Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract
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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.
KeywordsAtomic Force Microscope Imaging Chromatin Fiber Atomic Force Microscope Probe Chromatin Assembly Nucleosome Array
We are grateful to Assistant Professor CA Davey and Ms. MS Ong (Nanyang Technological University) for preparing the reconstituted 12-mer nucleosome array sample, Dr. FM Hameed and Professor GV Shivashankar (Mechanobiology Institute, Singapore) for helping prepare the chromatin sample from live cell nuclei, and Professor JF Marko (Northwestern University) for stimulating discussions. This work was supported by R144000192112 and R144000251112 from the Ministry of Education of Singapore (to JY) and from the Mechanobiology Institute at the National University of Singapore. Work at UC Berkeley was supported by GM057839 to (RH) from The National Institutes of Health.
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