scholarly journals The biogenesis and function of nucleosome arrays

2021 ◽  
Author(s):  
Ashish Kumar Singh ◽  
Tamás Schauer ◽  
Lena Pfaller ◽  
Tobias Straub ◽  
Felix Mueller-Planitz
Keyword(s):  
Dna Sequence ◽  
Chromatin Remodeling ◽  
Genome Engineering ◽  
Genotoxic Stress ◽  
Eukaryotic Cells ◽  
Complex Spaces ◽  
Nucleosome Density ◽  
Cryptic Transcription ◽  
And Function

AbstractNumerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shapes the nucleosome landscape. Precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery is strongly disruptive of evenly spaced nucleosomes, and proper nucleosome density and DNA sequence critical for their biogenesis. The INO80 remodeling complex spaces nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Spaced nucleosomes prevent cryptic transcription and protect cells against genotoxic stress such as DNA damage, recombination and transpositions. We derive a unifying model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.

scholarly journals The biogenesis and function of nucleosome arrays

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ashish Kumar Singh ◽  
Tamás Schauer ◽  
Lena Pfaller ◽  
Tobias Straub ◽  
Felix Mueller-Planitz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Sequence ◽  
Chromatin Remodeling ◽  
Genome Engineering ◽  
Genotoxic Stress ◽  
Eukaryotic Cells ◽  
Nucleosome Density ◽  
And Function

AbstractNumerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shape the nucleosome landscape. The precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery strongly disrupts evenly spaced nucleosomes. Proper nucleosome density and DNA sequence are critical for their biogenesis. The INO80 remodeling complex helps space nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Cells with irregularly spaced nucleosomes suffer from genotoxic stress including DNA damage, recombination and transpositions. We derive a model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.

scholarly journals Coordinated Hsp110 and Hsp104 Activities Power Protein Disaggregation in Saccharomyces cerevisiae

2017 ◽  
Vol 37 (11) ◽  
Author(s):  
Jayasankar Mohanakrishnan Kaimal ◽  
Ganapathi Kandasamy ◽  
Fabian Gasser ◽  
Claes Andréasson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Protein Aggregation ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Content Type ◽  
Dependent Protein ◽  
And Function ◽  
Cellular Dysfunction

ABSTRACT Protein aggregation is intimately associated with cellular stress and is accelerated during aging, disease, and cellular dysfunction. Yeast cells rely on the ATP-consuming chaperone Hsp104 to disaggregate proteins together with Hsp70. Hsp110s are ancient and abundant chaperones that form complexes with Hsp70. Here we provide in vivo data showing that the Saccharomyces cerevisiae Hsp110s Sse1 and Sse2 are essential for Hsp104-dependent protein disaggregation. Following heat shock, complexes of Hsp110 and Hsp70 are recruited to protein aggregates and function together with Hsp104 in the disaggregation process. In the absence of Hsp110, targeting of Hsp70 and Hsp104 to the aggregates is impaired, and the residual Hsp104 that still reaches the aggregates fails to disaggregate. Thus, coordinated activities of both Hsp104 and Hsp110 are required to reactivate aggregated proteins. These findings have important implications for the understanding of how eukaryotic cells manage misfolded and amyloid proteins.

scholarly journals Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
R Stefan Isaac ◽  
Fuguo Jiang ◽  
Jennifer A Doudna ◽  
Wendell A Lim ◽  
Geeta J Narlikar ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Dna Sequences ◽  
Surveillance System ◽  
Dynamic Properties ◽  
Eukaryotic Cells ◽  
Chromatin Remodelers ◽  
Nucleosomal Dna ◽  
Eukaryotic Chromatin ◽  
Versatile Tool

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.

scholarly journals Emergence of robust nucleosome patterns from an interplay of positioning mechanisms

2018 ◽  
Author(s):  
Johannes Nuebler ◽  
Michael Wolff ◽  
Benedikt Obermayer ◽  
Wolfram Möbius ◽  
Ulrich Gerland
Keyword(s):  
Dna Sequence ◽  
Chromatin Remodeling ◽  
Nucleosome Positioning ◽  
Eukaryotic Cells ◽  
Active Chromatin ◽  
Complex Interplay ◽  
Quantitative Characteristics ◽  
Characteristic Features ◽  
Species Specific ◽  
Effective Description

AbstractProper positioning of nucleosomes in eukaryotic cells is determined by a complex interplay of factors, including nucleosome-nucleosome interactions, DNA sequence, and active chromatin remodeling. Yet, characteristic features of nucleosome positioning, such as gene-averaged nucleosome patterns, are surprisingly robust across perturbations, conditions, and species. Here, we explore how this robustness arises despite the underlying complexity. We leverage mathematical models to show that a large class of positioning mechanisms merely affects the quantitative characteristics of qualitatively robust positioning patterns. We demonstrate how statistical positioning emerges as an effective description from the complex interplay of different positioning mechanisms, which ultimately only renormalize the model parameter quantifying the effective softness of nucleosomes. This renormalization can be species-specific, rationalizing a puzzling discrepancy between the effective nucleosome softness of S. pombe and S. cerevisiae. More generally, we establish a quantitative framework for dissecting the interplay of different nucleosome positioning determinants.

