Biology subjects

Zee, B. M.

Publications and source records attributed to Zee, B. M..

7 recordsLinked to original sources

bioRxiv2026

Elongin B orchestrates chromatin and transcriptional programs in H3K27M-mutant diffuse midline glioma

Recurrent driver mutations in genes encoding histone H3 (H3.3K27M and H3.1K27M) are observed in [~]80% of diffuse midline gliomas (DMG), which lead to aberrant gene regulation, yet the specific RNA polymerase II (Pol2) regulators that induce transcriptional dysregulation in DMG are not fully defined. We identified multiple regulators of Pol2 elongation as DMG genetic dependencies in a chromatin-focused CRISPR screen. Additional studies confirm that knockout (KO) of the Pol2 SIII complex gene elongin B (ELOB) inhibits DMG cell proliferation in tissue culture and tumor growth in xenograft models. Further genomic analyses reveal that ELOB binding sites are enriched in H3K27M oncohistones and that ELOB KO alters H3K27me3 and H3K27M incorporation at thousands of genomic regions, implicating ELOB in the maintenance of dysfunctional chromatin states in DMG. Correspondingly, PRO-seq and RNA-seq profiling reveal that ELOB loss disrupts Pol2 transcriptional activity and alters the expression of transcripts involved in metabolism, proliferation, and brain development. These findings suggest that Pol2 elongation factors like ELOB cooperate with H3K27M oncohistones to maintain the epigenetic and transcriptional landscape driving DMG malignancy.

cancer biology↗

bioRxiv2026

SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma

Diffuse midline gliomas (DMG) are aggressive pediatric brain tumors characterized by chromatin and transcriptional dysregulation induced by H3K27M mutations, with a median survival of 11-15 months. We identified multiple components of the SAGA and ATAC chromatin regulatory complexes as DMG genetic dependencies and found that genetic or pharmacological inhibition of the SAGA/ATAC-associated chromatin reader SGF29 reduces DMG proliferation in vitro and prolongs survival in xenograft models. Small molecule inhibitors targeting SAGA/ATAC-associated histone acetylation, ubiquitination, and methylation similarly suppress DMG growth. Integrative chromatin and transcriptomic profiling reveals that disruption of SAGA/ATAC through SGF29 knockout remodels the DMG chromatin landscape, producing distinct alterations at metabolic genes (loss of H3K9ac) and at embryonic/neurodevelopmental genes (redistribution of H3K4me3 and H3K27me3) and accompanying transcriptional changes. We further show that inhibition of the SAGA/ATAC-associated KAT2A/2B histone acetyltransferases represses cholesterol metabolism gene expression and that combined KAT2A/2B and cholesterol synthesis inhibition synergistically suppresses DMG growth in vitro and in a xenograft model. Together, these findings establish a mechanistic link between SAGA/ATAC-dependent chromatin regulation and the transcriptional and metabolic dysregulation underlying DMG malignancy. SignificanceWe demonstrate that SAGA/ATAC-dependent chromatin regulation sustains malignant transcription controlling cholesterol metabolism, proliferation, and cell fate in DMG, suggesting that a SAGA/ATAC-regulated epigenome-metabolome axis may be targeted to treat DMG.

cancer biology↗

bioRxiv2025

Expanding the Global Map of Protein Post Translational Modifications with Immunoaffinity Enrichment and nDIA Analysis on the Orbitrap Astral Mass Spectrometer

Post-translational modifications (PTMs) contribute greatly to the diversity of the human proteome by affecting protein structure, function, interactions, stability, localization, and more. The study of PTMs is essential to understand various cellular functions, disease mechanisms, and aid in the development of biomarkers and design of therapeutic targets. Owing to their diversity, dynamic nature, and low stoichiometry compared to unmodified proteome counterparts, the analysis of PTMs remains challenging. In this study, immunoaffinity enrichment of PTM peptides was combined with analysis using Data Dependent Acquisition (DDA) and narrow window Data Independent Acquisition (nDIA) on the Orbitrap Astral mass spectrometer (MS) as well as comparative analysis using the Orbitrap Fusion Lumos MS for Ubiquitination, Phosphorylation, Acetylation, Succinylation, and Methylation. Human cell line and mouse tissue samples at various input peptide amounts were immuno-enriched and mass spectrometry data was acquired on both instruments to assess depth of coverage and number of novel sites identified. The study identified a total of 88,731 unique ubiquitin sites, 64,397 phosphorylation sites (43,721 phosphoserine, 8,414 phosphothreonine and 12,262 phosphotyrosine), 11,629 acetylation, 5,272 succinylation and 1,461 mono-methylation sites. In half the acquisition time, nDIA analysis of immuno-enriched samples on Orbitrap Astral MS provided much greater depth of coverage for all PTMs compared to DDA analysis on Orbitrap Fusion Lumos MS, with up to 33-fold more PTM peptides identified and quantified. Overall, the data presented in this study demonstrates the need for enrichment for PTM detection and the utility of combining antibody-based peptide capture and nDIA on the Orbitrap Astral MS as powerful tools for discovery and profiling of protein post-translational modifications in cells and tissues.

biochemistry↗

bioRxiv2024

Site-specific quantification of the in vivo UFMylome reveals myosin modification in ALS

