Deciphering the Sox-Oct partner code by quantitative cooperativity measurements

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.

Figure 1.

(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).

Figure 2.

(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).

Figure 3.

(A) Sequences of the labeled Nanog element probes used (21). The Sox-binding sites are indicated in grey while the Oct-binding sites are indicated as underlined; (B) Representative EMSAs of different Sox proteins with Oct4 on canonical and compressed motif. The indicate mutants refer to amino acid position 57 of the HMG domain. (C) Cooperativity factors for various Sox mutants compared to their wild-type counterparts expressed as mean ± standard deviation. (D) Competitive EMSA analysis of showing that the Sox17EK-Oct4 complexes predominate Sox2-Oct4 complexes, whereas the Sox2-Oct4 complex clearly outcompetes Sox17-Oct4 (lanes 9 and 10). A N-and C-terminally extended Sox2 HMG domain (2L) comprising 109 amino acids (residues 33–141 of full length Sox2 protein) was used to distinguish the various complexes. The cartoon to the right symbolizes free DNA (black line), Sox2L (grey-filled circles), Sox17 and Sox17EK (grey empty circles) and Oct4 (black squares).