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Deciphering the Sox-Oct partner code by quantitative cooperativity measurements.

Ng CK, Li NX, Chee S, Prabhakar S, Kolatkar PR, Jauch R - Nucleic Acids Res. (2012)

Bottom Line: Sox5 and Sox18 show some cooperation on both elements, whereas Sox8 and Sox9 compete on both elements.Testing rationally mutated Sox proteins combined with structural modeling highlights critical amino acids for differential Sox-Oct4 partnerships and demonstrates that the cooperativity correlates with the efficiency in producing induced pluripotent stem cells.Our results suggest selective Sox-Oct partnerships in genome regulation and provide a toolset to study protein cooperation on DNA.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural Biochemistry, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore.

ABSTRACT
Several Sox-Oct transcription factor (TF) combinations have been shown to cooperate on diverse enhancers to determine cell fates. Here, we developed a method to quantify biochemically the Sox-Oct cooperation and assessed the pairing of the high-mobility group (HMG) domains of 11 Sox TFs with Oct4 on a series of composite DNA elements. This way, we clustered Sox proteins according to their dimerization preferences illustrating that Sox HMG domains evolved different propensities to cooperate with Oct4. Sox2, Sox14, Sox21 and Sox15 strongly cooperate on the canonical element but compete with Oct4 on a recently discovered compressed element. Sry also cooperates on the canonical element but binds additively to the compressed element. In contrast, Sox17 and Sox4 cooperate more strongly on the compressed than on the canonical element. Sox5 and Sox18 show some cooperation on both elements, whereas Sox8 and Sox9 compete on both elements. Testing rationally mutated Sox proteins combined with structural modeling highlights critical amino acids for differential Sox-Oct4 partnerships and demonstrates that the cooperativity correlates with the efficiency in producing induced pluripotent stem cells. Our results suggest selective Sox-Oct partnerships in genome regulation and provide a toolset to study protein cooperation on DNA.

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Related in: MedlinePlus

(A) Sequences of the idealized composite Sox-Oct-labeled probes used. The Sox-binding sites are indicated in orange while the Oct-binding sites are indicated in blue; (B) Bar plots showing cumulative mean cooperativity factors for 11 Sox HMG domains for elements shown in (A). Raw values and individual bar plots per element are shown in Supplementary Table S1 and Figure S1. To derive reliable omega values and to minimize errors in band quantification, the concentration of Sox HMG and the Oct4 POU was adjusted, such that the fractional contribution of each of the four microstates was at least 5%. If such conditions could not be established, that is, for maximally competitive binding excluding ternary complexes as seen on the plus1 element for most Sox HMGs or Sox2-Oct4 pairing on the compressed element, omega values were set to 0.01. Constitutive cooperativity was not observed in this study. (C) Heat map of cooperativity factors representing the different Sox-Oct4 dimers on the various DNA motifs. Log2-transformed mean cooperativity factors are expressed in a three-color gradient: red (competitive), white (additive binding) and blue (positive cooperativity). The matrix was hierarchically clustered using the heatmap.2 function in R with default parameters. Different categorizations were labeled as Clusters A–E and I–V. Each cooperativity factor was derived from at least 3 and maximally 30 replicates (see Supplementary Table S1). (D) Summary of the differential assembly dataset grouping Sox HMG domains exhibiting similar Oct4 cooperativity profiles. Candidate amino acids that likely explain the disparate Oct4 interactions at positions 57 and 64 are shown. (E) Differential assemblies of different Sox HMG members (50 nM) with the Oct4 POU protein (150 nM) were performed on compressed (left), canonical (center) and plus3 (right) element DNA. The cartoon to the left symbolizes free DNA (black line), Sox (blue circles) and Oct (orange squares).
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gks153-F2: (A) Sequences of the idealized composite Sox-Oct-labeled probes used. The Sox-binding sites are indicated in orange while the Oct-binding sites are indicated in blue; (B) Bar plots showing cumulative mean cooperativity factors for 11 Sox HMG domains for elements shown in (A). Raw values and individual bar plots per element are shown in Supplementary Table S1 and Figure S1. To derive reliable omega values and to minimize errors in band quantification, the concentration of Sox HMG and the Oct4 POU was adjusted, such that the fractional contribution of each of the four microstates was at least 5%. If such conditions could not be established, that is, for maximally competitive binding excluding ternary complexes as seen on the plus1 element for most Sox HMGs or Sox2-Oct4 pairing on the compressed element, omega values were set to 0.01. Constitutive cooperativity was not observed in this study. (C) Heat map of cooperativity factors representing the different Sox-Oct4 dimers on the various DNA motifs. Log2-transformed mean cooperativity factors are expressed in a three-color gradient: red (competitive), white (additive binding) and blue (positive cooperativity). The matrix was hierarchically clustered using the heatmap.2 function in R with default parameters. Different categorizations were labeled as Clusters A–E and I–V. Each cooperativity factor was derived from at least 3 and maximally 30 replicates (see Supplementary Table S1). (D) Summary of the differential assembly dataset grouping Sox HMG domains exhibiting similar Oct4 cooperativity profiles. Candidate amino acids that likely explain the disparate Oct4 interactions at positions 57 and 64 are shown. (E) Differential assemblies of different Sox HMG members (50 nM) with the Oct4 POU protein (150 nM) were performed on compressed (left), canonical (center) and plus3 (right) element DNA. The cartoon to the left symbolizes free DNA (black line), Sox (blue circles) and Oct (orange squares).

