In vivo formation of a human beta-globin locus control region core element requires binding sites for multiple factors including GATA-1, NF-E2, erythroid Kruppel-like factor, and Sp1.
The active elements of the beta-globin locus control region (LCR) are located within domains of unique chromatin structure. These nuclease hypersensitive sites (HSs) are characterized by high DNase I sensitivity, erythroid specificity, similar nucleosomal structure, and evolutionarily conserved clusters of cis-acting elements that are required for the formation and function of the core elements. To determine the requirements for HS core formation in the setting of nuclear chromatin, we constructed a series of artificial HS cores containing binding sites for GATA-1, NF-E2, and Sp1. In contrast to the results of previous in vitro experiments, we found that when constructs were stably integrated in mouse erythroleukemia cells the binding sites for NF-E2, GATA-1, or Sp1 alone or in any combination were unable to form core HS structures. We subsequently identified two new cis-acting elements from the LCR HS4 core that, when combined with the NF-E2, Sp1, and tandem inverted GATA elements, result in core structure formation. Both new cis-acting elements bind Sp1, and one binds erythroid Kruppel-like factor (EKLF). We conclude that in vivo beta-globin LCR HS core formation is more complex than previously thought and that several factors are required for this process to occur.
Goodwin
A J
AJ
Departments of Medicine and Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755.
McInerney
J M
JM
Glander
M A
MA
Pomerantz
O
O
Lowrey
C H
CH
D001483:Base Sequence; D001665:Binding Sites; D018076:DNA, Complementary; D003850:Deoxyribonuclease I / Q000378:metabolism; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019901:Locus Control Region; D008969:Molecular Sequence Data; D016296:Mutagenesis; D014157:Transcription Factors / Q000378:metabolism*
D018076:DNA, Complementary; D014157:Transcription Factors; D005914:Globins; D003850:Deoxyribonuclease I