Unique features of the apoptotic endonuclease DFF40/CAD relative to micrococcal nuclease as a structural probe for chromatinThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

2006 ◽  
Vol 84 (4) ◽  
pp. 405-410 ◽  
Author(s):  
Piotr Widlak ◽  
William T. Garrard
Keyword(s):  
Chromatin Structure ◽  
Recombinant Dna ◽  
Peer Review Process ◽  
Micrococcal Nuclease ◽  
Naked Dna ◽  
Dna Library ◽  
Double Stranded Dna ◽  
Structural Probe ◽  
Specific Sequences ◽  
Selection Of

The gold standard for studies of nucleosomal chromatin structure for the past 30 years has been the enzyme micrococcal nuclease (MNase). During the course of our studies on the elucidation of the mechanism of action of the apoptotic nuclease DNA fragmentation factor-40 / caspase-activated deoxyribonuclease (DFF40/CAD) on naked DNA and chromatin substrates, it became clear that this enzyme is superior in certain respects to MNase for studying several aspects of chromatin structure. Here we review our published results supporting this statement. Relative to MNase, we have found that DFF40/CAD has the following properties: (i) it does not cut within nucleosomes to generate subnucleosomal DNA fragments; (ii) it is more specific for the linker regions between nucleosomes; (iii) it lacks exonuclease activity; (iv) it is specific for double-stranded DNA and makes exclusively double-stranded breaks; and (v) it attacks histone-H1-containing chromatin more efficiently. Taken together, these facts explain why DFF40/CAD generates sharper oligonucleosomal DNA ladders compared with those generated by MNase. We therefore recommend the following uses for DFF40/CAD for chromatin research: nucleosome isolation, chromatin-remodeling assays, repeat length measurements, and nucleosome-positioning assays along specific sequences. Other uses include footprinting assays of transcription factor positions, shearing chromatin for immunopreciptitation experiments (ChIP), and shearing DNA for recombinant DNA library preparation or for shotgun cloning for sequencing.

scholarly journals Improving selectivity of DNA-RNA binding zinc finger using directed evolution

2019 ◽  
Author(s):  
Agata Agnieszka Sulej
Keyword(s):  
Directed Evolution ◽  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Amino Acid Residues ◽  
Double Stranded Dna ◽  
Dna Engineering ◽  
Materials Used ◽  
Specific Sequences ◽  
Selection Of

Abstract Objective Type C2H2 zinc fingers bind a variety of substrates among which, are specific sequences in the double-stranded DNA. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrid using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between modules of the zinc finger. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

scholarly journals Improving selectivity of DNA-RNA binding zinc finger using directed evolution

2019 ◽  
Author(s):  
Agata Agnieszka Sulej
Keyword(s):  
Directed Evolution ◽  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Amino Acid Residues ◽  
Double Stranded Dna ◽  
General Rules ◽  
Materials Used ◽  
Specific Sequences ◽  
Selection Of

Abstract Objective Type C2H2 zinc fingers bind a variety of substrates, specific sequences in the double-stranded DNA counting among them. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrids using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between the zinc finger modules. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of the modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

scholarly journals Improving selectivity of DNA-RNA binding zinc finger using directed evolution

2019 ◽  
Author(s):  
Agata Agnieszka Sulej
Keyword(s):  
Directed Evolution ◽  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Amino Acid Residues ◽  
Double Stranded Dna ◽  
General Rules ◽  
Materials Used ◽  
Specific Sequences ◽  
Selection Of

Abstract Objective Type C2H2 zinc fingers bind a variety of substrates, specific sequences in the double-stranded DNA counting among them. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrids using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between the zinc finger modules. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of the modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

scholarly journals Nucleosome distribution and linker DNA: connecting nuclear function to dynamic chromatin structureThis paper is one of a selection of papers published in a Special Issue entitled 31st Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (1) ◽  
pp. 24-34 ◽  
Author(s):  
Heather J. Szerlong ◽  
Jeffrey C. Hansen
Keyword(s):  
Chromatin Structure ◽  
Peer Review Process ◽  
Secondary Structures ◽  
Hierarchical Organization ◽  
Coding Region ◽  
Nucleosomal Array ◽  
Genome Wide ◽  
The Relationship ◽  
Selection Of

