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. Author manuscript; available in PMC: 2016 Oct 27.
Published in final edited form as: Cell Rep. 2016 Oct 11;17(3):809–820. doi: 10.1016/j.celrep.2016.09.046

Sirtuin 1 Promotes Deacetylation of Oct4 and Maintenance of Naïve Pluripotency

Eric O Williams 1, Amy K Taylor 1, Eric L Bell 1, Rachelle Lim 1, Daniel M Kim 1, Leonard Guarente 1,*
PMCID: PMC5081691  NIHMSID: NIHMS821474  PMID: 27732856

Abstract

The enhancer landscape is dramatically restructured as naïve preimplantation epiblasts transition to the post-implantation state of primed pluripotency. A key factor in this process is Otx2, which is upregulated during the early stages of this transition and ultimately recruits Oct4 to a different set of enhancers. In this study we discover that the acetylation status of Oct4 regulates the induction of the primed pluripotency gene network. Maintenance of the naïve state requires the NAD-dependent deacetylase, SirT1, which deacetylates Oct4. The activity of SirT1 is reduced during the naïve to primed transition; Oct4 becomes hyper-acetylated and binds to an Otx2 enhancer to induce Otx2 expression. Induction of Otx2 causes the reorganization of acetylated Oct4 and results in the induction of the primed pluripotency gene network. Regulation of Oct4 by SirT1 may link stem cell development to environmental conditions and provide strategies to manipulate epiblast cell state.

Graphical abstract

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Introduction

During embryogenesis, an exquisite program of ordered events enables a single zygote to develop into a multicellular organism with diverse cell types. A critical step in this process is the ability of pluripotent epiblast cells to coordinate the balance between self-renewal and lineage specification (Kunath et al., 2007; Niwa, 2007; Silva and Smith, 2008). This task is thought to be at least partially accomplished by the transition of these cells through at least two distinct pluripotent states (Brons et al., 2007; Tesar et al., 2007).

Pre-implantation epiblast cells exhibit “naïve” or ground state pluripotency. This state has been defined as fully unrestricted with the ability to contribute to all embryonic lineages (Nichols and Smith, 2009; Rossant, 2008). Embryonic stem cell lines derived from pre-implantation mouse blastocysts exhibit characteristics of naïve pluripotency (Evans and Kaufman, 1981; Nichols and Smith, 2011). These include a rounded morphology, the maintenance of self-renewal through Jak/Stat3 and Bmp4 (Matsuda et al., 1999; Niwa et al., 2009; Ying et al., 2003), single cell clonogenicity, and efficient contribution to chimeras (Han et al., 2010).

During embryo implantation, cells of the epiblast layer transition from the naïve state to the “primed” pluripotency state (Arnold and Robertson, 2009; Brons et al., 2007; Kojima et al., 2014; Tesar et al., 2007). While still capable of contributing to all three germ lineages, primed epiblast stem cells are thought to be more developmentally restricted than naïve embryonic stem cells and undergo X- chromosome inactivation (Bao et al., 2009; Guo et al., 2009; Heard, 2004; Silva et al., 2008). Indeed, chimeric contribution of pluripotent primed cells is significantly less efficient than that of pluripotent naïve cells (Brons et al., 2007; Han et al., 2010; Tesar et al., 2007). Primed cells can be cultured by obtaining epiblast cells of the early post-implantation mouse embryo (Nichols and Smith, 2011). The primed state can also be induced in vitro by the addition of Fgf and Activin A to naïve cells (Brons et al., 2007; Tesar et al., 2007; Thomson et al., 2011). In the primed state, cultured stem cells are morphologically flat and exhibit low single cell clonogenicity.

Interestingly, human embryonic stem cells derived from pre-implantation blastocysts resemble primed murine stem cells more than naïve cells (Brons et al., 2007; Rossant, 2008; Tesar et al., 2007). In addition, human induced pluripotent stem (IPS) cells also exhibit features of the primed state of pluripotency (Yamanaka et al., 2007; Yu et al., 2007), while mouse IPS cells naturally revert to the naïve state (Takahashi and Yamanaka, 2006). While the reasons for these mouse-human differences remain unclear, there are a number of practical advantages that make the naïve state a more desirable research tool. Pluripotent naïve stem cells are characterized by a more open chromatin structure (Murtha et al., 2015), allowing for more efficient genetic manipulation (Buecker et al., 2010; Zwaka and Thomson, 2003). In addition primed cells are prone to greater heterogeneity in gene expression (Bernemann et al., 2011; Gafni et al., 2013; Osafune et al., 2008), making it difficult to obtain unbiased lineage-specific specification. Lastly, naïve human embryonic stem cells are capable of contributing to cross-species chimeras (Gafni et al., 2013), a tool that will likely be useful for the creation of “humanized” animal models. Several attempts have been made to stably reprogram human embryonic stem cells to the naïve state using both genetic (Takashima et al., 2014; Wang et al., 2011) and chemical methodologies (Chan et al., 2013; Duggal et al., 2015; Gafni et al., 2013; Hanna et al., 2010; Theunissen et al., 2014; Valamehr et al., 2014; Ware et al., 2014). These studies have been met with varying levels of success, but the robustness of these protocols with regards to the long term culture of the naïve state is still being evaluated (Dodsworth et al., 2015).

