Publications by authors named "Prasanna R Kolatkar"

48 Publications

Transcription Factors: The Fulcrum Between Cell Development and Carcinogenesis.

Front Oncol 2021 14;11:681377. Epub 2021 Jun 14.

Diabetes Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar.

Higher eukaryotic development is a complex and tightly regulated process, whereby transcription factors (TFs) play a key role in controlling the gene regulatory networks. Dysregulation of these regulatory networks has also been associated with carcinogenesis. Transcription factors are key enablers of cancer stemness, which support the maintenance and function of cancer stem cells that are believed to act as seeds for cancer initiation, progression and metastasis, and treatment resistance. One key area of research is to understand how these factors interact and collaborate to define cellular fate during embryogenesis as well as during tumor development. This review focuses on understanding the role of TFs in cell development and cancer. The molecular mechanisms of cell fate decision are of key importance in efforts towards developing better protocols for directed differentiation of cells in research and medicine. We also discuss the dysregulation of TFs and their role in cancer progression and metastasis, exploring TF networks as direct or indirect targets for therapeutic intervention, as well as specific TFs' potential as biomarkers for predicting and monitoring treatment responses.
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http://dx.doi.org/10.3389/fonc.2021.681377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8236851PMC
June 2021

A Modelling Framework for Embedding-based Predictions for Compound-Viral Protein Activity.

Bioinformatics 2021 Feb 26. Epub 2021 Feb 26.

Qatar Computing Research Institute, Hamad Bin Khalifa University, Doha, 34110, Qatar.

Motivation: A global effort is underway to identify compounds for the treatment of COVID-19. Since de novo compound design is an extremely long, time-consuming, and expensive process, efforts are underway to discover existing compounds that can be repurposed for COVID-19 and new viral diseases.

Model: We propose a machine learning representation framework that uses deep learning induced vector embeddings of compounds and viral proteins as features to predict compound-viral protein activity. The prediction model in-turn uses a consensus framework to rank approved compounds against viral proteins of interest.

Results: Our consensus framework achieves a highmean Pearson correlation of 0.916, mean R2 of 0.840 and a low mean squared error of 0.313 for the task of compound-viral protein activity prediction on an independent test set. As a use case, we identify a ranked list of 47 compounds common to three main proteins of SARS-COV-2 virus (PL-PRO, 3CL-PRO and Spike protein) as potential targets including 21 antivirals, 15 anticancer, 5 antibiotics and 6 other investigationalhuman compounds.We performadditional molecular docking simulations to demonstrate thatmajority of these compounds have low binding energies and thus high binding affinity with the potential to be effective against the SARS-COV-2 virus.

Availability: All the source code and data is available at: https://github.com/raghvendra5688/Drug-Repurposing and https://dx.doi.org/10.17632/8rrwnbcgmx.3. We also implemented a web-server at: https://machinelearning-protein.qcri.org/index.html.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163000PMC
February 2021

Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies.

PLoS One 2020 6;15(11):e0241773. Epub 2020 Nov 6.

Neurological Disorder Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar.

Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241773PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647061PMC
January 2021

Probing the fibrillation of lysozyme by nanoscale-infrared spectroscopy.

J Biomol Struct Dyn 2021 Mar 10;39(4):1481-1490. Epub 2020 Mar 10.

Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Doha, Qatar.

Amyloid fibrillation is the root cause of several neuro as well as non-neurological disorders. Understanding the molecular basis of amyloid aggregate formation is crucial for deciphering various neurodegenerative diseases. In our study, we have examined the lysozyme fibrillation process using nano-infrared spectroscopy (nanoIR). NanoIR enabled us to investigate both structural and chemical characteristics of lysozyme fibrillar species concurrently. The spectroscopic results indicate that lysozyme transformed into a fibrillar structure having mainly parallel β-sheets, with almost no antiparallel β-sheets. Features such as protein stiffness have a good correlation with obtained secondary structural information showing the state of the protein within the fibrillation state. The structural and chemical details were compared with transmission electron microscopy (TEM) and circular dichroism (CD). We have utilized nanoIR and measured infrared spectra to characterize lysozyme amyloid fibril structures in terms of morphology, molecular structure, secondary structure content, stability, and size of the cross-β core. We have shown that the use of nanoIR can complement other biophysical studies to analyze the aggregation process and is particularly useful for studying proteins involved in aggregation to help in designing molecules against amyloid aggregation. Specifically, the nanoIR spectra afford higher resolution information and a characteristic fingerprint for determining states of aggregation.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2020.1734091DOI Listing
March 2021

Disruption of PHF21A causes syndromic intellectual disability with craniofacial anomalies, epilepsy, hypotonia, and neurobehavioral problems including autism.

Mol Autism 2019 22;10:35. Epub 2019 Oct 22.

21Department of Biology, Chungnam National University, Daejeon, Korea.

Background: has been associated with intellectual disability and craniofacial anomalies based on its deletion in the Potocki-Shaffer syndrome region at 11p11.2 and its disruption in three patients with balanced translocations. In addition, three patients with de novo truncating mutations in were reported recently. Here, we analyze genomic data from seven unrelated individuals with mutations in and provide detailed clinical descriptions, further expanding the phenotype associated with PHF21A haploinsufficiency.

