Investigating effects of histone tail modification on chromatin compaction in nucleosome arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

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Histone modifications at the grapevine VvOMT3 locus, which encodes an enzyme responsible for methoxypyrazine production in the berry

Functional Plant Biology ◽

10.1071/fp16434 ◽

2017 ◽

Vol 44 (7) ◽

pp. 655 ◽

Cited By ~ 3

Author(s):

Juri Battilana ◽

Jake D. Dunlevy ◽

Paul K. Boss

Keyword(s):

Histone Modifications ◽

Chromatin Immunoprecipitation ◽

Developmental Regulation ◽

Grape Variety ◽

Histone Tail ◽

Dicarbonyl Compound ◽

Grape Berries ◽

Allelic Differences ◽

Chromatin Arrangement ◽

Histone Tail Modification

Some herbaceous characters in wine are attributed to the presence of aroma compounds collectively known as methoxypyrazines (MPs). In grape berries their formation has been hypothesised to start from a reaction of two amino acids or an amino acid and an unknown 1,2-dicarbonyl compound, leading to the formation of hydroxypyrazine, which is then enzymatically methylated to form a MP. The enzyme responsible of the formation of 3-isobutyl-2-methoxypyrazine has been recently identified as VvOMT3 whose regulation is still not understood. The concentration of MPs in grapes is known to be influenced by development, environmental stimuli and most importantly grape variety. In order to investigate the chromatin arrangement of that region a chromatin immunoprecipitation analysis has been performed and putative differences in epigenetic regulation of VvOMT3 spatially between the skin and flesh tissues and also temporally during fruit development have been detected. There are also allelic differences in VvOMT3 histone modifications which are maintained in subsequent generations. This study provides evidence of histone tail modification of the VvOMT3 locus in grapevine, which may play a role in the spatial and developmental regulation of the expression of this gene.

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Detection of interactions between nucleosome arrays mediated by specific core histone tail domains

Methods ◽

10.1016/j.ymeth.2006.08.012 ◽

2007 ◽

Vol 41 (3) ◽

pp. 278-285 ◽

Cited By ~ 11

Author(s):

P KAN ◽

J HAYES

Keyword(s):

Core Histone ◽

Histone Tail ◽

Core Histone Tail ◽

Nucleosome Arrays

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The H3 Tail Domain Participates in Multiple Interactions during Folding and Self-Association of Nucleosome Arrays

Molecular and Cellular Biology ◽

10.1128/mcb.02181-06 ◽

2007 ◽

Vol 27 (6) ◽

pp. 2084-2091 ◽

Cited By ~ 81

Author(s):

Pu-Yeh Kan ◽

Xu Lu ◽

Jeffrey C. Hansen ◽

Jeffrey J. Hayes

Keyword(s):

Short Range ◽

Chromatin Condensation ◽

Cross Linking ◽

Tail Domain ◽

Histone Tail ◽

Linker Histones ◽

Complex Interactions ◽

Self Association ◽

Core Histone Tail ◽

Nucleosome Arrays

ABSTRACT The core histone tail domains play a central role in chromatin structure and epigenetic processes controlling gene expression. Although little is known regarding the molecular details of tail interactions, it is likely that they participate in both short-range and long-range interactions between nucleosomes. Previously, we demonstrated that the H3 tail domain participates in internucleosome interactions during MgCl2-dependent condensation of model nucleosome arrays. However, these studies did not distinguish whether these internucleosome interactions represented short-range intra-array or longer-range interarray interactions. To better understand the complex interactions of the H3 tail domain during chromatin condensation, we have developed a new site-directed cross-linking method to identify and quantify interarray interactions mediated by histone tail domains. Interarray cross-linking was undetectable under salt conditions that induced only local folding, but was detected concomitant with salt-dependent interarray oligomerization at higher MgCl2 concentrations. Interestingly, lysine-to-glutamine mutations in the H3 tail domain to mimic acetylation resulted in little or no reduction in interarray cross-linking. In contrast, binding of a linker histone caused a much greater enhancement of interarray interactions for unmodified H3 tails compared to “acetylated” H3 tails. Collectively these results indicate that H3 tail domain performs multiple functions during chromatin condensation via distinct molecular interactions that can be differentially regulated by acetylation or binding of linker histones.

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The Dynamic Influence of Linker Histone Saturation within the Poly-Nucleosome Array

10.1101/2020.09.20.305581 ◽

2020 ◽

Author(s):

Dustin C. Woods ◽

Francisco Rodríguez-Ropero ◽

Jeff Wereszczynski

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Long Range ◽

Chromatin Structure ◽

Linker Histone ◽

Linker Histones ◽

Chromatin Compaction ◽

Chromatin Fibers ◽

Dynamics Simulations ◽

Nucleosome Arrays

AbstractLinker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction.SignificanceLinker histones dynamically bind to DNA in chromatin fibers and serve as epigentic regulators. However, the extent to which they influence the gamut of chromatin architecture is still not well understood. Using molecular dynamics simulations, we studied compact octa-nucleosome arrays with and without the H1 linker histone to better understand the mechanisms dictating the structure of the chromatin fiber. Inclusion of H1 results in stabilization of the compact chromatin structure, while its removal results in a major conformational change towards an untwisted ladder-like state. The increased rigidity and correlations within the H1-bound array suggests that H1-saturated chromatin fibers are better suited to transferring long-range epigentic information.

