Structural variants involving MLLT10 fusion are associated with adverse outcomes in pediatric acute myeloid leukemia.

ABSTRACT: MLLT10 gene rearrangements with KMT2A occur in pediatric acute myeloid leukemia (AML) and confer poor prognosis, but the prognostic impact of MLLT10 in partnership with other genes is unknown. We conducted a retrospective study with 2080 children and young adults with AML registered on the Children's Oncology Group AAML0531 (NCT00372593) and AAML1031 trials (NCT01371981). Transcriptome profiling and/or karyotyping were performed to identify leukemia-associated fusions associated with prognosis. Collectively, 127 patients (6.1%) were identified with MLLT10 fusions: 104 (81.9%) with KMT2A::MLLT10, 13 (10.2%) with PICALM::MLLT10, and 10 (7.9%) X::MLLT10: (2 each of DDX3X and TEC), with 6 partners (DDX3Y, CEP164, SCN2B, TREH, NAP1L1, and XPO1) observed in single patients. Patients with MLLT10 (n = 127) demonstrated adverse outcomes, with 5-year event-free survival (EFS) of 18.6% vs 49% in patients without MLLT10 (n = 1953, P < .001), inferior 5-year overall survival (OS) of 38.2% vs 65.7% (P ≤ .001), and a higher relapse risk of 76% vs 38.6% (P < .001). Patients with KMT2A::MLLT10 had an EFS from study entry of 19.5% vs 12.7% (P = .628), and an OS from study entry of 40.4% vs 27.6% (P = .361) in those with other MLLT10 fusion partners. Patients with PICALM::MLLT10 had an EFS of 9.2% vs 20% in other MLLT10- without PICALM (X::MLLT10; P = .788). Patients with PICALM::MLLT10 and X::MLLT10 fusions exhibit a DNA hypermethylation signature resembling NUP98::NSD1 fusions, whereas patients with KMT2A::MLLT10 bear aberrations primarily affecting distal regulatory elements. Regardless of the fusion partner, patients with AML harboring MLLT10 fusions exhibit very high-risk features and should be prioritized for alternative therapeutic interventions.

Nucleosome assembly protein 1-like 1 (NAP1L1) in gastric cancer patients: a potential biomarker with diagnostic and prognostic utility.

BACKGROUND: The nucleosome assembly protein 1-like 1 (NAP1L1) is suggested to have an oncogenic role in several tumors based on its overexpression. However, its diagnostic and prognostic role in gastric cancer remains unclarified. This study aimed to evaluate the diagnostic and prognostic utility of NAP1L1 in gastric cancer patients. METHODS: A total of 85 patients [mean (SD) age: 60.9 (1.6) years, 49.4% were males] with newly-diagnosed gastric cancer and 40 healthy individuals [mean (SD) age: 60.7 (1.7) years, 52.5% were males] were included. Data on patient demographics (age, gender), TNM stages and tumor size, and the serum NAP1L1 levels were recorded. RESULTS: Serum NAP1L1 levels were significantly higher in gastric cancer patients than in control subjects [12 (9.5-13.8) vs. 1.8 (1.5-2.4) ng/mL, p < 0.001]. Also, certain tumor characteristics such as tumor size of >4 vs. <4 cm (p < 0.001), M1 vs. M0 stage (p < 0.001), N2 vs. N0 and N1 stage (p < 0.001), and T4 vs. lower T stage (p < 0.001) were associated with significantly higher serum NAP1L1 levels in gastric cancer patients. CONCLUSIONS: Our findings revealed for the first time that serum levels for NAP1L1 were overexpressed in the gastric cancer, as also correlated with the disease progression. NAP1L1 seems to be a potential biomarker for gastric cancer, providing clinically important information on early diagnosis and risk stratification.

This study aimed to investigate serum levels for nucleosome assembly protein 1-like 1 (NAP1L1) in patients with gastric cancer in relation to healthy controls and tumor pathology.It was demonstrated for the first time that serum levels for NAP1L1 were overexpressed in the gastric cancer, as also correlated with the disease progression.These findings seem to implicate the potential role of serum NAP1L1 as a distinct diagnostic and prognostic factor in patients with gastric cancer, offering clinically important information on early diagnosis and risk stratification.

Molecular basis of RanGTP-activated release of Histones H2A-H2B from Importin-9.

Imp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP is insufficient to release H2A-H2B. The resulting stable RanGTP·Imp9·H2A-H2B complex gains nucleosome assembly activity with H2A-H2B able to be deposited into an assembling nucleosome in vitro. Using hydrogen-deuterium exchange coupled with mass spectrometry (HDX), we show that Imp9 stabilizes H2A-H2B beyond the direct-binding site, like other histone chaperones. HDX also shows that binding of RanGTP releases H2A-H2B contacts at Imp9 HEAT repeats 4-5, but not 18-19. DNA- and histone-binding surfaces of H2A-H2B are exposed in the ternary complex, facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. Imp9 thus provides a connection between the nuclear import of H2A-H2B and its deposition into chromatin.

Dynamic Solution Structures of Whole Human NAP1 Dimer Bound to One and Two Histone H2A-H2B Heterodimers Obtained by Integrative Methods.

Nucleosome assembly protein 1 (NAP1) binds to histone H2A-H2B heterodimers, mediating their deposition on and eviction from the nucleosome. Human NAP1 (hNAP1) consists of a dimerization core domain and intrinsically disordered C-terminal acidic domain (CTAD), both of which are essential for H2A-H2B binding. Several structures of NAP1 proteins bound to H2A-H2B exhibit binding polymorphisms of the core domain, but the distinct structural roles of the core and CTAD domains remain elusive. Here, we have examined dynamic structures of the full-length hNAP1 dimer bound to one and two H2A-H2B heterodimers by integrative methods. Nuclear magnetic resonance (NMR) spectroscopy of full-length hNAP1 showed CTAD binding to H2A-H2B. Atomic force microscopy revealed that hNAP1 forms oligomers of tandem repeated dimers; therefore, we generated a stable dimeric hNAP1 mutant exhibiting the same H2A-H2B binding affinity as wild-type hNAP1. Size exclusion chromatography (SEC), multi-angle light scattering (MALS) and small angle X-ray scattering (SAXS), followed by modelling and molecular dynamics simulations, have been used to reveal the stepwise dynamic complex structures of hNAP1 binding to one and two H2A-H2B heterodimers. The first H2A-H2B dimer binds mainly to the core domain of hNAP1, while the second H2A-H2B binds dynamically to both CTADs. Based on our findings, we present a model of the eviction of H2A-H2B from nucleosomes by NAP1.

Genome-Wide Identification and Functional Analysis of NAP1 in Triticum aestivum.

As a main molecular chaperone of histone H2A-H2B, nucleosome assembly protein 1 (NAP1) has been widely researched in many species. However, there is little research investigating the function of NAP1 in Triticum aestivum. To understand the capabilities of the family of NAP1 genes in wheat and the relationship between TaNAP1 genes and plant viruses, we performed comprehensive genome-wide analysis and quantitative real-time polymerase chain reaction (qRT-PCR) for testing expression profiling under hormonal and viral stresses. Our results showed that TaNAP1 was expressed at different levels in different tissues, with higher expression in tissues with high meristematic capacity, such as roots. Furthermore, the TaNAP1 family may participate in plant defense mechanisms. This study provides a systematic analysis of the NAP1 gene family in wheat and lays the foundation for further studies on the function of TaNAP1 in the response of wheat plants to viral infection.

Histone chaperone Nap1 dismantles an H2A/H2B dimer from a partially unwrapped nucleosome.

DNA translocases, such as RNA polymerases, inevitably collide with nucleosomes on eukaryotic chromatin. Upon these collisions, histone chaperones are suggested to facilitate nucleosome disassembly and re-assembly. In this study, by performing in vitro transcription assays and molecular simulations, we found that partial unwrapping of a nucleosome by an RNA polymerase dramatically facilitates an H2A/H2B dimer dismantling from the nucleosome by Nucleosome Assembly Protein 1 (Nap1). Furthermore, the results uncovered molecular mechanisms of Nap1 functions in which the highly acidic C-terminal flexible tails of Nap1 contribute to the H2A/H2B binding by associating with the binding interface buried and not accessible to Nap1 globular domains, supporting the penetrating fuzzy binding mechanism seemingly shared across various histone chaperones. These findings have broad implications for the mechanisms by which histone chaperones process nucleosomes upon collisions with translocases in transcription, histone recycling and nucleosomal DNA repair.

Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage.

STUDY QUESTION: Can whole exome sequencing (WES) followed by trio bioinformatics analysis identify novel pathogenic genetic causes of first trimester euploid miscarriage? SUMMARY ANSWER: We identified genetic variants in six candidate genes that indicated plausible underlying causes of first-trimester euploid miscarriage. WHAT IS KNOWN ALREADY: Previous studies have identified several monogenic causes of Mendelian inheritance in euploid miscarriages. However, most of these studies are without trio analyses and lack cellular and animal models to validate the functional effect of putative pathogenic variants. STUDY DESIGN, SIZE, DURATION: Eight unexplained recurrent miscarriage (URM) couples and corresponding euploid miscarriages were included in our study for whole genome sequencing (WGS) and WES followed by trio bioinformatics analysis. Knock-in mice with Rry2 and Plxnb2 variants and immortalized human trophoblasts were utilized for functional study. Additional 113 unexplained miscarriages were included to identify the mutation prevalence of specific genes by multiplex PCR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole blood from URM couples and their <13 weeks gestation miscarriage products were both collected for WES, and all variants in selected genes were verified by Sanger sequencing. Different stage C57BL/6J wild-type mouse embryos were collected for immunofluorescence. Ryr2N1552S/+, Ryr2R137W/+, Plxnb2D1577E/+, and Plxnb2R465Q/+ point mutation mice were generated and backcrossed. Matrigel-coated transwell invasion assays and wound-healing assays were performed using HTR-8/SVneo cells transfected with PLXNB2 small-interfering RNA and negative control. Multiplex PCR was performed focusing on RYR2 and PLXNB2. MAIN RESULTS AND THE ROLE OF CHANCE: Six novel candidate genes, including ATP2A2, NAP1L1, RYR2, NRK, PLXNB2, and SSPO, were identified. Immunofluorescence staining showed that ATP2A2, NAP1L1, RyR2, and PLXNB2 were widely expressed from the zygote to the blastocyst stage in mouse embryos. Although compound heterozygous mice with Rry2 and Plxnb2 variants did not show embryonic lethality, the number of pups per litter was significantly reduced when backcrossing Ryr2N1552S/+ ♂ with Ryr2R137W/+ ♀ or Plxnb2D1577E/+ ♂ with Plxnb2R465Q/+ ♀ (P < 0.05), which were in accordance with the sequencing results of Family 2 and Family 3, and the proportion of Ryr2N1552S/+ offspring was significantly lower when Ryr2N1552S/+ female mice were backcrossed with Ryr2R137W/+ male mice (P < 0.05). Moreover, siRNA-mediated PLXNB2 knockdown inhibited the migratory and invasive abilities of immortalized human trophoblasts. Besides, additional 10 variants of RYR2 and PLXNB2 were detected in 113 unexplained euploid miscarriages by multiplex PCR. LIMITATIONS, REASONS FOR CAUTION: The relatively small number of samples is a limitation of our study which may result in the identification of variants in unique candidate genes with no definitive although plausible causal effect. Larger cohorts are needed to replicate these findings and additional functional research is needed to confirm the pathogenic effects of these variants. Moreover, the sequencing coverage restricted the detection of low-level parental mosaic variants. WIDER IMPLICATIONS OF THE FINDINGS: For first-trimester euploid miscarriage, variants in unique genes may be underlying genetic etiologies and WES on trio could be an ideal model to identify potential genetic causes, which could facilitate individualized precise diagnostic and therapeutic regimens in the future. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700604), National Natural Science Foundation of China (31900492, 82101784, 82171648), Basic Science Center Program of the National Natural Science Foundation of China (31988101), Key Research and Development Program of Shandong Province (2021LCZX02), Natural Science Foundation of Shandong Province (ZR2020QH051), Natural Science Foundation of Jiangsu Province (BK20200223), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201812154) and Young Scholars Program of Shandong University. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

A New Role of NAP1L1 in Megakaryocytes and Human Platelets.

Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD34+-derived megakaryocytes (MKs). NAP1L1 is expressed in PLTs and MKs. Co-IP experiments revealed that dihydrolipolylysine-residue acetyltransferase (DLAT encoded protein PDC-E2, ODP2) dynamically interacts with NAP1L1. PDC-E2 is part of the mitochondrial pyruvate-dehydrogenase (PDH) multi-enzyme complex, playing a crucial role in maintaining cellular respiration, and promoting ATP-synthesis via the respiratory chain. Since altered mitochondrial function is a hallmark of infectious syndromes, we analyzed PDH activity in PLTs from septic patients demonstrating increased activity, paralleling NAP1L1 expression levels. MKs PDH activity decreased following an LPS-challenge. Furthermore, overexpression of NAP1L1 significantly altered the ability of MKs to form proplatelet extensions, diminishing thrombopoiesis. These results indicate that NAP1L1 performs in other than nucleosome-assembly functions in PTLs and MKs, binding a key mitochondrial protein as a potential chaperone, and gatekeeper, influencing PDH activity and thrombopoiesis.

Mechanism of client selection by the protein quality-control factor UBE2O.

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.

Nucleosome Assembly Protein 1, Nap1, Is Required for the Growth, Development, and Pathogenicity of Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of rice blast, leading to significant reductions in rice and wheat productivity. Nap1 is a conserved protein in eukaryotes involved in diverse physiological processes, such as nucleosome assembly, histone shuttling between the nucleus and cytoplasm, transcriptional regulation, and the cell cycle. Here, we identified Nap1 and characterized its roles in fungal development and virulence in M. oryzae. MoNap1 is involved in aerial hyphal and conidiophore differentiation, sporulation, appressorium formation, plant penetration, and virulence. ΔMonap1 generated a small, elongated, and malformed appressorium with an abnormally organized septin ring on hydrophobic surfaces. ΔMonap1 was more sensitive to cell wall integrity stresses but more resistant to microtubule stresses. MoNap1 interacted with histones H2A and H2B and the B-type cyclin (Cyc1). Moreover, a nuclear export signal (NES) domain is necessary for Nap1's roles in the regulation of the growth and pathogenicity of M. oryzae. In summary, NAP1 is essential for the growth, appressorium formation, and pathogenicity of M. oryzae.

Genome-Wide Identification of NAP1 and Function Analysis in Moso Bamboo (Phyllostachys edulis).

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 (Phyllostachys edulis), characterized by extremely rapid growth and a long flowering cycle, we originally conducted a genome-wide analysis of the PheNAP1 gene. The phylogenetic relationship, gene expression pattern, DNA methylation, and histone modification were analyzed. Eventually, 12 PheNAP1 genes were recognized from the Phyllostachys edulis genome, divided into two sorts: the NRP subfamily (four members) and the NAP subfamily (eight members). Highly conserved motifs exist in each subfamily, which are distinct between subfamilies. PheNAP1 was distributed homogeneously on 10 out of 24 chromosomes, and gene duplication contributed significantly to the enhancement of the PheNAP1 gene in the genome. Cis-acting element analysis showed that PheNAP1 family genes are involved in light, hormone, and abiotic stress responses and may play an important role in the rapid growth and flowering. PheNAP1 exhibited the highest expression level in fast-growing shoots, indicating it is closely associated with the rapid growth of moso bamboo. Besides, PheNAP1 can rescue the early-flowering phenotype of nrp1-1 nrp2-2, and it affected the expression of genes related to the flowering pathway, like BSU1, suggesting the vital role that PheNAP1 may take in the flowering process of moso bamboo. In addition, histone modification results showed that PheNAP1 could bind to phosphorylation-, acetylation-, and methylation-modified histones to further regulate gene expression. A sketch appears: that PheNAP1 can accompany histones to regulate fast-growth- and flowering-related genes in moso bamboo. The consequences of this study enrich the understanding of the epigenetic regulation mechanism of bamboo plants and lays a foundation for further studies on the role of the NAP1 gene in Phyllostachys edulis and the function of chromatin regulation in forest growth and development.

Alternative polyadenylation mechanism links secreted phosphoprotein 1 gene to glioblastoma.

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.

NAP1L1 promotes tumor proliferation through HDGF/C-JUN signaling in ovarian cancer.

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.

Trametes robiniophila represses angiogenesis and tumor growth of lung cancer via strengthening let-7d-5p and targeting NAP1L1.

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 is a regulator of histone H1 acetylation.

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. Graphical Abstract.

NAP1L1 promotes proliferation and chemoresistance in glioma by inducing CCND1/CDK4/CDK6 expression through its interaction with HDGF and activation of c-Jun.

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.

NAP1L1 targeting suppresses the proliferation of nasopharyngeal carcinoma.

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.

NAP1L1 and NAP1L4 Binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein Is Bivalent and Requires Phosphorylation.

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 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, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCE Cellular 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 show 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.

Nucleosome assembly protein 1-like 1 (NAP1L1) in colon cancer patients: a potential biomarker with diagnostic and prognostic utility.

OBJECTIVE: To evaluate the diagnostic and prognostic utility of nucleosome assembly protein 1-like 1 (NAP1L1) in colon cancer patients. PATIENTS AND METHODS: 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. RESULTS: 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, p<0.001]. In colon cancer patients, serum NAP1L1 levels were significantly higher for tumor size of >4 cm vs. <4 cm [15(12-16) vs. 12(12-14) ng/mL), p<0.001] and for M1 vs. M0 stage [15(12-16) vs. 12(12-14) ng/mL), p<0.001]. Serum NAP1L1 levels were significantly higher in T4 stage tumors vs. T1, T2 and T3 stage tumors (p<0.001 for each), in T3 stage tumors vs. T1 and T2 stage tumors (p<0.001 for each) and in N2 stage tumors vs. N0 and N1 stage tumors (p<0.001 for each). CONCLUSIONS: 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.

NAP1-Related Protein 1 (NRP1) has multiple interaction modes for chaperoning histones H2A-H2B.

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.