Methods: Diagnostic trio whole exome sequencing, Sanger sequencing, use of GeneMatcher, targeted gene panel sequencing, and MiSeq sequencing techniques were used to identify and confirm variants. RT-qPCR was used to measure the normal expression pattern of in multiple human tissues including 13 different brain tissues. Protein-DNA modeling was performed to substantiate the pathogenicity of the missense mutation.

Results: We have identified seven heterozygous coding mutations, among which six are de novo (not maternal in one). Mutations include four frameshifts, one nonsense mutation in two patients, and one heterozygous missense mutation in the AT Hook domain, predicted to be deleterious and likely to cause loss of PHF21A function. We also found a new C-terminal domain composed of an intrinsically disordered region. This domain is truncated in six patients and thus likely to play an important role in the function of PHF21A, suggesting that haploinsufficiency is the likely underlying mechanism in the phenotype of seven patients. Our results extend the phenotypic spectrum of mutations by adding autism spectrum disorder, epilepsy, hypotonia, and neurobehavioral problems. Furthermore, is highly expressed in the human fetal brain, which is consistent with the neurodevelopmental phenotype.

Conclusion: Deleterious nonsense, frameshift, and missense mutations disrupting the AT Hook domain and/or an intrinsically disordered region in PHF21A were found to be associated with autism spectrum disorder, epilepsy, hypotonia, neurobehavioral problems, tapering fingers, clinodactyly, and syndactyly, in addition to intellectual disability and craniofacial anomalies. This suggests that is involved in autism spectrum disorder and intellectual disability, and its haploinsufficiency causes a diverse neurological phenotype.
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http://dx.doi.org/10.1186/s13229-019-0286-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805429PMC
June 2020

BCrystal: an interpretable sequence-based protein crystallization predictor.

Bioinformatics 2020 03;36(5):1429-1438

Data Analytics, Qatar Computing Research Institute, Hamad Bin Khalifa University, Doha 34110, Qatar.

Motivation: X-ray crystallography has facilitated the majority of protein structures determined to date. Sequence-based predictors that can accurately estimate protein crystallization propensities would be highly beneficial to overcome the high expenditure, large attrition rate, and to reduce the trial-and-error settings required for crystallization.

Results: In this study, we present a novel model, BCrystal, which uses an optimized gradient boosting machine (XGBoost) on sequence, structural and physio-chemical features extracted from the proteins of interest. BCrystal also provides explanations, highlighting the most important features for the predicted crystallization propensity of an individual protein using the SHAP algorithm. On three independent test sets, BCrystal outperforms state-of-the-art sequence-based methods by more than 12.5% in accuracy, 18% in recall and 0.253 in Matthew's correlation coefficient, with an average accuracy of 93.7%, recall of 96.63% and Matthew's correlation coefficient of 0.868. For relative solvent accessibility of exposed residues, we observed higher values to associate positively with protein crystallizability and the number of disordered regions, fraction of coils and tripeptide stretches that contain multiple histidines associate negatively with crystallizability. The higher accuracy of BCrystal enables it to accurately screen for sequence variants with enhanced crystallizability.

Availability And Implementation: Our BCrystal webserver is at https://machinelearning-protein.qcri.org/ and source code is available at https://github.com/raghvendra5688/BCrystal.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523644PMC
March 2020

Investigation of the Effect of PD-L1 Blockade on Triple Negative Breast Cancer Cells Using Fourier Transform Infrared Spectroscopy.

Vaccines (Basel) 2019 Sep 9;7(3). Epub 2019 Sep 9.

Cancer Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), P.O. Box 34110 Doha, Qatar.

Interactions between programmed death-1 (PD-1) with its ligand PD-L1 on tumor cells can antagonize T cell responses. Inhibiting these interactions using immune checkpoint inhibitors has shown promise in cancer immunotherapy. MDA-MB-231 is a triple negative breast cancer cell line that expresses PD-L1. In this study, we investigated the biochemical changes in MDA-MB-231 cells following treatment with atezolizumab, a specific PD-L1 blocker. Our readouts were Fourier Transform Infrared (FTIR) spectroscopy and flow cytometric analyses. Chemometrical analysis, such as principal component analysis (PCA), was applied to delineate the spectral differences. We were able to identify the chemical alterations in both protein and lipid structure of the treated cells. We found that there was a shift from random coil and α-helical structure to β-sheet conformation of PD-L1 on tumor cells due to atezolizumab treatment, which could hinder binding with its receptors on immune cells, ensuring sustained T cell activation for potent immune responses. This work provides novel information about the effects of atezolizumab at molecular and cellular levels. FTIR bio-spectroscopy, in combination with chemometric analyses, may expedite research and offer new approaches for cancer immunology.
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http://dx.doi.org/10.3390/vaccines7030109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789440PMC
September 2019

Sox2: A Regulatory Factor in Tumorigenesis and Metastasis.

