Pioneer transcription factors recognise and bind their target sequences in inaccessible chromatin to establish new transcriptional networks throughout development and cellular reprogramming. During this process, pioneer factors establish an accessible chromatin state to facilitate additional transcription factor binding, yet it remains unclear how different pioneer factors achieve this. Here, we discover that the pluripotency-associated pioneer factor OCT4 binds chromatin to shape accessibility, transcription factor co-binding, and regulatory element function in mouse embryonic stem cells. Chromatin accessibility at OCT4-bound sites requires the chromatin remodeller BRG1, which is recruited to these sites by OCT4 to support additional transcription factor binding and expression of the pluripotency-associated transcriptome. Furthermore, the requirement for BRG1 in shaping OCT4 binding reflects how these target sites are used during cellular reprogramming and early mouse development. Together this reveals a distinct requirement for a chromatin remodeller in promoting the activity of the pioneer factor OCT4 and regulating the pluripotency network.DOI:http://dx.doi.org/10.7554/eLife.22631.001 All cells in your body contain the same genetic information in the form of genes encoded within DNA. Yet, cells use this information in different ways so that the activities of individual genes within that DNA can vary from cell to cell. This allows identical cells to become different to each other and to adapt to changing circumstances.A group of proteins called transcription factors control the activity of certain genes by binding to specific sites on DNA. However, this isn’t a straightforward process because DNA in human and other animal cells is usually associated with structures called nucleosomes that can block access to the DNA. Pioneer transcription factors, such as OCT4, are a specific group of transcription factors that can attach to DNA in spite of the nucleosomes, but it’s not clear how this is possible. Once pioneer transcription factors attach to DNA they can help other transcription factors to bind alongside them.King et al. studied OCT4 in stem cells from mouse embryos to investigate how it is able to act as a pioneer transcription factor and control gene activity. The experiments show that several other transcription factors lose the ability to bind to DNA when OCT4 is absent. This leads to widespread changes in gene activity in the cells, which seems to be due to other transcription factors being unable to get past the nucleosomes to attach to the DNA.Further experiments showed that OCT4 needs a protein called BRG1 in order to act as a pioneer transcription factor. BRG1 is an enzyme that is able to move and remove (remodel) nucleosomes attached to DNA, suggesting that normal transcription factor binding requires this activity. The next challenge is to investigate whether BRG1, or similar enzymes, are also needed by other pioneer transcription factors that are required for normal gene activity and cell identity. This will be important because many enzymes that remodel nucleosomes are disrupted in human diseases like cancer where cells lose their normal identity.DOI:http://dx.doi.org/10.7554/eLife.22631.002 Transcription factors read DNA-encoded information to control gene transcription and define the complement of proteins a cell can produce. They achieve this by physically binding specific DNA sequence motifs in gene regulatory elements that ultimately control how RNA polymerases engage with and transcribe genes . In prokThis chromatin barrier therefore poses a significant challenge in establishing new gene regulatory elements when cells differentiate during development. Therefore, to overcome this obstacle, cells have evolved a specialised set of transcription factors that are able to recognise their cognate motifs even when nucleosomes are present . This isThe capacity of pioneer transcription factors to destabilise or reposition nucleosomes to facilitate chromatin accessibility at their target sites appears to be an essential feature of their activity. Initially, it was proposed that pioneer factors might achieve this by interacting specifically with nucleosomes to alter chromatin structure. For example, FoxA1 and FoxO1 structurally resemble histone H1, which has been proposed to lead to displacement of H1 and destabilisation of neighbouring nucleosomes . AlternaOne paradigm that has been used to study pioneer transcription factor function is the cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) by the Yamanaka transcription factors  . With thTo begin addressing this fundamental gap in our understanding, we have used mouse embryonic stem cells, which exist in an established pluripotent state, as a model system to dissect how the core pluripotency transcription factor OCT4 engages with target sites inside cells. In doing so we discover that OCT4 occupies sites that would otherwise be inaccessible and is required to shape the occupancy of additional pluripotency transcription factors. OCT4 achieves this by recruiting the chromatin remodelling factor BRG1 to support not only its own binding but also to stabilize further transcription factor binding events required to support pluripotency-associated gene regulation. This reveals that although OCT4 can engage with its target sites in inaccessible chromatin, the recruitment of a chromatin remodelling enzyme is a fundamental step in pluripotency transcription factor binding and gene expression. Intriguingly, OCT4-bound regulatory sites that require BRG1 are activated more slowly in response to cellular reprogramming and later during early development. Together these observations reveal that the capacity of OCT4 to cooperate with a chromatin remodeller is a key feature of its pioneering activity, and this is required to mature transcription factor binding and create functional gene regulatory elements.bona fide set of OCT4 binding events in mouse ESCs using chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq). We applied this approach to a conditional mouse ESC line where addition of a small molecule (doxycycline) leads to loss of OCT4 expression  which provides a genome-wide measure of chromatin accessibility  and examA defining feature of pioneer transcription factors is their capacity to support additional transcription factor occupancy, potentially through alteration of local chromatin structure. Given that removal of OCT4 had widespread effects on chromatin accessibility at its binding sites, we wondered whether its absence would result in defects in the binding of other pluripotency-associated transcription factors. To address this possibility, we used ChIP-seq to examine the binding of SOX2 and NANOG in the presence or absence of OCT4 . InteresUtf1 gene  tended to be significantly closer to OCT4 binding sites that displayed reduced chromatin accessibility (median distance 16.2 kb) than OCT4 binding sites that retained chromatin accessibility in the absence of OCT4 (median distance 125.4 kb)  . These s25.4 kb)  and thei25.4 kb) . TogetheSeveral modalities have been proposed to explain how pioneer factors facilitate chromatin accessibility at otherwise inaccessible regions of the genome. For example, pioneer transcription factors may bind their target motifs and evict or exclude nucleosomes through steric mechanisms  or they fl/flBrg1) (FDR < 0.05) to identify sites that experienced significant reductions in chromatin accessibility following BRG1 removal. This revealed that 45% of OCT4-bound sites showed significantly reduced ATAC-seq signal and these sites were also highly dependent on OCT4 for their accessibility  . Tamoxifg1fl/fl)   . When weg1fl/fl) . When weg1fl/fl)  and nuclg1fl/fl) . We thensibility . Howeversibility . Importasibility , revealiOur ATAC-seq experiments revealed an essential role for BRG1 in regulating chromatin accessibility at OCT4 target sites in ESCs. One could envisage several possible mechanisms by which BRG1 could cooperate with OCT4 to achieve this. For example, BRG1 could be actively recruited by OCT4 to target sites in order to create accessible chromatin, or it could alternatively function to broadly remodel the genome and in doing so indirectly support access of OCT4 to its target sites. Given that large scale proteomic studies have previously indicated that BRG1 may interact physically with OCT4 , and BRGfl/flBrg1 ESCs and condOCT4 ESCs (bona fide OCT4 binding sites (FDR < 0.05 and fold change >1.5 fold) of distal OCT4 targets following BRG1 removal (FDR < 0.05 and fold change >1.5 fold)  . Importa.5 fold) , yet res.5 fold) . This su.5 fold) . HoweverIn the absence of BRG1, the stable binding of pluripotency transcription factors OCT4, SOX2 and NANOG was disrupted at distal regulatory elements, suggesting that this may affect expression of the pluripotency-associated transcriptional network. Somewhat surprisingly, previous work has not supported a clear correlation between loss of BRG1 and reduced activity of OCT4 target genes , despiteFDR < 0.05 and fold change >1.5) with genes that significantly changed in expression following BRG1 removal, the overlap was low , OCT4 stably associates with a new set of previously inaccessible sites in a manner that appears to rely on co-binding of the EpiLC-specific transcription factor OTX2 . In the One of the central features of pioneer transcription factors is their ability to alter local chromatin structure, and this is closely linked to their capacity to support further transcription factor binding and enable the functional maturation of distal regulatory elements. Yet, in most instances the defined molecular mechanisms that support these activities have remained undetermined. While some pioneer factors, such as FoxA1, can stably bind their target sites and directly alter nucleosomal structure through steric disruption of histone:DNA contacts , the extcondOCT4 […] Reviewer #1:[…] The major outstanding issue in this study is mechanistically how do OCT4 and BRG1 render the chromatin accessible, what does accessibility really mean? The study uses ATAC-seq as a measure for chromatin accessibility, but as the paper describes OCT4-BRG1 cooperation it is maybe important to go one step further and analyse more precisely how this cooperation affects nucleosome positioning. Can the authors describe how loss of OCT4 or BRG1 affects nucleosome positioning? Can they show that OCT4 and BRG1 cooperate to generate a nucleosome-depleted region to which combinations of OCT4, SOX2 and Nanog bind? When either OCT4 or BRG1 are depleted does this affect nucleosome positioning in similar or different ways? According to the model of pioneer factors proposed in this paper, in absence of BRG1, OCT4 should bind regions occupied by nucleosomes that are displaced when BRG1 is present. Can the authors use the ATAC-seq data to map nucleosome positioning, if not perhaps they should perform Mnase mapping of nucleosome positioning in presence or absence of OCT4 and/or BRG1 to assess how positioning is altered and show that upon BRG1 recruitment critical nucleosomes are displaced to reveal the OCT4, SOX2 and Nanog binding sites.cond and Brg1fl/fl