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1.
EMBO Rep ; 22(4): e51298, 2021 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-33594776

RESUMEN

Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. Here, we identify heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, we observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, our findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling.


Asunto(s)
Proteínas de Drosophila , Animales , Proteínas Cromosómicas no Histona/genética , Drosophila/genética , Proteínas de Drosophila/genética , Heterocromatina , Homeostasis , Humanos , Receptores Notch/genética
2.
Proc Natl Acad Sci U S A ; 117(42): 26356-26365, 2020 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-33020270

RESUMEN

Understanding differences in DNA double-strand break (DSB) repair between tumor and normal tissues would provide a rationale for developing DNA repair-targeted cancer therapy. Here, using knock-in mouse models for measuring the efficiency of two DSB repair pathways, homologous recombination (HR) and nonhomologous end-joining (NHEJ), we demonstrated that both pathways are up-regulated in hepatocellular carcinoma (HCC) compared with adjacent normal tissues due to altered expression of DNA repair factors, including PARP1 and DNA-PKcs. Surprisingly, inhibiting PARP1 with olaparib abrogated HR repair in HCC. Mechanistically, inhibiting PARP1 suppressed the clearance of nucleosomes at DNA damage sites by blocking the recruitment of ALC1 to DSB sites, thereby inhibiting RPA2 and RAD51 recruitment. Importantly, combining olaparib with NU7441, a DNA-PKcs inhibitor that blocks NHEJ in HCC, synergistically suppressed HCC growth in both mice and HCC patient-derived-xenograft models. Our results suggest the combined inhibition of both HR and NHEJ as a potential therapy for HCC.


Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Cromonas/farmacología , Morfolinas/farmacología , Ftalazinas/farmacología , Piperazinas/farmacología , Animales , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Quimioterapia Combinada/métodos , Técnicas de Sustitución del Gen , Recombinación Homóloga , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Ratones Desnudos , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Reparación del ADN por Recombinación/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
3.
J Cell Sci ; 131(12)2018 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-29760279

RESUMEN

Dysregulation of the homeostatic balance of histone H3 di- and tri-methyl lysine 27 (H3K27me2/3) levels caused by the mis-sense mutation of histone H3 (H3K27M) is reported to be associated with various types of cancers. In this study, we found that reduction in H3K27me2/3 caused by H3.1K27M, a mutation of H3 variants found in patients with diffuse intrinsic pontine glioma (DIPG), dramatically attenuated the presence of 53BP1 (also known as TP53BP1) foci and the capability of non-homologous end joining (NHEJ) in human dermal fibroblasts. H3.1K27M mutant cells showed increased rates of genomic insertions/deletions and copy number variations, as well as an increase in p53-dependent apoptosis. We further showed that both hypo-H3K27me2/3 and H3.1K27M interacted with FANCD2, a central player in the choice of DNA repair pathway. H3.1K27M triggered the accumulation of FANCD2 on chromatin, suggesting an interaction between H3.1K27M and FANCD2. Interestingly, knockdown of FANCD2 in H3.1K27M cells recovered the number of 53BP1-positive foci, NHEJ efficiency and apoptosis rate. Although these findings in HDF cells may differ from the endogenous regulation of the H3.1K27M mutant in the specific tumor context of DIPG, our results suggest a new model by which H3K27me2/3 facilitates NHEJ and the maintenance of genome stability.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Cromatina/metabolismo , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Histonas/metabolismo , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/metabolismo , Línea Celular , Cromatina/genética , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Fibroblastos , Inestabilidad Genómica , Glioma/genética , Glioma/metabolismo , Células HEK293 , Histonas/genética , Humanos , Metilación , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo
4.
Nucleic Acids Res ; 45(3): 1144-1158, 2017 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-28180298

RESUMEN

Autophagy is an evolutionarily conserved cellular process that primarily participates in lysosome-mediated protein degradation. Although autophagy is a cytoplasmic event, how epigenetic pathways are involved in the regulation of autophagy remains incompletely understood. Here, we found that H2B monoubiquitination (H2Bub1) is down-regulated in cells under starvation conditions and that the decrease in H2Bub1 results in the activation of autophagy. We also identified that the deubiquitinase USP44 is responsible for the starvation-induced decrease in H2Bub1. Furthermore, the changes in H2Bub1 affect the transcription of genes involved in the regulation of autophagy. Therefore, this study reveals a novel epigenetic pathway for the regulation of autophagy through H2Bub1.


