Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly(ADP-ribosyl)ation.

Dynamically controlled posttranslational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating poly(ADP-ribose) polymerase-1 (PARP-1). JIL-1 phosphorylates the C terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch, consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch, leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1-dependent genes.

Related papers:

title: NAD+-Dependent Modulation of Chromatin Structure and Transcription by Nucleosome Binding Properties of PARP-1
abstract: PARP-1 is the most abundantly expressed member of a family of proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins. Herein, we describe previously uncharacterized nucleosome binding properties of PARP-1 that promote the formation of compact, transcriptionally repressed chromatin structures. PARP-1 binds in a specific manner to nucleosomes and modulates chromatin structure through NAD+-dependent automodification, without modifying core histones or promoting the disassembly of nucleosomes. The automodification activity of PARP-1 is potently stimulated by nucleosomes, causing the release of PARP-1 from chromatin. The NAD+-dependent activities of PARP-1 are reversed by PARG, a poly(ADP-ribose) glycohydrolase, and are inhibited by ATP. In vivo, PARP-1 incorporation is associated with transcriptionally repressed chromatin domains that are spatially distinct from both histone H1-repressed domains and actively transcribed regions. Thus, PARP-1 functions both as a structural component of chromatin and a modulator of chromatin structure through its intrinsic enzymatic activity.

title: Activator-induced spread of poly(ADP-ribose) polymerase promotes nucleosome loss at Hsp70.
abstract: Eukaryotic cells possess many transcriptionally regulated mechanisms to alleviate the nucleosome barrier. Dramatic changes to the chromatin structure of Drosophila melanogaster Hsp70 gene loci are dependent on the transcriptional activator, heat shock factor (HSF), and poly(ADP-ribose) polymerase (PARP). Here, we find that PARP is associated with the 5' end of Hsp70, and its enzymatic activity is rapidly induced by heat shock. This activation causes PARP to redistribute throughout Hsp70 loci and Poly(ADP-ribose) to concurrently accumulate in the wake of PARP's spread. HSF is necessary for both the activation of PARP's enzymatic activity and its redistribution. Upon heat shock, HSF triggers these PARP activities mechanistically by directing Tip60 acetylation of histone H2A lysine 5 at the 5' end of Hsp70, where inactive PARP resides before heat shock. This acetylation is critical for the activation and spread of PARP as well as for the rapid nucleosome loss over the Hsp70 loci.

title: Chromatin Loosening by Poly(ADP)-Ribose Polymerase (PARP) at Drosophila Puff Loci
abstract: Steroid response and stress-activated genes such ashsp70 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.

title: Reciprocal Binding of PARP-1 and Histone H1 at Promoters Specifies Transcriptional Outcomes
abstract: Nucleosome-binding proteins act to modulate the promoter chromatin architecture and transcription of target genes. We used genomic and gene-specific approaches to show that two such factors, histone H1 and poly(ADP-ribose) polymerase-1 (PARP-1), exhibit a reciprocal pattern of chromatin binding at many RNA polymerase II–transcribed promoters. PARP-1 was enriched and H1 was depleted at these promoters. This pattern of binding was associated with actively transcribed genes. Furthermore, we showed that PARP-1 acts to exclude H1 from a subset of PARP-1–stimulated promoters, suggesting a functional interplay between PARP-1 and H1 at the level of nucleosome binding. Thus, although H1 and PARP-1 have similar nucleosome-binding properties and effects on chromatin structure in vitro, they have distinct roles in determining gene expression outcomes in vivo.

title: Nucleosomal Core Histones Mediate Dynamic Regulation of Poly(ADP-ribose) Polymerase 1 Protein Binding to Chromatin and Induction of Its Enzymatic Activity*
abstract: 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.

