Author response: Non-canonical TAF complexes regulate active promoters in human embryonic stem cells

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew. https://doi.org/10.7554/eLife.00068.001 eLife digest Embryonic stem cells have two characteristic properties: they are able to differentiate into any type of cell, a property known as pluripotency, and they are able to replicate themselves indefinitely to produce an endless supply of new stem cells. Different genes code for the various proteins associated with these two properties, and understanding the behaviour and properties of stem cells in detail is a major challenge in developmental biology. In human embryonic stem cells that have not yet differentiated, the genes that code for the transcription factors involved in the self-renewal process are expressed, whereas the genes associated with differentiation are not active. However, if the expression of the genes for self-renewal is reduced, the process of differentiation will begin, and the embryonic stem cells will be able to produce any one of the 200 or so different types of cell found in the human body. All of this activity is orchestrated by proteins that oversee the transcription of specific regions of DNA into messenger RNA. Transcription is the first step in the process by which genes are expressed as proteins, and it cannot start until the relevant transcription factor binds to a stretch of DNA near the gene called the promoter. These transcription factors are complex structures that contain a central protein called TBP, which binds to the promoter, and 14 or so other proteins called TAFs. Maston et al. now report that the transcription machinery that regulates gene expression and self-renewal in human embryonic stem cells is different from that found in other types of cells, including embryonic stem cells taken from mice. In particular, they found that undifferentiated human embryonic stem cells contain only 6 of the 14 TAFs observed in other cells, although all 14 are present after differentiation. Moreover, for many active genes the transcription factors contained only two of these TAFs. There was also evidence for a new complex that contained the other four TAFs plus TBP. Maston et al. also demonstrated that the removal of just one of the six TAFs, or the addition of just one extra TAF, caused the process of differentiation to begin. This shows, they argue, that the unusual transcription machinery they have discovered is essential for the proper workings of human embryonic stem cells. https://doi.org/10.7554/eLife.00068.002 Introduction The specification of tissues and organs in development depends upon the spatially and temporally accurate execution of gene expression programs, much of which is regulated at the level of transcription. The factors involved in the accurate transcription of eukaryotic structural genes by RNA polymerase II can be classified into two groups. First, general (or basic) transcription factors (GTFs) are necessary and can be sufficient for accurate transcription initiation in vitro (for review, see Thomas and Chiang, 2006). These basic factors include RNA polymerase II itself and at least six GTFs: TFIID, TFIIA, TFIIB, TFIIE, TFIIF and TFIIH. The GTFs assemble on the core promoter in an ordered fashion to form a pre-initiation complex (PIC). Transcriptional activity is greatly stimulated by the second class of factors, promoter-specific activator proteins (activators). In general, activators are sequence-specific DNA-binding proteins whose recognition sites are typically present upstream of the core promoter. Activators work in large part by increasing PIC formation but can also act through other mechanisms, such as accelerating the rate of transcriptional elongation, promoting multiple rounds of transcription and directing chromatin modifications (reviewed in Green, 2005; Fuda et al., 2009; Weake and Workman, 2010). A long-held view of transcription activation is that specificity arises from the differential expression and activity of activators, which function through the common basic transcription machinery. However, it is now clear that the differential expression and use of basic transcription factors can also contribute to eukaryotic gene regulation (reviewed