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Purpose:Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Nucleosome assembly protein 1-like 5 (NAP1L5) is a protein-coding gene that encodes a protein similar to nucleosome assembly protein 1 (NAP1). It is a histone chaperone that plays an important role in gene transcription in organisms. However, the role of NAP1L5 in the pathogenesis of hepatocellular carcinoma remains to be elucidated.Method：We used QRT-PCR, immunohistochemistry and westernbolting to detect the expression of NAP1L5 in tissues and cell lines, and analyzed the relationship between its expression and clinicopathological features and prognosis. after up-regulating and down-regulating NAP1L5 in cell lines, we used a variety of methods to verify its regulatory effect on HCC, and verified it again in animal experiments, mass spectrometry analysis to find the possible binding protein (MYH9) and downstream signaling pathway (PI3K/AK/mTOR). And verify the relationship between them.Results: In this study, low expression of NAP1L5 was found in hepatocellular carcinoma, and the downregulation of NAP1L5 was related to shorter survival and disease-free survival. In addition, its expression is also related to the tumor size and recurrence of hepatocellular carcinoma. The overexpression and knockdown of NAP1L5 by plasmid and siRNA showed that NAP1L5 inhibited the proliferation, migration and invasion and induced apoptosis of hepatoma cells. In vivo experiments confirmed that NAP1L5 can inhibit the growth and metastasis of hepatocellular carcinoma cells. In the mechanistic study, we found that NAP1L5 affects the occurrence and development of hepatocellular carcinoma by regulating MYH9 to inhibit the PI3K/AKT/mTOR signaling pathway. Conclusion: As a functional tumor suppressor, NAP1L5 is expressed at low levels in HCC. NAP1L5 inhibits the PI3K/AKT/mTOR signaling pathway in hepatocellular carcinoma by regulating MYH9.It may be a new potential target for liver cancer treatment.

BACKGROUND: Secreted phosphoprotein 1 (SPP1), also known as osteopontin (OPN), is a multifunctional protein expressed in diverse normal tissues, and functionally is involved in cellular matrix and signaling processes. Many studies have linked SPP1 to pathophysiological conditions including cancer. OBJECTIVE: The aim of this study is to evaluate the 3’UTR length of SPP1 gene in glioblastoma cell line. METHODS: 3’ Rapid Amplification of cDNA End (3’-RACE) was used to determine the 3’ end of SPP1 gene. APAatlas data base, GEPIA web server, and miRcode were also used to extract related information and bioinformatic analysis part. RESULTS: In this study we show that SPP1 gene undergoes Alternative cleavage and Polyadenylation (APA) mechanism, by which it generates two 3’ termini, longer isoform and shorter isoform, in glioblastoma derived cell line, U87-MG. Further bioinformatic analysis reveals that SPP1 alternative 3’UTR (aUTR), which is absent in shorter isoform, is targeted by two families of microRNAs-miR-181abcd/4262 and miR-154/872. These miRNAs also target and perhaps negatively regulate NAP1L1 and ENAH genes that are involved in cell proliferation and cell polarity, respectively. Relative expression difference (RED), obtained from RNA-seq data of diverse normal tissues, representing APA usage appears to be negatively correlated with expression of NAP1L1 and ENAH, emphasizing co-expression of SPP1 longer isoform with these two genes, indicating miRNA sponge function of aUTR (longer 3’UTR). Bioinformatic analysis also shows that in normal brain tissue longer APA isoform of SPP1 is expressed; however shorter isoform appears to be expressed in cancer condition. CONCLUSION: Together, this study reveals that SPP1 APA isoforms have different pattern in normal and cancerous conditions, which can be considered as a diagnostic and prognostic marker in cancers.

Acetylation of histone H1 is generally considered to activate transcription, whereas deacetylation of H1 represses transcription. However, the precise mechanism of the acetylation is unknown. Here, using chromatography, we identified nucleosome assembly protein 1 (NAP-1) as having inhibitory activity against histone H1 acetylation by acetyltransferase p300. We found that native NAP-1 interacts with H1 in a Drosophila crude extract. We also found that it inhibits the deacetylation of histone H1 by histone deacetylase 1. The core histones in nucleosomes were acetylated in a GAL4–VP16 transcriptional activator-dependent manner in vitro. This acetylation was strongly repressed by hypoacetylated H1 but to a lesser extent by hyperacetylated H1. Consistent with these findings, a micrococcal nuclease assay indicated that hypoacetylated H1, which represses activator-dependent acetylation, was incorporated into chromatin, whereas hyperacetylated H1 was not. To determine the contribution of NAP-1 to transcriptional regulation in vivo, we compared NAP-1 knockdown (KD) with coactivator CREB-binding protein (CBP) KD using RNA sequencing in Drosophila Schneider 2 cells. Most genes were downregulated rather than upregulated by NAP-1 KD, and those downregulated genes were also downregulated by CBP KD. Our results suggest that NAP-1 plays a role in transcriptional regulation by fine-tuning the acetylation of histone H1.

The E2/E3 enzyme UBE2O ubiquitylates diverse clients to mediate important processes, including targeting unassembled 'orphan' proteins for quality control and clearing ribosomes during erythropoiesis. How quality-control factors, such as UBE2O, select clients on the basis of heterogeneous features is largely unknown. Here, we show that UBE2O client selection is regulated by ubiquitin binding and a cofactor, NAP1L1. Attaching a single ubiquitin onto a client enhances UBE2O binding and multi-mono-ubiquitylation. UBE2O also repurposes the histone chaperone NAP1L1 as an adapter to recruit a subset of clients. Cryo-EM structures of human UBE2O in complex with NAP1L1 reveal a malleable client recruitment interface that is autoinhibited by the intrinsically reactive UBC domain. Adding a ubiquitylated client identifies a distinct ubiquitin-binding SH3-like domain required for client selection. Our findings reveal how multivalency and a feed-forward mechanism drive the selection of protein quality-control clients.

NAP1L1 is a key regulator of embryonic neurogenesis but its role in lung cancer remains unexplored. In this study, we investigated the relationship between NAP1L1 expression and the clinicopathological parameters and prognosis of non-small cell lung cancer patients. To this end, the expression of NAP1L1 in tumor samples was evaluated by immunohistochemistry. NAP1L1 expression was significantly associated with reduced differentiation (P = 0.00014), higher pathological TNM stages (P < 0.00001), lymph node metastasis (P < 0.00001), intrapulmonary metastasis (P = 0.02955), lymphatic invasion (P = 0.00019), vascular invasion (P = 0.00008) and poorer prognosis (P = 0.0008) of patients with adenocarcinoma. Moreover, multivariate analyses using the Cox-proportional hazards model confirmed that NAP1L1 expression increased the risk of death after adjusting for other clinicopathological factors (HR = 2.46, 95% CI, 1.22-4.96). Furthermore, NAP1L1 expression was identified as an independent poor prognostic factor in patients with resectable stage I lung adenocarcinoma. NAP1L1-siRNA-treated lung adenocarcinoma-derived A549 cells showed significant suppression of proliferation, migration, and invasion abilities. These findings suggest that NAP1L1 may be a novel predictive and prognostic marker in lung adenocarcinoma, particularly in those with stage I of the disease.

The nucleosome assembly protein 1 (NAP1) family is the main histone chaperone of histone H2A-H2B. To explore the function of NAP1 family genes in moso bamboo (

Trametes robiniophila (Huaier) is available to refrain lung cancer (LC) cell progression, but its impact and mechanism on angiogenesis of LC are not proved. The study was to explore the potential mechanism of Huaier repressing angiogenesis and tumor growth in LC via strengthening let-7d-5p and targeting NAP1L1. Let-7d-5p and NAP1L1 expression was detected in LC tissues and cells (A549). Pretreatment of A549 cells was with Huaier. Transfection of changed let-7d-5p and NAP1L1 was to A549 cells to uncover their roles in LC cell progression with angiogenesis. Evaluation of the impact of let-7d-5p on angiogenesis in LC was in vitro in a mouse xenograft model. Identification of the targeting of let-7d-5p with NAP1L1 was clarified. The results clarified reduced let-7d-5p but elevated NAP1L1 were manifested in LC. Huaier restrained angiogenesis and tumor growth of LC in vivo and in vitro; Augmented let-7d-5p or declined NAP1L1 motivated the therapy of Huaier on LC; Let-7d-5p negatively modulated NAP1L1; Elevated NAP1L1 reversed the influence of enhancive let-7d-5p. These results strongly suggest that Huaier represses angiogenesis and tumor growth in LC via strengthening let-7d-5p and targeting NAP1L1. Huaier/let-7d-5p/NAP1L1 axis is supposed to be a promising target for the treatment of angiogenesis and tumor growth in LC via elevated let-7d-5p and targeted NAP1L1.

