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
During the development of the vertebrate nervous system, neural progenitors divide, generate progeny that exit mitosis, and then migrate to sites where they elaborate specific morphologies and synaptic connections. Mitotic exit in neurons is accompanied by an essential switch in ATP-dependent chromatin regulatory complexes from the neural progenitor Brg/Brm-associated factor (npBAF) to neuron-specific nBAF complexes that is in part driven by miR-9/9* and miR-124. Recapitulating this microRNA/chromatin switch in fibroblasts leads to their direct conversion to neurons. We have defined the kinetics of neuron-specific BAF complex assembly in the formation of induced neurons from mouse embryonic stem cells, human fibroblasts, and normal mouse neural differentiation and, using proteomic analysis, found that this switch also includes the removal of SS18 and its replacement by CREST at mitotic exit. We found that switching of chromatin remodeling mechanisms is highly correlated with a broad switch in the use of neurogenic transcription factors. Knock-down of SS18 in neural stem cells causes cell-cycle exit and failure to self-renew, whereas continued expression of SS18 in neurons blocks dendritic outgrowth, underlining the importance of subunit switching. Because dominant mutations in BAF subunits underlie widely different human neurologic diseases arising in different neuronal types, our studies suggest that the characteristics of these diseases must be interpreted in the context of the different BAF assemblies in neurons rather than a singular mammalian SWItch/sucrose nonfermentable (mSWI/SNF) complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Axônios/fisiologia , Células Ganglionares da Retina/fisiologia , Sinapses/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Comportamento Animal/fisiologia , Western Blotting , Diferenciação Celular/fisiologia , Células Cultivadas , DNA/genética , Eletroporação , Embrião não Mamífero , Feminino , Homeostase/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Microscopia Confocal , Plasmídeos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Retina/crescimento & desenvolvimento , Retina/fisiologia , Visão Ocular/fisiologia , Xenopus , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Hyperglycemia frequently induces apoptosis in endothelial cells and ultimately contributes to microvascular dysfunction in patients with diabetes mellitus (DM). Previous research reported that the expression of integrins as well as their ligands was elevated in the diseased vessels of DM patients. However, the association between integrins and hyperglycemia-induced cell death is still unclear. This research was designed to investigate the role played by integrin subunit ß5 (ITGB5) in hyperglycemia-induced endothelial cell apoptosis. METHODS: We used leptin receptor knockout (Lepr-KO) ( db / db ) mice as spontaneous diabetes animal model. Selective deletion of ITGB5 in endothelial cell was achieved by injecting vascular targeted adeno-associated virus via tail vein. Besides, we also applied small interfering RNA in vitro to study the mechanism of ITGB5 in regulating high glucose-induced cell apoptosis. RESULTS: ITGB5 and its ligand, fibronectin, were both upregulated after exposure to high glucose in vivo and in vitro . ITGB5 knockdown alleviated hyperglycemia-induced vascular endothelial cell apoptosis and microvascular rarefaction in vivo.In vitro analysis revealed that knockdown of either ITGB5 or fibronectin ameliorated high glucose-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). In addition, knockdown of ITGB5 inhibited fibronectin-induced HUVEC apoptosis, which indicated that the fibronectin-ITGB5 interaction participated in high glucose-induced endothelial cell apoptosis. By using RNA-sequencing technology and bioinformatic analysis, we identified Forkhead Box Protein O1 (FoxO1) as an important downstream target regulated by ITGB5. Moreover, we demonstrated that the excessive macroautophagy induced by high glucose can contribute to HUVEC apoptosis, which was regulated by the ITGB5-FoxO1 axis. CONCLUSION: The study revealed that high glucose-induced endothelial cell apoptosis was positively regulated by ITGB5, which suggested that ITGB5 could potentially be used to predict and treat DM-related vascular complications.
