Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.     

Oxygen tension modulates beta-globin switching in embryoid bodies

Little is known about the factors influencing the hemoglobin switch in vertebrates during development. Inasmuch as the mammalian conceptus is exposed to changing oxygen tensions in utero, we examined the effect of different oxygen concentrations on beta-globin switching. We used an in vitro model of mouse embryogenesis based on the differentiation of blastocyst-derived embryonic stem cells to embryoid bodies (EBs). Cultivation of EBs at increasing oxygen concentrations (starting at 1% O2) did not influence the temporal expression pattern of embryonic (betaH1) globin compared to the normoxic controls (20% O2). In contrast, when compared to normoxically grown EBs, expression of fetal/adult (betamaj) globin in EBs cultured at varying oxygen concentrations was delayed by about 2 days and persisted throughout differentiation. Quantitation of hemoglobin in EBs using a 2,7-diaminofluorene-based colorimetric assay revealed the appearence of hemoglobin in two waves, an early and a late one. This observation was verified by spectrophotometric analysis of hemoglobin within single EBs. These two waves might reflect the switch of erythropoiesis from yolk sac to fetal liver. Reduced oxygenation is known to activate the hypoxia-inducible factor-1 (HIF-1), which in turn specifically induces expression of a variety of genes among them erythropoietin (EPO). Although EBs increased EPO expression upon hypoxic exposure, the altered beta-globin appearance was not related to EPO levels as determined in EBs overexpressing EPO. Since mRNA from both mouse HIF-1alpha isoforms was detected in all EBs tested at different differentiation stages, we propose that HIF-1 modulates beta-globin expression during development.     

Expression of genes for human globin in the cell line K-562: an experimental model for the study of fetal erythropoiesis

Human leukemia K-562(S) cells are a useful model system to study the relationship between cell proliferation and induced erythroid differentiation. In these studies K-562(S) cells were cultured in alpha -medium, 10% fetal calf serum and induced to express erythroid genes by 75 microM hemin, 1.2 mM butyric acid or 1.5 ng/ml actinomycin D. Cell number was determined using a ZF Coulter Counter and the increase in the proportion of hemoglobin-containing cells was detected by a specific colorimetric reaction with benzidine. The characterization of the synthesized hemoglobins was performed by cellulose-acetate gel electrophoresis of post-mitochondrial supernatants. By cloning K-562(S) cells in semi-solid medium (O,33% agar) containing 75 microM hemin a variant cell line, denominated K-562(S6), have been isolated which does not undergo terminal cell division but does express human globin genes and accumulates on the average 12 pg of Hb/cell. K-562(S6) cells accumulate, upon exposure to 75 microM hemin, mostly Hb Gower 1 (zeta 2 epsilon 2) and low amounts of Hb X (epsilon 2 gamma 2) and Hb Portland (zeta 2 gamma 2), being suitable for studies focused on the expression of embryonic globin genes and on the molecular mechanisms controlling the switching from embryonic-type to fetal-type hemoglobin accumulation, when in the human embryo zeta and epsilon globin genes become less active, being sharply increased accumulation of alpha and gamma globin chains.     

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.     

IGF-I and vanadate stimulate Na/Pi-cotransport in OK cells by increasing type II Na/Pi-cotransporter protein stability

The nitric oxide (NO) signaling system, consisting of NO synthases, soluble guanylyl cyclase, and cGMP, plays a prominent role in salt handling and regulation of blood pressure. Soluble guanylyl cyclases are heme-containing heterodimers (alpha/beta). The alpha1/beta1 isoform has greater NO sensitivity than the alpha1/beta2. It has recently been shown that expression of the beta subunits is altered in the kidney of the Dahl salt-sensitive rat, ie, the beta1 subunit is decreased and the beta2 subunit increased. However, whether soluble guanylyl cyclase is linked to salt sensitivity is not known. In the present study, we investigated linkage of guanylyl cyclase genes to blood pressure. Alpha1 and beta1 gene loci for soluble guanylyl cyclase were mapped to rat chromosome 2, and the beta2 gene locus was mapped to rat chromosome 5 using fluorescent in situ metaphase hybridization. By use of a rat radiation hybrid panel, the gene loci were then further mapped with respect to known quantitative trait locus markers of salt-sensitive hypertension in the Dahl rat on chromosomes 2 and 5. Genes for alpha1 and beta1 were closely linked by two-point analysis to Na+,K+-ATPase alpha1 isoform (LOD of 15.1 and 14.0, respectively) and calmodulin-dependent protein kinase II-delta loci (LOD of 14.3 and 12.9, respectively), which have been previously shown to flank a quantitative trait locus for blood pressure in the Dahl rat. The alpha1 and beta1 genes were closely linked (LOD of 11.3; theta, 0.4). The beta2 gene locus was closely linked to the endothelin-2 (ET-2) locus (LOD of 13.0), which has been shown to cosegregate with blood pressure. We conclude that soluble guanylyl cyclase subunit loci, ie, alpha1, beta1, and beta2, are good candidates for genes controlling salt-sensitive hypertension in the Dahl rat.     

