RNF187 Facilitates Proliferation and Migration of Human Spermatogonial Stem Cells Through WDR77 Polyubiquitination

Abstract

The E3 ubiquitin ligase RNF187, also known as RING domain AP1 coactivator-1, is a member of the RING finger family. RNF187 is indispensable for the proliferation and migration of GC-1 cells derived from mouse spermatogonia and GC-2 cells derived from spermatocytes. However, it remains unclear whether RNF187 plays a crucial role in the self-renewal and migration of human spermatogonial stem cells (SSCs). In this study, we observed a positive correlation between RNF187 expression and the proliferation and migration of human SSCs. Through co-immunoprecipitation and mass spectrometry analyses, we identified WD repeat-containing protein 77 (WDR77) as an interacting partner of RNF187. Specifically, RNF187 recognises the K118 site of WDR77 through lysine 48-linked polyubiquitination, subsequently mediating its degradation via the ubiquitin-proteasome system (UPS). Further studies have revealed that decreased expression of WDR77 diminishes the symmetric dimethylation at H4R3 (H4R3me2s) catalysed by its interacting protein, the arginine methyltransferase PRMT5. This, in turn, relieves the transcriptional repression of early growth response protein 1 (EGR1), a positive regulator for human SSC maintenance. In conclusion, this study has unveiled a pivotal role for RNF187 in the proliferation and migration of human SSCs. This may provide a promising strategy for addressing non-obstructive azoospermia (NOA) caused by SSC dysfunction.

Keywords

EGR1 / RNF187 / spermatogonial stem cells / ubiquitin / WDR77

References

[1] A. Piechka, S. Sparanese, L. Witherspoon, F. Hach, and R. Flannigan, “Molecular Mechanisms of Cellular Dysfunction in Testes From Men With Non-obstructive Azoospermia,” Nature Reviews. Urology 21, no. 2 (2024): 67-90.

[2] N. E. Abdelaal, B. M. Tanga, M. Abdelgawad, et al., “Cellular Therapy via Spermatogonial Stem Cells for Treating Impaired Spermatogenesis, Non-Obstructive Azoospermia,” Cells 10, no. 7 (2021): 1779.

[3] H. Wei, Z. Wang, Y. Huang, et al., “DCAF2 Regulates the Proliferation and Differentiation of Mouse Progenitor Spermatogonia by Targeting p21 and Thymine DNA Glycosylase,” Cell Proliferation 57, no. 10 (2024): e13676.

[4] J. Zhao, K. Tang, G. Jiang, et al., “Dynamic Transcriptomic and Regulatory Networks Underpinning the Transition From Fetal Primordial Germ Cells to Spermatogonia in Mice,” Cell Proliferation 58, no. 2 (2024): e13755, https://doi.org/10.1111/cpr.13755.

[5] Z. Lin, P. J. Hsu, X. Xing, et al., “Mettl3−/Mettl14-Mediated mRNA N(6)-methyladenosine Modulates Murine Spermatogenesis,” Cell Research 27, no. 10 (2017): 1216-1230.

[6] L. Sun, Z. Lv, X. Chen, et al., “Splicing Factor SRSF1 Is Essential for Homing of Precursor Spermatogonial Stem Cells in Mice,” eLife 12 (2024): 12.

[7] Y. Yin, S. Cao, H. Fu, et al., “A Noncanonical Role of NOD-Like Receptor NLRP14 in PGCLC Differentiation and Spermatogenesis,” Proceedings of the National Academy of Sciences of the United States of America 117, no. 36 (2020): 22237-22248.

[8] S. J. van Wijk, S. Fulda, I. Dikic, and M. Heilemann, “Visualizing ubiquitination in mammalian cells,” EMBO Reports 20, no. 2 (2019): e46520, https://doi.org/10.15252/embr.201846520.

[9] D. Popovic, D. Vucic, and I. Dikic, “Ubiquitination in Disease Pathogenesis and Treatment,” Nature Medicine 20, no. 11 (2014): 1242-1253.

[10] N. Zheng and N. Shabek, “Ubiquitin Ligases: Structure, Function, and Regulation,” Annual Review of Biochemistry 86 (2017): 129-157.

[11] C. M. Pickart, “Mechanisms Underlying Ubiquitination,” Annual Review of Biochemistry 70 (2001): 503-533.

[12] G. Çetin, S. Klafack, M. Studencka-Turski, E. Krüger, and F. Ebstein, “The Ubiquitin-Proteasome System in Immune Cells,” Biomolecules 11, no. 1 (2021): 60.

[13] D. Han, L. Wang, S. Jiang, and Q. Yang, “The Ubiquitin-Proteasome System in Breast Cancer,” Trends in Molecular Medicine 29, no. 8 (2023): 599-621.

