Requirement of an E1A-sensitive Coactivator for Long-range Transactivation by the β-Globin Locus Control Region

Abstract

Four erythroid-specific DNase I-hypersensitive sites at the 5-end of the ␤-globin locus confer high-level transcription to the ␤-globin genes. To identify coactivators that mediate long-range transactivation by this locus control region (LCR), we assessed the influence of E1A, an inhibitor of the CBP/p300 histone acetylase, on LCR function. E1A strongly inhibited transactivation of A␥-and ␤-globin promoters by the HS2, HS2-HS3, and HS1-HS4 subregions of the LCR in human K562 and mouse erythroleukemia cells. Short-and long-range transactivation mediated by the LCR were equally sensitive to E1A. The E1A sensitivity was apparent in transient and stable transfection assays, and E1A inhibited expression of the endogenous ␥-globin genes. Only sites for NF-E2 within HS2 were required for E1A sensitivity in K562 cells, and E1A abolished transactivation mediated by the activation domain of NF-E2. E1A mutants defective in CBP/p300 binding only weakly inhibited HS2-mediated transactivation, whereas a mutant defective in retinoblastoma protein binding strongly inhibited transactivation. Expression of CBP/p300 potentiated HS2-mediated transactivation. Moreover, expression of GAL4-CBP strongly increased transactivation of a reporter containing HS2 with a GAL4 site substituted for the NF-E2 sites. Thus, we propose that a CBP/p300-containing coactivator complex is the E1A-sensitive factor important for LCR function.The ␤-globin locus control region (LCR) 1 is a complex genetic element necessary for high-level transcription of the ␤-globin genes (1-3). The LCR was defined initially by its ability to confer copy number-dependent and position-independent expression to ␤-globin transgenes (1). The LCR also confers strong, erythroid-specific expression to linked genes in transfection assays, consistent with an intrinsic transcriptional enhancer function. Mutational studies have shown that multiple recognition sites for trans-acting factors are necessary for enhancer activity in stable transfection assays and to overcome position effects in transgenic mice (4 -10).Tandem binding sites for the hematopoietic transcription factor NF-E2 within the HS2 subregion of the LCR are important for strong transactivation of ␤-globin promoters in multiple systems (6,(11)(12)(13)(14). NF-E2 binds to DNA as a heterodimer consisting of a 45-kDa hematopoietic subunit, p45 (15, 16), and an 18-kDa ubiquitous subunit, p18 (16,17). Besides NF-E2, additional proteins such as NRF1 (18,19), NRF2 (20), AP-1 (21), and Bach (22) proteins are known to interact with the NF-E2 sites, although the consequence of these interactions for LCR function is unresolved. The involvement of NF-E2 in functioning through these sites has been established in CB3 cells, murine erythroleukemia cells that lack p45 (23, 24). However, disruption of the murine p45 gene does not greatly impair ␤-globin synthesis (25), suggesting that there may be redundant factors functioning through the NF-E2 sites. The p45 gene disruption resulted in defective platelet formation, implicating NF-E2 ...

A WW Domain-binding Motif within the Activation Domain of the Hematopoietic Transcription Factor NF-E2 Is Essential for Establishment of a Tissue-specific Histone Modification Pattern

“…Because the purification was conducted under high stringency conditions, it is not surprising that only the high affinity leucine zipper-mediated p45/NF-E2-p18 interaction was preserved. Even though CBP/ p300 interacts with p45/NF-E2 with moderate affinity and high specificity in vitro, and the CBP/p300 inhibitor E1A abrogates NF-E2-mediated transactivation (50), endogenous p45/NF-E2 has not been co-purified with endogenous CBP/p300. A simple explanation for these results is that the high salt used to extract NF-E2 from nuclei dissociates NF-E2-co-regulator interactions.…”

“…An obvious step in transactivation in which the PXY-1 mutant might be compromised is CBP/p300 recruitment, because CBP/ p300 mediates NF-E2-dependent transactivation in transient transfection assays (49,50). CBP/p300 is also a mediator of GATA-1-dependent transactivation (60).…”

“…2A) strongly inhibits p45/NF-E2-mediated activation of endogenous ␤-globin transcription in CB3 cells (47,48). As p45/NF-E2 binds the transcriptional co-activators and histone acetyltransferases CBP and p300 in vitro (49,50), CBP/p300 might mediate NF-E2-dependent histone acetylation. The activation domain of p45/NF-E2 also interacts with WW domains (48,51), which consist of 38 -40 amino acids that bind PPXY motifs (52).…”

Transcriptional control of erythropoiesis: emerging mechanisms and principles

“…CBP acetylates GATA-1 (Hung et al, 1999) and increases NF-E2 DNA binding activity (Hung et al, 2001). Studies with E1A also indicated that CBP/p300 is crucial for LCRmediated transactivation (Forsberg et al, 1999a). CBP/ p300 acetylates EKLF, which enhances EKLF binding to the SWI/SNF complex (Zhang et al, 2001;Letting et al, 2003;Im et al, 2005;Kim et al, 2007).…”

“…Furthermore, CBP and p300 have distinct functions in T-cell lymphopoiesis (Kasper et al, 2006). CBP/p300 physically and functionally interact with many transcription factors, including GATA-1, p45/NF-E2 and EKLF (Blobel et al, 1998;Zhang and Bieker, 1998;Forsberg et al, 1999a). Studies with the CBP/p300 inhibitor adenoviral E1A provided evidence that CBP/ p300 facilitates GATA-1-mediated transcriptional activity and promotes erythroid differentiation (Blobel et al, 1998).…”