LCR-dependent gene expression in beta-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs

Yeast artificial chromosome (YAC) transgenesis is associated with a high frequency of deletions in the integrated transgenes. To determine the impact of these rearrangements on the ability to derive structure-function relationships using YACs, transgenic mice were generated with 248 or 155 kb beta-globin locus YACs. The transgenics were examined for structural integrity of the YAC using an approach of structural analysis that unambiguously demonstrates intactness of YAC transgene copies. Globin gene expression per copy of each integrated transgene and the profiles of globin gene expression during development were determined. Diverse deletion patterns were observed in one or more integrated YACs in all the 248 and most of the 155 kb transgenic lines we analyzed. However, when the structure of the major regulatory element of the beta-globin locus, the locus control region, was preserved, the genes of the beta-globin locus functioned normally and globin transgenes of both the 248 and 155 kb beta-YACs were expressed in a position-independent, copy number-dependent manner. Furthermore, the globin genes of both beta-YACs displayed normal developmental regulation. We conclude that YACs can be used for analysis of structure-function relationships of large genes or multigene loci in spite of the tendency for rearrangements and deletions of the integrated transgenes. However, detailed structural evidence for integrity and continuity of locus sequences is required for correct interpretation of functional data.     

Susceptibility to actively-induced murine experimental allergic encephalomyelitis is not linked to genes of the T cell receptor or CD3 complexes

The role of the T cell receptor (TCR) in the genetic control of susceptibility to autoimmune demyelinating diseases remains shrouded in controversy. We have used the CXD2 series of recombinant inbred lines (RIL) and a (B10.S/DvTe x SJL/J) x B10.S/DvTe backcross (BC1) population to test for linkage between susceptibility to actively-induced EAE and the different TCR and CD3 loci. The two populations were inoculated for induction of EAE, phenotyped for both clinical and histological parameters of disease, and genotyped using markers flanking the loci of interest in the CXD2 RIL and an SJL/J allele-specific TCR V beta assay in the BC1 mice. Comparisons between the CXD2 strain distribution pattern (SDP) for disease and the SDPs for the chromosomal regions containing the TCR alpha, beta, gamma, delta, and CD3 delta, epsilon, gamma and zeta loci showed no linkage to these loci. Additional tests between EAE susceptibility and several other immunologically important loci for which the SDPs were known also showed no linkage to the minor lymphocyte-stimulating antigen gene Mlsl, Hc, the gene encoding complement component C5, Cd8a, or Cd5. Furthermore, our data from the BC1 mice demonstrate that the Tcrb locus segregates independent of disease and does not modulate disease severity. We conclude that while autoreactive TCRs are undoubtedly necessary for disease pathogenesis, the principle non-MHC-linked loci controlling susceptibility to murine EAE in BALB/c mice are not linked to any of the individual TCR-CD3 complex genes. Similarly, the major disease genes in the SJL/J mouse are not linked to TCR V beta. Our data cannot, however, preclude the possibility that TCR/CD3 alleles are involved in epigenetic phenomena or susceptibility in other mouse strains or animal systems.     

Erythropoietin stimulates nuclear localization of diacylglycerol and protein kinase C beta II in B6SUt.EP cells

Erythropoietin (EPO) is a hormone, as well as a hematopoietic growth factor, that specifically regulates the proliferation and differentiation of erythroid progenitor cells. Although the membrane-bound receptor for EPO has no intrinsic kinase activity, it triggers the activation of protein kinases via phospholipases A2, C, and D. A cascade of serine and threonine kinases, including Raf-1, MAP kinase and protein kinase C (PKC) is activated following tyrosine phosphorylation. In this study, we have examined whether changes in nuclear PKC and 1,2-diacylglycerol (DAG) are induced following EPO treatment of the murine target cell line, B6SUt.EP. Western blot analysis using isoform-specific antibodies demonstrated the presence of PKC beta II, but not PKC alpha, beta I, gamma, epsilon, delta, eta, or zeta in the nuclei of cells stimulated with EPO. The increase in nuclear beta II levels was accompanied by an immediate rise in DAG mass levels with both of the increases peaking by 1 min. These rapid increases in nuclear DAG and PKC beta II expression suggest a mechanism for EPO-induced changes in gene expression necessary for cell proliferation.     

Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.