ABSTRACT
Rabenosyn (RBSN) is a conserved endosomal protein necessary for regulating internalized cargo. Here, we present clinical, genetic, cellular and biochemical evidence that two distinct RBSN missense variants are responsible for a novel Mendelian disorder consisting of progressive muscle weakness, facial dysmorphisms, ophthalmoplegia and intellectual disability. Using exome sequencing, we identified recessively acting germline alleles p.Arg180Gly and p.Gly183Arg, which are both situated in the FYVE domain of RBSN. We find that these variants abrogate binding to its cognate substrate phosphatidylinositol 3-phosphate (PI3P) and thus prevent its translocation to early endosomes. Although the endosomal recycling pathway was unaltered, mutant p.Gly183Arg patient fibroblasts show accumulation of cargo tagged for lysosomal degradation. Our results suggest that these variants are separation-of-function alleles, which cause a delay in endosomal maturation without affecting cargo recycling. We conclude that distinct germline mutations in RBSN cause non-overlapping phenotypes with specific and discrete endolysosomal cellular defects.
Subject(s)
Endosomes , Intellectual Disability , Vesicular Transport Proteins , Humans , Alleles , Endosomes/genetics , Endosomes/metabolism , Intellectual Disability/genetics , Lysosomes/genetics , Lysosomes/metabolism , Mutation , Protein Transport/genetics , Vesicular Transport Proteins/geneticsABSTRACT
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.
Subject(s)
Cell Transdifferentiation/genetics , Cellular Reprogramming/genetics , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/metabolism , POU Domain Factors/chemistry , POU Domain Factors/metabolism , Protein Multimerization , Amino Acid Substitution , Animals , Binding Sites , Cell Line , Embryonic Stem Cells , Enhancer Elements, Genetic , Epigenesis, Genetic , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mice , Models, Molecular , Nucleotide Motifs , Octamer Transcription Factors/chemistry , Octamer Transcription Factors/genetics , Octamer Transcription Factors/metabolism , POU Domain Factors/genetics , Promoter Regions, Genetic , Protein Binding , Protein Conformation , Protein Stability , TranscriptomeABSTRACT
mRNA decay is an essential and active process that allows cells to continuously adapt gene expression to internal and environmental cues. There are two mRNA degradation pathways: 3' to 5' and 5' to 3'. The DCPS protein is the scavenger mRNA decapping enzyme which functions in the last step of the 3' end mRNA decay pathway. We have identified a DCPS pathogenic mutation in a large family with three affected individuals presenting with a novel recessive syndrome consisting of craniofacial anomalies, intellectual disability and neuromuscular defects. Using patient's primary cells, we show that this homozygous splice mutation results in a DCPS loss-of-function allele. Diagnostic biochemical analyses using various m7G cap derivatives as substrates reveal no DCPS enzymatic activity in patient's cells. Our results implicate DCPS and more generally RNA catabolism, as a critical cellular process for neurological development, normal cognition and organismal homeostasis in humans.
Subject(s)
Abnormalities, Multiple/genetics , Endoribonucleases/genetics , Intellectual Disability/genetics , Muscle Hypotonia/genetics , Cells, Cultured , Child , Child, Preschool , Consanguinity , DNA Mutational Analysis , Endoribonucleases/deficiency , Genetic Association Studies , Humans , Male , Pedigree , RNA Splice Sites , SyndromeABSTRACT
In pluripotent cells, OCT4 associates with SOX2 to maintain pluripotency or with SOX17 to induce primitive endoderm commitment. The OCT4-SOX2 and OCT4-SOX17 combinations bind mutually exclusive to two distinct composite DNA elements, known as the "canonical" and "compressed" motifs, respectively. The structural basis for the OCT4-SOX17 cooperativity is unknown. Whereas SOX17 has been engineered to replace SOX2 in the pluripotency circuitry, all generated SOX2 mutants have failed to act like SOX17. From molecular simulations, we revealed the OCT4-SOX17 interaction interface and elucidated the SOX-dependent motif preference of OCT4. Moreover, we designed a SOX2 mutant that we predicted and confirmed experimentally to bind cooperatively with OCT4 to the compressed motif. Ultimately, we found a strong correlation between the experimental and calculated relative cooperative-binding free energies of 12 OCT4-SOX-DNA complexes. Therefore, we validated the OCT4-SOX interfaces and demonstrated that in silico design of DNA-binding cooperativity is suitable for altering transcriptional circuitries.
