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Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodeling1. While several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming2,3, the role of specific chromatin modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used shRNAs to target genes in DNA and histone methylation pathways, and have identified positive and negative modulators of iPSC generation. While inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase Ezh2, reduced reprogramming efficiency, suppression of SUV39H1, YY1, and Dot1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase Dot1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for Klf4 and c-Myc. Inhibition of Dot1L early in the reprogramming process is associated with a marked increase in two alternative factors, Nanog and Lin28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. Dot1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.
To examine the influence of chromatin modifiers on somatic cell reprogramming, we employed a loss-of-function approach to interrogate the role of 22 select genes in DNA and histone methylation pathways. We tested a pool of 3 hairpins for each of 22 target genes and observed knockdown efficiencies of >60% for 21 out of 22 targets (Supplementary Fig. 1). We infected fibroblasts differentiated from the H1 human embryonic stem cell (ESC) line (dH1fs) with shRNA pools, transduced them with reprogramming vectors expressing Oct4, Sox2, Klf4 and c-Myc (OSKM), and identified the resulting iPSCs by Tra-1-60 staining (Fig. 1a)4. Eight shRNA pools reduced reprogramming efficiency (Fig. 1b). Among the target genes were Pou5F1/Oct4 (included as a control), and Ehmt1 and SetDB1, two H3K9 methyltransferases whose histone mark is associated with transcriptional repression. The remaining five shRNA pools targeted components of polycomb repressive complexes (PRC), major mediators of gene silencing and heterochromatin formation5. Inhibition of PRC1 (Bmi1, Ring1) and PRC2 components (Ezh2, Eed, Suz12) significantly decreased reprogramming efficiency while having negligible effects on cell proliferation (Fig. 1c, Supplementary Fig. 2). This finding is of particular significance given that Ezh2 is necessary for fusion-based reprogramming6 and highlights the importance of transcriptional silencing of the somatic cell gene expression program during generation of iPSCs.
In contrast to genes whose functions appear to be required for reprogramming, inhibition of three genes enhanced reprogramming: YY1, Suv39H1, and Dot1L (Fig. 1b, 1d). YY1 is a context-dependent transcriptional activator or repressor7, whereas Suv39H1 is a histone H3K9 methyltransferase implicated in heterochromatin formation8. Interestingly, enzymes that modify H3K9 were associated with both inhibition and enhancement of reprogramming, which suggested that unraveling the mechanisms for their effects might be challenging. Thus, we focused on Dot1L, a histone H3 Lysine 79 methyltransferase that has not previously been studied in the context of reprogramming9. We utilized two hairpin vectors that resulted in the most significant downregulation of Dot1L and concomitant decrease in global H3K79 levels (Supplementary Fig. 3a, b). Fibroblasts expressing Dot1L shRNAs formed significantly more iPSC colonies when tested separately or in a context where they were fluorescently labeled and co-mixed with control cells (Fig 2a, Supplementary Fig. 4). This enhanced reprogramming phenotype could be reversed by overexpressing an shRNA-resistant wildtype Dot1L, but not a catalytically-inactive Dot1L, suggesting that inhibition of catalytic activity of Dot1L is key to enhance reprogramming10 (Fig. 2a). Our findings with dH1fs were applicable to other human fibroblasts, as IMR-90 and MRC-5 cells also exhibited 3-fold and 6-fold increases in reprogramming efficiency, respectively, upon Dot1L suppression (Supplementary Fig. 5). To validate our findings independently of shRNA-mediated knockdown, we utilized a recently discovered small molecule inhibitor of Dot1L catalytic activity. EPZ00477711 (referred to as iDot1L) abrogated H3K79 methylation at concentrations ranging from 1uM to 10uM and increased reprogramming efficiency 3–4 fold (Fig. 2b, Supplementary Fig. 6a, b). Combination of inhibitor treatment with Dot1L knockdown did not further increase reprogramming efficiency, reinforcing our previous observation that inhibition of Dot1L’s catalytic activity is key to reprogramming (Supplementary Fig. 6c). iPSCs generated through Dot1L inhibition exhibited characteristic ESC morphology, immunoreactivity for SSEA4, SSEA3, Tra-1-81, Oct4 and Nanog, and differentiated into all three embryonic germ layers in vitro and in teratomas (Supplementary Fig. 7 a–c). Therefore, iPSCs generated following Dot1L inhibition display all of the hallmarks of pluripotency.
