Biology subjects

Murphy, P. J.

Publications and source records attributed to Murphy, P. J..

14 recordsLinked to original sources

bioRxiv2026

Ablation and Seed-driven Restoration of an Alpha-Satellite Devoid Human Centromere Reveals Size Homeostasis Mechanisms

Human centromeres are epigenetically defined chromatin domains marked by nucleosomes containing the histone H3 variant CENP-A, which recruit the constitutive centromere-associated network (CCAN) to assemble functional kinetochores. Maintaining centromere function, including chromatin domain size and the ability to assemble the kinetochore, is essential for proper mitotic division across eukaryotes. In humans however, mechanistic studies of centromere establishment, maintenance, and size regulation have been hindered by the highly repetitive nature of canonical alphoid centromeres. Here, we develop a genetically tractable human neocentromere system which is devoid of repetitive DNA sequences. Using targeted genetic manipulation of a monoallelic naturally occurring neocentromere, we show that partial loss of centromeric chromatin triggers restoration of the residual CENP-A domain through seed-driven, sequence-independent expansion into adjacent naive chromatin. In contrast, creation of new domain boundaries without loss of centromeric chromatin mass results only in local boundary remodeling, without substantial domain expansion. These findings indicate that centromere formation proceeds through two mechanistically distinct steps, beginning with acquisition of a CENP-A seed and followed by regulated domain expansion to generate a mature centromere. More broadly, our results support a model in which centromeres transition between a maintenance state that preserves domain size and a restoration state that rebuilds centromeric chromatin following perturbation. Together, this study establishes a genetically tractable platform for dissecting the mechanisms governing human centromere formation, chromatin domain dynamics, and size homeostasis.

cell biology↗

bioRxiv2026

H2A.Z levels control the timing of major events at the maternal-zygotic transition

Across animals, early embryonic events are temporally tightly coordinated but the underlying mechanisms remain incompletely understood. In Drosophila, both global and nuclear levels of the histone variant H2Av rise progressively during the maternal-zygotic transition (MZT), but whether this increase is functionally important is unknown. We find that increased H2Av dosage expedites specific MZT events: the transition from nuclear cycle (NC) 13 to 14 occurs prematurely as does the turnover of thousands of maternal transcripts; in addition, a subset of genes is precociously expressed in the zygote. Reduced H2Av dosage has reciprocal effects. Comparable transcriptional shifts are observed in zebrafish embryos overexpressing the H2Av ortholog H2A.Z, suggesting H2Av/H2A.Z dosage as a conserved timer of early development. We also examined mutants with impaired H2Av sequestration on lipid droplets which exhibit increased nuclear H2Av levels but reduced cytoplasmic levels. Unexpectedly, nuclear H2Av abundance influences the timing of NC 13 but is not the main driver of transcriptome remodeling. In summary, we find that H2Av/H2A.Z levels are critical timers of early embryogenesis and that H2Av can act in part via a non-nuclear mechanism. Author summaryIn animals, the earliest stages of embryogenesis are initially driven by proteins and RNAs that the mother provides via the egg; later development is controlled by the embryos own genes. The switch from maternal to zygotic control is called the maternal-zygotic transition (MZT). This conserved process involves degradation of maternal mRNAs, activation of zygotic genes, cell cycle lengthening, and morphological remodeling. All these events require precise control, but how they are coordinated remains incompletely understood. Here we show that the levels of a specific histone, H2A.Z, provide a timer for the MZT. In Drosophila, H2A.Z levels - both in the embryo overall and in nuclei - increase during the MZT. When we ectopically increased H2A.Z levels, specific MZT events occurred prematurely. Reducing H2A.Z levels had the opposite effect. Using mutant flies in which H2A.Z levels are ectopically increased in the nucleus but reduced in the embryo as a whole, we uncovered both nuclear and non-nuclear roles for H2A.Z in the establishment of developmental timing. Moreover, we observed similar outcomes in genetically manipulated zebrafish embryos. Our results are consistent with an ancient mechanism in which H2A.Z abundance functions as a timer of early embryogenesis.

developmental biology↗

bioRxiv2025

Opposing effects of H2A.Z on memory, transcription and pathology in male and female Alzheimer's disease mice and patients

Alzheimers disease (AD) is a devastating neurodegenerative disease that disproportionately impacts women, but underlying mechanisms for sex-divergent outcomes are unknown. Here, we show that the histone variant H2A.Z is a novel sex-specific regulator of AD in human patients and AD model mice. Specifically, H2A.Z binding in chromatin declines in female and increases in male AD patients, indicating opposite patterns of AD-related H2A.Z dysregulation each sex. These sex differences were recapitulated in the 5xFAD model of AD, in which females accumulated H2A.Z at early disease stages and lost H2A.Z as the disease progressed, suggesting that H2A.Z occupancy shifts with advancing disease. Males showed no change in H2A.Z binding in early disease, but exhibited increased binding as disease progressed, albeit to a lesser extent than females. Consistent with sex-specific H2A.Z dysregulation, H2A.Z depletion produced sex-specific changes in gene expression, whereby H2A.Z was more repressive in female than in male mice and in 5xFAD than in WT males, suggesting that H2A.Zs role in transcription varies with sex and disease. Moreover, H2A.Z depletion improved memory and AD pathology in females, while impairing memory and worsening pathology in male mice. Together, these data suggest that H2A.Z is protective in males and detrimental in females with AD, with key implications for sex-specific therapeutic targeting of chromatin factors.