Curr Protein Pept Sci 2019 ;20(6):495-504

Diabetes Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), PO Box 34110, Doha, Qatar.

The transcription factor Sox2 plays an important role in various phases of embryonic development, including cell fate and differentiation. These key regulatory functions are facilitated by binding to specific DNA sequences in combination with partner proteins to exert their effects. Recently, overexpression and gene amplification of Sox2 has been associated with tumor aggression and metastasis in various cancer types, including breast, prostate, lung, ovarian and colon cancer. All the different roles for Sox2 involve complicated regulatory networks consisting of protein-protein and protein-nucleic acid interactions. Their involvement in the EMT modulation is possibly enabled by Wnt/ β-catenin and other signaling pathways. There are number of in vivo models which show Sox2 association with increased cancer aggressiveness, resistance to chemo-radiation therapy and decreased survival rate suggesting Sox2 as a therapeutic target. This review will focus on the different roles for Sox2 in metastasis and tumorigenesis. We will also review the mechanism of action underlying the cooperative Sox2- DNA/partner factors binding where Sox2 can be potentially explored for a therapeutic opportunity to treat cancers.
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http://dx.doi.org/10.2174/1389203720666190325102255DOI Listing
August 2019

DeepCrystal: a deep learning framework for sequence-based protein crystallization prediction.

Bioinformatics 2019 07;35(13):2216-2225

College of Science and Engineering, Hamad Bin Khalifa University, Doha, Qatar.

Motivation: Protein structure determination has primarily been performed using X-ray crystallography. To overcome the expensive cost, high attrition rate and series of trial-and-error settings, many in-silico methods have been developed to predict crystallization propensities of proteins based on their sequences. However, the majority of these methods build their predictors by extracting features from protein sequences, which is computationally expensive and can explode the feature space. We propose DeepCrystal, a deep learning framework for sequence-based protein crystallization prediction. It uses deep learning to identify proteins which can produce diffraction-quality crystals without the need to manually engineer additional biochemical and structural features from sequence. Our model is based on convolutional neural networks, which can exploit frequently occurring k-mers and sets of k-mers from the protein sequences to distinguish proteins that will result in diffraction-quality crystals from those that will not.

Results: Our model surpasses previous sequence-based protein crystallization predictors in terms of recall, F-score, accuracy and Matthew's correlation coefficient (MCC) on three independent test sets. DeepCrystal achieves an average improvement of 1.4, 12.1% in recall, when compared to its closest competitors, Crysalis II and Crysf, respectively. In addition, DeepCrystal attains an average improvement of 2.1, 6.0% for F-score, 1.9, 3.9% for accuracy and 3.8, 7.0% for MCC w.r.t. Crysalis II and Crysf on independent test sets.

Availability And Implementation: The standalone source code and models are available at https://github.com/elbasir/DeepCrystal and a web-server is also available at https://deeplearning-protein.qcri.org.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty953DOI Listing
July 2019

Identification of a Wells-Dawson polyoxometalate-based AP-2γ inhibitor with pro-apoptotic activity.

Biochem J 2018 06 11;475(11):1965-1977. Epub 2018 Jun 11.

Faculty of Health Sciences, University of Macau, Macau, China

AP-2 gamma (AP-2γ) is a transcription factor that plays pivotal roles in breast cancer biology. To search for small molecule inhibitors of AP-2γ, we performed a high-throughput fluorescence anisotropy screen and identified a polyoxometalate compound with Wells-Dawson structure K[PMoO] (Dawson-POM) that blocks the DNA-binding activity of AP-2γ. We showed that this blocking activity is due to the direct binding of Dawson-POM to AP-2γ. We also provided evidence to show that Dawson-POM decreases AP-2γ-dependent transcription similar to silencing the gene. Finally, we demonstrated that Dawson-POM contains anti-proliferative and pro-apoptotic effects in breast cancer cells. In summary, we identified the first small molecule inhibitor of AP-2γ and showed Dawson-POM-mediated inhibition of AP-2γ as a potential avenue for cancer therapy.
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http://dx.doi.org/10.1042/BCJ20170942DOI Listing
June 2018

An Integrative Developmental Genomics and Systems Biology Approach to Identify an In Vivo Sox Trio-Mediated Gene Regulatory Network in Murine Embryos.

Biomed Res Int 2017 28;2017:8932583. Epub 2017 May 28.

Department of Biology, Clarkson University, 8 Clarkson Avenue, Potsdam, NY 13699, USA.

Embryogenesis is an intricate process involving multiple genes and pathways. Some of the key transcription factors controlling specific cell types are the trio, namely, , , and , which play crucial roles in organogenesis working in a concerted manner. Much however still needs to be learned about their combinatorial roles during this process. A developmental genomics and systems biology approach offers to complement the reductionist methodology of current developmental biology and provide a more comprehensive and integrated view of the interrelationships of complex regulatory networks that occur during organogenesis. By combining cell type-specific transcriptome analysis and in vivo ChIP-Seq of the Sox trio using mouse embryos, we provide evidence for the direct control of and by the transcriptional trio in the murine model and by Morpholino knockdown in zebrafish and demonstrate the novel role of , , and in formation of , , and dependent tissues. Concurrently, a complete embryonic gene regulatory network has been generated, identifying a wide repertoire of genes involved and controlled by the Sox trio in the intricate process of normal embryogenesis.
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http://dx.doi.org/10.1155/2017/8932583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5467288PMC
March 2018

Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

FASEB J 2016 06 16;30(6):2411-25. Epub 2016 Mar 16.