Assuntos
Células Endoteliais , Hiperglicemia , Camundongos , Animais , Humanos , Células Endoteliais/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Fibronectinas , Macroautofagia , Cadeias beta de Integrinas , Apoptose/genética , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismoRESUMO
BACKGROUND: Immune checkpoint inhibitors (ICIs) have emerged as a revolutionary therapy in advanced squamous non-small cell lung cancer (sqNSCLC) and ushered a new era of immunotherapy. Despite of remarkable outcomes, a wide spectrum of immune-related adverse events (irAEs) was reported, among which cutaneous reactions were the most common. Cutaneous irAEs were mainly managed by glucocorticoids, whereas prolonged use of glucocorticoids may cuase kinds of side effects, especially in elderly paitients, and diminish the anti-tumor efficacy of ICIs, thus finding a safe and effective alternative approach to managing cutaneous irAEs is imperative. CASE SUMMARY: A 71-year-old man who was diagnosed with advanced sqNSCLC suffered from sporadic maculopapulars one week later after the fifth cycle of sintilimab treatment, and the skin lesions had been deteriorating rapidly. Skin biopsy revealed epidermal parakeratosis with a dense band-like lymphocytic infiltrate and acanthosis, indicating a diagnosis of immune-induced lichenoid dermatitis. Oral administration of traditional Chinese herbal formula modified Weiling decoction significantly alleviated the symptoms of the patient. The dosage of Weiling decoction were maintained for about three months without recurrence of cutaneous adverse reactions and any other side effects. The patient refused to receive further anti-tumor medication and stayed alive without disease progression at follow up. CONCLUSION: We present modified Weiling decoction successfully ameliorates immune-induced lichenoid dermatitis in a patient with sqNSCLC for the first time. This report indicates that Weiling decoction may be an effective and safe complementary or alternative approach for the treatment of cutaneous irAEs. Further investigation of the underling mechanism is required in the future.
Assuntos
Antineoplásicos Imunológicos , Carcinoma Pulmonar de Células não Pequenas , Dermatite , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neoplasias Pulmonares , Masculino , Humanos , Idoso , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Antineoplásicos Imunológicos/efeitos adversos , Pele/patologia , Dermatite/tratamento farmacológico , Dermatite/patologia , Estudos RetrospectivosRESUMO
RATIONALE: We report a rare case of hypertrophic cardiomyopathy (HCM) caused by a heterozygous variant in TTR gene. PATIENT CONCERNS: The proband had been vomiting without obvious inducement since the age of 27, accompanied by the expulsion of stomach contents. At the age of 28, she began to suddenly syncope. DIAGNOSIS: Cardiac magnetic resonance showed thickening of the right ventricular lateral wall and ventricular septum. The left ventricular diastolic function was limited. Targeted Sanger sequencing validates the presence of mutation p.Leu75Pro in TTR gene. INTERVENTIONS AND OUTCOMES: After admission to hospital for syncope, she was given metoprolol tablets 25 mg bid, spironolactone tablets 20 mg qd, and trimetazidine 20 mg tid. Her symptoms improved after taking the medicine. LESSONS: The results of this case show that HCM caused by TTR mutation is not easy to be identified and treatment is easy to be delayed. Therefore, high-risk patients with amyloidosis should be evaluated as soon as possible. Timely diagnosis of HCM caused by TTR mutation before irreversible organ damage is essential for proper treatment and better outcomes.
