Specific repression of beta-globin promoter activity by nuclear ferritin.

Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult beta-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the beta-globin promoter at -153 to -148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress beta-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the beta-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant beta-globin promoter for the -150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the -150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human beta-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases.

Use of somatic cell fusion to reprogram globin genes.

The developmental phenomenon of hemoglobin switching occurs in all classes of vertebrates and is due to differential regulation of divergent globin genes which are arranged in chromosomally clustered families. By fusing erythroid cells of different developmental programs, it has been shown that erythroid nuclei of either early or late developmental stage can be reprogrammed, i.e. the gene switch can be reversed in adult erythroid nuclei and/or prematurely-induced in fetal/embryonic erythroid nuclei. Experiments with heterokaryons demonstrate that the reprogramming is due to trans-acting factors that are developmental-stage-specific. These results suggest the feasibility of using fusisome-carried sets of nuclear factors to reprogram somatic cells.

Efficient 5' end labeling of dephosphorylated DNA.

Efficient 5'-end labeling of DNA is an important procedure in recombinant DNA technology. Prior to labeling, it is important to inactivate alkaline phosphatase, used in the dephosphorylation of the DNA, by using proteinase K. Removal of proteinase K is usually performed by extracting twice with chloroform:isoamyl alcohol. In this report we show that extracting the sample four times with chloroform results in more efficient removal of sodium dodecyl sulfate (SDS), an important constituent of proteinase K buffer, which allows a 25- to 40-fold increase in labeling efficiency compared with extracting twice or once with chloroform, respectively. Unremoved SDS inhibits efficient labeling, possibly by inhibiting the activity of the kinase.

Hemoglobin switching in Rana/Xenopus erythroid heterokaryons: factors mediating the metamorphic hemoglobin switch are conserved.

Hemoglobin switching, which occurs in all classes of vertebrates as well as in certain invertebrates, is due to developmental regulation of different globin genes which are typically arranged in clustered families. By fusing erythroid cells of different developmental programs, trans-acting factors that regulate this switch in gene expression have been detected [Ramseyer et al. (1989): Dev Biol 133:262-271]. Adult erythroid cells of one anuran species, Xenopus laevis, were fused with tadpole erythroid cells of another frog, Rana catesbeiana, creating developmental erythroid heterokaryons that synthesize adult Rana globin mRNA and hemoglobins. The results show that factors from adult Xenopus erythroid cells are capable of inducing adult Rana globin gene expression in the Rana tadpole erythroid cell nucleus. We have used the cross-induction of adult Rana hemoglobin synthesis in these adult Xenopus/Rana tadpole erythroid heterokaryons to address two practical questions, answers to which may be helpful in isolating developmental stage-specific globin gene regulatory proteins: 1) Are erythroblasts which are actively expressing globin mRNAs and hemoglobins richer in specific globin-inducing activities than other stages of erythroid cellular differentiation? 2) Do mature, circulating erythrocytes still have the activities necessary to mediate the cross-induction of Hb synthesis? The results reported here show that the answers to both questions are affirmative and show that quiescent, fully differentiated adult erythroid cells are still capable of expressing the trans-activator(s). These findings show that factors which mediate the metamorphic hemoglobin switch are conserved between these two genera of frogs.

The hemoglobins of the bullfrog, Rana catesbeiana. The cDNA-derived amino acid sequences of the alpha chains of adult hemoglobins B and C: their roles in deoxygenation-induced aggregation.

The adult bullfrog (Rana catesbeiana) has two major tetrameric hemoglobins, B and C, which share a common beta chain but have different alpha chains. Components B and C associate upon deoxygenation to form a complex of the form BC2, a trimer of tetramers that depends on contacts between the alpha B and alpha C chains. Nucleotide sequences of cDNA transcripts for these chains have been determined. Transcripts were identified by analysis of the amino acid compositions of the tryptic peptides of the components and by partial amino acid sequencing. These results, together with the amino acid sequence of the beta chain (Tam, L.-T., Gray, G. P., and Riggs, A. F. (1986) J. Biol. Chem. 261, 8290-8294), permit an analysis of the structures of the alpha 2 beta 2 tetramers of hemoglobins B and C. Molecular modeling suggests possible residues at the alpha B-alpha C interfaces in the BC2 trimer and additional alpha C-alpha C contacts that would form a closed ring of six alpha chain subunits that would further stabilize the BC2 trimer. Phylogenetic analysis of the alpha B sequence suggests that it may be a "tadpole" chain, the temporal expression of which has shifted from larva to adult.

Intracellular signals for developmental hemoglobin switching.

