The mRNA-binding protein AUF1 regulates the expression of many key players in cancer including proto-oncogenes, regulators of apoptosis and the cell cycle, and pro-inflammatory cytokines, principally ...by directing the decay kinetics of their encoded mRNAs. Most studies support an mRNA-destabilizing role for AUF1, although other findings suggest additional functions for this factor. In this review, we explore how changes in AUF1 isoform distribution, subcellular localization, and post-translational protein modifications can influence the metabolism of targeted mRNAs. However, several lines of evidence also support a role for AUF1 in the initiation and/or development of cancer. Many AUF1-targeted transcripts encode products that control pro- and anti-oncogenic processes. Also, overexpression of AUF1 enhances tumorigenesis in murine models, and AUF1 levels are enhanced in some tumors. Finally, signaling cascades that modulate AUF1 function are deregulated in some cancerous tissues. Together, these features suggest that AUF1 may play a prominent role in regulating the expression of many genes that can contribute to tumorigenic phenotypes, and that this post-transcriptional regulatory control point may be subverted by diverse mechanisms in neoplasia.
The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, ...and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.
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•Quantitative acetylomics reveals thousands of in vivo substrates of CBP/p300•CBP/p300-regulated sites display rapid deacetylation kinetics•Histone H2B is prominently acetylated by CBP/p300 at multiple sites•Rapid turnover of CBP/p300-catalyzed acetylation controls gene transcription
A comprehensive look at CBP/p300 acetylation reveals dynamic changes that shape the regulation of protein function and gene expression.
NUT midline carcinoma (NMC) is a rare, aggressive subtype of squamous carcinoma that is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two ...bromodomains, and NUT recruits the p300 histone acetyltransferse (HAT) to activate transcription of oncogenic target genes. BET selective bromodomain inhibitors have demonstrated on-target activity in NMC patients, but with limited efficacy. P300, like BRD4, contains a bromodomain. We show that combining selective p300/CBP and BET bromodomain inhibitors, GNE-781 and OTX015, respectively, induces cooperative depletion of MYC and synergistic inhibition of NMC growth. Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition. In three disseminated NMC xenograft models, NEO2734 provided greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or 'standard' chemotherapy. Our findings provide a strong rationale for clinical study of NEO2734 in NMC patients. Moreover, the synergistic inhibition of NMC growth by CBP/p300 and BET bromodomain inhibition lays the groundwork for greater mechanistic understanding of the interplay between p300 and BRD4-NUT that drives this cancer.
The histone acetyltransferase (HAT) enzymes p300 and CBP are closely related paralogs that serve as transcriptional coactivators and have been found to be dysregulated in cancer and other diseases. ...p300/CBP is a multidomain protein and possesses a highly conserved bromodomain that has been shown to bind acetylated Lys residues in both proteins and various small molecules, including I-CBP112 and CBP30. Here we show that the ligand I-CBP112 can stimulate nucleosome acetylation up to 3-fold while CBP30 does not. Activation of p300/CBP by I-CBP112 is not observed with the isolated histone H3 substrate but requires a nucleosome substrate. I-CBP112 does not impact nucleosome acetylation by the isolated p300 HAT domain, and the effects of I-CBP112 on p300/CBP can be neutralized by CBP30, suggesting that I-CBP112 likely allosterically activates p300/CBP through bromodomain interactions. Using mass spectrometry and Western blots, we have found that I-CBP112 particularly stimulates acetylation of Lys18 of histone H3 (H3K18) in nucleosomes, an established in vivo site of p300/CBP. In addition, we show that I-CBP112 enhances H3K18 acetylation in acute leukemia and prostate cancer cells in a concentration range commensurate with its antiproliferative effects. Our findings extend the known pharmacology of bromodomain ligands in the regulation of p300/CBP and suggest a novel approach to modulating histone acetylation in cancer.
p300 and CBP are highly related histone acetyltransferase (HAT) enzymes that regulate gene expression, and their dysregulation has been linked to cancer and other diseases. p300/CBP is composed of a ...number of domains including a HAT domain, which is inhibited by the small molecule A-485, and an acetyl-lysine binding bromodomain, which was recently found to be selectively antagonized by the small molecule I-CBP112. Here we show that the combination of I-CBP112 and A-485 can synergize to inhibit prostate cancer cell proliferation. We find that the combination confers a dramatic reduction in p300 chromatin occupancy compared to the individual effects of blocking either domain alone. Accompanying this loss of p300 on chromatin, combination treatment leads to the reduction of specific mRNAs including androgen-dependent and pro-oncogenic prostate genes such as KLK3 (PSA) and c-Myc. Consistent with p300 directly affecting gene expression, mRNAs that are significantly reduced by combination treatment also exhibit a strong reduction in p300 chromatin occupancy at their gene promoters. The relatively few mRNAs that are up-regulated upon combination treatment show no correlation with p300 occupancy. These studies provide support for the pharmacologic advantage of concurrent targeting of two domains within one key epigenetic modification enzyme.
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•Allosteric ligands of histone modifying enzymes pose a new drug targeting strategy.•KDM5A, p300/CBP, Gcn5, and PRMT3 have allosteric ligands that modulate function.•SGC707, an ...allosteric ligand of PRMT3, functions by inducing conformational changes.
Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings.
