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We propose that this 5' c-myc region be termed a 'dehancer' since this negative element has the opposite properties of a transcriptional enhancer. Key words: ...
The EMBO Journal vol.5 no.5 pp.899-904, 1986

A negative transcriptional control element located upstream of the murine c-myc gene

Elaine F.Remmers, Jian-Qing Yang and Kenneth B.Marcu Biochemistry Department, SUNY. at Stony Brook, Stony Brook, NY 11794, USA Communicated by G.Klein

We have investigated the nature of regulatory sequences within the vicinity of the murine c-myc locus by analyzing the expression of myc-chloramphenicol acetyl transferase (CAT) vectors transfected into a human lymphoblastoid cell line (BJAB) and a monkey fibroblast line (COS). CAT enzymatic assays and S1 nuclease protection experiments reveal that a negative element resides 428-1188 bp 5' of the first c-myc promoter, P1. This 760-bp segment of 5'-flanking cmyc DNA dramatically inhibits CAT gene expression in the pSV2CAT vector when placed in either orientation 1.7 kb 3' (and 3.2 kb 5' on the circular plasmid) from the SV40 promoter region. By employing this strategy, we were unable to identify an analogous DNA segment that is closer to or within the first c-myc exon. We propose that this 5' c-myc region be termed a 'dehancer' since this negative element has the opposite properties of a transcriptional enhancer. Key words: c-myc oncogene/CAT assay/SI nuclease mapping/ transcriptional dehancer element -

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Introduction Alterations in the structure and/or expression of the c-myc protooncogene as a consequence of chromosome translocation are well documented in most murine plasma cell tumors and human Burkitt lymphomas. The c-myc gene is broken in a number of these cases resulting in the loss of a large 5'-non-coding exon and associated 5'-flanking region from the transcribed gene (reviewed in Klein, 1983; Perry, 1983; Leder et al., 1983; Marcu et al., 1984; Klein and Klein, 1985). We have recently shown that a number of translocation sites in plasma cell tumors are clustered - 400 bp 5' of exon 1 and we have proposed that these clustered breakpoints define the location of a regulatory element upstream of c-myc (Yang et al., 1985). In this class of plasma cell tumors the relative usage of the two normal c-myc promoters (P1 and P2) is shifted in favor of the 5' promoter, P1 (Marcu et al., 1984; Yang et al., 1984, 1985). Similarly, viral insertions in spontaneous and induced murine T lymphomas frequently occur in the c-myc 5'-flanking region (Corcoran et al., 1984, 1985; Li et al., 1984; Selten et al., 1984; O'Donnell et al., 1985) and a shift in c-myc promoter usage has also recently been demonstrated in cells with proviruses integrated 5' of c-myc (Reicin et al., 1986). In this report, we have sought to determine whether these translocations and viral insertions act by disrupting a control element located upstream of c-myc. Hybrid gene constructs were prepared by fusing portions of the murine c-myc locus to the chloramphenicol acetyl transferase (CAT) gene. Based on transient expression of these myc-CAT hybrid genes in transfected BJAB cells, human lymphoblastoid © IRL Press Limited, Oxford, England

cells derived from a Burkitt-like lymphoma (Zech et al., 1976), we identified a negative regulatory element in the c-myc 5'-flanking region located 428-1188 bp 5' of the first c-myc promoter. Furthermore, this DNA segment inhibited expression from the SV40 enhancer-promoter driven CAT gene in the expression vector, pSV2CAT (Gorman et al., 1982), when positioned in either orientation 3' of the vector's polyadenylation site. We propose the term 'dehancer' for this negative regulatory region since it acts at a distance, in either orientation, and reduces expression from an enhancer-driven promoter to a level commensurate with enhancer removal.

