Podcasts about acf1

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Latest podcast episodes about acf1

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06
Effects of nucleosome remodeling factor ACF1 on in vivo chromatin organization

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06

Play Episode Listen Later Nov 27, 2015


Eukaryotic genomes make use of nucleosomes to considerably reduce their packaging volumes. As a consequence, the underlying DNA is rendered inaccessible. Cells make use of ATP-dependent remodeling factors to disrupt histone-DNA contacts and bring about access to the DNA. ACF1 is the largest regulatory subunit of two nucleosome remodeling factors, namely ACF and CHRAC. These complexes assemble, slide or evenly space nucleosomes on DNA with an ability to sense the linker lengths. However, roles of ACF1 in organizing nucleosomes in vivo and their physiological consequences are largely unclear. To understand the roles of ACF1 on chromatin organization, I compared nucleosome occupancy and transcription profiles in wild-type and ACF1-deficient Drosophila embryos. To further investigate and corroborate these chromatin changes, I performed genomewide mapping of ACF1 using chromatin immunoprecipitation. Nucleosome occupancy was mapped by subjecting DNA obtained from MNase-digested chromatin to deep sequencing and the occupancies were analyzed using advanced analog signal processing methods. We found discontinuous and discrete patches of regularly positioned nucleosomes in wild-type tissue, referred to as ‘regularity regions’. These regions span actively transcribing and silent chromatin domains and show associated variation in the linker lengths across them. A subset of these regions located at sides remote from the transcriptional start sites loses regularity upon ACF1 deletion and show presence of a novel DNA sequence motif. Analyzing nucleosome periodicity by autocorrelation function revealed that nucleosome linker length is longer in ACF1-deficient embryos. Despite profound quantifiable changes in the chromatin organization the RNA expression analyses did not show any major changes. Genomewide localization of ACF1 was studied using by chromatin immunoprecipitation. We observed a strong enrichment of ACF1 along active promoter regions, coinciding strikingly well with another remodeling factor, RSF-1. However, careful analyses using mutant tissues for both proteins demonstrated that the observed enrichments were in fact false positive. We define 3100 genomic sites as false positive ‘Phantom Peaks’ that tend to enrich in the ChIP-seq experiments. By comparing publicly accessible profiles and the Phantom regions, we showed that several ChIP-seq profiles of the epigenetic regulators show strong enrichment along the Phantom Peaks. In conclusion, we identify regions of regularly organized nucleosomes across the genome and show that a subset localized in silent chromatin regions is affected by ACF1 deletion. Moreover, we identified a class of false positive ChIP-seq peaks at active promoters. This list of Phantom Peaks can be used to assess potential false positive signal in a ChIP-seq profile, especially when mutant tissue is not available as a control.

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 04/06
Physical and functional interaction between DOM-B and ACF1

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 04/06

Play Episode Listen Later Aug 1, 2011


Mon, 1 Aug 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14010/ https://edoc.ub.uni-muenchen.de/14010/1/Vengadasalam_Sandra.pdf Vangadasalam, Sandra ddc:570, ddc:500, Fakultät für Biologie

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06
ATP-dependent Remodelling of Linker Histone-Containing Nucleosomal Fibres

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06

Play Episode Listen Later Jan 22, 2009


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.

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
Characterisation of CHRAC14 and CHRAC16, the two Histone Fold Subunits of the Chromatin Accessibility Complex

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06

Play Episode Listen Later Mar 22, 2006


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.

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 04/19
Charakterisierung von Chromatinfaktoren mit Hilfe von Antikörpern: Von der Fruchtfliege zum humanen Prostatakarzinom und zurück

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 04/19

Play Episode Listen Later Apr 7, 2005


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.