Understanding the molecular mechanisms driving the naïve to primed transition is crucial for comprehending the regulation of mammalian development and the optimization of experimental protocols for stem cell therapy. One important observation is the dramatic difference in the enhancer chromatin landscape between the naïve and primed states (Buecker et al., 2014; Factor et al., 2014; Sohni et al., 2015; Tesar et al., 2007; Yang et al., 2014; Yeom et al., 1996). Indeed, recent epigenomic analysis concluded that enhancer usage is the most distinguishing factor between these two states. Interestingly, enhancer usage changes are observed in both differentially expressed genes and also in genes with similar expression levels between these two states (Factor et al., 2014). An example is Oct4, a transcription factor whose expression levels promote self-renewal and inhibits differentiation of stem cells (Nichols et al., 1998; Radzisheuskaya et al., 2013). Oct4 enhancer activity shifts from a distal enhancer dependent on Oct4 itself in naïve cells to a proximal, Oct4-independent enhancer in primed cells without affecting the total expression level of the gene (Buecker et al., 2014; Yang et al., 2014).

Oct4 reorganization is driven by the up-regulation of the transcription factor, Otx2, early in the transition from naïve to primed states. Oct4 is required for the early upregulation of Otx2 and Otx2 is required for the recruitment of Oct4 to “primed” enhancers (Acampora et al., 2013; Buecker et al., 2014; Yang et al., 2014). Importantly, the induction of Otx2 expression by Oct4 is sufficient to induce the transition from the naive to the primed state (Buecker et al., 2014; Yang et al., 2014).

Here, we find that Oct4 is post-translationally modified by acetylation in mouse embryonic stem cells, and that Oct4 acetylation status is a key component of the naïve to primed transition. During this transition, activity of the NAD+ dependent deacetylase, SirT1, decreases and concomitantly, acetylation of Oct4 increases. Indeed, knocking out SirT1 in naïve ES cells increases Oct4 acetylation and results in the partial upregulation of the primed pluripotency network. Importantly, the hyper-acetylated Oct4 in SirT1 knockout ES cells displays increased occupancy at an Otx2 enhancer element, and thus induces Otx2 expression. Inactivation of SirT1 in wild type ES cells therefore provides a mechanism for the induction of Otx2 early in the naïve to primed transition. Lastly, knocking down Otx2 expression in SirT1 knock out cells reverses the induction of the primed network, thus linking the SirT1-Oct4-Otx2 axis to the naïve to primed transition of epiblasts.

Results

SirT1 Activity Decreases during the Transition from Naïve to Primed Pluripotency

Previous reports indicate that SirT1 is expressed at high levels in pluripotent stem cells and these levels decline considerably during lineage specification (Calvanese et al., 2010; McBurney et al., 2003a, 2003b; Sakamoto et al., 2004). We decided to use mouse embryoid bodies to model the differentiation that normally occurs in early post-implantation embryos (Desbaillets et al., 2000). Consistent with previous findings, we observed a decrease in SirT1 protein levels as pluripotent mouse embryonic stem cells transition from self-renewal (high Oct4 expression) towards lineage specification (low Oct4 expression) (Figure 1A).

Figure 1. SirT1 expression and activity in mouse embryonic stem cells.

Figure 1

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(A) SirT1 expression during embryoid body differentiation. Embryoid bodies were differentiated for 0, 2, 4, 6, 8, 10, and 12 days. Full length SirT1 protein is denoted with an arrow. SirT1 and Oct4 expression decline coordinately during differentiation.

(B) Immunocytochemistry of SirT1 and Oct4 in mouse embryonic stem cells grown in serum/LIF conditions. DAPI is blue, Oct4 is Red, and SirT1 is green. Row 1: wild type cells at 5X magnification (scale bar = 200um), Row 2: wild type cells at 63X magnification (scale bar = 15um), Row 3: SirT1 null cells at 63X magnification (scale bar = 15um). Yellow arrows highlight areas with relatively high protein levels of Oct4 and SirT1. White arrows highlight areas with relatively low protein levels of Oct4 and SirT1.

(C) SirT1 expression and activity during the transition from naïve pluripotency to primed EpiLCs. Wild type mouse embryonic stem cells were grown in naïve and primed EpiLC conditions. SirT1 and Oct4 protein levels are similar between naïve and primed EpiLC cells. Nanog protein levels are reduced in the EpiLC state. p53 acetylation at Lys379 is increased in EpiLCs.

(D) Immunocytochemistry of SirT1 and H3K4me3. DAPI is blue, SirT1 is red, H3K4me3 is green (scale bar = 5um).

Interestingly, the levels of SirT1 protein closely mimic that of Oct4, the master pluripotency transcription factor (Figure 1A). To determine the extent of SirT1/Oct4 co-regulation we performed immunocytochemistry in mouse embryonic stem cells grown in serum/LIF conditions. Mouse embryonic stem cells grown in serum/LIF exist in multiple stages of pluripotency and exhibit heterogeneous transcription factor expression (Torres-Padilla and Chambers, 2014; Ying et al., 2008). We identified colonies within our cultures with heterogeneous expression of Oct4. We observed that cells expressing the highest levels of Oct4 also have the highest levels of SirT1 expression (Figure 1B, yellow arrows). Conversely, cells with lower expression of Oct4 also have low levels of SirT1 (Figure 1B, white arrows), indicating a degree of co-regulation between SirT1 and Oct4. This may be a conserved mechanism between humans and mice as loss of Oct4 expression in human embryonic stem cells has also been reported to reduce SirT1 expression (Zhang et al., 2014). We also found that SirT1 is localized within the nucleus as evidenced by its colocalization with DAPI and the activating histone modification, (H3K4me3)(Heintzman et al., 2007) (Figure 1D). No SirT1 signal was detected in SirT1 null embryonic stem cells demonstrating the specificity of the antibody (Figure 1B).

