Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Dec 4.
Published in final edited form as: Cell Stem Cell. 2014 Dec 4;15(6):735–749. doi: 10.1016/j.stem.2014.10.016

SET7/9 methylation of the pluripotency factor LIN28A is a nucleolar localization mechanism that blocks let-7 biogenesis in human ESCs

Seung-Kyoon Kim 1,6, Hosuk Lee 1,6, Kyumin Han 1, Sang Cheol Kim 4, Yoonjung Choi 1, Sang-Wook Park 1, Geunu Bak 3, Younghoon Lee 3, Jung Kyoon Choi 2, Tae-Kyung Kim 5, Yong-Mahn Han 1,*, Daeyoup Lee 1,*
PMCID: PMC4258232  NIHMSID: NIHMS642184  PMID: 25479749

SUMMARY

LIN28 mediated processing of the miRNA let-7 has emerged as a multi-level program that control self-renewal in embryonic stem cells. LIN28A is believed to primarily act in the cytoplasm together with TUT4/7 to prevent final maturation of let-7 by Dicer, whereas LIN28B has been suggested to preferentially act on nuclear processing of let-7. Here, we find that SET7/9 mono-methylation in a putative nucleolar localization region of LIN28A increases its nuclear retention and protein stability. In the nucleoli of human embryonic stem cells (hESCs), methylated LIN28A sequesters pri-let-7 and blocks its’ processing independently of TUT4/7. The nuclear form of LIN28A regulates transcriptional changes in MYC-pathway targets, thereby maintaining stemness programs and inhibiting expression of early lineage-specific markers. These findings provide insight into the molecular mechanism underlying the post-translational methylation of nuclear LIN28A and its ability to modulate pluripotency by repressing let-7 miRNA expression in human ESCs.

INTRODUCTION

LIN28 was originally identified as a regulator of developmental timing in the nematode C. elegans (Ambros and Horvitz, 1984), and its expression is tightly regulated during animal development (Moss and Tang, 2003). Consistent with its role in development, LIN28A is highly expressed in ESCs and is one of four factors that convert human fibroblasts into induced pluripotent stem cells (iPSCs) (Yu et al., 2007b). Subsequent studies have revealed that LIN28A and its paralog LIN28B are also broadly involved in control of growth and metabolism, germ cell development, and cancer (Viswanathan et al., 2009; West et al., 2009; Zhu et al., 2011). Functionally, LIN28 proteins affect mRNA translation (Polesskaya et al., 2007) and specifically repress the processing of let-7 miRNAs into mature forms that mediate ESC differentiation, thereby modulating self-renewal and pluripotency (Heo et al., 2008; Newman et al., 2008; Rybak et al., 2008; Viswanathan et al., 2008). The pri-let-7 miRNAs are easily detectable in pluripotent ESCs, whereas mature let-7 miRNAs are undetectable, but undergo post-transcriptional induction during differentiation (Heo et al., 2008; Viswanathan et al., 2008), concomitant with suppression of self-renewal pathways (Melton et al., 2010; Yu et al., 2007a).

In the nucleus, LIN28A/B associate with the primary transcript of let-7 (pri-let-7) to block its Drosha-mediated processing to precursor let-7 (pre-let-7) (Newman et al., 2008; Viswanathan et al., 2008). Regulation of let-7 biogenesis involves direct interaction of LIN28A with the terminal loop region of let-7; this requires both the cold shock domain (CSD) and a cluster of two CCHC-type zinc finger domains of LIN28A (Nam et al., 2011; Piskounova et al., 2008). In the cytoplasm, the LIN28A protein directly interacts with pre-let-7 and recruits the terminal uridylyltransferase, TUT4/7 (Zcchc11/6), to induce oligo-uridylation of pre-let-7, which subsequently becomes targeted for degradation by the exoribonuclease, DIS3L2. This mechanism effectively blocks pre-let-7 processing by Dicer and facilitates its rapid decay (Chang et al., 2013; Hagan et al., 2009; Heo et al., 2009). In contrast, when LIN28A is absent in somatic cells, TUT2/4/7 mono-uridylate pre-let-7s, which enables Dicer processing and maturation of let-7 (Heo et al., 2012).

LIN28A/B proteins are differentially localized in cells in a variety of species, ranging from nematodes to mammals, with LIN28A being predominantly found in the cytoplasm with some distribution to the nucleus (Balzer and Moss, 2007; Chen and Carmichael, 2009; Heo et al., 2008; Piskounova et al., 2011). LIN28B is tagged by both a nuclear localization (NLS) and nucleolar localization signal (NoLS), and is abundant in the nucleolus (Piskounova et al., 2011). Consistently, LIN28B is believed to sequester pri-let-7 and block its processing by primarily acting in the nucleus. However, LIN28A/B are not co-expressed in mammalian cells with ubiquitous TUT4 expression, suggesting that LIN28A and LIN28B in the cytoplasm both employ TUT4/7 dependent mechanisms for let-7 degradation (Heo et al., 2012; Piskounova et al., 2011) Thus, LIN28A/B proteins may each decrease let-7 biogenesis via distinct mechanisms (Piskounova et al., 2011). Although many studies have examined the mechanism through which LIN28A regulates the let-7 miRNA in the cytoplasmic compartment, our understanding of how LIN28A functions in the nucleus is more limited. Notably, LIN28A is tagged by a region that is highly homologous with the NoLS of LIN28B.

Core pluripotency factors (e.g., OCT4, SOX2, NANOG, and/or LIN28A) tightly modulate the states of ESC transcriptionally and/or post-translationally (Boyer et al., 2005; Heo et al., 2008; Melton et al., 2010; Viswanathan et al., 2008). Although it is well known that the expression of these factors is coordinately modulated at the transcription level (Boyer et al., 2005), little is known about how post-translational modifications (PTMs; e.g., methylation, acetylation, phosphorylation, and ubiquitylation) may affect the subcellular localization, stability, protein-protein interactions, and/or functions of these factors. Recently, epigenetic modifiers have been shown to induce PTM of some pluripotency factors, thereby regulating their functions and contributing to pluripotency and self-renewal (Evans et al., 2007; Fang et al., 2014; Moretto-Zita et al., 2010; Wang et al., 2014; Wei et al., 2007). Whether PTMs of LIN28 proteins can affect function in hESCs remain unknown.

SET7/9 (SETD7) was originally identified as a protein methyltransferase that catalyzes mono-methylation of histone 3 (Nishioka et al., 2002; Strahl and Allis, 2000; Tao et al., 2011). However, it was later found to also regulate the activities of non-histone proteins, such as p53 (Chuikov et al., 2004), TAF10 (Kouskouti et al., 2004), DNMT1 (Estève et al., 2009), ERα (Subramanian et al., 2008), and E2F1 (Kontaki and Talianidis, 2010). Consensus target residue analysis identified a structurally conserved consensus recognition sequence in its substrates (K/R-S/T-K, with K being the target lysine residue) (Couture et al., 2006), and the authors proposed other putative target substrates among the non-histone protein pool in mammalian cells. Recently, SOX2 was identified as a SET7/9 substrate in mouse ESCs, targeting methylated SOX2 for degradation (Fang et al., 2014), however little is known about its function in hESCs. Similar to the high expression of LIN28A in skeletal muscle (Polesskaya et al., 2007), the expression of SET7/9 is gradually increased during skeletal muscle (Tao et al., 2011) as well as EB differentiation (Fang et al., 2014).

Here, we have examined the methylation of pluripotency factors and found that SET7/9 specifically mono-methylates LIN28A at lysine 135, which is in a region that is homologous with the NoLS of LIN28B. Our results further suggest that SET7/9-mediated methylation of LIN28A increases LIN28A protein stability and localization to the nucleus. Furthermore, we find that LIN28A methylation stimulates multimerization of LIN28A with let-7 miRNA. In human ESCs (hESCs), the methylated LIN28A inhibits processing of the let-7 miRNA by sequestering its transcripts to the nucleoli in a TUTase-independent manner, thereby regulating pluripotency and differentiation.