Genetic information in eukaryotes is managed by strategic hierarchical organization of chromatin structure. Primary chromatin structure describes an unfolded nucleosomal array, often referred to as “beads on a string”. Chromatin is compacted by the nonlinear rearrangement of nucleosomes to form stable secondary chromatin structures. Chromatin conformational transitions between primary and secondary structures are mediated by both nucleosome-stacking interactions and the intervening linker DNA. Chromatin model system studies find that the topography of secondary structures is sensitive to the spacing of nucleosomes within an array. Understanding the relationship between nucleosome spacing and higher order chromatin structure will likely yield important insights into the dynamic nature of secondary chromatin structure as it occurs in vivo. Genome-wide nucleosome mapping studies find the distance between nucleosomes varies, and regions of uniformly spaced nucleosomes are often interrupted by regions of nonuniform spacing. This type of organization is found at a subset of actively transcribed genes in which a nucleosome-depleted region near the transcription start site is directly adjacent to uniformly spaced nucleosomes in the coding region. Here, we evaluate secondary chromatin structure and discuss the structural and functional implications of variable nucleosome distributions in different organisms and at gene regulatory junctions.

Molecular dynamics of histone H1This paper is one of a selection of papers published in this Special Issue, entitled CSBMCB’s 51st Annual Meeting – Epigenetics and Chromatin Dynamics, and has undergone the Journal’s usual peer review process.

2009 ◽  
Vol 87 (1) ◽  
pp. 189-206 ◽  
Author(s):  
Nikhil Raghuram ◽  
Gustavo Carrero ◽  
John Th’ng ◽  
Michael J. Hendzel
Keyword(s):  
Chromatin Structure ◽  
Peer Review Process ◽  
Imaging Techniques ◽  
Histone H1 ◽  
Regulation Of Transcription ◽  
Chromatin Dynamics ◽  
Biochemical Mechanisms ◽  
Specific Regulation ◽  
Kinetics Of ◽  
Selection Of

The histone H1 family of nucleoproteins represents an important class of structural and architectural proteins that are responsible for maintaining and stabilizing higher-order chromatin structure. Essential for mammalian cell viability, they are responsible for gene-specific regulation of transcription and other DNA-dependent processes. In this review, we focus on the wealth of information gathered on the molecular kinetics of histone H1 molecules using novel imaging techniques, such as fluorescence recovery after photobleaching. These experiments have shed light on the effects of H1 phosphorylation and core histone acetylation in influencing chromatin structure and dynamics. We also delineate important concepts surrounding the C-terminal domain of H1, such as the intrinsic disorder hypothesis, and how it affects H1 function. Finally, we address the biochemical mechanisms behind low-affinity H1 binding.

Reuniting the contrasting functions of H2A.ZThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

2006 ◽  
Vol 84 (4) ◽  
pp. 528-535 ◽  
Author(s):  
Benoît Guillemette ◽  
Luc Gaudreau
Keyword(s):  
Chromatin Structure ◽  
Peer Review Process ◽  
Histone Variants ◽  
Molecular Techniques ◽  
Binding Assays ◽  
Post Translational Modifications ◽  
Genome Wide ◽  
On Chip ◽  
Inactive Chromatin ◽  
Selection Of

It is now well established that cells modify chromatin to set transcriptionally active or inactive regions. Such control of chromatin structure is essential for proper development of organisms. In addition to the growing number of histone post-translational modifications, cells can exchange canonical histones with different variants that can directly or indirectly change chromatin structure. Moreover, enzymatic complexes that can exchange specific histone variants within the nucleosome have now been identified. One such variant, H2A.Z, has recently been the focus of many studies. H2A.Z is highly conserved in evolution and has many different functions, while defining both active and inactive chromatin in different contexts. Advanced molecular techniques, such as genome-wide binding assays (chromatin immunoprecipitation on chip) have recently given researchers many clues as to how H2A.Z is targeted to chromatin and how it affects nuclear functions. We wish to review the recent literature and summarize our understanding of the mechanisms and functions of H2A.Z.

