Transitions in levels of H3 K27me3 have previously been associated with CTCF-binding and insulator activity, demarcating boundaries between transcriptionally-active and silent gene loci [35].
These classes are active promoters, and it has been shown that some paused promoters have insulator activity [ 36].
The mechanisms by which insulator activity can be regulated to orchestrate changes in the function and three-dimensional arrangement of the genome remain elusive.
However, poly(ADP-ribosyl)ation of K566 in CP190 promotes protein-protein interactions with other insulator proteins, association with the nuclear lamina, and insulator activity in vivo.
In the present study, we demonstrated that the functional region underlying insulator activity in our system comprises two units—a 2.5 kb fragment having constitutive blocker activity and a functional sequence regulating tissue specificity of the insulator activity.
Finally, we tested a 1.8 kb γ-satellite repeat that has recently been found to have strong insulator activity by the Larionov lab (personal communication, [40]).
We showed that the sequence identified in this series of experiments is a proper boundary sequence, since it had position-effect protection activity as well as insulator activity.
There is, however, a major difference between these two insulators in that the insulator activity of the H19/Igf2 ICR depends on parental origin [23], [24], [25], [26].
By contrast, forelimb DNA samples contained essentially no methylated residues (Figure 4), which is consistent with the absence of insulator activity in limbs.
Insulator activity is sometimes paired with barrier activity, which can prevent silencing of gene loci by inhibiting the spread of heterochromatin.
Enhancer blocking assays confirmed that −31 does indeed have insulator activity and supports our interpretation of −31 as defining the SIL/SCL regulatory boundary.
