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.DOI:http://dx.doi.org/10.7554/eLife.02042.001 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.DOI:http://dx.doi.org/10.7554/eLife.02042.002 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 . DespiteThrough 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 . NucleosAlthough the intrinsic structural preferences of DNA contribute substantially to nucleosome occupancy and positioning in vivo, this chromatin landscape is further manipulated by chromatin remodelers . Chromat. 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  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 . The K51osophila . In composophila .10.7554/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 . We found that under native conditions only 14% of Chd1 could be extracted . This leomal DNA . By usinomal DNA . By usinomal DNA . This teInitially 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 . FurtherSaccharomyces 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 .Chromatin remodelers are known to have a major role in the correct positioning and occupancy of nucleosomes . Therefocleosome . In S. ccleosome . Howevercleosome . TherefoThe 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 . Genes wTo 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 . NucleosThe mapping of MNase-digested mono-nucleosomes provides information regarding steady-state nucleosome occupancy and positioning, but does not yield insight regarding nucleosome dynamics. We used CATCH-IT  to measure nucleosome turnover . CATCH-IWe found that nucleosome turnover in the gene body correlated with increasing levels of elongating PolII . This isIn contrast to nucleosome turnover at the gene body, nucleosome turnover at the promoter was decreased upon the expression of K510R-Chd1. We sought to determine if this reduction correlated with PolII promoter density given that both Chd1 occupancy and loss in nucleosome occupancy was most pronounced at genes with high PolII promoter density. Genes with higher levels of PolII displayed increased nucleosome turnover surrounding the TSS , as mighS. cerevisiae PHO5 gene promoter indicated that Chd1 was required to remove the +1 nucleosome, which occludes the TSS, but no robust change in gene expression was observed in vivo without the synergistic loss of the chromatin remodeler ISWI  . The sigAs Chd1 modulates PolII release from promoters, we wondered whether this ATP-remodeling complex alleviates PolII stalling. Therefore, we calculated the stalling index in the presence of wild-type and mutant Chd1. We defined the stalling index as the ratio of total PolII density at the promoter (−100 to +300 bp) relative to the density of PolII Ser2phos within the gene body (+1 to +5 kb) . We founTo confirm that the main function of Chd1 is at processive genes, Chd1 binding to stalled or processive genes was addressed. As Chd1 binding scales with PolII density , only thThe Chd1-binding profile was plotted for processive genes over the promoter region to understand how Chd1 might be functioning to enable promoter clearance at processive genes . WildtypDrosophila indicated that at the more processive M1BP bound genes, PolII accumulated in front of the well-positioned +1 nucleosome  are found on either H3 or H4, the retention of the H3:H4 tetramer during transcription has been proposed to have a role in the maintenance of chromatin states throughout the genome . This suPiggybac expression vector with the CAG promoter driving N-terminal FLAG-tagged expression of Chd1  and puromycin resistance. The K510R mutation was generated by site directed mutagenesis and verified by sequencing. MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% FBS, 1% penicillin-streptomycin at 37°C and puromycin (1.5 µg/ml). Knockdown of endogenous Chd1 was achieved using vectors as previously described ; Chd1 ; γ-Tubulin .ChIP was performed as previously described  with the2, 0.1% Triton X-100, 1 mM DTT, 20% Glycerol) and then processed as previously described. Input and CATCH-IT DNAs were amplified using a whole-genome amplification kit (Sigma-Aldrich), followed by labeling using Cy3 and Cy5 heptamers according to the Roche Nimblegen labeling protocol. Labeled samples were hybridized together to mouse 2.1-million probe promoter arrays .CATCH-IT was performed as described previously with the following modifications . Nuclei Data have been submitted to the Gene Expression Omnibus under accession number GSE52349 . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent -driven nucleosome turnover at gene promoters in vertebrate cells. Accordingly, Chd1 arises as a key regulator of RNAPII activity at the level of pausing and elongation. Given the widespread occurrence of RNAPII pausing in metazoans and the ever-increasing appreciation of RNAPII elongation control in regulation of gene expression, the current manuscript is deemed of high impact for the gene expression and chromatin fields.eLife basically as it is.The manuscript is beautifully written, the data is of high quality and generated with cutting-edge technology and the conclusions are fully supported by data. In my mind, the manuscript is meritorious for publication in My only minor concern is the creation of the RNAPII 'stalling index' in Reviewer2:The authors are experts in chromatin biology, and have developed several assays that enable different attributes of promoter chromatin to be analyzed. Here, they explore the impact of Chd1 remodeler on promoter chromatin. This issue has been addressed previously in ES cells at lower resolution, where promoter association was observed, with Chd1 loss leading to the acquisition of heterochromatin.The paper starts with the application of methods developed in the Henikoff lab to examine both Chd1 and RNA PolII in MEFs. This involves the separate examination of larger and smaller fragments derived from limited MNase digestion of crosslinked chromatin, following chromatin IP (ChIP). The larger fragments represent linkage of the factor to nucleosomes, whereas the smaller represent the factor's footprint. Support is provided that smaller fragments with PolII better link to the TSS.For Chd1, the authors examine both an epitope-tagged WT and a catalytic mutant. With both Chd1 derivatives, localization to the +1 nuc was observed, however the catalytic mutant showed additional occupancy of proximal downstream nucleosomes. This data was nicely done and described. The authors then explored the impact on nucleosome occupancy and positioning following expression of the dominant-negative Chd1 derivative. Here, they claim a loss in nucleosome occupancy without a change in positioning. I find the effect very small overall, even when PolII levels are considered, but much more clear in the case where the highest quintile of Chd1 occupancy is examined. The authors note a difference in impact here in MEFs on the +1 nuc, vs in budding yeast where no change is observed following Chd1 loss, which I found interesting. I also found the correlation of Chd1 with 'open' promoters interesting. The authors then apply an alternative shRNA approach to knock down Chd1, and observe similar (and even somewhat stronger) results.As prior approaches provide steady-state measurements, the authors then apply the CATCH-IT technique to explore dynamics. First, they show that turnover is high at locations flanking the TSS. The authors then explore the impact of expression of the dominant negative. The authors emphasize a significant difference at the locations flanking the TSS, and in the gene body. However, this is really confined to the nucleosome directly on, or adjacent to, the TSS; here it is clear and interesting, however, regarding the gene body, I found the difference with the dominant negative apparent, but the scale/magnitude quite small.The next section shows the promoter/TSS affect with the dominant negative scales with PolII to distinguish between increased recruitment vs lack of escape, relative distribution of elongating PolII, and  showed support for a role in promoter escape. Stalling index analysis show increases with the catalytic mutant and a preference for processive genes, which correlated with wt Chd1 occupancy. I found this data quite interesting. I found the data showing the peak of dominant negative Chd1 occupancy 50 bp inside the nucleosome very interesting, and consistent with current models of DNA translocation. Likewise, the peaks of PolII preceding nucleosomes in the early gene body supports the authors interesting claim of utilizing Chd1 activity to help facilitate PolII passage and reassembly behind the polymerase.Overall, I found the work well designed, executed and interpreted, and I strongly support its publication.Reviewer3:This manuscript investigates the role of Chd1 in transcription. Using MNase-seq, PolII ChIP-seq and CATCH-IT in MEF cells expressing WT or a dominant negative form of Chd1, the authors conclude that Chd1 is a factor required for the RNAP to overcome the barrier of the +1 nucleosome and relieve stalling of RNAP, whereas in the gene body it suppresses nucleosome turnover. There are some potentially good ideas in this manuscript, but the data do not significantly extend what we know from previous work and the data suffers from insufficiently rigorous analysis.These results are similar to work from a number of labs , showing that Chd1 localizes at promoters and within gene bodies where it facilitates transcription by altering nucleosome occupancy and turnover/histone replacement, especially at highly active genes. In particular, the data is reminiscent of Radman-Livaja et al. (PLOS Genetics 2012), where it is shown in yeast that loss of Chd1 activity leads to slower turnover of nucleosomes over the promoter and faster turnover in gene bodies and near 3' ends.S. cerevisiae showing that Chd1 is a promoter-specific chromatin remodeler, and work in S. pombe  showed that Chd1 localized particularly to promoter regions where it helped remove nucleosomes near the transcription start site to enable transcription. Thus, I don't see significant novelty in the majority of conclusions presented and don't find compelling the suggested connection between Chd1 activity at promoters and the metazoan-specific phenomenon of promoter stalling of RNAP. On this point, the hypothesis that PolII is being blocked in Chd1 mutant cells by nucleosomes despite the overall occupancy of the +1 nucleosome being lower is not clear to me. I can see how PolII could be blocked by an increase in nucleosome occupancy or by more static nucleosomes, but i.e. not what is indicated by this data. What the authors show is reduced +1 occupancy and reduced +1 turnover, which is most simply explained by nucleosomes being removed and not efficiently replaced. There is no clear data that sheds light on how this lower occupancy or failure to re-assemble nucleosomes might inhibit PolII.The authors emphasize the idea that Chd1 does not remodel promoter nucleosomes in yeast, but I don't understand why (they cite a Science paper from the Owen-Hughes lab that focused on nucleosome positioning). In fact that paper showed lower nucleosome occupancy near promoters in remodeling deficient