The solitary long terminal repeats (LTRs) of ERV-9 endogenous retrovirus contain the U3, R, and U5 regions but no internal viral genes. They are middle repetitive DNAs present at 2,000 to 4,000 copies in primate genomes. Sequence analyses of the 5 boundary area of the erythroid -globin locus control region (-LCR) and the intron of the embryonic axin gene show that a solitary ERV-9 LTR has been stably integrated in the respective loci for at least 15 million years in the higher primates from orangutan to human. The solitary long terminal repeats (LTRs) of human endogenous retroviruses comprise approximately 5% of the human genome and belong to the category of middle repetitive DNAs characterized as retrotransposons (14,19,24,35). These solitary LTRs contain the U3 enhancer and promoter region, the transcribed R region whose 5Ј end marks the initiation site of retroviral RNA synthesis, and the U5 region (27) but no internal gag, pol, and env genes. During primate evolution, the LTRs were apparently self-replicated and inserted into various host chromosomal sites. The functional roles in the host genomes of these transposed, repetitive DNAs are not clear. The LTR retrotransposons have been suggested to be selfish DNAs that do not serve relevant host functions (8). However, recent findings indicate that the solo LTRs can provide enhancers and promoters for cis-linked genes and regulate host gene transcription (9,10,22,25,26,29).The human genome contains approximately 50 copies of the ERV-9 endogenous retrovirus and an additional 3,000 to 4,000 copies of solitary ERV-9 LTRs (15,17,19,35,36). Compared with the LTRs of other families of endogenous retroviruses, the ERV-9 LTRs exhibit an unusual sequence feature: the U3 regions contain from 5 to 17 tandem repeats of 37 to 41 bases (17, 18) with recurrent GATA (23), CCAAT (28), and CCACC (21) motifs potentially capable of binding to cognate transcription factors expressed in embryonic and hematopoietic cells.This suggests that the ERV-9 enhancer and promoter could be active in those cells.To gain further insight into the stability and functional significance of the ERV-9 LTRs, here we have mapped the erythroid -globin and embryonic axin gene loci in primates by using human primers in PCR. We found a solitary ERV-9 LTR that is conserved in identical locations in the 5Ј boundary area of the -globin gene locus and in the axin gene in the higher primates orangutan, gorilla, chimpanzee, and human, whose ancestors diverged over an evolutionary period of 15 million years (12). In the lower primates gibbon and monkey, whose ancestors diverged from the human ancestor 18 and 25 million years ago, respectively (12), the globin and axin LTRs are absent in the respective gene loci. However, other ERV-9 LTRs are detectable in the monkey genome. These results indicate that copies of the ERV-9 LTRs present in the lower primates were inserted into the globin and axin gene loci in the common ancestor of the higher primates 15 to 18 millions years ago and have remained stably integrate...
A 40-bp DNA, consisting of seven tandem GATA repeats, is located near the HS5 site in the 5' boundary area of the locus control region (LCR) of human beta-globin gene. This (GATA)(7) motif, named 5a, exhibits silencer activity in erythroid cells. In transfected, recombinant plasmids containing the chloramphenicol acetyltransferase (CAT) reporter gene, 5a repressed the activity of the cis-linked housekeeping phosphoglycerate kinase (pgk) promoter; 5a also repressed the activity of the cis-linked HS2 enhancer regardless of whether the CAT gene was driven by the pgk or the epsilon-globin promoter. Repression by 5a was most severe when 5a was spliced upstream of HS2 at a distance of less than 200 bases from the HS2 enhancer core. The silencer activity of 5a was independent of whether the component GATA motifs were in head to tail orientation as in the wild type 5a or in head to head or tail to tail orientation as in a mutant 5a. Band shift experiments show that the GATA-1 protein binds to both 5a and the mutant 5a and forms a large protein complex. Together, the results suggest that GATA-1 bound at 5a is a strong, proximal repressor of HS2 enhancer activity.
A classical notion regarding the expression of murine embryonic ζ- and adult α-globin genes holds that there is a switch in globin production from the embryonic to the adult form during fetal development. Our previous in situ hybridization studies challenged this view, since both ζ- and α-globin mRNAs can be detected simultaneously in the earliest erythrocyte populations. This finding raises the possibility that ζ-globin production might be wholly or partially redundant in embryos in which the adult α-globin is also expressed. To test this possibility, we created a null mutation of the ζ-globin gene using homologous recombination in embryonic stem cells. Many outbred mice homozygous for the ζ-null mutation were able to develop normally, undermining the notion that there is an absolute need for ζ-globin and indicating that α-globin alone can serve the survival needs of the fetus. Interestingly, insertion of the PGK-Neo cassette (used to create the null mutation) into the ζ-globin gene appears to influence the expression of the nearby α-globin genes, giving rise to reduced α-globin production and to an α-thalassemia–like syndrome. There is also evidence indicating the strong influence of genetic background on the ζ-null and α1-null phenotypes, both of which are much more severe in the 129/SvEv inbred genetic background. These quantitative differences can potentially be exploited to identify genes important for erythropoiesis.
A classical notion regarding the expression of murine embryonic ζ- and adult α-globin genes holds that there is a switch in globin production from the embryonic to the adult form during fetal development. Our previous in situ hybridization studies challenged this view, since both ζ- and α-globin mRNAs can be detected simultaneously in the earliest erythrocyte populations. This finding raises the possibility that ζ-globin production might be wholly or partially redundant in embryos in which the adult α-globin is also expressed. To test this possibility, we created a null mutation of the ζ-globin gene using homologous recombination in embryonic stem cells. Many outbred mice homozygous for the ζ-null mutation were able to develop normally, undermining the notion that there is an absolute need for ζ-globin and indicating that α-globin alone can serve the survival needs of the fetus. Interestingly, insertion of the PGK-Neo cassette (used to create the null mutation) into the ζ-globin gene appears to influence the expression of the nearby α-globin genes, giving rise to reduced α-globin production and to an α-thalassemia–like syndrome. There is also evidence indicating the strong influence of genetic background on the ζ-null and α1-null phenotypes, both of which are much more severe in the 129/SvEv inbred genetic background. These quantitative differences can potentially be exploited to identify genes important for erythropoiesis.
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