Abstract
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Accession numbers Data availability References Decision letter Author response Article and author information Metrics Abstract RNA polymerase II (PolII) transcribes RNA within a chromatin context, with nucleosomes acting as barriers to transcription. Despite these barriers, transcription through chromatin in vivo is highly efficient, suggesting the existence of factors that overcome this obstacle. To increase the resolution obtained by standard chromatin immunoprecipitation, we developed a novel strategy using micrococcal nuclease digestion of cross-linked chromatin. We find that the chromatin remodeler Chd1 is recruited to promoter proximal nucleosomes of genes undergoing active transcription, where Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover. The expression of a dominant negative form of Chd1 results in increased stalling of PolII past the entry site of the promoter proximal nucleosomes. We find that Chd1 evicts nucleosomes downstream of the promoter in order to overcome the nucleosomal barrier and enable PolII promoter escape, thus providing mechanistic insight into the role of Chd1 in transcription and pluripotency. https://doi.org/10.7554/eLife.02042.001 eLife digest DNA is tightly packaged in a material called chromatin inside the cell nucleus. To produce proteins this DNA must first be transcribed to produce a molecule of messenger RNA, which is then translated to make a protein. To assist with this process cells ‘unpack’ certain regions of the DNA so that enzymes that catalyze the different steps in this process can have access to the DNA. A protein called Chd1 is involved in the unpacking process in yeast, but its role in more complex animals is not clear. Now, Skene et al. have shown that this protein is needed to allow the enzyme that catalyzes the transcription of DNA—an enzyme called RNA polymerase II—to do its job. Chd1 acts to unpack the tightly packaged DNA from chromatin, thus allowing the transcription of the DNA to proceed. In the absence of Chd1 activity, RNA polymerase II stalls at the gene promoter—the region of DNA that starts the transcription of a particular gene. This work highlights how the packaging of DNA in the cell is highly dynamic and controls fundamental biological processes. Skene et al. modified a well-known genetic technique called ChIP-seq. Previous ChIP-seq protocols typically provided a blurry, low-resolution map of where proteins bound to chromatin. Skene et al. used an enzyme to ‘chew back’ the DNA to reveal the exact ‘footprints’ of the Chd1 protein and the RNA polymerase II enzyme on the chromatin in mice. It will be possible to adapt this new protocol to map the positions of other proteins, which will help to improve our understanding of the ways in which chromatin regulates access to DNA. https://doi.org/10.7554/eLife.02042.002 Introduction The eukaryotic genome is packaged into chromatin, with the basic unit being a nucleosome, consisting of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Chromatin limits the accessibility of DNA-binding factors to the underlying DNA sequence and presents a strong physical barrier that must be overcome by RNA polymerase II (PolII) during transcription. In vitro experiments have shown that transcription initiation is the most affected and that elongation is also highly inefficient unless nucleosomes are destabilized (Lorch et al., 1987; Hodges et al., 2009; Jin et al., 2010; Bintu et al., 2012). Despite this barrier, in vivo elongation rates through chromatin are comparable to rates on naked DNA, indicating that the cell has evolved mechanisms to overcome this chromatin barrier (Li et al., 2007; Ardehali and Lis, 2009; Singh and Padgett, 2009; Petesch and Lis, 2012). Through the combined action of PolII, elongation factors, nucleosome modifications and chromatin remodelers, the chromatin barrier to transcription is highly dynamic, with nucleosomal turnover correlating with gene expression (Deal et al., 2010). Nucleosomes are disrupted to allow efficient elongation and are reassembled in the wake of PolII to prevent cryptic initiation from intragenic sequences (Cheung et al., 2008; Petesch and Lis, 2008; Quan and Hartzog, 2010). In metazoans, PolII is often enriched immediately downstream of the transcription start site (TSS), raising the possibility that this is a significant barrier to promoter escape in the progression from transcriptional initiation to elongation, with an unknown mechanism by which this barrier is overcome (Guenther et al., 2007; Mavrich et al., 2008; Schones et al., 2008). Although the intrinsic structural preferences of DNA contribute substantially to nucleosome occupancy and positioning in vivo, this chromatin landscape is further manipulated by chromatin remodelers (Segal and Widom, 2009; Kaplan et al., 2010). Chromatin remodelers use the energy from ATP hydrolysis to evict, assemble, and slide nucleosomes in order to guide the proper nucleosome positioning at key sites, such as eukaryotic promoters, where they maintain the nucleosome-depleted region (NDR) and phased flanking nucleosomes (Hartley and Madhani, 2009; Gkikopoulos et al., 2011; Tolkunov et al., 2011). Chromatin remodelers share a common Snf2 helicase-like ATPase domain that is required for the disruption of histone–DNA contacts (Hota and Bartholomew, 2011; Petty and Pillus, 2013). Furthermore, they can be separated into two groups based upon their requirement for extra-nucleosomal DNA. First, ISWI, INO80/SWR1 and CHD require extra-nucleosomal DNA to remodel nucleosomes, whereby they are primarily involved in nucleosome assembly and generating equally spaced arrays by binding to stretches of exposed DNA and shifting nucleosomes into the gap (Whitehouse et al., 2003; McKnight et al., 2011; Udugama et al., 2011). Second, SWI/SNF and RSC remodelers slide or evict nucleosomes irrespective of extra-nucleosomal DNA (Whitehouse et al., 1999; Lorch et al., 2011).The potential role of remodelers in overcoming the nucleosomal barrier has not been fully investigated. Studies in yeast have suggested the main role for the chromatin remodeler Chd1 to be within the gene body, where Chd1 reassembles and positions nucleosomes in order to prevent cryptic initiation (Cheung et al., 2008; Gkikopoulos et al., 2011; Radman-Livaja et al., 2012; Smolle et al., 2012; Zentner et al., 2013). However, the promoter chromatin architecture is different in metazoans, with the TSS embedded in the nucleosome-depleted region, raising the possibility that this differing chromatin landscape contributes to metazoan PolII accumulating to high levels ahead of the first nucleosome (Mavrich et al., 2008; Schones et al., 2008; Rahl et al., 2010). Here, we show that mammalian Chd1 controls nucleosome dynamics at actively transcribed genes. We find that Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover at the promoter and is required to allow efficient PolII promoter escape, with PolII becoming stalled in the absence of Chd1 activity. Stalling with the loss of Chd1 activity implies that Chd1 is required to overcome the nucleosomal barrier to allow transcription within a chromatin context. In contrast, in the gene body, Chd1 reassembles nucleosomes and suppresses histone turnover. Interestingly, mammalian Chd1 is required for pluripotency of embryonic stem (ES) cells by maintaining euchromatin and is also required for efficient reprogramming, suggesting that Chd1 plays a key role in mammalian cellular identity (Gaspar-Maia et al., 2009). Through both positively and negatively impacting histone dynamics, Chd1 plays roles in transcription and in regulating pluripotency and reprogramming. Results Chd1 is recruited to the promoter and its ATPase activity is required for binding to extend into the gene body To study the role of mammalian Chd1, mouse embryonic fibroblasts (MEFs) were transfected with a transgene expressing FLAG-tagged wild-type mouse Chd1 or a mutant variant harboring the replacement of a conserved lysine to an arginine residue (K510R) in the ATP-binding site of the remodeler (Figure 1A). The K510R Chd1 mutation eliminates the catalytic activity without disrupting its ability to interact with other proteins, thereby generating a dominant negative (Corona et al., 2004). Mutation of this conserved lysine residue has been used to functionally investigate various chromatin remodelers including Brahma and ISWI in Drosophila (Figure 1A; Elfring et al., 1998; Deuring et al., 2000). In comparison to knocking down Chd1 expression, a dominant negative approach is less likely to enable redundant mechanisms to mask protein functions. Western analysis indicated that the FLAG-tagged proteins migrated at the expected size and were only moderately over-expressed relative to endogenous Chd1 in the MEFs (Figure 1B). Figure 1 with 2 supplements see all Download asset Open asset Chd1 is recruited to promoters with high PolII occupancy and requires ATPase activity to extend into the gene body. (A) Schematic of the domain structure of the full-length mouse Chd1 (1-1711) used to generate the N-terminal FLAG-tagged construct. A region corresponding to the Chd1 ATP-binding pocket is shown below and aligned to various homologues from Saccharomyces cerevisiae and Drosophila melanogaster. The arrow indicates the conserved lysine residue mutated to form the dominant negative. (B) MEFs stably expressing either FLAG-tagged wild-type Chd1 or the dominant negative K510R were subjected to western analysis with a two-fold dilution series. Untransfected MEFs were used as a reference. (C) A representative genome browser snapshot of ChIP-seq data (all fragment lengths) indicating the high occupancy of PolII, wildtype-Chd1 and K510R-Chd1 at gene promoters. PolII distribution was determined in cells expressing wildtype-Chd1 using the N20 antibody. Normalized counts are indicated on the y-axis. (D) Chd1 binding to the 5′ end of genes was determined by ChIP-seq. Here, all recovered DNA fragments, irrespective of length, were analyzed. Each row of the heatmap represents the binding pattern across the −0.5 kb to +2 kb region flanking the TSS. Genes were ranked by the level of PolII occupancy in the −100 to +300 bp region, as measured by ChIP-seq in the MEFs expressing wildtype Chd1, using the N20 antibody that binds to the N-terminus of the largest subunit of PolII. (E) Analyzing only the short DNA fragments recovered (35–75 bp) allows precise mapping of Chd1, indicating that wildtype Chd1 binding tracks into the gene body, whereas K510R-Chd1 accumulates just downstream of the TSS. The genome-wide average is shown across the 4 kb encompassing the TSS. https://doi.org/10.7554/eLife.02042.003 First, we determined the genomic localization of Chd1 using chromatin immunoprecipitation in combination with high throughput sequencing (ChIP-seq). We found that under native conditions only 14% of Chd1 could be extracted (Figure 1—figure supplement 1A). This led us to pursue a formaldehyde cross-linked ChIP strategy, which allows for harsher extraction conditions. Sonication is often used in ChIP to fragment the formaldehyde-cross-linked chromatin. This, however, only provides low resolution, as the fragment size is typically 100–300 bp in comparison to the footprint of either PolII or Chd1, which is 35 bp and 20–45 bp respectively (Samkurashvili and Luse, 1996; Ryan et al., 2011). We therefore used Micrococcal nuclease (MNase) digestion of cross-linked chromatin to increase the resolution of ChIP-seq. MNase digests unprotected DNA, leaving DNA fragments corresponding to mono-nucleosomes and sub-nucleosomal fragments, presumably protected by proteins cross-linked to the extra-nucleosomal DNA (Figure 1—figure supplement 1B). By using a modified Solexa library preparation protocol in combination with paired-end sequencing, we were able to map DNA fragments as small as ∼35 bp (Henikoff et al., 2011). By using MNase to fragment the chromatin and minimal sonication we were able to achieve essentially complete extraction of chromatin and associated proteins, thereby overcoming a significant limitation of native ChIP (Figure 1—figure supplement 1C). This technique is highly transferable, as it requires no special tool development and uses current sequencing technology. Initially analysis of all the recovered fragments was performed. As expected, wild-type Chd1 was recruited to the 5′ end of genes, similar to what has been shown for endogenous Chd1 in mouse ES cells (Figure 1C,D; Gaspar-Maia et al., 2009). Furthermore, the dominant negative K510R-Chd1 showed a broadly similar chromatin recruitment pattern, consistent with the mutation not affecting interactions with recruiting factors. In addition, we found that the enrichment of Chd1 was primarily just downstream of the TSS, with recruitment correlating with PolII occupancy of the promoter, as measured in the MEFs expressing wild-type Chd1 (Figure 1D). PolII has previously been shown to crosslink to nucleosomes (Koerber et al., 2009). Therefore, mapping the DNA recovered by immunoprecipitation of these cross-linked PolII:nucleosome complexes will not identify the exact binding site of PolII on DNA. To precisely map the position of PolII and Chd1, we mapped 35–75 bp fragments, which more closely corresponds to the footprint of PolII and Chd1 (Figure 1—figure supplement 2A). For PolII, there was a shift of the maximal occupancy towards the TSS, from +150 bp for all fragment sizes to a peak centered over the TSS, likely indicating that the larger fragments correspond to PolII crosslinked to the +1 nucleosome. Similarly, for both wild-type and K510R Chd1, larger size classes indicated Chd1 crosslinked to the well-positioned +1 nucleosome. Analysis of short fragments, however, indicated wild-type Chd1 was enriched over the beginning of the gene body with a steady decline from +500 bp. However, in the absence of ATPase activity, K510R-Chd1 density peaked downstream of the TSS at +60 bp, where it accumulated to higher levels than wild-type Chd1 and was depleted in the gene body (Figure 1E, Figure 1—figure supplement 2B). To confirm that mapping of short fragments was similar to that of all fragments, genes were ranked by PolII promoter occupancy as determined from all fragment lengths and separated into quintiles (Figure 1—figure supplement 2C). We find a correlation between Chd1 recruitment and PolII occupancy, as seen for all fragment lengths. Overall, this novel ChIP-seq strategy based on the MNase digestion of cross-linked chromatin and the mapping of short fragments allows precise mapping of the footprint of PolII and Chd1. We find that Chd1 binds just downstream of the TSS, correlates with PolII occupancy at the promoter and requires ATPase activity for the binding to extend into the gene body. Chd1 activity is required to maintain nucleosome occupancy within the promoter region and the gene body Chromatin remodelers are known to have a major role in the correct positioning and occupancy of nucleosomes (Gkikopoulos et al., 2011). Therefore we determined the nucleosome profile in cells expressing either wild-type or K510R Chd1 by mapping mono-nucleosome-sized fragments recovered from the sequencing of MNase digested, cross-linked chromatin. Cells expressing wild-type Chd1 display the classical metazoan nucleosome organization pattern, with a pronounced nucleosome depleted region (NDR) containing the TSS, flanked by well positioned nucleosomes, with the +1 nucleosome entry site ∼50 bp downstream of the TSS (Figure 2A; Mavrich et al., 2008; Schones et al., 2008). In addition, the position of the +1 nucleosome corresponded to the level of PolII promoter occupancy, with the 5′ edge progressively moving towards the TSS, to approximately +15 bp for genes with the lowest PolII occupancy, as previously indicated by ranking human nucleosome positions by steady-state RNA levels (Schones et al., 2008). The positioning of the nucleosomes was not affected by the loss of Chd1 activity, but the nucleosome occupancy was reduced both upstream and downstream of the TSS. In contrast to Saccharomyces cerevisiae, where deletion of Chd1 had no effect on the +1 nucleosome occupancy but only the surrounding nucleosomes, blocking Chd1 activity in MEFs affected promoter proximal nucleosomes including the +1 nucleosome (Gkikopoulos et al., 2011). In S. cerevisiae, the TSS is found just within the +1 nucleosome, thereby suggesting that pre-initiation complex formation requires loss of the +1 nucleosome (Rhee and Pugh, 2012). However, metazoans have a different promoter nuclear architecture with the TSS embedded in the nucleosome depleted region and the +1 nucleosome entry site ∼50 bp downstream (Mavrich et al., 2008; Schones et al., 2008). Therefore, loss of the mammalian +1 nucleosome occupancy observed here likely reflects a unique role of mammalian Chd1 because of differences to S. cerevisiae in promoter architecture and perhaps the mechanism of transcription through the promoter proximal chromatin. Decreased nucleosome occupancy was also apparent throughout the gene body including upstream of the transcriptional end site (TES). Figure 2 with 1 supplement see all Download asset Open asset Chd1 activity is required to maintain nucleosome occupancy in the promoter region and the gene body. Cross-linked chromatin was digested with MNase and the DNA fragments were subjected to paired-end sequencing. Mono-nucleosomal fragments (111–140 bp) were aligned relative to the TSS or TES. (A) Average nucleosome profile for all genes. Genes were also ranked and split into quintiles based on PolII promoter occupancy (all fragment sizes; density within −100 to +300 bp). The nucleosome map for the highest and lowest quintile is shown. (B) Nucleosome profiles for genes with either the highest or lowest quintile of wildtype-Chd1 binding (35–75 bp fragment sizes; density within 0 to +1 kb). Statistical significance was determined using the two sample Kolmogorov–Smirnov (KS) test on the average number of normalized counts within 5 kb downstream of the TSS or upstream of TES. All groups were highly statistically significant (p<1 × 10−9; TES of genes with the highest Chd1 at the promoter was significant p<3 × 10−4) with