title: JIL-1: a novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila.
abstract: We have cloned and characterized JIL-1, a novel tandem kinase in Drosophila that associates with the chromosomes throughout the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost 2-fold on the hypertranscribed male X chromosome compared to autosomes. Phylogenetic analysis suggests that JIL-1 together with human MSKs defines a separate family of tandem kinases. That JIL-1 is a functional kinase was demonstrated by autophosphorylation and phosphorylation of histone H3 in vitro. Based on these findings, we propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure.

title: The JIL-1 Tandem Kinase Mediates Histone H3 Phosphorylation and Is Required for Maintenance of Chromatin Structure in Drosophila
abstract: To analyze the function of the chromosomal kinase JIL-1, we generated an allelic series of hypomorphic and null mutations. JIL-1 is an essential kinase for viability, and reduced levels of JIL-1 kinase activity lead to a global change in chromatin structure. In JIL-1 hypomorphs, euchromatic regions of polytene chromosomes are severely reduced and the chromosome arms condensed. This is correlated with decreased levels of histone H3 Ser10 phosphorylation. These levels can be restored by a JIL-1 transgene placing JIL-1 directly in the pathway mediating histone H3 phosphorylation. We propose a model where JIL-1 kinase activity is required for maintaining the structure of the more open chromatin regions that facilitate gene transcription.

title: Phosphorylation of histone H3 at Ser10 facilitates RNA polymerase II release from promoter-proximal pausing in Drosophila.
abstract: The Drosophila JIL-1 kinase is known to phosphorylate histone H3 at Ser10 (H3S10) during interphase. This modification is associated with transcriptional activation, but its function is not well understood. Here we present evidence suggesting that JIl-1-mediated H3S10 phosphorylation is dependent on chromatin remodeling by the brahma complex and is required during early transcription elongation to release RNA polymerase II (Pol II) from promoter-proximal pausing. JIL-1 localizes to transcriptionally active regions and is required for activation of the E75A ecdysone-responsive and hsp70 heat-shock genes. The heat-shock transcription factor, the promoter-paused form of Pol II (Pol IIo(ser5)), and the pausing factor DSIF (DRB sensitivity-inducing factor) are still present at the hsp70 loci in JIL-1-null mutants, whereas levels of the elongating form of Pol II (Pol IIo(ser2)) and the P-TEFb kinase are dramatically reduced. These observations suggest that phosphorylation of H3S10 takes place after transcription initiation but prior to recruitment of P-TEFb and productive elongation. Western analyses of global levels of both forms of Pol II further suggest that JIL-1 plays a general role in early elongation of a broad range of genes. Taken together, the results introduce H3S10 phosphorylation by JIL-1 as a hallmark of early transcription elongation in Drosophila.

title: Phosphorylation of histone H3 correlates with transcriptionally active loci.
abstract: Posttranslational modifications of the N-terminal tails of the core histones within the nucleosome particle are thought to act as signals from the chromatin to the cell for various processes. The experiments presented here show that the acetylation of histones H3 and H4 in polytene chromosomes does not change during heat shock. In contrast, the global level of phosphorylated H3 decreased dramatically during a heat shock, with an observed increase in H3 phosphorylation at the heat shock loci. Additional experiments confirm that this change in phosphorylated H3 distribution is dependent on functional heat shock transcription factor activity. These experiments suggest that H3 phosphorylation has an important role in the induction of transcription during the heat shock response.

title: Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila
abstract: Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a `kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the `kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.

title: Drosophila Aurora B Kinase Is Required for Histone H3 Phosphorylation and Condensin Recruitment during Chromosome Condensation and to Organize the Central Spindle during Cytokinesis
abstract: Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila (Glover, D.M., M.H. Leibowitz, D.A. McLean, and H. Parry.  We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, M.A., A.V. Philp, D.M. Glover, and H.J. Bellen. . The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.

