Approaching DNA methylation at the nanoscale

Yi Cui, Purdue University

Abstract

Epigenetics involves a variety of biochemical modifications occurring on chromatin that are able to regulate and fine-tune genetic activities without altering the underlying DNA sequence. So far, four types of epigenetic modulation have been extensively studied: DNA methylation, histone post-translational modification, non-coding RNA, and nuclear organization. A fast-growing body of evidence suggests that epigenetic mechanism plays a fundamental role in physiology and pathology. Of the elucidated epigenetic processes, DNA methylation is the one under intense research due to its direct impact on gene expression, which dynamically bridges the microscale genotype and macroscale phenotype. However, to date our understanding of DNA methylation has primarily come from ensemble and end-point measurements using a population of cells or bulky samples. Unlike genetic aberrations (e.g., amplification, deletion, translocation, and mutation) that are rare to occur and resistant to reverse, events relating to epigenetic modifications take place in a time- and context-dependent manner, thus necessitating nanoscale tools to dissect epigenetic dynamics at a finer spatiotemporal resolution. In my Ph.D. work, a group of advanced single-molecule fluorescence tools, in addition to methodologies in bioengineering, nanotechnology, molecular biology, and bioinformatics, is implemented to approach the DNA methylation-related activities. After the introduction chapter, the second and third chapters of my dissertation center on elucidating the behavior of MBD3 protein within a single living cell, in order to infer the real-time dynamics of DNA methylation. Taking use of fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging (FLIM), Förster resonance energy transfer (FRET), and photon counting histogram (PCH), the in situ binding preference, dissociation kinetics, and stoichiometric transition of MBD3 were probed with unprecedented precision. Due to the substitution of two amino acids in its DNA-binding motif, MBD3 has evolved to differ from other MBD homologues in physicochemical properties as well as in biological functions. Consequently, its altered binding affinity and specificity with DNA methylation lead to unique molecular behaviors that can be sensed by single-molecule techniques. In this line of research, 1) the intracellular diffusion of MBD3 was explored to monitor active DNA demethylation and 2) the cell cycle-dependent activity of MBD3 was characterized. In these attempts my study establishes the dissociation of MBD3-DNA as a marking event in active DNA methylation, which might be involved in early oncogenesis. Moreover, the role of MBD3 in the DNA maintenance methylation is revealed for the first time – i.e., it constitutes a key balancing force to sustain the DNA methylation homeostasis. Considering its importance in DNA methylation, MBD3 was proposed to participate in brain cancer because the central nervous system possesses abundant DNA methylation marks and active methyl metabolism. Hence in the fourth chapter of my dissertation the regulatory landscape and clinical implication of MBD3 in human malignant glioma is studied. Utilizing whole-genome transcriptome microarray, the MBD3-mediated epigenetic regulation in glioma was profiled. The data uncover that MBD3 stands in the midst of an exquisite anti-glioma network via influencing a large number of transmembrane proteins and nuclear factors. By analyzing the differentially expressed genes upon depletion of MBD3, the MHC class II molecules were identified to be a group of downstream effectors activated by MBD3, which provides important insight into the anti-glioma immunity. Reactivation of tumor supper genes using DNA demethylating agents has proven to be effective in treating hematopoietic malignancies, but efficient delivery of these drugs into solid tumors is a challenging task. In the fifth chapter a polymeric nano-vector comprising PLGA and PEG is developed to improve the solubility, bioavailability, and therapeutic potency of the FDA-approved DNA demethylating agents – AZA and DAC. With equimolar drug content, the nano-conjugated AZA showed better anti-proliferative effect than free drug in treating xenografted breast cancer. This formulation can also be used to deliver a safe dosage of DAC for sensitizing the alkylation-resistant glioblastoma cells to temozolomide. The sixth chapter of my dissertation presents a light-controllable technique for loci-specific epigenome editing. The optogenetic proteins CRY2-CIB and PHYB-PIF are photosensitive pairs whose associations can be induced by blue and red light, respectively. Under proper illumination, CRY2 or PHYB is rapidly photoactivated to couple with CIB or PIF, and their optogenetic associations are reversible in the absence of the triggering light. The first half of this chapter elucidates the feasibility of using single-molecule fluorescence tools to characterize real-time optogenetic binding kinetics. In the second half, a pair of DNMT3A-CRY2-mCherry and TRF1-CIB1-EGFP fusion proteins was constructed to achieve selective de novo DNA methylation at subtelomeric CpGs. By varying the illumination conditions, the subtelomeric methylation can be incrementally manipulated. These optogenetic modules provide a powerful way to study DNA methylation and can be conveniently adapted for multiplexing epigenome editing and in vivo applications. (Abstract shortened by ProQuest.)

Degree

Ph.D.

Advisors

Irudayaraj, Purdue University.

Subject Area

Molecular biology|Genetics|Cellular biology|Biochemistry|Nanotechnology|Oncology

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