The use of ecological momentary assessment for family caregivers of adults with chronic conditions: A systematic review.

Background: Ecological momentary assessment (EMA) as a real-time data collection method can provide insight into the daily experiences of family caregivers. Purpose: This systematic review aimed to synthesize studies involving EMA completed by family caregivers of adults with chronic conditions. Methods: A systematic search was conducted within six databases for articles published from the inception of the database through September 2023. We extracted the characteristics of the included studies and data on EMA-specific methods to determine the quality of the included studies. Results: A total of 12 studies involving EMA completed by family caregivers of adults with chronic conditions were identified, with almost all studies focused on caregivers of persons with Alzheimer's or dementia-related conditions. The average compliance rate across the included studies was 75%, below the recommended rate. In addition, most of the included studies did not collect the family caregivers' daily activities and care contexts in their responses (i.e., affect, stress, well-being, care demand, and fatigue) to the EMA prompts. Discussion: This review showed that using EMA to collect information on family caregivers of adults with chronic health conditions appeared feasible and acceptable. However, the methodology or design of using EMA to collect caregiver information in this population is still preliminary. The limited number of existing studies that have used EMA to capture the daily experiences of family caregivers does not provide key information that could improve understanding of caregivers' emotional experiences and well-being in real-life situations. We identified gaps in the literature that warrant additional EMA studies for this population.

Gaining Wings to FLY: Using Drosophila Oogenesis as an Entry Point for Citizen Scientists in Laboratory Research.

Citizen science is a productive approach to include non-scientists in research efforts that impact particular issues or communities. In most cases, scientists at advanced career stages design high-quality, exciting projects that enable citizen contribution, a crowdsourcing process that drives discovery forward and engages communities. The challenges of having citizens design their own research with no or limited training and providing access to laboratory tools, reagents, and supplies have limited citizen science efforts. This leaves the incredible life experiences and immersion of citizens in communities that experience health disparities out of the research equation, thus hampering efforts to address community health needs with a full picture of the challenges that must be addressed. Here, we present a robust and reproducible approach that engages participants from Grade 5 through adult in research focused on defining how diet impacts disease signaling. We leverage the powerful genetics, cell biology, and biochemistry of Drosophila oogenesis to define how nutrients impact phenotypes associated with genetic mutants that are implicated in cancer and diabetes. Participants lead the project design and execution, flipping the top-down hierarchy of the prevailing scientific culture to co-create research projects and infuse the research with cultural and community relevance.

Selective CDK9 Inhibition by Natural Compound Toyocamycin in Cancer Cells.

Aberrant transcription in cancer cells involves the silencing of tumor suppressor genes (TSGs) and activation of oncogenes. Transcriptomic changes are associated with epigenomic alterations such as DNA-hypermethylation, histone deacetylation, and chromatin condensation in promoter regions of silenced TSGs. To discover novel drugs that trigger TSG reactivation in cancer cells, we used a GFP-reporter system whose expression is silenced by promoter DNA hypermethylation and histone deacetylation. After screening a natural product drug library, we identified that toyocamycin, an adenosine-analog, induces potent GFP reactivation and loss of clonogenicity in human colon cancer cells. Connectivity-mapping analysis revealed that toyocamycin produces a pharmacological signature mimicking cyclin-dependent kinase (CDK) inhibitors. RNA-sequencing revealed that the toyocamycin transcriptomic signature resembles that of a specific CDK9 inhibitor (HH1). Specific inhibition of RNA Pol II phosphorylation level and kinase assays confirmed that toyocamycin specifically inhibits CDK9 (IC50 = 79 nM) with a greater efficacy than other CDKs (IC50 values between 0.67 and 15 µM). Molecular docking showed that toyocamycin efficiently binds the CDK9 catalytic site in a conformation that differs from other CDKs, explained by the binding contribution of specific amino acids within the catalytic pocket and protein backbone. Altogether, we demonstrated that toyocamycin exhibits specific CDK9 inhibition in cancer cells, highlighting its potential for cancer chemotherapy.

The role of trauma, social support, and demography on veteran resilience.

