Mechanisms of RNF168 nucleosome recognition and ubiquitylation.

RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.

TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs.

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.

Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.

The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1-10. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11-14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks15,16. We further show that RING domains17 in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin18-22. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.

Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18.

The protein 53BP1 plays a central regulatory role in DNA double-strand break repair. 53BP1 relocates to chromatin by recognizing RNF168-mediated mono-ubiquitylation of histone H2A Lys15 in the nucleosome core particle dimethylated at histone H4 Lys20 (NCP-ubme). 53BP1 relocation is terminated by ubiquitin ligases RNF169 and RAD18 via unknown mechanisms. Using nuclear magnetic resonance (NMR) spectroscopy and biochemistry, we show that RNF169 bridges ubiquitin and histone surfaces, stabilizing a pre-existing ubiquitin orientation in NCP-ubme to form a high-affinity complex. This conformational selection mechanism contrasts with the low-affinity binding mode of 53BP1, and it ensures 53BP1 displacement by RNF169 from NCP-ubme. We also show that RAD18 binds tightly to NCP-ubme through a ubiquitin-binding domain that contacts ubiquitin and nucleosome surfaces accessed by 53BP1. Our work uncovers diverse ubiquitin recognition mechanisms in the nucleosome, explaining how RNF168, RNF169, and RAD18 regulate 53BP1 chromatin recruitment and how specificity can be achieved in the recognition of a ubiquitin-modified substrate.

Meliaceae genomes provide insights into wood development and limonoids biosynthesis.

Meliaceae is a useful plant family owing to its high-quality timber and its many limonoids that have pharmacological and biological activities. Although some genomes of Meliaceae species have been reported, many questions regarding their unique family features, namely wood quality and natural products, have not been answered. In this study, we provide the whole-genome sequence of Melia azedarach comprising 237.16 Mb with a contig N50 of 8.07 Mb, and an improved genome sequence of Azadirachta indica comprising 223.66 Mb with a contig N50 of 8.91 Mb. Moreover, genome skimming data, transcriptomes and other published genomes were comprehensively analysed to determine the genes and proteins that produce superior wood and valuable limonoids. Phylogenetic analysis of chloroplast genomes, single-copy gene families and single-nucleotide polymorphisms revealed that Meliaceae should be classified into two subfamilies: Cedreloideae and Melioideae. Although the Meliaceae species did not undergo additional whole-genome duplication events, the secondary wall biosynthetic genes of the woody Cedreloideae species, Toona sinensis, expanded significantly compared to those of A. indica and M. azedarach, especially in downstream transcription factors and cellulose/hemicellulose biosynthesis-related genes. Moreover, expanded special oxidosqualene cyclase catalogues can help diversify Sapindales skeletons, and the clustered genes that regulate terpene chain elongation, cyclization and modification would support their roles in limonoid biosynthesis. The expanded clans of terpene synthase, O-methyltransferase and cytochrome P450, which are mainly derived from tandem duplication, are responsible for the different limonoid classes among the species. These results are beneficial for further investigations of wood development and limonoid biosynthesis.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

Smoothened antagonist sonidegib affects the development of D. melanogaster larvae via suppression of epidermis formation.

Hedgehog (Hh) signaling is essential for the regulation of embryonic growth and development, the maintenance of stem cell autostasis, and tissue formation, whether in vertebrates or invertebrates. However, exploration into the Hh pathway antagonists in Drosophila or other pests of agricultural importance has been scant. In order to gain a better understanding of the potential utility of the antagonists in insect investigations, a conventional Hh antagonist, sonidegib, was used to evaluate the effects on the development of Drosophila larvae. The results showed that early instar larvae exposed to sonidegib exhibited new epidermal abnormalities and decreased motility after molting. Transcriptome analysis revealed that Sonidegib had a profound effect on chitin-based cuticle development throughout all stages of larvae. Physiological experiments revealed that sonidegib suppressed the epidermis formation and decreased the chitin content. The results of this study shed new light on the potential use of Hh antagonists in agricultural pest management.

Synergistic effects of chlorantraniliprole and camptothecin on physiological impairments, histopathological, biochemical changes, and genes responses in the larvae midgut of Spodoptera frugiperda.

