RESUMO
Heterochromatin protein 1 (HP1) isoforms are conserved non-histone chromatin-binding proteins that play an important role in packaging of heterochromatin and gene regulation. The functions of HP1 isoforms α and ß are influenced by the nuclear lamins. Cells expressing disease-causing lamin A rod domain mutants show depletion of HP1α and ß and E3 ubiquitin ligase HECW2 is upregulated in these cells. However, the role of HECW2 in the turnover of HP1 proteins has been hitherto unexplored. Here, we show that HECW2 interacts with HP1 isoforms α and ß but not HP1γ. Ectopic expression of HECW2 causes the ubiquitination of HP1α and ß, thereby targeting them for proteasomal degradation. Downregulation of HECW2 increases their steady-state levels indicating its role in their homeostatic regulation. Our results give important insights into the mechanism of proteasomal degradation of HP1 proteins in laminopathic cells.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Homólogo 5 da Proteína Cromobox , Humanos , Isoformas de Proteínas , UbiquitinaçãoRESUMO
Lamins constitute the major architectural proteins of the nuclear lamina that help in maintaining nuclear organization. Mutations in lamins are associated with diverse degenerative diseases, collectively termed laminopathies. HECW2, a HECT-type E3 ubiquitin ligase, is transcriptionally upregulated in HeLa cells expressing Emery-Dreifuss muscular dystrophy-causing-lamin A mutants. However, the role of HECW2 upregulation in mediating downstream effects in lamin mutant-expressing cells was previously unexplored. Here, we show that HECW2 interacts with two lamin A-binding proteins, proliferating cell nuclear antigen (PCNA), via a canonical PCNA-interacting protein (PIP) motif, and lamin B1. HECW2 mediates their ubiquitination and targets them for proteasomal degradation. Cells expressing lamin A mutants G232E and Q294P, in which HECW2 is upregulated, show increased proteasomal degradation of PCNA and lamin B1 most likely mediated by HECW2. Our findings establish HECW2 as an E3 ubiquitin ligase for PCNA and lamin B1 which regulates their levels in laminopathic cells. We also found that HECW2 interacts with wild-type lamin A and ubiquitinates it and this interaction is reduced in case of lamin mutants G232E and Q294P. Our findings suggest that interplay among HECW2, lamin A, PCNA, and lamin B1 determines their respective homeostatic levels in the cell and dysregulation of these interactions may contribute to the pathogenicity of laminopathies.
Assuntos
Lamina Tipo B/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Lamina Tipo A/genética , Lamina Tipo B/química , Lamina Tipo B/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mutação , Antígeno Nuclear de Célula em Proliferação/química , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , UbiquitinaçãoRESUMO
Lamins are key nuclear proteins which are important for maintaining nuclear structure and function. Mutations in lamins cause a spectrum of genetic diseases termed as laminopathies. RING finger containing E3 ubiquitin ligase, RNF123, is transcriptionally upregulated in cells expressing rod domain lamin A mutations. However, the functional relevance of RNF123 in laminopathic cells is not clear. Using a mass spectrometry-based approach, we identified lamins and lamin-binding proteins retinoblastoma protein (pRb), lamina-associated polypeptide 2α (LAP2α), and emerin as RNF123-interacting proteins. We determined that RNF123 mediated the ubiquitination of these proteins and caused the proteasomal degradation of pRb, LAP2α, and lamin B1. Furthermore, these proteins were also targeted for proteasomal degradation in cells expressing lamin A rod domain mutants G232E, Q294P, and R386K. Overexpression of RNF123 resulted in delayed transit through the S-phase which was alleviated by coexpression of pRb or LAP2α. Our findings imply that RNF123-mediated ubiquitination of lamin-binding proteins may contribute to disease-causing mechanisms in laminopathies by depletion of key nuclear proteins and defects in cell cycle kinetics.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Lamina Tipo B/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteína do Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Células HEK293 , Células HeLa , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lamina Tipo B/genética , Espectrometria de Massas , Proteínas de Membrana/genética , Mutação , Proteínas Nucleares/genética , Plasmídeos/química , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína do Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development.
