The c-myc protein represses the lambda 5 and TdT initiators (original) (raw)
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Stimulation of Myc transactivation by the TATA binding protein in promoter-reporter assays
2005
The c-Myc oncogenic transcription factor heterodimerizes with Max, binds specific DNA sites and regulates transcription. The role of Myc in transcriptional activation involves its binding to TRRAP and histone acetylases; however, Myc's ability to activate transcription in transient transfection assays is remarkably weak (2 to 5 fold) when compared to other transcription factors. Since a deletion Myc mutant D106-143 and a substitution mutant W135E that weakly binds TRRAP are still fully active in transient transfection reporter assays and the TATA binding protein (TBP) has been reported to directly bind Myc, we sought to determine the effect of TBP on Myc transactivation.
Genes & Development, 1994
The myc proto-oncogene family has been implicated in multiple cellular processes, including proliferation, differentiation, and apoptosis. The Myc proteins, as heterodimers with Max protein, have been shown to function as activators of transcription through an E-box DNA-binding element, CACGTG. We have now found that the c-Myc proteins regulate transcription through another, noncanonical, DNA sequence. The non-AUG-initiated form of the c-Myc protein, c-Myc 1, strongly and specifically activates transcription of the C/EBP sequences within the EFII enhancer element of the Rous sarcoma virus long terminal repeat. In contrast, comparable amounts of the AUG-initiated form, c-Myc 2, fail to significantly affect enhancer activity. However, both c-Myc proteins trans-activate the CACGTG sequence comparably. In addition, Myc/Max heterodimers, but not Max homodimers, bind to the EFII enhancer sequence in vitro. Finally, c-Myc 1 overexpression, but not c-Myc 2 overexpression, significantly inhi...
Molecular and cellular biology, 1989
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 ...
Independent regulation of transcription of the two strands of the c-myc gene
Molecular and Cellular Biology, 1987
Previously we demonstrated the existence of transcripts from the noncoding strand of a rearranged, truncated c-myc gene in murine plasmacytomas in which this oncogene is translocated to an immunoglobulin constant-region gene element (M. Dean, R. B. Kent, and G. E. Sonenshein, Nature [London] 305:443-446, 1983). Here we report on the transcription of the two strands of a normal, unrearranged c-myc gene. We examined the effects of gene rearrangements, growth state transitions, and differentiation on the relative levels of usage of the two strands. Transcription from intron 1 to exon 3 of the murine c-myc gene was studied in in vitro nuclear runoff assays. The level of transcription of the noncoding strand across this region of a germ line c-myc gene in a murine B-cell lymphoma line was comparable to the level observed in plasmacytomas with translocated c-myc genes. Rapid changes in transcription of the coding strand of the c-myc gene could be seen during growth arrest of WEHI 231 cell...
1999
We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library. Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells. To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed. This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites. Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244). The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region. When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited. Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common. Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression. Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-myc promoter. In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1. Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation. Mass.), G5E1BCAT (kindly provided by D. Dean, Washington University, St. Louis, Mo.), and c-myc CAT (25) plasmids were used as the reporter constructs in this study. The expression vector CMV-GAL4 construct was prepared by substituting the cytomegalovirus (CMV) promoter for the simian virus 40 early promoter of pSG424 (32) containing the GAL4 DNA binding domain (amino acids 1 to 147). Plasmid GALMBP1-338 was constructed by PCR amplification of MBP-1 cDNA (25) and cloned in frame with the GAL4 DNA binding domain into CMVGAL4 plasmid DNA. For MBP-1 deletion mutant proteins, desired fragments were generated by PCR amplification using sense and antisense oligonucleotides . Amplified fragments were digested with BamHI (5Ј end) and XbaI (3Ј end) and cloned in frame downstream of the GAL4 DNA binding domain of the CMVGAL4 vector. The mutant plasmids were analyzed by restriction enzyme digestion and DNA sequencing. pM3/3CGln (kindly provided by C. Sample, St. Jude Children's Research Hospital, Nashville, Tenn.) containing a DNA fragment encompassing the glutamine-rich activation domain from Epstein-Barr virus transcription factor EBNA3C (21) was inserted in frame into the GAL4 amino acid 1 to 147 sequence in the pM3 vector (33). 3CGln(MBP-1) was derived by in frame ligation of the DNA fragment encoding MBP1-47 to the downstream portion of the GAL4-3CGln sequence in pM3/3CGln at the SalI (5Ј end) and XbaI (3Ј end) sites. The resulting double-stranded plasmid DNAs were transformed into Escherichia coli DH5␣, and purified plasmid DNAs were used for in vitro transient expression assay.
