RESUMEN
Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.
Asunto(s)
Western Blotting/métodos , Electroforesis/métodos , Histonas/metabolismo , Urea/química , Acetilación , Animales , Línea Celular , Ratones , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Histone acetyltransferase enzymes (HATs) are important therapeutic targets, but there are few cell-based assays available for evaluating the pharmacodynamics of HAT inhibitors. Here we present the application of a FRET-based reporter, Histac, in live-cell studies of p300/CBP HAT inhibition, by both genetic and pharmacologic disruption. shRNA knockdown of p300/CBP led to increased Histac FRET, thus suggesting a role for p300/CBP in the acetylation of the histone H4 tail. Additionally, we describe a new p300/CBP HAT inhibitor, C107, and show that it can also increase cellular Histac FRET. Taken together, these studies provide a live-cell strategy for identifying and evaluating p300/CBP inhibitors.
Asunto(s)
Factores de Transcripción p300-CBP/antagonistas & inhibidores , Acetilación , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Dynamic acetylation of all lysine-4-trimethylated histone H3 is a complex phenomenon involved in Immediate-early gene induction in metazoan eukaryotes. Higher eukaryotes express repeated copies of three closely related H3 variants, inaccessible to genetic analysis. We demonstrate conservation of these phenomena in Dictyostelium which has three single-copy H3 variant genes. Although dynamic acetylation is targeted to two H3 variants which are K4-trimethylated, K9-acetylation is preferentially targeted to one. In cells lacking Set1 methyltransferase and any detectable K4-trimethylation, dynamic acetylation is lost demonstrating a direct link between the two. Gene replacement to express mutated H3 variants reveals a novel interaction between K4-trimethylation on different variants. Cells expressing only one variant show defects in growth, and in induction of a UV-inducible gene, demonstrating the functional importance of variant expression. These studies confirm that dynamic acetylation targeted to H3K4me3 arose early in evolution and reveal a very high level of specificity of histone variant utilization in a simple multicellular eukaryote.
Asunto(s)
Dictyostelium/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Acetilación/efectos de los fármacos , Sustitución de Aminoácidos , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Eliminación de Gen , Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Histonas/química , Histonas/genética , Ácidos Hidroxámicos/farmacología , MetilaciónRESUMEN
Histone modifications are reported to show different behaviors, associations, and functions in different genomic niches and organisms. We show here that rapid, continuous turnover of acetylation specifically targeted to all K4-trimethylated H3 tails (H3K4me3), but not to bulk histone H3 or H3 carrying other methylated lysines, is a common uniform characteristic of chromatin biology in higher eukaryotes, being precisely conserved in human, mouse, and Drosophila. Furthermore, dynamic acetylation targeted to H3K4me3 is mediated by the same lysine acetyltransferase, p300/cAMP response element binding (CREB)-binding protein (CBP), in both mouse and fly cells. RNA interference or chemical inhibition of p300/CBP using a newly discovered small molecule inhibitor, C646, blocks dynamic acetylation of H3K4me3 globally in mouse and fly cells, and locally across the promoter and start-site of inducible genes in the mouse, thereby disrupting RNA polymerase II association and the activation of these genes. Thus, rapid dynamic acetylation of all H3K4me3 mediated by p300/CBP is a general, evolutionarily conserved phenomenon playing an essential role in the induction of immediate-early (IE) genes. These studies indicate a more global function of p300/CBP in mediating rapid turnover of acetylation of all H3K4me3 across the nuclei of higher eukaryotes, rather than a tight promoter-restricted function targeted by complex formation with specific transcription factors.
Asunto(s)
Histonas/química , Histonas/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Sitios de Unión , Línea Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Evolución Molecular , Técnicas de Silenciamiento del Gen , Genes Inmediatos-Precoces , Genes fos , Genes jun , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Lisina/química , Ratones , Regiones Promotoras Genéticas , Especificidad de la Especie , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Factores de Transcripción p300-CBP/genéticaRESUMEN
The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. Although p300 inhibitors have been reported, a potent, selective, and readily available active-site-directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a K(i) of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anticancer target.
