Transcriptional regulation of the B-Globin locus: of mice and men

The aim of this thesis was to get more insight into the mechanism of transcriptional activation of the human B-globin locus. Chapter 1 serves as an introduction to chromatin. The different aspects of chromatin are briefly described. Chapter 2 reviews the work on the human B-globin locus over the last decade. This review aims to present some background information for the more. recent experiments that are described in chapters 3 to 6. Chapter 3 shows the results of individual hypersensitive site deletions of the p-globin LCR: the trans genes become subject to position effects and a novel position effect is described. In chapter 4 one of the parameters that is important in the activation of the globin genes is described: the frequency of LCR-gene . interactions. Chapter 5 presents evidence that the locus control region activates only one gene at a time. Finally in chapter 6 detailed analyses of the mouse p-globin locus is shown and the data suggest that the mechanism of gene activation is conserved during evolution. In concluding remarks I discuss some implications of our work and future experiments. For more detailed considerations of specific findings, the reader is referred to the discussion of chapters 3 to 6

The effect of distance on long-range chromatin interactions.

We have used gene competition to distinguish between possible mechanisms of transcriptional activation of the genes of the human beta-globin locus. The insertion of a second beta-globin gene at different points in the locus shows that the more proximal beta gene competes more effectively for activation by the locus control region (LCR). Reducing the relative distance between the genes and the LCR reduces the competitive advantage of the proximal gene, a result that supports activation by direct interaction between the LCR and the genes. Visualization of the primary transcripts shows that the level of transcription is proportional to the frequency of transcriptional periods and that such periods last approximately 8 min in vivo. We also find that the position of the beta-globin gene in the locus is important for correct developmental regulation

Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci

We have characterized mRNA expression and transcription of the mouse alpha- and beta-globin loci during development. S1 nuclease and primary transcript in situ hybridization analyses demonstrate that all seven murine globin genes (zeta, alpha1, alpha2, epsilony, betaH1, betamaj, and betamin) are transcribed during primitive erythropoiesis, however transcription of the zeta, epsilony, and betaH1 genes is restricted to the primitive erythroid lineage. Transcription of the betamaj and betamin genes in primitive cells is EKLF-dependent demonstrating EKLF activity in embryonic red cells. Novel kinetic analyses suggest that multigene expression in the beta locus occurs via alternating single-gene transcription whereas coinitiation cannot be ruled out in the alpha locus. Transcriptional activation of the individual murine beta genes in primitive cells correlates inversely with their distance from the locus control region, in contrast with the human beta locus in which the adult genes are only activated in definitive erythroid cells. The results suggest that the multigene expression mechanism of alternating transcription is evolutionarily conserved between mouse and human beta globin loci but that the timing of activation of the adult genes is altered, indicating important fundamental differences in globin gene switching