Mentions: Electrophoretic mobility shift assays (EMSAs) were carried out using forward strand 5′Cy5-labeled-dsDNA (Sigma Proligo, see Figures 2A and 3A). DNA probes were prepared by mixing equimolar amounts of complementary strands in 1 × annealing buffer (20 mM Tris–HCl, pH 8.0; 50 mM MgCl2; 50 mM KCl), heated to 95°C for 5 min and subsequently with 1°C/min ramping down to 4°C in a PCR block. Typical binding reactions contain 100 nM dsDNA with varying concentrations of both Sox and Oct4 proteins in a 1 × EMSA buffer [20 mM Tris–HCl pH 8.0, 0.1 mg/ml bovine serum albumin (Biorad), 50 µM ZnCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) Igepal CA630 and 2 mM β-mercaptoethanol] and were incubated for 1 hr at 4°C in the dark to reach binding equilibrium. Reactions were loaded into a pre-run 12% native 1 × Tris–glycine (25 mM Tris pH 8.3; 192 mM glycine) polyacrylamide gel, and DNA complexes were separated at 4°C for 30 min at 200 V. The bands were detected using a Typhoon 9140 PhosphorImager (Amersham Biosciences) and quantified using the ImageQuant TL software (Amersham Biosciences).


Deciphering the Sox-Oct partner code by quantitative cooperativity measurements.

Ng CK, Li NX, Chee S, Prabhakar S, Kolatkar PR, Jauch R - Nucleic Acids Res. (2012)

(A) Sequences of the idealized composite Sox-Oct-labeled probes used. The Sox-binding sites are indicated in orange while the Oct-binding sites are indicated in blue; (B) Bar plots showing cumulative mean cooperativity factors for 11 Sox HMG domains for elements shown in (A). Raw values and individual bar plots per element are shown in Supplementary Table S1 and Figure S1. To derive reliable omega values and to minimize errors in band quantification, the concentration of Sox HMG and the Oct4 POU was adjusted, such that the fractional contribution of each of the four microstates was at least 5%. If such conditions could not be established, that is, for maximally competitive binding excluding ternary complexes as seen on the plus1 element for most Sox HMGs or Sox2-Oct4 pairing on the compressed element, omega values were set to 0.01. Constitutive cooperativity was not observed in this study. (C) Heat map of cooperativity factors representing the different Sox-Oct4 dimers on the various DNA motifs. Log2-transformed mean cooperativity factors are expressed in a three-color gradient: red (competitive), white (additive binding) and blue (positive cooperativity). The matrix was hierarchically clustered using the heatmap.2 function in R with default parameters. Different categorizations were labeled as Clusters A–E and I–V. Each cooperativity factor was derived from at least 3 and maximally 30 replicates (see Supplementary Table S1). (D) Summary of the differential assembly dataset grouping Sox HMG domains exhibiting similar Oct4 cooperativity profiles. Candidate amino acids that likely explain the disparate Oct4 interactions at positions 57 and 64 are shown. (E) Differential assemblies of different Sox HMG members (50 nM) with the Oct4 POU protein (150 nM) were performed on compressed (left), canonical (center) and plus3 (right) element DNA. The cartoon to the left symbolizes free DNA (black line), Sox (blue circles) and Oct (orange squares).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367189&req=5

