RESUMEN
RNA Polymerase (Pol) I produces ribosomal (r)RNA, an essential component of the cellular protein synthetic machinery that drives cell growth, underlying many fundamental cellular processes. Extensive research into the mechanisms governing transcription by Pol I has revealed an intricate set of control mechanisms impinging upon rRNA production. Pol I-specific transcription factors guide Pol I to the rDNA promoter and contribute to multiple rounds of transcription initiation, promoter escape, elongation and termination. In addition, many accessory factors are now known to assist at each stage of this transcription cycle, some of which allow the integration of transcriptional activity with metabolic demands. The organisation and accessibility of rDNA chromatin also impinge upon Pol I output, and complex mechanisms ensure the appropriate maintenance of the epigenetic state of the nucleolar genome and its effective transcription by Pol I. The following review presents our current understanding of the components of the Pol I transcription machinery, their functions and regulation by associated factors, and the mechanisms operating to ensure the proper transcription of rDNA chromatin. The importance of such stringent control is demonstrated by the fact that deregulated Pol I transcription is a feature of cancer and other disorders characterised by abnormal translational capacity.
Asunto(s)
ADN Ribosómico/metabolismo , ARN Polimerasa I/genética , ARN Ribosómico/biosíntesis , Transcripción Genética , Animales , ADN Ribosómico/genética , Epigénesis Genética , Regulación de la Expresión Génica , Humanos , ARN Ribosómico/genéticaRESUMEN
The Wilms' tumor 1 protein WT1 is a transcriptional regulator that is involved in cell growth and differentiation. The transcriptional corepressor BASP1 interacts with WT1 and converts WT1 from a transcriptional activator to a repressor. Here, we demonstrate that the N-terminal myristoylation of BASP1 is required in order to elicit transcriptional repression at WT1 target genes. We show that myristoylated BASP1 binds to nuclear PIP2, which leads to the recruitment of PIP2 to the promoter regions of WT1-dependent target genes. BASP1's myristoylation and association with PIP2 are required for the interaction of BASP1 with HDAC1, which mediates the recruitment of HDAC1 to the promoter and elicits transcriptional repression. Our findings uncover a role for myristoylation in transcription, as well as a critical function for PIP2 in gene-specific transcriptional repression through the recruitment of histone deacetylase.
Asunto(s)
Núcleo Celular/metabolismo , Histona Desacetilasa 1/metabolismo , Lipoilación/fisiología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética/fisiología , Proteínas WT1/metabolismo , Núcleo Celular/genética , Histona Desacetilasa 1/genética , Humanos , Células K562 , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Fosfatidilinositol 4,5-Difosfato/genética , Regiones Promotoras Genéticas/fisiología , Unión Proteica , Proteínas Represoras/genética , Proteínas WT1/genéticaRESUMEN
The Wilms' tumour suppressor WT1 (Wilms' tumour 1) is a transcriptional regulator that plays a central role in organogenesis, and is mutated or aberrantly expressed in several childhood and adult malignancies. We previously identified BASP1 (brain acid-soluble protein 1) as a WT1 cofactor that suppresses the transcriptional activation function of WT1. In the present study we have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukaemia K562 cells. Our findings reveal that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We find that WT1 and BASP1 can divert the differentiation programme of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. WT1 and BASP1 co-operate to induce the differentiation of K562 cells to a neuronal-like morphology that exhibits extensive arborization, and the expression of several genes involved in neurite outgrowth and synapse formation. Functional analysis revealed the relevance of the transcriptional reprogramming and morphological changes, in that the cells elicited a response to the neurotransmitter ATP. Taken together, the results of the present study reveal that WT1 and BASP1 can divert the lineage potential of an established blood cell line towards a cell with neuronal characteristics.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/metabolismo , Proteínas WT1/metabolismo , Reprogramación Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de la Membrana/genética , Familia de Multigenes/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Activación Transcripcional/efectos de los fármacos , Proteínas WT1/genéticaRESUMEN
RNA polymerase (pol) III produces essential components of the biosynthetic machinery; therefore, its output is tightly coupled with the rate of cell growth and proliferation. In Saccharomyces cerevisiae, Maf1 is an essential mediator of pol III repression in response to starvation. We demonstrate that a Maf1 ortholog is also used to restrain pol III activity in mouse and human cells. Mammalian Maf1 represses pol III transcription in vitro and in transfected fibroblasts. Furthermore, genetic deletion of Maf1 elevates pol III transcript expression, thus confirming the role of endogenous Maf1 as an inhibitor of mammalian pol III output. Maf1 is detected at chromosomal pol III templates in rodent and human cells. It interacts with pol III as well as its associated initiation factor TFIIIB and is phosphorylated in a serum-sensitive manner in vivo. These aspects of Maf1 function have been conserved between yeast and mammals and are therefore likely to be of fundamental importance in controlling pol III transcriptional activity.
