RESUMEN
We show that the 3' boundary of the chicken beta-globin locus bears striking structural similarities to the 5' boundary. In erythroid cells a clear transition in DNase I sensitivity of chromatin at the 3' end of the locus is observed, the location of this transition is marked by a constitutive DNase I hypersensitive site (HS), and DNA spanning this site has the enhancer-blocking capacity of an insulator. This HS contains a binding site for the transcription factor CTCF. As in the case of the 5' insulator, the CTCF site is both necessary and sufficient for the enhancer-blocking activity of the 3' boundary. The position of this insulator is consistent with our proposal that it may function to maintain the distinct regulatory programs of the globin genes and their closely appended 3' neighbor, an odorant receptor gene. We conclude that both boundaries of the chicken beta-globin domain are capable of playing functionally similar roles and that the same protein is a necessary component of the molecular mechanism through which these boundaries are defined.
Asunto(s)
Cromatina/química , Globinas/química , Proteínas Represoras , Animales , Sitios de Unión , Factor de Unión a CCCTC , Pollos , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Globinas/genética , Globinas/metabolismo , Especificidad por Sustrato , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
H19 and Igf2 are linked and reciprocally imprinted genes. We demonstrate that the histones associated with the paternally inherited and unexpressed H19 allele are less acetylated than those associated with the maternal expressed allele. Cell growth in the presence of inhibitors of either histone deacetylase or DNA methylation activated the silent Igf2 allele, whereas derepression of the silent H19 allele required combined inhibition of DNA methylation and histone deacetylation. Our results indicate that histone acetylation as well as DNA methylation contribute to the somatic maintenance of H19 and Igf2 imprinting and that silencing of the imprinted alleles of these two genes is maintained via distinct mechanisms.
Asunto(s)
Metilación de ADN , Impresión Genómica , Histonas/metabolismo , Factor II del Crecimiento Similar a la Insulina/genética , Proteínas Musculares/genética , ARN no Traducido , Acetilación , Alelos , Animales , Células Cultivadas , Cromatina/metabolismo , Padre , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Madres , Nucleosomas/metabolismo , ARN Largo no Codificante , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The constitutive DNase I hypersensitive site at the 5' end of the chicken beta-globin locus marks the boundary of the active chromatin domain in erythroid cells. The DNA sequence containing this site has the properties of an insulator, as shown by its ability in stable transformation experiments to block enhancer-promoter interaction when it lies between the two, but not when it lies outside, and to protect against position effects in Drosophila. We now show that the chicken insulator can protect a stably integrated gene, which is otherwise subject to great variability of expression, from chromatin-mediated repression in cell culture. When the integrated reporter gene is surrounded by insulator elements, stably transformed cell lines display consistent enhancer-dependent expression levels, in accord with the strength of the enhancer. In the absence of insulators, long-term nonselective propagation of cells carrying the integrated reporter gene results in gradual extinction of the reporter's expression, with expression patterns from tandemly repeated inserted genes suggesting that the extinction of adjacent genes is coupled. We show that the uninsulated reporter genes, in addition to becoming transcriptionally inactive, lose several epigenetic hallmarks of active chromatin, including nuclease accessibility, DNA hypomethylation, and histone hyperacetylation during time in culture. Treatment with inhibitors of histone deacetylase or DNA methylation reverses the extinction of the uninsulated genes. Extinction is completely prevented by flanking the reporter construct with insulators. Furthermore, in contrast to the uninsulated reporter genes, chromatin over the insulated genes retains nuclease accessibility and histone hyperacetylation. However, there is no clear correlation between the presence of the insulators and the level of DNA methylation. This leads us to propose a model for the insulator's ability to protect against extinction in the transformed cell lines and to function as a chromatin boundary for the chicken beta-globin locus in normal erythroid cells.
