Podcasts about iswi

Desain fashion karya Mahasiswi ISWI Jakarta dan Garut Kulit, tampil di side event New York Fashion Week. Berkenalan dengan Namirah Zihniah, mahasiswa NYU yang menyutradarai film Cell Phone Cinema 3D.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.11.247080v1?rss=1 Authors: Gong, N. N., Chakravarti Dilley, L., Williams, C. E., Moscato, E. H., Szuperak, M., Wang, Q., Jensen, M., Girirajan, S., Tan, T. Y., Deardorff, M. A., Li, D., Song, Y., Kayser, M. S. Abstract: Sleep disruptions are among the most commonly-reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From a Drosophila screen of human NDD-associated risk genes, we identified the chromatin remodeler Imitation SWItch/SNF (ISWI) to be required for adult fly sleep. Loss of ISWI also results in disrupted circadian rhythms, memory, and social behavior, but ISWI acts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically, ISWI expression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the human ISWI homologs SMARCA1 and SMARCA5 differentially rescue adult phenotypes. We propose that sleep deficits are a primary phenotype of early developmental origin in NDDs, and point towards chromatin remodeling machinery as critical for sleep circuit formation. Copy rights belong to original authors. Visit the link for more info

Davide Corona, University of Palermo, Palermo - Italy speaks on "Chromatin Binding, Nucleosome Spacing and ncRNA-mediated Regulation of the Remodeling ATPase ISWI". This seminar has been recorded at University of Trieste by ICGEB Trieste

Eukaryotic genomes are condensed into a multilevel structure called chromatin which serves to organize and package the DNA, but at the same time needs to be flexible to permit regulated access to the stored information. ATP-dependent chromatin remodelling factors largely contribute to this dynamic nature of chromatin by catalysing processes such as the disruption of histone-DNA contacts, nucleosome repositioning and histone exchange. ATP-dependent remodelling has been well documented on a mononucleosomal level, but little is known about its regulation in a more physiological chromatin environment, where neighbouring nucleosomes and linker histones might interfere with the remodelling reaction. If and to what extent remodelling can work on chromatin bound by linker histones remains controversial, in spite of their high abundance and their strong influence on chromatin folding. We therefore investigated chromatin remodelling in the presence of linker histones H1 or H5 using regularly spaced, oligonucleosomal substrates reconstituted from purified components. Surprisingly, we found that both the remodelling complex ACF – consisting of the ATPase ISWI and the regulatory subunit ACF1 – and ISWI alone were able to catalyse the repositioning of entire chromatosomes (nucleosomes + H1). Linker histones inhibited their remodelling activity by only about 50%. In contrast, the related ATPase CHD1 remodelled chromatin only in the absence of linker histones, suggesting that linker histones allow remodelling by selected factors only. In addition, our data indicate that repositioning in the presence of H1 might be unidirectional. ACF1 is abundant during early Drosophila development, when H1 gradually replaces its early placeholder HMG-D. HMG-D binds to chromatin less tightly than H1 and unlike the latter, did not affect the remodelling activity of ACF in our assay. H1 was able to displace HMG-D from and bind to our reconstituted arrays without the help of cofactors. Strikingly, both H1 and HMG-D are more abundant in embryonic nuclei of acf1 null flies compared to the wild-type, raising the possibility that an ACF1-containing complex controls linker histone incorporation.

In eukaryotic nuclei, the DNA double helix is wound up and condensed into chromatin through the interaction with histones and further proteins. Several factors regulate the chromatin structure, allow unfolding or condensation of the chromatin fibre and permit or restrict access to DNA. One prominent class of chromosomal regulators is represented by ATP-dependent chromatin remodelling complexes, which use the energy derived from ATPhydrolysis to break or alter histone-DNA contacts. The ATP-utilising Chromatin Assembly and Remodelling Factor (ACF) and the Chromatin Accessibility Complex (CHRAC) are two closely related ATP-dependent chromatin remodelling factors. ACF consists of the ATPase ISWI and ACF1, a large protein that influences both the quality and efficiency of ISWI activity. CHRAC contains ISWI and ACF1 as well, but in addition the two small histone fold proteins CHRAC14 and CHRAC16. In this work, the CHRAC14 and CHRAC16 subunits are characterised both structurally and functionally. The generation of a bicistronic expression plasmid allowed the expression and purification of highly pure recombinant CHRAC14-CHRAC16 in stoichiometric amounts. The crystal structure of the CHRAC14-CHRAC16 complex was solved at a resolution of 2.4 Å and demonstrates that the two proteins interact with each other via their histone fold motifs, thereby closely resembling the structure of histones H2A-H2B and NFYB-NFYC, the histone fold subunits of nuclear factor Y (NF-Y). Rat monoclonal antibodies against CHRAC14 and CHRAC16 were raised and characterised, but due to their poor affinity, they turned out to be only of limited use for the analysis of the two proteins. CHRAC14-CHRAC16 interact with the N-terminus of ACF1, including the conserved WAC motif. They have a weak affinity for DNA, and studies with CHRAC14-CHRAC16 deletion variants revealed that their C-termini play important but distinct roles in DNA binding. Finally, CHRAC14-CHRAC16 facilitate ACF-dependent nucleosome mobilisation, and their ability to enhance ACF activity depends on both the interaction with the ACF1 N-terminus and the dynamic binding to DNA. In the light of profound similarities to the effects of HMGB1 (high mobility group box protein 1) on nucleosome sliding, these data imply that the CHRAC14-CHRAC16 subcomplex operates as a ‘DNA chaperone’ and assists ACF1 and ISWI during ATPdependent nucleosome remodelling by providing a transient DNA binding surface. This work provides the basis for further experiments to gain more insights into the mechanistic details of CHRAC-dependent nucleosome remodelling and to explore the roles of CHRAC in the living cell.