in Davidson, 2003; Hochheimer and Tjian, 2003). This notion is most dramatically illustrated by a variety of studies focused on the GTF TFIID, a multi-subunit complex composed of the TATA-box-binding protein (TBP) and a set of ∼14 TBP-associated factors (TAFs). One of the earliest clues about the differential function of TFIID came from studies in yeast demonstrating distinct classes of protein-coding genes that differ by their dependence on and recruitment of TAFs (Kuras et al., 2000; Li et al., 2000). Subsequently, similar classes of TAF-dependent and -independent genes were identified in mammalian cells (Raha et al., 2005; Tokusumi et al., 2007). Consistent with the existence of TAF-independent promoters, more recent studies have found that TAFs are depleted upon terminal differentiation of muscle (Deato and Tjian, 2007; Deato et al., 2008) and liver (D'Alessio et al., 2011). TFIID diversity is also promoted by tissue-specific variants of TAFs as well as TBP derivatives referred to as TBP-related factors (reviewed in D'Alessio et al., 2009; Müller et al., 2010). Human embryonic stem cells (hESCs) are a good example of a specialized cell type that is regulated by a unique transcriptional network. Two characteristic properties of hESCs, pluripotency, a capacity to differentiate into all fetal and adult cell lineages, and the ability to undergo symmetrical self-renewing divisions, are largely controlled at the transcriptional level (reviewed in Chen and Daley, 2008). In undifferentiated hESCs, pluripotency genes such as OCT4 (also called POU5F1), NANOG and SOX2 are expressed, whereas genes involved in differentiation are transcriptionally inactive (reviewed in Sun et al., 2006; Pan and Thomson, 2007). Decreased expression of pluripotency genes induces differentiation (Niwa et al., 2000), and thus proper transcriptional regulation is essential for self-renewal of undifferentiated hESCs. Despite intense efforts to identify hESC-specific activators involved in the transcriptional regulatory network of pluripotency, there has been relatively little analysis of GTFs in general and TFIID in particular. Here we find that both the composition and promoter occupancy patterns of hESC TAFs are highly unusual. We go on to show that this selective expression and use of TAFs establishes a transcriptional program required for hESC self-renewal. Results Undifferentiated hESCs express only a subset of TFIID TAFs In a search of published expression datasets (Abeyta et al., 2004), we found that several TAFs of the canonical TFIID complex were apparently not expressed in hESCs. To investigate this possibility, we analyzed expression of 13 TAFs by immunoblotting lysates from H9 cells, a well-characterized hESC line. As a control, we also analyzed TAF expression in HeLa cells, which have been extensively used to study TFIID composition and function. The immunoblot of Figure 1A shows, as expected, that all 13 TAFs were expressed in HeLa cells. By contrast, hESCs clearly expressed TAFs 2, 3, 5, 6, 7 and 11, whereas expression of TAFs 1, 4, 8, 9, 10, 12, and 13 was undetectable. Interestingly, TAF6 is expressed in both cell types, but the isoform present in H9 cells is predominantly the short delta form, whereas in HeLa cells, the major TAF6 isoform is the larger, alpha/beta form. The specificity of each TAF antibody was confirmed by RNA interference (RNAi)-mediated knockdown (Figure 1—figure supplements 1 and 2). We observed a similar TAF expression pattern in a second hESC line, H1 cells (Figure 1—figure supplement 3). Quantitative RT-PCR (qRT-PCR) analysis comparing TAF mRNA levels in HeLa and H9 cells correlated with the immunoblotting results (Figure 1B). Unlike the TAFs, all other GTFs analyzed were comparably expressed in HeLa and H9 cells (Figure 1C). Based upon these results we conclude that only six of the canonical TFIID TAFs are present in hESCs. Figure 1 with 3 supplements see all Download asset Open asset Undifferentiated hESCs express only a subset of TFIID TAFs. (A) Immunoblot analysis showing TAF levels in HeLa cells and H9 hESCs. β-actin (ACTB) was monitored as a loading control. (B) qRT-PCR analysis monitoring TAF expression in H9 cells relative to HeLa cells. A ratio of 1 (indicated by the red dotted line) indicates no difference in expression. Data are represented as mean ± SEM. (C) Immunoblot analysis showing levels of GTFs in HeLa cells and H9 hESCs. α-tubulin (TUBA) was monitored as a loading control. (D) Immunoblots showing TAF and TBP levels in H9 hESCs following induction of differentiation by retinoic acid treatment for 0, 3 or 6 days. OCT4 and NES were monitored as controls. https://doi.org/10.7554/eLife.00068.003 We next asked whether differentiation of hESCs results in a change in TAF composition. Toward this end, H9 cells were treated with retinoic acid to induce differentiation and TAF expression was analyzed by immunoblotting. Figure 1D shows, as expected, that following retinoic acid treatment, expression of the pluripotency factor OCT4 was lost and NES, a neuroectoderm marker, was induced. Significantly, TAFs 1, 4, 8, 9, 10, 12, and 13, which are not expressed in undifferentiated H9 cells, were induced following retinoic acid treatment. TAFs 2, 3, 5, 6, 7 and 11, which are expressed in undifferentiated H9 cells, were also present at a relatively constant level following retinoic acid treatment. hESCs have a non-canonical TBP-containing TAF complex To investigate whether the six hESC TAFs were associated in a stable complex, H9 cell nuclear extract was fractionated by sucrose gradient sedimentation and individual fractions analyzed for TAFs 2, 3, 5, 6, 7 and 11 by immunoblotting. The results of Figure 2A show that TAFs 2, 6, 7 and 11 co-sedimented with an apparent native molecular mass of ∼440 kDa. By contrast, TAFs 3 and 5 fractionated heterogeneously, and a substantial portion of both TAFs had an apparent molecular mass consistent with that of the free proteins (∼140 and ∼100 kDa, respectively). As expected, TBP, which is associated with multiple complexes involved in transcription by all three RNA polymerases, fractionated heterogeneously. Notably, however, a peak of TBP co-sedimented with TAFs 2, 6, 7 and 11. Figure 2 Download asset Open asset hESCs have a non-canonical TBP-containing TAF complex. (A) Sucrose gradient sedimentation. H9 cell nuclear extract was fractionated, and individual fractions were analyzed for TAFs by immunoblotting. Arrows indicate elution peaks of protein standards. (B) Co-immunoprecipitation analysis. Nuclear extracts from H9 cells were immunoprecipitated with an anti-TBP or control (anti-RAB2A) antibody and the immunoprecipitate was analyzed for TAFs and TBP by immunoblotting. https://doi.org/10.7554/eLife.00068.007 To provide additional evidence for a stable, multi-subunit TAF complex, and to determine whether TBP was a component, we performed co-immunoprecipitation experiments. TBP was immunoprecipitated from H9 cell nuclear extracts, and the immunoprecipitate was analyzed by immunoblotting for the six hESC TAFs. The results of Figure 2B show that TBP was stably associated with TAFs 2, 6, 7 and 11 but not TAFs 3 and 5. Collectively, these results indicate that H9 cells contain a non-canonical TAF complex composed of TBP and TAFs 2, 6, 7 and 11 but not TAFs 3 and 5. Two classes of active hESC genes based on TAF promoter occupancy The results of the expression analysis and biochemical experiments implied that the PIC formed on the promoters of active genes in hESCs would have an atypical TAF composition. To investigate this issue, we performed a series of chromatin immunoprecipitation (ChIP) experiments. In the first set of experiments we selected 10 transcriptionally active genes and performed ChIP analysis to monitor promoter occupancy by the six hESC TAFs. As a normalization standard, we also monitored occupancy of TBP and RNA polymerase II large subunit (POL2) on these 10 promoters. As expected, we found that TBP and POL2 were present at comparable levels at each of the 10 promoters (Figure 3A). However, the absolute level of TBP and POL2 bound to each promoter significantly varied among the 10 genes. Therefore, in this experiment and those presented below, TAF recruitment was normalized to the level of TBP occupancy. The ChIP results of Figure 3B revealed two groups of genes with distinct TAF promoter occupancy patterns. The first group, which we refer to as class I genes, were bound by TAFs 3 and 5 but not by TAFs 2, 6, 7 and 11, whereas the second group, class