Histone chaperones: assisting histone traffic and nucleosome dynamics.

The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

Essential role for centromeric factors following p53 loss and oncogenic transformation.

In mammals, centromere definition involves the histone variant CENP-A (centromere protein A), deposited by its chaperone, HJURP (Holliday junction recognition protein). Alterations in this process impair chromosome segregation and genome stability, which are also compromised by p53 inactivation in cancer. Here we found that CENP-A and HJURP are transcriptionally up-regulated in p53-null human tumors. Using an established mouse embryonic fibroblast (MEF) model combining p53 inactivation with E1A or HRas-V12 oncogene expression, we reproduced a similar up-regulation of HJURP and CENP-A. We delineate functional CDE/CHR motifs within the Hjurp and Cenpa promoters and demonstrate their roles in p53-mediated repression. To assess the importance of HJURP up-regulation in transformed murine and human cells, we used a CRISPR/Cas9 approach. Remarkably, depletion of HJURP leads to distinct outcomes depending on their p53 status. Functional p53 elicits a cell cycle arrest response, whereas, in p53-null transformed cells, the absence of arrest enables the loss of HJURP to induce severe aneuploidy and, ultimately, apoptotic cell death. We thus tested the impact of HJURP depletion in pre-established allograft tumors in mice and revealed a major block of tumor progression in vivo. We discuss a model in which an "epigenetic addiction" to the HJURP chaperone represents an Achilles' heel in p53-deficient transformed cells.

Cerebrospinal fluid transcripts may predict shunt surgery responses in normal pressure hydrocephalus.

Molecular biomarkers for neurodegenerative diseases are critical for advancing diagnosis and therapy. Normal pressure hydrocephalus (NPH) is a neurological disorder characterized by progressive neurodegeneration, gait impairment, urinary incontinence and cognitive decline. In contrast to most other neurodegenerative disorders, NPH symptoms can be improved by the placement of a ventricular shunt that drains excess CSF. A major challenge in NPH management is the identification of patients who benefit from shunt surgery. Here, we perform genome-wide RNA sequencing of extracellular vesicles in CSF of 42 NPH patients, and we identify genes and pathways whose expression levels correlate with gait, urinary or cognitive symptom improvement after shunt surgery. We describe a machine learning algorithm trained on these gene expression profiles to predict shunt surgery response with high accuracy. The transcriptomic signatures we identified may have important implications for improving NPH diagnosis and treatment and for understanding disease aetiology.

Examining Excess Mortality Among Critical Workers in Minnesota During 2020-2021: An Occupational Analysis.

Objectives. To understand the occupational risk associated with COVID-19 among civilian critical workers (aged 16-65 years) in Minnesota. Methods. We estimated excess mortality in 2020 to 2021 for critical occupations in different racial groups and vaccine rollout phases using death certificates and occupational employment rates for 2017 to 2021. Results. Excess mortality during the COVID-19 pandemic was higher for workers in critical occupations than for noncritical workers. Some critical occupations, such as transportation and logistics, construction, and food service, experienced higher excess mortality than did other critical occupations, such as health care, K-12 school staff, and agriculture. In almost all occupations investigated, workers of color experienced higher excess mortality than did White workers. Excess mortality in 2021 was greater than in 2020 across groups: occupations, vaccine eligibility tiers, and race/ethnicity. Conclusions. Although workers in critical occupations experienced greater excess mortality than did others, excess mortality among critical workers varied substantially by occupation and race. Public Health Implications. Analysis of mortality across occupations can be used to identify vulnerable populations, prioritize protective interventions for them, and develop targeted worker safety protocols to promote equitable health outcomes. (Am J Public Health. 2023;113(11):1219-1222. https://doi.org/10.2105/AJPH.2023.307395).

Opportunities and Challenges in Targeting the Proofreading Activity of SARS-CoV-2 Polymerase Complex.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. While the development of vaccines and the emergence of antiviral therapeutics is promising, alternative strategies to combat COVID-19 (and potential future pandemics) remain an unmet need. Coronaviruses feature a unique mechanism that may present opportunities for therapeutic intervention: the RNA polymerase complex of coronaviruses is distinct in its ability to proofread and remove mismatched nucleotides during genome replication and transcription. The proofreading activity has been linked to the exonuclease (ExoN) activity of non-structural protein 14 (NSP14). Here, we review the role of NSP14, and other NSPs, in SARS-CoV-2 replication and describe the assays that have been developed to assess the ExoN function. We also review the nucleoside analogs and non-nucleoside inhibitors known to interfere with the proofreading activity of NSP14. Although not yet validated, the potential use of non-nucleoside proofreading inhibitors in combination with chain-terminating nucleosides may be a promising avenue for the development of anti-CoV agents.