ATAC-seq datasets using the NucleoATAC analysis approach  to measure nucleosome signal in the presence or absence of OCT4/BRG1. In wild type cells, a nucleosome-depleted region is clearly evident at OCT4 binding sites, as demonstrated by the reduced nucleosome signal at the centre of the OCT4 peak compared to neighbouring regions  and compared this to nucleosome signal at OCT4-bound sites (see point 1). This analysis revealed that OCT4 and BRG1/BAF co-localise within a very narrow nucleosome-depleted region at the centre of OCT4 binding sites . As suggReviewer #2:The authors use an elegant system to show that OCT4 leads to increases in chromatin accessibility in a manner partly dependent on BRG1. This leads to co-binding of another key ESC TF – SOX2. The authors further demonstrate that OCT4-BRG1 crosstalk is important for proper ESC gene expression. The manuscript is well written, well organized and clearly focuses on an important event in stem cell biology. An important conclusion from the data is that some pioneer factors need chromatin remodelers to exert their 'pioneering' activity and maintain pluripotency. However, it seems that the main novelty here is the genome-wide experiments when combined with the control of OCT4 and BRG1 expression in ESCs. Previous studies (cited in the manuscript) already show some aspects that are also presented in this study .We thank the reviewer for their kind words regarding our manuscript and we agree that our study addresses an important event in stem cell biology. As pointed out by the reviewer, several previous studies have touched on aspects of OCT4 and BRG1 function related to our study. However, in many cases these studies have focused on a small number of genes, have been performed in different pluripotent cell types , and often come to differing conclusions making it difficult to grasp the scale, validity, and relevance of these often isolated observations. For example, it has remained elusive how OCT4 engages with the thousands of distal regulatory sites it binds in the ESC genome and how this is related to its proposed role as a pioneer transcription factor. In particular, previous studies examining the relationship between OCT4 binding and chromatin accessibility/nucleosome positioning have not addressed how OCT4 transitions inaccessible chromatin into functionally active and accessible regulatory elements required for gene regulation (see response to Point 5 for details on these studies). Furthermore, the link between BRG1 and OCT4 has largely stemmed from simple biochemical immunoprecipitation analysis , with the functional relevance of this proposed interaction remaining unclear.eLife.Here we have taken a systematic and genome-wide approach to address how OCT4 engages with the genome in mouse ESCs and demonstrate a defined role for BRG1 in shaping transcription factor binding, chromatin accessibility and gene regulatory function. We believe this encompasses an important step forward for understanding OCT4 function in ESCs. Furthermore, we provide new evidence that OCT4s function as a pioneer factor relies on an assisted loading type mechanism that utilizes the function of chromatin remodelling machines, which to our knowledge has not been previously proposed. We believe these important and novel discoveries will be well received and highly cited by the readership of 1) The authors limit their study to the first 24 Hrs. of Brg1 knockdown, which restricts the ability to observe its actual effects on the maintenance of pluripotency. The authors make the conclusion that Brg1-bound enhancers regulate the pluripotency network. The authors should explore how Brg1 knock down affects pluripotency over the several days necessary to observe or not maintenance of ESCs or evidence of differentiation.A central goal of our study was to understand in molecular detail, at the genome-scale, how OCT4 interacts with and functions on the genome to orchestrate the pluripotent ESC state, something that in our view is still poorly understood (which we now highlight in the subsection “BRG1 is required to create accessible chromatin at OCT4 target sites in ESCs”). In doing so, we have purposely limited our analysis to time points following genetic ablation of OCT4 and BRG1 that are sufficient to allow protein loss, but where cells still exist in a state that is morphologically and biochemically ESC-like . Our rat2) The authors use heatmaps throughout the manuscript. In the cases listed below, heatmaps are insufficient to support the claims raised by the authors. A quantitative analysis including statistics is needed.eLife to appreciate the striking reductions and trends in our ATAC-seq, ChIP-seq and RNA-seq datasets. Importantly, in parallel to these heatmap visualisations, we performed robust statistical analyses on all of the datasets presented in heatmaps in the original version of the manuscript. For example, all genomic experiments were performed in biological triplicate and differential transcription factor binding or accessibility was identified and statistically validated using the DiffBind package , with significance being achieved when a FDR < 0.05 and a fold change > 1.5-fold was realized . We acknowledge that these statistical measures were not highlighted sufficiently in the original submission, and have therefore included additional analyses and figures throughout the revised manuscript that provide a more detailed and quantitative interrogation of the genomic data. While these analyses have undoubtedly increased the statistical robustness of our study, we would like to stress that our interpretation of the data remains essentially unchanged from the original manuscript. Precise details of these additional analyses and figures are included below.We agree that heatmaps by themselves do not provide any quantitative or statistical support. However, they do provide an intuitive way for non-specialists who will read the manuscript in A) -43). Secondly, we identified genes that are significantly down-regulated in the OCT4-depleted ESCs . We then compared the OCT4 target sites for which the nearest gene shows such a significant reduction in expression (OCT4-dependent expression) with the OCT4 targets sites that show significant reductions in accessibility (OCT4-dependent accessibility). Through this analysis, we observe that the majority of significant reductions in gene expression are associated with significant reductions in chromatin accessibility at nearby OCT4 target sites (-147). Together these new statistical and quantitative analysis support our previous conclusions that OCT4 plays an essential role in determining regulatory element accessibility and function, which is important for the normal expression of nearby genes associated with the pluripotency transcriptional network.We have now carried out additional quantitative and statistical analysis to reinforce the observation that reductions in chromatin accessibility and transcription factor binding associated with the loss of OCT4 is linked to reduced expression of nearby genes see . This reet sites . As expeet sites . This niThe description and discussion of these new analyses is included in the last paragraph of the subsection “OCT4 supports transcription factor binding at distal regulatory elements to regulate pluripotency-associated genes”.B) To identify and quantify sites that have significant reductions in OCT4 binding following BRG1 removal we used the DiffBind package with a significant change in OCT4 binding being achieved when a FDR < 0.05 and a fold change > 1.5-fold was observed from biological triplicate experiments. This statistical analysis revealed that 60% of OCT4 bound distal sites showed significant reductions in OCT4 binding following removal of BRG1. In addition to stating this value in text, we have now also included a pie chart for this and similar statistical analysis of SOX2 and NANOG binding in However, we were conscious in reflection that one issue associated with using this binary segregation of affected/unaffected sites is that it relies on a somewhat arbitrary user defined thresholds (FDR < 0.05 and a fold change > 1.5-fold) which can lead to a range of affected sites that fall just below the fold change threshold being considered unaffected . In subsequent visualisation and analysis this can cloud one’s appreciation of the features associated with BRG1 dependency. To overcome this limitation and focus on the clearest BRG1 dependencies, in the revised manuscript we have stratified OCT4 targets into the 20% of sites that were least dependent on BRG1 (BRG1-independent) and compared these to the 20% of sites that were most dependent on BRG1 (BRG1-dependent). This now allows the reader to more clearly appreciate how BRG1 contributes to a series of features associated with OCT4 target sites in To clarify this for the reader we have highlighted the distinction between these two strategies in the figure legends and in the subsection “BRG1 supports transcription factor binding at distal gene regulatory elements”.C) We have now further explored the relationship between altered transcription factor binding and gene expression in the BRG1-depleted ESCs. In new analyses included in D) In 2 is not presented so there is no way to evaluate the authors' statement.3) Subsection “BRG1 is required to create accessible chromatin at OCT4 target sites in ESCs”. fl/fl and OCT4cond cells. This illustrates that chromatin accessibility at 76% of OCT4 targets sites relies on BRG1 . However, this is actually what we expected based on the heatmap visualisation, as it is clear that not all ATAC-seq signal at OCT4 target sites results directly from BRG1 activity. We suspect that this is due in part to transcription factor binding which is retained at some sites following BRG1 depletion  Subsection “The pioneer factor OCT4 binds distal regulatory sites in pluripotent cells that would otherwise be inaccessible”, first paragraph. The authors need to provide more detail in the text and figure legends regarding the called Oct4 peaks  as well As suggested by the reviewer we have now clarified the chromatin signature used to annotate OCT4 binding sites as either distal regulatory element or promoters. This description is now included in both the main text and in the figure legend . Briefly, this was achieved by examining the relative enrichment of H3K4me3  and H3K4me1  at OCT4 bound sites as previously described . To illustrate the effectiveness of these segregations, we have carried out additional metaplot visualisation of H3K4 modifications and calculated the distance to nearest annotated TSS for each class of site .Also, does motif analyses show an Oct4 motif enriched at these binding sites? What percentage of sites contain the motif, how strong is the enrichment of the motif?Our high confidence OCT4 interval set was generated by identifying OCT4 peaks (using DANPOS2) that lose OCT4 ChIP-seq signal after removal of OCT4. To our knowledge, no other previous OCT4 ChIP-seq analysis has included this important control that ensures that the OCT4 ChIP-seq signal is due to the protein and not due to other non-specific ChIP-seq signal. As suggested by the reviewer