LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

Zhen Sun,Jianlong Wang,Jin Zhang,Lang Chen,Yuqing Zhu,Tianyu Tan,Yuyan Xu,Cristina Correia,Li Zhang,Li Shen,Cheng Zhang,Hua Yu,Jing Zhao,Yu Feng,Xin Huang,George Q Daley,Ming Chen,Hu Li,Hongru Pan,Xianju Bi,Bo Gao,Anhua Lei,Hao Wu,Long Wang ,Xiaohua Shen,Qiming Sun

doi:10.1007/s13238-021-00864-5

Abstract

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

Highlights

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst of mouse early embryos and have the ability to differentiate into three germ layers and germ cells, but do not differentiate into trophectoderm and extraembryonic tissues, and are recognized as pluripotent
We inspected the endogenous retrovirus MERVL-Gag protein which was expressed predominantly at the 2-cell/4-cell stage and broadly reduced thereafter (Fig. S1B). This reciprocal expression pattern of ERV compared to LIN28A protein suggests that LIN28A may play a role in repressing ERV expression
LIN28 interacts with microRNA let-7 and mediates its processing and metabolism (Chang et al, 2013; Piskounova et al, 2011), and given that LIN28 interacts with rRNA and small nucleolar RNA (snoRNA), and snoRNAs are engaged in processing rRNA (Kiss, 2002), we examined mature rRNAs including 18S and 28S, and found that knockout cells had significantly reduced levels (Figs. 4B and S5G), and the defects can be rescued by overexpressing LIN28A (Fig. 4B)

Summary

Introduction

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst of mouse early embryos and have the ability to differentiate into three germ layers and germ cells, but do not differentiate into trophectoderm and extraembryonic tissues, and are recognized as pluripotent. The 2-cell state is characterized by activation of endogenous retrovirus or ERV genes which may act as alternative promoters or other regulatory elements for early developmental genes (Peaston et al, 2004). In cultured mouse ES cells, there is a rare and transient cell population with activated 2-cell marker genes including ERVs (Macfarlan et al, 2012). Understanding how ERVs are activated and repressed in the 2-cell state, and how these events influence zygotic genome activation and early embryo development are important questions that warrant further investigation. Other features beyond epigenetics such as translational or metabolic status of these 2-cell like cells, and how these features impact the 2-cell like cell fate has yet been fully explored (Eckersley-Maslin et al, 2016)

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Conclusion