title: Flytrap, a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster
abstract: Flytrap is a web-enabled relational database of transposable element insertions in Drosophila melanogaster. A green fluorescent protein (GFP) artificial exon carried by a transposable P-element is mobilized and inserted into a host gene intron creating a GFP fusion protein. The sequence of the tagged gene is determined by sequencing inverse-PCR products derived from genomic DNA. Flytrap contains two principle data types: micrographs of protein localization and a cellular component ontology, based on rules derived from the Gene Ontology consortium (http://www.geneontology.org), describing protein localization. Flytrap also has links to gene information contained in Flybase (http:// flybase.bio.indiana.edu). The system is designed to accept submissions of micrographs and descriptions from any type of tissue (e.g. wing imaginal disk, ovary) and at any stage of development. Insertion lines can be searched using a number of queries, including Berkeley Drosophila Genome Project (BDGP) numbers and protein localization. In addition, Flytrap provides online order forms linked to each insertion line so that users may request any line generated from this project. Flytrap may be accessed from the homepage at http://flytrap.med.

title: The Dynamic Alterations of H2AX Complex during DNA Repair Detected by a Proteomic Approach Reveal the Critical Roles of Ca2+/Calmodulin in the Ionizing Radiation-induced Cell Cycle Arrest*S
abstract: By using DNA nuclease digestion and a quantitative “dual tagging” proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non-irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca2+-binding proteins were found in the H2AX complex, the roles of Ca2+ were examined. The results indicate that Ca2+/CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.

title: Protein Structure Prediction Using Rosetta
abstract: Publisher Summary This chapter elaborates protein structure prediction using Rosetta. Double-blind assessments of protein structure prediction methods have indicated that the Rosetta algorithm is perhaps the most successful current method for de novo protein structure prediction. In the Rosetta method, short fragments of known proteins are assembled by a Monte Carlo strategy to yield native-like protein conformations. Using only sequence information, successful Rosetta predictions yield models with typical accuracies of 3–6 A˚ Cα root mean square deviation (RMSD) from the experimentally determined structures for contiguous segments of 60 or more residues. For each structure prediction, many short simulations starting from different random seeds are carried out to generate an ensemble of decoy structures that have both favorable local interactions and protein-like global properties. This set is then clustered by structural similarity to identify the broadest free energy minima. The effectiveness of conformation modification operators for energy function optimization is also described in this chapter.

title: PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta
abstract: PyRosetta is a stand-alone Python-based implementation of the Rosetta molecular modeling package that allows users to write custom structure prediction and design algorithms using the major Rosetta sampling and scoring functions. PyRosetta contains Python bindings to libraries that define Rosetta functions including those for accessing and manipulating protein structure, calculating energies and running Monte Carlo-based simulations. PyRosetta can be used in two ways: (i) interactively, using iPython and (ii) script-based, using Python scripting. Interactive mode contains a number of help features and is ideal for beginners while script-mode is best suited for algorithm development. PyRosetta has similar computational performance to Rosetta, can be easily scaled up for cluster applications and has been implemented for algorithms demonstrating protein docking, protein folding, loop modeling and design.


AVAILABILITY
PyRosetta is a stand-alone package available at http://www.pyrosetta.org under the Rosetta license which is free for academic and non-profit users. A tutorial, user's manual and sample scripts demonstrating usage are also available on the web site.

title: Increasing the precision of comparative models with YASARA NOVA—a self‐parameterizing force field
abstract: One of the conclusions drawn at the CASP4 meeting in Asilomar was that applying various force fields during refinement of template‐based models tends to move predictions in the wrong direction, away from the experimentally determined coordinates. We have derived an all‐atom force field aimed at protein and nucleotide optimization in vacuo (NOVA), which has been specifically designed to avoid this problem. NOVA resembles common molecular dynamics force fields but has been automatically parameterized with two major goals: (i) not to make high resolution X‐ray structures worse and (ii) to improve homology models built by WHAT IF. Force‐field parameters were not required to be physically correct; instead, they were optimized with random Monte Carlo moves in force‐field parameter space, each one evaluated by simulated annealing runs of a 50‐protein optimization set. Errors inherent to the approximate force‐field equation could thus be canceled by errors in force‐field parameters. Compared with the optimization set, the force field did equally well on an independent validation set and is shown to move in silico models closer to reality. It can be applied to modeling applications as well as X‐ray and NMR structure refinement. A new method to assign force‐field parameters based on molecular trees is also presented. A NOVA server is freely accessible at http://www.yasara.com/servers Proteins 2002;47:393–402.