RESULTS

LIN28A is mono-methylated at K135 by the SET7/9 lysine methyltransferase in hESCs

Preliminary in vitro methylation analysis using SET7/9 and the recombinant pluripotency factors showed that the SET7/9 protein was capable of methylating LIN28A (Figure S1A). Consistent with a recent report(Fang et al., 2014), SOX2 was also methylated by SET7/9, while OCT4 and NANOG were not methylated under the same condition. To investigate whether LIN28A is potentially regulated by lysine methylation, we tested in vitro methylation analysis using a variety of histone methyltransferases (HMTases). SET7/9 specifically methylated LIN28A (Figure S1B). To verify this finding, LIN28A was used to perform in vitro pull-down assays with HMTases (Figures S1C-E). Although several of the tested HMTases bound to the LIN28A protein, SET7/9 clearly showed the strongest enzymatic activity. Thus, we speculated that LIN28A may be functionally modified by lysine methylation.

To confirm the binding between LIN28A and SET7/9, we estimated their physical interaction using an immunoprecipitation assay in cultured cells. Endogenous or overexpressed LIN28A was immunoprecipitated from H9, NCCIT, and 293T cells, and subsequent immunoblotting with the relevant antibodies revealed that SET7/9 was immunoprecipitated with LIN28A in all three cell lines (Figures 1A, S1F, and S1G). LIN28A did not immunoprecipitate with the negative control IgG. Reverse immunoprecipitation of SET7/9 confirmed the identified interaction with LIN28A (Figure 1B).

Figure 1. LIN28A is associated with and methylated by SET7/9 in cultured cells and in vitro.

Figure 1

Open in a new tab

(A and B) Immunoprecipitation of endogenous LIN28A with SET7/9 from H9 cells was performed.

(C) Alignment of the consensus amino acid residues adjacent to lysines targeted for methylation by SET7/9. In each case, the target lysine is underlined and bolded, and the asterisk indicates the consensus SET7/9 recognition sequence, K/R-S/T-K.

(D) Detection of mono-methylated LIN28A from whole-cell extracts of 293T cells overexpressing FLAG-LIN28A wild type or the FLAG-LIN28A-K135R mutant. Samples were immunoblotted using the antibodies indicated on the right. β-Actin was used as a loading control.

(E) In vitro pull-down assays were carried out using GST-SET7/9 and 6×HIS-LIN28A or -K135R of full-length proteins.

(F) In vitro methyltransferase assay. Bacterially expressed recombinant full-length SET7/9 was incubated as indicated with full-length LIN28A or rC/H (positive control) plus [3H] SAM. Autoradiography (upper) and Ponceau S staining (bottom) were used to examine methylation and protein levels, respectively.

(G and H) In vitro methyltransferase assays were carried out with 6×HIS-LIN28A fragments, LIN28A (1-124), (125-209), (125-156), (157-209), and SET7/9 with rC/H used as a positive control (G), and with eleven full-length 6×HIS-LIN28A mutants bearing the K78R, K88R, K98R, K99R, K102R, K125R, K127R, K131R, K135R, K150R, and K153R substitutions, as described above. A nonspecific band is indicated by an asterisk (H).

See also Figures S1 and S2.

We next sought to identify the SET7/9-methylated residue(s) in LIN28A. We compared the amino acid sequence of LIN28A to the sequences surrounding the SET7/9 methylation sites of target substrates (Figure 1C). We found that the conserved lysine residue at position 135 and its neighboring residues of LIN28A showed strong similarities to the consensus SET7/9 recognition sequence (Couture et al., 2006). Interestingly, LIN28B does not contain the SET7/9 recognition sequence. When we aligned the full amino acid sequences of LIN28 proteins of various species, we found that the identified SET7/9 recognition sequence was evolutionarily conserved among the higher eukaryotes (Figure S2A).

To facilitate studies on the SET7/9-mediated mono-methylation of LIN28A at lysine 135 in cells, we generated polyclonal antibodies that specifically recognized unmethylated LIN28A-K135 or mono-methylated LIN28A-K135. Next, we transfected 293T cells with a construct encoding FLAG-tagged LIN28A wild-type or FLAG-LIN28A-K135R mutant (K135R; in which lysine 135 was replaced with arginine). Immunoblot analysis using the anti-LIN28A-K135-me1 (methylated) antibody revealed the presence of methylated LIN28A in extracts prepared from cells overexpressing LIN28A but not K135R (Figure 1D). We therefore conclude that LIN28A is methylated at lysine 135 in cells.

SET7/9 associates with and methylates LIN28A in vitro

To determine whether the interaction between LIN28A and SET7/9 in cells is direct, we generated a series of MAL, 6×HIS, and GST fusion proteins containing various truncated versions of LIN28A and SET7/9 (Figures S1D and S1E), and used them to perform pull-down assays. Our results revealed that the carboxyl terminus (amino acids 125–209) of LIN28A interacted with SET7/9 (Figures S1H and S2C). Both LIN28A and the K135R bound to SET7/9 in this assay (Figure 1E). These results suggest that the LIN28A and SET7/9 proteins interact directly.

We next examined whether SET7/9 could methylate LIN28A in vitro. When recombinant full-length LIN28A was incubated with recombinant SET7/9 in the presence of 3H-S-adenosine-methionine ([3H] SAM), we found that LIN28A was methylated (Figure 1F, lane 2). LIN28A was not methylated in the absence of the SET7/9 protein (Figure 1F, lane 3), indicating that LIN28A is indeed methylated by SET7/9 in vitro. As a positive control, recombinant human core histone (rC/H) was also shown to be methylated by SET7/9 under the same conditions, validating our in vitro methyltransferase assay (Figures 1F, lanes 4 and 5, and S2F).

Given that full-length LIN28A is methylated in cultured cells and in vitro by SET7/9, we next sought to confirm that K135 of LIN28A is a bona fide SET7/9 target. We purified 6×HIS-tagged proteins containing four different fragments of LIN28A and used them as substrates for an in vitro methyltransferase assay. Of the four tested fragments, LIN28A (125-209) and (125-156), which included K135, were methylated by SET7/9 (Figure 1G, lanes 4 and 6). In contrast, LIN28A (1–124) and (157-209), which did not include K135, showed no evidence of methylation (Figure 1G, lanes 2 and 8). As there are numerous lysine residues surrounding K135, we confirmed this result by running parallel experiments in which we substituted eleven lysines with non-methylatable arginines. A single point mutation at K135R, but not the other tested lysines, completely eliminated SET7/9-mediated methylation (Figure 1H, lane 19), providing additional evidence that SET7/9 methylates LIN28A at K135.

Since LIN28B does not have the SET7/9 target residue (Figure 1C), we performed in vitro methyltransferase assays using LIN28B to investigate the specificity of the SET7/9-induced methylation of LIN28A at K135. As expected, LIN28B was not methylated by SET7/9 (Figure S2D). In the context of a negative control, DOT1L (a H3K79 methyltransferase) was purified from a baculovirus expression system (Kim et al., 2013) and used in our methyltransferase assay. It failed to methylate LIN28A (Figure S2E). Collectively, these results indicate that the SET7/9-mediated methylation of LIN28A at K135 is specific.

SET7/9 stabilizes LIN28A via lysine methylation

SET7/9-mediated lysine methylation is known to regulate the stability of various K/R-S/T-K-containing targets. Therefore, we examined whether methylation by SET7/9 affects the stability of LIN28A in cells. Co-expression of SET7/9 in 293T cells up-regulated the expression of LIN28A but not K135R (Figure 2A), suggesting that the SET7/9-mediated methylation of LIN28A may increase its stability. Further supporting this notion, the co-expression of SET7/9 did not alter the mRNA levels of LIN28A (Figure 2B). To assess whether this apparent stabilization was associated with the methylation of LIN28A at K135, we investigated the effect of SET7/9 on the half-life of LIN28A and K135R using CHX, a protein synthesis inhibitor. We found that the SET7/9-mediated methylation of LIN28A promoted its stabilization and increased its half-life, whereas the half-life of K135R was reduced (Figure 2C). Furthermore, depletion of SET7/9 in H9 and NCCIT cells by transfection of small interfering RNA (siRNA) also affected stabilization of LIN28A (Figures 2D and 2E). Together, these data show that the SET7/9-mediated methylation of LIN28A at K135 increases the stability of LIN28A.