cells, in total agreement with the current manuscript. Furthermore, the Kornberg lab  has done nice work in Major concerns:1) There are a number of cases where strong conclusions are drawn based on non-quantitative analysis of normalized group–average profiles. Group averages can be dramatically shifted by a small subset of genes and are not a reliable way to draw generalizable conclusions about a population. In cases where the authors wish to say that Chd1 mutation 'increases' or 'decreases' signal, statistical evaluations should be performed and described clearly in the methods section. When the authors wish to say that two signals 'correlate' with each other, correlation analysis should be performed and Pearson/Spearman R squared or similar values should be presented.Drosophila that shows normal Ser2-P PolII (active elongation form) levels in Chd1 mutants (Armstrong and Tamkun labs).2) It is really hard to interpret the data on PolII ChIP in cells expressing the catalytically dead Chd1 mutant without knowing what the effects of blocking Chd1 activity are on gene expression. The authors should at the very least select some example genes where loss of Chd1 increases the PolII stalling index and measure the nascent RNA levels at that gene by run-on or RT-PCR of intronic regions. The interpretation of the authors is that PolII is being 'blocked' at the promoter by the presence of more stable nucleosomes, but without showing that there is actually less active elongation within the gene bodies, this conclusion is very weakly supported, especially in light of work in Drosophila. In particular, Dave Gilmour's recent EMBO paper clearly shows that there are different classes of genes that differ in the role of the +1 nucleosome. Genes with the most paused PolII lack a significant +1 nucleosome signal and are paused by separate factors (GAGA and NELF) whereas less stably paused, more processive genes have a strong +1 nucleosome signal that causes more transient pausing. Do the authors find something significantly different in the mammalian system? Can they show by heatmap the relationship between the level of pausing and the occupancy level or turnover of the +1 nucleosome and analyze this quantitatively to demonstrate better that the +1 nucleosome might be a static barrier to mammalian PolII?3) It is very surprising that the authors don't mention any of the considerable work done on the relationship between PolII and the +1 nucleosome in  As you will gather from the full reviews included below, there was a clear disparity in the initial assessment of your manuscript. Whereas Reviewers 1 and 2 observed the various strengths of your work and found it appropriate for publication in eLife, Reviewer 3 pointed out that several key observations into the role of Chd1 in nucleosome remodeling at distinct genomic locations described in your manuscript had been previously established in the literature. This led Reviewers 1 and 2 to re-review your manuscript to determine to what degree the novel aspects in your work constituted a significant advance in the field. After this process, Reviewers 1 and 2 agreed that overall the paper ranks as very good and that there is new information in the manuscript.Upon further discussion, we would like to encourage a resubmission of a revised manuscript that not only thoroughly addresses the technical and data interpretation concerns raised by Reviewer 3, but also is rewritten in a way that better emphasizes what was known and what is novel in this paper about the role of Chd1 in nucleosome position and exchange. We also encourage addition of any new available data that would further advance our understanding of the molecular mechanism of action of Chd1.Thank you for taking the time to thoughtfully review our manuscript. We have addressed the concerns in the revision and have included a detailed description in response to the comments below.We believe that our initial submission did not emphasize the novel nature of our method for high resolution mapping of Chd1 and PolII. This technique is based upon the MNase digestion of crosslinked chromatin, limited sonication for quantitative ChIP recovery, and the sequencing of protected fragments. The development of the technique was essential for identification of Chd1 binding to the surface of nucleosomes, as previously implicated in vitro. In addition, this technique is highly adaptable, only requiring a simple modification to the regularly used ChIP-seq protocol and uses existing sequencing platforms. In the resubmission, we have provided further details of the method as a resource for the scientific community.As detailed below, we have rewritten the manuscript to address previous work on the role of Chd1 in yeast. In brief, from the point of view of this study, yeast differ in two main ways: (1) yeast have the TSS embedded within the +1 nucleosome, whereas in metazoans the entry site for the +1 nucleosome is ∼50 bp downstream; (2) unlike the situation in yeast, the vast majority of metazoan genes exhibit high levels of PolII accumulation near the promoter. As such, we set out to investigate Chd1 function in mammals. Our motivation was spurred on by the observation that Chd1 is required for metazoan specific phenomena – maintenance of pluripotency and reprogramming, for which we provide possible mechanistic insights. Also, understanding the regulation of nucleosome turnover in mammals is vital, for example with the recent observation that the widely used anti-cancer drug doxorubicin increases turnover, consistent with the tumor suppressor role of mammalian Chd1.We find a number of unique changes to chromatin that are not found in yeast: (1) the +1 nucleosome occupancy is decreased in the absence of Chd1 activity; (2) Chd1 primarily drives histone turnover at the promoter, compared to the relatively weak effect seen in the gene body. Work in yeast has not yielded a clear link between changes in chromatin and transcription upon loss of Chd1. Here we show that Chd1 occupancy and activity is linked to transcription, with Chd1 responsible for essentially all of PolII-dependent histone turnover at the promoter and removes the barrier to PolII promoter escape.Reviewer 1:My only minor concern is the creation of the RNAPII 'stalling index' in, which is basically equivalent to the RNAPII 'pausing index' used by many reports in the literature. There is a minor technical difference in how Skene et al. calculated their index  which I don't mind, but I don't think there is need to introduce 'stalling' here, I'll be happy with 'pausing'.The reason that we defined the term as ‘stalling index’ is for the subtle differences in the definition of stalled vs paused polymerases, as defined by John Lis . In brief, paused polymerases specifically are elongation competent, whereas the concept of a stalled polymerase is an inclusive term for all polymerase density . Our metric makes no assumption concerning the elongation potential of the polymerase and therefore we favor the term ‘stalling index.’Reviewer 2:The authors are experts in chromatin biology, and have developed several assays that enable different attributes of promoter chromatin to be analyzed. Here, they explore the impact of Chd1 remodeler on promoter chromatin. This issue has been addressed previously in ES cells at lower resolution, where promoter association was observed, with Chd1 loss leading to the acquisition of heterochromatin.The paper starts with the application of methods developed in the Henikoff lab to examine both Chd1 and RNA PolII in MEFs. This involves the separate examination of larger and smaller fragments derived from limited MNase digestion of crosslinked chromatin, following chromatin IP (ChIP). The larger fragments represent linkage of the factor to nucleosomes, whereas the smaller represent the factor's footprint. Support is provided that smaller fragments with PolII better link to the TSS.As mentioned above, the work presented here actually uses a novel technique that was specifically developed to address the distribution of Chd1. Typically our lab has favored native ChIP conditions. However, in mammalian cells we found that under native ChIP conditions only 14% of Chd1; 10% of PolII and ∼25% of histone H3 could be extracted, raising concerns that perhaps native ChIP would only investigate a specific genomic compartment of Chd1 binding. In comparison, sonication is typically used to both fragment chromatin and solubilize proteins in crosslinking ChIP, which results in low resolution and variable recovery. Here, we present the novel combination of formaldehyde crosslinking, MNase digestion to yield protected fragments and limited sonication to solubilize near 100% of Chd1 and chromatin. Through this technique we are able to generate high resolution data of Chd1 and PolII distributions. This was vital to observe the site of action of Chd1 on nucleosomes, as had been previously indicated in vitro (PMID: 16086025 and 16518397) and the stalled PolII in front of nucleosomes upon loss of Chd1 activity. This highly transferable technique has broad appeal to the scientific community, as it requires no special tool generation and uses current sequencing technology. The manuscript has been expanded to emphasize the novel nature of this technique, and For Chd1, the authors examine both an epitope-tagged WT and a catalytic mutant. With both Chd1 derivatives, localization to the +1 nuc was observed, however the catalytic mutant showed additional occupancy of proximal downstream nucleosomes. This data was nicely done and described. The authors then explored the impact on nucleosome occupancy and positioning following expression of the dominant-negative Chd1 derivative. Here, they claim a loss in nucleosome occupancy without a change in positioning. I find the effect very small overall, even when PolII levels are considered, but much more clear in the case where the highest quintile of Chd1 occupancy is examined. The authors note a difference in impact here in MEFs on the +1 nuc, versus in budding yeast where no change is observed following Chd1 loss, which I found interesting.In the revision, we now emphasize this difference, especially as it implies a fundamental difference between yeast and metazoans in how transcription proceeds through chromatin at the promoter.I also found the correlation of Chd1 with 'open' promoters interesting. The authors then apply an alternative shRNA approach to knock down Chd1, and observe similar (and even somewhat stronger) results.As prior approaches provide steady-state measurements, the authors then apply the CATCH-IT technique to explore dynamics. First, they show that turnover is high at locations flanking the TSS. The authors then explore the impact of expression of the dominant negative. The authors emphasize a significant difference at the locations flanking the TSS, and in the gene body. However, this is really confined to the nucleosome directly on, or adjacent to, the TSS; here it is clear and interesting, however, regarding the gene body, I found the difference with the dominant negative apparent, but the scale/magnitude quite small.We agree that the gene body turnover difference is small in absolute magnitude, which