CUT&Tag Applied to Zebrafish Adult Tail Fins Reveals a Return of Embryonic H3K4me3 Patterns During Regeneration.

Regenerative potential is governed by a complex process of transcriptional reprogramming, involving chromatin reorganization and dynamics in transcription factor binding patterns throughout the genome. The degree to which chromatin and epigenetic changes contribute to this process remains partially understood. Here we provide a modified CUT&Tag protocol suitable for improved characterization and interrogation of epigenetic changes during adult fin regeneration in zebrafish. Our protocol generates data that recapitulates results from previously published ChIP-Seq methods, requires far fewer cells as input, and significantly improves signal to noise ratios. We deliver high-resolution enrichment maps for H3K4me3 of uninjured and regenerating fin tissues. During regeneration, we find that H3K4me3 levels increase over gene promoters which become transcriptionally active and genes which lose H3K4me3 become silenced. Interestingly, these epigenetic reprogramming events recapitulate the H3K4me3 patterns observed in developing fin folds of 24-hour old zebrafish embryos. Our results indicate that changes in genomic H3K4me3 patterns during fin regeneration occur in a manner consistent with reactivation of developmental programs, demonstrating CUT&Tag to be an effective tool for profiling chromatin landscapes in regenerating tissues.

Role of the histone variant H2A.Z.1 in memory, transcription, and alternative splicing is mediated by lysine modification.

Creating long-lasting memories requires learning-induced changes in gene expression, which are impacted by epigenetic modifications of DNA and associated histone proteins. Post-translational modifications (PTMs) of histones are key regulators of transcription, with different PTMs producing unique effects on gene activity and behavior. Although recent studies implicate histone variants as novel regulators of memory, effects of PTMs on the function of histone variants are rarely considered. We previously showed that the histone variant H2A.Z suppresses memory, but it is unclear if this role is impacted by H2A.Z acetylation, a PTM that is typically associated with positive effects on transcription and memory. To answer this question, we used a mutation approach to manipulate acetylation on H2A.Z without impacting acetylation of other histone types. Specifically, we used adeno-associated virus (AAV) constructs to overexpress mutated H2A.Z.1 isoforms that either mimic acetylation (acetyl-mimic) by replacing lysines 4, 7 and 11 with glutamine (KQ), or H2A.Z.1 with impaired acetylation (acetyl-defective) by replacing the same lysines with alanine (KA). Expressing the H2A.Z.1 acetyl-mimic (H2A.Z.1KQ) improved memory under weak learning conditions, whereas expressing the acetyl-defective H2A.Z.1KA generally impaired memory, indicating that the effect of H2A.Z.1 on memory depends on its acetylation status. RNA sequencing showed that H2A.Z.1KQ and H2A.Z.1KA uniquely impact the expression of different classes of genes in both females and males. Specifically, H2A.Z.1KA preferentially impacts genes involved in synaptic function, suggesting that acetyl-defective H2A.Z.1 impairs memory by altering synaptic regulation. Finally, we describe, for the first time, that H2A.Z is also involved in alternative splicing of neuronal genes, whereby H2A.Z depletion, as well as expression of H2A.Z.1 lysine mutants influence transcription and splicing of different gene targets, suggesting that H2A.Z.1 can impact behavior through effects on both splicing and gene expression. This is the first study to demonstrate that direct manipulation of H2A.Z post-translational modifications regulates memory, whereby acetylation adds another regulatory layer by which histone variants can fine tune higher brain functions through effects on gene expression and splicing.

Smad4 restricts injury-provoked biliary proliferation and carcinogenesis.

Cholangiocarcinoma (CCA) is a deadly and heterogeneous type of cancer characterized by a spectrum of epidemiologic associations as well as genetic and epigenetic alterations. We seek to understand how these features inter-relate in the earliest phase of cancer development and through the course of disease progression. For this, we studied murine models of liver injury integrating the most commonly occurring gene mutations of CCA - including Kras, Tp53, Arid1a and Smad4 - as well as murine hepatobiliary cancer models and derived primary cell lines based on these mutations. Among commonly mutated genes in CCA, we found that Smad4 functions uniquely to restrict reactive cholangiocyte expansion to liver injury through restraint of the proliferative response. Inactivation of Smad4 accelerates carcinogenesis, provoking pre-neoplastic biliary lesions and CCA development in an injury setting. Expression analyses of Smad4-perturbed reactive cholangiocytes and CCA lines demonstrated shared enriched pathways, including cell-cycle regulation, MYC signaling and oxidative phosphorylation, suggesting that Smad4 may act via these mechanisms to regulate cholangiocyte proliferation and progression to CCA. Overall, we showed that TGFß/SMAD4 signaling serves as a critical barrier restraining cholangiocyte expansion and malignant transformation in states of biliary injury.

Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis.

Aging is associated with a decline in stem cell functionality and number across the organism. In this study, we aimed to further unravel Muscle Stem Cells (MuSCs) aging by assessing how systemic factors influence MuSC fate decisions through long-term epigenetic landscape remodelling. As aging is intricately linked to a pro-inflammatory shift, we studied the epigenetic effects of inflammatory signals in MuSCs and measured decreased H4K20me1 levels. This loss disrupts MuSC quiescence, largely through epigenetic silencing of Notch target genes. In the setting of inflammatory signals or aging, the lack of Kmt5a and the subsequent absence of de novoH4K20me1 culminate in cell death by ferroptosis. Aged MuSCs manifest abnormal iron metabolism and reduced Gpx4 levels, resulting in the accumulation of intracellular iron, increased reactive oxygen species, genomic instability, and lipid peroxidation. We showed that ferroptosis is the predominant mode of cell death in aged MuSCs, with remarkably high levels of lipid peroxidation; a phenomenon we also observed in aged hematopoietic stem cells. Implementing preventative strategies to inhibit systemic inflammation prevented aged MuSC ferroptosis, preserving their numbers and regenerative capabilities. This intervention significantly enhanced aged muscle regeneration and strength recovery and extended both lifespan and healthspan in mice. This study delineates a previously underappreciated fate trajectory for stem cell aging, and offers meaningful insights into the treatment of age-related disorders.

A community-based advanced nurse practitioner-led integrated oncology care model for adults receiving oral anticancer medication: a pilot study.

Oral anti-cancer medications (OAMs) are being used increasingly within cancer care. OAMs offer the potential to improve patient convenience and increase hospital capacity. The clinical assessment for each cycle of OAMs requires specialist patient review often performed in hospital-based oncology units. Consequently, any potential improvement in patient expediency or increased hospital capacity that OAMs can offer is not realised. This study aimed to develop and pilot the specialist assessment of patients receiving OAMs by an Advanced Nurse Practitioner (ANP) in a community-based location.The primary aim of this pilot study was to assess the feasibility of a community-based ANP-led integrated oncology care model for adults receiving OAMs in Ireland who met the pre-specified eligibility criteria. The objectives were to determine the feasibility of a definitive trial of this intervention by measuring patient safety, acceptability to patients and staff and cost of the new model of care.This single-centre pilot study provided patient care (n = 37) to those receiving OAM therapies within a community setting for a 4-month period. Consent rate was high with no attrition other than for clinical reasons. There were 151 contacts with the sample during that time.Results demonstrated that the ANP-led intervention and new model for OAM care was safe, highly acceptable to patients and staff and that related healthcare costs could be captured. Based on the success of this pilot study, the authors conclude that a community-based ANP-led integrated oncology care model for adults receiving OAMs is feasible, and a definitive trial is warranted.Trial registration ISRCTN, ISRCTN10401455 . Registered 30 November 2020.

Anp32e protects against accumulation of H2A.Z at Sox motif containing promoters during zebrafish gastrulation.