Figure 2. SET7/9 increases the stability of LIN28A through lysine methylation.

Figure 2

Open in a new tab

(A) 293T cells were transfected with plasmids encoding FLAG-LIN28A or FLAG-LIN28A-K135R alone or together with HA-SET7/9, as indicated. Forty-eight hours post-transfection, whole-cell extracts were immunoblotted using the antibodies indicated on the right. β-Actin was used as a loading control. A representative result from three independent biological replicates is shown (left). The LIN28A signals were quantified and are given in arbitrary units of band intensity (right) compared to β-Actin. Error bars denote standard deviations obtained from three independent biological replicates.

(B) Real-time quantitative RT-PCR (RT-qPCR) was used to measure the mRNA levels of LIN28A andSET7/9, and the results were normalized with respect to the expression of GAPDH. Error bars denote standard deviations obtained from three independent biological replicates.

(C) 293T cells were transfected as indicated and cells were treated with 60 μg/ml of cycloheximide (CHX). Samples were immunoblotted using the antibodies indicated on the right. Quantification of the results (right) was obtained as described as in (A).

(D and E) Immunoblotting was performed as described above, using whole-cell extracts from single H9 and NCCIT cells.

Mono-methylated LIN28A is specifically localized in the nucleoli

Notably, K135 and its neighboring sequences in LIN28A are homologous with the NoLS of LIN28B (Figures S1H and S3A) (Piskounova et al., 2011). In order to determine whether methylated LIN28A preferentially localizes to either cellular compartment, we prepared nuclear and cytoplasmic fractions from either 293T cells (which do not endogenously express LIN28A), by exogenously expressing LIN28A or LIN28A-K135R or from H9 or NCCIT cells (which endogenously express LIN28A). These fractions were immunoblotted with the indicated antibodies (Figures 3A, 3B, and S3B). Consistent with the published data, we found that LIN28A (the cytoplasmic form) was abundant in the cytoplasm of all tested cells. A smaller portion localized to the nucleus, largely the methylated form (the nuclear form) (Figures 3A, lane 5 and 3B, lane 1 and 2). Intriguingly, the methylated form of LIN28A specifically localized to both the nucleoli and the nucleoplasm of H9 cells (Figure 3C). These data were further confirmed by immunofluorescence assays in H9, NCCIT, and 293T cells, wherein abundant mono-methylated LIN28A was detected along with SET7/9 in the nucleus; this was especially evident in the nucleoli (Figures 3D, S3C, and S3D).

Figure 3. Methylated LIN28A is mainly located in the nucleus.

Figure 3

Open in a new tab

(A) 293T cells were transfected with control vectors or vectors encoding FLAG-LIN28A or FLAG-LIN28A-K135R. Nuclear and cytoplasmic fractions were immunoblotted using the antibodies indicated on the right. H3 was used as a nuclear marker, while α-tubulin was used as a cytoplasmic marker. The LIN28A signals were quantified and are given in arbitrary units of band intensity (right). Error bars denote standard deviations obtained from three independent biological replicates.

(B) Nuclear or cytoplasmic fractions from H9 cells were immunoblotted for endogenous LIN28A using the antibodies indicated on the right. H3 was used as a nuclear marker and α-tubulin was used as a cytoplasmic marker.

(C) Nuclear fractions from H9 cells were further fractionated to nucleolar or nucleoplasmic subfractions, and immunoblotting was performed using the antibodies indicated on the right. Fibrillarin was used as a nucleolar marker.

(D) Immunofluorescent detection of endogenous LIN28A, LIN28A-me1, fibrillarin, and SET7/9 in H9 cells. Scale bars, 400 and 50 μm (for colony formation).

(E) Immunoblotting was performed as described above, using nuclear and cytoplasmic fractions from H9 cells subjected to siRNA-mediated knockdown of EGFP control or SET7/9 (left). Immunofluorescent analysis of H9 cells subjected to siRNA-mediated knockdown as indicated (right). Scale bar, 400 μm.

(F) The signal intensity of LIN28A and SET7/9 shown in (E) were quantified and are given in arbitrary units (n=8). Pairs of cells with nuclei of the same scan width (as determined with DAPI staining) were used for measurements. The nucleus (Nuc) and cytoplasm (Cyt) are indicated.

See also Figure S3.

To further support our proposal that the nuclear localization of mono-methylated LIN28A is due to SET7/9-mediated methylation, we subjected SET7/9 to siRNA-mediated knockdown and used immunoblotting and immunofluorescence assays to show that the specific nuclear localization pattern of the nuclear form of LIN28A was diminished in these cells (Figure 3E). The signal intensity levels of LIN28A and SET7/9 were also quantified in control- and SET7/9-siRNA treated H9 cells (Figure 3F). Taken together, these data suggest that K135-mono-methylated LIN28A may function in a manner similar to the NoLS of LIN28B. Sequence alignment of the LIN28 homologs further supported this idea (Figure S3A).

Nuclear LIN28A blocks pri-let-7 maturation/processing in the nucleus

Compared to the localization of LIN28B (Piskounova et al., 2011), methylation of LIN28A appears to be an exclusive signal that simultaneously increases LIN28A protein stability and directs subcellular localization (Figures 2, 3, and S3). Based on these results, we hypothesized that mono-methylated LIN28A may function within the nucleus to regulate the processing of pri-let-7-1 miRNA in a TUTase-independent manner (similar to the parallel function of LIN28B) and explored whether nuclear LIN28A can inhibit miRNA processing. Co-transfection of human pri-let-7a-1 and let-7aBS-Luc with either FLAG-LIN28A or FLAG-K135R led to accumulation of pri-let-7a-1 RNAs and luciferase mRNAs, which was accompanied by a corresponding accumulation in the levels of mature let-7a-1 RNAs (Figure 4A). Notably, substantially more pri-let-7a-1 RNAs were accumulated by the nuclear form compared to the cytoplasmic form of LIN28A (Figure 4A, lanes 4 and 5), which is consistent with the differences in their nuclear levels (Figure 3A). The levels of mature let-7 miRNAs, however, were somewhat decreased also by cytoplasmic form of LIN28A (Figures 4A, lane 5 and S4D, lane 4) due to its activity in the cytoplasm to block the processing of let-7. As a control, the expression of AGO2, which is a catalytic component of the RISC (RNA-induced silencing complex) known to repress miRNA-mediated translation (Heo et al., 2008), triggered a ~ 2-fold decrease of luciferase mRNA levels (Figure 4A, lane 6). Similar results were obtained with another let-7 family member, pri-let-7g (Figures S4C and S4D), but not with the negative control, miR-16-1 (Figure 4B), indicating that the nuclear LIN28A specifically targets let-7 family members.

Figure 4. Methylated LIN28A is a truly active form that blocks the processing of pri-let-7 miRNAs.

Figure 4

Open in a new tab

(A) 293T cells were co-transfected with human pri-let-7a-1 and the protein expression vectors, FLAG-LIN28A, FLAG-LIN28A-K135R, or FLAG-AGO2 along with the renilla (pRL-CMV) and firefly (let-7aBS-Luc, a luciferase construct containing three binding sites of let-7a-1 in its 3’UTR that can be targeted by mature let-7a-1) luciferase genes, as indicated. RT-qPCR was used to measure the relative expression levels of let-7aBS-Luc, pri-let-7a-1, and mature let-7a-1. The firefly luciferase signal was normalized with respect to that from renilla, and each value was normalized with respect to that of GAPDH. The U6 snRNA was used as a reference for the mature let-7a-1 levels. Error bars denote standard deviations obtained from three independent biological replicates.

(B) Vectors were co-transfected as indicated into 293T cells, and RT-qPCR was used to evaluate the relative expression levels of miR-16BS-Luc (containing one binding site for miR-16), pri-miR-16-1, and mature miR-16-1. The values were normalized as described in (A). Error bars denote standard deviations obtained from three independent biological replicates.

