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Cell Stem Cell. Author manuscript; available in PMC 2012 Jan 7.
Published in final edited form as:
Cell Stem Cell. 2011 Jan 7; 8(1): 96–105.
doi:  10.1016/j.stem.2010.12.001
PMCID: PMC3220622

Reprogramming factor expression induces rapid and widespread targeted chromatin remodeling


Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency-related gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarifies the temporal order of certain events during reprogramming.


Exposure to ectopic transcription factors has been established as a robust way to shift somatic cells towards alternative somatic states and to pluripotency (Graf and Enver, 2009). Ectopic expression of four transcription factors, Oct4, Sox2, Klf4, and c-Myc (OSKM), is capable of directing cells from any tissue towards the formation of induced pluripotent stem (iPS) cells in mouse and human (Hanna et al., 2010). Fully reprogrammed iPS cells can contribute to all germ layers and can form complete, fertile mice by tetraploid embryo complementation (Hanna et al., 2010). Moreover, iPS cells are similar to their embryo-derived counterparts on a molecular level, indicating a genome-wide cascade of transcriptional and epigenetic changes that lead to a stable, newly acquired state (Mikkelsen et al., 2008).

Despite the remarkable fidelity that governs the transition to pluripotency, the overall frequency in which it occurs within induced populations is low and requires an extended latency of one or several weeks (Jaenisch and Young, 2008). Previous studies and the general reprogramming timeline suggest a requirement for secondary or stochastic events through which certain cells acquire unique advantages that permit transition to pluripotency (Hanna et al., 2009; Jaenisch and Young, 2008; Meissner et al., 2007; Yamanaka, 2009). Therefore, the ectopic expression of the current set of embryonic factors appears insufficient to completely reset the somatic nucleus alone and the mechanism of action likely includes the activation of additional yet unidentified downstream effectors.

Recent evidence suggests that certain phases of the reprogramming process may be more coordinated than previously assumed. This includes live imaging analysis that demonstrates conserved transitions within reprogramming populations (Smith et al., 2010). Transcriptional profiling and RNAi screening in clonally reprogramming populations have demonstrated robust silencing of somatic transcription factors and effectors as well as activation of critical epithelial markers that govern the most immediate definitive transition from fibroblast towards a “primed” or reprogramming amenable state; the output of somatic factor repression or intermediate stabilizing signaling factors have demonstrated improved iPS cell colony generation that suggests that this phase is an essential early step (Samavarchi-Tehrani et al., 2010). Despite recent progress, the global nature and scale of these early events as well as their impact on transcriptional and epigenetic landscapes remain unknown.

To gain more insight into the early events during reprogramming, we assayed genome-scale gene expression, DNA methylation, and chromatin state (marked by H3K4me1, me2 and me3, H3K27me3 and H3K36me3) in populations of induced fibroblasts that have undergone a discrete number of divisions. We find that dynamic transcription within the reprogramming population is limited and functionally dominated by silencing of somatic factors. In contrast to the relative rarity of transcription changes, we found that euchromatin-associated H3K4 methylation is a predominant global early activating response and occurs in the absence of transcriptional activation at corresponding loci and this is further supported by absence of gene body H3K36me3 or promoter RNA PolII enrichment. Interestingly, these targets include the promoters of many essential pluripotency related genes, and describe a coherent shift in cellular identity. We observe highly localized, coordinated depletion of repressive chromatin (H3K27me3) exclusively at promoters where H3K4 methylation is gained. Finally, this targeted remodeling extends to enhancers across the genome, which transition dramatically from the somatic state and represents a higher order level of cell state transition. Taken together, our results suggest that early transcriptional dynamics are largely dependent on pre-existing, accessible chromatin, and that ectopic factor induction initiates a concerted change in target chromatin through which pluripotent targets are primed for subsequent activation.