Patient Portal Use, Perceptions of Electronic Health Record Value, and Self-Rated Primary Care Quality Among Older Adults: Cross-sectional Survey.

BACKGROUND: Older adults are increasingly accessing information and communicating using patient-facing portals available through their providers' electronic health record (EHR). Most theories of technology acceptance and use suggest that patients' overall satisfaction with care should be independent of their chosen level of portal engagement. However, achieving expected benefits of portal use depends on demonstrated support from providers to meet these expectations. This is especially true among older adults, who may require more guidance. However, little is known about whether misalignment of expectations around technology-facilitated care is associated with lower perceptions of care quality. OBJECTIVE: The aims of this study were to analyze whether older adults' assessment of primary care quality differs across levels of patient portal engagement and whether perceptions of how well their provider uses the EHR to support care moderates this relationship. METHODS: We conducted a cross-sectional survey analysis of 158 older adults over the age of 65 (average age 71.4 years) across Michigan using a 13-measure composite of self-assessed health care quality. Portal use was categorized as none, moderate (use of 1-3 functionalities), or extensive (use of 4-7 functionalities). EHR value perception was measured by asking respondents how they felt their doctor's EHR use improved the patient-provider relationship. RESULTS: Moderate portal users, compared to those who were extensive users, had lower estimated care quality (-0.214 on 4-point scale; P=.03). Differences between extensive portal users and nonportal users were not significant. Quality perception was only particularly low among moderate portal users with low EHR value perception; those with high EHR value perception rated quality similarly to other portal user groups. CONCLUSIONS: Older adults who are moderate portal users are the least satisfied with their care, and the most sensitive to perceptions of how well their provider uses the EHR to support the relationship. Encouraging portal use without compromising perceptions of quality requires thinking beyond patient-focused education. Achieving value from use of patient-facing technologies with older adults is contingent upon matched organizational investments that support technology-enabled care delivery. Providers and staff need policies and practices that demonstrate technology adeptness. Older adults may need more tailored signaling and accommodation for technology to be maximally impactful.

A specific function for the histone chaperone NASP to fine-tune a reservoir of soluble H3-H4 in the histone supply chain.

Proper genome packaging requires coordination of both DNA and histone metabolism. While histone gene transcription and RNA processing adequately provide for scheduled needs, how histone supply adjusts to unexpected changes in demand remains unknown. Here, we reveal that the histone chaperone nuclear autoantigenic sperm protein (NASP) protects a reservoir of soluble histones H3-H4. The importance of NASP is revealed upon histone overload, engagement of the reservoir during acute replication stress, and perturbation of Asf1 activity. The reservoir can be fine-tuned, increasing or decreasing depending on the level of NASP. Our data suggest that NASP does so by balancing the activity of the heat shock proteins Hsc70 and Hsp90 to direct H3-H4 for degradation by chaperone-mediated autophagy. These insights into NASP function and the existence of a tunable reservoir in mammalian cells demonstrate that contingency is integrated into the histone supply chain to respond to unexpected changes in demand.

PP32 and SET/TAF-Iß proteins regulate the acetylation of newly synthesized histone H4.

Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iß. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iß protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iß depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iß and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4.

Contrasting Effectiveness and Cost-Effectiveness of Colorectal Cancer Screening Under Commercial Insurance vs. Medicare.