Assuntos
Coração/fisiologia , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Mutação , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Núcleo Celular/metabolismo , Sequência Conservada , DNA Complementar/genética , Expressão Gênica , Coração/embriologia , Humanos , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Peixe-ZebraRESUMO
Cyclin D3 is important for muscle development and regeneration, and is involved in post-mitotic arrest of muscle cells. Cyclin D3 also has cell-cycle-independent functions such as regulation of specific genes in other tissues. Ectopic expression of cyclin D3 in myoblasts, where it is normally undetectable, promotes muscle gene expression and faster differentiation kinetics upon serum depletion. In the present study, we investigated the mechanistic role of cyclin D3 in muscle gene regulation. We initially showed by mutational analysis that a stable and functional cyclin D3 was required for promoting muscle differentiation. Using chromatin immunoprecipitation assays, we demonstrated that expression of cyclin D3 in undifferentiated myoblasts altered histone epigenetic marks at promoters of muscle-specific genes like MyoD, Pax7, myogenin and muscle creatine kinase but not non-muscle genes. Cyclin D3 expression also reduced the mRNA levels of certain epigenetic modifier genes. Our data suggest that epigenetic modulation of muscle-specific genes in cyclin-D3-expressing myoblasts may be responsible for faster differentiation kinetics upon serum depletion. Our results have implications for a regulatory role for cyclin D3 in muscle-specific gene activation.
Assuntos
Creatina Quinase Forma MM/genética , Ciclina D3/genética , Células Musculares/metabolismo , Proteína MyoD/genética , Miogenina/genética , Fator de Transcrição PAX7/genética , Regiões Promotoras Genéticas/genética , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Ciclina D3/metabolismo , Regulação da Expressão Gênica/genética , Camundongos , Células Musculares/citologia , RNA Mensageiro/genética , Regeneração/genéticaRESUMO
Lamins are major structural proteins present in the nuclei of metazoan cells and contribute significantly to nuclear organization and function. The expression of different types of lamins is developmentally regulated and lamin A is detectable in most differentiated tissues. Although the proximal promoter of the mammalian lamin A gene has been characterized, the tissue-specific regulatory elements of the gene have not been identified. In this study, we have cloned and functionally characterized a 2.99 kb segment upstream of exon 1 in the zebrafish lamin A gene. This fragment was able to drive GFP expression in several tissues of the developing embryo at 14-72 h post fertilization in stable transgenic lines. Deletion fragments of the 2.99 kb promoter were analyzed by microinjection into zebrafish embryos in transient assays as well as by luciferase reporter assays in cultured cells. A minimal promoter segment of 1.24 kb conferred tissue-specific expression of GFP in the zebrafish embryo as well as in a myoblast cell line. An 86 bp fragment of this 1.24 kb segment was able to activate a heterologous promoter in myoblasts. Mutational analysis revealed the importance of muscle-specific regulatory motifs in the promoter. Our results have important implications for understanding the tissue-specific regulation and functions of the lamin A gene.
Assuntos
Lamina Tipo A/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , DNA , Primers do DNA , Microscopia Confocal , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Peixe-Zebra/embriologiaRESUMO
The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome-lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin-chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina-chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.
RESUMO
BACKGROUND: Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. METHODOLOGY AND PRINCIPAL FINDINGS: We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna(+/-) embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as ß-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna(+/-) cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna(+/-) embryonic stem cells. CONCLUSIONS: We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development.
Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Animais , Regulação para Baixo , Corpos Embrioides/metabolismo , Corpos Embrioides/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Endoderma/fisiologia , Regulação da Expressão Gênica/genética , Haploinsuficiência , Heterozigoto , Camundongos , Neurônios/metabolismo , Neurônios/fisiologia , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismoRESUMO
BACKGROUND: The nuclear lamina is a key determinant of nuclear architecture, integrity and functionality in metazoan nuclei. Mutations in the human lamin A gene lead to highly debilitating genetic diseases termed as laminopathies. Expression of lamin A mutations or reduction in levels of endogenous A-type lamins leads to nuclear defects such as abnormal nuclear morphology and disorganization of heterochromatin. This is accompanied by increased proteasomal degradation of certain nuclear proteins such as emerin, nesprin-1α, retinoblastoma protein and heterochromatin protein 1 (HP1). However, the pathways of proteasomal degradation have not been well characterized. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanisms underlying the degradation of HP1 proteins upon lamin misexpression, we analyzed the effects of shRNA-mediated knock-down of lamins A and C in HeLa cells. Cells with reduced levels of expression of lamins A and C exhibited proteasomal degradation of HP1α and HP1ß but not HP1γ. Since specific ubiquitin ligases are upregulated in lamin A/C knock-down cells, further studies were carried out with one of these ligases, RNF123, which has a putative HP1-binding motif. Ectopic expression of GFP-tagged RNF123 directly resulted in degradation of HP1α and HP1ß. Mutational analysis showed that the canonical HP1-binding pentapeptide motif PXVXL in the N-terminus of RNF123 was required for binding to HP1 proteins and targeting them for degradation. The role of endogenous RNF123 in the degradation of HP1 isoforms was confirmed by RNF123 RNAi experiments. Furthermore, FRAP analysis suggested that HP1ß was displaced from chromatin in laminopathic cells. CONCLUSIONS/SIGNIFICANCE: Our data support a role for RNF123 ubiquitin ligase in the degradation of HP1α and HP1ß upon lamin A/C knock-down. Hence lamin misexpression can cause degradation of mislocalized proteins involved in key nuclear processes by induction of specific components of the ubiquitin-proteasome system.
Assuntos
Proteínas Cromossômicas não Histona , Lamina Tipo A/genética , Ubiquitina-Proteína Ligases , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Lâmina Nuclear/genética , Lâmina Nuclear/metabolismo , Ligação Proteica , Proteólise , RNA Interferente Pequeno , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Lamins are the major structural components of the nucleus and mutations in the human lamin A gene cause a number of genetic diseases collectively termed laminopathies. At the cellular level, lamin A mutations cause aberrant nuclear morphology and defects in nuclear functions such as the response to DNA damage. We have investigated the mechanism of depletion of a key damage sensor, ATR (Ataxia-telangiectasia-mutated-and-Rad3-related) kinase, in HeLa cells expressing lamin A mutants or lamin A shRNA. The degradation of ATR kinase in these cells was through the proteasomal pathway as it was reversed by the proteasomal inhibitor MG132. Expression of lamin A mutants or shRNA led to transcriptional activation of three ubiquitin ligase components, namely, RNF123 (ring finger protein 123), HECW2 (HECT domain ligase W2) and the F-box protein FBXW10. Ectopic expression of RNF123, HECW2 or FBXW10 directly resulted in proteasomal degradation of ATR kinase and the ring domain of RNF123 was required for this degradation. However, these ligases did not alter the stability of DNA-dependent protein kinase, which is not depleted upon lamin misexpression. Although degradation of ATR kinase was reversed by MG132, it was not affected by the nuclear export inhibitor, leptomycin B, suggesting that ATR kinase is degraded within the nucleus. Our findings indicate that lamin misexpression can lead to deleterious effects on the stability of the key DNA damage sensor, ATR kinase by upregulation of specific components of the ubiquitination pathway.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Expressão Gênica , Lamina Tipo A/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima , Proteínas Mutadas de Ataxia Telangiectasia , Núcleo Celular/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lamina Tipo A/metabolismo , Leupeptinas/farmacologia , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Desnaturação Proteica , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Differentiation of skeletal muscle myoblasts involves activation of muscle-specific markers such as MyoD, Myf5, MRF4, and myogenin, followed by exit from the cell cycle, expression of structural proteins, and fusion into multinucleated myotubes. Cyclin D3 is upregulated during muscle differentiation, and expression of cyclin D3 in proliferating myoblasts causes early activation of myogenesis. In this study, we have identified the genes activated by cyclin D3 expression in C2C12 myoblasts and differentiated cells by real-time PCR analysis. Cyclin D3 expression induced faster differentiation kinetics and increase in levels of myogenic genes such as MyoD, Myf5, and myogenin at an early stage during the differentiation process, although long-term myogenic differentiation was not affected. Transcript levels of the transcription factor Pax7 that is expressed in muscle progenitors were enhanced by cyclin D3 expression in myoblasts. Components of a histone methyltransferase complex recruited by Pax7 to myogenic gene promoters were also regulated by cyclin D3. Further, the Pax7 promoter was upregulated in myoblasts expressing cyclin D3. Myoblasts that expressed cyclin D3 showed moderately higher levels of the cyclin-dependent kinase inhibitor p21 and were stalled in G2/M phase of the cell cycle. Our findings suggest that cyclin D3 primes myoblasts for differentiation by enhancing muscle specific gene expression and cell cycle exit.
