Transcription changes within and between four promoter transcription classes of genes in ESCs and MEFs

ESCs have an unusually open chromatin structure and a high level of global transcription, which have been proposed to be the basis for ESC pluripotency (Efroni et al. 2008). We too observed that ESCs have a slightly greater number of active genes (85%) as compared with MEFs (80%) (Fig. 3A). Furthermore, we calculated that 28% of genes are significantly more transcriptionally active in ESCs than in MEFs, whereas only 16% have significantly higher nascent transcriptional activity in MEFs than in ESCs, supporting heightened transcriptional activity and complexity as a distinguishable feature of ESCs (see also the Supplemental Material; Supplemental Fig. S1).

We further classified genes based on the density of transcriptionally engaged Pol II in both the gene body and the promoter-proximal region to derive four major categories (Fig. 3A; Supplemental Fig.S5): (1) class I (not paused, transcribed): active transcription in the gene body without a significant enrichment of density at the promoter; (2) class II (paused, transcribed): active transcription in the gene body with significantly higher 5′ pause density; (3) class III (paused, not transcribed): no significant transcription in the body of the gene, but with a significantly higher 5′ pause density; and (4) class IV (not paused, not transcribed): inactive transcription without a 5′ peak. Additionally, the presence or absence of a divergent polymerase peak near the TSS was determined for each gene to assess the relationship between divergent transcription and transcription in the sense direction. In total, ~39% and 34% of all mappable RefSeq genes have paused Pol II in ESCs and MEFs, respectively, and ~76% and 67% exhibit peaks of divergent polymerase in ESCs and MEFs, respectively (Fig. 3A). Although ESCs have a higher number of genes classified as paused, the proportions of transcriptionally active genes where pausing is a rate-limiting step (class II) or where it does not appear to be so (class I) are almost equally divided in both ESCs and MEFs, indicating that neither cell type possesses a skewed bias toward transcription regulation via one mechanism or the other.

About half of the transcriptionally active genes in ESCs and MEFs have significantly higher 5′ levels of Pol II, suggesting that promoter-proximal pausing is a common rate-limiting step during transcription. In contrast, very few (<2%) of transcriptionally inactive genes have paused Pol II (class III) (Fig. 3A), bolstering the view that pausing is associated more with transcribed, rather than strictly nontranscribed, genes (Core et al. 2008). (Note, however, that the sensitivity and low background of GRO-seq forces us to call genes as transcribed that would be considered inactive in a microarray assay.) The majority of transcriptionally active, but not inactive, genes also has RNA polymerase that is engaged in divergent transcription (Fig. 3A).

Strikingly, GO analysis has revealed that class I and class II genes are each enriched for distinct categories of gene function. Class I, and not class II, genes in ESCs are significantly enriched for genes that play roles in multicellular organismal development, cell adhesion, and intracellular signaling cascade (Fig. 3B). In contrast, class II (and not class I) genes in ESCs are significantly enriched for genes that function in response to extracellular or intracellular stimuli, such as translation, cell cycle, DNA damage, and modification-dependent catabolic processes. The GO terms enriched for both class I and class II genes were the same in MEFs (Fig. 3C). These results indicate that different cellular processes use distinct mechanisms when regulating transcription, and imply that the transcription activators that are associated with a particular cellular process act at the same step in transcription regulation.

A majority of genes in both ESCs and MEFs are in the same promoter-transcription class (Fig. 4A), but often exhibit substantial and quantitative differences in their relative amounts of polymerases in the promoter-proximal pause or the gene body region (Fig. 4B, panels a–c). Additionally, we found that many genes change their promoter transcription classification in MEFs compared with ESCs, indicating that they have undergone regulated changes that alter their rate-limiting step.

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Figure 4.

