Non-canonical MLL1 activity regulates centromeric phase separation and genome stability.

Epigenetic dysregulation is a prominent feature in cancer, as exemplified by frequent mutations in chromatin regulators, including the MLL/KMT2 family of histone methyltransferases. Although MLL1/KMT2A activity on H3K4 methylation is well documented, their non-canonical activities remain mostly unexplored. Here we show that MLL1/KMT2A methylates Borealin K143 in the intrinsically disordered region essential for liquid-liquid phase separation of the chromosome passenger complex (CPC). The co-crystal structure highlights the distinct binding mode of the MLL1 SET domain with Borealin K143. Inhibiting MLL1 activity or mutating Borealin K143 to arginine perturbs CPC phase separation, reduces Aurora kinase B activity, and impairs the resolution of erroneous kinetochore-microtubule attachments and sister-chromatid cohesion. They significantly increase chromosome instability and aneuploidy in a subset of hepatocellular carcinoma, resulting in growth inhibition. These results demonstrate a non-redundant function of MLL1 in regulating inner centromere liquid condensates and genome stability via a non-canonical enzymatic activity.

A neuron-specific microexon ablates the novel DNA-binding function of a histone H3K4me0 reader PHF21A.

How cell-type-specific chromatin landscapes emerge and progress during metazoan ontogenesis remains an important question. Transcription factors are expressed in a cell-type-specific manner and recruit chromatin-regulatory machinery to specific genomic loci. In contrast, chromatin-regulatory proteins are expressed broadly and are assumed to exert the same intrinsic function across cell types. However, human genetics studies have revealed an unexpected vulnerability of neurodevelopment to chromatin factor mutations with unknown mechanisms. Here, we report that 14 chromatin regulators undergo evolutionary-conserved neuron-specific splicing events involving microexons. Of the 14 chromatin regulators, two are integral components of a histone H3K4 demethylase complex; the catalytic subunit LSD1 and an H3K4me0-reader protein PHF21A adopt neuron-specific forms. We found that canonical PHF21A (PHF21A-c) binds to DNA by AT-hook motif, and the neuronal counterpart PHF21A-n lacks this DNA-binding function yet maintains H3K4me0 recognition intact. In-vitro reconstitution of the canonical and neuronal PHF21A-LSD1 complexes identified the neuronal complex as a hypomorphic H3K4 demethylating machinery with reduced nucleosome engagement. Furthermore, an autism-associated PHF21A missense mutation, 1285 G>A, at the last nucleotide of the common exon immediately upstream of the neuronal microexon led to impaired splicing of PHF21A -n. Thus, ubiquitous chromatin regulatory complexes exert unique intrinsic functions in neurons via alternative splicing of their subunits and potentially contribute to faithful human brain development.

Iditarod, a Drosophila homolog of the Irisin precursor FNDC5, is critical for exercise performance and cardiac autophagy.

Mammalian FNDC5 encodes a protein precursor of Irisin, which is important for exercise-dependent regulation of whole-body metabolism. In a genetic screen in Drosophila, we identified Iditarod (Idit), which shows substantial protein homology to mouse and human FNDC5, as a regulator of autophagy acting downstream of Atg1/Atg13. Physiologically, Idit-deficient flies showed reduced exercise performance and defective cold resistance, which were rescued by exogenous expression of Idit. Exercise training increased endurance in wild-type flies, but not in Idit-deficient flies. Conversely, Idit is induced upon exercise training, and transgenic expression of Idit in wild-type flies increased endurance to the level of exercise trained flies. Finally, Idit deficiency prevented both exercise-induced increase in cardiac Atg8 and exercise-induced cardiac stress resistance, suggesting that cardiac autophagy may be an additional mechanism by which Idit is involved in the adaptive response to exercise. Our work suggests an ancient role of an Iditarod/Irisin/FNDC5 family of proteins in autophagy, exercise physiology, and cold adaptation, conserved throughout metazoan species.

Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase.