neuroscience↗

bioRxiv2025

CUT&Tag Identifies Repetitive Genomic Loci that are Excluded from ChIP Assays

Determining the genomic localization of chromatin features is an essential aspect of investigating gene expression control, and ChIP-Seq has long been the gold standard technique for interrogating chromatin landscapes. Recently, the development of alternative methods, such as CUT&Tag, have provided researchers with alternative strategies that eliminate the need for chromatin purification, and allow for in situ investigation of histone modifications and chromatin bound factors. Mindful of technical differences, we set out to investigate whether distinct chromatin modifications were equally compatible with these different chromatin interrogation techniques. We found that ChIP-Seq and CUT&Tag performed similarly for modifications known to reside at gene regulatory regions, such as promoters and enhancers, but major differences were observed when we assessed enrichment over heterochromatin-associated loci. Unlike ChIP-Seq, CUT&Tag detects robust levels of H3K9me3 at a substantial number of repetitive elements, with especially high sensitivity over evolutionarily young retrotransposons. IAPEz-int elements for example, exhibited underrepresentation in mouse ChIP-Seq datasets but strong enrichment using CUT&Tag. Additionally, we identified several euchromatin-associated proteins that co-purify with repetitive loci and are similarly depleted when applying ChIP-based methods. This study reveals that our current knowledge of chromatin states across the heterochromatin portions of the mammalian genome is extensively incomplete, largely due to limitations of ChIP-Seq. We also demonstrate that newer in situ chromatin fragmentation-based techniques, such as CUT&Tag and CUT&RUN, are more suitable for studying chromatin modifications over repetitive elements and retrotransposons. HighlightsIn situ fragmentation overcomes biases produced by ChIP-Seq. Heterochromatic regions of the genome are lost to the insoluble pellet during ChIP-Seq. CUT&Tag allows for mapping chromatin features at young repetitive elements. Euchromatin-associated regulatory factors co-purify with insoluble heterochromatin.

genomics↗

bioRxiv2023

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. HighlightsO_LIAn early-developmental time course of zebrafish chromatin accessibility is achieved using an integrated UMAP analysis of datasets from two separate published studies. C_LIO_LICUT&Tag sequencing is used to characterize the genomic localization for the histone chaperone ANP32E. C_LIO_LIChanges in Anp32e enrichment coincide with opposing changes in H2A.Z enrichment during zebrafish gastrulation. C_LIO_LIPrecociously expressed genes in embryos lacking Anp32e are disproportionately Sox-marked and may represent H2A.Z-mediated developmental accelerations. C_LI

developmental biology↗

bioRxiv2022

Medial accumbens shell spiny projection neurons encode relative reward preference

Medial nucleus accumbens shell (mNAcSh) is a critical brain region for driving motivated behaviors. Despite this well-established role, the underlying reward processing of individual neurons, circuits and cell-types within mNAcSh remains largely unknown. Here, we leverage deep brain 2-photon calcium imaging through endoscopic lenses to record mNAcSh spiny projection neuron (SPN) ensemble responses to rewards of different concentrations and to reward-predictive cues across cue-reward learning. Reward responses were found to be heterogeneous and particularly differentiated based on reward concentration and cell type. A large subpopulation of reward-excited enkephalinergic SPNs were found to be specifically recruited during consumption of high concentration, unpreferred reward. A major enkephalinergic efferent projection from mNAcSh to ventral pallidum (VP) was also found to be recruited to high concentration but unpreferred reward and to causally drive low positive reward preference. Enkephalin and dynorphinergic SPNs in mNAcSh distinctly represent rewards of different preference and propagate distinct signals through efferent projections to drive consummatory behavior.

neuroscience↗

bioRxiv2022

Competition for H2A.Z between genes and repetitive elements establishes response to anti-viral immune activation

Activation of endogenous retroviruses leads to widespread transcriptional reprograming, affecting innate immune activation, metabolic control, and development. We find that the histone variant H2A.Z plays a central role in orchestrating these responses. Stimulating retroviral expression in zebrafish embryos causes H2A.Z to exit developmental gene promoters, which become silent, and to accumulate specifically at primed repetitive elements, which are pre-marked by H3K27ac and H3K9me3. Remarkably, this rewiring is greatly influenced by total H2A.Z abundance, and developmental consequences of retrovirus activation are mitigated by H2A.Z over-expression. Our results uncover mechanisms whereby H2A.Z levels determine sensitivity to retroviral activation, and repetitive elements function as a nuclear sink to dramatically influence total transcriptional output. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/486614v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@275f37org.highwire.dtl.DTLVardef@dc2d9aorg.highwire.dtl.DTLVardef@135f599org.highwire.dtl.DTLVardef@18f290c_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2021