Subject(s)
HMGB Proteins/chemistry , Octamer Transcription Factor-3/chemistry , SOXB1 Transcription Factors/chemistry , SOXF Transcription Factors/chemistry , Stem Cells/physiology , Amino Acid Sequence , Animals , Base Sequence , Cell Differentiation , Consensus Sequence , DNA/chemistry , HMGB Proteins/genetics , Hydrophobic and Hydrophilic Interactions , Mice , Molecular Dynamics Simulation , Molecular Sequence Data , Octamer Transcription Factor-3/genetics , Protein Binding , Protein Interaction Domains and Motifs , SOXB1 Transcription Factors/genetics , SOXF Transcription Factors/genetics , ThermodynamicsABSTRACT
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 ß-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.
Subject(s)
Induced Pluripotent Stem Cells/physiology , Octamer Transcription Factor-3/metabolism , SOXF Transcription Factors/metabolism , Alkaline Phosphatase/metabolism , Animals , Cell Culture Techniques , Cellular Reprogramming/genetics , Cellular Reprogramming/physiology , HMGB Proteins/genetics , HMGB Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Mice , Mice, SCID , Octamer Transcription Factor-3/genetics , Point Mutation , SOXF Transcription Factors/genetics , Transcriptional Activation , beta Catenin/metabolismABSTRACT
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.
Subject(s)
Algorithms , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Animals , Chromatin Immunoprecipitation , High-Throughput Nucleotide Sequencing , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Mice , Mutation , Protein Binding , Receptors, Estrogen/metabolism , Sequence Analysis, DNA , ThermodynamicsABSTRACT
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.
Subject(s)
Embryo, Mammalian/embryology , Endoderm/embryology , Enhancer Elements, Genetic/physiology , Gene Expression Regulation, Developmental/physiology , HMGB Proteins/metabolism , Octamer Transcription Factor-3/metabolism , SOXB1 Transcription Factors/metabolism , SOXF Transcription Factors/metabolism , Amino Acid Motifs , Animals , Binding Sites , Cell Differentiation/physiology , Cell Line , Embryo, Mammalian/cytology , Endoderm/cytology , HMGB Proteins/genetics , Mice , Octamer Transcription Factor-3/genetics , SOXB1 Transcription Factors/genetics , SOXF Transcription Factors/genetics , Transcription, Genetic/physiologyABSTRACT
Terminally differentiated cells can be reprogrammed to pluripotency by the forced expression of Oct4, Sox2, Klf4 and c-Myc. However, it remains unknown how this leads to the multitude of epigenetic changes observed during the reprogramming process. Interestingly, Oct4 is the only factor that cannot be replaced by other members of the same family to induce pluripotency. To understand the unique role of Oct4 in reprogramming, we determined the structure of its POU domain bound to DNA. We show that the linker between the two DNA-binding domains is structured as an α-helix and exposed to the protein's surface, in contrast to the unstructured linker of Oct1. Point mutations in this α-helix alter or abolish the reprogramming activity of Oct4, but do not affect its other fundamental properties. On the basis of mass spectrometry studies of the interactome of wild-type and mutant Oct4, we propose that the linker functions as a protein-protein interaction interface and plays a crucial role during reprogramming by recruiting key epigenetic players to Oct4 target genes. Thus, we provide molecular insights to explain how Oct4 contributes to the reprogramming process.
