Rincón-Arano Héctor, Furlan-Magaril Mayra, Recillas-Targa Félix
Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 México, D.F., México.
Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):14044-9. doi: 10.1073/pnas.0704999104. Epub 2007 Aug 21.
Epigenetic silencing of genes relocated near telomeres, termed telomeric position effect, has been extensively studied in yeast and more recently in vertebrates. However, protection of a transgene against telomeric position effects by chromatin insulators has not yet been addressed. In this work we investigated the capacity of the chicken beta-globin insulator cHS4 to shield a transgene against silencing by telomeric heterochromatin. Using telomeric repeats, we targeted transgene integration into telomeres of the chicken cell line HD3. When the chicken cHS4 insulator is incorporated to the transgene, we observe a sustained gene expression of single-copy integrants that can be maintained for >100 days of continuous culture. However, uninsulated single-copy clones showed an accelerated gene expression extinction profile. Unexpectedly, telomeric silencing was not reversed with trichostatin A or nicotidamine. In contrast, significant reactivation was obtained with 5-aza-2'-deoxycytidine, consistent with the subtelomeric DNA methylation status. Strikingly, insulated transgenes integrated into telomeric regions were enriched in histone methylation, such as H3K4me2 and H3K79me2, but not in histone acetylation. Furthermore, the cHS4 insulator counteracts telomeric position effects in an upstream stimulatory factor-independent manner. Our results suggest that this insulator has the capacity to adapt to different chromatin propagation signals in distinct insertional epigenome environments.
基因迁移至端粒附近后的表观遗传沉默，即端粒位置效应，已在酵母中得到广泛研究，最近在脊椎动物中也有相关研究。然而，染色质绝缘子对转基因免受端粒位置效应影响的保护作用尚未得到探讨。在这项工作中，我们研究了鸡β-珠蛋白绝缘子cHS4保护转基因免受端粒异染色质沉默的能力。利用端粒重复序列，我们将转基因整合靶向到鸡细胞系HD3的端粒中。当鸡cHS4绝缘子被整合到转基因中时，我们观察到单拷贝整合体的基因表达持续存在，这种表达在连续培养超过100天时仍可维持。然而，未受绝缘子保护的单拷贝克隆显示出基因表达加速消失的情况。出乎意料的是，曲古抑菌素A或烟酰胺并不能逆转端粒沉默。相反，5-氮杂-2'-脱氧胞苷能使基因显著重新激活，这与亚端粒DNA甲基化状态一致。引人注目的是，整合到端粒区域的受绝缘子保护的转基因富含组蛋白甲基化，如H3K4me2和H3K79me2，但组蛋白乙酰化水平不高。此外，cHS4绝缘子以上游刺激因子非依赖的方式抵消端粒位置效应。我们的结果表明，这种绝缘子有能力在不同的插入表观基因组环境中适应不同的染色质传播信号。