Histone modifications, largely regulated by histone acetyltransferases (HAT) and histone deacetylases, have been recognized as major regulatory mechanisms governing human diseases, including cancer. ...Despite significant effort and recent advances, the mechanism by which the HAT and transcriptional coactivator p300 mediates tumorigenesis remains unclear. Here, we use a genetic and chemical approach to identify the microphthalmia-associated transcription factor (MITF) as a critical downstream target of p300 driving human melanoma growth. Direct transcriptional control of MITF by p300-dependent histone acetylation within proximal gene regulatory regions was coupled to cellular proliferation, suggesting a significant growth regulatory axis. Further analysis revealed forkhead box M1 (FOXM1) as a key effector of the p300-MITF axis driving cell growth that is selectively activated in human melanomas. Targeted chemical inhibition of p300 acetyltransferase activity using a potent and selective catalytic p300/CBP inhibitor demonstrated significant growth inhibitory effects in melanoma cells expressing high levels of MITF. Collectively, these data confirm the critical role of the p300-MITF-FOXM1 axis in melanoma and support p300 as a promising novel epigenetic therapeutic target in human melanoma. SIGNIFICANCE: These results show that MITF is a major downstream target of p300 in human melanoma whose expression is predictive of melanoma response to small-molecule inhibition of p300 HAT activity.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein ...expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3′-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD+ binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.The glycolytic enzyme GAPDH is a non-canonical RNA-binding protein.
A dimer interface mutation impairs the two-step binding of GAPDH to AU-rich elements from TNF-α mRNA and leads to RNA structural changes.
Dimer and tetramer interface residues are important for GAPDH-RNA binding.
We propose a novel model for GAPDH binding to AU-rich RNA via the oligomeric interfaces.
The ongoing Russian invasion of Ukraine marks a critical juncture in a series of events posing severe threat to the health of Ukrainian citizens. While recent reports reveal higher rates of PTSD in ...Ukrainian refugees following Russia's invasion - data for Ukrainians remaining at the warfront is inherently difficult to access. A primarily elderly demographic, Ukrainians in previously Russian-occupied areas near the front (UPROANF) are at particular risk.
Data was sourced from screening questionnaires administered between March 2022 and July 2023 by mobile health clinics providing services to UPROANF.
Previously occupied villages in Eastern and Southern Ukraine.
UPROANF attending clinics completed voluntary self-report surveys reporting demographics, prior health diagnoses, and PTSD symptom severity (
= 450; Mean
= 53.66; 72.0% female).
Participants were exposed to Russian occupation of Ukrainian villages.
The PTSD Checklist for the DSM-V (PCL-5) with recommended diagnostic threshold (i.e. 31) was utilized to assess PTSD prevalence and symptom severity. ANCOVA was used to examine hypothesized positive associations between (1) HTN and (2) loneliness and PTSD symptoms (cumulative and by symptom cluster).
Between 47.8% and 51.33% screened positive for PTSD. Though cumulative PTSD symptoms did not differ based on HTN diagnostic status, those with HTN reported significantly higher PTSD re-experiencing symptoms (
= 1.25,
= 0.60,
= .046). Loneliness was significantly associated with more severe cumulative PTSD symptoms (
= 1.29,
= 0.31,
< .001), re-experiencing (
= 0.47,
= 0.12,
< .001), avoidance (
= .18,
= 0.08,
= .038), and hypervigilance (
= 0.29,
= 0.13,
= .036).
PTSD prevalence was higher than other war-exposed populations. Findings highlight the urgent mental health burden among UPROANF, emphasizing the need for integrated care models addressing both trauma and physical health. Given the significance of loneliness as a risk factor, findings suggest the potential for group-based, mind-body interventions to holistically address the physical, mental, and social needs of this highly traumatized, underserved population.
AU-rich element RNA-binding protein 1 (AUF1) regulates the stability and/or translational efficiency of diverse mRNA targets, including many encoding products controlling the cell cycle, apoptosis, ...and inflammation by associating with AU-rich elements residing in their 3′-untranslated regions. Previous biochemical studies showed that optimal AUF1 binding requires 33–34 nucleotides with a strong preference for U-rich RNA despite observations that few AUF1-associated cellular mRNAs contain such extended U-rich domains. Using the smallest AUF1 isoform (p37AUF1) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37AUF1 RNP complexes within a larger RNA context. In particular, p37AUF1 binding to short AU-rich RNA targets was significantly stabilized by interactions with a 3′-purine residue and largely base-independent but non-ionic contacts 5′ of the AU-rich site. RNP stabilization by the upstream RNA domain was associated with an enhanced negative change in heat capacity consistent with conformational changes in protein and/or RNA components, and fluorescence resonance energy transfer-based assays demonstrated that these contacts were required for p37AUF1 to remodel local RNA structure. Finally, reporter mRNAs containing minimal high affinity p37AUF1 target sequences associated with AUF1 and were destabilized in a p37AUF1-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs but also suggest how AUF1 binding could regulate protein and/or microRNA binding events at adjacent sites.
Background: AUF1 post-transcriptionally regulates mRNA targets.
Results: Short AU-rich sequences nucleate AUF1 ribonucleoproteins but require non-ionic contacts with flanking RNA to stabilize complexes and manipulate local RNA structure.
Conclusion: AUF1 uses several molecular determinants to bind and remodel RNA targets.
Significance: This model explains AUF1 interactome diversity and predicts allosteric effects on protein and microRNA trans-factor binding to proximal sites.
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