Assuntos
Pontos de Checagem do Ciclo Celular , Diferenciação Celular/genética , Ciclina D3/metabolismo , Fator de Transcrição PAX7/metabolismo , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Células Musculares/fisiologia , Desenvolvimento Muscular/genética , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Miogenina/biossíntese , Fator de Transcrição PAX7/genética , Regiões Promotoras GenéticasRESUMO
Lamins are major structural proteins of the nucleus and are essential for nuclear integrity and organization of nuclear functions. Mutations in the human lamin genes lead to highly degenerative genetic diseases that affect a number of different tissues such as muscle, adipose or neuronal tissues, or cause premature ageing syndromes. New findings on the role of lamins in cellular signalling pathways, as well as in ubiquitin-mediated proteasomal degradation, have given important insights into possible mechanisms of pathogenesis.
Assuntos
Doenças Genéticas Inatas/etiologia , Laminas/metabolismo , Lâmina Nuclear/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Doenças Genéticas Inatas/metabolismo , Humanos , Laminas/genética , Mutação de Sentido Incorreto , Transdução de SinaisRESUMO
BACKGROUND: Lamins are major structural proteins of the nucleus and contribute to the organization of various nuclear functions. Mutations in the human lamin A gene cause a number of highly degenerative diseases, collectively termed as laminopathies. Cells expressing lamin mutations exhibit abnormal nuclear morphology and altered heterochromatin organization; however, the mechanisms responsible for these defects are not well understood. METHODOLOGY AND PRINCIPAL FINDINGS: The lamin A rod domain mutants G232E, Q294P and R386K are either diffusely distributed or form large aggregates in the nucleoplasm, resulting in aberrant nuclear morphology in various cell types. We examined the effects of these lamin mutants on the distribution of heterochromatin protein 1 (HP1) isoforms. HeLa cells expressing these mutants showed a heterogeneous pattern of HP1alpha and beta depletion but without altering HP1gamma levels. Changes in HP1alpha and beta were not observed in cells expressing wild-type lamin A or mutant R482L, which assembled normally at the nuclear rim. Treatment with proteasomal inhibitors led to restoration of levels of HP1 isoforms and also resulted in stable association of lamin mutants with the nuclear periphery, rim localization of the inner nuclear membrane lamin-binding protein emerin and partial improvement of nuclear morphology. A comparison of the stability of HP1 isoforms indicated that HP1alpha and beta displayed increased turnover and higher basal levels of ubiquitination than HP1gamma. Transcript analysis of components of the ubiquitination pathway showed that a specific F-box protein, FBXW10 was induced several-fold in cells expressing lamin mutants. Importantly, ectopic expression of FBXW10 in HeLa cells led to depletion of HP1alpha and beta without alteration of HP1gamma levels. CONCLUSIONS: Mislocalized lamins can induce ubiquitin-mediated proteasomal degradation of certain HP1 isoforms by activation of FBXW10, a member of the F-box family of proteins that is involved in E3 ubiquitin ligase activity.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas F-Box/metabolismo , Lamina Tipo A/química , Lamina Tipo A/metabolismo , Proteínas Mutantes/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Biomarcadores/metabolismo , Homólogo 5 da Proteína Cromobox , Células HeLa , Histonas/metabolismo , Humanos , Lamina Tipo A/genética , Leupeptinas/farmacologia , Proteínas de Membrana/metabolismo , Metilação/efeitos dos fármacos , Proteínas Mutantes/química , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Inibidores de Proteassoma , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Solubilidade/efeitos dos fármacos , TransfecçãoRESUMO
