CHARACTERIZATION OF STRUCTURE AND FUNCTION OF HISTONES IN EMBRYONIC STEM CELLS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It has been suggested that chromatin-involved epigenetic mechanisms affect the accessibility of effector proteins and regulate important downstream cellular activities via modifications of chromatin structure resulting from combinatorial covalent modifications of histones.Despite its significance, comprehensive characterization of modifications on mammalian histones and their function remains a challenge because of its complexity especially the overwhelming number of modifications on them.In this project, firstly, we will separate the histones by chromatography first and then (1) apply topdown FTICR/ECD/MS strategy to directly characterize modifications on H1, H2, H4 and (2) apply our screening strategy and middle-down FTICR/ECD/MS technique to comprehensively characterize the modifications, we will perturb the biological system by gene deletion and RNA interference techniques and then employ our mass spectrometry strategies to characterize all the induced changes in modification due to the perturbation.Mass spectrometry will play a central and indispensable role in this project. There are no other alternative methods by which we can comprehensively characterize all the modifications in order to elucidate their biological functions.