CFSE labeling enables enrichment of cells that have undergone discrete numbers of cell divisions

To further elucidate critical early steps in the reprogramming process we investigated responses to reprogramming factor expression in cells that had undergone no cell division and cells that had divided 1, 2 or more than 3 times. Using inducible (OSKM) secondary mouse embryonic fibroblasts (MEFs) we could ensure rapid and homogenous induction of the four factors as described previously (Mikkelsen et al., 2008; Wernig et al., 2008). We isolated doxycycline-induced cells that had undergone a defined number of cell divisions by combining the live stain CFSE (Carboxyfluorescein succinimidyl ester) and a serum pulsing protocol. Four distinct fractions were enriched based upon their mean proliferative number in a manner that ensures that proliferation is the predominant experimental variable (Figure 1A). All cells were collected in an arrested (serum-starved) state except the final sample, which was allowed to divide continuously under factor induction. We confirmed that the relative fluorescence intensity remains unchanged in the serum starved control compared to a serum starved, doxycycline-induced population that remains exposed to the reprogramming factors for 96 hours and experiences minimal or no cell division (Figure 1A). Importantly, CFSE labeled cells that proliferated continuously for 96 hours (with a mean fluorescence reduction to ~4,600 A.U. indicating 3 or more divisions), show highly similar global transcriptional attributes to populations that had not undergone CFSE labeling or serum withdrawal, demonstrating that this protocol does not interfere with the general reprogramming process (Figure S1A,B).

Figure 1
Global transcriptional and epigenetic dynamics during early induction of reprogramming factors

Transcriptional dynamics of early reprogramming populations are limited to sites with pre-existing H3K4 trimethylation

We next used our discrete cell populations to investigate the early gene expression and chromatin dynamics induced by the four factors. Global mRNA expression profiles revealed continuous trends across populations and a primary response to factor induction that operates almost exclusively within accessible H3K4me3 chromatin (Figure 1B, 97%, Fisher’s exact test p < 10−16). Upregulated (2-fold, t test p < 0.05) targets are predominantly associated with promoter histone H3K4me3 in MEFs prior to induction, and moreover are enriched 2.2 fold for loci that are H3K4me3 within ES cells (Figure 1B). Repressed genes (2-fold, t test p < 0.05) were enriched for H3K4me3 only or H3K4me3/H3K27me3 (bivalent) promoters in MEFs, but enriched 2.8 fold for bivalent states in pluripotent cells (Figure 1B). Both activated and repressed gene sets exhibited preferential promoter binding for the induced factors, with an asymmetric bias for enhanced expression among c-Myc regulated targets (9.5-fold increased likelihood, Fisher’s exact text p < 10−16) consistent with its function in the transition to transcriptional elongation as opposed to Pol II recruitment/initiation (Figure 1C) (Rahl et al., 2010). These observations indicate that early expression changes mediated by factor induction are in large part constrained by pre-existing chromatin and may only operate at promoters that are already in an open and accessible state. Moreover, these changes occur immediately and gradually increase with additional cell divisions (Figure S1C,D). These data suggest that in the earliest phase of reprogramming, fibroblast identity is predominantly perturbed by transcriptional silencing of somatic targets and not the activation of pluripotency-associated targets of the reprogramming factors.

Activating chromatin marks are targeted to the promoters of pluripotent genes prior to transcriptional activation

Next we wanted to investigate the consequences of ectopic factor activity at the chromatin level to compare the dynamics of functional epigenetic markers to the more limited observations that could be made when measuring transcriptional output alone. We generated genome-wide chromatin maps for the three independent methylation marks on H3K4 (mono-, di- and tri-methylation) as well as for H3K27 trimethylation and H3K36 trimethylation across the isolated populations using ChIP-Seq (Mikkelsen et al., 2007). We then focused our initial query towards H3K4me2, because it is a general marker of both promoter and enhancer regions and is broadly amenable to genome-wide analysis (as opposed to tri-methylation which is exclusive to promoters) (Bernstein et al., 2005; Heintzman et al., 2007). H3K27me3 was chosen as a marker associated with transcriptional silencing, in particular of developmental transcription factors (Bernstein et al., 2006; Lee et al., 2006; Mikkelsen et al., 2007). Comparison with previously published data sets confirms that our serum starvation protocol does not induce significant chromatin changes in the MEFs (Figure S1E,F) and ChIP followed by quantitative PCR for representative loci confirms the trends observed in our ChIP-Seq results (Figure S1G).

