The role of c-myb during the maturation of murine CFU-E.

Proper expression of the c-myb proto-oncogene is essential for definitive, but not primitive erythropoiesis. To examine the role of c-myb during adult erythropoiesis, we incubated purified murine colony-forming units (CFU-E) with a c-myb-specific antisense oligodeoxynucleotide (AS-oligo) in order to diminish expression levels. By western blot analysis, c-myb expression was reduced during the first seven hours of AS-oligo treatment as compared to untreated cells. We then quantitated the amount of heme synthesized in CFU-E treated with c-myb AS-oligo, a random sequence oligo or no oligo. No significant differences were seen in the amount of heme synthesized during 42 hours of erythroid culture with either high levels (1 U/mL) or physiological levels (20 mU/mL) of Epo. In contrast, CFU-E treated with an AS-oligo directed toward mRNA encoding the first enzyme of the heme biosynthetic pathway in erythroid cells (d-aminolevulinate synthase) demonstrated a 65% reduction in the amount of heme synthesized. We conclude that the major role of c-myb during hematopoiesis must be in progenitor cells antecedent to the CFU-E stage and may possibly involve the establishment of the genetic program directing the formation of red blood cells.

Multiple copies of the ALA-D gene are located at the Lv locus in Mus domesticus mice.

Incremental differences in delta-aminolevulinate dehydratase (ALA-D; the second enzyme of the heme biosynthetic pathway) activity among inbred mouse strains can be attributed to variation in the number of copies of the ALA-D gene. We have cloned and characterized the Lv locus from an inbred mouse strain (DBA/2J) that has three times the normal ALA-D activity levels. The entire 12-kb ALA-D gene plus 16 kb of flanking DNA are found in 28-kb tandemly repeating units. We used the derived nucleotide sequence surrounding the internal junction of the repeats to survey wild-caught mice and demonstrate that multiple copies of the ALA-D gene occur in 7 of 24 worldwide locations of Mus domesticus mice. Data are consistent with a model that high lead (Pb) in the environment may be providing a selective advantage to mice harboring multiple copies of the ALA-D gene, since the enzyme is potently inhibited by lead.

Novel regulation of delta-aminolevulinate synthase in the rat harderian gland.

The mode of expression of delta-aminolevulinate synthase (ALAS), as well as that of mRNAs for other heme pathway enzymes, was examined in the rat Harderian gland. Northern blot and in situ hybridization analyses demonstrated that the non-specific ALAS (ALAS-N) mRNA is highly expressed in this tissue, whereas the erythroid-specific ALAS (ALAS-E) mRNA is not. Immunoblot analysis of ALAS also confirmed this finding at the protein level. ALAS-N mRNA was maximally induced in the Harderian gland and was not increased further by treatment of animals with 2-allyl-2-isopropylacetamide (AIA). The levels of mRNAs for other heme pathway enzymes, i.e., delta-aminolevulinate dehydratase, porphobilinogen deaminase, uroporphyrinogen decarboxylase, and coproporphyrinogen oxidase, also were increased markedly in the Harderian gland and not influenced by AIA treatment. The level of ferrochelatase (FeC) mRNA in the gland was, however, lower than that in the liver. The gland contained an extremely high level of protoporphyrin, while heme was undetectable. Microsomal heme oxygenase-1 (HO-1) mRNA levels were significantly higher in the Harderian gland than in the liver. When isolated glands were incubated with hemin in vitro in organ cultures, the level of HO-1 mRNA was increased, whereas the ALAS-N mRNA level was not. These findings indicate that markedly elevated levels of protoporphyrin and extremely low levels of heme in the Harderian gland are the results of both decreased expression of FeC and markedly increased expression of ALAS-N and HO-1. The constitutive expression of the ALAS-N gene in the Harderian gland suggests a novel transcriptional control mechanism of this gene.

Genetic regulation of delta-aminolevulinate dehydratase during erythropoiesis.

In an effort to understand how the heme biosynthetic pathway is uniquely regulated in erythroid cells, we examined the structure of the gene encoding murine delta-aminolevulinate dehydratase (ALAD; EC4.2.1.24), which is the second enzyme of the pathway. The gene contains two first exons, named 1A and 1B, which are alternatively spliced to exon 2, where the coding region begins. Each first exon has its own promoter. The promoter driving exon 1A expression is TATA-less and contains many GC boxes. In contrast, the exon 1B promoter bears regulatory sequences similar to those found for beta-globin and other erythroid-specific genes. Tissue distribution studies reveal that ALAD mRNA containing axon 1A is ubiquitous, whereas mRNA containing axon 1B is found only in erythroid tissues. This finding, together with our further observation that GATA-1 mRNA levels increase 3-fold during maturation of murine erythroid progenitor cells, may help explain simultaneous 3-fold increases in exon 1B expression. The unexpected result that axon 1A expression also increases 3-fold during CFU-E maturation may be attributable to the action of NF-E2, since there is a potential binding site in a position analogous to the NF-E2 site in the locus control region of the beta-globin gene cluster.

Isolation of a novel receptor tyrosine kinase cDNA expressed by developing erythroid progenitors.

Activation of distinct receptor tyrosine kinases (RTK), such as the products of the c-fms and c-kit proto-oncogenes, profoundly affects hematopoietic development. We have isolated a novel RTK cDNA, called met-related kinase (MRK), which is expressed on early erythroid progenitors. MRK is also expressed in many hematopoietic cell lines, and is not lineage restricted. Several regions within the catalytic domain of MRK have amino acid similarity to the c-met proto-oncogene and v-sea oncogene. Specific polyclonal anti-MRK antisera detects an 84-Kd polypeptide in COS cells transfected with an expression vector containing the MRK cDNA. Further studies of this novel RTK will provide potential insight into its role during hematopoiesis.

Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis.

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.

Roles of erythropoietin, insulin-like growth factor 1, and unidentified serum factors in promoting maturation of purified murine erythroid colony-forming units.

We have used 75% to 90% pure murine erythroid colony-forming units (CFU-E) to delineate the processes and factors underlying their maturation. These CFU-E form 32 cell colonies and are drawn from what we term generation I of a six-generation long maturation sequence (Landschulz et al, Blood 79:2749, 1992). Applying assays of 59Fe-heme biosynthesis and colony numbers as measures of maturation and analyses of DNA degradation as an index of programmed cell death, we find that (1) erythropoietin (Epo) enhances maturation throughout most of its course; (2) Epo first seems able to forestall DNA degradation when CFU-E reach generation II; (3) the processes that Epo elicits thereafter start to persist when Epo is withdrawn; (4) insulin-like growth factor I (IGF-I) also forestalls DNA breakdown, but later loses effectiveness; (5) IGF-I adds little to maturation when Epo levels are high, but when Epo levels are low, enhances it substantially; and (6) for maturation to be entirely optimal, an unidentified serum factor(s) is probably required when Epo levels are high and is certainly needed when Epo levels are like those in normal animals. Quantitatively, about 40% of optimal in vitro erythropoiesis at normal Epo levels depends on Epo alone, another 30% or less on the addition of IGF-I, and the remaining 30% or more on the addition of unidentified serum factor(s). Applied together, these three or more factors lead to two-thirds of the maximum maturation realized with saturating Epo levels. Because we also find that heme accumulated in CFU-E culture can closely approach levels in red blood cells, we suppose that our conclusions apply as well to CFU-E maturation in vivo.

Sequential activation of genes for heme pathway enzymes during erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells.

Changes in the level of transcripts encoding enzymes of the heme biosynthetic pathway as well as those encoding ubiquitous proteins were examined in murine Friend virus-transformed erythroleukemia cells during erythroid cell differentiation induced by chemicals including dimethyl sulfoxide (DMSO). Early changes following DMSO treatment were marked decreases in mRNAs for three ubiquitous proteins, i.e., a 70 kDa heat shock protein (less than 6 h), heme oxygenase and nonspecific delta-aminolevulinate synthase (ALAS) (less than 12 h). These changes were followed by sequential increases in mRNAs for enzymes in the heme biosynthetic pathway. Namely, mRNAs for the erythroid-specific ALAS, delta-aminolevulinate dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase started to increase at 12, 18, 18-24 and 24 h, respectively. Nuclear runoff studies revealed that these changes are largely transcriptional. Treatments with other inducers of erythroid differentiation, e.g., hexamethylene bisacetamide, n-butyric acid and N'-methylnicotinamide, also showed similar effects on mRNAs as those following DMSO. These findings suggest that both suppression of ubiquitous genes and activation of heme pathway enzyme genes are associated with erythroid differentiation, and the former occurs preceding changes in the latter.

Isolation and characterization of a rat delta-aminolevulinate dehydratase processed pseudogene.

Southern blot analysis of genomic DNA from different strains of rat indicated that there were multiple copies of the gene encoding the second enzyme of the heme biosynthetic pathway, delta-aminolevulinate dehydratase (ALA-D). Two types of genomic clones were isolated from a Sprague-Dawley rat library. One appears to be the expressed gene, whereas the nucleotide sequence of the other suggests that it contains an ALA-D processed pseudogene because (1) there are no introns, (2) there are multiple mutations that alter the predicted amino acid sequence of ALA-D and cause premature termination, (3) there is a 3' polyadenylated tract, and (4) there is an 8-bp direct repeat flanking the gene. The rat genome is unusual in this respect since ALA-D pseudogenes have not been detected in Southern blot analyses of other mammals, including human, gorilla, chimpanzee, orangutan, rabbit, mouse, and Chinese hamster.

Isolation of a rat liver delta-aminolevulinate dehydrase (ALAD) cDNA clone: evidence for unequal ALAD gene dosage among inbred mouse strains.

We have isolated several cDNA clones encoding delta-aminolevulinate dehydrase [ALAD; porphobilinogen synthase; 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24], the second enzyme in the heme biosynthetic pathway. We used a rabbit polyclonal antibody developed against the purified 35-kDa subunit of rat liver ALAD to screen a lambda gt11 cDNA expression library constructed from rat liver mRNA. A prototype clone (ALAD-1) contained a 680-base-pair insert and expressed a 140-kDa beta-galactosidase gene cDNA insert fusion protein immunoreactive with both polyclonal and monoclonal anti-ALAD. Identity of ALAD-1 was verified by hydridization to ALAD mRNA that had been enriched via immunopurification of rat liver polysomes with anti-ALAD. Intensity of such hybridization to a 1500-nucleotide-long mRNA was approximately equal to 200-fold greater than that realized with whole liver mRNA, a result consistent with the degree of immunoenrichment of ALAD mRNA found independently by analysis of cell-free translation products. A second ALAD cDNA clone (ALAD-3) was isolated when the rat liver cDNA expression library was rescreened with ALAD-1. The identity of both ALAD cDNA clones was established by correspondence between their nucleotide sequence and the reported amino-terminal protein sequence of bovine ALAD. Hybridization of ALAD cDNA to mouse genomic DNA indicates that the previously unexplained incremental differences in ALAD enzymatic activity among inbred mouse strains has arisen through alterations in ALAD gene dose.