RESUMEN
The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Chaperonas de Histonas/metabolismo , Cinetocoros/metabolismo , Factores de Elongación Transcripcional/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Centrómero/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Proteómica , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/genéticaRESUMEN
Proper genome packaging requires coordination of both DNA and histone metabolism. While histone gene transcription and RNA processing adequately provide for scheduled needs, how histone supply adjusts to unexpected changes in demand remains unknown. Here, we reveal that the histone chaperone nuclear autoantigenic sperm protein (NASP) protects a reservoir of soluble histones H3-H4. The importance of NASP is revealed upon histone overload, engagement of the reservoir during acute replication stress, and perturbation of Asf1 activity. The reservoir can be fine-tuned, increasing or decreasing depending on the level of NASP. Our data suggest that NASP does so by balancing the activity of the heat shock proteins Hsc70 and Hsp90 to direct H3-H4 for degradation by chaperone-mediated autophagy. These insights into NASP function and the existence of a tunable reservoir in mammalian cells demonstrate that contingency is integrated into the histone supply chain to respond to unexpected changes in demand.
Asunto(s)
Autoantígenos/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Autofagia , Células HEK293 , Células HeLa , Humanos , Solubilidad , Células Tumorales CultivadasRESUMEN
Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity.
Asunto(s)
Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Replicación del ADN , Desoxirribonucleasas/metabolismo , Células HeLa , Chaperonas de Histonas/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iß. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iß protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iß depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iß and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Western Blotting , Proteínas de Unión al ADN , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Chaperonas de Histonas/genética , Histonas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Lisina/genética , Lisina/metabolismo , Espectrometría de Masas , Proteínas Nucleares , Unión Proteica , Proteómica , Interferencia de ARN , Proteínas de Unión al ARN , Factores de Transcripción/genéticaRESUMEN
Correct chromosome segregation requires a unique chromatin environment at centromeres and in their vicinity. Here, we address how the deposition of canonical H2A and H2A.Z histone variants is controlled at pericentric heterochromatin (PHC). Whereas in euchromatin newly synthesized H2A and H2A.Z are deposited throughout the cell cycle, we reveal two discrete waves of deposition at PHC - during mid to late S phase in a replication-dependent manner for H2A and during G1 phase for H2A.Z. This G1 cell cycle restriction is lost when heterochromatin features are altered, leading to the accumulation of H2A.Z at the domain. Interestingly, compromising PHC integrity also impacts upon neighboring centric chromatin, increasing the amount of centromeric CENP-A without changing the timing of its deposition. We conclude that the higher-order chromatin structure at the pericentric domain influences dynamics at the nucleosomal level within centromeric chromatin. The two different modes of rearrangement of the PHC during the cell cycle provide distinct opportunities to replenish one or the other H2A variant, highlighting PHC integrity as a potential signal to regulate the deposition timing and stoichiometry of histone variants at the centromere.
Asunto(s)
Ciclo Celular , Histonas/metabolismo , Células 3T3 , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Centrómero/genética , Proteína A Centromérica , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Heterocromatina , Histonas/genética , Ratones , Complejos Multiproteicos/metabolismoRESUMEN
OBJECTIVE: To determine in a guinea pig model the factors of invasiveness of a bipolar electrode implanted in the horizontal semicircular canal (HSC) and to evaluate the consequences on hearing of electrical stimulation of the ampullary nerve. DESIGN: Sixteen guinea pigs divided into four groups underwent surgical opening of the HSC of one ear as follows: control (group 1), cyanoacrylate glue application on the HSC opening (group 2), electrode implantation with cyanoacrylate glue on the HSC opening (group 3), and electrode implantation with electrical stimulation (1 hr/day) for 9 days (group 4). Auditory brainstem responses were recorded before and after surgery and after electrical stimulation. The effectiveness of electrical stimulation in producing a horizontal vestibulo-ocular reflex was evaluated by recording eye movement with video-oculography. RESULTS: Group 1 animals showed hearing loss, and in group 2, sealing the HSC opening with cyanoacrylate glue preserved the hearing thresholds. After electrode implantation, seven of the eight animals showed hearing loss compared with preoperative values. Electrical stimulation did not induce additional hearing loss. CONCLUSION: Electrode implantation at the canal level entailed a risk of hearing loss in an animal model, but electrical stimulation of the horizontal ampullary nerve did not further alter hearing function.
