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

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.

DUX: One Transcription Factor Controls 2-Cell-like Fate

“…ChIP-seq data on p53 further showed the direct binding of P53 in the Dux locus. In addition, Lin28 or Dppa2/4 deletion-mediated Dux activation is associated with p53 activation [11,28]. In contrast, in vivo data reveal that p53-knockout embryos are still positive for anti-DUX antibody staining, indicating that Dux activation is more complicated in in vivo settings [28].…”

“…Recently, LIN28, an RNA-binding protein, was identified as able to repress Dux by an epigenetic program (Figure 2A). H3K9me3 levels were decreased at Dux and its downstream targets, and thus de-repressed Dux expression in Lin28 knockout cells [11]. However, the mechanisms underlying how Lin28 regulates H3K9me3 remain elusive.…”

“…Thus, differences in ribosome biogenesis can cause specialized mRNA subsets to be translated, which indirectly determine cell identity. Recent studies indicate that the inhibition of rRNA biogenesis can promote the state transition from ESCs to 2CLCs [11,13]. CX-5461, an RNA Pol I inhibitor, can decrease rRNA biogenesis and disrupt the nucleolar integrity and formation of peri-nucleolar heterochromatin (PNH).…”

“…Upon the disruption of nucleolar integrity, the loss of NCL/TRIM28 complex from PNH causes changes in epigenetic modification, leading to the release of Dux (Figure 4) [13]. Transcription factor LIN28 has been reported to bind to RNAs in the nucleolus, and its loss impeded ribosome assembly and recapitulated CX-5461-induced 2CLCs molecular phenotypes [11]. In addition, rRNA repression will activate p53, an effector of DNA-damage response pathway [34].…”

“…A transcriptome analysis of mouse preimplantation embryos revealed that the activation of unique transcripts takes place at the 2-cell stage but is undetectable at any other stages. These transcripts include, but are not limited to, zinc finger and SCAN domain containing 4 (Zscan4) [9][10][11][12][13][14], zinc finger protein 352 (Zfp352) [9], 2-cell-stage, variable group, member 1/3 (Tcstv1/3) [9], predicted gene 4340 (Gm4340) [11], TD and POZ domain containing 1-5 (Tdpoz1-5) [14] and procollagen-proline, 2-oxoglutarate 4-dioxygenase, 2 of 13 alpha II polypeptide (P4ha2) [10], most of them able to generate chimaeric transcripts linked to murine endogenous retrovirus with leucine tRNA primer (MERVL) element. The MERVL-one class of endogenous retroviral elements (ERVs) and hundreds of genes driven by the 5 LTR of MERVL are upregulated specifically at the 2-cell stage.…”