Search

We have previously identified the region containing transcriptional enhancer in mouse immunoglobulin heavy chain (IgH) gene as an initiation region of DNA replication. The octamer sequence therein has been found to be essential for initiation of DNA replication as well as for transcription. Octamer-binding protein family, including Oct (OTF)-1, 2A, and 2B, bound to the element in double-stranded form. Here we identified in various cells novel proteins which bound to the upper strand of the octamer sequence in the IgH gene. Three proteins of 35, 60, and 125 KDa recognized the sequences. The proteins, which were not recognized by an anti-Oct-1 antibody, may be involved in regulation of the initiation of DNA replication in the IgH gene.

The cellular oncogene c-myc encodes a nuclear protein that is considered to play a role in cell proliferation. In this report, the region upstream from the transcriptional promoter of the c-myc gene was examined for regulatory activity on its expression during cell cycle. Plasmids which contain the upstream region of human c-myc gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected to rat 3Y1 cells together with pSV2Hg (containing the hygromycin resistance gene linked to the SV40 promoter). Stably transformed cell lines were obtained by hygromycin selection. In random culture, the cells possessing CAT gene preceeded by the upstream region of the c-myc gene, including the HindIII-PstI [myc(H-P)] region, showed strong CAT activity. The myc(H-P) region contains a c-myc protein complex binding site. On the other hand, the cells carrying a similar myc-CAT construct, but without the myc(H-P) region, showed very low levels of CAT expression. These cell lines were then synchronized by serum starvation and their CAT expression was examined by Northern blotting. The expression became maximal between G1 and S phases of the cell cycle, in correspondence with the increase of endogenous c-myc expression. CAT expression of the cells containing the CAT gene linked to the SV40 enhancer/ promoter was less affected by cell cycle, neither was the expression of a housekeeping gene, the hypoxanthine phosphoribosyl transferase (HPRT). These results suggest that the myc(H-P) region is important for cell cycle dependent regulation of c-myc expression.

We have reported that a novel c-Myc binding protein, AMY-1, stimulated the transcription activity of c-Myc and was translocated from cytoplasm to nuclei in a c-Myc-dependent manner. Here, the role of AMY-1 in cell differentiation was examined. AMY-1 expression was up-regulated after differentiation induction of human K562 cells to erythrocyte cells by AraC, while c-Myc expression was rapidly down-regulated. K562 cell lines expressing exogenous AMY-1 were established, and these cells expressed a high level of epsilon-globin mRNA, a marker gene necessary for erythrocyte cell differentiation, without differentiation induction. The addition of AraC rapidly initiated differentiation in these cell lines, which continued to differentiate to erythrocyte cells possessing a high level of hemoglobin even after the decrease in AMY-1 expression. These results suggest that AMY-1 is a trigger for K562 cells to differentiate to erythrocyte cells and that AMY-1 may have a function independent of or different from c-Myc.

Two cDNAs encoding mouse MSSP and Scr3 were cloned. Both proteins, like those in humans, share significant homology of the region near the N-terminus containing the RNA-binding protein, RNP, while little homology exists near the C-terminal region of the proteins. Expression of mouse MSSP and Scr3 in mouse tissues were examined by Northern blot hybridization and both mRNAs were ubiquitously expressed in all the tissues except for testis, where the smaller sizes of MSSP mRNAs, but not Scr3 mRNA, were highly expressed. Then, the genomic DNA of mouse MSSP was cloned. Mouse genomic MSSP gene was comprised of at least 13 exons over the region spanning 60 kb. Furthermore, a chromosome location of human MSSP gene was identified at 2q24 by FISH mapping.

The mouse p53 gene, spreading over 16 kb, was divided into 10 segments of approximately 1,000 bp and subcloned into pUC19. The clones were transfected into mouse L cells together with an expression vector of the hygromycin B- or blasticidin S-resistance gene. The transfected cells were cultured in the drug-containing medium to establish cell lines resistant to the drug. Among the established lines, a high percentage of the cells originally transfected with pp53(H-R), possessing the HindIII-EcoRI fragment located downstream from the last coding exon of the p53 gene, harbored plasmid DNAs in an episomal state. The plasmid DNAs recovered from the 53HR cell-lines, transfected with pp53(H-R), were indistinguishable in structure from the original plasmids used for transfection, as assayed by Southern blotting and back-transformation to bacteria. The plasmids in episome of the cells replicated once per cell cycle in S phase in concert with chromosomal DNA. In the HindIII-EcoRI fragment (i.e., the p53(H-R) fragment) which showed a highly efficient replication activity, there exists a putative sequence for p53 recognition near the HindIII site. A DNA bending region with the clusters of AT tracts also exists near the EcoRI site of the same p53(H-R) fragment. In a transient replication system, pp53(H-R) autonomously replicated in episome of transfected cells, while mutant plasmids lacking either the p53 recognition sequence or the bending region did not. The results suggest that the HindIII-EcoRI region downstream from the p53 gene contains an activity of cellular DNA replication and both the p53 recognition sequence and the bending region are necessary for the replication activity.