title: A charged and contoured surface on the nucleosome regulates chromatin compaction
abstract: Local nucleosome-nucleosome interactions in cis drive chromatin folding, whereas interactions in trans lead to fiber-fiber oligomerization. Here we show that peptides derived from the histone H4 tail and Kaposi's sarcoma herpesvirus LANA protein can replace the endogenous H4 tail, resulting in array folding and oligomerization. Neutralization of a LANA binding site on the histone surface enhanced rather than abolished nucleosome-nucleosome interactions. We maintain that the contoured nucleosome surface is centrally involved in regulating chromatin condensation.

title: Translating the Histone Code
abstract: Chromatin, the physiological template of all eukaryotic genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on histone amino termini, thereby regulating access to the underlying DNA. Distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states. The combinatorial nature of histone amino-terminal modifications thus reveals a “histone code” that considerably extends the information potential of the genetic code. We propose that this epigenetic marking system represents a fundamental regulatory mechanism that has an impact on most, if not all, chromatin-templated processes, with far-reaching consequences for cell fate decisions and both normal and pathological development.

title: Transcriptional regulation in the context of chromatin structure.
abstract: A wide variety of histone-like proteins can be assembled into nucleosomal structures. Core and linker histone variants, proteins of the histone-fold and winged-helix families can all contribute to the local differentiation of functional chromosomal domains. It is very difficult to disrupt core histone interactions within a nucleosome in vivo. Histones H3 and H4 do not exchange out of chromatin outside S-phase. Histones H2A and H2B do exchange out of chromatin, but do so predominantly during transcription. This confers stability on the nucleosome during the cell cycle. Linker histones have a much less stable association with nucleosomal DNA, allowing for reversible activation of transcription. A distinct feature of histone interactions with nucleosomal DNA is the exposure of DNA on the surface of the nucleosome. One side of DNA is occluded on the histone surface, but the other is exposed and potentially accessible to other regulatory proteins. A major contributory factor to the functional specialization of chromatin is the capacity to target nucleosome modification and disruption.

title: The FlyBase database of the Drosophila genome projects and community literature.
abstract: FlyBase (http://flybase.bio.indiana.edu/) provides an integrated view of the fundamental genomic and genetic data on the major genetic model Drosophila melanogaster and related species. FlyBase has primary responsibility for the continual reannotation of the D. melanogaster genome. The ultimate goal of the reannotation effort is to decorate the euchromatic sequence of the genome with as much biological information as is available from the community and from the major genome project centers. A complete revision of the annotations of the now-finished euchromatic genomic sequence has been completed. There are many points of entry to the genome within FlyBase, most notably through maps, gene products and ontologies, structured phenotypic and gene expression data, and anatomy.

title: The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin.
abstract: Activation and repression of transcription in eukaryotes involve changes in the chromatin fiber that can be accomplished by covalent modification of the histone tails or the replacement of the canonical histones with other variants. Here we show that the histone H2A variant of Drosophila melanogaster, H2Av, localizes to the centromeric heterochromatin, and it is recruited to an ectopic heterochromatin site formed by a transgene array. His2Av behaves genetically as a PcG gene and mutations in His2Av suppress position effect variegation (PEV), suggesting that this histone variant is required for euchromatic silencing and heterochromatin formation. His2Av mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric region. H2Av accumulation or histone H4 Lys 12 acetylation is not affected by mutations in Su(var)3-9 or Su(var)2-5. The results suggest an ordered cascade of events leading to the establishment of heterochromatin and requiring the recruitment of the histone H2Av variant followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment can take place.