We next assessed Dot1L inhibition in murine reprogramming. iDot1L treatment led to 3-fold enhancement of reprogramming of Oct4-GFP MEFs (Fig. 2c). Reprogramming of tail-tip fibroblasts (TTFs) derived from a conditional knockout Dot1L mouse strain yielded significantly more iPSC colonies upon deletion of Dot1L12 (Supplementary Fig. 8a). Cre-mediated excision of both floxed Dot1L alleles in iPSC clones derived from homozygous TTFs was confirmed by genomic PCR (Supplementary Fig. 8b). Dot1L inhibition also increased reprogramming efficiency of MEFs and peripheral blood cells derived from an inducible secondary iPSC mouse strain13 (Supplementary Fig. 8c, d). Taken together, these results demonstrate that Dot1L inhibition enhances reprogramming of both mouse and human cells.
We next examined the cellular mechanisms by which Dot1L inhibition promotes reprogramming. Dot1L inhibition affected neither retroviral transgene expression nor cellular proliferation (Supplementary Fig. 9 a–c). Although previous studies indicated that Dot1L null cells have increased apoptosis and accumulation of cells in G2 phase9, we failed to observe a significant increase in apoptosis or change in the cell cycle profile of Dot1L-inhibited fibroblasts (Supplementary Fig. 9d, e). In human iPSC clones derived from shDot1L fibroblasts, Dot1L inhibition was no longer evident, reflecting the known silencing of retroviruses that occurs during reprogramming (Supplementary Fig. 10a). qPCR analysis revealed that the silencing occurred by day 15 after OSKM transduction (Supplementary Fig. 10b, c). To define the crucial time window for Dot1L inhibition, we treated fibroblasts with iDot1L at 1-week intervals during reprogramming. iDot1L treatment in either the first or second week was sufficient to enhance reprogramming, whereas treatment in the third week or a 5-day pretreatment had no effect (Supplementary Fig. 10d, e). Immunofluorescence analysis revealed significantly greater numbers of Tra-1-60-positive cell clusters on day 10 and day 14 in shDot1L cultures (Supplementary Fig. 11a, b), indicating that the emergence of iPSCs is accelerated upon Dot1L inhibition. When we extended the reprogramming experiments by 10 more days, shDot1L cells still yielded more iPSC colonies than controls (Supplementary Fig. 11c). Taken together, these findings indicate that Dot1L inhibition acts in early to middle stages to accelerate and increase the efficiency of the reprogramming process.
To assess whether Dot1L inhibition could replace any of the reprogramming factors, we infected control and Dot1L-inhibited fibroblasts with 3 factors, omitting one factor at a time. In the absence of Oct4 or Sox2 no iPSC colonies emerged (Fig. 2d). When we omitted either Klf4 or c-Myc, Dot1L-inhibited fibroblasts gave rise to robust numbers of Tra-1-60 positive colonies, while control cells generated very few colonies, as reported previously4 (Fig. 2d–f, Supplementary Fig. 12a). Importantly, Dot1L-inhibited fibroblasts transduced with only Oct4 and Sox2 gave rise to Tra-1-60-positive colonies, whereas control fibroblasts did not (Fig. 2d–f). These two-factor iPSCs exhibited typical ESC morphology, silenced the reprogramming vectors and had all of the hallmarks of pluripotency as gauged by endogenous pluripotency factor expression and the ability to form all three embryonic germ layers in vitro and in teratomas (Supplementary Fig. 7a–c, 12b). PCR on genomic DNA isolated from expanded colonies confirmed the absence of integrated Klf4 and c-Myc transgenes (Supplementary Fig. 12c). Thus, we were able to generate two-factor iPSCs either by suppression of Dot1L expression or chemical inhibition of its methyltransferase activity.
