Single molecule fluorescence spectroscopy and microscopy in epigenetics
Abstract
Epigenetics, defined as heritable changes in phenotype or gene expression not related to changes in DNA sequences, play a fundamental role in the development and differentiation of cells and has become a hallmark of diagnostic and prevention strategies in addition to its impact on basic science. Predominant epigenetic modifications are DNA methylation and histone modifications which are thought to affect genome regulation and function. However, the question of how histone modifications influence cellular development and differentiation is largely unknown. My thesis work develops advanced single molecule fluorescence spectroscopy and microscopy tools to evaluate histone modifications in vitro and in vivo to provide a new dimension to field of epigenetics to avail the possibility to explore epigenetic mechanism and phenomenon in living cells. The first part of my thesis focuses on the development of multiparameter single molecule fluorescence techniques including fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), fluorescence lifetime imaging, fluorescence resonance energy transfer (FLIM-FRET), photon counting histogram and fluorescence lifetime correlation spectroscopy (FLCS). It allows simultaneous monitoring of diffusion time, direct concentration, fluorescence lifetime, fluorescence intensity as well as the imaging of the fluorescence molecules. The platform established here not only opens up new opportunities in studying biomolecule interaction and phenomenon at single molecule sensitivity but also provides new insights in biomedical research. In the third and the fourth chapter of this dissertation, donor-acceptor FRET pairs are used to investigate histone modification patterns in single nucleosomes as well as in fixed cells using multiparameter single molecule fluorescence techniques. The composition of histone H2A variant H2A.Z/Htz1p is examined through PCH. We find that yeast nucleosomes containing Htz1p are primarily comprised of two copies of Htz1p, H4 K12ac and H3 K4me3 but not H3 K36me3 and these patterns are conserved in mammalian cells. Finally, we develop a live cell antibody delivery method to target histone modifications and core proteins with high efficiency. This allowed the possibility to explore transcription activation and repression dynamics associated with specific histone modification in live cells. It is also found that transcription activation associated histone modification (H4 K12ac and H3 K4me3) diffuses much faster compared to transcription repression associated histone modification (H3 K27me3 and H3 K9me2). Combing those powerful techniques, quantification of epigenetic patterns and dynamics in mononucleosome and live cells could help us understand epigenetic patterns in individual cells and characterization of multiprotein machineries that are involved in histone variant exchange which could potentially revolutionize our understanding of gene regulation.
Degree
Ph.D.
Advisors
Irudayaraj, Purdue University.
Subject Area
Biomedical engineering|Biophysics
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