Hypersensitive site 4 of the human beta globin locus control region.

The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2.     

The beta-globin dominant control region: hypersensitive site 2.

The Dominant Control Region (DCR) of the human beta-globin gene locus consists of four strong hypersensitive sites (HSS) upstream of the epsilon-globin gene. Addition of these sites confers copy number dependent expression on the human beta-globin gene in murine erythroleukaemia cells and transgenic mice, at levels comparable with the endogenous mouse globin genes. We have shown previously that a 1.9 kb fragment comprising HSS 2 accounts for 40-50% of the full effect of the DCR. In this paper we describe a deletional analysis of HSS 2. We show that a 225 bp fragment is sufficient to direct high levels of expression of the human beta-globin gene which is copy number dependent and integration site independent. This 225 bp fragment overlaps the major region that is hypersensitive 'in vivo'. DNase I footprinting shows the presence of four binding sites for the erythroid specific protein NF-E1; the three other footprinted regions display a remarkable redundancy of the sequence GGTGG and bind a number of proteins including Sp1 and the CACC box protein. The significance of these results for the regulation of globin gene expression is discussed.     

DNA methylation pattern and restriction endonuclease accessibility in chromatin of a germ-line specific gene, the rainbow trout protamine gene.

The chromatin structure of a germ-line specific gene, the TPG-3 gene, one of the rainbow trout protamine genes was analyzed in various tissues. The protamine genes are expressed in early stage testis but not in late stage testis, liver or erythrocyte. Five potential CpG methylation sites in the coding and flanking regions of the TPG-3 protamine gene were monitored in early and late stage testis, nucleoprotamine, liver and erythrocyte. In all cases the patterns of methylation were identical with only one CpG site at position -740 being methylated. Thus, the methylation pattern of this protamine gene remained the same independently of the expression of the gene. Two Msp I sites at positions -293 and/or -275 and +155 were accessible to the enzyme in the TPG-3 chromatin of early stage testis. Since the Msp I site at position -293 and/or -275 was also present in the TPG-3 chromatin of liver, only the site at position +155 within the transcribed region correlated with the expression of the protamine gene.