Asunto(s)
Benzoatos/química , Inhibidores Enzimáticos/química , Histona Acetiltransferasas/antagonistas & inhibidores , Pirazoles/química , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Acetilación , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Benzoatos/farmacología , Sitios de Unión , Unión Competitiva , Dominio Catalítico , Línea Celular Tumoral , Simulación por Computador , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Histona Acetiltransferasas/metabolismo , Ligandos , Ratones , Pirazoles/farmacología , Pirazolonas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The DNA of eukaryotic genomes is highly packaged by its organisation into chromatin, the fundamental repeating unit of which is the nucleosome core particle, consisting of 147 base pairs of DNA wrapped around an octamer of two copies each of the four core histone proteins H2A, H2B, H3 and H4 (K. Luger, A.W. Mader, R.K. Richmond, D.F. Sargent, T.J. Richmond, Crystal structure of the nucleosome core particle at 2.8 A resolution, Nature 389 (1997) 251-260 [1] and references therein). Accessibility of DNA within chromatin is a central factor that affects DNA-dependent nuclear function such as transcription, replication, recombination and repair. To integrate complex signalling networks associated with these events, many protein and multi-protein complexes associate transiently with nucleosomes. One class of such are the High-Mobility Group (HMG) proteins which are architectural DNA and nucleosome-binding proteins that may be subdivided into three families; HMGA (HMGI/Y/C), HMGB (HMG1/2) and HMGN (HMG14/17). The structure of chromatin and nucleosomes can be altered, both locally and globally, by interaction with such architectural proteins thereby influencing accessibility of DNA. This chapter deals with the HMGN protein family, specifically their post-translational modification as part of regulatory networks. We focus particularly on HMGN1, the most extensively studied family member to date, and to a lesser extent on HMGN2. We critically evaluate evidence for the role of post-translational modification of these proteins in response to different signals, exploring the sites and potential significance of such modification.
Asunto(s)
Cromatina/metabolismo , Proteínas HMGN/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Proteínas HMGN/química , Humanos , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
The combination of chromatin immunoprecipitation (ChIP) with microarray analysis (ChIP-chip) or high-throughput sequencing (ChIP-seq and ChIP-SAGE) has provided maps of a wide variety of site-specific histone modifications across mammalian genomes in various cell types. Although distinct genomic regions and functional elements have been strongly associated with specific histone modifications, an overwhelming number of combinatorial patterns have also been observed across the genome. While peaks of enrichment in ChIP-chip and ChIP-seq data may suggest stable and predictive 'landmarks' across the genomic landscape, studies from transcribed genes indicate a more dynamic model of how these data may be interpreted. In light of such studies, which show highly dynamic methylation, acetylation and phosphorylation of histone H3 during gene transcription, we consider the extent to which genome-wide maps of chromatin state could be interpreted as 'snapshots' of heterogeneous profiles deriving from dynamic modification processes. Rather than acting as static 'epigenetic' landmarks, histone modifications may function as dynamic and transient operational marks supporting specific steps in diverse processes throughout the mammalian genome.
Asunto(s)
Epigénesis Genética , Genoma , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Animales , Centrómero/metabolismo , Cromatina/metabolismo , Islas de CpG , Metilación de ADN , Silenciador del Gen , Humanos , Regiones Promotoras Genéticas , Telómero/metabolismoRESUMEN
Understanding the function of histone modifications across inducible genes in mammalian cells requires quantitative, comparative analysis of their fate during gene activation and identification of enzymes responsible. We produced high-resolution comparative maps of the distribution and dynamics of H3K4me3, H3K36me3, H3K79me2 and H3K9ac across c-fos and c-jun upon gene induction in murine fibroblasts. In unstimulated cells, continuous turnover of H3K9 acetylation occurs on all K4-trimethylated histone H3 tails; distribution of both modifications coincides across promoter and 5' part of the coding region. In contrast, K36- and K79-methylated H3 tails, which are not dynamically acetylated, are restricted to the coding regions of these genes. Upon stimulation, transcription-dependent increases in H3K4 and H3K36 trimethylation are seen across coding regions, peaking at 5' and 3' ends, respectively. Addressing molecular mechanisms involved, we find that Huntingtin-interacting protein HYPB/Setd2 is responsible for virtually all global and transcription-dependent H3K36 trimethylation, but not H3K36-mono- or dimethylation, in these cells. These studies reveal four distinct layers of histone modification across inducible mammalian genes and show that HYPB/Setd2 is responsible for H3K36 trimethylation throughout the mouse nucleus.
