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The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. Here we provide multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, we found that the organization of nucleoplasmic omega speckles depends on ISWI function. Our findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA., Author Summary Chromatin structure and function are regulated by the concerted activity of covalent modifiers of chromatin, nucleosome remodeling factors, and several emerging classes of non-coding RNAs. ISWI is an evolutionarily conserved ATP-dependent chromatin remodeler playing essential roles in chromosome condensation, gene expression, and DNA replication. ISWI activity has been involved in a variety of nuclear functions including telomere silencing, stem cell renewal, neural morphogenesis, and epigenetic reprogramming. Using an in vivo assay to identify factors regulating ISWI activity in the model system Drosophila melanogaster, we recovered a genetic interaction between ISWI and hsrω. The hsrω gene encodes multiple non-coding RNAs (ncRNAs), of which the >10 kb nuclear hsrω-n RNA, with functional homolog in mammals, is essential for the assembly and organization of hnRNP-containing nucleoplasmic omega speckles. These special nuclear compartments play essential roles in the storage/sequestration of hnRNP family and other proteins, thus playing important roles in mRNA maturation and other regulatory processes. Here we show that the hsrω-n ncRNA interacts in vivo and in vitro with ISWI to directly regulate its ATPase activity. We also provide in vivo data showing that omega speckle nuclear organization depends on ISWI function, highlighting a novel role for chromatin remodelers in nuclear speckles organization.

This article talks about the link between migration and cultural translation within the framework of contemporary immigrant writing - with particular reference to African and Indian literature.

Dynamic regulation of histone modifications is critical during development, and aberrant activity of chromatin-modifying enzymes has been associated with diseases such as cancer. Histone demethylases have been shown to play a key role in eukaryotic gene transcription; however, little is known about how their activities are coordinated in vivo to regulate specific biological processes. In Drosophila, two enzymes, dLsd1 (Drosophila ortholog of lysine-specific demethylase 1) and Lid (little imaginal discs), demethylate histone H3 at Lys 4 (H3K4), a residue whose methylation is associated with actively transcribed genes. Our studies show that compound mutation of Lid and dLsd1 results in increased H3K4 methylation levels. However, unexpectedly, Lid mutations strongly suppress dLsd1 mutant phenotypes. Investigation of the basis for this antagonism revealed that Lid opposes the functions of dLsd1 and the histone methyltransferase Su(var)3–9 in promoting heterochromatin spreading at heterochromatin–euchromatin boundaries. Moreover, our data reveal a novel role for dLsd1 in Notch signaling in Drosophila, and a complex network of interactions between dLsd1, Lid, and Notch signaling at euchromatic genes. These findings illustrate the complexity of functional interplay between histone demethylases in vivo, providing insights into the epigenetic regulation of heterochromatin/euchromatin boundaries by Lid and dLsd1 and showing their involvement in Notch pathway-specific control of gene expression in euchromatin.

Poly ADP-ribosylation (PARylation) is a posttranslational protein modification catalyzed by poly -ADP-ribose polymerases (PARPs). Poly ADP-ribose metabolism is involved in a wide range of biological processes, such as maintenance of genome stability, transcriptional regulation, energy metabolism, and programed cell death. Recently, chromatin components, including histones, have been shown to be targets of PARylation. Unlike mammals, which have several PARP-encoded genes, the model organism Drosophila melanogaster has only one PARP gene, highly related to mammalian PARP1. These features make flies a great model system to study PARP biology. Commercially available antibodies recognizing this covalent modification have made possible the development of immunofluorescence approaches to study PARylation of chromatin components. Here, we present a protocol to immunostain polytene chromosomes of the model system D. melanogaster.

Telomeres are specialized structures at the end of eukaryotic chromosomes that are required to preserve genome integrity, chromosome stability and nuclear architecture. Telomere maintenance and function are established epigenetically in several eukaryotes. However, the exact chromatin enzymatic modifications regulating telomere homeostasis are poorly understood. In Drosophila melanogaster, telomere length and stability are maintained through the retrotransposition of specialized telomeric sequences and by the specific loading of protecting capping proteins, respectively. Here, we show that the loss of the essential and evolutionarily conserved histone deacetylase Rpd3, the homolog of mammalian HDAC1, causes aberrant telomeric fusions on polytene chromosome ends. Remarkably, these telomere fusion defects are associated with a marked decrease of histone H4 acetylation, as well as an accumulation of heterochromatic epigenetic marks at telomeres, including histone H3K9 trimethylation and the heterochromatic protein HP2. Our work suggests that Drosophila telomere structure is epigenetically regulated by the histone deacetylase Rpd3.