emphasizes that the biggest effect by far of mammalian Chd1 is at promoter proximal nucleosomes. This is fundamentally different from what is seen in yeast, where suppression of turnover in the gene body appears to be the primary role of yChd1 . We then extend this observation of mammalian Chd1 driving turnover and show that is responsible for essentially all of PolII directed nucleosome turnover, indicating its fundamental role in transcription through promoter proximal chromatin. The text has been modified to emphasize this novel role of Chd1, not evident from studies in yeast.Reviewer 3:This manuscript investigates the role of Chd1 in transcription. Using MNase-seq, PolII ChIP-seq and CATCH-IT in MEF cells expressing WT or a dominant negative form of Chd1, the authors conclude that Chd1 is a factor required for the RNAP to overcome the barrier of the +1 nucleosome and relieve stalling of RNAP, whereas in the gene body it suppresses nucleosome turnover. There are some potentially good ideas in this manuscript, but the data do not significantly extend what we know from previous work and the data suffers from insufficiently rigorous analysis.These results are similar to work from a number of labs , showing that Chd1 localizes at promoters and within gene bodies where it facilitates transcription by altering nucleosome occupancy and turnover/histone replacement, especially at highly active genes. In particular, the data is reminiscent of Radman-Livaja et al. (PLOS Genetics 2012), where it is shown in yeast that loss of Chd1 activity leads to slower turnover of nucleosomes over the promoter and faster turnover in gene bodies and near 3' ends.We are not disputing the importance of work that has been done with regards to Chd1 function in yeast. There are, however, key differences between our results in mammals and yeast, which likely reflects differences in promoter function and chromatin architecture. In addition, our work presents the novel finding that Chd1 functions to overcome the nucleosomal barrier to transcription, which has not been previously shown.In yeast, studies have focused on Chd1 suppressing histone turnover in the gene body , which we also observed in mammals, and further show that Chd1 recruitment to the gene body closely follows elongating PolII density. There are, however, fundamental differences in both transcription and chromatin context at the promoter between mammals and yeast. First, in yeast the transcription start site is embedded within the +1 nucleosome (PMID: 22258509), implying that the +1 nucleosome must be mobilized prior to pre-initiation complex formation. In contrast, in mammals the TSS is embedded within the nucleosome depleted region and the +1 nucleosome entry site found on average ∼50 bp downstream. Second, in mammals the vast majority of genes display PolII accumulation at the promoter region , whereas in yeast the PolII density is more evenly distributed along the length of the gene . This suggests a fundamental difference in the mechanism of PolII transit through the promoter proximal nucleosomes, which is supported by the identification of specific role of mammalian Chd1 at the promoter in driving turnover. In contrast to yeast where the magnitude of change in turnover at the promoter is approximately the same as in the gene body , the absence of mammalian Chd1 activity leads to a much greater reduction in promoter histone turnover. We present the novel finding that mammalian Chd1 is responsible for almost all PolII dependent turnover at the promoter and loss of this activity results in further accumulation of PolII at the promoter, a metazoan specific phenomenon. The previous yeast studies have not identified a consequence for transcription upon the loss of turnover at the promoter in the absence of Chd1. As such, our study identifies novel roles of Chd1 in terms of transcription related chromatin changes and places Chd1 as vital in the manipulation of metazoan specific chromatin structure.The text has been altered to include the yeast studies describing the role of Chd1 in turnover and emphasize that mammalian Chd1 is primarily functioning to drive turnover at the promoter in contrast to yeast (Results section entitled “Chd1 has opposing roles in regulating nucleosome turnover at the promoter and the gene body”).The authors emphasize the idea that Chd1 does not remodel promoter nucleosomes in yeast, but I don't understand why (they cite a Science paper from the Owen-Hughes lab that focused on nucleosome positioning). In fact that paper showed lower nucleosome occupancy near promoters in remodeling deficient cells, in total agreement with the current manuscript.This study  has done nice work in S. cerevisiae showing that Chd1 is a promoter-specific chromatin remodeler, and work in S. pombe  showed that Chd1 localized particularly to promoter regions where it helped remove nucleosomes near the transcription start site to enable transcription.These studies did not to identify a genome-wide link between Chd1 dependent chromatin changes and the changes in transcription. Here we have identified a unique role of mammalian Chd1 in regulating promoter nucleosome nuclear architecture in order to facilitate PolII promoter escape. These yeast studies have been acknowledged and discussed (In the Results section entitled “Chd1 activity is required for efficient promoter escape by PolII”).PHO5 promoter and indicated that Chd1 remodeling activity removed the +1 nucleosome in vitro . This is in contrast to the Owen-Hughes genome-wide analysis referred to above indicating no role for Chd1 in remodeling the +1 nucleosome (PMID: 21940898). This raises the possibility that Chd1 has a unique role at the PHO5 promoter. Additionally, the Kornberg study admits that in vivo PHO5 activation was robust in the chd1Δ strain, but required synergistic loss of ISWI to compromise gene activation. This raises the possibility of a separable function of Chd1 remodeling the yeast +1 nucleosome and ISWI-dependent transcription. In contrast, here we find absence of Chd1 activity alone has a genome-wide effect on metazoan-specific chromatin architecture and provide evidence for its vital role in overcoming the chromatin barrier to promoter escape via a novel high resolution mapping strategy.The Ehrensberger study (PMID: 21646535) investigated the chromatin structure at the S.pombe (PMID: 17510629) indicated that mutant strains displayed increased nucleosome density at Chd1-bound promoters, the opposite of what we observe here. This may reflect the unusually short 7 bp average linker length of S.pombe, which is very different from that of mammals, which is ∼50 bp. Moreover, the resolution (a single 500 bp Eurogentec microarray probe per promoter) was too low to draw distinctions between promoters and flanking nucleosomes, and only half of Chd1 bound promoters showed a change. The authors went on to look for changes in steady state RNA levels and found that 580 genes were upregulated and 238 genes were downregulated, suggesting that most genes were activated by the absence of Chd1. There was statistically insignificant overlap between the genes that showed increased nucleosome occupancy at the promoter upon loss of Chd1 and the expression changes . Therefore, it is important to separate steady state occupancy measurements from turnover. We find that a unique role of mammalian Chd1 is to drive PolII-dependent nucleosome turnover at the promoter, indicating the presence of more static nucleosomes at the promoter region in the absence of Chd1 activity. In the absence of Chd1 activity, we find that the barrier to PolII promoter escape is increased consistent with a more static chromatin barrier. The text has been altered to clarify this point (third paragraph of the Discussion).The reviewer presumes that nucleosome occupancy is correlated with turnover. However, work in yeast has shown that the highest turnover is at the promoter despite low nucleosome occupancy (PMID: 17347438). In Majorconcerns:1) There are a number of cases where strong conclusions are drawn based on non-quantitative analysis of normalized group-average profiles. Group averages can be dramatically shifted by a small subset of genes and are not a reliable way to draw generalizable conclusions about a population. In cases where the authors wish to say that Chd1 mutation 'increases' or 'decreases' signal, statistical evaluations should be performed and described clearly in the methods section. When the authors wish to say that two signals 'correlate' with each other, correlation analysis should be performed and Pearson/Spearman R squared or similar values should be presented.Statistical analysis has been performed and details included in the corresponding figure legends.2) It is really hard to interpret the data on PolII ChIP in cells expressing the catalytically dead Chd1 mutant without knowing what the effects of blocking Chd1 activity are on gene expression. The authors should at the very least select some example genes where loss of Chd1 increases the PolII stalling index and measure the nascent RNA levels at that gene by run-on or RT-PCR of intronic regions.We are interested in events occurring during transcription, whereas analysis of nascent chains can be complicated by co-transcriptional processing events. Therefore analyzing events that occur at the level of PolII are more relevant to the Chd1-related processes that we are studying here rather than analyzing the RNA produced downstream, especially as Chd1 has been proposed to interact with splicing factors (PMID: 18042460). In any case there is a close association between GRO-seq and PolII-Ser2-phos ChIP (PMID: 23062713), so nascent chain analysis is unlikely to provide any further insight. Therefore we favor our approach to measure ongoing transcription by genome-wide mapping of elongating PolII. Moreover we show that this reduction in elongating PolII density is more pronounced at genes with higher levels of Chd1 occupancy and is concomitant with an accumulation of PolII at the promoter, likely indicating a direct effect.The interpretation of the authors is that PolII is being 'blocked' at the promoter by the presence of more stable nucleosomes, but without showing that there is actually less active elongation within the gene bodies, this conclusion is very weakly supported, especially in light of work in Drosophila that shows normal Ser2-P PolII (active elongation form) levels in Chd1 mutants (Armstrong and Tamkun labs).Drosophila polytene chromosomes ”. Therefore, our observation of a decrease in genome-wide elongating PolII is not at all inconsistent with the results of this study.This previous observation of no change in Ser2-P PolII was based upon immunofluorescence of 3) It is very surprising that the authors don't mention any of the considerable work done on the relationship between PolII and the +1 nucleosome in Drosophila. In particular, Dave Gilmour's recent EMBO paper clearly shows that there are different classes of genes that differ in the role of the +1 nucleosome. Genes with the most paused PolII lack a significant +1 nucleosome signal and are paused by separate factors (GAGA and NELF) whereas less stably paused, more processive genes have a strong +1 nucleosome signal that causes more transient pausing. Do the authors find something significantly different in the mammalian system? Can they show by heatmap the relationship between the level of pausing and the occupancy level or turnover of the +1 nucleosome and analyze this quantitatively to demonstrate better that the +1 nucleosome might be a static barrier tomammalian PolII?Here we are investigating nucleosome dependent effects on PolII transit. NELF controls promoter proximal pausing, which occurs ∼30 bp downstream of the TSS, before the +1 nucleosome is encountered. We find that chromatin remodeler Chd1 preferentially operates on highly processive genes, which are independent of NELF regulation, to remove the nucleosome barrier to PolII. We have now altered the manuscript to include this work (last paragraph of the Results). We have also analyzed the data and find a similar situation in mammals, with processive genes having higher promoter proximal nucleosome occupancy and further show that the +1 nucleosome is more dynamic at processive genes . This su


Tristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described.ZFP36) by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates ZFP36 expression by two mechanisms: by binding the ZFP36 promoter directly through ETS-binding site (+ 883 to +905 bp) and by inducing expression of EGR-1, which in turn increases ZFP36 expression through sequences located between -111 and -103 bp.Our results demonstrate the induction of the gene coding TTP (EGF activates TTP expression via ELK-1 and EGR-1 transcription factors. ZFP36 encodes for tristetraprolin . The gene product is the prototype of the tandem CCCH zinc finger protein family, called TIS11, which includes four structurally and sequentially related proteins - TTP, BRF-1, BRF-2 and ZFP36L3 [Gene The activity of TTP is regulated predominantly via its phosphorylation by MAP kinases (in particular p38 and ERK1/2), kinases downstream of MAPKs  or AKT in response to pro-inflammatory cytokines , LPS and anti-inflammatory factors . The TTP protein sequence contains several key serine/threonine residues, the modification of which was previously shown to determine the ability of TTP to recruit the mRNA-degradation machinery, bind transcripts, be shuttled to P-bodies or stress granules and its nuclear/cytoplasmic localization and protein stability -12.Zfp36 gene promoter has been characterized, with the identification of a conserved proximal EGR-1 transcription factor binding site, AP2, SP1, TTP promoter element 1, STAT6 and SMAD biding sites and a functional GAS element. The first intron has also been shown to play a key role in mitogen-induced expression of Zfp36 [Less is known about the regulation of TTP expression. Both mRNA and protein are induced rapidly after stimulation of the cells with pro- and anti-inflammatory factors  ,13-18. Tof Zfp36 .EGR-1 and FOS seem to have the most important function in the regulation of the immediate-early cell response and widen the spectrum of ELK-1-regulated genes [The ELK-1 transcription factor is a representative member of ETS protein family characterized by the presence of the evolutionary conserved ETS domain stabilized by three key tryptophan residues and responsible for the interaction with DNA . The ELKed genes ,25. The ed genes .Stimulation of the human breast cancer MCF-7 cell line with EGF resulted in a rapid induction of TTP expression, the maximal effect being observed 30 minutes after EGF treatment. The ERK1/2 pathway inhibitor, U0126, inhibited this process Figure . In ordeZFP36 promoter fragment (-488 to +905 bp), further abbreviated as ZFP36. The promoter fragment included the first exon, intron and the upstream promoter sequence of ZFP36 gene. This promoter fragment was activated after EGF treatment, and this activation was blocked by the ERK1/2 pathway inhibitor, U0126  or repression (EN) domains fused in frame with C-terminus of ELK-1, the transcription factor acts independently of MAPK activation while preserving the DNA binding specificity [ZFP36 promoter than phosphorylated form of ELK-1. Dose-dependent regulation of activation/repression of ZFP36 promoter by Elk-VP16 or Elk-EN suggests that observed regulation is specific.Having shown that ELK-1 is activated by ERK1/2 in response to EGF stimulation, we checked whether the promoter of cificity . ZFP36 iZFP36 promoter to EGF. In MCF-7 as well as ELK-1 transfected HEK293 cells the EGF-dependent activation of the promoter was blocked by U0126. This made us conclude, that ELK-1 can be engaged in the regulation of ZFP36 promoter after EGF stimulation. These findings were further supported by the results obtained by qPCR. In ELK-1-limited HEK293 cells the stimulation of TTP expression by EGF is not observed (data not shown).In the mock-transfected MCF-7 cells, EGF stimulation caused an increase in promoter activity, which was completely abolished by the inhibition of ERK1/2 Figure . The traZFP36 promoter by ELK-1 we designed a series of truncation mutants. Deletion of two regions, -293 to -103 bp and +744 to +905 bp, has resulted in a substantial decrease in promoter activation by Elk-VP16 , namely EBS2 and EBS3 and one AP-1 binding site Figure . We intrn Figure , despiten Figure . Only th6 Figure . Compari6 Figure . ImportaF Figure . Taken tZFP36 promoter . Despite high degree (90%) of conservation of EBS6 sequence among analyzed species  which potentially can bind transcription factors from the ETS family and EBS6 sequence which can potentially interact with EGR-1 Figure . We haves Figure , its muty Figure . These rZFP36. Removing of both of them resulted in a loss of dose-dependent regulation of ZFP36 promoter by Elk-VP16  did not result in a loss of dose-dependent responsiveness to Elk-VP16 we decided to check whether deletion of both regions (mutant R5) will abolish this regulation. The results indicate that both investigated regions (-293 to -103 bp and +744 to +905 bp) are jointly needed for the regulation of 6 Figure .To confirm the binding of EGR-1 to the sequence located -293 to -103 bp and the binding of ELK-1 to the sequence located +744 to +905 bp chromatin immunoprecipitation was performed. The lysates from MCF-7 cells were immunoprecipitated with anti-EGR-1, anti-ELK-1 or nonspecific antibody. By PCR with primers flanking the investigated sequences, the levels of immunoprecipitated promoter sequences was analyzed. We have observed increased level of -293 to -103 bp amplicon after immunoprecipitation with anti-EGR-1 antibody, in comparison to the level of template immunoprecipitated with anti-ELK-1 or nonspecific IgG  and its exclusive receptor c-ErbB1. Elevated levels of c-ErbB1 and HER-2 in breast cancer is correlated with high disease recurrence rates and decreased patient survival [EGF regulates p Figure . TTP wassurvival . Upregulsurvival -44. Thus2. MCF-7 cells in Minimal Eagle's Medium (MEM) (Sigma) supplemented with 10% FBS and bovine insuline (10 ug/ml), HEK293, HeLa and HepG2 in Dulbecco's modified Eagle's Minimum Essential Medium  with 1 g/L D-glucose supplemented with 10% FBS.Adenocarcinoma cell line MCF-7 (ATCC No HTB-22), human embryonic kidney epithelium cell line HEK293 (ATCC No CRL-1573), human epithelium cell line HeLa (ATCC No CCL-2) and human hepatoma cell line (HepG2 No CRL-10741) were cultured at 37°C and 5% COCells were stimulated with EGF (20 ng/ml) or PMA (100 ng/ml) (R&D). When applied, the inhibitor of MEK1/2, U0126 (10 μM)  was added to the medium 30 min prior stimulation. Pre-designed oligonucleotide Silencer siRNA targeted to EGR-1 (Cat#16810) and control one (Cat# 4611) were supplied by Ambion.Total RNA isolation and northern blot analysis was performed as described previously .ZFP36 promoter fragment (-488 to +905 bp) was generated by two step PCR, using total DNA isolated from MCF-7 cells. The first round PCR was carried out with the primer forward: 5' GTCTTCCCTCCCTTCCTCAC 3' and reverse 5' GTCAGGGCTCAGCGACAG 3'. Then the second round was performed with nested primers introducing SacI and BglII restriction sites: forward: 5' TCGAGCTCTTCCTCACCCTGTCTATC 3' and reverse: 5' TCAGATCTCAGGAGGCTCTGCGGAAATG 3'. The introduced restriction sites were used during cloning of PCR product to pGL2-Basic reporter vector (Promega). The set of deletion mutants was prepared using pZFP36 as a template. The following forward primers with restriction site for SacI or NheI were used in PCR reactions:pEF1/Myc-His/lacZ is a control vector containing the gene for β-galactosidase (Invitrogen). pElk-VP16, pElk-EN were described before . pZFP36 GCTAGCCAGTTTCCTTCTACAAGCCTCAG 3'F1: 5' GCTAGCCGCGTCCGGGAAG 3'F3, R5: 5' GAGCTCGCGTCCGGGAAGC 3'F5: 5' GAGCTCGGCCCCGGCCCCGG 3F6: 5' The set of promoter constructs lacking different 3'-terminal sequences were generated with the following reverse primers:R01: 5' TCAGATCTGGAACTAGAGCTTGCCAG 3'R0, R5: 5' TCAGATCTAGAGTTGGAGGTTCTGAG 3'R1: 5' CTTAAGCACGCGTCGGGATCTC 3'R2: 5' CTTAAGTTTGAGCGAAGAGCCGGGTG 3'R4, F6, F5: 5' GCTAGCCTCGTAGATGGCAGTCAG 3'The constructs containing mutations in selected transcription factors' binding sites were generated using QuikChange XL Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer's procedure. The sequences were changed as follows:EBS2: CCCCCACCCC to CCCCCAgCtgEBS3: CCGGGGGCG to CCatGGGCGAP-1: CAAGCCTCAG to CAAGCCatgGETS3: ATCCGGAA to tgCaGccAETS4: ACCCATTTCC to ACCCATggCCEBS6: GGGTGGGGCG to GGGTaccGCGThe point mutant ETS5 was created by means of deletion mutation after a PCR reaction performed with a reverse primer 5' TCAGATCTTGCGGAAATGGGTCGGT 3'.ZFP36 promoter fragments and 10 ng of pEF1/Myc-His/lacZ. For some experiments indicated amounts of pElk-VP16 or pElk-EN were used. The amount of DNA per well was equalized using mock DNA (pcDNA3). Luciferase assays were carried out using the dual light reporter gene assay system (Tropix) according to the manufacturer's procedure. Luciferase activity was measured 24 hrs after transfection or at indicated time point after stimulation. β-galactosidase activity was measured to normalize the efficiency of transfection. All experiments were repeated at least three times in duplicates.Transient transfection experiments were carried out using Lipofectamine 2000 reagent (Invitrogen) in 12-well plate. Total amount of 1.6 μg of DNA per each well was used, including 0.4 μg of reporter vectors with Western blot was carried out using Immobilon Western chemiluminescent HRP substrate (Millipore) and anti-phospho ERK1/2 , anti-ELK-1 (Santa Cruz), anti- phospho p38 , anti-phospho JNK (Abcam), anti-c-FOS (Santa Cruz), anti-EGR-1 (Abcam), anti-GAPDH (Abcam), anti-phospho Ser383 ELK-1 (Santa Cruz), anti-ERK, anti-p38, anti-JNK .Was carried out using Lipofectamine 2000 reagent (Invitrogen) in 12-well plate. Total amount of 600 ng plasmid DNA and 50 pmol siRNA oligonucleotides per each well was used.Chromatin immunoprecipitation was carried out as described before  using anMF, PT, AB, AS constructs generation, reporter gene assays. AS qPCR. PT, JK, MW, KS western blots. AK chromatin immunoprecipitation, conception and interpretation of data. MF, PT, AK writing of manuscript. All authors drafted, read and approved the manuscript.