We have detected trans-acting factors that regulate developmental hemoglobin switching by fusing erythroid cells of different developmental programs. Adult erythroid cells of one anuran species, Xenopus laevis, were fused with tadpole erythroid cells of another frog, Rana catesbeiana. In a second set of experiments, dimethyl sulfoxide-induced murine erythroleukemia cells, which express only adult mouse globins, were fused with Rana tadpole erythroid cells, which express only embryonic and fetal-like globins. Adult Rana globin gene expression was detected in both sets of transient heterokaryons at 6 hr after fusion. Dot blots and Northern blots of total RNA from the heterokaryons contained material that reacted with an adult Rana alpha-globin probe; newly synthesized adult Rana hemoglobin tetramers were detected with native polyacrylamide gel electrophoresis. These results show that developmental stage-specific transacting factors for globin genes can function across vertebrate classes (mammalia to amphibia) and suggest that the mechanisms that regulate developmental hemoglobin switching are highly conserved.

Erythroid heterokaryons: a system for investigating the functional role of trans-acting factors in developmental hemoglobin switching.

We have shown that erythroid cells from widely divergent species such as amphibians and mammals can be efficiently fused using either calcium phosphate bridges or polyethylene glycol. Transient heterokaryons of adult mouse erythroid (MEL) cells and Rana catesbeiana (bullfrog) tadpole erythroid cells produce adult Rana alpha globin mRNA and adult Rana hemoglobin (Hb) tetramers. Rana tadpole/adult Xenopus erythroid heterokaryons also exhibit this switch to adult Rana globin gene expression. These results indicate that trans-acting factors--and the globin gene regulatory mechanism of which they are a part--are conserved in vertebrate phylogeny. We also wish to know whether the reciprocal Hb switch occurs in each of these two types of heterokaryons, i.e., whether embryonic or fetal globin genes are reactivated in the adult nucleus. Experiments to answer these questions are in progress and are briefly discussed. The influence of stage of erythroid differentiation of the larval and adult donor cells on the cross-inductions is also being explored. These types of experiments should indicate which cells will be the best sources of stimulatory and inhibitory factors that are globin-gene specific. This system may be useful as an in situ assay for the function of purified trans-factors, which could be encapsulated within RBC ghosts and delivered via cell fusion.

Formation of transient polykaryons by fusion of erythrocytes of different developmental programs.

We report here two methods of fusing erythroid cells from bullfrogs (Rana catesbeiana), using polyethylene glycol or calcium phosphate, which yield masses of polykaryons in which the cytoplasms and nuclei of tadpole and adult frog erythroid cells are intermixed. The masses of fused cells carry out protein synthesis in culture, including the assembly of normal hemoglobin (Hb) tetramers. In these polykaryons there is reactivation of the expression of specific Hbs that have previously been "turned off" in vivo as the result of either a developmental Hb switch or normal cellular differentiation and RBC maturation. For example, the products of fusion of tadpole erythroblasts with adult frog mature RBCs synthesize adult Hb, whereas neither cell population alone does so. Recent experiments have taken advantage of a Hb-expression polymorphism that we discovered in this species, such that some tadpoles have greatly reduced expression of one of the larval Hbs (Hb Td-4). Fusion of erythroblasts from such tadpoles with RBC from frogs that had expressed Hb Td-4 when they were tadpoles produces polykaryons that synthesize Hb Td-4, indicating there is a trans factor that stimulates Td-4 expression. Heterospecific erythroid cell polykaryons can be constructed in an analogous manner, facilitating the study of trans-acting factors that regulate specific globin gene expression during development.

Determination of hemoglobin expression patterns in erythroid cells of Rana catesbeiana tadpoles.

1. Rana catesbeiana (bullfrog) tadpoles are heterogeneous in the relative amounts of four major tadpole hemoglobins (Hbs), as well as in the relative amounts of two tadpole red blood cell types in the peripheral blood. 2. Previous work has shown that this heterogeneity is present at all stages of larval development and growth. 3. Although some tadpoles lack one of the Hbs in their peripheral blood (i.e. the electrophoretically slowest form, Td-4), the missing Hb can be found in the erythropoietic organ from which it emanates (the kidneys), indicating that the heterogeneity results from quantitative differences in gene expression. 4. We wished to know whether this in vivo regulation is subject to external environmental perturbation and report that tadpoles of known Hb phenotypes regenerate precisely the pre-anemia Hb profile during early as well as late stages of recovery from phenylhydrazine-induced anemia. 5. These and other results indicate that the in vivo mechanism for regulating the pattern of Hb expression has become firmly determined in the erythropoietic system by the earliest larval stage of development.

Circulating epoxide hydrolase immunodeterminants in rats bearing hyperplastic nodules induced by 2-acetylaminofluorene.

