Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells.
A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.
Ninios
Yiannis P
YP
Institute of Biology, National Centre for Scientific Research Demokritos, 153 10 Aghia Paraskevi, Attikis, Greece.
Sekeri-Pataryas
Kalliope E
KE
Sourlingas
Thomae G
TG
D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D049109:Cell Proliferation / Q000187:drug effects; D005591:Chemical Fractionation; D002843:Chromatin / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D047468:Immunoprecipitation; D007938:Leukemia / Q000201:enzymology*; D000075223:Poly-ADP-Ribose Binding Proteins; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects
D051017:Apoptosis Regulatory Proteins; D002843:Chromatin; D006657:Histones; D006877:Hydroxamic Acids; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; C109664:caspase-activated DNase inhibitor; C012589:trichostatin A; C113696:DFFB protein, human; D003851:Deoxyribonucleases; D053148:Caspase 3; D053453:Caspase 9