title: c-Myc creates an activation loop by transcriptionally repressing its own functional inhibitor, hMad4, in young fibroblasts, a loop lost in replicatively senescent fibroblasts.
abstract: c-Myc transcriptional activity in cells is dampened by the Mad family of transcriptional repressors. The expression of one member, hMad4, is increased in growth-arrested states such as quiescence or replicative senescence; hMad4 mRNA levels in replicatively senescent fibroblasts are about twice those seen in their young contact-inhibited quiescent counterparts. Moreover, the repression of hMad4 transcription following serum stimulation observed in quiescent young fibroblasts is lost in senescent cells. This loss results in persistent expression of hMad4, which leads to an inability to switch from an hMad4/Max complex to a c-Myc/Max complex on selected c-Myc target genes following serum stimulation. We have located an initiator element (Inr), a candidate for Miz-1 binding, in the hMad4 promoter. In reporter assays, Miz-1 enhances reporter GFP expression; this enhancement is inhibited by co-expressing c-Myc. Thus hMad4, as does its murine counterpart, contains the Inr element through which Miz-1 activates its expression; but this action is inhibited in the presence of c-Myc. This inhibition may explain the down-regulation of hMad4, corresponding to the up-regulation of c-Myc, in young serum-starved quiescent fibroblasts upon serum stimulation. However, this reciprocal change does not occur in replicatively senescent fibroblasts upon serum stimulation; instead, hMad4 persists in the presence of high levels of c-Myc activation. Our results suggest that: (1) replicative senescence-specific factors may block c-Myc inhibition of Miz-1 activation of hMad4 expression; and (2) the continual presence of hMad4 protein may transcriptionally repress selected c-Myc target genes, whose functions are key to the signaling pathways leading to apoptosis inhibition and permanent exit of cell cycle traverse in normal human fibroblasts.
authors.affiliation: The Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, The Sir Mortimer B. Davis-Jewish General Hospital, and Department of Medicine, McGill University, Montréal, Québec, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Marcotte
authors.forename: Jen Mou
authors.initials: JM
authors.lastname: Chen
authors.forename: Sylvain
authors.initials: S
authors.lastname: Huard
authors.forename: Eugenia
authors.initials: E
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D015152:Blotting, Northern; D002460:Cell Line; D049109:Cell Proliferation; D002469:Cell Separation; D016922:Cellular Senescence; D047369:Chromatin Immunoprecipitation; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013947:Thymosin / Q000031:analogs & derivatives / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D039561:Eukaryotic Initiation Factor-4E; D049452:Green Fluorescent Proteins; D051741:Kruppel-Like Transcription Factors; D011498:Protein Precursors; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D013947:Thymosin; D014157:Transcription Factors; C484347:MXD4 protein, human; C107961:ZBTB17 protein, human; C040782:prothymosin alpha