RESUMO
The consolidation of unambiguous cell fate commitment relies on the ability of transcription factors (TFs) to exert tissue-specific regulation of complex genetic networks. However, the mechanisms by which TFs establish such precise control over gene expression have remained elusive-especially in instances in which a single TF operates in two or more discrete cellular systems. In this study, we demonstrate that ß cell-specific functions of NKX2.2 are driven by the highly conserved NK2-specific domain (SD). Mutation of the endogenous NKX2.2 SD prevents the developmental progression of ß cell precursors into mature, insulin-expressing ß cells, resulting in overt neonatal diabetes. Within the adult ß cell, the SD stimulates ß cell performance through the activation and repression of a subset of NKX2.2-regulated transcripts critical for ß cell function. These irregularities in ß cell gene expression may be mediated via SD-contingent interactions with components of chromatin remodelers and the nuclear pore complex. However, in stark contrast to these pancreatic phenotypes, the SD is entirely dispensable for the development of NKX2.2-dependent cell types within the CNS. Together, these results reveal a previously undetermined mechanism through which NKX2.2 directs disparate transcriptional programs in the pancreas versus neuroepithelium.
Assuntos
Proteínas de Homeodomínio , Células Secretoras de Insulina , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteína Homeobox Nkx-2.2 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Diferenciação Celular , Proteínas de Peixe-Zebra/genéticaRESUMO
Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.
Assuntos
Processamento Alternativo , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/metabolismo , Proteínas Repressoras/metabolismo , Animais , DNA/metabolismo , Células-Tronco Embrionárias/citologia , Genes Homeobox , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Isoformas de Proteínas/metabolismoRESUMO
Transcription factor over-expression is a proven method for reprogramming cells to a desired cell type for regenerative medicine and therapeutic discovery. However, a general method for the identification of reprogramming factors to create an arbitrary cell type is an open problem. Here we examine the success rate of methods and data for differentiation by testing the ability of nine computational methods (CellNet, GarNet, EBseq, AME, DREME, HOMER, KMAC, diffTF and DeepAccess) to discover and rank candidate factors for eight target cell types with known reprogramming solutions. We compare methods that use gene expression, biological networks and chromatin accessibility data, and comprehensively test parameter and preprocessing of input data to optimize performance. We find the best factor identification methods can identify an average of 50-60% of reprogramming factors within the top ten candidates, and methods that use chromatin accessibility perform the best. Among the chromatin accessibility methods, complex methods DeepAccess and diffTF have higher correlation with the ranked significance of transcription factor candidates within reprogramming protocols for differentiation. We provide evidence that AME and diffTF are optimal methods for transcription factor recovery that will allow for systematic prioritization of transcription factor candidates to aid in the design of new reprogramming protocols.
Assuntos
Reprogramação Celular , Cromatina , Diferenciação Celular/genética , Reprogramação Celular/genética , Cromatina/genética , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether the pace of sequential activation of HOX genes, the 'HOX clock', is controlled by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cell-derived axial progenitors differentiating into diverse spinal cord motor neuron subtypes. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking the FGF pathway stalled induction of HOX genes, while a precocious increase of FGF, alone or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The pacing of the HOX clock is thus dynamically regulated by exposure to secreted cues. Its manipulation by extrinsic factors provides synchronized access to multiple human neuronal subtypes of distinct rostro-caudal identities for basic and translational applications.This article has an associated 'The people behind the papers' interview.
Assuntos
Relógios Circadianos , Proteínas de Homeodomínio/metabolismo , Neurônios Motores/metabolismo , Células-Tronco Pluripotentes/metabolismo , Benzamidas/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular , Relógios Circadianos/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/farmacologia , Proteínas de Homeodomínio/genética , Humanos , Neurônios Motores/citologia , Células-Tronco Pluripotentes/citologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Although Hox genes encode for conserved transcription factors (TFs), they are further divided into anterior, central and posterior groups based on their DNA-binding domain similarity. The posterior Hox group expanded in the deuterostome clade and patterns caudal and distal structures. We aimed to address how similar Hox TFs diverge to induce different positional identities. We studied Hox TF DNA-binding and regulatory activity during an in vitro motor neuron differentiation system that recapitulates embryonic development. We found diversity in the genomic binding profiles of different Hox TFs, even among the posterior group paralogs that share similar DNA-binding domains. These differences in genomic binding were explained by differing abilities to bind to previously inaccessible sites. For example, the posterior group HOXC9 had a greater ability to bind occluded sites than the posterior HOXC10, producing different binding patterns and driving differential gene expression programs. From these results, we propose that the differential abilities of posterior Hox TFs to bind to previously inaccessible chromatin drive patterning diversification.This article has an associated 'The people behind the papers' interview.
