Search

The v-abl oncogene of Abelson murine leukemia virus encodes a deregulated form of the cellular nonreceptor tyrosine kinase. v-Abl activates c-myc transcription, and c-Myc is an essential downstream component in the v-Abl transformation program. To explore the mechanism by which v-Abl activates c-myc transcription, a cotransfection assay was developed. We show that transactivation of a c-myc promoter by v-Abl requires the SH1 (tyrosine kinase) and SH2 domains of v-Abl; the C-terminal domains are not required for transactivation. The assay also identified the E2F site in the c-myc promoter as a v-Abl-responsive element. In addition, multimerized E2F sites were shown to be sufficient to confer v-Abl-dependent activation on a minimal promoter. This is the first identification of a v-Abl response element for transcriptional activation. v-Abl tyrosine kinase-dependent changes in proteins binding the c-myc E2F site were also demonstrated, including induction of a complex containing DP1, p107, cyclin A, and cdk2. Identification of v-Abl-dependent changes in E2F-binding proteins provides an important link between v-Abl, transcription, cell cycle regulation, and control of cellular growth.

Activation of the murine c-myc promoter by murine c-Myb protein was examined in several cell lines by using a transient expression system in which Myb expression vectors activate the c-myc promoter linked to a chloramphenicol acetyltransferase reporter gene or a genomic beta-globin gene. S1 nuclease protection analyses confirmed that the induction of c-myc by c-Myb was transcriptional and affected both P1 and P2 start sites in a murine T-cell line, EL4, and a myelomonocytic line, WEHI-3. Mutational analyses of the c-myc promoter revealed that two distinct regions could confer Myb responsiveness in two T-cell lines, a distal site upstream of P1 and a proximal site within the first noncoding exon. In contrast, only the proximal site was required for other cell lineages examined. Five separate Myb-binding sites were located in this proximal site and found to be important for c-Myb trans activation. DNA binding was necessary for c-myc activation, as shown by the loss of function associated with mutation of Myb's DNA-binding domain and by trans-dominant repressor activity of the DNA binding, trans-activation-defective mutant. The involvement of additional protein factors was addressed by inhibiting protein synthesis with cycloheximide in a conditional expression system in which the activity of presynthesized Myb was under the control of estrogen. These experiments indicate that de novo synthesis of additional proteins was not necessary for c-myc trans activation. Together these data reveal two cell lineage-dependent pathways by which c-Myb regulates c-myc; however, both pathways are mechanistically indistinguishable in that direct DNA binding by Myb is required for activating c-myc whereas neither de novo protein synthesis nor other labile proteins are necessary.

M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antigen receptor (TCR) alpha-chain genes in vitro and thus represents a dynamic system for studying TCR assembly. In an effort to investigate whether the TCR alpha locus is accessible for V(D)J rearrangement events, we examined M14T cells for the presence of germ line TCR alpha transcripts. Several unrearranged V alpha segments were found to be transcriptionally active in M14T cells. By comparison, germ line V alpha transcripts are absent in nonlymphoid and pro-T-cell lines and barely detectable in mature T-cell lines, suggesting that this phenomenon is likely stage and tissue specific. We demonstrate a perfect correlation between transcriptionally active V alpha segments and their involvement in ongoing V alpha-to-J alpha rearrangements. In addition, data suggesting that the unrearranged J alpha locus is also transcriptionally active in the M14T line are presented. Furthermore, the recombination-activating genes RAG-1 and RAG-2 are differentially expressed, with RAG-2 detectable only by polymerase chain reaction, implying that very low levels of one of these gene products are sufficient to complement the other to facilitate VJ rearrangements. These findings provide the first direct evidence for an accessibility model of antigen receptor rearrangement in T cells.

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 c-myc DNA dramatically inhibits CAT gene expression in the pSV2CAT vector when placed in either orientation approximately 1.7 kb 3' (and approximately 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.

Chemically induced differentiation of mouse erythroleukemia (MEL) cells leads to complex changes in c-myc mRNA levels. Within 1-2 hr after the addition of the inducer hexamethylene bisacetamide (HMBA), c-myc mRNA levels decrease 10- to 20-fold and remain low until 12-24 hr, at which time the mRNA reaccumulates to its original level. Thereafter as the cells undergo terminal differentiation, c-myc mRNA again declines to a low level. We have investigated the regulation of these changes by measuring c-myc gene transcription and mRNA turnover. We find that the early rapid decline in c-myc mRNA is due to an increase in the block to elongation of transcription within the c-myc first exon. Effective c-myc transcription is then restored after 2 hr of HMBA treatment to the level present in uninduced cells and is maintained throughout the remainder of the differentiation program. These results demonstrate that, except for the rapid decline in c-myc mRNA immediately following inducer treatment, all subsequent regulation of message levels occurs through post-transcriptional mechanisms. Studies of c-myc mRNA turnover suggest that some post-transcriptional regulation is nuclear.