Asunto(s)
Autofagia/genética , Epigénesis Genética , Histonas/metabolismo , Ubiquitinación/genética , Animales , Proteínas Relacionadas con la Autofagia/antagonistas & inhibidores , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Diferenciación Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Regulación hacia Abajo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Histonas/química , Histonas/genética , Humanos , Ratones , Modelos Biológicos , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa , Proteasas Ubiquitina-Específicas/antagonistas & inhibidores , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismo , ADN Metiltransferasa 3B
5.
Proc Natl Acad Sci U S A ; 112(45): 13988-93, 2015 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-26508632

RESUMEN

Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.


Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Drosophila/genética , Epigénesis Genética/fisiología , Heterocromatina/fisiología , Aprendizaje/fisiología , Animales , Secuencia de Bases , Western Blotting , Ensamble y Desensamble de Cromatina/genética , Inmunoprecipitación de Cromatina , Drosophila/fisiología , Heterocromatina/genética , Inmunoprecipitación , Datos de Secuencia Molecular , Octoxinol , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/anatomía & histología , Glándulas Salivales/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN
6.
J Biol Chem ; 291(9): 4684-97, 2016 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-26710852

RESUMEN

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules.


Asunto(s)
Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Mitosis , Huso Acromático/enzimología , Acetilación/efectos de los fármacos , Sustitución de Aminoácidos , Resistencia a Medicamentos , Células HeLa , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/genética , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Microscopía Fluorescente , Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Mutación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estabilidad Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Imagen de Lapso de Tiempo , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacología
8.
J Cell Sci ; 125(Pt 22): 5369-78, 2012 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-22956542

RESUMEN

Core histone modifications play an important role in chromatin remodeling and transcriptional regulation. Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. Herein, we demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster resulted in a reduction in body size. Further genetic studies showed that Hdac3 counteracted the organ overgrowth induced by overexpression of insulin receptor (InR), phosphoinositide 3-kinase (PI3K) or S6 kinase (S6K), and the growth regulation by Hdac3 was mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the overexpression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, resulted in changes in body size. Furthermore, we found that H4K16ac was modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracted the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, our studies indicated that Hdac3 served as an important regulator of the PI3K pathway and revealed a novel link between histone acetylation and growth control.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Especificidad de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Acetilación , Animales , Tamaño Corporal , Tamaño de la Célula , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/citología , Drosophila melanogaster/ultraestructura , Femenino , Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/deficiencia , Insulina/metabolismo , Masculino , Proteínas Nucleares/metabolismo , Receptor de Insulina/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Transcripción Genética
9.
Nat Genet ; 37(12): 1361-6, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16258543

RESUMEN

Drosophila melanogaster heterochromatin protein 1 (HP1a or HP1) is believed to be involved in active transcription, transcriptional gene silencing and the formation of heterochromatin. But little is known about the function of HP1 during development. Using a Gal4-induced RNA interference system, we showed that conditional depletion of HP1 in transgenic flies resulted in preferential lethality in male flies. Cytological analysis of mitotic chromosomes showed that HP1 depletion caused sex-biased chromosomal defects, including telomere fusions. The global levels of specific histone modifications, particularly the hallmarks of active chromatin, were preferentially increased in males as well. Expression analysis showed that approximately twice as many genes were specifically regulated by HP1 in males than in females. Furthermore, HP1-regulated genes showed greater enrichment for HP1 binding in males. Taken together, these results indicate that HP1 modulates chromosomal integrity, histone modifications and transcription in a sex-specific manner.


Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Cromatina/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Genes de Insecto , Genes Letales , Humanos , Masculino , Mitosis/genética , Datos de Secuencia Molecular , Factores Sexuales , Telómero/metabolismo , Transcripción Genética
10.
J Biol Chem ; 287(49): 41469-80, 2012 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-22982396

RESUMEN

It is well established that the small GTPase Ras promotes tumor initiation by activating at least three different mediators: Raf, PI3K, and Ras-like (Ral) guanine nucleotide exchange factors. However, the exact mechanisms that underlie these different Ras signaling pathways, which are involved in tumor progression, remain to be elucidated. In this study, we report that the Ras-PI3K pathway, but not Raf or the Ral guanine nucleotide exchange factors, specifically targets the acetylation of H3 at lysine 56 (H3K56ac), thereby regulating tumor cell activity. We demonstrate that the Ras-PI3K-induced reduction in H3K56ac is associated with the proliferation and migration of tumor cells by targeting the transcription of tumor-associated genes. The depletion of the histone deacetyltransferases Sirt1 and Sirt2 rescues the Ras-PI3K-induced decrease in H3K56ac, gene transcription, tumor cell proliferation, and tumor cell migration. Furthermore, we demonstrate that the Ras-PI3K-AKT pathway regulates H3K56ac via the MDM2-dependent degradation of CREB-binding protein/p300. Taken together, the results of this study demonstrate that the Ras-PI3K signaling pathway targets specific epigenetic modifications in tumor cells.


Asunto(s)
Histonas/química , Lisina/química , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas ras/metabolismo , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/metabolismo , Transducción de Señal
11.
J Biol Chem ; 286(11): 9020-30, 2011 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-21205821

RESUMEN

The turnover of tumor suppressor p53 is critical for its role in various cellular events. However, the pathway that regulates the turnover of the Drosophila melanogaster DMP53 is largely unknown. Here, we provide evidence for the first time that the E2 ligase, Drosophila homolog of Rad6 (dRad6/Dhr6), plays an important role in the regulation of DMP53 turnover. Depletion of dRad6 results in DMP53 accumulation, whereas overexpression of dRad6 causes enhanced DMP53 degradation. We show that dRad6 specifically interacts with DMP53 at the transcriptional activation domain and regulates DMP53 ubiquitination. Loss of dRad6 function in transgenic flies leads to lethalities and altered morphogenesis. The dRad6-induced defects in cell proliferation and apoptosis are found to be DMP53-dependent. The loss of dRad6 induces an accumulation of DMP53 that enhances the activation of apoptotic genes and leads to apoptosis in the presence of stress stimuli. In contrast to that, the E3 ligase is the primary factor that regulates p53 turnover in mammals, and this work demonstrates that the E2 ligase dRad6 is critical for the control of DMP53 degradation in Drosophila.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Proteínas de Drosophila/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Morfogénesis/fisiología , Estructura Terciaria de Proteína , Proteína p53 Supresora de Tumor/genética , Enzimas Ubiquitina-Conjugadoras/genética
12.
Cell Death Differ ; 27(5): 1660-1676, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31685978

RESUMEN

Cancer cells reprogram their energy metabolic system from the mitochondrial oxidative phosphorylation (OXPHOS) pathway to a glucose-dependent aerobic glycolysis pathway. This metabolic reprogramming phenomenon is known as the Warburg effect, a significant hallmark of cancer. However, the detailed mechanisms underlying this event or triggering this reprogramming remain largely unclear. Here, we found that histone H2B monoubiquitination (H2Bub1) negatively regulates the Warburg effect and tumorigenesis in human lung cancer cells (H1299 and A549 cell lines) likely through controlling the expression of multiple mitochondrial respiratory genes, which are essential for OXPHOS. Moreover, our work also suggested that pyruvate kinase M2 (PKM2), the rate-limiting enzyme of glycolysis, can directly interact with H2B in vivo and in vitro and negatively regulate the level of H2Bub1. The inhibition of cell proliferation and nude mice xenograft of human lung cancer cells induced by PKM2 knockdown can be partially rescued through lowering H2Bub1 levels, which indicates that the oncogenic function of PKM2 is achieved, at least partially, through the control of H2Bub1. Furthermore, PKM2 and H2Bub1 levels are negatively correlated in cancer specimens. Therefore, these findings not only provide a novel mechanism triggering the Warburg effect that is mediated through an epigenetic pathway (H2Bub1) but also reveal a novel metabolic regulator (PKM2) for the epigenetic mark H2Bub1. Thus, the PKM2-H2Bub1 axis may become a promising cancer therapeutic target.


Asunto(s)
Epigénesis Genética , Histonas/metabolismo , Ubiquitinación , Efecto Warburg en Oncología , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Respiración de la Célula/genética , Células HEK293 , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/genética , Simulación del Acoplamiento Molecular , Unión Proteica , Hormonas Tiroideas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión a Hormona Tiroide
13.
Zhongguo Zhong Yao Za Zhi ; 33(18): 2109-12, 2008 Sep.
Artículo en Zh | MEDLINE | ID: mdl-19160797

RESUMEN

OBJECTIVE: To investigate the effects of morroniside on H2O2-induced apoptosis in nerve cells. METHOD: Human neuroblastoma cell line SH-SY5Y cells were pre-incubaed with morroniside (1, 10, and 100 micromol x L(-1)) for 24 h prior to exposure to H2O2 (500 micromol x L(-1)) for 18 h. The activity of reactive SOD was measured by a biochemical assay. The expression of caspase-3, caspase-9, Bcl-2 and Bax was determined by Wastern blotting method. RESULT: Pretreatment of the cells with morroniside (10 and 100 micromol x L(-1)) increasd SOD activity by 14% (P<0.01) and 11% (P<0.05) in comparison with cells exposed only to H2O2. Morroniside (1, 10, 100 micromol x L(-1)) lowered caspase-3 level by 31% (P<0.01), 103% (P<0.001) and 95% (P<0.001), decreased caspase-9 content by 71% (P<0.001), 132% (P<0.001) and 37% (P<0.05), and increasd Bcl-1 level by 88% (P<0.01), 121% (P<0.001) and 60% (P<0.01) respectively but no significant change occurred in Bax level in comparison with cells exposed only to H2O2. CONCLUSION: Morroniside has neuroprotection effect against H2O2-induced oxidation injury in nerve cell.


Asunto(s)
Apoptosis/efectos de los fármacos , Glicósidos/farmacología , Peróxido de Hidrógeno/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Oxidantes/farmacología , Animales , Western Blotting , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/metabolismo
14.
Mol Cell Biol ; 24(18): 8210-20, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15340080

RESUMEN

The heterochromatic domains of Drosophila melanogaster (pericentric heterochromatin, telomeres, and the fourth chromosome) are characterized by histone hypoacetylation, high levels of histone H3 methylated on lysine 9 (H3-mK9), and association with heterochromatin protein 1 (HP1). While the specific interaction of HP1 with both H3-mK9 and histone methyltransferases suggests a mechanism for the maintenance of heterochromatin, it leaves open the question of how heterochromatin formation is targeted to specific domains. Expression characteristics of reporter transgenes inserted at different sites in the fourth chromosome define a minimum of three euchromatic and three heterochromatic domains, interspersed. Here we searched for cis-acting DNA sequence determinants that specify heterochromatic domains. Genetic screens for a switch in phenotype demonstrate that local deletions or duplications of 5 to 80 kb of DNA flanking a transposon reporter can lead to the loss or acquisition of variegation, pointing to short-range cis-acting determinants for silencing. This silencing is dependent on HP1. A switch in transgene expression correlates with a switch in chromatin structure, judged by nuclease accessibility. Mapping data implicate the 1360 transposon as a target for heterochromatin formation. We propose that heterochromatin formation is initiated at dispersed repetitive elements along the fourth chromosome and spreads for approximately 10 kb or until encountering competition from a euchromatic determinant.