Asunto(s)
Terapia por Estimulación Eléctrica/métodos , Electrodos Implantados , Pérdida Auditiva/prevención & control , Canales Semicirculares/fisiología , Enfermedades Vestibulares/terapia , Nervio Vestibular/fisiología , Animales , Umbral Auditivo/fisiología , Cianoacrilatos , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Movimientos Oculares/fisiología , Cobayas , Audición/fisiología , Masculino , Complicaciones Posoperatorias/prevención & control , Reflejo Vestibuloocular/fisiología , Canales Semicirculares/inervación , Enfermedades Vestibulares/cirugíaRESUMEN
Central vestibular neurons receive substantial inputs from the contralateral labyrinth through inhibitory and excitatory brainstem commissural pathways. The functional organization of these pathways was studied by a multi-methodological approach in isolated frog whole brains. Retrogradely labeled vestibular commissural neurons were primarily located in the superior vestibular nucleus in rhombomeres 2/3 and the medial and descending vestibular nucleus in rhombomeres 5-7. Restricted projections to contralateral vestibular areas, without collaterals to other classical vestibular targets, indicate that vestibular commissural neurons form a feedforward push-pull circuitry. Electrical stimulation of the contralateral coplanar semicircular canal nerve evoked in canal-related second-order vestibular neurons (2 degrees VN) commissural IPSPs (approximately 70%) and EPSPs (approximately 30%) with mainly (approximately 70%) disynaptic onset latencies. The dynamics of commissural responses to electrical pulse trains suggests mediation predominantly by tonic vestibular neurons that activate in all tonic 2 degrees VN large-amplitude IPSPs with a reversal potential of -74 mV. In contrast, phasic 2 degrees VN exhibited either nonreversible, small-amplitude IPSPs (approximately 40%) of likely dendritic origin or large-amplitude commissural EPSPs (approximately 60%). IPSPs with disynaptic onset latencies were exclusively GABAergic (mainly GABA(A) receptor-mediated) but not glycinergic, compatible with the presence of GABA-immunopositive (approximately 20%) and the absence of glycine-immunopositive vestibular commissural neurons. In contrast, IPSPs with longer, oligosynaptic onset latencies were GABAergic and glycinergic, indicating that both pharmacological types of local inhibitory neurons were activated by excitatory commissural fibers. Conservation of major morpho-physiological and pharmacological features of the vestibular commissural pathway suggests that this phylogenetically old circuitry plays an essential role for the processing of bilateral angular head acceleration signals in vertebrates.
Asunto(s)
Lateralidad Funcional/fisiología , Vías Nerviosas/fisiología , Equilibrio Postural/fisiología , Rana esculenta/fisiología , Transmisión Sináptica/fisiología , Núcleos Vestibulares/fisiología , Animales , Evolución Biológica , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/fisiología , Glicina/metabolismo , Movimientos de la Cabeza/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Inhibición Neural/fisiología , Vías Nerviosas/citología , Neuronas/fisiología , Filogenia , Rana esculenta/anatomía & histología , Tiempo de Reacción/fisiología , Receptores de GABA-A/metabolismo , Canales Semicirculares/fisiología , Especificidad de la Especie , Sinapsis/fisiología , Nervio Vestibular/fisiología , Núcleos Vestibulares/citología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
BACKGROUND: Maintaining a proper supply of soluble histones throughout the cell cycle is important to ensure chromatin and genome stability. Following their synthesis, histones undergo a series of maturation steps to prepare them for deposition onto chromatin. RESULTS: Here, we identify the lysine demethylase JMJD1B as a novel player in the maturation cascade that contributes to regulate histone provision. We find that depletion of JMJD1B increases the protein levels of the histone chaperone tNASP leading to an accumulation of newly synthesized histones H3 and H4 at early steps of the histone maturation cascade, which perturbs chromatin assembly. Furthermore, we find a high rate of JMJD1B mutations in cancer patients, and a correlation with genomic instability. CONCLUSIONS: Our data support a role for JMJD1B in fine-tuning histone supply to maintain genome integrity, opening novel avenues for cancer therapeutics.