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.

We have examined the contributions of transcriptional and post-transcriptional control mechanisms for the regulated activation of the c-myc proto-oncogene in regenerating kidney tissue in vivo. The c-myc gene is activated in kidney tubule cells which are induced to proliferate as a consequence of folic acid-induced renal injury in vivo. We show that c-myc transcriptional initiation is induced in a co-ordinate fashion with a strong block to transcriptional elongation within the gene's first exon. Therefore, transcription is not effectively induced throughout the c-myc gene in these regenerating renal cells. In contrast, initiation of c-fos gene transcription was induced while the degree of transcriptional blockage within the gene's first exon remained constant. The steady-state levels of myc transcripts, originating from the gene's P1 and P2 start sites, accumulated to very high levels within 4-6 hr which were maintained for at least 24 hr after folic acid treatment. We conclude that post-transcriptional control mechanisms are largely responsible for the dramatic induction of c-myc mRNAs in regenerating kidney tissue in vivo.

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

Accumulation of unusually high amounts of larger-than-normal c-myc mRNAs occurs in two mouse plasmacytomas, TEPC 1165 and TEPC 2027. Southern blot and DNA sequence analyses showed that both tumors have undergone translocations of immunoglobulin heavy chain loci to positions 5' of the c-myc gene promotors resulting in removal of DNA sequences encoding a negative transcriptional regulatory element. In contrast to other mouse plasmacytomas, TEPC 1165 and TEPC 2027 rearranged myc genes show increased transcription, partially explaining their abundance of myc RNA. Similar to other mouse plasmacytomas, the abundance of myc RNA in TEPC 1165 and TEPC 2027 is also influenced by increased stability of structurally atypical myc RNAs. Two myc mRNAs are found in TEPC 2027, a 2.4 kb species including all 3 myc exons and a 4.0 kb species with the 3 exons plus the first intron. The two major myc mRNAs in TEPC 1165, 3.0 and 3.9 kb species, also include all three myc exons plus portions of the first intron. S1 nuclease protection analyses show that the 5' initiation and 3' untranslated (UT) regions of the unusual TEPC 1165 RNAs are normal showing that the size differences arise solely from inclusion of first intron sequences in the large myc RNAs. DNA sequence analysis showed that the presence of first intron sequences in the large myc RNAs is due to mutations affecting the splice donor region at the 3' end of exon 1 in both tumors. SDS-PAGE analysis of immunoprecipitated TEPC 1165 and TEPC 2027 myc proteins showed them to be of normal electrophoretic mobility but no more abundant than in a pre-B cell line 18-81 that contains at least 10 fold less myc RNA. The 4.0 kb myc mRNA of TEPC 2027 is atypically stable while the 2.4 kb myc mRNA undergoes normal rapid turnover within the same cell, demonstrating that the presence of first intron sequences in the large myc RNA stabilizes it despite the presence of 3' UT and putative exon 1 destabilizing sequences. These results show that myc intron 1 sequences can counteract the effect of 3' UT region destabilizing sequences in myc RNA and suggest that the increased myc RNA stability noted in TEPC 1165 and TEPC 2027 is largely due to the presence of the intron 1 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

In plasmacytoma cells producing IgG, IgA, or IgM immunoglobulin heavy chains, the large precursors of the heavy chain messenger RNA's contain nucleotide sequences that specify only the expressed class of constant region. This indicates that the switch from one class of heavy chain to another during B cell ontogeny does not occur by altered processing of a complex gene transcript.

We have identified a positive modulator within the c-myc first exon downstream of the gene's transcription initiation sites, P1 and P2. We introduced myc-CAT (chloramphenicol acetyltransferase) hybrid genes into three cell lines (BJAB, COS and HeLa) and measured their expression by either CAT enzymatic activity, S1 nuclease protection or by a nuclear 'run-on' transcription assay. Removal of 46 bp from the 3' end of the first exon results in a decrease of myc-CAT expression and P2 activity. A 438-bp exon 1 segment, lacking the normal myc promoters, efficiently drives the expression of SV40 early promoters. We find that this first exon segment efficiently functions as a positive modulator only in its sense orientation, 3' of a nearby promoter. The positive effects of the myc first exon and the SV40 enhancer are complementary.

We present a detailed analysis of strand-specific transcription in different regions of the murine c-myc locus. In normal and transformed cell lines, RNA polymerase II directed transcription occurs in the sense and anti-sense direction. Three noncontiguous regions show a high level of transcription in the anti-sense orientation: upstream of the first exon, within the first intron and in the 3' part of the gene (intron 2 and exon 3). In a cell line carrying a c-myc amplification (54c12), anti-sense transcription is not uniformly increased throughout the locus and is differentially affected by inhibition of protein synthesis. These results suggest that anti-sense transcription in various parts of the locus is independently regulated. In the sense orientation, transcriptional activity is higher in the first exon than in the rest of the gene indicating that transcription pauses near the 3' end of the first exon. The extent of this intragenic pausing varies among different cell lines and is most severe in cells with a c-myc amplification. Transcription initiation and pausing are both negatively regulated by labile proteins.