To assess SirT1 expression during the naïve to prime transition we employed the previously described EpiLC differentiation protocol (Buecker et al., 2014; Hayashi et al., 2011; Yang et al., 2014). Mouse embryonic stem cells were grown under serum-free 2i LIF conditions (MEK/ERK inhibitor, GSK3β inhibitor, and recombinant mouse LIF) to promote the naïve state. Primed pluripotency was induced by stimulating with Fgf2 and Activin A. Within 48 hours, naïve embryonic stem cells transition to primed epiblast-like cells (EpiLCs). Consistent with previous studies we found little to no change in Oct4 expression, but dramatically reduced expression of Nanog in the primed state (Silva et al., 2009) (Figure 1C). Genome wide transcriptional analysis of naïve ES cells and EpiLCs revealed significant overlap with previously characterized primed pluripotency datasets (Kim et al., 2013; Buecker et al., 2014) (Figure S1 A-D). While SirT1 levels remain unchanged between the two states, SirT1 activity was significantly reduced in primed EpiLCs. p53, a canonical target of SirT1 deacetylation (Vaziri et al., 2001), was found to be hyper-acetylated at Lysine 379 in EpiLCs (Figure 1C), indicating a reduction in SirT1 activity during the transition to primed pluripotency. Quantification of p53 acetylation levels revealed a 1.67-1.95 fold increase in EpiLCs (Figure S1E). To confirm SirT1 dependence of p53 acetylation in ES cells we tested p53 acetylation in SirT1 null and wild type ES cells grown in naïve conditions (2i +LIF). p53 was found to be 1.63-2.11 fold more acetylated in SirT1 null ES cells (Figure S1F). To rule out the possibility that decreased HDAC1 levels in EpiLC and SirT1 null ES cell could be causing increased p53 acetylation at lysine 379 (Ito et al., 2002), we performed western blots on HDAC1. We found no difference in HDAC1 protein levels between wild type ES cells, SirT1 null ES cells, and EpiLCs (Figure S1G).

SirT1 Interacts with and Deacetylates Oct4 in the Naïve State

To gain insights into the function of SirT1 in naïve pluripotency, we assessed SirT1 occupancy at Oct4 enhancer domains. The regulation of Oct4 expression within the naïve and primed states is well characterized. Activation of the Oct4 distal enhancer is associated with the promotion of the naïve state, while Oct4 proximal enhancer activity is associated with primed pluripotency (Tesar et al., 2007; Theunissen et al., 2014; Yeom et al., 1996) (Figure 2A). ChIP qPCR analysis of these enhancer regions revealed an enrichment of SirT1 protein within the distal enhancer of Oct4 in naïve ES cells (Figure 2B). Interestingly, the enrichment of SirT1 was strongest at region 3, which was previously characterized to have strong Oct4/Sox2 binding (Chew et al., 2005). Indeed, ChIP qPCR of Oct4 and Sox2 revealed similar occupancy patterns as SirT1 (Figure 2C).

Figure 2. SirT1 deacetylates Oct4 and binds to the Oct4 distal enhancer.

Figure 2

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(A) Genomic structure of the mouse Oct4 gene. The distal enhancer is highlighted in orange and the proximal enhancer is highlighted in blue. The locations of nine qPCR amplicons used in qPCR analysis are identified.

(B) SirT1 ChIP-qPCR of the proximal and distal enhancers of Oct4. Wild type ES cells are represented in blue and SirT1 null ES cells are represented in red. Error bars represent SEM.

(C) Oct4 and Sox2 ChIP-qPCR of the proximal and distal enhancer of Oct4. All samples are from wild type ES cells. Oct4 ChIP is represented in brown and Sox2 ChIP is represented in black. Error bars represent SEM.

(D) Co-immunoprecipitation of SirT1 and Oct4 and analysis of acetylated Oct4. Top 2 rows: pull down with SirT1 (or IgG) antibodies in wild type and SirT1 null ES cells. Western blot with SirT1 antibody in the 1st row and Oct4 antibody in the 2nd row. Middle two rows: pull down with Oct4 (or IgG) antibodies in wild type and SirT1 null ES cells. Western blot with SirT1 antibody in the 3rd row and Oct4 antibody in the 4th row. Bottom two rows: pull down with Oct4 (or IgG) antibodies in wild type and SirT1 null ES cells. Western blot with pan-acetyl lysine antibody in the 5th row and Oct4 antibody in the 6th row.

(E) Acetylation of Oct4 in naïve ES cells and primed EpiLCs. Pull down with Oct4 (or IgG) antibodies in wild type naïve ES cells grown in naïve conditions and wild type primed EpiLCs. Western blot with pan-acetyl lysine antibody on the top row and Oct4 antibody on the bottom row.

(F) Deacetylation of Oct4 in HEK 293T cells. HEK cells were co-transfected with Myc tagged-Oct4, SirT1, and SirT1355A. Western blot of inputs for SirT1, Oct4, and β-actin are on the left and myc-immunopurified samples are on the right. Myc-immunopurified samples were blotted with antibodies against pan-acetyl lysine and Oct4, as shown.

To determine if SirT1 physically interacts with the Oct4 complex, we performed co-immunoprecipitation experiments. Lysates prepared from mouse embryonic stem cells grown in naïve conditions were immunopurified with a SirT1 antibody. Immunoblotting with an Oct4 antibody revealed Oct4 co-immunoprecipitation (Figure 2D). SirT1-Oct4 co-immunoprecipitation was not observed in SirT1 null cells, verifying the specificity of the interaction (Figure 2D). Conversely, immunopurification of Oct4 yielded the co-immunoprecipitation of SirT1. This signal was also abolished in SirT1 null cells (Figure 2D).

To determine if Oct4 is a target of SirT1 mediated deacetylation, we used a pan-acetyl lysine antibody to assess Oct4 acetylation in Oct4 immunopurified lysates. This analysis revealed that Oct4 is hyper-acetylated in SirT1 null cells compared to wild type cells (Figure 2D). Quantification of Oct4 acetylation levels revealed a 1.45 fold increase in the SirT1 null ES cells (Figure S2A). Furthermore, Oct4 hyper-acetylation was observed in wild type ES cells treated with the SirT1 inhibitor, Ex-527 (Figure S2C-D)(Napper et al., 2005; Solomon et al., 2006). Consistent with the observed reduction in SirT1 activity in primed EpiLCs (Figure 1C), we find Oct4 acetylation levels increase during the transition to primed EpiLCs (Figure 2E). Quantification of Oct4 acetylation levels revealed a 1.39-1.64 fold increase in acetylation in EpiLCs (Figure S2B). To rule out the possibility that HATs or other deacetylases present in the primed state result in the increase Oct4 acetylation, we assessed whether SirT1 could deacetylate Oct4 in transfected HEK 293T cells. Myc-tagged Oct4, SirT1, and catalytically inactive SirT1H355A (Rodgers et al., 2005) were co-transfected. Co-expression of SirT1, but not catalytically inactive SirT1H355A, reduced Oct4 acetylation in transfected HEK 293T cells (Figure 2F). Collectively, these results suggest that Oct4 is a direct target of SirT1 mediated deacetylation in naïve cells, and that Oct4 acetylation increases in EpiLCs due to decreased activity of SirT1.

Loss of SirT1 in the Naïve State induces Partial Activation of the Primed Pluripotency Network Morphologically, SirT1 knockout ES cells were found to adopt a slightly flatter morphology when grown in 2i LIF but were indistinguishable from wild type cells when grown in serum with MEFs (data not shown). The cell-autonomous growth rate of SirT1 knockout ES cells was found to be slightly slower than wild type cells (Figure S3A), but no observable differences were identified in apoptosis or cell cycle progression (Figure S3B-D).

Clonogenicity is a distinguishing factor between the naïve and primed states. Naïve ES cells are able to form colonies from single ES cells while single primed cells either differentiate or undergo apoptosis (Brons et al., 2007; Tesar et al., 2007). We found that SirT1 knock out ES cells exhibited similar clonogenicity as wild type when grown in naïve conditions. However, SirT1 knockout ES cells lost clonogenicity faster than wild type ES cells during the transition from the naïve to primed state (Figure S3E). In addition, loss of SirT1 inhibited the reprogramming of primed cells to the naïve state as measured by clonogenicity (Figure S3F). To determine if the SirT1-Oct4 interaction might play a role in the regulation of pluripotency networks, we performed RNAseq transcriptome analysis in SirT1 null embryonic stem cells. Wild type and SirT1 null embryonic stem cells were grown in naïve conditions. In addition, wild type cells were differentiated into EpiLCs in order to define gene networks induced during the naïve to prime transition. We identified 2616 genes as significantly differentially expressed between the naïve and primed EpiLC state. These “primed” genes exhibited extensive overlap with previously published data sets (Buecker et al., 2014) (Kim et al., 2013) (Figure S1A-D). Comparison of wild type-naïve and SirT1 null embryonic stem cells revealed 1535 differentially expressed genes. 931 of these genes overlap with the differential expression observed during the transition from wild type naïve ES cells to wild type primed EpiLCs. Similar overlap was also observed between the expression changes induced by loss of SirT1 and previously published data sets comparing the naïve and primed states (Buecker et al., 2014; Kim et al., 2013) (Figure 3A). The positive correlation between the gene network induced during the transition from naïve to primed EpiLCs and the expression changes caused by the loss of SirT1 had a Pearson product-moment correlation coefficient of R=0.669 (Figure 3B).

Figure 3. Loss of SirT1 in naïve ES cells induces the primed pluripotency gene network.

Figure 3

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(A) Overlap of genes changed in SirT1 null cells and primed datasets. Green circles contain all genes with significant differential expression between SirT1 null cells grown in naïve conditions and wild type cells grown in naïve conditions. Purple circles contain all genes with significant differential expression in primed vs naïve data sets. Top row: Purple circle contains differential expression generated by comparing our wild type differentiated EpiLCs and wild type naïve ES cells. Middle row: Primed data set is defined by Buecker et al., 2014. Data was generated by RNAseq and compares differentiated EpiLC and naïve ES cells. Bottom row: Primed data set is defined by Kim et al., 2013. Data was generated from microarrays comparing mouse epiblast stem cells and mouse embryonic stem cells.

(B) Correlation of expression changes in SirT1 null ES cells and primed EpiLCs. All genes exhibiting significant differential expression in differentiated wild type EpiLC/wild type naïve ES cells are plotted against the gene expression changes comparing SirT1 null/wild type naïve cells. Pearson product-moment correlation coefficient R=0.669 (n=2616).

