Characterization Of The Lcr Dependent Activation Of The Beta-Globin Genes

The activity of the β-globin locus is regulated by the locus control region (LCR) which in humans and mice is comprised of six DNaseI hypersensitive sites (HSs) located upstream of the β-like globin genes. Hispanic thalassemia, a naturally occurring deletion of the LCR plus 25kb upstream results in the failure to activate the β-globin locus at the levels of chromatin structure, transcription and replication. In order to examine how the HSs interact to regulate the endogenous β-globin locus, we have utilized homologous recombination for the mutational analysis of the endogenous murine β-globin LCR in embryonic stem cells, followed by the generation of mice. Previously we reported that deletion of the endogenous LCR by homologous recombination (ΔLCR) does not completely silence expression of the β-like genes, and has no measurable effect on nuclease sensitivity, promoter hypersensitive site formation or core histone hyperacetylation. Thus the LCR provides a necessary enhancer-like activity. In addition, while loss of the LCR leads to only a slight decrease in pre-initiation complex formation and Pol II binding to the promoter, there is a significant decrease in downstream polymerase and this correlates with a basal level of ser-5 phosphorylation of Pol II. To determine if the decrease in downstream Pol II observed along Δthe LCR allele is due to decreased release of polymerase from the promoter, or downstream polymerase pausing, KMnO4 in vivo foot-printing was done.