we have further characterised this interval set by performing motif enrichment analysis  at our 15,920 OCT4 peaks using the MEME suite of tools. As expected, this analysis revealed a striking enrichment of the OCT4:SOX2 dual motif sequence, in addition to significant enrichments for the highly similar OCT2 motif and two de novo OCT4 motifs. These motifs are enriched at the peak centre , and 60% of peaks contained at least one of these motifs. This is comparable with other studies for transcription factor motifs and binding . We have included these new analyses in a new supplementary figure  and desc5) Subsection “The pioneer factor OCT4 binds distal regulatory sites in pluripotent cells that would otherwise be inaccessible”. The reference to "modest chromatin alterations" is ambiguous; a more specific description or quantitative comparison between these data and the cited papers is needed.In this line of the text we are referring to previous studies that have proposed a role for OCT4 in promoting alterations to chromatin but which were limited in their scope, technical approach, rigor or interpretation. For example, Shakya et al. (2015) performed histone H3 ChIP-qPCR at a single OCT4 target (the POU5F1 enhancer) and observed depletion of H3 signal after OCT4 over-expression in differentiated cells. You et al. (2011) observed increased nucleosome occupancy (as measured by M.CviPI digestion efficiency) at two OCT4 targets after OCT4 siRNA in NCCIT embryonic carcinoma cells. In both of these cases, the effects on chromatin accessibility/nucleosome occupancy are appreciable and significant, but these studies are limited in that they examine individual loci and are not directly comparable to our study in ESCs.Alternatively, two studies have generated genome-wide datasets examining the role of OCT4 in determining chromatin accessibility using different approaches. Chen et al. (2014) perform restriction endonuclease digestion (RED)-seq 5 days after knocking down OCT4 with siRNA in mouse ESCs and observed reduced chromatin accessibility at regulatory elements genome-wide. However, ESCs cells depleted of OCT4 for extended periods of time (i.e. 5 days) differentiate , meaning that these observations are of little relevance to understanding the precise contribution of OCT4 to maintenance of the pluripotent state that is the focus of our study. Furthermore, these experiments were only performed in biological singlicate, meaning that interpretations drawn from any comparison with this data would lack experimental, statistical, and quantitative rigor. Lu et al. (2016) perform DNase-seq in the early mouse embryo after ablating OCT4 expression with siRNA at the 8-cell stage. However, as described in the original manuscript, knockdown of OCT4 results in reduced DNase-seq signal at both OCT4-bound and OCT4-negative regulatory elements. We therefore find it difficult to interpret whether the observed effects are driven by OCT4 or are secondary effects associated with alterations in cellular trajectory that would result from removal of OCT4 at this early developmental stage. For these reasons, we find it difficult to make meaningful and quantitative comparisons of these studies with our data. In fact, these studies exemplify a lack of clarity in the current understanding of OCT4s function in shaping chromatin structure and accessibility in the pluripotent state, which was one of the central motivations for our detailed and systematic genome-wide study in ESCs.Nevertheless, in order to better reflect the conclusions drawn by these previous studies, we have rephrased the sentence originally highlighted by the reviewer with the following text, although do not describe each in detail each for the sake of brevity.“This is supported by previous studies describing a role for OCT4 in maintaining nucleosome-depleted regions and/or chromatin accessibility at individual loci in pluripotent cells  or genome-wide .”6) Subsection “BRG1 is required to create accessible chromatin at OCT4 target sites in ESCs”. In fl/fl ESCs before and after tamoxifen treatment and have visualised these data by heat mapping as suggested by the reviewer. We have also included genomic snapshots for this BRG1 ChIP-seq data in fl/fl ESCs with tamoxifen results in dramatic reduction in BRG1 ChIP-seq signal at OCT4 target sites. Furthermore, these OCT4 target sites are ranked in the same order as in We have now performed BRG1 ChIP-seq in the Brg12=0.33) and not 'very good' or 'high' as stated in the main text and the legend, respectively. This should be more carefully phrased. Also, the authors should reconcile the reason for the weak correlation.7) Subsection “OCT4 establishes chromatin accessibility by recruiting BRG1 to chromatin”. 2), which represents the goodness of fit between the two variables and Pearson correlation coefficient (cor) to quantify the degree to which these two variables are related. Therefore, the Pearson correlation is 0.57 which we believe represents a good correlation between these two variables given that we are comparing two different genomic assays (ChIP-seq and ATAC-seq) that have very different inherent features . Furthermore, we have now performed ChIP-seq for an additional BAF subunit (SS18), and this has revealed similar trend but with an appreciably higher correlation (cor = 0.80). We suspect this may reflect the better signal-to-noise of the SS18 ChIP-seq compared to the BRG1 ChIP-seq. We have included this new analysis in the manuscript (In nuscript  and have8) Subsection “BRG1 supports transcription factor binding at distal gene regulatory elements”. In cond ESCs, which is described and characterised in Throughout the manuscript we have used a single OCT4 peak set based on the OCT4 ChIP-seq performed in the OCT49) Subsections “BRG1 supports transcription factor binding at distal gene regulatory elements” and “Gene expression defects in the absence of BRG1 are linked to altered transcription factor binding”. What is meant by "functionally mature" TF binding events?We have used this term to describe the process by which the initial sampling or binding of OCT4 to inaccessible chromatin is ultimately resolved into the formation of distal regulatory elements with a full complement of transcription factor binding and gene regulatory capacity. Importantly our work demonstrates a clear role for BRG1/BAF in the functional maturation of OCT4 target sites in ESCs. We have clarified this point at various points throughout the manuscript to better reflect this .10) Subsection “BRG1-dependency reveals distinct modes of OCT4 function at distal regulatory elements during reprogramming and development”, last paragraph. In In Reviewer #3:[…] 1) From the presented experiments it seems that the BRG1/BAF complex is as much as a "pioneer" factor as is OCT4. In fact, the Schöler lab has shown  that the BAF complex is binding before OCT4 and Sox2 to the ES specific genes. Thus, the authors need to revise their ideas of OCT4 being a pioneer factor throughout the whole manuscript. #From our reading of the Singhal et al. (2010) study we do not find evidence to support the BAF complex functioning as a pioneer factor at target sites prior to OCT4 and SOX2 binding. In fact, binding of the BAF complex was not examined in this study (by ChIP or otherwise). Instead, what the Singhal et al. study demonstrates is that overexpression of BRG1/BAF with the reprogramming factors OCT4, KLF, and SOX2 (OKS) in mouse embryonic fibroblasts increases the efficiency of reprogramming and iPSC formation (now described in the second paragraph of the Discussion). While this clearly indicates that BRG1 is important for the efficiency of OCT4-driven reprogramming, it does not suggest that BRG1 functions as a pioneer factor. This term is usually reserved to describe sequence-specific transcription factors that are capable of binding inaccessible chromatin to establish new functional regulatory elements and drive gene expression . Based on our observations it seems more plausible that the iPSC reprogramming process is potentiated by BAF through its recruitment to regulatory sites via OCT4 in a temporally coincident manner. Indeed, similar conclusions were arrived at in a recently published study examining BRG1 binding during reprogramming , which we now cite in the subsection “OCT4 establishes chromatin accessibility by recruiting BRG1 to chromatin”. In our view, these observations would exclude BRG1/BAF from being considered as a classical pioneer transcription factor, but instead are consistent with OCT4 recruiting BRG1 to distal regulatory elements as part of an assisted loading mechanism for pioneer transcription factor binding and function as suggested in our original submission.2) The authors should carry out ChIP-seq with a second subunit of the BAF complex to be able to show that the effects they see with BRG1 are indeed due to the BAF ATP dependent remodelling complex(es).As suggested by the reviewer we have now performed ChIP-seq for SS18, which forms part of the BAF remodelling complex in ESCs and other cell types , before and after removal of OCT4. In agreement with our BRG1 ChIP-seq analysis, SS18 binding is largely dependent upon OCT4 at OCT4 bound distal regulatory sites . This pr3) In the OCT4 knock down experiments the authors carry out ATAC-seq, Oct4, Sox2, Nanog and Brg1 ChIP-seqs, however in the BRG1 knock down conditions they carry out ATAC-seq, Oct4, Sox2, and Brg1 ChIP-seqs, but not Nonog ChIP-seq. Why?fl/fl ESCs before and after tamoxifen treatment. The effect on NANOG binding following removal of BRG1 is highly similar to SOX2, with sites relying on BRG1 for OCT4 binding displaying reductions NANOG occupancy and sites that retain OCT4 binding displaying maintenance or even increased in NANOG occupancy . However, OCT4 can bind to its target sites on nucleosomal DNA independently of SOX2 , suggesting that binding with SOX2 may function to stabilise OCT4 binding as opposed to directing its initial binding. Consistent with these in vitro observations, our in vivo experiments suggest that SOX2 or NANOG are not sufficient to recruit BRG1 as SOX2 and/or NANOG are retained at some target sites following depletion of OCT4, yet BRG1 binding is either completely lost or substantially reduced .10.7554/4) To resolve these issues, the authors should carry out time scale experiments to test which factor(s) is leaving the first from the Oct4 occupied sites after Oct4 knock-down.We agree that our study, and previous studies, lack the fine scale temporal resolution necessary to determine the kinetics with which altered chromatin accessibility, transcription factor binding and BRG1 chromatin occupancy occur following OCT4 removal. While we are immensely interested in these important questions, the current genetic ablation systems that are available are relatively slow  and asynchronous amongst the cell population. This presents a major technical barrier towards effectively assigning order of events in the type of kinetic experiments proposed by the reviewer. We envisage that addressing these important questions will require the development of rapid and synchronous protein ablations systems, likely degron based, and