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air-water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.5 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including a smaller amount of protein required and avoiding protein denaturation at the air-water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the nonheme diiron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

Development of an atmospheric plasma jet device for versatile treatment of electron microscope sample grids.

Atmospheric-pressure plasmas have been widely applied for surface modification and biomedical treatment because of their ability to generate highly reactive radicals and charged particles. In negative-stain electron microscopy (Neg-EM) and cryogenic electron microscopy (cryo-EM), plasmas have been used to generate hydrophilic surfaces and eliminate surface contaminants to embed specimens onto grids. In addition, plasma treatment is a prerequisite for negative-stain and Quantifoil grids, whose surfaces are coated with hydrophobic amorphous carbon. Although the conventional glow discharge system has been used successfully in this purpose, there has been no further effort to take an advantage from the recent progress in the plasma field. Here, we developed a nonthermal atmospheric plasma jet system as an alternative tool for treatment of surfaces. The low-temperature plasma is a nonequilibrium system that has been widely used in biomedical area. Unlike conventional glow discharge systems, the plasma jet system successfully cleans and introduces hydrophilicity on the grid surface in the ambient environment without a vacuum. Therefore, we anticipate that the plasma jet system will have numerous benefits, such as convenience and versatility, as well as having potential applications in surface modification for both negative-stain and cryo-EM grid treatment.

Regulation of MLL1 Methyltransferase Activity in Two Distinct Nucleosome Binding Modes.

Cryo-EM structures of the KMT2A/MLL1 core complex bound on nucleosome core particles (NCPs) suggest unusual rotational dynamics of the MLL1 complex approaching its physiological substrate. However, the functional implication of such dynamics remains unclear. Here, we show that the MLL1 core complex also shows high rotational dynamics bound on the NCP carrying the catalytically inert histone H3 lysine 4 to methionine (K4M) mutation. There are two major binding modes of the MLL1 complex on the NCPK4M. Importantly, disruption of only one of the binding modes compromised the overall MLL1 activity in an NCP-specific manner. We propose that the MLL1 core complex probably exists in an equilibrium of poised and active binding modes. The high rotational dynamics of the MLL1 complex on the NCP is a feature that can be exploited for loci-specific regulation of H3K4 methylation in higher eukaryotes.

Mechanism for DPY30 and ASH2L intrinsically disordered regions to modulate the MLL/SET1 activity on chromatin.

Recent cryo-EM structures show the highly dynamic nature of the MLL1-NCP (nucleosome core particle) interaction. Functional implication and regulation of such dynamics remain unclear. Here we show that DPY30 and the intrinsically disordered regions (IDRs) of ASH2L work together in restricting the rotational dynamics of the MLL1 complex on the NCP. We show that DPY30 binding to ASH2L leads to stabilization and integration of ASH2L IDRs into the MLL1 complex and establishes new ASH2L-NCP contacts. The significance of ASH2L-DPY30 interactions is demonstrated by requirement of both ASH2L IDRs and DPY30 for dramatic increase of processivity and activity of the MLL1 complex. This DPY30 and ASH2L-IDR dependent regulation is NCP-specific and applies to all members of the MLL/SET1 family of enzymes. We further show that DPY30 is causal for de novo establishment of H3K4me3 in ESCs. Our study provides a paradigm of how H3K4me3 is regulated on chromatin and how H3K4me3 heterogeneity can be modulated by ASH2L IDR interacting proteins.

Dismantling and Rebuilding the Trisulfide Cofactor Demonstrates Its Essential Role in Human Sulfide Quinone Oxidoreductase.

Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in sulfide clearance, coupling H2S oxidation to coenzyme Q reduction. Recent structures of human SQOR revealed a sulfur atom bridging the SQOR active site cysteines in a trisulfide configuration. Here, we assessed the importance of this cofactor using kinetic, crystallographic, and computational modeling approaches. Cyanolysis of SQOR proceeds via formation of an intense charge transfer complex that subsequently decays to eliminate thiocyanate. We captured a disulfanyl-methanimido thioate intermediate in the SQOR crystal structure, revealing how cyanolysis leads to reversible loss of SQOR activity that is restored in the presence of sulfide. Computational modeling and MD simulations revealed an ∼105-fold rate enhancement for nucleophilic addition of sulfide into the trisulfide versus a disulfide cofactor. The cysteine trisulfide in SQOR is thus critical for activity and provides a significant catalytic advantage over a cysteine disulfide.

Insights on the regulation of the MLL/SET1 family histone methyltransferases.

In eukaryotes, histone H3K4 methylation by the MLL/SET1 family histone methyltransferases is enriched at transcription regulatory elements including gene promoters and enhancers. The level of H3K4 methylation is highly correlated with transcription activation and is one of the most frequently used histone post-translational modifications to predict transcriptional outcome. Recently, it has been shown that rearrangement of the cellular landscape of H3K4 mono-methylation at distal enhancers precedes cell fate transition and is used for identification of novel regulatory elements for development and disease progression. Similarly, broad H3K4 tri-methylation regions have also been used to predict intrinsic tumor suppression properties of regulator regions in a variety of cellular models. Understanding the regulation for how H3K4 methylation is deposited and regulated is of paramount importance. In this review, we will discuss new findings on how the MLL/SET1 family enzymes are regulated on chromatin and their potential functional and regulatory implications. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.

An H3K9 methylation-dependent protein interaction regulates the non-enzymatic functions of a putative histone demethylase.

H3K9 methylation (H3K9me) specifies the establishment and maintenance of transcriptionally silent epigenetic states or heterochromatin. The enzymatic erasure of histone modifications is widely assumed to be the primary mechanism that reverses epigenetic silencing. Here, we reveal an inversion of this paradigm where a putative histone demethylase Epe1 in fission yeast, has a non-enzymatic function that opposes heterochromatin assembly. Mutations within the putative catalytic JmjC domain of Epe1 disrupt its interaction with Swi6HP1 suggesting that this domain might have other functions besides enzymatic activity. The C-terminus of Epe1 directly interacts with Swi6HP1, and H3K9 methylation stimulates this protein-protein interaction in vitro and in vivo. Expressing the Epe1 C-terminus is sufficient to disrupt heterochromatin by outcompeting the histone deacetylase, Clr3 from sites of heterochromatin formation. Our results underscore how histone modifying proteins that resemble enzymes have non-catalytic functions that regulate the assembly of epigenetic complexes in cells.

A cell's identity depends on which of its genes are active. One way for cells to control this process is to change how accessible their genes are to the molecular machinery that switches them on and off. Special proteins called histones determine how accessible genes are by altering how loosely or tightly DNA is packed together. Histones can be modified by enzymes, which are proteins that add or remove specific chemical 'tags'. These tags regulate how accessible genes are and provide cells with a memory of gene activity. For example, a protein found in yeast called Epe1 helps reactivate large groups of genes after cell division, effectively 're-setting' the yeast's genome and eliminating past memories of the genes being inactive. For a long time, Epe1 was thought to do this by removing methyl groups, a 'tag' that indicates a gene is inactive, from histones ­ that is, by acting like an enzyme. However, no direct evidence to support this hypothesis has been found. Raiymbek et al. therefore set out to determine exactly how Epe1 worked, and whether or not it did indeed behave like an enzyme. Initial experiments testing mutant versions of Epe1 in yeast cells showed that the changes expected to stop Epe1 from removing methyl groups instead prevented the protein from 'homing' to the sections of DNA it normally activates. Detailed microscope imaging, using live yeast cells engineered to produce proteins with fluorescent markers, revealed that this inability to 'home' was due to a loss of interaction with Epe1's main partner, a protein called Swi6. This protein recognizes and binds histones that have methyl tags. Swi6 also acts as a docking site for proteins involved in deactivating genes in close proximity to these histones. Further biochemical studies revealed how the interaction between Epe1 and Swi6 can help in gene reactivation. The methyl tag on histones in inactive regions of the genome inadvertently helps Epe1 interact more efficiently with Swi6. Then, Epe1 can simply block every other protein that binds to Swi6 from participating in gene deactivation. This observation contrasts with the prevailing view where the active removal of methyl tags by proteins such as Epe1 switches genes from an inactive to an active state. This work shows for the first time that Epe1 influences the state of the genome through a process that does not involve enzyme activity. In other words, although the protein may 'moonlight' as an enzyme, its main job uses a completely different mechanism. More broadly, these results increase the understanding of the many different ways that gene activity, and ultimately cell identity, can be controlled.

