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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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(A) Alignment of amino acid sequence of all mouse Sox-high-mobility group (HMG) domains shaded with BOXSHADE. The Sox subfamilies are indicated to the right. The numbering corresponds to the HMG convention (29). α-Helices are marked with a red bar. The Phe-Met wedge is indicated with an orange bar below the alignment. DNA interacting residues are marked by black empty circles while Sox-Oct interacting residues are marked by blue empty circles. Highly conserved and similar sequences are shaded in black or gray. (B) A phylogenetic tree calculated using PROML (http://caps.ncbs.res.in/iws/proml.html). This simplified tree largely corresponds to the more exhaustive phylogenetic analysis of Sox factors. Sox subgroups (29) and the amino acids found at position 57 of the HMG domains are indicated in single letter codes. Electrostatic surface maps of representing Sox members were calculated as described (26). Positively and negatively charged regions were represented in red and blue patches, respectively. Homology models for Sox HMGs were generated using I-TASSER (28) and surface patches that differ for Sox groups are boxed. (C) Illustration of how the microstates of the DNA complexes were quantified using the ImageQuant TL software. The cy5-labeled dsDNA migrated differently on native gel depending on how the proteins and DNA associate. Thus, the fractional contribution of the microstates of the free DNA (f0), Sox-DNA (f1), Oct4-DNA (f2) and ternary complex (f3) can be quantified. (D) Schematic diagram highlighting the approach to calculate the cooperativity of TF pairs on composite DNA elements. Boltzmann weights of the respective complexes are denoted as b_D, b_DP1, b_DP2 and b_DP1P2 and scaled so that the b_D = 1. [P1] and [P2] are the concentrations of the free proteins. The cooperativity factor omega does not depend on the concentration of the reactants but solely on the relative ratios of the four microstates represented by their fractional contributions measured in (C) (see main text and alternate derivation of the equation in the ‘Materials and Methods’ section).
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gks153-F1: (A) Alignment of amino acid sequence of all mouse Sox-high-mobility group (HMG) domains shaded with BOXSHADE. The Sox subfamilies are indicated to the right. The numbering corresponds to the HMG convention (29). α-Helices are marked with a red bar. The Phe-Met wedge is indicated with an orange bar below the alignment. DNA interacting residues are marked by black empty circles while Sox-Oct interacting residues are marked by blue empty circles. Highly conserved and similar sequences are shaded in black or gray. (B) A phylogenetic tree calculated using PROML (http://caps.ncbs.res.in/iws/proml.html). This simplified tree largely corresponds to the more exhaustive phylogenetic analysis of Sox factors. Sox subgroups (29) and the amino acids found at position 57 of the HMG domains are indicated in single letter codes. Electrostatic surface maps of representing Sox members were calculated as described (26). Positively and negatively charged regions were represented in red and blue patches, respectively. Homology models for Sox HMGs were generated using I-TASSER (28) and surface patches that differ for Sox groups are boxed. (C) Illustration of how the microstates of the DNA complexes were quantified using the ImageQuant TL software. The cy5-labeled dsDNA migrated differently on native gel depending on how the proteins and DNA associate. Thus, the fractional contribution of the microstates of the free DNA (f0), Sox-DNA (f1), Oct4-DNA (f2) and ternary complex (f3) can be quantified. (D) Schematic diagram highlighting the approach to calculate the cooperativity of TF pairs on composite DNA elements. Boltzmann weights of the respective complexes are denoted as b_D, b_DP1, b_DP2 and b_DP1P2 and scaled so that the b_D = 1. [P1] and [P2] are the concentrations of the free proteins. The cooperativity factor omega does not depend on the concentration of the reactants but solely on the relative ratios of the four microstates represented by their fractional contributions measured in (C) (see main text and alternate derivation of the equation in the ‘Materials and Methods’ section).

Mentions: The 80 amino acid HMG domain of Sox proteins is highly conserved for all paralogs (Figure 1A). In accordance with similar DNA sequence preferences for all Sox proteins (2), amino acids that contact DNA bases are nearly invariant for all Sox proteins. However, protein contact interfaces as defined in structural studies on Sox2 and Oct1 show some disparity (30) (highlighted as blue empty circles). As an extension of earlier work on Sox2 and Sox17, we generated homology models for all Sox families and inspected the electrostatic charge distribution on the van der Waals’ surface (Figure 1B). The protein surface of Sox proteins facing Oct4 when bound to canonical sox-oct motifs show pronounced differences distinguishing Sox families. The SoxC, E and F groups contain an acidic patch at this interface, the SoxB and SoxG groups are highly basic and the SoxD group is largely neutral. We have recently shown that residue 57 (numbering according to HMG conventions), which is causing the disparate electrostatic pattern of the Sox families, is critical for the effective dimerization of Sox2 with Oct4 on pluripotency enhancers (19). To understand how these structural differences affect Sox-Oct partnerships, we developed a quantitative method to study TF cooperation and analysed the interaction of 11 Sox proteins with Oct4.Figure 1.