Results c-myc 5' DNA contains a negative element To study the control of c-myc expression, pM(Bg)CAT was constructed (Figure IA). This plasmid contains 1.7 kb of the c-myc locus including 5'-flanking DNA and the entire non-coding cmyc first exon (Stanton et al., 1984) inserted into the vector pCAT(AEP), which has the CAT gene but lacks enhancer and promoter elements. This portion of the c-myc locus was sufficient to drive expression of the CAT gene in transiently transfected BJAB cells (Figure 2A and Table I). S1 nuclease protection of RNA isolated from stably transfected HeLa cells indicated that the majority of CAT transcripts from this vector initiate from the normal myc second promoter (J.-Q.Yang, E.F.Remmers and K.B.Marcu, submitted). As an initial step to identify the location(s) of regulatory elements within this 1.7-kb portion of the c-myc locus in pM(Bg)CAT, we deleted 760 bp from the 5' end of the c-myc insert by removing the DNA segment 5' of the SmaI site in pM(Bg)CAT [see pM(Sm-Bg)CAT in Figure 1]. We chose to delete the myc DNA 5' of this site because it closely corresponds to the site of clustered 5' translocations in plasma cell tumors and frequent viral insertions in T lymphomas. Removal of this 5'-flanking DNA in pM(Sm-Bg)CAT increases expression of the CAT gene in the BJAB lymphoblastoid cells by 3-fold (Figure 2A and Table I). 5' c-myc negative control element can act upon a heterologous promoter To obtain further evidence that a negative regulatory element is within the 760-bp BglII-SmaI 5'-flanking c-myc fragment, we assessed its ability to repress expression from a heterologous gene's promoter. As a stringent test for this idea, the BglH -SnaI fragment was inserted in both orientations 3' of the polyadenylation site of the well-characterized CAT expression vector, pSV2CAT (Gorman et al., 1982) [see pSV2CAT(Bg-Sm 3') and pSV2CAT(Sm-Bg 3') in Figure IB]. Results presented in Figure 2 and Table I show that the 760-bp BglII -SmaI fragment effectively inhibited expression (30- to 45-fold less than pSV2CAT) from the SV40 promoter-enhancer driven CAT gene in each of these two vectors in BJAB cells. As a control for this experiment and to screen for other potentially analogous elements, we inserted other portions of the 1.7-kb myc BglII segment (both -

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E.F.Remmers, J.-Q.Yang and K.B.Marcu

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Fig. 1. myc-CAT vector constructions. (A) A 1.7-kb BglII fragment containing the c-myc first exon and 5'-flanking DNA was inserted in pCAT(AEP) as indicated for pM(Bg)CAT. pM(Sm-Bg)CAT was prepared by deletion of myc sequences 5' of the SnaI site. (B) The indicated c-myc fragments were inserted in the BamHI site of pSV2CAT. Restriction enzyme sites are labeled as follows: Ac, AccI; Bm, BamHI; Bg, BglIl; Hd, HindIII; Nd, NdeI; RI, EcoRI; Ss, SstI; Xh, XhoI.

orientations of a 546-bp SamI -BamHI fragment and a 460-bp HindIll -Bglll fragment) into the same site 3' of the CAT gene in pSV2CAT (see Figure iB). These vectors [pSV2CAT(SmBm 3'), pSV2CAT(Bm-Sm 3'), pSV2CAT(Hd-Bg 3') and pSV2CAT(Bg-Hd 3')] possessed the same CAT activity as the original pSV2CAT vector (Figure 2 and Table I). We conclude from these data that a negative transcriptional element is located within the 760-bp BglII -SmaI fragment which is 428 bp 5' of the first c-myc promoter. To demonstrate directly that attachment of the BglII -SmaI fragment 3' of pSV2CAT resulted in the reduction of CAT RNAs, as shown indirectly by CAT enzymatic activity, we determined the presence of CAT transcripts in transiently transfected COS cells by an Si nuclease protection assay. COS cells were used to increase the sensitivity of this analysis since plasmids which contain the SV40 origin of replication are amplified in these cells (Gluzman, 1981). Total RNAs prepared from pSV2CAT-transfected cells generated two SI nuclease-resistant bands with a 452 nucleotide 5' end-labeled Sp -RI* probe (see Figure 3). These SI-protected bands correspond to the two start sites of the SV40 early promoter region, SVE2 and SVE, (also referred to as the SV40 early UP and DO RNAs) (Ghosh and Lebowitz, 1981; Ghosh et al., 1981) of the pSV2CAT vector. Neither of these bands are apparent with RNAs isolated from pSV2CAT (Bg-Sm 3')- or pSV2CAT(AE)-transfected cells. An SV40-rabbit ,B-globin expression vector, pSV2flG, was co900

transfected along with each of these CAT vectors as an internal reference control for RNA integrity and transfection efficiency. S1 nuclease analysis with a 5' end-labeled SV2fG probe revealed the expected SVE2- and SVE1-initiated RNAs in each of the transfected samples (see Figure 3). To determine if the copy numbers of each of the CAT vectors in the transfected COS cells were similar, Hirt supernatants (Hirt, 1967) were prepared. The yields of the various CAT plasmid DNAs, analysed by Southern blotting, could not account for the difference in RNA levels or CAT expression (data not shown). The CAT assay data described above was obtained using a different cell line (BJAB) than used for the RNA analysis (COS). Although these cell lines cannot be compared directly, CAT assays of lysates prepared from transfected COS cells also demonstrated the strong negative effect of the Bgmll-SSmaI fragment 3' of pSV2CAT as seen in BJAB cells (data not shown). Discussion We have demonstrated that a negative control element is located 428-1188 bp 5' of the murine c-myc gene. This element is likely to inhibit gene expression at the transcriptional level and not at the post-transcriptional level since it is located outside the c-myc gene's transcription unit. Enhancer elements have the opposite effect on gene expression. Enhancers can be located at a distance upstream, downstream, or within a gene and can increase transcription from their normal or heterologous promoters in-

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