Erythroid overexpression of C/EBPgamma in transgenic mice affects gamma-globin expression and fetal liver erythropoiesis

The CCAAT boxes of the beta-like globin genes interact with three proteins: NF-Y, GATA-1 and NFE-6. We demonstrate that NFE-6 contains C/EBPgamma, and address its role in globin gene regulation by erythroid overexpression of C/EBPgamma, and a dominant-negative form C/EBPgammaDeltaB, in mice. Elevated levels of C/EBPgamma, but not C/EBPgammaDeltaB, increase expression of the (fetal) gamma-globin relative to the (adult) beta-globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPgamma and C/EBPgammaDeltaB levels are further increased in homozygous transgenics. We suggest that targeted expression of dominant-negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice.     

The GCN4 bZIP can bind to noncognate gene regulatory sequences

We show that a minimalist basic region/leucine zipper (bZIP) hybrid, comprising the yeast GCN4 basic region and C/EBP leucine zipper, can target mammalian and other gene regulatory sequences naturally targeted by other bZIP and basic/helix-loop-helix (bHLH) proteins. We previously reported that this hybrid, wt bZIP, is capable of sequence-specific, high-affinity binding of DNA comparable to that of native GCN4 to the cognate AP-1 and CRE DNA sites. In this work, we used DNase I footprinting and electrophoretic mobility shift assay to show that wt bZIP can also specifically target noncognate gene regulatory sequences: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (Xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and the E-box (Enhancer box, 5'-CACGTG). Although wt bZIP still targets AP-1 with strongest affinity, both DNA-binding specificity and affinity are maintained with wt bZIP binding to noncognate gene regulatory sequences: the dissociation constant for wt bZIP in complex with AP-1 is 13 nM, while that for C/EBP is 120 nM, XRE1 240 nM, and E-box and HRE are in the microM range. These results demonstrate that the bZIP possesses the versatility to bind various sequences with varying affinities, illustrating the potential to fine-tune a designed protein's affinity for its DNA target. Thus, the bZIP scaffold may be a powerful tool in design of small, alpha-helical proteins with desired DNA recognition properties.     

The bZIP targets overlapping DNA subsites within a half-site, resulting in increased binding affinities

We previously reported that the wt bZIP, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes GCN4 cognate site AP-1 (TGACTCA) but also selectively targets noncognate DNA sites, in particular the C/EBP site (TTGCGCAA). In this work, we used electrophoretic mobility shift assay and DNase I footprinting to investigate the factors driving the high affinity between the wt bZIP and the C/EBP site. We found that on each strand of the C/EBP site, the wt bZIP recognizes two 4 bp subsites, TTGC and TGCG, which overlap to form the effective 5 bp half-site (TTGCG). The affinity of the wt bZIP for the overall 5 bp half-site is >or=10-fold stronger than that for either 4 bp subsite. Our results suggest that interactions of the wt bZIP with both subsites contribute to the strong affinity at the overall 5 bp half-site and, consequently, the C/EBP site. Accordingly, we propose that the wt bZIP undergoes conformational changes to slide between the two overlapping subsites on the same DNA strand and establish sequence-selective contacts with the different subsites. The proposed binding mechanism expands our understanding of what constitutes an actual DNA target site in protein-DNA interactions.