[14] N. Li, Q. Zhou, Z. Yi, H. Zhang, and D. Zhou, “Ubiquitin Protein E3 Ligase ASB9 Suppresses Proliferation and Promotes Apoptosis in Human Spermatogonial Stem Cell Line by Inducing HIF1AN Degradation,” Biological Research 56, no. 1 (2023): 4.

[15] A. Chakraborty, M. E. Diefenbacher, A. Mylona, O. Kassel, and A. Behrens, “The E3 Ubiquitin Ligase Trim7 Mediates c-Jun/AP-1 Activation by Ras Signalling,” Nature Communications 6 (2015): 6782.

[16] C. C. Davies, A. Chakraborty, M. E. Diefenbacher, M. Skehel, and A. Behrens, “Arginine Methylation of the c-Jun Coactivator RACO-1 Is Required for c-Jun/AP-1 Activation,” EMBO Journal 32, no. 11 (2013): 1556-1567.

[17] L. Zhang, J. Chen, J. Yong, L. Qiao, L. Xu, and C. Liu, “An Essential Role of RNF187 in Notch1 Mediated Metastasis of Hepatocellular Carcinoma,” Journal of Experimental & Clinical Cancer Research 38, no. 1 (2019): 384.

[18] S. L. Yu, J. C. Wu, P. F. Liu, et al., “Up-Regulation of RNF187 Induces Hepatocellular Carcinoma Cell Epithelial to Mesenchymal Transitions,” Oncotarget 8, no. 60 (2017): 101876-101886.

[19] Z. Fu, W. Yu, H. Wang, and X. Chen, “Overexpression of RNF187 Induces Cell EMT and Apoptosis Resistance in NSCLC,” Journal of Cellular Physiology 234, no. 8 (2019): 14161-14169.

[20] W. B. Wan, K. Wu, K. Peng, et al., “High Level of RNF187 Contributes to the Progression and Drug Resistance of Osteosarcoma,” Journal of Cancer 11, no. 6 (2020): 1351-1358.

[21] Z. Wang, Q. Kong, P. Su, et al., “Regulation of Hippo Signaling and Triple Negative Breast Cancer Progression by an Ubiquitin Ligase RNF187,” Oncogene 9, no. 3 (2020): 36.

[22] M. Rengasamy, F. Zhang, A. Vashisht, et al., “The PRMT5/WDR77 Complex Regulates Alternative Splicing Through ZNF326 in Breast Cancer,” Nucleic Acids Research 45, no. 19 (2017): 11106-11120.

[23] X. Lu, C. Zhang, L. Zhu, et al., “TBL2 Promotes Tumorigenesis via PRMT5/WDR77-Mediated AKT Activation in Breast Cancer,” Adv Sci (Weinh) 11, no. 47 (2024): e2400160.

[24] Z. Gu, F. Zhang, Z. Q. Wang, W. Ma, R. E. Davis, and Z. Wang, “The p44/wdr77-Dependent Cellular Proliferation Process During Lung Development Is Reactivated in Lung Cancer,” Oncogene 32, no. 15 (2013): 1888-1900.

[25] H. Liang, M. L. Fisher, C. Wu, C. Ballon, X. Sun, and A. A. Mills, “PRMT5/WDR77 Enhances the Proliferation of Squamous Cell Carcinoma via the ΔNp63α-p21 Axis,” Cancers (Basel) 16, no. 22 (2024): 3789.

[26] H. Yuan, L. Zhao, Y. Yuan, et al., “HBx Represses WDR77 to Enhance HBV Replication by DDB1-Mediated WDR77 Degradation in the Liver,” Theranostics 11, no. 17 (2021): 8362-8378.

[27] J. J. Liang, Z. Wang, L. Chiriboga, et al., “The Expression and Function of Androgen Receptor Coactivator p44 and Protein Arginine Methyltransferase 5 in the Developing Testis and Testicular Tumors,” Journal of Urology 177, no. 5 (2007): 1918-1922.

[28] M. M. Roshan, H. Azizi, M. A. Majelan, and A. N. Tabar, “Sox9 Downregulation in Non-obstructive Azoospermia by UTF1 and Mediator Role of POU5F1,” BMC Research Notes 17, no. 1 (2024): 77.

[29] Y. Zheng, L. J. Phillips, R. Hartman, J. An, and C. T. Dann, “Ectopic POU5F1 in the Male Germ Lineage Disrupts Differentiation and Spermatogenesis in Mice,” Reproduction 152, no. 4 (2016): 363-377.

[30] Y. Zheng, A. Thomas, C. M. Schmidt, and C. T. Dann, “Quantitative Detection of Human Spermatogonia for Optimization of Spermatogonial Stem Cell Culture,” Human Reproduction 29, no. 11 (2014): 2497-2511.