A negative transcriptional control element located upstream of the murine c-myc gene
The EMBO Journal
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.
A Triplex-forming Sequence from the Human c-MYC Promoter Interferes with DNA Transcription
Journal of Biological Chemistry, 2007
Naturally occurring DNA sequences that are able to form unusual DNA structures have been shown to be mutagenic, and in some cases the mutagenesis induced by these sequences is enhanced by their transcription. It is possible that transcription-coupled DNA repair induced at sites of transcription arrest might be involved in this mutagenesis. Thus, it is of interest to determine whether there are correlations between the mutagenic effects of such noncanonical DNA structures and their ability to arrest transcription. We have studied T7 RNA polymerase transcription through the sequence from the nucleasesensitive element of the human c-MYC promoter, which is mutagenic in mammalian cells (Wang, G., and Vasquez, K. M. (2004) Proc. Natl. Acad. Sci. U. S. . This element has two mirror-symmetric homopurine-homopyrimidine blocks that potentially can form either DNA triplex (H-DNA) or quadruplex structures. We detected truncated transcription products indicating partial transcription arrest within and closely downstream of the element. The arrest required negative supercoiling and was much more pronounced when the pyrimidine-rich strand of the element served as the template. The exact positions of arrest sites downstream from the element depended upon the downstream flanking sequences. We made various nucleotide substitutions in the wild-type sequence from the c-MYC nuclease-sensitive element that specifically destabilize either the triplex or the quadruplex structure. When these substitutions were ranked for their effects on transcription, the results implicated the triplex structure in the transcription arrest. We suggest that transcriptioninduced triplex formation enhances pre-existing weak transcription pause sites within the flanking sequences by creating steric obstacles for the transcription machinery.
Activation by c-Myc of transcription by RNA polymerases I, II and III
Biochemical Society symposium, 2006
The proto-oncogene product c-Myc can induce cell growth and proliferation. It regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. We have found that c-Myc can also activate transcription by RNA polymerases I and III, thereby stimulating production of rRNA and tRNA. As such, c-Myc may possess the unprecedented capacity to induce expression of all ribosomal components. This may explain its potent ability to drive cell growth, which depends on the accumulation of ribosomes. The activation of RNA polymerase II transcription by c-Myc is often inefficient, but its induction of rRNA and tRNA genes can be very strong in comparison. We will describe what is known about the mechanisms used by c-Myc to activate transcription by RNA polymerases I and II.
Nucleic Acids Research, 1988
We have studied the transcriptional activation of translocated c-myc genes in murine plasmacytomas in which the translocation juncture occurs within the first intron of c-myc and juxtaposes c-myc with the immunoglobulin CK gene segment. It has been widely suggested that a novel transcriptional enhancer element located near the C. gene segment might activate the translocated c-myc gene. We have carried out an extensive search for such an element and find no significant transcriptional enhancer activity in a 22 kb region encompasing the translocation junction, Co(gene segment and regions 3' of Co.. We also find that the cryptic promoter region of the translocated c-myc gene is a very weak promoter of transcription. Despite this evidence against the presence of strong transcriptional regulatory elements, the translocated c-myc gene locus is transcribed at high rates that are 25->100% of that measured for the highly active immunoglobulin genes in murine plasmacytomas. These data suggest the presence of a novel type of strong activator of transcription in the murine heavy chain locus.
Transcription Factor Binding and Induced Transcription Alter Chromosomal c-myc Replicator Activity
Molecular and Cellular Biology, 2004
The observation that transcriptionally active genes generally replicate early in S phase and observations of the interaction between transcription factors and replication proteins support the thesis that promoter elements may have a role in DNA replication. To test the relationship between transcription and replication we constructed HeLa cell lines in which inducible green fluorescent protein (GFP)-encoding genes replaced the proximal ∼820-bp promoter region of the c-myc gene. Without the presence of an inducer, basal expression occurred from the GFP gene in either orientation and origin activity was restored to the mutant c-myc replicator. In contrast, replication initiation was repressed upon induction of transcription. When basal or induced transcription complexes were slowed by the presence of α-amanitin, origin activity depended on the orientation of the transcription unit. To test mechanistically whether basal transcription or transcription factor binding was sufficient for r...