To gain insights into the molecular mechanisms of how Dot1L inhibition promotes reprogramming and replaces Klf4 we performed global gene-expression analyses on control and shDot1L fibroblasts prior to and 6 days after OSKM and OSM transduction along with cells that were treated with iDot1L. Relatively few genes were differentially expressed in shDot1L cells on Day 6 of reprogramming (22 up, 23 down; Supplementary Table 3). Inhibitor treated cells exhibited broader gene expression changes (405 up and 175 down; Supplementary Table 3), presumably due to more complete inhibition of K79me2 levels (Fig. 3a). In the absence of Klf4, 94 genes were differentially upregulated in shDot1L cells; intersection of this set of genes with the set differentially upregulated in 4-factor reprogramming of Dot1L-inhibited cells yielded only 5 common genes (Fig. 3a, b). We were particularly intrigued to find Nanog and Lin28 upregulated in all three instances of Dot1L inhibition, because these two genes are part of the core pluripotency network of human ES cells14,15 and can reprogram human fibroblasts into iPSCs when used in combination with Oct4 and Sox216.
We explored the possibility that Nanog and Lin28 upregulation might account for the enhanced reprogramming observed following Dot1L inhibition, and validated their upregulation in shDot1L fibroblasts upon OSM or OS transduction (Supplementary Fig. 13a, b). Interestingly, at this early time-point Rex1 and Dnmt3b, two other well-characterized pluripotency genes, were not upregulated suggesting that Dot1L inhibition does not broadly upregulate the pluripotency network. Suppression of either Nanog or Lin28 abrogated the 2-factor (OS) reprogramming of shDot1L fibroblasts, indicating the essential roles of Nanog and Lin28 in this process (Fig. 3c, Supplementary Fig. 13c). Dot1L inhibition also led to increased Nanog expression in the context of Oct4, Sox2 and Lin28 (OSL) and Lin28 expression in the context of Oct4, Sox2 and Nanog (OSN) (Supplementary Fig. 14a). Furthermore, Dot1L inhibition significantly increased the efficiency of three-factor reprogramming in the context of OSN and OSL (Supplementary Fig. 14b). Finally, inclusion of Nanog and Lin28 in the OSKM reprogramming cocktail did not confer any additional enhancement to shDot1L cells (Fig. 4d, Supplementary Fig. 14c). Taken together, these data implicate Nanog and Lin28 in the enhancement of reprogramming and replacement of Klf4 and c-Myc with Dot1L inhibition.
To gain insight into the genome-wide chromatin changes that are facilitated by Dot1L inhibition, we performed Chromatin ImmunoPrecipitation followed by DNA sequencing (ChIP-seq) for H3K79me2 and H3K27me3 in human ES cells as well as fibroblasts undergoing reprogramming, with or without iDot1L treatment (Supplementary Fig. 15). In both ES cells and fibroblasts, H3K79me2 is positively associated with transcriptionally active genes and negatively associated with genes marked by H3K27me3 (Supplementary Fig. 16a-c). ES cell specific genes marked by H3K79 included pluripotency factors, a subset of their downstream targets, and genes involved in epithelial cell adhesion such as CDH1 (280 genes; Supplementary Fig. 17a, b and Supplementary Table 4, 5). In contrast, in fibroblasts, genes marked by H3K79me2 were significantly enriched in genes induced during the epithelial to mesenchymal transition (EMT) (377 genes, Supplementary Fig. 17a).