Asunto(s)
Inestabilidad Genómica , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Procesamiento Proteico-Postraduccional , Células HeLa , Código de Histonas , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , MutaciónRESUMEN
Central vestibular neurons process head movement-related sensory signals over a wide dynamic range. In the isolated frog whole brain, second-order vestibular neurons were identified by monosynaptic responses after electrical stimulation of individual semicircular canal nerve branches. Neurons were classified as tonic or phasic vestibular neurons based on their different discharge patterns in response to positive current steps. With increasing frequency of sinusoidally modulated current injections, up to 100 Hz, there was a concomitant decrease in the impedance of tonic vestibular neurons. Subthreshold responses as well as spike discharge showed classical low-pass filter-like characteristics with corner frequencies ranging from 5 to 20 Hz. In contrast, the impedance of phasic vestibular neurons was relatively constant over a wider range of frequencies or showed a resonance at approximately 40 Hz. Above spike threshold, single spikes of phasic neurons were synchronized with the sinusoidal stimulation between approximately 20 and 50 Hz, thus showing characteristic bandpass filter-like properties. Both the subthreshold resonance and bandpass filter-like discharge pattern depend on the activation of an I(D) potassium conductance. External current or synaptic stimulation that produced impedance increases (i.e., depolarization in tonic or hyperpolarization in phasic neurons) had opposite and complementary effects on the responses of the two types of neurons. Thus, membrane depolarization by current steps or repetitive synaptic excitation amplified synaptic inputs in tonic vestibular neurons and reduced them in phasic neurons. These differential, opposite membrane response properties render the two neuronal types particularly suitable for either integration (tonic neurons) or signal detection (phasic neurons), respectively, and dampens variations of the resting membrane potential in the latter.
Asunto(s)
Canales de Potasio con Entrada de Voltaje/metabolismo , Rombencéfalo/fisiología , Canales Semicirculares/fisiología , Transmisión Sináptica/fisiología , Vestíbulo del Laberinto/fisiología , 4-Aminopiridina/farmacología , Animales , Tronco Encefálico , Impedancia Eléctrica , Técnicas In Vitro , Canal de Potasio Kv.1.1/análisis , Potenciales de la Membrana/fisiología , Neuronas/química , Neuronas/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Rana temporaria , Temperatura , Nervio Vestibular/fisiología , Vestíbulo del Laberinto/citologíaRESUMEN
Small-conductance Ca(2+)-activated potassium (SK) channels are heteromeric complexes of SK alpha-subunits and calmodulin that modulate membrane excitability, are responsible for part of the after-hyperpolarization (AHP) following action potentials, and thus control the firing patterns and excitability of most central neurons. An engineered knockout allele for the SK2 subunit has previously been reported. The hippocampal neurons of these mice lacked the medium latency component of the AHP, but the animals were not described as presenting any overt behavioral phenotype. In this report, we describe a deletion in the 5' region of the Kcnn2 gene encoding the SK2 subunit in the mouse neurological frissonnant (fri) mutant. The frissonnant mutant phenotype is characterized by constant rapid tremor and locomotor instability. It has been suggested, based merely on its phenotype, as a potential model for human Parkinson disease. We used a positional cloning strategy to identify the mutation underlying the frissonnant phenotype. We narrowed the genetic disease interval and identified a 3,441-bp deletion in the Kcnn2 gene, one of the three candidate genes present in the interval. Expression studies showed complete absence of normal Kcnn2 transcripts while some tissue-specific abnormal truncated variants were detected. Intracellular electrophysiological recordings of central vestibular neurons revealed permanent alterations of the AHP and firing behavior that might cause the tremor and associated locomotor deficits. Thus, the fri mutation suggests a new, potentially important physiological role, which had not been described, for the SK2 subunit of small-conductance Ca(2+)-activated potassium channels.
