Biology subjects

Glass, K. C.

Publications and source records attributed to Glass, K. C..

10 recordsLinked to original sources

bioRxiv2026

lncRNA MANCR isoforms selectively mediate multiple levels of epigenomic and P53-responsive transcriptional control in triple negative breast cancer

Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are frequently dysregulated in cancer. The mitotically associated lncRNA MANCR is highly expressed in aggressive cancers and contributes to genomic instability in triple-negative breast cancer (TNBC), but the molecular mechanisms underlying its activity remain poorly defined. Here we integrate computational and experimental approaches to examine the structure and regulatory interactions of MANCR isoforms. Analysis of transcriptomic datasets revealed tumor-type-specific expression patterns for seven MANCR isoforms in breast cancer cell lines. Computational prediction of RNA secondary structures identified conserved structural features across isoforms, suggesting potential functional specialization. We identify p53 as a MANCR-interacting protein through computational docking and RNA immunoprecipitation sequencing (RIP-seq) and demonstrate that MANCR depletion reduces p53-dependent transcriptional activity. Chromatin isolation by RNA purification sequencing (ChIRP-seq) revealed 1, 250 genomic regions associated with MANCR, including enrichment of p53 consensus motifs and GC-rich sequence elements. Motif analysis further identified candidate sequence features associated with MANCR-occupied chromatin regions. Computational prediction of RNA-miRNA interactions identified multiple potential miRNA binding sites across MANCR isoforms, including miR-6756-5p, which targets the androgen receptor (AR). Consistent with this prediction, AR expression decreased following MANCR knockdown in TNBC cells. Together, these results suggest that MANCR isoforms may contribute to transcriptional regulation in TNBC through interactions with chromatin, p53 signaling pathways, and potential miRNA regulatory networks. One Sentence SummaryMitotically-associated lncRNA (MANCR) is prevalent in aggressive cancers interacting with DNA, P53, and miRNAs, to mediate multiple levels of epigenetic transcriptional control in triple negative breast cancer.

cancer biology↗

bioRxiv2026

Cryo-EM Structure of Human ATAD2B Reveals a Hexameric Organization Contributes to ATPase Activity and Substrate Coordination

ATPase family AAA+ domain-containing protein 2B (ATAD2B) is a poorly characterized member of the ATAD2-like protein family, which contains a unique combination of tandem AAA+ ATPase domains with a C-terminal bromodomain. In humans, ATAD2B is dysregulated in several disease states including cancer and respiratory disorders, yet despite its promise as a therapeutic target, little is known about its molecular function. Here, we report the first high-resolution cryo-EM structure of human ATAD2B at 3.0 [A], revealing a two-tiered hexameric assembly with a shallow spiral staircase architecture. Structural analysis uncovers conserved AAA+ ATPase features, including nucleotide coordination at inter-subunit interfaces, inter-subunit signaling (ISS) gate loops, and pore loops that engage a substrate within the central channel. Biochemical assays demonstrate that ATAD2B is an active enzyme with an ATP hydrolysis rate of 0.34 ATP/hexamer/sec. Furthermore, the integrity of the hexameric complex is stabilized through unique knob-hole interactions, a linker arm that extends between the AAA2 and bromodomain, and an N-terminal linker domain (LD). These findings establish ATAD2B as a functional AAA+ ATPase and provide mechanistic insight into its enzymatic activities, laying the foundation for understanding its role in chromatin-associated processes.

biochemistry↗

bioRxiv2025

Breaking Barriers: Transitioning from X-ray Crystallography to Cryo-EM for Structural Studies of ATAD2B

Cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling near-atomic resolution of large macromolecular complexes without the need for crystallization. Here, we describe our laboratorys transition from X-ray crystallography to single-particle cryo-EM to investigate the ATPase family AAA+ domain-containing protein 2B (ATAD2B), a chromatin regulator implicated in epigenetic signaling. We outline the challenges encountered during protein expression, purification, and sample preparation, including co-purification of the chaperonin GroEL, and strategies employed to overcome these obstacles. Our workflow highlights critical steps in sample optimization, grid vitrification, and data processing using CryoSPARC, cisTEM, and Topaz, as well as computational requirements for high-resolution reconstructions. We also discuss model building, refinement, and validation approaches, emphasizing best practices for new cryo-EM users. This work provides practical insights for structural biologists adopting cryo-EM, particularly for large, flexible protein complexes, and underscores the importance of integrated approaches combining biochemical, computational, and imaging strategies.

biochemistry↗

bioRxiv2024

The CECR2 bromodomain displays distinct binding modes to select for acetylated histone proteins versus non-histone ligands.