Among the 348 genes that showed reduced H3K79me2 six days after OSKM expression, we likewise found a significant enrichment of gene sets associated with the induction of a mesenchymal state, including Snai2, TGF-β2 and TGFBR1 (Supplementary Fig. 18a)17,18. Only a few of these genes showed decreased expression at Day 6 (12 out of 348), but the vast majority of them lacked this mark in the pluripotent state (272 out of the 348 devoid of H3K79me2 in ESCs), suggesting they were destined for transcriptional silencing during reprogramming. This finding prompted us to ask whether Dot1L inhibition results in the removal of K79me2 from such fibroblast-specific, EMT-associated genes. Upon Dot1L inhibitor treatment, K79me2 levels were reduced on almost all loci, with the exception of a subset comprised mostly of housekeeping genes that also had high levels of K79me2 in ES cells (Supplementary Fig. 19a). Strikingly, the genes that lost proportionally the most K79me2 in inhibitor-treated fibroblasts during reprogramming (8-fold or more) were again highly enriched in genes induced in EMT (Fig. 19b). Mesenchymal master regulators such as Snai1/2, Zeb1/2, and Tgfb2 were among these genes (Fig. 4a)19. In the presence of the Dot1L inhibitor, these regulators were more strongly repressed during reprogramming, while epithelial genes such as E-cadherin and Occludin were more robustly upregulated (Fig. 4b). The extinction of fibroblast gene expression was accompanied by increased deposition of the repressive H3K27me3 mark on the majority of fibroblast specific regulators examined (Supplementary Fig. 20). In contrast, K27me3 was depleted to a greater extent on Sox2 and E-cadherin promoters, reflecting their activation during reprogramming. Finally, the H3K27me3 status of master regulators of other lineages, such as Olig2, MyoD1, Nkx2-1 and Gata4, remained unchanged upon Dot1L inhibitor treatment, suggesting that the deposition of H3K27me3 was specific to fibroblast specific regulators.
To test the functional importance of downregulation of mesenchymal regulators in the Dot1L-mediated enhancement of reprogramming, we overexpressed Twist1, Snail1 and Zeb1 or added soluble TGF-β2 to cells undergoing reprogramming in the presence of the Dot1L inhibitor. All of these perturbations significantly counteracted the enhancement observed with Dot1L inhibition (Fig. 4c). Interestingly, expression of these factors also abrogated the iDot1L-mediated upregulation of Nanog and Lin28, suggesting that the effect of Dot1L inhibition on these two pluripotency genes is likely to be indirect (Fig. 4d, e). Conversely, we tested whether destabilization of the mesenchymal state by inhibition of TGF-β signaling would be redundant with Dot1L inhibition. A small molecule inhibitor of TGF-β signaling (SB431542) increased reprogramming efficiency, but in combination with the Dot1L inhibitor, showed no significant further increase in iPSC colonies (Supplementary Fig. 21). Taken together these data indicate that in fibroblasts, downregulation of the mesenchymal gene expression program is critical to enhancement of reprogramming by Dot1L inhibition.
Our loss of function survey indicates that chromatin-modifying enzymes play critical roles for both reactivating silenced loci as well as reinstating closed domains of heterochromatin during the global epigenetic remodeling of differentiated cells to pluripotency, thus implicating specific enzymes as facilitators or barriers to cell fate transitions. Dot1L inhibition appears to enhance reprogramming at least in part by facilitating loss of K79me2 from fibroblast genes whose silencing is required for reprogramming (Supplementary Fig. 22). Interestingly, Klf4, which can be replaced by Dot1L inhibition, has been shown to facilitate MET by inducing E-cadherin expression20. Persistent K79me2 at the fibroblast master regulators during the initial phases of reprogramming seems to prevent shutdown of these genes, thus hindering the acquisition of an epithelial phenotype concomitant with delayed activation of Nanog and Lin28. In this regard H3K79me2 acts as a barrier to efficient repression of the somatic program by the reprogramming factors. This notion is consistent with the role of Dot1 in yeast, where it antagonizes gene repression21. As reprogramming of blood cells is also enhanced by Dot1L inhibition, we speculate that Dot1L inhibition may enhance reprogramming in a broad range of cell types by facilitating the silencing of lineage-specific programs of gene expression. Finally, our results also demonstrate that specific chromatin modifiers can be modulated to generate iPSCs more efficiently and with fewer exogenously introduced transcription factors.