Targets of regulation in the transition from ESCs to MEFs. (A) The percentages of genes from each promoter transcription class that change class from ESCs to MEFs are presented above each straight arrow. The circular arrows represent the number of genes that do not change classification. (B) Representative GRO-seq plots comparing ESCs (top) versus MEFs (bottom) of genes that maintain (panels a–c) or switch (panels d–i) their promoter transcription class. (C) Comparisons of the average GRO-seq densities in the bodies of class I and class II genes in ESCs and MEFs. Oct4 (dot) and Nanog (star), two core pluripotency factors that switched from class II in ESCs to class I in MEFs, are presented to show the difference in the gene body densities between two cell types. Actn1, which is significantly up-regulated in MEFs and switched from class I in ESCs to class II in MEFs, is indicated with an X. The middle line in each box plot indicates the median value, the top and bottom edges of the box plot are the 75th and 25th percentiles, and the small horizontal bars denote the 95th and fifth percentiles. (***) P-value < 0.0001 by Mann-Whitney test.

Twenty-five percent of the paused, transcribed genes (class II) in ESCs transition to transcribed, not paused (class I) in MEFs (Figs. 4A,B, panels d–f). Notably, in ESCs, the genes encoding the core pluripotency transcription factors—e.g., OCT4 and NANOG (Jaenisch and Young 2008; Chambers and Tomlinson 2009)—are expressed at high levels, yet possess higher densities of engaged Pol II at their 5′ end (class II genes) (Fig. 4B, panels e,f), indicating that pausing remains a rate-limiting step for transcription of these genes even when they are highly expressed. In MEFs, the core pluripotency transcription factors are dramatically down-regulated (Fig. 2C), with Oct4 and Nanog showing extremely low Pol II on the body of the gene or the pause region that is barely above the background level (among the lowest percentile expression levels of class I genes) (Fig. 4C). Notably, class II genes generally showed much higher transcriptional activity than class I genes (P < 0.0001 by Mann-Whitney test) (Fig. 4C). Among the genes that transitioned from class II in ESCs to class I in MEFs, ~50% were significantly down-regulated in MEFs and ~11% were significantly up-regulated in MEFs. This up-regulated transition is expected for genes activated in MEFs that are no longer rate-limited in the transition of paused Pol II to productively elongating states. Importantly, these data also indicate that pausing is not strictly a repressive mechanism, and that a reduction in pausing can accompany a reduction in gene expression. These findings agree with studies in Drosophila cell culture, where a function of paused Pol II is to prevent nucleosomes from binding key promoter sequences and thereby repressing transcription (Gilchrist et al. 2008, 2010).

Transcribed, nonpaused genes (class I) transition readily to paused, transcribed (class II) (Figs. 4A,B, panel g), as expected, if escape from the pause to productive elongation becomes rate limiting. Among the 11% of class I genes in ESCs that switched to class II in MEFs, 49% and nearly 19% became significantly up-regulated and down-regulated, respectively, in MEFs. Thus, changes in gene expression during differentiation are accompanied by regulatory changes that can eliminate or generate pausing as a rate-limiting step.

Roughly 15% of completely silent genes (class IV) in ESCs become transcribed, not paused (class I) in MEFs and vice versa (Fig. 4A), representing genes regulated at or prior to Pol II initiation (Fig. 4B, panels h,i). Interestingly, paused, transcribed genes (class II) rarely transition to completely inactive (class IV), and, correspondingly, inactive genes rarely transition to paused and transcribed, at least in these cell types. Therefore, paused genes appear to be designed to be active and tunable in both cell types, rather than to transition between a paused and an inactive state or vice versa. The paused Pol II at the 5′ end of the gene may offer the advantage of maintaining the transcriptional activity of promoters that must be responsive to a myriad of cellular signaling pathways during differentiation.