title: The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development.
abstract: 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.

title: Loss of poly(ADP-ribose) glycohydrolase causes progressive neurodegeneration in Drosophila melanogaster
abstract: Poly(ADP-ribosyl)ation has been suggested to be involved in regulation of DNA repair, transcription, centrosome duplication, and chromosome stability. However, the regulation of degradation of poly(ADP-ribose) and its significance are not well understood. Here we report a loss-of-function mutant Drosophila with regard to poly(ADP-ribose) glycohydrolase, a major hydrolyzing enzyme of poly(ADP-ribose). The mutant lacks the conserved catalytic domain of poly(ADP-ribose) glycohydrolase, and exhibits lethality in the larval stages at the normal development temperature of 25°C. However, one-fourth of the mutants progress to the adult stage at 29°C but showed progressive neurodegeneration with reduced locomotor activity and a short lifespan. In association with this, extensive accumulation of poly(ADP-ribose) could be detected in the central nervous system. These results suggest that poly(ADP-ribose) metabolism is required for maintenance of the normal function of neuronal cells. The phenotypes observed in the parg mutant might be useful to understand neurodegenerative conditions such as the Alzheimer's and Parkinson's diseases that are caused by abnormal accumulation of substances in nervous tissue.

title: GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of drosophila
abstract: We have screened a collection of approximately 400 GAL4 enhancer trap lines for useful patterns of expression in the embryo, larval brain, imaginal discs, and ovary using a UAS‐lacZ reporter construct. Although similar patterns of expression have previously been reported in the original P[lacZ] enhancer trap screens, these lines are useful for directing ectopic expression of genes in discrete patterns during these stages. In addition, we have identified some unique patterns of expression that have not been previously reported.

title: GFP-tagged balancer chromosomes for Drosophila melanogaster
abstract: We constructed green fluorescent protein (GFP)-expressing balancer chromosomes for each of the three major chromosomes of Drosophila melanogaster. Expression of GFP in these chromosomes is driven indirectly by a Kruppel (Kr) promoter, via the yeast GAL4-UAS regulatory system. GFP fluorescence can be seen in embryos as early as the germ band extension stage, and can also be seen in larvae, pupae, and adults. We show the patterns of GFP expression of these balancers and demonstrate the use of the balancers to identify homozygous progeny.

title: Uncoupling of the transactivation and transrepression functions of PARP1 protein
abstract: 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 Zn-finger. 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.

title: DNA double-strand break-induced phosphorylation of Drosophila histone variant H2Av helps prevent radiation-induced apoptosis.
abstract: The response of eukaryotic cells to the formation of a double-strand break (DSB) in chromosomal DNA is highly conserved. One of the earliest responses to DSB formation is phosphorylation of the C-terminal tail of H2A histones located in nucleosomes near the break. Histone variant H2AX and core histone H2A are phosphorylated in mammals and budding yeast, respectively. We demonstrate the DSB-induced phosphorylation of histone variant H2Av in Drosophila melanogaster. H2Av is a member of the H2AZ family of histone variants. Ser137 within an SQ motif located near the C- terminus of H2Av was phosphorylated in response to gamma-irradiation in both tissue culture cells and larvae. Phosphorylation was detected within 1 min of irradiation and detectable after only 0.3 Gy of radiation exposure. Photochemically induced DSBs, but not general oxidative damage or UV-induced nicking of DNA, caused H2Av phosphorylation, suggesting that phosphorylation is DSB specific. Imaginal disc cells from Drosophila expressing a mutant allele of H2Av with its C-terminal tail deleted, and therefore unable to be phosphorylated, were more sensitive to radiation-induced apoptosis than were wildtype controls, suggesting that phosphorylation of H2Av is important for repair of radiation-induced DSBs. These observations suggest that in addition to providing the function of an H2AZ histone, H2Av is also the functional homolog in Drosophila of H2AX.

