Chromatin immunofractionation

Fig. lA-D. Immunofractionation of chromatin digested by micrococcal nuclease for various extents of time on an anti-poly(ADP-ribose) Sepharose column. HeLa nuclei from 10 cells were digested with micrococcal nuclease [50 units/10 nuclei at 37°C for 1 min (A), 5 min (B), 10 min (C), and 20 min (D)]. Soluble chromatin was subsequently prepared and immunofractionated on an anti-poly(ADP-ribose) Sepharose column. The columns were loaded and washed with phosphate buffered saline. Transmittance at 254 nm was continuously monitored. When no further UV-absorbing material was eluted, 6 M guanidinium hydrochloride was added to elute-bound material. The insets in each panel represent 1/50 of the DNA extracted from the pooled unbound U) and bound (B) peak fractions subjected to electrophoresis on 1.5% agarose. The gel was stained with ethidium bromide. (Taken from [10])... 

In the data shown in Fig. 1, soluble chromatin was applied to a poly(ADP-ribose) antibody column in PBS, and washed extensively. In earlier studies, nucleosomes were labeled in vitro with NAD prior to immunofractionation. In the experiments described [1-3], no in vitro synthesis of exogenous poly(ADP-ribosylation) was performed and absorption to the antibody column relied upon the presence of in vivo synthesized poly(ADP-ribose) at the sites in chromatin where it presumably exists naturally. This laboratory has recently shown the natural presence of 15 units of poly(ADP-ribose) attached to histone HI isolated from intact, unlabeled cells using the same antibody [5]. 

Fig. 2A-D. Dot-blot and Southern blot analyses of immunofractionated chromatin. The DNA extracted from immunofractionated chromatin was subjected to electrophoresis on 1.5% agarose and blotted to nitrocellulose by the method of Southern or spotted directly to nitrocellulose. The membranes were probed for active (A, B) and inactive gene (C, D) sequences. Unfractionated total T), unbound (U), and bound B) antibody column fractions are shown for DNA derived from chromatin digested for 2.5 (A, C) and 20 min (B, D) with micrococcal nuclease. (Taken from [10])...

Different approaches were taken to obtain a representative view of the relative abundance of active and inactive genes in poly(ADP-ribosylated) and unmodified chromatin using dot-blot hybridization analysis of DNA derived from bound or unbound material [10]. In one approach, immunofractionated nucleosomes were obtained from either short or long digests with micrococal nuclease. No in vitro poly(ADP-ribosylation) was performed with these nucleosomal preparations prior to immunofractionation. 

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