Asunto(s)
ARN Polimerasa III/metabolismo , Proteínas Represoras/fisiología , Transcripción Genética , Animales , Células Madre Embrionarias/metabolismo , Células HeLa , Humanos , Ratones , Fosforilación , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , TransfecciónRESUMEN
Normally, cell cycle progression is tightly coupled to the accumulation of cell mass; however, the mechanisms whereby proliferation and cell growth are linked are poorly understood. We have identified cyclin (Cyc)D2, a G(1) cyclin implicated in mediating S phase entry, as a potential regulator of hypertrophic growth in adult post mitotic myocardium. To examine the role of CycD2 and its downstream targets, we subjected CycD2-null mice to mechanical stress. Hypertrophic growth in response to transverse aortic constriction was attenuated in CycD2-null compared with wild-type mice. Blocking the increase in CycD2 in response to hypertrophic agonists prevented phosphorylation of CycD2-target Rb (retinoblastoma gene product) in vitro, and mice deficient for Rb had potentiated hypertrophic growth. Hypertrophic growth requires new protein synthesis and transcription of tRNA genes by RNA polymerase (pol) III, which increases with hypertrophic signals. This load-induced increase in RNA pol III activity is augmented in Rb-deficient hearts. Rb binds and represses Brf-1 and TATA box binding protein (TBP), subunits of RNA pol III-specific transcription factor B, in adult myocardium under basal conditions. However, this association is disrupted in response to transverse aortic constriction. RNA pol III activity is unchanged in CycD2(-/-) myocardium after transverse aortic constriction, and there is no dissociation of TBP from Rb. These investigations identify an essential role for the CycD2-Rb pathway as a governor of cardiac myocyte enlargement in response to biomechanical stress and, more fundamentally, as a regulator of the load-induced activation of RNA pol III.
Asunto(s)
Cardiomegalia/metabolismo , Ciclinas/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/fisiología , ARN Polimerasa III/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Edad , Animales , Cardiomegalia/patología , Tamaño de la Célula , Células Cultivadas , Ciclina D2 , Ciclinas/genética , Modelos Animales de Enfermedad , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F3/metabolismo , Factor de Transcripción E2F4/metabolismo , Factor de Transcripción E2F5/metabolismo , Ratones , Ratones Mutantes , Miocardio/citología , Miocitos Cardíacos/citología , Fosforilación , Ratas , Proteína de Retinoblastoma/genética , Transducción de Señal/fisiología , Estrés MecánicoRESUMEN
RNA polymerase (pol) III manufactures transfer RNAs, 5S ribosomal RNA and several other untranslated RNA molecules that are essential components of the biosynthetic process. Accordingly, transcription by pol III is closely coupled to cellular growth rate. In mammals, stringent regulation of pol III output is achieved through the concerted action of various mechanisms that target the essential pol III-specific transcription factor TFIIIB. Positive regulators of growth, including ERK and c-Myc, directly bind and activate TFIIIB, thus increasing pol III output when growth demands are high. In contrast, TFIIIB is inactive when bound by RB. Growth stimulation leads to RB hyperphosphorylation, which alleviates this repression. These TFIIIB-directed mechanisms regulate pol III transcription in proliferating fibroblasts, and this is likely to contribute to the tight coordination of cell growth with division. Recent evidence indicates that these same pol III-regulatory mechanisms operate in terminally differentiated cells, where growth occurs in the absence of division, leading to hypertrophic enlargement. This cell division-independent regulation of pol III transcription, and hence biosynthetic capacity, is consistent with a direct involvement of these proteins in controlling cell growth. ERK-mediated induction of expression of the TFIIIB subunit Brf1 was identified as an additional mechanism for raising pol III output in terminally differentiated cardiomyocytes. Brf1 levels are limiting for pol III transcription in resting cardiomyocytes and so hypertrophic stimuli induce Brf1 expression as part of the pol III response in this context. The complex network of strategies that couple pol III transcription with cell growth suggest that stringent control of this system is of fundamental importance.
Asunto(s)
Proliferación Celular , ARN Polimerasa III/metabolismo , Transcripción Genética , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa III/genética , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismoRESUMEN
The proto-oncogene product c-Myc can induce cell growth and proliferation. It regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. We have found that c-Myc can also activate transcription by RNA polymerases I and III, thereby stimulating production of rRNA and tRNA. As such, c-Myc may possess the unprecedented capacity to induce expression of all ribosomal components. This may explain its potent ability to drive cell growth, which depends on the accumulation of ribosomes. The activation of RNA polymerase II transcription by c-Myc is often inefficient, but its induction of rRNA and tRNA genes can be very strong in comparison. We will describe what is known about the mechanisms used by c-Myc to activate transcription by RNA polymerases I and II.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Activación Transcripcional , Animales , ARN Polimerasas Dirigidas por ADN/genética , Humanos , Ratones , Modelos Biológicos , Proto-Oncogenes Mas , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN Ribosómico/genética , ARN de Transferencia/genéticaRESUMEN
The cell division-independent growth of terminally differentiated cardiomyocytes is commonly associated with cardiovascular disease. We demonstrate that it is accompanied by a substantial rise in transcription by RNA polymerase (pol) III, which produces essential components of the biosynthetic apparatus, including 5S rRNA and tRNAs. This increase in transcription is achieved by changes in both the activity and level of the essential pol III-specific transcription factor TFIIIB. Erk and c-Myc, which directly activate TFIIIB in proliferating fibroblasts, also induce pol III transcription in growing cardiomyocytes. Furthermore, hypertrophic stimulation increases expression of the essential TFIIIB subunit Brf1, an effect not seen when fibroblasts proliferate. Erk mediates this induction of Brf1 expression and therefore contributes in at least two ways to pol III transcriptional activation during hypertrophy. Increased production of tRNA and 5S rRNA will contribute to the enhanced translational capacity required to sustain hypertrophic growth.
Asunto(s)
Cardiomegalia/metabolismo , Aumento de la Célula , Miocitos Cardíacos/metabolismo , ARN Polimerasa III/metabolismo , Transcripción Genética , Animales , Cardiomegalia/patología , Células Cultivadas , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Ratones Transgénicos , Miocitos Cardíacos/patología , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa III/genética , ARN Ribosómico 5S/metabolismo , ARN de Transferencia/metabolismo , Ratas , Factor de Transcripción TFIIIB/metabolismoRESUMEN
RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes.