ORCID

https://orcid.org/0000-0001-5266-6734

Date of Award

Spring 5-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The human gut microbiota is home to tens of trillions of microbes belonging to all three domains of life. The structure and expressed functions of this community have myriad effects on host physiology, metabolism, and immune function. My studies focused on a facet of host-microbial interactions and mutualism that has not been explored to a significant degree in part because of the absence of suitable tools: namely, if, when, and how the gut microbiota produces durable effects on host biology through its impact on the epigenome. To address this area, I turned to gnotobiotic mice and developed a variety of experimental, methodological, and computational approaches to characterize the chromatin landscape of various host cell populations. I selected small populations of T cells likely to be exposed to the gut microbiota and its products, principally TCR αβ+ and TCR γδ+ small intestinal intraepithelial lymphocytes (IELs). I also chose to study circulating CD4+ and CD8+ T cells, with the advantage that these populations can be isolated from donors without using highly invasive techniques. I designed a series of approaches to enrich, isolate, and purify each of these cell populations from single animals, and to subsequently compare their chromatin landscape within and across mice using a recently described Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq), focusing on enhancer and super-enhancer loci within the mouse genome. These analyses revealed a conserved set of super-enhancer loci between αβ and γδ IELs, including super-enhancers near genes responsible for phospholipid binding and T cell receptor signaling. In comparing C57BL/6J male mice reared under germ-free (GF) conditions to age- and sex-matched conventionally raised (CONV-R) mice (i.e., animals that acquired microbes from their environment beginning at birth), I was able to directly assess the impact of colonization on chromatin ‘state’ in these different purified cell populations and to identify colonizationdependent effects in IELs on enhancers associated with genes involved in a number of metabolic and signaling pathways. I then compared the results to GF mice that had been colonized following the end of the weaning period with an intact cecal microbiota from a CONV-R C57BL/6J donor. The resulting conventionalized (CONV-D) animals allowed me to identify modifications to host chromatin landscape that are ‘induced’ following the suckling-weaning transition and, in doing so, ascertain whether there were developmental windows that could constrain the durable effects of the microbiota on chromatin accessibility. In doing so, I observed changes in chromatin accessibility with colonization that may reveal a functional maturation of IEL populations that is related to the timing of exposure to the microbiota during postnatal development. My thesis project involved development of an elaborate, multi-faceted computational pipeline for the analysis of these novel, large datasets, including the prediction and characterization of putative enhancers and super-enhancers, proximally associated genes, and metabolic pathways influenced by those elements. As a whole, this work defines the impact of gut microbial colonization on host chromatin landscape and provides an analytical toolkit for further studies.

Language

English (en)

Chair and Committee

Jeffrey I. Gordon

Committee Members

Barak Cohen, Gautam Dantas, Todd Druley, Daniel Goldberg,

Comments

Permanent URL: https://doi.org/10.7936/K77M06C1

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