Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

DNA constructs

Luciferase reporter plasmids. Mouse or human Oct4 promoters that cover 2.3 (mouse) or 2.5 kb (human) fragments upstream of ATG were cloned into pGL3 vector (Promega); 10 repeats of upstream activation sequence (UAS) element were inserted in the 5′-end of the Oct4 promoter to serve as a positive control (39). Human NANOG luciferase reporter pNANOG-Luc was obtained from Addgene (Addgene #25900).

Mutated mOct4-Luc reporter plasmids. Site-directed mutagenesis was performed using PCR approach described previously (40). Briefly, the wild-type mOct4-Luc reporter was first amplified with primers that introduced the T to C mutation. The amplified fragment was then treated with DpnI to remove the original template plasmids; and the remaining product was used for transformation to obtain mutated reporter plasmids M-6, M-11, M-17, M-20 and M-25. Next, these mutated reporters were used as template for further amplification; primers used in this step were designed to anneal to DNA adjacent to the −120 to −104-bp region while carrying the target sequences of selected TALE-VP64s at 5′. The amplified fragments were then treated with DpnI as described above, yielding reporter plasmids that carried the relocated target sequences from −120 to −104 bp in mouse Oct4 promoter.

Fuw-tetO-TALE-VP64 plasmids. An nuclear localization signal (NLS)-VP64-HA fragment was assembled using PCR approach and inserted into the PspXI and HpaI sites in the Fuw-tetO vector, which was modified from the plasmid FUW-tetO-hOCT4 (Addgene #20726) by destroying the BsmBI site and introducing BsrGI, PspXI and HpaI sites enclosed by the EcoRI site (41). The DNA fragment coding LacZ flanked by TALE N- and C-terminals was released from the plasmid pTAL1 (Addgene #31031) (42) and inserted into the BsrGI and PspXI sites of the Fuw-tetO-NLS-VP64-HA to generate a Fuw-tetO-TALE(LacZ)-VP64 scaffold vector. Various 17-bp sequences preceded by a T were selected to be the candidate TALE target sequences (42). Corresponding TALE repeat arrays were then assembled using the Fuw-tetO-TALE(LacZ)-VP64 scaffold vector using the previously described Golden Gate cloning method (Supplementary Figure S1) (42,43).

Fuw-tetO-TALE-KRAB plasmids. The KRAB domain was amplified from pLV-tTRKRAB (Addgene #12249) and inserted into the PspXI and HpaI sites of Fuw-tetO-TALE-VP64s to replace VP64s.

dCas9-VP64/KRAB plasmids. The H840A mutation was introduced into the hCas9_D10A plasmid (Addgene # 41816) to produce a catalytically inactive Cas9 (dCas9) (14,19). The full-length dCas9, dCas9 with deletion of N-terminal RuvC1 domain (ΔN) or C-terminal HNH domain (ΔC) were amplified using PCR approach and inserted into BsrGI and PspXI sites of the modified Fuw-tetO vector, followed by insertion of VP64/KRAB.

sgRNA constructs. The 20-bp sequences that precede NGG, the protospacer adjacent motif (PAM) required for sgRNA targeting (44), were selected as candidate sgRNA target sequences. To generate an sgRNA, a pair of 26-mer oligos containing sgRNA target sequences were synthesized. They were annealed and then inserted into the BsmBI site in the sgRNA expression vector MLM3636 (Addgene # 43860) (Supplementary Figure S5A) (44).

shRNA constructs. Two shRNAs were designed to target separated 19-bp sequences in p300 coding sequence using WI siRNA selection program http://jura.wi.mit.edu/bioc/siRNAext/. They were p300 shRNA1: 5′-GCACGACTTACCAGATGAA and p300 shRNA2: 5′-GCCTCAAACTACAATAAAT. Synthesized oligonucleotides were cloned into pSUPER.puro (BglII and HindIII sites; Oligoengine).

Vectors containing p300, JMJD3 and JMJD2B were obtained from Addgene (Addgene #10717, #24167 and #24181).

Cell culture

HEK293T and NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS) (Life Technologies).

