Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis.

Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

According to the histone code hypothesis, histone variants and modified histones provide binding sites for proteins that change the chromatin state to either active or repressed. Here, we identify histone variants that regulate the targeting and enzymatic activity of poly(ADP-ribose) polymerase 1 (PARP1), a chromatin regulator in higher eukaryotes. We demonstrate that PARP1 is targeted to chromatin by association with the histone H2A variant (H2Av)-the Drosophila homolog of the mammalian histone H2A variants H2Az/H2Ax-and that subsequent phosphorylation of H2Av leads to PARP1 activation. This two-step mechanism of PARP1 activation controls transcription at specific loci in a signal-dependent manner. Our study establishes the mechanism through which histone variants and changes in the histone modification code control chromatin-directed PARP1 activity and the transcriptional activation of target genes.poly(ADP-ribosyl)ation | poly(ADP-ribose) glycohydrolase | nucleosome | Hsp70

Poly(ADP ribose) polymerase 1 (PARP1) is a nuclear protein that regulates chromatin remodeling and transcription as well as DNA repair and genome stability pathways. Recent studies have revealed a paradoxical dual role of PARP1 protein in transcription. Specifically, although PARP1 controls transcriptional activation of a subset of genes that are heat shock-or hormone-dependent, it also directly inactivates transcription, establishes heterochromatin domains, and silences retrotransposable elements. However, the domains required for these disparate functions are currently unknown. In this paper, we report the discovery of a previously undescribed mutation in the Drosophila Parp locus. We show that the mutants express a deletion mutant of PARP1 protein with an altered DNA binding domain that carries only the second Znfinger. We demonstrate that this alteration specifically excludes PARP1 protein from heterochromatin and makes PARP1 unable to maintain repression of retrotransposable elements. By characterizing the biological activity of this unique PARP1 mutant protein isoform, we have uncoupled the transactivation and transrepression functions of this protein.chromatin | poly(ADP ribose) polymerase | transcription P oly(ADP ribose) polymerase 1 (PARP1) protein has been known for decades as a nuclear protein that recognizes and binds nicks and ends of DNA and catalyses poly(ADP ribose) (pADPr) synthesis (1). The basic enzymatic reactions catalyzed by PARP1 involve transferring ADPr from nicotinamide-adenine dinucleotide to either a protein acceptor or an existing pADPr chain, the average length of which is 80 or more residues (2). PARP1 protein can modify numerous chromatin proteins in vivo and in vitro (3). A key role of PARP1 was shown in DNA repair and apoptosis (3), where PARP works as a trigger between the DNA repair (4) and apoptotic pathways (5). PARP1 enzymatic activity has also been shown to be required for normal assembly of higher order chromatin structures and for transcriptional activation (6). Moreover, it has been shown that PARP1 regulates the transcription of these genes by inducing chromatin loosening at targeted genetic loci (6, 7). Finally, PARP1 establishes silent chromatin domains and represses retrotransposable elements (8).The characterization of deletion mutants of PARP that distinguish among the varied functions of this protein is essential to establish a more complete understanding of PARP1 protein biology. At present, however, we have identified neither the mechanism of PARP protein targeting to specific chromatin domains nor the mechanism of local PARP activation. Closing these gaps in our current knowledge is complicated because the presence of 18 paralogous PARP proteins (9) in mammals most likely results in corresponding functional redundancies. The Drosophila genome (8, 10, 11) encodes only a single nuclear PARP (PARP1), making this animal an invaluable model system for the study of PARP functions.The PARP1 protein has three functionally defined domains conserved from human to Droso...

The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events. INTRODUCTIONAs points of structural linkage between the extracellular matrix (ECM) and the intracellular cytoskeleton, focal adhesions possess a complex function. For example, during migration, cells must rapidly break down and reform adhesions with the ECM, providing force for propulsion (Lauffenburger and Horwitz, 1996). At mitotic entry, cultured cells round up and decrease adhesion to the ECM; at mitotic exit, basal attachments reassemble and contribute to the force generation required for efficient progress through cytokinesis and reentry into G 1 . In interphase cells, the formation of novel focal adhesion-ECM interactions can specify cellular differentiation by activating specific signaling cascades culminating in the induction of differentiationpromoting transcription factors, and in parallel enforce removal from the cell cycle (Boudreau and Bissell, 1998). In many cell types, sustained loss of adhesion is a sufficient stimulus to induce apoptosis (anoikis) (Frisch and Francis, 1994), a surveillance mechanism against cancer, inhibiting the formation of micrometastases. Hence, one frequent effect of oncogenic transformation is the circumvention of the adhesion-viability coupling, leading to acquisition by cancer cells of the ability to grow in an anchorage-independent manner (Schwartz, 1997). Based on these diverse biological roles, there has been considerable research effort directed at elucidating the signaling role of focal adhesion-associated proteins (Schlaepfer et al., 1999).HEF1, p130Cas, and Efs/Sin define the Cas family of proteins Bouton et al., 2001). In interphase cells, Cas proteins predominantly localize to focal adhesions. During initial integrin engagement, induced by cell a...