We have used transgenic mice to study the influence of position of the human globin genes relative to the locus control region (LCR) on their expression pattern during development. The LCR, which is located 5' of the globin gene cluster, is normally required for the activation of all the genes. When the human 13-globin gene is linked as a single gene to the LCR it is activated prematurely in the embryonic yolk sac. We show that the correct timing of [] gene activation is restored when it is placed farther from the LCR than a competing human ~,-or ~-globin gene. Correct timing is not restored when 13 is the globin gene closest to the LCR. Similarly, the human ~,-globin gene is silenced earlier when present farthest from the LCR. On the basis of this result, we propose a model of developmental gene control based on stage-specific elements immediately flanking the genes and on polarity in the locus. We suggest that the difference in relative distance to the LCR, which is a consequence of the ordered arrangement of the genes, results in nonreciprocal competition between the genes for activation by the LCR.

We have introduced a human beta-globin minilocus, containing the recently described dominant control region (DCR), the beta-globin or Thy-1 gene, and a thymidine kinase (tk)-neoR gene into erythroid and non-erythroid cells. Analysis of the transcription levels of the genes shows that the DCR directs high levels of human beta-globin, Thy-1 and tk-neo expression independent of integration sites in an erythroid-specific manner. The presence of the DNAasel hypersensitive sites at the 5' end of the locus is required for this effect on the homologous and heterologous gene. An analysis of the DCR chromatin in transfected mouse erythroleukemic cells suggests that the formation of the hypersensitive sites in this region precedes beta-globin gene expression.

The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2.

Using the dominant control region (DCR) sequences that flank the p-globin gene locus, we have been able to achieve high-level expression of the human a-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of the endogenous mouse a-globin genes. Transgenic fetuses with high-copy numbers of the transgene suffer severe anemia and die before birth. Using a construct with both the human a-and p-globin genes and the p-globin DCR, live mice with low-copy numbers were obtained. Both human globin genes are expressed at high levels in adult red cells to give human hemoglobin HbA in amounts equal to or greater than endogenous mouse hemoglobin. Expression of HbA in murine red cells is not accompanied by any increase in mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). However, these transgenic mice tend to have an increased number of reticulocytes in peripheral blood, consistent with some degree of hemolysis. Metabolic labeling experiments showed balanced mouse globin synthesis, but imbalanced human globin synthesis, with an a/p biosynthetic ratio of -0.6. Thus, these mice have mild anemia. These results are discussed with relation to the coordinate regulation of a-and P-globin synthesis in erythroid tissues.

We show that VP16 is phosphorylated by cellular kinases in vivo and in vitro and map the major sites of phosphorylation to be on serines towards the C–terminus, downstream of position 370 in both cases. Deletion of the acidic activation domain had no effect on phosphorylation, refining the sites to between position 370 and 411. Within VP16, the C‐terminal boundary for complex formation with Oct‐1 and HCF lies at position 388, and between 370 and 388 lies one serine, at position 375. This is a consensus casein kinase II (CKII) site and, using purified wild‐type and mutant proteins, we show that it is the main CKII site in the body of the N‐terminal complex‐forming region. This site is also phosphorylated in nuclear extracts. Although other sites, mainly Ser411, are also phosphorylated by nuclear kinase(s), the single substitution of Ser375 to alanine abolishes CKII phosphorylation in vitro and virtually eliminates complex formation. This serine lies in a surface‐exposed region of VP16 and, although complex formation is disrupted, other activities of the mutant are unaffected. Ser375 is also required in vivo where substitution to alanine abolishes transactivation, while replacement with threonine restores normal levels of activity.