OBJECTIVES: Most cost-effectiveness analyses of colorectal cancer (CRC) screening assume Medicare payment rates and a lifetime horizon. Our aims were to examine the implications of differential payment levels and time horizons for commercial insurers vs. Medicare on the cost-effectiveness of CRC screening. METHODS: We used our validated Markov cohort simulation of CRC screening in the average risk US population to examine CRC screening at ages 50-64 under commercial insurance, and at ages 65-80 under Medicare, using a health-care sector perspective. Model outcomes included discounted quality-adjusted life-years (QALYs) and costs per person, and incremental cost/QALY gained. RESULTS: Lifetime costs/person were 20-44% higher when assuming commercial payment rates rather than Medicare rates for people under 65. Most of the substantial clinical benefit of screening at ages 50-64 was realized at ages ≥65. For commercial payers with a time horizon of ages 50-64, fecal occult blood testing (FOBT) and fecal immunochemical testing (FIT) were cost-effective (<$61,000/QALY gained), but colonoscopy was costly (>$185,000/QALY gained). Medicare experienced substantial clinical benefits and cost-savings from screening done at ages <65, even if screening was not continued. Among those previously screened, continuing FOBT and FIT under Medicare was cost-saving and continuing colonoscopy was highly cost-effective (<$30,000/QALY gained), and initiating any screening in those previously unscreened was highly effective and cost-saving. CONCLUSIONS: Modeling suggests that CRC screening is highly cost-effective over a lifetime even when considering higher payment rates by commercial payers vs. Medicare. Screening may appear relatively costly for commercial payers if only a time horizon of ages 50-64 is considered, but it is predicted to yield substantial clinical and economic benefits that accrue primarily at ages ≥65 under Medicare.

Methylation of histone H3 lysine 9 occurs during translation.

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.

Histone lysine methylation and chromatin replication.

In eukaryotic organisms, the replication of the DNA sequence and its organization into chromatin are critical to maintain genome integrity. Chromatin components, such as histone variants and histone post-translational modifications, along with the higher-order chromatin structure, impact several DNA metabolic processes, including replication, transcription, and repair. In this review we focus on lysine methylation and the relationships between this histone mark and chromatin replication. We first describe studies implicating lysine methylation in regulating early steps in the replication process. We then discuss chromatin reassembly following replication fork passage, where the incorporation of a combination of newly synthesized histones and parental histones can impact the inheritance of lysine methylation marks on the daughter strands. Finally, we elaborate on how the inheritance of lysine methylation can impact maintenance of the chromatin landscape, using heterochromatin as a model chromatin domain, and we discuss the potential mechanisms involved in this process.

Subfunctionalization via adaptive evolution influenced by genomic context: the case of histone chaperones ASF1a and ASF1b.

Gene duplication is regarded as the main source of adaptive functional novelty in eukaryotes. Processes such as neo- and subfunctionalization impact the evolution of paralogous proteins where functional divergence is frequently key to retain the gene copies. Here, we examined antisilencing function 1 (ASF1), a conserved eukaryotic H3-H4 histone chaperone, involved in histone dynamics during replication, transcription, and DNA repair. Although yeast feature a single ASF1 protein, two paralogs exist in most vertebrates, termed ASF1a and ASF1b, with distinct cellular roles in mammals. To explain this division of tasks, we integrated evolutionary and comparative genomic analyses with biochemical and structural approaches. First, we show that a duplication event at the ancestor of jawed vertebrates, followed by ASF1a relocation into an intron of the minichromosome maintenance complex component 9 (MCM9) gene at the ancestor of tetrapods, provided a different genomic environment for each paralog with marked differences of GC content and DNA replication timing. Second, we found signatures of positive selection in the N- and C-terminal regions of ASF1a and ASF1b. Third, we demonstrate that regions outside the primary interaction surface are key for the preferential interactions of the human paralogs with distinct H3-H4 chaperones. On the basis of these data, we propose that ASF1 experienced subfunctionalization shaped by the adaptation of the genes to their respective genomic context, reflecting a case of genomic context-driven escape from adaptive conflict.

Colorectal testing utilization and payments in a large cohort of commercially insured US adults.

