The c-myc Insulator Element and Matrix Attachment Regions Definethe c-myc ChromosomalDomain

Abstract

Insulator elements and matrix attachment regions are essential for the organization of genetic information within the nucleus. By comparing the pattern of histone modifications at the mouse and human c-myc alleles, we identified an evolutionarily conserved boundary at which the c-myc transcription unit is separated from the flanking condensed chromatin enriched in lysine 9-methylated histone H3. This region harbors the c-myc insulator element (MINE), which contains at least two physically separable, functional activities: enhancer-blocking activity and barrier activity. The enhancer-blocking activity is mediated by CTCF. Chromatin immunoprecipitation assays demonstrate that CTCF is constitutively bound at the insulator and at the promoter region independent of the transcriptional status of c-myc. This result supports an architectural role of CTCF rather than a regulatory role in transcription. An additional higher-order nuclear organization of the c-myc locus is provided by matrix attachment regions (MARs) that define a domain larger than 160 kb. The MARs of the c-myc domain do not act to prevent the association of flanking regions with lysine 9-methylated histones, suggesting that they do not function as barrier elements.

ACKNOWLEDGMENTS

This work was supported by a Research Scholar Grant from the American Cancer Society to A.K. (RSG-01-163-01-GMC). W.H.S. was supported by a grant from the NIH (CA82459).

We are especially grateful to Jim Moon and Brad Nelson (Virginia Mason Research Center) for generously providing and preparing chromatin from CTLL2 cells; Corty Thienes (Fred Hutchinson Cancer Research Center) for help with MAR assays; Mark Groudine, Susan Parkhurst, and Mike Bulger (Fred Hutchinson Cancer Research Center) for critical reading of the manuscript, and all members of the Groudine lab for discussion.