Nucleosome assembly protein 1-like 1 (NAP1L1) is significantly involved in the development of various cancers. However, its role in the molecular mechanism of nasopharyngeal carcinoma (NPC) remains undetermined. In this study, we detected the upregulated expression of NAP1L1 mRNA and protein levels by quantitative polymerase chain reaction and Western blot analysis in NPC cell lines. Results of the immunohistochemistry analysis of NPC tissue biopsies showed that upregulated NAP1L1 protein expression promoted NPC progression and negatively correlated with poor prognosis in NPC patients. Suppression of NAP1L1 expression by small interfering RNA (siRNA) or small hairpin RNA (shRNA) methods significantly decreased cell proliferation in vivo and in vitro. Mechanism analysis revealed that the regulation of cell growth was enriched by Gene Set Enrichment Analysis based on RNA sequencing data. Cell cycle-induced genes CCND1 and E2F1 were downregulated in NAP1L1 knockdown NPC cells. Reduced NAP1L1 suppressed the recruitment of hepatoma-derived growth factor (HDGF) and decreased its expression. Knockdown of HDGF reduced the expression of c-JUN, a key oncogenic transcription factor that can induce the expression of cyclin D1 (CCND1), reducing cell cycle progression and suppressing cell growth in NPC. Transfecting HDGF or c-JUN could reverse the growth-suppressive effects in NAP1L1-downregulated NPC cells. The data obtained in this study suggest that NAP1L1 acts as a potential oncogene by activating HDGF/c-JUN/CCND1 signaling in NPC.

The prognosis of glioma is poor as its pathogenesis and mechanisms underlying cisplatin chemoresistance remain unclear. Nucleosome assembly protein 1 like 1 (NAP1L1) is regarded as a hallmark of malignant tumors. However, the role of NAP1L1 in glioma remains unknown. In this study, we aimed to investigate the molecular functions of NAP1L1 in glioma and its involvement in cisplatin chemoresistance, if any. NAP1L1 was found to be upregulated in samples from The Cancer Genome Atlas (TCGA) database. Immunohistochemistry indicated that NAP1L1 and hepatoma-derived growth factor (HDGF) were enhanced in glioma as compared to the para-tumor tissues. High expressions of NAP1L1 and HDGF were positively correlated with the WHO grade, KPS, Ki-67 index, and recurrence. Moreover, NAP1L1 expression was also positively correlated with the HDGF expression in glioma tissues. Functional studies suggested that knocking down NAP1L1 could significantly inhibit glioma cell proliferation both in vitro and in vivo, as well as enhance the sensitivity of glioma cells to cisplatin (cDDP) in vitro. Mechanistically, NAP1L1 could interact with HDGF at the protein level and they co-localize in the cytoplasm. HDGF knockdown in NAP1L1-overexpressing glioma cells significantly inhibited cell proliferation. Furthermore, HDGF could interact with c-Jun, an oncogenic transcription factor, which eventually induced the expressions of cell cycle promoters, CCND1/CDK4/CDK6. This finding suggested that NAP1L1 could interact with HDGF, and the latter recruited c-Jun, a key oncogenic transcription factor, that further induced CCND1/CDK4/CDK6 expression, thereby promoting proliferation and chemoresistance in glioma cells. High expression of NAP1L1 in glioma tissues indicated shorter overall survival in glioma patients.

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last two decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins, but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication; ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD; iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD; iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD and HVD phosphorylation is mediated by CK2 kinase; v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication.IMPORTANCECellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We showed that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

Background Nucleosome assembly protein 1-like 1 (NAP1L1) is highly expressed in various types of cancer and plays an important role in carcinogenesis, but its specific role in tumor development and progression remains largely unknown. In this study, we suggest the potential of NAP1L1 as a prognostic biomarker and therapeutic target for the treatment of ovarian cancer (OC). Methods In our study, a tissue microarray (TMA) slide containing specimens from 149 patients with OC and 11 normal ovarian tissues underwent immunohistochemistry (IHC) to analyze the correlation between NAP1L1 expression and clinicopathological features. Loss-of- function experiments were performed by transfecting siRNA and following lentiviral gene transduction into SKOV3 and OVCAR3 cells. Cell proliferation and the cell cycle were assessed by the Cell Counting Kit-8, EDU assay, flow cytometry, colony formation assay, and Western blot analysis. In addition, co-immunoprecipitation (Co-IP) and immunofluorescence assays were performed to confirm the relationship between NAP1L1 and its potential targets in SKOV3/OVCAR3 cells. Results High expression of NAP1L1 was closely related to poor clinical outcomes in OC patients. After knocking down NAP1L1 by siRNA or shRNA, both SKOV3 and OVCAR3 cells showed inhibition of cell proliferation, blocking of the G1/S phase, and increased apoptosis in vitro. Mechanism analysis indicated that NAP1L1 interacted with hepatoma-derived growth factor (HDGF) and they were co-localized in the cytoplasm. Furthermore, HDGF can interact with jun proto-oncogene (C-JUN), an oncogenic transformation factor that induces the expression of cyclin D1 (CCND1). Overexpressed HDGF in NAP1L1 knockdown OC cells not only increased the expression of C-JUN and CCND1, but it also reversed the suppressive effects of si-NAP1L1 on cell proliferation. Conclusions Our data demonstrated that NAP1L1 could act as a prognostic biomarker in OC and can interact with HDGF to mediate the proliferation of OC, and this process of triggered proliferation may contribute to the activation of HDGF/C-JUN signaling in OC cells.

Nucleosome Assembly Protein 1 (NAP1) family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes. In this study, we determined the crystal structure of Arabidopsis NAP1-Related Protein 1 (NRP1) complexed with H2A-H2B and uncovered a previously unknown interaction mechanism in histone chaperoning. Both in vitro binding and in vivo plant rescue assays proved that interaction mediated by the N-terminal α-helix (αN) domain is essential for NRP1 function. In addition, the C-terminal acidic domain (CTAD) of NRP1 binds to H2A-H2B through a conserved mode similar to other histone chaperones. We further extended previous knowledge of the NAP1-conserved earmuff domain by mapping the amino acids of NRP1 involved in association with H2A-H2B. Finally, we showed that H2A-H2B interactions mediated by αN, earmuff, and CTAD domains are all required for the effective chaperone activity of NRP1. Collectively, our results reveal multiple interaction modes of a NAP1 family histone chaperone and shed light on how histone chaperones shield H2A-H2B from nonspecific interaction with DNA.

To evaluate the diagnostic and prognostic utility of nucleosome assembly protein 1-like 1 (NAP1L1) in colon cancer patients.A total of 95 patients with colon cancer [mean (SD) age: 61.0 (1.7) years, 58.9% were males] and 50 healthy individuals [mean (SD) age: 61.0 (2.3) years, 52.0% were males] were included in this prospective multicenter study. Data on patient demographics (age, gender) and serum NAP1L1 levels were recorded in both control and colon cancer groups. In colon cancer patients, serum NAP1L1 levels were further analyzed with respect to TNM stages and tumor size.Serum NAP1L1 levels were significantly higher in colon cancer patients as compared with control subjects [median (min-max) 14(12-16) vs. 2(1-2) ng/mL, p0.001]. In colon cancer patients, serum NAP1L1 levels were significantly higher for tumor size of4 cm vs.4 cm [15(12-16) vs. 12(12-14) ng/mL), p0.001] and for M1 vs. M0 stage [15(12-16) vs. 12(12-14) ng/mL), p0.001]. Serum NAP1L1 levels were significantly higher in T4 stage tumors vs. T1, T2 and T3 stage tumors (p0.001 for each), in T3 stage tumors vs. T1 and T2 stage tumors (p0.001 for each) and in N2 stage tumors vs. N0 and N1 stage tumors (p0.001 for each).Our findings revealed for the first time the substantial rise in serum NAP1L1 levels among colon cancer patients as compared to controls and as correlated with the disease progression. Accordingly, NAP1L1 seems to be a potential biomarker for colon cancer, offering clinically important information on early diagnosis and risk stratification.

