Correction of sickle cell disease in transgenic mouse models by gene therapy.

Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.

Regulation of hemoglobin synthesis and proliferation of differentiating erythroid cells by heme-regulated eIF-2alpha kinase.

Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2alpha kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2alpha. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta-globin in engrafted mice.

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human beta-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked beta-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human beta-globin and the green fluorescent protein in 20-95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of beta-locus control region hypersensitive site 2 alone, human beta-globin mRNA expression levels ranged from 0.15% to 20% with human beta-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Upregulation of protein synthesis initiation factor eIF-4E is an early event during colon carcinogenesis.

A general increase in protein synthesis and a specific increase in the synthesis of growth-promoting proteins are necessary for mitogenesis. Regulation of protein synthesis, as well as preferential translation of some mRNAs coding for growth promoting proteins (e.g. cyclin D1), involves the essential protein synthesis initiation factor eIF-4E. This factor is induced by various oncoproteins, and, when overexpressed, it can transform cultured cells. In this report we explore the roles of eIF-4E in human neoplastic disorders of the colon and in the regulation of general and specific protein synthesis. We find that eIF-4E is increased in colon adenomas and carcinomas, and this increase is accompanied in most but not all cases by elevation of cyclin D1 levels. While general protein synthesis is increased by eIF-4E overexpression in cultured cells, only a small proportion of proteins is preferentially upregulated by eIF-4E, as revealed by two-dimensional gel electrophoresis. These results are consistent with the view that eIF-4E plays a role in carcinogenesis by increasing general protein synthesis and by preferentially upregulating a subset of putative growth promoting proteins. Our results, taken together with the recent findings that c-myc transcription is negatively regulated by APC and our earlier data on transcriptional activation of eIF-4E expression by c-Myc suggest that eIF-4E is a downstream target of the APC/beta-catenin/Tcf-4 pathway, and is strongly involved in colon tumorigenesis.

The effects of pyrroloquinoline quinone on heme-regulated eIF-2alpha kinase and eIF-2B activities in eukaryotic protein synthesis.

Pyrroloquinoline quinone (PQQ), a novel cofactor of biological redox processes, is ubiquitous in animal cells. We have examined the effects of PQQ on protein synthesis. PQQ inhibits protein synthesis in hemin-supplemented rabbit reticulocyte lysates. This inhibition is characterized by increased phosphorylation of eIF-2alpha and by diminished guanine nucleotide exchange activity of eIF-2B. The increased eIF-2alpha phosphorylation is the result of activation by PQQ of the heme-regulated eIF-2alpha kinase (HRI). The addition of 10 microM PQQ completely inhibits the increase in protein synthesis that occurs on the addition of hemin (20 microM) to heme-deficient lysates, whereas a lower concentration of PQQ (100 nM) causes a very slight stimulation of protein synthesis. The increased eIF-2alpha phosphorylation that occurs at high concentrations of PQQ inhibits eIF-2B activity, presumably due to formation of a 15S complex [eIF-2(alphaP).eIF-2B] in which eIF-2B becomes non-functional. Low concentrations of PQQ (0.1-1 microM) do not affect eIF-2alpha phosphorylation, but rather enhance the guanine nucleotide exchange activity of eIF-2B in reticulocyte lysates. In Chinese hamster ovary cell extract which is devoid of significant eIF-2alpha kinase activity, addition of both low and high concentrations of PQQ results in an increase in eIF-2B activity. The addition of PQQ to reticulocyte lysates activates HRI whereas addition of PQQ to purified HRI in vitro inhibits the autokinase and eIF-2alpha kinase activity of the HRI; the inhibition of purified HRI by PQQ is observed both in the presence and absence of hemin. These findings suggest that PQQ inhibits purified HRI by acting as an oxidant whereas in lysates in which PQQ is readily reduced, the PQQ acts as a reductant and increases the activities of both HRI and eIF-2B.

Transient inhibition of protein synthesis induces expression of proto-oncogenes and stimulates resting cells to enter the cell cycle.