Entry into productive elongation is a general mechanism of regulating transcription in differentiation

Mouse genes with higher transcriptional activity in the gene body also have higher levels of Pol II density at the 5′ end when either ESCs or MEFs are analyzed. This can be seen from examples of individual genes (Fig. 4B, panels a–c) and in the genome-wide analysis (Supplemental Fig. S6A). However, the pausing index (the ratio of the GRO-seq density at the 5′ end of the gene to that in the gene body) decreases with increasing gene activity, as seen with individual genes (Fig. 4B, panel c) and in the genome-wide analysis (Supplemental Fig. S6B).

The transition of ESCs to MEFs may involve more than one regulatory event. Nonetheless, by examining the changes in Pol II distribution on all genes between cell types, we can infer whether the net regulatory changes have had major effects on rates of initiation or escape of paused Pol II to productive elongation (Fig. 5A). For example, if the density of Pol II on the body of the gene increases much more than the density on the promoter (i.e., the pausing index decreases with an increase in expression), then we can infer the greater regulatory effect is on the rate of pause escape. If an increase in Pol II density on the gene body is accompanied by similar increases in both paused and gene body Pol II (i.e., no changes in the pausing index), then we can infer the major change is on the rate of initiation (Core et al. 2008).

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Figure 5.

Regulation of gene expression by changing the efficiency of entry into productive elongation. (A) Illustrations depicting types of transcriptional activity change in MEFs (increase [1] and decrease [2]) relative to ESCs representing quadrants 1 and 2, as in B and C, are provided. Pause index is defined as the ratio of pause peak density to gene body density. The fold changes of pause peak GRO-seq density (B) or pausing index (C) versus gene body GRO-seq density in MEFs relative to ESCs are plotted for all mappable genes. The R-value, determined by Pearson's correlation, is presented within the plot. Contour lines by decile are shown in the heat map scale at the right.

Overall, we found that changes in the gene body transcription activity in MEFs relative to ESCs are accompanied by qualitatively (i.e., of the same sign) similar changes in GRO-seq density at the 5′ end, consistent with rates of recruitment and initiation indeed contributing to the overall activity of a gene (Fig. 5B). Nevertheless, as transcription levels increase, the relative rate of release of paused Pol II into productive elongation increases to a greater extent than entry into the pause site, and, correspondingly, as transcription levels decrease, the relative rate of release of paused Pol II into productive elongation decreases to a greater extent than entry into the pause site (Fig. 5C). These findings support the hypothesis that full-length transcription has a significant component of control at the level of escape of paused Pol II into productive elongation.

Developmental regulatory genes are transcribed at a modest level in ESCs and are not enriched for pausing

Because ESCs are pluripotent, the promoters of genes that regulate and execute lineage specification are expected to be poised for activation but repressed in ESCs by repressive protein complexes PRC1 and PRC2 (Ku et al. 2008). We evaluated whether such developmental regulatory genes have paused Pol II and assessed their level of expression. We found that, although transcriptional activity is detectable in ESCs at many of these master regulators of lineage specification, only a small portion of these active genes display significantly higher GRO-seq-measured densities at the 5′ end (class II) (Fig. 6A).

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Figure 6.

Developmental regulators of many lineages are transcribed but not paused in ESCs. (A) The promoter transcription classes and GRO-seq levels of known regulators associated with lineage specification are shown for ESCs and MEFs. The GRO-seq density levels in the body of the gene (from the lowest to the highest, 10%, 25%, 50%, 75%, and 100%, as ranked by the gene body density) is indicated by heat map for class I (green) and class II (orange) genes. The lists of developmental regulators are compiled from Mikkelsen et al. (2007), Stock et al. (2007), and Marson et al. (2008). (B) The changes in the GRO-seq gene body density for developmental controllers and markers involved in mesenchymal lineages (left) or all lineages except for mesenchymal lineages (right) are compared between ESCs and MEFs. Significance of changes in expression levels between cell types are P < 0.05 for mesenchymal and P < 0.01 for nonmesenchymal lineage controllers by Mann-Whitney test.

