G-band expression and megabase fragmentations in apoptosis.
Apoptosis seems characterized by a cascade of megabase to 200-bp fragmentations and by a commitment to perish at the initial level. How that could be achieved seems unclear. Preferential cleavage of transcriptionally active chromatin by apoptotic nuclease activity has long been suggested. We show here the manifestation of self-inflicted G-banding patterns in mitotic chromosomes, or G-band expression, occurring concurrently with a pattern of megabase fragmentations in two apoptotic systems that we have established in human Chang liver cells using (a) staurosporine and (b) vanadyl(4) prepulsing. We further show that rare-cutting NotI and MluI restriction endonucleases with C-G dinucleotide sequence specificity had produced similar G-bandings and megabase fragmentations cascading down to the 200-bp ladder fragmentation that were also associated with the expression of characteristic apoptotic morphologies by the digested cells. CpG-specific methylation using the methylase SssI abolished the DNA fragmentation cascade, G-banding, and apoptotic expressions induced by NotI and MluI, implicating endonuclease cleavage of active chromatin, where CpG islands are concentrated, as the initiating event. Reproducing the G-bandings and megabase fragmentations by directly applying NotI and MluI endonucleases to fixed chromosomes and extracted genomic DNA, respectively, further confirmed the notion of endonucleolytic cleavage of active chromatin as the causation. Nuclease-digested light G-band regions of chromosomes appeared to be the chromosome sites providing the megabase fragments. Transcriptionally active genes of the genome are known to be preferentially cleaved by nuclease activity and are established as being concentrated in the light G-bandings that correspond to R-bandings, which are also known to be the sites of more frequent cytogenetic breakpoints. Manifestation of self-inflicted G-banding patterns (G-banding expression) in apoptosis would then imply cleavage of the transcriptionally active genes in every light G-band site of every chromosome in the genome. This must be suicidal.
Chen
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Department of Anatomy, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore.
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D017209:Apoptosis; D001426:Bacterial Proteins; D002453:Cell Cycle; D002460:Cell Line; D002875:Chromosomes; D053938:DNA Fragmentation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D008938:Mitosis; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
D001426:Bacterial Proteins; D014638:Vanadates; C035041:endodeoxyribonuclease MluI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases; D019311:Staurosporine