RESUMO
mSWI/SNF or BAF chromatin regulatory complexes are dosage-sensitive regulators of human neural development frequently mutated in autism spectrum disorders and intellectual disability. Cell cycle exit and differentiation of neural stem/progenitor cells is accompanied by BAF subunit switching to generate neuron-specific nBAF complexes. We manipulated the timing of BAF subunit exchange in vivo and found that early loss of the npBAF subunit BAF53a stalls the cell cycle to disrupt neurogenesis. Loss of BAF53a results in decreased chromatin accessibility at specific neural transcription factor binding sites, including the pioneer factors SOX2 and ASCL1, due to Polycomb accumulation. This results in repression of cell cycle genes, thereby blocking cell cycle progression and differentiation. Cell cycle block upon Baf53a deletion could be rescued by premature expression of the nBAF subunit BAF53b but not by other major drivers of proliferation or differentiation. WNT, EGF, bFGF, SOX2, c-MYC, or PAX6 all fail to maintain proliferation in the absence of BAF53a, highlighting a novel mechanism underlying neural progenitor cell cycle exit in the continued presence of extrinsic proliferative cues.
Assuntos
Actinas/metabolismo , Ciclo Celular/genética , Córtex Cerebelar/embriologia , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Actinas/genética , Animais , Sítios de Ligação/genética , Células Cultivadas , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Deleção de Genes , Genes cdc/genética , Camundongos , Neurogênese/genética , Proteínas do Grupo Polycomb/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Alternative splicing is a key step in eukaryotic gene expression that allows for the production of multiple transcript and protein isoforms from the same gene. Even though splicing is perturbed in many diseases, we currently lack insights into regulatory mechanisms promoting its precision and efficiency. We analyze high-throughput mutagenesis data obtained for an alternatively spliced exon in the proto-oncogene RON and determine the functional units that control this splicing event. Using mathematical modeling of distinct splicing mechanisms, we show that alternative splicing is based in RON on a so-called "exon definition" mechanism. Here, the recognition of the adjacent exons by the spliceosome is required for removal of an intron. We use our model to analyze the differences between the exon and intron definition scenarios and find that exon definition prevents the accumulation of deleterious, partially spliced retention products during alternative splicing regulation. Furthermore, it modularizes splicing control, as multiple regulatory inputs are integrated into a common net input, irrespective of the location and nature of the corresponding cis-regulatory elements in the pre-messenger RNA. Our analysis suggests that exon definition promotes robust and reliable splicing outcomes in RON splicing.
Assuntos
Processamento Alternativo , Proto-Oncogenes , Éxons/genética , Íntrons/genéticaRESUMO
Mechanisms controlling the proliferative activity of neural stem and progenitor cells (NSPCs) have a pivotal role to ensure life-long neurogenesis in the mammalian brain. How metabolic programs are coupled with NSPC activity remains unknown. Here we show that fatty acid synthase (Fasn), the key enzyme of de novo lipogenesis, is highly active in adult NSPCs and that conditional deletion of Fasn in mouse NSPCs impairs adult neurogenesis. The rate of de novo lipid synthesis and subsequent proliferation of NSPCs is regulated by Spot14, a gene previously implicated in lipid metabolism, that we found to be selectively expressed in low proliferating adult NSPCs. Spot14 reduces the availability of malonyl-CoA, which is an essential substrate for Fasn to fuel lipogenesis. Thus, we identify here a functional coupling between the regulation of lipid metabolism and adult NSPC proliferation.
Assuntos
Células-Tronco Adultas/metabolismo , Ácido Graxo Sintases/metabolismo , Lipogênese , Células-Tronco Neurais/metabolismo , Células-Tronco Adultas/citologia , Animais , Proliferação de Células , Giro Denteado/metabolismo , Ácido Graxo Sintases/deficiência , Ácido Graxo Sintases/genética , Perfilação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Malonil Coenzima A/metabolismo , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/citologia , Neurogênese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The dynamics of quantum phase transitions pose one of the most challenging problems in modern many-body physics. Here, we study a prototypical example in a clean and well-controlled ultracold atom setup by observing the emergence of coherence when crossing the Mott insulator to superfluid quantum phase transition. In the 1D Bose-Hubbard model, we find perfect agreement between experimental observations and numerical simulations for the resulting coherence length. We, thereby, perform a largely certified analog quantum simulation of this strongly correlated system reaching beyond the regime of free quasiparticles. Experimentally, we additionally explore the emergence of coherence in higher dimensions, where no classical simulations are available, as well as for negative temperatures. For intermediate quench velocities, we observe a power-law behavior of the coherence length, reminiscent of the Kibble-Zurek mechanism. However, we find nonuniversal exponents that cannot be captured by this mechanism or any other known model.
