title: Enhancer blocking by chicken beta-globin 5'-HS4: role of enhancer strength and insulator nucleosome depletion.
abstract: The 5'-HS4 chicken beta-globin insulator functions as a positional enhancer blocker on chromatinized episomes in human cells, blocking the HS2 enhancer of the human beta-globin locus control region from activating a downstream epsilon-globin gene. 5'-HS4 interrupted formation of a domain of histone H3 and H4 acetylation encompassing the 6-kb minilocus and inhibited transfer of RNA polymerase from the enhancer to the gene promoter. We found that the enhancer blocking phenotype was amplified when the insulated locus contained a weakened HS2 enhancer in which clustered point mutations eliminated interaction of the transcription factor GATA-1. The GATA-1 mutation compromised recruitment of histone acetyltransferases and RNA polymerase II to HS2. Enhancer blocking correlated with a significant depletion of nucleosomes in the core region of the insulator as revealed by micrococcal nuclease and DNase I digestion studies. Nucleosome depletion at 5'-HS4 was dependent on interaction of the insulator protein CCCTC-binding factor (CTCF) and was required for enhancer blocking. These findings provide evidence that a domain of active chromatin is formed by spreading from an enhancer to a target gene and can be blocked by a nucleosome-free gap in an insulator.
authors.affiliation: Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.forename: Aeri
authors.initials: A
authors.lastname: Kim
authors.forename: Sang-Hyun
authors.initials: SH
authors.lastname: Song
authors.forename: Ann
authors.initials: A
authors.lastname: Dean
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D004742:Enhancer Elements, Genetic; D050982:GATA1 Transcription Factor / Q000235:genetics; D005914:Globins / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D008836:Micrococcal Nuclease / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary
chemical_list: D050982:GATA1 Transcription Factor; D006657:Histones; D009707:Nucleosomes; D005914:Globins; D008836:Micrococcal Nuclease