Browsing by Author "Berryhill, Christine A."

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    Global lysine methylome profiling using systematically characterized affinity reagents
    (Springer Nature, 2023-01-07) Berryhill, Christine A.; Hanquier, Jocelyne N.; Doud, Emma H.; Cordeiro‑Spinetti, Eric; Dickson, Bradley M.; Rothbart, Scott B.; Mosley, Amber L.; Cornett, Evan M.; Biochemistry and Molecular Biology, School of Medicine
    Lysine methylation modulates the function of histone and non-histone proteins, and the enzymes that add or remove lysine methylation—lysine methyltransferases (KMTs) and lysine demethylases (KDMs), respectively—are frequently mutated and dysregulated in human diseases. Identification of lysine methylation sites proteome-wide has been a critical barrier to identifying the non-histone substrates of KMTs and KDMs and for studying functions of non-histone lysine methylation. Detection of lysine methylation by mass spectrometry (MS) typically relies on the enrichment of methylated peptides by pan-methyllysine antibodies. In this study, we use peptide microarrays to show that pan-methyllysine antibodies have sequence bias, and we evaluate how the differential selectivity of these reagents impacts the detection of methylated peptides in MS-based workflows. We discovered that most commercially available pan-Kme antibodies have an in vitro sequence bias, and multiple enrichment approaches provide the most comprehensive coverage of the lysine methylome. Overall, global lysine methylation proteomics with multiple characterized pan-methyllysine antibodies resulted in the detection of 5089 lysine methylation sites on 2751 proteins from two human cell lines, nearly doubling the number of reported lysine methylation sites in the human proteome.
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    Identification of nonhistone substrates of the lysine methyltransferase PRDM9
    (Elsevier, 2023) Hanquier, Jocelyne N.; Sanders, Kenidi; Berryhill, Christine A.; Sahoo, Firoj K.; Hudmon, Andy; Vilseck, Jonah Z.; Cornett, Evan M.; Biochemistry and Molecular Biology, School of Medicine
    Lysine methylation is a dynamic, posttranslational mark that regulates the function of histone and nonhistone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate nonhistone proteins. In this work, we investigate the substrate selectivity of the KMT PRDM9 to identify both potential histone and nonhistone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on nonhistone proteins. Using lysine-oriented peptide libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative nonhistone substrates, which were tested by peptide spot array, and a subset was further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the nonhistone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.
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    Protein Thermal Stability Changes Induced by the Global Methylation Inhibitor 3-Deazaneplanocin A (DZNep)
    (MDPI, 2024-07-09) Berryhill, Christine A.; Doud, Emma H.; Hanquier, Jocelyne N.; Smith-Kinnaman, Whitney R.; McCourry, Devon L.; Mosley, Amber L.; Cornett, Evan M.; Biochemistry and Molecular Biology, School of Medicine
    DZNep (3-deazaneplanocin A) is commonly used to reduce lysine methylation. DZNep inhibits S-adenosyl-l-homocysteine hydrolase (AHCY), preventing the conversion of S-adenosyl-l-homocysteine (SAH) into L-homocysteine. As a result, the SAM-to-SAH ratio decreases, an indicator of the methylation potential within a cell. Many studies have characterized the impact of DZNep on histone lysine methylation or in specific cell or disease contexts, but there has yet to be a study looking at the potential downstream impact of DZNep treatment on proteins other than histones. Recently, protein thermal stability has provided a new dimension for studying the mechanism of action of small-molecule inhibitors. In addition to ligand binding, post-translational modifications and protein–protein interactions impact thermal stability. Here, we sought to characterize the protein thermal stability changes induced by DZNep treatment in HEK293T cells using the Protein Integral Solubility Alteration (PISA) assay. DZNep treatment altered the thermal stability of 135 proteins, with over half previously reported to be methylated at lysine residues. In addition to thermal stability, we identify changes in transcript and protein abundance after DZNep treatment to distinguish between direct and indirect impacts on thermal stability. Nearly one-third of the proteins with altered thermal stability had no changes at the transcript or protein level. Of these thermally altered proteins, CDK6 had a stabilized methylated peptide, while its unmethylated counterpart was unaltered. Multiple methyltransferases were among the proteins with thermal stability alteration, including DNMT1, potentially due to changes in the SAM/SAH levels. This study systematically evaluates DZNep’s impact on the transcriptome, the proteome, and the thermal stability of proteins.
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    Quantitative analysis of non-histone lysine methylation sites and lysine demethylases in breast cancer cell lines
    (bioRxiv, 2024-09-22) Berryhill, Christine A.; Evans, Taylor N.; Doud, Emma H.; Smith-Kinnaman, Whitney R.; Hanquier, Jocelyne N.; Mosley, Amber L.; Cornett, Evan M.; Biochemistry and Molecular Biology, School of Medicine
    Growing evidence shows that lysine methylation is a widespread protein post-translational modification that regulates protein function on histone and non-histone proteins. Numerous studies have demonstrated that dysregulation of lysine methylation mediators contributes to cancer growth and chemotherapeutic resistance. While changes in histone methylation are well documented with extensive analytical techniques available, there is a lack of high-throughput methods to reproducibly quantify changes in the abundances of the mediators of lysine methylation and non-histone lysine methylation (Kme) simultaneously across multiple samples. Recent studies by our group and others have demonstrated that antibody enrichment is not required to detect lysine methylation, prompting us to investigate the use of Tandem Mass Tag (TMT) labeling for global Kme quantification sans antibody enrichment in four different breast cancer cell lines (MCF-7, MDA-MB-231, HCC1806, and MCF10A). To improve the quantification of KDMs, we incorporated a lysine demethylase (KDM) isobaric trigger channel, which enabled 96% of all KDMs to be quantified while simultaneously quantifying 326 Kme sites. Overall, 142 differentially abundant Kme sites and eight differentially abundant KDMs were identified between the four cell lines, revealing cell line-specific patterning.