Subject(s)
Cell Differentiation , Cellular Reprogramming , DNA/metabolism , Embryonic Stem Cells/cytology , Fibroblasts/cytology , Octamer Transcription Factor-3/chemistry , Pluripotent Stem Cells/cytology , Amino Acid Sequence , Animals , Blotting, Western , Cells, Cultured , Crystallography, X-Ray , DNA/genetics , Electrophoretic Mobility Shift Assay , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Fibroblasts/metabolism , Humans , Kruppel-Like Factor 4 , Luciferases/metabolism , Mice , Molecular Sequence Data , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Transcription factors (TF) often bind in heterodimeric complexes with each TF recognizing a specific neighboring cis element in the regulatory region of the genome. Comprehension of this DNA motif grammar is opaque, yet recent developments have allowed the interrogation of genome-wide TF binding sites. We reasoned that within this data novel motif grammars could be identified that controlled distinct biological programs. For this purpose, we developed a novel motif-discovery tool termed fexcom that systematically interrogates ChIP-seq data to discover spatially constrained TF-TF composite motifs occurring over short DNA distances. We applied this to the extensive ChIP-seq data available from mouse embryonic stem cells (ESCs). In addition to the well-known and most prevalent sox-oct motif, we also discovered a novel constrained spacer motif for Esrrb and Sox2 with a gap of between 2 and 8 bps that Essrb and Sox2 cobind in a selective fashion. Through the use of knockdown experiments, we argue that the Esrrb-Sox2 complex is an arbiter of gene expression differences between ESCs and epiblast stem cells (EpiSC). A number of genes downregulated upon dual Esrrb/Sox2 knockdown (e.g., Klf4, Klf5, Jam2, Pecam1) are similarly downregulated in the ESC to EpiSC transition and contain the esrrb-sox motif. The prototypical Esrrb-Sox2 target gene, containing an esrrb-sox element conserved throughout eutherian and metatherian mammals, is Nr0b1. Through positive regulation of this transcriptional repressor, we argue the Esrrb-Sox2 complex promotes the ESC state through inhibition of the EpiSC transcriptional program and the same trio may also function to maintain trophoblast stem cells.
Subject(s)
DNA/metabolism , Embryonic Stem Cells/metabolism , Germ Layers/metabolism , Receptors, Estrogen/metabolism , SOXB1 Transcription Factors/metabolism , Transcription, Genetic , Algorithms , Animals , Base Sequence , Chromatin Immunoprecipitation , DAX-1 Orphan Nuclear Receptor/genetics , DAX-1 Orphan Nuclear Receptor/metabolism , DNA/genetics , Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , Germ Layers/cytology , Germ Layers/growth & development , Kruppel-Like Factor 4 , Mice , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Receptors, Estrogen/genetics , SOXB1 Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Octamer Transcription Factor-3/metabolism , SOX Transcription Factors/chemistry , SOX Transcription Factors/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , DNA/metabolism , Dimerization , Mice , Molecular Sequence Data , Mutation , Protein Binding , Protein Interaction Domains and Motifs , SOX Transcription Factors/genetics , Sequence AlignmentABSTRACT
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.
Subject(s)
DNA/chemistry , Macromolecular Substances/chemistry , SOXC Transcription Factors/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Conserved Sequence , Crystallography, X-Ray , Electrophoretic Mobility Shift Assay , Enhancer Elements, Genetic , HMGB Proteins/chemistry , Laminin/genetics , Mice , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , SOXF Transcription Factors/chemistryABSTRACT
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.
Subject(s)
DNA/metabolism , Embryonic Stem Cells/metabolism , HMGB Proteins/genetics , Octamer Transcription Factor-3/metabolism , SOXB1 Transcription Factors/genetics , SOXF Transcription Factors/genetics , Amino Acid Motifs/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Differentiation , Computer Simulation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/cytology , Endoderm/cytology , Endoderm/metabolism , HMGB Proteins/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding/genetics , Protein Engineering , Protein Multimerization , SOXB1 Transcription Factors/metabolism , SOXF Transcription Factors/metabolism , Sequence AlignmentSubject(s)
DNA/chemistry , Inverted Repeat Sequences , Octamer Transcription Factor-6/chemistry , Recombinant Proteins/chemistry , Amino Acid Sequence , Animals , Crystallography, X-Ray , Mice , Molecular Sequence Data , Octamer Transcription Factor-6/biosynthesis , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/biosynthesisABSTRACT
The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 angstrom resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 angstrom, alpha = beta = gamma = 90 degrees. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit.