Department of Biological Sciences, National University of Singapore, Singapore;

Trocarin D (TroD), a venom prothrombin activator from Tropidechis carinatus, shares similar structure and function with blood coagulation factor Xa [Tropidechis carinatus FX (TrFX) a]. Their distinct physiologic roles are due to their distinct expression patterns. The genes of TroD and TrFX are highly similar, except for promoter and intron 1, indicating that TroD has probably evolved by duplication of FX, the plasma counterpart. The promoter insertion in TroD accounts for the elevated but not venom gland-specific expression. Here we examined the roles of 3 insertions and 2 deletions in intron 1 of TroD in the regulation of expression using luciferase as a reporter. By systematic deletions, we showed that a 209 bp region within the second insertion silences expression in mammalian and unmilked venom gland cells. Through bioinformatics analysis, we identified 5 AG-rich motifs in this region. All except the 5th motif are important for silencing function. YY1, Sp3 and HMGB2 were identified to bind these AG-rich motifs and silence gene expression in mammalian cells. Similar AG-rich motif clusters are also found in other toxin genes but not in their physiologic counterparts. Thus, AG-rich motifs contribute to regulation of expression of TroD, and probably other toxin genes.-Han, S. X., Kwong, S., Ge, R., Kolatkar, P. R., Woods, A. E., Blanchet, G., Kini, R. M. Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.
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http://dx.doi.org/10.1096/fj.201600213RDOI Listing
June 2016

Structure and decoy-mediated inhibition of the SOX18/Prox1-DNA interaction.

Nucleic Acids Res 2016 05 2;44(8):3922-35. Epub 2016 Mar 2.

Genome Regulation Laboratory, Drug Discovery Pipeline, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

The transcription factor (TF) SOX18 drives lymphatic vessel development in both embryogenesis and tumour-induced neo-lymphangiogenesis. Genetic disruption of Sox18 in a mouse model protects from tumour metastasis and established the SOX18 protein as a molecular target. Here, we report the crystal structure of the SOX18 DNA binding high-mobility group (HMG) box bound to a DNA element regulating Prox1 transcription. The crystals diffracted to 1.75Å presenting the highest resolution structure of a SOX/DNA complex presently available revealing water structure, structural adjustments at the DNA contact interface and non-canonical conformations of the DNA backbone. To explore alternatives to challenging small molecule approaches for targeting the DNA-binding activity of SOX18, we designed a set of five decoys based on modified Prox1-DNA. Four decoys potently inhibited DNA binding of SOX18 in vitro and did not interact with non-SOX TFs. Serum stability, nuclease resistance and thermal denaturation assays demonstrated that a decoy circularized with a hexaethylene glycol linker and terminal phosphorothioate modifications is most stable. This SOX decoy also interfered with the expression of a luciferase reporter under control of a SOX18-dependent VCAM1 promoter in COS7 cells. Collectively, we propose SOX decoys as potential strategy for inhibiting SOX18 activity to disrupt tumour-induced neo-lymphangiogenesis.
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http://dx.doi.org/10.1093/nar/gkw130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856986PMC
May 2016

Crystallization and X-ray diffraction analysis of the HMG domain of the chondrogenesis master regulator Sox9 in complex with a ChIP-Seq-identified DNA element.

Acta Crystallogr F Struct Biol Commun 2015 Nov 30;71(Pt 11):1437-41. Epub 2015 Oct 30.

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

Sox9 is a fundamental sex-determining gene and the master regulator of chondrogenesis, and is involved in the development of various vital organs such as testes, kidney, heart and brain, and in skeletal development. Similar to other known Sox transcription factors, Sox9 recognizes and binds DNA with the consensus sequence C(T/A)TTG(T/A)(T/A) through the highly conserved HMG domain. Nonetheless, the molecular basis of the functional specificity of Sox9 in key developmental processes is still unclear. As an initial step towards a mechanistic understanding of Sox9 transcriptional regulation, the current work describes the details of the purification of the mouse Sox9 HMG domain (mSox9HMG), its crystallization in complex with a ChIP-Seq-identified FOXP2 promoter DNA element and the X-ray diffraction data analysis of this complex. The mSox9HMG-FOXP2 promoter DNA complex was crystallized by the hanging-drop vapour-diffusion method using 20% PEG 3350 in 200 mM sodium/potassium phosphate with 100 mM bis-tris propane at pH 8.5. The crystals diffracted to 2.7 Å resolution and the complex crystallized in the tetragonal space group P41212, with unit-cell parameters a = b = 99.49, c = 45.89 Å. Crystal-packing parameters revealed that asymmetric unit contained one mSox9HMG-FOXP2 promoter DNA complex with an estimated solvent content of 64%.
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http://dx.doi.org/10.1107/S2053230X1501969XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631595PMC
November 2015

Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

Acta Crystallogr F Struct Biol Commun 2014 Oct 25;70(Pt 10):1357-61. Epub 2014 Sep 25.

Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore.

Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188079PMC
http://dx.doi.org/10.1107/S2053230X14017415DOI Listing
October 2014

The SOX transcription factors as key players in pluripotent stem cells.

Stem Cells Dev 2014 Nov 26;23(22):2687-99. Epub 2014 Sep 26.

Qatar Biomedical Research Institute , Qatar Foundation, Education City, Doha, Qatar .

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs), and induced PSCs (iPSCs) are able to self-renew and differentiate into a multitude of specialized cellular lineages. In these cells, the pluripotential identity is maintained by a group of transcription factors (TFs). Among these factors, SOX TFs play an essential role, not only in regulating pluripotency but also in mediating self-renewal and differentiation. Some SOX TFs are highly expressed in undifferentiated PSCs, while others are upregulated upon differentiation to promote specific lineage differentiation. Further roles of SOX factors in pluripotency are highlighted through their critical involvement in iPSCs generation. To perform these multiple functions and activities, SOX TFs are strongly associated with a complex regulatory network(s) that involves the binding of SOX factors to variant trans-acting partners to activate or suppress specific genes. Although, SOX2 has attracted special attention as a critical factor in maintaining PSCs characteristics and as an integral component that is required to reprogram somatic cells into pluripotency, new reports widely appreciated that other SOX TFs, such as SOX1, SOX3, or reengineered SOX7 and SOX17, can compensate for the absence of SOX2 and thus play a fundamental role during the reprogramming process and maintaining pluripotency. These findings indicate that the recent progress has greatly expanded our knowledge about the role of SOX factors in PSCs. Thus, in this review we summarize what is currently known about the roles of SOX factors in PSCs and their role in somatic cell reprogramming. Also, we intend to provide an update on their relationship with other factors in regulating the characteristics and early differentiation of PSCs.
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http://dx.doi.org/10.1089/scd.2014.0297DOI Listing
November 2014

Structure of liganded T-state haemoglobin from cat (Felis silvestris catus), a low oxygen-affinity species, in two different crystal forms.

Acta Crystallogr D Biol Crystallogr 2014 Jul 29;70(Pt 7):1898-906. Epub 2014 Jun 29.

Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India.

Haemoglobin (Hb) is an iron-containing metalloprotein which plays a major role in the transportation of oxygen from the lungs to tissues and of carbon dioxide back to the lungs. Hb is in equilibrium between low-affinity tense (T) and high-affinity relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express `high' or `low' oxygen-affinity Hbs. Although Hbs from the former group have been studied extensively, a more limited number of structural studies have been performed for low oxygen-affinity Hbs. Here, the crystal structure of low oxygen-affinity cat methaemoglobin (metHb) has been solved at 2.0 and 2.4 Å resolution in two different crystal forms. Even though both structures are fully liganded, they unusually adopt a T-state-like quaternary conformation but with several localized R-like tertiary-structural and quaternary-structural features. The study provides atomic-level insights into the ligand-binding properties of this Hb, including its low cooperativity, blunt response to allosteric effectors and low affinity for oxygen, as well as further contributing to the mechanism underlying Hb allostery.
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http://dx.doi.org/10.1107/S139900471400916XDOI Listing
July 2014

Sox transcription factors require selective interactions with Oct4 and specific transactivation functions to mediate reprogramming.

Stem Cells 2013 Dec;31(12):2632-46

Stem Cell and Developmental biology, Genome Institute of Singapore, Singapore, Singapore.

The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs, but Sox4, Sox5, Sox6, Sox8, Sox9, Sox11, Sox12, Sox13, and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover, the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming, albeit at low efficiency. By molecular dissection of the C-terminus of Sox17, we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK, we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2.
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http://dx.doi.org/10.1002/stem.1522DOI Listing
December 2013

HIV-1 Tat protein induces viral internalization through Env-mediated interactions in dose-dependent manner.

AIDS 2013 Sep;27(15):2355-64

aDepartment of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institutet, Stockholm, Sweden bLaboratory of Structural Biochemistry, Genome Institute of Singapore, Singapore cDepartment of Molecular and Cellular Biology, University of California, Davis, California, USA.

Objective: To study the dose-dependent manner of HIV-1 Tat-induced effects on viral replication, internalization and spread, and to directly observe these effects on soluble Env immunogens and virus-like particles.

Design: In order to determine the manner through which Tat affects viral replication, we incubated cells, virions and soluble Env spikes with Tat at different concentrations, and directly visualized the effects of such incubation.

Methods: Cell-based infectivity assays were carried out to assay Tat dose-dependency of viral infectivity. Transmission electron microscopy of virus-like particles and soluble gp140 immunogens incubated with Tat at various concentrations was performed to directly observe Tat-induced effects.