(C) RT-qPCR showing changes in the levels of endogenous primary (upper) or mature (bottom) let-7a miRNAs and control miRNA upon knockdown of the LIN28A, SET7/9, or TUT4 in H9 cells. GAPDH and the U6 snRNA were used as references for the primary and mature miRNAs, respectively.

See also Figure S4 and Table S1.

To explore whether the nuclear LIN28A is capable of inhibiting the processing of endogenous let-7 miRNAs, we measured the levels of all mature let-7 miRNAs in 293T cells expressing FLAG-LIN28A or FLAG-K135R (Figure S4E). All of the tested endogenous pri-let-7 miRNAs showed considerably more up-regulation in cells overexpressing LIN28A versus K135R mutant, whereas the levels of endogenous mature miR-16 and miR-21 were unaffected (Figure S4F, upper). The nuclear LIN28A-induced decreases in the levels of mature let-7 miRNAs were accompanied by corresponding accumulations of pri-let-7 (Figure S4F, bottom). Next, we examined whether the nuclear LIN28A is an endogenous inhibitor of miRNA biogenesis in hESCs. We used siRNAs targeting LIN28A or SET7/9 to knockdown the expression of endogenous LIN28A or SET7/9 in H9 hESCs (Figures 4C and S4G). Knockdown of LIN28A led to a remarkable increase of mature let-7 miRNAs in H9 cells, while levels of other negative control miRNAs were unchanged (Figure 4C, bottom). Mature let-7 miRNAs likely accumulated due to the long half-lives of mature miRNAs (Kim, 2005). The accumulation of mature let-7 miRNAs in LIN28A-knockdown cells was accompanied by a corresponding decrease in pri-let-7 (Figure 4C, upper). Similar results were obtained in SET7/9-knockdown cells, in which decreased LIN28A methylation was associated with up-regulation of mature let-7 (Figures 4C and S4F, green). A smaller increase in the most mature let-7 miRNAs was observed in TUT4-depleted cells compared to LIN28A- or SET7/9-depleted cells, while pri-let-7 miRNAs were unaffected (Figure 4C, purple) (Hagan et al., 2009; Heo et al., 2009). Together, these results strongly suggest that the nuclear LIN28A (i.e., the active form) blocks the processing of pri-let-7 in the nucleus.

Nuclear LIN28A is efficiently multimerized on primary let-7 miRNA

To further investigate the mechanism through which nuclear LIN28A blocks let-7 processing, we compared the relative binding abilities of the nuclear and cytoplasmic forms of LIN28A to let-7 RNA (Figures 5A-5C). Previous reports suggest that multiple molecules of LIN28A assemble on let-7 RNA to fulfill its maximum inhibitory function on let-7 biogenesis (Desjardins et al., 2014). We carried out EMSA with pri- or pre-let-7a-1. Surprisingly, we found increased multimeric binding of the nuclear LIN28A on pri-let-7a-1, especially as a trimer, compared to the cytoplasmic form (Figure 5B). The faster formation of multimeric complexes by the nuclear LIN28A was accompanied by a corresponding decrease in monomer (Figure 5B, 60 and 80 nM). The average ratio of the trimer band of the nuclear LIN28A over that of the dimer (2.49-fold) was much higher than that of cytoplasmic form (0.61-fold) (Figures 5B, S5A, and S5B), suggesting much faster binding of the nuclear LIN28A to pri-let-7. We also verified that both nuclear and cytoplasmic forms of LIN28A bind to pre-let-7a-1 with similar affinities (Figures 5C, S5C, and S5D). Together with the above-described data (Figures 2-4), these assays strongly indicate that multiple assembly of the nuclear LIN28A on pri-let-7 miRNA may efficiently isolate and stabilize LIN28A-RNA complexes, thereby more efficiently inhibiting its biogenesis in the nucleoli.

Figure 5. Pri-let-7 may have a preference for the nuclear form of LIN28A.

Figure 5

Open in a new tab

(A) Coomassie blue staining of purified FLAG-LIN28A and FLAG-LIN28A-K135R proteins (left). Purified FLAG-LIN28A and FLAG-LIN28A-K135R proteins were immunoblotted using the indicated antibodies (right).

(B and C) EMSA of pri- (B) or pre-let-7a-1 miRNA (C) with purified FLAG-LIN28A or FLAG-LIN28A-K135R. Unbound, monomer, dimer, and trimer indicate the multimeric binding of purified proteins to pri-let-7a-1 miRNA. In bottom panel, schematic diagram of EMSA with pri- (B) or pre-let-7a-1 (C) are shown. Unbound, monomer, dimer, and trimer fractions are indicated by blue, red, green, and purple bar, respectively. Each fraction was quantitated from three independent biological replicates.

(D and E) RNA Immunoprecipitation (RIP) assay. Nuclear and cytoplasmic fractions were prepared from 293T cells transfected with FLAG-LIN28A or FLAG-LIN28A-K135R (D) or H9 (E), and RIP analyses were carried out with the indicated antibodies. All signals were normalized with respect to the input signal. Error bars denote standard deviations obtained from three biological replicates, each consisting of three qPCR reactions.

See also Figure S5.

To gain further insight into our evolving model, in which the nuclear LIN28A multimerizes to and blocks the processing of pri-let-7, we next investigated the association of RNAs with the nuclear LIN28A in cells. Endogenous or overexpressed LIN28A was targeted for RIP analyses using antibodies against control IgG, LIN28A (detecting both methylated and unmethylated LIN28A), LIN28A-K135-me1, or LIN28A-K135-me0, and the relative levels of pri-let-7a-1 copurified with LIN28A were quantified. Our data revealed that the nuclear LIN28A binds to pri-let-7a-1 RNA in the nucleus, whereas the cytoplasmic LIN28A does not (Figures 5D and 5E). Substantially more pri-let-7a-1 associated with the nuclear LIN28A using whole cell extracts (Figure S5E). The anti-LIN28A and anti-LIN28A-K135-mel antibodies detected 2.8- and 5.6-fold enrichments, respectively, of pri-let-7a-1 associated with the nuclear LIN28A in the nucleus (Figure 5D), and 20.0-and 20.8-fold enrichments, respectively, of pri-let-7a-1 in the nucleus and cytoplasm (Figure 5E). The fold enrichments between LIN28A-me0 and LIN28A-K135-me0 signals did not significantly differ between the two proteins, suggesting that most of the methylated LIN28A was bound to pri-let-7a-1 in the nucleus. Based on these findings, together with the above data (Figures 2-4), we strongly suggest that nuclear form of LIN28A directly targets pri-let-7 miRNA to modulate its processing.

Nuclear LIN28A and SET7/9 modulate stem cell pluripotency and differentiation by regulating let-7 maturation

Consistent with the previous report (Viswanathan et al., 2008), we found that increased levels of mature let-7 miRNA in ESCs were accompanied by decreased pri-let-7 (Figure 4C). We next evaluated the importance of LIN28A and SET7/9 in let-7-mediated regulation of pluripotency in H9 cells. Transfection with LIN28A- or SET7/9-siRNA reduced protein levels of LIN28A or SET7/9 respectively (Figures S6A and S6B). Interestingly, LIN28A knockdown did not affect expression of representative pluripotency genes, NANOG and SOX2, compared to cells transfected with control EGFP-siRNA (Figure S6B).