Epigenetic regulation of chromatin states is crucial for proper gene expression programs and progression during development, but precise mechanisms by which epigenetic factors influence differentiation remain poorly understood. Here we find that the histone variant H2A.Z accumulates at Sox motif-containing promoters during zebrafish gastrulation while neighboring genes become transcriptionally active. These changes coincide with reduced expression of anp32e, the H2A.Z histone removal chaperone, suggesting that loss of Anp32e may lead to increases in H2A.Z binding during differentiation. Remarkably, genetic removal of Anp32e in embryos leads to H2A.Z accumulation prior to gastrulation and developmental genes become precociously active. Accordingly, H2A.Z accumulation occurs most extensively at Sox motif-associated genes, including many which are normally activated following gastrulation. Altogether, our results provide compelling evidence for a mechanism in which Anp32e preferentially restricts H2A.Z accumulation at Sox motifs to regulate the initial phases of developmental differentiation in zebrafish.

The chromatin source-sink hypothesis: a shared mode of chromatin-mediated regulations.

We propose that several chromatin-mediated regulatory processes are dominated by source-sink relationships in which factors operate as 'sources' to produce or provide a resource and compete with each other to occupy separate 'sinks'. In this model, large portions of genomic DNA operate as 'sinks', which are filled by 'sources', such as available histone variants, covalent modifications to histones, the readers of these modifications and non-coding RNAs. Competing occupation for the sinks by different sources leads to distinct states of genomic equilibrium in differentiated cells. During dynamic developmental events, such as sexual reproduction, we propose that dramatic and rapid reconfiguration of source-sink relationships modifies chromatin states. We envision that re-routing of sources could occur by altering the dimensions of the sink, by reconfiguration of existing sink occupation or by varying the size of the source, providing a central mechanism to explain a plethora of epigenetic phenomena, which contribute to phenotypic variegation, zygotic genome activation and nucleolar dominance.

Competition for H2A.Z underlies the developmental impacts of repetitive element de-repression.

The histone variant H2A.Z is central to early embryonic development, determining transcriptional competency through chromatin regulation of gene promoters and enhancers. In addition to genic loci, we find that H2A.Z resides at a subset of evolutionarily young repetitive elements, including DNA transposons, long interspersed nuclear elements and long terminal repeats, during early zebrafish development. Moreover, increases in H2A.Z occur when repetitive elements become transcriptionally active. Acquisition of H2A.Z corresponds with a reduction in the levels of the repressive histone modification H3K9me3 and a moderate increase in chromatin accessibility. Notably, however, de-repression of repetitive elements also leads to a significant reduction in H2A.Z over non-repetitive genic loci. Genic loss of H2A.Z is accompanied by transcriptional silencing at adjacent coding sequences, but remarkably, these impacts are mitigated by augmentation of total H2A.Z protein via transgenic overexpression. Our study reveals that levels of H2A.Z protein determine embryonic sensitivity to de-repression of repetitive elements, that repetitive elements can function as a nuclear sink for epigenetic factors and that competition for H2A.Z greatly influences overall transcriptional output during development. These findings uncover general mechanisms in which counteractive biological processes underlie phenotypic outcomes.

Exploring non-participation in colorectal cancer screening: A systematic review of qualitative studies.

INTRODUCTION: Worldwide, colorectal cancer is a major public health issue. Despite the existence of screening programmes in many countries, global uptake remains low. This meta-ethnography aimed to analyse qualitative literature to explore attitudes towards colorectal cancer screening and reasons for non-participation in eligible people that do not participate when invited. METHODS: Systematic searches were conducted in five databases in May 2021. Critical appraisal of included studies was performed using the CASP checklist for qualitative studies. FINDINGS: Thirteen studies were included. Three main themes and eight sub-themes were developed across studies: (1) Differences in motivation, with non-participants expressing a lack of knowledge and varying levels of intention to participate but not feeling screening was personally necessary; (2) Active aversion to screening expressed by fear, discomfort, disgust or not wanting to know; and (3) Contextual barriers of the healthcare system such as practical constraints or poor relationships with healthcare professionals. CONCLUSION: Findings suggest multiple pathways to non-participation including ambivalence, aversion to the process and consequences of screening or lack of support. Persuasive messages and prompts to action to target ambivalence, reassurance regarding the screening procedures to target negative reactions, and increased support from healthcare professionals may be beneficial in increasing screening uptake.