There is evidence that resting cells are able to produce molecules with antiproliferative activity, some of which behave as short-lived repressor proteins. We suggest that transient inhibition of protein synthesis in resting cells would lead to a decrease in the levels of these negative growth regulators and might, therefore, promote mitogenic responses. We report that treatment of resting (serum-deprived) NIH 3T3 cells with cyclocheximide (CH) or puromycin induces expression of c-fos, c-jun and c-myc proto-oncogenes in a manner similar to that of platelet-derived growth factor (PDGF). Actinomycin D (Act D) abrogates the induction of proto-oncogene expression. Transient inhibition of protein synthesis by CH or puromycin also induces the resting NIH 3T3 and C3H 1OT1/2 cells to enter the cell cycle. Inhibition of new RNA or protein synthesis abolishes the proliferative response. These findings show that control mechanisms at both transcriptional and translational levels are operative in the resting cells treated with protein synthesis inhibitors. Cell fusion experiments with resting and serum-stimulated NIH 3T3 cells revealed that brief pre-incubation of resting cells with either PDGF, CH or puromycin abrogates their ability to suppress the onset of DNA synthesis in the nuclei of stimulated cells in heterodikaryons. However, the abrogative effect of PDGF disappeared in the presence of Act D, whereas the effects of protein synthesis inhibitors did not, indicating their independence of the induction of transcription. The data suggest that the observed effects of protein synthesis inhibitors are connected with elimination of some short-lived negative growth regulators, since a brief translational arrest is sufficient for the resumption of DNA synthesis in the nuclei of stimulated cells blocked by resting cells in heterodikaryons.

Eukaryotic translation initiation factor 4E regulates expression of cyclin D1 at transcriptional and post-transcriptional levels.

Regulation of the cell cycle is orchestrated by cyclins and cyclin-dependent kinases. We have demonstrated previously that overexpression of eukaryotic translation initiation factor 4E (eIF-4E) in NIH 3T3 cells growing in 10% fetal calf serum leads to highly elevated levels of cyclin D1 protein without significant increase in cyclin D1 mRNA levels, suggesting that a post-transcriptional mechanism is involved. (Rosenwald, I. B., Lazaris-Karatzas, A., Sonenberg, N., and Schmidt, E. V. (1993) Mol. Cell. Biol. 13, 7358-7363). In the present research, we did not find any significant effect of eIF-4E on polysomal distribution of cyclin D1 mRNA. However, the total amount of cyclin D1 mRNA associated with polysomes was significantly increased by eIF-4E overexpression. Further, we determined that the levels of both cyclin D1 protein and mRNA are increased in serum-deprived cells overexpressing eIF-4E. Nuclear run-on experiments demonstrated that the rate of the cyclin D1 transcription is not down-regulated in serum-deprived cells overexpressing eIF-4E. Thus, elevated levels of eIF-4E may lead to increased transcription of the cyclin D1 gene, and this effect becomes visible when serum deprivation down-regulates the rate of cyclin D1 mRNA synthesis in control cells. However, artificial overexpression of cyclin D1 mRNA in serum-deprived cells in the absence of eIF-4E overexpression did not cause the elevation of cyclin D1 protein, and this overexpressed cyclin D1 mRNA accumulated in the nucleus, suggesting that one post-transcriptional role of eIF-4E is to transport cyclin D1 mRNA from the nucleus to cytoplasmic polysomes.

Regulation of protein synthesis by heme-regulated eIF-2 alpha kinase.

Protein synthesis is regulated by the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha) in a variety of cells. At present, there are two distinct mammalian eIF-2 alpha kinases that have been cloned, the double-stranded-RNA-dependent eIF-2 alpha kinase (PKR) and the heme-regulated eIF-2 alpha kinase (HRI). HRI is activated under conditions of heme deficiency in immature erythroid cells, and its activity is inhibited by heme. The high levels of HRI in reticulocytes indicate that its major physiological role is the regulation of protein synthesis, particularly of hemoglobin, according to the concentration of heme in these cells.