Mouse ESCs (E14) were cultured as previously described (45). Briefly, cells were cultured on gelatin-coated dishes in DMEM medium, supplemented with 15% heat-inactivated FBS (Life Technologies), 0.1 mM β-mercaptoethanol (Life Technologies), 2 mM L-glutamine, 0.1 mM MEM non-essential amino acid (Life Technologies) and 1000 U/ml of leukemia inhibitory factor (LIF) (Life Technologies). The culture medium was refreshed every day and the cells were passaged every two days.

Human ESCs (H1) were cultured as previously described (46). Briefly, they were cultured feeder-free on Matrigel (BD Biosciences). Medium containing 20% knockout serum replacement, 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol and 4 ng/ml basic fibroblast growth factor (bFGF) (Life Technologies) was conditioned by mouse embryonic fibroblast. Additional 8 ng/ml bFGF was added freshly to conditioned medium for human ESC culture. Medium was changed daily and cells were subcultured with 1 mg/ml collagenase IV (Life technologies) every 5–7 days.

Transfection

HEK293T cells were seeded into 12-well plates at a density of 2.4 × 10⁵ cells/well one day before transfection. Unless otherwise stated, 1.6 µg TALE-VP64 plasmids (or 0.8 µg dCas9-VP64 + 0.8 µg of sgRNA) were used for Lipofectamine 2000 (Life Technologies) transfection in each well following the manufacturer’s instruction. When more than one TALE-VP64 or sgRNA was examined, the amount for each TALE-VP64 or sgRNA plasmid equaled to 1.6 µg (TALE-VP64) or 0.8 µg (sgRNA) divided equally by the numbers of plasmids. Cells were grown for an additional 48 h before harvesting for qRT-PCR analysis or immunoblotting. The estimated transfection efficiency was around 83.1% using 1.6 µg pEGFP-N1 plasmid.

For transfection in NIH3T3, 1 × 10⁵ cells were seeded into each well of 12-well plates one day before transfection. A mixture of 0.8 µg of FUW-M2rtTA and 0.8 µg TALE-VP64 (or 0.4 µg dCas9-VP64 + 0.4 µg of sgRNA) plasmids were used for transfection in each well. Similarly, when more than one TALE-VP64 or sgRNA was used, the amount of individual plasmids was divided equally as described above. Cells were grown in the presence of 1 µg/ml doxycycline (Dox) for an additional 48 h before harvesting for qRT-PCR analysis. The estimated transfection efficiency was ∼78.8% using the plasmid pEGFP-N1.

RNA extraction, reverse transcription and quantitative real-time PCR

Total RNA was extracted from cell samples using TRIzol reagent (Life Technologies) and reverse-transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) in an ABI7900HT Real Time PCR system. Measured transcript was normalized to Gapdh and samples were run in triplicate. Primers used for qRT-PCR analysis were provided in Supplementary Table S1.

Western blot

Cells were collected by trypsinization and washed with phosphate buffered saline (PBS). Samples were then incubated in lysis buffer [50 mM Tris, 0.5% NP40, 1 mM EDTA, 10% glycerol, 400 mM sodium chloride and Protease Inhibitor Cocktail (Roche)] and cleared by centrifuging at 20 000 × g, 4°C for 30 min. Protein concentration was determined with BCA Protein Assay Reagent (Thermo Scientific). From each sample, 10 μg protein were resolved by SDS/PAGE and subsequently transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with 5% non-fat dry milk in PBST buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.46 mM KH₂PO₄, 0.05% Tween-20) for 1 h at room temperature and incubated with Oct3/4 or GAPDH antibodies (Santa Cruz) overnight. Membrane were then washed three times with PBST buffer and incubated with rabbit anti-goat-HRP or goat anti-mouse-HRP (GE technology) at a dilution of 1:20 000 in PBST for 1 h at room temperature. Signals were detected using the Amersham ECL Select Western Blotting Detection Kit (GE Health Care Life Sciences) and exposed to Super RX-N film (Fuji).

Dual luciferase reporter assay

HEK293T cells were seeded into 96-well plates at a density of 2 × 10⁴ cells/well one day before transfection. Cells were transfected with 100 ng TALE-VP64 plasmid (or 50 ng dCas9-VP64 + 50 ng sgRNA), 100 ng corresponding reporter plasmids and 10 ng Renilla (Promega) using Lipofectamine 2000 according to the manufacturer’s instructions. When more than one TALE-VP64 or sgRNA was used, the amount of individual plasmids was divided equally as described above.

To examine the effect of Dox-inducible expression, 50 ng FUW-M2rtTA and 50 ng pLV-tTRKRAB were co-transfected with 12.5 ng TALE-VP64, 50 ng of the corresponding reporter plasmids and 10 ng Renilla into the HEK293T cells in each well of 96-well plates. Cells were then maintained in the presence or absence of 1 µg/ml Dox for 2 days before analysis.

To examine the transcriptional repression by TALE-KRAB or dCas9-KRAB, mouse ESCs E14 were seeded into 24-well plates at a density of 8 × 10⁴ cells/well 6 h before transfection. The cells were then transfected with 200 ng TALE-KRAB plasmid (or 100 ng dCas9-KRAB + 100 ng sgRNA), 200 ng FUW-M2rtTA, 200 ng corresponding reporter plasmids and 20 ng Renilla (Promega). The estimated transfection efficiency was around 74.9% using the plasmid pEGFP-N1.

Two days after transfection, luciferase reporter assays were carried out using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. Relative luciferase activity was measured using a GLOMA20/20 Luminometer (Promega). The activity of the firefly luciferase was normalized with that of Renilla luciferase.

Statistical analysis

Statistical significance was determined using unpaired two sample Student’s T-test. P < 0.05 was considered as significant. Data were shown as mean ± SEM (n = 3).

In vitro methylation assay

In vitro methylation of mOct4-Luc and hOCT4-Luc reporter plasmids was performed using CpG methyltransferase M.SssI (New England Biolabs) as previously described (31). Briefly, 45 µg of reporter plasmid were incubated overnight with 45 units of M.SssI enzyme. The methylation status of the plasmid DNA was then confirmed by digestion with MspI and HpaII (New England Biolabs). The effect of TALE-VP64s on the in vitro methylated reporter was then examined by luciferase assay as described above.

Bisulfite sequencing analysis

The genomic DNA from cell samples was extracted using PureLink™ Genomic DNA Mini Kit (Life Technologies). For each sample, 300 ng of genomic DNA was used for bisulfite conversion using the EZ DNA Methylation-Gold™ Kit (Zymo Research Corporation) in accordance with the manufacturer’s instructions. The promoter sequence was amplified using HotStarTaq Plus DNA Polymerase (QIAGEN) with the following primers: mouse Oct4: 5′-ATGGGTTGAAATATTGGGTTTATTTA and 5′-CCACCCTCTAACCTTAACCTCTAAC; human OCT4 (region 1): 5′-AAGTTTTTGTGGGGGATTTGTAT and 5′-CCACCCACTAACCTTAACCTCTA; human OCT4 (region 2): 5′-GAGAGAGGGGTTGAGTAGTTTT and 5′-CACTAACCCCACTCCAACCTAAA. Amplified PCR products were then ligated into the pGEM-T Easy vector (Promega) and sequenced with M13 forward primer.

TALE-VP64s activated mouse and human Oct4 promoters by targeting a non-conserved proximal region

We constructed a scaffold vector to assemble TALEs, NLS, VP64 and HA-tag under the control of Dox-inducible tetO-CMV fusion promoter (Figure 1A, upper panel) (41). Based on this vector, we assembled different TALE DNA-binding domains using previously described Golden Gate cloning method (Supplementary Figure S1) (42,43). In total, we generated 26 TALE-VP64s, each recognizing a 17-bp sequence on various regions across 2.3-kb upstream genomic region of mouse Oct4 gene (Figure 1B; Supplementary Figure S2) (27). These TALE-VP64s hereinafter were referred to as mO1–mO26. Four of the mO TALE-VP64s targeted the conserved DE region CR4, eight recognized the PE CR2, four bound to the proximal promoter CR1 region and the remaining 10 located in the non-conserved regions upstream of CR4 (mO1–mO4) or between CR1 and CR2 (mO17–mO22) (Figure 1B; Supplementary Figure S2).