RESUMO
New neurons are generated throughout life in distinct regions of the mammalian brain. This process, called adult neurogenesis, has been implicated in physiological brain function, and failing or altered neurogenesis has been associated with a number of neuropsychiatric diseases. Here, we provide an overview of the mechanisms governing the neurogenic process in the adult brain and describe how new neurons may contribute to brain function in health and disease.
Assuntos
Células-Tronco Adultas/fisiologia , Encéfalo/crescimento & desenvolvimento , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Adulto , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Humanos , Camundongos , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologiaRESUMO
The 3' untranslated regions (3' UTRs) of transcripts serve as important hubs for posttranscriptional gene expression regulation. Here, we find that the exonisation of intergenic Alu elements introduced new terminal exons and polyadenylation sites during human genome evolution. While Alu exonisation from introns has been described previously, we shed light on a novel mechanism to create alternative 3' UTRs, thereby opening opportunities for differential posttranscriptional regulation. On the mechanistic level, we show that intergenic Alu exonisation can compete both with alternative splicing and polyadenylation in the upstream gene. Notably, the Alu-derived isoforms are often expressed in a tissue-specific manner, and the Alu-derived 3' UTRs can alter mRNA stability. In summary, we demonstrate that intergenic elements can affect processing of preceding genes, and elucidate how intergenic Alu exonisation can contribute to tissue-specific posttranscriptional regulation by expanding the repertoire of 3' UTRs.
Assuntos
Regiões 3' não Traduzidas , Elementos Alu , Evolução Molecular , Éxons , Linhagem Celular , DNA/metabolismo , DNA Intergênico/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Íntrons , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Poliadenilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA/metabolismo , Splicing de RNA , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AFRESUMO
Metabolism has emerged as a key regulator of stem cell behavior. Mitochondria are crucial metabolic organelles that are important for differentiated cells, yet considered less so for stem cells. However, recent studies have shown that mitochondria influence stem cell maintenance and fate decisions, inviting a revised look at this topic. In this review, we cover the current literature addressing the role of mitochondrial metabolism in mouse and human neural stem cells (NSCs) in the embryonic and adult brain. We summarize how mitochondria are implicated in fate regulation and how substrate oxidation affects NSC quiescence. We further explore single-cell RNA sequencing (scRNA-seq) data for metabolic signatures of adult NSCs, highlight emerging technologies reporting on metabolic signatures, and discuss mitochondrial metabolism in other stem cells.
Assuntos
Células-Tronco Adultas , Células-Tronco Neurais , Humanos , Camundongos , Animais , Células-Tronco Neurais/metabolismo , Diferenciação Celular/fisiologia , Mitocôndrias/metabolismo , Células-Tronco Adultas/metabolismo , OxirreduçãoRESUMO
Target deconvolution of small molecule hits from phenotypic screens presents a major challenge. Many screens have been conducted to find inhibitors for the Hedgehog signaling pathway - a developmental pathway with many implications in health and disease - yielding many hits but only few identified cellular targets. We here present a strategy for target identification based on Proteolysis-Targeting Chimeras (PROTACs), combined with label-free quantitative proteomics. We develop a PROTAC based on Hedgehog Pathway Inhibitor-1 (HPI-1), a phenotypic screen hit with unknown cellular target. Using this Hedgehog Pathway PROTAC (HPP) we identify and validate BET bromodomains as the cellular targets of HPI-1. Furthermore, we find that HPP-9 is a long-acting Hedgehog pathway inhibitor through prolonged BET bromodomain degradation. Collectively, we provide a powerful PROTAC-based approach for target deconvolution, that answers the longstanding question of the cellular target of HPI-1 and yields a PROTAC that acts on the Hedgehog pathway.
