: bHLH proteins contact DNA with residues in their basic regions (indicated in green in Figure 2a). Although each E-box half-site is contacted by a bHLH monomer, there are also extensive contacts across the E-box, i.e. with the other half-site, and most frequently these occur on the opposite strand of DNA (Figure 2b and c) (20,21). For instance, residue 13 in the basic region often contacts the base at position 4 of the CANNTG E-box (Figure 2b). This suggests that residue 13 may contribute to half-site specificity on the opposite side of the E-box.Figure 2.


Using a structural and logics systems approach to infer bHLH-DNA binding specificity determinants.

De Masi F, Grove CA, Vedenko A, Alibés A, Gisselbrecht SS, Serrano L, Bulyk ML, Walhout AJ - Nucleic Acids Res. (2011)

Analysis of bHLH residue/DNA-contact frequency. (a) Canonical structure of a bHLH monomer contacting the CANNTG E-box. The E-box nucleotides on each DNA strand are indicated in blue, the bHLH basic region is shown in green and bHLH helices 1 and 2 are indicated in red and cyan, respectively. Residues 1, 15 and 50 indicate the start of the basic region, helices 1 and 2, respectively. (b) Plot indicating the frequency of contacts between bHLH residues and the bases in or flanking the E-box based on seven co-crystal structures. (c) As in (b) but indicating contacts between bHLH residues and the DNA backbone. P, phosphate; dR, deoxyribose. bHLH residue consensus numbering scheme based on Atchley et al. (33).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113581&req=5

Figure 2: Analysis of bHLH residue/DNA-contact frequency. (a) Canonical structure of a bHLH monomer contacting the CANNTG E-box. The E-box nucleotides on each DNA strand are indicated in blue, the bHLH basic region is shown in green and bHLH helices 1 and 2 are indicated in red and cyan, respectively. Residues 1, 15 and 50 indicate the start of the basic region, helices 1 and 2, respectively. (b) Plot indicating the frequency of contacts between bHLH residues and the bases in or flanking the E-box based on seven co-crystal structures. (c) As in (b) but indicating contacts between bHLH residues and the DNA backbone. P, phosphate; dR, deoxyribose. bHLH residue consensus numbering scheme based on Atchley et al. (33).
Mentions: bHLH proteins contact DNA with residues in their basic regions (indicated in green in Figure 2a). Although each E-box half-site is contacted by a bHLH monomer, there are also extensive contacts across the E-box, i.e. with the other half-site, and most frequently these occur on the opposite strand of DNA (Figure 2b and c) (20,21). For instance, residue 13 in the basic region often contacts the base at position 4 of the CANNTG E-box (Figure 2b). This suggests that residue 13 may contribute to half-site specificity on the opposite side of the E-box.Figure 2.

Bottom Line: These TFs bind E-box (CANNTG) and E-box-like sequences.Validation experiments using mutant bHLH proteins provide support for our inferences.Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Genetics, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Numerous efforts are underway to determine gene regulatory networks that describe physical relationships between transcription factors (TFs) and their target DNA sequences. Members of paralogous TF families typically recognize similar DNA sequences. Knowledge of the molecular determinants of protein-DNA recognition by paralogous TFs is of central importance for understanding how small differences in DNA specificities can dictate target gene selection. Previously, we determined the in vitro DNA binding specificities of 19 Caenorhabditis elegans basic helix-loop-helix (bHLH) dimers using protein binding microarrays. These TFs bind E-box (CANNTG) and E-box-like sequences. Here, we combine these data with logics, bHLH-DNA co-crystal structures and computational modeling to infer which bHLH monomer can interact with which CAN E-box half-site and we identify a critical residue in the protein that dictates this specificity. Validation experiments using mutant bHLH proteins provide support for our inferences. Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.

Show MeSH