Asunto(s)
Conducta Animal/fisiología , Eliminación de Secuencia , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Potenciales de Acción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Mapeo Cromosómico , Cartilla de ADN/genética , Fenómenos Electrofisiológicos , Femenino , Expresión Génica , Haplotipos , Hibridación in Situ , Hígado/metabolismo , Locomoción/genética , Locomoción/fisiología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Mutantes , Datos de Secuencia Molecular , Fenotipo , Homología de Secuencia de Aminoácido , Temblor/genética , Temblor/fisiopatologíaRESUMEN
In the last decade, numerous studies have investigated synaptic transmission changes in various auditory nuclei after unilateral cochlear injury. However, few data are available concerning the potential effect of electrical stimulation of the deafferented auditory nerve on the inhibitory neurotransmission in these nuclei. We report here for the first time the effect of chronic electrical stimulation of the deafferented auditory nerve on alpha1 subunit of the glycinergic receptor (GlyRalpha1) and glutamic acid decarboxylase (GAD)67 expression in the central nucleus of inferior colliculus (CIC). Adult rats were unilaterally cochleectomized by intracochlear neomycin sulphate injection. Fifteen days later, the ipsilateral auditory nerve was chronically stimulated either 4, 8 or 22 h daily, for 5 days using intracochlear bipolar electrodes. GlyRalpha1 and GAD67 mRNA and protein were quantified in the CIC using in situ hybridization and immunohistofluorescence methods. Our data showed that as after surgical ablation, GlyRalpha1 and GAD67 expression were strongly decreased in the contralateral CIC after unilateral chemical cochleectomy. Most importantly, these postlesional down-modulations were significantly reversed by chronic electrical stimulation of the deafferented auditory nerve. This recovery, however, did not persist for more than 5 days after the cessation of the deafferented auditory nerve electrical stimulation. Thus, downregulations of GlyRalpha1 and GAD67 may be involved both in the increased excitability observed in the CIC after unilateral deafness and consequently in the tinnitus frequently observed in unilateral adult deaf patients. Electrical stimulation of the deafferented auditory nerve in patients may be a potential new approach for treating tinnitus with unilateral hearing loss.
Asunto(s)
Percepción Auditiva/fisiología , Implantes Cocleares , Sordera/terapia , Terapia por Estimulación Eléctrica/métodos , Colículos Inferiores/fisiología , Inhibición Neural/fisiología , Animales , Vías Auditivas/anatomía & histología , Vías Auditivas/fisiología , Biomarcadores/metabolismo , Sordera/fisiopatología , Regulación hacia Abajo/fisiología , Lateralidad Funcional/fisiología , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Glicina/metabolismo , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Transmisión Sináptica/fisiología , Factores de Tiempo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We report the first investigation of whether unilateral labyrinthectomy in adult rats affects the expression of two amino acid transporters, vesicular glutamate transporter 2 (VGLUT2) and vesicular inhibitory amino acid transporter (VIAAT) and of chloride cotransporters (KCC1, KCC2 and NKCC1) in the intact and deafferented medial vestibular nuclei (MVN). In situ hybridization with specific radioactive oligonucleotide probes and immunofluorescent methods were used in normal and unilaterally labyrinthectomized rats at various times following the lesion: 5 h, and 1, 3 and 8 days. In normal animals, several brainstem regions including the lateral, medial, superior and inferior vestibular nuclei contained VGLUT2, VIAAT and KCC2 mRNA. In contrast, no or a very faint labeling was observed with KCC1 and NKCC1 probes. In unilaterally lesioned rats, there was no asymmetry between the two MVN with any of the oligonucleotide probes at any time after the lesion. Similarly, there were no differences in the intensity of MVN labeling between controls and lesioned animals. Finally, no over-expression of the cotransporter KCC1 and NKCC1 was found in ipsilateral or controlateral MVN in lesioned rats at any time. Immunohistochemical experiments gave similar conclusions. Our findings suggest that the recovery of the resting discharge of the deafferented MVN neurons, and consequently the functional compensation of the deficits, are not dependent on changes in the expression of amino acid transporters (VIAAT, VGLUT2), and chloride cotransporters (KCC1, KCC2 and NKCC1) or on their mRNAs.