[31] N. Kossack, N. Terwort, J. Wistuba, et al., “A Combined Approach Facilitates the Reliable Detection of Human Spermatogonia In Vitro,” Human Reproduction 28, no. 11 (2013): 3012-3025, https://doi.org/10.1093/humrep/det336.

[32] X. Yu, B. Xu, T. Gao, et al., “E3 Ubiquitin Ligase RNF187 Promotes Growth of Spermatogonia via Lysine 48-Linked Polyubiquitination-Mediated Degradation of KRT36/KRT84,” FASEB Journal 37, no. 10 (2023): e23217.

[33] B. Y. Xu, X. L. Yu, W. X. Gao, et al., “RNF187 Governs the Maintenance of Mouse GC-2 Cell Development by Facilitating Histone H3 Ubiquitination at K57/80,” Asian Journal of Andrology 26, no. 3 (2024): 272-281.

[34] Z. Xu, C. Lv, J. Gao, et al., “LncRNA ACVR2B-as1 Interacts With ALDOA to Regulate the Self-Renewal and Apoptosis of Human Spermatogonial Stem Cells by Controlling Glycolysis Activity,” Cellular and Molecular Life Sciences 81, no. 1 (2024): 391.

[35] J. Gao, Z. Xu, W. Song, et al., “USP11 Regulates Proliferation and Apoptosis of Human Spermatogonial Stem Cells via HOXC5-Mediated Canonical WNT/β-Catenin Signaling Pathway,” Cellular and Molecular Life Sciences 81, no. 1 (2024): 211.

[36] J. Yu, C. Shen, M. Lin, et al., “BMI1 Promotes Spermatogonial Stem Cell Maintenance by Epigenetically Repressing Wnt10b/β-Catenin Signaling,” International Journal of Biological Sciences 18, no. 7 (2022): 2807-2820.

[37] H. Zhou, C. Shen, Y. Guo, X. Huang, B. Zheng, and Y. Wu, “The Plasminogen Receptor Directs Maintenance of Spermatogonial Stem Cells by Targeting BMI1,” Molecular Biology Reports 49, no. 6 (2022): 4469-4478.

[38] Y. Liu, X. Yu, A. Huang, et al., “INTS7-ABCD3 Interaction Stimulates the Proliferation and Osteoblastic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells by Suppressing Oxidative Stress,” Frontiers in Physiology 12 (2021): 758607.

[39] K. Zhang, J. Xu, Y. Ding, et al., “BMI1 Promotes Spermatogonia Proliferation Through Epigenetic Repression of Ptprm,” Biochemical and Biophysical Research Communications 583 (2021): 169-177.

[40] J. Yu, Y. Wu, H. Li, et al., “BMI1 Drives Steroidogenesis Through Epigenetically Repressing the p38 MAPK Pathway,” Frontiers in Cell and Development Biology 9 (2021): 665089.

[41] S. K. Subash and P. G. Kumar, “Spermatogonial Stem Cells: A Story of Self-Renewal and Differentiation,” Front Biosci (Landmark Ed) 26, no. 1 (2021): 163-205.

[42] L. Simon, G. C. Ekman, T. Garcia, et al., “ETV5 Regulates Sertoli Cell Chemokines Involved in Mouse Stem/Progenitor Spermatogonia Maintenance,” Stem Cells 28, no. 10 (2010): 1882-1892, https://doi.org/10.1002/stem.508.

[43] M. Tracz and W. Bialek, “Beyond K48 and K63: Non-canonical Protein Ubiquitination,” Cellular & Molecular Biology Letters 26, no. 1 (2021): 1.

[44] C. Wang, X. Tan, D. Tang, et al., “GPS-Uber: A Hybrid-Learning Framework for Prediction of General and E3-Specific Lysine Ubiquitination Sites,” Briefings in Bioinformatics 23, no. 2 (2022): bbab574.

[45] H. Qi, X. Shi, M. Yu, et al., “Sirtuin 7-Mediated Deacetylation of WD Repeat Domain 77 (WDR77) Suppresses Cancer Cell Growth by Reducing WDR77/PRMT5 Transmethylase Complex Activity,” Journal of Biological Chemistry 293, no. 46 (2018): 17769-17779.

[46] J. A. Mäkelä and R. M. Hobbs, “Molecular Regulation of Spermatogonial Stem Cell Renewal and Differentiation,” Reproduction 158, no. 5 (2019): R169-r187.

[47] R. S. Mahla, N. Reddy, and S. Goel, “Spermatogonial Stem Cells (SSCs) in Buffalo (Bubalus bubalis) Testis,” PLoS One 7, no. 4 (2012): e36020.

[48] M. Kanatsu-Shinohara, I. Onoyama, K. I. Nakayama, and T. Shinohara, “Skp1-Cullin-F-Box (SCF)-type Ubiquitin Ligase FBXW7 Negatively Regulates Spermatogonial Stem Cell Self-Renewal,” Proceedings of the National Academy of Sciences of the United States of America 111, no. 24 (2014): 8826-8831.

