Structural insights into the human NuA4/TIP60 acetyltransferase and chromatin remodeling complex.

The human NuA4/TIP60 co-activator complex, a fusion of the yeast SWR1 and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4/H2A/H2A.Z to regulate gene expression and maintain genome stability. Our cryo-electron microscopy studies show that, within the NuA4/TIP60 complex, the EP400 subunit serves as a scaffold holding the different functional modules in specific positions, creating a unique arrangement of the ARP module. EP400 interacts with the TRRAP subunit using a footprint that overlaps with that of the SAGA acetyltransferase complex, preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.

Risk Variants Associated With Normal Pressure Hydrocephalus: Genome-Wide Association Study in the FinnGen Cohort.

BACKGROUND AND OBJECTIVES: Large-scale genome-wide studies of chronic hydrocephalus have been lacking. We conducted a genome-wide association study (GWAS) in normal pressure hydrocephalus (NPH). METHODS: We used a case-control study design implementing FinnGen data containing 473,691 Finns with genotypes and nationwide health records. Patients with NPH were selected based on ICD-10 G91.2 diagnosis. To select patients with idiopathic NPH (iNPH) for sensitivity analysis, we excluded patients with a potentially known etiology of the condition using an algorithm on their disease history. The controls were the remaining non-hydrocephalic participants. For a replication analysis, the NPH cohort from UK Biobank (UKBB) was used. RESULTS: We included 1,522 patients with NPH (mean age 72.2 years, 53% women) and 451,091 controls (mean age 60.5 years, 44% women). In the GWAS comparing patients with NPH with the controls, we identified 6 gene regions significantly (p < 5.0e-8) associated with NPH that replicated in a meta-analysis with UKBB (NPH n = 173). The top loci near the following genes were rs7962263, SLCO1A2 (odds ratio [OR] 0.71, 95% CI 0.65-0.78, p = 1.0e-14); rs798495, AMZ1/GNA12 (OR 1.29, 95% CI 1.20-1.39, p = 2.9e-12); rs10828247, MLLT10 (OR 0.77, 95% CI 0.71-0.83, p = 1.5e-11); rs561699566 and rs371919113, CDCA2 (OR 0.76, 95% CI 0.70-0.82, p = 1.5e-11); rs56023709, C16orf95 (OR 1.24, 95% CI 1.16-1.33, p = 3.0e-9); and rs62434144, PLEKHG1 (OR 1.23, 95% CI 1.14-1.32, p = 1.4e-8). In the sensitivity analysis comparing only patients with iNPH (n = 1,055) with the controls (n = 451,091), 4 top loci near the following genes remained significant: rs7962263, SLCO1A2 (OR 0.70, 95% CI 0.63-0.78, p = 2.1e-11); rs10828247, MLLT10 (OR 0.74, 95% CI 0.62-0.82, p = 4.6e-10); rs798511, AMZ1/GNA12 (OR 1.28, 95% CI 1.17-1.39, p = 1.7e-8); and rs56023709, C16orf95 (OR 1.28, 95% CI 1.17-1.39, p = 1.7e-8). DISCUSSION: We identified 6 loci significantly associated with NPH in the thus far largest GWAS in chronic hydrocephalus. The genes near the top loci have previously been associated with blood-brain barrier and blood-CSF barrier function and with increased lateral brain ventricle volume. The effect sizes and allele frequencies remained similar in NPH and iNPH cohorts, indicating the identified loci are risk determinants for iNPH and likely not explained by associations with other etiologies. However, the exact role of these loci is still unknown, warranting further studies.

ASXLs binding to the PHD2/3 fingers of MLL4 provides a mechanism for the recruitment of BAP1 to active enhancers.