Asunto(s)
Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Animales , Northern Blotting , Línea Celular , Proteínas de Drosophila/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Immunoblotting , Inmunoprecipitación , Lisina/metabolismo , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Activación Transcripcional , TransfecciónRESUMEN
Stably enhanced histone acetylation has long been regarded as a condition of transcriptionally active genes. Recent papers suggest a more dynamic model, with rapid turnover of acetylation observed at nontranscribing "poised" genes and shown to be an important determinant of transcriptional efficiency upon gene induction. Are these "special cases," restricted to specific genes and specific types of histone modifications, or could the entire panoply of histone modifications associated with transcription now be revisited with a much more dynamic perspective?
Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Transcripción Genética/genética , Acetilación , Animales , Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Nucleosomas/genética , Nucleosomas/metabolismo , Activación TranscripcionalAsunto(s)
Cromatina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Saccharomyces cerevisiae/genética , Dominio Catalítico , Inmunoprecipitación de Cromatina , Histonas/metabolismo , Sistema de Señalización de MAP Quinasas , Análisis de Secuencia por Matrices de Oligonucleótidos , Presión Osmótica , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4)-methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9-methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9-methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.
Asunto(s)
Núcleo Celular/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Acetilación/efectos de los fármacos , Animales , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Histonas/química , Ácidos Hidroxámicos/farmacología , Lisina/química , Metilación/efectos de los fármacos , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Proto-Oncogénicas c-jun/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Phosphorylation of histone H3 is implicated in transcriptional activation and chromosome condensation, but its immediate molecular function has remained obscure. By affinity chromatography of nuclear extracts against modified H3 tail peptides, we identified 14-3-3 isoforms as proteins that bind these tails in a strictly phosphorylation-dependent manner. Acetylation of lysines 9 and 14 does not impede 14-3-3 binding to serine 10-phosphorylated H3 tails. In vivo, 14-3-3 is inducibly recruited to c-fos and c-jun nucleosomes upon gene activation, concomitant with H3 phosphoacetylation. We have determined the structures of 14-3-3zeta complexed with serine 10-phosphorylated or phosphoacetylated H3 peptides. These reveal a distinct mode of 14-3-3/phosphopeptide binding and provide a structural understanding for the lack of effect of acetylation at lysines 9 and 14 on this interaction. 14-3-3 isoforms thus represent a class of proteins that mediate the effect of histone phosphorylation at inducible genes.