scholarly journals Improving selectivity of DNA–RNA binding zinc finger using directed evolution

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Agata A. Sulej
Keyword(s):  
Directed Evolution ◽  
Zinc Finger ◽  
Rna Binding ◽  
Zinc Fingers ◽  
Amino Acid Residues ◽  
Double Stranded Dna ◽  
General Rules ◽  
Materials Used ◽  
Specific Sequences ◽  
Selection Of

Abstract Objective Type C2H2 zinc fingers bind a variety of substrates, specific sequences in the double-stranded DNA counting among them. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA–RNA hybrids using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between the zinc finger modules. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of the modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA–RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

The nucleosome: a little variation goes a long wayThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

2006 ◽  
Vol 84 (4) ◽  
pp. 505-507 ◽  
Author(s):  
Emily Bernstein ◽  
Sandra B. Hake
Keyword(s):  
Peer Review Process ◽  
Gene Activation ◽  
Epigenetic Inheritance ◽  
Histone Variants ◽  
Post Translational Modifications ◽  
Chromatin Compaction ◽  
Cellular Processes ◽  
Chromatin Template ◽  
Mitosis And Meiosis ◽  
Selection Of

Changes in the overall structure of chromatin are essential for the proper regulation of cellular processes, including gene activation and silencing, DNA repair, chromosome segregation during mitosis and meiosis, X chromosome inactivation in female mammals, and chromatin compaction during apoptosis. Such alterations of the chromatin template occur through at least 3 interrelated mechanisms: post-translational modifications of histones, ATP-dependent chromatin remodeling, and the incorporation (or replacement) of specialized histone variants into chromatin. Of these mechanisms, the exchange of variants into and out of chromatin is the least well understood. However, the exchange of conventional histones for variant histones has distinct and profound consequences within the cell. This review focuses on the growing number of mammalian histone variants, their particular biological functions and unique features, and how they may affect the structure of the nucleosome. We propose that a given nucleosome might not consist of heterotypic variants, but rather, that only specific histone variants come together to form a homotypic nucleosome, a hypothesis that we refer to as the nucleosome code. Such nucleosomes might in turn participate in marking specific chromatin domains that may contribute to epigenetic inheritance.

Major histocompatibility complex class I genes in murine fibrosarcoma IC9 are down regulated at the level of the chromatin structure

1989 ◽  
Vol 9 (7) ◽  
pp. 3136-3142
Author(s):  
U Maschek ◽  
W Pülm ◽  
S Segal ◽  
G J Hämmerling
Keyword(s):  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Chromatin Structure ◽  
Dnase I ◽  
Class I ◽  
Micrococcal Nuclease ◽  
Complex Class ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Nuclear Extracts

The fibrosarcoma IC9 is deficient in the expression of the major histocompatibility complex class I genes Kb, Kk, and Dk and expresses only the Db molecule. Because class I deficiency may enable tumor cells to escape the immune response by cytotoxic T lymphocytes, we investigated why the class I genes are not expressed. Expression of the silent class I genes could not be induced, but all known DNA-binding factors specific for class I genes could be detected in nuclear extracts of IC9 cells. After cloning of the silent Kb gene from the IC9 cells and subsequent transfection of this cloned Kb gene into LTK- and IC9 cells, normal Kb antigens were expressed on the cell surface of both cell lines. Digestion of the chromatin of IC9 cells with micrococcal nuclease and DNase I showed a decreased nuclease sensitivity of the silent class I genes in comparison with active genes and the absence of DNase I hypersensitive sites in the promoter region of the silent Dk gene. These findings demonstrate that class I expression is turned off by a cis-acting regulatory mechanism at the level of the chromatin structure.

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