The human methyltransferase and transcriptional coactivator MLL4 and its paralog MLL3 are frequently mutated in cancer. MLL4 and MLL3 monomethylate histone H3K4 and contain a set of uncharacterized PHD fingers. Here, we report a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL4 and MLL3 that bind to ASXL2, a component of the Polycomb repressive H2AK119 deubiquitinase (PR-DUB) complex. The structure of MLL4 PHD2/3 in complex with the MLL-binding helix (MBH) of ASXL2 and mutational analyses reveal the molecular mechanism which is conserved in homologous ASXL1 and ASXL3. The native interaction of the Trithorax MLL3/4 complexes with the PR-DUB complex in vivo depends solely on MBH of ASXL1/2, coupling the two histone modifying activities. ChIP-seq analysis in embryonic stem cells demonstrates that MBH of ASXL1/2 is required for the deubiquitinase BAP1 recruitment to MLL4-bound active enhancers. Our findings suggest an ASXL1/2-dependent functional link between the MLL3/4 and PR-DUB complexes.

Cerebral Amyloidosis in Individuals with Subjective Cognitive Decline: From Genetic Predisposition to Actual Cerebrospinal Fluid Measurements.

The possible relationship between Subjective Cognitive Decline (SCD) and dementia needs further investigation. In the present study, we explored the association between specific biomarkers of Alzheimer's Disease (AD), amyloid-beta 42 (Aß42) and Tau with the odds of SCD using data from two ongoing studies. In total, 849 cognitively normal (CN) individuals were included in our analyses. Among the participants, 107 had available results regarding cerebrospinal fluid (CSF) Aß42 and Tau, while 742 had available genetic data to construct polygenic risk scores (PRSs) reflecting their genetic predisposition for CSF Aß42 and plasma total Tau levels. The associations between AD biomarkers and SCD were tested using logistic regression models adjusted for possible confounders such as age, sex, education, depression, and baseline cognitive test scores. Abnormal values of CSF Aß42 were related to 2.5-fold higher odds of SCD, while higher polygenic loading for Aß42 was associated with 1.6-fold higher odds of SCD. CSF Tau, as well as polygenic loading for total Tau, were not associated with SCD. Thus, only cerebral amyloidosis appears to be related to SCD status, either in the form of polygenic risk or actual CSF measurements. The temporal sequence of amyloidosis being followed by tauopathy may partially explain our findings.

Walking time and genetic predisposition for Alzheimer's disease: Results from the HELIAD study.

Objective: Our study aimed to explore whether physical condition might affect the association between genetic predisposition for Alzheimer's Disease (AD) and AD incidence. Methods: The sample of participants consisted of 561 community-dwelling adults over 64 years old, without baseline dementia (508 cognitively normal and 53 with mild cognitive impairment), deriving from the HELIAD, an ongoing longitudinal study with follow-up evaluations every 3 years. Physical condition was assessed at baseline through walking time (WT), while a Polygenic Risk Score for late onset AD (PRS-AD) was used to estimate genetic predisposition. The association between WT and PRS-AD with AD incidence was evaluated with Cox proportional hazard models adjusted for age, sex, education years, global cognition score and APOE ε-4 genotype. Then, the association between WT and AD incidence was investigated after stratifying participants by low and high PRS-AD. Finally, we examined the association between PRS-AD and AD incidence after stratifying participants by WT. Results: Both WT and PRS-AD were connected with increased AD incidence (p < 0.05), after adjustments. In stratified analyses, in the slow WT group participants with a greater genetic risk had a 2.5-fold higher risk of developing AD compared to participants with lower genetic risk (p = 0.047). No association was observed in the fast WT group or when participants were stratified based on PRS-AD. Conclusions: Genetic predisposition for AD is more closely related to AD incidence in the group of older adults with slow WT. Hence, physical condition might be a modifier in the relationship of genetic predisposition with AD incidence.

Surface-Enhanced Raman Spectroscopy (SERS) for Identifying Traces of Adenine in Organic-Bearing Extraterrestrial Dust Analogs Captured in the Tanpopo Aerogel after Hypervelocity Impacts.