UFMylation is a ubiquitin-like protein post-translational modification of Ubiquitin Fold Modifier 1 (UFM1) applied to substrate proteins. The UFMylation system is important for normal development and plays a critical role in a variety of cellular processes including regulating telomere length, stress responses and protein quality control. Here, we describe the development of an antibody-based enrichment approach to immunoprecipitate in vivo remnant UFMylated peptides and identification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). We used this approach to identify >200 UFMylation sites from various mouse tissues revealing extensive modification in skeletal muscle. Furthermore, we show that UFMylation is increased in skeletal muscle biopsies from people living with Amyotrophic Lateral Sclerosis (plwALS). Quantification of UFMylation sites in these participant biopsies with multiplexed isotopic labeling and LC-MS/MS reveal prominent increases in myosin UFMylation. Finally, in silico modelling suggest UFMylation of myosin directly adjacent to the ATP-binding site may regulate stability and/or function. Our data suggest that although UFMylation is not as widespread as ubiquitylation, its in vivo status is more complex than previously thought.

biochemistry↗

bioRxiv2023

Cell state-dependent chromatin targeting in NUT carcinoma

Aberrant transcriptional programming and chromatin dysregulation are common to most cancers. Whether by deranged cell signaling or environmental insult, the resulting oncogenic phenotype is typically manifested in transcriptional changes characteristic of undifferentiated cell growth. Here we analyze targeting of an oncogenic fusion protein, BRD4-NUT, composed of two normally independent chromatin regulators. The fusion causes the formation of large hyperacetylated genomic regions or megadomains, mis-regulation of c-MYC, and an aggressive carcinoma of squamous cell origin. Our previous work revealed largely distinct megadomain locations in different NUT carcinoma patient cell lines. To assess whether this was due to variations in individual genome sequences or epigenetic cell state, we expressed BRD4-NUT in a human stem cell model and found that megadomains formed in dissimilar patterns when comparing cells in the pluripotent state with the same cell line following induction along a mesodermal lineage. Thus, our work implicates initial cell state as the critical factor in the locations of BRD4-NUT megadomains. These results, together with our analysis of c-MYC protein-protein interactions in a patient cell line, are consistent with a cascade of chromatin misregulation underlying NUT carcinoma.

genomics↗

bioRxiv2022

Variant Polycomb complexes in Drosophila consistent with ancient functional diversity

Polycomb group (PcG) mutants were first identified in Drosophila based on their failure to maintain proper Hox gene repression during development. The proteins encoded by the corresponding fly genes mainly assemble into one of two discrete Polycomb Repressive Complexes: PRC1 or PRC2. However, biochemical analyses in mammals have revealed alternative forms of PRC2, and multiple distinct types of non-canonical or variant PRC1. Through a series of proteomic analyses, we identify analogous PRC2 and variant PRC1 complexes in Drosophila, as well as a broader repertoire of interactions implicated in early development. Our data provide strong support for the ancient diversity of PcG complexes, and a framework for future analysis in a longstanding and versatile genetic system.

developmental biology↗

bioRxiv2022

cMYC protein interactions and chromatin association in NUT carcinoma

The oncogene cMYC (HGNC:7553) is a critical genomic target of the BRD4-NUT (B4N) protein that defines many of the NUTM1-rearrangement cancer subtypes in NUT carcinoma (NC). Previously, we reported that B4N interacts with the EP300 lysine acetyltransferase (KAT3B) and creates hyperacetylated "megadomains" that activate downstream genes such as cMYC. Here we ask how misregulated cMYC in turn interacts with protein partners and target genes in patient-derived NC797 cells, and whether these interactions change in response to B4N inactivation. We used CRISPR-Cas9 mediated knock-in of a BioTAP affinity tag to analyze cMYC protein expressed from its normal chromosomal context. This allowed us to implement a crosslinking purification method termed BioTAP-XL to preserve cMYC integrity and chromatin association for genomic and mass spectrometry-based proteomic analyses. We found that in the NC797 cell line, cMYC interacts primarily with the NuA4 KAT5 lysine acetyltransferase complex, with interactions that are maintained despite a decrease in cMYC levels after JQ1 treatment. We propose that a cascade of aberrant acetyltransferase activities in NC797 cells, first via EP300 recruitment by B4N to mis-regulate cMYC, and then by KAT5 interaction with the resulting overexpressed cMYC protein, drive NC cell proliferation and blockade to differentiation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/489219v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@82ec09org.highwire.dtl.DTLVardef@1566d31org.highwire.dtl.DTLVardef@11ab4dborg.highwire.dtl.DTLVardef@57b741_HPS_FORMAT_FIGEXP M_FIG C_FIG Simple SummaryA longstanding goal in biology is to understand how protein interactions influence and reflect cellular disease states. MYC is a critical regulator of cellular proliferation that is mis-regulated in many cancers, including those with NUTM1-rearrangements featured in this issue. NUT carcinoma cells are dependent on MYC expression, which blocks differentiation. Using a crosslinking approach to identify MYC protein interactions in a NUTM1-rearranged patient cell line, we found that MYC interacts primarily with subunits of the NuA4 lysine acetyltransferase (KAT5), one of two KAT complexes previously discovered as MYC interactors in non-NUTM1 cell lines.

cancer biology↗