shRNAs were designed using the RNAi Codex22. 97-mer oligonucleotides (Supplementary Table 1) were PCR amplified and cloned into MSCV-PM23 vector. Reprogramming assays were carried out with either retroviral4 or lentiviral16 reprogramming vectors. dH1f cells were previously described4. For gene expression analyses, total RNA was extracted from two or three independent culture plates for each condition and transcriptional profiling was performed using Affymetrix U133A microarrays. ChIP-seq was performed as described with slight modifications12.
shRNAs were designed using the RNAi Codex22. 97-mer oligos (Supplementary Table 1) were amplified with the following primer pair: Forward: GATGGCTGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG, Reverse: GTCTAGAGGAATTCCGAGGCAGTAGGC. PCR products were gel purified, digested with EcoRI and XhoI and ligated into the MSCV-PM vector. Clones were verified by sequencing. shRNA targeting the firefly luciferase was used as a control23. Nanog shRNA was previously described24.
293T cells were plated at a density of 2.5 × 106 cells per 10-cm dish. The next day, cells were transfected with 2.5 μg viral vector, 2.25 μg Gag-Pol vector and 0.25 μg VSV-G plasmid using 20 μl Fugene 6 (Roche Applied Science) in 400 μl DMEM per plate. Supernatant was collected 48 h and 72 h post-transfection and filtered through 45 μm pore size filters. For concentration, viral supernatants were mixed with PEG3350 solution (Sigma P3640, dissolved in PBS, 10% final concentration) and left overnight at 4°C. The next day, supernatants were centrifuged at 2500 rpm for 20minutes, and the pellets were re-suspended in PBS. Titering was performed on 293Ts. For shRNA infections, 500 μl of unconcentrated viral supernatant was used to infect 25,000 cells in the presence of 10ug/ml protamine sulfate. For fluorescent labeling of dh1fs, we used lentiviruses PRRL-GFP (Addgene #12252) and FUdGW-Tomato (Addgene #22771).
dH1f cells were first infected with shRNA viruses at high MOI to ensure all cells received at least one vector (Gauged by puromycin resistance of parallel infected wells). 25,000 shRNA-infected dH1f cells were then plated per well in 12-well plates and infected overnight with either retroviral (MOI 2.5)25 or lentiviral (Addgene #21162, 21164; 100–200 μl supernatant)26 reprogramming factors. For 2-factor reprogramming, Oct4 and Sox2 viruses were used at an MOI of 5. 6 days later, cells were trypsinized and re-plated 1:4 or 1:6 onto 6-well plates. Medium was changed to hES medium daily until Day 21 when plates were fixed. Small molecule inhibitor of Dot1L, EPZ004777 (a gift from Epizyme, Inc., Cambridge, MA) was dissolved in DMSO as a 10mM stock and was added at the indicated concentrations. For Dot1L rescue experiments, an MSCV-based retroviral vector encoding human Dot1L with or without mutations in the SAM binding site (gifts of Y. Zhang) were mutagenized at the shRNA target site using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). In certain experiments, Nanog and Lin28 expression was achieved using lentiviruses (Addgene #21163). IMR-90 and MRC5 human diploid fibroblasts were purchased from ATCC and 50000 cells were used in reprogramming experiments. SB431542 (Stemgent) was used at a final concentration of 2 μM. TGF-beta2 (R&D Systems) was added daily at a concentration of 2ng/ml. Twist1 (Addgene #1783), Snai1 (Addgene #23347) and Zeb1 (a gift of R.A.Weinberg) were overexpressed using retro- or lentiviruses. Statistical analysis was performed using a Student’s t-test.