scholarly journals Homotypic cooperativity and collective binding are determinants of bHLH specificity and function

2019 ◽  
Vol 116 (32) ◽  
pp. 16143-16152 ◽  
Author(s):  
Christian A. Shively ◽  
Jiayue Liu ◽  
Xuhua Chen ◽  
Kaiser Loell ◽  
Robi D. Mitra
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Dna Binding Proteins ◽  
Eukaryotic Cells ◽  
Sequence Information ◽  
Genome Wide ◽  
Important Open Problem ◽  
And Function

Eukaryotic cells express transcription factor (TF) paralogues that bind to nearly identical DNA sequences in vitro but bind at different genomic loci and perform different functions in vivo. Predicting how 2 paralogous TFs bind in vivo using DNA sequence alone is an important open problem. Here, we analyzed 2 yeast bHLH TFs, Cbf1p and Tye7p, which have highly similar binding preferences in vitro, yet bind at almost completely nonoverlapping target loci in vivo. We dissected the determinants of specificity for these 2 proteins by making a number of chimeric TFs in which we swapped different domains of Cbf1p and Tye7p and determined the effects on in vivo binding and cellular function. From these experiments, we learned that the Cbf1p dimer achieves its specificity by binding cooperatively with other Cbf1p dimers bound nearby. In contrast, we found that Tye7p achieves its specificity by binding cooperatively with 3 other DNA-binding proteins, Gcr1p, Gcr2p, and Rap1p. Remarkably, most promoters (63%) that are bound by Tye7p do not contain a consensus Tye7p binding site. Using this information, we were able to build simple models to accurately discriminate bound and unbound genomic loci for both Cbf1p and Tye7p. We then successfully reprogrammed the human bHLH NPAS2 to bind Cbf1p in vivo targets and a Tye7p target intergenic region to be bound by Cbf1p. These results demonstrate that the genome-wide binding targets of paralogous TFs can be discriminated using sequence information, and provide lessons about TF specificity that can be applied across the phylogenetic tree.

scholarly journals The Yeast Chromatin Remodeler RSC Complex Facilitates End Joining Repair of DNA Double-Strand Breaks

2005 ◽  
Vol 25 (10) ◽  
pp. 3934-3944 ◽  
Author(s):  
Eun Yong Shim ◽  
Jia-Lin Ma ◽  
Ji-Hyun Oum ◽  
Yvonne Yanez ◽  
Sang Eun Lee
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Immunoprecipitation ◽  
Genotoxic Stress ◽  
Double Strand Breaks ◽  
Cell Cycle Checkpoints ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks

ABSTRACT Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Nonhomologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal truncation of RSC8 impairs NHEJ of a chromosome DSB created by HO endonuclease in vivo. rsc30Δ maintains a robust level of homologous recombination and the damage-induced cell cycle checkpoints. By chromatin immunoprecipitation, we show recruitment of RSC to a chromosome DSB with kinetics congruent with its involvement in NHEJ. Recruitment of RSC to a DSB depends on Mre11, Rsc30, and yKu70 proteins. Rsc1p and Rsc2p, two other RSC subunits, physically interact with yKu80p and Mre11p. The interaction of Rsc1p with Mre11p appears to be vital for survival from genotoxic stress. These results suggest that chromatin remodeling by RSC is important for NHEJ.

scholarly journals Efficiency of SpCas9 and AsCpf1 (Cas12a) programmable nucleases at genomic safe harbor loci in HEK293 cells

2020 ◽  
Vol 49 ◽  
Author(s):  
S. V. Pavlova ◽  
E. A. Elisaphenko ◽  
L. Sh. Shayakhmetova ◽  
S. P. Medvedev
Keyword(s):  
Genome Engineering ◽  
Hek293 Cells ◽  
Open Chromatin ◽  
Safe Harbor ◽  
Dna Breaks ◽  
Guide Rna ◽  
Nucleosome Density ◽  
Low Efficiency ◽  
Hek293ft Cell

Rationale: The development of eukaryote genome engineering tools based on CRISPR-Cas programmable bacterial nucleases systems opens wide horizons for gene therapies, human disease cell modeling, as well as investigation into manifestation of disease phenotypes and visualization of cellular processes. The safety and approximation of experiments both at the cellular and organismal levels depend on the accuracy of introducing double-stranded breaks into the target DNA regions. The search for new variants of more accurate CRISPR-Cas nucleases and evaluation of their ability to hydrolyze nucleosome DNA in vivo is considered a critical task for the development of the genome engineering technologies.Aim: To analyze the activity of the programmable nuclease AsCpf1 (Cas12a), with low level of off-target activity, in the human genome loci that are safe for the introduction of transgenic constructs (“safe harbor”) and to compare its efficiency with that of the widely used SpCas9 nuclease in HEK293 cells.Materials and methods: We performed the bioinformatics analysis of the association between target regions with nucleosomes and other proteins in the safe harbor loci AAVS1 and GSH-Ch1 and the transcriptionally inactive gene MYBPC3 (cardiac myosin binding protein 3) based on ATAC-seq data for the HEK293FT cells obtained from the NCBI SRA database. Plasmids encoding SpCas9 and AsCpf1 nucleases and guide RNA to the target regions were constructed and transfected into the HEK293FT cells. Events in the target regions of the HEK293FT cell genome were studied in the sequenograms with the TIDE algorithm.Results: The results of the ATAC-seq experiments for HEK293FT cells have shown that the AAVS1 locus can be referred as open chromatin with a low nucleosome density, while the GSH-Ch1 locus can be attributed to closed chromatin. In HEK293FT cells, the cardiac MYBPC3 gene has intermediate chromatin density. Assessment of the efficiency of introducing breaks into the studied HEK293FT cell chromatin loci by nucleases has shown that SpCas9 is able to cope with chromatin of any nucleosome density, while AsCpf1 can effectively introduce DNA breaks only at loci with open chromatin, such as AAVS1 and MYBPC3. Editing events occur at a very low rate at the GSH-Ch1 locus with a high nucleosome density.Conclusion: We have found low efficiency of the AsCpf1 nuclease in the genomic safe harbor locus GSH-Ch1, which is characterized by a high nucleosome density. When planning an experiment on AsCpf1 nuclease genome editing, the epigenetic chromatin landscape and the nucleosome density should be considered, as well as chromatin opening substances should be used.

scholarly journals Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

2021 ◽  
Author(s):  
Nour J Abdulhay ◽  
Laura J Hsieh ◽  
Colin P McNally ◽  
Mythili Ketavarapu ◽  
Sivakanthan Kasinathan ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Single Molecule ◽  
Single Fiber ◽  
Chromatin Remodelers ◽  
Linker Length ◽  
Dna Accessibility ◽  
Nucleosome Density ◽  
Regulatory Effects

ABSTRACTATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.

Osseointegration interface of calcified bone and endosseous implants

1992 ◽  
Vol 50 (2) ◽  
pp. 1096-1097
Author(s):  
K.E. Krizan ◽  
J.E. Laffoon ◽  
M.J. Buckley
Keyword(s):  
Structure And Function ◽  
Titanium Implants ◽  
En Bloc ◽  
Endosseous Implants ◽  
Ultrastructural Studies ◽  
The Past ◽  
Cacodylate Buffer ◽  
One Year ◽  
And Function

With increase use of tissue-integrated prostheses in recent years it is a goal to understand what is happening at the interface between haversion bone and bulk metal. This study uses electron microscopy (EM) techniques to establish parameters for osseointegration (structure and function between bone and nonload-carrying implants) in an animal model. In the past the interface has been evaluated extensively with light microscopy methods. Today researchers are using the EM for ultrastructural studies of the bone tissue and implant responses to an in vivo environment. Under general anesthesia nine adult mongrel dogs received three Brånemark (Nobelpharma) 3.75 × 7 mm titanium implants surgical placed in their left zygomatic arch. After a one year healing period the animals were injected with a routine bone marker (oxytetracycline), euthanized and perfused via aortic cannulation with 3% glutaraldehyde in 0.1M cacodylate buffer pH 7.2. Implants were retrieved en bloc, harvest radiographs made (Fig. 1), and routinely embedded in plastic. Tissue and implants were cut into 300 micron thick wafers, longitudinally to the implant with an Isomet saw and diamond wafering blade [Beuhler] until the center of the implant was reached.

The Hierarchic Order of Intermediate Filament Structure and Assembly

1985 ◽  
Vol 43 ◽  
pp. 722-725
Author(s):  
U. Aebi ◽  
E.C. Glavaris ◽  
R. Eichner
Keyword(s):  
Tissue Specificity ◽  
Structural Model ◽  
Eukaryotic Cells ◽  
Cdna Sequencing ◽  
Filament Structure ◽  
Keratin Filaments ◽  
Isoelectric Points ◽  
Immunological Crossreactivity

Five different classes of intermediate-sized filaments (IFs) have been identified in differentiated eukaryotic cells: vimentin in mesenchymal cells, desmin in muscle cells, neurofilaments in nerve cells, glial filaments in glial cells and keratin filaments in epithelial cells. Despite their tissue specificity, all IFs share several common attributes, including immunological crossreactivity, similar morphology (e.g. about 10 nm diameter - hence ‘10-nm filaments’) and the ability to reassemble in vitro from denatured subunits into filaments virtually indistinguishable from those observed in vivo. Further more, despite their proteinchemical heterogeneity (their MWs range from 40 kDa to 200 kDa and their isoelectric points from about 5 to 8), protein and cDNA sequencing of several IF polypeptides (for refs, see 1,2) have provided the framework for a common structural model of all IF subunits.

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