Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells.

Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs, and Prms. Here, by studying spermatogenesis in the absence of a spermatid-specific histone variant, H2A.L.2, we discover a fundamental mechanism involved in the transformation of nucleosomes into nucleoprotamines. H2A.L.2 is synthesized at the same time as TPs and enables their loading onto the nucleosomes. TPs do not displace histones but rather drive the recruitment and processing of Prms, which are themselves responsible for histone eviction. Altogether, the incorporation of H2A.L.2 initiates and orchestrates a series of successive transitional states that ultimately shift to the fully compacted genome of the mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should be revisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms.

NPM3 as a novel oncogenic factor and poor prognostic marker contributes to cell proliferation and migration in lung adenocarcinoma.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide, and despite recent advances in targeted therapies and immunotherapies, the clinical benefit remains limited. Therefore, there is an urgent need to further investigate the molecular mechanisms underlying lung cancer. The aim of this study was to investigate the expression and function of NPM3 in the tumor microenvironment of lung adenocarcinoma (LUAD). METHODS: We utilized bioinformatics tools and databases, including UALCAN, GEPIA2, HPA, and Sangerbox, to analyze NPM3 expression in LUAD samples and its association with prognosis and mutational landscape. NPM3 expression in various cell types was assessed at the single cell level using the TISCH database. We also used algorithms such as TIMER and EPIC to explore the crosstalk between NPM3 expression and immune features. KEGG enrichment analysis was performed to identify potential signaling pathways of NPM3. Finally, we employed siRNA knockdown strategy to investigate the effect of NPM3 on LUAD cell proliferation and migration in vitro. RESULTS: NPM3 was significantly upregulated in LUAD tissues and was strongly associated with poor prognosis and TP53 gene mutations. Single-cell sequencing analysis revealed that NPM3 was expressed in immune cells (dendritic cells and monocytes/macrophages) in the tumor microenvironment. Moreover, NPM3 expression was negatively associated with immune B cell and CD4 T cell infiltration, as well as with several immune-related genes (including CCL22, CXCR2, CX3CR1, CCR6, HLA-DOA, HLA-DQA2). KEGG enrichment analysis indicated that NPM3 expression was associated with cell cycle, CAMs, and NSCLC pathway genes. Finally, in vitro experiments showed that NPM3 knockdown inhibited LUAD cell proliferation and migration in NCI-H1299 and SPC-A1 cells, and suppressed the expression of CCNA2 and MAD2L1. CONCLUSION: Elevated NPM3 expression predicts poor clinical outcome and an immunosuppressive microenvironment in LUAD tissues. NPM3 promotes LUAD progression by promoting cell proliferation and migration, and targeting NPM3 may represent a novel therapeutic strategy for LUAD.

Nucleophosmin3 carried by small extracellular vesicles contribute to white adipose tissue browning.

BACKGROUND: Browning of white adipose tissue (WAT) is a particularly appealing target for therapeutics in the treatment of obesity and related metabolic diseases. Although small extracellular vesicles (sEVs) released from adipose tissue (sEVs-AT) have emerged as novel player that regulate systemic metabolism by connecting different organs, the role of specific contents in sEVs-AT played in WAT browning has not been clarified. RESULTS: We revealed Nucleophosmin3 (NPM3), which was mainly transferred by sEVs derived from brown adipose tissue (sEVs-BAT), was served as a batokine that could induce WAT browning by regulating the stability of PRDM16 mRNA. sEVs-BAT enhanced the expressions of browning related genes in 3T3-L1 preadipocytes and WAT while knocking down of NPM3 in BAT impaired sEVs-BAT mediated WAT browning and weight loss in obesity. CONCLUSION: These data provided new insight into the role of NPM3 in regulating the browning of WAT. Our study indicated that a supplement of sEVs-BAT might represent a promising therapeutic strategy to promote thermogenesis and energy expenditure in the future.

Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.

Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

Characterization of the AGR2-NPM3 axis uncovers the AGR2 involvement in PD-L1 regulation in colorectal cancer.

Despite extensive research, the molecular role of AGR2 in the progression and metastasis of colorectal cancer (CRC) has not been fully characterized. We used quantitative mass spectrometry (SWATH MS) to identify differentially expressed proteins in paired CRC cell models of the SW480 and SW620 cell lines in response to AGR2 protein level manipulation. Relying on the results from SWATH MS and subsequent immunochemical validation, we selected NMP3 as the top candidate protein associated with AGR2 in CRC tumour cells in our screen. RT‒qPCR and immunochemical analysis confirmed the involvement of AGR2-mediated regulation of NPM3 at the transcriptional and posttranscriptional levels. Since PD-L1 is a constituent of the NPM3 regulatory axis, we aimed to correlate the changes in PD-L1 to the differential expression of AGR2 in our cell models. We found that AGR2 positively regulates PD-L1 levels in both SW480 and SW620 cell lines; additionally, several different CRC patient transcriptome cohorts confirmed the association of AGR2 with PD-L1. Our work reveals a new AGR2-NPM3 regulatory axis and the involvement of AGR2 in the regulation of PD-L1, which paves the way for the association of AGR2 with immune evasion in CRC cells.

Pumilio1 regulates NPM3/NPM1 axis to promote PD-L1-mediated immune escape in gastric cancer.

Abnormal regulation of RNA binding proteins (RBPs) plays an essential role in tumorigenesis and progression, but their functions and mechanisms remain largely elusive. Previously, we reported that Pumilio 1 (PUM1), a RBP, could regulate glycolysis metabolism and promote the progression of gastric cancer (GC). However, the role of PUM1 in tumor immune regulation remains largely elusive. In this study, we report that PUM1 induces immune escape through posttranscriptional regulation of PD-L1 in GC. We used multiplexed immunohistochemistry to analyze the correlation between PUM1 expression and immune microenvironment in GC. The effect of PUM1 deficiency on tumor killing of T cells was examined in vitro and in vivo. The molecular mechanism of PUM1 was evaluated via RNA immunoprecipitation, chromatin immunoprecipitation, Western blot, co-immunoprecipitation, and RNA stability assays. Clinically, elevated PUM1 expression is associated with high-expression of PD-L1, lack of CD8+ T cell infiltration and poor prognosis in GC patients. PUM1 positively regulates PD-L1 expression and PUM1 reduction enhances T cell killing of tumors. Mechanistically, PUM1 directly binds to nucleophosmin/nucleoplasmin 3 (NPM3) mRNA and stabilizes NPM3. NPM3 interacts with NPM1 to promote NPM1 translocation into the nucleus and increase the transcription of PD-L1. PUM1 inhibits the anti-tumor activity of T cells through the PUM1/NPM3/PD-L1 axis. In summary, this study reveals the critical post-transcriptional effect of PUM1 in the modulation of PD-L1-dependent GC immune escape, thus provides a novel indicator and potential therapeutic target for cancer immunotherapy.

NPM3 as an unfavorable prognostic biomarker involved in oncogenic pathways of lung adenocarcinoma via MYC translational activation.

BACKGROUND: The nucleoplasmin/nucleophosmin (NPM) family was previously regarded as a critical regulator during disease development, and its mediation in carcinogenesis has achieved intensive attention recently. However, the clinical importance and functional mechanism of NPM3 in lung adenocarcinoma (LUAD) have not been reported yet. OBJECTIVE: This study aimed to investigate the role and clinical significance of NPM3 in the development and progression of LUAD, including the underlying mechanisms. METHOD: The expression of NPM3 in pan-cancer was analyzed via GEPIA. The effect of NPM3 on prognosis was analyzed by the Kaplan-Meier plotter and the PrognoScan database. In vitro, cell transfection, RT-qPCR, CCK-8 assay, and wound healing assay were employed to examine the role of NPM3 in A549 and H1299 cells. Gene set enrichment analysis (GSEA) was performed using the R software package to analyze the tumor hallmark pathway and KEGG pathway of NPM3. The transcription factors of NPM3 were predicted based on the ChIP-Atlas database. Dual-luciferase reporter assay was applied to verify the transcriptional regulatory factor of the NPM3 promoter region. RESULTS: The NPM3 expression was found to be markedly higher in the LUAD tumor group than the normal group and to be positively correlated with poor prognosis, tumor stages, and radiation therapy. In vitro, the knockdown of NPM3 greatly inhibited the proliferation and migration of A549 and H1299 cells. Mechanistically, GSEA predicted that NPM3 activated the oncogenic pathways. Further, the NPM3 expression was found to be positively correlated with cell cycle, DNA replication, G2M checkpoint, HYPOXIA, MTORC1 signaling, glycolysis, and MYC targets. Besides, MYC targeted the promoter region of NPM3 and contributed to the enhanced expression of NPM3 in LUAD. CONCLUSION: The overexpression of NPM3 is an unfavorable prognostic biomarker participating in oncogenic pathways of LUAD via MYC translational activation and it contributes to tumor progression. Thus, NPM3 could be a novel target for LUAD therapy.

Pan-cancer analysis of the prognostic and immunological role of nucleophosmin/nucleoplasmin 3 (NPM3) and its potential significance in lung adenocarcinoma.

Background: Nucleophosmin/nucleoplasmin 3 (NPM3), a member of the NPM protein family, is widely expressed in various human tissues. Although previous studies identified elevated NPM3 expression in several cancers, a systematic pan-cancer analysis remains lacking. In this study, we conducted a comprehensive analysis of NPM3 to determine its role in tumorigenesis and tumor development. Methods: Using data from The Cancer Genome Atlas (TCGA) and various bioinformatics analysis tools, we conducted a pan-cancer analysis of NPM3. Additionally, we collected gene expression and clinical data from 890 patients with lung adenocarcinoma (LUAD) from TCGA and the Gene Expression Omnibus database. We performed Cox regression analyses to explore the independent prognostic value of NPM3 expression in LUAD and plotted a nomogram to predict patient survival. We also used real-time quantitative polymerase chain reaction (RT-qPCR) to examine the expression levels of NPM3 in seven pairs of LUAD and paraneoplastic tissue samples. Results: NPM3 expression was significantly increased in 20 types of cancer and was associated with poor prognosis in five types (P < 0.05). NPM3 expression was negatively correlated with DNA methylation and positively correlated with copy number variation. NPM3 was also significantly associated with immune cell infiltration in various cancers. Cox regression analyses revealed that NPM3 expression could serve as an independent prognostic marker of LUAD. Moreover, our nomogram demonstrated good predictive ability for the prognosis of patients with LUAD. Finally, the high expression of NPM3 in LUAD was verified using RT-qPCR. Conclusion: NPM3 is a promising biomarker for predicting pan-cancer prognosis and immunotherapeutic efficacy.