SYNERGISTIC REGULATION OF HUMAN BETA-GLOBIN GENE SWITCHING BY LOCUS-CONTROL REGION ELEMENTS HS3 AND HS4

Proper tissue- and developmental stage-specific transcriptional contro l over the five genes of the human beta-globin locus is elicited in pa rt by the locus control region (LCR), but the molecular mechanisms tha t dictate this determined pattern of gene expression during human deve lopment are still controversial. By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chro mosome (YAC) bearing the entire human beta-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addresse d the function of LCR hypersensitive site (HS) elements 3 and 4 in hum an beta-globin gene switching. The experiments revealed a number of un expected properties that are directly attributable to LCR function. fi rst, deletion of either HS3 or HS4 core elements from an otherwise int act YAC results in catastrophic disruption of globin gene expression a t all erythroid developmental stages, despite the presence of all othe r HS elements in the YAC transgenes. If HS3 is used to replace HS4, ge ne expression is normal at all developmental stages. Conversely, inser tion of the HS4 element in place of HS3 results in significant express ion changes at every developmental stage, indicating that individual L CR HS elements play distinct roles in stage-specific beta-type globin gene activation. Although the HS4 duplication leads to alteration in t he levels of epsilon- and gamma-globin mRNAs during embryonic erythrop oiesis, total beta-type globin mRNA synthesis is balanced, thereby lea ding to the conclusion that all of the human beta-locus genes are comp etitively regulated. In summary, the human beta-globin HS elements app ear to form a single, synergistic functional entity called the LCR, an d HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.