Asunto(s)
Proteínas 14-3-3/metabolismo , Histonas/metabolismo , Proteínas 14-3-3/química , Acetilación , Secuencia de Aminoácidos , Animales , Línea Celular , Cristalografía por Rayos X , Células HeLa , Histonas/química , Histonas/aislamiento & purificación , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Nucleosomas/genética , Nucleosomas/metabolismo , Fosforilación , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Alineación de Secuencia , Serina/química , Serina/metabolismoRESUMEN
ERK and p38 MAP kinases, acting through the downstream mitogen- and stress-activated kinase 1/2 (MSK1/2), elicit histone H3 phosphorylation on a subfraction of nucleosomes--including those at Fos and Jun--concomitant with gene induction. S10 and S28 on the H3 tail have both been shown to be phospho-acceptors in vivo. Both phospho-epitopes appear with similar time-courses and both occur on H3 tails that are highly sensitive to TSA-induced hyperacetylation, similarities which might suggest that MSK1/2 phosphorylates both sites on the same H3 tails. Indeed, on recombinant histone octamers in vitro, MSK1 efficiently phosphorylates both sites on the same H3 tail. However, sequential immunoprecipitation studies show that antibodies against phosphorylated S10-H3 recover virtually all this epitope without depletion of phosphorylated S28-H3, and vice versa, indicating that the two phospho-epitopes are not located on the same H3 tail in vivo. Confocal immunocytochemistry confirms the clear physical separation of the two phospho-epitopes in the intact mouse nucleus. Finally, we used transfection-based experiments to test models that might explain such differential targeting. Overexpression and delocalisation of MSK1 does not result in the breakdown of targeting in vivo despite the fact that the ectopic kinase is fully activated by external stimuli. These studies reveal a remarkable level of targeting of S10 and S28 phosphorylation to distinct H3 tails within chromatin in the interphase mouse nucleus. Possible models for such exquisite targeting are discussed.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Proteína Quinasa 11 Activada por Mitógenos/fisiología , Proteína Quinasa 8 Activada por Mitógenos/fisiología , Acetilación , Animales , Anisomicina/farmacología , Línea Celular , Núcleo Celular/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Ácidos Hidroxámicos/farmacología , Interfase/fisiología , Ratones , Fosforilación , Serina/metabolismo , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
Genome-wide and gene-specific changes in histone H3 phosphorylation during heat shock have recently been described using two well-established experimental models, the "puffing" of heat shock loci in Drosophila polytene chromosomes and the induction of hsp70 mRNA transcripts in cultured mouse cells. Despite conservation of the molecular participants and overall stress response in these two organisms, some striking differences have emerged. Here, we summarize accounts of heat shock-modulated histone phosphorylation in Drosophila and mouse cells highlighting these differences. In addition, we describe a further complexity of this response in cultured mouse cells that becomes apparent when the nucleosomal response, referring to histone H3 and HMGN1 phosphorylation, is monitored through the cell cycle. This suggests that some heat shock-induced effects in mouse cells may be indirect and arise as a secondary consequence of the effect of heat shock on the cell cycle, complicating comparisons between the fly and mouse systems.
Asunto(s)
Ciclo Celular/fisiología , Cromosomas/fisiología , Respuesta al Choque Térmico/fisiología , Histonas/genética , Histonas/metabolismo , Animales , Ciclo Celular/genética , Línea Celular , Cromatina/genética , Cromatina/fisiología , Cromosomas/genética , Drosophila , Regulación de la Expresión Génica , Genes de Insecto , Proteína HMGN1/genética , Proteína HMGN1/fisiología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Ratones , Nucleosomas/fisiología , Fosforilación , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
A question of major current interest is whether histone modification at a given gene correlates simply with transcriptional status or if distinctive modifications appear depending on how that gene is activated. The stress-inducible gene Hsp70 is activated by heat shock or by sodium arsenite. Heat shock produces acetylation of histone H4 at Hsp70 chromatin, whereas arsenite produces both H4 acetylation and H3 phosphorylation at the gene. Histone H3 remains markedly hypoacetylated at Hsp70 under these conditions. Arsenite, but not heat shock, requires signaling via p38 MAP kinase for Hsp70 induction and histone H3 phosphorylation. However, independently of p38 MAP kinase, both stresses strongly activate the transcription factor Hsf1. Using Hsf1-/- cells, we show that this factor is responsible for targeting histone H4 acetylation to Hsp70 chromatin. We establish here that histone modifications at inducible genes are not simply a reflection of transcriptional activity, but are strictly dependent on the stimulus used for induction.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Histonas/metabolismo , Factores de Transcripción/metabolismo , Animales , Arsenitos/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/metabolismo , Fibroblastos/citología , Fibroblastos/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Calor , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Compuestos de Sodio/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Mitogen-activated protein kinase (MAPK) pathways regulate eukaryotic gene expression in response to extracellular stimuli. MAPKs and their downstream kinases phosphorylate transcription factors, co-regulators and chromatin proteins to initiate transcriptional changes. However, the spatial context in which the MAPKs operate in transcription complexes is poorly understood. Recent findings in budding yeast show that MAPKs can form integral components of transcription complexes and have novel structural functions in addition to phosphorylating local substrates. Hog1p MAPK is stably recruited to target promoters by specific transcription factors in response to osmotic stress, and acts as both a structural adaptor and enzymatic activator driving the assembly and activation of the transcription complex. We review the evidence that suggests a similar bifunctional role for MAPKs in mammalian transcription complexes.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/fisiología , Transcripción Genética , Activación Enzimática , Modelos Biológicos , FosforilaciónRESUMEN
The phosphorylation and acetylation (phosphoacetylation) of histone H3 tails concomitant with gene activation is now well established and has been observed at several inducible genes. However, two aspects of this response have been controversial. The first relates to the identity of the kinase that phosphorylates histone H3. Experiments with Coffin-Lowry cells purporting to show that Rsk2 was the histone H3 kinase have proven to be irreproducible. The second relates to the proposition that histone H3 phosphorylation and acetylation are 'synergistic and coupled' in mammalian cells. But here too, some of the experiments have not been reproducible and some of the key statements contaminated by issues of antibody specificity. More recent studies indicate that H3 phosphorylation and acetylation are independently targeted to the same histone H3 tail.
