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Molecular and Cellular Biology, May 2007, p. 3499-3510, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.02170-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Analysis of the H19ICR Insulator{triangledown}

Young Soo Yoon,1,2 Sangkyun Jeong,1 Qi Rong,1 Kye-Yoon Park,1 Jae Hoon Chung,2 and Karl Pfeifer1*

Laboratory of Mammalian Genes and Development, NICHD, NIH, Bethesda, Maryland 20892,1 Department of Biological Sciences, KAIST, Taejon, South Korea2

Received 20 November 2006/ Returned for modification 20 December 2006/ Accepted 15 February 2007

Transcriptional insulators are specialized cis-acting elements that protect promoters from inappropriate activation by distal enhancers. The H19 imprinting control region (ICR) functions as a CTCF-dependent, methylation-sensitive transcriptional insulator. We analyzed several insertional mutations and demonstrate that the ICR can function as a methylation-regulated maternal chromosome-specific insulator in novel chromosomal contexts. We used chromosome conformation capture and chromatin immunoprecipitation assays to investigate the configuration of cis-acting elements at these several insertion sites. By comparing maternal and paternal organizations on wild-type and mutant chromosomes, we hoped to identify mechanisms for ICR insulator function. We found that promoter and enhancer elements invariably associate to form DNA loop domains at transcriptionally active loci. Conversely, active insulators always prevent these promoter-enhancer interactions. Instead, the ICR insulator forms novel loop domains by associating with the blocked promoters and enhancers. We propose that these associations are fundamental to insulator function.

* Corresponding author. Mailing address: NICHD, Bldg. 6B—2B206, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 451-2017. Fax: (301) 402-0543. E-mail: pfeiferk{at}mail.nih.gov

{triangledown} Published ahead of print on 5 March 2007.

Molecular and Cellular Biology, May 2007, p. 3499-3510, Vol. 27, No. 9
0270-7306/07/$08.00+0     doi:10.1128/MCB.02170-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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