Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
Genetics. 2012 Dec;192(4):1281-93. doi: 10.1534/genetics.112.143743. Epub 2012 Sep 28.
Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.
调控序列可以影响侧翼基因的远距离表达，X 染色体失活是顺式作用表观遗传基因调控的一个经典例子。针对 Mus musculus Hprt 基因座的基因敲入提供了一个独特的机会，可以在相同的基因组环境中分析沉默向不同的人类 DNA 序列的传播。通过具有非随机 X 染色体失活的雌性中来自失活 X 染色体的基因表达缺失以及人类转基因启动子 DNA 甲基化，证明了四个基因敲入构建体（包括超过 195 kb 的细菌人工染色体 [BAC] 整合）的 X 染色体失活。我们进一步利用启动子 DNA 甲基化来评估包含 >1.5 Mb DNA 的 74 个人类报告基因构建体的失活状态。在所检查的 47 个基因中，只有 PHB 基因表现出雌性 DNA 低甲基化，接近男性水平，并且通过证明失活 X 染色体上的基因表达验证了 X 染色体失活的逃逸。PHB 的整合导致女性中侧翼 HPRT 启动子的 DNA 甲基化降低，这表明存在一个显性顺式作用逃逸元件。与启动子无关的 CpG 岛的女性特异性 DNA 高甲基化暗示了 X 染色体失活过程中广泛施加的 DNA 甲基化；然而，当整合到失活 X 染色体上的相同位置时，转基因显示出不同的积累 DNA 甲基化的能力，这表明存在其他顺式作用序列效应。由于分析的人类转基因中只有一个逃避了 X 染色体失活，我们得出结论，允许从失活 X 染色体持续表达的元件在人类基因组中是罕见的。