this remains an important challenge for future studies.5) The analyses of Oct4 or Brg1 regulated genes in We thank the reviewer for raising this important point that we have now clarified with the support by several new analyses. In the absence of BRG1, our observations demonstrate that normal binding of pluripotency transcription factors OCT4, SOX2 and NANOG was disrupted at OCT4-bound distal regulatory elements, suggesting that this may affect expression of the pluripotency-associated transcriptional network. Somewhat surprisingly, previous work did not support a clear correlation between loss of BRG1 and the activity of OCT4 target genes , despite our observations that BRG1 plays an important role in transcription factor binding at many target sites associated with pluripotency-associated genes. However, we also observed that the effect on OCT4, SOX2 and NANOG binding following BRG1 removal varied in magnitude between individual sites with some sites actually displaying increases in transcription factor binding . We therIs it possible that Oct4 (or BRG1) are not regulating the analysed nearby genes?We used a simple approach based on coupling OCT4-bound distal regulatory elements with the closest TSS in order to examine changes in gene expression with altered chromatin accessibility and transcription factor binding. Therefore, this set of relationships will inevitably not represent the complete set of target genes affected by OCT4 bound distal regulatory elements and will also encompass false positives. However, in the absence of experimentally validated and high resolution genome-wide enhancer-promoter interaction maps we believe this is a reasonable approach that is likely to represent many functionally relevant relationships. Similar approaches have previously been used in genomic studies to broadly examine the function of distal regulatory elements in gene regulation .


Polyglutamine (polyQ) diseases are hereditary neurodegenerative disorders caused by an abnormal expansion of a trinucleotide CAG repeat in the coding region of their respective associated genes. PolyQ diseases mainly display progressive degeneration of the brain and spinal cord. Nine polyQ diseases are known, including Huntington’s disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and six forms of spinocerebellar ataxia (SCA). HD is the best characterized polyQ disease. Many studies have reported that transcriptional dysregulation and post-translational disruptions, which may interact with each other, are central features of polyQ diseases. Post-translational modifications, such as the acetylation of histones, are closely associated with the regulation of the transcriptional activity. A number of groups have studied the interactions between the polyQ proteins and transcription factors. Pharmacological drugs or genetic manipulations aimed at correcting the dysregulation have been confirmed to be effective in the treatment of polyQ diseases in many animal and cellular models. For example, histone deaceylase inhibitors have been demonstrated to have beneficial effects in cases of HD, SBMA, DRPLA, and SCA3. In this review, we describe the transcriptional and post-translational dysregulation in polyQ diseases with special focus on HD, and we summarize and comment on potential treatment approaches targeting disruption of transcription and post-translation processes in these diseases. Nine genetic neurodegenerative diseases are caused by the expansion of CAG repeats in seemingly unrelated genes. Spinal and bulbar muscular atrophy (SBMA) reveals an X-linked pattern of inheritance while others are inherited in an autosomal dominant fashion. Neurodegenerative diseases are a group of devastating disorders presenting as movement and/or cognitive impairment affecting a certain population of neurons within the central nervous system. PolyQ diseases mainly display progressive degeneration of the brain and spine, and in the case of SCA7, the macula and retina. These diseases have a striking genotype–phenotype correlation. The length of the CAG repeat is strongly positively correlated with disease severity and time of disease onset. As these diseases progress, the extensive neurodegeneration results in cell apoptosis.Some groups have emphasized the regulatory roles of polyQ protein and different transcription factors, coactivators, or transcriptional regulators. Post-translational protein modifications can affect transcription and also play a major role in the disease pathogenesis. However, the mechanisms underlying transcriptional and post-transcriptional dysregulation in polyQ diseases remain incomplete. Several studies have concentrated on pharmacological interventions in several animal models of polyQ diseases. However, further studies are needed before these treatments can be used clinically.In this review, we focus on the transcriptional and post-translational dysregulation in polyQ diseases. We present the existing evidence for the regulation of transcription factors and co-activators in polyQ diseases and further discuss the role of post-translational regulation in polyQ pathogenesis. We mainly discuss the regulation of Huntington’s disease (HD), which is the best-known polyQ disease. Finally, we summarize the latest therapeutic strategies for polyQ diseases, based on selective targeting of the transcriptional and post-translational dysfunctions. Challenges and further directions regarding these novel approaches will be discussed in the last part of the review.Huntington’s disease is a fatal hereditary neurodegenerative disorder characterized by progressive motor and cognitive deficits. The pathogenesis is associated with an abnormal expansion in the number of glutamine residues located in the amino (N) terminus of huntingtin (Htt), a very large protein found mainly in the cytoplasm . PolyQ eMany studies have shown that the nuclear effects of mHtt might be associated with its interaction with a number of transcriptional regulators, including the cAMP response element-binding protein (CREB), CREB binding protein (CBP), repressor element 1 (RE1)-silencing transcription factor (REST), and other factors .Table 1).Until now, scientists have failed to find any effective treatments for HD . HoweverFigure 1).cAMP response element-binding protein is a member of the basic leucine zipper family of transcription factors and regulates several neuroprotective processes. CREB is phosphorylated at serine 133 (Ser133), and then recruits with transcriptional co-activators CBP and p300 to activate transcription . SeveralHowever, Drosophila model , is depleted from its normal nuclear location and is present in polyQ aggregates in HD cell models, HD transgenic mice, and the human HD postmortem brain . Overexpla model , whereasla model . Howeverla model . Jiang emHtt inhibits the acetyltransferase activity of p300, p300/CBP-associated factor (P/CAF), as well as CBP . HoweverTransducers of regulated CREB activity (TORCs) expression decreases in a HD cellular model, in the striatum of a transgenic mice model, and in postmortem HD striatal tissue. TORCs overexpression increases CREB mRNA and protein levels, as well as the resistance of striatal cells to 3-NP toxicity. mHtt interferes with the TORC1-CREB interaction to repress BDNF transcription .Inhibiting phosphodiesterases (PDEs), which are enzymes responsible for cAMP/cGMP degradation, is responsible for increasing CREB phosphorylation and thus playing a neuroprotective role in HD. Rolipram, a PDE type 4 (PDE4) inhibitor, increases the levels of activated CREB and BDNF in the striatal spiny neurons of the R6/2 HD mouse model . The potIn conclusion, phosphorylation of CREB was repressed, while overexpression of CBP rescued polyQ-induced toxicity. Inhibiting PDE has been confirmed by several groups to play a therapeutic role in HD.Figure 1).(RE1)-silencing transcription factor, also known as neuron-restrictive silencing factor (NRSF), is a repressor of neuronal gene transcription in non-neuronal lineages that binds to a 21-bp DNA element, RE-1/neuron restrictive silencer element (NRSE), thereby activating its silencing activity. In the presence of wHtt, REST is tethered in the cytoplasm, but REST translocates to the nucleus in the presence of mHtt where it represses transcription of its target genes, including the BDNF gene . REST/NRmiR-124, which is upregulated by REST, is downregulated and then increases BDNF protein levels, playing a neuroprotective role in the striatum and the cortex of the R6/2 HD mouse model, a “truncated” Htt mouse model of HD .Several groups have confirmed that the REST/NRSF-RE1/NRSE system might be acting as a pharmaceutical target. Transfection of REST decoy oligonucleotides in a cellular model of HD restores expression of REST target genes, such as BDNF . OverexpIn brief, REST and its target gene transcription play important roles in the progression of HD. Molecules able to affect REST/NRSF nuclear translocation, DNA binding or, more generally, formation of the REST/NRSF transcriptional complex, may be regarded as potential drug design targets for HD.Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is a transcriptional coactivator that regulates several metabolic processes, including mitochondrial biogenesis and oxidative biogenesis. Reduced full-length PGC-1α expression has been observed in transgenic mouse models of HD and in postmortem brain tissue from HD patients , in muscOverexpression of PPARGC1A, the gene encoding for PGC-1α, rescues HD neurological phenotypes and neurodegeneration . The PPAThe PGC-1α signaling pathway is now increasingly being recognized as an important therapeutic target for HD. Treatment with rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma (PPARγ), could prevent the reduction in PGC-1α and rescue BDNF deficiency. These effects might be related to a beneficial role in rescuing motor function in the HD mouse brain . The PPAOne study revealed that exosomes from adipose-derived stem cells decrease mHtt aggregates in R6/2 HD mice-derived neuronal cells, which might be related to upregulation of PGC-1α and phosphorylated CREB . SimilarIn conclusion, PGC-1α expression decreases in several HD models, while several PPARGC1A SNPs are related to earlier or delayed onset of HD. Several potential treatments associated with preventing the reduction in PGC-1α may be helpful in the intervention of HD.Specificity protein 1 (sp1) contains three zinc-finger motifs and its C-terminal domain interacts with other transcription factors in a synergistic manner to control gene expression in a temporal and spatial manner .Several groups have reported the neuroprotective role of sp1. Dunah and his group demonstrated that co-expression of sp1 and TAFII130 decreases transcriptional dysfunction of the dopamine D2 gene caused by mHtt, as well as protects cultured wild-type and HD transgenic mice striatal cells from mHtt-induced cellular toxicity . Chen-PlDrosophila and mouse models of HD  is a family of DNA-binding proteins that are important transcription factors involved in immune and inflammatory responses, as well as in cell survival and apoptosis. Loss of Htt function and the HD mutation slows the rate of