c-Myc-induced extrachromosomal elements carry active chromatin.
Murine Pre-B lymphocytes with experimentally activated MycER show both chromosomal and extrachromosomal gene amplification. In this report, we have elucidated the size, structure, and functional components of c-Myc-induced extrachromosomal elements (EEs). Scanning electron microscopy revealed that EEs isolated from MycER-activated Pre-B+ cells are an average of 10 times larger than EEs isolated from non-MycER-activated control Pre-B- cells. We demonstrate that these large c-Myc-induced EEs are associated with histone proteins, whereas EEs of non-MycER-activated Pre B- cells are not. Immunohistochemistry and Western blot analyses using pan-histone-specific, histone H3 phosphorylation-specific, and histone H4 acetylation-specific antibodies indicate that a significant proportion of EEs analyzed from MycER-activated cells harbors transcriptionally competent and/or active chromatin. Moreover, these large, c-Myc-induced EEs carry genes. Whereas the total genetic make-up of these c-Myc-induced EEs is unknown, we found that 30.2% of them contain the dihydrofolate reductase (DHFR) gene, whereas cyclin C (CCNC) was absent. In addition, 50% of these c-Myc-activated Pre-B+ EEs incorporated bromodeoxyuridine (BrdU), identifying them as genetic structures that self-propagate. In contrast, EEs isolated from non-Myc-activated cells neither carry the DHFR gene nor incorporate BrdU, suggesting that c-Myc deregulation generates a new class of EEs.
Smith
Greg
G
Manitoba Institute of Cell Biology, CancerCare Manitoba, the Genomic Center for Cancer Research and Diagnosis, Winnipeg, Manitoba, Canada.
Taylor-Kashton
Cheryl
C
Dushnicky
Len
L
Symons
Stephen
S
Wright
Jim
J
Mai
Sabine
S
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D002875:Chromosomes / Q000648:ultrastructure*; D056745:Cyclin C; D016213:Cyclins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D040342:Genetic Structures; D006657:Histones / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008954:Models, Biological; D010766:Phosphorylation; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000378:metabolism; D016622:Silver Staining; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic
D000963:Antimetabolites; C503674:CCNC protein, human; C503675:Ccnc protein, mouse; D002843:Chromatin; D056745:Cyclin C; D016213:Cyclins; D006657:Histones; D016271:Proto-Oncogene Proteins c-myc; D013762:Tetrahydrofolate Dehydrogenase; D001973:Bromodeoxyuridine