Epoxide hydrolase immunodeterminants (EH-immunodeterminants), as measured by a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA), were detected in the sera of rats bearing hyperplastic liver nodules induced by feeding 2-acetylaminofluorene (2-AAF), while they were low or absent (less than 0.05 microgram/ml) in the sera of rats fed a basal diet. The levels of EH-immunodeterminants ranged from 0.2 microgram/ml to 2 microgram/ml of serum and correlated (P less than 0.01) with the nodular status of the liver. Rats with predominantly small nodules (less than 3 mm) had circulating levels of antigen from 0.15 to 0.35 microgram/ml while those with large nodules (greater than 3 mm) had values ranging from 0.4 to 2.0 micrograms/ml serum and the serum levels for rats containing both small and large nodules were found to be in the intermediate range (0.2-0.8 microgram/ml). EH-immunodeterminants were also released into tissue culture medium from incubated hyperplastic nodule slices and the levels of the immunodeterminants released from nodules were about 5-fold higher than that released from incubated control liver.

Hemoglobin switching across vertebrate classes: exchange of developmental signals by cell fusion.

Our aim is to obtain evidence for trans-acting factors that regulate developmental hemoglobin (Hb) switching. Our approach is to fuse erythroid cells that have different developmental programs, allowing the trans-acting factors from the adult cell to have access to the nucleus of the fetal or embryonic cell and vice-versa. After cell fusion, the heteropolykaryons are cultured for six hours, and globin gene expression is assayed at two levels: (1) at the level of mRNAs on dot blots hybridized with globin-specific cDNA probes, and (2) at the level of fully-formed Hb tetramers separated by native polyacrylamide gel electrophoresis (PAGE). Since the donor erythroid cells are from different species, it is easy to determine which globin gene products are from which nucleus. And since there is no nuclear fusion for at least twelve hours, the Hb switching that occurs is due to regulation in trans. Our results show that developmental Hb switching occurs in mouse-frog erythroid cell polykaryons. When DMSO-induced murine erythroleukemia (MEL) cells (which express only adult mouse Hbs) are fused with Rana tadpole RBCs (which express only embryonic and fetal-like Hbs), the resultant heteropolykaryons express adult frog globin mRNA and adult frog Hbs. We conclude that there are developmental stage-specific trans-acting factors for Hb switching, that trans-acting factors from adult mouse erythroid cells can induce expression of adult frog globin genes in a tadpole RBC nucleus, and that Hb switching mechanisms are conserved across vertebrate classes.

In vivo regulation of hemoglobin phenotypes of developing Rana catesbeiana.

We have examined the effects of phenylhydrazine-induced anemia on the in vivo synthesis of specific hemoglobins at larval, metamorphic, and post-metamorphic stages of the bullfrog Rana catesbeiana, and have found that at all stages the animals qualitatively and quantitatively regenerate their pre-anemia hemoglobin profiles, with one exception: Animals approaching or undergoing the metamorphic hemoglobin switch synthesize only adult hemoglobin during recovery from anemia. We conclude that the ontogenetic progression of hemoglobins in R. catesbeiana is regulated at the level of differentiation of distinct erythroid cell lines, each committed to expressing a particular hemoglobin phenotype; this regulation is unperturbed by anemia.

Embryonic and larval hemoglobins during the early development of the bullfrog, Rana catesbeiana.

The main hemoglobin (Hb) found in Shumway (embryonic) stage 25 bullfrogs is that which we have designated Td-4. The other major tadpole Hbs (Td-1, 2, and 3) predominate during Taylor and Kollros (larval) stages I-XVIII. We propose that Td-4 is an embryonic Hb, whereas Td-1, 2, and 3 are larval (fetal-like) Hbs. Embryonic Hb Td-4 continues to be synthesized during the larval stages. During the larval period, the average peripheral blood Hb profile changes very little with morphological stage or general growth. However, there is great heterogeneity in the embryonic:larval Hb ratio among individual tadpoles of a given stage or weight, apparently due to differential Hb and red cell production by the two active erythropoietic sites, mesonephric kidneys (Td-4), and liver (Td-1, 2, 3).

Erythrocyte differentiation during the metamorphic hemoglobin switch of Rana catesbeiana.

Anurans (frogs and toads) switch from tadpole to adult hemoglobin synthesis during metamorphosis. A number of workers have attempted to determine whether tadpole and adult Hb types are expressed in the same or different erythroid cells during the switch. If the different Hb types are found in different cells during the transition, the switch in globin gene expression occurs at an early stage of cellular differentiation. Previous studies, in which immunocytochemical techniques were used to approach this question, are in conflict in regard to the metamorphic Hb switch of the North American bullfrog Rana catesbeiana. We have purified newly differentiating erythroid cells from the blood of metamorphosing tadpoles by using Percoll gradients. These new cells have an immature morphology, are very active in the synthesis of adult Hb, and contain no detectable tadpole Hb. The tadpole cells have no detectable adult Hb, are synthetically inactive, increase in density during the switch, and are then cleared from the circulation. Thus, only adult Hb expression is detected in newly differentiating erythroid cells during metamorphosis.