Assuntos
Diferenciação Celular , Cromatina/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Neurônios Motores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Proteínas de Homeodomínio/genética , Camundongos , Neurônios Motores/citologia , Fatores de Transcrição/genéticaRESUMO
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a method for the genome-wide de novo discovery of chromatin interactions. Existing computational methods typically fail to detect weak or dynamic interactions because they use a peak-calling step that ignores paired-end linkage information. We have developed a novel computational method called Chromatin Interaction Discovery (CID) to overcome this limitation with an unbiased clustering approach for interaction discovery. CID outperforms existing chromatin interaction detection methods with improved sensitivity, replicate consistency, and concordance with other chromatin interaction datasets. In addition, CID also outperforms other methods in discovering chromatin interactions from HiChIP data. We expect that the CID method will be valuable in characterizing 3D chromatin interactions and in understanding the functional consequences of disease-associated distal genetic variations.
Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/química , Cromatina/metabolismo , Biologia Computacional/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Conjuntos de Dados como Assunto , Etiquetas de Sequências Expressas , Humanos , Ligação ProteicaRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1A4V). Moreover, we found that the administration of TUDCA in an hSOD1G93A mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.
Assuntos
Neurônios Motores/citologia , Células-Tronco/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Humanos , Indóis/farmacologia , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Mutação , Células-Tronco/efeitos dos fármacos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Ácido Tauroquenodesoxicólico/farmacologiaRESUMO
Giant axonal neuropathy (GAN) is a progressive neurodegenerative disease caused by autosomal recessive mutations in the GAN gene resulting in a loss of a ubiquitously expressed protein, gigaxonin. Gene replacement therapy is a promising strategy for treatment of the disease; however, the effectiveness and safety of gigaxonin reintroduction have not been tested in human GAN nerve cells. Here we report the derivation of induced pluripotent stem cells (iPSCs) from three GAN patients with different GAN mutations. Motor neurons differentiated from GAN iPSCs exhibit accumulation of neurofilament (NF-L) and peripherin (PRPH) protein and formation of PRPH aggregates, the key pathological phenotypes observed in patients. Introduction of gigaxonin either using a lentiviral vector or as a stable transgene resulted in normalization of NEFL and PRPH levels in GAN neurons and disappearance of PRPH aggregates. Importantly, overexpression of gigaxonin had no adverse effect on survival of GAN neurons, supporting the feasibility of gene replacement therapy. Our findings demonstrate that GAN iPSCs provide a novel model for studying human GAN neuropathologies and for the development and testing of new therapies in relevant cell types.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Neuropatia Axonal Gigante/genética , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Filamentos Intermediários/genética , Neurônios Motores/metabolismo , Axônios , Diferenciação Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Terapia Genética/métodos , Vetores Genéticos/genética , Neuropatia Axonal Gigante/terapia , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Cariotipagem , Lentivirus/genética , Neurônios Motores/citologia , Mutação , FenótipoRESUMO
Regulatory proteins can bind to different sets of genomic targets in various cell types or conditions. To reliably characterize such condition-specific regulatory binding we introduce MultiGPS, an integrated machine learning approach for the analysis of multiple related ChIP-seq experiments. MultiGPS is based on a generalized Expectation Maximization framework that shares information across multiple experiments for binding event discovery. We demonstrate that our framework enables the simultaneous modeling of sparse condition-specific binding changes, sequence dependence, and replicate-specific noise sources. MultiGPS encourages consistency in reported binding event locations across multiple-condition ChIP-seq datasets and provides accurate estimation of ChIP enrichment levels at each event. MultiGPS's multi-experiment modeling approach thus provides a reliable platform for detecting differential binding enrichment across experimental conditions. We demonstrate the advantages of MultiGPS with an analysis of Cdx2 binding in three distinct developmental contexts. By accurately characterizing condition-specific Cdx2 binding, MultiGPS enables novel insight into the mechanistic basis of Cdx2 site selectivity. Specifically, the condition-specific Cdx2 sites characterized by MultiGPS are highly associated with pre-existing genomic context, suggesting that such sites are pre-determined by cell-specific regulatory architecture. However, MultiGPS-defined condition-independent sites are not predicted by pre-existing regulatory signals, suggesting that Cdx2 can bind to a subset of locations regardless of genomic environment. A summary of this paper appears in the proceedings of the RECOMB 2014 conference, April 2-5.
Assuntos
Biologia Computacional/métodos , Proteínas de Homeodomínio/química , Motivos de Aminoácidos , Animais , Inteligência Artificial , Teorema de Bayes , Sítios de Ligação , Fator de Transcrição CDX2 , Linhagem Celular , Imunoprecipitação da Cromatina , Análise por Conglomerados , Células-Tronco Embrionárias/citologia , Genoma , Proteínas de Homeodomínio/metabolismo , Camundongos , Ligação Proteica , Análise de Sequência de DNARESUMO
Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1, and column-specific markers that mirror those observed in vivo in human embryonic spinal cord. They also exhibited spontaneous and induced activity, and projected axons toward muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1(+)/LHX3(-)). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays.
