Functional analysis of Scratch2 domains: implications in the evolution of Snail transcriptional repressors

The Snail superfamily of transcription factors have a modular organization and their similarities and divergences are the basis for subdividing the superfamily into the Snail1/2 and Scratch families. As it is generally accepted that the Snail and Scratch families originated through gene duplication, understanding the functional contribution of each module could provide us with further insight about the molecular and functional evolution of the Snail superfamily. Thus, in this work, we investigated the function of the SNAG and SCRATCH domains in chicken Scratch2. Through evolutionary comparison analysis we identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. Similar to members of the Snail1/2 families, Scratch2-mediated transcriptional repression requires SNAG and nuclear localization requires the zinc-finger domain. We also identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. HINGE is highly conserved in amniotes. Single mutations of the conserved Tyrosine and Serine residues of HINGE downregulated Scratch2-mediated transcriptional repression. This effect depended on the presence of the SCRATCH domain

Functional analysis of Scratch2 domains: implications in the evolution of Snail transcriptional repressors

The Snail superfamily of transcription factors have a modular organization and their similarities and divergences are the basis for subdividing the superfamily into the Snail1/2 and Scratch families. As it is generally accepted that the Snail and Scratch families originated through gene duplication, understanding the functional contribution of each module could provide us with further insight about the molecular and functional evolution of the Snail superfamily. Thus, in this work, we investigated the function of the SNAG and SCRATCH domains in chicken Scratch2. Through evolutionary comparison analysis we identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. Similar to members of the Snail1/2 families, Scratch2-mediated transcriptional repression requires SNAG and nuclear localization requires the zinc-finger domain. We also identified a novel HINGE domain that lies between the SNAG and SCRATCH domain. HINGE is highly conserved in amniotes. Single mutations of the conserved Tyrosine and Serine residues of HINGE downregulated Scratch2-mediated transcriptional repression. This effect depended on the presence of the SCRATCH domain

Gene expression study in neural cells derived from induced pluripotent stem cells of individuals with Autism Spectrum Disorder

O Transtorno do Espectro Autista (TEA) é um transtorno neuropsiquiátrico grave caracterizado por comprometimento da capacidade de interação social e comunicação e pela presença de interesses, atividades e comportamentos repetitivos e restritos. O TEA apresenta alta heterogeneidade genética e pode ser enquadrado em diferentes modelos de herança, o que torna difícil apontar os fatores genéticos associados ao transtorno e compreender de que forma eles interagem e ocasionam, em última instância, sua manifestação clínica. Ainda, fenômenos como penetrância incompleta e discordância entre gêmeos monozigóticos são frequentemente observados em famílias com indivíduos afetados, tornando ainda mais complexo o entendimento do envolvimento de cada um dos fatores genéticos na etiologia do TEA. Uma forma de contornar esses problemas é a utilização de abordagens transcriptômicas no estudo desse transtorno. Nesse contexto, a busca por variantes patogênicas deixa de ser o principal foco do estudo e a atenção volta-se para a investigação de vias de sinalização e processos biológicos que estejam desregulados durante o processo de diferenciação neuronal e que sejam compartilhados pelos indivíduos afetados. Usando como principal ferramenta o sequenciamento do transcriptoma de células progenitoras neurais (NPC) e neurônios derivados de células-tronco pluripotentes induzidas (iPSC), identificamos uma série de alterações transcricionais importantes entre pacientes e controles, sugerindo que, a despeito da grande heterogeneidade genética associada ao transtorno, essas alterações se refletem em processos biológicos comuns. Nossos resultados são consistentes com os obtidos em trabalhos anteriores realizados com outros modelos de estudo e as linhagens celulares geradas a partir de nosso protocolo parecem refletir de forma fidedigna o perfil de expressão do cérebro fetal. Ainda, em um caso particular incluído neste estudo, investigamos as consequências transcricionais e funcionais de uma duplicação na região 17p13.3 - alteração previamente implicada em outros casos de autismo - nas células neuronais deste paciente, contribuindo para a compreensão do papel desta duplicação na patofisiologia do TEA. Dessa forma, nosso trabalho reforça a validade do uso de células neurais derivadas de iPSC e de abordagens transcricionais para o estudo de transtornos do neurodesenvolvimentoAutism Spectrum Disorder (ASD) is a severe neuropsychiatry disorder characterized by impaired social interaction and communication, restricted interests and repetitive behaviors. ASD is highly genetically heterogeneous and can be caused by different inheritance models, which hampers the identification of the genetic factors associated to the disorder. Incomplete penetrance and discordance between monozygotic twins are often observed in families of affected individuals, making it even more complex to understand the involvement of genetic factors in ASD. Applying transcriptomic approaches to study this disorder is an interesting way to overcome these problems. In this context, searching for pathogenic variants is no longer the study\'s main focus. Instead, one aims to investigate signaling pathways and biological process that are deregulated during neuronal differentiation and shared by affected individuals. Based on the transcriptome sequencing of neural progenitor cells (NPC) and neurons derived from induced pluripotent stem cells (iPSC), we were able to identify a series of important transcriptional alterations between patients and controls, which suggest that despite the great genetic heterogeneity associated to ASD, those alterations are reflected in common biological processes. Our results are consistent with those obtained in previous studies performed with other models and the lineages generated by our protocol seem to reliably reflect fetal brain expression profile. Moreover, in a particular case included in this study, we investigated transcriptional and functional consequences of a duplication located at 17p13.3 - an alteration previously found in other autism cases - in the neuronal cells of this patient, contributing towards a better comprehension of the role of this duplication in ASD pathophysiology. Hence, our work reinforces the validity of the use of neural cell derived from iPSC and transcriptional approaches in the studies of neurodevelopmental disorder