Subject(s)
DNA , Inverted Repeat Sequences , Smad1 Protein/chemistry , Animals , Base Sequence , Bone Morphogenetic Proteins/metabolism , Crystallography, X-Ray , DNA/genetics , DNA/metabolism , Mice , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Smad1 Protein/genetics , Smad1 Protein/metabolism , X-Ray DiffractionABSTRACT
Specific wiring of gene-regulatory networks is likely to underlie much of the phenotypic difference between species, but the extent of lineage-specific regulatory architecture remains poorly understood. The essential vertebrate transcriptional repressor REST (RE1-Silencing Transcription Factor) targets many neural genes during development of the preimplantation embryo and the central nervous system, through its cognate DNA motif, the RE1 (Repressor Element 1). Here we present a comparative genomic analysis of REST recruitment in multiple species by integrating both sequence and experimental data. We use an accurate, experimentally validated Position-Specific Scoring Matrix method to identify REST binding sites in multiply aligned vertebrate genomes, allowing us to infer the evolutionary origin of each of 1,298 human RE1 elements. We validate these findings using experimental data of REST binding across the whole genomes of human and mouse. We show that one-third of human RE1s are unique to primates: These sites recruit REST in vivo, target neural genes, and are under purifying evolutionary selection. We observe a consistent and significant trend for more ancient RE1s to have higher affinity for REST than lineage-specific sites and to be more proximal to target genes. Our results lead us to propose a model where new transcription factor binding sites are constantly generated throughout the genome; thereafter, refinement of their sequence and location consolidates this remodeling of networks governing neural gene regulation.
Subject(s)
DNA-Binding Proteins/genetics , Gene Regulatory Networks , Nerve Tissue Proteins/genetics , Repressor Proteins/genetics , Vertebrates/genetics , Animals , Base Sequence , Co-Repressor Proteins , Genome , Genome, Human , Humans , Molecular Sequence Data , Protein Binding , Sequence AlignmentABSTRACT
Sox17 regulates endodermal lineage commitment and is thought to function antagonistically to the pluripotency determinant Sox2. To investigate the biochemical basis for the distinct functions of Sox2 and Sox17, we solved the crystal structure of the high mobility group domain of Sox17 bound to a DNA element derived from the Lama1 enhancer using crystals diffracting to 2.7 A resolution. Sox17 targets the minor groove and bends the DNA by approximately 80 degrees . The DNA architecture closely resembles the one seen for Sox2/DNA structures, suggesting that the degree of bending is conserved between both proteins and nucleotide substitutions have only marginal effects on the bending topology. Accordingly, affinities of Sox2 and Sox17 for the Lama1 element were found to be identical. However, when the Oct1 contact interface of Sox2 is compared with the corresponding region of Sox17, a significantly altered charge distribution is observed, suggesting differential co-factor recruitment that may explain their biological distinctiveness.
Subject(s)
HMGB Proteins/chemistry , SOXF Transcription Factors/chemistry , Amino Acid Sequence , Circular Dichroism , Crystallography, X-Ray , DNA/metabolism , Electrophoretic Mobility Shift Assay , Laminin/genetics , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Sox17 is a member of the SRY-related high-mobility group (HMG) of transcription factors that have been shown to direct endodermal differentiation in early mammalian development. The LAMA1 gene encoding the alpha-chain of laminin-1 has been reported to be directly bound and regulated by Sox17. This paper describes the details of initial crystallization attempts with the HMG domain of mouse Sox17 (mSox17-HMG) with a 16-mer DNA element derived from the LAMA1 enhancer and optimization strategies to obtain a better diffracting crystal. The best diffracting crystal was obtained in a condition containing 0.1 M Tris-HCl pH 7.4, 0.2 M MgCl(2), 30% PEG 3350 using the hanging-drop vapour-diffusion method. A highly redundant in-house data set was collected to 2.75 A resolution with 99% completeness. The presence of the mSox17-HMG-DNA complex within the crystals was confirmed and Matthews analysis indicated the presence of one complex per asymmetric unit.
Subject(s)
DNA/chemistry , HMGB Proteins/chemistry , SOXF Transcription Factors/chemistry , Animals , Base Sequence , Cloning, Molecular , Crystallization , Crystallography, X-Ray , DNA/metabolism , HMGB Proteins/isolation & purification , HMGB Proteins/metabolism , Mice , Molecular Sequence Data , Protein Structure, Tertiary , SOXF Transcription Factors/isolation & purification , SOXF Transcription Factors/metabolism , X-Ray DiffractionABSTRACT
The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.