Raman spectroscopy is a non-destructive analytical technique for characterizing organic and inorganic materials with spatial resolution in the micrometer range. This makes it a method of choice for space-mission sample characterization, whether on return or in situ. To enhance its sensitivity, we use signal amplification via interaction with plasmonic silver-based colloids, which corresponds to surface-enhanced Raman scattering (SERS). In this study, we focus on the analysis of biomolecules of prebiotic interest on extraterrestrial dust trapped in silica aerogel, jointly with the Japanese Tanpopo mission. The aim is twofold: to prepare samples as close as possible to the real ones, and to optimize analysis by SERS for this specific context. Serpentinite was chosen as the inorganic matrix and adenine as the target biomolecule. The dust was projected at high velocity into the trapping aerogel and then mechanically extracted. A quantitative study shows effective detection even for adenine doping from a 5·10-9mol/L solution. After the dust has been expelled from the aerogel using a solvent, SERS mapping enables unambiguous adenine detection over the entire dust surface. This study shows the potential of SERS as a key technique not only for return samples, but also for upcoming new explorations.

The Alzheimer's disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons.

Bridging Integrator 1 (BIN1) is the second most important Alzheimer's disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.

DNA damage remodels the MITF interactome to increase melanoma genomic instability.

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.

Genetic Predisposition for White Matter Hyperintensities and Risk of Mild Cognitive Impairment and Alzheimer's Disease: Results from the HELIAD Study.

The present study investigated the association of genetic predisposition for white matter hyperintensities (WMHs) with incident amnestic mild cognitive impairment (aMCI) or Alzheimer's disease (AD), as well as whether such an association was influenced by age, sex, and cognitive reserve. Overall, 537 individuals without aMCI or dementia at baseline were included. Among them, 62 individuals developed aMCI/AD at follow up. Genetic propensity to WMH was estimated using a polygenic risk score for WMHs (PRS WMH). The association of PRS WMH with aMCI/AD incidence was examined using COX models. A higher PRS WMH was associated with a 47.2% higher aMCI/AD incidence (p = 0.015) in the fully adjusted model. Subgroup analyses showed significant results in the older age group, in which individuals with a higher genetic predisposition for WMHs had a 3.4-fold higher risk for developing aMCI/AD at follow up (p < 0.001), as well as in the lower cognitive reserve (CR, proxied by education years) group, in which individuals with a higher genetic predisposition for WMHs had an over 2-fold higher risk (p = 0.013). Genetic predisposition for WMHs was associated with aMCI/AD incidence, particularly in the group of participants with a low CR. Thus, CR might be a modifier in the relationship between genetic predisposition for WMHs and incident aMCI/AD.

Amino Acid Polymerization on Silica Surfaces.

The polymerization of unactivated amino acids (AAs) is an important topic because of its applications in various fields including industrial medicinal chemistry and prebiotic chemistry. Silica as a promoter for this reaction, is of great interest owing to its large abundance and low cost. The amide/peptide bond synthesis on silica has been largely demonstrated but suffers from a lack of knowledge regarding its reaction mechanism, the key parameters, and surface features that influence AA adsorption and reactivity, the selectivity of the reaction product, the role of water in the reaction, etc. The present review addresses these problems by summarizing experimental and modeling results from the literature and attempts to rationalize some apparent divergences in published results. After briefly presenting the main types of silica surface sites and other relevant macroscopic features, we discuss the different deposition procedures of AAs, whose importance is often neglected. We address the possible AA adsorption mechanisms including covalent grafting and H-bonding and show that they are highly dependent on silanol types and density. We then consider how the adsorption mechanisms determine the occurrence and outcome of AA condensation (formation of cyclic dimers or of long linear chains), and outline some recent results that suggest significant polymerization selectivity in systems containing several AAs, as well as the formation of specific elements of secondary structure in the growing polypeptide chains.

The first genome-wide association study in the Argentinian and Chilean populations identifies shared genetics with Europeans in Alzheimer's disease.

