Supplementary Material for: Direct Evidence of Allele-Specific Binding of CTCF and MeCP2 to <b><i>Tsix</i></b> in a HPRT-Deficient Female F<sub>1</sub> Hybrid Mouse Cell Line

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 <i>(Xist)</i> and <i>Tsix</i>, the antisense counterpart of <i>Xist</i>. The regulatory elements control X chromosome inactivation (XCI) via the formation of DNA-DNA and DNA-protein complexes with <i>cis</i>- and <i>trans</i>-acting factors. However, the process-dependent regulation of <i>Xist/Tsix</i> by these elements in each XCI process remains largely unknown. In this study, a 6-thioguanine-resistant female F<sub>1</sub> 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 <i>Mus spretus</i> 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 <i>Tsix</i> on the active X chromosome (Xa) was not expressed in somatic cells despite the fact that CTCF, which ensures <i>Tsix</i> expression in embryonic stem cells, was still bound to the 5′ end of <i>Tsix</i> on Xa, implying that CTCF may function differently during each XCI process and its <i>trans</i>-activating activity for <i>Tsix</i> expression may be lost in the maintenance process. In addition, the monoallelic expression of <i>Tsix</i> 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 <i>Tsix</i>. The HOBMSKI2 mouse line is a versatile and useful resource for studying the molecular mechanism of the XCI process.

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