II genes, were bound by all six hESC TAFs. Figure 3 Download asset Open asset Two classes of hESC genes based on TAF promoter occupancy. (A) Recruitment of TBP and POL2 to the promoters of 10 transcriptionally active genes (GAPDH, INTS6, MORF4L2, SLC25A3, TBP, TP53, FSCN1, OCT4, PCNA, SFPQ), each represented by a data point, were monitored by ChIP in H9 cells. For each gene, enrichment of TBP or POL2 binding to the promoter was normalized to a no antibody control and for non-specific recruitment at a control locus. (B) ChIP analysis monitoring TAF recruitment to the promoters of the 10 genes in H9 cells. TAF recruitment is specified relative to TBP recruitment (which was set to 1), after normalizing to a no antibody control and for non-specific recruitment to a control gene desert locus. Data are represented as mean ± SD. https://doi.org/10.7554/eLife.00068.008 To support this conclusion, we also compared TAF occupancy in HeLa and H9 cells across seven class I genes that are transcriptionally active in both cell types (Figure 4A). As a control, we first analyzed a representative subset of TAFs that are expressed in HeLa but not H9 cells. The ChIP analysis of Figure 4B shows that TAFs 1, 8 and 9 were readily detected on the promoters of genes in HeLa cells but, as expected, not H9 cells. Next, we analyzed the six TAFs that are expressed in both HeLa and hESCs. Figure 4C shows that in both cell types TAFs 3 and 5 were recruited to the promoters of the seven genes. By contrast we found that TAFs 2, 6, 7 and 11 were bound to the promoters of the seven genes in HeLa but not in H9 cells (Figure 4C). These results indicate that on the same transcriptionally active gene the TAF composition is strikingly different in HeLa and H9 cells. Figure 4 Download asset Open asset Comparison of TAF promoter occupancy on an identical set of transcriptionally active genes in HeLa and H9 cells. (A) Recruitment of TBP and POL2 to the promoters of seven class I genes (EEF1A1, FOS, GAPDH, SLC25A3, TBP, TLE4 and TP53) were monitored by ChIP in HeLa and H9 cells. Data were normalized as described in Figure 3A. (B) ChIP analysis monitoring recruitment of TAFs 1, 8 and 9 to the promoters of the seven class I genes in HeLa and H9 cells, as described in (A). Data are represented as mean ± SD. (C) ChIP analysis monitoring recruitment of TAFs 2, 3, 5, 6, 7 and 11 to the promoters of the seven class I genes in HeLa and H9 cells, as described in (A). Data are represented as mean ± SD. https://doi.org/10.7554/eLife.00068.009 We next performed a series of RNAi experiments to determine the relationship between TAF occupancy and transcriptional activity. The qRT-PCR results of Figure 5A show that siRNA-mediated knockdown of TAFs 3 and 5 in H9 cells (Figure 5—figure supplement 1) greatly reduced expression of both class I and II genes. By contrast, siRNA-mediated knockdown of TAFs 2, 6, 7 and 11 decreased expression of class II, but did not affect expression of class I genes (Figure 5B). Comparable results were obtained with a second, unrelated siRNA directed against each of the six TAFs (Figure 5—figure supplement 2). Collectively, these results establish a strong relationship between TAF occupancy and transcriptional activity in hESCs. Figure 5 with 2 supplements see all Download asset Open asset A strong relationship between TAF occupancy and transcriptional activity in hESCs. (A) qRT-PCR analysis monitoring expression of class I and II genes in H9 TAF3 or TAF5 knockdown (KD) cells. Normalized Ct values were analyzed after subtracting the signal obtained with the control RN18S1 shRNA (see ‘Materials and methods’). Data are represented as mean ± SEM. (B) qRT-PCR analysis monitoring expression of class I and II genes in H9 TAF2, 6, 7, or 11 KD cells. Data are represented as mean ± SEM. https://doi.org/10.7554/eLife.00068.010 Global ChIP-chip analysis of TAF occupancy in hESCs To confirm and extend the ChIP results, we performed global ChIP-chip analyses. In these experiments we monitored, in parallel, promoter occupancy of TAFs 3 and 5, TAFs 7 and 11 (as representative members of the TBP/TAF 2,6,7,11 complex), TBP and POL2. The overall results are summarized in Figure 6. We first defined a group of ∼3600 high-confidence actively transcribed genes