Chromatin ribosylation

The four core histones, H2A, H2B, H3, H4 and their variants, and the linker histone HI subtypes are susceptible to a wide range of post-synthetic modifications, including acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation (Figs. 1 and 2). In this chapter, the four latter modifications and their functions in chromatin structure and function are presented. 

Histone ADP-ribosylation was first reported in 1968 [290]. Poly(ADP-ribosylation) has been implicated in several nuclear processes, including DNA replication, repair and recombination [291-294]. Histone HI and the four core histones are modified by adenosine diphospho (ADP) ribosylation which involves the transfer of the ADP-ribose moiety of NAD" " to the histone acceptor (Figs. 1 and 2). HI is the principle poly(ADP-ribosylated) histone, while core histones are ADP-ribosylated to a minor extent [295-297]. HI is modified at Glu residues 2, 14 (or 15), and 116 (or 117) and at Lys located at the C-terminus [25,298,299]. Poly(ADP-ribosylated) HI is associated with dynamically acetylated core histones [295]. There is conflicting results as to whether poly(ADP-ribosylation) of HI promotes chromatin decondensation [300-304]. 

The enzyme catalyzing the addition of ADP-ribose units onto the histones and itself is poly(ADP-ribose) polymerase or synthetase. Poly(ADP-ribose) polymerase is a nuclear, DNA-dependent enzyme that is stimulated by DNA breaks [302]. This property of the enzyme would target its action to sites that have DNA strand breaks (regions of the genome involved in replication, repair, recombination). The enzyme is associated with chromatin areas and perichromatin regions in interphase Chinese hamster ovary cells [312]. Degradation of the ADP-ribose polymer is catalyzed by the nuclear enzyme poly(ADP-ribose) glycohydrolase and ADP-ribosyl protein lyase. 

As described above, histones are much more than passive structural players within chromatin. Dynamic post-translational modifications of these proteins confer specialized chemical proprieties to chromatin of both informational and structural nature with important functional implications. The highly conserved sites for acetylation, methylation, phosphorylation, ADP-ribosylation, and ubiquitination events on histone tails appear to orchestrate functional activities that range from transcriptional activation and repression to DNA repair and recombination. 

Chromatin fibers from control or poly(ADP-ribosyljated CE nuclei In vitro poly(ADP-ribosyl)ated fibers... 

Poly(ADP-ribosyl)ation induces decondensation of chromatin structure which remains significantly decondensed even in the presence of Mg ions ... 

Each type of histone has variant forms, because certain amino acid side chains are enzymatically modified by methylation, ADP-ribosylation, phosphorylation, gly-cosylation, or acetylation. Such modifications affect the net electric charge, shape, and other properties of histones, as well as the structural and functional properties of the chromatin, and they play a role in the regulation of transcription (Chapter 28). 

Figure 7.4 Activation of PARP-1 by DNA breaks. PARP-1 is composed of three domains, DNA binding, automodification and catalytic (NAD+ binding) domains (1). In cells, PARP-1 localizes to nucleoli and actively transcribed regions of chromatin by interacting with RNA. When PARP-1 binds to DNA breaks, PARP-1 initiates the poly(ADP-ribosyl)ation reaction by using NAD+ as its substrate (2). PARP-1 itself is the main target of the poly(ADP-ribosyl)ation reaction. ADP-ribose polymers are formed on the automodification domain of PARP-1 (automodification). As a consequence of automodification, PARP-1 dissociates from DNA breaks (3). When cells are committed to apoptosis, PARP-1 is specifically cleaved by an apoptosisspecilic protease, caspase-3, resulting in the formation of a 24kDa N-terminal and 89 kDa C-terminal fragments (4). (see Color Plate 7)...

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. 

Poly(ADP-ribose) Polymerase Structure and Molecular Cloning of the Gene ADP-ribosylation and Chromatin Structure... 

Poly(ADP-ribose) polymerase is activated by DNA strand breaks and this property is central to its regulatory role in cell metabolism. The enzyme is tightly bound to chromatin where it ADP-ribosylates chromosomal proteins including histones, nonhistones, and itself (automodification), and its activity influences and is influenced by chromatin structural changes. These properties point to a funda-... 

Figure 3. Proposed pleiotropic functions carried out by nuclear ADP-ribosylation reactions. Events such as cellular proliferation, differentiation, transformation, and DNA damage caused by external agents (e.g., ionizing radiation, drugs) involve changes in the integrity of DNA and/or chromatin architecture (a) which activate the poly(ADP-ribose) polymerase to catalyze the ADP-ribosylation of nuclear proteins predominantly at the expense of cytoplasmic NAD (b). The consequences of protein ADP-ribosylation are a decrease in cellular NAD content, alterations in chromatin structure, and possibly also the activity of various enzymes involved in chromatin function (c). This tripartite system operates, either wholly or partly, to ameliorate the activation of the polymerase by modulating the repair of DNA strand breaks, thereby affecting those processes which initially triggered the activation of the enzyme (d). Pr, protein NAm, nicotinamide (ADPR) , poly(ADP-ribose). (From Gaal and Pearson, 1986).

Figure 5. Poly(ADP-ribosyl)ation-dependent histone shuttle on DNA. Step (1) shows a poly(ADP-ribose) polymerase molecule bound to chromatin. Step (2) auto(ADP-ri-bosyDation of the polymerase attracts histones from the DNA so they become noncovalently bound to the polymeric ADP-ribose chains attached to the polymerase. Step (3) indicates an extreme case where the local DNA has been completely denuded of histones. Step (4) upon degradation of the poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase the histones reassociate with the DNA. (From Realini and Althaus, 1992).

It now becomes easier to envisage how nuclear ADP-ribosylation can influence so many major nuclear processes. Any initial relaxation of chromatin structure caused by DNA strand breaks would be further emphasized by the ensuing ADP-ribosylation of the activated polymerase. This would allow access to other enzymes and regulatory molecules involved, for example, in DNA repair, cell differentiation, gene expression, etc. 

PARP-1 is a signaling enzyme that transfers ADP-ribose groups from NAD to itself and other nuclear proteins in response to detecting DNA damage. Poly(ADP)ribosylation of histones has been shown to loosen chromatin (de Murcia et al, 1986), indicating that damage detection by PARP-1 leads to increased access to the site of damage for other repair... 

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