movement of NF-κB from the synapse to the nucleus . The mamThe NF-κB pathway is also associated with neurodegeneration in non-neuronal HD cells. Enhanced activation of RelA is observed in astrocytes of a HD mouse model and in patients with HD . mHtt inA non-coding SNP, rs13102260: G > A, in a NF-κB binding site modulates the binding of NF-κB and reduces transcriptional activity of the Htt gene promoter .The NF-κB pathway inhibitor EVP4593 protects medium spinal neurons in the striatum of YAC128 mice against glutamine toxicity . AnotherIn conclusion, RelA is down-regulated in HD neuronal cells, while enhanced activation of RelA level is observed in HD non-neuronal cells. Inhibiting the NF-κB pathway might also be helpful in the treatment of HD.Many groups have confirmed the role of PTM in the pathogenesis of HD, such as acetylation, phosphorylation, methylation, ubiquitination, and small ubiquitin modifier protein (SUMO) modification , mainly Acetylation has been best characterized, and a large number of groups have focused on the therapeutic role of histone deacetylase (HDAC) inhibitors. Several groups assumed early on that altered histone acetylation might contribute to HD when scientists discovered that histone acetyltransferases (HAT), such as CBP, were recruited into mHtt aggregates, and an HDAC inhibitor increased histone acetylation; thus, improving the phenotype in several animal models of HD .Lysine residues  in the 17 amino acids of the N-terminal Htt are significantly acetylated, which retards fibril formation, promotes globular aggregation, and induces binding to lipids . Acetylain vivo and in vitro models. These models simulate the human HD pathology more closely. However, few clinical outcomes have been found, possibly because of the slow progression of HD.Different types of HDAC inhibitors can be used to increase acetylation, which is beneficial in the treatment of several human diseases . SeveralRGFP966, a benzamide-type HDAC inhibitor, shows direct beneficial effects on HD disease phenotypes in N171-82Q transgenic mice . In the Overexpression of sirtuin1 (Sirt1), a non-classical type of HDAC inhibitor, improves motor function, reduces brain atrophy, and attenuates mHtt-mediated metabolic abnormalities in both fragmented and full-length HD mouse models . The SirDrosophila and R6/2 mice models, as well as in HEK293 cells  and prevent striatal neurodegeneration caused by mHtt . The levArginine methylation, an important part of PTM, is very important in the pathogenesis of HD. The compensation of protein arginine methyltransferase 5, an enzyme mediating arginine demethylation, reverses the toxic effect of mHtt in HD pathogenesis .+1 protein, a frameshift mutation form of ubiquitin, accumulates in the brain of postmortem HD patients. It was found that UBB+1 induces aggregate formation of expanded polyQ in SH-SY-5Y neuroblastoma cells  residues in ubiquitin and alters the levels of toxic proteins. Ubiquitination involves the concerted actions of three enzymes: ubiquitin-activating (E1), ubiquitin-conjugating (E2), and a number of ubiquitin-ligating (E3) enzymes. mHtt aggregation has been reported in the cortex and striatum of the postmortem HD brain, consistent with the expression of ubiquitin . Ubiquitma cells . Althougproteins . The mHtproteins . Absenceproteins . WWP1, aproteins . E2-25K proteins . A deficproteins . The levproteins . One stuproteins .Some studies have focused on the relationship between SUMO modification and the development of HD. SUMO modification at K6 and K9 in mHtt is reduced in mutant HD cell models . OverexpSCA1 is an autosomal dominant progressive neurodegenerative disease characterized by ataxia, ophthalmoparesis, and a variable degree of motor weakness. Symptoms usually begin during the third or fourth decade of life and the disease gradually worsens, often resulting in complete disability and death 10–20 years after onset. Loss of Purkinje neurons within the cerebellum is found in patients with SCA1, which might be important in the development of the disease .SCA1 is caused by a CAG expansion encoding a polyQ expansion in the protein ataxin-1. Sp1 is an important transcription factor in HD that interacts with ataxin-1 and the interaction decreases when Atxn1 contains an expanded polyQ tract .PolyQ expansion of mutant ataxin-1 increases the interaction with poly-Q tract-binding protein-1 (PQBP-1) which, in turn, stimulates PQBP-1 binding to RNA polymerase II (Pol II) and reduces Pol II phosphorylation and transcription . TransgeAtaxin-1 binds HDAC leading to strong transcriptional repression. The complete loss of HDAC results in progressive neurodegeneration in Purkinje cells, the most relevant cell type in SCA1 . UbiquitSCA2 is characterized by slowly progressive cerebellar ataxia and dysarthria with ocular findings (such as nystagmus and slow saccades). Excessive CAG repeats in the exon 1 of the ATXN2 gene in patients with SCA2 cause expansion of a polyQ domain in the ataxin-2 protein . Ataxin-Transcription of ataxin-2 can be activated by the interaction between ataxin-2 and the krüppel-associated box-containing zinc-finger transcriptional regulator, which participates in transcriptional repression of RNA polymerase as well as binding and splicing of RNA .Drosophila with SCA3  suppresses polyQ toxicity by alleviating the cellular level of CBP and improving histone acetylation in a Drosophila model of SCA3 , a CACNA1A gene, influences P/Q-type channel function and is associated with the dominantly inherited conditions of migraine, epilepsy, and episodic and progressive ataxia called SCA6 . Few stu-/- mice .SCA7 is a neurodegenerative disease primarily affecting the brainstem, retina, and Purkinje cells of the cerebellum . The disCBP and p53 play roles in the pathogenesis of SCA7. Similar to ataxin-3, mutant ataxin-7 regulates activation of p53. in vivo and in vitro  .The HDAC inhibitor sodium butyrate improves motor impairment and ameliorates the neurological phenotype in the SBMA mouse model . Sirt1, Arginine methylation also takes part in the development of SBMA. Protein arginine methyltransferase 6 (PRMT6) acts as a co-activator of the AR and the interactions between PRMT6 and AR are significantly enhanced in cell and fly models of SBMA. The inhibitor of PRMT6 can reduce toxicity of the mutant AR .SUMOylation plays a role in the pathogenesis of SBMA. In both cell and mouse models of SBMA, decreasing SUMOylation restores mutant AR function and dissociates the pathogenic role of AR dysfunction .Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant progressive neurodegenerative disease that results in intellectual deterioration and various motor deficits, including ataxia, choreothetosis and myoclonus. DRPLA is caused by an abnormal expansion of CAG repeats within the atrophin-1 protein . Sodium Transcriptional disruption and post-translational modifications have been well-established as important pathological processes in polyglutamine diseases. Transcriptional disruption may interact with post-translational modifications. For example, the HAT CBP also acts as a CREB-binding protein and can take part in transcriptional dysfunction in HD, whereas post-translational modification affects transcriptional regulation, mutant ataxin-3 and mutant ataxin-7 could cause phosphorylation and ubiquitination of p53, resulting in p53 transcriptional regulation . In addiAs dysfunction of transcription and post-translational modifications has been found in polyQ diseases during the last decades, huge efforts have been made by researchers. Beneficial effects have been observed in various preclinical polyQ disease models, through corrections in transcriptional dysregulation and post-translational modifications. PDE inhibitors and HDAC inhibitors are the most widely studied agents. A large number of studies have confirmed their therapeutic value in polyQ diseases, while the precise mechanism of action has yet to be determined. Safety and efficacy of HDAC inhibitors and PDE inhibitors are currently being tested. However, the results are not as expected. Vast problems need to be solved. The specificity of compounds altering transcription remains an important concern. Numerous cell-based and whole-animal studies have shown that the compounds have diverse biological activities that impact physiological and developmental functions. This problem has recently been reviewed for HDAC inhibitors . The difCX contributed to drafting and revising the manuscript. SZ contributed to drafting and modifying the table. XD and SM contributed to drafting and modifying the figure. SC contributed to drafting and revising the manuscript. All of the authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Manduca sexta, and a 140-bp region in the moricin promoter contains binding sites for additional transcription factors. In this study, we identified three forkhead (Fkh)-binding sites in the 140-bp region of the moricin promoter and several Fkh-binding sites in the lysozyme promoter, and demonstrated that Fkh-binding sites are required for activation of both moricin and lysozyme promoters by Fkh factors. In addition, we found that Fkh mRNA was undetectable in Drosophila S2 cells, and M. sexta Fkh (MsFkh) interacted with Relish-Rel-homology domain (RHD) but not with Dorsal-RHD. Dual luciferase assays with moricin mutant promoters showed that co-expression of MsFkh with Relish-RHD did not have an additive effect on the activity of moricin promoter, suggesting that MsFkh and Relish regulate moricin activation independently. Our results suggest that insect AMPs can be activated by Fkh factors under non-infectious conditions, which may be important for protection of insects from microbial infection during molting and metamorphosis.Antimicrobial peptides (AMPs) play an important role in defense against microbial infections in insects. Expression of AMPs is regulated mainly by NF-κB factors Dorsal, Dif and Relish. Our previous study showed that both NF-κB and GATA-1 factors are required for activation of moricin promoter in the tobacco hornworm,  Synthesis of small cationic antimicrobial peptides (AMPs) in the fat body of insects is an important defense mechanism of humoral immune responses against microbial infection9. Expression of insect AMPs is regulated mainly by the evolutionarily conserved Toll and immune deficiency (IMD) pathways13. The Toll pathway defends against Gram-positive bacteria, fungi and viruses15, whereas the IMD pathway acts against Gram-negative bacteria16. Both the Toll and IMD pathways activate the Rel/NF-κB family of transcription factors, including Dorsal, Dif 17 and Relish18. These NF-κB factors contain N-terminal Rel-homology domain (RHD) that is required for DNA binding and dimerization19.In insects, innate immunity is the first line of defense against pathogens and it is composed of both humoral and cellular immune responsesManduca sexta, we found that both NF-κB and GATA-1 binding sites are crucial for activation of moricin promoter, and we also identified a 140-bp region (between −240 and −100 bp) in the moricin promoter, designated as moricin promoter activating element (MPAE), which contains binding sites for additional