Assuntos
Amiloidose , Cardiomiopatia Hipertrófica , Septo Interventricular , Feminino , Humanos , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Ventrículos do Coração/patologia , Síncope , Septo Interventricular/patologia , AdultoRESUMO
OBJECTIVE: To establish a community-based management model for heart failure patients under the professional guidance of upper first-class hospital staff. METHODS: Two hundreds heart failure (New York Heart Function II-IV) patients aged from 35 to 85 in two communities of Chengdu city were divided into two groups by cluster randomization: the management group and the control group. The community hospital doctors were trained for the evaluation and management of heart failure according standardized guidelines by upper first-class hospital doctors, and responsible for the management of patients in the management group. Meanwhile, the management group patients also received self-care education. Patients in control group were treated by community doctors without special training. Data including the community hospital doctors' knowledge rate of heart failure, positive diagnosis rate, and the rate for standardized medication for heart failure; the patients' knowledge rate of heart failure, the rate of drug compliance, the rate of standardized drug taken for heart failure, the rate of self-care in daily-life, the quality of life, the incidence of cardiovascular events, hospitalization time and cost were compared between the two groups. RESULTS: The community hospital doctors' knowledge rate of heart failure, the related knowledge for prevention and treatment on the causes of heart failure, the positive diagnosis rate, and the rate for standardized medication for heart failure [ß receptor blocker 77.3% (17/22); angiotensin-converting enzyme inhibitors 63.6% (14/22)] were significantly higher than doctors in the control group (all P < 0.05). There were 96 in the management group and 97 in the control group. Data were similar between the two groups at baseline. After (18.5 ± 0.5) months, the patient's knowledge rate of heart failure [100% (96/96) vs. 71.1% (69/97)], the rate of drug compliance [78.1% (75/96) vs. 13.4% (13/97)], the rate of standardized drug taken for heart failure[ß receptor blocker: 75.0% (72/96) vs. 8.2% (8/97); angiotensin-converting enzyme inhibitors: 60.4% (58/96)vs. 10.3% (10/97)], and the rate of self-care in daily-life [salt and food restriction:88.5% (85/96) vs. 29.9% (23/97); blood pressure monitoring: 83.3% (80/96) vs. 56.7% (55/97); weight monitoring:78.1% (75/96) vs. 13.4% (13/97)] were all significantly higher in the management group than in control group. For patients with New York Heart Function III-IV, the score of the LiHFe questionnaire (43.7 ± 9.2 vs. 49.5 ± 11.3), the incidence of cardiovascular events [63.3% (19/30) vs. 90.3% (28/31)], the days of hospitalization [(8.2 ± 3.2)days vs. (13.9 ± 10.9) days], and the cost for hospitalization [(2873.3 ± 401.6) Yuan vs. (4525.8 ± 6417.8) Yuan] were all significantly lower in the management group (n = 30) than in the control group (n = 31) (all P < 0.05). CONCLUSIONS: The community-based management model for heart failure patients in the community level is effective to improve the management and outcome in this cohort.
Assuntos
Medicina Comunitária/organização & administração , Insuficiência Cardíaca/terapia , Hospitais Gerais , Doença Crônica , Insuficiência Cardíaca/prevenção & controle , Humanos , Resultado do TratamentoRESUMO
Background: Rifaximin, a rifamycin antibiotic, is widely used to treat infectious diarrhea but not commonly used in H. pylori eradication. With its potential advantages of the agent, some studies were conducted on this topic. The aim of this study is to assess effectiveness and safety of rifaximin-containing regimens and to evaluate whether they are alternative choices for H. pylori eradication. Methods: Scientific databases including PubMed, EMbase and Cochrane Library were used to identify clinical trials on rifaximin-containing regimens published from January 2000 to October 2021. Review Manager 5.4 and STATA12 were adopted for the systematic review. Results: In this study, totally 1025 patients were included from 3 randomized controlled and 9 single-arm studies. It showed that the differences in effectiveness and safety between rifaximin-containing and first-line regimens were not statistically significant in randomized controlled trials. However, the results of the single-arm trials indicated that the eradication and adverse drug reaction rate varied suggesting data instability (r=38.1%-85.4%, r ADR 0.00-67.5% by ITT analysis). Among them, the eradication rate of pediatric patients (r=85.4% by ITT analysis) was higher than that of adult patients (r=38.1-74.5% by ITT analysis). Meanwhile, in all adult subgroups (triple or quadruple, with or without amoxicillin, different duration and rifaximin dose), the results did not show sufficient effectiveness as all the eradication rates did not meet the minimum ideal or ideal target. Conclusion: Taken together, rifaximin-containing regimens should not be recommended for H. pylori eradication as they cannot achieve the eradication rate desired.