title: Preparation of nucleosome core particle from recombinant histones.
abstract: Publisher Summary This chapter describes the preparation method of nucleosome core particles (NCPs) from recombinant histones. The ability to make defined NCPs, or arrays of nucleosomes, from histone proteins expressed in bacteria has several advantages over previously used methods using histones isolated from natural sources. The chapter discusses protocols for the overexpression and purification of histones H2A, H2B, H3, and H4, both as full-length proteins and as corresponding trypsin-resistant globular domains. The chapter presents a method for the refolding and purification of a histone octamer from denatured recombinant histone proteins together with a protocol for the assembly and purification of a nucleosome core particle using 146 base pairs of DNA. The purity and homogeneity of the final core-particle preparation are assessed by a high-resolution gel shift assay. The chapter presents a flow chart that describes the procedures involved in the preparation of synthetic nucleosomes.

title: Phosphorylation of SU(VAR)3–9 by the Chromosomal Kinase JIL-1
abstract: The histone methyltransferase SU(VAR)3–9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR)3–9's activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR)3–9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR)3–9 N-terminus. Among several other proteins known to bind Su(VAR)3–9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR)3–9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR)3–9 and JIL-1.

title: Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4.
abstract: The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, we assembled nucleosome arrays containing albumin enhancer sequences and compacted them with linker histone. HNF3 and GATA-4, but not NF-1, C/EBP, and GAL4-AH, bound their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events.

title: Structural Basis for DNA Damage–Dependent Poly(ADP-ribosyl)ation by Human PARP-1
abstract: Dissecting DNA Repair Covalent modification of proteins can be a crucial regulatory event. Poly(ADP-ribose) polymerase 1 (PARP-1) (ADP, adenosine diphosphate) synthesizes poly(ADP-ribose), which is attached to and regulates proteins involved in DNA repair. Langelier et al. (p. 728; see the Perspective by Gagné et al.) use x-ray crystallography and biochemical analysis to demonstrate how PARP-1 detects DNA damage and how the interaction with DNA is coupled to poly(ADP-ribosyl)ation activity. An enzyme that binds to damaged DNA undergoes a structural reorganization that enhances its catalytic activity. Poly(ADP-ribose) polymerase–1 (PARP-1) (ADP, adenosine diphosphate) has a modular domain architecture that couples DNA damage detection to poly(ADP-ribosyl)ation activity through a poorly understood mechanism. Here, we report the crystal structure of a DNA double-strand break in complex with human PARP-1 domains essential for activation (Zn1, Zn3, WGR-CAT). PARP-1 engages DNA as a monomer, and the interaction with DNA damage organizes PARP-1 domains into a collapsed conformation that can explain the strong preference for automodification. The Zn1, Zn3, and WGR domains collectively bind to DNA, forming a network of interdomain contacts that links the DNA damage interface to the catalytic domain (CAT). The DNA damage–induced conformation of PARP-1 results in structural distortions that destabilize the CAT. Our results suggest that an increase in CAT protein dynamics underlies the DNA-dependent activation mechanism of PARP-1.

title: I-TASSER: a unified platform for automated protein structure and function prediction
abstract: The iterative threading assembly refinement (I-TASSER) server is an integrated platform for automated protein structure and function prediction based on the sequence-to-structure-to-function paradigm. Starting from an amino acid sequence, I-TASSER first generates three-dimensional (3D) atomic models from multiple threading alignments and iterative structural assembly simulations. The function of the protein is then inferred by structurally matching the 3D models with other known proteins. The output from a typical server run contains full-length secondary and tertiary structure predictions, and functional annotations on ligand-binding sites, Enzyme Commission numbers and Gene Ontology terms. An estimate of accuracy of the predictions is provided based on the confidence score of the modeling. This protocol provides new insights and guidelines for designing of online server systems for the state-of-the-art protein structure and function predictions. The server is available at http://zhanglab.ccmb.med.umich.edu/I-TASSER.