Assuntos
Extremidades/inervação , Neurônios Motores/fisiologia , Células-Tronco Neurais/fisiologia , Animais , Axônios/fisiologia , Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Galinha , DNA Complementar/biossíntese , DNA Complementar/genética , Feminino , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Proteínas com Homeodomínio LIM/genética , Masculino , Camundongos , Neurônios Motores/metabolismo , Células-Tronco Neurais/metabolismo , Técnicas de Patch-Clamp , Complexo de Inativação Induzido por RNA , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Medula Espinal/citologia , Medula Espinal/embriologia , Transplante de Células-Tronco/métodos , Fatores de Transcrição/genéticaRESUMO
The study of developmentally regulated transcription factors by chromatin immunoprecipitation and deep sequencing (ChIP-seq) faces two major obstacles: availability of ChIP-grade antibodies and access to sufficient number of cells. We describe versatile genome-wide analysis of transcription-factor binding sites by combining directed differentiation of embryonic stem cells and inducible expression of tagged proteins. We demonstrate its utility by mapping DNA-binding sites of transcription factors involved in motor neuron specification.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Análise de Sequência de DNARESUMO
Giant axonal neuropathy (GAN) is a rare pediatric neurodegenerative disease. It is best known for the "giant" axons caused by accumulations of intermediate filaments. The disease is progressive, with onset around age 3 years and death by the third decade of life. GAN results from recessive mutations in the GAN gene encoding gigaxonin, and our analysis of all reported mutations shows that they are distributed throughout the protein structure. Precisely how these mutations cause the disease remains to be determined. In addition to changes in peripheral nerves that are similar to those seen in neuropathies such as Charcot-Marie-Tooth type 2, GAN patients exhibit a wide range of central nervous system signs. These features, corroborated by degeneration of central tracts apparent from postmortem pathology, indicate that GAN is also a progressive neurodegenerative disease. To reflect this phenotype more precisely, we therefore propose that the disease should be more appropriately referred to as "giant axonal neurodegeneration."
Assuntos
Neuropatia Axonal Gigante/etiologia , Neuropatia Axonal Gigante/patologia , Animais , Proteínas do Citoesqueleto/metabolismo , Progressão da Doença , Neuropatia Axonal Gigante/genética , Neuropatia Axonal Gigante/fisiopatologia , Humanos , Filamentos Intermediários/patologia , Mutação/genéticaRESUMO
Aging is a major risk factor in amyotrophic lateral sclerosis (ALS) and other adult-onset neurodegenerative disorders. Whereas young neurons are capable of buffering disease-causing stresses, mature neurons lose this ability and degenerate over time. We hypothesized that the resilience of young motor neurons could be restored by re-expression of the embryonic motor neuron selector transcription factors ISL1 and LHX3. We found that viral re-expression of ISL1 and LHX3 reactivates aspects of the youthful gene expression program in mature motor neurons and alleviates key disease-relevant phenotypes in the SOD1G93A mouse model of ALS. Our results suggest that redeployment of lineage-specific neuronal selector transcription factors can be an effective strategy to attenuate age-dependent phenotypes in neurodegenerative disease.
RESUMO
Formation of functional motor circuits relies on the ability of distinct spinal motor neuron subtypes to project their axons with high precision to appropriate muscle targets. While guidance cues contributing to motor axon pathfinding have been identified, the intracellular pathways underlying subtype-specific responses to these cues remain poorly understood. In particular, it remains controversial whether responses to axon guidance cues depend on axonal protein synthesis. Using a growth cone collapse assay, we demonstrate that mouse embryonic stem cell-derived spinal motor neurons (ES-MNs) respond to ephrin-A5, Sema3f, and Sema3a in a concentration-dependent manner. At low doses, ES-MNs exhibit segmental or subtype-specific responses, while this selectivity is lost at higher concentrations. Response to high doses of semaphorins and to all doses of ephrin-A5 is protein synthesis independent. In contrast, using microfluidic devices and stripe assays, we show that growth cone collapse and guidance at low concentrations of semaphorins rely on local protein synthesis in the axonal compartment. Similar bimodal response to low and high concentrations of guidance cues is observed in human ES-MNs, pointing to a general mechanism by which neurons increase their repertoire of responses to the limited set of guidance cues involved in neural circuit formation.