Results: Treating virus with exogenous Tat increased infectivity in a dose-dependent manner. In the presence of anti-Tat antibodies, virus replication and spread were repressed, postulating Tat contributions to disease progression. When CXCR4 coreceptors were blocked, Tat treatment overcame the inhibition relative to absence of Tat treatment. Similarly, syncytium formation between chronically infected and uninfected target cells was also increased by exogenous Tat treatment. Inhibiting the CD4 receptor for virus entry abolished syncytium formation and Tat treatment was unable to overcome CD4 dependency. We show that Tat reduces virus infectivity at higher Tat concentrations through Env interactions resulting in viral aggregation.

Conclusion: Treating virions or chronically infected cells with exogenous Tat could enhance virus infectivity and spread through coreceptor tropism switch or through another undetermined mechanism. The aggregation potential of Tat suggests a mechanism of negative-feedback regulation of viral replication, providing another regulative function to control viral replication.
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http://dx.doi.org/10.1097/01.aids.0000432452.83604.59DOI Listing
September 2013

Putative role of Tat-Env interaction in HIV infection.

AIDS 2013 Sep;27(15):2345-54

aDivision of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden bDepartment of Molecular and Cellular Biology, University of California, Davis, California cNovartis Vaccines and Diagnostics Inc., Cambridge, Massachusetts, USA dLaboratory of Structural Biochemistry, Genome Institute of Singapore, Singapore, Singapore eProtein Sciences Corporation, Meriden, Connecticut, USA.

Objective: To study the complex formed between Tat protein and Env soluble trimeric immunogen, and compare with previously determined structures of Env native trimers and Env-CD4m complexes.

Design: The soluble Env trimer was used to mimic the spike glycoprotein on the virus surface for the study. To overcome limitations of other structural determination methods, cryoelectron microscopy was employed to image the complex, and single particle reconstruction was utilized to reconstruct the structure of the complex from collected micrographs. Molecular modeling of gp120-Tat was performed to provide atomic coordinates for docking.

Methods: Images were preprocessed by multivariate statistical analysis to identify principal components of variation then submitted for reconstruction. Reconstructed structures were docked with modeled gp120-Tat atomic coordinates to study the positions of crucial epitopes.

Results: Analysis of the Env-Tat complex demonstrated an intermediate structure between Env native trimers and Env-CD4m structures. Docking results indicate that the CD4-binding site and the V3 loop are exposed in the Env-Tat complex. The integrin-binding sequence in Tat was also exposed in Env-Tat docking.

Conclusion: The intermediate structure induced by Tat-interaction with Env could potentially provide an explanation for increased virus infection in the presence of Tat protein. Consequently, exposure of CD4-binding sites and a putative integrin-binding sequence on Tat in the complex may provide a new avenue for rational design of an effective HIV vaccine.
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http://dx.doi.org/10.1097/01.aids.0000432453.60733.b2DOI Listing
September 2013

TherMos: Estimating protein-DNA binding energies from in vivo binding profiles.

Nucleic Acids Res 2013 Jun 16;41(11):5555-68. Epub 2013 Apr 16.

Computational and Systems Biology, Genome Institute of Singapore, 60 Biopolis St, Singapore 138672, Singapore.

Accurately characterizing transcription factor (TF)-DNA affinity is a central goal of regulatory genomics. Although thermodynamics provides the most natural language for describing the continuous range of TF-DNA affinity, traditional motif discovery algorithms focus instead on classification paradigms that aim to discriminate 'bound' and 'unbound' sequences. Moreover, these algorithms do not directly model the distribution of tags in ChIP-seq data. Here, we present a new algorithm named Thermodynamic Modeling of ChIP-seq (TherMos), which directly estimates a position-specific binding energy matrix (PSEM) from ChIP-seq/exo tag profiles. In cross-validation tests on seven genome-wide TF-DNA binding profiles, one of which we generated via ChIP-seq on a complex developing tissue, TherMos predicted quantitative TF-DNA binding with greater accuracy than five well-known algorithms. We experimentally validated TherMos binding energy models for Klf4 and Esrrb, using a novel protocol to measure PSEMs in vitro. Strikingly, our measurements revealed strong non-additivity at multiple positions within the two PSEMs. Among the algorithms tested, only TherMos was able to model the entire binding energy landscape of Klf4 and Esrrb. Our study reveals new insights into the energetics of TF-DNA binding in vivo and provides an accurate first-principles approach to binding energy inference from ChIP-seq and ChIP-exo data.
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http://dx.doi.org/10.1093/nar/gkt250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675472PMC
June 2013

Oct4 switches partnering from Sox2 to Sox17 to reinterpret the enhancer code and specify endoderm.

EMBO J 2013 Apr 8;32(7):938-53. Epub 2013 Mar 8.

Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore.

