Loading of DNA-Binding Factors to an Erythroid Enhancer

Abstract

3The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage-and erythroid lineage-specific expression of the human ␣-like globin genes, the embryonic and the adult ␣2/␣/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3-NA, modulates the capability of HS-40 to activate the embryonic -globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. The interplay among the multiple nuclear factor-DNA complexes formed at the enhancers (enhanceosomes) and their cis-linked promoters (polymerase II [pol II] preinitiation complexes) is essential for the regulation of many eukaryotic genes (reviewed in references 6 and 9). Several modes of actions have been proposed for the enhanceosome function. Some enhanceosomes, such as the locus control region (LCR) of the human ␤-globin locus (reviewed in references 16, 20, and 22) may set up and/or maintain an active chromatin state of a gene or locus domain, thus allowing the formation of active pol II preinitiation complex. On the other hand, enhanceosomes could also facilitate the assembly of active pol II preinitiation complex by direct interaction with, or recruitment of, coactivators and different basal transcription factors (reference 6 and references therein).HS-40 is an element located at 40 kb upstream of the human ␣-globin locus (Fig. 1). Genetic and molecular data have indicated that it is essential for transcriptional regulation of the human embryonic -and adult ␣-globin promoters during erythroid development (21). Most of the ␣-globin gene cluster maintains an open chromatin structure in both erythroid and nonerythroid cells, possibly due to the transcription of certain ubiquitous genes (54). Further remodeling and modification of the chromatin structure must occur in erythroid cells, however, since several new HS sites appear, including those at the HS-40 element and the promoter regions of the -and ␣-globin genes (54, 59). These latter sites apparently result from erythroid lineage-specific binding of nuclear factors to the above transcriptional regulatory elements, as demonstrated by previous genomic footprint analysis of 60) and of the ␣-globin upstream promoters (47).HS-40 acts as a classical enhancer in transient transfection assays (44,46,60), and it confers appropriate developmental control of the human -globin promoter activity in transgenic mice (19,23,45,56). In vitro and in vivo binding studies have shown that the HS-40 enhanceosome consists mainly of six DNA sequence motifs that are bound with nuclear factors in an erythroid lineage-specific manner: two NF-E2/AP1 motifs (5Ј-NA and 3Ј-NA), three GATA-1 motifs (b, c, and d), and a GT motif (26,50,60) (Fig. 1A). Of these, the NF-E2/AP1 motifs could be recognized by the erythroid-enriched factor NF-E2, the ubiquitous AP1, the homodimers of small Maf family, or...

Erythroid Gene Suppression by NF-κB

“…Plasmids-The reporter constructs pHS40-597GH, pHS40-␣590GH, and their mutated versions have been described previously (8,9). The constructions of pHS40-␣590Luc and pHS40-597Luc were done in the following way.…”

A role for Rab27b in NF-E2-dependent pathways of platelet formation

“…Chromatin immunoprecipitation (ChIP) is a proven and useful approach to assess transcriptional regulation by NF-E2 and other factors in many contexts 40,41 and we used this method to determine if NF-E2 is recruited to any of its putative binding sites in the Rab27b locus. To establish specificity in this PCR-based assay, we first performed ChIP using dimethyl sulfoxide (DMSO)-treated murine erythroleukemia (MEL) cells, which activate NF-E2-dependent ␤-globin gene expression, and PCR primers specific for DNase I hypersensitive site 2 (HS2) within the ␤-globin locus control region.…”

Smooth Muscle α-Actin Gene Requires Two E-Boxes for Proper Expression In Vivo and Is a Target of Class I Basic Helix-Loop-Helix Proteins

“…However, inasmuch as these studies were performed in 3T3 fibroblasts, it is possible that the results are more reflective of regulation in myofibroblasts than in SMCs. For this reason and because several studies have shown differences in transcriptional regulation between episomal reporter genes and chromosomal genes 17 and also because we 16 and others 18 have demonstrated that protein binding to oligonucleotide probes in EMSAs does not necessarily correlate with binding in vivo in the context of intact chromatin, we wanted to determine whether E2A proteins and SRF were specifically bound to the endogenous SM ␣-actin promoter in SMCs. We addressed this question using ChIP assays.…”