Browsing by Subject "Methylation"

In the following document, I will describe two distinct strategies that poliovirus

(PV) deploys to manage host antiviral responses. In the first section, I report on a role of

the constitutive repressor of eIF2α phosphorylation (CReP) in translation of PV and the

endoplasmic reticulum (ER)-resident chaperone binding immunoglobulin protein (BiP)

at the ER. Functional, proximity-dependent labeling and cell fractionation studies

revealed that CReP, through binding of the eukaryotic translation initiation factor eIF2α,

anchors translation initiation machinery at the ER and enables protein synthesis in this

compartment. This ER site was protected from the suppression of cytoplasmic protein

synthesis by acute stress responses. I propose that partitioning of translation initiation

machinery at the ER enables cells to maintain active translation of PV during stress.

In the second section, I report that PV 2A protease cleaves all three members of

the YTHDF protein family, cytosolic N6-methyladenosine (m6A) ‘readers’ that regulate

target mRNA fate. These cleavages occurred early during infection, and preemptive

YTHDF3 depletion enhanced viral replication. This corresponded with diminished type-

I interferon (IFN) receptor (IFNAR) expression and IFN-stimulated gene induction,

while IFN production was not significantly changed. I propose that 2A protease cleaves

YTHDF proteins, in part, to interfere with IFNAR expression and antagonize the host

antiviral response.

Low-dose ionizing radiation (LDIR) exposure (under 10.0 centigray (cGy)) from man-made sources, such as diagnostic imaging, predominates in the US population and comprises nearly 50% of an average individual's yearly radiation exposure (Ullrich, Brooks et al. 2009). The increase in such exposures has led to public and government alarm about the impact of LDIR on human health (Ullrich, Brooks et al. 2009). Besides the mutational effects of radiation exposure, there is concern it might also result in modifications of the epigenome. Such aberrations can disrupt normal development and are involved in the progression of numerous diseases, including cancer (Gasser and Li 2011). High doses of radiation (>100.0 cGy) have been shown to cause epigenetic disruption (Kaup, Grandjean et al. 2006; Tamminga, Koturbash et al. 2008; Ilnytskyy, Koturbash et al. 2009), which is necessary for the persistence of radiation-induced genomic instability (Rugo, Mutamba et al. 2011); however, it is presently unclear to what extent LDIR in vivo alters the epigenome.

The viable yellow agouti (Avy) mouse was used here to characterize the dose-dependent epigenetic response to LDIR. The Avy mouse is a unique biological model that functions as a biosensor for environmentally induced epigenetic changes and disease susceptibility due to the presence of a metastable epiallele that modulates coat color (Waterland and Jirtle 2003). Pregnant dams were whole-body exposed to one of five doses of X-ray radiation ranging from 0-10.0 cGy on gestational day 4.5. Using a phantom mouse model, the intrauterine doses were estimated to be 0.0 cGy, 0.4 cGy, 0.7 cGy, 1.4 cGy, 3.0 cGy, and 7.6 cGy, respectively. At weaning, offspring coat colors were assessed and tissues were collected for methylation analysis. First, methylation changes at CpG sites in the Avy and Cdk activator binding protein (CabpIAP) metastable epialleles and at intracisternal a particle (IAP) elements across the genome were quantified using Sequenom technology. Second, three imprinted genes, Peg3, Nnat, and H19, were assessed for methylation changes in differentially methylated regions (DMRs) that regulate their parent-of-origin monoallelic expression using Sequenom technology. Lastly, it was postulated that the epigenetic changes at the Avy locus could be counteracted with dietary alterations. To test this hypothesis, female mice were placed on an antioxidant-supplemented diet prior to pregnancy and throughout gestation and lactation. Pregnant dams were irradiated with 3.0 cGy of whole-body X-rays. Offspring coat colors were assessed and methylation changes at the Avy allele were measured with the Sequenom platform.

Herein, I demonstrate that in utero LDIR exposure induced epigenetic changes in the Avy mouse in a dose-dependent and sex-specific manner. Acute, whole-body exposure to 0.7 cGy, 1.4 cGy, 3.0 cGy or 7.6 cGy X-rays significantly shifted offspring coat color distribution toward pseudoagouti. Acute exposure to 1.4 cGy, 3.0 cGy, and 7.6 cGy significantly increased methylation at multiple CpG sites in the Avy metastable epiallele in male offspring, but not female offspring. Methylation changes at DMRs in Nnat, Peg3, and H19 also occurred in a dose-dependent manner. Furthermore, inhibition of the phenotypic and Avy methylation changes with an antioxidant-supplemented diet suggests that the mechanisms to induce epigenetic changes are mediated by oxidative stress. These results demonstrate that relevant, low doses of radiation can elicit epigenetic changes that lead to a persistent phenotype, but can be mitigated with dietary supplementation. The successful completion of this project has resulted in the first in vivo epigenetic characterization of LDIR exposure and will contribute to the development of more relevant risk assessment strategies for protecting human populations.