Die Organisation der DNA in Nukleosomen hat einen großen Einfluss auf die Regulation von grundliegenden Prozessen wie Transkription, Replikation oder Reparatur der DNA im Zellkern. Um die hinderliche Natur des Chromatins bei diesen fundamentalen Prozessen zu überwinden, existieren mehrere verschiedene Chromatin modifizierende Proteinkomplexe im Zellkern. Chromatin Remodelling Komplexe nützen die Energie der ATP-Hydrolyse um die Position der Nukleosomen so zu verändern, dass verschiedene Abschnitte der DNA für die Interaktion mit regulierenden Faktoren zugänglich werden. Ein Klasse solcher Remodelling Faktoren beinhalten die ATPase ISWI als katalytische Untereinheit. Das Protein wurde zuerst in Drosophila entdeckt und die drei verschiedenen ISWI enthaltenden Komplexe, nämlich NURF, ACF und CHRAC, wurden ausführlich in diesem Modellorganismus untersucht. Homolog zur Fruchtfliege existieren sehr ähnliche Protein Komplexe beim Menschen. Wir haben das humane ISWI mit den Isoformen Snf2h und Snf2L im Prostatakarzinom untersucht. In einem Tissue Microarray wurden Gewebeproben mit Hilfe von polyklonalen Antikörpern gegen ISWI gefärbt. Es folgte ein quantitativer Vergleich der Färbungsintensitäten im Karzinomgewebe sowie in gutartigem Gewebe der Prostata durch Anwendung von digitaler Bildanalyse. Das Ergebnis war eine signifikant stärkere Färbung im neoplastischen Gewebe. Eine Anreicherung von ISWI in Krebszellen ist besonders interessant im Kontext der bekannten Funktionen des Proteins für DNA-Replikation, Zellproliferation und Regulation der Chromatinstruktur. In einem zweiten Projekt sind wir zum Modell der Fruchtfliege zurückgekehrt und entwickelten monoklonale Antikörper gegen Toutatis, das zu einer Proteinfamilie gehört, die auch einige bekannte Interaktionspartner von ISWI umfasst. Die Proteine dieser Familie haben vermutlich eine regulatorische Funktion in den Remodelling Komplexen, denn am Beispiel von Acf1 wurde gezeigt, dass sie die nukleosomale Bindung sowie die Effizienz und Richtung der Mobilisierung von Nukleosomen modifizieren. Unsere Antikörper wurden etabliert, um Toutatis enthaltende Komplexe durch Western Blot Analyse von gereinigten Drosophila-Extrakten und Immunfluoreszenz zu charakterisieren. Mit diesen Methoden fanden wir eine Koelution von Toutatis mit der ATPase Brahma und dem Strukturprotein Spectrin alpha sowie eine Lokalisation in der Lamina des Zellkerns. Ein mögliches Zusammenspiel dieser Proteine in einem neuen Chromatin Remodelling Komplex mit einer Beteiligung an der DNA-Reparatur wird diskutiert.

