Polynucleosomes relaxation

Niedergang C, Ittel ME, de Murcia G, Pouyet J, Mandel P (1985) Kinetics of nucleosomal histone HI hyper (ADP-ribosyl)ation and polynucleosomes relaxation. This volume... 

Kinetics of Nucleosomal Histone HI Hyper(ADP-Ribosylation) and Polynucleosomes Relaxation... 

It appears from our investigations that the polynucleosome relaxation process is closely correlated with the generation of ADP-ribosylated forms of histone HI. The kinetics as followed by histone HI modification, electron microscopic visualization. 

Poirer, G.G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C. and Mandel, P. (1982) Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure. Proc. Natl. Acad. Sci. USA, 79, 3423-3427. 

When rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADPR) polymerase and examined by electron microscopy a relaxation of their native zigzag structure was observed, even at high ionic strengths they showed a close resemblance to chromatin depleted of histones HI. The relaxed state of poly(ADP-ribosylated) polynucleosomes was also confirmed by sedimentation velocity analysis [19, 20]. Locally relaxed regions can also be generated within poly-nucleosome chains by the activity of their intrinsic poly(ADPR) polymerase and appeared to be correlated with the formation of hyper(ADP-ribosylated) forms of histone HI and an increase of DNA polymerase activity [21]. The posttranslational transitory modifications of histones are potential modulators of chromatin stmcture. This may be involved in DNA transcription, replication, and repair. 

Poirier GG, De Murda G, Jongstra-Bilen J, Niedergang C, Mandel P (1982) Poly(ADP-ribosyla-tion) of polynucleosomes causes relaxation of chromatin structure. Proc Natl Acad Sd USA 79 3423-3427... 

Concomitantly, structures resulting from the poly(ADP-ribosyl)ation of native chromatin, chromatin-Hl and core particles were examined by electron microscopy. We found, as described earlier for pancreatic chromatin [6, 7], that calf thymus chromatin adopts a more relaxed conformation upon poly(ADP-ribosyl)ation by purified calf thymus poly(ADP-ribose) polymerase free of its DNA (Fig. 3a,d). It was also found that this chromatin exhibited a lower sedimentation velocity as compared to control chromatin [6]. And recently, it has been shown that DNA polymerase a activity is more than twofold higher in the presence of pancreatic polynucleosomes ADP-ribosylated as compared to control polynucleosomes [16]. In striking contrast, no ultrastructural effect was observed when chromatin depleted of histone HI was poly(ADP-ribosyl)ated (Fig. 3b, e). 

Poly(ADP-Ribose) Glycohydrolase Activity Causes Recondensation of Relaxed Poly(ADP-Ribosyl)ated Polynucleosomes... 

Previous studies have shown that at NAD concentrations above 10 jtxM, histone HI is the major histone acceptor of ADP-ribose in pancreatic nucleosomes [11, 12]. Burzio et al. [13] have also observed that the ADP-ribosylation of histone HI decreases its affinity for DNA. Furthermore, it was recently shown that under conditions of extensive ADP-ribosylation, using exogenous purified poly(ADP-ribose) polymerase and 1 mM NAD, the polynucleosomal architecture is fully relaxed [4]. Histone HI has been found to be the main histone acceptor of ADP-ribose and to be modified as hyper(ADP-ribosylated) forms [4]. The mechanism of the relaxation process is not known, but it has been shown with the endogenous poly(ADP-ribose) polymerase to be dependent on the concentration of NAD and correlated with the hyper(ADP-ribosylation) of histone HI [5]. 

In this paper we investigate further the correlation between histone HI ADP-ribosylation and chromatin relaxation by performing a time course study. Polynucleosomes which have been poly(ADP-ribosylated) using the purified calf thymus enzyme, and histone HI modifications as well as changes in the polynucleosomes morphology have been analyzed at different time intervals. The template capacity of the poly(ADP-ribosylated) nucleosomes has also been determined along the ADP-ribosylation time course. 

The concentration of 200 iM NAD was chosen in order to slow down the reaction rate. Indeed, polynucleosomes ADP-ribosylated with 1 mM NAD were almost fully relaxed after 3 min [4] and histone HI appeared hyper(ADP-ribosylated) in less than 1 min. At a NAD concentration of 200 iM, incorporation of radioactivity into ADP-ribose was roughly linear for up to 15 min of reaction time. 

The time course of ADP-ribosylation of polynucleosomes in the presence of 200 ijM NAD is shown in Fig. 2A. Control nucleosomes adopt a native, condensed conformation and as the ADP-ribosylation reaction proceeds, individual nucleosomes coimected by DNA filaments begin to be visible either at the extremities or within the poly-nucleosomal chains. This decondensation phenomenon is rapidly amplified leading to an almost fully relaxed conformation for 10 to 15 min. It is noteworthy that the relaxation appears complete at the same time as the HI complex of poly(ADP-ribosylated) histone HI becomes predominant. 

Sedimentation coefficient determinations [4,21] on polynucleosomes ADP-ribosylated in the presence of 200 fjM NAD for different time intervals, gave values of S20 w ing from 51.5 0.4 S for control nucleosomes to 44.9 0.2 S for fully relaxed nucleosomes (Fig. 3). The time course was in good agreement with the relaxation process visualized by electron microscopy. 

In order to investigate the modification of DNA accessibility in chromatin structures relaxed by ADP-ribosylation, the DNA polymerase a activity has been determined [22]. As shown in Table 1, this activity is more than twofold higher in polynucleosomes ADP-ribosylated for 25 min than in control polynucleosomes. Moreover, this increase in template capacity is correlated with the ADP-ribosylation induced relaxation time course as demonstrated by electron microscopy and sedimentation velocity analysis. 

However, the ADP-ribosylation in vivo of other chromatin components, histones [27, 28], or nonhistone proteins [29], may also contribute to the relaxation process. Histone H2B has been shown to be ADP-ribosylated in isolated polynucleosomes at very low NAD concentrations [27] and also in hepatoma cells in vivo [28]. But, in contrast to HI, the interactions of H2B with DNA remain unchanged after ADP-ribosylation [13]. 

---