OBJECTIVES: Screening decreases colorectal cancer (CRC) mortality. The national press has scrutinized colonoscopy charges. Little systematic evidence exists on colorectal testing and payments among commercially insured persons. Our aim was to characterize outpatient colorectal testing utilization and payments among commercially insured US adults. METHODS: We conducted an observational cohort study of outpatient colorectal test utilization rates, indications, and payments among 21 million 18-64-year-old employees and dependants with noncapitated group health insurance provided by 160 self-insured employers in the 2009 Truven MarketScan Databases. RESULTS: Colonoscopy was the predominant colorectal test. Among 50-64-year olds, 12% underwent colonoscopy in 1 year. Most fecal tests and colonoscopies were associated with screening/surveillance indications. Testing rates were higher in women, and increased with age. Mean payments for fecal occult blood and immunochemical tests were $5 and $21, respectively. Colonoscopy payments varied between and within sites of service. Mean payments for diagnostic colonoscopy in an office, outpatient hospital facility, and ambulatory surgical center were $586 (s.d. $259), $1,400 (s.d. $681), and $1,074 (s.d. $549), respectively. Anesthesia and pathology services accompanied 35 and 52% of colonoscopies, with mean payments of $494 (s.d. $354) and $272 (s.d. $284), respectively. Mean payments for the most prevalent colonoscopy codes were 1.4- to 1.9-fold the average Medicare payments. CONCLUSIONS: Most outpatient colorectal testing among commercially insured adults was associated with screening or surveillance. Payments varied widely across sites of service, and payments for anesthesia and pathology services contributed substantially to total payments. Cost-effectiveness analyses of CRC screening have relied on Medicare payments as proxies for costs, but cost-effectiveness may differ when analyzed from the perspectives of Medicare or commercial insurers.

Physician practice competition and prices paid by private insurers for office visits.

IMPORTANCE: Physician practice consolidation could promote higher-quality care but may also create greater economic market power that could lead to higher prices for physician services. OBJECTIVE: To assess the relationship between physician competition and prices paid by private preferred provider organizations (PPOs) for 10 types of office visits in 10 prominent specialties. DESIGN AND SETTING: Retrospective study in 1058 US counties in urbanized areas, representing all 50 states, examining the relationship between measured physician competition and prices paid for office visits in 2010 and the relationship between changes in competition and prices between 2003 and 2010, using regression analysis to control for possible confounding factors. EXPOSURES: Variation in the mean Hirschman-Herfindahl Index (HHI) of physician practices within a county by specialty (HHIs range from 0, representing maximally competitive markets, to 10,000 in markets served by a single [monopoly] practice). MAIN OUTCOMES AND MEASURES: Mean price paid by county to physicians in each specialty by private PPOs for intermediate office visits with established patients (Current Procedural Terminology [CPT] code 99213) and a price index measuring the county-weighted mean price for 10 types of office visits with new and established patients (CPT codes 99201-99205, 99211-99215) relative to national mean prices. RESULTS: In 2010, across all specialties studied, HHIs were 3 to 4 times higher in the 90th-percentile county than the 10th-percentile county (eg, for family practice: 10th percentile HHI = 1023 and 90th percentile HHI = 3629). Depending on specialty, mean price for a CPT code 99213 visit was between $70 and $75. After adjustment for potential confounders, depending on specialty, prices at the 90th-percentile HHI were between $5.85 (orthopedics; 95% CI, $3.46-$8.24) and $11.67 (internal medicine; 95% CI, $9.13-$14.21) higher than at the 10th percentile. Including all types of office visits, price indexes at the 90th-percentile HHI were 8.3% (orthopedics; 95% CI, 5.0%-11.6%) to 16.1% (internal medicine; 95% CI, 12.8%-19.5%) higher. Between 2003 and 2010, there were larger price increases in areas that were less competitive in 2002 than in initially more competitive areas. CONCLUSIONS AND RELEVANCE: More competition among physicians is related to lower prices paid by private PPOs for office visits. These results may inform work on policies that influence practice competition.

Label-free duplex SAMDI-MS screen reveals novel SARS-CoV-2 3CLpro inhibitors.

The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome conoravirus 2 (SARS-CoV-2) remains a promising therapeutic target to combat COVID-19. Our group recently described a novel duplexed biochemical assay that combines self-assembled monolayers of alkanethiolates on gold with matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS) to simultaneously measure 3CLpro and human rhinovirus 3C protease activities. This study describes applying the assay for the completion of a high-throughput duplexed screen of 300,000 diverse, drug-like small molecules in 3 days. The hits were confirmed and evaluated in dose response analyses against recombinant 3CLpro, HRV3C, and the human Cathepsin L proteases. The 3CLpro specific inhibitors were further assessed for activity in cellular cytotoxicity and anti-viral assays. Structure activity relationship studies informed on structural features required for activity and selectivity to 3CLpro over HRV3C. These results will guide the optimization of 3CLpro selective inhibitors to combat COVID-19 along with antiviral compounds against coronaviruses and rhinoviruses.

Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry.

Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid-metabolizing enzymes.

High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease.

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)-ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose-response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.

Label-Free Screening of SARS-CoV-2 NSP14 Exonuclease Activity Using SAMDI Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.