CTCF-dependent insulation of Hoxb13 and the heterochronic control of tail length

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.