Compared with MEFs, which are of mesenchymal lineage, ESCs express a more diverse range of lineage specifiers. In general, the transcription level of these developmental regulators is clearly higher in ESCs compared with MEFs when genes related to mesenchymal lineage are excluded (Fig. 6B, right), albeit expressed at a modest level compared with the average GRO-seq density of all active genes (Fig. 6A). Interestingly, neuroectodermal or neuronal lineage-controlling master transcription factors such as Olig1, Olig2, and Nestin are transcribed at the highest levels relative to other lineages in ESCs. In contrast, genes controlling mesenchymal lineage establishment are consistently up-regulated in MEFs relative to ESCs (Fig. 6B, left). Together, our findings demonstrate that master regulators or markers of many different lineages are broadly transcribed at a modest level in ESCs, but mostly do not possess a significant level of paused Pol II.

RNA polymerase at genes whose promoters have either activating or repressing histone modifications

Promoters usually have adjacent nucleosomes that bear either an H3K4me3 activation mark or an H3K27me3-repressive mark. Comparison of GRO-seq with ChIP analysis of histone methylation marks shows that the genes that harbor H3K4me3 near the promoter (Ku et al. 2008) display significant levels of transcriptionally competent RNA polymerase at the promoter in ESCs, as measured by GRO-seq (Fig. 7A). In contrast, genes with only H3K27me3 show very little GRO-seq activity. The metagene profiles of Pol II and H3K4me3 ChIP-seq densities in genes with different GRO-seq signal levels (deciles) show that the level of transcriptionally competent polymerase correlates quantitatively with the amount of promoter-associated Pol II and H3K4me3 marks (Fig. 7B).

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Figure 7.

Regulation of distinct steps in transcription at polycomb target genes. (A) GRO-seq metagene profiles of all genes (purple), the subset of genes marked with H3K4me3 but not H3K27me3 (green), or the subset with H3K27me3 but not H3K4me3 (orange) near the promoters in ESCs (Ku et al. 2008). (B) The composite profiles of GRO-seq, Pol II (8WG16) ChIP-seq (Seila et al. 2008), and H3K4me3 ChIP-seq (Mikkelsen et al. 2007) are shown for genes whose level of expression in the gene bodies are in the top 10% (purple), middle 10% (green), and bottom 10% (orange) of active genes in ESCs, as determined by GRO-seq. The Y-axis is reads per kilobase per million. For ChIP-seq data, the forward and reverse reads are plotted above and below the horizontal axis, respectively. The midpoint between peaks of forward and reverse read density corresponds to the in vivo binding site, as described in Seila et al. (2008). (C) GRO-seq metagene profiles for all genes (purple) and H3K4me3/H3K27me3 bivalent genes (gray) (Ku et al. 2008). (D) Bivalent genes were subclassified into those that have PRC1 (cyan) or not (squash), based on Ku et al. (2008). The composite profiles for each subclass are plotted for GRO-seq, Pol II ChIP-seq, and H3K4me3 ChIP-seq, as in B. (E) The average GRO-seq densities of genes targeted by core pluripotency transcription factors with (sea green; n = 381) or without (red; n = 2,838) PRC2 component SUZ12 co-occupancy in ESCs. The gene lists were taken from Marson et al. (2008).

Transcription of genes with bivalent chromatin domains is regulated distinctly by PRC2 and PRC1

Determining the presence and status of Pol II at bivalent genes is important for understanding the mechanisms that govern their transcription. We found that bivalent genes that are targets of the PRC2 complex, but not PRC1, retain promoter-proximal engaged Pol II that is more tightly confined to a region near the TSS and also display dramatically reduced levels of elongating Pol II. These results suggest that transcription at bivalent genes bound by PRC2 is regulated after the initiation step but very early in elongation. Although PRC2 activity has been shown to function in recruiting PRC1 and in DNA looping, no connections to distinct steps in transcription have been made previously (Simon and Kingston 2009).

