Developmentally regulated and erythroid-specific expression of the human embryonic beta-globin gene in transgenic mice.

Transgenic mice have proven to be an effective expression system for studying developmental control of the human fetal and adult beta-globin genes. In the current work we are interested in developing the transgenic mouse system for the study of the human embryonic beta-globin gene, epsilon. An epsilon-globin gene construction (HSII,I epsilon) containing the human epsilon-globin gene with 0.2 kb of 3' flanking sequence and 13.7 kb of extended 5' flanking region including the erythroid-specific DNase I super-hypersensitive sites HSI and HSII was made. This construction was injected into fertilized mouse ova, and its expression was analyzed in peripheral blood, brain, and liver samples of 13.5 day transgenic fetuses. Fetuses carrying intact copies of the transgene expressed human epsilon-globin mRNA in their peripheral blood. Levels of expression of human epsilon-globin mRNA in these transgenic mice ranged from 2% to 26% per gene copy of the endogenous mouse embryonic epsilon y-globin mRNA level. Furthermore, the human epsilon-globin transgene was expressed specifically in peripheral blood but not in brain or in liver which is an adult erythroid tissue at this stage. Thus, the HSII,I, epsilon transgene was expressed in an erythroid-specific and embryonic stage-specific manner in the transgenic mice. A human epsilon-globin gene construction that did not contain the distal upstream flanking region which includes the HSI and HSII sites, was not expressed in the embryos of transgenic mice. These data indicate that the human epsilon-globin gene with 5' flanking region extending to include DNase I super-hypersensitive sites HSI and HSII is sufficient for the developmentally specific activation of the human epsilon-globin gene in erythroid tissue of transgenic mice.     

DNase I hypersensitive regions correlate with a site-specific endogenous nuclease activity on the r-chromatin of Tetrahymena

A novel nuclease activity have been detected at three specific sites in the chromatin of the spacer region flanking the 5'-end of the ribosomal RNA gene from Tetrahymena. The endogenous nuclease does not function catalytically in vitro, but is in analogy with the DNA topoisomerases activated by strong denaturants to cleave DNA at specific sites. The endogenous cleavages have been mapped at positions +50, -650 and -1100 relative to the 5'-end of the pre-35S rRNA. The endogenous cleavage sites are associated with micrococcal nuclease hypersensitive sites and DNase I hypersensitive regions. Thus, a single well-defined micrococcal nuclease hypersensitive site is found approximately 130 bp upstream from each of the endogenous cleavages. Clusters of defined sites, the majority of which fall within the 130 bp regions defined by vicinal micrococcal nuclease and endogenous cleavages, constitute the DNase I hypersensitive regions.     

Function of the upstream hypersensitive sites of the chicken beta-globin gene cluster in mice.

We have shown previously that the chicken beta A-globin gene, with its 3' enhancer, is expressed in a copy number-dependent manner in transgenic mice. The expression level was low but increased approximately 6-fold upon inclusion of 11 kb of upstream DNA containing four DNase I hypersensitive sites. To study the effect of the individual upstream hypersensitive sites on transgene expression, we produced lines of mice in which the individual upstream sites were linked to the beta A gene and enhancer. RNA levels were measured in blood from adult animals. With each of these four constructs, the level of transgene RNA per DNA copy varied over a > 20-fold range. These data suggest that addition of a hypersensitive site to the beta A-globin/enhancer region abrogates its position independent expression. The average beta A-globin expression per copy in the lines carrying an upstream site was comparable with that in lines without an upstream site. Thus, no single upstream hypersensitive site accounts for the higher level of beta A-globin expression seen in mice containing the complete upstream region. We had shown previously that control of the chicken beta-globin cluster is distributed between at least two regions, the beta A/epsilon enhancer and the upstream region. Our current results suggest that the control mediated by the upstream DNA is itself distributed and is not due to a single hypersensitive site.     

Alternative sites of transcription initiation upstream of the canonical cap site in human gamma-globin and beta-globin genes

Using S1 mapping and primer extension analysis, we have identified a number of human kappa-globin and beta-globin 5' RNA termini originating in the 200 bp upstream of the canonical mRNA cap sites. Upstream initiation sites have previously been reported for the human epsilon-globin gene (4,5) and the present work indicates that this is a general feature of the human beta-type globin genes. We have attempted to identify features common to such sites between the three genes. One site 170 bp upstream of the major beta-globin cap site and a site 1400 bp upstream of the major epsilon-globin cap site are located near putative PolIII promoter sequences and may therefore be transcribed by this enzyme. Alternative initiation sites located 200 bp and 50-100 bp upstream of the epsilon-globin and kappa-globin cap sites respectively are located within S1 hypersensitive regions of chromatin.     

Identification and sequence analysis of the 5' end of the major chicken vitellogenin gene.

We have precisely determined the positions of the first three exons for the major chicken vitellogenin gene (VTG II) by a combination of S1 nuclease protection, primer extension and DNA sequencing experiments. In addition, we have determined the nucleotide sequences of the 5' flanking nuclease hypersensitive sites that we have previously shown are induced during the estrogen mediated activation of the VTG II gene in liver (1). One of these sites is found to be nearly identical to the enhancer core sequence of SV40. A computer assisted analysis of the DNA sequences upstream from the VTG II gene has revealed four short (7 to 9 base pair) sequence elements that are present in similar positions flanking the other major estrogen inducible gene for liver, very low density apolipoprotein II (apoVLDL II). For VTG II, these sequences are located between two of the induced nuclease hypersensitive sites that are liver specific. Sequences homologous to one element, located approximately 100 base pairs upstream from the mRNA cap sites of the VTG II and apoVLDL II genes, are also observed for three estrogen inducible genes that are expressed in the oviduct, although for each of these genes the sequence falls further upstream, between -220 and -200. We suggest that these conserved sequences may be important in mediating the tissue specific responses of these genes to estrogen.     

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

The human β-like globin genes are arranged as a clusterof five genes (ε, Gγ, Aγ, δ and β) in the order of theirtemporal expression. The human embryonic ε-globin geneis expressed in the blood island of the embryonic yolk sacand is silenced completel     

A 46 base pair enhancer sequence within the locus activating region is required for induced expression of the gamma-globin gene during erythroid differentiation.

The locus activating region (LAR), contained within 30 kb of chromatin flanking the human beta-globin gene cluster, has recently been shown to be essential for high level beta-globin gene expression. To determine the effect of fragments containing LAR sequences on globin gene expression, mRNA from a marked gamma-globin gene linked to LAR fragments was assayed in stably transfected K562 erythroleukemia cells. DNaseI hypersensitive site II (HS II), located 10.9 kb upstream of the epsilon-globin gene, was required for high level gamma-globin gene expression. We also showed that a 46 bp enhancer element within HS II was necessary and sufficient for the increased gamma-globin gene expression observed with hemin induced erythroid maturation of K562 cells. These results localize a distant regulatory element important for activation of globin genes during human erythroid cell maturation.     

Nuclease hypersensitivity in the beta-globin gene region of K562 cells

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.