RESUMO
AIMS: An improved left ventricular ejection fraction (HFiEF) was observed across heart failure (HF) patients with a reduced or mid-range ejection fraction (HFrEF or HFmrEF, respectively). We postulated that HFiEF patients are clinically distinct from non-HFiEF patients. METHODS AND RESULTS: A total of 447 patients hospitalized due to a clinical diagnosis of HF (LVEF <50% at baseline) were enrolled from September 2017 to September 2019. Echocardiogram re-evaluation was conducted repeatedly over 6 months of follow-up after discharge. The primary endpoint included the composite of HF hospitalization and all-cause mortality. Subjects (n = 184) with HFiEF (defined as an absolute LVEF improvement≥10%) were compared with 263 non-HFiEF (defined by <10% improvement in LVEF) subjects. Multivariable Cox regression was performed and identified younger age, smaller left ventricular end diastolic dimension (LVEDD), beta-blocker use, AF ablation and cardiac resynchronization therapy (CRT) as independent predictors of HFiEF. According to Kaplan-Meier analysis, HFiEF subjects had lower cardiac composite outcomes (P = 0.002) and all-cause mortality (P = 0.003) than non-HFiEF subjects. Multivariate Cox survival analysis revealed that non-HFiEF (compared with HFiEF) was an independent predictor of both the primary endpoints (HR = 0.679, 95% CI: 0.451-0.907, P = 0.012), which was driven by all-cause mortality (HR = 0.504, 95% CI: 0.256-0.991, P = 0.047). CONCLUSIONS: These data confirm that compared with non-HFiEF, HFiEF is a distinct HF phenotype with favourable clinical outcomes.
Assuntos
Insuficiência Cardíaca , Insuficiência Cardíaca/diagnóstico , Humanos , Prognóstico , Estudos Retrospectivos , Volume Sistólico , Função Ventricular EsquerdaRESUMO
Disruptions in the ubiquitin protein ligase E3A (UBE3A) gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium- and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. BK antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases.
Assuntos
Síndrome de Angelman/metabolismo , Canais de Cálcio Tipo N/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Síndrome de Angelman/fisiopatologia , Animais , Epilepsia/metabolismo , Humanos , Camundongos , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organoides , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Potássio/uso terapêutico , Convulsões/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Sr(2+) triggers neurotransmitter release similar to Ca(2+), but less efficiently. We now show that in synaptotagmin 1 knockout mice, the fast component of both Ca(2+)- and Sr(2+)-induced release is selectively impaired, suggesting that both cations partly act by binding to synaptotagmin 1. Both the C(2)A and the C(2)B domain of synaptotagmin 1 bind Ca(2+) in phospholipid complexes, but only the C(2)B domain forms Sr(2+)/phospholipid complexes; therefore, Sr(2+) binding to the C(2)B domain is sufficient to trigger fast release, although with decreased efficacy. Ca(2+) induces binding of the synaptotagmin C(2) domains to SNARE proteins, whereas Sr(2+) even at high concentrations does not. Thus, triggering of the fast component of release by Sr(2+) as a Ca(2+) agonist involves the formation of synaptotagmin/phospholipid complexes, but does not require stimulated SNARE binding.
Assuntos
Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio , Exocitose/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Estrôncio/metabolismo , Membranas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Proteínas de Transporte Vesicular , Animais , Animais Recém-Nascidos , Bário/metabolismo , Bário/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Quelantes/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Substâncias Macromoleculares , Glicoproteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/efeitos dos fármacos , Células PC12 , Fosfolipídeos/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Ratos , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Proteínas SNARE , Estrôncio/farmacologia , Membranas Sinápticas/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Sinaptotagmina I , SinaptotagminasRESUMO
OBJECTIVE: To compare the safety and efficiency of pacing at right ventricular outflow versus right ventricular apex. METHOD: Patients were divided into two groups: pacing at ventricular cardiac apex (common pacing group, n = 22) and pacing at right ventricular outflow tract (uncommon pacing group, n = 18). RESULTS: Impedance and amplitude of R-wave were similar during implantation between the two groups (all P > 0.05). The pacing threshold and electric current were significant higher in uncommon group than those in common pacing group (all P < 0.05), however, these differences disappeared at 1 month post pacemaker implantation (all P > 0.05). The mean QRS duration tended to be shorter in uncommon pacing group compared to that in common pacing group (P > 0.05). There was no pacemaker associated adverse effect in both groups. CONCLUSION: The safety and efficiency of pacing at right ventricular outflow was similar as those of pacing at right ventricular apex.
