ABSTRACT
Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/ultrastructure , DNA-Binding Proteins/genetics , Helix-Loop-Helix Motifs/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/ultrastructure , Amino Acid Sequence , Basic Helix-Loop-Helix Transcription Factors/chemistry , Binding Sites/genetics , DNA-Binding Proteins/chemistry , Homeodomain Proteins/chemistry , Humans , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/ultrastructure , Sequence Homology, Amino Acid , Signal Transduction/genetics , Transcription Factor HES-1ABSTRACT
The basic helix-loop-helix (bHLH) transcription factor TWIST1 is essential to embryonic development, and hijacking of its function contributes to the development of numerous cancer types. It forms either a homodimer or a heterodimeric complex with an E2A or HAND partner. These functionally distinct complexes display sometimes antagonistic functions during development, so that alterations in the balance between them lead to pronounced morphological alterations, as observed in mice and in Saethre-Chotzen syndrome patients. We, here, describe the structures of TWIST1 bHLH-DNA complexes produced in silico through molecular dynamics simulations. We highlight the determinant role of the interhelical loops in maintaining the TWIST1-DNA complex structures and provide a structural explanation for the loss of function associated with several TWIST1 mutations/insertions observed in Saethre-Chotzen syndrome patients. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:27.
Subject(s)
Helix-Loop-Helix Motifs , Nuclear Proteins/chemistry , Twist-Related Protein 1/chemistry , Acrocephalosyndactylia/genetics , Amino Acid Sequence , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/ultrastructure , Crystallography, X-Ray , DNA/chemistry , Humans , Mice , Molecular Dynamics Simulation , Mutation , MyoD Protein/chemistry , MyoD Protein/ultrastructure , Nuclear Proteins/genetics , Protein Binding/genetics , Protein Multimerization , Sequence Alignment , Transcription Factor 3/chemistry , Transcription Factor 3/ultrastructure , Twist-Related Protein 1/geneticsABSTRACT
The temporal and spatial patterning involved in the specification, differentiation, and myelination by oligodendroglia is coordinated in part by the activation and repression of various transcriptional programs. Olig2 is a basic helix-loop-helix transcription factor necessary for oligodendroglial development and expressed continuously throughout the lineage. Despite evidence for the critical role of Olig2 in oligodendroglial specification and differentiation, the function for Olig2 during later stages of oligodendroglial development, namely, the transition into mature oligodendrocytes (OLs) and the formation of the myelin sheath, remains unclear. To address the possibility for a stage-specific role, we deleted Olig2 in oligodendrocyte precursor cells (OPCs) under the control of the CNPase-promoter or in immature OLs under the inducible proteolipid protein promoter. As expected, ablation of Olig2 in OPCs significantly inhibits differentiation, resulting in hypomyelination. However, deletion of the Olig2 gene in immature OLs significantly enhances the maturation process and accelerates the kinetics of myelination/remyelination. Underlying the stage-specific roles for Olig2 is the compensatory expression and function of Olig1, a transcription factor that promotes OL maturation and (re)myelination. Olig1 expression is significantly reduced upon Olig2 deletion in OPCs but is dramatically increased by nearly threefold when deleted in immature OLs. By enforcing expression of Olig1 into OPCs in a null Olig2 background, we demonstrate that overexpression of Olig1 is sufficient to rescue the differentiation phenotype and partially compensates for the Olig2 deletion in vitro. Our results suggest a stage-specific regulatory role for Olig2, mediated by Olig1 that conveys opposing functions on the differentiation and maturation of oligodendrocytes.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/deficiency , Cell Differentiation/physiology , Nerve Tissue Proteins/deficiency , Oligodendroglia/physiology , 2',3'-Cyclic-Nucleotide Phosphodiesterases/genetics , 2',3'-Cyclic-Nucleotide Phosphodiesterases/metabolism , Animals , Animals, Newborn , Arabidopsis Proteins/metabolism , Autophagy-Related Proteins , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/ultrastructure , Bromodeoxyuridine/metabolism , Cell Differentiation/drug effects , Cuprizone/toxicity , Demyelinating Diseases/chemically induced , Demyelinating Diseases/metabolism , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Green Fluorescent Proteins/genetics , In Situ Nick-End Labeling , Intracellular Signaling Peptides and Proteins/metabolism , Intramolecular Transferases/metabolism , Male , Mice , Mice, Transgenic , Microscopy, Electron, Transmission , Monoamine Oxidase Inhibitors/toxicity , Myelin Basic Protein/metabolism , Myelin Proteolipid Protein/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/ultrastructure , Oligodendrocyte Transcription Factor 2 , Oligodendroglia/drug effects , Transfection , ran GTP-Binding Protein/metabolismABSTRACT
At the diplotene stage of meiotic prophase, the nucleus of mouse preovulatory oocytes contains multiple interchromatin granule clusters (IGC). These nuclear compartments are universal and evolutionary conserved and enriched in pre-mRNA splicing factors. Nowadays, IGCs are believed to play an important role in gene expression events and contain different molecular components that allow coupling of many processes from transcription to mRNA export. We obtained the data on the distributions of poly(A)+RNA, hnRNPS A/B, and NXF1/TAP factor of mRNA export. These factors were found to associate with IGCs of mouse preovulatory oocytes. In the present study, we have demonstrated for the first time the dynamics of large IGCs after specific phosphorilation of SR-proteins with okadaic acid, an inhibitor of protein phosphatases. Using electron microscopy, conventional fluorescent and confocal microscopies, as well as microinjections of olygonucleotide probes in mouse oocytes, some features of structural organization and molecular compositions of IGCs in the nuclei of mouse oocyte from antral follicles were established. Possible roles of IGCs in pre-mRNA metabolism and the participation of these structures in mRNA export are discussed.