Surprisingly, H3K4me2 peaks exhibit dramatic changes at over 1,500 genes and continuously increase with successive cell divisions (Figure 1D). The results highlight two striking findings. First, H3K4me2 target loci do not correspond to observed changes in gene expression (Figure 1E, Chi square test p > 0.1). Furthermore, changes in H3K4me2 are apparent even in populations that have not yet divided based on CFSE intensity (Mann-Whitney U test p < 10−16). Notably, these regions are strongly enriched for pluripotency and developmentally regulated targets, such as Sall4, Lin28, and Fgf4, none of which will become transcriptionally active until later stages of iPS cell formation. These results provide novel insights into the reprogramming process and describe an unexpected chromatin remodeling response to the reprogramming factors that precedes transcriptional activation of ES cell exclusive genes (Figure S2A). We confirmed this observation with the transcriptionally-associated histone mark H3K36me3, which exhibits no enrichment at identified loci across the early reprogramming phase or outside of pluripotent cell types, and by RNA PolII occupancy at representative promoters did not yield significant enrichment compared to established iPS cell lines (Figure S2B,C). This suggests that complete chromatin remodeling to transcriptional initiation is either unstable or not yet established during this early phase.

For further analysis, we subdivided loci that gain H3K4me2 during early reprogramming into two classes: a set of “de novo” H3K4me2 loci which have essentially undetectable H3K4me2 levels in MEFs and a set of “enhanced” H3K4me2 loci whose H3K4me2 signals increase by a minimum of 2.5-fold relative to the MEF control (Figure 2A,B). In both cases, the chromatin changes are reproducible across the target loci and consecutively increase in magnitude with cell divisions, suggestive of a reproducible, coordinated process (Figure 2C). A third class of promoters was less represented but exhibited a loss of promoter H3K4me2 that correlates with transcriptionally silenced somatic determinants such as Postn (Figure 2D, 1.75 fold decrease in expression, n~110 genes, Mann-Whitney U Test p < 0.02). Overall, the changes in promoter H3K4me2 occur rapidly and are primarily targeted to a set of loci that function in early development or as active mediators of pluripotency, including epigenetic reprogramming of the endogenous Sox2, Klf4 and c-Myc promoters themselves (Figure S2D,E). Moreover, promoters gaining H3K4me2 are significantly enriched for targets of Oct4, and Sox2 (Figure 2E, Fisher’s exact test p < 0.0009 and 0.00039 for Oct4 and Sox2 respectively).

Figure 2
H3K4 dimethylation increases at pluripotency related genes and is lost in repressed somatic targets

We next investigated the positioning of the related histone marks H3K4me1 and H3K4me3 to explore potential overlaps with H3K4me2. Surprisingly, we find that H3K4me2 is exclusive within the de novo promoter set, which is devoid of all forms of H3K4 methylation in MEF controls and does not gain H3K4me1 or H3K4me3 concurrently with H3K4me2 (Figure 2F). Alternatively, the “enhanced” promoter set, which exhibits both H3K4me2 and H3K4me3 within control populations, coordinately increases both marks as induced populations continue to proliferate (Figure 2F). These data emphasize the value of H3K4me2 as a dynamic mark across promoters because it detects nascent histone modification at de novo promoters, which are under-enriched for these marks in MEFs, as well as increased representation of pre-existing chromatin modifications within enhanced promoters that are augmented by ectopic factor activity. Additionally, within pluripotent cells, H3K4me3 is enriched at the vast majority of genes that gain H3K4me2 within the early reprogramming phase. These H3K4me2 exclusive promoters may therefore imply a decoupled and transiently stable chromatin mechanism that precedes complete remodeling and gene activation.