TM enrichment pathway analysis revealed Hltf regulates cell cycle, cell adhesion, and TGF-beta receptor signaling. Hltf's most important role is in the G2/M transition of the cell cycle (p  =  4.672e-7) with an emphasis on transcript availability of major components in chromosome cohesion and condensation. Hltf null brains have reduced transcript levels for Rad21/Scc1, histone H3.3, Cap-E/Smc2, Cap-G/G2, and Aurora B kinase. The loss of Hltf in its yeast Rad5-like role in DNA damage repair is accompanied by down regulation of Cflar, a critical inhibitor of TNFRSF6-mediated apoptosis, and increased (p<0.0001) active caspase-3, an indicator of intrinsic triggering of apoptosis in null brains. Hltf also regulates Smad7/Bambi/Tgf-beta/Bmp5/Wnt10b signaling in brain. ChIP confirmed Hltf binding to consensus sequences in predicted (promoter Scgb3a1 gene) and previously unidentified (P-element on chromosome 7) targets. This study is the first to provide a comprehensive view of Hltf targets in brain. Moreover, it reveals how silencing Hltf disrupts cell cycle progression, and attenuates DNA damage repair. HLTF participates in transcription, chromatin remodeling, DNA damage repair, and tumor suppression. Aside from being expressed in mouse brain during embryonic and postnatal development, little is known about Hltf's functional importance. Splice variant quantification of wild-type neonatal (6-8 hour postpartum) brain gave a ratio of 5:1 for Hltf isoform 1 (exons 1-25) to isoform 2 . Western analysis showed a close correlation between mRNA and protein expression. Complete loss of Hltf caused encephalomalacia with increased apoptosis, and reduced viability. Sixty-four percent of Hltf null mice died, 48% within 12-24 hours of birth. An RNA-Seq snapshot of the neonatal brain transcriptome showed 341 of 20,000 transcripts were altered (p < 0.05) - 95 up regulated and 246 down regulated. MetaCore As a mammalian DNA damage response gene, HLTF maintains genome stability by promoting the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA) via its ubiquitin ligase activity Knocking out either USP7 Santa Cruz Biotechnology, Inc.  was the source of Protein A/G PLUS-Agarose (sc-2003) beads, goat anti-mouse IgG-HRP (sc-2005), donkey anti-goat IgG HRP (sc-2033) and mouse brain extract (sc-24879). GE HealthCare  is the source of donkey anti-rabbit IgG HRP (NA934V). Ready Gel Tris-HCl precast polyacrylamide gels , and Immuno-Star Western Kit (170–5070) were purchased from Bio-Rad . PerkinElmer Life Science Products  was the source of Kodak Film (NEF596). Sudan Black B (199664) was purchased from Sigma-Aldrich . The Caspase 3 Assay Kit (ab39401) was purchased from Abcam .Antibodies for NeuN (MAB377) were purchased from Millipore . Antibodies to HLTF - N-terminus, residues 164–300, and residues 600–700 - were purchased from Santa Cruz Biotechnology, Inc. (sc-27542X), Sigma-Aldrich (HPA015284), and Abcam (ab17984), respectively. Invitrogen Corp.  was the source of Alexa-conjugated antibodies - Alexa Fluor 488 chicken anti-rabbit IgG (A21441) and Alexa Fluor 594 chicken anti-mouse IgG (A21201) – and prolong gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI).Mouse brain extract was prepared with the Active Motif  nuclear extract kit (#40010). DNeasy Blood & Tissue Kit (69506) was purchased from Qiagen  for isolation of genomic DNA from tail biopsies. Expand Long Template PCR System Buffer (11681842001) and PCR nucleotide mix (11814362001) were purchased from Roche Applied Science . PCR primers  were synFor RNA-seq, Otogenetics Corporation  used the following reagents: Ribo-Zero rRNA Removal Kit from Epicentre , an Illumina company; NEBNext mRNA Sample Prep kit (E6110) and NEBNext reagents (E6040) from New England Biolabs .Microscopy, chromatin immunoprecipitation (ChIP), and Western analyses were performed as previously detailed Hltf knockout mice were developed in collaboration with genOway . All studies were conducted in accord with the NIH Guidelines for the Care and Use of Laboratory Animals, as reviewed and approved by the Animal Care and Use Committee at Texas Tech University Health Sciences Center . TTUHSC’s IACUC specifically approved this study. All efforts were made to minimize pain and suffering.Hltf locus was analyzed approximately 8.5-kb on each side of exon 1 for GC content, repeats and secondary structure. For promoter analysis, web-based tools  were used for finding putative cis-regulatory elements in genomic sequences. Predictions were based on the integration of binding site predictions generated with high-quality transcription factor models, and cross-species comparison filtering (phylogenic footprinting). Consensus (K-K/R-X-K/R) nuclear localization sequence (NLS) based on in silico analysis with PredictNLS through the PredictProtein server was used to identify a single monopartite NLS at position 380  encoded by exons 10–11. Primo-analysis was used to lower the probability of random PCR priming by comparing primer sequences with the mouse transcriptome.The Sequences encoding the NLS were targeted by inserting a FRT-neomycin-FRT-LoxP selection cassette downstream of exon 12, and a LoxP site upstream of exon 11 . After hPCR screening reactions  were useIndividual samples [2 brains/sample × 3 biological replicates for test and control mice  = 6 total samples] were flash frozen and sent to Otogenetics Corp.  for RNA-seq assays. Total RNA was isolated; its integrity and purity were assessed using Agilent Bioanalyzer and OD260/280 . Each saRNA-seq in conjunction with 3SEQ/transcriptome was used to quantify expression levels by comparing sequencing reads against a reference transcriptome. Each transcript was quantified by calculating its RPKM (reads per kilobase of transcript per million mapped reads) enabling direct comparisons of expression levels among transcripts and across experimental conditions. RPKM and total read counts were reported for each gene.Otogenetics via DNAnexus provided an unbiased gene expression analysis report of RNA-seq; alternative splicing analysis of Hltf; mutation/RNA-editing analysis and parallel comparison of expression profiles between null and control samples. FPKM (fragments per kilobase of transcript per million mapped reads) were mapped against mm9 with Tophat (V1.3.3) to obtain.bam mapping files that were input into Cuffdiff (V 1.3.0) for comparison between two conditions  and three replicates for each condition. Using a free trial, data were imported to MetaCore™ for pathway analysis . Standard enrichment parameters  were used.p<0.05 significance level) using GraphPad Prism V.6.0b (GraphPad Software). The p value for the difference between elevations caused by the presence or absence of Hltf expression was p  = 0.0003. Regression analyses of expression level measured by RPKM for control vs. Hltf null values were achieved with GraphPad Prism V.6.0b.Caspase 3 assays were performed on whole brain extract according to the manufacturer’s protocol. Triplicate values were evaluated by ANCOVA (Hltf is located on chromosome 3qA3 and extends over 60.5 kb. The cDNA sequence (NM_009210) was used to establish the 25-exon/24-intron organization of the Hltf gene. To generate an Hltf knockout mouse model, three different targeting strategies were attempted: 1) exon 1 to eliminate the ATG; 2) exons 4–6 to eliminate the HIRAN  are unaffected (p>0.05) by deletion of Hltf. However, transcript availability of Rad21/Scc1– that encodes one of two major cohesin subunits  that holThe loss of Hltf in its yeast Rad5-like role in DNA damage repair - characterized by increased apoptosis  – is accNewborn Hltf null mice and their littermate controls display a suckling reflex and drink immediately after birth. However, two of three nulls do not survive. This finding correlates with the fact that two out of three nulls are hypoglycemic (data not shown). Hypoglycemia coincides with altered mRNA levels for solute carriers (Slc) that are normally enriched in the blood brain barrier , S4, andThe strategy for the development of the Hltf null mouse was developed in collaboration with genOway, and utilized the NMD mRNA quality control mechanism The phenotype of our Hltf null mouse differs from a model in vitro, and cellular loss of either protein increases the frequency of chromosomal abnormalities following DNA damage. In addition to its ubiquitin ligase activity for PCNA, HLTF displays protein-clearing activity at the stalled replication fork. Results from RNA-seq analysis of brains in newborn Hltf null mice indicate the loss of Hltf compromises the DNA-damage avoidance pathway in mouse brain, and modulates mutagenesis by regulating key participants in the G2/M phase of the cell cycle, some of which contribute to DNA double-strand break repair.HLTF and SHPRH (SNF2 histone-linker PHD-finger RING-finger helicase) are the two human Rad5-related proteins Cell division consists of duplication of the DNA content of chromosomes followed by segregation of the chromosomes into daughter cells The negative impact of Hltf deletion on cell cycle is not limited to cohesin. It extends to condensin’s role in the compaction of sister chromatids. The condensin complex consists of the two Smc subunits Smc2 and Smc4, and three non-Smc subunits . KnockdoThe non-Smc subunits of condensin I and Histone H3 are Aurora B substrates. Aurora B globally phosphorylates Histone H3 on Ser10, and colocalizes with the phosphorylated isoform (H3Ser10ph), which is required for chromosome condensation and segregation. Abrogated expression (RNAi) of Aurora B produced a concomitant reduction in global phosphorylation of Histone H3 in vivo models that will include the experimental manipulation of the uterine environment.Loss of Hltf expression results in apoptosis  rather tin utero and later stages of neural development into adulthood. Low-level Hltf expression was detected by in situ hybridization from E11.5-E18.5 Hltf/Bmp5 signaling establishes a link between brain development It is clear deletion of Hltf alters the extracellular environment of the brain and as well as its chemosensing ability. For example, reduced (p<0.011) mRNA for Slc7a11, a gene that encodes the cysteine/L-glutamine exchanger xCT, would result in loss of neuroprotection due to reduced cellular glutathione export In conclusion, the complete loss of Hltf gene product  means all genetic and epigenetic functions are lost at the molecular level from cells of Hltf null mice. Beginning with Hltf null brain, because of its compelling morphology, RNA-seq revealed global insights into Hltf-regulatory networks. The most significant finding are: 1) Hltf regulates genes that encode key components of cohesin/condensin complexes that are essential for genome stability; and 2) the absence of Hltf triggers an apoptotic response. Taken together, these finding expand our understanding of Hltf signaling in development and disease.Table S1DNAnexus Alternative Splicing analyses quantified the usage for each exon by mapping read counts to the spliceome. Mapping coordinates from the spliceome were converted to mapping coordinates to the genome. Mappings of the same read to the same genomic location were combined, and their posterior probabilities summed. Exon quantification was performed to show the relative expression level of all known Hltf exons in brain.(XLSX)Click here for additional data file.Table S2DNAnexus Alternative Splicing analyses quantified the usage for each possible splice junction in control RNA-seq samples. This resulted in the comparison of different known splice products in addition to the identification of new splice products shown here as Hltf exon-skip events in brain.(XLSX)Click here for additional data file.Table S3RPKM values were used to identify differentially expressed genes. Genes whose expression was decreased in Hltf null mouse brain compared with controls are identified here.(XLSX)Click here for additional data file.Table S4RPKM values were used to identify differentially expressed genes. Genes whose expression was increased in Hltf null mouse brain compared with controls are identified here.(XLSX)Click here for additional data file.