Upper extremity weakness: A novel risk factor for non-cardiovascular mortality among community-dwelling older adults.

BACKGROUND: Aging-associated upper extremity weakness has been shown to be associated with adverse health outcomes in older adults, but less is known about the association between impaired upper extremity function and cause-specific mortalities. METHODS: Among the 5512 prospective community-based longitudinal Cardiovascular Health Study participants, 1438 had difficulty with one of the three upper extremity functions of lifting, reaching, or gripping. We assembled a propensity score-matched cohort in which 1126 pairs of participants with and without difficulty with upper extremity function, balanced on 62 baseline characteristics including geriatric and functional variables such as physical and cognitive function. Hazard ratios (HRs) and 95% confidence intervals (CIs) for all-cause and cause-specific mortalities associated with upper extremity weakness were estimated in the matched cohort. RESULTS: Matched participants had a mean age of 73.1 years, 72.5% were women, and 17.0% African American. During 23 years of follow-up, all-cause mortality occurred in 83.7% (942/1126) and 81.2% (914/1126) of participants with and without upper extremity weakness, respectively (HR, 1.11; 95% CI, 1.01-1.22; p = 0.023). Upper extremity weakness was associated with a higher risk of non-cardiovascular mortality, occurring in 595 (52.8%) and 553 (49.1%) of participants, respectively (HR, 1.17; 95% CI, 1.04-1.31; p = 0.010), but had no association with cardiovascular mortality (30.8% vs 32.1% in those with and without upper extremity weakness, respectively; HR, 1.03; 95% CI, 0.89-1.19; p = 0.70). CONCLUSION: Among community-dwelling older adults, upper extremity weakness had a weak, albeit independent, significant association with all-cause mortality, which was primarily driven by a higher risk of non-cardiovascular mortality. Future studies need to replicate these findings and understand the underlying reasons for the observed associations.

Exploring barriers and enablers to the delivery of Making Every Contact Count brief behavioural interventions in Ireland: A cross-sectional survey study.

OBJECTIVES: The public health impact of the Irish Making Every Contact Count (MECC) brief intervention programme is dependent on delivery by health care professionals. We aimed to identify enablers and modifiable barriers to MECC intervention delivery to optimize MECC implementation. DESIGN: Online cross-sectional survey design. METHODS: Health care professionals (n = 4050) who completed MECC eLearning were invited to complete an online survey based on the Theoretical Domains Framework (TDF). Multiple regression analysis identified predictors of MECC delivery (logistic regression to predict delivery or not; linear regression to predict frequency of delivery). Data were visualized using Confidence Interval-Based Estimates of Relevance (CIBER). RESULTS: Seventy-nine per cent of participants (n = 283/357) had delivered a MECC intervention. In the multiple logistic regression (Nagelkerke's R2  = .34), the significant enablers of intervention delivery were 'professional role' (OR = 1.86 [1.10, 3.15]) and 'intentions/goals' (OR = 4.75 [1.97, 11.45]); significant barriers included 'optimistic beliefs about consequences' (OR = .41 [.18, .94]) and 'negative emotions' (OR = .50 [.32, .77]). In the multiple linear regression (R2  = .29), the significant enablers of frequency of MECC delivery were 'intentions/goals' (b = 10.16, p = .02) and professional role (b = 6.72, p = .03); the significant barriers were 'negative emotions' (b = -4.74, p = .04) and 'barriers to prioritisation' (b = -5.00, p = .01). CIBER analyses suggested six predictive domains with substantial room for improvement: 'intentions and goals', 'barriers to prioritisation', 'environmental resources', 'beliefs about capabilities', 'negative emotions' and 'skills'. CONCLUSION: Implementation interventions to enhance MECC delivery should target intentions and goals, beliefs about capabilities, negative emotions, environmental resources, skills and barriers to prioritization.

The chromatin source-sink hypothesis: a shared mode of chromatin-mediated regulations.

