NF-E2 disrupts chromatin structure at human beta-globin locus control region hypersensitive site 2 in vitro.
The human beta-globin locus control region (LCR) is responsible for forming an active chromatin structure extending over the 100-kb locus, allowing expression of the beta-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HS1 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 core 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 more 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 not 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.
Armstrong
J A
JA
Regulatory Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Emerson
B M
BM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000502:physiology* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004330:Drosophila; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005914:Globins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D046911:Macromolecular Substances; D050985:MafK Transcription Factor; D051379:Mice; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D009687:Nuclear Proteins / Q000378:metabolism; D009707:Nucleosomes / Q000502:physiology / Q000648:ultrastructure; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002843:Chromatin; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; C494695:MAFK protein, human; D046911:Macromolecular Substances; D050985:MafK Transcription Factor; C494696:Mafk protein, mouse; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; C494693:Nfe2 protein, mouse; D009687:Nuclear Proteins; D009707:Nucleosomes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D005914:Globins; D003850:Deoxyribonuclease I