Steviol is a natural diterpenoid glycoside isolated fromFor this purpose, we made use of lymphocyte cultures and the analysis of their CD3Our results showed that Steviol reduces the number of lymphocytes due to falls of CD4

The nucleosome, the fundamental gene-packing unit comprising an octameric histone protein core wrapped around by DNA, has a flexible structure that enables dynamic gene regulation mechanisms. Histone lysine acetylation at H3K56 removes a positive charge from the histone core where it interacts with the termini of the nucleosomal DNA and acts as a critical gene regulatory signal that is implicated in transcription initiation and elongation. The predominant proposal on the biophysical role of H3K56 acetylation (H3K56ac) is that weakened electrostatic interaction between DNA termini and the histone core results in facilitated opening and subsequent disassembly of the nucleosome. However, this effect alone is too weak to account for the strong coupling between H3K56ac and its regulatory outcomes. Here we utilized a semi-synthetically modified nucleosome with H3K56ac in order to address this discrepancy. Based on the results, we propose an innovative mechanism by which the charge neutralization effect of H3K56ac is significantly amplified via protein binding. We employed three-color single-molecule fluorescence resonance energy transfer (smFRET) to monitor the opening rate of nucleosomal DNA termini induced by binding of histone chaperone Nap1. We observed an elevated opening rate upon H3K56ac by 5.9 folds which is far larger than 1.5 folds as previously reported for the spontaneous opening dynamics in the absence of Nap1. Our proposed mechanism successfully reconciles this discrepancy based on that DNA opening for Nap1 binding must be larger than the average spontaneous opening. This is a novel mechanism that can explain how a small biophysical effect of histone acetylation results in a significant change in protein binding rate.

Eukaryotes have evolved a specific strategy to package DNA. The nucleosome is a 147-base-pair DNA segment wrapped around histone core proteins that plays important roles regulating DNA-dependent biosynthesis and gene expression. Chromatin remodeling complexes (RSC, Remodel the Structure of Chromatin) hydrolyze ATP to perturb DNA-histone contacts, leading to nucleosome sliding and ejection. Here, we utilized tethered particle motion (TPM) experiments to investigate the mechanism of RSC-mediated nucleosome remodeling in detail. We observed ATP-dependent RSC-mediated DNA looping and nucleosome ejection along individual mononucleosomes and dinucleosomes. We found that nucleosome assembly protein 1 (Nap1) enhanced RSC-mediated nucleosome ejection in a two-step disassembly manner from dinucleosomes but not from mononucleosomes. Based on this work, we provide an entire reaction scheme for the RSC-mediated nucleosome remodeling process that includes DNA looping, nucleosome ejection, the influence of adjacent nucleosomes, and the coordinated action between Nap1 and RSC.

Nucleosome assembly proteins (Naps) influence chromatin dynamics by directly binding to histones. Here we provide a comprehensive structural and biochemical analysis of a Nap protein from Caenorhabditis elegans (CeNap1). CeNap1 naturally lacks the acidic N-terminal tail and has a short C-terminal tail compared to many other Nap proteins. Comparison of CeNap1 with full length and tail-less constructs of Saccharomyces cerevisiae Nap1 uncovers the role of these tails in self-association, histone binding, and Nap competition with DNA for H2A-H2B. We find that the presence of tails influences the stoichiometry of H2A-H2B binding and is required to complete the interactions between H2A-H2B and DNA. The absolute stoichiometry of the Nap protein and H2A-H2B complex is 2:1 or 2:2, with only a very small population of higher-order oligomers occurring at 150 mM NaCl. We also show that H3-H4 binds differently than H2A-H2B and that an (H3-H4)2 tetramer can simultaneously bind two Nap2 protein homodimers.

Long non-coding RNAs (lncRNAs) show great potential as therapeutic targets in many diseases including hepatocellular carcinoma (HCC). Here, we aimed to investigate the clinical significance and function of lncRNA CDKN2B antisense RNA 1 (CDKN2B-AS1) in HCC. Here, we identified a novel oncogenic lncRNA CDKN2B-AS1, which was highly expressed in HCC and positively associated with large tumor size, microvascular invasion, high tumor grade, advanced tumor stage and reduced survival of HCC patients. CDKN2B-AS1 knockdown inhibited cell proliferation, migration and invasion, and induced G1 arrest and apoptosis of HCC cells in vitro, and CDKN2B-AS1 silencing suppressed tumor growth and metastasis of HCC in vivo. In accordance, CDKN2B-AS1 overexpression accelerated HCC cell growth and metastasis. Mechanistically, CDKN2B-AS1 promoted nucleosome assembly protein 1 like 1 (NAP1L1) expression by sponging let-7c-5p, thereby activated PI3K/AKT/mTOR signaling in HCC cells. Notably, NAP1L1 restoration abolished the effects of CDKN2B-AS1 silencing on HCC cell growth and metastasis. CDKN2B-AS1, an oncogenic lncRNA of HCC, promoted NAP1L1-mediated PI3K/AKT/mTOR signaling by acting as a molecular sponge of let-7c-5p. Our findings indicate that CDKN2B-AS1 may be a potential prognostic biomarker and a candidate target for HCC therapy.

Background Despite the fact that miRNAs play pivotal roles in various human malignancies, their molecular mechanisms influencing RCC are poorly understood. Methods The expression of miRNAs from RCC and paired normal renal specimens was analysed by a combined computational and experimental approach using two published datasets and qRT-PCR assays. The functional role of these miRNAs was further identified by overexpression and inhibition assays in vivo and in vitro. Western blots, luciferase assays, and chromatin immunoprecipitation were performed to investigate the potential mechanisms of these miRNAs. Results Bioinformatics analysis and qRT-PCR revealed that miR-532-5p was one of the most heavily downregulated miRNAs. Overexpression of miR-532-5p inhibited RCC cell proliferation, while knockdown of miR-532-5p promoted cell proliferation. Mechanistic analyses indicated that miR-532-5p directly targets KRAS and NAP1L1. Interestingly, ETS1 suppressed the transcription of miR-532-5p by directly binding a special region of its promoter. Moreover, high levels of ETS1, as an oncogene in RCC, were significantly associated with poor survival in a large cohort of RCC specimens. Conclusions Our work presents a road map for the prediction and validation of a miR-532-5p/KRAS-NAP1L1/P-ERK/ETS1 axis feedback loop regulating cell proliferation, which could potentially provide better therapeutic avenues for treating RCC.

Hepatocellular carcinoma (HCC) is a major leading cause of cancer mortality worldwide. PRDI-BF1 and RIZ homology domain containing 8 (PRDM8) is a key regulator in neural development and testis steroidogenesis; however, its role in liver carcinogenesis remains to be investigated. In this study, PRDM8 was found to be down-regulated in HCC, which was linked with shorter recurrence-free survival. Lentiviral-based overexpression and knockdown approaches showed that PRDM8 inhibited HCC cell proliferation, migration, and invasion. PRDM8 caused G1/S cell cycle arrest and induced apoptosis. An in vivo tumor model confirmed the antitumor role of PRDM8 in HCC growth and metastasis. Mechanistic study showed that PRDM8 suppressed the PI3K/AKT/mTOR signaling cascade through the regulation of nucleosome assembly protein 1-like 1 (NAP1L1). Conclusion: PRDM8 as a functional tumor suppressor is frequently down-regulated in HCC. Through regulating NAP1L1, PRDM8 inhibits PI3K/AKT/mTOR signaling in HCC. PRDM8 is a potential target for therapies of HCC. (Hepatology 2018).