gks153-F2: (A) Sequences of the idealized composite Sox-Oct-labeled probes used. The Sox-binding sites are indicated in orange while the Oct-binding sites are indicated in blue; (B) Bar plots showing cumulative mean cooperativity factors for 11 Sox HMG domains for elements shown in (A). Raw values and individual bar plots per element are shown in Supplementary Table S1 and Figure S1. To derive reliable omega values and to minimize errors in band quantification, the concentration of Sox HMG and the Oct4 POU was adjusted, such that the fractional contribution of each of the four microstates was at least 5%. If such conditions could not be established, that is, for maximally competitive binding excluding ternary complexes as seen on the plus1 element for most Sox HMGs or Sox2-Oct4 pairing on the compressed element, omega values were set to 0.01. Constitutive cooperativity was not observed in this study. (C) Heat map of cooperativity factors representing the different Sox-Oct4 dimers on the various DNA motifs. Log2-transformed mean cooperativity factors are expressed in a three-color gradient: red (competitive), white (additive binding) and blue (positive cooperativity). The matrix was hierarchically clustered using the heatmap.2 function in R with default parameters. Different categorizations were labeled as Clusters A–E and I–V. Each cooperativity factor was derived from at least 3 and maximally 30 replicates (see Supplementary Table S1). (D) Summary of the differential assembly dataset grouping Sox HMG domains exhibiting similar Oct4 cooperativity profiles. Candidate amino acids that likely explain the disparate Oct4 interactions at positions 57 and 64 are shown. (E) Differential assemblies of different Sox HMG members (50 nM) with the Oct4 POU protein (150 nM) were performed on compressed (left), canonical (center) and plus3 (right) element DNA. The cartoon to the left symbolizes free DNA (black line), Sox (blue circles) and Oct (orange squares).
Mentions: Electrophoretic mobility shift assays (EMSAs) were carried out using forward strand 5′Cy5-labeled-dsDNA (Sigma Proligo, see Figures 2A and 3A). DNA probes were prepared by mixing equimolar amounts of complementary strands in 1 × annealing buffer (20 mM Tris–HCl, pH 8.0; 50 mM MgCl2; 50 mM KCl), heated to 95°C for 5 min and subsequently with 1°C/min ramping down to 4°C in a PCR block. Typical binding reactions contain 100 nM dsDNA with varying concentrations of both Sox and Oct4 proteins in a 1 × EMSA buffer [20 mM Tris–HCl pH 8.0, 0.1 mg/ml bovine serum albumin (Biorad), 50 µM ZnCl2, 100 mM KCl, 10% (v/v) glycerol, 0.1% (v/v) Igepal CA630 and 2 mM β-mercaptoethanol] and were incubated for 1 hr at 4°C in the dark to reach binding equilibrium. Reactions were loaded into a pre-run 12% native 1 × Tris–glycine (25 mM Tris pH 8.3; 192 mM glycine) polyacrylamide gel, and DNA complexes were separated at 4°C for 30 min at 200 V. The bands were detected using a Typhoon 9140 PhosphorImager (Amersham Biosciences) and quantified using the ImageQuant TL software (Amersham Biosciences).

Bottom Line: Sox5 and Sox18 show some cooperation on both elements, whereas Sox8 and Sox9 compete on both elements.Testing rationally mutated Sox proteins combined with structural modeling highlights critical amino acids for differential Sox-Oct4 partnerships and demonstrates that the cooperativity correlates with the efficiency in producing induced pluripotent stem cells.Our results suggest selective Sox-Oct partnerships in genome regulation and provide a toolset to study protein cooperation on DNA.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural Biochemistry, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore.

ABSTRACT
Several Sox-Oct transcription factor (TF) combinations have been shown to cooperate on diverse enhancers to determine cell fates. Here, we developed a method to quantify biochemically the Sox-Oct cooperation and assessed the pairing of the high-mobility group (HMG) domains of 11 Sox TFs with Oct4 on a series of composite DNA elements. This way, we clustered Sox proteins according to their dimerization preferences illustrating that Sox HMG domains evolved different propensities to cooperate with Oct4. Sox2, Sox14, Sox21 and Sox15 strongly cooperate on the canonical element but compete with Oct4 on a recently discovered compressed element. Sry also cooperates on the canonical element but binds additively to the compressed element. In contrast, Sox17 and Sox4 cooperate more strongly on the compressed than on the canonical element. Sox5 and Sox18 show some cooperation on both elements, whereas Sox8 and Sox9 compete on both elements. Testing rationally mutated Sox proteins combined with structural modeling highlights critical amino acids for differential Sox-Oct4 partnerships and demonstrates that the cooperativity correlates with the efficiency in producing induced pluripotent stem cells. Our results suggest selective Sox-Oct partnerships in genome regulation and provide a toolset to study protein cooperation on DNA.

Show MeSH
Related in: MedlinePlus