INTRODUCTION: Genome-wide association studies (GWAS) are fundamental for identifying loci associated with diseases. However, they require replication in other ethnicities. METHODS: We performed GWAS on sporadic Alzheimer's disease (AD) including 539 patients and 854 controls from Argentina and Chile. We combined our results with those from the European Alzheimer and Dementia Biobank (EADB) in a meta-analysis and tested their genetic risk score (GRS) performance in this admixed population. RESULTS: We detected apolipoprotein E ε4 as the single genome-wide significant signal (odds ratio  = 2.93 [2.37-3.63], P = 2.6 × 10-23 ). The meta-analysis with EADB summary statistics revealed four new loci reaching GWAS significance. Functional annotations of these loci implicated endosome/lysosomal function. Finally, the AD-GRS presented a similar performance in these populations, despite the score diminished when the Native American ancestry rose. DISCUSSION: We report the first GWAS on AD in a population from South America. It shows shared genetics modulating AD risk between the European and these admixed populations. HIGHLIGHTS: This is the first genome-wide association study on Alzheimer's disease (AD) in a population sample from Argentina and Chile. Trans-ethnic meta-analysis reveals four new loci involving lysosomal function in AD. This is the first independent replication for TREM2L, IGH-gene-cluster, and ADAM17 loci. A genetic risk score (GRS) developed in Europeans performed well in this population. The higher the Native American ancestry the lower the GRS values.

The Identification of Nuclear FMRP Isoform Iso6 Partners.

A deficiency of FMRP, a canonical RNA-binding protein, causes the development of Fragile X Syndrome (FXS), which is characterised by multiple phenotypes, including neurodevelopmental disorders, intellectual disability, and autism. Due to the alternative splicing of the encoding FMR1 gene, multiple FMRP isoforms are produced consisting of full-length predominantly cytoplasmic (i.e., iso1) isoforms involved in translation and truncated nuclear (i.e., iso6) isoforms with orphan functions. However, we recently implicated nuclear FMRP isoforms in DNA damage response, showing that they negatively regulate the accumulation of anaphase DNA genomic instability bridges. This finding provided evidence that the cytoplasmic and nuclear functions of FMRP are uncoupled played by respective cytoplasmic and nuclear isoforms, potentially involving specific interactions. While interaction partners of cytoplasmic FMRP have been reported, the identity of nuclear FMRP isoform partners remains to be established. Using affinity purification coupled with mass spectrometry, we mapped the nuclear interactome of the FMRP isoform iso6 in U2OS. In doing so, we found FMRP nuclear interaction partners to be involved in RNA processing, pre-mRNA splicing, ribosome biogenesis, DNA replication and damage response, chromatin remodeling and chromosome segregation. By comparing interactions between nuclear iso6 and cytoplasmic iso1, we report a set of partners that bind specifically to the nuclear isoforms, mainly proteins involved in DNA-associated processes and proteasomal proteins, which is consistent with our finding that proteasome targets the nuclear FMRP iso6. The specific interactions with the nuclear isoform 6 are regulated by replication stress, while those with the cytoplasmic isoform 1 are largely insensitive to such stress, further supporting a specific role of nuclear isoforms in DNA damage response induced by replicative stress, potentially regulated by the proteasome.

High-Content Screening of Synaptic Density Modulators in Primary Neuronal Cultures.

The synapse, which represents the structural and functional basis of neuronal communication, is one of the first elements affected in several neurodegenerative diseases. To better understand the potential role of gene expression in synapse loss, we developed an original high-content screening (HCS) model capable of quantitatively assessing the impact of gene silencing on synaptic density. Our approach is based on a model of primary neuronal cultures (PNCs) from the neonatal rat hippocampus, whose mature synapses are visualized by the relative localization of the presynaptic protein Synaptophysin with the postsynaptic protein Homer1. The heterogeneity of PNCs and the small sizes of the synaptic structures pose technical challenges associated with the level of automation necessary for HCS studies. We overcame these technical challenges, automated the processes of image analysis and data analysis, and carried out tests under real-world conditions to demonstrate the robustness of the model developed. In this article, we describe the screening of a custom library of 198 shRNAs in PNCs in the 384-well plate format. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of primary hippocampal rat neurons in 384-well plates Basic Protocol 2: Lentiviral shRNA transduction of primary neuronal culture in 384-well plates Basic Protocol 3: Immunostaining of the neuronal network and synaptic markers in 384-well plates Basic Protocol 4: Image acquisition using a high-throughput reader Basic Protocol 5: Image segmentation and analysis Basic Protocol 6: Synaptic density analysis.

