NF-E2 disrupts chromatin structure at human β-globin locus control region hypersensitive site 2 in vitro

Jennifer A. Armstrong, Beverly M. Emerson

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Abstract

The human β-globin locus control region (LCR) is responsible for forming an active chromatin structure extending over the 100-kb locus, allowing expression of the β-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HSI to -4). DNase I hypersensitive sites are thought to represent nucleosome-free regions of DNA which are bound by trans-acting factors. Of the four hypersensitive sites only HS2 acts as a transcriptional enhancer. In this study, we examine the binding of an erythroid protein to its site within HS2 in chromatin in vitro. NF-E2 is a transcriptional activator consisting of two subunits, the hematopoietic cell-specific p45 and the ubiquitous DNA-binding subunit, p18. NF-E2 binds two tandem AP1-like sites in HS2 which form the cure of its enhancer activity. In this study, we show that when bound to in vitro- reconstituted chromatin, NF-E2 forms a DNase I hypersensitive site at HS2 similar to the site observed in vivo. Moreover, NF-E2 binding in vitro results in a disruption of nucleosome structure which can be detected 200 bp away. Although NF-E2 can disrupt nucleosomes when added to preformed chromatin, the disruption is inure pronounced when NF-E2 is added to DNA prior to chromatin assembly. Interestingly, the hematopoietic cell-specific subunit, p45, is necessary for binding to chromatin but nut to naked DNA. Interaction of NF-E2 with its site in chromatin-reconstituted HS2 allows a second erythroid factor, GATA-1, to bind its nearby sites. Lastly, nucleosome disruption by NF-E2 is an ATP-dependent process, suggesting the involvement of energy-dependent nucleosome remodeling factors.

Original languageEnglish (US)
Pages (from-to)5634-5644
Number of pages11
JournalMolecular and Cellular Biology
Volume16
Issue number10
StatePublished - Sep 30 1996

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ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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