Assuntos
Estimulação Cardíaca Artificial/métodos , Eletrocardiografia , Ventrículos do Coração/fisiopatologia , Adulto , Idoso , Débito Cardíaco , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Função Ventricular DireitaRESUMO
The neuronal SNARE complex formed by synaptobrevin, syntaxin and SNAP-25 plays a central role in Ca2+-triggered neurotransmitter release. The SNARE complex contains several potential Ca2+-binding sites on the surface, suggesting that the SNAREs may be involved directly in Ca2+-binding during release. Indeed, overexpression of SNAP-25 bearing mutations in two putative Ca2+ ligands (E170A/Q177A) causes a decrease in the Ca2+-cooperativity of exocytosis in chromaffin cells. To test whether the SNARE complex might function in Ca2+-sensing, we analyzed its Ca2+-binding properties using transverse relaxation optimized spectroscopy (TROSY)-based NMR methods. Several Ca2+-binding sites are found on the surface of the SNARE complex, but most of them are not specific for Ca2+ and all have very low affinity. Moreover, we find that the E170A/Q177A SNAP-25 mutation does not alter interactions between the SNAREs and the Ca2+ sensor synaptotagmin 1, but severely impairs SNARE complex assembly. These results suggest that the SNAREs do not act directly as Ca2+ receptors but SNARE complex assembly is coupled tightly to Ca2+-sensing during neurotransmitter release.
Assuntos
Técnicas Biossensoriais , Cálcio/metabolismo , Neurônios/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE , Sinaptotagmina I , Sinaptotagminas , Proteínas de Transporte Vesicular/genéticaRESUMO
The development of the vertebrate nervous system requires a switch of ATP-dependent chromatin remodeling mechanisms, which occurs by substituting subunits within these complexes near cell cycle exit. This switching involves a triple negative genetic circuitry in which REST represses miR-9 and miR-124, which in turn repress BAF53a, which in turn repress the homologous neuron-specific BAF53b. Recapitulation of this microRNA/chromatin switch in human fibroblasts converts them to neurons. The genes involved in this fate-determining chromatin switch play genetically dominant roles in several human neurologic diseases suggesting that they are rate-limiting for aspects of human neural development. We review how this switch in ATP-dependent chromatin complexes might interface with traditional ideas about neural determination and reprogramming.
Assuntos
Diferenciação Celular/genética , Fibroblastos/citologia , MicroRNAs/genética , Neurônios/citologia , Reprogramação Celular , Cromatina/genética , Montagem e Desmontagem da Cromatina , Humanos , MicroRNAs/metabolismo , NeurogêneseRESUMO
Somatic progenitors suppress differentiation to maintain tissue self-renewal. The mammalian SWI/SNF chromatin-remodeling complex regulates nucleosome packaging to control differentiation in embryonic and adult stem cells. Catalytic Brg1 and Brm subunits are required for these processes; however, the roles of SWI/SNF regulatory subunits are not fully understood. Here, we show that ACTL6a/BAF53A modulates the SWI/SNF complex to suppress differentiation in epidermis. Conditional loss of ACTL6a resulted in terminal differentiation, cell-cycle exit, and hypoplasia, whereas ectopic expression of ACTL6a promoted the progenitor state. A significant portion of genes regulated by ACTL6a were found to also be targets of KLF4, a known activator of epidermal differentiation. Mechanistically, we show that ACTL6a prevents SWI/SNF complex binding to promoters of KLF4 and other differentiation genes and that SWI/SNF catalytic subunits are required for full induction of KLF4 targets. Thus, ACTL6a controls the epidermal progenitor state by sequestering SWI/SNF to prevent activation of differentiation programs.