transcription factors required for activation of moricin promoter20. The purpose of this study is to identify the additional transcription factor(s) that can bind to the MPAE region, investigate whether the new factor(s) can also activate other AMP genes, and whether the new factor(s) acts independently or cooperatively with NF-κB factors. Sequence analysis of the 140-bp MPAE region showed a binding site for silk gland factor-1 (SGF-1), a transcription factor in the silk worm Bombyx mori that is homologous to Drosophila melanogaster forkhead (Fkh). SGF-1 can bind in vitro to a cis-element located in the promoter region of sericin-1, which encodes a silk protein in the middle silk gland (MSG) cells23. Further analysis showed that there are three Fkh-binding sites in the 140-bp MPAE region  superfamily proteins24. Members of the Fox family proteins consist of a conserved long DNA binding domain connected to a pair of loops or “wings” via a small β-sheet25. The long DNA binding domain, also known as winged-helix or forkhead domain, is about 90 residues and composed of three α-helices with a helix–turn–helix core, thus, the Fox family proteins are also known as winged helix transcription factors26. Fox proteins have been identified in eukaryotic organisms from yeast to human, and they play important roles in various biological processes, including cellular differentiation, development, metabolism, insulin signaling, immune regulation, cancer development, and aging33. Forkhead gene A (FoxA) was first discovered in D. melanogaster since mutation in this gene resulted in fork-headed structure in the embryos with defects in the anterior and posterior gut formation34. Later, cDNA encoding hepatocyte nuclear factor 3α (HNF3α), also known as FoxA1, was cloned in rat35. The central 90-residue DNA-binding domain (forkhead domain) of D. melanogaster FoxA and human FoxA1 shows shockingly high (>85%) identity36. Since the discovery of Drosophila FoxA, hundreds of Fox genes have been identified in various species. There are at least 4 Fox genes in yeast, 16 in D. melanogaster, 15 in Caenorhabditis elegans, 44 in mice, and 50 in humans39. The genome of Aedes aegypti contains eighteen loci that encode putative Fox factors and six of them are involved in reproduction40. Based on phylogenetic analysis, metazoan Fox family is divided into 19 subfamilies (FoxA-S)24. Human Fox subfamilies are classified into two classes based on the sequence homology within and beyond the forkhead domain: Fox A-G, I-L and Q are class 1, Fox H and M-P are class 2 Fox proteins24.Forkhead transcription factors belong to the Fox  is involved in early patterning in embryo41, Biniou (FoxF) plays a role in visceral mesoderm42, sloppy paired 1 & 2 (FoxG) are involved in early embryo segmentation44, domina (FoxN) is involved in vitality and fertility45, and FoxO regulates the insulin signaling in the brain and fat body46. Drosophila FoxO is also involved in innate immunity and expression of AMPs48. Forkhead (Fkh) is the founding member of the FoxO family and it is activated upon TOR inhibition by rapamycin48. However, functions of Fox transcription factors in regulation of AMPs in other insect species have yet to be reported.In M. sexta moricin and lysozyme genes, cloned Fkh genes from both M. sexta and D. melanogaster, and demonstrated activation of AMP gene promoters by Fkh factors. We also found that Fkh mRNA was undetectable in Drosophila S2 cells, which may account for low activity of some AMP promoters in S2 cells. In addition, co-expression study showed that M. sexta Fkh (MsFkh) interacted with Relish (Rel2)-RHD, but did not show any interaction with Dorsal-RHD. However, co-expression of MsFkh and MsRelish-RHD did not have an additive effect on the activity of moricin promoter compared to expression of MsFkh or MsRelish-RHD alone, suggesting that MsFkh and MsRelish regulate AMP gene expression independently. Activation of AMPs by Fkh factors under non-infectious conditions is particularly important for insects during molting and metamorphosis to protect them from microbial infections.In this study, we identified Fkh-binding sites in the promoters of M. sexta moricin, which was responsible for activation of moricin and D. melanogaster drosomycin promoters in S. frugiperda Sf9 cells20. To identify nuclear factor(s) in Sf9 cells that can bind to the MPAE region, we divided the 140-bp MPAE region into three fragments (MPAE-1 to 3)  in the three MPAE fragments, and found that there is an SGF-1 binding site in MPAE-3 . Fkh is a member of the FoxO family proteins and FoxO binds to the consensus sequence of (T/C)(G/A)AAACAA49. We found an ATAAACA sequence in both MPAE-2 and MPAE-3, and an ATAAAGA sequence in MPAE-1  Fig. , and perE-3 Fig. . SGF-1 iE-1 Fig. . Thus, wATAAACA sequence, competed with binding of the labeled MPAE-3 to nuclear proteins of Sf9 cells and the competition by MPAE-3b was dose-dependent  with 10-bp overlapping region (MPAE-3a to 3c) Fig.  for compM. sexta Fkh (MsFkh), we first cloned MsFkh cDNA and analyzed tissue distribution and induced expression of MsFkh mRNA. Real-time PCR results showed that MsFkh mRNA was highly expressed in the hemocytes of the fifth- instar M. sexta naïve larvae compared to fat body, midgut and other tissues , but was not induced in the midgut by any of the microorganisms tested  is not expressed in S2 cells (Flybase), we performed real-time PCR to determined transcripts of several Drosophila Fox genes in S2 cells, including DmFkh, FoxK long and short isoforms, jumeau (jumu) and dFoxO. The results showed that mRNAs of FoxK, jumu and dFoxO, but not DmFkh, were detected in S2 cells, with higher transcript level of FoxK and jumu than dFoxO  in S2 cells. To test whether overexpression of DmFkh in S2 cells can activate expression of AMP genes, DmFkh was also cloned.Since MPAE did not bind to nuclear proteins in S2 cells Fig. , and DrooxO Fig. , confirmM. sexta moricin and lysozyme promoters by forkhead factors, MsFkh and DmFkh were over-expressed in S2 cells, and dual luciferase assays were performed with several truncated moricin and lysozyme promoters. MsFkh is 355 residues long with a theoretical molecular weight of 39.5 kDa and pI of 9, while DmFkh (Forkhead isoform A) is 510 residues with molecular weight of 54.3 kDa and pI of 8.7. The full length MsFkh shows 96% identity in amino acid sequence to B. mori SGF-1 (Genbank accession no. NP_001037329.1), 95% identity to Helicoverpa armigera (Genbank accession no. AAW56613.1) and Spodoptera exigua (Genbank accession no. ACA30303.1) forkhead domain transcription factors, but only 42% identity to the full length DmFkh. However, the forkhead domains of MsFkh and DmFkh proteins share 98% identity. Dual luciferase results showed that both MsFkh and DmFkh activated M. sexta moricin, lysozyme, defensin-1, defensin-3 and attacin-2 promoters, but did not activate M. sexta cecropin, attacin-1, or defensin-2 promoter  was activated by Fkh factors to a similarly high level as the 242-bp truncated moricin promoter (Mor-242), but further truncation of the 242-bp moricin promoter significantly decreased the activity of the truncated promoters   Fig. , suggest80) Fig. , indicatM. sexta moricin promoter and the 345-bp region of lysozyme promoter that are responsible for activation by Fkh factors, we first analyzed the Fkh-binding sites in the 140-bp MPAE and the 345-bp region of lysozyme promoter. Three and four Fkh-binding sites with the core sequence of AAACA were predicted within the 140 bp MPAE of moricin and 345 bp of lysozyme promoters, respectively  transcription factor has been reported as a negative regulator of Drosophila IMD pathway downstream of, or parallel to Relish57; Dorsal interacting protein 3 (Dip3) can bind to the RHD of Dorsal and Relish via its BESS domain, and it functions in both dorsoventral patterning and immune response58. In Drosophila, it has been reported that activation of AMPs can be achieved by the transcription factor FoxO independent of the immune signaling pathways47, and induction of two AMPs, diptericin and metchnikowin, after downregulation of TOR by rapamycin is regulated by the transcription factor forkhead (Fkh)48. FoxO is an important regulator of stress, metabolism and aging62, and a key transcription factor in the insulin signaling pathway64; whereas Fkh is the founding member of the FoxO family. However, very little is known about regulation of AMPs in other insect species by the FoxO family transcription factors.Synthesis of AMPs is a major defense mechanism against infection in insects52. For example, moricin and gloverin have been identified only in the lepidopteran insects. We have previously shown that M. sexta moricin is regulated by NF-κB factor Relish and GATA-1 factor65, and it can also be activated by unidentified nuclear factor(s) that bind to the MPAE region of moricin promoter20. In this study, we identified three Fkh-binding sites in the MPAE region and demonstrated that Fkh-binding sites 2 and 3 played an equally important role in activation of moricin promoter by Fkh factor. We also identified four Fkh-binding sites in M. sexta lysozyme promoter and all four Fkh-binding sites are important for activation of lysozyme promoter by Fkh factor. Since the consensus sequence for FoxO is about 8 bp [(T/C)(G/A)AAACAA]49 with a core sequence of AAACA, there are many predicted/potential Fkh-binding sites in promoters. Thus, we used truncated promoters first to narrow the length of promoters and then focused on potential active Fkh-binding sites in the moricin and lysozyme promoters with the core sequence of AAACA, as the core sequence may be crucial for binding of Fkh factor. Among the three and four Fkh-binding sites in the Mor-242 and Lyz-345 truncated promoters, all six Fkh-binding sites but the binding site 1 in the Mor-242 contains the AAACA core sequence  did not decrease the activity of Mor-242 promoter as significantly as did mutation of Fkh-binding site 2 or 3, suggesting that the core sequence of AAACA is crucial for activation of genes by Fkh factor. In addition to moricin and lysozyme promoters, Fkh factor also activated M. sexta defensin-1, defensin-3 and attacin-2 promoters. To our knowledge, this is the first report about activation of AMPs by Fkh factors in a lepidopteran insect. Activation of AMPs by Fkh factor under non-infectious conditions may be important for insects during molting and metamorphosis, since insects at these particular developmental stages are vulnerable to infection, and induced expression of AMPs by Fkh factor may protect insects from microbial infection. We observed that DmFkh stimulated the activity of some AMP promoters to a higher level than MsFkh did in S2 cells. This may be because MsFkh is not completely compatible in Drosophila S2 cells or the longer DmFkh (510 residues) may contain some other domains/motifs that can help Fkh to activate AMP gene promoters.Insects can synthesize a variety of AMPs, some AMPs are common to most insects, whereas some other AMPs are found only in certain insect speciesce Figs.  and 5B. M. sexta moricin, we previously found that both NF-κB and GATA-1 factors are required for activation of moricin