Amyloid-Beta Peptides Trigger Premature Functional and Gene Expression Alterations in Human-Induced Neurons.

Alzheimer's disease (AD) is the most prevalent cause of dementia in the elderly, characterized by the presence of amyloid-beta (Aß) plaques, neurofibrillary tangles, neuroinflammation, synapse loss and neurodegeneration in the brain. The amyloid cascade hypothesis postulates that deposition of Aß peptides is the causative agent of AD pathology, but we still lack comprehensive understanding of the molecular mechanisms connecting Aß peptides to neuronal dysfunctions in AD. In this work, we investigate the early effects of Aß peptide accumulation on the functional properties and gene expression profiles of human-induced neurons (hiNs). We show that hiNs acutely exposed to low concentrations of both cell-secreted Aß peptides or synthetic Aß1-42 exhibit alterations in the frequency of calcium transients suggestive of increased neuronal excitability. Using single-cell RNA sequencing, we also show that cell-secreted Aß up-regulates the expression of several synapse-related genes and down-regulates the expression of genes associated with metabolic stress mainly in glutamatergic neurons and, to a lesser degree, in GABAergic neurons and astrocytes. These neuronal alterations correlate with activation of the SEMA5, EPHA and NECTIN signaling pathways, which are important regulators of synaptic plasticity. Altogether, our findings indicate that slight elevations in Aß concentrations are sufficient to elicit transcriptional changes in human neurons, which can contribute to early alterations in neural network activity.

Acetylation reprograms MITF target selectivity and residence time.

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.

Step by step: towards a better understanding of the genetic architecture of Alzheimer's disease.

Alzheimer's disease (AD) is considered to have a large genetic component. Our knowledge of this component has progressed over the last 10 years, thanks notably to the advent of genome-wide association studies and the establishment of large consortia that make it possible to analyze hundreds of thousands of cases and controls. The characterization of dozens of chromosomal regions associated with the risk of developing AD and (in some loci) the causal genes responsible for the observed disease signal has confirmed the involvement of major pathophysiological pathways (such as amyloid precursor protein metabolism) and opened up new perspectives (such as the central role of microglia and inflammation). Furthermore, large-scale sequencing projects are starting to reveal the major impact of rare variants - even in genes like APOE - on the AD risk. This increasingly comprehensive knowledge is now being disseminated through translational research; in particular, the development of genetic risk/polygenic risk scores is helping to identify the subpopulations more at risk or less at risk of developing AD. Although it is difficult to assess the efforts still needed to comprehensively characterize the genetic component of AD, several lines of research can be improved or initiated. Ultimately, genetics (in combination with other biomarkers) might help to redefine the boundaries and relationships between various neurodegenerative diseases.

DNA damage-induced interaction between a lineage addiction oncogenic transcription factor and the MRN complex shapes a tissue-specific DNA Damage Response and cancer predisposition.

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA Damage Response (DDR) programs. However, some cells, in skin for example, are normally exposed to high levels of DNA damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Here we show, using melanoma as a model, that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a non-transcriptional role in shaping the DDR. On exposure to DNA damaging agents, MITF is phosphorylated by ATM/DNA-PKcs, and unexpectedly its interactome is dramatically remodelled; most transcription (co)factors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks, and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement, high MITF levels are associated with increased SNV burden in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of ATM/DNA-PKcs-phosphorylated MITF. Our data suggest that a non-transcriptional function of a lineage-restricted transcription factor contributes to a tissue-specialised modulation of the DDR that can impact cancer initiation.

Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation.

BACKGROUND: Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. RESULTS: We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. CONCLUSION: Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.