RESUMEN
The Drosophila male-specific lethal (MSL) dosage compensation complex increases transcript levels on the single male X chromosome to equal the transcript levels in XX females. However, it is not known how the MSL complex is linked to its DNA recognition elements, the critical first step in dosage compensation. Here, we demonstrate that a previously uncharacterized zinc finger protein, CLAMP (chromatin-linked adaptor for MSL proteins), functions as the first link between the MSL complex and the X chromosome. CLAMP directly binds to the MSL complex DNA recognition elements and is required for the recruitment of the MSL complex. The discovery of CLAMP identifies a key factor required for the chromosome-specific targeting of dosage compensation, providing new insights into how subnuclear domains of coordinate gene regulation are formed within metazoan genomes.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Compensación de Dosificación (Genética) , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Cromosoma X/genética , Cromosoma X/metabolismo , Animales , Línea Celular , Femenino , Masculino , Unión ProteicaRESUMEN
The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. Here we use global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome.
Asunto(s)
Cromosomas de Insectos/genética , Compensación de Dosificación (Genética)/genética , Drosophila melanogaster/genética , Transcripción Genética , Cromosoma X/genética , Acetilación , Animales , Línea Celular , Cromosomas de Insectos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Genes de Insecto/genética , Genes Ligados a X/genética , Histonas/química , Histonas/metabolismo , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Cromosoma X/metabolismoRESUMEN
Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Drosophila melanogaster/genética , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/metabolismo , Desoxirribonucleasa I/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Exones/genética , Regulación de la Expresión Génica/genética , Genes de Insecto/genética , Genoma de los Insectos/genética , Histonas/química , Histonas/metabolismo , Masculino , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 1 , ARN/análisis , ARN/genética , Análisis de Secuencia , Transcripción Genética/genéticaRESUMEN
BACKGROUND: Hepatocyte nuclear factor 4α (HNF4α), a liver-specific transcription factor, plays a significant role in liver-specific functions. However, its functions are poorly understood in the regulation of the inflammatory response. In order to obtain a genomic view of HNF4α in this context, microarray analysis was used to probe the expression profile of an inflammatory response induced by cytokine stimulation in a model of HNF4α knock-down in HepG2 cells. RESULTS: The expression of over five thousand genes in HepG2 cells is significantly changed with the dramatic reduction of HNF4α concentration compared to the cells with native levels of HNF4α. Over two thirds (71%) of genes that exhibit differential expression in response to cytokine treatment also reveal differential expression in response to HNF4α knock-down. In addition, we found that a number of HNF4α target genes may be indirectly mediated by an ETS-domain transcription factor ELK1, a nuclear target of mitogen-activated protein kinase (MAPK). CONCLUSION: The results indicate that HNF4α has an extensive impact on the regulation of a large number of the liver-specific genes. HNF4α may play a role in regulating the cytokine-induced inflammatory response. This study presents a novel function for HNF4α, acting not only as a global player in many cellular processes, but also as one of the components of inflammatory response in the liver.
Asunto(s)
Perfilación de la Expresión Génica , Factor Nuclear 4 del Hepatocito/genética , Inflamación/genética , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Citocinas/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Hígado/metabolismo , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
DNA shape variation and the associated variation in minor groove electrostatic potential are widely exploited by proteins for DNA recognition. Here we show that the hydroxyl radical cleavage pattern is a quantitative measure of DNA backbone solvent accessibility, minor groove width, and minor groove electrostatic potential, at single nucleotide resolution. We introduce maps of DNA shape and electrostatic potential as tools for understanding how proteins recognize binding sites in a genome. These maps reveal periodic structural signals in yeast and Drosophila genomic DNA sequences that are associated with positioned nucleosomes.