Spodoptera frugiperda is an economically important agricultural pest and poses a serious threat to food security globally. Its management is gravely challenged by its high polyphagous nature, strong migratory ability, and massive fecundity. Chlorantraniliprole (CHL) is widely utilized in controlling S. frugiperda, its intensive application and over-reliance pose adverse health risks, development of resistance, toxicity to beneficial insects, natural enemies, and environmental contamination. To address S. frugiperda resistance to CHL and its inherent challenges, this study explores the synergistic effects of camptothecin (CPT) with CHL in its management. The binary mixed adversely induced the larvae weight and mortality when compared to single-treated. CHL + CPT (1:20 mg/L) had the highest larvae mortality of (73.80 %) with a high antagonistic factor (0.90), while (1:10 mg/L) with (66.10%) mortality exhibited a high synergistic factor (1.43). Further, CHL + CPT (1:10 mg/L) considerably altered the midgut epithelial cell, peritrophic membrane, microvilli, basement membrane, and regenerative cells. For biochemical analysis, CHL + CPT (1:10 mg/L) significantly decreased glutathione-S-transferase (1-chloro-2,4-dinitrobenzene CDNB) and cytochrome P450 (7-ethoxycoumarin O-deethylation) activities in the midgut in a dose and time dependent manner. Based on RNA-Seq analysis, a total of 4,373 differentially expressed genes (DEGs) were identified from the three treatments. CPT vs CK (Control) had 1694 (968 up-, 726 down-regulated), CHL vs CK with 1771 (978 up-, 793 down-regulated), and CHL + CPT vs CK had 908 (394 up-, 514 down-regulated) DEGs. The enrichment analysis disclosed significant pathways such as metabolism of xenobiotics by cytochrome P450, glutathione metabolism, TOLL and IMD (Immune Deficiency) signaling pathway, longevity regulating pathway. This study provides basis to expatiate on the molecular toxicological mechanism of CHL + CPT in management of fall armyworm.

Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ.

The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.

A rare germline CDKN2A variant (47T>G; p16-L16R) predisposes carriers to pancreatic cancer by reducing cell cycle inhibition.

Germline mutations in CDKN2A, encoding the tumor suppressor p16, are responsible for a large proportion of familial melanoma cases and also increase risk of pancreatic cancer. We identified four families through pancreatic cancer probands that were affected by both cancers. These families bore a germline missense variant of CDKN2A (47T>G), encoding a p16-L16R mutant protein associated with high cancer occurrence. Here, we investigated the biological significance of this variant. When transfected into p16-null pancreatic cancer cells, p16-L16R was expressed at lower levels than wild-type (WT) p16. In addition, p16-L16R was unable to bind CDK4 or CDK6 compared with WT p16, as shown by coimmunoprecipitation assays and also was impaired in its ability to inhibit the cell cycle, as demonstrated by flow cytometry analyses. In silico molecular modeling predicted that the L16R mutation prevents normal protein folding, consistent with the observed reduction in expression/stability and diminished function of this mutant protein. We isolated normal dermal fibroblasts from members of the families expressing WT or L16R proteins to investigate the impact of endogenous p16-L16R mutant protein on cell growth. In culture, p16-L16R fibroblasts grew at a faster rate, and most survived until later passages than p16-WT fibroblasts. Further, western blotting demonstrated that p16 protein was detected at lower levels in p16-L16R than in p16-WT fibroblasts. Together, these results suggest that the presence of a CDKN2A (47T>G) mutant allele contributes to an increased risk of pancreatic cancer as a result of reduced p16 protein levels and diminished p16 tumor suppressor function.

Synergistic effects of botanical curcumin-induced programmed cell death on the management of Spodoptera litura Fabricius with avermectin.

Chemical pesticides and adjuvants have caused many negative effects. Botanical compounds provide solutions for the development of environment friendly pesticides and the management of increasing pest resistance. Curcumin, a natural polyphenol, showed synergistic effects on avermectin upon the destructive agricultural pest, Spodoptera litura. However, the botanical synergist and its relevant mechanisms remain unclear. In the article, curcumin significantly enhanced the growth inhibition and midgut structural damage of avermectin on the larvae of S. litura, and the synergistic effects were confirmed with pot experiments. There were only a few influences on the gene expression of avermectin targets, while apoptotic and autophagic related genes and proteins were accumulated in the avermectin/curcumin mixed regent (0.013/0.0013 µg/mL) treated group. Moreover, the potential mechanism was explored with an in vitro model, insect Spodoptera frugiperda Sf9 cell line. Morphology observation featured the damage on cells and Hoechst33258 staining revealed the fragments of DNA after treating with the avermectin/curcumin mixed regent (10/1 µg/mL). Dansylcadaverine and LysoTracker staining, as well as the gene expressions, supposed that curcumin exhibited autophagy inducing effects and the mixed regent possessed a higher ability to induce apoptosis and autophagy. All these results suggested that the synergistic effects of curcumin on the pest management of avermectin potentially mainly derived from the enhancement of programed cell death. It provides new sights for the application of natural compounds in integrated pest management and enriches examples of synergistic mechanisms.

Proteomic profiling for ovarian development and azadirachtin exposure in Spodoptera litura during metamorphosis from pupae to adults.

