RESUMO
Androgen receptor (AR) is a critical transcription factor that regulates various target genes and contributes to the pathophysiology of prostate cancer hormone dependently. Here, we identify amyloid precursor protein (APP) as a primary androgen target through chromatin immunoprecipitation (ChIP) combined with genome tiling array analysis (ChIP-chip). ChIP-treated DNA were obtained from prostate cancer LNCaP cells with R1881 or vehicle treatment using AR or acetylated histone H3 antibodies. Ligand-dependent AR binding was further enriched by PCR subtraction. Using chromosome 21/22 arrays, we identified APP as one of the androgen-regulated genes with adjacent functional AR binding sites. APP expression is androgen-inducible in LNCaP cells and APP immunoreactivity was correlated with poor prognosis in patients with prostate cancer. Gain-of-function and loss-of-function studies revealed that APP promotes the tumor growth of prostate cancer. The present study reveals a novel APP-mediated pathway responsible for the androgen-dependent growth of prostate cancer. Our findings will indicate that APP could be a potential molecular target for the diagnosis and treatment of prostate cancer.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Neoplasias Hormônio-Dependentes/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Receptores de Superfície Celular/genética , Acetilação , Idoso , Precursor de Proteína beta-Amiloide/biossíntese , Androgênios/fisiologia , Animais , Sítios de Ligação , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Processos de Crescimento Celular/fisiologia , Imunoprecipitação da Cromatina , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Nexinas de Proteases , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Androgênicos/metabolismo , Receptores de Superfície Celular/biossínteseRESUMO
TP53 (tumor protein p53; p53) regulates its target genes under various cellular stresses. By combining chromatin immunoprecipitation with oligonucleotide microarrays, we have mapped binding sites of p53 (p53-BS) in the genome of HCT116 human colon carcinoma cells, along with those of acetylated H3, acetylated H4, and methylated H3-K4. We analyzed a 30-Mb portion of the human genome selected as a representative model by the ENCODE Consortium. In the region, we found 37 p53-BS, of which the p53-binding motif was present in 32 (86%). Acetylated histone H3 and H4 were detected at 14 (38%) and 33 (89%) of the p53-BS, respectively. A significant portion (58%) of H4 acetylation in the p53-BS was not accompanied by H3 acetylation. Acetyl H3 were preferentially located at the 5' and 3' ends of genes, whereas acetyl H4 were distributed widely across the genome. These results provide novel insights into how p53 binding coordinates with histone modification in human.
Assuntos
Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico , DNA/genética , DNA/metabolismo , Genes p53 , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Ativação TranscricionalRESUMO
DNA methylation is an epigenetic mark crucial in regulation of gene expression. Aberrant DNA methylation causes silencing of tumor suppressor genes and promotes chromosomal instability in human cancers. Most of previous studies for DNA methylation have focused on limited genomic regions, such as selected genes or promoter CpG islands (CGIs) containing recognition sites of methylation-sensitive restriction enzymes. Here, we describe a method for high-resolution analysis of DNA methylation using oligonucleotide tiling arrays. The input material is methylated DNA immunoprecipitated with anti-methylcytosine antibodies. We examined the ENCODE region ( approximately 1% of human genome) in three human colorectal cancer cell lines and identified over 700 candidate methylated sites (CMS), where 24 of 25 CMS selected randomly were subsequently verified by bisulfite sequencing. CMS were enriched in the 5' regulatory regions and the 3' regions of genes. We also compared DNA methylation patterns with histone H3 and H4 acetylation patterns in the HOXA cluster region. Our analysis revealed no acetylated histones in the hypermethylated region, demonstrating reciprocal relationship between DNA methylation and histone H3 and H4 acetylation. Our method recognizes DNA methylation with little bias by genomic location and, therefore, is useful for comprehensive high-resolution analysis of DNA methylation providing new findings in the epigenomics.
Assuntos
Metilação de DNA , Predisposição Genética para Doença/genética , Genoma Humano/genética , Acetilação , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sítios de Ligação/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Biologia Computacional/métodos , Decitabina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Análise em Microsséries/métodos , Sondas de Oligonucleotídeos , Análise de Sequência de DNA/métodosRESUMO
Various functions are integrated into a single chromosome molecule. The genomic approach (ChIP-chip) we introduce here is a very powerful tool to study dynamic changes of the structure and function of the chromosome at the level of protein-DNA interaction as precisely as possible without prejudice or bias. This technology opens up the way to understand how local protein-protein or protein-DNA interactions lead to the dynamic changes of chromosome structure and how various chromosomal functions are connected to make a network for the faithful maintenance of the genome.