Iva B. Zovkic

Histone variants are critical components of neuronal chromatin that are emerging as key regulators of long-term memory formation. We previously showed that depleting the macrodomain-containing histone variant macroH2A1 (mH2A1) in the mouse hippocampus impairs long-term memory, establishing this histone as essential for memory consolidation. However, mH2A1 undergoes alternative splicing to generate two isoforms, mH2A1.1 and mH2A1.2, which differ by a single exon within the macrodomain. Though mH2A1.1 and mH2A1.2 have been reported to regulate unique molecular processes in non-neuronal cells, distinct functional contributions of hippocampal mH2A1.1 and mH2A1.2 to long-term memory formation in the adult brain are unknown. Here, we characterized genomic localization of mH2A1 splice isoforms in the mouse hippocampus and evaluated how isoform-specific knockdown impacts hippocampal transcription and memory. Although both isoforms localize to and regulate memory-relevant genes, their depletion affected largely non-overlapping gene sets, and only loss of mH2A1.1 impaired long-term memory. Notably, mH2A1.1 depletion increased expression of several genes that negatively regulate memory formation, including the well-established memory suppressor calcineurin. Thus, under normal conditions, mH2A1.1 may promote memory by repressing transcriptional programs that constrain plasticity. Together, these findings reveal isoform-specific functions of mH2A1 in the hippocampus and identify alternative splicing of mH2A1 as a key epigenetic mechanism that fine-tunes neural chromatin composition to enable long-term memory formation.

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.

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.1^KQ) improved memory under weak learning conditions, whereas expressing the acetyl-defective H2A.Z.1^KA generally impaired memory, indicating that the effect of H2A.Z.1 on memory depends on its acetylation status. RNA sequencing showed that H2A.Z.1^KQ and H2A.Z.1^KA uniquely impact the expression of different classes of genes in both females and males. Specifically, H2A.Z.1^KA 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.

Histone variants H2A.Z and H3.3 are epigenetic regulators of memory, but roles of other variants are not well characterized. macroH2A (mH2A) is a structurally unique histone that contains a globular macrodomain connected to the histone region by an unstructured linker. Here we assessed if mH2A regulates memory and if this role varies for the two mH2A-encoding genes,H2afy(mH2A1) andH2afy2(mH2A2). We show that fear memory is impaired in mH2A1, but not in mH2A2-deficient mice, whereas both groups were impaired in a non-aversive spatial memory task. However, impairment was larger for mH2A1- deficient mice, indicating a preferential role for mH2A1 over mH2A2 in memory. Accordingly, mH2A1 depletion in the mouse hippocampus resulted in more extensive transcriptional de-repression compared to mH2A2 depletion. mH2A1-depleted mice failed to induce a normal transcriptional response to fear conditioning, suggesting that mH2A1 depletion impairs memory by altering transcription. Using chromatin immunoprecipitation (ChIP) sequencing, we found that both mH2A proteins are enriched on transcriptionally repressed genes, but only mH2A1 occupancy was dynamically modified during learning, displaying reduced occupancy on upregulated genes after training. These data identify mH2A as a regulator of memory and suggest that mH2A1 supports memory by repressing spurious transcription and promoting learning-induced transcriptional activation.

Background: Circulating autoantibodies and sex-dependent discrepancy in prevalence are unexplained phenomena of Alzheimer’s disease (AD). Using the 3xTg-AD mouse model, we reported that adult males show early manifestations of systemic autoimmunity, increased emotional reactivity, enhanced expression of the histone variant macroH2A1 in the cerebral cortex, and loss of plaque/tangle pathology. Conversely, adult females display less severe autoimmunity and retain their AD-like phenotype. This study examines the link between immunity and other traits of the current 3xTg-AD model. Methods: Young 3xTg-AD and wild-type mice drank a sucrose-laced 0.4 mg/ml solution of the immunosuppressant cyclophosphamide on weekends for 5 months. After behavioral phenotyping at 2 and 6 months of age, we assessed organ mass, serologic markers of autoimmunity, molecular markers of early AD pathology, and expression of genes associated with neurodegeneration. Results: Chronic immunosuppression prevented hematocrit drop and reduced soluble Aβ in 3xTg-AD males while normalizing the expression of histone variant macroH2A1 in 3xTg-AD females. This treatment also reduced hepatosplenomegaly, lowered autoantibody levels, and increased the effector T cell population while decreasing the proportion of regulatory T cells in both sexes. Exposure to cyclophosphamide, however, neither prevented reduced brain mass and BDNF expression nor normalized increased tau and anxiety-related behaviors. Conclusion: The results suggest that systemic autoimmunity increases soluble Aβ production and affects transcriptional regulation of macroH2A1 in a sex-related manner. Despite the complexity of multisystem interactions, 3xTg-AD mice can be a useful in vivo model for exploring the regulatory role of autoimmunity in the etiology of AD-like neurodegenerative disorders.