Azadirachtin is one of the most successful botanical pesticides in agricultural pest control. To build a repertoire of proteins and pathways in response to azadirachtin exposure during ovarian development, iTRAQ-based comparative proteomic was conducted. 1423 and 1686 proteins were identified as differentially accumulated proteins (DAPs) by comparing the protein abundance in adult ovary with that in pupal ovary under normal and azadirachtin exposure condition, respectively. Bioinformatics analysis indicated that pupae-to-adult transition requires proteins related to proteasome and branched chain amino acids (BCAAs) degradation for ovary development. Azadirachtin exposure strongly affected glycosylation-related pathway. And proteins related to vitamin B6 synthesis were necessary for ovary development under normal and AZA-exposure condition. RNAi assays confirmed the essential roles of DAPs related to glycosylation and vitamin B6 synthesis in moth growth and ovary development. The results enhance our understanding of the molecular regulatory network for ovary development and provide valuable resources for using AZA-responsive proteins to develop novel bio-rational insecticides.

Rotenone-induced necrosis in insect cells via the cytoplasmic membrane damage and mitochondrial dysfunction.

Rotenone, a selective inhibitor of mitochondrial complex I, has been extensively studied on kinds of neuron and neuroblast in Parkinson's disease. However, little is known about the potential mechanism of this promising botanical insecticide upon insect cells. In the article, cell proliferation of two Lepidoptera cell lines, Spodoptera litura SL-1 cells and Spodoptera frugiperda Sf9 cells, were all inhibited by rotenone in a time- and dose-dependent manner. Typical necrotic characteristics of cell morphology and ultrastructure, such as plasma membrane collapses and organelle lyses, were all observed by transmission electron microscope and scanning electron microscope. Moreover, irregular DNA degradation was also detected by DNA gel electrophoresis and Hoechst 33258 staining, while the typical apoptotic feature, DNA ladder, hadn't been observed. Flow cytometric analysis showed that rotenone-induced cell death of Sf9 and SL-1 cells accompanied with the plasma membrane potential depolarization and mitochondrial membrane potential reduction. Furthermore, the activity of Na+-K+-ATPase was detected in our study. In conclusion, rotenone could cause necrosis but not apoptosis in insect cells through a mitochondrial- and plasmic membrane-dependent pattern, which shed a light on the rotenone-induced cytotoxicity on insects.

Natural harmine negatively regulates the developmental signaling network of Drosophila melanogaster (Drosophilidae: Diptera) in vivo.

The widely distributed ß-carboline alkaloids exhibit promising psychopharmacological and biochemical effects. Harmine, a natural ß-carboline, can inhibit insect growth and development with unclear mechanisms. In this study, harmine (at 0-200 mg/L) showed a dose-dependent inhibitory effect on the pupal weight, length, height, pupation rate and eclosion rate of fruit flies Drosophila melanogaster, which was similar to the inhibition induced by the well-known botanical insect growth regulator azadirachtin. Moreover, the expression levels of major regulators from the developmental signaling network were down-regulated during the pupal stage except Numb, Fringe, Yorkie and Pten. The Notch, Wnt, Hedgehog and TGF-ß pathways mainly played vital roles in coping with harmine exposure in pupae stage, while the Hippo, Hedgehog and TGF-ß elements were involved in the sex differences. Notch, Hippo, Hedgehog, Dpp and Armadillo were proved to be suppressed in the developmental inhibition with fly mutants, while Numb and Punt were increased by harmine. In conclusion, harmine significantly inhibited the development of Drosophila by negatively affecting their developmental signaling network during different stages. Our results establish a preliminary understanding of the developmental signaling network subjected to botanical component-induced growth inhibition and lay the groundwork for further application.

Curcumin-induced autophagy and nucleophagy in Spodoptera frugiperda Sf9 insect cells occur via PI3K/AKT/TOR pathways.

Compounds from plants or microbes are important resources for new natural pesticides against a wide variety of pests. The growing attention on the role of autophagy (type II cell death) in regulation of insect toxicology has propelled researchers to investigate autophagic cell death pathways. Our previous study proved that the cytotoxic effect of curcumin in Spodoptera frugiperda cells is regulated by autophagy. However, the signaling pathways and molecular mechanisms had not been determined. The current study elucidates curcumin inhibition of survival signaling by blocking the activation of PI3K/AKT/TOR pathways to induce autophagy in S. frugiperda cells. The result demonstrates that nucleophagy associated with cell death following the curcumin treatment. Following the curcumin treatment, Atg8/LC3 immunostaining in both nucleus and cytoplasm was markedly increased. Further, messenger RNA expression level of Atg8 and Atg1 genes regulation by curcumin was examined using quantitative reverse transcription polymerase chain reaction, and the result exhibited increased level of expression after curcumin treatment in a time-dependent manner. Our current study provides new insights to the autophagy occurring via PI3K/AKT/TOR pathways in S. frugiperda Sf9 insect cells induced by curcumin. Taken together, our results show for the first time that curcumin induced nucleophagy in lepidopteron insect cell line.