The dynamic gain of H3K4 methylation occurs without promoter wide changes in somatically defined, repressive H3K27me3 when inspected across the entirety of target promoters (Figure S3A; Kolmogorov-Smirnov test p > 0.1). The retention of somatic heterochromatin marks at the same promoters highlights a possible barrier that prevents gene activation and suggests that repressive marks might be less dynamic than H3K4me2.

Repressive H3K27me3 is lost specifically at sites where H3K4 methylation is gained

We next investigated the positional context of H3K4me2 to explore possible epigenetic or genetic determinants of the early response to ectopic factor induction. Enhanced H3K4me2 peaks occur directly at transcription start sites (TSS) in two distinct promoter classes: those that will ultimately be activated at the iPS cell stage and those that are not activated but rather transition to a poised bivalent state (Figure 3A, Figure S3B). The positional gain of H3K4me2 is targeted to the TSS and does not display the bimodality seen in ES/iPS cells that is associated with nucleosome depletion at the site of initiation (Figure 3B, shaded region). We also examined chromatin changes at the subset of promoters with H3K27me3 in MEFs. Here, we found that positional gain of H3K4me2 is accompanied by a corresponding depletion of H3K27me3 that exhibits strong negative correlation (Figure 3C, Student’s t test p < 0.01). Remarkably, this H3K27me3 reduction is only present within the punctate boundaries of a sharply gained H3K4me2 peak and does not spread to the surrounding regions, which retain somatic levels of facultative, inhibitory heterochromatin as in the starting state.

Figure 3
Chromatin remodeling and genetic determinants define the early reprogramming phase

We also generated genome-wide DNA methylation data from the 0, 1 and >3 division populations and compared them to control and ES cell promoters. As expected, the majority of regions exhibiting dramatic H3K4me2 gain displayed promoter hypomethylation in all states (Figure 3B). Moreover, promoters with the most dramatic shifts in chromatin state generally exhibit higher CpG density and preferentially enrich for CpG islands (82%, Fisher’s exact test p < 10−33). DNA methylation data confirmed that these regions were consistently hypomethylated across populations, including in the starting fibroblast state, an expected epigenetic landscape that is generally characteristic of CpG islands. Additionally, it is interesting to note that regions with depletion of H3K4me2 were frequently associated with transcriptional repression and a vast majority (95%, Fisher’s exact test p < 10−41) corresponded to non-CpG island promoters at which H3K4 methylation status is often predictive of transcriptional activity. Taken together, these data suggest that the plasticity of somatic chromatin to changes by reprogramming factors is most amenable within certain boundaries in part governed by genetic determinants, such as CpG density and the regulatory motifs for reprogramming factors themselves.