How regulatory information is encoded in the genome is poorly understood and poses a challenge when studying biological processes. We demonstrate here that genomic redistribution of Oct4 by alternative partnering with Sox2 and Sox17 is a fundamental regulatory event of endodermal specification. We show that Sox17 partners with Oct4 and binds to a unique 'compressed' Sox/Oct motif that earmarks endodermal genes. This is in contrast to the pluripotent state where Oct4 selectively partners with Sox2 at 'canonical' binding sites. The distinct selection of binding sites by alternative Sox/Oct partnering is underscored by our demonstration that rationally point-mutated Sox17 partners with Oct4 on pluripotency genes earmarked by the canonical Sox/Oct motif. In an endodermal differentiation assay, we demonstrate that the compressed motif is required for proper expression of endodermal genes. Evidently, Oct4 drives alternative developmental programs by switching Sox partners that affects enhancer selection, leading to either an endodermal or pluripotent cell fate. This work provides insights in understanding cell fate transcriptional regulation by highlighting the direct link between the DNA sequence of an enhancer and a developmental outcome.
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http://dx.doi.org/10.1038/emboj.2013.31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616284PMC
April 2013

Cloning, purification and preliminary X-ray data analysis of the human ID2 homodimer.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Nov 30;68(Pt 11):1354-8. Epub 2012 Oct 30.

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

The ID proteins are named for their role as inhibitors of DNA binding and differentiation. They contain a helix-loop-helix (HLH) domain but lack a basic DNA-binding domain. In complex with basic HLH (bHLH) transcription factors, gene expression is regulated by DNA-binding inactivation. Although the HLH domain is highly conserved and shares a similar topology, the IDs preferentially bind class I bHLH-group members such as E47 (TCF3) but not the class III bHLH member Myc. A structure of an ID protein could potentially shed light on its mechanism. Owing to their short half-lives in vivo and reported in vitro instability, this paper describes the strategies that went into expressing sufficient soluble and stable ID2 to finally obtain diffraction-quality crystals. A 2.1 Å resolution data set was collected from a crystal belonging to space group P3(1)21 with unit-cell parameters a=b=51.622, c=111.474 Å, α=β=90, γ=120° that was obtained by hanging-drop vapour diffusion in a precipitant solution consisting of 0.1 M MES pH 6.5, 2.0 M potassium acetate. The solvent content was consistent with the presence of one or two molecules in the asymmetric unit.
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http://dx.doi.org/10.1107/S174430911203895XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515380PMC
November 2012

A divalent ion is crucial in the structure and dominant-negative function of ID proteins, a class of helix-loop-helix transcription regulators.

PLoS One 2012 30;7(10):e48591. Epub 2012 Oct 30.

Laboratory for Structural Biochemistry, Genome Institute of Singapore, Singapore, Singapore.

Inhibitors of DNA binding and differentiation (ID) proteins, a dominant-negative group of helix-loop-helix (HLH) transcription regulators, are well-characterized key players in cellular fate determination during development in mammals as well as Drosophila. Although not oncogenes themselves, their upregulation by various oncogenic proteins (such as Ras, Myc) and their inhibitory effects on cell cycle proteins (such as pRb) hint at their possible roles in tumorigenesis. Furthermore, their potency as inhibitors of cellular differentiation, through their heterodimerization with subsequent inactivation of the ubiquitous E proteins, suggest possible novel roles in engineering induced pluripotent stem cells (iPSCs). We present the high-resolution 2.1Å crystal structure of ID2 (HLH domain), coupled with novel biochemical insights in the presence of a divalent ion, possibly calcium (Ca2+), in the loop of ID proteins, which appear to be crucial for the structure and activity of ID proteins. These new insights will pave the way for new rational drug designs, in addition to current synthetic peptide options, against this potent player in tumorigenesis as well as more efficient ways for stem cells reprogramming.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048591PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484135PMC
May 2013

Structural analysis and dimerization profile of the SCAN domain of the pluripotency factor Zfp206.

Nucleic Acids Res 2012 Sep 26;40(17):8721-32. Epub 2012 Jun 26.

Laboratory for Structural Biochemistry, Stem Cell and Developmental Biology, Genome Institute of Singapore, Genome, 60 Biopolis Street, Singapore.

Zfp206 (also named as Zscan10) belongs to the subfamily of C(2)H(2) zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 Å crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C(2)H(2) transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development.
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http://dx.doi.org/10.1093/nar/gks611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458555PMC
September 2012

Crystal optimization and preliminary diffraction data analysis of the SCAN domain of Zfp206.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Apr 27;68(Pt 4):443-7. Epub 2012 Mar 27.

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

Zfp206 (also named Zscan10) is a transcription factor that plays an important role in maintaining the pluripotent state of embryonic stem cells. Zfp206 is a member of the SCAN-domain family of C(2)H(2) zinc-finger transcription factors. The SCAN domain is a highly conserved motif of 84 residues which mediates the self-association of and heterodimerization between SCAN-domain family transcription factors. The SCAN domain may therefore be the key to the selective oligomerization of and may combinatorially enhance the regulatory versatility of C(2)H(2) zinc fingers. This paper describes crystallization attempts with the SCAN domain of Zfp206 (Zfp206SCAN) and optimization strategies to obtain diffraction-quality crystals. The best diffracting crystal was grown in a solution consisting of 0.3 M ammonium sulfate, 0.1 M Tris-HCl pH 8.6, 25% PEG 3350, 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) using the hanging-drop vapour-diffusion technique. Optimized crystals diffracted to 1.85 Å resolution and belonged to space group I422, with unit-cell parameters a = 67.57, c = 87.54 Å. A Matthews analysis indicated the presence of one Zfp206SCAN molecule per asymmetric unit.
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http://dx.doi.org/10.1107/S1744309112006070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325816PMC
April 2012

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

Nucleic Acids Res 2012 Jun 16;40(11):4933-41. Epub 2012 Feb 16.