Let-7 miRNA can participate in down-regulation of two MYC family members, MYC and MYCN, an activity that may reduce cell “stemness” (Melton et al., 2010; Smith et al., 2010). MYC and MYCN have shared functions in embryonic development and cellular growth (Malynn et al., 2000), and have largely overlapping target genes (Chen et al., 2008). To assess whether the SET7/9-mediated methylation of LIN28A affects stemness through regulation of let-7, we performed genome wide transcriptional expression profiling and then analyzed the expression of MYC-pathway targets (Ji et al., 2011) in LIN28A-, SET7/9-, or TUT4-depleted H9 cells (Figure 6 and Tables S3-S5). Genes activated by MYC-pathway were significantly down-regulated upon LIN28A knockdown whereas those repressed by MYC-pathway were significantly up-regulated upon LIN28A knockdown (Figure 6A, left). Similar results were obtained in SET7/9-knockdown cells in which LIN28A methylation was associated with stemness through down-regulation of mature let-7 (Figure 6A, right). The moderate changes of MYC-pathway targets between the expression changes were observed upon TUT4 knockdown compared to LIN28A and SET7/9 knockdown (Figure 6B), which is consistent with the differences in their abilities to regulate mature let-7 levels (Figure 4C). Simultaneously, the expression changes of MYC-pathway positive (red dots) or negative (blue dots) targets induced by LIN28A and SET7/9, by LIN28A and TUT4, and by SET7/9 and TUT4 showed same patterns that red or blue dots was down or up in both conditions as well as a positive correlation was observed between them (Figures 6C and 6D). Taken together, these proteins appear to have shared functions in regulating the MYC-pathway.

Figure 6. Expression profiles in LIN28A-, SET7/9-, and TUT4-depleted hESCs.

Figure 6

Open in a new tab

(A and B) Box plots showing the expression change (log2 ratio) of the MYC-pathway targets (Ji et al., 2011) in comparison with all genes in LIN28A-, SET7/9- (A), or TUT4-depleted hESCs (B). (p=0.0 and p=0.0037, p=0.0 and p=0.0033, and p=0.0 or p=0.043 for the MYC-pathway positive and negative genes in LIN28A, SET7/9, or TUT4-depleted cells, respectively).

(C and D) Scatter plot comparing the expression change (log2 ratio) of the MYC-pathway targets (red and blue dots indicating the positive and negative genes, respectively) induced by each experiment as described in (A and B).

(E) Box plots showing the expression change (log2 ratio) of the MYC-pathway targets in comparison with all genes in LIN28A-depleted hESCs with transfection of LIN28A-siR or K135R-siR at the same time. (p=0.0 and p=0.195, and p=0.0 and p=0.397 for the MYC-pathway positive and negative genes LIN28A-siR or K135R-siR, respectively). The increase of the MYC-pathway positive genes is more distinct (p=0.03) in LIN28A-siR than in K135R-siR (middle).

See also Figures S6, S7, and Tables S3-S7.

To further determine the specific contribution of LIN28A methylation to transcriptional changes, we expressed nuclear LIN28A-siR or cytoplasmic K135R-siR, resistant to LIN28A-siRNA due to the silent mutations in target sequences (Figure S6D and S6E), in H9 cells which were then subjected to knockdown of endogenous LIN28A. The expression of the nuclear or cytoplasmic LIN28A caused the activation of the MYC-pathway positive targets and the repression of the MYC-pathway negative targets (Figure 6E, Table S6, and S7). However, the MYC-pathway positive genes were activated to a higher degree (p=0.03) by the nuclear LIN28A than by the cytoplasmic LIN28A (Figure 6E, middle), suggesting that the trimerization of the nuclear LIN28A on pri-let-7 may facilitate transcriptional changes through maximum inhibition of let-7 processing. The repression of positive and activation of negative genes upon K135R-siR expression was milder than expected, although the contribution of LIN28A methylation in transcriptional changes was more significant. This may be due to the additional inhibitory effect of cytoplasmic LIN28A in TUTase-dependent let-7 processing in overexpressed condition. Furthermore, the overall level of H3K4 methylations and the occupancy of H3K4 mono-methylation for the transcriptional regulation of MYC-pathway targets were not severely affected upon SET7/9 knockdown (Figure S7) and the SET7/9 knockdown closely mimics LIN28A knockdown (Figures 4C, 6A, 7A, and 7C). Thus, we suggest that SET7/9 seems to mostly induce the transcriptional changes of MYC-pathway targets through LIN28A, although we cannot rule out other functions of SET7/9 beyond the regulation of LIN28A.

Figure 7. Nuclear LIN28A is critical for regulating stem cell maintenance of hESCs.

Figure 7

Open in a new tab

(A and B) The expression levels of representative early lineage markers in LIN28A-, SET7/9, and TUT4-depleted hESCs (A) and in LIN28A-depleted hESCs, followed by transfection with control vector, LIN28A-siR, or K135R-siR (B), as determined by microarray analysis. The fold-change values (log2) and expression profiles of the top-scoring genes are shown.

(C and D) Immunofluorescent detection of endogenous TRA-1-60, SSEA-4, OCT4, SOX2, and alkaline phosphatase staining in H9 cells subjected to siRNA-mediated knockdown of control, LIN28A or SET7/9 (C) and LIN28A-depletion with expression of control vector, LIN28A-siR or K135R-siR at the same time (D). Scale bar, 50 μm. See also Figure S6.

To further support this observation, we evaluated the expression levels of early lineage markers and observed that knockdown of LIN28A or SET7/9 increased the expression of representative early lineage markers while TUT4 knockdown moderately increased these markers (r = 0.40 ~ 0.95) (Figure 7A), which is consistent with the above data (Figure 4C). The expression of nuclear LIN28A efficiently inhibited expression of early lineage markers (r = 0.52) compared to the cytoplasmic form (Figure 7B), suggesting that LIN28A methylation has a specific function in pluripotency. A comparative analysis confirmed that expression patterns observed in our microarray analyses were reliable (Figure S6C). These data were further confirmed by immunofluorescence assays in H9 cells, where moderate but consistent reductions in expression of ES-specific surface antigens (TRA-1-60 and SSEA-4) and pluripotency marker, ALP were observed, while the control marker (SOX2) was unaffected upon LIN28A and SET7/9 knockdown (Figure 7C). The observed marginal reduction in OCT4 confirms previous reports suggesting that OCT4 expression is post-transcriptionally decreased by LIN28A depletion (Qiu et al., 2010). Those markers were not significantly affected upon TUT4 knockdown (Figure 7C, bottom). Notably, LIN28A-siR expression efficiently sustained the pluripotency and suppressed differentiation of hESCs subjected to LIN28A-depletion compared to vector or K135R-siR expression (Figure 7D). Collectively, our findings suggest that LIN28A and SET7/9 play key functions in regulating pluripotency in hESCs via modulation of the let-7 miRNA.

DISCUSSION

In recent years it has become clear that histones are not the sole physiological substrates of the histone-modifying enzymes, and that post-translational modifications, alone or together with other specific modifications of non-histone proteins may play equally critical roles in diverse intracellular pathways (Freiman and Tjian, 2003; Gu and Roeder, 1997; Turner, 2002). This was further supported by the “protein code” hypothesis, which was based on the “histone code” hypothesis (Jenuwein and Allis, 2001; Strahl and Allis, 2000). Here, we reveal that LIN28A is specifically mono-methylated at K135 by SET7/9, an exclusive H3K4 mono-methyltransferase. This SET7/9-mediated methylation significantly contributes to the protein stability, the multimeric binding to pri-let-7, and subcellular localization of LIN28A to the nucleus. In pluripotent cells, we suggest that the LIN28A methylation signal in the nucleus might be a prerequisite for maximally inhibiting the processing of let-7 miRNA, thereby modulating pluripotency and differentiation. Together, these results provide a novel mechanism through which methylation regulates the function of LIN28A in hESCs.