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Reconstitution of recombinant chromatin establishes a requirement for histone-tail modifications during chromatin assembly and transcription

Genes & Development ◽

10.1101/gad.937401 ◽

2001 ◽

Vol 15 (21) ◽

pp. 2837-2851

Author(s):

Alejandra Loyola ◽

Gary LeRoy ◽

Yuh-Hwa Wang ◽

Danny Reinberg

Keyword(s):

Rna Polymerase Ii ◽

Histone H3 ◽

Dependent Manner ◽

Histone Tail ◽

Histone Tails ◽

Transcription System ◽

Nuclear Extracts ◽

Chromatin Acetylation ◽

Rna Polymerase Ii Transcription ◽

Nucleosome Arrays

The human ISWI-containing factor RSF (remodeling andspacing factor) was found to mediate nucleosome deposition and, in the presence of ATP, generate regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation was impaired in directing transcription. Histone-tail modifications were found to regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 was found to increase transcription. Methylation of the histone H3 tail by Suv39H1 was found to repress transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system was refractory to the effect imposed by acetylation and methylation.

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Epigenetic Control of a Local Chromatin Landscape

International Journal of Molecular Sciences ◽

10.3390/ijms21030943 ◽

2020 ◽

Vol 21 (3) ◽

pp. 943 ◽

Cited By ~ 4

Author(s):

Anna M. Chiarella ◽

Dongbo Lu ◽

Nathaniel A. Hathaway

Keyword(s):

Gene Expression ◽

Eukaryotic Cell ◽

Histone Tail ◽

Cellular Processes ◽

Technological Advances ◽

Specific Manner ◽

Chromatin Biology ◽

Influence Gene Expression ◽

Histone Tail Modification ◽

Made In

Proper regulation of the chromatin landscape is essential for maintaining eukaryotic cell identity and diverse cellular processes. The importance of the epigenome comes, in part, from the ability to influence gene expression through patterns in DNA methylation, histone tail modification, and chromatin architecture. Decades of research have associated this process of chromatin regulation and gene expression with human diseased states. With the goal of understanding how chromatin dysregulation contributes to disease, as well as preventing or reversing this type of dysregulation, a multidisciplinary effort has been launched to control the epigenome. Chemicals that alter the epigenome have been used in labs and in clinics since the 1970s, but more recently there has been a shift in this effort towards manipulating the chromatin landscape in a locus-specific manner. This review will provide an overview of chromatin biology to set the stage for the type of control being discussed, evaluate the recent technological advances made in controlling specific regions of chromatin, and consider the translational applications of these works.

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Acetylation Mimics within Individual Core Histone Tail Domains Indicate Distinct Roles in Regulating the Stability of Higher-Order Chromatin Structure

Molecular and Cellular Biology ◽

10.1128/mcb.01245-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 227-236 ◽

Cited By ~ 118

Author(s):

Xiaodong Wang ◽

Jeffrey J. Hayes

Keyword(s):

Chromatin Structure ◽

Higher Order ◽

Core Histone ◽

Lysine Acetylation ◽

Tail Domain ◽

Histone Tail ◽

Self Association ◽

Core Histone Tail ◽

The Stability ◽

Nucleosome Arrays

ABSTRACT Nucleosome arrays undergo salt-dependent self-association into large oligomers in a process thought to recapitulate essential aspects of higher-order tertiary chromatin structure formation. Lysine acetylation within the core histone tail domains inhibits self-association, an effect likely related to its role in facilitating transcription. As acetylation of specific tail domains may encode distinct functions, we investigated biochemical and self-association properties of model nucleosome arrays containing combinations of native and mutant core histones with lysine-to-glutamine substitutions to mimic acetylation. Acetylation mimics within the tail domains of H2B and H4 caused the largest inhibition of array self-association, while modification of the H3 tail uniquely affected the stability of DNA wrapping within individual nucleosomes. In addition, the effect of acetylation mimics on array self-association is inconsistent with a simple charge neutralization mechanism. For example, acetylation mimics within the H2A tail can have either a positive or negative effect on self-association, dependent upon the acetylation state of the other tails and nucleosomal repeat length. Finally, we demonstrate that glutamine substitutions and lysine acetylation within the H4 tail domain have identical effects on nucleosome array self-association. Our results indicate that acetylation of specific tail domains plays distinct roles in the regulation of chromatin structure.

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