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Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.
Almouzni,
Nuclear assembly, structure, and function: the use of Xenopus in vitro systems.
1993, Pubmed,
Xenbase
Almouzni,
Nuclear assembly, structure, and function: the use of Xenopus in vitro systems.
1993,
Pubmed
,
Xenbase
Bennink,
Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers.
2001,
Pubmed
,
Xenbase
Cui,
Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure.
2000,
Pubmed
Davey,
Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution.
2002,
Pubmed
,
Xenbase
Eltsov,
Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ.
2008,
Pubmed
Fakan,
The functional architecture of the nucleus as analysed by ultrastructural cytochemistry.
2004,
Pubmed
Fischle,
Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation.
2005,
Pubmed
,
Xenbase
Freedman,
Xenopus egg extracts increase dynamics of histone H1 on sperm chromatin.
2010,
Pubmed
,
Xenbase
Hameed,
Probing structural stability of chromatin assembly sorted from living cells.
2009,
Pubmed
Hirano,
A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro.
1994,
Pubmed
,
Xenbase
Hirano,
Cell cycle control of higher-order chromatin assembly around naked DNA in vitro.
1991,
Pubmed
,
Xenbase
Hirano,
Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts.
1993,
Pubmed
,
Xenbase
Horowitz,
The three-dimensional architecture of chromatin in situ: electron tomography reveals fibers composed of a continuously variable zig-zag nucleosomal ribbon.
1994,
Pubmed
Horowitz-Scherer,
Organization of interphase chromatin.
2006,
Pubmed
Huynh,
A method for the in vitro reconstitution of a defined "30 nm" chromatin fibre containing stoichiometric amounts of the linker histone.
2005,
Pubmed
Kireeva,
Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure.
2004,
Pubmed
Ladoux,
Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy.
2000,
Pubmed
,
Xenbase
Laskey,
Assembly of SV40 chromatin in a cell-free system from Xenopus eggs.
1977,
Pubmed
,
Xenbase
Losada,
Identification of Xenopus SMC protein complexes required for sister chromatid cohesion.
1998,
Pubmed
,
Xenbase
Luger,
Crystal structure of the nucleosome core particle at 2.8 A resolution.
1997,
Pubmed
Lusser,
Strategies for the reconstitution of chromatin.
2004,
Pubmed
MacCallum,
ISWI remodeling complexes in Xenopus egg extracts: identification as major chromosomal components that are regulated by INCENP-aurora B.
2002,
Pubmed
,
Xenbase
Maresca,
Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.
2006,
Pubmed
,
Xenbase
Maresca,
Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts.
2005,
Pubmed
,
Xenbase
McDowall,
Cryo-electron microscopy of vitrified chromosomes in situ.
1986,
Pubmed
Noll,
Subunit structure of chromatin.
1974,
Pubmed
Paulson,
The structure of histone-depleted metaphase chromosomes.
1977,
Pubmed
Robinson,
EM measurements define the dimensions of the "30-nm" chromatin fiber: evidence for a compact, interdigitated structure.
2006,
Pubmed
Ruberti,
Mechanism of chromatin assembly in Xenopus oocytes.
1986,
Pubmed
,
Xenbase
Sandaltzopoulos,
A solid-phase approach for the analysis of reconstituted chromatin.
1999,
Pubmed
,
Xenbase
Thoma,
Influence of histone H1 on chromatin structure.
1977,
Pubmed
Thoma,
Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin.
1979,
Pubmed
Wang,
Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin.
2002,
Pubmed
Wignall,
The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts.
2003,
Pubmed
,
Xenbase
Woodcock,
Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length.
1994,
Pubmed
Woodcock,
Chromatin higher-order structure and dynamics.
2010,
Pubmed
Yan,
Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics.
2007,
Pubmed
,
Xenbase
Zheng,
Salt-dependent intra- and internucleosomal interactions of the H3 tail domain in a model oligonucleosomal array.
2005,
Pubmed
,
Xenbase
