RESUMO
Regulation of chromatin plays fundamental roles in the development of the brain. Haploinsufficiency of the chromatin remodeling enzyme CHD7 causes CHARGE syndrome, a genetic disorder that affects the development of the cerebellum. However, how CHD7 controls chromatin states in the cerebellum remains incompletely understood. Using conditional knockout of CHD7 in granule cell precursors in the mouse cerebellum, we find that CHD7 robustly promotes chromatin accessibility, active histone modifications, and RNA polymerase recruitment at enhancers. In vivo profiling of genome architecture reveals that CHD7 concordantly regulates epigenomic modifications associated with enhancer activation and gene expression of topologically-interacting genes. Genome and gene ontology studies show that CHD7-regulated enhancers are associated with genes that control brain tissue morphogenesis. Accordingly, conditional knockout of CHD7 triggers a striking phenotype of cerebellar polymicrogyria, which we have also found in a case of CHARGE syndrome. Finally, we uncover a CHD7-dependent switch in the preferred orientation of granule cell precursor division in the developing cerebellum, providing a potential cellular basis for the cerebellar polymicrogyria phenotype upon loss of CHD7. Collectively, our findings define epigenomic regulation by CHD7 in granule cell precursors and identify abnormal cerebellar patterning upon CHD7 depletion, with potential implications for our understanding of CHARGE syndrome.
Assuntos
Síndrome CHARGE/genética , Cerebelo/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Polimicrogiria/genética , Animais , Síndrome CHARGE/patologia , Divisão Celular/genética , Cerebelo/patologia , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Epigênese Genética , Código das Histonas , Humanos , Lactente , Camundongos , Camundongos Knockout , Mutação , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Polimicrogiria/patologia , RNA-SeqRESUMO
Compensation among paralogous transcription factors (TFs) confers genetic robustness of cellular processes, but how TFs dynamically respond to paralog depletion on a genome-wide scale in vivo remains incompletely understood. Using single and double conditional knockout of myocyte enhancer factor 2 (MEF2) family TFs in granule neurons of the mouse cerebellum, we find that MEF2A and MEF2D play functionally redundant roles in cerebellar-dependent motor learning. Although both TFs are highly expressed in granule neurons, transcriptomic analyses show MEF2D is the predominant genomic regulator of gene expression in vivo. Strikingly, genome-wide occupancy analyses reveal upon depletion of MEF2D, MEF2A occupancy robustly increases at a subset of sites normally bound to MEF2D. Importantly, sites experiencing compensatory MEF2A occupancy are concentrated within open chromatin and undergo functional compensation for genomic activation and gene expression. Finally, motor activity induces a switch from non-compensatory to compensatory MEF2-dependent gene regulation. These studies uncover genome-wide functional interdependency between paralogous TFs in the brain.
Assuntos
Cerebelo/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Neurônios/metabolismo , Animais , Cerebelo/citologia , Cromatina/genética , Estudo de Associação Genômica Ampla , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Neurônios/citologiaRESUMO
Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional KO of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal male and female mouse brain. In laser capture microdissection followed by RNA-Seq, designed to profile gene expression specifically in the external granule layer of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursors in vivo Remarkably, bioinformatics analyses reveal that SnoN-regulated genes contain binding sites for the transcription factors N-myc and Pax6, which promote the proliferation and differentiation of granule neuron precursors, respectively. Accordingly, we uncover novel physical interactions of SnoN with N-myc and Pax6 in cells. In behavior analyses, conditional KO of SnoN impairs cerebellar-dependent learning in a delayed eye-blink conditioning paradigm, suggesting that SnoN-regulation of granule neuron precursor proliferation bears functional consequences at the organismal level. Our findings define a novel function and mechanism for the major transcriptional regulator SnoN in the control of granule neuron precursor proliferation in the mammalian brain.SIGNIFICANCE STATEMENT This study reports the discovery that the transcriptional regulator SnoN plays a crucial role in the proliferation of cerebellar granule neuron precursors in the postnatal mouse brain. Conditional KO of SnoN in granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cycle exit specifically at later stages of cerebellar development, with biological consequences of impaired cerebellar-dependent learning. Genomics and bioinformatics analyses reveal that SnoN promotes the expression of cell proliferation genes and concomitantly represses cell differentiation genes in vivo Although SnoN has been implicated in distinct aspects of the development of postmitotic neurons, this study identifies a novel function for SnoN in neuronal precursors in the mammalian brain.