Background: Historically, resilience has often been conceptualized as the sustained lack of symptoms following trauma exposure. In line with a novel conceptualization of resilience as being dynamic over lifespan, determined by interacting biological and environmental factors, we examined the VA Mid-Atlantic Post Deployment Mental Health Repository (PDMH) comprised of 3876 US Military Veterans with and without PTSD diagnoses. Methods: We performed regression modelling to study the relationship between resilience (measured with Connor Davidson Resilience Scale; CD-RISC), posttraumatic stress disorder (PTSD) severity (Davidson Trauma Scale; DTS), social support (Medical Outcome Study Social Support Survey; MOSSS), combat exposure (Combat Exposure Scale; CES), childhood trauma (Trauma Life Events Questionnaire; TLEQ), and demographic factors. CD-RISC was positively correlated with years of education and negatively correlated with DTS, CES and TLEQ scores. Results: We found an interaction between CD-RISC and CES in predicting PTSD severity (Davidson Trauma Scale). Specifically, high resilience predicted lower PTSD symptom severity than low resilience, this relationship was amplified with increasing levels of combat exposure. Structural equation modelling (SEM) identified an optimal latent variable that represents resilience and relationships between latent variables for resilience, trauma, and illness. We derived a resilience latent variable composed of age, education level, MOSSS and race. Conclusions: Our results support a conceptualization of resilience as a multifactorial determinant that coexists with PTSD, a state rather than trait variable, and can be quantified by biological and behavioural metrics. HIGHLIGHTS: • Historically, resilience has often been conceptualized as the sustained lack of symptoms following trauma exposure.• We examined the VA Mid-Atlantic Post Deployment Mental Health Repository (PDMH) comprised of 3876 US Military Veterans.• We found an interaction effect between CD-RISC and CES in predicting PTSD severity (Davidson Trauma Scale).

Antecedentes: Históricamente, la resiliencia a menudo se ha conceptualizado como la ausencia sostenida de síntomas después de la exposición al trauma. En línea con una novedosa conceptualización de la resiliencia como un fenómeno dinámico a lo largo de la vida, determinada por la interacción de factores biológicos y ambientales, examinamos el Repositorio de salud mental post-despliegue VA Mid-Atlantic (PDMH por sus siglas en ingles) compuesto por 3.876 veteranos militares de EE.UU. con y sin diagnósticos de TEPT.Métodos: Realizamos modelos de regresión para estudiar la relación entre resiliencia (medida con la Escala de resiliencia de Connor Davidson; CD-RISC por sus siglas en ingles), gravedad del trastorno de estrés postraumático (TEPT) (con Escala de Trauma de Davidson; DTS por sus siglas en ingles), apoyo social (Encuesta de Estudio de Resultados Médicos - Apoyo Social; MOSSS por sus siglas en ingles), exposición al combate (Escala de exposición al combate; CES por sus siglas en ingles), trauma infantil (Cuestionario de Eventos de vida traumáticos; TLEQ por sus siglas en ingles), y factores demográficos. CD-RISC se correlacionó positivamente con años de educación y se correlacionó negativamente con los puntajes de DTS, CES y TLEQ.Resultados: Encontramos una interacción entre CD-RISC y CES en la predicción de la gravedad del TEPT (Escala de trauma de Davidson). Específicamente, una alta resiliencia predijo menor gravedad de los síntomas de TEPT que una baja resiliencia, esta relación fue amplificada con niveles crecientes de exposición al combate. El modelo de ecuaciones estructurales (SEM por sus siglas en ingles) identificó una variable latente óptima que representa la resiliencia y las relaciones entre las variables latentes de resiliencia, trauma y enfermedad. Derivamos una variable latente de resiliencia compuesta por edad, nivel educativo, MOSSS y raza.Conclusiones: Nuestros resultados apoyan una conceptualización de la resiliencia como un determinante multifactorial que coexiste con el TEPT, una variable de estado más que de rasgo, y puede ser cuantificada con mediciones biológicas y conductuales.