Enhancer signatures are driven from somatic towards an ES cell-like state

The activity of reprogramming factors on target chromatin is not restricted to the promoter regions and operates similarly within intergenic regions (Figure 4A, S4A). Non-promoter intervals enriched for H3K4me2 have been correlated to functional enhancers genome-wide, the patterns of which are remarkably variable across cell type and have been used as a high information content signature of a given cell state (Heintzman et al., 2007). We thus reasoned that non-promoter H3K4me2 elements that differ between MEFs and iPS cells could provide further insight into the early dynamics of reprogramming. Unlike promoter elements, which predominantly gain H3K4me2, epigenetic signatures of enhancers are gained and lost as reprogramming populations shift away from the somatic state (Figure 4B). Moreover, enhancer dynamics are shifted rapidly, a majority of intergenic H3K4me2 dynamics occur on or before a single cell division (54% gained, 66% lost) and progress continuously with division number (Figure S4B). Of the 11,228 H3K4me2 enhancers identified in the reprogramming populations, 46% are shared with ES cells and 8,407 somatic exclusive enhancer regions are depleted (Figure 4B). Intergenic analysis of additional H3K4 methylation marks confirm the canonical architecture of enhancer elements, with strong overlap of H3K4me1 and H3K4me2 and relative lack of promoter exclusive H3K4me3 (Figure 4C). Moreover, reprogramming induced enhancer signatures appear to acquire H3K4 methylation sequentially, first gaining H3K4me1 followed by H3K4me2 (Figure 4C). From this context, examination of the epigenetic changes within intergenic regions provide a unique opportunity to model enhancer dynamics; moreover, genome wide characterization of H3K4me2 confirm its value as a highly informative epigenetic mark, being present in disparate promoter and intergenic contexts where H3K4me1 or H3K4me3 are mutually exclusive (Figure S4D). Intergenic shifts in H3K4me2 enrichment thus serve as a unique barcode for cellular identity and sensitively delineate the epigenetic displacement caused by reprogramming factor induction.

Figure 4
Global epigenetic dynamics during the early stage of reprogramming factor induction extends beyond target promoter regions to putative enhancers

We incorporated genome-scale DNA methylation maps of ES cells and MEFs (Meissner et al., 2008) with those generated for our induced populations for use in our analysis of intergenic H3K4me2. Genomic intervals that display rapid gain of H3K4me2 tended to exhibit relatively lower DNA methylation levels in MEFs (Figure 4D, left panel). In contrast, ES cell enhancer elements that are not activated after 96 hours of factor induction have significantly higher DNA methylation levels in MEFs (Figure 4D, right panel, Student’s t test p < 10−32). Interestingly, the MEF exclusive enhancers that are lost during reprogramming display complete hypermethylation within ES cells, but not within induced populations (Figure S4C). This suggests that ES-like DNA methylation patterns are not fully established until later stages of reprogramming. The failure to redistribute heterochromatin to somatic intergenic H3K4me2 enhancers may, in part, account for the instability/elasticity of reprogramming populations, which may traverse back towards a fibroblast-like state upon premature removal of ectopic factor expression (Samavarchi-Tehrani et al., 2010).

The sensitivity of H3K4me2 enhancement to DNA methylation is consistent with a model where DNA methylation and associated repressive chromatin structures limit the accessibility of these elements to nuclear reprogramming (Mikkelsen et al., 2008). Newly activated enhancers that are covered by genome-scale CpG methylation assays exhibit lower methylation levels at the site of H3K4me2 gain and are generally hypomethylated in starting fibroblasts (Figure 4D). The boundary of modification is confined by heterochromatin, here as dictated by CpG methylation, and is resistant to change. These data corroborate changes in promoter histone methylation, where H3K4me2 gain is restricted to sites of high CpG density, which are generally hypomethylated (Meissner et al., 2008), and uniquely amenable to rapid epigenetic reconfiguration (Xu et al., 2009).


To further advance our understanding of the transcription factor mediated reprogramming process we isolated clonally induced cells that had undergone defined cell divisions for genomic characterization. Our data demonstrate a robust trend within the early reprogramming population towards a primed epigenetic state that clearly precedes transcriptional activation and complete reprogramming. In addition to suggesting an early coordinated response, our data highlight transcriptional measurement as an incomplete descriptor of the cellular response to reprogramming factor induction. Importantly, gain of H3K4 methylation includes a broader array of notable targets such as key pluripotency and early development genes. As we report, these are particularly enriched for CpG island containing promoters. Moreover, at sites where H3K4me2 is dynamic, somatic heterochromatin (marked by H3K27me3) is depleted exclusively within the CpG island context but continues to be present in the periphery. Reestablishment of H3K27me3 at bivalent promoters is not observed and must pertain to a later phase of iPS cell generation (Pereira et al., 2010).

