Chan H, Hartung S, Breindl M
Department of Biology, San Diego State University, California 92182.
Mol Cell Biol. 1991 Jan;11(1):47-54. doi: 10.1128/mcb.11.1.47-54.1991.
We have studied the role of DNA methylation in repression of the murine alpha 1 type I collagen (COL1A1) gene in Mov13 fibroblasts. In Mov13 mice, a retroviral provirus has inserted into the first intron of the COL1A1 gene and blocks its expression at the level of transcriptional initiation. We found that regulatory sequences in the COL1A1 promoter region that are involved in the tissue-specific regulation of the gene are unmethylated in collagen-expressing wild-type fibroblasts and methylated in Mov13 fibroblasts, confirming and extending earlier observations. To directly assess the role of DNA methylation in the repression of COL1A1 gene transcription, we treated Mov13 fibroblasts with the demethylating agent 5-azacytidine. This treatment resulted in a demethylation of the COL1A1 regulatory sequences but failed to activate transcription of the COL1A1 gene. Moreover, the 5-azacytidine treatment induced a transcription-competent chromatin structure in the retroviral sequences but not in the COL1A1 promoter. In DNA transfection and microinjection experiments, we found that the provirus interfered with transcriptional activity of the COL1A1 promoter in Mov13 fibroblasts but not in Xenopus laevis oocytes. In contrast, the wild-type COL1A1 promoter was transcriptionally active in Mov13 fibroblasts. These experiments showed that the COL1A1 promoter is potentially transcriptionally active in the presence of proviral sequences and that Mov13 fibroblasts contain the trans-acting factors required for efficient COL1A1 gene expression. Our results indicate that the provirus insertion in Mov13 can inactivate COL1A1 gene expression at several levels. It prevents the developmentally regulated establishment of a transcription-competent methylation pattern and chromatin structure of the COL1A1 domain and, in the absence of DNA methylation, appears to suppress the COL1A1 promoter in a cell-specific manner, presumably by assuming a dominant chromatin structure that may be incompatible with transcriptional activity of flanking cellular sequences.
我们研究了DNA甲基化在Mov13成纤维细胞中对小鼠α1 I型胶原（COL1A1）基因抑制的作用。在Mov13小鼠中，一种逆转录病毒前病毒插入到COL1A1基因的第一个内含子中，并在转录起始水平阻断其表达。我们发现，COL1A1启动子区域中参与该基因组织特异性调控的调控序列在表达胶原的野生型成纤维细胞中未甲基化，而在Mov13成纤维细胞中甲基化，这证实并扩展了早期的观察结果。为了直接评估DNA甲基化在COL1A1基因转录抑制中的作用，我们用去甲基化剂5-氮杂胞苷处理Mov13成纤维细胞。这种处理导致COL1A1调控序列去甲基化，但未能激活COL1A1基因的转录。此外，5-氮杂胞苷处理在逆转录病毒序列中诱导了具有转录活性的染色质结构，但在COL1A1启动子中未诱导。在DNA转染和显微注射实验中，我们发现前病毒干扰了Mov13成纤维细胞中COL1A1启动子的转录活性，但在非洲爪蟾卵母细胞中没有。相反，野生型COL1A1启动子在Mov13成纤维细胞中具有转录活性。这些实验表明，在存在前病毒序列的情况下，COL1A1启动子具有潜在的转录活性，并且Mov13成纤维细胞含有有效表达COL1A1基因所需的反式作用因子。我们的结果表明，Mov13中的前病毒插入可在多个水平上使COL1A1基因表达失活。它阻止了COL1A1结构域具有转录活性的甲基化模式和染色质结构在发育过程中的建立，并且在没有DNA甲基化的情况下，似乎以细胞特异性方式抑制COL1A1启动子，可能是通过形成一种与侧翼细胞序列的转录活性不相容的显性染色质结构。