Total RNA was extracted from two or three independent culture plates for each condition with an RNeasy Mini kit (Qiagen). Synthesis of cRNA from total RNA and hybridization/scanning of microarrays were performed with Affymetrix GeneChip products (HGU133A) as described in the GeneChip manual. Normalization of the raw gene expression data, quality control checks, and subsequent analyses were done with the open-source R-project statistical software (R Development Core Team, 2007)(http://www.r-project.org/) together with Bioconductor packages. Raw data files (.CEL) were converted into probe set values by RMA normalization. Genes were selected at a threshold of Log Ratio 0.4. The microarray data have been deposited in National Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE29253.
Total RNA was extracted using RNeasy Mini kit coupled with RNase-free DNase set (Qiagen) and reverse transcribed with Hexanucleotide Mix (Roche). The resulting cDNAs were used for PCR using SYBR-Green Master PCR mix (Applied Biosystem) in triplicates. All quantitations were normalized to an endogenous Beta-Actin control. The relative quantitation value for each target gene compared to the calibrator for that target is expressed as 2−(Ct-Cc) (Ct and Cc are the mean threshold cycle differences after normalizing to Beta-Actin). List of primers can be found in supplementary table 2.
Immunostaining of reprogramming plates were performed as described27. Briefly, cells were fixed with 4% p-formaldehyde and stained with biotin-anti-Tra-1-60 (eBioscience, #13-8863-82, 1:250) and streptavidin horseradish peroxidase (Biolegend, #405210, 1:500) diluted in PBS (3%), FCS (0.3%) Triton X-100. Staining was developed with the Vector labs DAB kit (#SK-4100), and iPSC colonies quantified with ImageJ software. For the characterization of shDot1l-iPS cells, we picked single colonies onto MEF coated 96-well plates. The plates were fixed for 20 min with 4% p-formaldehyde/PBS (+/+), washed several times with PBS (+/+) and incubated overnight at 4°C with primary antibody and Hoechst diluted in 3% donkey serum/3% BSA Fraction VII/0.01% Triton X-100/PBS (+/+); Hoechst, Invitrogen #H3570 (1:20,000), Tra-1-81/A488 (BD #560174), SSEA-4/A647 (BD #560219), Tra-1-60/A647 (BD #560122), Nanog, rabbit polyclonal (Abcam #ab21624), Oct4, rabbit polyclonal (Abcam #ab19857). For Nanog and Oct4, donkey anti-rabbit IgG/A555 (Molecular Probes #A31572) secondary antibody was used. After several washes with PBS (+/+), images were acquired using a BD Pathway 435 imager equipped with a ×10 objective.
iPSCs grown on MEFs were harvested with Collagenease IV (1 mg/ml in DMEM/F12). Cell clumps from one 6-well plate were resuspended in 50 μl DMEM/F12, 100 μl collagen I (Invitrogen-#A1064401) and 150 μl hESC-qualified matrigel (BD Biosciences#354277). Cell clumps were then injected into the hind limb femoral muscles (100 μl suspension per leg) of Rag2 γ/c mice. After 6–8 weeks, teratomas were harvested and fixed in Bouin’s solution overnight. Samples were then embedded in paraffin, and sections were stained with hematoxylin/eosin (Rodent Histopathology Core, Harvard Medical School, Boston, MA, USA).
Embryoid body differentiation was performed as described28. To check for the presence of the reprogramming transgenes, genomic DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen) and PCR was performed with specific primers to the endogenous or the viral trangenes4.
