Multiple Nucleosome Positioning Sites Regulate the CTCF-Mediated Insulator Function of the H19 Imprinting Control Region†

Abstract

The 5′ region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.

We gratefully acknowledge valuable advice during the course of this work from Andreas Reik, Andy P. Feinberg, Gary Franklin, Sandy Morse, Rainer Renkawitz, Alan Wolffe, and Örjan Wrange.

This work was supported by grants from the Natural Science Research Council (NFR), the Swedish Cancer Research Foundation (RmC/CF), the Swedish Pediatric Cancer Foundation (BCF), and the Lundberg Foundation to R.O. and intramural NIAID NIH Research Funding to V.L.