The effects of individual mO TALE-VP64s were examined by luciferase assay in HEK293T cells. Each mO was co-transfected with a luciferase reporter under the control of 2.3 kb mouse Oct4 promoter (mOct4-Luc) (Figure 1A, lower panel). Ten repeats of UAS elements were also inserted into the reporter plasmid (Figure 1A, lower panel) (39); and Gal4-VP64, which could target the UAS region, was included to serve as a positive control. As expected, mO TALE-VP64s targeting different regions of Oct4 promoter exhibited various effects in activating the mOct4-Luc reporter (Figure 1B). Yet it was surprising that mO5–mO16, which targeted the conserved enhancer regions (CR2 and CR4) that were known to be bound by ESC TFs such as OCT4 itself, SOX2, NANOG and KLF4 for maintaining the transcription of Oct4 gene (47), showed only moderate activity (Figure 1B). In contrast, mO21, which targeted a sequence outside of the conserved regions, at −120 to −104-bp upstream of the TSS (27), exhibited exceptionally high activity in boosting the transcription (Figure 1B). This transcriptional activation by mO21 was well correlated with the presence of Dox (Figure 1C). To examine whether these TALEs could mediate gene repression, we fused the TALE domains in mO21 and mO22 to a transcriptional repressor KRAB (9,48). These mO21^KRAB and mO22^KRAB fusion proteins were then examined in mouse ESCs, where the transcription of mOct4-Luc reporter could be maintained at a high level. Indeed, luciferase assay showed that mO21^KRAB and mO22^KRAB repressed the transcription of mOct4-Luc reporter by ∼80% and 30%, respectively (Figure 1D). Collectively, these indicated that optimized TALE-VP64s could modulate the transcription of mouse Oct4 promoter efficiently by targeting the non-conserved −120 to −104-bp region.

To examine whether the activity of a TALE-VP64 depends on its target position, we selected five TALE-VP64s mO6, mO11, mO17, mO20 and mO25 and analyzed the positional effect by moving their target sequences from the original locations into the observed optimal region at −120 to −104 bp. First, 5′ T in the target sequences of mO6, mO11, mO17, mO20 and mO25 were mutated into C to abolish the recognition by TALEs to their original target sites. The mutated reporter constructs were named M-6, M-11, M-17, M-20 and M-25. Next, the original mO21 target sequence at the −120 to −104-bp region in the plasmids M-6, M-11, M-17, M-20 and M-25 was replaced with the target sequences of mO6, mO11, mO17, mO20 and mO25. This generated the reporter constructs MR-6, MR-11, MR-17, MR-20 and MR-25 which carried the re-located target sequences. Relocation of the target sequences into the proposed optimal region dramatically increased the transcriptional activation induced by these TALE-VP64s (Figure 1E), suggesting that the target positions on the promoter region influenced the activity of TALE-VP64s.

Based on this result, we designed eight TALE-VP64s targeting the non-conserved region as well as CR1 in human OCT4 promoter (hO1–hO8) (Figure 1F; Supplementary Figure S2). Similarly, an hOCT4-Luc reporter plasmid was constructed to examine the activity of individual hO TALE-VP64s. Interestingly, hO3 and hO4 that targeted the −113 to −80-bp region upstream of the TSS exhibited significantly higher activity than other TALE-VP64s in the luciferase assay (Figure 1F; Supplementary Figure S2). Besides OCT4, we also generated four TALE-VP64s (N1–N4) targeting DNA regions close to the TSS in human NANOG promoter (Supplementary Figure S2). We found that TALE-VP64 (N3) which targeted from −96 to −80-bp upstream of the NANOG TSS showed the highest activity in the luciferase assay using hNANOG-Luc reporter (Figure 1G). Together with the result obtained with mO1–mO26, these data suggested that a positional effect of the engineered TFs TALE-VP64s might exist independent of genomic and epigenetic contexts.