Assuntos
Axônios/fisiologia , Sinais (Psicologia) , Neurônios Motores/fisiologia , Biossíntese de Proteínas/fisiologia , Animais , Axônios/metabolismo , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Efrina-A5/administração & dosagem , Efrina-A5/fisiologia , Cones de Crescimento/patologia , Cones de Crescimento/fisiologia , Humanos , Masculino , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/fisiologia , Camundongos , Neurônios Motores/classificação , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/fisiologia , Semaforina-3A , Transdução de Sinais/fisiologia , Medula Espinal/citologia , Medula Espinal/fisiologiaRESUMO
We present a method, LineageProgram, that uses the developmental lineage relationship of observed gene expression measurements to improve the learning of developmentally relevant cellular states and expression programs. We find that incorporating lineage information allows us to significantly improve both the predictive power and interpretability of expression programs that are derived from expression measurements from in vitro differentiation experiments. The lineage tree of a differentiation experiment is a tree graph whose nodes describe all of the unique expression states in the input expression measurements, and edges describe the experimental perturbations applied to cells. Our method, LineageProgram, is based on a log-linear model with parameters that reflect changes along the lineage tree. Regularization with L(1) that based methods controls the parameters in three distinct ways: the number of genes change between two cellular states, the number of unique cellular states, and the number of underlying factors responsible for changes in cell state. The model is estimated with proximal operators to quickly discover a small number of key cell states and gene sets. Comparisons with existing factorization, techniques, such as singular value decomposition and non-negative matrix factorization show that our method provides higher predictive power in held, out tests while inducing sparse and biologically relevant gene sets.
Assuntos
Algoritmos , Diferenciação Celular , Biologia Computacional/métodos , Animais , Modelos Lineares , Camundongos , Estudos de Validação como AssuntoRESUMO
Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/ß affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3α/ß signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog ( 4 ) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/ß. Finally, we used our chemical probe pair ( 2 and 4 ) to demonstrate that inhibition of CDKL5 and/or GSK3α/ß promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.
RESUMO
Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a compound that has advanced to phase II clinical trials and is a known inhibitor of several cyclin-dependent kinases (CDKs) and cyclin-dependent kinase-like kinases (CDKLs). We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/ß affinity. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/ß signaling and solved a co-crystal structure of analog 2 bound to human CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/ß, making it a suitable negative control. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3α/ß promotes the survival of human motor neurons exposed to endoplasmic reticulum stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.
Assuntos
Quinase 3 da Glicogênio Sintase , Transdução de Sinais , Humanos , Transdução de Sinais/fisiologia , Neurônios , Quinases Ciclina-Dependentes , Proteínas Serina-Treonina QuinasesRESUMO
Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, ELAVL3, and ELAVL4) can increase (i) 3'UTR length, (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR-mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense "self" dsRNAs to preemptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.
Assuntos
Infecção por Zika virus , Zika virus , Humanos , Regiões 3' não Traduzidas/genética , RNA de Cadeia Dupla , Doenças Neuroinflamatórias , Inflamação , Receptores de Reconhecimento de Padrão/genética , NeurôniosRESUMO
Neurons born in the embryo can undergo a protracted period of maturation lasting well into postnatal life. How gene expression changes are regulated during maturation and whether they can be recapitulated in cultured neurons remains poorly understood. Here, we show that mouse motor neurons exhibit pervasive changes in gene expression and accessibility of associated regulatory regions from embryonic till juvenile age. While motifs of selector transcription factors, ISL1 and LHX3, are enriched in nascent regulatory regions, motifs of NFI factors, activity-dependent factors, and hormone receptors become more prominent in maturation-dependent enhancers. Notably, stem cell-derived motor neurons recapitulate ~40% of the maturation expression program in vitro, with neural activity playing only a modest role as a late-stage modulator. Thus, the genetic maturation program consists of a core hardwired subprogram that is correctly executed in vitro and an extrinsically-controlled subprogram that is dependent on the in vivo context of the maturing organism.
Assuntos
Neurônios Motores , Neurogênese , Animais , Hormônios/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Neurônios Motores/metabolismo , Neurogênese/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Proper assembly and function of the nervous system requires the generation of a uniquely diverse population of neurons expressing a cell-type-specific combination of effector genes that collectively define neuronal morphology, connectivity, and function. How countless partially overlapping but cell-type-specific patterns of gene expression are controlled at the genomic level remains poorly understood. Here we show that neuronal genes are associated with highly complex gene regulatory systems composed of independent cell-type- and cell-stage-specific regulatory elements that reside in expanded non-coding genomic domains. Mapping enhancer-promoter interactions revealed that motor neuron enhancers are broadly distributed across the large chromatin domains. This distributed regulatory architecture is not a unique property of motor neurons but is employed throughout the nervous system. The number of regulatory elements increased dramatically during the transition from invertebrates to vertebrates, suggesting that acquisition of new enhancers might be a fundamental process underlying the evolutionary increase in cellular complexity.