[49] D. Zhou, X. Wang, Z. Liu, et al., “The Expression Characteristics of FBXW7 in Human Testis Suggest Its Function Is Different From That in Mice,” Tissue & Cell 62 (2020): 101315, https://doi.org/10.1016/j.tice.2019.101315.

[50] S. Zhou, J. Dong, M. Xiong, et al., “UHRF1 Interacts With snRNAs and Regulates Alternative Splicing in Mouse Spermatogonial Stem Cells,” Stem Cell Reports 17, no. 8 (2022): 1859-1873.

[51] P. Dai, X. Wang, L. T. Gou, et al., “A Translation-Activating Function of MIWI/piRNA During Mouse Spermiogenesis,” Cell 179, no. 7 (2019): 1566-1581.e1516.

[52] L. T. Gou, P. Dai, J. H. Yang, et al., “Pachytene piRNAs Instruct Massive mRNA Elimination During Late Spermiogenesis,” Cell Research 25, no. 2 (2015): 266.

[53] K. L. Fok, R. Bose, K. Sheng, et al., “Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response,” Endocrinology 158, no. 11 (2017): 4000-4016.

[54] E. Wu, W. He, C. Wu, et al., “HSPA8 Acts as an Amyloidase to Suppress Necroptosis by Inhibiting and Reversing Functional Amyloid Formation,” Cell Research 33, no. 11 (2023): 851-866.

[55] H. Huang, H. Li, T. Zhao, et al., “TSPAN1-Elevated FAM110A Promotes Pancreatic Cancer Progression by Transcriptionally Regulating HIST1H2BK,” Journal of Cancer 13, no. 3 (2022): 906-917.

[56] H. Guo, H. Wu, Y. Hou, et al., “Oat β-D-Glucan Ameliorates Type II Diabetes Through TLR4/PI3K/AKT Mediated Metabolic Axis,” International Journal of Biological Macromolecules 249 (2023): 126039.

[57] H. S. Noh, H. P. Lee, D. W. Kim, et al., “A cDNA Microarray Analysis of Gene Expression Profiles in Rat Hippocampus Following a Ketogenic Diet,” Brain Research. Molecular Brain Research 129, no. 1-2 (2004): 80-87.

[58] M. Ma, X. Chen, L. Lu, et al., “Identification of Crucial Genes Related to Postmenopausal Osteoporosis Using Gene Expression Profiling,” Aging Clinical and Experimental Research 28, no. 6 (2016): 1067-1074.

[59] L. J. Leandro-García, S. Leskelä, I. Landa, et al., “Tumoral and Tissue-Specific Expression of the Major Human Beta-Tubulin Isotypes,” Cytoskeleton (Hoboken) 67, no. 4 (2010): 214-223.

[60] E. Bittermann, Z. Abdelhamed, R. P. Liegel, et al., “Differential Requirements of Tubulin Genes in Mammalian Forebrain Development,” PLoS Genetics 15, no. 8 (2019): e1008243.

[61] Y. Wang, Y. Li, J. Feng, et al., “Mydgf Promotes Cardiomyocyte Proliferation and Neonatal Heart Regeneration,” Theranostics 10, no. 20 (2020): 9100-9112.

[62] J. Li, P. Wang, Z. Xie, et al., “TRAF4 Positively Regulates the Osteogenic Differentiation of Mesenchymal Stem Cells by Acting as an E3 Ubiquitin Ligase to Degrade Smurf2,” Cell Death and Differentiation 26, no. 12 (2019): 2652-2666.

[63] R. Liu, J. Gao, Y. Yang, et al., “PHD Finger Protein 1 (PHF1) is a Novel Reader for Histone H4R3 Symmetric Dimethylation and Coordinates With PRMT5-WDR77/CRL4B Complex to Promote Tumorigenesis,” Nucleic Acids Research 46, no. 13 (2018): 6608-6626.

[64] X. Zhao, Z. Huang, Y. Chen, et al., “MAGEB2-Mediated Degradation of EGR1 Regulates the Proliferation and Apoptosis of Human Spermatogonial Stem Cell Lines,” Stem Cells International 2023 (2023): 3610466.

[65] C. Chiaro, D. L. Lazarova, and M. Bordonaro, “Tcf3 and Cell Cycle Factors Contribute to Butyrate Resistance in Colorectal Cancer Cells,” Biochemical and Biophysical Research Communications 428, no. 1 (2012): 121-126.

[66] D. Zhou, J. Fan, Z. Liu, et al., “TCF3 Regulates the Proliferation and Apoptosis of Human Spermatogonial Stem Cells by Targeting PODXL,” Frontiers in Cell and Development Biology 9 (2021): 695545.

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