RESUMEN
Mesenchymal stem cells' differentiation into several lineages is coordinated by a complex of transcription factors and co-regulators which bind to specific gene promoters. The Chromatin-Related Mesenchymal Modulator, CHD9 demonstrated in vitro its ability for remodeling activity to reposition nucleosomes in an ATP-dependent manner. Epigenetically, CHD9 binds with modified H3-(K9me2/3 and K27me3). Previously, we presented a role for CHD9 with RNA Polymerase II (Pol II)-dependent transcription of tissue specific genes. Far less is known about CHD9 function in RNA Polymerase I (Pol I) related transcription of the ribosomal locus that also drives specific cell fate. We here describe a new form, the nucleolar CHD9 (n-CHD9) that is dynamically associated with Pol I, fibrillarin, and upstream binding factor (UBF) in the nucleoli, as shown by imaging and molecular approaches. Inhibitors of transcription disorganized the nucleolar compartment of transcription sites where rDNA is actively transcribed. Collectively, these findings link n-CHD9 with RNA pol I transcription in fibrillar centers. Using chromatin immunoprecipitation (ChIP) and tilling arrays (ChIP- chip), we find an association of n-CHD9 with Pol I related to rRNA biogenesis. Our new findings support the role for CHD9 in chromatin regulation and association with rDNA genes, in addition to its already known function in transcription control of tissue specific genes.
Asunto(s)
Diferenciación Celular/genética , ADN Ribosómico/genética , Células Madre Mesenquimatosas/citología , Transactivadores/genética , Animales , Células COS , Linaje de la Célula , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Chlorocebus aethiops , Cromatina/genética , ADN Helicasas , Regulación de la Expresión Génica , Genes de ARNr , Células Madre Mesenquimatosas/metabolismo , Ratones , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , Ribosomas/genética , Transactivadores/metabolismoRESUMEN
Mutations of lamin A/C (LMNA) cause a wide range of human disorders, including progeria, lipodystrophy, neuropathies and autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD). EDMD is also caused by X-linked recessive loss-of-function mutations of emerin, another component of the inner nuclear lamina that directly interacts with LMNA. One model for disease pathogenesis of LMNA and emerin mutations is cell-specific perturbations of the mRNA transcriptome in terminally differentiated cells. To test this model, we studied 125 human muscle biopsies from 13 diagnostic groups (125 U133A, 125 U133B microarrays), including EDMD patients with LMNA and emerin mutations. A Visual and Statistical Data Analyzer (VISDA) algorithm was used to statistically model cluster hierarchy, resulting in a tree of phenotypic classifications. Validations of the diagnostic tree included permutations of U133A and U133B arrays, and use of two probe set algorithms (MAS5.0 and MBEI). This showed that the two nuclear envelope defects (EDMD LMNA, EDMD emerin) were highly related disorders and were also related to fascioscapulohumeral muscular dystrophy (FSHD). FSHD has recently been hypothesized to involve abnormal interactions of chromatin with the nuclear envelope. To identify disease-specific transcripts for EDMD, we applied a leave-one-out (LOO) cross-validation approach using LMNA patient muscle as a test data set, with reverse transcription-polymerase chain reaction (RT-PCR) validations in both LMNA and emerin patient muscle. A high proportion of top-ranked and validated transcripts were components of the same transcriptional regulatory pathway involving Rb1 and MyoD during muscle regeneration (CRI-1, CREBBP, Nap1L1, ECREBBP/p300), where each was specifically upregulated in EDMD. Using a muscle regeneration time series (27 time points) we develop a transcriptional model for downstream consequences of LMNA and emerin mutations. We propose that key interactions between the nuclear envelope and Rb and MyoD fail in EDMD at the point of myoblast exit from the cell cycle, leading to poorly coordinated phosphorylation and acetylation steps. Our data is consistent with mutations of nuclear lamina components leading to destabilization of the transcriptome in differentiated cells.
Asunto(s)
Lamina Tipo A/genética , Músculo Esquelético/fisiología , Distrofias Musculares/genética , Membrana Nuclear/patología , Regeneración/genética , Biopsia , Niño , Dermatoglifia del ADN , Perfilación de la Expresión Génica/métodos , Humanos , Proteínas de la Membrana/genética , Modelos Estadísticos , Músculo Esquelético/patología , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patología , Mutación , Proteína MioD/metabolismo , Proteínas Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Unión Proteica , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Timopoyetinas/genética , Transcripción GenéticaRESUMEN
The tumor suppressor p53 and its close relative p73 are activated in response to DNA damage resulting in either cell cycle arrest or apoptosis. Here, we show that DNA damage induces the acetylation of p73 by the acetyltransferase p300. Inhibiting the enzymatic activity of p300 hampers apoptosis in a p53(-/-) background. Furthermore, a nonacetylatable p73 is defective in activating transcription of the proapoptotic p53AIP1 gene but retains an intact ability to regulate other targets such as p21. Finally, p300-mediated acetylation of p73 requires the protooncogene c-abl. Our results suggest that DNA damage-induced acetylation potentiates the apoptotic function of p73 by enhancing the ability of p73 to selectively activate the transcription of proapoptotic target genes.