The cat eye syndrome chromosome region candidate 2 (CECR2) protein is an epigenetic regulator involved in chromatin remodeling and transcriptional control. The CECR2 bromodomain (CECR2-BRD) plays a pivotal role in directing the activity of CECR2 through its capacity to recognize and bind acetylated lysine residues on histone proteins. This study elucidates the binding specificity and structural mechanisms of CECR2-BRD interactions with both histone and non-histone ligands, employing techniques such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, and a high-throughput peptide assay. The CECR2-BRD selectively binds acetylated histone H3 and H4 ligands, exhibiting a preference for multi-acetylated over mono-acetylated targets. The highest affinity was observed for tetra-acetylated histone H4. Neighboring post-translational modifications, including methylation and phosphorylation, modulate acetyllysine recognition, with significant effects observed for histone H3 ligands. Additionally, this study explored the interaction of the CECR2-BRD with the acetylated RelA subunit of NF-{kappa}B, a pivotal transcription factor in inflammatory signaling. Dysregulated NF-{kappa}B signaling is implicated in numerous pathologies, including cancer progression, with acetylation of RelA at lysine 310 (K310ac) being critical for its transcriptional activity. Recent evidence linking the CECR2-BRD to RelA suggests it plays a role in inflammatory and metastatic pathways, underscoring the need to understand the molecular basis of this interaction. We found the CECR2-BRD binds to acetylated RelA with micromolar affinity, and uses a distinctive binding mode to recognize this non-histone ligand. These results provide new insight on the role of CECR2 in regulating NF-{kappa}B-mediated inflammatory pathways. Functional mutagenesis of critical residues, such as Asn514 and Asp464, highlight their roles in ligand specificity and binding dynamics. Notably, the CECR2-BRD remained monomeric in solution and exhibited differential conformational responses upon ligand binding, suggesting adaptive recognition mechanisms. Furthermore, the CECR2-BRD exclusively interacts with nucleosome substrates containing multi-acetylated histones, emphasizing its role in transcriptional activation within euchromatic regions. These findings position the CECR2-BRD as a key chromatin reader and a promising therapeutic target for modulating transcriptional and inflammatory processes, particularly through the development of selective bromodomain inhibitors. HIGHLIGHTSO_LIThe CECR2 bromodomain recognizes a range of combinatorial PTMs on the histone H3 and H4 N-terminal tails. C_LIO_LIThe CECR2 bromodomain binds to an acetylated RelA ligand with micromolar affinity. C_LIO_LINMR perturbation studies delineate the distinct binding modes driving CECR2-BRD recognition of histone versus non-histone ligands. C_LIO_LISite-directed mutagenesis reveals the specificity determinants of CECR2-BRD ligand binding. C_LIO_LIThe bromodomain of CECR2 exhibits a strong interaction with multi-acetylated nucleosomes. C_LI

biochemistry↗

bioRxiv2022

Regulation of ATAD2B bromodomain binding activity by the histone code

The ATPase family AAA+ domain containing 2 (ATAD2) protein, and its paralog ATAD2B, have a C-terminal bromodomain that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2 and ATAD2B bromodomains to select acetylated lysine among multiple histone post-translational modifications. Isothermal titration calorimetry experiments revealed that the ATAD2 and ATAD2B bromodomains selectively recognize distinct patterns of acetylated lysine residues on the N-terminal tails of histone proteins. Adjacent methylation or phosphorylation marks were found to either enhance or weaken the recognition of acetylated lysine by the ATAD2/B bromodomains. Complementary structural studies provide mechanistic insights into how residues within the bromodomain binding pocket coordinate the acetyllysine group in the context of adjacent post- translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands, either as onco mutations or as histone variants, impact the recognition of an adjacent acetylated lysine residue. In summary, our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2 and ATAD2B bromodomains, resulting in distinct binding modes of the two bromodomains. KEY POINTSO_LIMultiple independent ATAD2 gene duplication events are evident during metazoan evolution, indicating expansion of functionality in the ATAD2 gene family and suggesting distinct functions for ATAD2 and ATAD2B. C_LIO_LIHigh-resolution structures of the ATAD2 and ATAD2B bromodomains in complex with their histone ligands demonstrate how multiple post-translational modifications are coordinated. C_LIO_LIRecognition of different subsets acetylated histone ligands by the ATAD2 and ATAD2B bromodomains is driven by unique features within the binding pockets of these paralogous proteins. C_LIO_LIOnco-histone mutations and histone variants that change the amino acid sequence of the histone tails modulate the ATAD2 and ATAD2B bromodomain activity. C_LIO_LIThis study demonstrates how the combinatorial activity of multiple post- translational modifications forms a histone code and influences the recognition of acetylated lysine by bromodomain-containing proteins. C_LI

biochemistry↗

bioRxiv2022

Structural insights into acetylated histone ligand recognition by the BDP1 bromodomain of Plasmodium falciparum