Publisher Correction: Cryo-EM structure of the human MLL1 core complex bound to the nucleosome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cryo-EM structure of the human MLL1 core complex bound to the nucleosome.

Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

Concurrent activation of growth factor and nutrient arms of mTORC1 induces oxidative liver injury.

mTORC1 is a protein kinase important for metabolism and is regulated by growth factor and nutrient signaling pathways, mediated by the Rheb and Rag GTPases, respectively. Here we provide the first animal model in which both pathways were upregulated through concurrent mutations in their GTPase-activating proteins, Tsc1 and Depdc5. Unlike former models that induced limited mTORC1 upregulation, hepatic deletion of both Tsc1 and Depdc5 (DKO) produced strong, synergistic activation of the mTORC1 pathway and provoked pronounced and widespread hepatocyte damage, leading to externally visible liver failure phenotypes, such as jaundice and systemic growth defects. The transcriptome profile of DKO was different from single knockout mutants but similar to those of diseased human livers with severe hepatitis and mouse livers challenged with oxidative stress-inducing chemicals. In addition, DKO liver cells exhibited prominent molecular pathologies associated with excessive endoplasmic reticulum (ER) stress, oxidative stress, DNA damage and inflammation. Although DKO liver pathologies were ameliorated by mTORC1 inhibition, ER stress suppression unexpectedly aggravated them, suggesting that ER stress signaling is not the major conduit of how hyperactive mTORC1 produces liver damage. Interestingly, superoxide scavengers N-acetylcysteine (NAC) and Tempol, chemicals that reduce oxidative stress, were able to recover liver phenotypes, indicating that mTORC1 hyperactivation induced liver damage mainly through oxidative stress pathways. Our study provides a new model of unregulated mTORC1 activation through concomitant upregulation of growth factor and nutrient signaling axes and shows that mTORC1 hyperactivation alone can provoke oxidative tissue injury.

A Catalytic Trisulfide in Human Sulfide Quinone Oxidoreductase Catalyzes Coenzyme A Persulfide Synthesis and Inhibits Butyrate Oxidation.

Mitochondrial sulfide quinone oxidoreductase (SQR) catalyzes the oxidation of H2S to glutathione persulfide with concomitant reduction of CoQ10. We report herein that the promiscuous activity of human SQR supported the conversion of CoA to CoA-SSH (CoA-persulfide), a potent inhibitor of butyryl-CoA dehydrogenase, and revealed a molecular link between sulfide and butyrate metabolism, which are known to interact. Three different CoQ1-bound crystal structures furnished insights into how diverse substrates access human SQR, and provided snapshots of the reaction coordinate. Unexpectedly, the active site cysteines in SQR are configured in a bridging trisulfide at the start and end of the catalytic cycle, and the presence of sulfane sulfur was confirmed biochemically. Importantly, our study leads to a mechanistic proposal for human SQR in which sulfide addition to the trisulfide cofactor eliminates 201Cys-SSH, forming an intense charge-transfer complex with flavin adenine dinucleotide, and 379Cys-SSH, which transfers sulfur to an external acceptor.

MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase.

Soluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions. The maximum catalytic activity of hydroxylase (MMOH) is achieved through the interplay of its regulatory protein (MMOB) and reductase. An additional auxiliary protein, MMOD, functions as an inhibitor of MMOH; however, its inhibitory mechanism remains unknown. Here, we report the crystal structure of the MMOH-MMOD complex from Methylosinus sporium strain 5 (2.6 Å). Its structure illustrates that MMOD associates with the canyon region of MMOH where MMOB binds. Although MMOD and MMOB recognize the same binding site, each binding component triggers different conformational changes toward MMOH, which then respectively lead to the inhibition and activation of MMOH. Particularly, MMOD binding perturbs the di-iron geometry by inducing two major MMOH conformational changes, i.e., MMOH ß subunit disorganization and subsequent His147 dissociation with Fe1 coordination. Furthermore, 1,6-hexanediol, a mimic of the products of sMMO, reveals the substrate access route.

Structures of CENP-C cupin domains at regional centromeres reveal unique patterns of dimerization and recruitment functions for the inner pocket.

The successful assembly and regulation of the kinetochore are critical for the equal and accurate segregation of genetic material during the cell cycle. CENP-C (centromere protein C), a conserved inner kinetochore component, has been broadly characterized as a scaffolding protein and is required for the recruitment of multiple kinetochore proteins to the centromere. At its C terminus, CENP-C harbors a conserved cupin domain that has an established role in protein dimerization. Although the crystal structure of the Saccharomyces cerevisiae Mif2CENP-C cupin domain has been determined, centromeric organization and kinetochore composition vary greatly between S. cerevisiae (point centromere) and other eukaryotes (regional centromere). Therefore, whether the structural and functional role of the cupin domain is conserved throughout evolution requires investigation. Here, we report the crystal structures of the Schizosaccharomyces pombe and Drosophila melanogaster CENP-C cupin domains at 2.52 and 1.81 Å resolutions, respectively. Although the central jelly roll architecture is conserved among the three determined CENP-C cupin domain structures, the cupin domains from organisms with regional centromeres contain additional structural features that aid in dimerization. Moreover, we found that the S. pombe Cnp3CENP-C jelly roll fold harbors an inner binding pocket that is used to recruit the meiosis-specific protein Moa1. In summary, our results unveil the evolutionarily conserved and unique features of the CENP-C cupin domain and uncover the mechanism by which it functions as a recruitment factor.

S-3-Carboxypropyl-l-cysteine specifically inhibits cystathionine γ-lyase-dependent hydrogen sulfide synthesis.

Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 µm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine ß-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80-90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.

Mis16 Switches Function from a Histone H4 Chaperone to a CENP-ACnp1-Specific Assembly Factor through Eic1 Interaction.

The Mis18 complex, composed of Mis16, Eic1, and Mis18 in fission yeast, selectively deposits the centromere-specific histone H3 variant, CENP-ACnp1, at centromeres. How the intact Mis18 holo-complex oligomerizes and how Mis16, a well-known ubiquitous histone H4 chaperone, plays a centromere-specific role in the Mis18 holo-complex, remain unclear. Here, we report the stoichiometry of the intact Mis18 holo-complex as (Mis16)2:(Eic1)2:(Mis18)4 using analytical ultracentrifugation. We further determine the crystal structure of Schizosaccharomyces pombe Mis16 in complex with the C-terminal portion of Eic1 (Eic1-CT). Notably, Mis16 accommodates Eic1-CT through the binding pocket normally occupied by histone H4, indicating that Eic1 and H4 compete for the same binding site, providing a mechanism for Mis16 to switch its binding partner from histone H4 to Eic1. Thus, our analyses not only determine the stoichiometry of the intact Mis18 holo-complex but also uncover the molecular mechanism by which Mis16 plays a centromere-specific role through Eic1 association.

Integrative Structural Investigation on the Architecture of Human Importin4_Histone H3/H4_Asf1a Complex and Its Histone H3 Tail Binding.

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The cross-linking mass spectrometry data showed that the C-terminal region of Importin4 was extensively cross-linked with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic patch in Importin4 accommodates the histone H3 tail, and that histone H3 Lys14 contributes to the interaction with Importin4. In addition, we show that Asf1a modulates the binding of histone H3/H4 to Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.

Structure-based nuclear import mechanism of histones H3 and H4 mediated by Kap123.

Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kluyveromyces lactis Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.