(C) Western blot analysis of canonical primed pluripotency markers in SirT1 knockout ES cells. Tamoxifen inducible SirT1 knockout cell lines are indicated in lanes 3, 4, 5, and 6. Addition of 1uM tamoxifen induces loss of catalytic exon 4 of SirT1 for these lines and can be observed by the downward shift in SirT1 immunoreactivity (black arrow). Lane 8 contains SirT1 null ES cells. Quantification of the average band intensity for each marker is indicated (average SirT1 knock out intensity/average wild type intensity). All quantification measurements are normalized to β-actin.

Consistent with a shift towards primed pluripotency in SirT1 null ES cells, our RNAseq analysis revealed changes in the expression of important markers of primed pluripotency. These include the downregulation of Nanog and Klf2 (Silva et al., 2009; Yeo et al., 2014), upregulation of Fgf5 and Otx2 (Acampora et al., 2013; Buecker et al., 2014; Nichols and Smith, 2009; Yang et al., 2014), and the maintenance of Oct4 expression levels between the two states. The expression changes of primed markers were confirmed by quantitative RT-PCR (Figure S4A). In addition, western blots were performed in both SirT1 null and homozygous SirT1ΔEx4 ES cells. To acutely remove exon 4 of SirT1, a domain required for SirT1 deacetylase activity (Cheng et al., 2003), we employed a tamoxifen-inducible cre. Using fluorescent reporters, we estimate that 90-95% of cells excise exon 4 of SirT1 (data not shown). In all samples with SirT1 deleted, there were reductions in Nanog and Klf2 proteins and increases in Fgf5 and Otx2 proteins (Figure 3C). These data indicate that loss of SirT1 induces gene expression changes consistent with the induction of the primed pluripotency gene network.

Loss of SirT1 causes Oct4 Reorganization at Naïve and Primed Enhancers

The shift from naïve to primed pluripotency is characterized by a dramatic reorganization of Oct4 occupancy at enhancer regions (Buecker et al., 2014; Factor et al., 2014; Yang et al., 2014). To test if loss of SirT1 and subsequent increased Oct4 acetylation affects Oct4 occupancy at naïve enhancers we performed ChIP qPCR in wild type and SirT1 knock out embryonic stem cells grown in naïve conditions (2i-LIF). We found that Oct4 occupancy at the Oct4 naïve enhancer (region 3 of the distal enhancer) was significantly reduced in SirT1 knock out ES cells and wild type ES cells treated with the SirT1 inhibitor, Ex-527 (Figure 4A, left). To determine if decreased Oct4 binding affects Oct4 distal enhancer activity, we performed Oct4 enhancer luciferase reporter assays in wild type and SirT1 knock out ES cells (Tesar et al., 2007; Yeom et al., 1996). Consistent with previous findings, the distal enhancer of Oct4 was more active in the naïve state than the primed state. Moreover, the distal enhancer of Oct4 was less active in SirT1 knock out ES cells grown in naïve conditions (Figure 4B, Figure S4B), indicating that loss of Oct4 binding results in decreased Oct4 distal enhancer activity. Under conditions of primed pluripotency, loss of Oct4 distal enhancer activity is compensated by increased activity of the proximal enhancer, thus maintaining equivalent expression levels of Oct4 (Tesar et al., 2007). Likewise, the proximal enhancer activity of Oct4 was increased in SirT1 knock out cells (Figure 4B), thus helping to maintain the levels of Oct4 expression (Figure 3C, Figure 4C). In support of these data, loss of SirT1 also facilitated changes in activities of the distal and proximal enhancers during the naïve to primed transition, and inhibited the reverse changes during the primed to naïve transition (Figure S4B).

Figure 4. Loss of SirT1 alters expression of transcripts regulated by Oct4 in an Otx2 dependent manner.

Figure 4

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(A) ChIP-qPCR of the proximal and distal enhancers of Oct4. Wild type ES cells (blue), SirT1 knock out ES cells (red) and wild type ES cells treated with 1uM Ex-527 for 48 hours (purple) were analyzed after IP with Oct4 antibody. Left: Oct4 enhancer regions (as described in Figure 2A). Right: Otx2 enhancer regions. Student t-tests with a p value less than 0.05 are indicated. Error bars represent SEM.

(B) Luciferase activity of the Oct4 distal and proximal enhancer. Wild type (blue) and SirT1 knock out (red) ES cells grown in naïve or primed conditions after transfection with luciferase reporter constructs driven by the distal (left) or proximal (right) Oct4 enhancer. Figure S4B contains additional differentiation time points for this experiment. Student t-tests with a p value less than 0.05 are indicated. Error bars represent SEM.

(C) Relative expression of primed pluripotency markers. Average FPKM (fragment per kilobase of exon per million fragments mapped) values in wild type cells grown in naïve conditions, wild type differentiated EpiLCs, SirT1 null ES cells grown in naïve conditions, and SirT1 null and Otx2 knockdown ES cells grown in naïve conditions. Error bars represent SEM.

(D) Expression of genes activated or repressed by Oct4. Genes activated or repressed by Oct4 were defined by changes in Oct4 occupancy in the EpiLC state (Buecker et al., 2014) and changes in RNA expression in the EpiLC state. Left: Green circles indicate the fold activation in RNA expression within SirT1 knockout cells (compared to naïve wild type ES cells). Purple circles indicate the fold repression in RNA expression within SirT1 knockout ES cells (compared to naïve wild type ES cells). Right: Green circles indicate the fold activation in RNA expression within SirT1 knockout, Otx2 knock down ES cells (compared to naïve wild type ES cells). Purple circles indicate the fold repression in RNA expression within SirT1 knockout, Otx2 knock down ES cells (compared to naïve wild type ES cells).