Chromatin Patterns Distinguish Breast Tumor Subtypes and Disease Progression in Association with ANP32E levels

Despite highly advanced diagnosis and treatment strategies, breast cancer patient outcomes vary extensively, even among individuals with the same diagnosis. Thus, a better understanding of the unique molecular characteristics that underlie tumor trajectories and responses to therapy remains a central goal. We report that chromatin patterns represent an important characteristic, capable of stratifying tumor identity and progression. We find that patterns of chromatin accessibility can be classified into 3 major groups, representing Basal-like tumors, hormone receptor (HR)-expressing tumors, and invasive lobular Luminal-A tumors. Major chromatin differences occur throughout the genome at motifs for the transcription factor FOXA1 in HR-positive tumors, and motifs for SOX9 in Basal-like tumors. A large portion of lobular Luminal-A tumors display a chromatin signature defined by accessibility at FOXA1 binding motifs, distinguishing them from others within this subtype. Expression of the histone chaperone ANP32E is inversely correlated with tumor progression and chromatin accessibility 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. Our results indicate that ANP32E may function through chromatin state regulation to control breast cancer differentiation and tumor plasticity.

cancer biology↗

bioRxiv2021

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

BackgroundCell 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. ResultsTo overcome these challenges, we adapted the chromatin profiling methods 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 from mid gastrula stage embryos. Using this data, we identify a conserved subset of developmental genes that are enriched in both H3K4me3 and H3K27me3 during gastrulation, provide evidence for an evolving H2A.Z landscape during embryo development, and demonstrate the increased effectiveness of CUT&RUN for detecting protein enrichment at repetitive sequences. ConclusionsOur 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.

genomics↗

bioRxiv2021

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 how developmental gene poising is established de novo 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. Impact StatementDe novo polycomb domains are formed in zebrafish early embryos by focal histone H2Aub1 deposition by Rnf2-PRC1 - which imposes transcription silencing - followed by subsequent recruitment of Aebp2-PRC2 and H3K27me3 deposition.

molecular biology↗

bioRxiv2020

Genome-wide Chromatin Accessibility is Restricted by ANP32E

Genome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how control of genomic H2A.Z localization by ANP32E contributes to chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that through antagonism of H2A.Z, ANP32E restricts genome-wide DNA access. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.

genetics↗

bioRxiv2019

Paternal cigarette smoke alters DNA methylation in sperm and gene expression in offspring brain

Paternal cigarette smoke (CS) exposure is associated with increased risk of behavioral disorders and cancer in offspring, but the mechanism has not been identified. This study used mouse models to evaluate: 1) what impact paternal CS exposure has on sperm DNA methylation (DNAme), 2) whether sperm DNAme changes persist after CS exposure ends, 3) the degree to which DNAme and gene expression changes occur in offspring and 4) the mechanism underlying impacts of CS exposure. We demonstrate that CS exposure induces sperm DNAme changes that are partially corrected within 28 days of removal from CS exposure. Additionally, paternal smoking causes changes in neural DNAme and gene expression in offspring. Remarkably, the effects of CS exposure are largely recapitulated in oxidative stress-compromised Nrf2-/- mice and their offspring, independent of paternal smoking. These results demonstrate that paternal CS exposure impacts offspring phenotype and that oxidative stress underlies CS induced heritable epigenetic changes.

genetics↗

bioRxiv2018

Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan

Polycomb group proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model to comprehensively study Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the recruitment of H3K27me3 and Polycomb group protein Rnf2. However, in absence of Ezh2, only minor changes in global H3K4me3 levels and gene and protein expression occurred. These changes were mainly due to local deregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important only after the body plan is formed to maintain spatially restricted transcriptional profiles of Polycomb-targeted transcription factors.

developmental biology↗

bioRxiv2017

Placeholder nucleosomes underlie germline-to-embryo DNA methylation reprogramming

The fate and function of epigenetic marks during the germline-to-embryo transition is a key issue in developmental biology, with relevance to stem cell programming and trans-generational inheritance. In zebrafish, DNA methylation (DNAme) patterns are programmed in transcriptionally-quiescent cleavage embryos; remarkably, paternally-inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal. Here we provide the mechanism, by demonstrating that Placeholder nucleosomes, containing histone H2A variant H2A.Z(FV) and H3K4me1, occupy virtually all regions lacking DNAme in both sperm and cleavage embryos, and resides at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with Placeholder become either active (H3K4me3) or silent (H3K4me3/K27me3). Importantly, functional perturbation causing Placeholder loss confers DNAme accumulation, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally-quiescent gametic and embryonic stages, an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNAme, poising parental genes for either gene-specific activation or facultative repression.\n\nHighlights: four, 85 characters eachO_LIPlaceholder nucleosomes bear H3K4me and the histone variant H2A.Z(FV)\nC_LIO_LIPlaceholders occupy all DNA hypomethylated loci in zebrafish sperm and early embryos\nC_LIO_LIAt ZGA, Placeholders resolve into either active or poised (bivalent) nucleosomes\nC_LIO_LIPlaceholder localization excludes DNAme, and regulates embryonic transcription\nC_LI

developmental biology↗