Hansen R S, Ellis N A, Gartler S M
Department of Medicine, University of Washington, Seattle 98195.
Mol Cell Biol. 1988 Nov;8(11):4692-9. doi: 10.1128/mcb.8.11.4692-4699.1988.
X8/6T2, a hamster-human hybrid cell line which contains an inactive human X chromosome, was treated with 5-azacytidine and selected for derepression of hypoxanthine-guanine phosphoribosyltransferase. Clones were examined for coreactivation of the phosphoglycerate kinase gene (Pgk). Of 68 of these hybrids, approximately 20% expressed measurable human phosphoglycerate kinase (PGK) activity. A 600-base-pair region of the Pgk 5' CpG cluster was examined for the methylation status of eight CCGG sites (site 1 being 5'-most) in a number of PGK-negative and PGK-positive cell lines. The inactive X chromosome is normally methylated at all eight sites, and this was also true for the majority of X8/6T2 cells. However, several PGK-negative hybrids were demethylated in the site 3 to site 6 region. PGK activity correlated with demethylation at both sites 6 and 7. The data for PGK-positive and -negative hybrids indicate that demethylation at or near site 7 was necessary for reactivation of Pgk. Chromatin sensitivity to MspI digestion in the nuclei of male lymphoblastoid cells and several PGK-positive and PGK-negative hybrids was examined. PGK-positive cell lines were hypersensitive to digestion, while PGK-negative hybrids were resistant. Cleavage at sites 6 and 7 was observed in all PGK-positive cell lines at each MspI concentration examined. Sites 7 and 8 were less accessible to digestion than site 6. Cleavage in the site 2 to site 5 region was observable at the lowest MspI concentration. In most PGK-positive hybrids, a nonspecific endogenous nuclease detected the presence of a hypersensitive region spanning at least 450 base pairs, bounded at the 3' end near HpaII site 6. Nuclease hypersensitivity appears to be related to promoter activity, because sites 7 and 8 are in transcribed regions of the gene. These data indicate that specific sites within the CpG cluster have a dominant controlling influence over the Pgk promoter conformation and the transcriptional activation of Pgk.
X8/6T2是一种仓鼠 - 人类杂交细胞系，含有一条失活的人类X染色体，用5 - 氮杂胞苷处理后，筛选次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶去抑制的细胞。检测克隆中磷酸甘油酸激酶基因（Pgk）的共激活情况。在这些杂交细胞中，68个中有约20%表达可检测到的人类磷酸甘油酸激酶（PGK）活性。在一些PGK阴性和PGK阳性细胞系中，检测了Pgk 5' CpG簇的一个600碱基对区域中8个CCGG位点（位点1是最靠近5'端的）的甲基化状态。失活的X染色体通常在所有8个位点都发生甲基化，大多数X8/6T2细胞也是如此。然而，几个PGK阴性杂交细胞在位点3到位点6区域发生了去甲基化。PGK活性与位点6和位点7的去甲基化相关。PGK阳性和阴性杂交细胞的数据表明，位点7处或其附近的去甲基化对于Pgk的重新激活是必要的。检测了雄性淋巴母细胞以及几个PGK阳性和PGK阴性杂交细胞的细胞核中染色质对MspI消化的敏感性。PGK阳性细胞系对消化高度敏感，而PGK阴性杂交细胞具有抗性。在所检测的每个MspI浓度下，所有PGK阳性细胞系在位点6和位点7都观察到了切割。位点7和位点8比位点6更不易被消化。在最低MspI浓度下，在位点2到位点5区域可观察到切割。在大多数PGK阳性杂交细胞中，一种非特异性内源性核酸酶检测到存在一个超敏区域，至少跨越450个碱基对，在3'端靠近HpaII位点6处界定。核酸酶超敏性似乎与启动子活性相关，因为位点7和位点8在基因的转录区域。这些数据表明，CpG簇内的特定位点对Pgk启动子构象和Pgk的转录激活具有主要控制作用。