ChIP-seq was performed as described with slight modifications12. 300000 cells were fixed at room temperature in PBS 1% formalin (v/v) for 10 minutes with gentle agitation. Fixation was stopped by the addition of glycine (125 mM final concentration) and agitation for 5 min at room temperature. Fixed cells were washed twice in ice-cold PBS, resuspended in 100μl of SDS lysis buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl, pH 8.1). Chromatin was sheared by sonication to about 100–500bp fragments using bioruptor (diagenode, Denville, NJ) and diluted ten fold with dilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1,167 mM NaCl). Antibodies against specific histone modifications was added to sonicated chromatin solution and incubated at 4°C overnight with gentle agitation. The antibodies used were anti-H3K27me3 (Millipore 07–449) and anti-H3K79me2 (abcam 3594). Immune complexes were collected by incubation with 20ul of Protein A/G agarose beads (Millipore) for an hour at 4 degree with gentle agitation. Precipitates were washed sequentially with ice cold low salt wash (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris-HCl, pH8.1, 150mM NaCl), high salt wash (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl wash (0.25M LiCl, 1% IGEPAL CA-630, 1% deoxycholic acid, 1mM EDTA, 10mM Tris-HCl, pH 8.1) and TE wash (1mM EDTA, 10mM Tris-HCl, pH 8.1) for 5 mins each at 4°C with gentle agitation. Samples were centrifuged briefly in between washes to collect the beads. Immunoprecipitated DNA was eluted by incubating beads wtih 150ul elution buffer (1%SDS, 0.1 M NaHCO3) with gentle agitation for 15mins at room temperature. Elution was repeated once and eluates were combined, sodium chloride (final concentration of 0.2M) were added to the eluate and eluates were incubated at 65°C overnight to reverse crosslinking. DNA was purified using PCR purification spin column (Qiagen). For ChIP sequencing, ChIP DNA libraries were made following Illumina ChIP-seq library preparation kit and subjected to Solexa sequencing (Illumina) at Center for Cancer Computational Biology, Dana Faber Cancer Institute. Sequencing was performed on Illumina HiSeq2000. The reads were aligned to the human genome hg18 using Bowtie29 and the reads that mapped to multiple locations in the genome were discarded. We quantified the histone modification level as the number of reads per million per kb in a window of interest. The window was 1 Kb upstream to 1 Kb downstream from the Transcription Start Site (TSS) for K27me3 and 1 Kb upstream to 2 Kb downstream of the TSS for K79me2. To determine the significance of signal at a gene, an empirical background model was estimated. Genes that showed interesting pattern of histone methylation change were identified using iCanPlot (www.icanplot.org). Geneset Overlap Analysis was performed by finding the overlap of a set of genes of our interest with the genesets in the collections c2.all, c3.all and c5.all in MSigDB (Total number of genesets in these collections is 5562)30. Hypergeometric test, with Bonferroni correction for multiple hypothesis testing, was performed to generate the p-values associated with gene set overlap analysis. ChIP-Seq data have been deposited at the NCBI Gene Expression Omnibus with accession number GSE35791.
We thank G. Hu and S.J. Elledge for providing the MSCV-PM vector, K. Ng and M.W. Lensch for teratoma injections and assessment and S. Loewer for discussions. We also thank Ed Olhava and Epizyme Inc. for synthesizing and providing the Dot1L inhibitor, EPZ004777. G.Q.D. is an investigator of the Howard Hughes Medical Institute. Research was funded by grants from the US National Institutes of Health (NIH) to S.A.A. (CA140575) and G.Q.D., and the CHB Stem Cell Program.
Author contributionsT.T.O. performed project planning, experimental work, data interpretation and preparation of the manuscript. N.K., A.C, N.Z., J.U. and O.B.M. performed experimental work. P.C. and A.U.S. participated in data analysis. K.M.B. and S.A.A. provided critical materials and participated in the preparation of the manuscript. P.B.G. and E.S.L., participated in data acquisition, data interpretation and preparation of the manuscript. G.Q.D supervised research and participated in project planning, data interpretation and preparation of the manuscript.