The K135 and its surrounding residues within LIN28A have sequential homology to the NoLS of LIN28B (Figures 1, S1H, and S3A) (Piskounova et al., 2011), suggesting that LIN28A/B may share a specific role in the nucleoli (Figures 3 and 4). The SET7/9-mediated methylation of LIN28A may allow this region to function in a manner similar to the NoLS of LIN28B and to act as a switch which causes nuclear retention of LIN28A, explaining why a methylated LIN28A is found in the nucleoli of ESCs. This LIN28A methylation signal in the nucleus stimulates the faster formation of methylation-related multimeric binding which may function as an active nuclear retention signal, especially in the nucleoli that may efficiently isolate and protect LIN28A/let-7 from degradation, thereby stabilizing it. Notably, the microprocessor components DGCR8 and Drosha are absent in nucleoli (Piskounova et al., 2011), suggesting that sequestration of pri-let-7 to this cellular compartment has evolved as a mechanism to withhold a pool of nuclear pri-let-7 from processing. Interestingly, overexpressed FLAG-LIN28A was remarkably methylated by endogenous SET7/9 in 293T cells (Figures 1D, 2A, S4A and S4B). This finding led us to speculate that the previously reported ability of LIN28A to block pri-let-7 processing in the nucleus of 293T cells (Heo et al., 2008; Viswanathan et al., 2008) may reflect the function of methylated LIN28A. For the sophisticated regulation of coding or noncoding RNA stability/processing (Shyh-Chang and Daley, 2013), we suggest that a limited quantity of LIN28A may be retained in the nucleus via the methylation signal (Figures 3 and S3). If the quantitative balance between LIN28A and its target RNAs is disrupted in the nucleus, diverse cellular processes that may affect stemness might be triggered, such as off-target effects through binding to non-specific RNAs, inappropriate RNA splicing/processing, and microRNA-directed transcriptional gene silencing (Benhamed et al., 2012; Shyh-Chang and Daley, 2013). Collectively, the delicate methylation-mediated regulation of the quantity and quality of LIN28A in the nucleus appears to be necessary to specifically control the stability and processing of RNAs, including let-7 in ESCs.

The methyltransferase of LIN28A, SET7/9 is located in the nucleus and surprisingly the methylated nuclear LIN28A seems to rapidly form the multimeric complex on pri-let-7 miRNA rather than the unmethylated cytoplasmic LIN28A (Figures 3 and 5) which may be transported to the nucleus though a methylation independent mechanism. Our group and others have found that multimeric binding appears to be correlated with efficient sequestration of pri-let-7 in nucleoli and maximum inhibition of its processing (Figure 4) (Desjardins et al., 2014). Thus, we speculated about the presence of a binding partner (or complex) and shuttle system of the nuclear LIN28A between nucleoli and nucleoplasm, and the LIN28A-pri-let-7-multimeric complex may rapidly enter the nucleoli through a shuttle system to significantly inhibit the microprocessor-mediated cleavage of pri-let-7 (Desjardins et al., 2014; Viswanathan et al., 2008). Indeed, the multimeric assembly may indicate the presence of multiple binding sites on the multimeric complex (Desjardins et al., 2014). A binding partner may specifically recognize that nuclear LIN28A belongs to the multimeric complex and rapidly take it to the nucleoli faster than the LIN28A mono- or di-meric complex. Further studies are required to examine the potential mechanism(s) responsible for the specific recognition of the nuclear LIN28A by a binding partner (or together with its shuttle system), and the fate as well as role of the multimeric complex in the nucleoli of ESCs for the dynamic functional regulation of let-7 miRNAs to understand the various regulatory pathways of pluripotency.

Induction of early lineage markers in LIN28A-knockdown cells was milder than expected (Figures 7A). This may be due to the modest changes in the levels of pluripotency factors (Figures 7B and S6B), and suggests that induction of lineage-specific markers may be predominantly dependent on let-7-mediated differentiation during this period. Therefore, induction of mature let-7 appears to occur via direct action of nuclear LIN28A on pri-let-7 biogenesis and cytoplasmic LIN28A on pre-let-7 miRNA biogenesis, rather than indirect effects of differentiation mediated by other factors. Indeed, several known target genes of let-7 (e.g., RAS, MYCN, CDC25A, and CCND2) were moderately but consistently affected during early stages of differentiation (r = 0.98) (Figure S6F).

Our present work and other reports (Heo et al., 2008; Newman et al., 2008; Rybak et al., 2008; Viswanathan et al., 2008) reveal that LIN28A functions in both the nucleus and cytoplasm to block let-7 processing. Although we show that the nuclear LIN28A and LIN28B can bind to both pri- and pre-let-7 RNAs in figures 5B and 5C and other reports (Heo et al., 2008; Viswanathan et al., 2008), and it remains formally possible that the nuclear LIN28A/B may localize in the cytoplasm to efficiently utilize TUTase in certain contexts or cell types (Guo et al., 2006; Heo et al., 2008), the nuclear LIN28A/B mostly functions in the nucleus to sequester and inhibit the processing of pri-let-7 miRNAs via a TUTase-independent mechanism (Piskounova et al., 2011). Indeed, pri-let-7 miRNAs seem to be a better binding substrate of the nuclear LIN28A (Figures 5B-E). Thus, the LIN28-mediated regulation of pri-let-7 in the nucleus may be a critical blockading step in the let-7 miRNA-processing pathway. Intriguingly, the expressions of LIN28A/B are mutually exclusive in diverse cells (Piskounova et al., 2011) and compensatory redundancy has been observed between them (Wilbert et al., 2012). Thus, it appears likely that nuclear LIN28A may take over the function of LIN28B in certain types of cells, such as embryonic cells, certain differentiated cells, and the various cancer cells that exhibit depletion of LIN28B.

EXPERIMENTAL PROCEDURES

Detailed procedures can be found in the Supplemental Experimental Procedures.

In vitro methyltransferase assays

In vitro methyltransferase assays were performed with recombinant proteins. Protein preparation is described in the Supplemental Experimental Procedures.

Cell lines

H9 hESCs were cultured in ESC medium and grown on mitomycin C (Sigma-Aldrich, St. Louis, MO)-treated mouse embryonic fibroblast. NCCIT and 293T cells were cultured in RPMI 1640 (Gibco, Grand Island, NY) and DMEM (Hyclone, South Logan, Utah). All cells were maintained at 37°C, 5% CO2.

Electrophoretic mobility shift assay (EMSA)

EMSA was performed as described (Piskounova et al., 2008). Briefly, purified nuclear FLAG-LIN28A or FLAG-LIN28A-K135R proteins were reacted with 1.3nM of 5’-end 32P-labeled pri- or pre-let-7a-1 RNA within binding buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10 mM β-mercaptoethanol and 5% glycerol) and incubated 40 min at room temperature (RT). Bound complexes were resolved 5% native polyacrylamide gels at 200V, 4℃ for 2 hours. The gels were dried, exposed, and detected by FLA-7000 (GE Healthcare). Band intensities were quantified with Image J software and were used to calculate the percentage of bound fractions.

Immunofluorescence

Immunofluorescence was performed as described (Piskounova et al., 2011) with minor modification. Cells were fixed in 4% formaldehyde (HT5011, Sigma-Aldrich, St. Louis, MO) at room temperature (RT) for 20 min, washed three times with PBST (PBS containing 0.1% Tween 20; P7949, Sigma-Aldrich, St. Louis, MO) for 15 min, permeabilized with 0.1% Triton X-100 (T8532, Sigma-Aldrich, St. Louis, MO) in PBS, and blocked with blocking solution (3% fetal bovine serum; A9647, Sigma-Aldrich, St. Louis, MO) at RT for 1 h. Cells were reacted with primary antibodies against LIN28A, LIN28A-K135-me1, SET7/9, and Fibrillarin (diluted 1:200 with blocking solution) at 4°C overnight and washed six times with PBST. After washing, the cells were incubated with Alexa-488- or -594-conjugated secondary antibodies (Invitrogen, Eugene, OR; diluted 1:300) in the dark at RT for 1 hr, and then washed six times with PBST. During the washing step, DNA was counterstained with DAPI (4′-6-diamidino-2-phenylindole; D5942, Sigma-Aldrich, St. Louis, MO). Finally, fluorescence images were captured on a Zeiss LSM 510 confocal microscope equipped with argon and helium-neon lasers (Carl Zeiss, Germany).

Alkaline phosphatase activity was measured using an alkaline phosphatase detection kit (M8168, Sigma-Aldrich, St. Louis, MO) according to the manufacturer's guidelines, with minor modifications. Briefly, dissociated single H9 and NCCIT cells were plated in 4-well plates, incubated overnight, and transfected with 100 nM of LIN28A, SET7/9, or 60 nM of control siRNA. After three days of siRNA transfection, the cells were fixed in citrate-acetone-formaldehyde solution for 1 min at RT, incubated in AP staining solution (Naphthol/Fast Red violet) for 15 min in the dark, and washed. Images were obtained under a microscope (Olympus, Japan).