Retroviral transfer of a human beta-globin/delta-globin hybrid gene linked to beta locus control region hypersensitive site 2 aimed at the gene therapy of sickle cell disease.

Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I hypersensitive site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients.

Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.

Regulation of heme-regulated eIF-2 alpha kinase and its expression in erythroid cells.

In this article we focus first on the molecular mechanisms controlling the activity of the heme-regulated translational inhibitor, HRI, in erythroid cells. Then we discuss the tissue-specific expression of HRI. The experimental evidence obtained to date indicates that the major physiological role of HRI is in adjusting the synthesis of globin to the availability of heme.

Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) in Saccharomyces cerevisiae by the GCN2 protein kinase stimulates the translation of GCN4 mRNA. The protein kinases heme-regulated inhibitor of translation (HRI) and double-stranded RNA-dependent eIF-2 alpha protein kinase (dsRNA-PK) inhibit initiation of translation in mammalian cells by phosphorylating Ser-51 of eIF-2 alpha. We show that HRI and dsRNA-PK phosphorylate yeast eIF-2 alpha in vitro and in vivo and functionally substitute for GCN2 protein to stimulate GCN4 translation in yeast. In addition, high-level expression of either mammalian kinase in yeast decreases the growth rate, a finding analogous to the inhibition of total protein synthesis by these kinases in mammalian cells. Phosphorylation of eIF-2 alpha inhibits initiation in mammalian cells by sequestering eIF-2B, the factor required for exchange of GTP for GDP on eIF-2. Mutations in the GCN3 gene, encoding a subunit of the yeast eIF-2B complex, eliminate the effects of HRI and dsRNA-PK on global and GCN4-specific translation in yeast. These results provide further in vivo evidence that phosphorylation of eIF-2 alpha inhibits translation by impairing eIF-2B function and identify GCN3 as a regulatory subunit of eIF-2B. These results also suggest that GCN4 translational control will be a good model system to study how mammalian eIF-2 alpha kinases are modulated by environmental signals and viral regulatory factors.

Recycling and phosphorylation of eukaryotic initiation factor 2 on 60S subunits of 80S initiation complexes and polysomes.

Phosphorylation of the alpha-subunit (38 kDa) of eukaryotic initiation factor 2 (eIF-2 alpha) regulates initiation of protein synthesis in eukaryotic cells. This phosphorylation is enhanced in cycloheximide-treated heme-deficient reticulocyte lysates in which polysomes are maintained. In early heme deficiency prior to polysome disaggregation, eIF-2(alpha P) accumulates primarily on the 60S subunits of polysomes. Further, isolated polysomes contain eIF-2 alpha that is efficiently phosphorylated in vitro by heme-regulated inhibitor (HRI). Immunoblot analysis of eIF-2 distribution in sucrose gradients of actively protein-synthesizing lysates indicates that eIF-2 is distributed at low levels throughout the polysome profiles. These findings suggest that polysome-bound eIF-2 alpha is a target of HRI under physiological conditions. The presence of eIF-2 on the 60S subunits of polysomes is incompatible with the conventional model in which eIF-2 is recycled during the joining of the 48S preinitiation complex and the 60S subunit to form the 80S initiation complex. A modified model is presented with emphasis on the translocation of eIF-2 from the 40S ribosomal subunit of the 48S preinitiation complex (eIF-2.GTP.Met-tRNA(f).40S.mRNA) to the 60S subunit of the 80S initiation complex.

Effects of hemin and porphyrin compounds on intersubunit disulfide formation of heme-regulated eIF-2 alpha kinase and the regulation of protein synthesis in reticulocyte lysates.

