Mammalian tail length is controlled by several genetic determinants, among which are Hox13 genes, whose function is to terminate the body axis. Accordingly, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks posterior genes between Hoxb9 and Hoxb13 , two genes separated by a ca. 70 kb large DNA segment containing a high number of CTCF sites, potentially isolating Hoxb13 from the rest of the cluster and thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes. These defects were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant Embryonic Stem (ES) cells grown as pseudoembryos in vitro, which allowed us to examine in detail the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB , and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations were already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.

中文翻译：

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Hoxb13 的 CTCF 依赖性绝缘和尾长的异时控制


哺乳动物的尾巴长度受多种遗传决定因素控制，其中包括 Hox13 基因，其功能是终止体轴。因此，这些基因转录激活的精确时间可能会影响体长。与其他 Hox 簇不同，HoxB 在 Hoxb9 和 Hoxb13 之间缺乏后基因，这两个基因由一个包含大量 CTCF 位点的约 70 kb 大 DNA 片段隔开，可能将 Hoxb13 与簇的其余部分隔离开来，从而延迟其对树干延伸的负面影响。我们删除了间隔区 DNA 以诱导 Hoxb13 在生理浓度下的潜在异时功能获得，并观察到尾部缩短以及其他异常表型。这些缺陷都是通过灭活 Hoxb13 in-cis 和缺失来挽救的。在体外作为假胚胎生长的突变胚胎干细胞 （ES） 细胞中观察到类似的功能增益，这使我们能够详细检查 CTCF 位点的数量和方向在 DNA 垫片绝缘活性中的重要性。包含所有 CTCF 位点的短盒足以将 Hoxb13 与 HoxB 的其余部分隔离，并且对该 CTCF 盒的额外修改表明，收敛方向的两个 CTCF 位点已经能够在这些情况下显着延迟 Hoxb13 激活。我们讨论了基因组距离与 CTCF 位点的数量和方向在防止 Hoxb13 在躯干伸展过程中过早被激活从而调节尾部长度方面的相对重要性。