NF-E2 Disrupts Chromatin Structure at Human β-Globin Locus Control Region Hypersensitive Site 2 In Vitro

Abstract

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.

Context-dependent regulation of GATA-1 by friend of GATA-1

“…At sites where GATA-1 binding does not require FOG-1, such as HS3, neighboring proteins might provide an activity to render the local chromatin configuration permissive to GATA-1 binding. For example, previous studies have shown that GATA-1 binding to chromatinized templates in vitro is facilitated by prior binding and recruitment of remodeling activity by the transcription factor NF-E2 (44).…”

Chromatin Remodeling and the Control of Gene Expression

“…NURF activity is abundant in chromatin reconstitution extracts derived from Drosophila embryos (91,92) that are increasingly employed for analyses of the activities of sequencespecific transcription factors in a native chromatin context (87,(93)(94)(95)(96)(97). In these crude extracts, a number of transcription factors (GAGA factor, HSF, GAL4, Sp1, NF-B, NF-E2, and lac repressor) have been reported to participate in nucleosome disordering and, in several cases, to stimulate transcription manyfold on chromatinized templates.…”

Heterogeneous nuclear ribonucleoprotein C1/C2, MeCP1, and SWI/SNF form a chromatin remodeling complex at the β-globin locus control region

“…However, deletion of the LCR DNA sequences severely reduces the transcription of the ␤-like genes, but the DNase hypersensitivity and polymerase II association with promoters of the remaining ␤-globin genes persists (17)(18)(19). Several biochemical studies in cell lines suggest that the MARE sequences of HS2, apart from their involvement in the enhancement of transcription, also take part in the alteration of the chromatin structure of HS2 and a linked promoter (20)(21)(22)(23). These studies suggest the possible association of the LCR with specific chromatin remodeling activities, although such a LCR-specific chromatin remodeling activity has not been isolated.…”