Son J, Min N Y, Choi J-H, Ko Y J, Liang W, Rhee S, Lee K-H
School of Life Sciences, College of Natural Science, Chung-Ang University, Seoul, Republic of Korea.
Cytogenet Genome Res. 2012;138(1):11-8. doi: 10.1159/000341503. Epub 2012 Aug 10.
Mammalian dosage compensation requires silencing of one of the two X chromosomes in females and is controlled by the X inactivation center (Xic). Xic contains many of the regulatory elements for the mutual interplay of X-inactive specific transcript (Xist) and Tsix, the antisense counterpart of Xist. The regulatory elements control X chromosome inactivation (XCI) via the formation of DNA-DNA and DNA-protein complexes with cis- and trans-acting factors. However, the process-dependent regulation of Xist/Tsix by these elements in each XCI process remains largely unknown. In this study, a 6-thioguanine-resistant female F(1) hybrid mouse cell line (designated HOBMSKI2) was constructed from a cross between a female HPRT-deficient transgenic mouse (designated BM3) and a male wild type Mus spretus mouse (designated MS), which enabled the direct discrimination of both allele-specific expression of X-linked genes and allele-specific binding of proteins associated with XCI due to DNA polymorphisms between BM3 and MS. Using this cell line, we found that Tsix on the active X chromosome (Xa) was not expressed in somatic cells despite the fact that CTCF, which ensures Tsix expression in embryonic stem cells, was still bound to the 5' end of Tsix on Xa, implying that CTCF may function differently during each XCI process and its trans-activating activity for Tsix expression may be lost in the maintenance process. In addition, the monoallelic expression of Tsix on Xa was inhibited by epigenetic modification of the chromatin in the maintenance process, which was mediated by protein complexes recruited by MeCP2. The results indicate the value of HOBMSKI2 in directly detecting the allele-specific binding of CTCF and MeCP2 to the 5' end of Tsix. The HOBMSKI2 mouse line is a versatile and useful resource for studying the molecular mechanism of the XCI process.
哺乳动物的剂量补偿需要使雌性两条X染色体中的一条失活，这一过程由X染色体失活中心（Xic）控制。Xic包含许多调控元件，用于X染色体失活特异性转录本（Xist）与其反义转录本Tsix之间的相互作用。这些调控元件通过与顺式和反式作用因子形成DNA-DNA和DNA-蛋白质复合物来控制X染色体失活（XCI）。然而，在每个XCI过程中，这些元件对Xist/Tsix的过程依赖性调控仍 largely未知。在本研究中，从雌性次黄嘌呤磷酸核糖转移酶（HPRT）缺陷型转基因小鼠（命名为BM3）与雄性野生型西班牙小鼠（命名为MS）的杂交后代中构建了一种6-硫鸟嘌呤抗性雌性F(1)杂交小鼠细胞系（命名为HOBMSKI2），这使得能够因BM3和MS之间的DNA多态性而直接区分X连锁基因的等位基因特异性表达以及与XCI相关的蛋白质的等位基因特异性结合。利用该细胞系，我们发现尽管在胚胎干细胞中确保Tsix表达的CCCTC结合因子（CTCF）仍与活性X染色体（Xa）上Tsix的5'端结合，但Xa上的Tsix在体细胞中并不表达，这意味着CTCF在每个XCI过程中的功能可能不同，其对Tsix表达的反式激活活性可能在维持过程中丧失。此外，在维持过程中，Xa上Tsix的单等位基因表达受到染色质表观遗传修饰的抑制，这是由甲基化CpG结合蛋白2（MeCP2）招募的蛋白质复合物介导的。结果表明HOBMSKI2在直接检测CTCF和MeCP2与Tsix 5'端的等位基因特异性结合方面的价值。HOBMSKI2小鼠品系是研究XCI过程分子机制的一种通用且有用的资源。
