Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes.

High-level beta-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1-HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb beta-globin locus yeast artificial chromosome and measuring the activity of beta-globin genes in GT6m beta-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the beta-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of epsilon- and gamma-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, gamma-globin gene expression was significantly reduced while beta-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal epsilon- and gamma-globin gene expression during embryonic erythropoiesis and for gamma-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression.

The histone deacetylase inhibitor, trichostatin A, reactivates the developmentally silenced gamma globin expression in somatic cell hybrids and induces gamma gene expression in adult BFUe cultures.

Somatic cell hybrids that have undergone globin gene switching and developmental silencing of gamma globin expression were treated with the histone deacetylase inhibitor trichostatin A (TSA). Culture of the post-switch hybrids in the presence of TSA reactivated gamma globin expression and concommitantly downregulated beta globin expression, as determined by both mRNA quantitation and immunofluorescent quantitation of gamma globin expressing cells. In contrast, similar treatment of pre-switch hybrids, which were expressing predominantly gamma globin and only small levels of beta globin, had no effect on the relative gamma or beta globin gene expression. In addition, trichostatin A induced gamma gene expression in adult BFUe cultures in a maturation-independent fashion. The results provide direct evidence that inhibition of HDAC activity can alter expression from the human beta globin locus in the adult stage of development.

Activation of the beta-like globin genes in transgenic mice is dependent on the presence of the beta-locus control region.

The beta-globin locus control region (LCR) is a powerful regulatory element required for high-level globin gene expression. We have generated transgenic mouse lines carrying a beta-globin locus yeast artificial chromosome lacking the LCR to determine if the LCR is required for globin gene activation. beta-Globin gene expression was analyzed by RNase protection, but no detectable levels of epsilon-, gamma- and beta-globin gene transcripts were produced at any stage of development. These findings suggest that the presence of the LCR is a minimum requirement for globin gene expression. Next, we tested whether the LCR is necessary to activate globin gene expression in a gamma-globin promoter mutant that causes hereditary persistence of fetal hemoglobin (HPFH). beta-YAC transgenic mice carrying the -117 HPFH mutation and a HS3 core deletion that specifically abolishes gamma-globin gene expression during definitive erythropoiesis were produced to test whether the -117 (A)gamma promoter is activated in the absence of interaction with the LCR. In four transgenic mouse lines, gamma-globin gene expression was absent in adult erythrocytes, suggesting that an interaction between the gamma-globin gene promoter and the LCR is required for gamma gene activation even when the promoter contains an HPFH mutation.