RESUMEN
BTB (Bric-a-brack, Tramtrack and Broad Complex) is a diverse group of protein-protein interaction domains found within metazoan proteins. Transcription factors contain a dimerizing BTB subtype with a characteristic N-terminal extension. The Tramtrack group (TTK) is a distinct type of BTB domain, which can multimerize. Single-particle cryo-EM microscopy revealed that the TTK-type BTB domains assemble into a hexameric structure consisting of three canonical BTB dimers connected through a previously uncharacterized interface. We demonstrated that the TTK-type BTB domains are found only in Arthropods and have undergone lineage-specific expansion in modern insects. The Drosophila genome encodes 24 transcription factors with TTK-type BTB domains, whereas only four have nonTTKtype BTB domains. Yeast two-hybrid analysis revealed that the TTK-type BTB domains have an unusually broad potential for heteromeric associations presumably through dimer-dimer interaction interface. Thus, the TTK-type BTB domains are a structurally and functionally distinct group of protein domains specific to Arthropodan transcription factors.
RESUMEN
Dermal papilla cells (DPCs) play roles in key functions of the epidermis such as hair generation. The use of human induced pluripotent cells (hiPSCs) makes it possible to obtain DP-like cells and study the molecular mechanisms of DPC development during embryogenesis. In this work, we studied the phenotypic trajectory of hiPSCs during their differentiation into DP-like cells and evaluated the epithelial-mesenchymal interaction potential of the resulting cell line. Specifically, we differentiated hiPSCs into neural progenitor cells (NPCs) and subsequently into DP-like cells. Analysis of bulk RNA-seq data during this process enabled us to observe gene expression dynamics during five stages of dermal differentiation. Furthermore, functional assays (organoids in both collagen gels and hanging drop cultures and tubulogenesis assays) revealed that the dermal cell lines we generated could interact with epidermal cells.
Asunto(s)
Células Epidérmicas , Células-Madre Neurales , Humanos , Diferenciación Celular , Organoides , BioensayoRESUMEN
Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. We found that a deletion of the 3'UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, we detected more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain. Importantly, deletion of the 3'UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3'UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Regiones no Traducidas 3'/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Memoria a Largo Plazo/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Multipotent mesenchymal stem/stromal cells (MSC) are one of the crucial regulators of regeneration and tissue repair and possess an intrinsic program from self-organization mediated by condensation, migration and self-patterning. The ability to self-organize has been successfully exploited in tissue engineering approaches using cell sheets (CS) and their modifications. In this study, we used CS as a model of human MSC spontaneous self-organization to demonstrate its structural, transcriptomic impact and multipotent stromal cell commitment. We used CS formation to visualize MSC self-organization and evaluated the role of the Rho-GTPase pathway in spontaneous condensation, resulting in a significant anisotropy of the cell density within the construct. Differentiation assays were carried out using conventional protocols, and microdissection and RNA-sequencing were applied to establish putative targets behind the observed phenomena. The differentiation of MSC to bone and cartilage, but not to adipocytes in CS, occurred more effectively than in the monolayer. RNA-sequencing indicated transcriptional shifts involving the activation of the Rho-GTPase pathway and repression of SREBP, which was concordant with the lack of adipogenesis in CS. Eventually, we used an inhibitory analysis to validate our findings and suggested a model where the self-organization of MSC defined their commitment and cell fate via ROCK1/2 and SREBP as major effectors under the putative switching control of AMP kinase.
RESUMEN
In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. Here, we show that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. We obtained a crystal structure of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. We speculate that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.