The bivalent genes that are occupied by both the PRC2 and the PRC1 complexes display much less promoter-proximal Pol II, suggesting a preinitiation block to earlier steps in transcription at these genes. This is consistent with the ability of PRC1 to compact nucleosomes (Francis et al. 2004), interfere with specific functions of the transcriptional apparatus, or both (Breiling et al. 2001; Dellino et al. 2004). The stronger repression of bivalent genes through the additional activity of PRC1 is also evident in that these genes show more robust retention of repressive chromatin through differentiation than PRC1⁻,2⁺ bivalent genes (Ku et al. 2008). Nonetheless, these genes are not completely silent. We found that both bivalent gene classes exhibit significant levels of productive elongation that are higher than at PRC-bound genes with H3K27me3 but not H3K4me3 (Fig. 7, cf. A and C).

The transcriptional activities of most genes are proportional to the levels of H3K4me3 on their promoter regions (Pokholok et al. 2005). In marked contrast, the transcriptional activity and divergent transcription of both classes of bivalent genes can vary widely, but the amount of H3K4me3 on the promoter remains at a level that is almost equal to the genome-wide average (Fig. 7D). This constant presence of H3K4me3 modifications across the promoter region may suggest that bivalent genes are poised for further activation. Recently, H3K4me3 modifications have been shown to evoke a dynamic cycle of histone acetylation and deacetylation at the promoters of inactive genes, which may facilitate the cross-talk between different histone modifications to prepare for activation (Wang et al. 2009). In addition, the high affinity of the TAF3 subunit of TFIID for the H3K4me3-modified histone tail may assist activation for bivalent genes that are highly enriched for CpG islands but lacking a TATA box (Vermeulen et al. 2007).

Many developmental regulatory genes are targets of PRC2 and PRC1 in ESCs, and most are transcribed at a modest level but do not feature a significant peak of paused Pol II. Interestingly, another study has shown that these genes retain a high level of Ser5-phosphorylated Pol II at the promoter relative to the gene body (Stock et al. 2007). Although paused Pol II is Ser5-phosphorylated, these promoters could possibly contain a form of Pol II that either has not fully entered elongation or is backtracked and unable to elongate in a run-on assay.

Most developmental regulatory genes are not highly expressed in ESCs; however, we do note that regulators of multiple lineages show some “promiscuous transcription,” and this shares some similarities with what has been observed in the hematopoietic system (Hu et al. 1997; Miyamoto et al. 2002). Interestingly, the regulators of neural and neuroectodermal lineages are among the highly expressed regulators in ESCs, supporting the hypothesis that ESCs in culture have an “aptitude” for neural differentiation (Hemmati-Brivanlou and Melton 1997; Ying et al. 2003).

Nuclear run-on assay

GRO-seq experiments were carried out as described previously (Core et al. 2008). Briefly, nuclei from 5 × 10⁶ cells were isolated, run-on-transcribed with Br-UTP and other NTPs, and base-hydrolyzed to yield nascent RNAs with an average size of roughly 100 nt. Br-UTP-incorporated nascent transcripts were purified after three rounds of serial Br-UTP enrichment steps. In order to empirically determine the sensitivity of our assay in detecting rare nascent transcripts in the reaction and the purity of Br-UTP-incorporated nascent transcripts over UTP-containing transcripts, we included spiking controls incorporated with or without Br-UTP at known concentrations in the nuclear run-on reaction. Nascent RNAs were prepared for Illumina sequencing.

We thank Steven Petesch, Katherine Munson, and the Lis labroatory members for critical reading of the manuscript; Hojoong Kwak for help in heat map analysis; and Peter Schweitzer and Tom Stelick at Cornell Core DNA sequencing facility and Illumina, Inc., for help with Solexa sequencing. This work was supported by GM25232 and HG004845 from the National Institutes of Health (to J.T.L.), Cornell Institutional grants from the New York Stem Cell Science (to J.T.L. and J.C.S.), and a post-doctoral fellowship from the American Cancer Society (I.M.M.).