Identification and Characterization of Histone H3 Lysine 23 Methylation in Germline Chromatin

Eukaryotic DNA and histone proteins are packaged into chromatin that can occur in two cytologically distinct states: heterochromatin, which is highly compacted and transcriptionally repressive; or euchromatin, which is relatively open and transcriptionally permissive. Increasing experimental evidence suggest that the structure and function of chromatin is governed, in part, by histone post-translational modifications (PTMs), but their characterization is generally protracted because of the biological complexities associated with higher eukaryotes. Moreover, recent studies have shown that euchromatin and heterochromatin represent the extremes of a continuum of physiological chromatin states, which may be encoded for by distinct combinations of histone PTMs. These states also have transcription-independent roles during interphase, mitosis, and meiosis that contribute to, among other activities, maintaining genomic stability. To screen for novel heterochromatin associated histone PTMs, we took advantage of the nuclear dimorphism afforded by the single-celled ciliate Tetrahymena thermophila. This model organism has two physically separable nuclei with its cytoplasm: a heterochromatic micronucleus (germline genome), and a euchromatic macronucleus (somatic genome). In this dissertation, I present our work identifying and characterizing histone H3 lysine 23 methylation (H3K23me) in the micronucleus. We report that H3K23 trimethylation (H3K23me3) is a micronucleus-specific heterochromatin modification that becomes particularly enriched during meiosis. Loss of H3K23me3, through deletion of its putative methyltransferase Ezl3p, causes mislocalization of meiosis-induced DNA double-strand breaks to heterochromatin, and decreases progeny viability. We extend the significance of H3K23me3 to higher eukaryotes by showing its meiotic upregulation is conserved in germlines of C. elegans and mice. In summary, our results suggest that the evolutionary conserved H3K23me3 plays an important role in protecting meiotic, germline heterochromatin from programmed DSBs

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymenas germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.</p

Human Fecal Bile Acid Analysis after Investigational Microbiota-Based Live Biotherapeutic Delivery for Recurrent <i>Clostridioides difficile</i> Infection

Microbiome-based therapeutics are increasingly evaluated as a strategy to reduce recurrent Clostridioides difficile infection (rCDI), with proposed mechanisms including restoration of the microbiota and microbiota-mediated functions, such as bile acid (BA) metabolism. This study reports a quantitative and sensitive assay for targeted metabolomic assessment, and the application of the assay to profile BA composition in a Phase 2 trial of the investigational microbiota-based live biotherapeutic RBX2660 for reduction of rCDI. A liquid chromatography tandem mass spectrometry method was developed to extract and quantify 35 BAs from 113 participant stool samples from 27 RBX2660-treated rCDI participants in the double-blinded, placebo-controlled clinical trial. The results demonstrate a high-confidence assay as represented by sensitivity, linearity, accuracy, and precision. Furthermore, the assay enabled the observation of primary BAs as the dominant BA species at baseline in stool samples from clinical trial participants, consistent with the expected loss of commensals after broad-spectrum antibiotic treatment. After RBX2660 administration, there was a significant drop in primary BAs concurrent with increased secondary BAs that sustained through 24 months post-RBX2660. Taken together, we describe a robust assay that demonstrates altered BA metabolism in rCDI patients treated with RBX2660 administration

Diet-Induced Circadian Enhancer Remodeling Synchronizes Opposing Hepatic Lipid Metabolic Processes

Overnutrition disrupts circadian metabolic rhythms by mechanisms that are not well understood. Here, we show that diet-induced obesity (DIO) causes massive remodeling of circadian enhancer activity in mouse liver, triggering synchronous high-amplitude circadian rhythms of both fatty acid (FA) synthesis and oxidation. SREBP expression was rhythmically induced by DIO, leading to circadian FA synthesis and, surprisingly, FA oxidation (FAO). DIO similarly caused a high-amplitude circadian rhythm of PPARα, which was also required for FAO. Provision of a pharmacological activator of PPARα abrogated the requirement of SREBP for FAO (but not FA synthesis), suggesting that SREBP indirectly controls FAO via production of endogenous PPARα ligands. The high-amplitude rhythm of PPARα imparted time-of-day-dependent responsiveness to lipid-lowering drugs. Thus, acquisition of rhythmicity for non-core clock components PPARα and SREBP1 remodels metabolic gene transcription in response to overnutrition and enables a chronopharmacological approach to metabolic disorders

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum. DOI: http://dx.doi.org/10.7554/eLife.19090.00