The replication of genomic DNA is limited to a single round per cell cycle. The first component, which recognises and remains bound to origins from recognition until activation and replication elongation, is the origin recognition complex. How origin recognition complex (ORC) proteins remain associated with chromatin throughout the cell cycle is not yet completely understood. Several genome-wide studies have undoubtedly demonstrated that RNA polymerase II (RNAP-II) binding sites overlap with replication origins and with the binding sites of the replication components. RNAP-II is no longer merely associated with transcription elongation. Several reports have demonstrated that RNAP-II molecules affect chromatin structure, transcription, mRNA processing, recombination and DNA repair, among others. Most of these activities have been reported to directly depend on the interaction of proteins with the C-terminal domain (CTD) of RNAP-II. Two-dimensional gels results and ChIP analysis presented herein suggest that stalled RNAP-II molecules bound to the rDNA chromatin participate in the anchoring of ORC proteins to origins during the G1 and S-phases. The results show that in the absence of RNAP-II, Orc1p, Orc2p and Cdc6p do not bind to origins. Moreover, co-immunoprecipitation experiments suggest that Ser2P-CTD and hypophosphorylated RNAP-II interact with Orc1p. In the context of rDNA, cryptic transcription by RNAP-II did not negatively interfere with DNA replication. However, the results indicate that RNAP-II is not necessary to maintain the binding of ORCs to the origins during metaphase. These findings highlight for the first time the potential importance of stalled RNAP-II in the regulation of DNA replication. In contrast, in S. cerevisiae, ORIs have been found in restricted regions that are referred as autonomously replicating sequences (ARSs) Replication originates at multiple and specific locations in the genome that define starting sites, which are called replication origins (ORIs) A report demonstrated that in HeLa cells, the ORC is localised to specific sites that frequently overlap with RNA polymerase II-associated sequences Drosophila, Xenopus and mammalian cells, transcriptional elements at replication initiation sites have been shown to stimulate replication In virus, yeast, S. cerevisiae consists of several hundred repeats that contain sequences encoding the 35S and 5S rRNA genes, which are separated by two intergenic non-coding regions (IGS1 and IGS2)  . Every rnd IGS2)  [35],  from Invitrogen containing 1.42 M 2-mercaptoethanol) and boiled for 5 minutes. The supernatants were saved at −80°C for analysis by western blot. Aliquots of 10 µl were loaded in each case. For SDS–PAGE gel electrophoresis, the total cell extract was loaded onto a 4–12% or 3–8% gradient polyacrylamide gel according to NuPAGE (Invitrogen) specifications. The gels were transferred to Hybond-P membranes (Amersham-Bioscience) in Tris-glycine buffer without SDS. The membranes were blocked with 5% milk in PBS and 0.1% Tween-20 (Sigma). After probing with antibodies, the membranes were developed using ECL-Plus (Amersham).Exponentially growing cells  The fission yeast S. pombe contains multiple copies of rRNA genes in two clusters at both ends of chromosome III. The ARSs and RFBs are shown. ChIP analysis using the 4H8 antibody against RNAP-II (green line). The values are expressed as the mean, n = 2. Human ribosomal RNA genes are arranged as tandem repeats clusters at the middle of the short arms of chromosomes 13, 14, 15, 21 and 22. Human CDC27 pseudogene and the consensus-binding site for p53 are shown. (b) ChIP analysis of Rap1p to the rDNA in mutant rpb1–1 cells at 25°C followed by incubation at 37°C, thus inactivating RNAP-II. Rap1p was tagged with 9-MYC epitope, and anti-MYC antibody was used. The location of the primers is shown. Values are expressed as the mean. n = 2.(TIFF)Click here for additional data file.Figure S2ChIP analysis of the rpb1–1 ts strain growing at 25°C or shifted at 37°C for 30 minutes. Diagram of the rDNA with the locations of the replication barrier (RFBs), the replication origins (ARSs) and sites for restriction enzymes. Theoretical schemes and 2D gels of chromatin digested with BglII (a), StuI (b and c) and SphI (d). Results of quantification are represented in histograms (c).(TIFF)Click here for additional data file.Figure S3Schematic diagram of one rDNA unit. ChIP analysis of Ser7P-CTD and hypophosphorylated RNAP-II (8GW16) using a wild type strain within rDNA IGS regions. Mean ± S.E.M. n = 3.(TIFF)Click here for additional data file.Figure S4Hypothetical model representing replication of the rDNA locus. (a) Model representing the possible interaction between Orc1p and stalled RNAP-II complexes bound at the rDNA ARS element. The green circles represent the post-translationally modified CTD tail of the largest subunit of RNAP-II (Rpb1p). Stalled RNAP-II mediates the chromatin interaction between IGS1-IGS2 (TIFF)Click here for additional data file.


Other domains of BCPs are responsible for binding partner proteins to form regulatory complexes. Once these complexes are assembled, BCPs alter chromosomal states and regulate gene expression. Some BCP complexes bind nucleosomes, are involved in basal transcription regulation, and influence the transcription of many genes. However, most BCPs are involved in targeting. For example, some BCPs function as a recruitment platform or scaffold through their Brds-binding targeting sites. Others are recruited to form a complex to bind the targeting sites of their partners. The regulation mediated by these proteins is especially critical during normal and abnormal development. Mutant BCPs or dysfunctional BCP-containing complexes are implicated in the initiation and development of neurodevelopmental disorders. However, the pathogenic molecular mechanisms are not fully understood. In this review, we focus on the roles of regulatory BCPs associated with neurodevelopmental disorders, including mental retardation, Fragile X syndrome (FRX), Williams syndrome (WS), Rett syndrome and Rubinstein-Taybi syndrome (RTS). A better understanding of the molecular pathogenesis, based upon the roles of BCPs, will lead to screening of targets for the treatment of neurodevelopmental disorders.Neurodevelopmental disorders are classified as diseases that cause abnormal functions of the brain or central nervous system. Children with neurodevelopmental disorders show impaired language and speech abilities, learning and memory damage, and poor motor skills. However, we still know very little about the molecular etiology of these disorders. Recent evidence implicates the bromodomain-containing proteins (BCPs) in the initiation and development of neurodevelopmental disorders. BCPs have a particular domain, the bromodomain (Brd), which was originally identified as specifically binding acetyl-lysine residues at the N-terminus of histone proteins  Neurodevelopmental disorders are complicated diseases. Studies are currently underway to understand their regulatory mechanisms, to explore better treatments and, ultimately, to prevent their onset.Chromatin modification traits are associated with the pathogenic characteristics of many neurodevelopmental disorders. Bromodomain-containing proteins (BCPs), which contain a bromodomain (Brd), represent a family of proteins found in many chromatin-associated proteins and in a range of transcription factors. A hydrophobic pocket formed by the ZA loop and BC loop is responsible for acetyl-lysine recognition and specific binding, the accuracy of which could be improved by sequence and acetyl-lysine modification of target sites. Thus, several inhibitors targeting these interactions have been designed for potential clinical application.BCPs play pivotal roles in transcription regulation. Some BCPs participate in initiation and regulation of basal transcription by binding nucleosomes. However, as targeting proteins, some BCPs can recruit their binding substrates and bind their target through the Brds as the ‘reader’ or a recruitment platform for modular protein complexes. They regulate gene transcription by both activation and repression. Conversely, some BCPs can be recruited by other transcription factors (such as p53), and function as modifying enzymes. Consequently, this affects the transcription of downstream genes of other transcription factors.Abnormal BCPs or BCP-containing complexes in the nervous system have been shown to cause neurodevelopmental disorders, including neural tube defects , mental retardation , Williams syndrome , Rett syndrome , Rett syndrome and Rubinstein-Taybi syndrome , and Fragile X syndrome .Although the available inhibitors are not efficient enough to be used clinically, for example, by simply increasing histone acetyltransferase (HAT) activity, recent evidence has provided the starting point to investigate BCP’s role in nervous system development and neurodevelopmental disorders.Neurodevelopmental disorders are a group of diseases characterized by impairment of the growth and development of the brain or central nervous system. Most neurodevelopmental disorders affect behavior, resulting in economic and mental problems, not only for the patients, but also for their families and society. The most common forms of these disorders include mental retardation, autism, bipolar disorder, schizophrenia, attention-deficit/hyperactivity disorder, and learning and memory impairment ,2.Understanding the regulatory mechanisms of these diseases is vital for developing better treatments and for preventing their onset. Neurodevelopmental