DNA 5' to the human r-globin gene exhibits unique patterns of DNase I-hypersensitive sites (DHS) in three human erythroleukemic cell lines which represent the embryonic (K562), fetal (HEL), and adult (KMOE) stages of erythroid development. We have mapped 10 r-globin DHS in K562 cells, in which the r-globin gene is maximally active. Major sites are located -11.7, -10.5, -6.5, -2.2 kilobase pairs (kbp) and -200 base pairs (bp) upstream of the gene and directly over the major cap site. Minor sites are located -5.5, -4.5, and -1.48 kbp and -900 bp upstream of the cap site. In HEL cells, in which the r-globin gene is expressed at extremely low levels, the -11.7-, -10.5-, -5.5-, -4.5-, and -2.2-kbp DHS are no longer detectable; the -200-bp site is approximately 300-fold less sensitive to DNase I; and the -1.48-kbp, -900-bp, and major cap site DHS are 3-to 4-fold less sensitive. Only the DHS located -6.5 kbp relative to the major cap site is detectable at all three stages of erythroid development, including KMOE cells in which r-globin synthesis is undetectable. We suggest that this site may be implicated in maintaining the entire j8-globin cluster in an active chromatin conformation. The five DHS downstream of the -6.5-kbp element possess associated promoters. Thus two distinct types of DHS exist-promoter positive and promoter negative. In HEL cells, all the upstream promoters are inactivated, although the -1.48-kbp and -900-and -200-bp DHS are still present. This suggests that the maintenance of DHS and regulation of their associated promoters occur by independent mechanisms. The inactivation of the upstream promoters in HEL cells while the major cap site remains active represents a unique pattern of expression and suggests that HEL cells possess regulatory factors which specifically down regulate the e-globin upstream promoters.The human p-like globin genes extend over approximately 60 kilobase pairs (kbp) of chromosome 11. The genes are arranged 5-,E-G'Y_A-y-5.i_33, in the same order as they are developmentally expressed. The E-globin gene is expressed up to 10 weeks gestation and is then silenced for the remainder of the lifetime of the individual. This gene possesses 12 known DNase I-hypersensitive sites (DHS) between the major cap site and -15 kbp upstream of the gene (16,36, 41). Five of these DHS contain upstream promoters located -4.5, -2.2, and -1.48 kbp and -900 and -200 base pairs (bp) from the major cap site (1, 2; Fig. 1). The -2.2-kbp promoter, located within an Alu repetitive element, is transcribed by PolIll (12) in the opposite direction from the E-globin gene, giving rise to 350-bp transcripts which are nonpolyadenylated, nucleus confined, and detectable in vivo only when the e-globin gene is fully active (5). The remainder of the upstream promoters give rise to transcripts which extend through the gene and are polyadenylated. In the human erythroleukemia cell line K562 or in normal embryonic red blood cells, 10 to 15% of E-globin transcripts originate from the upstream sites (2). With the exception...
We have identified a short, highly repetitive element within intron -1 of the human c-Ha-ras gene. This element was found to be transcribed in both orientations and to be homologous to heterogeneous nonpolyadenylated transcripts. The repetitive element blocked transcriptional readthrough from a strong upstream viral promoter but allowed unimpaired readthrough from the c-Has-ras promoter. We suggest that it may serve to prevent excessive transcription into the coding region of the gene under such circumstances as viral insertion.Elevated expression of the c-Ha-ras gene is associated with many important tumor types, such as breast (28), colon (13), and gastric (4). In addition, elevated expression of the normal gene under the control of a retrovirus long terminal repeat has been shown to induce tumorigenic transformation of 3T3 cells (7), and activation of the c-Ha-ras oncogene in an avian nephroblastoma has been attributed to proviral insertion (30).The potential importance of c-Ha-ras transcriptional deregulation in tumor progression led us to examine the nature of the c-Ha-ras promoter and the factors which regulate its expression (20), and we have recently identified a bidirectional c-Ha-ras promoter whose 3' end (the donor splice site) is located 1,105 base pairs (bp) upstream of the ATG codon. The direct promoter contains 18 initiation sites between -196 and -16 bp relative to the donor splice site (D). The reverse promoter contains nine initiation sites between -248 and -278 bp relative to D. A 43-bp element between -243 and -196 up regulates activity of the direct promoter by 20-to 40-fold (20; summarized in Fig. lc).A highly repetitive, transcribed element in c-Ha-ras intron -1. To confirm that the far upstream exon -1 promoter gives rise to mature 1,400-bp c-Ha-ras mRNA, we performed Northern (RNA) blot analysis, using probes derived from exon -1 and the coding exon 1. Total RNA was prepared from EJ bladder carcinoma cells (23) and the human erythroleukemia cell line K562 (26). Probes derived both from exon -1 and exon 1 detected a major 1,400-bp c-Ha-ras mRNA species and a minor species of approximately 5.5 kilobase pairs (kb) (Fig. la). To determine whether the larger RNA species represented a splicing intermediary, a 1.16-kb Sau3A fragment containing intron -1 was used as a hybridization probe. Surprisingly, this probe hybridized to highly repetitive heterogeneous RNA species ranging in size from approximately 7 kb to 800 bp. We proceeded to localize the DNA sequence coding for repetitive RNA by using a series of probes derived from intron -1. The 837-bp XmaIII-Sau3A probe (containing the 3' end of intron -1 and coding exon 1) hybridizes to c-Ha-ras 1,400-bp mRNA, whereas the 305-bp XmaIII probe * Corresponding author. detects the repetitive species. A 130-bp XmaIII-SstII fragment from the extreme 5' end of intron -1 also hybridizes to repetitive RNA, whereas the 170-bp SstII-XmaIII probe does not. The repetitive RNA species are mainly represented in the nonpolyadenylated RNA fraction. Using multiple ...
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