Deciphering the Sox-Oct partner code by quantitative cooperativity measurements

Ng, Calista Keow Leng; Li, Noel X.; Chee, Sheena; Prabhakar, Shyam; Kolatkar, Prasanna R.; Jauch, Ralf

doi:10.1093/nar/gks153

Cited by 85 publications

(120 citation statements)

References 35 publications

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“…Next, we extended the analysis and included a total of six TFs belonging to POU classes I, II, III, V, and VI (Figs 1E and F, and EV1B). We quantified the cooperativity of the homodimeric Oct–Oct and heterodimeric Sox–Oct binding of the POU proteins to the MORE ( OctOct ) and SoxOct elements using previously derived equations 27, 40. Cooperativity represents the ratio of the equilibrium binding constant for the binding of a factor to free DNA to the binding of DNA in the presence of another protein (Sox2 for heterodimers and another POU factor for homodimers).…”

Section: Resultsmentioning

confidence: 99%

“…EMSAs were performed essentially as described 27 using 100 nM of 5′Cy5‐labeled SoxOct or MORE dsDNA mixed with 100–500 nM of Oct POUs and 20–100 nM of Sox2 HMG (79‐aa protein). Reaction mixtures were incubated for 1 hr on ice in the dark.…”

Section: Methodsmentioning

confidence: 99%

“…Cooperativity factors (ω) were calculated after quantifying the fraction of bound DNA in EMSAs using the below equations. Equations were derived using Boltzmann weights and principles of statistical mechanisms as detailed in 27, 40.…”

Section: Methodsmentioning

confidence: 99%

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Changing POU dimerization preferences converts Oct6 into a pluripotency inducer

Jerabek

et al. 2016

EMBO Reports

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The transcription factor Oct4 is a core component of molecular cocktails inducing pluripotent stem cells (iPSCs), while other members of the POU family cannot replace Oct4 with comparable efficiency. Rather, group III POU factors such as Oct6 induce neural lineages. Here, we sought to identify molecular features determining the differential DNA‐binding and reprogramming activity of Oct4 and Oct6. In enhancers of pluripotency genes, Oct4 cooperates with Sox2 on heterodimeric SoxOct elements. By re‐analyzing ChIP‐Seq data and performing dimerization assays, we found that Oct6 homodimerizes on palindromic OctOct more cooperatively and more stably than Oct4. Using structural and biochemical analyses, we identified a single amino acid directing binding to the respective DNA elements. A change in this amino acid decreases the ability of Oct4 to generate iPSCs, while the reverse mutation in Oct6 does not augment its reprogramming activity. Yet, with two additional amino acid exchanges, Oct6 acquires the ability to generate iPSCs and maintain pluripotency. Together, we demonstrate that cell type‐specific POU factor function is determined by select residues that affect DNA‐dependent dimerization.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Changing POU dimerization preferences converts Oct6 into a pluripotency inducer

Jerabek

et al. 2016

EMBO Reports

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123

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“…For example, one could assay the binding of two proteins, A and B, by generating libraries that have variable binding sites for each and also have variable spacings between them. If the EMSA gel has four bands, one for each protein bound alone as well as the unbound and doubly bound DNA (e.g., Ng et al 2012), one could obtain the specificity profile for each protein as well as the cooperativity as a function of the spacer DNA all in one experiment. While limited to a few thousand variants in any one reaction, by performing sets of reactions with different components varied, one can expect to get a very thorough view of the binding of the protein complexes to DNA.…”

Section: Lac Repressor Specificitymentioning

confidence: 99%

High-Resolution Specificity from DNA Sequencing Highlights Alternative Modes of Lac Repressor Binding

Zuo¹,

Stormo

2014

Genetics

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Knowing the specificity of transcription factors is critical to understanding regulatory networks in cells. The lac repressoroperator system has been studied for many years, but not with high-throughput methods capable of determining specificity comprehensively. Details of its binding interaction and its selection of an asymmetric binding site have been controversial. We employed a new method to accurately determine relative binding affinities to thousands of sequences simultaneously, requiring only sequencing of bound and unbound fractions. An analysis of 2560 different DNA sequence variants, including both base changes and variations in operator length, provides a detailed view of lac repressor sequence specificity. We find that the protein can bind with nearly equal affinities to operators of three different lengths, but the sequence preference changes depending on the length, demonstrating alternative modes of interaction between the protein and DNA. The wild-type operator has an odd length, causing the two monomers to bind in alternative modes, making the asymmetric operator the preferred binding site. We tested two other members of the LacI/ GalR protein family and find that neither can bind with high affinity to sites with alternative lengths or shows evidence of alternative binding modes. A further comparison with known and predicted motifs suggests that the lac repressor may be unique in this ability and that this may contribute to its selection.T HE lactose regulatory system established the paradigm of a trans-acting factor binding to a cis-acting element to regulate the expression of the adjacent gene in response to an environmental signal (Jacob and Monod 1961). Many aspects of the lac repressor protein have been studied extensively (reviewed in Lewis 2005). Our primary interest is in the DNA binding specificity of the lac repressor. Measurements of affinity changes due to operator sequence variation, by base replacement, by the use of base analogs, or by changing the length of the operator, have been performed almost since the operator sequence was first determined (Goeddel et al. 1978;Sadler et al. 1983;Betz et al. 1986;Sartorius et al. 1989;Lehming et al. 1990;Sasmor and Betz 1990;Frank et al. 1997;Spronk et al. 1999;Falcon and Matthews 2001;Kalodimos et al. 2002Kalodimos et al. , 2004bDaber and Lewis 2009). But those analyses all measured binding affinity to only a few sequences.The lac repressor has not, to our knowledge, been analyzed by current high-throughput methods that can determine specificity over thousands, or even millions, of sequences in parallel (Stormo and Zhao 2010), such as protein-binding microarrays (PBM) (Berger et al. 2006;Gordan et al. 2013), SELEX-seq [or HT-SELEX (Zhao et al. 2009;Zykovich et al. 2009;Jolma et al. 2010;Wong et al. 2011)], bacterial one-hybrid (B1H) (Meng et al. 2005;Noyes et al. 2008;Christensen et al. 2011), and mechanically induced trapping of molecular interactions (MITOMI) (Maerkl and Quake 2007). While those methods offer an expansive overvie...

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“…The complexity of an organ development is manifested through spatiotemporal expression of genes involved in development, which is tightly regulated to a large extent by the combination of transcription factors in multi-protein complexes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). In these processes, the binding affinity of transcription factors to their genetic elements, which is crucial for transcription activity, is modulated by cooperative binding: low inherent binding affinity of the individual factors is largely enhanced when they present together by their synergistic action.…”

Section: Introductionmentioning

confidence: 99%

Examining Cooperative Binding of Sox2 on DC5 Regulatory Element Upon Complex Formation with Pax6 Through Excess Electron Transfer Assay

Saha¹

2018

Molecular Recognition of DNA Double Helix

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Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein-DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, Br U-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.

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Deciphering the Sox-Oct partner code by quantitative cooperativity measurements

Cited by 85 publications

References 35 publications

Changing POU dimerization preferences converts Oct6 into a pluripotency inducer

Changing POU dimerization preferences converts Oct6 into a pluripotency inducer

High-Resolution Specificity from DNA Sequencing Highlights Alternative Modes of Lac Repressor Binding

Examining Cooperative Binding of Sox2 on DC5 Regulatory Element Upon Complex Formation with Pax6 Through Excess Electron Transfer Assay

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