Chromatin dynamics and the regulation of B-glohin gene expression

The human B-globin locus is frequently used as a model system to study mechanisms controlling tissue-specific and developmentally regulated gene expression. Much of the recent progress in understanding the regulation of B-globin gene expression has come from a better knowledge of the process of transcription. Proper transcriptional regulation of the human B-globin genes occurs, at least in part, through specific interactions of regulatory traus-acting proteins to defined cis-regulatory sequences that include promoters, enhancers, silencers, and elements of the locus control region

The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene

The beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been described. Expression of the beta-like globin genes is absolutely dependent on the presence of the LCR. The developmental expression pattern of the genes in the cluster is achieved through competition of the promoters for the activating function of the LCR. Transgenic mice experiments suggest that subtle changes in the transcription factor environment lead to the successive silencing of the embryonic epsilon-globin and fetal gamma-globin promoters, resulting in the almost exclusive transcription of the beta-globin gene in adult erythropoiesis. In this paper, we have asked the question whether the LCR and its individual hypersensitive sites 5'HS1-4 can activate a basic promoter in the absence of any other globin sequences. We have employed a minimal promoter derived from the mouse Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The results show that the muLCR and 5'HS3 direct erythroid-specific, embryonic expression of this construct, while 5'HS1, 5'HS2 and 5'HS4 are inactive at any stage of development. Expression of the muLCR and 5'HS3 transgenes is repressed during fetal stages of development. The transgenes are in an inactive chromatin conformation and the lacZ gene is not transcribed, as shown by in situ hybridization. These data are compatible with the hypothesis that the LCR requires the presence of an active promoter to adopt an open chromatin conformation and with models proposing progressive heterochromatization during embryogenesis. The results suggest that the presence of a beta-globin gene is required for LCR function as conditions become more stringent during development

Intergenic transcription and transinduction of the human β-globin locus

We have identified novel nuclear transcripts in the human β-globin locus using nuclear run-on analysis in erythroid cell lines and in situ hybridization analysis of erythroid tissue. These transcripts extend across the LCR and intergenic regions but are undetectable in nonerythroid cells. Surprisingly, transient transfection of a β-globin gene (ε, γ, or β) induces transcription of the LCR and intergenic regions from the chromosomal β-globin locus in nonerythroid cell lines. The β-globin genes themselves, however, remain transcriptionally silent. Induction is dependent on transcription of the globin gene in the transfected plasmid but does not require protein expression. Using in situ hybridization analysis, we show that the plasmid colocalizes with the endogenous β-globin locus providing insight into the mechanism of transinduction

The role of EKLF in human β-globin gene competition

We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human β-globin genes in compound EKLF knockout/human β- locus transgenic mice. EKLF affects only the adult mouse β-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human ε and γ-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of γ- and β-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of γ- to β-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active β genes with a reciprocal increase in the number of transcriptionally active γ genes. β-gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the β promoter. There is a further increase in the number of transcriptionally active γ genes and accompanying γ gene promoter chromatin alterations. These results indicate that EKLF plays a major role in γ- and β-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the β-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription