Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.     

Nuclear factor 1 is a component of the nuclear matrix

Chicken histone H5 is an H1-like linker histone that is expressed only in nucleated erythrocytes. The histone H5 promoter has binding sites for Sp1 (a high affinity site) and UPE-binding protein, while the 3′ erythroid-specific enhancer has binding sites for Sp1 (one moderate and three weak affinity), GATA-1, and NF1. In this study we investigated whether trans-acting factors that bind to the chicken histone H5 promoter or enhancer are associated with adult chicken immature and mature erythrocyte nuclear matrices. We show that NF1, but not Sp1, GATA-1, or UPE-binding protein, is associated with the internal nuclear matrices of these erythroid cells. Further, we found that a subset of the NF1 family of proteins is bound to the mature erythrocyte nuclear matrix. These results suggest that in chicken erythrocytes NF1 may mediate an interaction between the histone H5 enhancer and the erythroid internal nuclear matrix. NF1 was also present in the internal nuclear matrices of chicken liver and trout liver. The observations of this study provide evidence that NF1 may have a role in a variety of cell types in targeting specific DNA sequences to the nuclear matrix. © 1994 Wiley-Liss, Inc.     

Targeting of Sp1 to a non-Sp1 site in the human neurofilament (H) promoter via an intermediary DNA-binding protein.

The human neurofilament (H) promoter contains multiple binding sites for nuclear proteins including a Proximal (Prox) site centered around the sequence GGTTGGACC and an adjacent pyrimidine (Pyr) tract site centered around the sequence CCCTCCTCCCC. Surprisingly binding to a probe containing the Prox/Pyr region of the NF(H) promoter was competed in gel shifts by an oligonucleotide containing only an Sp1 binding site (GGGGCGGGG). Supershift assays with a polyclonal anti-Sp1 antisera confirmed that Sp1 was part of the complex formed with the Prox/Pyr probe. However neither bacterially expressed Sp1 516C or vaccinia virus expressed full-length Sp1 778C bound to the Prox or Pyr sequences in DNase I footprints or gel shift assays. Gel shift competitions and supershift assays with probes containing either Prox or Pyr tract sites alone demonstrated targeting of Sp1 to the Prox binding site and identified a non-Sp1 containing complex which contains a Prox binding protein. Adding exogenous Sp1 to a HeLa nuclear extract enhanced the Sp1-containing complex but had no effect on the Prox complex. These studies show that Sp1 can be targeted to a non-Sp1 site in the human NF(H) promoter through protein/protein interactions with a distinct sequence specific DNA-binding protein.     

The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites.

The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity.     

Repression of enhancer activity by the adenovirus E1A tumor protein(s) is mediated through a novel HeLa cell nuclear factor.

We have examined the mechanism for the host cell-dependent repression of enhancer activity by the adenovirus early region 1A (E1A) proteins. The enhancer used in this study, from the human BK virus P2, functions efficiently in cis to activate expression from the adenovirus major late promoter in the human kidney cell line, 293, and in a monkey kidney cell line, MK2. In addition, enhancer activity can be stimulated by the E1A gene products in these cells. However, cis-enhancer activity is repressed in the HeLa cell line, and we demonstrate here that further repression can be induced by the E1A proteins. We show that the binding site for the negative regulatory factor involved in cis-repression, designated BK virus enhancer factor 1 (BEF-1), is also required for E1A-induced repression. Using gel mobility retardation assays, we demonstrated a 4-fold increase in active BEF-1 in nuclear extracts containing the E1A proteins. However, the E1A proteins did not change the binding pattern or the strength of binding of BEF-1 to its target sequence. BEF-1 was identified as a 98-kDa nuclear factor, and phosphorylation was shown to be important for DNA binding. Three potential nuclear factor 1 (NF-1) sites are present in the BEF-1-binding site. Using a known NF-1 site as competitor DNA in a gel mobility retardation assay, we provide evidence that BEF-1 may be a newly identified NF-1 family member. In addition, the sequence TGA present in the repressor-binding site was shown to be essential for high affinity binding of BEF-1. Overall, our data demonstrate that an enhancer can be repressed by the trans-activation of a negative regulatory factor.     

Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms.

We describe the purification to near homogeneity of proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer. Proteins binding to this site produce four protein-DNA complexes which are distinguished by their mobility in gel retardation assays and their elution properties in an anion exchange column. DNA affinity-purified preparations of three chromatographically separated pools, containing different subsets of the four complexes, each contained three polypeptides of 42.5, 44, and 45 kilodaltons (kDa). UV crosslinking of protein to enhancer DNA demonstrated that site C2-binding activities in the three different pools bound DNA through proteins of similar sizes (about 45 kDa), even though the protein-DNA complexes formed by these binding activities were quite distinct. Gel exclusion chromatography and equilibrium binding analyses indicated that the distinct protein-DNA complexes were due to different oligomeric forms of the individual subunits and that a larger multimeric form bound with high affinity to the heavy-chain enhancer site C2, while a smaller species had a much lower affinity for heavy-chain enhancer sequences. Purified protein has been used to map high-affinity binding sites for site C2-binding proteins within an immunoglobulin heavy-chain promoter and at site KE3 in the kappa light-chain enhancer.