An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.

Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.     

Competition between HMG-I(Y), HMG-1 and histone H1 on four-way junction DNA.

High mobility group proteins HMG-I(Y) and HMG-1, as well as histone H1, all share the common property of binding to four-way junction DNA (4H), a synthetic substrate commonly used to study proteins involved in recognizing and resolving Holliday-type junctions formed during in vivo genetic recombination events. The structure of 4H has also been hypothesized to mimic the DNA crossovers occurring at, or near, the entrance and exit sites on the nucleosome. Furthermore, upon binding to either duplex DNA or chromatin, all three of these nuclear proteins share the ability to significantly alter the structure of bound substrates. In order to further elucidate their substrate binding abilities, electrophoretic mobility shift assays were employed to investigate the relative binding capabilities of HMG-I(Y), HMG-1 and H1 to 4H in vitro. Data indicate a definite hierarchy of binding preference by these proteins for 4H, with HMG-I(Y) having the highest affinity (Kd approximately 6.5 nM) when compared with either H1 (Kd approximately 16 nM) or HMG-1 (Kd approximately 80 nM). Competition/titration assays demonstrated that all three proteins bind most tightly to the same site on 4H. Hydroxyl radical footprinting identified the strongest site for binding of HMG-I(Y), and presumably for the other proteins as well, to be at the center of 4H. Together these in vitro results demonstrate that HMG-I(Y) and H1 are co-dominant over HMG-1 for binding to the central crossover region of 4H and suggest that in vivo both of these proteins may exert a dominant effect over HMG-1 in recognizing and binding to altered DNA structures, such as Holliday junctions, that have conformations similar to 4H.     

The HMG-I(Y) A.T-hook peptide motif confers DNA-binding specificity to a structured chimeric protein.

Chromosomal translocations involving genes coding for members of the HMG-I(Y) family of "high mobility group" non-histone chromatin proteins (HMG-I, HMG-Y, and HMG-IC) have been observed in numerous types of human tumors. Many of these gene rearrangements result in the creation of chimeric proteins in which the DNA-binding domains of the HMG-I(Y) proteins, the so-called A.T-hook motifs, have been fused to heterologous peptide sequences. Although little is known about either the structure or biophysical properties of these naturally occurring fusion proteins, the suggestion has been made that such chimeras have probably assumed an altered in vivo DNA-binding specificity due to the presence of the A.T-hook motifs. To investigate this possibility, we performed in vitro "domain-swap" experiments using a model protein fusion system in which a single A. T-hook peptide was exchanged for a corresponding length peptide in the well characterized "B-box" DNA-binding domain of the HMG-1 non-histone chromatin protein. Here we report that chimeric A. T-hook/B-box hybrids exhibit in vitro DNA-binding characteristics resembling those of wild type HMG-I(Y) protein, rather than the HMG-1 protein. These results strongly suggest that the chimeric fusion proteins produced in human tumors as a result of HMG-I(Y) gene chromosomal translocations also retain A.T-hook-imparted DNA-binding properties in vivo.