the exception of the TSS and TES of genes with the lowest PolII density and the TSS of genes with the lowest Chd1 density which showed no significant change. https://doi.org/10.7554/eLife.02042.006 The decrease in nucleosome occupancy was most apparent at genes with the highest PolII levels at the promoter, as expected given that Chd1 occupancy correlates with PolII promoter occupancy, with no statistically significant change at genes with low PolII levels at the promoter (Figure 2A). Genes were ranked by wildtype-Chd1 binding within the beginning of the gene to more directly test the role of Chd1 in maintaining nucleosome occupancy (Figure 2B). Genes with the highest recruitment of Chd1 exhibited the most dramatic loss of nucleosomes at the promoter upon expression of K510R-Chd1, consistent with a direct role of Chd1. Additionally, genes with the lowest recruitment of wild-type Chd1 displayed a markedly different nucleosome landscape, with unphased nucleosomes and the absence of a pronounced NDR (Figure 2B). Despite this relatively flat profile at the TSS being insensitive to the expression of K510R-Chd1, it suggests that the ‘open’ and ‘closed’ promoter architecture, as defined by the existence of an NDR at the TSS, can be categorized based on recruitment of remodelers such as Chd1 (Cairns, 2009). The loss of nucleosome occupancy within the last 1 kb of the gene body was independent of the level of Chd1 recruitment to the promoter (Figure 2B). In contrast, the loss of nucleosome occupancy in the gene body correlated with level of Chd1 binding in the gene body (Figure 2—figure supplement 1A). This indicates that Chd1 action within the distal part of the gene body is independent of its recruitment to the promoter. To establish whether the effects of the K510R mutant can be mimicked by reduced levels of Chd1, endogenous levels of Chd1 were reduced using a short-hairpin RNA (Figure 2—figure supplement 1B). Nucleosome occupancy was reduced at both the promoter and the gene body upon knockdown of Chd1, as seen with the expression of K510R-Chd1 (Figure 2—figure supplement 1C). To determine the concordance between knockdown and expression of the dominant negative, genes were ranked by transgenic wild-type Chd1 occupancy. Genes with the highest Chd1 occupancy were most affected by knockdown, suggesting that knockdown of Chd1 phenocopied the expression of the dominant negative. Overall, loss of Chd1 activity resulted in reduced nucleosome occupancy at both the promoter and the gene body confirming that Chd1 is involved in defining the chromatin landscape. Chd1 has opposing roles in regulating nucleosome turnover at the promoter and the gene body The mapping of MNase-digested mono-nucleosomes provides information regarding steady-state nucleosome occupancy and but not insight regarding nucleosome We used of to and identify to nucleosome turnover (Deal et al., 2010). the of proteins and the of nucleosomes. results showed that histone turnover was most at the nucleosomes flanking either of the promoter and progressively towards the gene body (Figure We observed in nucleosome turnover upon the expression of First, nucleosome turnover was reduced on either of the TSS (Figure Second, nucleosome turnover was increased within the gene body, from bp downstream of the TSS and also in the gene body upstream of the TES (Figure Figure supplement 1A). results opposing roles for Chd1 in regulating nucleosome turnover at the promoter and the gene body. We found that the role of Chd1 in regulating nucleosome turnover at the promoter and the gene body is independent of gene (Figure supplement 1B). This is in contrast to yeast, where a study indicated Chd1 genes from nucleosome replacement in the gene body et al., 2012). In yeast, the of the effect on nucleosome turnover was approximately at the promoter and the gene body et al., 2012; Smolle et al., 2012). However, we find that the action of mammalian Chd1 on nucleosome turnover is at the promoter, which a different of Chd1 in Figure with 1 supplement see all Download asset Open asset Chd1 activity has opposing effects on nucleosome turnover at the promoter and the gene body. Nucleosome turnover is over the promoter but increased over the gene body. The genome-wide average nucleosome turnover was using at both the (A) TSS and (B) TES. (C) Nucleosome turnover correlates with PolII density and is increased in cells expressing Genes were ranked by the density of PolII within the last kb of the gene body. The average density of PolII was with a of genes the average within