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Regulación de la Expresión Génica/fisiología , Simportadores/inmunología , Núcleos Vestibulares/metabolismo , Vestíbulo del Laberinto/lesiones , Sistemas de Transporte de Aminoácidos/genética , Animales , Modelos Animales de Enfermedad , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Simportadores de Cloruro de Sodio-Potasio , Miembro 2 de la Familia de Transportadores de Soluto 12 , Simportadores/genética , Factores de Tiempo , Proteína 2 de Transporte Vesicular de Glutamato , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/genética , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismo , Núcleos Vestibulares/citología , Cotransportadores de K ClRESUMEN
Facial nerve axotomy is a good model for studying neuronal plasticity and regeneration in the peripheral nervous system. In the present study, we investigated the effect of axotomy on the different subunits of GABA(A) and GABA(B) receptors of facial motoneurons. The facial nerve trunk was unilaterally sectioned and operated rats were sacrificed at 1, 3, 8, 30, and 60 days later. mRNAs coding for alpha1, beta2, and gamma2 of GABA(A) receptors and for GABA(1B) and GABA(B2) receptors were down-regulated by axotomy. This decrease began as soon as 1 or 3 days after axotomy, and the minimum was 8 days post-lesion; the mRNA levels remained lower than normal at day post-lesion 60. The abundance of mRNAs coding for the three other alpha2, beta1, and beta3 facial subunits of GABA(A) receptors and for the pre-synaptic GABA(B1A) subunit remained unchanged during the period 1-8 days post-lesion. Immunohistochemistry using specific antibodies against alpha1, gamma2 subunits of GABA(A) and against GABA(B2) subunits confirmed this down-regulation. Colchicine treatment and blockade of action potential by tetrodotoxin significantly decreased GABA(A)alpha1 immunoreactivity in the axotomized facial nucleus after 7 days. Finally, muscle destruction by cardiotoxin or facial palsy induced by botulinum toxin failed to change GABA(A)alpha1 subunit expression. Our data demonstrate that axotomy strongly reduced the amounts of alpha1, beta2, and gamma2 subunits of GABA(A) receptors and B(1B) and B(2) subunits of GABA(B) receptors in the axotomized facial motoneurons. The loss of GABA(A)alpha1 subunit was most probably induced by both the loss of trophic factors transported from the periphery and a positive injury signal. It also seems to be dependent on activity disruption.
Asunto(s)
Regulación hacia Abajo/fisiología , Nervio Facial/citología , Neuronas Motoras/metabolismo , Receptores de GABA/metabolismo , Animales , Autorradiografía , Transporte Axonal/efectos de los fármacos , Axotomía , Toxinas Botulínicas/farmacología , Recuento de Células/métodos , Proteínas Cardiotóxicas de Elápidos/farmacología , Colchicina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Lateralidad Funcional , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Receptores de GABA/clasificación , Receptores de GABA/genética , Tetrodotoxina/farmacología , Factores de TiempoRESUMEN
BACKGROUND: Breast cancer is a heterogeneous disease with different molecular subtypes that have varying responses to therapy. An ongoing challenge in breast cancer research is to distinguish high-risk patients from good prognosis patients. This is particularly difficult in the low-grade, ER-positive luminal A tumors, where robust diagnostic tools to aid clinical treatment decisions are lacking. Recent data implicating chromatin regulators in cancer initiation and progression offers a promising avenue to develop new tools to help guide clinical decisions. METHODS: Here we exploit a published transcriptome dataset and an independent validation cohort to correlate the mRNA expression of selected chromatin regulators with respect to the four intrinsic breast cancer molecular subtypes. We then perform univariate and multivariate analyses to compare the prognostic value of a panel of chromatin regulators to Ki67, a currently utilized proliferation marker. RESULTS: Unsupervised hierarchical clustering revealed a gene cluster containing several histone chaperones and histone variants highly-expressed in the proliferative subtypes (basal-like, HER2-positive, luminal B) but not in the luminal A subtype. Several chromatin regulators, including the histone chaperones CAF-1 (subunits p150 and p60), ASF1b, and HJURP, and the centromeric histone variant CENP-A, associated with local and metastatic relapse and poor patient outcome. Importantly, we find that HJURP can discriminate favorable and unfavorable outcome within the luminal A subtype, outperforming the currently utilized proliferation marker Ki67, as an independent prognostic marker for luminal A patients. CONCLUSIONS: The integration of chromatin regulators as clinical biomarkers, in particular the histone chaperone HJURP, will help guide patient substratification and treatment options for low-risk luminal A breast carcinoma patients.