LHPP-Mediated Histidine Dephosphorylation Suppresses the Self-Renewal of Mouse Embryonic Stem Cells.

Rong Mu Xia,Xiu Qin Xu,Xue Min Cai,Dong Bo Yao

doi:10.3389/fcell.2021.638815

Abstract

Self-renewal of embryonic stem cells (ESCs) is orchestrated by a vast number of genes at the transcriptional and translational levels. However, the molecular mechanisms of post-translational regulatory factors in ESC self-renewal remain unclear. Histidine phosphorylation, also known as hidden phosphorylation, cannot be detected by conventional experimental methods. A recent study defined phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) as a histidine phosphatase, which regulates various biological behaviors in cells via histidine dephosphorylation. In this study, the doxycycline (DOX)-induced hLHPP-overexpressing mouse ESCs and mouse LHPP silenced mESCs were constructed. Quantitative polymerase chain reaction (qPCR), western blotting analysis, immunofluorescence, Flow cytometry, colony formation assays, alkaline phosphatase (AP) and bromodeoxyuridine (Brdu) staining were performed. We found that the histidine phosphorylation level was strikingly reduced following LHPP overexpression. Besides, the expression of Oct4 and Lefty1, indispensable genes in the process of ESCs self-renewal, was significantly down-regulated, while markers related to the differentiation were markedly elevated. Moreover, LHPP-mediated histidine dephosphorylation induced G0/G1 phase arrest in mESCs, suggesting LHPP was implicated in cell proliferation and cell cycle. Conversely, silencing of Lhpp promoted the self-renewal of mESCs and reversed the RA induced increased expression of genes associated with differentiation. Mechanistically, our findings suggested that the enzymatic active site of LHPP was the cysteine residue at position 226, not 53. LHPP-mediated histidine dephosphorylation lowered the expression levels of β-catenin and the cell cycle-related genes CDK4 and CyclinD1, while it up-regulated the cell cycle suppressor genes P21 and P27. Taken together, our findings reveal that LHPP-mediated histidine dephosphorylation plays a role in the self-renewal of ESCs. LHPP-mediated histidine dephosphorylation inhibited the self-renewal of ESCs by negatively regulating the Wnt/β-catenin pathway and downstream cell cycle-related genes, providing a new perspective and regulatory target for ESCs self-renewal.

Highlights

Embryonic stem cells (ESCs), with the attributes of pluripotency and self-renewal, have the ability to differentiate into multicell lineages (Dogan, 2018)
Our findings suggested that Lhpp expression was upregulated during mouse ESCs differentiation
Via construction of DOX-induced LHPP-overexpressing and LHPP-silenced mouse ESC lines, we further found that Lhpp overexpression contributed to the down-regulation of histidine phosphorylation

Summary

Introduction

Embryonic stem cells (ESCs), with the attributes of pluripotency and self-renewal, have the ability to differentiate into multicell lineages (Dogan, 2018). Previous work has indicated that transcriptional and post-transcriptional modifications are important for the regulation of ESCs self-renewal (Li et al, 2014). It has been reported that Oct and NANOG recruit LSD1 and bind to promoters of development-related genes, thereby controlling ESCs self-renewal by modulating H3K4 and H3K27 methylation (Adamo et al, 2011; Whyte et al, 2012). Zinc finger protein ZC3H13 maintains ESCs self-renewal via m6A modifications of mRNA (Wen et al, 2018). Foxp1mediated ESCs-specific splicing switches play a crucial role in maintaining self-renewal (Gabut et al, 2011)

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Results

Conclusion