NIARD (non-immunoglobulin-associated rearranging DNA) is located on mouse chromosome 15 at the break point of a commonly observed translocation event involving chromosomes 15 and 12 in murine plasmacytomas. The human cellular analogue of the v-myc oncogene of avian myelocytomatosis virus, strain MC-29, is known to reside on the distal end of human chromosome 8 and has been observed to translocate to chromosome 14 in Burkitt lymphomas. Using a cDNA clone specific for the transcript of the human c-myc gene (H c-myc), we show that the mouse c-myc (M c-myc) gene is contained within NIARD. NIARD-associated chromosome translocations occurred 1.3-2 kilobases (kb) 5' of the mouse c-myc gene where NIARD recombines with the switch region of the C(alpha) immunoglobulin gene in various murine plasmacytomas. The mouse c-myc encoding region within NIARD spanned <2.4 kb of DNA and expressed a low level of a 2.3-kb polyadenylylated RNA in BALB/c spleen. Increased (10- to 20-fold) levels of rearranged mouse c-myc transcripts (i.e., approximately 1.8-2.1 kb) were observed in plasmacytomas that have NIARD-associated chromosome translocations. Human c-myc and NIARD probes detected DNA rearrangements of human c-myc in four of seven Burkitt lymphomas. DNA sequences adjacent to the human c-myc gene recombined with the C(mu) immunoglobulin gene locus on chromosome 14 in several Burkitt lymphomas. The activation of the c-myc oncogene by chromosome translocation implicates its involvement in B-cell oncogenesis.

We have identified sequences in the 5' flanking region of the murine c-myc gene's P1 and P2 transcription initiation sites which form specific complexes with nuclear factors of murine and human origin and are also required for normal P1 and P2 usage. Four nuclear factor binding sites were identified within 400 bp 5' of P1 (5'Mf, 5'Mg1, 5'Mg2, and 5'Mg3) and two others within 100 bp 5' of P2 (ME1a1 and ME1a2). The Sp1 transcription factor bound to 5'Mg1 and 5'Mg3 with high affinity and with low affinity to 5'Mg2, ME1a1 and ME1a2 which also bound with high affinity to other factors in crude nuclear extracts. Deletion mutagenesis of sequences 5' of the P1 initiation site revealed that 109 bp encompassing 5'Mg3 and a TATA sequence were sufficient for P1 usage. The ME1a1 binding site 5' of P2 was necessary for maximal P2 activity and the loss of this sequence resulted in enhanced P1 usage. These findings demonstrate that the P1 and P2 initiation sites are independently regulated and that the ME1a1 binding site plays a central role in the normal usage of the c-myc promoters.

To assess possible alterations of c-myc transcriptional control in murine B-cell tumors, we have investigated the pattern of DNaseI hypersensitive sites in the gene's putative regulatory region and within the gene in a variety of genomic contexts. A number of such sites were found in several cell types, but none of these was detectable in a gene which was shown to be transcriptionally silent by the criterion of elongation of nascent transcripts in isolated nuclei. These results differ from those of a previous study, in which a DNaseI-hypersensitive site approximately 2 kb upstream of the gene was proposed to be associated with negative regulation of c-myc transcription in human cells. An analysis of DNA sequences presented here reveals that this region is highly homologous between mouse and human, suggesting that these upstream hypersensitive sites do not reflect species-specific regulatory elements. We also present data indicating that this hypersensitive site distinguishes the c-myc alleles in translocation-positive plasma cell tumors which lack c-myc rearrangement. Furthermore, we report the existence of hypersensitive sites within the gene. One of these appears to be associated with cryptic promoters that are employed only when the normal promoters are lost as a consequence of chromosome translocation. These results are discussed in the context of c-myc translocation and gene breakage and with respect to possible stage-specific regulation of the gene's transcriptional competence.

Comparative nucleotide sequence analysis of a rearranged c-myc gene in a murine plasmacytoma and c-myc cDNA from normal spleen reveals that chromosomal translocation in the plasmacytoma breaks the c-myc gene within the first exon or intron. In the plasmacytoma truncated c-myc RNAs initiate from newly exposed promoter sites. Nevertheless, the myc polypeptide produced in the plasmacytoma is probably the same as that from the intact c-myc gene because the exon lost by breakage and translocation is non-coding. The second and third exons of the mouse c-myc gene are substantially conserved in the v-myc gene of the avian retrovirus, MC29.