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Autoantígenos/metabolismo , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína A Centromérica , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Análisis por Conglomerados , Estudios de Cohortes , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Análisis Multivariante , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Modelos de Riesgos Proporcionales , ARN Mensajero/genética , ARN Mensajero/metabolismo , Resultado del TratamientoRESUMEN
Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction-recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Autoantígenos/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/fisiología , Complejos Multiproteicos/fisiología , Proteínas de Xenopus/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Centrómero/metabolismo , Centrómero/ultraestructura , Proteína A Centromérica , Cromatina/metabolismo , Cromatina/ultraestructura , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/fisiología , Humanos , Interfase , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
HP1 enrichment at pericentric heterochromatin is considered important for centromere function. Although HP1 binding to H3K9me3 can explain its accumulation at pericentric heterochromatin, how it is initially targeted there remains unclear. Here, in mouse cells, we reveal the presence of long nuclear noncoding transcripts corresponding to major satellite repeats at the periphery of pericentric heterochromatin. Furthermore, we find that major transcripts in the forward orientation specifically associate with SUMO-modified HP1 proteins. We identified this modification as SUMO-1 and mapped it in the hinge domain of HP1α. Notably, the hinge domain and its SUMOylation proved critical to promote the initial targeting of HP1α to pericentric domains using de novo localization assays, whereas they are dispensable for maintenance of HP1 domains. We propose that SUMO-HP1, through a specific association with major forward transcript, is guided at the pericentric heterochromatin domain to seed further HP1 localization.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/metabolismo , Sumoilación , Animales , Centrómero/metabolismo , Homólogo de la Proteína Chromobox 5 , Ratones , Estructura Terciaria de Proteína , ARN , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
In the last decade, numerous studies have investigated molecular changes in excitatory glutamatergic receptors in axotomized motoneurons, but few data are available concerning the modulation of inhibitory amino acid receptors. We report here the effect of axotomy on the expression of glycine receptors, gephyrin, vesicular inhibitory amino acid transporter (VIAAT) and synapsin I in rat facial motor neurons as demonstrated by in situ hybridization and immunohistochemistry. The facial nerve trunk was sectioned unilaterally and rats were killed 1, 3, 8, 30 or 60 days after surgery. We investigated the mechanisms underlying the changes in production of these proteins following axotomy by perfusing the facial nerve with colchicine or tetrodotoxin, and injecting cardiotoxin or botulinum toxin independently and unilaterally into the whisker pads of normal rats. Animals were killed 8 days later and processed for immunohistochemistry. The abundance of GlyR subunits and gephyrin fell sharply in the axotomized facial nucleus. This decrease began 1 day after axotomy and was lowest at 8 days, with protein levels returning to normal by day 60. Abnormal synapsin immunolabelling was also observed between days 8 and 60 after axotomy but we detected no change in VIAAT immunoreactivity. The effect of colchicine was similar to, but weaker than, that of axotomy. In contrast, tetrodotoxin, cardiotoxin and botulinum toxin had no significant effect. Thus, axotomy-induced changes probably resulted from a loss of trophic factor transported from the periphery or a positive injury signal, or both. They did not seem to depend on the disruption of activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Nervio Facial/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Glicina/metabolismo , Animales , Axotomía , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Regulación de la Expresión Génica/fisiología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Neuronas Motoras/metabolismo , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Ratas , Ratas Long-Evans , Receptores de Glicina/biosíntesis , Receptores de Glicina/genéticaRESUMEN
