Probing structural stability of chromatin assembly sorted from living cells.
Post-translational modifications of the histone tails and other chromatin binding proteins affect the stability of chromatin structure. In this study, we have purified chromatin from live cell nuclei using a fluorescence activated cell sorter (FACS) and studied the structural stability of this self-assembled structure. Using total internal reflection fluorescence (TIRF) microscopy, we map the effect of covalent modifications on the interaction of histone-DNA complex, by measuring the dissociation rates of histones from the chromatin fiber in the presence of different salt concentrations. Dynamic force spectroscopy (DFS) experiments were carried out to measure the structural disintegration of large chromatin globules under force. The characteristic rupture of multiple linkages in the large chromatin globules show differences in the stiffness of the higher order structure of chromatin with altered epigenetic states. Our studies reveal a direct correlation between histone modifications and the structural stability of higher order chromatin assembly.
Hameed
Feroz M
FM
National Centre for Biological Sciences, TIFR, Bellary Road, Bangalore 560065, India.
Soni
G V
GV
Krishnamurthy
H
H
Shivashankar
G V
GV
D002467:Cell Nucleus / Q000737:chemistry; D002469:Cell Separation; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D004247:DNA / Q000737:chemistry / Q000648:ultrastructure; D005434:Flow Cytometry; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000648:ultrastructure; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D011499:Protein Processing, Post-Translational; D013057:Spectrum Analysis
D002843:Chromatin; D006657:Histones; D004247:DNA