Regulation of gene expression takes place in the nucleus in a highly structured and condensed nucleoprotein environment, called chromatin (Felsenfeld and Groudine, 2003; Khorasanizadeh, 2004; Vaquero et al., 2003). A broad group of factors regulates the properties of chromatin; e.g. by covalently modifying histones and / or by ATP-dependent chromatin remodeling, thereby allowing or preventing gene expression. The mammalian genome contains hundreds of gene copies encoding precursor ribosomal RNA and the transcription of these genes is highly regulated with respect to cellular metabolism (Grummt, 2003). However, even in actively growing cells, only a subset of the rRNA genes are actively transcribed, exhibiting an accessible chromatin conformation (Conconi et al., 1989). In a chromatin context, the activation of rDNA genes involves the transcription termination factor TTF-I (Längst et al., 1998; Längst et al., 1997a). However, the silenced rDNA gene fraction remains in an inaccessible heterochromatic state throughout the cell cycle (Conconi et al., 1989). Until recently, the onset of silencing and the mechanisms that maintain the inactive state of rRNA genes were less understood. Recent studies, including the work presented in this thesis, provide insights into the molecular mechanism of ribosomal RNA gene silencing (Lawrence et al., 2004; Németh et al., 2004; Santoro and Grummt, 2001; Santoro et al., 2002; Strohner et al., 2004; Zhou et al., 2002). Accumulating evidence indicates that the combined action of chromatin modifying mechanisms such as chromatin remodeling, histone modification and DNA methylation contribute to the process of rRNA gene silencing. Here I present data demonstrating an active role of the chromatin remodeling complex NoRC in rDNA gene silencing and propose dual functions of TTF-I in rDNA regulation in chromatin, namely involvement in both activation and silencing of rDNA transcription. 4.1 NoRC, a novel chromatin remodeling complex In this doctoral study, a novel protein complex, composed of the nucleolar protein Tip5 and the ATPase Snf2h, was purified using convential chromatography and affinity purification methods. A detailed chromatin remodeling analysis revealed that this complex is able to induce mononucleosome movement in an ATP and histone H4 tail dependent fashion. Finally, this Tip5-Snf2h complex was termed NoRC (nucleolar remodeling complex), a novel member of the ISWI family of ATP-dependent chromatin remodeling complexes (Strohner et al., 2001). To dissect its functions, the NoRC complex was reconstituted from its recombinant subunits Tip5 and Snf2h, using the baculo virus driven expression system. Reconstitution confirmed the direct interaction between Tip5 and Snf2h. Furthermore, recombinant and cellular NoRC display similar sizes in gel filtration columns. Recombinant NoRC exhibits chromatin stimulated ATPase activity and mobilizes nucleosomes in an energy-dependent manner. Both activities are histone H4 tail dependent. NoRC and its subunits Tip5 and Snf2h were compared in different DNA / Nucleosome binding assays. NoRC shows preferred binding to structured (bent) DNA, e.g. a region within the mouse rDNA promoter, and interacts with mononucleosomes in electrophoretic mobility shift assays (EMSA). While no stable interaction with core nucleosomes could be detected in EMSA, ATPase assays and DNase I protection assays noticeably pinpointed to NoRC / nucleosome interactions with both nucleosomal and protruding linker DNA. 4.2 NoRC specifically represses rDNA transcription in chromatin The functional consequences of the Tip5 / TTF-I interaction were assessed and the influence on chromatin structure of the rDNA promoter in an in vitro system was determined. Tip5 in NoRC interacts with the N-terminal part of full length TTF-I and unmasks its DNA binding site. This interaction is required both for binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter in chromatin. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome. To elucidate a potential role of NoRC in rDNA transcriptional regulation, we used an in vitro transcription system with an rDNA minigene reconstituted into chromatin. These studies revealed a specific function for NoRC in rDNA transcriptional repression on chromatin templates. In contrast, NoRC had no effect on DNA transcription. Transcription experiments were then performed with chromatin templates reconstituted from recombinant histones lacking individual histone tails. The results indicate that NoRC-mediated rDNA gene repression is dependent on the histone H4 tail, suggesting an involvement of chromatin remodeling. Further transcription experiments revealed that NoRC-mediated repression occurs prior to preinitiation complex formation and does not affect activated rDNA genes. NoRC stably associates with the silenced gene, and these early steps of rDNA repression do not depend on DNA and histone modifications (Strohner et al., 2004). NoRC showed preferred binding to a structured (bent) region within the mouse rDNA promoter. Methylation of a single CpG dinucleotide within this region abrogated rDNA transcription in chromatin (Santoro and Grummt, 2001), but did not influence DNA binding of NoRC. Furthermore, nucleosomal DNA is less methylated than free DNA, but chromatin remodeling enhances methylation. The results suggest an important role for the chromatin remodeling complex NoRC in the establishment of rDNA silencing. NoRC then contributes to maintenance of the silenced state throughout the cell cycle by interacting with DNA and histone modifying enzymes. Transcriptional repression by chromatin remodeling factors seems to be a common mechanism to stably inhibit gene expression.