An early event in Oct4 reorganization is the induction of Otx2, a transcription factor whose overexpression is sufficient to induce the primed pluripotency network. Oct4 expression is required for Otx2 induction and Oct4 binding increases at an Otx2 enhancer in the primed state (Buecker et al., 2014; Yang et al., 2014). We thus tested whether loss of SirT1 resulted in increased binding of Oct4 at this enhancer (enhancer 1). Indeed, increased Oct4 binding was observed in SirT1 knockout ES cells and wild type ES cells treated with the SirT1 inhibitor, Ex-527 (Figure 4A, right), consistent with the increased expression of Otx2 described above (Figure 3C, Figure 4C). SirT1 itself was not found to be enriched at this Otx2 enhancer (Figure S4C).

Otx2 directly binds to Oct4 in the primed state and is required for Oct4 reorganization (Yang et al., 2014). If the upregulation of Otx2 in SirT1 knockout cells is sufficient to induce Oct4 reorganization, we expect those genes that are activated or repressed by Oct4 will be upregulated or downregulated in the context of SirT1 loss. To define genes whose transcripts are activated or repressed by Oct4, we used previously reported Oct4 ChIPseq data in the naïve and EpiLC state (Buecker et al., 2014) and our RNAseq expression data in the wild type naïve and primed EpiLC states. Genes activated by Oct4 were defined by increased Oct4 occupancy and increased expression in the primed EpiLC state or decreased Oct4 occupancy and decreased EpiLC expression in the primed EpiLC state. Genes repressed by Oct4 were defined by increased occupancy and decreased expression in the primed EpiLC state or decreased occupancy and increased expression in the primed EpiLC state. We found that genes that were activated or repressed by Oct4 exhibited directionally concordant expression in SirT1 knock out cells (Figure 4D, left). These results indicate that loss of SirT1 deacetylase activity may drive naïve cells toward the primed state by promoting Oct4 binding at the Otx2 promoter.

Knockdown of Otx2 Restores the Naïve Transcription Network in SirT1 Knockout ES Cells

To test whether the induction of Otx2 is required for the transition to primed pluripotency networks in the SirT1 knock out ES cells, we employed a short hairpin RNA (shRNA) mediated silencing approach to downregulate Otx2. Wild type and SirT1 knock out ES cells were infected with lentivirus expressing Otx2 targeting shRNA or scrambled controls. Three Otx2 constructs consistently reduced Otx2 expression levels in SirT1 knock out cells (Figure S5A). RNAseq analysis on one of these shRNA lines (Otx2-3) also confirmed that Otx2 expression was restored to wild type levels in SirT1 null ES cells (Figure 4C).

Downregulation of Otx2 to wildtype levels in SirT1 knockout ES cells was sufficient to rescue the expression levels of canonical markers of primed pluripotency. Genes such as Nanog and Klf2 which are downregulated in the primed state and SirT1 knock out ES cells were restored to wild type levels (Figure 4C). Conversely, Fgf5, which is induced in the primed state and SirT1 knockout ES cells, was also restored to wild type levels. These expression changes were confirmed by western blot analysis (Figure S5B). Numerous other genes showed a similar pattern of expression (Figure S4A). In addition, the distal and proximal enhancer activity of the Oct4 promoter returned to wild type naïve levels in SirT1 knock out Otx2 knockdown ES cells (Figure S5C).

Our previous analysis revealed that loss of SirT1 induced transcriptional networks associated with the primed state (Figure 3A-B). We used this transcriptome data to study the role of Otx2 by first grouping transcripts that were downregulated or upregulated in wild type primed EpiLCs compared to wild type naïve ES cells (Figure 5A-B). Next we compared the relative expression of SirT1 knock out naïve ES cells, which clearly displayed a shift in the direction of primed wild type EpiLCs (Figure 5A-B). We then tested the hypothesis that upregulation of Otx2 was responsible for this shift by knocking down Otx2 in SirT1 knock out naive ES cells and analyzing the expression of these transcripts. There was a clear normalization toward the wild type naïve transcriptome profile in these cells (Figure 5A-B), indicating that the upregulation of Otx2 per se helps drive the shift toward the primed state in SirT1 knockout ES cells.

Figure 5. Otx2 knock down in SirT1 null ES cells rescues induction of primed EpiLC gene network.

Figure 5

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(A) Heat map comparing relative expression of genes up or down regulated in EpiLC state. Genes were chosen based on their significant differential expression between the naïve ES cell and primed EpiLCs state. The relative expression of these genes were compared between wild type ES cells grown in naïve conditions, wild type EpiLCs, SirT1 null ES cells grown in naïve conditions, and SirT1 null and Otx2 knock down ES cells grown in naïve conditions. Blue signal indicates low relative expression. Red signal indicates high relative expression.

(B) Boxplots of expression changes in SirT1 knockout and SirT1 knockout Otx2 knockdown for genes up or down regulated during the naïve to primed EpiLC transition. Genes exhibiting up or down regulation during the naïve and primed EpiLC states were defined in (A). Up regulated genes were defined as exhibiting significant increased expression in the naïve state. Down regulated genes were define as exhibiting significant decreased expression in the naïve state.

(C) The role of SirT1, Oct4, and Otx2 during the naïve to prime transition. In the naïve state, SirT1 deacetylates Oct4 at naïve enhancers (left panels). Naïve enhancers are defined as enhancers which are active in the naïve state. During the naïve to primed transition, SirT1 becomes inactive, resulting in the acetylation of Oct4. Acetylated Oct4 binds an Otx2 enhancer and drives Otx2 expression (middle panels). In the primed state Otx2 binds acetylated Oct4 and directs it to primed enhancers. Primed enhancers are defined as enhancers which are active in the primed state.