Asunto(s)
Acetiltransferasas/metabolismo , Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Fosfotransferasas/metabolismo , Acetilación , Animales , Histona Acetiltransferasas , Mamíferos/genética , Mamíferos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosforilación , Transducción de Señal/fisiología , Activación TranscripcionalRESUMEN
That signalling pathways, particularly the mitogen-activated protein kinase cascades, elicit modification of chromatin proteins such as histone H3 by phosphorylation and/or acetylation concomitant with gene activation is now well established. The picture that is emerging is one of a complex and dynamic pattern of multiple modifications at the H3 tail. Here, we review the inducible gene systems where H3 modifications have been reported and re-evaluate the controversy as to the kinase(s) that phosphorylates it as well as the proposed coupling between H3 phosphorylation and acetylation.
Asunto(s)
Histonas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Acetilación , Animales , Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Histonas/química , Humanos , Sistema de Señalización de MAP Quinasas , Fosforilación , Transducción de Señal , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
Cells respond to mitogenic or stress stimuli by the rapid induction of immediate-early (IE) genes, which occurs concomitantly with the phosphorylation of histone H3 and the high-mobility-group protein HMG-14. In mammalian cells this response is mediated via ERK and p38 MAP kinase pathways, but the identity of the downstream kinase that phosphorylates histone H3 has been contentious. One study, based on Coffin- Lowry cells defective in RSK2, reported that RSK2 was the histone H3 kinase, while a second study, based on the efficiency of RSKs and MSKs as in vitro histone H3 kinases, and their relative susceptibility to kinase inhibitors, suggested that MSKs were responsible. We show here that the histone H3 phosphorylation response is normal in Coffin-Lowry cells. Further more, we show that histone H3 and HMG-14 phosphorylation is severely reduced or abolished in mice lacking MSK1 and MSK2. We also show that, despite this, histone H3 acetylation is unimpaired in these cells and that IE genes can be induced, although at a reduced efficiency. We conclude that MSKs are the major kinases for histone H3 and HMG-14 in response to mitogenic and stress stimuli in fibroblasts.
Asunto(s)
Proteína HMGN1/metabolismo , Histonas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Acetilación , Animales , Anisomicina/farmacología , Células Cultivadas , Genes fos , Genes jun , Ratones , Ratones Noqueados , Mitógenos/farmacología , Mitosis , Nucleosomas/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas 90-kDa/deficiencia , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Estrés Fisiológico , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Switching mechanisms that control genes could be viewed either as stable binary switches, in which genes exist in 'on' or 'off' states; or as quantitative rheostat-like switches, in which the rate of transcription is continuously variable and coupled directly to the strength of intracellular signalling events. Here, we discuss the biological need for quantitative gene regulation and, using mitogen-activated protein kinase (MAPK)-controlled transcription as a model, assess the evidence for its existence and postulate mechanisms by which it might occur.