Abstract 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 summary In 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 embryo’s 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.

Mapping epigenetic features is essential for investigating chromatin regulation and gene expression control. We recently found that crosslinking- and sonication-based techniques preferentially exclude insoluble chromatin, restricting the investigation of large portions of eukaryotic genomes. Here, we present a protocol for overcoming this issue. We describe steps for chromatin fractionation, DNA extraction, and preparation of next-generation sequencing (NGS) libraries. This strategy can identify underrepresented heterochromatic regions, including repetitive elements and centromeres, permitting functional investigation of regions that are otherwise undetectable.For complete details on the use and execution of this protocol, please refer to Park et al.1

ABSTRACT 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 naïve 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.

Epigenetic reprogramming of chromatin, mediated through modifications to canonical histones and the incorporation of histone variants, plays a central role in the establishment of cell identity and regulation of gene expression programs. Such epigenetic programming becomes particularly important during the development of highly specialized cell types such as germ cells-sperm and oocytes, and stem cells within the blastula of early embryos. Determining patterns of epigenetic modifications within these cell types can be particularly challenging due to technical limitations associated with limited starting materials. Here we present a brief overview of studies focused on epigenetic reprogramming during the transition from sperm to blastula stage embryos and compare between several diverse model systems including Drosophila, zebrafish and mammals. Furthermore, we discuss and compare prevailing genomic profiling approaches, such as ChIP-Seq, CUT&Tag, and CUT&RUN, for performing genome-wide epigenomic analysis.

Alzheimer’s 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.Z’s 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. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45, PJT-156414, PJT-496194

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.

Zebrafish has become a preeminent model for developmental biology research, largely due to the ease of transgenesis. Despite widespread usage of transgenic lines, mapping of transgene insertion sites is rare, which raises complications involving potential local chromatin influences on transgene expression, off-target effects, and issues with allelic variation. To address these shortcomings, we introduce TransTag, a simple and efficient method utilizing Tn5 transposase-mediated tagmentation, for the streamlined identification of Tol2-based transgene insertion sites in zebrafish. TransTag is straightforward to perform and can identify insertion sites without the need for the alignment of raw sequencing data. We also provide a detailed protocol for TransTag, a step-by-step guide for data analysis, and a user-friendly Shiny app, making transgene mapping achievable at a low cost for researchers without programming expertise. Altogether, TransTag emerges as a valuable tool to enhance the precision and utility of transgenesis studies by providing essential chromosome-specific information on transgene locations.

New in situ chromatin profiling methods, such as CUT&Tag, have streamlined studies of chromatin features by eliminating the need for up-front purification, but we find that some features are not equally detectable when comparing with previous methods. ChIP-Seq and CUT&Tag identify similar chromatin enrichment profiles for genic loci, such as promoters, but major differences are detected at heterochromatin-associated regions. Unlike ChIP-Seq, CUT&Tag detects robust levels of H3K9me3 over a substantial number of repetitive elements, with especially high sensitivity over evolutionarily young retrotransposons. For example, mouse IAPEz-int elements exhibit strong enrichment using CUT&Tag but underrepresentation using ChIP-Seq. Additionally, several euchromatin-associated proteins, such as RUNX1, co-purify with insoluble heterochromatin in ChIP studies, but are detectible at repetitive elements when applying in situ fragmentation methods. Our study reveals that the current understanding of chromatin states is extensively incomplete, and newer in situ chromatin fragmentation-based techniques are preferred for investigating repetitive elements and retrotransposons.

Aging is characterized by a decline in the functionality and number of stem cells across the organism. In this study, we uncovered a mechanism by which systemic inflammation drives muscle stem cell (MuSC) aging through epigenetic erosion. We demonstrate that age-related inflammation decreases monomethylation of H4K20 in MuSCs, disrupting their quiescence and inducing ferroptosis, a form of iron-dependent cell death. Our findings show that inflammatory signals downregulate Kmt5a, the enzyme responsible for depositing H4K20me1, leading to the epigenetic silencing of anti-ferroptosis genes. This results in aberrant iron metabolism, increased reactive oxygen species levels and lipid peroxidation in aged MuSCs. Notably, long-term inhibition of systemic inflammation that is initiated at 12 months of age effectively prevents ferroptosis, preserves MuSC numbers and enhances muscle regeneration and functional recovery. These findings reveal an epigenetic switch that links chronic inflammation to MuSC aging and ferroptosis, offering potential therapeutic strategies for combating age-related muscle degeneration. Blanc et al. uncover how chronic inflammation triggers an epigenetic switch in aged muscle stem cells, leading to iron accumulation and cell death by ferroptosis—offering insights into muscle aging and potential paths for regenerative therapies.

A valuable and widely utilized approach to study gene regulatory networks is through transgenic lines. However, detailed information of transgene insertion sites is often not available, complicating the interpretation of experimental results involving transgenes. Here, we present a protocol for mapping the insertion sites of zebrafish Tol2 transgenes, using the recently developed tagmentation-based TransTag technique. We describe detailed steps for preparing sequencing libraries and analyzing raw reads. This protocol can be used to robustly identify transgene insertion sites in zebrafish. For complete details on the use and execution of this protocol, please refer to Meng et al.1.

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 TGFβ/SMAD4 signaling serves as a critical barrier restraining cholangiocyte expansion and malignant transformation in states of biliary injury.

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 only partially understood. Here we provide a modified CUT Tag protocol suitable for improved characterization and interrogation of changes in chromatin modifications 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 reprogramming events recapitulate the H3K4me3 patterns observed in developing fin folds of 24-h 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.

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 novo H4K20me1 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.

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.

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.

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.

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 vari-ables 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 rea-sons for the observed associations.

Murphy, Patrick MSN, RN-BC; Carter-Templeton, Heather PhD, RN-BC, FAANEditor(s): Alexander, Susan DNP, ANP-BC, ADM-BC Author Information

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.

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 Statement De 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.