Furthermore, we generated TALE-VP64s targeting several other genes, including human SOX2, KLF4, c-MYC and CDH1 (or E-Cadherin) (Supplementary Figure S2). Individual TALE-VP64s were transfected into HEK293T cells and the expression of targeted genes was examined using qRT-PCR. We found that the activation of endogenous genes using individual TALE-VP64s was relatively inefficient. The mRNA level induced by TALE-VP64s targeting KLF4 and CDH1 was around 6-fold and 11-fold; while that in c-MYC and SOX2 genes was only around 3-fold (Supplementary Figure S3). Although the most efficient TALE-VP64s against KLF4, c-MYC and CDH1 lied within a similar region as that in mO21, hO3, hO4 and N3 (Supplementary Figure S3A–C), TALE-VP64 targeting a similar region in CDH1 (E2) and SOX2 promoter (S2) exhibited low activity or no obvious gene activation (Supplementary Figure S3C, D). These suggested that the ability of TALE-VP64s to activate endogenous genes might be also determined by other factors, such as intrinsic epigenetic modification.

TALE-VP64s activated silenced Oct4 gene expression in mouse and human somatic cells

In order to activate silenced Oct4 genes in somatic cells, which are constantly masked by DNA methylation on CpG di-nucleotides in their promoter regions (45), we examined whether TALE-VP64s could activate mOct4-Luc reporter methylated in vitro by Escherichia coli CpG methyltransferase M.SssI. Recent studies have reported that TALE repeats use different codes to discriminate between cytosine (C) and methylated cytosine (mC) (49,50). Compared to HD, which specifically recognizes unmethylated cytosine (C), the code NG (previously known to bind to thymidine) or N* (lacking the residue at position 13 and recognizing both cytosine and thymidine) were found to bind mC with higher affinity (49,50). With the current Golden Gate cloning system (42), we generated a series of mO21 variants, which carried NG, instead of the original HD, to target the potential mC at the fourth, eighth or both nucleotides (Figure 2A). The abilities of these mO21 variants to activate either unmethylated (Figure 2B, upper panel) or in vitro methylated mOct4-Luc reporters (Figure 2B, lower panel; Supplementary Figure S4A) were examined using luciferase assays. We found that mO21*—which carried NG targeting mC at both the fourth and eighth positions—exhibited higher activity in initiating transcription from the methylated mOct4-Luc reporter (Figure 2B), compared with the original mO21 as well as other variants which carried one NG or irrelevant TALE code NI(A) at the fourth and eighth positions (Figure 2A and B). The overall transcriptional activity of mO21 variants was lowered using methylated reporter (Figure 2B), which was probably due to the transcriptional repression associated with DNA methylation. Non-optimal mO21 variants could still activate the reporters, although with reduced activity (Figure 2B). This observation might be explained by the nature that TALE domains could tolerate one or two mismatches (37).

Afterwards, we transfected mO21 and its variants into mouse fibroblast NIH3T3 cells, and examined the level of endogenous Oct4 transcription using qRT-PCR. Three-fold up-regulation of Oct4 mRNA induced by mO21* was observed, whereas mO21 showed only marginal activity (Figure 2C, upper panel). In support of this observation, bisulfite sequencing analysis showed that two CpG within the mO21 target sequence were indeed methylated (Figure 2C, lower panel). This suggested that using NG instead of HD for targeting mC is more suitable for activating a silenced gene.

Next, we generated a variant of hO3, named hO3*, by targeting the potential mC in its target sequence with NG (Figure 2D). Similar to mO21*, the hO3* was more effective in activating the transcription of in vitro methylated hOCT4-Luc reporter, compared to the original hO3 (Figure 2E). Moreover, qRT-PCR analysis showed that hO3* could up-regulate the endogenous OCT4 transcription 2.5-fold, whereas hO3 showed no obvious enhancement (Figure 2F). Another TALE-VP64 hO4, which targeted a region containing no CpG sites, could also induce endogenous OCT4 transcription, but to a lesser extent than hO3* (Figure 2F).

To achieve a higher up-regulation of endogenous Oct4 genes, we further examined the combinatory effect of multiple TALE-VP64s that targeted different regions in the same promoter. TALE-VP64 mOs that targeted CR1, CR2, CR4 and non-conserved regions were combined separately and transfected into NIH3T3 cells (Figure 2G). In addition, two bigger pools of mOs were examined, targeting multiple regions of CR1, CR2, CR4 and non-conserved regions. The highest activity (around 30-fold up-regulation of endogenous Oct4 transcription; Figure 2G) was observed with a combination of nine effective TALE-VP64s (mO10, 11, 16, 17, 19, 20, 21*, 22 and 25) (individual relative luciferase activities >3, Figure 1B). Next, the combinatory effects of hO TALE-VP64s were also examined. Indeed, the pool of all eight hOs showed a significant synergistic effect in activating the transcription of endogenous OCT4 gene (Figure 2H). Removal of individual hOs from the combination showed that hO1 and hO3* contributed to the combinatory activity of the pool, whereas hO2 might have minor inhibitory effect (Figure 2H). The highest activity (∼20-fold) was achieved using combinations of hOs with numbers 1, 3*, 4 and 6, or 1, 3*, 5, 7 and 8 (Figure 2H). More importantly, we detected OCT4 protein in these multiple TALE-VP64-transfected HEK293T cells using a specific antibody described previously (Figure 2I) (46). Collectively, TALE-VP64s did indeed activate endogenous OCT4 genes and produce mature proteins.

sgRNA-guided dCas9-VP64s activated silenced Oct4 genes

To investigate the potential of sgRNA-mediated dCas9-TFs for modulating expression of endogenous Oct4 genes, we generated dCas9-TFs by fusing dCas9 protein containing two mutations in the RuvC1 and HNH nuclease domains (D10A and H840A) to VP64 or KRAB (Figure 3A) (14). Based on the aforementioned result with TALE-VP64s, we generated eight sgRNAs targeting different 20-bp DNA sequences on either template (T) or non-template (NT) strands in mouse Oct4 promoter around CR1 and the nearby non-conserved region (Figure 3B; Supplementary Figure S5). These sgRNAs were cloned by annealing 26-nt oligo pairs, followed by insertion into an sgRNA scaffold vector (Supplementary Figure S5A) (44). Each sgRNA was then co-transfected with dCas9-VP64 and mOct4-Luc reporter for luciferase assay. We found that five out of the eight sgRNAs significantly activated the transcription of mOct4-Luc reporter (Figure 3B). Interestingly, the most effective sgRNAs T2 and NT3 targeted regions ranging from −147 to −89-bp upstream of the TSS (Figure 3B; Supplementary Figure S5B), which was similar to the highly effective TALE-VP64s. Deletion of either RuvC1 or HNH nuclease domains, each of which is responsible for binding to one strand of target DNA (14), abolished transcriptional activation mediated by dCas9-VP64 or transcriptional repression mediated by dCas9-KRAB (Figure 3C).

Similar to TALE-VP64s, when multiple sgRNAs were applied in combination, synergistic effects were observed (Figure 3D). The combinations of sgRNAs, NT1 to NT4 or T1, T2, NT2 and NT3, further boosted the transcription of mOct4-Luc reporter about 2-fold, compared with the most potent single sgRNA T2 (Figure 3D). Moreover, we found that the combination of multiple competent sgRNAs could induce endogenous Oct4 mRNA ∼9-fold in NIH3T3 cells, whereas individual sgRNAs showed marginal or no increase (Figure 3E). This indicated that combined sgRNAs could mediate dCas9-VP64 to activate silenced Oct4 genes in mouse somatic cells.

Next, we generated seven sgRNAs targeting CR1 as well as the non-conserved region in human OCT4 promoter (Figure 3F; Supplementary Figure S5B). Luciferase assay showed that four out of the seven sgRNAs (H1–H4) could activate the transcription of hOCT4-Luc reporter (Figure 3F). Of these four sgRNAs, H4—which targeted −142 to −123-bp-region upstream of the TSS—exhibited the greatest activity. On the other hand, sgRNAs H5, H6 and H7, which targeted the −85 to −14-bp region, induced marginal transcriptional activation (Figure 3F). This was consistent with the result using sgRNAs targeting the mouse Oct4 promoter, where T3, T4 and NT1, targeting the −87 to −30-bp region, showed significantly lower activity, compared with T2 and NT3, which targeted the −147 to −128-bp and −108 to −89-bp regions (Figure 3B; Supplementary Figure S5B). These results suggested that dCas9-VP64 might function with optimal activity when being recruited to the region around −147 to −89-bp upstream of the TSS, thus it further clarified the proximal TSS region for sgRNA targeting reported in Mali et al.’s study (37). The minor difference in the optimal targeting locations between TALE-VP64s and sgRNA/dCas9-VP64s might be caused by their size and structural difference.

