Cytosine methylation is a common epigenetic regulation that affects how genes are expressed or silenced. Histone H3 lysine 4 trimethylation (H3K4me3) is a particular histone modification that was found to antagonize DNA methylation in Arabidopsis thaliana. The Jacobsen Lab has previously discovered that targeting of H3K4me3 by SET DOMAIN GENE 2 (SDG2) with an artificial zinc finger (ZF) contributes to the removal of CG methylation in FLOWERING WAGENINGEN (FWA) promoters and off-target sites. H3K4me3 reader proteins EBS, AL1, and AL2, and demethylases REPRESSOR OF SILENCING 1 (ROS1) and DEMETER-like 2 (DML2) collectively serve to repress methylation at H3K4me3-deposited regions. However, these findings warrant further exploration of additional intermediates that are involved in this demethylation process. In this study, McrBC-qPCR was conducted to quantify methylation levels in a region, and BS-PCR was utilized to determine the methylation status of a single nucleotide point. Y2H assays analyzed the interactions between various demethylases and H3K4me3 reader proteins. These findings provide an understanding of H3K4me3-mediated antagonism of DNA methylation and general insight into epigenetic controls within the bounds of histone marks and DNA methylation.

The immunosuppressive microenvironment of immunologically “cold” tumors poses a major challenge to generating proper host immune responses against cancer. RNA-LNP vaccination represents a promising immunotherapy platform that could circumvent this obstacle by harnessing the body’s own immune system to fight cancer. Our lab is interested in improving existing immunotherapies, such as mRNA vaccines, to target these immunologically “cold” tumors with a novel RNA adjuvant. We aim to activate antigen-presenting cells, induce a more robust innate immune response, and improve the efficacy of cancer vaccines. We hypothesized that our RNA adjuvant would induce innate immune activation through the engagement of key pattern recognition receptors (PRRs). We investigated whether Toll-like receptor 3 (TLR3) and mitochondrial antiviral signaling protein (MAVS) are crucial PRRs for the innate immune activity of our RNA adjuvant. We treated bone marrow-derived macrophages with our RNA adjuvant and induced robust innate immune responses as measured by qPCR of cytokine transcripts. We found TLR3 and MAVS to be critical for innate immune signaling, as loss of both PRRs resulted in abrogation of innate immune activation following treatment with our RNA adjuvant in vitro. We aim to utilize this RNA adjuvant in combination with mRNA vaccines to improve innate immune activation, increase recruitment of cytotoxic T cells to the tumor microenvironment, and facilitate an anti-tumor response against immunologically “cold” tumors.

Brown adipose tissue (BAT) is distinguished by its ability to expend energy through thermogenesis, a process with considerable therapeutic potential for metabolic disorders. Although glycogen represents a relatively minor energy reserve in brown adipocytes compared with liver and muscle, glycogen turnover increases sharply during metabolic transitions and is required for full thermogenic activation. The rate-limiting enzyme glycogen synthase 1 (GYS1) catalyzes glycogen chain elongation. Adipose-specific deletion of Gys1 impairs adaptive thermogenesis, highlighting the importance of GYS1 in coordinating glucose and lipid metabolism. However, the mechanisms regulating GYS1 in BAT remain poorly defined. To address this gap, we used an unbiased proximity-labeling strategy in brown adipocytes to map the GYS1 interactome. We fused GYS1 to the biotin ligase TurboID and identified 425 putative interactors through mass spectrometry. These included canonical glycogen-handling proteins such as glycogenin and glycogen phosphorylase, validating the approach. Gene ontology analysis uncovered broader enrichment in cytoskeletal remodeling, vesicle trafficking, and transcriptional regulation, suggesting previously unrecognized roles for GYS1. Notably, we identified several novel candidate interactors, including CAST, PDAP1, and CCDC102A. Together, these findings define the most comprehensive GYS1 interactome reported to date and provide new insights into the molecular mechanisms linking glycogen metabolism to thermogenic function in BAT.

This abstract has been withheld from publication.

The UHRF1 protein is an epigenetic regulator that links DNA methylation and histone modification. The upregulation of UHRF1 is found in several cancer types in humans, making it a possible therapeutic target. This study investigates the cloning and identification of a UHRF1 paralogue in S. purpuratus. The methods for cloning used PCR with primers to amplify a region containing UHRF1 from S. purpuratus DNA. Further cloning was performed using vector ligation and infecting E. coli with the UHRF1. The vector was later released from E. Coli using a restriction digest. Verification of successful cloning was accomplished using gel electrophoresis in order to compare the sizes of PCR, ligation, and restriction digest fragments to their expected lengths. The identity of the UHRF1 paralogue was identified using Echinobase, megaBLAST, and Interpro. A phylogenetic tree analysis was also created to identify amino acid sequence similarity. The results of this study indicate a successful PCR replication of the target UHRF1 sequence. The identification of S. purpuratus UHRF1 found that it shared similar domains and phylogenetic heritage to paralogues in other deuterosomes. This study presents an opportunity to evaluate the viability for the S. purpuratus UHRF1 protein as a target to evaluate upregulation and effects from drug targets. Future steps in this research will allow for the confirmation of paralogue similarity and may utilize UHRF1 to contribute guidance to cancer therapies.