to −0.5 kb relative to the TES correlation The for was and is shown (D) Chd1 is recruited to genes with actively PolII. Genes were ranked as (C) and the average ChIP-seq for FLAG-tagged wildtype Chd1 (35–75 bp fragment within the correlation We found that nucleosome turnover in the gene body correlated with levels of PolII (Figure This is consistent with suggesting that there is of the complete octamer at high transcriptional rates than at et al., and Bintu et al., 2011). nucleosome turnover was highly at low levels of PolII indicating that there is significant in the mechanism of PolII through nucleosomes at low rates of transcription. Nucleosome turnover in the gene body was increased in cells expressing K510R-Chd1, with the increase approximately at all of PolII, suggesting an by Chd1 in the of nucleosome turnover irrespective of transcription (Figure We to further the of Chd1 action in the gene body by potential recruitment As previously the loss of nucleosome occupancy within the distal regions of the gene body not with Chd1 recruitment to the promoter. This suggests Chd1 recruitment to the promoter and the gene body. We found that the level of both wildtype and K510R-Chd1 recruitment to the gene body positively correlated with PolII density within the gene body (Figure these that occupancy of Chd1 at the promoter and the gene body correlates with the of PolII in the genomic The of correlation between Chd1 occupancy and of nucleosome turnover in the mutant suggests that Chd1 is not required for turnover at high transcription Overall, these results that Chd1 is recruited to actively chromatin where it is involved in the of nucleosome turnover within the gene body. Chd1 is responsible for most of PolII-directed nucleosome turnover around promoters In contrast to nucleosome turnover at the gene body, nucleosome turnover at the promoter was upon the expression of We to determine this correlated with PolII promoter density given that both Chd1 occupancy and loss in nucleosome occupancy was most pronounced at genes with high PolII promoter Genes with higher levels of PolII displayed increased nucleosome turnover surrounding the TSS (Figure as have been expected from the correlation between steady-state RNA levels and nucleosome turnover (Deal et al., 2010; et al., 2013). of the promoter region high in the absence of Chd1 activity, but was markedly reduced within the promoter proximal region to bp) encompassing the nucleosome, the TSS and the +1 and +2 nucleosomes (Figure We determined the average within the promoter proximal region as a of PolII occupancy (Figure low to PolII we found that cells expressing wildtype-Chd1 displayed a correlation between PolII density and nucleosome with turnover a at high levels of PolII. In contrast, expression of the dominant negative Chd1 resulted in the of turnover to levels for all levels of PolII occupancy. of endogenous Chd1 resulted in a in nucleosome turnover at the promoter (Figure supplement consistent with either knockdown or redundant factors. The loss of nucleosome turnover in the K510R-Chd1 mutant suggests that Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover within the promoter proximal In by nucleosome dynamics in combination with steady-state nucleosome occupancy, our results that Chd1 is responsible for PolII-directed nucleosome turnover at promoters but suppresses nucleosome turnover within gene Despite these of Chd1 in nucleosome turnover we find that Chd1 is required to maintain nucleosome occupancy both around the promoter and within the gene body. Figure 4 with 1 supplement see all Download asset Open asset Chd1 activity is responsible for PolII-directed nucleosome turnover at the promoter. (A) data as a heatmap for the kb surrounding the TSS. Genes were ranked by the level of PolII occupancy at the promoter (all fragment sizes; density within −100 to +300 bp). (B) Nucleosome turnover is reduced over the promoter proximal region in cells expressing The in between cells expressing wildtype and K510R-Chd1 at the TSS bp is The genome-wide average nucleosome occupancy is shown for for cells expressing (C) Chd1 is required for PolII-directed turnover at the promoter proximal Genes were ranked by PolII promoter density in cells expressing wildtype-Chd1 and the average in the promoter proximal region with a of genes. Chd1 activity is required for efficient promoter escape by PolII Previous work in yeast has not a between the loss of Chd1 and the on transcription. In yeast, approximately of Chd1 bound promoters showed a change in nucleosomal