Harmine induced apoptosis in Spodoptera frugiperda Sf9 cells by activating the endogenous apoptotic pathways and inhibiting DNA topoisomerase I activity.

Harmine, a useful botanical compound, has demonstrated insecticidal activity against some pests. However, harmine's mechanism of action has not been thoroughly elucidated to date. To preliminarily explore harmine's insecticidal mechanisms, the cytotoxicity of harmine against Spodoptera frugiperda Sf9 cells was comprehensively investigated. Our results indicated that harmine induced apoptosis in Sf9 cells, as evidenced by cellular and nuclear morphological changes, DNA laddering and increases in caspase-3-like activities. In addition, activation of the mitochondrial apoptotic pathway by harmine was confirmed by the generation of ROS, opening of mitochondrial permeability transition pores (MPTPs), increase in cytosolic Ca2+, changes in mRNA expression levels of genes involved in the mitochondrial apoptotic pathway and increase and release of Cytochrome c. Furthermore, lysosomal membrane permeabilization, release of cathepsin L from the lysosome into the cytosol and cleavage of caspase-3 were also triggered, which indicated that lysosomes were involved in this physiological process. Moreover, the effect of harmine on DNA topoisomerase I activity was investigated by in vivo and molecular docking experiments. These data not only verified that harmine induced apoptosis via comprehensive activation of the mitochondrial and lysosomal pathways and inhibition of DNA topoisomerase I activity in Sf9 cells but also revealed a mechanism of harmine insecticidal functions for pest control.

Natural ß-carboline alkaloids regulate the PI3K/Akt/mTOR pathway and induce autophagy in insect Sf9 cells.

The ß-carboline alkaloids are a large group of naturally occurring and synthetic indole alkaloids with remarkable pharmacological properties. Furthermore, these alkaloids have also been reported to be effective agents for controlling many pests and plant pathogenic nematodes. However, studies on these potential insecticidal components are scarce. The previous finding that these bioactive compounds can induce programmed cell death in cancer cell lines provided a new insight for exploration of their toxicological mechanisms on insects. In the present study, the cytotoxicity of five natural harmala alkaloids was measured, and the autophagy-inducing effect was confirmed in the Spodoptera frugiperda Sf9 cultured cell line. The results demonstrated that these alkaloids inhibited the proliferation of Sf9 cells in a dose- and time-dependent manner, and the unsaturated ß-carboline alkaloids, harmine and harmol, exhibited stronger autophagy induction activity based on monodansylcadaverineand LysoTracker Red staining. Many autophagy-related genes were increased after ß-carbolines treatment at the RNA level, and the protein expression of Sf-Atg8 was also confirmed to increase after treatment. In addition, the primary autophagic signaling pathway, the PI3K/Akt/mTOR pathway, was altered during the procedure. Furthermore, experiments with special inhibitors and activators were performed to confirm the effect of ß-carbolines on this pathway. The results suggested that the PI3K/Akt/mTOR pathway primarily regulated harmine-induced autophagy in insect cells, and this finding may potentially benefit the application of these promising bioactivity components.

Proteomic Profiling Analysis of Male Infertility in Spodoptera Litura Larvae Challenged with Azadirachtin and its Potential-Regulated Pathways in the Following Stages.

Biopesticides are considered as an alternative to synthetic pesticide with a focus on increasing agricultural productivity as well as maintaining the ecosystem. Prior to application, its potential mechanism should be clearly addressed. Here, the effects of azadirachtin on the reproductive behavior in male Spodoptera litura (Fabricius) are determined. To further explore its molecular mechanism, an iTRAQ (isobaric tags for relative and absolute quantitation) based approach is applied to identify the differentially expressed proteins regulated by azadirachtin at two developmental stages. The results demonstrate that many proteins in the pathway of focal adhesion are regulated to exert influences in detachment of cell attachment, the loss of cell-cell interactions and inducing apoptosis at pupal stage, and many proteins in adenosine monophosphate-activated protein kinase pathway are also changed at the adult stage after azadirachtin-treatment as larvae. Moreover, based on their important roles, it is suggested that some proteins, such as ACTB-G1, ste20-related adaptor protein alpha, and regulatory-associated protein of mTOR (mTORC1) could serve as potential target proteins of azadirachtin to induce male infertility. The results of this study could provide evidence to illuminate the mechanism of male infertility induced by azadirachtin and potential targets for the development of environmentally friendly pesticides.

Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106.

Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)(2) tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)(2). We show that the Rtt106-(H3-H4)(2) interaction is important for gene silencing and the DNA damage response.