We propose that several chromatin-mediated regulatory processes are dominated by source-sink relationships in which factors operate as 'sources' to produce or provide a resource and compete with each other to occupy separate 'sinks'. In this model, large portions of genomic DNA operate as 'sinks', which are filled by 'sources', such as available histone variants, covalent modifications to histones, the readers of these modifications and non-coding RNAs. Competing occupation for the sinks by different sources leads to distinct states of genomic equilibrium in differentiated cells. During dynamic developmental events, such as sexual reproduction, we propose that dramatic and rapid reconfiguration of source-sink relationships modifies chromatin states. We envision that re-routing of sources could occur by altering the dimensions of the sink, by reconfiguration of existing sink occupation or by varying the size of the source, providing a central mechanism to explain a plethora of epigenetic phenomena, which contribute to phenotypic variegation, zygotic genome activation and nucleolar dominance.

Anp32e protects against accumulation of H2A.Z at Sox motif containing promoters during zebrafish gastrulation.

Epigenetic regulation of chromatin states is crucial for proper gene expression programs and progression during development, but precise mechanisms by which epigenetic factors influence differentiation remain poorly understood. Here we find that the histone variant H2A.Z accumulates at Sox motif-containing promoters during zebrafish gastrulation while neighboring genes become transcriptionally active. These changes coincide with reduced expression of anp32e, the H2A.Z histone removal chaperone, suggesting that loss of Anp32e may lead to increases in H2A.Z during differentiation. Remarkably, genetic removal of Anp32e in embryos leads to H2A.Z accumulation prior to gastrulation, and precocious developmental transcription of Sox motif associated genes. Altogether, our results provide compelling evidence for a mechanism in which Anp32e restricts H2A.Z accumulation at Sox motif-containing promoters, and subsequent down-regulation of Anp32e enables temporal up-regulation of Sox motif associated genes.

Sensing Magnetic Field and Intermolecular Interactions in Diamagnetic Solution Using Residual Dipolar Couplings of Zephycandidine.

An unusual residual dipolar coupling of methylene protons was recorded in NMR spectra because aromatic zephycandidine has preferential orientation at the external magnetic field. The observed splitting contains contribution from the dipole-dipole D-coupling and the anisotropic component of J-coupling. Absolute values of the anisotropy of magnetic susceptibility |Δχax| are larger for protic solvents because of the hydrogen-bonding compared to aprotic solvents for which polar and dispersion forces are more important. The energy barrier for the reorientation due to hydrogen-bonding is 1.22 kJ/mol in methanol-d4, 0.85 kJ/mol in ethanol-d6 and 0.87 kJ/mol in acetic acid-d6. In dimethyl sulfoxide-d6, 1.08 kJ/mol corresponds to the interaction of solvent lone pair electrons with π-electrons of zephycandidine. This energy barrier decreases for acetone-d6 which has smaller electric dipole moment. In acetonitrile-d3, there is no energy barrier which suggests solvent ordering around the solute due to the solvent-solvent interactions. The largest absolute values of the magnetic anisotropy are observed for aromatic benezene-d6 and tolune-d8 which have their own preferential orientation and enhance the order in the solution. The magnetic anisotropy of "isolated" zephycandidine, not hindered by intermolecular interaction could be estimated from the correlation between Δχax and cohesion energy density.

Stakeholders' perceptions of a community-based advanced nurse practitioner-led integrated oncology care model for adults receiving oral anti-cancer medication-A qualitative study.

Oral anti-cancer medications (OAMs) are increasingly prescribed in oncology, and although administered at home, ongoing monitoring generally requires the patient to attend an acute hospital. With the requirement to provide safe yet convenient care and to increase hospital capacity, the potential exists to transition this cohort of patients to the community to be assessed by oncology health care professionals (HCPs). The onset of COVID-19 facilitated this planned transition. OBJECTIVE: The primary objective was to understand stakeholders' perceptions of a community-based advanced nurse practitioner (ANP)-led integrated OAM care model for adults. METHODS: Qualitative data from interviews and focus groups were obtained from 33 individuals; either service users who attended ANP-led OAM clinics or stakeholders involved in OAM care. Data were subsequently analysed using thematic analysis. RESULTS: Four themes were identified and included reflection on pre-COVID-19 system, role of ANP in current OAM care, importance of robust communication and infrastructural requirements for transition to an integrated OAM care model. CONCLUSION: This study demonstrated that patients and HCPs perceived the proposal positively. They identified the ANP as the appropriate HCP to care for this cohort and the importance of communication and strategic planning for transitioning this model of care to the community setting. CLINICAL TRIAL REGISTRATION: ISRCTN10401455.

NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization.

Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.

Synthesis of (+)-(R)-Tiruchanduramine.

The absolute stereochemistry of the marine alkaloid (+)-(R)-tiruchanduramine was established via a convergent total synthesis in six steps and 15.5% overall yield from Fmoc-D-Dab(Boc)-OH.

Establishment of developmental gene silencing by ordered polycomb complex recruitment in early zebrafish embryos.

Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal the order of chromatin states in establishing developmental gene poising in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with 'Placeholder' nucleosomes (bearing H2A.Z, H3K4me1, and H3K27ac), and DNA hypomethylation. Silencing is initiated by the recruitment of polycomb repressive complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 - establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos.

Identification of chromatin states during zebrafish gastrulation using CUT&RUN and CUT&Tag.

BACKGROUND: Cell fate decisions are governed by interactions between sequence-specific transcription factors and a dynamic chromatin landscape. Zebrafish offer a powerful system for probing the mechanisms that drive these cell fate choices, especially in the context of early embryogenesis. However, technical challenges associated with conventional methods for chromatin profiling have slowed progress toward understanding the exact relationships between chromatin changes, transcription factor binding, and cellular differentiation during zebrafish embryogenesis. RESULTS: To overcome these challenges, we adapted the chromatin profiling methods Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and CUT&Tag for use in zebrafish and applied these methods to generate high-resolution enrichment maps for H3K4me3, H3K27me3, H3K9me3, RNA polymerase II, and the histone variant H2A.Z using tissue isolated from whole, mid-gastrula stage embryos. Using this data, we identify a subset of genes that may be bivalently regulated during both zebrafish and mouse gastrulation, provide evidence for an evolving H2A.Z landscape during embryo development, and demonstrate the effectiveness of CUT&RUN for detecting H3K9me3 enrichment at repetitive sequences. CONCLUSIONS: Our results demonstrate the power of combining CUT&RUN and CUT&Tag methods with the strengths of the zebrafish system to define emerging chromatin landscapes in the context of vertebrate embryogenesis.

Subtype-Independent ANP32E Reduction During Breast Cancer Progression in Accordance with Chromatin Relaxation.

BACKGROUND: Chromatin state provides a clear decipherable blueprint for maintenance of transcriptional patterns, exemplifying a mitotically stable form of cellular programming in dividing cells. In this regard, genomic studies of chromatin states within cancerous tissues have the potential to uncover novel aspects of tumor biology and unique mechanisms associated with disease phenotypes and outcomes. The degree to which chromatin state differences occur in accordance with breast cancer features has not been established. METHODS: We applied a series of unsupervised computational methods to identify chromatin and molecular differences associated with discrete physiologies across human breast cancer tumors. RESULTS: Chromatin patterns alone are capable of stratifying tumors in association with cancer subtype and disease progression. Major differences occur at DNA motifs for the transcription factor FOXA1, in hormone receptor-positive tumors, and motifs for SOX9 in Basal-like tumors. We find that one potential driver of this effect, the histone chaperone ANP32E, is inversely correlated with tumor progression and relaxation of chromatin at FOXA1 binding sites. Tumors with high levels of ANP32E exhibit an immune response and proliferative gene expression signature, whereas tumors with low ANP32E levels appear programmed for differentiation. CONCLUSIONS: Our results indicate that ANP32E may function through chromatin state regulation to control breast cancer differentiation and tumor plasticity. This study sets a precedent for future computational studies of chromatin changes in carcinogenesis.