Drosophila lamin C (LamC) is a developmentally regulated component of the nuclear lamina. The lamC gene is situated in the fifth intron of the essential gene tout velu (ttv). We carried out genetic analysis of lamC during development. Phenotypic analyses of RNAi-mediated downregulation of lamC expression as well as targeted misexpression of lamin C suggest a role for lamC in cell survival. Of particular interest in the context of laminopathies is the caspase-dependent apoptosis induced by the overexpression of lamin C. Interestingly, misexpression of lamin C in the central nervous system, where it is not normally expressed, did not affect organization of the nuclear lamina. lamC mutant alleles suppressed position effect variegation normally displayed at near-centromeric and telomeric regions. Further, both downregulation and misexpression of lamin C affected the distribution of heterochromatin protein 1. Our results suggest that Drosophila lamC has a tissue-specific role during development and is required for chromatin organization.
Assuntos
Cromatina/metabolismo , Drosophila melanogaster/metabolismo , Lamina Tipo A/genética , Animais , Apoptose , Efeitos da Posição Cromossômica/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação para Baixo/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos/genética , Loci Gênicos/genética , Larva/citologia , Larva/metabolismo , Mutação/genética , Especificidade de Órgãos/genética , Fenótipo , Transporte Proteico , Interferência de RNA , Asas de Animais/citologia , Asas de Animais/metabolismoRESUMO
Pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. During mitosis, nuclear speckles are disassembled by metaphase and reassembled in telophase in structures termed mitotic interchromatin granules (MIGs). We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during interphase and dispersed in metaphase. In telophase, GFP-SC35 was highly enriched within telophase nuclei and also detected in MIGs. Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of cells with inhibitors of cyclin-dependent kinases (cdks) caused changes in the organization of nuclear compartments such as nuclear speckles and nucleoli, with corresponding changes in the mobility of GFP-SC35 and GFP-fibrillarin. Our results suggest that the dynamics of SC35 are significantly influenced by the organization of the compartment in which it is localized during the cell cycle.
Assuntos
Compartimento Celular , Interfase , Mitose , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/genética , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-ArgininaRESUMO
A generic feature in all intracellular biochemical processes is the time required to complete the whole sequence of reactions to yield any observable quantity--from gene expression to circadian rhythms. This widespread phenomenon points towards the importance of time delay in biological functions. Theoretically time delay is known to be the source of instability, and has been attributed to lead to oscillations or transient dynamics in several biological functions. Negative feedback loops, common in biochemical pathways, have been shown to provide stability and withstand considerable variations and random perturbations of biochemical parameters. The interaction of these two opposing factors--of instability and homeostasis--are features that are widespread in intracellular processes. To test the effect of these divergent forces in the dynamics of gene expression, we have designed and constructed simple negatively auto-regulated gene circuits consisting of a basic regulator and transcriptional repressor module, and compared it with one, which has delayed repression. We show, both theoretically and experimentally, that delayed repression induces transient increase and heterogeneity in gene expression before the gain of stability effected by the negative feedback. This design, therefore, seems to be suitable for conferring both stability and variability in cells required for adaptive response to a noisy environment.