promoter20. We also found that M. sexta Dorsal can interact with Relish (Rel2), and Dorsal/Relish heterodimers serve as negative regulators to prevent over-activation of M. sexta AMP genes65. We found that MsFkh interacted with MsRel2-RHD. In the Mor-242 promoter, which contains both NF-κB and Fkh binding sites, overexpression of MsFkh factor alone activated the promoter activity to a significantly higher level compared to overexpression of MsRel2-RHD alone, suggesting that Fkh factor plays an important role in activation of moricin under non-infectious conditions. In the mutant Mor-242 promoters in which the Fkh-binding sites were mutated, co-expression of MsFkh and MsRel2-RHD activated the activity of Fkh-binding site mutant promoters to a similar high level as that activated by MsRel2-RHD alone, indicating that MsFkh and MsRel2 regulate moricin activation independently. This result also suggests that although MsFkh can interact with MsRelish to form heterodimers, homodimers of MsRelish may be predominant. This may be because the distance between Fkh and NF-κB binding sites in the moricin promoter , jumu and dFoxO were detected. Nuclear proteins from S2 cells did not bind to MPAE region, further supporting that DmFkh is not expressed (or was expressed at an undetectable level) in S2 cells. This information is important when performing promoter reporter assays for Fkh factor in S2 cells, as stress conditions may not activate the promoters in S2 cells due to lack of endogenous Fkh factor. In future, we will investigate activation of AMPs under non-infectious conditions via Fkh factor and maybe other members of the FoxO family and whether enhanced expression of AMPs by FoxO family members can protect insects from microbial infection during molting and/or metamorphosis.We also confirmed that M. sexta eggs from Carolina Biological Supplies  and reared larvae to fifth-instar on an artificial diet at 25 °C66 for all the experiments. D. melanogaster Schneider S2 cells were obtained from American Type Culture Collection (ATCC), and Spodoptera frugiperda Sf9 cells were from Invitrogen . Cells were maintained at 27 °C in Insect Cell Culture Media  supplemented with 1% penicillin-streptomycin solution  and 10% heat-inactivated fetal bovine serum .We purchased 8 cells) and homogenized in 500 μl of 1 × Cytoplasmic Lysis Buffer with 27-gauge needle and centrifuged at 8,000 × g for 20 min at 4 °C. The supernatants containing the cytosolic proteins were transferred to fresh tubes and stored at −80 °C for later use, whereas the pellets were resuspended in 100 μl of ice-cold Nuclear Extraction Buffer containing 0.5 mM DTT and 1/1000 Protease Inhibitor Cocktail. The nuclei were disrupted using 27-gauge needle, the mixture was incubated at 4 °C for 60 min with gentle agitation, and then centrifuged at 16,000 × g for 5 min at 4 °C. These supernatants containing nuclear proteins were removed to fresh tubes and stored at −80 °C for later use.Cytosolic and nuclear proteins were isolated from S2 and Sf9 cells using the Nuclear Extraction Kit  following the manufacturer’s instructions. Briefly, S2 and Sf9 cells with 70–80% confluency were collected  following the manufacturer’s instructions. Genomic DNA fragments from the 140-bp MPAE region of moricin promoter  were synthesized with or without covalently linked biotins by Integrated DNA Technologies (IDT) . EMSA was performed in 20 μl reactions containing 2 μl of biotinylated DNA (20 fmole) with 3 μg of cytosolic or nuclear proteins from S2 or Sf9 cells, in the absence or presence of unlabeled DNAs . The mixtures were incubated at room temperature for 30 min, separated on 6% polyacrylamide gel  pre-run in 0.5 × TBE at 100 V for 60 min. The gels were then transferred to nylon membrane pre-soaked in 0.5 × TBE at 380 mA for 30 min in ice-cold 0.5 × TBE, and the membranes were crosslinked for 1 min using a UV-light crosslinking instrument. The biotinylated DNA was detected by Streptavidin-Horseradish Peroxidase conjugated anti-mouse antibody  by chemiluminescence using ECL Chemiluminescence Detection Kit , and the membranes were exposed to films and scanned by Typhoon FLA7000 .Electrophoretic mobility shift assay (EMSA) was performed using the LightShiftD. melanogaster forkhead (transcript variant A) (DmFkh) in the database (Genbank accession numbers: J03177.1 and NM_079818.3) with identical open reading frame (ORF) of 1530 bp, which encodes 510 amino acid of DmFkh. To identify M. sexta forkhead, we used the ORF of DmFkh to blast the M. sexta database (http://agripestbase.org/manduca/?q=blast) and obtained one sequence , which is significantly similar to DmFkh . This M. sexta cDNA sequence contains an ORF of 1065 bp, encoding a protein of 355 amino acids with a forkhead domain. We named this protein M. sexta Fkh (MsFkh).There are two cDNA sequences for MsFkh mRNA, day 2 fifth-instar M. sexta naïve larvae were dissected. Hemocytes, fat body, midgut, epidermis and testis were collected and washed 3 times in anti-coagulant (AC) saline . Total RNAs were extracted from these tissues with TRIzol® Reagent  and cDNAs were prepared from total RNAs (1 μg for each sample) using moloney murine leukemia virus (M-MLV) reverse transcriptase  with an anchor-oligo(dT)18 primer following the manufacturer’s instructions as described previously65.To determine tissue distribution of MsFkh transcript in M. sexta larvae, day 2 fifth-instar naïve larvae were injected with heat-killed Staphylococcus aureus, Bacillus subtilis, Escherichia coli strain XL1-blue, Serratia marcescens (each at 5 × 107 cells/larva), or Saccharomyces cerevisiae (107 cells/larva), or with water as a control. Hemocytes, fat body and midgut were collected separately at 24 h post-injection for total RNA extraction and cDNA preparation as described previously67. Total RNA and cDNA were also prepared from Drosophila S2 cells. Briefly, S2 cells (5 × 106 cells) were collected in 1 ml of TRIzol® Reagent  and homogenized using hand held pestle and mixer . Then, 200 μl of chloroform were added, and the mixture was vortexed and centrifuged at 12000 × g at 4 °C for 15 min. The top aqueous phase (200 μl) was transferred to fresh Eppendorf tubes and isopropanol (500 μl) was added. RNA was precipitated by centrifugation at 12,000 × g at 4 °C for 10 min. The RNA pellets were washed with chilled 70% ethanol, air dried and re-suspended in 50 μl of nuclease free water and stored at −80 °C for later use.To determine induced expression of MsFkh  in different tissues of M. sexta larvae and several Drosophila Fox genes in S2 cells, including DmFkh (Genbank accession no. NP_524542.1) , forkhead box K (FoxK) long and short isoforms (Genbank accession no. AY787838) (primers DmFoxK-(L + S)-N and DmFoxK-(L + S)-R for both isoforms, primers DmFoxK-(L)-N and DmFoxK-(L)-R for long isoform only, amplicon sizes of 150 bp), which are also known as forkhead transcription factor long isoform (mnf-l) and short isoform (mnf-s), jumu (Genbank accession no. NM_079578.3)  and dFoxO (Genbank accession no. NM_206483.3) , in 20 μl reactions containing 10 μl 2 × SYBR® GreenERTM qPCR SuperMix Universal , 4 μl H2O, 4 μl diluted cDNA template, and 1 μl forward and reverse primers (10 pmol each), and M. sexta ribosomal protein S3 (rpS3)  or D. melanogaster ribosomal protein 49 (rp49)  gene was used as an internal standard to normalize the amount of RNA template. Real-time PCR program was 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min and the dissociation curve analysis. Data from three replicas of each sample were analyzed by the ABI 7500 SDS software (Applied Biosystems) using a comparative method (2CT−∆∆)69. These experiments were repeated with three different biological samples.Real-time PCR was performed for 65. To construct V5-tagged Fkh into pAC5.1/V5-His A expression vector, cDNA fragments encoding MsFkh (residues 1–355) and DmFkh (residues 1–510) were amplified by PCR using forward and reverse primers (Table Kpn I and DmFkh-F-Kpn I) contain a Kpn I site and the reverse primers (MsFkh-R-Not I and DmFkh-R-Not I) contain a Not I site in-frame fused to V5 tag and stop codon of pAC5.1/V5-His A expression vector. PCR reactions were performed with the following conditions: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, Tm-5 °C for 30 s, 72 °C for 30 s to 4 min, followed by a final extension at 72 °C for 10 min. The PCR products were recovered by agarose gel electrophoresis-Wizard® SV Gel and PCR Clean-Up System  and subcloned into T-Easy vectors . Recombinant T-vectors were purified using PureYield™ Plasmid Miniprep System  according to the manufacturer’s instruction and digested with Kpn I/Not I restriction enzymes, and DNA fragments were recovered and inserted into Kpn I/Not I digested pAC5.1/V5-His A expression vector using T4 DNA ligase . Recombinant plasmids were then purified and sequenced by an Applied Biosystems 3730 DNA Analyzer in the DNA Sequencing and Genotyping Facility at University of Missouri – Kansas City, and used to transfect S2 cells.Recombinant MsRelish-RHD (MsRel2-RHD) and MsDorsal-RHD (MsDl-RHD) with a Flag-tag in pAC5.1/V5-His A expression vectors were already constructed as described previouslyrs Table . Forward65. To construct Fkh-binding site mutation promoters, site-directed mutagenesis was performed using the truncated M. sexta moricin-242 (242 bp) and lysozyme-345 (345 bp) promoters as templates. Primers with specific mutation sites were designed for each mutated promoter and listed in Table EcoR I, Nde I and Bam HI were engineered in the mutant Fkh-binding site 1, 2 and 3 of the moricin-242 promoter, respectively. To generate Mormut-1&2, Mormut-1&3, Mormut-2&3 and Mormut-1,2&3 mutant promoters, site-directed mutagenesis was performed using the mutant promoter as the template with the second pairs of primers. For example, to generate Mormut-1&2 promoter, Mormut-1 was used as the template with MPAE-Fkh-2 primers, and to obtain Mormut-1,2&3 promoter, Mormut-1&2 was used as the template with MPAE-Fkh-3 primers. PCR program was 95 °C for 3 min, and then 17 cycles of 95 °C for 1 min, 55 °C for 2 min, 68 °C for 15 min, followed by a final extension of 68 °C for 30 min. The PCR products were recovered, digested with Dpn I, and then transformed into competent E. coli XL1 Blue cells. The mutant reporter plasmids were then purified and sequenced by an Applied Biosystems 3730 DNA Analyzer in the DNA Sequencing and Genotyping Facility at University of Missouri – Kansas City, and used for transient transfection in S2 cells.The moricin and lysozyme truncated promoters were constructed as described previouslyTM transfection reagent  was used for transient transfection according to the manufacturer’s instructions. After overnight transfection, S2 cells were centrifuged and resuspended in complete growth medium to induce protein expression for 48 h. Protein expression in cell culture media and cell extracts were analyzed by immunoblotting.For DNA transfection, S2 cells were placed overnight to 70% confluence prior to transfection in serum-free medium . GenCarrier-1M. sexta moricin, lysozyme, cecropin, defensin-1, defensin-2, defensin-3, attacin-1, and attacin-2  in the GloMax® Multi Microplate