Otx2 is thought to promote the primed state through the binding and genomic reorganization of Oct4. To test if Otx2 induction is required for Oct4 reorganization in SirT1 knock out ES cells, we analyzed the expression of genes regulated by Oct4 during the naïve to EpiLC transition. We found that suppression of Otx2 induction in SirT1 knock out ES cells prevented the activation or suppression of these Oct4 regulated genes (Figure 4D, right), further supporting the role of Otx2 induction in SirT1 knockout ES cells as a causative factor underlying the shift to the prime state.

Discussion

In this report, we describe a close association between SirT1 and Oct4 in embryonic stem cells, both physically and functionally. First, SirT1 and Oct4 are highly expressed in pluripotent embryonic stem cells and are coordinately turned off during cell differentiation. Second, SirT1 and Oct4 co-immunoprecipitate, and cells lacking SirT1 have hyper-acetylated Oct4, suggesting that Oct4 is a substrate for deacetylation by SirT1 in stem cells. Indeed, expression of SirT1, but not a catalytically inactive mutant results in Oct4 deacetylation in HEK293 cells. Third, SirT1 and Oct4 co-occupy the same distal enhancer region in the Oct4 promoter, along with Sox2. In order to probe the functional significance of the SirT1-Oct4 interaction, we took a cue from the fact that SirT1 activity declines during the transition from naive to primed stem cells, and queried whether SirT1 helps to maintain cells in the naïve state. Importantly, knocking out SirT1 in naïve cells phenocopies the transition to primed cells, as indicated by the increase in expression of the primed cell-induced proteins, Otx2 and Fgf5, and a decrease in the primed cell-repressed proteins, Nanog, and Klf2. At the genome-wide level, the transcriptome of naïve cells is globally shifted toward that of primed cells by knocking out SirT1, further indicating that this Sirtuin helps maintain the naïve state. At the molecular level, enhancer sites bound by Oct4 also shift in cells lacking SirT1 to mimic what happens in primed cells. At the Oct4 promoter, Oct4 binding shifts from the distal to the proximal enhancer, as does the relative activities of these two regions. In addition, Oct4 binding to the Otx2 enhancer is greatly elevated by knocking out SirT1, corresponding to a large increase in Otx2 expression.

What is the mechanism by which SirT1 maintains cells in the naïve state? A key step in driving cells toward the primed state is induction of Otx2, which interacts with Oct4 to alter the enhancers bound by this pluripotency factor and to generate the primed transcriptome (Buecker et al., 2014; Yang et al., 2014). We tested whether induction of Otx2 in SirT1-lacking cells is a key determinant of priming by using shRNA to knock down Otx2 back to the low levels normally observed in wild type naïve cells. This intervention normalized the transcriptome of SirT1-lacking cells back toward that of naïve cells, strongly indicating that induction of Otx2 is required for inducing the primed phenotype in SirT1-lacking cells. Since the acetylation status of Oct4 is also increased in cells lacking SirT1 or in normal primed cells, we suggest a model in which acetylation of Oct4 may facilitate its binding to an Otx2 enhancer to increase its expression, followed by the redistribution of Oct4-Otx2 to enhancer sites to generate the primed transcriptome (Figure 5C). In addition, acetylated Oct4 may be a better binding partner for induced Otx2 during priming. Consistent with this model, naïve reprogramming efficiency is reduced in SirT1 null cells. We conclude that the inactivation of SirT1 is a driver of Otx2 induction and possibly the binding of Otx2 and Oct4 in the transition from the naïve to the primed state (Figure 5C).

Our findings are consistent with previous studies, which indicate a role for SirT1 in induced pluripotency (Mu et al., 2015). Loss of SirT1 decreased reprogramming efficiency of mouse embryonic fibroblasts, an effect that was shown to involve the SirT1 dependent deacetylation of Sox2. While acetylation of both Sox2 (Mu et al., 2015) and Oct4 increase in ES cells lacking SirT1, only Oct4 occupancy of the Otx2 enhancer was significantly elevated, consistent with the model that acetylation of Oct4 may be driving the naïve to primed transition. However, an important role for Sox2 in this process is certainly possible.

What is the logic in having SirT1 as an important factor in maintaining the naïve state of embryonic stem cells? It is clear that naïve cells are more robust than primed cells, both in their growth rate and in their ability to contribute to the differentiated cell population in injected blastocycts (Brons et al., 2007; Dodsworth et al., 2015; Silva et al., 2008; Tesar et al., 2007). Indeed, we found that knocking out SirT1 also slowed the growth of naïve cells and by some assays altered their morphology to resemble primed cells. One key may rest on the fact that SirT1 generally promotes cell survival, for example by deacetylating and down-regulating p53 and thus inhibiting programmed cell death. SirT1 is also critical in DNA repair, aiding both single strand and double strand repair by multiple mechanisms. We suggest that as precursors for all the cells of the developed animal, naïve cells must be robust in their potential for duplication in a pluripotent state and capacity for differentiation by combining extremely high fidelity of genome maintenance and rapid cell division. These features may be favored by high SirT1 activity in naïve cells. Our findings in this report indicate that, surprisingly, these benefits are coupled to the cell fate decision itself between naïve and primed cells by the mechanism in Figure 5C. This coupling may be rationalized because it would allow culling of cells with low SirT1 activity from the naïve pool and prevent failures in quality control early in development from being incorporated into the composition of the fully developed animal.