The regions upstream from the human N-myc gene were examined for both transcription and replication. The plasmids containing several N-myc regions linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected to human neuroblastoma IMR32 cells. The results indicated that the region spanning from the PstI to the BamHI site (N-mycP-B; about 800 bp adjacent to the RNA start site), possessed enhancer activity. Various segments of the N-myc gene, overlapping the transcriptional regulatory regions, were subcloned into pUC19, and the plasmids were transfected to IMR32 cells together with a hygromycin B resistance-expression vector. The transfected cells were cultured in the presence of hygromycin B to establish cell lines resistant to the drug. Among the cell lines obtained, only the cells co-transfected with pN-mycP-B, the region of which contains enhancer activity described above, harboured the replicated pN-mycB-P in an episomal state. The results of BrdU labelling followed by CsCl isopycnic centrifugation suggested that the pN-mycP-B replicated once per cell cycle (in S phase) in concord with the replication of chromosomal DNA. In an in vitro DNA replication system using the nuclear extract from IMR32 cells, pN-mycP-B functioned as an template DNA of replication. Deletions up to 200 bp in the N-mycP-B region did not abolish the replicating activity, regardless of the positions of deletion, but deletions of longer than 300 bp did. These results suggest that the replicating activity of the fragment upstream of the human N-myc gene requires certain length, as well as the specific sequences not yet determined.

A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 ori, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp70) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. The segments of ARS activity contained the sequences previously identified as binding sequences for a c-myc protein complex (T. Taira, Y. Negishi, F. Kihara, S. M. M. Iguchi-Ariga, and H. Ariga, Biochem. Biophys. Acta 1130:166-174, 1992). Mutations introduced within the c-myc protein complex binding sequences abolished the ARS activity. Moreover, the ARS plasmids stably replicated at episomal state for a long time in established cell lines. The results suggest that the promoter region of the human hsp70 gene plays a role in DNA replication as well as in transcription.

DNA bending is considered to be important in DNA replication, transcription, and recombination. Several DNA bending regions reported so far are placed relatively near an origin of DNA replication or transcriptional regulatory regions. We have identified a DNA bending region in the human c-myc gene; it is located in the third exon, about 80 bases downstream of the coding region. This region includes a polyA addition signal. Deletion analysis of DNA showed that the sequence necessary for bending was about 130 base pairs long and possessed ten sets of adenine/thymine (AT) clusters. The bending region is specifically bound by proteins in HeLa cell nuclear extracts; the proteins seem to have a specific affinity for bent structures.

The myc gene family, including c- and N-myc, is believed to play a critical role in the regulation of cell cycle and proliferation. The function of N-myc, in contrast to c-myc, is not clearly understood. Here we report that, using a N-myc expression vector in CAT and S1 nuclease protection assays, the N-myc protein trans-represses expression of the human N-myc, the mouse N-myc, and the human MHC class 1 antigen genes. The analysis of N-myc deletion mutants showed that the N-terminal region where there are amino acid sequences highly conserved in the myc family and the central region where the acidic amino acids are concentrated, are necessary for trans-repression activity. Also, the region near the C-terminus is relevant for DNA-protein and protein-protein interaction; in particular, the basic region, helix-loop-helix, and leucine zipper are important for activity.

We have previously reported that c-myc protein, or protein(s) complexed with c-myc protein, binds to the region upstream of the first exon of the c-myc gene and that this region contains an origin of cellular DNA replication (ori) and also a transcriptional enhancer. Here we show by Southwestern blotting that c-myc protein binds directly to a 7 bp sequence within the above region. Furthermore, we show that the c-myc protein binding sequences are indispensable for both ori and enhancer functions, but that additional sequences are required for maximal ori and enhancer activities. Thus, c-myc protein is a sequence specific factor which is apparently used both in initiation of DNA replication and in regulation of RNA transcription.

In mammals and other vertebrates, cytosine methylation in CpG sites is often negatively correlated with gene activity. Because methylation of the promoter region is most crucial for this effect, the simplest hypothesis is that CpG methylation interferes with the binding of specific transcription factors. We have examined this hypothesis with two different transcription factor-binding sites that contain a CpG dinucleotide, namely the cAMP-responsive element (CRE; 5'-TGACGTCA) and the Sp1-binding site (5'-GTGAGGCGGTGAGACT). We have reported previously that CpG methylation of the Sp1-binding site affected neither factor binding nor transcription in HeLa cells, which may be related to the fact that Sp1 is typically associated with promoters of housekeeping genes. In contrast, CREs are often associated with promoters of cell type-specific genes. A synthetic oligonucleotide containing two tandem CREs derived from the gene encoding the human glycoprotein hormone alpha-subunit was cloned upstream of a reporter gene. Transcription of this gene was dependent on the CRE sequences in both PC12 and HeLa cells. Bandshift and methylation interference assays show that similar, if not the same, factor(s) bind to the CRE in both cell lines, even though induction by cAMP was only observed in PC12 cells. CpG methylation of the CRE consensus sequences (TGACGTCA) resulted in loss of specific factor binding, as well as loss of transcriptional activity in vitro and in vivo, in both cell types. This suggests that the inactivity of methylated promoters can, at least in some cases, be explained by their inability to bind specific transcription factors.