Our results provide a novel and sensitive measurement of the somatic response to transcription factor activity, which displays a greater trend towards promoter associated H3K4 methylated euchromatin, which may represent a critical step towards transcriptional activation. The continuous behavior of this trend as populations divide clearly demonstrates unique underlying activity that is likely to utilize the endogenous epigenetic machinery. The unexpected genome-wide extent of these events, including putative enhancer activation, appears mostly limited by sequence context and is most likely to occur within CpG islands in which reprogramming factor regulatory motifs are present. The scope through which promoters and enhancers are modified supports a deterministic model for the initial reprogramming response, as the global events are at expected targets and occur at a detectable frequency similar to what is observed within pluripotent populations. This is further consistent with more recent image-based data (Smith et al., 2010) and provides a novel interpretation for the epigenetic response to factor induction, in which genome-wide remodeling occurs within the majority of cells in the induced population, as opposed to selectively within an exclusive subpopulation that will contribute iPS cell progeny (Yamanaka, 2009). The immediate and progressive accumulation of euchromatin-associated marks at ES cell specific promoters and enhancers suggests that a detectable majority of cells in which the factors are induced undergo a certain level of epigenetic reprogramming even in the absence of cell division; these events are immeasurable by expression profiling alone and have to date been largely overlooked.

Moreover, as these events precede detectable transcription, it is likely that the chromatin dynamics observed at the endogenous loci are a critical initial step in the transition to molecular pluripotency. It is intriguing that the promoter dynamics observed are initially restricted to areas of high CpG density and especially CpG islands, whereas peripheral chromatin retains its original, somatic pattern. CpG islands are noted for their plasticity and responsiveness to transcription factor activity (Ramirez-Carrozzi et al., 2009). The periphery of these regions behave inversely – they are less CpG rich and more susceptible to DNA methylation and/or extended H3K27me3 spreading, marks that may stably maintain heterochromatin domains in restricted cell types and may require transcriptional activation to be completely depleted. Notably, it is in these regions where somatic epigenetic artifacts might be observed in iPS cell characterization studies and a likely explanation could be that these regions are generally less responsive to chromatin remodeling. In our model, the type of mark, the developmental history of its acquisition, and its distribution along target promoter elements all contribute to the response observed. At CpG dense, hypomethylated transcription start sites, factor binding is sufficient to induce the rapid redistribution of H3K4me2 marks at the promoter that may signal or prime that locus for transcriptional activation. This principle is recapitulated at enhancer sites, where H3K4me2 gain is restricted to somatically hypomethylated regions. As discussed earlier, factor induction alone is not sufficient for complete reprogramming. Instead, the process likely depends on the presence of further chromatin remodeling complexes or transcriptional recruitment elements that may be unavailable in somatic cells.

In conclusion, our data argue for an orchestrated response that yields an epigenetically definable intermediate state in the earliest stages of the reprogramming timeline. However it cannot as of yet be ascertained if the continuation to full pluripotency is predetermined by existing effectors within a select subpopulation or by stochastic activation of these players in iPS cell forming lineages. It is also likely that these epigenetic reprogramming events describe the limiting effect of the four factors (OSKM) themselves on a reprogramming population where only a select subset will progress to endogenous target activation; transition through this phase towards complete reprogramming likely involves additional factors. Regardless, continued dissection of the reprogramming process promises for a comprehensive identification of a sufficient factor set for complete and safe somatic to pluripotent reprogramming.