title: Atomistic modeling of protein-DNA interaction specificity: progress and applications.
abstract: An accurate, predictive understanding of protein-DNA binding specificity is crucial for the successful design and engineering of novel protein-DNA binding complexes. In this review, we summarize recent studies that use atomistic representations of interfaces to predict protein-DNA binding specificity computationally. Although methods with limited structural flexibility have proven successful at recapitulating consensus binding sequences from wild-type complex structures, conformational flexibility is likely important for design and template-based modeling, where non-native conformations need to be sampled and accurately scored. A successful application of such computational modeling techniques in the construction of the TAL-DNA complex structure is discussed. With continued improvements in energy functions, solvation models, and conformational sampling, we are optimistic that reliable and large-scale protein-DNA binding prediction and engineering is a goal within reach.

title: Expanding molecular modeling and design tools to non‐natural sidechains
abstract: Protein–protein interactions encode the wiring diagram of cellular signaling pathways and their deregulations underlie a variety of diseases, such as cancer. Inhibiting protein–protein interactions with peptide derivatives is a promising way to develop new biological and therapeutic tools. Here, we develop a general framework to computationally handle hundreds of non‐natural amino acid sidechains and predict the effect of inserting them into peptides or proteins. We first generate all structural files (pdb and mol2), as well as parameters and topologies for standard molecular mechanics software (CHARMM and Gromacs). Accurate predictions of rotamer probabilities are provided using a novel combined knowledge and physics based strategy. Non‐natural sidechains are useful to increase peptide ligand binding affinity. Our results obtained on non‐natural mutants of a BCL9 peptide targeting beta‐catenin show very good correlation between predicted and experimental binding free‐energies, indicating that such predictions can be used to design new inhibitors. Data generated in this work, as well as PyMOL and UCSF Chimera plug‐ins for user‐friendly visualization of non‐natural sidechains, are all available at http://www.swisssidechain.ch. Our results enable researchers to rapidly and efficiently work with hundreds of non‐natural sidechains.

title: A smoothed backbone-dependent rotamer library for proteins derived from adaptive kernel density estimates and regressions.
abstract: Rotamer libraries are used in protein structure determination, prediction, and design. The backbone-dependent rotamer library consists of rotamer frequencies, mean dihedral angles, and variances as a function of the backbone dihedral angles. Structure prediction and design methods that employ backbone flexibility would strongly benefit from smoothly varying probabilities and angles. A new version of the backbone-dependent rotamer library has been developed using adaptive kernel density estimates for the rotamer frequencies and adaptive kernel regression for the mean dihedral angles and variances. This formulation allows for evaluation of the rotamer probabilities, mean angles, and variances as a smooth and continuous function of phi and psi. Continuous probability density estimates for the nonrotameric degrees of freedom of amides, carboxylates, and aromatic side chains have been modeled as a function of the backbone dihedrals and rotamers of the remaining degrees of freedom. New backbone-dependent rotamer libraries at varying levels of smoothing are available from http://dunbrack.fccc.edu.

title: SCWRL and MolIDE: computer programs for side-chain conformation prediction and homology modeling
abstract: SCWRL and MolIDE are software applications for prediction of protein structures. SCWRL is designed specifically for the task of prediction of side-chain conformations given a fixed backbone usually obtained from an experimental structure determined by X-ray crystallography or NMR. SCWRL is a command-line program that typically runs in a few seconds. MolIDE provides a graphical interface for basic comparative (homology) modeling using SCWRL and other programs. MolIDE takes an input target sequence and uses PSI-BLAST to identify and align templates for comparative modeling of the target. The sequence alignment to any template can be manually modified within a graphical window of the target–template alignment and visualization of the alignment on the template structure. MolIDE builds the model of the target structure on the basis of the template backbone, predicted side-chain conformations with SCWRL and a loop-modeling program for insertion–deletion regions with user-selected sequence segments. SCWRL and MolIDE can be obtained at http://dunbrack.fccc.edu/Software.php.