X chromosome inactivation (XCI) is an essential mechanism for gene dosage compensation between male and female cells in mammals. The Okinawa spiny rat (Tokudaia muenninki) is a native rodent in Japan with XX/XY sex chromosomes, like most mammals; however, the X chromosome has acquired a neo-X region (Xp) by fusion with an autosome. We previously reported that dosage compensation has not yet evolved in the neo-X region; however, X-inactive-specific transcript (Xist) RNA (long non-coding RNA required for the initiation of XCI) is partially localized in the region. Here, we show that the neo-X region represents an early chromosomal state in the acquisition of XCI by analyses of heterochromatin and Barr body formation. We found no evidence for heterochromatin formation in the neo-X region by R-banding by acridine orange (RBA) assays and immunostaining of H3K27me3. Double-immunostaining of H3K27me3 and HP1, a component of the Barr body, revealed that the entire ancestral X chromosome region (Xq) showed a bipartite folded structure. By contrast, HP1 was not localized to the neo-X region. However, BAC-FISH revealed that the signals of genes on the neo-X region of the inactive X chromosome were concentrated in a narrow region. These findings indicated that although the neo-X region of the inactive X chromosome does not form a complete Barr body structure (e.g., it lacks HP1), it forms a slightly condensed structure. These findings combined with the previously reported partial binding of Xist RNA suggest that the neo-X region exhibits incomplete inactivation. This may represent an early chromosomal state in the acquisition of the XCI mechanism.

中文翻译：

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在冲绳刺鼠 (Tokudaia muenninki) 中，Neo-X 不形成巴尔体，但显示出稍微压缩的结构。

X染色体失活（XCI）是哺乳动物雄性和雌性细胞之间基因剂量补偿的重要机制。冲绳刺鼠 (Tokudaia muenninki) 是日本本土啮齿动物，与大多数哺乳动物一样，具有 XX/XY 性染色体；然而，X 染色体通过与常染色体融合获得了新 X 区域 (Xp)。我们之前报道过neo-X区域的剂量补偿尚未进化；然而，X-失活特异性转录物 (Xist) RNA（启动 XCI 所需的长非编码 RNA）部分位于该区域。在这里，我们通过异染色质和巴尔体形成的分析表明，neo-X 区域代表了获得 XCI 的早期染色体状态。通过吖啶橙 (RBA) 测定和 H3K27me3 免疫染色的 R 显带，我们没有发现 Neo-X 区域异染色质形成的证据。对 Barr 小体的组成部分 H3K27me3 和 HP1 进行双重免疫染色，结果显示整个祖先 X 染色体区域 (Xq) 呈现二分折叠结构。相比之下，HP1 并不局限于 neo-X 区域。然而，BAC-FISH显示，失活X染色体的neo-X区域上的基因信号集中在一个狭窄的区域。这些发现表明，虽然失活X染色体的neo-X区域没有形成完整的巴尔体结构（例如，它缺乏HP1），但它形成了一个稍微浓缩的结构。这些发现与之前报道的 Xist RNA 部分结合相结合表明 neo-X 区域表现出不完全失活。这可能代表获得 XCI 机制的早期染色体状态。