We investigated whether the production of the sixteen subunits of the GABA(A) receptors and of the different variants of GABA Breceptors are modulated in rat medial vestibular nuclei (MVN) following unilateral labyrinthectomy. Specific alpha1-6, beta1-3, gamma1-3 and delta GABA(A) and GABA(B) B1 and B2receptor radioactive oligonucleotides were used for in situ hybridization to probe sections of rat vestibular nuclei. Specific antibodies against alpha1, beta2, beta3 and gamma2 subunits of GABA(A) receptors and against GABA( B)receptors were also used to detect a potential protein expression modulation. No asymmetry was observed by autoradiography in the intact and deafferented MVN at any time (5 h to 8 days) following the lesion and for any of the oligonucleotide probes used. Also, no difference in the alpha1, beta2, beta3 and gamma2 of the GABA(A) and in the GABA(B) receptor immunohistochemical signal could be detected between the intact and deafferented vestibular nuclei at any time following the lesion. Our data suggest that GABA(A) and GABA Breceptor density changes most probably were not involved in the early stage of the vestibular compensation process, i.e., in the restoration of a normal resting discharge of the deafferented vestibular neurons and consequently in the recovery of a normal posture and eye position.
Asunto(s)
Plasticidad Neuronal/fisiología , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Nervio Vestibular/metabolismo , Núcleos Vestibulares/metabolismo , Vestíbulo del Laberinto/lesiones , Adaptación Fisiológica/fisiología , Animales , Especificidad de Anticuerpos/fisiología , Desnervación , Oído Interno/fisiología , Oído Interno/cirugía , Técnica del Anticuerpo Fluorescente , Lateralidad Funcional/fisiología , Hibridación in Situ , Masculino , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Receptores de GABA-A/genética , Receptores de GABA-B/genética , Nervio Vestibular/fisiopatología , Núcleos Vestibulares/fisiopatologíaRESUMEN
We investigated whether the expression of glycinergic receptor (GLYR) subunits of gephyrin and of their mRNAs in the medial vestibular nuclei are affected following unilateral labyrinthectomy. Specific radioactive oligonucleotide probes recognizing the sequences encoding alpha1-3 and ss subunits of GLYR and the anchoring protein gephyrin were used to probe sections of vestibular nuclei. Signals in these in situ hybridization experiments were detected with film or by emulsion photography. Animals were killed at various times following the lesion: 5 h, 1, 3, 8, 30 and 60 days. Specific monoclonal GLYR and gephyrin antibodies were also used to determine GLYR and gephyrin immunoreactivity in control and operated rats (5 h, 1, 3 and 8 days post-lesion). In normal animals, several brainstem regions including the lateral, medial, superior and inferior vestibular nuclei contained mRNAs for gephyrin and the alpha1 and beta subunits of GLYR, and expressed the GLYR and gephyrin polypeptides. In unilaterally labyrinthectomized rats, no asymmetry was detected on autoradiographs between the two medial vestibular nuclei with any of the oligonucleotide probes used, or at any time following the lesion. No difference in the immunofluorescence staining was observed between the intact and deafferented medial vestibular nuclei of lesioned animals or between the vestibular nuclei of lesioned and controls rats. Thus, deafferentation of the vestibular nuclei did not affect the expression of gephyrin, of the various GLYR subunits, or of their mRNAs in the deafferented and intact medial vestibular nuclei. It is therefore unlikely that GLYR and gephyrin modulation contribute significantly to the recovery of the resting discharge of the deafferented medial vestibular neurons and consequently to the restoration of a normal posture and eye position.