We questioned the significance of haplotype structure in gene regulation by testing whether individual single nucleotide polymorphisms (SNPs) within a gene promoter region [interleukin-1-beta (IL1B)] might affect promoter function and, if so, whether function was dependent on haplotype context. We sequenced genomic DNA from 25 individuals of diverse ethnicity, focusing on exons and upstream flanking regions of genes of the cluster. We identified four IL1B promoter region SNPs that were active in transient transfection reporter gene assays. To substantiate allelic differences found in reporter gene assays, we also examined nuclear protein binding to promoter sequence oligonucleotides containing different alleles of the SNPs. The effect of individual SNPs on reporter gene transcription varied according to which alleles of the three other SNPs were present in the promoter construct. The SNP patterns that influenced function reflected common haplotypes that occur in the population, suggesting functionally significant interactions between SNPs according to haplotype context. Of the haplotypes that include the four functional IL1B promoter SNPs (-3737, -1464, -511, -31), the four haplotypes that showed different contextual effects on SNP function accounted for >98% of the estimated haplotypes in Caucasian and African-American populations. This finding underlines the importance of understanding the haplotype structure of populations used for genetic studies and may be especially important in the functional analysis of genetic variation across gene regulatory regions.

In heme deficiency, protein synthesis in reticulocytes is inhibited by activation of heme-regulated ␣-subunit of eukaryotic initiation factor-2␣ (eIF-2␣) kinase (HRI). Previous studies indicate that HRI contains two distinct heme-binding sites per HRI monomer. To study the role of the N terminus in the heme regulation of HRI, two N-terminally truncated mutants, Met2 and Met3 (deletion of the first 103 and 130 amino acids, respectively), were prepared. Met2 and Met3 underwent autophosphorylation and phosphorylated eIF-2␣ with a specific activity of approximately 50% of that of the wild type HRI. These mutants were significantly less sensitive to heme regulation both in vivo and in vitro. In addition, the heme contents of purified Met2 and Met3 HRI were less than 5% of that of the wild type HRI. These results indicated that the N terminus was important but was not the only domain involved in the heme-binding and heme regulation of HRI. Heme binding of the individual HRI domains showed that both N terminus and kinase insertion were able to bind hemin, whereas the C terminus and the catalytic domains were not. Thus, both the N terminus and the kinase insertion, which are unique to HRI, are involved in the heme binding and the heme regulation of HRI.

In heme-deficient reticulocytes, protein synthesis is inhibited due to the activation of heme-regulated eIF2alpha kinase (HRI). Activation of HRI is accompanied by its phosphorylation. We have investigated the role of autophosphorylation in the formation of active and stable HRI. Two autophosphorylated species of recombinant HRI expressed in Escherichia coli were resolved by SDS-PAGE. Both species of HRI were multiply autophosphorylated on serine, threonine, and to a lesser degree also tyrosine residues. Species II HRI exhibited a much higher extent of autophosphorylation and thus migrates slower in SDS-PAGE than species I HRI. Similarly, HRI naturally present in reticulocytes also exhibited these species with different degrees of phosphorylation. Importantly, in heme-deficient intact reticulocytes, inactive species I HRI was converted completely into species II. We further separated and characterized these two species biochemically. We found that species I was inactive and had a tendency to aggregate while the more extensively autophosphorylated species II was an active heme-regulated eIF2alpha kinase and stable homodimer. Our results strongly suggest that autophosphorylation regulates HRI in a two-stage mechanism. In the first stage, autophosphorylation of newly synthesized HRI stabilizes species I HRI against aggregation. Although species I is an active autokinase, it is still without eIF2alpha kinase activity. Additional multiple autophosphorylation in the second stage is required for the formation of stable dimeric HRI (species II) with eIF2alpha kinase activity that is regulated by heme.

The regulation of protein synthesis by the availability of heme in reticulocytes is well established. However, the mechanism by which heme regulates translational initiation is not clear. In this study, we have examined the heme regulation directly on the homogeneous heme-regulated eIF-2A kinase (HRI), which is activated during heme deficiency. We found that HRI purified as a hemoprotein with the characteristic Soret band of hemoprotein at 424 nm. This HRI was an active autokinase and eIF-2A kinase, and its kinase activities were inhibited by submicromolar concentrations of hemin with an apparent K i of 0.5 µM. Homogeneous HRI was a homodimer, and its activities could not be inhibited by incubation with purified inactive K199R HRI in vitro. Our results suggest that there are two distinct types of hemebinding sites in the HRI homodimer. The binding of heme to the first site is stable, while the binding of heme to the second site is responsible for the rapid downregulation of HRI activity by heme. These results indicate that HRI binds heme and serves as a sensor of the availability of heme to coordinate the balanced synthesis of globins and heme in erythroid cells.Keywords : protein kinase ; heme ; hemoprotein ; translation ; initiation.Protein synthesis in reticulocytes is regulated by the availability of heme. During heme deficiency, protein synthesis is inhibited at the level of initiation, as a result of the activation of the heme-regulated eukaryotic initiation factor 2A (eIF-2A) kinase (HRI) (reviewed in [1Ϫ3]). Phosphorylation of the A subunit of eIF-2 at the Ser51 residue by activated HRI results in the formation of the eIF-2(AP)/eIF-2B complex and renders eIF-2B non-functional. eIF-2B is required for the exchange of GTP for GDP bound to eIF-2 in the recycling of eIF-2 for another round of initiation. Since eIF-2B is limiting, phosphorylation of a fraction of eIF-2 is sufficient to shut-off protein synthesis.In addition to HRI, there are two other eIF-2A kinases that have been studied extensively (reviewed in [4]); these are human and mouse double-stranded RNA-dependent eIF-2A kinase (PKR), and yeast and Drosophila [5] GCN2 protein kinase. These three eIF-2A kinases share extensive homology in the kinase catalytic domains [5Ϫ8], and phosphorylate eIF-2A at Ser51 [9,10]. However, the regulatory mechanisms of these three eIF-2A kinases are very different, involving heme for HRI, dsRNA for PKR and the condition of amino acid starvation for GCN2. Both HRI and PKR can functionally substitute for GCN2 in the GCN4 translational control in yeast [11]. The autophosphorylation, eIF-2A phosphorylation and the inhibition of protein synthesis by purified HRI is inhibited by incubation with hemin [28]. In addition, the binding of hemin to a highly purified HRI has been demonstrated [29]. We have shown that hemin promotes intersubunit disulfide-bond formation in HRI [30], and that this disulfide-bond formation in HRI correlates with the maintenance of protein synthesis, the reversal of the inhibition of protein synthesi...