Poly-ADP-ribosylation is a post-translational modification catalyzed by PARP enzymes with roles in transcription and chromatin biology. Here we show that distinct macrodomains, including those of histone macroH2A1.1, are recruited to sites of PARP1 activation induced by laser-generated DNA damage. Chemical PARP1 inhibitors, PARP1 knockdown and mutation of ADP-ribose-binding residues in macroH2A1.1 abrogate macrodomain recruitment. Notably, histone macroH2A1.1 senses PARP1 activation, transiently compacts chromatin, reduces the recruitment of DNA damage factor Ku70-Ku80 and alters gamma-H2AX patterns, whereas the splice variant macroH2A1.2, which is deficient in poly-ADP-ribose binding, does not mediate chromatin rearrangements upon PARP1 activation. The structure of the macroH2A1.1 macrodomain in complex with ADP-ribose establishes a poly-ADP-ribose cap-binding function and reveals conformational changes in the macrodomain upon ligand binding. We thus identify macrodomains as modules that directly sense PARP activation in vivo and establish macroH2A histones as dynamic regulators of chromatin plasticity.

Argonaute (Ago) proteins mediate silencing of nucleic acid targets by small RNAs. In fission yeast, Ago1, Tas3 and Chp1 assemble into a RITS complex, which silences transcription near centromeres. Here we describe a repetitive motif within Tas3, termed the 'Argonaute hook', that is conserved from yeast to humans and binds Ago proteins through their PIWI domains in vitro and in vivo. Site-directed mutation of key residues in the motif disrupts Ago binding and heterochromatic silencing in vivo. Unexpectedly, a PIWI domain pocket that binds the 5' end of the short interfering RNA guide strand is required for direct binding of the Ago hook. Moreover, wild-type but not mutant Ago hook peptides derepress microRNA-mediated translational silencing of a target messenger RNA. Proteins containing the conserved Ago hook may thus be important regulatory components of effector complexes in RNA interference.

Mono-ADP-ribosylation is a reversible posttranslational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. For the latter, both the enzymes and their targets have largely remained elusive, mainly due to the lack of specific techniques to study this reaction. The recent discovery of the macro domain, a protein module that interacts selectively with ADP-ribose, prompted us to investigate whether this interaction can be extended to the identification of ADP-ribosylated proteins. Here, we report that macro domains can indeed be used as selective baits for high-affinity purification of mono-ADP-ribosylated proteins, which can then be identified by mass spectrometry. Using this approach, we have identified a series of cellular targets of ADP-ribosylation reactions catalyzed by cellular ADP-ribosyltransferases and toxins. These proteins include most of the known targets of ADP-ribosylation, indicating the validity of this method, and a large number of other proteins, which now need to be individually validated. This represents an important step toward the discovery of new ADP-ribosyltransferase targets and an understanding of the physiological role and the pharmacological potential of this protein modification.ADP-ribosylation ͉ ADP-ribosyltransferase ͉ NAD ͉ toxin ͉ posttranslational modification

The macro module is a globular protein domain of about 25 kDa that is evolutionarily conserved in organisms from viruses, bacteria, yeast to humans. It is generally part of proteins that have wide-ranging (and yet to be discovered) cellular functions. There are several examples of macro domains associated with modules showing homology to poly-ADP-ribosyl-polymerases. Many macro domains, including those of the human histone macroH2A1.1, bind NAD metabolites such as ADP-ribose, suggesting that macro domains may function in the recognition of this and related molecules. The presence of a metabolite-binding function in a repressive chromatin component opens new potential connections between chromosome structure, gene silencing and cellular metabolism. Current evidence suggests that macro domains also represent a novel tool for studying NAD metabolites and may be an attractive drug target for the treatment of diseases.