Assuntos
Antineoplásicos , Proteínas Hedgehog , Quimera de Direcionamento de Proteólise , Domínios Proteicos , ProteóliseRESUMO
We have experimentally studied few-body impurity systems consisting of a single fermionic atom and a small bosonic field on the sites of an optical lattice. Quantum phase revival spectroscopy has allowed us to accurately measure the absolute strength of Bose-Fermi interactions as a function of the interspecies scattering length. Furthermore, we observe the modification of Bose-Bose interactions that is induced by the interacting fermion. Because of an interference between Bose-Bose and Bose-Fermi phase dynamics, we can infer the mean fermionic filling of the mixture and quantify its increase (decrease) when the lattice is loaded with attractive (repulsive) interspecies interactions.
RESUMO
The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Repressão Epigenética/fisiologia , Regulação da Expressão Gênica/fisiologia , Complexos Multiproteicos/fisiologia , Proteínas do Grupo Polycomb/fisiologia , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Repressão Epigenética/genética , Edição de Genes , Regulação da Expressão Gênica/genética , Genes Homeobox , Genoma , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Mutação com Perda de Função , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.
Assuntos
Sítios de Ligação , Metilação de DNA , DNA/metabolismo , Ensaios de Triagem em Larga Escala , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Animais , Cromatina , Desmetilação do DNA , Replicação do DNA , Epigenômica , Expressão Gênica , Camundongos , Nucleossomos , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Células Sf9 , Fatores de Transcrição/metabolismoRESUMO
The human Immunodeficiency Centromeric Instability Facial Anomalies (ICF) 4 syndrome is a severe disease with increased mortality caused by mutation in the LSH gene. Although LSH belongs to a family of chromatin remodeling proteins, it remains unknown how LSH mediates its function on chromatin in vivo. Here, we use chemical-induced proximity to rapidly recruit LSH to an engineered locus and find that LSH specifically induces macroH2A1.2 and macroH2A2 deposition in an ATP-dependent manner. Tethering of LSH induces transcriptional repression and silencing is dependent on macroH2A deposition. Loss of LSH decreases macroH2A enrichment at repeat sequences and results in transcriptional reactivation. Likewise, reduction of macroH2A by siRNA interference mimicks transcriptional reactivation. ChIP-seq analysis confirmed that LSH is a major regulator of genome-wide macroH2A distribution. Tethering of ICF4 mutations fails to induce macroH2A deposition and ICF4 patient cells display reduced macroH2A deposition and transcriptional reactivation supporting a pathogenic role for altered marcoH2A deposition. We propose that LSH is a major chromatin modulator of the histone variant macroH2A and that its ability to insert marcoH2A into chromatin and transcriptionally silence is disturbed in the ICF4 syndrome.
Assuntos
DNA Helicases/metabolismo , Histonas/metabolismo , Doenças da Imunodeficiência Primária/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , DNA Helicases/genética , Regulação para Baixo , Feminino , Histonas/genética , Humanos , Masculino , Camundongos , Doenças da Imunodeficiência Primária/enzimologia , Doenças da Imunodeficiência Primária/genética , Transcrição GênicaRESUMO
Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.