Assuntos
Regulação da Expressão Gênica , Expressão Gênica , Redes Reguladoras de Genes , Ritmo Circadiano/genética , Simulação por Computador , Retroalimentação Fisiológica , Proteínas de Fluorescência Verde/genética , Homeostase/genética , Cinética , Modelos Genéticos , Transdução de Sinais/genéticaRESUMO
Lamins are the major architectural proteins of the nucleus and are essential for nuclear integrity and assembly. Lamins are also involved in the organization of nuclear functions such as DNA replication, transcription, and repair. Mutations in the human lamin genes lead to highly debilitating genetic diseases that affect a number of different tissues such as muscle, adipose, and neuronal tissues, or cause premature aging syndromes. The observed interactions of lamins with inner nuclear membrane proteins, chromatin, and various regulatory factors have given important insights into the role of lamins in cellular processes and tissue-specific signaling pathways.
Assuntos
Núcleo Celular/metabolismo , Predisposição Genética para Doença/genética , Laminas/metabolismo , Lâmina Nuclear/metabolismo , Transdução de Sinais/genética , Animais , Diferenciação Celular/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Regulação da Expressão Gênica/genética , Humanos , Laminas/genética , Mutação/genética , Lâmina Nuclear/genética , Lâmina Nuclear/ultraestrutura , Transcrição Gênica/genéticaRESUMO
Lamin A/C is a major component of the nuclear lamina. An intact nuclear lamina has been proposed to be necessary for muscle differentiation. Cyclin D3 is known to be upregulated in differentiated muscle cells and to form insoluble complexes with cell-cycle regulatory factors in these cells. We have examined the possibility of direct binding interactions between lamin A/C and cyclin D3 by in vitro binding assays and co-immunoprecipitation studies with muscle cells. Our results indicate that cyclin D3 binds specifically to amino acid residues 383-474 of lamin A/C and associates with lamin A/C in muscle cells. The identification of cyclin D3 as a novel binding partner of lamin A/C has important implications for a role for lamin A/C in muscle differentiation.
Assuntos
Ciclinas/metabolismo , Lamina Tipo A/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Ciclina D3 , Ciclinas/genética , Lamina Tipo A/genética , Camundongos , Ligação ProteicaRESUMO
Lamins are the major structural proteins of the nucleus in an animal cell. In addition to being essential for nuclear integrity and assembly, lamins are involved in the organization of nuclear processes such as DNA replication, transcription and repair. Mutations in the human lamin A gene lead to highly debilitating genetic disorders that primarily affect muscle, adipose, bone or neuronal tissues and also cause premature ageing syndromes. Mutant lamins alter nuclear integrity and hinder signalling pathways involved in muscle differentiation and adipocyte differentiation, suggesting tissue-specific roles for lamins. Furthermore, cells expressing mutant lamins are impaired in their response to DNA damaging agents. Recent reports indicate that certain lamin mutations act in a dominant negative manner to cause nuclear defects and cellular toxicity, and suggest a possible role for aberrant lamins in normal ageing processes.
Assuntos
Núcleo Celular/ultraestrutura , Doenças Genéticas Inatas/genética , Laminas/genética , Adipócitos/citologia , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Reparo do DNA/genética , Replicação do DNA/fisiologia , Regulação da Expressão Gênica , Doenças Genéticas Inatas/metabolismo , Humanos , Laminas/fisiologia , Laminas/ultraestrutura , Modelos Animais , Modelos Biológicos , MutaçãoRESUMO
Lamin A is an important component of nuclear architecture in mammalian cells. Mutations in the human lamin A gene lead to highly degenerative disorders that affect specific tissues. In studies directed towards understanding the mode of regulation of the lamin A promoter, we have identified an essential GT motif at -55 position by reporter gene assays and mutational analysis. Binding of this sequence to Sp transcription factors has been observed in electrophoretic mobility shift assays and by chromatin immunoprecipitation studies. Further functional analysis by co-expression of recombinant proteins and ChIP assays has shown an important regulatory role for CREB-binding protein in promoter activation, which is mediated by the GT motif.