To study the mechanism by which heme regulates the heme-regulated eIF-2 alpha kinase (HRI), the effects of various protoporphyrin IX (PP) compounds on the kinase activities and intersubunit disulfide formation of HRI and on protein synthesis in reticulocyte lysates were examined. Hemin and cobalt protoporphyrin (CoPP) are more effective than ZnPP, NiPP, SnPP, and metal-free PP in promoting intersubunit disulfide bond formation in HRI, in inhibiting the autokinase and eIF-2 alpha kinase activities of HRI, in inhibiting phosphorylation of eIF-2 alpha in rabbit reticulocytes, in maintaining protein synthesis, and in reversing the inhibition of protein synthesis in heme deficiency. There is an apparent correlation of in vitro intersubunit disulfide formation of HRI and the regulation of HRI kinase activities and protein synthesis by these porphyrin compounds. HRI in the reticulocyte lysate can be cross-linked by 1,6-bismaleimidohexane (bis-NEM). The formation of bis-NEM cross-linked dimers in lysates is prevented completely by N-ethylmaleimide (NEM) which alkylates free sulfhydryl groups and is diminished by hemin and CoPP. These results support the view that HRI in hemin-supplemented lysates is in equilibrium between the noncovalently linked dimer and the disulfide-linked dimer. The molecular size of HRI in control, hemin-supplemented, or NEM-treated hemin-supplemented lysates is identical to that of purified HRI; activation of HRI and changes in its thiol status do not significantly affect its molecular size.

Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase of rabbit reticulocytes: homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2 alpha kinase.

We have cloned the cDNA of the heme-regulated eIF-2 alpha kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2 alpha kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of approximately 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2 alpha kinase. This observation suggests that GCN2 protein kinase may be an eIF-2 alpha kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.

Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.

We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases.

Disulfide bond formation in the regulation of eIF-2 alpha kinase by heme.

The inhibition of the autophosphorylation of the heme-regulated eukaryotic initiation factor (eIF)-2 alpha kinase (HRI) by hemin is very similar to that produced by thiol oxidation by diamide. The results obtained from the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unphosphorylated and phosphorylated HRI under reducing and nonreducing conditions indicate that hemin promotes disulfide formation in HRI. Hemin-promoted disulfide formation in HRI occurs under quasi-physiological conditions, i.e. 30 degrees C, 10 min at hemin concentrations of 5-10 microM. Under nondenaturing conditions, unphosphorylated HRI, phosphorylated HRI, hemin-treated unphosphorylated HRI, and hemin-treated prephosphorylated HRI are all eluted identically on Sephacryl S-300 column chromatography with an apparent molecular mass of 290,000 daltons. It appears, therefore, that the disulfide formation promoted by hemin occurs within the unit of 290,000 daltons. In addition, hemin treatment of phosphorylated HRI results in the appearance of a disulfide-linked form of higher molecular mass when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. A similar high molecular mass form is observed when HRI is treated with 1,6-bismaleimidohexane, a double sulfhydryl cross-linker agent, and the autophosphorylation of HRI and the phosphorylation of eIF-2 alpha by HRI are greatly diminished; these effects are similar to the effects of hemin on HRI. We conclude that disulfide formation by hemin provides a likely mechanism by which hemin prevents the activation and inhibits the activity of HRI.

An erythroid-specific, developmental-stage-independent enhancer far upstream of the human "beta-like globin" genes.

We have identified an erythroid-specific enhancer element far upstream of the human "beta-like globin" genes, at 10.2-11.0 kilobases 5' of the embryonic epsilon-globin gene, and thus at 53-54 kilobases 5' of the adult beta-globin gene. It is capable of enhancing the expression of a cis-linked test gene by up to 300-fold. This enhancer element is apparently developmental-stage-independent, as it is functional at the embryonic and the adult developmental stages in erythroid cells that are expressing the respective beta-like globin genes. The enhancer and globin promoter sequences work in synergy and are capable of conferring on a cis-linked gene the high transcriptional efficiency (enhancer function), erythroid specificity (enhancer and promoter functions), and developmental-stage specificity (promoter function) that are characteristic of the in vivo transcription of the beta-like globin genes in erythroid cells.