Asunto(s)
Drosophila melanogaster/genética , Heterocromatina/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Mapeo Cromosómico , Cromosomas/genética , ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Anomalías del Ojo/genética , Femenino , Silenciador del Gen , Genes de Insecto , Genes Reporteros , Prueba de Complementación Genética , Heterocromatina/metabolismo , Masculino , Mutación , Fenotipo , Eliminación de Secuencia , Secuencias Repetidas en Tándem
15.
Nucleic Acids Res ; 33(9): 2852-8, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15905474

RESUMEN

Heterochromatin protein 1 (HP1) was originally described as a non-histone chromosomal protein and is required for transcriptional gene silencing and the formation of heterochromatin. Although it is localized primarily at pericentric heterochromatin, a scattered distribution over a large number of euchromatic loci is also evident. Here, we provide evidence that Drosophila HP1 is essential for the maintenance of active transcription of euchromatic genes functionally involved in cell-cycle progression, including those required for DNA replication and mitosis. Depletion of HP1 in proliferating embryonic cells caused aberrant progression of the cell cycle at S phase and G2/M phase, linked to aberrant chromosome segregation, cytokinesis, and an increase in apoptosis. The chromosomal distribution of Aurora B, and the level of phosphorylation of histone H3 serine 10 were also altered in the absence of HP1. Using chromatin immunoprecipitation analysis, we further demonstrate that the promoters of a number of cell-cycle regulator genes are bound to HP1, supporting a direct role for HP1 in their active transcription. Overall, our data suggest that HP1 is essential for the maintenance of cell-cycle progression and the transcription of cell-cycle regulatory genes. The results also support the view that HP1 is a positive regulator of transcription in euchromatin.


Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Animales , Ciclo Celular , Proteínas de Ciclo Celular/biosíntesis , Células Cultivadas , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Eucromatina/genética , Histonas/metabolismo , Fosforilación , Interferencia de ARN , Transcripción Genética
16.
Cancer Lett ; 400: 18-29, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28455245

RESUMEN

Lung cancer is one of the most lethal cancers due to its highly metastatic spreading. The motility of lung cancer cells is regulated by paracrine factors, such as TGF-ß, in the tumor microenvironment through the induction of epithelial-to-mesenchymal transition (EMT). The stability of microtubules is reported to be associated with the EMT process and the migration of cancer cells. Here, we observed that RCC1 domain-containing protein 1 (RCCD1) is highly expressed in non-small cell lung cancer (NSCLC) patients with poor prognosis, and RCCD1 is much higher expressed in tumor tissues compared with adjacent normal tissues. Depletion of RCCD1 using siRNAs significantly inhibits the migration of lung cancer cells. Subsequent studies reveal that the loss of RCCD1 results in upregulation of acetylated α-tubulin levels and stabilizes cytoskeletal microtubules. Mechanistically, we observed that RCCD1 modulates the stability of microtubules through interacting with JMJD5. Furthermore, RCCD1 depletion significantly attenuates the TGF-ß-induced EMT process, as assessed by altered expression of epithelial and mesenchymal markers (Occludin, Vimentin and Snail), and inhibits TGF-ß-induced cell migration. Collectively, these findings support RCCD1 as a novel regulator of TGF-ß-induced EMT in NSCLC.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Células A549 , Acetilación , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras/genética , Movimiento Celular/efectos de los fármacos , Biología Computacional , Bases de Datos Genéticas , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Células HEK293 , Histona Demetilasas/metabolismo , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas de la Membrana/genética , Microtúbulos/patología , Invasividad Neoplásica , Pronóstico , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transfección , Factor de Crecimiento Transformador beta1/farmacología , Tubulina (Proteína)/metabolismo
17.
Mol Cell Biol ; 37(6)2017 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-28031328

RESUMEN

RAD6, an E2 ubiquitin-conjugating enzyme, is a key node for determining different DNA damage repair pathways, controlling both the error-prone and the error-free DNA damage repair pathways through differential regulation of the ubiquitination of the proliferating cell nuclear antigen (PCNA) protein. However, whether other pathways are involved in the RAD6-mediated regulation of DNA damage repair is still unclear. To deeply understand the molecular mechanisms of RAD6 in DNA damage repair, we performed a proteomic analysis and identified the changes of the protein-protein interaction (PPI) networks of RAD6 before and after X-ray irradiation. Furthermore, our study indicated that a proteasome-related event is likely involved in the DNA damage repair process. Moreover, we found that RAD6 promotes proteasome activity and nuclear translocation by enhancing the degradation of PSMF1 and the lamin B receptor (LBR). Therefore, we provide a novel pathway that is employed by RAD6 in response to DNA damage.