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

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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http://dx.doi.org/10.1093/nar/gks153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367189PMC
June 2012

The crystal structure of the Sox4 HMG domain-DNA complex suggests a mechanism for positional interdependence in DNA recognition.

Biochem J 2012 Apr;443(1):39-47

Laboratory for Structural Biochemistry, Genome Institute of Singapore, 60 Biopolis St, Singapore 138672.

It has recently been proposed that the sequence preferences of DNA-binding TFs (transcription factors) can be well described by models that include the positional interdependence of the nucleotides of the target sites. Such binding models allow for multiple motifs to be invoked, such as principal and secondary motifs differing at two or more nucleotide positions. However, the structural mechanisms underlying the accommodation of such variant motifs by TFs remain elusive. In the present study we examine the crystal structure of the HMG (high-mobility group) domain of Sox4 [Sry (sex-determining region on the Y chromosome)-related HMG box 4] bound to DNA. By comparing this structure with previously solved structures of Sox17 and Sox2, we observed subtle conformational differences at the DNA-binding interface. Furthermore, using quantitative electrophoretic mobility-shift assays we validated the positional interdependence of two nucleotides and the presence of a secondary Sox motif in the affinity landscape of Sox4. These results suggest that a concerted rearrangement of two interface amino acids enables Sox4 to accommodate primary and secondary motifs. The structural adaptations lead to altered dinucleotide preferences that mutually reinforce each other. These analyses underline the complexity of the DNA recognition by TFs and provide an experimental validation for the conceptual framework of positional interdependence and secondary binding motifs.
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http://dx.doi.org/10.1042/BJ20111768DOI Listing
April 2012

Structural basis for the cooperative DNA recognition by Smad4 MH1 dimers.

Nucleic Acids Res 2011 Oct 30;39(18):8213-22. Epub 2011 Jun 30.

Laboratory for Structural Biochemistry, Genome Institute of Singapore, Singapore-138672.

Smad proteins form multimeric complexes consisting of the 'common partner' Smad4 and receptor regulated R-Smads on clustered DNA binding sites. Deciphering how pathway specific Smad complexes multimerize on DNA to regulate gene expression is critical for a better understanding of the cis-regulatory logic of TGF-β and BMP signaling. To this end, we solved the crystal structure of the dimeric Smad4 MH1 domain bound to a palindromic Smad binding element. Surprisingly, the Smad4 MH1 forms a constitutive dimer on the SBE DNA without exhibiting any direct protein-protein interactions suggesting a DNA mediated indirect readout mechanism. However, the R-Smads Smad1, Smad2 and Smad3 homodimerize with substantially decreased efficiency despite pronounced structural similarities to Smad4. Therefore, intricate variations in the DNA structure induced by different Smads and/or variant energetic profiles likely contribute to their propensity to dimerize on DNA. Indeed, competitive binding assays revealed that the Smad4/R-Smad heterodimers predominate under equilibrium conditions while R-Smad homodimers are least favored. Together, we present the structural basis for DNA recognition by Smad4 and demonstrate that Smad4 constitutively homo- and heterodimerizes on DNA in contrast to its R-Smad partner proteins by a mechanism independent of direct protein contacts.
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http://dx.doi.org/10.1093/nar/gkr500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185416PMC
October 2011

Conversion of Sox17 into a pluripotency reprogramming factor by reengineering its association with Oct4 on DNA.

Stem Cells 2011 Jun;29(6):940-51

Laboratory for Structural Biochemistry and Genome Institute of Singapore, Singapore, Singapore.

Very few proteins are capable to induce pluripotent stem (iPS) cells and their biochemical uniqueness remains unexplained. For example, Sox2 cooperates with other transcription factors to generate iPS cells, but Sox17, despite binding to similar DNA sequences, cannot. Here, we show that Sox2 and Sox17 exhibit inverse heterodimerization preferences with Oct4 on the canonical versus a newly identified compressed sox/oct motif. We can swap the cooperativity profiles of Sox2 and Sox17 by exchanging single amino acids at the Oct4 interaction interface resulting in Sox2KE and Sox17EK proteins. The reengineered Sox17EK now promotes reprogramming of somatic cells to iPS, whereas Sox2KE has lost this potential. Consistently, when Sox2KE is overexpressed in embryonic stem cells it forces endoderm differentiation similar to wild-type Sox17. Together, we demonstrate that strategic point mutations that facilitate Sox/Oct4 dimer formation on variant DNA motifs lead to a dramatic swap of the bioactivities of Sox2 and Sox17.
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http://dx.doi.org/10.1002/stem.639DOI Listing
June 2011
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