Asunto(s)
Metilación de ADN , Globinas/genética , Histonas/metabolismo , Acetilación , Animales , Línea Celular , Pollos , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , ADN Complementario/química , ADN Complementario/genética , Elementos de Facilitación Genéticos , Células Precursoras Eritroides/metabolismo , Expresión Génica , Genes Reporteros , Histonas/química , Modelos Biológicos , Regiones Promotoras Genéticas , Receptores de Interleucina-2/genética , Transcripción Genética , TransfecciónRESUMEN
The ability of transcription factors to gain access to their sites in chromatin requires the disruption or displacement of nucleosomes covering the promoter, signalled by the generation of a nuclease hypersensitive site. We characterise here the alterations in nucleosome structure caused by binding of the erythroid factor GATA-1 to a nucleosome carrying GATA-1 sites. DNase I and micrococcal nuclease probes show that GATA-1 binding causes extensive, cooperative breakage of the histone/DNA contacts to generate a complex very similar to that formed by the factor with free DNA. The only region which differs is confined to about 50 bp surrounding the nucleosome dyad axis which appears to be the domain of residual contact between the DNA and histone octamer. Despite considerable breakage of the histone/DNA contacts, the complex is completely stable in solution, and disruption of the nucleosome is entirely reversible: it is regenerated quantitatively upon removal of the transcription factor. Moreover, the histone 2A/2B component of the octamer does not exchange to external competitor. We suggest that formation of this complex may be a step in the generation of a fully hypersensitive site in vivo over regulatory elements containing GATA family binding sites.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Pollos , Cromatina/química , ADN/química , Huella de ADN , Desoxirribonucleasa I/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Regulación de la Expresión Génica/genética , Histonas/química , Nucleasa Microcócica/metabolismo , Modelos Moleculares , Oligodesoxirribonucleótidos/metabolismo , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Proteínas Recombinantes/metabolismoAsunto(s)
Cromatina/genética , Transcripción Genética , Animales , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Globinas/genética , Humanos , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Biochemical characterization of cGATA-1, a key transcription factor in the regulation of globin expression in chickens, has been precluded by the unavailability of appreciable amounts of the pure protein. Purification directly from embryonic red blood cells has been limited by the difficulty in obtaining large quantities of the starting material, and previous attempts at bacterial expression have consistently yielded truncated product. To solve these problems, we have taken two approaches to the expression of cGATA-1. First, we were able to produce efficient expression from baculovirus-infected insect cells. Second, by altering the codon usage in cDNA encoding the protein's carboxy-terminal region, we obtained good expression of full-length protein in Escherichia coli. These preparations should prove useful in biochemical and structural studies of the factor. Additionally, we describe a primer extension/PCR-based method which can be used to synthesize extended regions of DNA sequence for gene construction.
Asunto(s)
Codón , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Dedos de Zinc , Animales , Baculoviridae , Secuencia de Bases , Proteínas de Unión al ADN/química , Desoxirribonucleasa EcoRI/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Vectores Genéticos , Datos de Secuencia Molecular , Proteínas Nucleares/química , Mapeo Restrictivo , Spodoptera , Factores de Transcripción/químicaRESUMEN
Recent studies suggest that enhancers may increase the accessibility of chromatin to transcription factors. To test the effects of a viral enhancer on chromatin accessibility, we have inserted minigenes with or without the polyomavirus enhancer into the third exon of the hypoxanthine phosphoribosyltransferase (HPRT) gene by homologous recombination and have prepared high-resolution maps of gene accessibility by using a novel polymerase chain reaction assay for DNase I sensitivity. In its native state, we find that the HPRT gene has low sensitivity to DNase I in fibrosarcoma cells. Insertion of the polyomavirus enhancer and neo reporter gene into exon 3 confers altered HPRT DNase I sensitivity for several kilobases on either side of the enhancer. The changes in DNase I sensitivity peak near the enhancer and decline with distance from the enhancer. The increase in HPRT DNase I sensitivity persisted when the tk promoter was deleted from the inserted construct but disappeared when the enhancer was deleted. These experiments identify the polyomavirus enhancer as a cis-acting initiator of chromatin accessibility.
Asunto(s)
Cromatina/metabolismo , Elementos de Facilitación Genéticos , Hipoxantina Fosforribosiltransferasa/genética , Poliomavirus/genética , Integración Viral/genética , Secuencia de Bases , Línea Celular , Desoxirribonucleasa I , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fos and junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.
Asunto(s)
División Celular , Senescencia Celular/fisiología , Genes fos , Genes jun , Genes myc , Genes p53 , Genes ras , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/biosíntesis , Secuencia de Bases , Línea Celular , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Histonas/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Cinética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Factores de TiempoRESUMEN
Previous studies have implicated histone H5 dephosphorylation as a causal factor in genetic inactivation and chromatin condensation during erythroid senescence in adult chickens. We show that histone H5 phosphorylation declines in two stages as various cohorts of erythroid cells senesce in chick embryos. The first decline occurs between 5 and 6 days and coincides with the senescence of primitive erythrocytes. The second decline in H5 phosphorylation occurs between 17 and 19 days of chicken development, when the definitive erythrocytes undergo senescence and chromatin condensation. These results point to a role for histone dephosphorylation during the programmed senescence of erythroid cells.