The participation of poly(ADP-ribosyl)ated histone H1 in oligonucleosomal condensation.

The chromatin-associated enzyme poly(ADP-Rib) polymerase causes an NAD-dependent crosslinking of modified oligonucleosomes, as demonstrated by electrophoretic and sedimentation analysis [Butt, T. R. and Smulson, M. (1980) Biochemistry, 19, 5235-5242]. It was speculated that poly(ADP-ribosyl)ation of histone H1 and subsequent formation through crosslinking to an H1 dimer may be an important component of this phenomenon. To study this process, a method of complexing histone H1 to chromatin was required that promoted the restoration of accurate poly(ADP-ribosyl)ation of this histone. Previously we have established that two histone H1 molecules are crosslinked by a chain of poly(ADP-Rib) 15 or 16 units in length. In the current study, we made use of the ability of oligonucleosomes, reconstituted with H1, to carry out the synthesis of the poly(ADP-Rib)-H1 complex in order to monitor the accuracy of reconstitution. It appears that a specific distance and juxtaposition of adjacent H1 molecules along the polynucleosome fiber is required for the enzymatic synthesis of this modified histone complex. We established that a controlled trypsin digestion of oligonucleosomes removed H1 histone with minimal perturbation of other nuclear proteins associated with chromatin. In addition, poly(ADP-Rib) polymerase was partially removed from chromatin by this procedure. Subsequently, methods utilizing gradient salt dialysis have been employed to reconstitute both the polymerase and histone H1 to the depleted oligonucleosomes. The reassociation of H1 (and polymerase) to specific binding sites within oligonucleosomes was accomplished by the above procedures. Poly(ADP-Rib)--H1-dimer synthesis was not observed in depleted oligonucleosomes, but this capacity was found to be partially restored in the reconstituted chromatin. Similarly, the ability of NAD to promote crosslinking of nucleosomes was restored in the reconstituted samples. These results provide a basis for further studies on how the poly(ADP-ribosyl)ation of histones alters the structure of chromatin.