based upon co-occupancy of both TBP and POL2 at the transcription start-site (Figure 6—figure supplements 1–3). The vast majority of active genes had promoter-bound TAF3 and TAF5 (Figure 6A). Significantly, a smaller fraction of active genes had promoter-bound TAF7 or TAF11, and there was substantial overlap between TAF7- and TAF11-bound genes (Figure 6B). As expected, the vast majority of genes bound by TAFs 7 and 11 were also bound by TAFs 3 and 5 (Figure 6C). Representative examples of promoter occupancy maps for two class I (SLC25A3, CCNB2) and class II (SFPQ, UCHL1) genes are shown in Figure 6D. Figure 6 with 3 supplements see all Download asset Open asset Global ChIP-chip analysis of TAF occupancy. (A) Venn diagram showing the overlap between TBP-, POL2-, TAF3- and TAF5-bound genes. (B) Venn diagram showing the overlap between TBP-, POL2-, TAF7- and TAF11-bound genes. (C) Venn diagram showing the overlap between TAF3- and TAF5-bound genes and TAF7- and TAF11-bound genes. (D) Representative maps showing TAF3, TAF5, TAF7, TAF11, TBP and POL2 occupancy at the promoters of class I (SLC25A3 and CCNB2) and class II (SFPQ and UCHL1) genes. (E) Differences between class I and II genes with respect to promoter H3K4me3. (F) Differences between class I and II genes with respect to average number of alternative promoters per ChIP-enriched site. (G) Representative promoter occupancy maps for two class II genes with alternative promoters, IFRD1 and CSF2RA. https://doi.org/10.7554/eLife.00068.013 To validate the ChIP-chip results, we selected a representative set of 44 genes and performed directed ChIP experiments using promoter-specific primer pairs. These validation experiments, which are shown in Figure 7, confirmed the predicted TAF occupancy patterns for ≥85% of the genes tested. For example, consistent with the ChIP-chip results, there was no significant binding (i.e., no enrichment relative to the no antibody negative control) of TBP or TAFs 3, 5, 7 or 11 to a group of inactive promoters predicted by the ChIP-chip analyses to not be bound by these factors (Figure 7A). Most importantly, Figure 7B shows that TAF7 occupancy validated at 24 of 27 predicted sites (88.9%), TAF11 occupancy at 27 of 28 predicted sites (96.4%), TAF3 occupancy at 36 of 39 predicted sites (92.3%), and TAF5 occupancy at 39 of 39 predicted sites (100.0%). Figure 7 Download asset Open asset Validation of ChIP-chip results by directed ChIP experiments using promoter-specific primer pairs. (A) ChIP analysis monitoring binding of TBP and TAFs 3, 5, 7 and 11 to a representative set of promoters that, based on ChIP-chip analyses, were predicted not to be bound by these factors. The results were normalized to a no antibody control (which was set to 1). Data are represented as mean ± SEM. For four genes (THOC1, CBWD3, RBM39 and ZNF260), the inactive promoter (A2) was analyzed in (A), and the active promoter (A1) was analyzed in (B) and (C). (B) ChIP analysis monitoring binding of TAFs 3, 5, 7 and 11 to the promoters of a representative set of 47 genes predicted by the ChIP-chip analyses to be bound by some or all of the factors. Data are normalized to TBP. Cutoff for a ‘positive’ is >0.4-fold enrichment vs TBP (red line). Data are represented as mean ± SEM. (C) ChIP analysis monitoring binding of TAF2 and TAF6 to 19 promoters that are bound by TAF7 and TAF11. Data are represented as mean ± SEM. https://doi.org/10.7554/eLife.00068.017 Figure 7B also shows that the overlap between TAF7- and TAF11-bound genes was higher than that predicted by the ChIP-chip analyses. Specifically, the results show that TAF7 was present at 7 of 7 predicted TAF11-only sites, and TAF11 was present at 5 of 6 predicted TAF7-only sites, indicating that TAF7 and 11 co-occupancy was 12 of 13 Moreover, as expected, genes bound by TAFs 7 and 11 were also by TAFs 2 and 6 (Figure For example, of the promoters analyzed that were bound by TAF7 and TAF11, TAF2 was present at and TAF6 was present at of these promoters. Collectively, the ChIP-chip analyses, in with the results described confirm the existence of two groups of genes in hESCs whose promoters are bound either only by TAFs 3 and 5 I or by all six hESC TAFs II we analyzed the ChIP-chip in to studies in hESCs (Abeyta et