Consistently, combination of these sgRNAs showed a synergistic effect in activating the hOCT4-Luc reporter (Figure 3F). Among various combinations examined, the pool of H1–H4 showed the highest activity (Figure 3F). This pool of sgRNAs was also able to elevate endogenous OCT4 transcription around 17-fold (Figure 3G) and induced OCT4 protein in transfected HEK293T cells, though at a lower level compared to that in human ESCs (Figure 3H). Interestingly, we also observed transcriptional synergy when sgRNAs (H3 and H4) and TALE-VP64s (hO1, 3*, 4 and 6) were applied simutanously (Figure 3I), suggesting that these engineered TFs could be used in combination for activating gene expression.

TALE-VP64-induced activation of endogenous OCT4 was transient

Next, we examined whether the observed up-regulation of endogenous Oct4 gene could be stably maintained. HEK293T cells transfected with single or combinations of hO TALE-VP64s were sub-cultured every 2 days and maintained for three passages. Specific primers amplifying a common fragment in all hO TALE-VP64s showed that the overall expression of TALE-VP64s decreased quickly after the second passage (Supplementary Figure S6). In accordance with the decrease of TALE-VP64 expression, we observed that the initial up-regulation of endogenous OCT4 transcript induced by the combined hO TALE-VP64s also decreased rapidly and was undetectable after three passages (Figure 4A). This indicated that the up-regulation of endogenous OCT4 transcription was dependent on the exogenous TALE-VP64s, and OCT4 transcriptional activation could not be maintained when the ectopic expression of hO TALE-VP64s decreased due to instability of plasmids. Bisulfite sequencing analysis on these TALE-VP64-transfected HEK293T cells consistently showed that genomic DNA in OCT4 promoter remained to be hypermethylated, even when its transcription was elevated by the exogenous TALE-VP64s (Figure 4B). Beside the region that contains hO3 target sequence (Figure 2D), analysis on another region covering the TSS showed a similar unchanged DNA hypermethylation pattern (Figure 4B, region 2). This result suggested that transient up-regulation of endogenous OCT4 transcription did not reverse the DNA methylation status and thus could not establish sustainable OCT4 expression. Similarly to TALE-VP64s, we found that the dCas9-VP64-mediated activation of OCT4 gene also decreased rapidly in subsequent passages (Supplementary Figure S7). These results suggested that neither TALE-VP64 nor dCas9-VP64-induced activation of OCT4 gene could reverse its epigenetic repression and achieve stable expression in the long term.

Furthermore, we examined whether TALE-VP64-induced activation of OCT4 gene could activate other pluripotency genes, including downstream targets of OCT4 protein in ESCs. However, qRT-PCR analysis of the HEK293T cells transfected with single or combined hO TALE-VP64s showed that activation of OCT4 had no effect on the transcriptional activation of SOX2, KLF4, NANOG, c-MYC and CDH1 genes, which work in the same pluripotency network (Figure 4C). This is consistent with the previous notion that endogenous pluripotency genes could not be activated directly by forced expression of TFs during reprogramming (30,51); instead, they were probably up-regulated via an indirect path by multiple factors, after the global epigenetic status had been significantly modified.

