Homology-dependent gene silencing was shown to be used to inhibit Stellate gene expression in the D. melanogaster germline, ensuring male fertility. dsRNA-mediated silencing may provide a basis for negative autogenous control of gene expression. The related surveillance system is implicated to control expression of retrotransposons in the germline.

Steroid response and stress-activated genes such as hsp70 undergo puffing in Drosophila larval salivary glands, a local loosening of polytene chromatin structure associated with gene induction. We find that puffs acquire elevated levels of adenosine diphosphate (ADP)-ribose modified proteins and that poly(ADP)-ribose polymerase (PARP) is required to produce normal-sized puffs and normal amounts of Hsp70 after heat exposure. We propose that chromosomal PARP molecules become activated by developmental or environmental cues and strip nearby chromatin proteins off DNA to generate a puff. Such local loosening may facilitate transcription and may transiently make protein complexes more accessible to modification, promoting chromatin remodeling during development.

Poly(ADP-ribose) polymerase (PARP) is a major NAD-dependent modifying enzyme that mediates important steps in DNA repair, transcription, and apoptosis, but its role during development is poorly understood. We found that a single Drosophila Parp gene spans more than 150 kb of transposon-rich centromeric heterochromatin and produces several differentially spliced transcripts, including a novel isoform, PARP-e, predicted to encode a protein lacking enzymatic activity. An insertion mutation near the upstream promoter for Parp-e disrupts all Parp expression. Heterochromatic but not euchromatic sequences become hypersensitive to micrococcal nuclease, nucleoli fail to form, and transcript levels of the copia retrotransposon are elevated more than 50-fold; the variegated expression of certain transgenes is dominantly enhanced. Larval lethality can be rescued and PARP activity restored by expressing a cDNA encoding PARP-e. We propose that PARP-e autoregulates Parp transcription by influencing the chromatin structure of its heterochromatic environment. Our results indicate that Parp plays a fundamental role organizing the structure of Drosophila chromatin.

FoxA proteins are pioneer transcription factors, among the first to bind chromatin domains in development and enable gene activity. The Fox DNA-binding domain structurally resembles linker histone and binds nucleosomes stably. Using fluorescence recovery after photobleaching, we found that FoxA1 and FoxA2 move much more slowly in nuclei than other transcription factor types, including c-Myc, GATA-4, NF-1, and HMGB1. We find that slower nuclear mobility correlates with high nonspecific nucleosome binding, and point mutations that disrupt nonspecific binding markedly increase nuclear mobility. FoxA's distinct nuclear mobility is consistent with its pioneer activity in chromatin.Supplemental material is available at http://www.genesdev.org.

Poly(ADP-ribose) polymerase 1 protein (PARP1) mediates chromatin loosening and activates the transcription of inducible genes, but the mechanism of PARP1 regulation in chromatin is poorly understood. We have found that PARP1 interaction with chromatin is dynamic and that PARP1 is exchanged continuously between chromatin and nucleoplasm, as well as between chromatin domains. Specifically, the PARP1 protein preferentially interacts with nucleosomal particles, and although the nucleosomal linker DNA is not necessary for this interaction, we have shown that the core histones, H3 and H4, are critical for PARP1 binding. We have also demonstrated that the histones H3 and H4 interact preferentially with the C-terminal portion of PARP1 protein and that the N-terminal domain of PARP1 negatively regulates these interactions. Finally, we have found that interaction with the N-terminal tail of the H4 histone triggers PARP1 enzymatic activity. Therefore, our data collectively suggests a model in which both the regulation of PARP1 protein binding to chromatin and the enzymatic activation of PARP1 protein depend on the dynamics of nucleosomal core histone mediation.Eukaryotic chromatin organization involves the fundamental nucleosomal unit, which consists of four core histones plus a linker histone (1). Recently, it has been shown that the activity of transcription complexes at nucleosomes is regulated by the PARP1 3 protein (2, 3). Notwithstanding these findings, major gaps in our present understanding exist that involve the mechanism by which the PARP1 protein binds to specific chromatin domains and the mechanism by which the local PARP1 protein is activated in response to developmental and environmental stimuli.After histones, PARP1 is the most abundant nuclear protein (4). The distribution of PARP1 in chromatin is broad and occurs in regions characterized by distinct cell types (2, 3, 5). Nevertheless, exactly how the PARP1 enzyme interacts with chromatin in vivo has not been thoroughly investigated, and the molecular basis for PARP1 binding to chromatin remains poorly understood. Although zinc fingers within the PARP1 protein contribute to DNA binding in vitro, they specifically recognize damaged DNA (6) and therefore do not contribute to the association of PARP1 with intact chromatin. Moreover, a PARP1 paralog, PARP2, that has no zinc fingers and no direct DNA binding capability, nevertheless exhibits a pattern of chromatin association similar to PARP1 and is able to partially complement PARP1 functions in a PARP1 null mutant (7-9). This suggests that PARP1 and PARP2 both bind chromatin indirectly, through an interaction with one or more DNA-binding proteins.A key aim of this study is to determine the specific mechanisms by which PARP1 protein associates with chromatin in vivo. Considerable evidence now suggests that PARP1 interacts with chromatin by binding to histones (10). For example, histones H1, H2A, and H2B are efficient targets for PARP1 binding in vitro (11) and are enzymatically modified by . This idea is, howev...

The biological functions of poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) are not well understood. However, it is known that hnRNPs are involved in the regulation of alternative splicing for many genes, including the Ddc gene in Drosophila. Therefore, we first confirmed that poly(ADP-ribose) (pADPr) interacts with two Drosophila hnRNPs, Squid/hrp40 and Hrb98DE/hrp38, and that this function is regulated by Poly(ADP-ribose) Polymerase 1 (PARP1) and Poly(ADP-ribose) Glycohydrolase (PARG) in vivo. These findings then provided a basis for analyzing the role of pADPr binding to these two hnRNPs in terms of alternative splicing regulation. Our results showed that Parg null mutation does cause poly(ADP-ribosyl)ation of Squid and hrp38 protein, as well as their dissociation from active chromatin. Our data also indicated that pADPr binding to hnRNPs inhibits the RNA-binding ability of hnRNPs. Following that, we demonstrated that poly(ADP-ribosyl)ation of Squid and hrp38 proteins inhibits splicing of the intron in the Hsrω-RC transcript, but enhances splicing of the intron in the Ddc pre-mRNA. Taken together, these findings suggest that poly(ADP-ribosyl)ation regulates the interaction between hnRNPs and RNA and thus modulates the splicing pathways.

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.

Cell growth and differentiation during developmental processes require the activation of many inducible genes. However, eukaryotic chromatin, which consists of DNA and histones, becomes a natural barrier impeding access to the functional transcription machinery. To break through the chromatin barrier, eukaryotic organisms have evolved the strategy of using Poly(ADP-ribose) polymerase 1 (PARP1) to modulate chromatin structure and initiate the steps leading to gene expression control. As a structural protein in chromatin, enzymatically silent PARP1 inhibits transcription by contributing to the condensation of chromatin, which creates a barrier against gene transcription. However, once activated by environmental stimuli and developmental signals, PARP1 can modify itself and other chromatin-associated proteins, thereby loosening chromatin to facilitate gene transcription. Here we discuss the roles of PARP1 in transcriptional control during development.