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

Mammalian tail length is controlled by several genetic determinants, among which are 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 clusters, lacks posterior genes between and , two genes separated by a ca. 70 kb large DNA segment containing a high number of CTCF sites, potentially isolating 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 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes. These defects were all rescued by inactivating 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 from the rest of , and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations were already capable of importantly delaying activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing to be activated too early during trunk extension and hence to modulate tail length.