CONSERVED E-BOX 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 lo cus 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 re gion, called hypersensitive site 2 (HS2), functions as a strong enhanc er. Matches to the consensus binding sites for basic helix-loop-helix (bHLH) proteins (E boxes) are well conserved within the HS2 core. We s how that mutations of the HS2 core that alter an invariant E box cause a 3.5-fold reduction in enhancement of expression of an E-globin repo rter gene in transiently transfected K562 cells, both before and after induction. Mutations of the HS2 core that alter a less-highly conserv ed E box cause a more modest reduction in enhancement. Footprint analy sis shows binding of erythroid nuclear proteins in vitro to the invari ant E box as well as an adjacent CAC/GTG box. Probes containing the E box regions form sequence-specific complexes with proteins from both K 562 and MEL nuclear extracts; these are disrupted by the same mutation s that decrease enhancement, Some of these latter complexes contain kn own bHLH proteins, as revealed by specific loss of individual complexe s when treated with antibodies against TAL1 and USF. Interaction betwe en the E boxes and the bHLH proteins, as well as other binding protein s, could account for the role of these sites in enhancement by HS2.