Next we examined whether activation of other pluripotency genes has an effect on OCT4 transcription. With the TALE-VP64s targeting SOX2, KLF4, NANOG, c-MYC and CDH1, which were generated previously (Supplementary Figure S2), we identified combinations that could increase the transcription level of endogenous SOX2 by around 26-fold (S1, S2 and S3), KLF4 by around 9-fold (K2, K3 and K4), and CDH1 by around 27-fold (E2, E3 and E4) (Figure 4D and Supplementary Figure S8A). Similar to OCT4 activation, activation of SOX2, KLF4 and CDH1 showed no obvious effect on transcription of other pluripotency genes (Figure 4D). Combinations of TALE-VP64s to NANOG and c-MYC induced relatively low transcriptional activation of their endogenous genes (∼2- and 4-fold, respectively) (Supplementary Figure S8), which might be explained by its stringent epigenetic repression (NANOG) or the high basal level of the endogenous gene (c-MYC).

p300 enhanced the activation of endogenous OCT4 induced by TALE-VP64s and dCas9-VP64s

Histone H3 lysine 27 acetylation (H3K27ac) and histone H3 lysine 4 trimethylation (H3K4me3) are known epigenetic modifications for maintaining the Oct4 locus in an active or permissive status in pluripotent cells; whereas histone H3 lysine 9 and 27 trimethylation (H3K9me3 and H3K27me3) mediate epigenetic repression and are associated with Oct4 gene silencing in somatic cells. Hence, we examined whether introducing epigenetic modification enzymes that catalyse active epigenetic marks could facilitate the activation of silenced OCT4 genes in the presence of corresponding TALE-VP64s or sgRNA/dCas9-VP64s. We transfected H3K27 demethylase JMJD3 (52), H3K9 demethylase JMJD2B (53) or histone acetyltransferase p300 (54) individually with either hO3* or the combination of hO1, 3*, 4 and 6 into HEK293T cells. Analysis by qRT-PCR showed that p300 significantly enhanced the endogenous OCT4 transcription induced by these TALE-VP64s, either in a single form (Figure 5A) or in a combination (Figure 5B). Further analysis of these epigenetic modifiers using hOCT4-Luc reporter plasmid showed that they did not enhance the TALE-VP64-induced activation of OCT4 reporter (Supplementary Figure S9). This suggested that the enhancement effect of p300 on activating transcription was dependent on its epigenetic modification function in a chromatin context.

Next, we examined whether these active epigenetic modifiers could also enhance the transcription activation mediated by dCas9-VP64s. sgRNAs H3 and H4, which targeted the human OCT4 promoter, were co-transfected with dCas9-VP64 and JMJD3, or JMJD2B, or p300 in HEK293T cells. Analysis by qRT-PCR at 48 h post-transfection showed that p300 also enhanced the transcriptional activation induced by sgRNA-guided dCas9-VP64 (Figure 5C). Importantly, when the endogenous p300 was depleted using specific shRNAs, we observed a reduction of OCT4 mRNA in the presence of TALE-VP64s or sgRNAs/dCas-VP64s (Figure 5D), suggesting that p300 was indeed involved in the activation of endogenous OCT4 transcription induced by the engineered TFs. We also observed that the alteration of p300 level did not influence the expression of TALE-VP64s (Figure 5E), eliminating the possibility that p300 enhanced the activation of OCT4 transcription indirectly through modulating the level of engineered TFs. Taken together, these data indicated that p300 could facilitate the activation of endogenous OCT4 gene mediated by TALE-VP64s or sgRNA/dCas9-VP64s.

Since TALEs and sgRNAs can tolerate one or two mismatches (37), they may target undesired DNA sequences due to close similarity of the sequences and cause off-target effect. To assess if p300 also enhanced activation of undesired genes due to off-target effect, we carried out genome wide search and identified six genes which carry two-mismatch, and 29 genes carrying three-mismatch of hO3* target sequence in their proximal promoters (maximum 250-bp upstream of the TSSs) (Supplementary Table S2). Complete match or one-mismatch sequences could not be found within proximal region in any promoters. We selected 20 potential off-target genes and analyzed their expression in the HEK293T cells that had been transfected with hO3* or combination of hO1, 3*, 4, 6, in the presence or absence of p300. With the valid expression data of thirteen genes out of the twenty, we found that other than OCT4, all the mRNA levels remained unchanged in cells transfected with these TALE-VP64s and p300 (Supplementary Figure S10). This suggested that off target effect is not a primary concern for the TALE-VP64s-induced activation of endogenous OCT4.

Supplementary Data

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Supplementary Materials