disorders are thought to be caused by (a combination of) genetic and environmental factors. Epigenetic modification is affected by environmental factors, such as drugs, nutrition, toxicities, and mental stress, and its traits are in line with the pathogenic characteristics of neurodevelopmental disorders. Epigenetic modification of chromatin provides a dynamic platform for regulating the expression of target genes via acetylation, phosphorylation, methylation, ubiquitination, and sumoylation . Among sIn the present review, we focus on the roles of BCPs in the pathogenesis of neurodevelopmental disorders. BCPs represent a family of proteins found in many chromatin-associated proteins or in nearly all known nuclear HATs, and contain particular Brds. The importance of BCPs is underscored by the fact that genetic alterations of these genes and the subtle balance of aberrant acetylation are strongly linked to complicated diseases, such as cancer and neurodevelopmental disorders Table . Here, wDrosophila brahma (BRM) protein, is a highly evolutionarily conserved domain. More than 75 human BCPs have been identified in the National Center for Biotechnology Information (NCBI) protein database using Brd sequences as query sequences [The Brd, first identified in the equences ,21. WithThe spatial structure of Brd results in recognition and binding of target regions. Other critical domains in BCPs, the amino acid sequences and posttranslational modifications of target sites combine to subtly and accurately regulate both the chromatin state and the transcription of target genes.et al. identified a larger domain of about 110 residues [et al. determined that Brd1 of BRD2 forms an intact homodimer, in the crystal and also in solution. Brd1 recognizes acetylated H4K12, whereas the dimer interface of Brd1 binds to hypoacetylated H4K8 [IID, TAF1, and BRDT. Self-dimerization may help to avoid erasing histone codes during mitosis [Originally, Brd was described as an approximately 60 amino acid domain forming two α-helices. After analyzing the Brds of several BCPs, Jeanmougin residues . Based oresidues ,24. Brdsted H4K8 . Brd2 ofted H4K8 . Other B mitosis  or might mitosis .Brds can specifically bind target sites, and the other domains in BCPs contribute to recruit partner proteins or play other critical roles. For example, in p300/CBP, these other domains include three cysteine-histidine (CH) rich domains , a KIX domain, and an ADA2-homology domain , and die at E11.5, with a smaller embryonic size and neural tube defects (NTDs) [cyclin A promoter [IID and SWI/SNF complexes. The available evidence suggests that BRD2 plays an essential role as an integrator of transcription and chromatin structure during mammalian embryogenesis and neurogenesis.BRD2  is an important transcriptional modulator that is expressed in brain vesicles, the neural tube, the spinal cord and dorsal root ganglia. At the cellular level, BRD2 is localized to the nucleus in proliferating cells and to the cytoplasm in differentiating neuronal precursors . BRD2−/−s (NTDs) ,55  ID: 300659) (Table BRWD3 (unpublished data). Research into the causative mechanism is underway. BRWD3 is assumed to be involved in modulation of the JAK/STAT signaling pathway [BRWD3 contains two Brds and nine WD repeats. The WD repeat domains are responsible for interaction with other proteins to regulate cellular events, such as cell cycle, signaling transduction, and apoptosis. Truncation mutants of 9) Table . The aff pathway . NotablyHATs play important roles in regulating gene expression during brain development and memory formation. Aberrant BCPs, as HATs, contribute to neurodevelopmental disorders.Gcn5 may be associated with increasing risk of NTDs in humans [in vitro and in vivo, and double mutants of Gcn5 and p53 mouse embryos survive longer than Gcn5 null mice. Deletion of p53 cannot rescue the embryonic lethality caused by the mutant of Gcn5. When the HAT activity of GCN5 is abnormal, cranial neural tube closure in mice is defective [GCN5 is a transcriptional co-activator with HAT activity . Mutant n humans  , which is characterized by mild to severe mental retardation, craniofacial defects, short stature, skeletal abnormalities, broad big toes, and broad thumbs ,15,63  is a microdeletion or contiguous gene deletion syndrome characterized by hemizygous deletion of 1.5 to 1.8 Mb on chromosome 7q11.23 [WSTF including the nucleosome assembly complex (WINAC) is another SWI/SNF-type complex that has ATP-dependent chromatin-remodeling activity. It interacts with the vitamin D receptor (VDR) through the Williams syndrome transcription factor . WINAC is a key complex for repressing and activating transcription , and is  7q11.23 . The fre 7q11.23 ,80. The  7q11.23 . DNA seqDeregulation of methylation and BCPs may alter complex networks of gene expression and brain function, contributing to neurodevelopmental disorders.Brain development is a complicated process, involving neuron proliferation, differentiation, migration, communication, and apoptosis. Genetic deficits and negative environmental exposures may lead to abnormal neurological development. The characteristics of neurodevelopmental disorders strongly coincide with the traits of dysfunctional epigenetic modification at the chromatin level, alone or in combination. Here, we use Rett syndrome (OMIM ID: 312750) as an example to elucidate the contribution of aberrant methylation and dysfunction of BCPs to neurodevelopmental disorders.Bdnf) in resting neurons. Overexpression of Bdnf can rescue a subset of RTS-like phenotypes [Rett syndrome, a progressive childhood neurodevelopmental disorder, is characterized by stereotypies and mental retardation, and autistic behavior in females. The affected individuals show neurodevelopmental defects, such as common hand movements, aberrant gait, and seizures . Rett syenotypes . In summThe growing number of identified BCPs and the links between some of these members and neurodevelopmental disorders underscore the importance of this class of proteins. BCPs, which are ubiquitous and evolutionarily conserved, play pivotal roles in chromatin modifications, remodeling, and transcriptional regulation. Remarkably, all HATs contain Brds, but not all BCPs are HATs. Some of them are now considered as modulators of chromatin remodeling (such as BAZ1A and CECR2), while some are transcriptional regulators (such as BRD2 and BRWD3) (Table In general, BCPs bind to target sites through their Brds, and they regulate transcription together with their binding partners. BCPs not only ‘read’ the histone code , but alsFurthermore, histone acetylation can improve the memory and learning ability through direct control of epigenetic suppression of gene expression . This exAsh1: Absent, small, or homeotic disc1; ATAD: ATPase family AAA domain-containing protein; BAZ1A: Bromodomain adjacent zinc finger, 1A; BAZ1B: Bromodomain adjacent zinc finger, 1B; BCPs: Bromodomain-containing proteins; Brd1: The first bromodomain protein gene; Brd2: The second Brd protein gene; BRM: Brahma; BET: Bromodomain and the extra terminal domain; BPTF: BRD and PHD finger-containing transcription factor; Brd: Bromodomain; CBP: CREB-binding protein; DRG: Dorsal root ganglia; FRX: Fragile X syndrome; HAT domain: Histone acetyltransferase domain; HDACs: Histone deacetylases; LAP finger: Leukemia-associated-protein finger; MeCP2: Methyl CpG binding protein 2; MLL: Mixed-lineage leukemia; NTDs: Neural tube defects; OMIM: Online Mendelian Inheritance in Man; PBAF: Polybromo BRG1-associated factor; PCAF: P300/CREB-binding protein-associated factor; PDE-4: Phospodiesterase-4; P-TEFb: Positive transcription elongation factor b; RTS: Rubinstein-Taybi syndrome; SAGA: Spt-Ada-Gcn5 acetyltransferase; SAHA: Suberoylanilide hydroxamic acid; SNP: Single nucleotide polymorphism; TAF1: TATA-binding protein-associated factor-1; TAZ: Transcription adaptor putative zinc finger; TFs: Transcription factors; Tg: Transgenic; TPA: Tetradecanoyl phorbol acetate; VDR: Vitamin D receptor; VPA: Valproic acid; WD: Tryptophan-aspartate; WS: Williams syndrome; TFIID: transcription initiation factor.The authors declare that they have no competing interests.JL, GZ and XG made substantial contributions to design of this study, and JL drafted and manuscript, and GZ and XG revised the manuscript. All authors contributed to writing the manuscript. All authors read and approved the final manuscript.JL focuses on developmental biology and pathogenesis of complicated diseases. Recently, his laboratory has identified SNP loci that are positively associated with mental retardation in the Qinba mountain region, northwest China. Both loci rs7049509 and rs12689192 are near the second Brd domain in the BRWD3 gene. After investigating the functional domains and phosphorylation sites of BRWD3, the group is currently studying the molecular mechanism of neurodevelopment based upon BRWD3. GZ is a full professor, the director of Key Laboratory of Resource Biology and Biotechnology in western China. XG is a full professor, and the vice-director of the Institute of Population and Health. Since its inception in 1995, the Institute of Population and Health has been devoted to the study of neurodevelopmental disorders, such as mental retardation and Fragile X syndrome. The authors’ research teams have carried out investigations on the pathogenesis of neurodevelopmental disorders in the Qinba mountain area, where the incidence is about 3%. In addition to environmental factors, genetic deficits are critical. Susceptible loci and several genes have been identified using gene scanning methods.


Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes.The regulation of variant gene expression in  Plasmodium falciparum maintains its persistence in the vertebrate host at least in part by expression of several variant proteins at the surface of infected erythrocytes. The most important variant antigen is PfEMP-1  which is encoded by approximately 60 members of the var multigene family var genes are expressed per single parasite The human malaria parasite rif family has approximately 160 different intact members in the 3D7 strain annotated in the PlasmoDB databank. Recently, we rif transcription switches apparently faster than transcription of var genes, at least in vitro. Similar to var genes, rif gene transcription and silencing is dependent on the histone deacetylase PfSir2A rif gene loci are associated to histone 3 lysine 9 trimethylation as are other virulence-associated multigene families var, rif, and stevor seem to be controlled by a common factor and overexpression of artificial loci containing a rif promoter suppressed the transcription of var loci var genes, recently gained further support The multigene var gene which is actively transcribed was associated to histones carrying an acetyl group at the lysine 9 of histone 3 (H3K9) and Chookajorn and colleagues var locus active in early trophozoite stage and silenced in schizont stage  was associated with a dimethylated lysine 4 in histone 3 At the molecular level, Lopez-Rubio and colleagues P. falciparum, we addressed the question if dynamic histone modifications which are decisive in var regulation are also associated with active or silent rif loci. For this, the transcription of rif genes was monitored in consecutive cultures and compared to the histone modifications found for each locus in the same parasites.Given the observation of similar histone landmarks in ChIP-on-Chip experiments Var gene loci in their active state are associated with class 3 histones (H3) carrying an acetylation mark at their lysine 9 (H3K9ac), while silenced var loci associate with H3K9me3 rif loci are associated with the same epigenetic landmarks as var loci, we first performed a qRT-PCR-based rif transcript analysis in parasites previously selected for cytoadherence in CHO-CD36, since in this phenotype a number of rif loci were found active with rapid switching after a small number of reinvasions and given the large number of rif genes the analysis was restricted to these loci rif genes after 10 and 20 reinvasions after the last panning procedure. As expected, a number of rif genes were actively transcribed while others were silenced or transcribed at background levels . Noteworthy, the overall intensity of immunoprecipitation varied between loci. In order to show if predominant H3K9-acetylation - expressed as a higher ratio H3K9ac/H3K9me3 – statistically correlated with higher transcript abundance, we divided the H3K9ac/H3K9me3 ratios after 20 reinvasions by the H3K9ac/H3K9me3 ratios after 10 reinvasions and plotted them against the coefficients of relative transcript quantity from each locus after 20 and 10 reinvasions (2 = 0.5299). The extreme outliers were not considered in the analysis (Pf080138 and PFB0040c). A significant correlation of H3K9ac/H3K9me3 and transcript quantities was also observed when the data were analyzed separately for the 10 and 20 reinvasion material  were reselected for cytoadherence on CHO-CD36 cells. After two selection rounds, trophozoites at 20–24 h post reinvasion showed most transcripts from the PFI0025c locus and minor quantities from loci PFD1240w, PFD0070c and PF100397  rif loci which are actively transcribed in trophozoites (20–24 h post reinvasion) and/or schizonts (30–34 h post reinvasion), we cultivated parasites previously cytoadherence-selected on CHO-CD36 (25 reinvasions after selection), synchronized them and harvested chromatin 20 and 30 h post reinvasion and analyzed for their modification at H3K9 and H3K4 by ChIP. In trophozoites, we detected relatively low rif transcription at the tested loci (below 0.5 relative units), while in schizonts more transcripts accumulated (rif loci showed a tendency to slightly increased acetylation over trimethylation when transcripts were detected (above 0.1 relative units) and there was a significant correlation of transcript quantity with H3K9ac/H3K9me3 ratios in schizonts but not in trophozoites (rif PFB0040c (silent in trophozoites) showed increased acetylation at H3K9 when compared to trophozoites, and the strongly transcribed PF100397 showed accentuated acetylation. The only locus which did not follow the pattern was PFD0070c, which showed very few transcripts in trophozoites and in schizonts, although its locus was strongly acetylated at H3K9. The loci with no detectable transcription in trophozoites or in schizonts followed the pattern of either stronger trimethylation or lower levels of any modification  when we plotted the transcript quantities against the H3K4me2/H3 enrichment ratio. The silent loci PFL2640, PFD1010w, PF080138 and the weakly transcribed locus PFB0040c were not significantly marked  Posttranslational modification (PTM) of histones is a well-known mechanism that is related to the control of gene expression in eukaryotes. It is believed that a “histone code” orchestrates the accessibility of transcription factors to promoter regions through modification of histone tails by methyl, acetyl and phosphate groups, and proteins as ubiquitin and SUMO as reviewed by Berger rif gene transcription activation and silencing, we performed a detailed analysis of a number of rif genes. Since the selective upregulation of rif genes by phenotypic selection is not yet possible as it is for var genes e.g. rif genes and the frequent transcriptional activity of the herein tested loci In order to elucidate mechanisms responsible for var gene locus activity seem at least partially also functional for rif loci: When a significant presence of transcripts for any tested locus was observed, H3K9 acetylation dominated. This mark has not been identified before for active rif genes. Inversely, in the absence of transcripts, loci were either associated with strong tri-methylation of H3K9, in accordance to data published by Lopez-Rubio and colleagues var loci, the modifications are dynamic var genes, one would perhaps expect higher acetylation in “on” rif genes of which oligos amplify in the 5′ region of the gene – in proximity to the promoter region which is supposed to carry most of the acetyl residues rif genes are much shorter than the sometimes 12 kb-var genes. It is possible that the short central 500 bp range in which the oligos were allocated in the different rif genes may not allow to discriminate these positional effects, taking also in account that the immunoprecipitated genomic DNA fragments generated during the ChIP procedure had around 400 bp (data not shown). In order to address the question of positional effects of priming sites in genes analyzed by ChIP, we compared signal differences in the loci PFI0025c and PF100397, using additional specific oligo pairs in the coding sequence. For this experiment, material from the experiment in rif gene  is distant from transcribed rif genes and no specific pattern of transcript quantity and genomic context  following instructions of the manufacturer. The washed RNA pellet was briefly dried at RT and dissolved in water and stored at −80°C until use. About 5 µg of total RNA was used for cDNA synthesis. Briefly, total RNA was treated 3 times with DNAse I (Fermentas) prior to synthesis to prevent genomic DNA contamination. The treated RNA was reversed transcribed using MuLV-Reverted aid transcriptase reverse (Fermentas) and random hexamer primers following the manufacturers' instructions.9 parasites were crosslinked with 1% formaldehyde and RBC were lysed in PBS supplemented with saponin 0.1%. Following saponin lysis, parasites were resuspended in lysis buffer , supplemented with 1× Protease Inhibitor Cocktail . After 30 minutes of incubation, parasites were lysed using a Dounce Homogenisator (100 strokes), centrifuged for 10 minutes at 14.000 rpm/4°C, and then sonicated using a cell disruptor (Unique Ultrasonic) until resulting DNA fragments had 200–500 bp. At this stage, 10% of the material (“input”) was retrieved and submitted directly to the reversion of crosslinking (see below). For immunoprecipitations, sonicated chromatin was diluted in ChIP buffer . Chromatin diluted in ChIP buffer was pre-cleared in 50 µL/mL of a DNA sperm salmon/protein A-sepharose slurry for 2 h/4°C. Afterwards, chromatin was incubated with 1∶250 of anti-H3K9ac, H3K9me3, H3K4me2 (Upstate) and H3 (Abcam). For negative controls (mock samples) we used an anti-IgG mouse, and for calibration of the overall chromatin quantity, similar amounts of material from the “input” fraction were employed. The chromatin-immune complexes were then washed under stringent conditions. Following this, crosslink was reverted by incubation at 65°C for 4 h and 0.2 M NaCl. DNA was extracted twice with Phenol/Chloroform/isoamylalcohol, precipitated and resuspended in 20–50 µl sterile water. The results presented are the average of two or three independent immunoprecipitations from different extracts of parasites . The significance of differences between signals was evaluated by Student's T test (two tailed) using GraphPad Prism 5.ChIP was conducted as described in rif transcript sequences and only oligos with perfect primer-to-target specificity were considered. Later, oligonucleotide pairs were selected for global target specificity by ePCR using the 3D7 genome sequence as template. Importantly, the primer performance in qPCRs differed no more than 1 Ct between oligonucleotide pairs used herein and the internal control oligonucleotide pair. PCRs were performed in triplicates and equal amounts of input DNA (0.05–0.5 µg) per imunoprecipitated sample and mock immunoprecipitations (unrelated IgG) were analyzed together with samples. Mock samples presented CTs>35, which was considered as unamplified targets. All rif oligos in each immunoprecipitation were calibrated with the input DNA signal using the formula 2−ΔCt = (CtoligoAb−Ctoligoinput), where Ab is the respective antibody used for ChIP. The plotted enrichment was calculated as the ratio of immunoprecipitated Ab oligo signal/immunoprecipitated H3 oligo signal. The internal control transcript used for calibration throughout the experiments was locus seryl t-RNA transferase (PlasmoDB No. PF07.0073), previously shown as reliable  and D, schizonts 30–36 h p.i. from the same reinvasion cycle as in D. E: Material from trophozoites 20–24 h p.i. freshly repanned over CHO-CD36.(TIF)Click here for additional data file.Figure S2Position of oligos used in qPCR and ChIP in the deduced transcript sequence of analyzed rif targets and their genomic context. A) The black bars above and the numbers below the scheme of each rif coding region indicate the initial position of the amplification of the primer. The numbers at the end of each gene indicate the size of the respective coding regions. The genes were divided in the figure according to the rif promoter classification proposed by Joannin et al 2008 C and T means telomeric and centromeric position, respectively. Rif A type genes are believed to encode IRBC-surface displayed RIFINs while type B RIFINs are probably localized to Maurer's clefts rif loci, extracted from http://plasmodb.org (6/15/2011).(PPTX)Click here for additional data file.Figure S3Relative quantities of rif transcripts and chromatin enrichment for H3K9ac, H3K9me3 and H3K4me2 (poised mark) in trophozoite and schizont stages for selected rif loci. A: relative transcript amounts of chosen rif targets in trophozoites and schizonts in 3D7 parasites of the same reinvasion, calculated as above. Chromatin enrichment of H3K9ac and H3K9me3 for trophozoites (B) and schizonts (C). The chromatin enrichment was normalized using the qPCR data from ChIP with anti-H3 and 10% of the input. Significant differences between H3K9 trimethylation and acetylation are depicted by single asterisks, and significant H3K4me2 modification (compared to the H3K9me3 control) are indicated by two asterisks .(TIF)Click here for additional data file.Figure S4Raw results from experiment in  showing the influence of primer localization on the qPCR signal after ChIP at two rif loci. Immunoprecipitated material from the experiment in (PPTX)Click here for additional data file.