RESUMO
The technique of chromatin immunoprecipitation (ChIP) is a powerful method for identifying in vivo DNA binding sites of transcription factors and for studying chromatin modifications. Unfortunately, the large number of cells needed for the standard ChIP protocol has hindered the analysis of many biologically interesting cell populations that are difficult to obtain in large numbers. New ChIP methods involving the use of carrier chromatin have been developed that allow the one-gene-at-a-time analysis of very small numbers of cells. However such methods are not useful if the resultant sample will be applied to genomic microarrays or used in ChIP-sequencing assays. Therefore, we have miniaturized the ChIP protocol such that as few as 10,000 cells (without the addition of carrier reagents) can be used to obtain enough sample material to analyze the entire human genome. We demonstrate the reproducibility of this MicroChIP technique using 2.1 million feature high-density oligonucleotide arrays and antibodies to RNA polymerase II and to histone H3 trimethylated on lysine 27 or lysine 9.
Assuntos
Imunoprecipitação da Cromatina/métodos , Genoma Humano , Genômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Anticorpos , Cromatina/genética , Cromatina/imunologia , Imunoprecipitação da Cromatina/normas , Metilação de DNA , Genômica/normas , Histonas/genética , Histonas/imunologia , Humanos , Miniaturização , Análise de Sequência com Séries de Oligonucleotídeos/normas , RNA Polimerase II/genética , RNA Polimerase II/imunologia , Reprodutibilidade dos Testes , Fatores de Transcrição/genéticaRESUMO
Liver cancer is very common worldwide and the rates of hepatocellular carcinoma (HCC) have increased by over 70% in the last 2 decades in the US. Late diagnosis, because of the lack of clinical symptoms, and decreased hepatic function, because of underlying hepatic disease, lead to the extremely high mortality rates associated with HCC. Clearly, the identification of markers that are expressed early in the development of HCC and that are easily detected in high-risk patients would aid in early diagnosis and increased survival. We present the cloning and characterization of a novel gene, CRG-L2 (Cancer related gene-Liver 2), which displays high expression in murine and human hepatocellular carcinomas. Using in situ hybridization, we show that CRG-L2 mRNA levels are increased early during the development of liver tumors in C3H/HeJ mice, and that in normal tissues CRG-L2 mRNA is restricted to the murine testis and human placenta. Its restricted expression in normal tissues and unique early upregulation during tumor development make CRG-L2 an excellent candidate as a new clinical marker of HCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Hepáticas/patologia , Proteínas/metabolismo , Sequência de Bases , Primers do DNA , DNA Complementar , Humanos , Hibridização In Situ , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso , RNA Mensageiro/genética , Regulação para CimaRESUMO
Methylation of cytosine in the context of CpG dinucleotides is an epigenetic phenomenon in eukaryotes that plays important roles in genome function and transcription regulation. Aberrant changes in DNA methylation is an important feature of several human diseases such as cancer and neurological disorders. These discoveries have opened a new field of new therapies and diagnostics. During recent years, there has been a revolution in DNA methylation analysis technologies. This article focuses on methods with which to study DNA methylation that employ protein domains that specifically recognize either 5-methyl-cytosine in the CpG context or nonmethylated DNA, and methods developed for the detection of 5-hydroxymethylcytosine, the recently described epigenetic mark known as the sixth base of the epigenome.
Assuntos
5-Metilcitosina/metabolismo , Biomarcadores/metabolismo , Citosina/análogos & derivados , Metilação de DNA/genética , Reação em Cadeia da Polimerase/métodos , Domínios e Motivos de Interação entre Proteínas/genética , Ilhas de CpG/genética , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Primers do DNA/genética , Humanos , Imunoprecipitação/métodos , Sensibilidade e Especificidade , SulfitosRESUMO
There is widespread interest in efficient characterization of differences between tumor and normal samples. Here, we show an effective methodology for genome-scale characterization of tumors. Using matched normal and tumor samples from liver cancer patients, as well as non-cancer-related normal liver tissue, we first determined changes in gene expression as monitored on RNA expression arrays. We identified several hundred mRNAs that were consistently changed in the tumor samples. To characterize the mechanisms responsible for creation of the tumor-specific transcriptome, we performed chromatin immunoprecipitation on microarray experiments to assay binding of RNA polymerase II, H3me3K27, and H3me3K9 and DNA methylation in 25,000 promoter regions. These experiments identified changes in active and silenced regions of the genome in the tumor cells. Finally, we used a "virtual comparative genomic hybridization" method to identify copy number alterations in the tumor samples. Through comparison of RNA polymerase II binding, chromatin structure, DNA methylation, and copy number changes, we suggest that the major contributor to creation of the liver tumor transcriptome was changes in gene copy number.
Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Fígado/fisiologia , Idoso , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Criopreservação , Feminino , Deleção de Genes , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA/genética , RNA/isolamento & purificação , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Transcrição GênicaRESUMO
We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent versus differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a preinitiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl-cytosine, and POLR2A. We found that (1) the percentage of low expressed genes bound by POLR2A, H3me3K27, H3me3K9, or 5-methyl-cytosine is similar in all 12 cell types, regardless of differentiation or neoplastic state; (2) a gene is generally repressed by only one mechanism; and (3) distinct classes of genes are repressed by certain mechanisms. We further characterized two transitioning cell populations, 3T3 cells progressing from G0/G1 into S phase and mES cells differentiating into embryoid bodies. We found that the transient regulation through the cell cycle was achieved predominantly by changes in the recruitment of the general transcriptional machinery or by post-POLR2A recruitment mechanisms. In contrast, changes in chromatin silencing were critical for the permanent changes in gene expression in cells undergoing differentiation.