In this study, we have isolated ER-binding fragments by the CpG-GBS method. These ER-binding CpG islands contained perfect, imperfect, and/or half-palindromic EREs and had the capacity to bind to the ER in vitro. The transcript could be detected in estrogen target cells when the ER-binding CpG islands were used as probes. This high frequency of detection of the associated transcripts is remarkable and may be fortified by the use of CpG islands for the GBS method in combination with the use of ER-positive cell lines MCF-7, HOS-TE85, and HEC-1 for the Northern blot analysis. While we found estrogen-responsive genes associated with perfect palindromic EREs of EB1 and EB9, it has been reported that an imperfect or multiple half-palindromic ERE could be functional as an estrogen-dependent enhancer in vivo (2
). Therefore, imperfect or multiple half-palindromic EREs of these fragments might also be functional in vivo.
COX7RP cDNA, related to cytox VIIa, was isolated from a human placental cDNA library. The perfect palindromic ERE found in the putative first intron of the gene was shown to have an estrogen-dependent enhancer activity in a CAT assay when it was inserted at the 5′ upstream position. A number of enhancers are known to exist in introns (22
), including an example of an ERE in mouse hepatocyte growth factor gene (30
). Recently, Segade et al. reported a new subunit of cytox VIIa, SIG81, from the mouse (45
). They indicated that SIG81 localized in the mitochondrial fraction and that the fraction contained some cytochrome oxidase c
activity. They also deduced the human SIG81 sequence by connecting the available expressed sequence tag sequences. The C-terminal portion (56 to 113 amino acids) of the assembled sequence described in that report is identical to the corresponding portion (57 to 114 amino acids) of the COX7RP sequence described here. The cytox VIIa protein is composed of an N-terminal mitochondrial presequence (17
) and a C-terminal mature cytox VII protein region (43
). COX7RP cDNA also had a mature-protein sequence in its C-terminal region that is well conserved with cytox VIIa, but its N-terminal region was different from cytox VIIa.
oxidase is a complex enzyme composed of 13 subunits in mammals, of which three (cytox I, II, and III) are encoded by the mitochondrial genome and the others are encoded by the nuclear genome (21
). Interestingly, it has been reported that type II subunit in GH4
rat pituitary tumor cells (54
) and type III subunit in the rat hippocampus (3
) are regulated by estrogen. However, no ERE-like sequence was found in the mitochondrial genome, and no estradiol binding activity was detected in the mitochondria (20
). cytox VII is a nuclear subunit, but little is known about the coordinate synthesis of the various subunits. It has also been postulated that the mitochondrial mRNA levels of cytox I, II, and III could be affected by increased levels of the nuclear subunits (3
). Thus, estrogen might modulate the transcription of some nuclear subunits and then affect the synthesis or stabilization of the mitochondrial subunit mRNAs. Indeed, the nuclear subunits appear to modulate the entire enzymatic activity by regulating the holoenzyme assembly in yeast (11
). We have shown here that COX7RP mRNA was up-regulated by estrogen in MCF-7 cells. It is possible that this COX7RP represents a regulatory subunit of cytochrome c
oxidase and mediates the higher level of energy production in target cells by estrogen.
Recently, mutations in the mitochondrial cytox I and II genes were found to segregate at a higher frequency with Alzheimer’s disease (AD) than with other neurodegenerative and metabolic diseases (9
). These mutations cause a decrease of cytochrome c
oxidase activity and increased production of reactive oxygen species. It has also been suggested that estrogen plays an important role in memory and learning and even in the treatment of AD (50
). If the decrease of cytochrome c
oxidase activity underlies the pathogenesis of AD, it is possible that the mechanism of estrogen action in the treatment of AD is associated with the modulation of cytox I and II and/or COX7RP by estrogen.
A novel cDNA, EBAG9, was isolated from the MCF-7 cDNA library by using EB9 as a probe. A perfect palindromic ERE of EB9 was not included in the 5′ untranslated region of EBAG9 but was located in the far-upstream region of the genomic EBAG9 fragment. Thus, this ERE may be located in the 5′-flanking region of this gene, just like in the Xenopus
vitellogenin A2 gene (23
) and the rat prolactin gene (33
). To confirm this, the transcription initiation site of EBAG9 must be identified. This perfect palindromic ERE was shown to have an estrogen-dependent enhancer activity when it was placed in the upstream region of the β-globin promoter. The mRNA was up-regulated by estrogen in MCF-7 cells, and the up-regulation was dependent on the RNA synthesis but not on new protein synthesis, indicating that the transcriptional activation could take place via the perfect palindromic ERE. The tissue distribution of this mRNA was almost ubiquitous (data not shown), but significant homology was not found in the DNA or protein database. Although the functional association between estrogen and this gene remains unclear, it seems to be a novel estrogen target gene in vivo.
Several methods for isolation of the target gene of transcription factors have been reported. Differential or subtractive hybridization methods (32
) are useful for isolation of any regulatory gene, including direct as well as indirect target genes. DNA binding sites of transcription factors can be isolated from native chromatin by immunoprecipitation (15
). Among these methods, the GBS method (18
) can be applicable if the recombinant protein of the transcription factor is available in certain amounts. In this method, target elements could be isolated, depending on their binding activities but not on the expression levels of the associated genes. However, the GBS cloning method would not be suitable when the binding of the transcription factor is weak or the binding requires heterodimers or cofactors. The use of CpG islands for isolation of the target elements has a significant advantage over the use of whole genomic fragments, because CpG islands are enriched with active genes (8
). Furthermore, most of CpG islands exist as single copies in the genome. Thus, when the CpG islands were isolated as ER-binding fragments, they could be used efficiently as probes for Northern and Southern blotting for detection of their associated genes. Of course, it must be noted that CpG islands include a maximum of 60% of the human gene, so the target elements which are not associated with the CpG islands cannot be isolated by this strategy.
In this report, we have shown that the efficiency of cDNA screening can be increased when the target elements are isolated from a CpG island library by the GBS method. A random appearance of the 10-bp sequence of one perfect palindromic ERE may be 1 in about every 1,000 kb. Therefore, by calculation, the human genome would contain a few thousand ERE sequences. If the recognition sequence of the transcription factor is 6 to 8 bp long, the human genome could contain more than 100,000 recognition sequences. Most of these sequences would be nonspecific, which makes the application of the GBS method difficult. The human CpG island library contains 45,000 copies of the gene, and the average insert size is 1 kb. Thus, the CpG island library would contain only 50 ERE sequences, and these ERE sequences could be suitable for further analysis.
While this paper was being prepared, a report by Shago and Giguere (46
) which described the use of CpG island-enriched DNA for the isolation of retinoic acid-responsive genes appeared. They enriched the CpG island fraction by restriction enzymes containing CpG within their recognition sequences and selected retinoic acid-responsive elements from the fraction by utilizing a combination of PCR amplification and gel mobility shift assay. Although the procedures are different from ours, the results of their study are consistent with ours in defining the importance of the use of CpG islands for isolation of the target elements of transcription factors.