al., et al., 2005; et al., for that class I and class II genes and found two significant (Figure 1). First, the promoters of class I genes had 4 than those of class II genes (Figure the fraction of genes with alternative promoters based upon see ‘Materials and was significantly higher for class II than for class I genes (Figure Representative examples of promoter occupancy maps for two class II genes with alternative promoters are shown in Figure 1 analysis of ChIP-chip data TBP POL2 I genes vs TBP II genes vs TBP I vs promoters promoters occupancy The composition of hESC TAFs is required for proper regulation of gene expression and of the undifferentiated we analyzed whether the unusual composition of TAFs was for the characteristic ability of hESCs to an undifferentiated and self-renew. Figure shows that knockdown of each of the six hESC TAFs (Figure supplement 1) induced as by a decreased of To confirm this conclusion, we also whether hESC TAFs would induce differentiation by expression of a set of differentiation NES and Figure shows that of each hESC TAF in to of these differentiation Comparable results were obtained with a second, unrelated shRNA or siRNA directed against each of the six TAFs (Figure supplements 2 and 3). the induction of differentiation following knockdown of hESC TAFs was also by decreased expression of the pluripotency genes NANOG (Figure and Figure supplement and OCT4 (Figure 5 and Figure 5—figure supplement 2). Thus, the hESC TAFs are required to the undifferentiated Figure 8 with 4 supplements see all Download asset Open asset The composition of hESC TAFs is required for of the undifferentiated (A) of H9 TAF knockdown (KD) with Data are represented as mean ± SD. (B) qRT-PCR analysis monitoring expression of differentiation NES and in H9 cells treated with a TAF are relative to those obtained with a control which was set to Data are represented as mean ± SEM. (C) qRT-PCR analysis monitoring expression of NANOG in H9 cells treated with a TAF are relative to those obtained with a control which was set to Data are represented as mean ± SEM. (D) qRT-PCR analysis monitoring expression in H9 cells with a expressing as a control, of was monitored following expression is specified relative to that obtained with the which was set to Data are represented as mean ± SEM. (E) of H9 ectopically expressing as a control, Data are represented as mean ± SD. (F) qRT-PCR monitoring expression of differentiation NES and in H9 cells ectopically expressing are relative to those obtained in H9 cells expressing which was set to Data are represented as mean ± SEM. (G) qRT-PCR monitoring expression of class I and II genes in H9 cells ectopically expressing Data are represented as mean ± SEM. Immunoblot analysis showing OCT4 levels in H9 cells TAFs, TBP or ChIP analysis monitoring recruitment of TBP, POL2 and to the OCT4 promoter in H9 cells ectopically expressing or Data are represented as mean ± SD. of the protein shown are In a set of experiments, we TAF composition by ectopically expressing a TAF that is not present in hESCs. We found that expression of (Figure in as by a decreased number of (Figure the induction of differentiation (Figure and decreased expression of NANOG (Figure Interestingly, Figure shows that expression decreased expression of several class II genes, including as OCT4, whereas expression of class I genes was either or in some expression of several other TAFs not present in hESCs also in of expression of OCT4, a class II gene, but not a class I gene (Figure By contrast, expression of TBP or TAFs that are present in hESCs did not affect OCT4 To investigate the for the decreased OCT4 we performed ChIP analysis. Figure shows that, following expression of TBP and POL2 were no recruited to the OCT4 promoter. Collectively, the results of Figure 8 show that the composition of hESC TAFs results in of gene expression and induction of differentiation. Discussion In this report we have shown that the general transcription machinery of hESCs is highly unusual in that approximately of the canonical TFIID TAFs are not expressed. The of TAFs was confirmed by including immunoblotting (Figure 1A and Figure 1—figure supplement qRT-PCR (Figure and ChIP (Figure studies have tissue-specific TAF variants a