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to up-regulate and down-regulate certain genes required for invasion and replication. The P. falciparum genome encodes six bromodomaincontaining proteins, of which Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and the 8 human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

biophysics↗

bioRxiv2022

Functional networks of the human bromodomain-containing proteins

BackgroundBromodomains are a structurally conserved epigenetic reader domain that bind to acetylated lysine residues in both histone and non-histone proteins. Bromodomain-containing proteins (BRD proteins) function are established scaffolds in the assembly of multi-protein complexes to regulate diverse biological processes. BRD proteins have been classified based on biological and functional similarity, however the functions of many BRD proteins remains unknown. PPI network analysis is useful for revealing organizational roles, identifying functional clusters, and predicting function for BRD proteins. ResultsWe used available data to construct protein-protein interaction networks (PPINs) to study the properties of the human bromodomain protein family. The network properties of the BRD PPIN establishes that the BRD proteins serve as hub proteins that are enriched near the global center to form an inter-connected PPIN. We identified dense subgraphs formed by BRD proteins and find that different BRD proteins share topological similarity and functional associations. We explored the functional relationships through clustering and Hallmark pathway gene set enrichment analysis and identify potential biological roles for different BRD proteins. ConclusionsIn our network analysis we confirmed that BRD proteins are conserved central nodes in the human PPI network and function as scaffolds to form distinctive functional clusters. Overall, this study provides detailed insight into the predictive functions of BRD proteins in the context of functional complexes and biological pathways.

bioinformatics↗

bioRxiv2021

Coordination of di-acetylated histone ligands by the ATAD2 bromodomain

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four -helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket, but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.

biochemistry↗

bioRxiv2020

The BRPF1 bromodomain is a molecular reader of di-acetyllysine

Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex, which is associated with chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of chromatin reader domains including two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) domain. BRPF1 is known to recruit the MOZ HAT complex to chromatin by recognizing acetylated lysine residues on the N-terminal histone tail region through its bromodomain. However, histone proteins can contain several acetylation modifications on their N-terminus, and it is unknown how additional marks influence bromodomain recruitment to chromatin. Here, we identify the BRPF1 bromodomain as a selective reader of di-acetyllysine modifications on histone H4. We used ITC assays to characterize the binding of di-acetylated histone ligands to the BRPF1 bromodomain and found that the domain binds preferentially to histone peptides H4K5acK8ac and H4K5acK12ac. Analytical ultracentrifugation (AUC) experiments revealed that the monomeric state of the BRPF1 bromodomain coordinates di-acetylated histone ligands. NMR chemical shift perturbation studies, along with binding and mutational analyses, revealed non-canonical regions of the bromodomain-binding pocket that are important for histone tail recognition. Together, our findings provide critical information on how the combinatorial action of post-translational modifications can modulate BRPF1 bromodomain binding and specificity.

biochemistry↗

bioRxiv2018

Structural insights into the recognition of mono- and di-acetyllysine by the ATAD2B bromodomain

Bromodomains are chromatin reader modules that recognize acetylated lysine. Different bromodomains exhibit a preference for specific patterns of lysine acetylation marks on core and variant histone proteins, however, the functional relationships that exist between histone acetyllysine ligands and bromodomain recognition remain poorly understood. In this study, we examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain selects for mono- and di-acetylated histones, and structural analysis identified key residues in the acetyllysine binding pocket that dictate ligand binding specificity. The X-ray crystal structure of the ATAD2B bromodomain in complex with an ATAD2 bromodomain inhibitor was solved at 2.4 [A] resolution. This structure demonstrated that critical contacts required for bromodomain inhibitor coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized a variant of the ATAD2B bromodomain that through alternative splicing loses critical amino acids required for histone ligand and inhibitor coordination. Altogether our results outline the structural and functional features of the ATAD2B bromodomain and identify a novel mechanism important for regulating the interaction of the ATAD2B protein with chromatin. HIGHLIGHTSO_LIThe ATAD2B bromodomain recognizes mono- and di-acetylated histone ligands. C_LIO_LIChemical shift perturbations outline the ATAD2B bromodomain acetyllysine binding pocket. C_LIO_LIAn ATAD2B bromodomain-inhibitor complex reveals important binding contacts. C_LIO_LIAn alternate splice variant in the ATAD2B bromodomain abolishes histone and inhibitor binding. C_LI

biochemistry↗