Cell cycle dependent alterations of chromatin structure in situ as revealed by the accessibility of the nuclear protein AF-2 to monoclonal antibodies.
We have recently described a novel nuclear antigen, AF-2, which is related to cell cycle dependent alterations of chromatin structure. We show by two parameter flow cytometry on a cell by cell basis that the antigen is accessible to specific monoclonal antibodies only in mitotic and postmitotic early G1-phase cells. The evaluation of nuclease susceptibility and AF-2 antigen accessibility reveals different subcompartments of the G1-phase of the cell cycle with distinct chromatin conformations. Digestion with DNase I seems to alter the chromatin structure according to concentration and this is reflected by an increase of the antigen accessibility. Chromatin in the more condensed early G1-phase is specifically digested by lower concentrations of the enzyme than chromatin in later stages of interphase. Chromatin from cells in the late-G1, S-, and G2-phases shows a higher relative resistance to DNase I and a reduced accessibility of the AF-2 antigen to monoclonal antibodies. Nuclease S1 has a similar effect on chromatin topology, as revealed by the reaction with anti-AF-2 antibodies, without digestion of detectable amounts of DNA. The antigen becomes available to the antibodies in almost all cells by digestion with high concentrations of DNase I or Nuclease S1.
Pfeffer
U
U
Laboratory of Molecular Biology, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
Di Vinci
A
A
Geido
E
E
Vidali
G
G
Giaretti
W
W
D000911:Antibodies, Monoclonal; D002453:Cell Cycle; D002843:Chromatin / Q000648:ultrastructure*; D004273:DNA, Neoplasm / Q000032:analysis; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002843:Chromatin; D004273:DNA, Neoplasm; D009687:Nuclear Proteins