RESUMO
We conducted a genomewide screen for prostate cancer-susceptibility genes on the basis of data from 98 families from the United States and Canada that had three or more verified diagnoses of prostate cancer among first- and second-degree relatives. We found a statistically significant excess of markers for which affected relatives exhibited modest amounts of excess allele-sharing; however, no single chromosomal region contained markers with excess allele-sharing of sufficient magnitude to indicate unequivocal evidence of linkage. Positive linkage signals of nominal statistical significance were found in two regions (5p-q and 12p) that have been identified as weakly positive in other data sets and in region 19p, which has not been identified previously. All these signals were considerably stronger for analyses restricted to families with mean age at onset below the median than for analyses of families with mean age at onset above the median. The data provided little support for any of the putative prostate cancer-susceptibility genes identified in other linkage studies.
Assuntos
Heterogeneidade Genética , Predisposição Genética para Doença/genética , Neoplasias da Próstata/genética , Idade de Início , Idoso , Alelos , Canadá , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 5/genética , Ligação Genética/genética , Marcadores Genéticos/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/epidemiologia , Grupos Raciais/genética , Estatísticas não Paramétricas , Estados UnidosRESUMO
We have utilized the Escherichia coli lac repressor-operator system to test whether protein binding can interfere with de novo DNA methylation in mammalian cells. We find that a DNA binding protein can protect sites on the episome as well as in the genome from the de novo methylation activity of Dnmt3a. Transcriptional machinery moving through the binding sites does not affect the de novo methylation of these sites, and it does not affect the binding protein protection of these sites from de novo methylation. This study and previous studies provide a possible mechanism for the observation that an Sp1 site can serve as a cis-acting signal for demethylation and for preventing de novo methylation of the CpG island upstream of the mouse adenine phosphoribosyltransferase (Aprt) gene. These findings also support the hypothesis that protein binding may play a crucial role in changes of CpG methylation pattern in mammalian cells.
Assuntos
Proteínas de Bactérias/metabolismo , Metilação de DNA , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Plasmídeos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Repressores Lac , Plasmídeos/genética , Ligação Proteica , Proteínas Repressoras/genéticaRESUMO
In the present study, we utilize the well-characterized Escherichia coli lac repressor/operator system to demonstrate that protein binding can lead to demethylation at the binding sites in the chromosome. Similar to the findings using the episome, we found that the presence of LacI in the cells can lead to demethylation of methylated lacO in the chromosome and the LacI inhibitor, isopropyl-beta-D-thiogalactopyranoside (IPTG), can prevent demethylation of the methylated lacO. The lacO sites become progressively more demethylated over time with the presence of LacI, supporting the role of protein occupancy in demethylation targeting. These results validate our earlier conclusions using a stable episomal system, and establish for the first time that protein binding can specify sites of demethylation in the chromosome.
Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Humanos/metabolismo , Metilação de DNA , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , Southern Blotting , Linhagem Celular , Cromossomos Humanos/genética , Metilação de DNA/efeitos dos fármacos , Genoma Humano , Humanos , Isopropiltiogalactosídeo/farmacologia , Óperon Lac/genética , Repressores Lac , Ligação Proteica/efeitos dos fármacos , Recombinação Genética/genética , Proteínas Repressoras/genética , Fatores de Tempo , TransfecçãoRESUMO
It has recently been shown that in Xenopus, DNA demethylation at promoter regions may involve protein-DNA interactions, based on the specificity of the demethylated sites. Utilizing a stable episomal system in human cells, we recently mapped the sites and dissected the steps of demethylation at oriP sites bound by EBNA1 protein. Although it is clear that protein binding is required for demethylation of the oriP sites, it is uncertain whether this is a unique feature of the replication origin or whether it is a general phenomenon for all DNA sequences to which sequence-specific proteins are bound. In the present study, we utilize the well-defined Escherichia coli lac repressor/operator system in human cells to determine whether protein binding to methylated DNA, in a region that is neither a replication origin nor a promoter, can also lead to demethylation of the binding sites. We found that demethylation specified by protein binding is not unique to the replication origin or to the promoter. We also found that transcriptional activity does not influence demethylation of the lac operator. Isopropyl-beta-D-thiogalactopyranoside (IPTG), an inhibitor of the lac repressor, can prevent demethylation of the lac operator DNA sites and can modulate demethylation of the lac operator by affecting the binding affinity of the lac repressor. Using this system, a titration of protein binding can be done. This titration permits one to infer that protein binding site occupancy is the determinant of demethylation at DNA sites and permits a determination of how this process progresses over time.