Assuntos
Encéfalo/citologia , Proliferação de Células , Cerebelo/fisiologia , Células-Tronco Neurais/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Animais , Comportamento Animal , Piscadela/fisiologia , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular/genética , Cerebelo/citologia , Biologia Computacional , Grânulos Citoplasmáticos/fisiologia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Genes myc/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/fisiologiaRESUMO
Activity-dependent transcription influences neuronal connectivity, but the roles and mechanisms of inactivation of activity-dependent genes have remained poorly understood. Genome-wide analyses in the mouse cerebellum revealed that the nucleosome remodeling and deacetylase (NuRD) complex deposits the histone variant H2A.z at promoters of activity-dependent genes, thereby triggering their inactivation. Purification of translating messenger RNAs from synchronously developing granule neurons (Sync-TRAP) showed that conditional knockout of the core NuRD subunit Chd4 impairs inactivation of activity-dependent genes when neurons undergo dendrite pruning. Chd4 knockout or expression of NuRD-regulated activity genes impairs dendrite pruning. Imaging of behaving mice revealed hyperresponsivity of granule neurons to sensorimotor stimuli upon Chd4 knockout. Our findings define an epigenetic mechanism that inactivates activity-dependent transcription and regulates dendrite patterning and sensorimotor encoding in the brain.
Assuntos
Cerebelo/fisiologia , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Dendritos/fisiologia , Inativação Gênica , Neurônios/fisiologia , Animais , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Camundongos Knockout , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins.
Assuntos
Sistema Nervoso Central/enzimologia , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Animais , Isótopos de Carbono , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Marcação por Isótopo , Cinética , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/líquido cefalorraquidiano , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ratos , Ratos Transgênicos , Proteínas Recombinantes/líquido cefalorraquidiano , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/líquido cefalorraquidiano , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Espectrometria de Massas em TandemRESUMO
BACKGROUND: The number of elderly patients undergoing chronic haemodialysis (HD) in the nursing home (NH) setting is growing; however, little published data exists on this group of patients. METHODS: We describe our experience with 271 patients undergoing staff-assisted HD in the NH setting from 1 January 2001 to 30 June 2004. Acceptance into the programme required that the patients were mentally responsive, haemodynamically stable without sepsis and not be considered terminal or in hospice. RESULTS: Mean age at entry was 70.5+/-12.1 years, 53% were female, 54% were white and 34% black. Main causes of end-stage renal disease (ESRD) were diabetes mellitus (DM, 48%) and hypertension (HTN, 25%). Comorbid conditions included HTN-90%, DM-65%, coronary artery disease-54%, congestive heart failure-59%, cerebrovascular accident-31%, and 40% could not ambulate. The average time on chronic dialysis prior to entering the nursing programme was 18+/-27 months, and the median time was 4 months (range: 0.1-191 months). The average time in the NH programme was 2.9+/-3.6 months (median: 1.6 months, range: 0.1-24 months). During the study period 42% of the patients died, 37% were discharged from the NH, 4.4% withdrew from dialysis, and 16% remained active in the programme. Patient survival from entry into the NH programme was 82% at 1 month, 64% at 3 months, 38% at 6 months and 26% at 12 months (median survival of 4.1 months). However, the patient survival from initiation of chronic dialysis was 75% at 6 months, 66% at 12 months and 38% at 60 months with a median survival of 3.4 years. When evaluating patients based on the duration of chronic dialysis prior to entering the NH programme we found that established HD patients (on HD>or=12 months prior to programme entry) had fewer myocardial infarctions (15 vs 26%, P=0.05), more amputations (19 vs 8%, P=0.01), higher creatinine (6.7 vs 4.7 mg/dl, P<0.01), haemoglobin (11.1 vs 10.5 g/dl, P<0.01) and albumin (3.2 vs 3.0 g/dl, P=0.09) compared with new HD patients (on HDAssuntos
Falência Renal Crônica/terapia
, Casas de Saúde/estatística & dados numéricos
, Diálise Renal/estatística & dados numéricos
, Instituições de Cuidados Especializados de Enfermagem/estatística & dados numéricos
, Adulto
, Idoso
, Idoso de 80 Anos ou mais
, Complicações do Diabetes/complicações
, Feminino
, Humanos
, Hipertensão/complicações
, Falência Renal Crônica/complicações
, Falência Renal Crônica/etiologia
, Masculino
, Pessoa de Meia-Idade
, Estudos Retrospectivos
, Taxa de Sobrevida
, Resultado do Tratamento