The evolutionarily conserved ATP-dependent nucleosome remodelling factor ISWI can space nucleosomes affecting a variety of nuclear processes. In Drosophila, loss of ISWI leads to global transcriptional defects and to dramatic alterations in higher-order chromatin structure, especially on the male X chromosome. In order to understand if chromatin condensation and gene expression defects, observed in ISWI mutants, are directly correlated with ISWI nucleosome spacing activity, we conducted a genome-wide survey of ISWI binding and nucleosome positioning in wild-type and ISWI mutant chromatin. Our analysis revealed that ISWI binds both genic and intergenic regions. Remarkably, we found that ISWI binds genes near their promoters causing specific alterations in nucleosome positioning at the level of the Transcription Start Site, providing an important insights in understanding ISWI role in higher eukaryote transcriptional regulation. Interestingly, differences in nucleosome spacing, between wild-type and ISWI mutant chromatin, tend to accumulate on the X chromosome for all ISWI-bound genes analysed. Our study shows how in higher eukaryotes the activity of the evolutionarily conserved nucleosome remodelling factor ISWI regulates gene expression and chromosome organization genome-wide.

The eukaryotic genome is a highly organized nucleoprotein structure comprising of DNA, histones, non-histone proteins, and RNAs, referred to as chromatin. The chromatin exists as a dynamic entity, shuttling between the open and closed forms at specific nuclear regions and loci based on the requirement of the cell. This dynamicity is essential for the various DNA-templated phenomena like transcription, replication, and repair and is achieved through the activity of ATP-dependent chromatin remodeling complexes and covalent modifiers of chromatin. A growing body of data indicates that chromatin enzymatic activities are finely and specifically regulated by a variety of small molecules derived from the intermediary metabolism. This review tries to summarize the work conducted in many laboratories and on different model organisms showing how ATP-dependent chromatin remodeling complexes are regulated by small molecules and metabolites such as adenosine triphosphate (ATP), acetyl coenzyme A (AcCoA), S-adenosyl methionine (SAM), nicotinamide adenine dinucleotide (NAD), and inositol polyphosphates (IPs).

ISWI is an evolutionarily conserved ATP-dependent chromatin remodeling factor playing central roles in DNA replication, RNA transcription, and chromosome organization. The variety of biological functions dependent on ISWI suggests that its activity could be highly regulated. Our group has previously isolated and characterized new cellular activities that positively regulate ISWI in Drosophila melanogaster. To identify factors that antagonize ISWI activity we developed a novel in vivo eye-based assay to screen for genetic suppressors of ISWI. Our screen revealed that ISWI interacts with an evolutionarily conserved network of cellular and nuclear factors that escaped previous genetic and biochemical analyses.

Background Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. Methodology/Principal Findings Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. Conclusions/Significance Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

In the quest for a mathematical measure able to capture and shed light on the dual notions of information and complexity in biosequences, Hazen et al. have introduced the notion of Functional Information (FI for short). It is also the result of earlier considerations and findings by Szostak and Carothers et al. Based on the experiments by Charoters et al., regarding FI in RNA binding activities, we decided to study the relation existing between FI and classic measures of complexity applied on protein-DNA interactions on a genome-wide scale. Using classic complexity measures, i.e, Shannon entropy and Kolmogorov Complexity as both estimated by data compression, we found that FI applied to protein-DNA interactions is genuinely different from them. Such a fact, together with the non-triviality of the biological function considered, contributes to the establishment of FI as a novel and useful measure of biocomplexity. Remarkably, we also found a relationship, on a genome-wide scale, between the redundancy of a genomic region and its ability to interact with a protein. This latter finding justifies even more some principles for the design of motif discovery algorithms. Finally, our experiments bring to light methodological limitations of Linguistic Complexity measures, i.e., a class of measures that is a function of the vocabulary richness of a sequence. Indeed, due to the technology and associated statistical preprocessing procedures used to conduct our studies, i.e., genome-wide ChIP-chip experiments, that class of measures cannot give any statistically significant indication about complexity and function. A serious limitation due to the widespread use of the technology. References J.M. Carothers, S.C. Oestreich, J.H. Davis, and J.W. Szostack. Informational complexity and functional activity of RNA structures. J. AM. CHEM. SOC., 126 (2004), pp. 5130-5137. R.M. Hazen, P.L. Griffin, J.M. Carothers, and J.W. Szostak. Functional Information and the emergence of biocomplexity. Proc. of Nat. Acad. Sci, 104 (2007), pp. 8574-8581. J.W. Szostak. Functional Information: molecular messages, Nature, 423 (2003).

