Genome Architecture of the Human β-Globin Locus Affects Developmental Regulation of Gene Expression

To test the role of gene order in globin gene expression, mutant human -globin locus yeast artificial chromosome constructs were used, each having one additional globin gene encoding a “marked” transcript ( m , m ,o r m ) integrated at different locations within the locus. When a m -globin gene was placed between the locus control region (LCR) and the -globin gene, m -globin expression dominated primitive and definitive erythropoiesis; only m -globin mRNA was detected during the fetal and adult definitive stages of erythropoiesis. When an Am -globin gene was placed at the same location, Am -globin was expressed during embryonic erythropoiesis and the fetal liver stage of definitive erythropoiesis but was silenced during the adult stage. The downstream wild-type -globin genes were not expressed. When an m -globin gene was placed between the and -globin genes, it remained silent during embryonic erythropoiesis; only the LCR-proximal wild-type -globin gene was expressed. Placement of a m -globin gene upstream of the G -globin gene resulted in expression of m -globin in embryonic cells and in a significant decrease in expression of the downstream wild-type -globin gene. These results indicate that distance from the LCR, an inherent property of spatial gene order, is a major determinant of temporal gene expression during development. The human -globin locus consists of five functional globin genes, which are expressed during ontogeny in the order in which they lie on the chromosome, 5-e- G - A ---3. During development, two switches occur in globin gene expression, from embryonic to fetal, coinciding with the transition from embryonic to definitive erythropoiesis, and from fetal to adult around the perinatal period. High-level expression of the -like globin genes is dependent upon both gene-proximal and -distal cis-acting regulatory elements. The major distal regulatory element is the locus control region (LCR). It contains five DNase I-hypersensitive sites (5HS1 to -5) (42) and is located 6 to 22 kb upstream of the e-globin gene. Functions of LCRs include tissue-specific enhancement of gene expression, chromatin-opening activity, insulation of the locus from the effects of surrounding chromatin, and determination of replication timing and choice of origin used (26, 27). It is currently assumed that LCR sequences, together with their cognate transcription factors and coactivators, form an active site “hub” that interacts with gene-proximal elements and their bound transcription factors to form a transcriptosome specific for the globin gene(s) expressed at each developmental stage (8, 20, 26, 34). Less-conserved sequences between the HS cores also may function in globin gene regulation (19), perhaps by acting to constrain the complex conformation. Because the spatial arrangement of the -like globin genes parallels their temporal expression pattern, it has been proposed that gene order or proximity to the LCR may be a determinant of the order of globin gene expression during development. Previous studies have established an effect of either gene order or distance from the LCR (10, 18, 39, 44). Data from transgenic mice produced with - and -globin genes linked at various distances to the LCR demonstrated that proximity to the LCR increased the probability of interaction with the LCR (18). The effect of gene order on globin gene developmental regulation was tested by producing transgenic mice containing two tandemly arranged -o r-globin or - and -globin genes linked to a LCR (39). These studies showed that the trans-acting environment is the primary determinant for correct developmental control but that proximity to the LCR also is involved in regulation. The effect of distance from the LCR was analyzed using a larger 70-kb -globin locus in two linked cosmids (10). When the e-globin gene was replaced with a marked -globin ( m ) gene, -globin expression was sustained throughout development. In contrast, when the m -globin gene was placed upstream of the -globin gene, m -globin gene expression accounted for 75% of total -globin gene expression ( m wt ). Inversion of the whole locus relative to the LCR in -globin locus yeast artificial chromosome (-YAC) transgenic mice resulted in -globin gene expression throughout development, with no e-globin gene expression and limited -globin gene expression during primitive erythropoiesis, again suggesting that proximity to the LCR is a determinant of globin gene expression (44). In this study we sought to further test the role of spatial gene order in the control of globin gene expression using -YAC transgenic mice carrying constructs containing one additional globin gene encoding a “marked” transcript (e m , m ,o r m )