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) Alignment of amino acid sequence of all mouse Sox-high-mobility group (HMG) domains shaded with BOXSHADE. The Sox subfamilies are indicated to the right. The numbering corresponds to the HMG convention (29). α-Helices are marked with a red bar. The Phe-Met wedge is indicated with an orange bar below the alignment. DNA interacting residues are marked by black empty circles while Sox-Oct interacting residues are marked by blue empty circles. Highly conserved and similar sequences are shaded in black or gray. (B) A phylogenetic tree calculated using PROML (http://caps.ncbs.res.in/iws/proml.html). This simplified tree largely corresponds to the more exhaustive phylogenetic analysis of Sox factors. Sox subgroups (29) and the amino acids found at position 57 of the HMG domains are indicated in single letter codes. Electrostatic surface maps of representing Sox members were calculated as described (26). Positively and negatively charged regions were represented in red and blue patches, respectively. Homology models for Sox HMGs were generated using I-TASSER (28) and surface patches that differ for Sox groups are boxed. (C) Illustration of how the microstates of the DNA complexes were quantified using the ImageQuant TL software. The cy5-labeled dsDNA migrated differently on native gel depending on how the proteins and DNA associate. Thus, the fractional contribution of the microstates of the free DNA (f0), Sox-DNA (f1), Oct4-DNA (f2) and ternary complex (f3) can be quantified. (D) Schematic diagram highlighting the approach to calculate the cooperativity of TF pairs on composite DNA elements. Boltzmann weights of the respective complexes are denoted as b_D, b_DP1, b_DP2 and b_DP1P2 and scaled so that the b_D = 1. [P1] and [P2] are the concentrations of the free proteins. The cooperativity factor omega does not depend on the concentration of the reactants but solely on the relative ratios of the four microstates represented by their fractional contributions measured in (C) (see main text and alternate derivation of the equation in the ‘Materials and Methods’ section).
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gks153-F1: (A) Alignment of amino acid sequence of all mouse Sox-high-mobility group (HMG) domains shaded with BOXSHADE. The Sox subfamilies are indicated to the right. The numbering corresponds to the HMG convention (29). α-Helices are marked with a red bar. The Phe-Met wedge is indicated with an orange bar below the alignment. DNA interacting residues are marked by black empty circles while Sox-Oct interacting residues are marked by blue empty circles. Highly conserved and similar sequences are shaded in black or gray. (B) A phylogenetic tree calculated using PROML (http://caps.ncbs.res.in/iws/proml.html). This simplified tree largely corresponds to the more exhaustive phylogenetic analysis of Sox factors. Sox subgroups (29) and the amino acids found at position 57 of the HMG domains are indicated in single letter codes. Electrostatic surface maps of representing Sox members were calculated as described (26). Positively and negatively charged regions were represented in red and blue patches, respectively. Homology models for Sox HMGs were generated using I-TASSER (28) and surface patches that differ for Sox groups are boxed. (C) Illustration of how the microstates of the DNA complexes were quantified using the ImageQuant TL software. The cy5-labeled dsDNA migrated differently on native gel depending on how the proteins and DNA associate. Thus, the fractional contribution of the microstates of the free DNA (f0), Sox-DNA (f1), Oct4-DNA (f2) and ternary complex (f3) can be quantified. (D) Schematic diagram highlighting the approach to calculate the cooperativity of TF pairs on composite DNA elements. Boltzmann weights of the respective complexes are denoted as b_D, b_DP1, b_DP2 and b_DP1P2 and scaled so that the b_D = 1. [P1] and [P2] are the concentrations of the free proteins. The cooperativity factor omega does not depend on the concentration of the reactants but solely on the relative ratios of the four microstates represented by their fractional contributions measured in (C) (see main text and alternate derivation of the equation in the ‘Materials and Methods’ section).
Mentions: The 80 amino acid HMG domain of Sox proteins is highly conserved for all paralogs (Figure 1A). In accordance with similar DNA sequence preferences for all Sox proteins (2), amino acids that contact DNA bases are nearly invariant for all Sox proteins. However, protein contact interfaces as defined in structural studies on Sox2 and Oct1 show some disparity (30) (highlighted as blue empty circles). As an extension of earlier work on Sox2 and Sox17, we generated homology models for all Sox families and inspected the electrostatic charge distribution on the van der Waals’ surface (Figure 1B). The protein surface of Sox proteins facing Oct4 when bound to canonical sox-oct motifs show pronounced differences distinguishing Sox families. The SoxC, E and F groups contain an acidic patch at this interface, the SoxB and SoxG groups are highly basic and the SoxD group is largely neutral. We have recently shown that residue 57 (numbering according to HMG conventions), which is causing the disparate electrostatic pattern of the Sox families, is critical for the effective dimerization of Sox2 with Oct4 on pluripotency enhancers (19). To understand how these structural differences affect Sox-Oct partnerships, we developed a quantitative method to study TF cooperation and analysed the interaction of 11 Sox proteins with Oct4.Figure 1.

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