Asunto(s)
Daño del ADN , Complejo de la Endopetidasa Proteasomal/metabolismo , Mapas de Interacción de Proteínas , Proteómica/métodos , Enzimas Ubiquitina-Conjugadoras/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Reparación del ADN/efectos de la radiación , Regulación hacia Abajo/genética , Regulación hacia Abajo/efectos de la radiación , Células HEK293 , Humanos , Modelos Biológicos , Proteínas/metabolismo , Proteolisis/efectos de la radiación , Receptores Citoplasmáticos y Nucleares/metabolismo , Ubiquitina/metabolismo , Rayos X , Receptor de Lamina B
18.
Cell Cycle ; 15(21): 2980-2991, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27715397

RESUMEN

Microtubules play essential roles in mitosis, cell migration, and intracellular trafficking. Drugs that target microtubules have demonstrated great clinical success in cancer treatment due to their capacity to impair microtubule dynamics in both mitotic and interphase stages. In a previous report, we demonstrated that JMJD5 associated with mitotic spindle and was required for proper mitosis. However, it remains elusive whether JMJD5 could regulate the stability of cytoskeletal microtubules and whether it affects the efficacy of microtubule-targeting agents. In this study, we find that JMJD5 localizes not only to the nucleus, a fraction of it also localizes to the cytoplasm. JMJD5 depletion decreases the acetylation and detyrosination of α-tubulin, both of which are markers of microtubule stability. In addition, microtubules in JMJD5-depleted cells are more sensitive to nocodazole-induced depolymerization, whereas JMJD5 overexpression increases α-tubulin detyrosination and enhances the resistance of microtubules to nocodazole. Mechanistic studies revealed that JMJD5 regulates MAP1B protein levels and that MAP1B overexpression rescued the microtubule destabilization induced by JMJD5 depletion. Furthermore, JMJD5 depletion significantly promoted apoptosis in cancer cells treated with the microtubule-targeting anti-cancer drugs vinblastine or colchicine. Together, these findings suggest that JMJD5 is required to regulate the stability of cytoskeletal microtubules and that JMJD5 depletion increases the susceptibility of cancer cells to microtubule-destabilizing agents.


Asunto(s)
Antineoplásicos/farmacología , Eliminación de Gen , Histona Demetilasas/metabolismo , Microtúbulos/metabolismo , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo
19.
Adv Sci (Weinh) ; 3(8): 1500393, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27818907

RESUMEN

Stem-cell-derived hepatocyte transplantation is considered as a potential method for the therapy of acute and chronic liver failure. However, the low efficiency of differentiation into mature and functional hepatocytes remains a major challenge for clinical applications. By using polyethyleneimine-modified silica nanoparticles, this study develops a system for sustained delivery of growth factors, leading to induce hepatocyte-like cells (iHeps) from mouse embryonic stem cells (mESCs) and improve the expression of endoderm and hepatocyte-specific genes and proteins significantly, thus producing a higher population of functional hepatocytes in vitro. When transplanted into liver-injured mice after four weeks, mESC-derived definitive endoderm cells treated with this delivery system show higher integration efficiency into the host liver, differentiated into iHeps in vivo and significantly restored the injured liver. Therefore, these findings reveal the multiple advantages of functionalized nanoparticles to serve as efficient delivery platforms to promote stem cell differentiation in the regenerative medicine.

20.
Mol Cell Biol ; 35(2): 406-16, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25384975

RESUMEN

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.


Asunto(s)
Autofagia/genética , Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/metabolismo , Reparación del ADN por Recombinación/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Línea Celular , Homólogo de la Proteína Chromobox 5 , Humanos
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