Surprising phenotypic diversity of cancer-associated mutations of Gly 34 in the histone H3 tail.

Sequencing of cancer genomes has identified recurrent somatic mutations in histones, termed oncohistones, which are frequently poorly understood. Previously we showed that fission yeast expressing only the H3.3G34R mutant identified in aggressive pediatric glioma had reduced H3K36 trimethylation and acetylation, increased genomic instability and replicative stress, and defective homology-dependent DNA damage repair. Here we show that surprisingly distinct phenotypes result from G34V (also in glioma) and G34W (giant cell tumors of bone) mutations, differentially affecting H3K36 modifications, subtelomeric silencing, genomic stability; sensitivity to irradiation, alkylating agents, and hydroxyurea; and influencing DNA repair. In cancer, only 1 of 30 alleles encoding H3 is mutated. Whilst co-expression of wild-type H3 rescues most G34 mutant phenotypes, G34R causes dominant hydroxyurea sensitivity, homologous recombination defects, and dominant subtelomeric silencing. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in H3 and highlight the utility of genetically tractable systems for their analysis.

A pharmacokinetic-pharmacodynamic model for the MET tyrosine kinase inhibitor, savolitinib, to explore target inhibition requirements for anti-tumour activity.

BACKGROUND AND PURPOSE: Savolitinib (AZD6094, HMPL-504, volitinib) is an oral, potent, and highly MET receptor TK inhibitor. This series of studies aimed to develop a pharmacokinetic-pharmacodynamic (PK/PD) model to link inhibition of MET phosphorylation (pMET) by savolitinib with anti-tumour activity. EXPERIMENTAL APPROACH: Cell line-derived xenograft (CDX) experiments using human lung cancer (EBC-1) and gastric cancer (MKN-45) cells were conducted in athymic nude mice using a variety of doses and schedules of savolitinib. Tumour pMET changes and growth inhibition were calculated after 28 days. Population PK/PD techniques were used to construct a PK/PD model for savolitinib. KEY RESULTS: Savolitinib showed dose- and dose frequency-dependent anti-tumour activity in the CDX models, with more frequent, lower dosing schedules (e.g., twice daily) being more effective than intermittent, higher dosing schedules (e.g., 4 days on/3 days off or 2 days on/5 days off). There was a clear exposure-response relationship, with maximal suppression of pMET of >90%. Data from additional CDX and patient-derived xenograft (PDX) models overlapped, allowing calculation of a single EC50 of 0.38 ng·ml-1 . Tumour growth modelling demonstrated that prolonged, high levels of pMET inhibition (>90%) were required for tumour stasis and regression in the models. CONCLUSION AND IMPLICATIONS: High and persistent levels of MET inhibition by savolitinib were needed for optimal monotherapy anti-tumour activity in preclinical models. The modelling framework developed here can be used to translate tumour growth inhibition from the mouse to human and thus guide choice of clinical dose and schedule.

Discordant Effects of Putative Lysine Acetyltransferase Inhibitors in Biochemical and Living Systems.

Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo.

Two factor authentication: Asf1 mediates crosstalk between H3 K14 and K56 acetylation.

The ability of histone chaperone Anti-silencing factor 1 (Asf1) to direct acetylation of lysine 56 of histone H3 (H3K56ac) represents an important regulatory step in genome replication and DNA repair. In Saccharomyces cerevisiae, Asf1 interacts functionally with a second chaperone, Vps75, and the lysine acetyltransferase (KAT) Rtt109. Both Asf1 and Vps75 can increase the specificity of histone acetylation by Rtt109, but neither alter selectivity. However, changes in acetylation selectivity have been observed in histones extracted from cells, which contain a plethora of post-translational modifications. In the present study, we use a series of singly acetylated histones to test the hypothesis that histone pre-acetylation and histone chaperones function together to drive preferential acetylation of H3K56. We show that pre-acetylated H3K14ac/H4 functions with Asf1 to drive specific acetylation of H3K56 by Rtt109-Vps75. Additionally, we identified an exosite containing an acidic patch in Asf1 and show that mutations to this region alter Asf1-mediated crosstalk that changes Rtt109-Vps75 selectivity. Our proposed mechanism suggests that Gcn5 acetylates H3K14, recruiting remodeler complexes, allowing for the Asf1-H3K14ac/H4 complex to be acetylated at H3K56 by Rtt109-Vps75. This mechanism explains the conflicting biochemical data and the genetic links between Rtt109, Vps75, Gcn5 and Asf1 in the acetylation of H3K56.

Author Correction: Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The central role of disgust in disorders of food avoidance.

BACKGROUND: Individuals with extreme food avoidance such as Avoidant Restrictive Food Intake Disorder (ARFID) experience impairing physical and mental health consequences from nutrition of insufficient variety or/and quantity. Identifying mechanisms contributing to food avoidance is essential to develop effective interventions. Anxiety figures prominently in theoretical models of food avoidance; however, there is limited evidence that repeated exposures to foods increases approach behavior in ARFID. Studying disgust, and relationships between disgust and anxiety, may offer novel insights, as disgust is functionally associated with avoidance of contamination from pathogens (as may occur via ingestion) and is largely resistant to extinction. METHOD: This exploratory, cross-sectional study included data from 1,644 adults who completed an online questionnaire. Participant responses were used to measure ARFID classification, picky eating, sensory sensitivity, disgust, and anxiety. Structural equation modeling tested a measurement model of latent disgust and anxiety factors as measured by self-reported frequency of disgust and anxiety reactions. Mediational models were used to explore causal ordering. RESULTS: A latent disgust factor was more strongly related to severity of picky eating (B ≈ 0.4) and ARFID classification (B ≈ 0.6) than the latent anxiety factor (B ≈ 0.1). Disgust partially mediated the association between anxiety and picky eating and fully mediated the association between anxiety and ARFID. Models testing the reverse causal ordering demonstrated poorer fit. Findings suggest anxiety may be associated with food avoidance in part due to increased disgust. CONCLUSIONS: Disgust may play a prominent role in food avoidance. Findings may inform novel approaches to treatment.

Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic "immuno-oncogenes" that modulate the immune microenvironment of cancer.Muscle-invasive bladder cancer (MIBC) is a potentially lethal disease. Here the authors characterize diverse genetic alterations in MIBC that convergently lead to constitutive activation of PPARgamma/RXRalpha and result in immunosurveillance escape by inhibiting CD8+ T-cell recruitment.

The Clinical Significance of Posterior Insular Volume in Adolescent Anorexia Nervosa.

OBJECTIVE: The diagnostic criterion disturbance in the experience of the body remains a poorly understood and persistent feature of anorexia nervosa (AN). Increased sophistication in understanding the structure of the insular cortex-a neural structure that receives and integrates visceral sensations with action and meaning-may elucidate the nature of this disturbance. We explored age, weight status, illness severity, and self-reported body dissatisfaction associations with insular cortex volume. METHODS: Structural magnetic resonance imaging data were collected from 21 adolescents with a history of AN and 20 age-, sex-, and body mass index-matched controls. Insular cortical volumes (bilateral anterior and posterior regions) were identified using manual tracing. RESULTS: Volumes of the right posterior insula demonstrated the following: (a) a significant age by clinical status interaction (ß = -0.018 [0.008]; t = 2.32, p = .02) and (b) larger volumes were associated with longer duration of illness (r = 0.48, p < .04). In contrast, smaller volumes of the right anterior insula were associated with longer duration of illness (r = -0.50, p < .03). The associations of insular volume with body dissatisfaction were of moderate effect size and also of opposite direction, but a statistical trend in right posterior (r = 0.40, p < .10 in right posterior; r = -0.49, p < .04 in right anterior). CONCLUSIONS: In this exploratory study, findings of atypical structure of the right posterior insular cortex point to the importance of future work investigating the role of visceral afferent signaling in understanding disturbance in body experience in AN.

Histone H3G34R mutation causes replication stress, homologous recombination defects and genomic instability in S. pombe.

Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3.3. H3.3-G34R/V mutants are common in tumors with mutations in p53 and ATRX, an H3.3-specific chromatin remodeler. To gain insight into the role of H3-G34R, we generated fission yeast that express only the mutant histone H3. H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation, and mutants show partial transcriptional overlap with set2 deletions. H3-G34R mutants exhibit genomic instability and increased replication stress, including slowed replication fork restart, although DNA replication checkpoints are functional. H3-G34R mutants are defective for DNA damage repair by homologous recombination (HR), and have altered HR protein dynamics in both damaged and untreated cells. These data suggest H3-G34R slows resolution of HR-mediated repair and that unresolved replication intermediates impair chromosome segregation. This analysis of H3-G34R mutant fission yeast provides mechanistic insight into how G34R mutation may promote genomic instability in glioma.

Genome Sequences of Five Streptomyces Bacteriophages Forming Cluster BG.

Cluster BG of the actinobacteriophage was formed upon discovery of five novel bacteriophages isolated by enrichment from their host, Streptomyces griseus subsp. griseus strain ATCC 10137. Four members of this cluster (BabyGotBac, Maih, TP1605, and YDN12) share over 89% average nucleotide identity, while the other (Xkcd426) has only 72% similarity to other cluster members.

Impact of a High-fat Diet on Tissue Acyl-CoA and Histone Acetylation Levels.

Cellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of Acly in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. Butyryl-CoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity in vitro This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver.

Interaction with the DNA Repair Protein Thymine DNA Glycosylase Regulates Histone Acetylation by p300.

How protein-protein interactions regulate and alter histone modifications is a major unanswered question in epigenetics. The histone acetyltransferase p300 binds thymine DNA glycosylase (TDG); utilizing mass spectrometry to measure site-specific changes in histone acetylation, we found that the absence of TDG in mouse embryonic fibroblasts leads to a reduction in the rate of histone acetylation. We demonstrate that TDG interacts with the CH3 domain of p300 to allosterically promote p300 activity to specific lysines on histone H3 (K18 and K23). However, when TDG concentrations approach those of histones, TDG acts as a competitive inhibitor of p300 histone acetylation. These results suggest a mechanism for how histone acetylation is fine-tuned via interaction with other proteins, while also highlighting a connection between regulators of two important biological processes: histone acetylation and DNA repair/demethylation.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch.

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

Acquired savolitinib resistance in non-small cell lung cancer arises via multiple mechanisms that converge on MET-independent mTOR and MYC activation.

Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. The receptor tyrosine kinase MET has been implicated as an oncogene in numerous cancer subtypes, including non-small cell lung cancer (NSCLC). Here we explore the therapeutic potential of savolitinib (volitinib, AZD6094, HMPL-504), a potent and selective MET inhibitor, in NSCLC. In vitro, savolitinib inhibits MET phosphorylation with nanomolar potency, which correlates with blockade of PI3K/AKT and MAPK signaling as well as MYC down-regulation. In vivo, savolitinib causes inhibition of these pathways and significantly decreases growth of MET-dependent xenografts. To understand resistance mechanisms, we generated savolitinib resistance in MET-amplified NSCLC cell lines and analyzed individual clones. We found that upregulation of MYC and constitutive mTOR pathway activation is a conserved feature of resistant clones that can be overcome by knockdown of MYC or dual mTORC1/2 inhibition. Lastly, we demonstrate that mechanisms of resistance are heterogeneous, arising via a switch to EGFR dependence or by a requirement for PIM signaling. This work demonstrates the efficacy of savolitinib in NSCLC and characterizes acquired resistance, identifying both known and novel mechanisms that may inform combination strategies in the clinic.

Modulation of p300/CBP Acetylation of Nucleosomes by Bromodomain Ligand I-CBP112.

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.

Quantitative Measurement of Histone Tail Acetylation Reveals Stage-Specific Regulation and Response to Environmental Changes during Drosophila Development.

Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as γ-irradiation altered histone acetylation patterns. Strikingly, γ-irradiation dramatically increased the level of acetylation at H3K18, a site linked to DNA repair via nonhomologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in the level of H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation.