IU Indianapolis ScholarWorks :: Browsing by Author "Cornett, Evan M."

Browsing by Author "Cornett, Evan M."

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Functional and Quantitative Mass Spectrometry-Based Approaches for Mapping the Lysine Methylome
(2024-09) Berryhill, Christine Annette; Cornett, Evan M.; Aoki, Scott T.; Georgiadis, Millie M.; Mosley, Amber L.; Turchi, John J.
Proteins are frequently modified with small chemical tags, or modifications, that play a key role in controlling their functions within the cell. One modification, lysine methylation, is found on thousands of human proteins and is added and removed by lysine methyltransferases (KMTs) and lysine demethylases (KDMs), respectively. Recognition of methylated lysines by specific reader proteins regulates downstream processes. Lysine methylation, KMTs, KDMs, and reader proteins therefore create a signaling network. Components of lysine methylation signaling networks are frequently dysregulated in human disease, but current methods to detect lysine methylation present barriers for understanding the impact an awry signaling network has on lysine methylation. In this study, we investigated the use of mass spectrometry (MS)-based proteomics to better detect and quantify both lysine methylation sites and methyl regulators across multiple samples. We investigated the sequence bias of commercially available pan-methyllysine antibodies using both a lysine-oriented peptide library and immunoprecipitation mass spectrometry. Our results showed that most antibodies have a preference for certain sequences. Furthermore, we observed that unenriched samples obtained the same number of identified lysine methylation sites as enriched samples. Following the establishment of an efficient and quantitative MS-based proteomics approach, we applied it to profile both lysine methylation and KDMs within breast cancer cell lines. Studies have repeatedly shown that components of the lysine methylation signaling network are overexpressed within breast cancer. Indeed, we characterized distinct lysine methylation and KDM patterns across the cell lines, suggesting the existence of different lysine methylation signaling. Given the ability to quantitatively profile lysine methylation, this work also characterized the impact of a compound known to disrupt the lysine methylation signaling network, 3-deazanplanocin A. The observed transcript, protein, and lysine methylation site abundance changes highlight how dysregulation of methyl mediators impacts lysine methylation and cellular signaling. Overall, we developed a reproducible pipeline that promises to enable a deeper understanding of how a dysregulated lysine methylation landscape influences cellular signaling and associated phenotypes.
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.
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.
Lysine Methylation Regulators Moonlighting outside the Epigenome
(Elsevier, 2019-09-19) Cornett, Evan M.; Ferry, Laure; Defossez, Pierre-Antoine; Rothbart, Scott B.; Biochemistry and Molecular Biology, School of Medicine
Landmark discoveries made nearly two decades ago identified known transcriptional regulators as histone lysine methyltransferases; since then the field of lysine methylation signaling has been dominated by studies of how this small chemical posttranslational modification regulates gene expression and other chromatin-based processes. However, recent advances in mass spectrometry-based proteomics have revealed that histones are just a subset of the thousands of eukaryotic proteins marked by lysine methylation. As the writers, erasers, and readers of histone lysine methylation are emerging as a promising therapeutic target class for cancer and other diseases, a key challenge for the field is to define the full spectrum of activities for these proteins. Here we summarize recent discoveries implicating non-histone lysine methylation as a major regulator of diverse cellular processes. We further discuss recent technological innovations that are enabling the expanded study of lysine methylation signaling. Collectively, these findings are shaping our understanding of the fundamental mechanisms of non-histone protein regulation through this dynamic and multi-functional posttranslational modification.
A physical basis for quantitative ChIP-sequencing
(Elsevier, 2020-11-20) Dickson, Bradley M.; Tiedemann, Rochelle L.; Chomiak, Alison A.; Cornett, Evan M.; Vaughan, Robert M.; Rothbart, Scott B.; Biochemistry and Molecular Biology, School of Medicine
ChIP followed by next-generation sequencing (ChIP-Seq) is a key technique for mapping the distribution of histone posttranslational modifications (PTMs) and chromatin-associated factors across genomes. There is a perceived challenge to define a quantitative scale for ChIP-Seq data, and as such, several approaches making use of exogenous additives, or "spike-ins," have recently been developed. Herein, we report on the development of a quantitative, physical model defining ChIP-Seq. The quantitative scale on which ChIP-Seq results should be compared emerges from the model. To test the model and demonstrate the quantitative scale, we examine the impacts of an EZH2 inhibitor through the lens of ChIP-Seq. We report a significant increase in immunoprecipitation of presumed off-target histone PTMs after inhibitor treatment, a trend predicted by the model but contrary to spike-in-based indications. Our work also identifies a sensitivity issue in spike-in normalization that has not been considered in the literature, placing limitations on its utility and trustworthiness. We call our new approach the sans-spike-in method for quantitative ChIP-sequencing (siQ-ChIP). A number of changes in community practice of ChIP-Seq, data reporting, and analysis are motivated by this work.
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.
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.
A Read/Write Mechanism Connects p300 Bromodomain Function to H2A.Z Acetylation
(Elsevier, 2019-11-22) Colino-Sanguino, Yolanda; Cornett, Evan M.; Moulder, David; Smith, Grady C.; Hrit, Joel; Cordeiro-Spinetti, Eric; Vaughan, Robert M.; Krajewski, Krzysztof; Rothbart, Scott B.; Clark, Susan J.; Valdés-Mora, Fátima; Biochemistry and Molecular Biology, School of Medicine
Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active regulatory regions associated with gene expression. Although the Tip60 complex is proposed to acetylate H2A.Z, functional studies suggest additional enzymes are involved. Here, we show that p300 acetylates H2A.Z at multiple lysines. In contrast, we found that although Tip60 does not efficiently acetylate H2A.Z in vitro, genetic inhibition of Tip60 reduces H2A.Zac in cells. Importantly, we found that interaction between the p300-bromodomain and H4 acetylation (H4ac) enhances p300-driven H2A.Zac. Indeed, H2A.Zac and H4ac show high genomic overlap, especially at active promoters. We also reveal unique chromatin features and transcriptional states at enhancers correlating with co-occurrence or exclusivity of H4ac and H2A.Zac. We propose that differential H4 and H2A.Z acetylation signatures can also define the enhancer state. In conclusion, we show both Tip60 and p300 contribute to H2A.Zac and reveal molecular mechanisms of writer/reader crosstalk between H2A.Z and H4 acetylation through p300.
Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling
(American Association for the Advancement of Science, 2024) Chomiak, Alison A.; Tiedemann, Rochelle L.; Liu, Yanqing; Kong, Xiangqian; Cui, Ying; Wiseman, Ashley K.; Thurlow, Kate E.; Cornett, Evan M.; Topper, Michael J.; Baylin, Stephen B.; Rothbart, Scott B.; Biochemistry and Molecular Biology, School of Medicine
DNA methyltransferase inhibitor (DNMTi) efficacy in solid tumors is limited. Colon cancer cells exposed to DNMTi accumulate lysine-27 trimethylation on histone H3 (H3K27me3). We propose this Enhancer of Zeste Homolog 2 (EZH2)-dependent repressive modification limits DNMTi efficacy. Here, we show that low-dose DNMTi treatment sensitizes colon cancer cells to select EZH2 inhibitors (EZH2is). Integrative epigenomic analysis reveals that DNMTi-induced H3K27me3 accumulates at genomic regions poised with EZH2. Notably, combined EZH2i and DNMTi alters the epigenomic landscape to transcriptionally up-regulate the calcium-induced nuclear factor of activated T cells (NFAT):activating protein 1 (AP-1) signaling pathway. Blocking this pathway limits transcriptional activating effects of these drugs, including transposable element and innate immune response gene expression involved in viral defense. Analysis of primary human colon cancer specimens reveals positive correlations between DNMTi-, innate immune response-, and calcium signaling-associated transcription profiles. Collectively, we show that compensatory EZH2 activity limits DNMTi efficacy in colon cancer and link NFAT:AP-1 signaling to epigenetic therapy-induced viral mimicry.
Sex-dependent differential chromatin state changes in adipocytes across different fat depots during obesity
(2023-08) Chaisakhon, Natthamon; Roh, Hyun Cheol; Mosley, Amber L.; Cornett, Evan M.
Adipose tissue exhibits a high degree of plasticity and undergoes significant cellular remodeling in response to nutritional availability. While adipose tissue remodeling displays sexual dimorphism, its underlying molecular mechanisms remain poorly understood. In this study, we investigate the role of epigenetic modifications and gene expression in sexual dimorphism of adipose tissue in mice during obesity. We perform CUT&Tag (Cleavage Under Targets and Tagmentation) technique in conjunction with RNA-sequencing with female adipocytes from different fat depots and compare them to male mice. We find significant changes in H3K27ac, H3K27me3, H3K9me3 and RNA-seq after high fat diet (HFD) feeding. The changes are functionally characterized by a reduction in cellular metabolism and an elevation in cytoskeletal organization. Interestingly, these changes commonly occur in both visceral and subcutaneous fat depots with minor differences in females, which distinguishes them from males that exhibit differential responses between depots. Surprisingly, the chromatin states and expression profiles of female subcutaneous fat depots closely resemble those of male visceral adipose tissues during obesity. These results suggest that distinct responses across fat depots in male and females may contribute to different susceptibilities to metabolic diseases between the sexes. These findings enhance our understanding of the molecular mechanisms underlying adipocyte dysfunction during obesity and highlights the interplay between sex, adipose tissue depots, and metabolic responses.

Browsing by Author "Cornett, Evan M."

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