CFSE labeling and enrichment for proliferative cohorts

Mouse E13.5 fibroblasts were generated by blastocyst injection with doxycycline inducible Oct4, Sox2, Klf4, and c-Myc primary iPS cells as previously described. Cells were passaged several times and serum starved with 0.5% FBS containing medium for 18 h before CFSE labeling. Cells were labeled with CFSE in 5×106 cell batches with 5 uM cellTrace CFSE® (Invitrogen) in PBS according to the manufacturers protocol and plated at 1×106 cells per 10 cm dish in 0.5% FBS for an additional 12 hours before the induction of OSKM reprogramming factors. Factors were induced with 2 ug/ml doxycycline supplemented medium in either 0.5% or 15% FBS to control the relative number of proliferation for 96 hours (see Figure 1A). In brief: our “no division” cohort was cultured exclusively in 0.5% FBS containing medium while each successive proliferative cohort was cultured in 15% FBS containing medium containing doxycycline medium for 24 hours, 48 hours, and 96 hours. After serum pulsing, cells were switched back into 0.5% FBS medium to quell further division; all samples were cultured in doxycycline-supplemented medium for the entire 96 hours. The relative proliferative number for each cohort was ascertained using a BD LSR II fluorescent cytometer against an uninduced, serum starved control. RNA was collected using TRIzol (Invitrogen) and cells were crosslinked using 1% formaldehyde.

ChIP-seq library preparation and RRBS

Generation of genome wide sequencing libraries were performed as described using ~500,000 crosslinked samples as available input for a given antibody targeting a covalent histone mark. Sample sonication, Chromatin Immunoprecipitation and library generation were performed as described. RRBS libraries were performed on standardized 100 ng of genomic DNA isolated by proteinase K digestion and phenol:chloroform extraction as previously described (Gu et al., 2010). A refined protocol with available antibodies and lot numbers used in this document are available as supplementary information.


Gene expression profiles were acquired with Affymetrix Mouse Genome 430 2.0 Arrays and Robust Multi-Array (RMA)-normalized using GenePattern (http://www.broadinstitute.org/cancer/software/genepattern/). ChIP libraries were sequenced using the Illumina Genome Analyzer and mapped to the mouse mm8 genome as previously described (Mikkelsen et al., 2007). Description of enrichment calculations, statistical analyses, and normalizations are available as supplementary information. OSKM factor enrichment was performed using previously published data and analysis (Kim et al., 2008; Marson et al., 2008).

Research Highlights

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    Ectopic Oct4, Sox2, Klf4 and c-Myc induce H3K4 methylation at developmental promoters
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    Promoter H3K4 methylation precedes transcriptional activation
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    Localized depletion of H3K27me3 occurs within CpG island promoters gaining H3K4me2
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    H3K4me2 enhancer signatures are gained and lost in response to OSKM factor induction

Supplementary Material




We would like to thank Tarjei Mikkelsen for critical reading of the manuscript. We would like to apologize to author’s whose primary work we didn’t cite due to space restrictions. B.E.B. is an early career scientist of the HHMI. AM is a New Investigator of the Massachusetts Life Science Center (MLSC) and Pew Scholar. This work was funded by the MLSC.