Histone acetylation plays essential roles in cell cycle progression, DNA repair, gene expression and silencing. Although the knowledge regarding the roles of acetylation of histone lysine residues is rapidly growing, very little is known about the biochemical pathways providing the nucleus with metabolites necessary for physiological chromatin acetylation. Here, we show that mutations in the scheggia (sea)-encoded Sea protein, the Drosophila ortholog of the human mitochondrial citrate carrier Solute carrier 25 A1 (SLC25A1), impair citrate transport from mitochondria to the cytosol. Interestingly, inhibition of sea expression results in extensive chromosome breakage in mitotic cells and induces an ATR-dependent cell cycle arrest associated with a dramatic reduction of global histone acetylation. Notably, loss of SLC25A1 in short interfering RNA (siRNA)-treated human primary fibroblasts also leads to chromosome breaks and histone acetylation defects, suggesting an evolutionary conserved role for Sea/SLC25A1 in the regulation of chromosome integrity. This study therefore provides an intriguing and unexpected link between intermediary metabolism and epigenetic control of genome stability.

The positioning of nucleosomes along chromatin has been implicated in the regulation of gene expression in eukaryotic cells, because packaging DNA into nucleosomes affects sequence accessibility. In this paper we propose a new model (called MLM) for the identification of nucleosomes and linker regions across DNA, consisting in a thresholding technique based on cut-set conditions. For this purpose we have defined a method to generate synthetic microarray data fully inspired from the approach that has been used by Yuan et al. Results have shown a good recognition rate on synthetic data, moreover, the $MLM$ shows a good agreement with the recently published method based on Hidden Markov Model when tested on Saccharomyces cerevisiae chromosomes microarray data.

Imitation SWI (ISWI) and other ATP-dependent chromatin-remodeling factors play key roles in transcription and other processes by altering the structure and positioning of nucleosomes. Recent studies have also implicated ISWI in the regulation of higher-order chromatin structure, but its role in this process remains poorly understood. To clarify the role of ISWI in vivo, we examined defects in chromosome structure and gene expression resulting from the loss of Iswi function in Drosophila. Consistent with a broad role in transcriptional regulation, the expression of a large number of genes is altered in Iswi mutant larvae. The expression of a dominant-negative form of ISWI leads to dramatic alterations in higher-order chromatin structure, including the apparent decondensation of both mitotic and polytene chromosomes. The loss of ISWI function does not cause obvious defects in nucleosome assembly, but results in a significant reduction in the level of histone H1 associated with chromatin in vivo. These findings suggest that ISWI plays a global role in chromatin compaction in vivo by promoting the association of the linker histone H1 with chromatin., Author Summary Chromatin-remodeling factors such as ISWI play a role in transcription and other nuclear processes by altering the structure and positioning of nucleosomes (the protein–DNA complexes that organize chromatin). Recent studies have suggested that chromatin-remodeling factors can also influence higher-order chromatin structure, but how they do this is not well understood. Using Drosophila melanogaster as a model organism, we investigated the role of ISWI in gene expression and the regulation of chromosome structure in higher eukaryotes. Loss of ISWI alters the expression of a large number of genes. The loss of ISWI function also causes dramatic alterations in higher-order chromatin structure—including the decondensation of mitotic and polytene chromosomes—accompanied by a striking reduction in the amount of the linker histone H1 associated with chromatin. Based on these findings, we propose that ISWI plays a global role in chromosome compaction by promoting the association of a linker histone with chromatin., ISWI is a chromatin-remodeling factor that recruits linker histone H1 into chromatin, thus altering the structure and positioning of nucleosomes and the degree of chromatin compaction.

Eukaryotic DNA is packaged into a highly compact and dynamic structure called chromatin. While this packaging allows the cell to organize a large and complex genome in the nucleus, it can also block the access of transcription factors and other proteins to DNA. Nucleosomes are the fundamental repeating units of eukaryotic chromatin. Nucleosome position can be regulated in vivo by multi-subunit chromatin remodeling complexes, and their position can influence gene expression in eukaryotic cells. Alterations in chromatin structure, and hence in nucleosome organization, can result in a variety of diseases, including cancer, highlighting the need to achieve a better understanding of the molecular processes modulating chromatin dynamics. In this paper, we present a new method, Multi-Layer Model (MLM), that can be successfully used to identify genome wide nucleosome positions starting from microarray data. This new approach allows a significant reduction in computational time as well as a better structural view of the input data when compared to a Hidden Markov Model HMM recently used for the same purpose. In order to evaluate the accuracy of the method the $MLM$ has been validated on synthetic data and compared with the HMM on real microarray data of the Saccharomyces cerevisiae. By the way, although the MLM is faster than the other methods devoted to the same purpose when tested on real data, the processing of large microarray data (more than 10^4 base pairs) is a real case where the MLM becomes computationally demanding making essential the use of high performance computing architectures. To this purpose, two parallel implementations of the MLM are described and analysed.