Early Post-translational Modification of CNEP-A Influences Conformation of Centromeric Chromatin.

Bailey, Aaron Oakley, Department of Cell Biology, University of Virginia
Hunt, Donald F., Department of Cell Biology, University of Virginia

Centromeres are maintained throughout the cell cycle, but during mitosis these sites direct kinetochore assembly and enable the accurate segregation of the duplicated chromosomes to daughter cells. Since the DNA sequences at centromeric sites are neither necessary nor required for forming centromeres, the best candidate for establishing centromere identity is presence of the specialized H3 variant CENP-A. The CENP-A loading pathway describes the process in which complexes of new CENP-A are recruited to existing centromeres, maintaining accurate localization through DNA replication and then inherited by daughter cells in mitosis. The presence of CENP-A is highly conserved and required for eukaryotic life to exist. We sought to use mass spectrometry to detect CENP-A PTMs which may be important for the progression of the CENP-A loading pathway. Our initial attempts to identify post-translational modifications on CENP-A were not successful due to an anomalous biochemical property of CENP-A which prevented efficient detection by mass spectrometry. We developed a generic assay to directly test protein solubility, finding that CENP-A becomes insoluble in our conventional sample preparation. We utilized this assay to screen for ideal buffer components and developed a detergent replacement sample preparation strategy which utilizes an acid-cleavable surfactant to maintain CENP-A solubility during the protease digestion steps required for mass spectrometry. We successfully applied our tailored sample preparation to the analysis of posttranslational modifications (PTMs) of CENP-A and proteins associated with CENP-A at multiple cell cycle time points and cellular compartments. We discovered multiple previously-unknown modifications on CENP-A including simultaneous phosphorylation of serines S16 and S18, as well as N-terminal trimethylation. Our in vitro reaction data demonstrates that this latter PTM is catalyzed specifically by N-terminal RCC1 Methyltransferase. We additionally identified the predominant combinatorial modifications states of centromeric histones H3 and H4, finding that monomethylation of H4K20 is a specific mark enriched at centromeric loci. Lastly, we discovered several previously-unreported serine phosphorylations on the CENP-A-bound form of the specific chaperone HJURP. We use a limited survey of biophysical techniques to test for the structural and functional consequences of phosphorylation on S16/S18 located on the CENP-A N- terminal tail. We found that phosphorylation causes tails to become more compact and that at millimolar-range concentrations permitted non-covalent tail-tail interactions. Our analytical centrifugation measurements resolved that polynucleosome arrays assembled using a phospho-mimetic S16D/S18D CENP-A preferentially form intra-array interactions. This behavior is distinct from that exhibited by the unmodified wild type CENP-A-containing arrays which readily form inter-array interactions and become oligomerized. Our calculations suggest that high concentrations of S16/S18- phosphorylated CENP-A in chromatin causes tail-tail associations on adjacent CENP-A nucleosomes and results in local chromatin compaction. We propose a model in which S16/S18 phosphorylation is involved in guiding the higher-order organization of centromeric chromatin structure important for normal kinetochore function.

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PHD (Doctor of Philosophy)
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