Conserved E boxes function as part of the enhancer in hypersensitive site 2 of the beta-globin locus control region. Role of basic helix-loop-helix proteins.
The human beta-globin gene cluster is regulated in part by a distal locus control region that is required for opening a chromatin domain in erythroid cells and enhancing expression of the beta-like globin genes at the correct developmental stages. One part of the locus control region, called hypersensitive site 2 (HS2), functions as a strong enhancer. Matches to the consensus binding sites for basic helix-loop-helix (bHLH) proteins (E boxes) are well conserved within the HS2 core. We show that mutations of the HS2 core that alter an invariant E box cause a 3.5-fold reduction in enhancement of expression of an epsilon-globin reporter gene in transiently transfected K562 cells, both before and after induction. Mutations of the HS2 core that alter a less-highly conserved E box cause a more modest reduction in enhancement. Footprint analysis shows binding of erythroid nuclear proteins in vitro to the invariant E box as well as an adjacent CAC/GTG box. Probes containing the E box regions form sequence-specific complexes with proteins from both K562 and MEL nuclear extracts; these are disrupted by the same mutations that decrease enhancement. Some of these latter complexes contain known bHLH proteins, as revealed by specific loss of individual complexes when treated with antibodies against TAL1 and USF. Interaction between the E boxes and the bHLH proteins, as well as other binding proteins, could account for the role of these sites in enhancement by HS2.
Elnitski
L
L
Department of Biochemistry, The Pennsylvania State University, University Park 16802, USA.
Miller
W
W
Hardison
R
R
D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000235:genetics*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D014157:Transcription Factors / Q000378:metabolism; D051720:Upstream Stimulatory Factors
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D005914:Globins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497181:USF1 protein, human; C067143:TAL1 protein, human