NAP1L1 has been shown to function in the life cycle of several DNA virus and retrovirus. However, whether NAP1L1 regulates hepatitis C virus (HCV), a positive-stranded RNA virus, remain to be elucidated. In this study, we identified NAP1L1 as a novel binding partner for HCV NS3. Our results suggest that NAP1L1 contributes to HCV entry, but not viral replication. These findings provide mechanistic insight into the role of NAP1L1 in HCV life cycle and extend the role of NAP1L1 to RNA virus.

Nucleosomes, the basic units of chromatin, package and regulate expression of eukaryotic genomes. Although the structure of the intact nucleosome is well characterized, little is known about structures of partially unwrapped, transient intermediates. In this study, we present nine cryo-EM structures of distinct conformations of nucleosome and subnucleosome particles. These structures show that initial DNA breathing induces conformational changes in the histone octamer, particularly in histone H3, that propagate through the nucleosome and prevent symmetrical DNA opening. Rearrangements in the H2A-H2B dimer strengthen interaction with the unwrapping DNA and promote nucleosome stability. In agreement with this, cross-linked H2A-H2B that cannot accommodate unwrapping of the DNA is not stably maintained in the nucleosome. H2A-H2B release and DNA unwrapping occur simultaneously, indicating that DNA is essential in stabilizing the dimer in the nucleosome. Our structures reveal intrinsic nucleosomal plasticity that is required for nucleosome stability and might be exploited by extrinsic protein factors.

Nuclear factor-κB (NF-κB) participates in apoptosis signaling pathway under various pathophysiological conditions. It exerts transcriptional control on the anti-apoptotic Bcl-2 family, such as Bcl-2, Bcl-xl, and Mcl-1, which act on the mitochondrial outer membrane. Previously, we described that NF-κB is negatively regulated by diacylglycerol kinase ζ (DGKζ), an enzyme that phosphorylates a lipid second messenger diacylglycerol. DGKζ downregulation enhances inhibitors of NF-κB α (IκBα) degradation and p65 subunit phosphorylation, leading to enhanced NF-κB transcriptional activity. Transcriptional machinery is tightly regulated by assembly/disassembly and modification of nucleosomal components. Of those, the human NAP1-like protein (NAP1L) family functions in the transport, assembly/disassembly of nucleosome core particles. We previously identified NAP1L1 and NAP1L4 as novel DGKζ binding partners, but the mechanism by which NAP1Ls are involved in NF-κB signaling pathway remains unclear. Here we show that knockdown of NAP1L1 suppresses IκBα degradation and nuclear transport of p65 subunit after treatment with TNF-α stimulation, leading to attenuation of the NF-κB transcriptional activity, whereas NAP1L4 knockdown remains silent. Moreover, ChIP assay reveals that NAP1L1 knockdown attenuates p65 binding to the Mcl-1 promoter after TNF-α stimulation. This attenuation leads to reduced expression of anti-apoptotic Mcl-1, thereby decreasing the mitochondrial membrane potential and subsequent apoptosis after treatment with TNF-α and CHX. Collectively, results of this study suggest that NAP1L1 downregulation renders the cell vulnerable to apoptotic cell death through attenuation of NF-κB transcriptional activity on the anti-apoptotic Mcl-1 gene.

Nap1l1 gene encodes a tissue specific nucleosome assembly protein and is essential for tissue development. Here, we report the generation and characterization of a nap1l1 transgenic reporter in zebrafish model. We showed that a 5-kilobase (kb) genomic fragment immediately upstream of the nap1l1 gene transcription initiation site is capable of targeting the nucleic enhanced green fluorescence protein (EGFP) expression initially to central nervous system and subsequently to lateral line neuromasts, cardiomyocytes, and paraxial vessels, where the endogenous nap1l1 normally expresses with only a few exception. In adulthood, zebrafish nap1l1 promoter-driving nEGFP is predominantly expressed in lateral line system, liver, and ovary, but not in heart. Therefore, this novel transgenic reporter line, Tg(nap1l1:nEGFP)zs102, would be a valuable tool for studying the development and regeneration of lateral line system and also for investigating cardiac development.

Nucleosome assembly protein 1-like (Nap1l) family plays numerous biological roles including nucleosome assembly, transcriptional regulation, and cell cycle progression. However, the tissue specific in vivo functions of the Nap1l family members remain largely unknown. In this study, we finished the complete expression patterns of nap1l1 and nap1l4a in zebrafish embryos by whole-mount in situ hybridization. We observed maternal existence of nap1l1 transcript and that its zygotic expression is abundant and not spatially restricted at 6 somite stage, while nap1l4a mRNA is not detectable until 6 somite stage when it is weakly transcribed throughout the embryo. At 24 h post-fertilization (hpf), nap1l1 is predominantly expressed in the central nervous system, neural tube, ventral mesoderm, branchial arches, and pectoral fins, while nap1l4a mRNA is throughout the embryo, enriched in the eyes, tectum, and myotomes. As the embryo develops, nap1l1 expression maintains throughout the head, with gradually enriched in the tectum, olfactory vesicle, lens, optic cups, heart, branchial arches, pectoral fins, axial vasculature, pronephros, and lateral line neuromasts, whereas nap1l4a expression is weak in the tectum, branchial arches, and pectoral fins. Overall, these expression analyses provide a valuable basis for the functional study of nap1l family in zebrafish development.

Background Apart the gene-regulatory functions as docking sites for histone ‘readers’, some histone modifications could directly affect nucleosome structure. The H2BK34-ubiquitylation deposited by MOF-MSL complex, increases nucleosome dynamics in vitro and promotes donation of one H2A/H2B dimer to histone acceptors. Methods We evaluated temperature-depended stability of H2BK34-ubiquitylated nucleosomes under ‘physiological’ ionic conditions in the presence or absence of histone acceptor, and examined assembly and disassembly of ubiquitylated nucleosomes in vitro by recombinant mouse NAP1. Results H2BK34ub modification is sufficient to promote selective eviction of only one H2A/H2B dimer independently of histone-binding agents. Despite the robust H2A/H2B dimer-displacement effect of mNAP1 with the H2BK34ub (but not unmodified) nucleosomes, NAP1 could assemble symmetrically- or asymmetrically ubiquitylated nucleosomes under ‘physiological’ conditions in vitro. Conclusions and general significance The increased mobility of one nucleosomal H2A/H2B dimer is an intrinsic nucleosome destabilizing property of H2BK34 ubiquitylation that has the intranucleosome bases. The ability of NAP to reasonably efficiently assemble H2BK34-ubiquitylated nucleosomes supposes a potential mechanism for deposition/distribution of H2BK34ub mark in the MOF-MSL independent manner (for example, during histone dimer exchange upon transcription elongation).

In eukaryotes, nucleosome assembly is crucial for genome integrity. The histone chaperone NAP1 plays an important role in histone folding, storage, and transport, as well as histone exchange and nucleosome assembly. At present, the molecular basis of these activities is not fully understood. We have solved high-resolution crystal structures of Caenorhabditis elegans NAP1 (ceNAP1) in complex with its cognate substrates: the C. elegans H2A-H2B dimer (ceH2A-H2B) and the H2A.Z-H2B dimer (ceH2A.Z-H2B). Our structural and biochemical data reveals the acidic concave surface is relevant to tetramerization, and uncovers how a ceNAP1 homodimer uses its concave surface to asymmetrically recognize a ceH2A-H2B or ceH2A.Z-H2B heterodimer. Intriguingly, an "acidic strip" within the concave surface of ceNAP1 is crucial for binding histones, including H2A-H2B, H3-H4, and histone variants. Thus, our results provide insight into the molecular mechanisms of NAP1 histone chaperone activity.

The p53 tumor suppressor regulates expression of genes involved in various stress responses. Upon genotoxic stress, p53 induces target genes regulating cell cycle arrest for survival or apoptosis. Nevertheless, detailed mechanisms of how p53 selectively regulates these opposing outcomes remain unclear. For this study, we investigated p53 regulatory mechanisms exerted by nucleosome assembly protein 1-like 1 (NAP1L1) and NAP1L4, both of which are identified as DGKζ-interacting proteins. Here we demonstrate that, under normal conditions, NAP1L1 knockdown decreases Lys320 acetylation of p53 with attenuated proarrest p21 expression, whereas NAP1L4 knockdown increases Lys320 acetylation with enhanced p21 expression. These conditions lead respectively to facilitation and suppression of cell growth. Under genotoxic stress conditions, NAP1L1 knockdown increases Lys382 acetylation with enhanced proapoptotic Bax levels, thereby facilitating cell death. By contrast, NAP1L4 knockdown decreases Lys382 acetylation with attenuated Bax levels, thereby suppressing apoptosis. These results suggest that NAP1L1 and NAP1L4 regulate cell fate by controlling the expression of p53-responsive proarrest and proapoptotic genes through selective modulation of p53 acetylation at specific sites during normal homeostasis and in stress-induced responses.

Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes. Currently, only nine of the ∼80 eukaryotic r-proteins are known to be guarded by such chaperones. In search of new dedicated r-protein chaperones, we performed a tandem-affinity purification based screen and looked for factors co-enriched with individual small subunit r-proteins. We report the identification of Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively. Both factors promote the solubility of their r-protein clients in vitro. While Tsr4 is specific for Rps2, Nap1 has several interaction partners including Rps6 and two other r-proteins. Tsr4 binds co- translationally to the essential, eukaryote-specific N-terminal extension of Rps2, whereas Nap1 interacts with a large, mostly eukaryote-specific binding surface of Rps6. Mutation of the essential Tsr4 and deletion of the non-essential Nap1 both enhance the 40S synthesis defects of the corresponding r-protein mutants. Our findings highlight that the acquisition of eukaryote-specific domains in r-proteins was accompanied by the co-evolution of proteins specialized to protect these domains and emphasize the critical role of r-protein chaperones for the synthesis of eukaryotic ribosomes.

The organization of the membrane cytoskeleton of red blood cells allows them to tolerate constant exposure to mechanical stresses. We demonstrated that the proper organization of the membrane cytoskeleton of red blood cells requires Ttll4. Red blood cells from Ttll4-knockout mice showed an abnormal arrangement of the membrane cytoskeleton., Mammalian red blood cells (RBCs) circulate through blood vessels, including capillaries, for tens of days under high mechanical stress. RBCs tolerate this mechanical stress while maintaining their shape because of their elastic membrane skeleton. This membrane skeleton consists of spectrin-actin lattices arranged as quasi-hexagonal units beneath the plasma membrane. In this study, we found that the organization of the RBC cytoskeleton requires tubulin tyrosine ligase–like 4 (Ttll4). RBCs from Ttll4-knockout mice showed larger average diameters in smear test. Based on the rate of hemolysis, Ttll4-knockout RBCs showed greater vulnerability to phenylhydrazine-induced oxidative stress than did wild-type RBCs. Ultrastructural analyses revealed the macromolecular aggregation of cytoskeletal components in RBCs of Ttll4-knockout mice. Immunoprecipitation using the anti-glutamylation antibody GT335 revealed nucleosome assembly protein 1 (NAP1) to be the sole target of TTLL4 in the RBCs, and NAP1 glutamylation was completely lost in Ttll4-knockout RBCs. In wild-type RBCs, the amount of glutamylated NAP1 in the membrane was nearly double that in the cytosol. Furthermore, the absence of TTLL4-dependent glutamylation of NAP1 weakened the binding of NAP1 to the RBC membrane. Taken together, these data demonstrate that Ttll4 is required for proper cytoskeletal organization in RBCs.

Nucleosomes impose physical barriers to DNA-templated processes, playing important roles in eukaryotic gene regulation. DNA is packaged into nucleosomes by histone proteins mainly through strong electrostatic interactions that can be modulated by various post-translational histone modifications. Investigating the dynamics of histone dissociation from the nucleosome and how it is altered upon histone modifications is important for understanding eukaryotic gene regulation mechanisms. In particular, histone H2A-H2B dimer displacement in the nucleosome is one of the most important and earliest steps of histone dissociation. Two conflicting hypotheses on the requirement for dimer displacement are that nucleosomal DNA needs to be unwrapped before a dimer can displace and that a dimer can displace without DNA unwrapping. In order to test the hypotheses, we employed three-color single-molecule FRET and monitored in a time-resolved manner the early kinetics of H2AH2B dimer dissociation triggered by high salt concentration and by histone chaperone Nap1. The results reveal that dimer displacement requires DNA unwrapping in the vast majority of the nucleosomes in the salt-induced case, while dimer displacement precedes DNA unwrapping in >60% of the nucleosomes in the Nap1-mediated case. We also found that acetylation at histone H4K16 or H3K56 affects the kinetics of Nap1-mediated dimer dissociation and facilitates the process both kinetically and thermodynamically. On the basis of these results, we suggest a mechanism by which histone chaperone facilitates H2A-H2B dimer displacement from the histone core without requiring another factor to unwrap the nucleosomal DNA.

The SWI/SNF and RSC family of ATP-dependent chromatin remodelers disassembles nucleosomes by moving nucleosomes into the vicinity of adjoining nucleosomes. We found that the histone chaperone Nap1 efficiently promotes disassembly of adjacent nucleosomes with which RSC collides and not the disassembly of nucleosomes mobilized by RSC. Nap1 is specific to RSC, as it does not target SWI/SNF, its paralog in Saccharomyces cerevisiae. Extensive mutational analysis of Nap1 has revealed that Nap1 affinity for histones H2A-H2B and H3-H4 and its ability to displace histones from DNA are required for Nap1 to enhance RSC-mediated disassembly. Other histone chaperones, such as Vps75, that also bind histones are not able to enhance RSC-mediated disassembly. Our study suggests a mechanism by which Nap1 is recruited to actively transcribed regions and assists in the passage of the transcription complex through chromatin, and it provides a novel mechanism for the coordinated action of RSC and Nap1.

Separase is a caspase-like cysteine protease that is best known for its essential role during the metaphase-to-anaphase transition when it cleaves the cohesin ring complex that keeps the sister chromatids together. Another important function of separase is to regulate the process of centriole separation, known as centriole disengagement, at the end of mitosis. We used proximity-dependent biotin identification (BioID) to expand our knowledge on the identity of separase's proximity interactors. We show that separase BioID labeled two domains at the mother centriole: an area underneath the centriolar appendages and another at the proximal end of the mother centriole. BioID analysis identified more than 200 proximity interactors of separase, one being the Alström Syndrome Protein 1 (ALMS1) at the base of centrioles. Other proximity interactors are the histone chaperons NAP1L1 and NAP1L4, which localize to the spindle poles during mitosis and the spindle assembly checkpoint proteins BUBR1, SKA1 and SKA3 that reside at kinetochores in early mitosis. Finally, we show that depletion of BUBR1 homolog from Caenorhabditis elegans delayed the recruitment of separase to mitotic chromosomes, and eventually anaphase onset.

Telomere elongation is a function that all eukaryote cells must accomplish in order to guarantee, first, the stability of the end of the chromosomes and second, to protect the genetic information from the inevitable terminal erosion. The targeted transposition of the telomere transposons HeT-A, TART and TAHRE perform this function in Drosophila, while the telomerase mechanism elongates the telomeres in most eukaryotes. In order to integrate telomere maintenance together with cell cycle and metabolism, different components of the cell interact, regulate, and control the proteins involved in telomere elongation. Different partners of the telomerase mechanism have already been described, but in contrast, very few proteins have been related with assisting the telomere transposons of Drosophila. Here, we describe for the first time, the implication of NAP-1 (Nucleosome assembly protein 1), a histone chaperone that has been involved in nuclear transport, transcription regulation, and chromatin remodeling, in telomere biology. We find that Nap-1 and HeT-A Gag, one of the major components of the Drosophila telomeres, are part of the same protein complex. We also demonstrate that their close interaction is necessary to guarantee telomere stability in dividing cells. We further show that NAP-1 regulates the transcription of the HeT-A retrotransposon, pointing to a positive regulatory role of NAP-1 in telomere expression. All these results facilitate the understanding of the transposon telomere maintenance mechanism, as well as the integration of telomere biology with the rest of the cell metabolism., This work was supported by a grant from the Spanish Ministry of Science and Innovation BFU2009-08318/BMC to E.C.

International audience; Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

Summary The +1 nucleosome of yeast genes, within which reside transcription start sites, is characterized by histone acetylation, by the displacement of an H2A-H2B dimer, and by a persistent association with the RSC chromatin-remodeling complex. Here we demonstrate the interrelationship of these characteristics and the conversion of a nucleosome to the +1 state in vitro. Contrary to expectation, acetylation performs an inhibitory role, preventing the removal of a nucleosome by RSC. Inhibition is due to both enhanced RSC-histone interaction and diminished histone-chaperone interaction. Acetylation does not prevent all RSC activity, because stably bound RSC removes an H2A-H2B dimer on a timescale of seconds in an irreversible manner.

Alphaviruses are widely distributed in both hemispheres and circulate between mosquitoes and amplifying vertebrate hosts. Geographically separated alphaviruses have adapted to replication in particular organisms. The accumulating data suggest that this adaptation is determined not only by changes in their glycoproteins but also by the amino acid sequence of the hypervariable domain (HVD) of the alphavirus nsP3 protein. We performed a detailed investigation of chikungunya virus (CHIKV) nsP3 HVD interactions with host factors and their roles in viral replication in vertebrate and mosquito cells. The results demonstrate that CHIKV HVD is intrinsically disordered and binds several distinctive cellular proteins. These host factors include two members of the G3BP family and their mosquito homolog Rin, two members of the NAP1 family, and several SH3 domain-containing proteins. Interaction with G3BP proteins or Rin is an absolute requirement for CHIKV replication, although it is insufficient to solely drive it in either vertebrate or mosquito cells. To achieve a detectable level of virus replication, HVD needs to bind members of at least one more protein family in addition to G3BPs. Interaction with NAP1L1 and NAP1L4 plays a more proviral role in vertebrate cells, while binding of SH3 domain-containing proteins to a proline-rich fragment of HVD is more critical for virus replication in the cells of mosquito origin. Modifications of binding sites in CHIKV HVD allow manipulation of the cell specificity of CHIKV replication. Similar changes may be introduced into HVDs of other alphaviruses to alter their replication in particular cells or tissues.IMPORTANCE Alphaviruses utilize a broad spectrum of cellular factors for efficient formation and function of replication complexes (RCs). Our data demonstrate for the first time that the hypervariable domain (HVD) of chikungunya virus nonstructural protein 3 (nsP3) is intrinsically disordered. It binds at least 3 families of cellular proteins, which play an indispensable role in viral RNA replication. The proteins of each family demonstrate functional redundancy. We provide a detailed map of the binding sites on CHIKV nsP3 HVD and show that mutations in these sites or the replacement of CHIKV HVD by heterologous HVD change cell specificity of viral replication. Such manipulations with alphavirus HVDs open an opportunity for development of new irreversibly attenuated vaccine candidates. To date, the disordered protein fragments have been identified in the nonstructural proteins of many other viruses. They may also interact with a variety of cellular factors that determine critical aspects of virus-host interactions.

Background In mammals, nucleostemin (NS), a nucleolar GTPase, is involved in stem cell proliferation, embryogenesis and ribosome biogenesis. Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) has previously been shown to be essential for plant growth and development. However, the role of NSN1 in cell proliferation is largely unknown. Results Using nsn1, a loss-of-function mutant of Arabidopsis NSN1, we investigated the function of NSN1 in plant cell proliferation and cell cycle regulation. Morphologically, nsn1 exhibited developmental defects in both leaves and roots, producing severely reduced vegetative organs with a much smaller number of cells than those in the wild type. Dynamic analysis of leaf and root growth revealed a lower cell proliferation rate and slower cell division in nsn1. Consistently, the transcriptional levels of key cell cycle genes, including those regulating the transition of G1-S and G2-M, were reduced drastically in nsn1. The introduction of CYCLIN B1::GUS into nsn1 resulted in confined expression of GUS in both the leaf primordia and root meristem, indicating that cell proliferation was hampered by the mutation of NSN1. Upon subjection to treatment with bleomycin and methyl methanesulfonate (MMS), nsn1 plants exhibited hypersensitivity to the genotoxic agents. In the nucleus, NSN1 interacted with nucleosome assembly protein1 (AtNAP1;1), a highly conserved histone chaperone functioning in cell proliferation. Notably, the N-terminal conserved domains of Arabidopsis NSN1 were critical for the physical interaction. Conclusions As a conserved homolog of mammalian nucleostemin, Arabidopsis NSN1 plays pivotal roles in embryogenesis and ribosome biogenesis. In this study, NSN1 was found to function as a positive regulator in cell cycle progression. The interaction between NSN1 and histone chaperone AtNAP1;1, and the high resemblance in sensitivity to genotoxics between nsn1 and atnap1;1 imply the indispensability of the two nuclear proteins for cell cycle regulation. This work provides an insight into the delicate control of cell proliferation through the cooperation of a GTP-binding protein with a nucleosome assembly/disassembly protein in Arabidopsis. Electronic supplementary material The online version of this article (10.1186/s12870-018-1289-2) contains supplementary material, which is available to authorized users.

Nucleosome assembly protein 1 (NAP1) binds both the (H3-H4)2 tetramer and two H2A-H2B dimers, mediating their sequential deposition on DNA. NAP1 contains a C-terminal acidic domain (CTAD) and a core domain that promotes dimer formation. Here, we have investigated the roles of the core domain and CTAD of human NAP1 in binding to H2A-H2B and H3-H4 by isothermal calorimetry and native mass spectrometry and compared them with the roles of yeast NAP1. We show that the hNAP1 and yNAP1 dimers bind H2A-H2B by two different modes: a strong endothermic interaction and a weak exothermic interaction. A mutant hNAP1, but not yNAP1, dimer lacking CTAD loses the exothermic interaction and shows greatly reduced H2A-H2B binding activity. The isolated CTAD of hNAP1 binds H2A-H2B only exothermically with relatively stronger binding as compared with the exothermic interaction observed for the full-length hNAP1 dimer. Thus, the two CTADs in the hNAP1 dimer seem to provide binding assistance for the strong endothermic interaction of the core domain with H2A-H2B. By contrast, in the relatively weaker binding of hNAP1 to H3-H4 as compared with yNAP1, CTAD of hNAP1 has no significant role. To our knowledge, this is the first distinct role identified for the hNAP1 CTAD.

Background/Aims: To investigate whether nucleosome assembly protein 1-like 1 (Nap1l1) regulates the proliferation of induced pluripotent stem cells (iPSC) and the potential mechanisms. Methods: Nap1l1-knockdown-iPSC and Nap1l1-overexpression-iPSC were constructed by transfection of lentiviral particles. The proliferation of iPSC was detected by MTT analysis, and cell cycle was analyzed by flow cytometry. Results: Nap1l1 overexpression promoted iPSC proliferation and induced G2/M transition compared to their control iPSC while Nap1l1-knockdown-iPSC dramatically displayed the reduced proliferation and accumulated G2/M phase cells. Further analysis showed that Nap1l1 overexpression in iPSC increased the expression of cyclin B1, downregulated the expression of p21 and p27, while knockdown of Nap1l1 showed the opposite effects. In addition, overexpression of Nap1l1 promoted the phosphorylation of AKT and ERK in iPSC, while knockdown of Nap1l1 inhibited the effects. However, these effects displayed in Nap1l1-overexpression-iPSC were greatly suppressed by the inhibition of AKT or ERK signaling. Conclusions: The results indicate that Nap1l1 promotes the proliferation of iPSC attributable to G2/M transition caused by downregulation of p27 and p21, and upregulation of cyclin B1, the activation of AKT or ERK is involved in the process. The present study has revealed a novel molecular mechanism involved in the proliferation of iPSC.

The role of disturbed chromatin remodeling in the pathogenesis of intellectual disability (ID) is well established and illustrated by de novo mutations found in a plethora of genes encoding for proteins of the epigenetic regulatory machinery. We describe mutations in the “SET nuclear proto-oncogene” (SET), encoding a component of the “inhibitor of histone acetyltransferases” (INHAT) complex, involved in transcriptional silencing. Using whole exome sequencing, four patients were identified with de novo mutations in the SET gene. Additionally, an affected mother and child were detected who carried a frameshift variant in SET. Four patients were found in literature. The de novo mutations in patients affected all four known SET mRNA transcripts. LoF mutations in SET are exceedingly rare in the normal population and, if present, affect only one transcript. The pivotal role of SET in neurogenesis is evident from in vitro and animal models. SET interacts with numerous proteins involved in histone modification, including proteins encoded by known autosomal dominant ID genes, that is, EP300, CREBBP, SETBP1, KMT2A, RAC1, and CTCF. Our study identifies SET as a new component of epigenetic regulatory modules underlying human cognitive disorders, and as a first member of the Nucleosome Assembly Protein (NAP) family implicated in ID.

Nucleosome assembly protein 1 (NAP1) is a histone chaperone that exchanges histone H2A-H2B dimer from chromatin templates. Studies with yeast NAP1 (yNAP1) have revealed its existence as multiple oligomeric species in solution. Here, rat NAP1 (rNAP1), which is 98% identical to the human NAP1 (hNAP1) was used as a model to characterize the oligomeric structures of this protein in higher eukaryotes. Gel filtration chromatography and Dynamic light scattering of recombinant rNAP1 indicated that the protein exists as a complex mixture of multimeric species even at 500 mM ionic strength. The solution-state complexity remains unchanged even at higher ionic strengths. Equilibrium unfolding (ΔG 14.6 kcal mol− 1) shows that rNAP1, both dimeric and oligomeric, follow the two-state model of unfolding with no detectable intermediates. Homology modelling suggests that rat and yeast NAP1 share an overall similar structure with conserved domains. However, dissimilar substitutions like threonine and lysine with glycine in the β-hairpin involved in oligomerization, possibly leads to the observed differences in the oligomerization propensity of the two proteins. Molecular dynamic simulation (MDS) of the two structures also revealed that rNAP1 dimer is more stable owing to the extensive hydrogen bonding in comparison to yNAP1. Further, in vitro kinase assay showed that the phosphorylation of rNAP1 favors oligomerization with no effect on its histone binding capacity. Our results clearly suggest that there are differences in the in-solution behavior of rNAP1 compared to yNAP1 which may have in vivo functional implications for the regulation of these complexes during chromatin assembly and rearrangement.

Chromatin is the natural form of DNA in the eukaryotic nucleus and is the substrate for diverse biological phenomena. The functional analysis of these processes ideally would be carried out with nucleosomal templates that are assembled with customized core histones, DNA sequences, and chromosomal proteins. Here we report a simple, reliable, and versatile method for the ATP-dependent assembly of evenly spaced nucleosome arrays. This minimal chromatin assembly system comprises the Drosophila nucleoplasmin-like protein (dNLP) histone chaperone, the imitation switch (ISWI) ATP-driven motor protein, core histones, template DNA, and ATP. The dNLP and ISWI components were synthesized in bacteria, and each protein could be purified in a single step by affinity chromatography. We show that the dNLP-ISWI system can be used with different DNA sequences, linear or circular DNA, bulk genomic DNA, recombinant or native Drosophila core histones, native human histones, the linker histone H1, the non-histone chromosomal protein HMGN2, and the core histone variants H3.3 and H2A.V. The dNLP-ISWI system should be accessible to a wide range of researchers and enable the assembly of customized chromatin with specifically desired DNA sequences, core histones, and other chromosomal proteins.

Summary: The precise function and role of nucleosome assembly protein 1-like 1 (Nap1l1) in brain development are unclear. Here, we find that Nap1l1 knockdown decreases neural progenitor cell (NPC) proliferation and induces premature neuronal differentiation during cortical development. A similar deficiency in embryonic neurogenesis was observed in Nap1l1 knockout (KO) mice, which were generated using the CRISPR-Cas9 system. RNA sequencing (RNA-seq) analysis indicates that Ras-associated domain family member 10 (RassF10) may be the downstream target of Nap1l1. Furthermore, we found that Nap1l1 regulates RassF10 expression by promoting SETD1A-mediated H3K4 trimethylation at the RassF10 promoter. Nap1l1 KO defects may be rescued by RassF10 overexpression, suggesting that Nap1l1 controls NPC differentiation through RassF10. Our findings reveal an essential role for the Nap1l1 histone chaperone in cortical neurogenesis during early embryonic brain development. : Nap1l1 plays essential roles in embryonic neurogenesis, including the proliferation and differentiation of neural progenitors. Qiao et al. find that Nap1l1 regulates RassF10 through SETD1A-mediated H3K4me3 of the RassF10 promoter. Keywords: neural progenitor cell, neural differentiation and proliferation, cortex development, gain of function, loss of function, histone chaperone, Nap1l1, RassF10, H3K4me3

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA hepatotropic virus. Despite cellular defenses, HCV is able to replicate in hepatocytes and to establish a chronic infection that could lead to severe complications and hepatocellular carcinoma. An important player in subverting the host response to HCV infection is the viral nonstructural protein NS5A, which, in addition to its role in replication and assembly, targets several pathways involved in the cellular response to viral infection. Several unbiased screens identified nucleosome assembly protein 1-like 1 (NAP1L1) as an interaction partner of HCV NS5A. Here we confirmed this interaction and mapped it to the C terminus of NS5A of both genotype 1 and 2. NS5A sequesters NAP1L1 in the cytoplasm, blocking its nuclear translocation. However, only NS5A from genotype 2 HCV, and not that from genotype 1, targets NAP1L1 for proteosome-mediated degradation. NAP1L1 is a nuclear chaperone involved in chromatin remodeling, and we demonstrated the NAP1L1-dependent regulation of specific pathways involved in cellular responses to viral infection and cell survival. Among those, we showed that lack of NAP1L1 leads to a decrease of RELA protein levels and a strong defect of IRF3 TBK1/IKKε-mediated phosphorylation, leading to inefficient RIG-I and Toll-like receptor 3 (TLR3) responses. Hence, HCV is able to modulate the host cell environment by targeting NAP1L1 through NS5A. IMPORTANCE Viruses have evolved to replicate and to overcome antiviral countermeasures of the infected cell. Hepatitis C virus is capable of establishing a lifelong chronic infection in the liver, which could develop into cirrhosis and cancer. Chronic viruses are particularly able to interfere with the cellular antiviral pathways by several different mechanisms. In this study, we identified a novel cellular target of the viral nonstructural protein NS5A and demonstrated its role in antiviral signaling. This factor, called nucleosome assembly protein 1-like 1 (NAP1L1), is a nuclear chaperone involved in the remodeling of chromatin during transcription. When it is depleted, specific signaling pathways leading to antiviral effectors are affected. Therefore, we provide evidence for both a novel strategy of virus evasion from cellular immunity and a novel role for a cellular protein, which has not been described to date.

The essential histone chaperone FACT plays a critical role in DNA replication, repair, and transcription, primarily by binding to histone H2A-H2B dimers and regulating their assembly into nucleosomes. While FACT histone chaperone activity has been extensively studied, the exact nature of the H2A and H2B residues important for FACT binding remains controversial. In this study, we characterized the functions of residues in the histone H2A and H2B acidic patch, which is important for binding many chromatin-associated factors. We found that mutations in essential acidic patch residues cause a defect in histone occupancy in yeast, even though most of these histone mutants are expressed normally in yeast and form stable nucleosomes in vitro. Instead, we show that two acidic patch residues, H2B L109 and H2A E57, are important for histone binding to FACT in vivo. We systematically screened mutants in other H2A and H2B residues previously suspected to be important for FACT binding and confirmed the importance of H2B M62 using an in-vivo FACT-binding assay. Furthermore, we show that, like deletion mutants in FACT subunits, an H2A E57 and H2B M62 double mutant is lethal in yeast. In summary, we show that residues in the nucleosome acidic patch promote histone occupancy and are important for FACT binding to H2A-H2B dimers in yeast.

The DNA of metazoan somatic cells is packaged into a compact nucleoprotein complex termed chromatin (Van Holde 1988; Wolffe 1998). Chromatin fiber is comprised of highly conserved repetitive units (nucleosomes) that contain an octamer of four core histones and 145–147 base pairs (bp) of DNA wrapped around the octamer in 1.65 turns of a left-handed superhelix (Luger et al. 1997). Nucleosomes are assembled in vivo in an ATP-dependent fashion through a concerted and sequential action of core histone chaperones and motor proteins that belong to the Snf2 family of DNA-dependent ATPases (Gorbalenya and Koonin 1993; Eisen et al. 1995; Mello and Almouzni 2001; Akey and Luger 2003; Haushalter and Kadonaga 2003; Piatti et al. 2011; Torigoe et al. 2013). For instance, Drosophila ACF/CHRAC can mediate chromatin assembly in conjunction with histone chaperone NAP-1 (Varga-Weisz et al. 1997; Ito et al. 1999). Other known ATP-dependent chromatin assembly factors include RSF, CHD1, ATRX, and ToRC/NoRC (LeRoy et al. 1998, 2000; Lusser et al. 2005; Lewis et al. 2010; Emelyanov et al. 2012). The most abundant chromatin component in male germline cells is protamines—small positively charged arginine- and cysteine-rich proteins (Balhorn 2007). During spermiogenesis, protamines replace 85%–95% of DNA-bound histones in the nucleus to achieve a higher density of sperm nuclear DNA (Ward and Coffey 1991; Rathke et al. 2014). Crystalline-like sperm chromatin structure is sixfold more compact than metaphase chromosomes and renders sperm DNA enzymatically inert (Balhorn 1982). At fertilization, the oocyte remodels the condensed sperm chromatin into a transcriptionally competent chromatin of the male pronucleus (McLay and Clarke 2003). During, this process, protamines are expelled and replaced with oocyte-supplied histones, which are then organized into nucleosomes. Sperm chromatin remodeling (SCR) is controlled by biochemical activities in the early oocyte, but components of these activities remain largely unknown (McLay and Clarke 2003). However various protein factors have been implicated in SCR, including core histone chaperones from Xenopus (Nucleoplasmin, N1, HIRA, and TAF-I) and Drosophila (NAP-1, p22, DF31, HIRA, and Yemanuclein) (Dilworth et al. 1987; Philpott and Leno 1992; Kawasaki et al. 1994; Crevel and Cotterill 1995; Ito et al. 1996a; Matsumoto et al. 1999; Loppin et al. 2001; Ray-Gallet et al. 2002; Orsi et al. 2013). In mammals, members of nucleoplasmin/nucleophosmin family proteins (NPM1–3) function in sperm chromatin decondensation in vitro (Okuwaki et al. 2012). In addition, Npm2 knockout female mice exhibit fertility defects consistent with a role of NPM2 in nuclear and nucleolar chromatin organization (Burns et al. 2003). It was also suggested that sperm chromatin decondensation is ATP-dependent (Wright and Longo 1988). Drosophila melanogaster sperm cells contain two major protamines (A and B) encoded by male-specific transcripts Mst35Ba and Mst35Bb, respectively (Ashburner et al. 1999; Jayaramaiah Raja and Renkawitz-Pohl 2005). The Drosophila maternal effect mutant sesame (ssm) prevents male pronucleus formation (Loppin et al. 2001). ssm encodes the histone variant H3.3-specific chaperone HIRA (Tagami et al. 2004), postulated to be required for replication-independent deposition of histones in the male pronucleus during sperm decondensation (Loppin et al. 2005; Bonnefoy et al. 2007). In eggs from homozygous ssm females, maternal histones are not deposited in the chromatin of male pronuclei, preventing normal mitosis and resulting in the development of gynogenetic haploid embryos and embryonic stage lethality. A similar phenotype is observed in null mutants of the gene encoding ATP-dependent chromatin assembly factor CHD1 (Konev et al. 2007). Thus, CHD1 and HIRA act cooperatively and are required for nucleosome assembly during SCR. Intriguingly, protamines are efficiently expelled from the DNA of nascent male pronuclei in Chd1 and ssm eggs (Jayaramaiah Raja and Renkawitz-Pohl 2005; Konev et al. 2007), suggesting that protamine removal and histone deposition are functionally distinct steps. In this study, we used a biochemical approach to identify specific protein components of the Drosophila egg machinery that promote the dissociation of protamine–DNA complexes of sperm chromatin. These factors turn out to be two known core histone chaperones (NAP-1 and NLP), a homolog of mammalian nucleophosmin, and a novel Drosophila histone chaperone (TAP/p32). These putative “protamine chaperones” facilitate SCR independently of CHD1 and HIRA, which mediate nucleosome assembly in nascent male pronuclei. Of note, TAP/p32 is specifically required to expel Drosophila protamine B from sperm chromatin in vitro, whereas NAP-1, NLP, and nucleophosmin share roles in removal of protamine A. We also provide in vivo evidence that TAP/p32 functions in Drosophila egg SCR. In conclusion, we characterized protein factors that mediate the first obligatory step of SCR (protamine dissociation) and reconstituted the complete SCR reaction (reorganization of protamine-containing sperm chromatin into core histone-containing nucleosome arrays) in a purified defined system in vitro.

Low efficiency of cardiomyocyte differentiation from induced pluripotent stem cells (iPSCs) hinders the clinical application of iPSC technology for cardiac repair strategy. Recently, we screened out nucleosome assembly protein 1-like 1 (Nap1l1), which was downregulated during the differentiation of P19CL6 cells into cardiomyocytes. Here, we attempted to study the role of Nap1l1 in cardiomyogenesis of iPSC. Nap1l1 was downregulated during the differentiation of iPSC. Knockdown of Nap1l1 dramatically enhanced the differentiation of iPSC into functional cardiomyocytes while overexpression of Nap1l1 sharply lowered the differentiation. Moreover, although Nap1l1-knockdown had little effect on endoderm differentiation, the Nap1l1 modulation significantly accelerated mesoderm development. Re-expressing Nap1l1 in Nap1l1-knockdown-iPSC rescued the effects of Nap1l1. Inducibly overexpressing Nap1l1 at early stage of differentiation greatly inhibited mesoderm induction and cardiogenesis of iPSC. However, mesoderm stem cells (Flk-1-positive cells) originated from Nap1l1-knockdown- or -overexpression-iPSC showed no difference in further cardiomyocyte differentiation compared with that of control-iPSC. Further study revealed that Nap1l1-overexpression increased γ-secretase activity and the expression of Notch intracellular domain (NICD) and downstream genes during the differentiation of iPSC. γ-Secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) greatly suppressed the production of NICD and abolished the inhibitory effects of Nap1l1-overexpression on mesoderm induction and cardiogenesis. These findings demonstrate that downregulation of Nap1l1 significantly enhances mesodermal induction and subsequent cardiogenesis of murine iPSC via inhibition of γ-secretase-regulated Notch signaling, which would facilitate the application of iPSC in heart diseases. Stem Cells 2014;32:1759–1773

Histone chaperones function in chromatin assembly and disassembly, suggesting they have important regulatory roles in transcription elongation. The Saccharomyces cerevisiae proteins Nap1 and Vps75 are structurally related, evolutionarily conserved histone chaperones. We showed that Nap1 genetically interacts with several transcription elongation factors and that both Nap1 and Vps75 interact with the RNA polymerase II kinase, CTK1. Loss of NAP1 or VPS75 suppressed cryptic transcription within the open reading frame (ORF) observed when strains are deleted for the kinase CTK1. Loss of the histone acetyltransferase Rtt109 also suppressed ctk1-dependent cryptic transcription. Vps75 regulates Rtt109 function, suggesting that they function together in this process. Histone H3 K9 was found to be the important lysine that is acetylated by Rtt109 during ctk1-dependent cryptic transcription. We showed that both Vps75 and Nap1 regulate the relative level of H3 K9 acetylation in the STE11 ORF. This supports a model in which Nap1, like Vps75, directly regulates Rtt109 activity or regulates the assembly of acetylated chromatin. Although Nap1 and Vps75 share many similarities, due to their distinct interactions with SET2, Nap1 and Vps75 may also play separate roles during transcription elongation. This work sheds further light on the importance of histone chaperones as general regulators of transcription elongation.