To wit SirT1 may be suitable as a factor that maintains fidelity as well as the naïve state of embryonic stem cells because it responds to environment cues, including food availability and stress. It is possible that unfavorable conditions during early stages of development can repress SirT1 activity and impose a developmental checkpoint. For example, toxic environmental agents could trigger DNA damage, activation of PARPs, depletion of NAD and inactivation of SirT1, thereby imposing a roadblock to continued embryonic development. While speculative, such a mechanism would explain why SirT1 may be an ideal coupler of the naïve state to favorable conditions in embryonic stem cells.

The decline in SirT1 activity in the transition of naïve to primed wild type stem cells appears to occur post-translationally. Possible mechanisms include the induction of a SirT1 inhibitor and a reduction in NAD levels, although the known protein inhibitor of SirT1, DBC1, is not induced during this transition. In this case of NAD, we have recently observed that addition of an NAD precursor can facilitate the conversion of primed cells into naïve cells and inhibit the reverse process. In summary, we have shown that SirT1 is an important factor in maintaining the naïve state of embryonic stem cells and inhibiting their conversion into primed cells. This control of cell state may increase the potential of naïve cells to contribute to the body composition with high fidelity and couple their maintenance to favorable environmental conditions.

Experimental Procedures

Cell Culture, Differentiation Assays, and Otx2 knockdown

Naïve mESCs were cultured in N2B27 media containing 2i inhibitors (PD0325901 and CHIR99021) and LIF. ES cells grown in MEF-serum conditions where grown on irradiated DR4 MEFs in media containing 15% FBS and LIF. Embryoid bodies were formed in 10cm Costar Ultra Low Attachment (Corning) plates.

Differentiation of naïve mESC to primed EpiLCs was performed as previously described (Buecker et al., 2014; Hayashi et al., 2011). mESC were passaged 3-5 times in N2B27 2i+LIF and transferred to fibronectin coated plates. Differentiating cells were stimulated with 12 ng/ml Fgf2 (Invitrogen), 20ng/ml Activin A (Sigma), and 1% KOSR (Invitrogen) for 48 hours. For Otx2 knockdown, ES cells were infected for 24 hours with lentivirus expressing shRNAs targeting Otx2 or scrambled control, and then switched to selection media containing 1ug/ml of puromycin for 3-4 days.

Immunocytochemistry

ES cells were grown on coverslips and fixed with fresh 4% paraformaldehyde (Sigma) for 15 minutes. For antigen retrieval, cells were incubated in PBS with 0.25% triton X-100 for 10 minutes. Primary antibody was applied to cells overnight at 4C. See Table S1 for antibody information.

Western Blot Assays

Whole cell lysates were collected from ES cells, EpiLCs, and transfected HEK 293T cells in RIPA buffer. HEK 293T cells were transfected by calcium phosphate using the following plasmids: pCAG-Myc-Oct4-IP (Addgene 13460), pAD-Track Flag-SIRT1 (Addgene 8438), and pAD-Track Flag-SIRT1 H355A (Addgene 8439). Co-IPs were performed using Pierce’s direct co-IP kit. See Table S1 for antibody information.

RNAseq

RNA was extracted with Trizol and purified using Direct-zol RNA miniprep kit (Zymo Research). RNA-seq libraries were sequenced using the Illumina Hi-seq 2000 platform. FASTQ files were aligned to the NCBI37/mm9 reference genome using the Tophat aligner. The Cuffdiff tool from the Cufflinks package was used to detect differential expression between datasets. Significant differential expression was defined as genes with Cuffdiff q values less than 0.05 and greater than 2 fold differential expression.

Chromatin Immunoprecipitation qPCR

Chromatin immunoprecipitations were performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling). All samples were normalized to input for qPCR analysis. See Table S1 for oligo and antibody information.

Oct4 Enhancer Luciferase Reporter Assay

Mouse Oct4 proximal and distal enhancers were cloned into pGL3 as previously characterized (Tesar et al., 2007). Constructs were co-transfected with pRL-TK (Renilla) using Xfect mESC transfection reagent (Clontech) and incubated for 48 hours. Luciferase activity was assessed using the Dual-Glo Luciferase

Statistical methods

Unpaired two-tailed Student’s t-tests were used to assess statistical significance for experiments involving quantitative PCR, flow cytometry, luciferase assays, cell growth analysis, and clonogenicity assays. Differential expression within RNAseq datasets was defined as genes with Cuffdiff q values less than 0.05 and greater than 2 fold differential expression. Statistical significance of correlated gene expression profiles was assessed by Pearson correlation.

For more detailed descriptions see Supplemental Experimental Materials.

Supplementary Material

1
2

Acknowledgements

We thank the Jaenisch lab for luciferase constructs and Michael Williams for bioinformatics and statistical advice. This work was supported by grants from NIH and The Glenn Foundation for Medical Research (LG) and the American Cancer Society (EW). LG is a founder of Elysium Health and consults for GSK, Segterra, and Chronos (Oxford).

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.

Accession Numbers

The GEO accession number for the RNA-seq data reported in this paper is GSE81494.

Author Contribution

Conceptualization, E.O.W. and L.G.; Methodology, E.O.W., E.L.B, and L.G.; Formal Analysis, E.O.W.; Investigation, E.O.W, A.K.T, R.L., and D.M.K.; Writing – Original Draft, E.O.W. and L.G.; Writing – Review & Editing, E.O.W. and L.G.; Funding Acquisition, E.O.W. and L.G.; Supervision, E.O.W. and L.G.

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