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The structural basis for the oligomerization of the N-terminal domain of SATB1.

Wang Z, Yang X, Chu X, Zhang J, Zhou H, Shen Y, Long J - Nucleic Acids Res. (2012)

Bottom Line: Our results also reveal that SATB1 can form a tetramer through its N-terminal domain.Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets.Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, China.

ABSTRACT
Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene expression regulator essential for T-cell development and breast cancer tumor growth and metastasis. The oligomerization of the N-terminal domain of SATB1 is critical for its biological function. We determined the crystal structure of the N-terminal domain of SATB1. Surprisingly, this domain resembles a ubiquitin domain instead of the previously proposed PDZ domain. Our results also reveal that SATB1 can form a tetramer through its N-terminal domain. The tetramerization of SATB1 plays an essential role in its binding to highly specialized DNA sequences. Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets. Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.

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Oligomeric assembly of wild-type and SATB1 mutants measured by analytical ultracentrifugation. (A) c(S) distributions from SV runs for SATB1 (7.5 μM, dotted black line; 15 μM, solid black line), EFH–AAA (7.5 µM, dotted cyan line; 15 μM, solid cyan line) and KWN–AAA (7.5 μM, dotted red line; 15 μM, solid red line). Representative SE profiles for SATB1 (B) and KWN–AAA (C) derived from a global fit MW ∼344.4 kDa (1σ confidence interval 342.6–345.8) and ∼174.5 kDa (1σ confidence interval 174.1–174.9), indicating that wild-type SATB1 and the KWN–AAA mutant assemble into a tetramer and dimer in solution, respectively.
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gkr1284-F3: Oligomeric assembly of wild-type and SATB1 mutants measured by analytical ultracentrifugation. (A) c(S) distributions from SV runs for SATB1 (7.5 μM, dotted black line; 15 μM, solid black line), EFH–AAA (7.5 µM, dotted cyan line; 15 μM, solid cyan line) and KWN–AAA (7.5 μM, dotted red line; 15 μM, solid red line). Representative SE profiles for SATB1 (B) and KWN–AAA (C) derived from a global fit MW ∼344.4 kDa (1σ confidence interval 342.6–345.8) and ∼174.5 kDa (1σ confidence interval 174.1–174.9), indicating that wild-type SATB1 and the KWN–AAA mutant assemble into a tetramer and dimer in solution, respectively.

Mentions: To investigate the oligomerization of SATB1 in solution, we mutated residues within the two interfaces to test the ULD domain-mediated oligomerization state of full-length SATB1. The first mutant was designed to disrupt the EFH interface-mediated ULD dimer by replacing the 97E98F162H motif with an ‘AAA’ cassette, and the second mutant was designed to disrupt the KWN interface-mediated ULD dimer by substituting the 136K137W138N motif with an ‘AAA’ cassette (referred to as EFH–AAA and KWN–AAA, respectively) (Figure 1A). Wild-type SATB1 and the two mutants were purified to homogeneity by the protocol summarized in Supplementary Figure S7 (Figure 3A, inset) and analyzed by SV analysis. Wild-type SATB1 exhibited a narrow sedimentation coefficient distribution in continuous size distribution analysis (black lines in Figure 3A) (28), suggesting that it is mono-disperse, and it is estimated to be a stable tetramer. SE analysis of analytical ultracentrifugation further confirmed that wide-type full-length SATB1 assembled into a tetramer with a molecular mass of ∼344.4 kDa (Figure 3B and Supplementary Figures S8A–S8C). The KWN–AAA mutant also exhibited a mono-disperse sedimentation coefficient distribution (red lines in Figure 3A), suggesting that it is a dimer. The dimeric state of the KWN–AAA mutant in solution was confirmed by SE analysis, yielding a calculated molecular weight of ∼174.5 kDa (Figure 3B and Supplementary Figures S8D–S8F). In sharp contrast, the EFH–AAA mutant exhibited a wide range of sedimentation coefficients in the c(S) distribution (cyan lines in Figure 3A) indicating that it exists as multiple forms of oligomers including dimmers, tetramers and even high-order form. These data suggest that this mutation drastically interferes with the oligomerization of SATB1 and that the EFH–AAA mutant does not have a unique form of oligomers, which is consistent with its elution profile by size exclusion chromatography (Supplementary Figure S9). Taken together, we conclude that both dimers in the ULD tetramer contribute to the oligomerization of full-length SATB1.Figure 3.


