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Molecular and Cellular Biology, April 2007, p. 2636-2647, Vol. 27, No. 7
0270-7306/07/$08.00+0     doi:10.1128/MCB.02036-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Allele-Specific Binding of CTCF to the Multipartite Imprinting Control Region KvDMR1{triangledown}

Galina V. Fitzpatrick,1 Elena M. Pugacheva,2 Jong-Yeon Shin,1 Ziedulla Abdullaev,2 Youwen Yang,1,{dagger} Kavita Khatod,1 Victor V. Lobanenkov,2* and Michael J. Higgins1*

Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263,1 and Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 208922

Received 31 October 2006/ Returned for modification 12 December 2006/ Accepted 11 January 2007

Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of the Kcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.

* Corresponding author. Mailing address for Michael J. Higgins: Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-3582. Fax: (716) 845-1579. E-mail: michael.higgins{at}roswellpark.org. Mailing address for Victor V. Lobanenkov: Section of Molecular Pathology, Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852. Phone: (301) 435-1690. Fax: (301) 402-0077. E-mail: vlobanenkov{at}niad.nih.gov.

{triangledown} Published ahead of print on 22 January 2007.

{dagger} Present address: School of Medicine, University of South Hampton, South Hampton SO16 6YD, United Kingdom.

Molecular and Cellular Biology, April 2007, p. 2636-2647, Vol. 27, No. 7
0270-7306/07/$08.00+0     doi:10.1128/MCB.02036-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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