Assuntos
Actinas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Actinas/genética , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Cellular differentiation and reprogramming are accompanied by changes in replication timing and 3D organization of large-scale (400 to 800 Kb) chromosomal domains ('replication domains'), but few gene products have been identified whose disruption affects these properties. RESULTS: Here we show that deletion of esBAF chromatin-remodeling complex components BAF250a and Brg1, but not BAF53a, disrupts replication timing at specific replication domains. Also, BAF250a-deficient fibroblasts reprogrammed to a pluripotency-like state failed to reprogram replication timing in many of these same domains. About half of the replication domains affected by Brg1 loss were also affected by BAF250a loss, but a much larger set of domains was affected by BAF250a loss. esBAF binding in the affected replication domains was dependent upon BAF250a but, most affected domains did not contain genes whose transcription was affected by loss of esBAF. CONCLUSIONS: Loss of specific esBAF complex subunits alters replication timing of select replication domains in pluripotent cells.
RESUMO
Numerous transcriptional regulators of neurogenesis have been identified in the developing and adult brain, but how neurogenic fate is programmed at the epigenetic level remains poorly defined. Here, we report that the transcription factor Pax6 directly interacts with the Brg1-containing BAF complex in adult neural progenitors. Deletion of either Brg1 or Pax6 in the subependymal zone (SEZ) causes the progeny of adult neural stem cells to convert to the ependymal lineage within the SEZ while migrating neuroblasts convert to different glial lineages en route to or in the olfactory bulb (OB). Genome-wide analyses reveal that the majority of genes downregulated in the Brg1 null SEZ and OB contain Pax6 binding sites and are also downregulated in Pax6 null SEZ and OB. Downstream of the Pax6-BAF complex, we find that Sox11, Nfib, and Pou3f4 form a transcriptional cross-regulatory network that drives neurogenesis and can convert postnatal glia into neurons. Taken together, elements of our work identify a tripartite effector network activated by Pax6-BAF that programs neuronal fate.
Assuntos
Células-Tronco Adultas/metabolismo , Proteínas do Olho/metabolismo , Redes Reguladoras de Genes , Proteínas de Homeodomínio/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Células-Tronco Adultas/citologia , Animais , Regulação para Baixo , Camundongos , Células-Tronco Neurais/citologia , Fator de Transcrição PAX6 , Fatores de Transcrição/genéticaRESUMO
Trans-SNAP receptor (SNARE, where SNAP is defined as soluble NSF attachment protein, and NSF is defined as N-ethylmaleimide-sensitive factor) complexes catalyze synaptic vesicle fusion and bind complexin, but the function of complexin binding to SNARE complexes remains unclear. Here we show that in neuronal synapses, complexin simultaneously suppressed spontaneous fusion and activated fast calcium ion-evoked fusion. The dual function of complexin required SNARE binding and also involved distinct amino-terminal sequences of complexin that localize to the point where trans-SNARE complexes insert into the fusing membranes, suggesting that complexin controls the force that trans-SNARE complexes apply onto the fusing membranes. Consistent with this hypothesis, a mutation in the membrane insertion sequence of the v-SNARE synaptobrevin/vesicle-associated membrane protein (VAMP) phenocopied the complexin loss-of-function state without impairing complexin binding to SNARE complexes. Thus, complexin probably activates and clamps the force transfer from assembled trans-SNARE complexes onto fusing membranes.
Assuntos
Fusão de Membrana , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Proteínas SNARE/metabolismo , Sinapses/fisiologia , Potenciais de Ação , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores , Camundongos , Camundongos Knockout , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/química , Vesículas Sinápticas/fisiologia , Sinaptotagminas/metabolismoRESUMO
Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.
Assuntos
Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Sítios de Ligação/fisiologia , Ligação Competitiva/fisiologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular , Células Cultivadas , Sistema Nervoso Central/metabolismo , Humanos , Substâncias Macromoleculares/metabolismo , Camundongos , Modelos Biológicos , Ratos , Proteínas SNARE/metabolismo , Fatores de TempoRESUMO
Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin.