Abstract Study question Whether and how METTL5 regulates germ cell development during spermatogenesis. Summary answer METTL5 is required for spermiogenesis. Loss of METTL5 resulted in teratozoospermia and male infertility via reduced translation of acrosome and flagellum formation proteins. What is known already The roles of m6A modifications on mRNA in spermatogenesis have been extensively studied. It was reported that METTL5 knockout mice showed the brain development defect and sterility of 16-week male mice. However, the detailed mechanism of METTL5 affecting male fertility remains elusive. Study design, size, duration Mettl5 KO mice were kindly gifted from Prof. Shuibin Lin of The First Affiliated Hospital of Sun Yat-sen University. Heterozygotes of Mettl5 mice were used to generate Mettl5 homozygous knockout mice. The phenotype of KO mice was assessed. Ribosomal sequencing, proteomics analysis and further validation of protein translation were performed to explore the mechanism. Participants/materials, setting, methods Fertility assessment, sperm parameter analyses, sperm nuclear morphology analysis, Transmission electron microscopy (TEM), tissue Collection and histological analysis, protein extraction and western blot analysis, immunofluorescence studies, cDNA synthesis and qRT-PCR, in vitro fertilization (IVF) were used in this study. Main results and the role of chance Here we reported that methyltransferase-like 5 (METTL5) is involved in spermiogenesis as a methyltransferase mediating m6A modification on rRNA. Mettl5 knockout mice were infertile with teratozoospermia. The acrosome in the sperm head was absent with reduced sperm motility. Furthermore, the fertilization ability of sperm in the IVF experiment failed. Mechanistically, the level of rRNA m6A modification was significantly decreased in the testes of Mettl5 KO mice. The translational efficiency and protein levels of acrosome and flagellum formation proteins such as FSIP2, ODF2, GK2, PGK2, and AKAP4 were significantly reduced when METTL5 was depleted. Limitations, reasons for caution The METTL5 mutation in the patient with teratozoospermia was not examined in the present study Wider implications of the findings The rRNA m6A modification is also involved in regulating spermatogenesis by METTL5. This study highlights the critical role of rRNA epigenetic modifications during spermatogenesis and provides novel theoretical explanations for the m6A modifications. Trial registration number Not applicable
Silver staining is an excellent technique for detecting proteins that are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein silver staining technology has higher sensitivity and is suitable for the detection of low-concentration proteins compared to other staining techniques including the Coomassie brilliant blue detection method. The present study was conducted to enhance the detection ability of the protein staining method. Herein, we modified the recipe of silver staining, a very reproducible method, by adding AMP, PVP, Tween-80, and xylene to enhance the detection ability of protein staining. Furthermore, the particle size and potentiometer were used to detect the particle size and potential difference of the silver ions in the prepared dyeing materials, and then, the morphology, transparency, and size of the dyed silver particles in different dyeing solutions were studied using a transmission electron microscopy (TEM). The obtained results revealed that the use of 0.5% of AMP, PVP, Tween-80, and xylene improved the staining ability of protein silver staining, compared with the original method. Furthermore, 0.5% AMP, 0.5% PVP, 0.5% Tween-80 reagents significantly influenced the morphology, size, potential, and dispersion of silver ions. These results suggested a new idea for further improving the detection ability of protein silver staining.
Significance The AE9a protein (alternative splicing at exon 9) is often used to model t(8;21) leukemia. Our study demonstrates that increased oncogene dosage is a critical parameter of AE9a transformation, likely as a result of impaired transcriptional regulation of AML1-ETO target genes. This insight could assist in identifying those downstream genes most critical for t(8;21)-associated transformation.
Abstract The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is fused by reciprocal translocation to a diverse group of partner genes in both acute myeloid and acute lymphoid leukemia (AML, ALL). As a result of the t(4;11)(q21;q23), MLL fuses to AF4 (AFF1), one of the most common MLL fusion partner proteins. Unlike several other MLL fusions that are frequently found in both AML and ALL, MLL-AF4 is almost exclusively associated with B-cell ALL with a pro-B immunophenotype. It accounts for 10-15% of ALL cases and confers a poor prognosis. Although many MLL-fusion leukemia models have been established, it has not been possible to generate a t(4;11) pro-B leukemia model that accurately recapitulates the human disease, hampering research into the molecular mechanisms that underlie the development of this subtype of leukemia. Here we present a faithful model of t(4;11) pro-B-ALL that fully recapitulates the immunophenotypic and molecular aspects of the human disease. Human hematopoietic CD34+ cells transduced with a modified MLL-AF4 fusion gene successfully initiate ALL in NSG mice with high penetrance. The leukemia cells have a CD19+CD34+ pro-B immunophenotype and are CD10(-), a common feature in MLL-AF4 patients. The effect of the oncogene is species-specific, as retroviral transduction and transplantation of murine hematopoietic cells results in only AML using either lymphoid or myeloid conditions. An MLL-AF4 specific gene signature derived from patients is significantly enriched in our model cells, as shown by RNA-Seq, and the model samples group tightly with MLL-AF4 patients, even when compared with other MLL-fusion leukemia samples in unsupervised hierarchical clustering analysis. Interestingly, using gene profiles of normal pro-B and pre-B cells as reference, our MLL-AF4 leukemia cells show strong enrichment for pro-B genes, while instead, pre-B but not pro-B genes are overrepresented in our MLL-AF9 B-ALL leukemia cells. This differential developmental stage blockage of MLL-fusions is also reflected in patient samples. More strikingly, in accordance with the distinct lineage bias of MLL-fusions observed in the clinic, human cells expressing MLL-AF4 have a strong predilection for the lymphoid lineage and a demonstrated resistance to lineage redirection in response to myeloid signals compared to human cells expressing MLL-AF9. This difference in lineage predisposition of MLL-AF4 compared to MLL-AF9 can be attributed to differential effects on lineage-specific gene expression. Under myeloid-priming conditions, phenotypically (CD33+CD19-) and morphologically myeloid MLL-AF4 cells are still able to initiate pro-B ALL in immunodeficient mice, while only AML is generated by MLL-AF9 myeloid cells. Accordingly, an active lymphoid molecular program with lower expression of critical myeloid genes is observed in MLL-AF4 myeloid cells compared to MLL-AF9 myeloid cells. Interestingly, we find that the polycomb gene BMI1, which was reported to be critical to prevent lymphoid priming in normal hematopoiesis, is expressed at significantly lower levels in MLL-AF4 than in MLL-AF9 myeloid cells. Reintroduction of BMI1 into MLL-AF4 cells enables AML generation with variable penetrance, while control vector transduced cells always result in B-ALL. Our results demonstrate that lineage fate in response to MLL-fusion protein expression involves a complex interplay of oncogene, intra- and extra-cellular microenvironmental cues. In addition, our data clearly demonstrate the species specificity associated with the t(4;11). This in vivo model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis. This abstract is also presented as Poster A06. Citation Format: Shan Lin, Roger T. Luo, Mark Wunderlich, Joseph J. Kaberlein, Ahmad Rayes, John Anastasi, Maureen M. O'Brien, James C. Mulloy, Michael J. Thirman. A novel MLL-AF4 in vivo model phenocopies t(4;11) pro-B ALL and reveals a lymphoid lineage bias of the fusion protein in human cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR01.
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