Assuntos
Processamento Alternativo/genética , Mutagênese/genética , Neoplasias/genética , Receptores Proteína Tirosina Quinases/genética , Sequência de Bases , Sítios de Ligação , Éxons/genética , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Íntrons/genética , Modelos Lineares , Células MCF-7 , Mutação/genética , Proto-Oncogene Mas , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de RNARESUMO
Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Epigênese Genética , Edição de Genes , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Inativação Gênica , Células HEK293 , Heterocromatina/metabolismo , Humanos , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras GenéticasRESUMO
Trithorax-group proteins and their mammalian homologs, including those in BAF (mSWI/SNF) complexes, are known to oppose the activity of Polycomb repressive complexes (PRCs). This opposition underlies the tumor-suppressive role of BAF subunits and is expected to contribute to neurodevelopmental disorders. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease-associated mutations in BAF subunits induce genome-wide increases in PRC deposition and activity. We show that point mutations in SMARCA4 (also known as BRG1) mapping to the ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction is ATP dependent, consistent with a transient eviction mechanism. Using a new chemical-induced proximity assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, thereby establishing a new mechanism for the widespread BAF-PRC opposition underlying development and disease.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Cromatina/genética , DNA Helicases/genética , Proteínas Nucleares/genética , Mutação Puntual/genética , Proteínas do Grupo Polycomb/genética , Fatores de Transcrição/genética , Trifosfato de Adenosina/genética , Animais , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Camundongos , Transtornos do Neurodesenvolvimento/genéticaRESUMO
The opposition between Polycomb repressive complexes (PRCs) and BAF (mSWI/SNF) complexes has a critical role in both development and disease. Mutations in the genes encoding BAF subunits contribute to more than 20% of human malignancies, yet the underlying mechanisms remain unclear, owing largely to a lack of assays to assess BAF function in living cells. To address this, we have developed a widely applicable recruitment assay system through which we find that BAF opposes PRC by rapid, ATP-dependent eviction, leading to the formation of accessible chromatin. The reversal of this process results in reassembly of facultative heterochromatin. Surprisingly, BAF-mediated PRC eviction occurs in the absence of RNA polymerase II (Pol II) occupancy, transcription, and replication. Further, we find that tumor-suppressor and oncogenic mutant BAF complexes have different effects on PRC eviction. The results of these studies define a mechanistic sequence underlying the resolution and formation of facultative heterochromatin, and they demonstrate that BAF opposes PRC on a minute-by-minute basis to provide epigenetic plasticity.
Assuntos
Carcinogênese/genética , Proteínas de Ligação a DNA/genética , Heterocromatina/genética , Proteínas Nucleares/genética , Proteínas do Grupo Polycomb/genética , Cromatina/genética , Replicação do DNA/genética , Epigênese Genética/genética , Humanos , Mutação/genética , RNA Polimerase II/genética , Transcrição Gênica/genéticaRESUMO
Demyelinating diseases are characterized by a loss of oligodendrocytes leading to axonal degeneration and impaired brain function. Current strategies used for the treatment of demyelinating disease such as multiple sclerosis largely rely on modulation of the immune system. Only limited treatment options are available for treating the later stages of the disease, and these treatments require regenerative therapies to ameliorate the consequences of oligodendrocyte loss and axonal impairment. Directed differentiation of adult hippocampal neural stem/progenitor cells (NSPCs) into oligodendrocytes may represent an endogenous source of glial cells for cell-replacement strategies aiming to treat demyelinating disease. Here, we show that Ascl1-mediated conversion of hippocampal NSPCs into mature oligodendrocytes enhances remyelination in a diphtheria-toxin (DT)-inducible, genetic model for demyelination. These findings highlight the potential of targeting hippocampal NSPCs for the treatment of demyelinated lesions in the adult brain.
Assuntos
Lesões Encefálicas/patologia , Doenças Desmielinizantes/patologia , Hipocampo/citologia , Bainha de Mielina/metabolismo , Células-Tronco Neurais/citologia , Neurogênese , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doenças Desmielinizantes/etiologia , Toxina Diftérica/toxicidade , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismoRESUMO
Proliferation of neural stem/progenitor cells (NSPCs) in the adult brain is tightly controlled to prevent exhaustion and to ensure proper neurogenesis. Several extrinsic stimuli affect NSPC regulation. However, the lack of unique markers led to controversial results regarding the in vivo behavior of NSPCs to different stimuli. We recently identified SPOT14, which controls NSPC proliferation through regulation of de novo lipogenesis, selectively in low-proliferating NSPCs. Whether SPOT14-expressing (SPOT14+) NSPCs react in vivo to neurogenic regulators is not known. We show that aging is accompanied by a marked disappearance of SPOT14+ NSPCs, whereas running, a positive neurogenic stimulus, increases proliferation of SPOT14+ NSPCs. Furthermore, transient depletion of highly proliferative cells recruits SPOT14+ NSPCs into the proliferative pool. Additionally, we have established endogenous SPOT14 protein staining, reflecting transgenic SPOT14-GFP expression. Thus, our data identify SPOT14 as a potent marker for adult NSPCs that react dynamically to positive and negative neurogenic regulators.