Point mutations in the active site of isocitrate dehydrogenases 1 and 2 (\textit{IDH}) occur in the majority of WHO grade II and III gliomas, resulting in a unique milieu of signaling and metabolism. IDH1/2 active site mutations confer a gain-of-function activity to the enzyme, which results in the production of the oncometabolite D-2-hydroxyglutarate (D-2HG). D-2HG accumulation in turn promotes tumor formation through competitive inhibition of $\alpha$-ketoglutarate dependent ($\alpha$-KG) enzymes. Inhibition of $\alpha$-KG-dependent enzymes, such as histone demethylases and DNA demethylases, is sufficient to induce tumor-promoting epigenetic changes, but can also impose situational constraints on cell proliferation. To develop better therapies for mutant IDH1-bearing gliomas, it is essential to determine whether the epigenetic changes induced by the mutant IDH proteins actively require the mutation after tumor formation. Furthermore, it is imperative to decode the molecular mechanisms that promote tumor cells’ fitness under IDH mutation-dependent constraints in representative models. Here, we describe and characterize CRISPR-Cas9 based isogenic cell line models using patient-derived IDH1$^{R132H/WT}$ glioma cell lines. We uncover that these models show persistent DNA hypermethylation in CpG loci of the glioma CpG island methylator phenotype even after D-2HG production has been abolished. We also report a genome wide pattern of DNA demethylation in CpG sites outside of CpG islands, which reflect the acquisition of a G-CIMP-low like state after loss of D-2HG production. Then, using these cell line tools, we performed an unbiased sub-genomic CRISPR-library screening to identify genes whose functions supported the growth of glioma cells bearing endogenous IDH1 mutations. This work thus provides new patient derived models for exploring novel therapeutic opportunities for IDH1 mutant tumors, and uncovers the extent to which IDH mutation linked hypermethylation profiles in glioma depend upon D-2HG production from the IDH mutation.

Background

Results

Conclusions

Glioma development and progression are driven by complex genetic alterations, including point mutations and gain or loss of genomic copy number, as well as epigenetic aberrations, including DNA methylation and histone modifications. However, the molecular mechanisms underlying the causes and effects of these alterations are poorly understood, and improved treatments are greatly needed. Here, we report a comprehensive evaluation of the recurrent genomic alterations in gliomas and further dissect the molecular effects of the most frequently-occurring genomic events. First, we performed a multifaceted genomic analysis to identify genes targeted by copy number alteration in glioblastoma, the most aggressive malignant glioma. We identify EGFR negative regulator, ERRFI1, as a glioblastoma-targeted gene within the minimal region of deletion in 1p36.23. Furthermore, we demonstrate that Aurora-A kinase substrate, TACC3, displays gain of copy number on 4p16.3 and is overexpressed in a grade-specific pattern. Next, using a gene targeting approach, we knocked-in a single copy of the most frequently observed point mutation in gliomas, IDH1R132H/WT, into a human cancer cell line. We show that heterozygous expression of the IDH1R132H allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Together, these data provide insight on genetic and epigenetic alterations which drive human gliomas.

If nothing else, the 2019 Coronavirus pandemic has made it abundantly clear that understanding the mechanisms of infectious disease is imperative to the survival of our species. While the last fifty years of developments in molecular biology has accelerated our ability to study microbial pathogens, limitations in pathogen tropism, microbial survival in laboratory conditions, uneven sampling of human cohorts across geographical and socioeconomic lines, and heterogeneous complexity during human infection have limited our ability to study complex mechanisms of human susceptibility to infectious disease. In this work, I build on recent developments in utilizing High-throughput Human in vitro Susceptibility Testing (Hi-HOST) to not only (a) identify novel sites in the human genome that contribute to natural variation in infectious disease susceptibility based on highly quantifiable cellular phenotypes, but (b) use these sites as a springboard to understand the entire, complex host-pathogen interaction. From this perspective, I paired the model pathogen Salmonella enterica and the Hi-HOST system to identify that natural variation in the mammalian gene arhgef26 contributes to susceptibility to Salmonella invasion. I used this finding as a starting point to fully explore the role of ARHGEF26 during infection, redefining its role in invasion, inflammation, and its interaction with host and bacterial proteins during the process. Similarly, I used prior Hi-HOST findings that methionine metabolism influences the host response to Salmonella enterica serovar Typhimurium (S. Typhimurium) as a launching point to investigate the impacts of host and bacterial metabolism on the virulence of S. Typhimurium. I found that the metabolite methylthioadenosine is a potent inhibitor of S. Typhimurium type III secretion, motility, and invasion. Finally, I mechanistically explain some of these findings by linking methionine metabolism to DNA methylation using a novel approach to integrate the Salmonella Typhimurium methylome and transcriptome. In sum, these findings demonstrate the ability for cellular GWAS to serve as a launching point to understand complex host-pathogen interactions.