Quantitative RT-PCR

RNA samples were transcribed with the Improm Kit (A3802, Promega, Madison, WI) according to the manufacturer's guidelines. Real-time PCR analysis was performed with 2X h-Taq real-time mix (Solgent, Daejeon, Korea) and 20X Evagreen (Biotium, 31000). PCR reactions were performed on CFX96 (Bio-Rad Laboratories, Hercules, CA, USA) with the primers (Tables S1 and S2) listed in Supplemental Experimental Procedures.

Microarray

Total RNAs were hybridized to whole-human gene expression microarray (Agilent) in accordance with the manufacturer's instruction. Original data are available in the NCBI Gene Expression Omnibus (accession number GSE53038).

Supplementary Material

1
2
3
4
5
6
7

ACKNOWLEDGMENTS

We thank Drs. Ryan D. Mohan and Tamaki Suganuma for critical readings and discussions. We wish to acknowledge V. Narry Kim for kindly providing the pRL-CMV, pGL3-CMV-let-7a binding sites, pGL3-CMV-miR-16 binding site, and pCK-FLAG-AGO2 constructs. This work was supported by grants from the Stem Cell Research Program (2011–0019509 and 2012M 3A9B 4027953), the Basic Science Research Program (NRF-2012R1A6A3A01038981), the KAIST Future Systems Healthcare Project, and the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (2011-0031955). This work was also funded by The Welch foundation I-1786, The Klingenstein Fund, and NIH-NINDS R01NS085418 (T.-K.K).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AUTHOR CONTRIBUTIONS

S-K. Kim and D. Lee designed the concepts and experiments. S-K. Kim performed most of the experiments. H. Lee performed EMSA experiment and mainly assisted in the research. K. Han, Y. Choi, S-W. Park, G. Bak, SC Park and Y. Lee assisted in the research. S-K. Kim, SC. Kim, and JK. Choi analyzed the microarray data. JK. Choi and T-K. Kim commented on the manuscript. S-K. Kim wrote the manuscript under the technical supervision and mentorship of Y-M. Han and D. Lee. All authors have read and approved the final manuscript.

SUPPLEMENTAL INFORMATION

The Supplemental Information, which includes Supplemental Experimental Procedures, 7 figures and 7 tables, can be found with this article online at http://.

The authors declare that no competing interest exists.

REFERENCES

  1. Ambros V, Horvitz HR. Heterochronic mutants of the nematode Caenorhabditis elegans. Science. 1984;226:409–416. doi: 10.1126/science.6494891. [DOI] [PubMed] [Google Scholar]
  2. Balzer E, Moss EG. Localization of the developmental timing regulator Lin28 to mRNP complexes, P-bodies and stress granules. RNA Biol. 2007;4:16–25. doi: 10.4161/rna.4.1.4364. [DOI] [PubMed] [Google Scholar]
  3. Benhamed M, Herbig U, Ye T, Dejean A, Bischof O. Senescence is an endogenous trigger for microRNA-directed transcriptional gene silencing in human cells. Nat Cell Biol. 2012;14:266–275. doi: 10.1038/ncb2443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell. 2005;122:947–956. doi: 10.1016/j.cell.2005.08.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang H-M, Triboulet R, Thornton JE, Gregory RI. A role for the Perlman syndrome exonuclease Dis3l2 in the Lin28-let-7 pathway. Nature. 2013;497:244–248. doi: 10.1038/nature12119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen L-L, Carmichael GG. Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells: functional role of a nuclear noncoding RNA. Mol Cell. 2009;35:467–478. doi: 10.1016/j.molcel.2009.06.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell. 2008;133:1106–1117. doi: 10.1016/j.cell.2008.04.043. [DOI] [PubMed] [Google Scholar]
  8. Chuikov S, Kurash JK, Wilson JR, Xiao B, Justin N, Ivanov GS, McKinney K, Tempst P, Prives C, Gamblin SJ, et al. Regulation of p53 activity through lysine methylation. Nature. 2004;432:353–360. doi: 10.1038/nature03117. [DOI] [PubMed] [Google Scholar]
  9. Couture J-F, Collazo E, Hauk G, Trievel RC. Structural basis for the methylation site specificity of SET7/9. Nat Struct Mol Biol. 2006;13:140–146. doi: 10.1038/nsmb1045. [DOI] [PubMed] [Google Scholar]
  10. Desjardins A, Bouvette J, Legault P. Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors. Nucleic Acids Res. 2014 doi: 10.1093/nar/gkt1391. doi:10.1093/nar/gkt1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Estève P-O, Chin HG, Benner J, Feehery GR, Samaranayake M, Horwitz GA, Jacobsen SE, Pradhan S. Regulation of DNMT1 stability through SET7-mediated lysine methylation in mammalian cells. Proc Natl Acad Sci USA. 2009;106:5076–5081. doi: 10.1073/pnas.0810362106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Evans PM, Zhang W, Chen X, Yang J, Bhakat KK, Liu C. Krüppel-like factor 4 is acetylated by p300 and regulates gene transcription via modulation of histone acetylation. J Biol Chem282. 2007:33994–34002. doi: 10.1074/jbc.M701847200. [DOI] [PubMed] [Google Scholar]
  13. Fang L, Zhang L, Wei W, Jin X, Wang P, Tong Y, Li J, Du JX, Wong J. A Methylation-Phosphorylation Switch Determines Sox2 Stability and Function in ESC Maintenance or Differentiation. Mol Cell. 2014 doi: 10.1016/j.molcel.2014.06.018. [DOI] [PubMed] [Google Scholar]
  14. Freiman RN, Tjian R. Regulating the regulators: lysine modifications make their mark. Cell. 2003;112:11–17. doi: 10.1016/s0092-8674(02)01278-3. [DOI] [PubMed] [Google Scholar]
  15. Gu W, Roeder RG. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell. 1997;90:595–606. doi: 10.1016/s0092-8674(00)80521-8. [DOI] [PubMed] [Google Scholar]
  16. Guo Y, Chen Y, Ito H, Watanabe A, Ge X, Kodama T, Aburatani H. Identification and characterization of lin-28 homolog B (LIN28B) in human hepatocellular carcinoma. Gene. 2006;384:51–61. doi: 10.1016/j.gene.2006.07.011. [DOI] [PubMed] [Google Scholar]
  17. Hagan JP, Piskounova E, Gregory RI. Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells. Nat Struct Mol Biol. 2009;16:1021–1025. doi: 10.1038/nsmb.1676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heo I, Ha M, Lim J, Yoon M-J, Park J-E, Kwon SC, Chang H, Kim VN. Mono-uridylation of pre-microRNA as a key step in the biogenesis of group ii let-7 microRNAs. Cell. 2012;151:521–532. doi: 10.1016/j.cell.2012.09.022. [DOI] [PubMed] [Google Scholar]
  19. Heo I, Joo C, Cho J, Ha M, Han J, Kim VN. Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA. Mol Cell. 2008;32:276–284. doi: 10.1016/j.molcel.2008.09.014. [DOI] [PubMed] [Google Scholar]
  20. Heo I, Joo C, Kim Y-K, Ha M, Yoon M-J, Cho J, Yeom K-H, Han J, Kim VN. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell. 2009;138:696–708. doi: 10.1016/j.cell.2009.08.002. [DOI] [PubMed] [Google Scholar]
  21. Jenuwein T, Allis CD. Translating the histone code. Science. 2001;293:1074–1080. doi: 10.1126/science.1063127. [DOI] [PubMed] [Google Scholar]
  22. Ji H, Wu G, Zhan X, Nolan A, Koh C, De Marzo A, Doan HM, Fan J, Cheadle C, Fallahi M, et al. Cell-type independent MYC target genes reveal a primordial signature involved in biomass accumulation. PLoS One. 2011;6:e26057. doi: 10.1371/journal.pone.0026057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim S-K, Jung I, Lee H, Kang K, Kim M, Jeong K, Kwon CS, Han Y-M, Kim YS, Kim D, et al. Human histone H3K79 methyltransferase DOT1L protein binds actively transcribing RNA polymerase II to regulate gene expression. J Biol Chem. 2013;287:39698–39709. doi: 10.1074/jbc.M112.384057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kim VN. MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol. 2005;6:376–385. doi: 10.1038/nrm1644. [DOI] [PubMed] [Google Scholar]
  25. Kontaki H, Talianidis I. Lysine methylation regulates E2F1-induced cell death. Mol Cell. 2010;39:152–160. doi: 10.1016/j.molcel.2010.06.006. [DOI] [PubMed] [Google Scholar]
  26. Kouskouti A, Scheer E, Staub A, Tora L, Talianidis I. Gene-specific modulation of TAF10 function by SET9-mediated methylation. Mol Cell. 2004;14:175–182. doi: 10.1016/s1097-2765(04)00182-0. [DOI] [PubMed] [Google Scholar]
  27. Malynn BA, de Alboran IM, O'Hagan RC, Bronson R, Davidson L, DePinho RA, Alt FW. N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation. Genes Dev. 2000;14:1390–1399. [PMC free article] [PubMed] [Google Scholar]
  28. Melton C, Judson RL, Blelloch R. Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature. 2010;463:621–626. doi: 10.1038/nature08725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moretto-Zita M, Jin H, Shen Z, Zhao T, Briggs SP, Xu Y. Phosphorylation stabilizes Nanog by promoting its interaction with Pin1. Proc Natl Acad Sci USA. 2010;107:13312–13317. doi: 10.1073/pnas.1005847107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moss EG, Tang L. Conservation of the heterochronic regulator Lin-28, its developmental expression and microRNA complementary sites. Dev Biol. 2003;258:432–442. doi: 10.1016/s0012-1606(03)00126-x. [DOI] [PubMed] [Google Scholar]
  31. Nam Y, Chen C, Gregory RI, Chou JJ, Sliz P. Molecular basis for interaction of let-7 microRNAs with Lin28. Cell. 2011;147:1080–1091. doi: 10.1016/j.cell.2011.10.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Newman MA, Thomson JM, Hammond SM. Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing. RNA. 2008;14:1539–1549. doi: 10.1261/rna.1155108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nishioka K, Chuikov S, Sarma K, Erdjument-Bromage H, Allis CD, Tempst P, Reinberg D. Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev. 2002;16:479–489. doi: 10.1101/gad.967202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Piskounova E, Polytarchou C, Thornton JE, LaPierre RJ, Pothoulakis C, Hagan JP, Iliopoulos D, Gregory RI. Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms. Cell. 2011;147:1066–1079. doi: 10.1016/j.cell.2011.10.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Piskounova E, Viswanathan SR, Janas M, LaPierre RJ, Daley GQ, Sliz P, Gregory RI. Determinants of microRNA processing inhibition by the developmentally regulated RNA-binding protein Lin28. J Biol Chem. 2008;283:21310–21314. doi: 10.1074/jbc.C800108200. [DOI] [PubMed] [Google Scholar]
  36. Polesskaya A, Cuvellier S, Naguibneva I, Duquet A, Moss EG, Harel-Bellan A. Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency. Genes Dev. 2007;21:1125–1138. doi: 10.1101/gad.415007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Qiu C, Ma Y, Wang J, Peng S, Huang Y. Lin28-mediated post-transcriptional regulation of Oct4 expression in human embryonic stem cells. Nucleic Acids Res. 2010;38:1240–1248. doi: 10.1093/nar/gkp1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rybak A, Fuchs H, Smirnova L, Brandt C, Pohl EE, Nitsch R, Wulczyn FG. A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment. Nat Cell Biol. 2008;10:987–993. doi: 10.1038/ncb1759. [DOI] [PubMed] [Google Scholar]
  39. Shyh-Chang N, Daley GQ. Lin28: primal regulator of growth and metabolism in stem cells. Cell Stem Cell. 2013;12:395–406. doi: 10.1016/j.stem.2013.03.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Smith KN, Singh AM, Dalton S. Myc represses primitive endoderm differentiation in pluripotent stem cells. Cell Stem Cell. 2010;7:343–354. doi: 10.1016/j.stem.2010.06.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Strahl BD, Allis CD. The language of covalent histone modifications. Nature. 2000;403:41–45. doi: 10.1038/47412. [DOI] [PubMed] [Google Scholar]
  42. Subramanian K, Jia D, Kapoor-Vazirani P, Powell DR, Collins RE, Sharma D, Peng J, Cheng X, Vertino PM. Regulation of estrogen receptor α by the SET7 lysine methyltransferase. Mol Cell. 2008;30:336–347. doi: 10.1016/j.molcel.2008.03.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tao Y, Neppl RL, Huang Z-P, Chen J, Tang R-H, Cao R, Zhang Y, Jin S-W, Wang D-Z. The histone methyltransferase Set7/9 promotes myoblast differentiation and myofibril assembly. J Cell Biol. 2011;194:551–565. doi: 10.1083/jcb.201010090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Turner BM. Cellular memory and the histone code. Cell. 2002;111:285–291. doi: 10.1016/s0092-8674(02)01080-2. [DOI] [PubMed] [Google Scholar]
  45. Viswanathan SR, Daley GQ, Gregory RI. Selective blockade of microRNA processing by Lin28. Science. 2008;320:97–100. doi: 10.1126/science.1154040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Viswanathan SR, Powers JT, Einhorn W, Hoshida Y, Ng TL, Toffanin S, O'Sullivan M, Lu J, Phillips LA, Lockhart VL, et al. Lin28 promotes transformation and is associated with advanced human malignancies. Nat Genet. 2009;41:843–848. doi: 10.1038/ng.392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang L.-x., Wang J, Qu T.-t, Zhang Y, Shen Y.-f. Reversible acetylation of Lin28 mediated by PCAF and SIRT1. Biochim Biophys Acta. 2014;1843:1188–1195. doi: 10.1016/j.bbamcr.2014.03.001. [DOI] [PubMed] [Google Scholar]
  48. Wei F, Schöler HR, Atchison ML. Sumoylation of Oct4 enhances its stability, DNA binding, and transactivation. J Biol Chem. 2007;282:21551–21560. doi: 10.1074/jbc.M611041200. [DOI] [PubMed] [Google Scholar]
  49. West JA, Viswanathan SR, Yabuuchi A, Cunniff K, Takeuchi A, Park I-H, Sero JE, Zhu H, Perez-Atayde A, Frazier AL, et al. A role for Lin28 in primordial germ-cell development and germ-cell malignancy. Nature. 2009;460:909–913. doi: 10.1038/nature08210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wilbert ML, Huelga SC, Kapeli K, Stark TJ, Liang TY, Chen SX, Yan BY, Nathanson JL, Hutt KR, Lovci MT, et al. LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance. Mol Cell. 2012;48:195–206. doi: 10.1016/j.molcel.2012.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, Huang Y, Hu X, Su F, Lieberman J, et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell. 2007a;131:1109–1123. doi: 10.1016/j.cell.2007.10.054. [DOI] [PubMed] [Google Scholar]
  52. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007b;318:1917–1920. doi: 10.1126/science.1151526. [DOI] [PubMed] [Google Scholar]
  53. Zhu H, Shyh-Chang N, Segrè AV, Shinoda G, Shah SP, Einhorn WS, Takeuchi A, Engreitz JM, Hagan JP, Kharas MG. The Lin28/let-7 axis regulates glucose metabolism. Cell. 2011;147:81–94. doi: 10.1016/j.cell.2011.08.033. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS642184-supplement-1.docx (149.8KB, docx)
2
NIHMS642184-supplement-2.pdf (6.4MB, pdf)
3
NIHMS642184-supplement-3.xlsx (31.3KB, xlsx)
4
NIHMS642184-supplement-4.xlsx (17.6KB, xlsx)
5
NIHMS642184-supplement-5.xlsx (21.6KB, xlsx)
6
NIHMS642184-supplement-6.xlsx (29.1KB, xlsx)
7
NIHMS642184-supplement-7.xlsx (25.5KB, xlsx)

RESOURCES