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1. Bernstein BE, Kamal M, Lindblad-Toh K, Bekiranov S, Bailey DK, Huebert DJ, McMahon S, Karlsson EK, Kulbokas EJ, 3rd, Gingeras TR, et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell. 2005;120:169–181. [PubMed]
2. Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K, et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006;125:315–326. [PubMed]
3. Boland MJ, Hazen JL, Nazor KL, Rodriguez AR, Gifford W, Martin G, Kupriyanov S, Baldwin KK. Adult mice generated from induced pluripotent stem cells. Nature. 2009;461:91–94. [PubMed]
4. Gu H, Bock C, Mikkelsen TS, Jager N, Smith ZD, Tomazou E, Gnirke A, Lander ES, Meissner A. Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution. Nat Methods. 2010;7:133–136. [PMC free article] [PubMed]
5. Hanna J, Saha K, Pando B, van Zon J, Lengner CJ, Creyghton MP, van Oudenaarden A, Jaenisch R. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature. 2009;462:595–601. [PMC free article] [PubMed]
6. Hanna JH, Saha K, Jaenisch R. Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues. Cell. 2010;143:508–525. [PMC free article] [PubMed]
7. Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, Barrera LO, Van Calcar S, Qu C, Ching KA, et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet. 2007;39:311–318. [PubMed]
8. Jaenisch R, Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell. 2008;132:567–582. [PMC free article] [PubMed]
9. Kim J, Chu J, Shen X, Wang J, Orkin SH. An extended transcriptional network for pluripotency of embryonic stem cells. Cell. 2008;132:1049–1061. [PMC free article] [PubMed]
10. Lee T, Jenner R, Boyer L, Guenther M, Levine S, Kumar R, Chevalier B, Johnstone S, Cole M, Isono K, et al. Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells. Cell. 2006;125:301–313. [PMC free article] [PubMed]
11. Marson A, Levine SS, Cole MF, Frampton GM, Brambrink T, Johnstone S, Guenther MG, Johnston WK, Wernig M, Newman J, et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell. 2008;134:521–533. [PMC free article] [PubMed]
12. Meissner A, Mikkelsen TS, Gu H, Wernig M, Hanna J, Sivachenko A, Zhang X, Bernstein BE, Nusbaum C, Jaffe DB, et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature. 2008;454:766–770. [PMC free article] [PubMed]
13. Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol. 2007;25:1177–1181. [PubMed]
14. Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, Bernstein BE, Jaenisch R, Lander ES, Meissner A. Dissecting direct reprogramming through integrative genomic analysis. Nature. 2008;454:49–55. [PMC free article] [PubMed]
15. Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. 2007;448:553–560. [PMC free article] [PubMed]
16. Pereira CF, Piccolo FM, Tsubouchi T, Sauer S, Ryan NK, Bruno L, Landeira D, Santos J, Banito A, Gil J, et al. ESCs require PRC2 to direct the successful reprogramming of differentiated cells toward pluripotency. Cell stem cell. 2010;6:547–556. [PubMed]
17. Rahl PB, Lin CY, Seila AC, Flynn RA, McCuine S, Burge CB, Sharp PA, Young RA. c-Myc regulates transcriptional pause release. Cell. 2010;141:432–445. [PMC free article] [PubMed]
18. Ramirez-Carrozzi VR, Braas D, Bhatt DM, Cheng CS, Hong C, Doty KR, Black JC, Hoffmann A, Carey M, Smale ST. A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell. 2009;138:114–128. [PMC free article] [PubMed]
19. Samavarchi-Tehrani P, Golipour A, David L, Sung H, Beyer T, Datti A, Woltjen K, Nagy A, Wrana J. Functional Genomics Reveals a BMP-Driven Mesenchymal-to-Epithelial Transition in the Initiation of Somatic Cell Reprogramming. Cell stem cell. 2010 [PubMed]
20. Smith ZD, Nachman I, Regev A, Meissner A. Dynamic single-cell imaging of direct reprogramming reveals an early specifying event. Nat Biotechnol. 2010;28:521–526. [PMC free article] [PubMed]
21. Wernig M, Lengner CJ, Hanna J, Lodato MA, Steine E, Foreman R, Staerk J, Markoulaki S, Jaenisch R. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol. 2008;26:916–924. [PMC free article] [PubMed]
22. Xu J, Watts JA, Pope SD, Gadue P, Kamps M, Plath K, Zaret KS, Smale ST. Transcriptional competence and the active marking of tissue-specific enhancers by defined transcription factors in embryonic and induced pluripotent stem cells. Genes Dev. 2009;23:2824–2838. [PMC free article] [PubMed]
23. Yamanaka S. Elite and stochastic models for induced pluripotent stem cell generation. Nature. 2009;460:49–52. [PubMed]
24. Zhao XY, Li W, Lv Z, Liu L, Tong M, Hai T, Hao J, Guo CL, Ma QW, Wang L, et al. iPS cells produce viable mice through tetraploid complementation. Nature. 2009;461:86–90. [PubMed]
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