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Published online 23 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 4 1354-1362
© 2004 Oxford University Press

CTCF-dependent enhancer blockers at the upstream region of the chicken {alpha}-globin gene domain

Viviana Valadez-Graham, Sergey V. Razin1 and Félix Recillas-Targa*

Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, México D.F. 04510, México and 1 Laboratory of Structural and Functional Organization of Chromosomes, Institute of Gene Biology of the Russian Academy of Sciences, Vavilov Str. 34/5, 119334 Moscow, Russia

*To whom correspondence should be addressed. Tel: +52 55 5622 5674; Fax: +52 55 5622 5630; Email: frecilla{at}ifc.unam.mx

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5' flanking region of the chicken {alpha}-globin domain, 11–16 kb upstream of the embryonic {alpha}-type {pi} gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two {alpha}-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific {alpha}-globin gene domain located in a permanently open chromatin area.


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