The structural basis for the oligomerization of the N-terminal domain of SATB1.

Wang Z, Yang X, Chu X, Zhang J, Zhou H, Shen Y, Long J - Nucleic Acids Res. (2012)

Oligomeric assembly of wild-type and SATB1 mutants measured by analytical ultracentrifugation. (A) c(S) distributions from SV runs for SATB1 (7.5 μM, dotted black line; 15 μM, solid black line), EFH–AAA (7.5 µM, dotted cyan line; 15 μM, solid cyan line) and KWN–AAA (7.5 μM, dotted red line; 15 μM, solid red line). Representative SE profiles for SATB1 (B) and KWN–AAA (C) derived from a global fit MW ∼344.4 kDa (1σ confidence interval 342.6–345.8) and ∼174.5 kDa (1σ confidence interval 174.1–174.9), indicating that wild-type SATB1 and the KWN–AAA mutant assemble into a tetramer and dimer in solution, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351170&req=5

gkr1284-F3: Oligomeric assembly of wild-type and SATB1 mutants measured by analytical ultracentrifugation. (A) c(S) distributions from SV runs for SATB1 (7.5 μM, dotted black line; 15 μM, solid black line), EFH–AAA (7.5 µM, dotted cyan line; 15 μM, solid cyan line) and KWN–AAA (7.5 μM, dotted red line; 15 μM, solid red line). Representative SE profiles for SATB1 (B) and KWN–AAA (C) derived from a global fit MW ∼344.4 kDa (1σ confidence interval 342.6–345.8) and ∼174.5 kDa (1σ confidence interval 174.1–174.9), indicating that wild-type SATB1 and the KWN–AAA mutant assemble into a tetramer and dimer in solution, respectively.
Mentions: To investigate the oligomerization of SATB1 in solution, we mutated residues within the two interfaces to test the ULD domain-mediated oligomerization state of full-length SATB1. The first mutant was designed to disrupt the EFH interface-mediated ULD dimer by replacing the 97E98F162H motif with an ‘AAA’ cassette, and the second mutant was designed to disrupt the KWN interface-mediated ULD dimer by substituting the 136K137W138N motif with an ‘AAA’ cassette (referred to as EFH–AAA and KWN–AAA, respectively) (Figure 1A). Wild-type SATB1 and the two mutants were purified to homogeneity by the protocol summarized in Supplementary Figure S7 (Figure 3A, inset) and analyzed by SV analysis. Wild-type SATB1 exhibited a narrow sedimentation coefficient distribution in continuous size distribution analysis (black lines in Figure 3A) (28), suggesting that it is mono-disperse, and it is estimated to be a stable tetramer. SE analysis of analytical ultracentrifugation further confirmed that wide-type full-length SATB1 assembled into a tetramer with a molecular mass of ∼344.4 kDa (Figure 3B and Supplementary Figures S8A–S8C). The KWN–AAA mutant also exhibited a mono-disperse sedimentation coefficient distribution (red lines in Figure 3A), suggesting that it is a dimer. The dimeric state of the KWN–AAA mutant in solution was confirmed by SE analysis, yielding a calculated molecular weight of ∼174.5 kDa (Figure 3B and Supplementary Figures S8D–S8F). In sharp contrast, the EFH–AAA mutant exhibited a wide range of sedimentation coefficients in the c(S) distribution (cyan lines in Figure 3A) indicating that it exists as multiple forms of oligomers including dimmers, tetramers and even high-order form. These data suggest that this mutation drastically interferes with the oligomerization of SATB1 and that the EFH–AAA mutant does not have a unique form of oligomers, which is consistent with its elution profile by size exclusion chromatography (Supplementary Figure S9). Taken together, we conclude that both dimers in the ULD tetramer contribute to the oligomerization of full-length SATB1.Figure 3.

Bottom Line: Our results also reveal that SATB1 can form a tetramer through its N-terminal domain.Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets.Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, China.

ABSTRACT
Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene expression regulator essential for T-cell development and breast cancer tumor growth and metastasis. The oligomerization of the N-terminal domain of SATB1 is critical for its biological function. We determined the crystal structure of the N-terminal domain of SATB1. Surprisingly, this domain resembles a ubiquitin domain instead of the previously proposed PDZ domain. Our results also reveal that SATB1 can form a tetramer through its N-terminal domain. The tetramerization of SATB1 plays an essential role in its binding to highly specialized DNA sequences. Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets. Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.

Show MeSH
Related in: MedlinePlus