Luminometer (Promega). Relative luciferase activity (RLA) from S2 cells co-transfected with empty pAC5.1/V5-His A and pGL3B (empty reporter vector) was used as the calibrator. Relative luciferase activity (RLA) was obtained as the ratio of firefly luciferase activity to renilla luciferase activity70. These assays were performed in quadruplet and three independent experiments were repeated.Dual-luciferase reporter assays in S2 cells were performed in 96-well culture plates with recombinant pAC5.1/V5-His A expression plasmid (0.3 μg), pGL3B (empty vector) or different pGL3B firefly luciferase reporter plasmids from the promoters of 6 cells/well) expressing MsFkh, MsRel2-RHD and MsDorsal-RHD proteins were prepared as described previously12. Cell culture media (10 μl each) and cell extracts  were separated on 10%, 12%, or 15% SDS-PAGE and proteins were transferred to nitrocellulose membranes . Anti-Flag M2 antibody  and anti-V5 antibody  were used as primary antibodies, horseradish peroxidase-conjugate anti-mouse antibody  was used as secondary antibody for chemiluminescence using ECL Chemiluminescence Detection Kit , and membranes were exposed to films and scanned by Typhoon FLA7000 , while alkaline phosphatase (AP)-conjugate anti-mouse antibody  was used as secondary antibody for color development using AP-conjugate color development Kit .For immunoblotting analysis, cell extracts from S2 cells . Captured proteins were eluted with 30 μl of sample buffer mixed with 0.1% bromphenol blue, heated to 95 °C for 3 minutes, centrifuged at 12,000 × g for 1 min, and the supernatants were loaded onto SDS-PAGE for immunoblotting analysis as described previously12.Co-immunoprecipitation (Co-IP) assays were performed using cell extracts from S2 cells overexpressing MsFkh, MsRel2-RHD and MsDorsal-RHD proteins. Cell extracts were mixed and Co-IP was performed as described previouslyMsFkh mRNA in different tissues, or MsFkh mRNA in hemocytes, fat body or midgut by different treatments . Statistical significance was calculated by one way ANOVA followed by a Tukey’s multiple comparison tests using GraphPad Prism for comparisons of lls Fig. , or relalls Fig.  and 7, aSupplementary Info


Escherichia coli. In order to identify the whole set of genes under the direct control of Lrp, we performed Genomic SELEX screening and identified a total of 314 Lrp-binding sites on the E. coli genome. As a result, the regulation target of Lrp was predicted to expand from the hitherto identified genes for amino acid metabolism to a set of novel target genes for utilization of amino acids for protein synthesis, including tRNAs, aminoacyl-tRNA synthases and rRNAs. Northern blot analysis indicated alteration of mRNA levels for at least some novel targets, including the aminoacyl-tRNA synthetase genes. Phenotype MicroArray of the lrp mutant indicated significant alteration in utilization of amino acids and peptides, whilst metabolome analysis showed variations in the concentration of amino acids in the lrp mutant. From these two datasets we realized a reverse correlation between amino acid levels and cell growth rate: fast-growing cells contain low-level amino acids, whilst a high level of amino acids exists in slow-growing cells. Taken together, we propose that Lrp is a global regulator of transcription of a large number of the genes involved in not only amino acid transport and metabolism, but also amino acid utilization.Leucine-responsive regulatory protein (Lrp) is a transcriptional regulator for the genes involved in transport, biosynthesis and catabolism of amino acids in  Leucine-responsive regulatory protein (Lrp) is known as a global regulator of the genes for transport, biosynthesis and catabolism of amino acids to establish their balance needed for protein synthesis. After Genomic SELEX screening, however, we identified that Lrp not only controls the production of amino acids, but also the utilization pathway of amino acids by regulating the genes for tRNAs, aminoacyl-tRNA synthetases and rRNAs. Phenotype MicroArray and metabolome analyses indicated Lrp-mediated correlation between the intracellular levels of amino acids and their utilization for protein synthesis: the intracellular levels are low for amino acids that are efficiently used for protein synthesis, allowing fast cell growth, but cell growth is low even in the presence of high levels of amino acids that are not so much used for protein synthesis. Here, we also identified another expanded role of Lrp in regulation of a set of transcription factors, each playing a regulatory role in the control of a specific metabolism pathway or physiological response to a specific nutritional condition. Lrp stays on the top of this hierarchic network of transcription factors. Overall, we propose an expanded role for Lrp in controlling the production and utilization of amino acids – the key metabolites of cell construction.Escherichia coli Lrp of 164 aa in size consists of three functional domains: an N-terminal 40  % domain containing the helix–turn–helix motif of DNA binding, the next 40  % of the middle domain responsible for transcription activation and an overlapping C-terminal domain required for the response to Leu (Leucine-responsive regulatory protein (Lrp) belongs to the widely distributed Lrp–AsnC family of small, basic transcription factors. e to Leu . Lrp forE. coli as a regulatory protein involved in the control of the transport of branched-chain amino acids , suggesting that Lrp plays a regulatory role in transport and metabolism of not only Leu, but also some other amino acids. The number of regulation targets of Lrp has further increased concomitant with the advance of genome expression monitoring systems. Proteome analysis suggested the alteration of levels of a total of 25 proteins in the lrp mutant  sigma subunits and a total of ∼300 species of transcription factors, we developed the Genomic SELEX screening system E. coli DH5α was used for plasmid amplification. E. coli BL21 was used for Lrp expression. E. coli BW25113 (W3110 lacIqrrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78) (lrp single-gene deletion mutant of BW25113) . Cells were grown in M9/glucose medium at 30 °C under aeration with constant shaking at 150 r.p.m. Cell growth was monitored by measuring OD600.BADLD78)  and JW08E. coli K-12 W3110 was PCR-amplified and inserted into pET21α between NdeI and NotI so as to fuse to the C-terminal His-tag. The expression of His-tagged Lrp was performed in E. coli BL21. Lrp was affinity-purified according to the standard procedure  and incubated for 30 min at 37 °C. The DNA–Lrp mixture was treated with anti-Lrp antibody, and DNA fragments recovered from the complexes were PCR-amplified and subjected to next cycle of SELEX for enrichment of Lrp-bound DNA fragments.The Genomic SELEX method was carried out as described previously   . Fluctua600 0.5) was filtered under vacuum through a 0.4 μm pore size filter. Cells on the membrane filter were immediately washed with MilliQ water to remove extracellular components and then quickly immersed in 2 ml methanol containing 2.5 μM each of the internal standards, methionine sulfone, MES and d-camphor 10-sulfonic acid. Dishes containing filters were sonicated for 30 s to resuspend the cells. A 1.6 ml aliquot of the cell suspension was transferred to a tube, and mixed with 1.6 ml chloroform and 0.64 ml MilliQ water. After vortexing and centrifugation, the aqueous layer was recovered and clarified using Ultrafree-MC ultrafilter devices for Metabolome Analysis UFC3LCCNB-HMT (Millipore). After drying up, materials attached on the filter were dissolved in 25 μl MilliQ water and subjected to capillary electrophoresis time-of-flight MS (CE-TOF-MS) analysis.Samples for intracellular metabolite measurements were processed as described previously -MS sprayer kit (Agilent Technologies). The system was controlled by Agilent G2201AA ChemStation software for CE. Data acquisition was performed by Analyst QS 7222 software for Agilent TOF (Applied Biosystems and MDSSciex). Instrumental conditions for separations and detections of metabolites were as follows. The cationic metabolites were separated on a fused silica capillary (50 μm × 100 cm) using 1 M formic acid as the electrolyte with the voltage set at 30 kV. A solution of 50  % (v/v) methanol/water was delivered as the sheath liquid at a flow rate of 10 ml minE. coli BW25113 and JW0872 were grown overnight at 30 °C in M9/glucose (0.2  %). Cells were washed with IF-0 (inoculating fluid), and then resuspended in IF-0 for PM plates 1 and 2, in IF-0 containing 20 mM sodium succinate and 2 mM ferric citrate for PM plates 3–8, and in IF-10 containing 2.0 g tryptone, 1.0 g yeast extract and 1.0 g NaCl l–1 at a density corresponding to 85  % transmittance (OD420∼0.12) using a 20 mm diameter tube. Tetrazolium violet was added at the final concentration of 0.01  %. The suspensions were then inoculated into the appropriate microplates PM1–10 for bacteria (Biolog) at a volume of 0.1 ml per well. The microplates were placed in an OmniLog instrument at 30 °C and monitored by OmniLog reader (Biolog) for colour change in the wells at 15 min intervals up to 72 h. Kinetic data were analysed with OmniLog-PM software. Each strain was tested at least twice.The PM assay was performed essentially according to the published methods (E. coli cells (OD600 0.5) by the hot phenol method. RNA purity was checked by electrophoresis on 1.5  % agarose gel in the presence of formaldehyde followed by staining with methylene blue. Northern blot analysis was performed essentially as described previously (Taq DNA polymerase (TaKaRa). Total RNAs (1 mg) were incubated in formaldehyde-MOPS gel-loading buffer for 10 min at 65 °C for denaturation, subjected to electrophoresis on formaldehyde-containing 1.5  % agarose gel and then transferred to a nylon membrane (Roche). Hybridization was performed with a DIG easy Hyb system (Roche) at 50 °C overnight with a DIG-labelled probe. For detection of the DIG-labelled probe, the membrane was treated with anti-DIG-AP Fab fragments and CDP-Star (Roche), and the image was scanned with a LAS-4000 IR multi-colour imager (Fuji Film).Total RNAs were extracted from exponentially growing E. coli genome fragments of 200–300 bp in length and Lrp-bound DNA fragments were affinity-isolated. As the specificity of target recognition of Lrp is known to change toward different directions, depending on the species of interacting amino acid effector  and a maximum of 296 (type A 156 plus type B 140)  . In ordee B 140)  and, as in vitro estimation by SELEX and in vivo measurement by ChIP-chip. The binding in vivo of Lrp should be interfered by competitive binding by other DNA-binding proteins. In addition, the intracellular conditions were different from in vitro SELEX conditions, altogether influencing the Lrp–DNA interaction modes. Amongst the total of 296 candidate genes under the direct control of Lrp, 114 were related to the metabolism of amino acids , 96 species of nitrogen source (PM plate 3), 96 species of phosphorus and sulfur sources (PM plate 4), 96 species of nutrient supplement (PM plate 5), 288 chemicals as peptide nitrogen source (PM plates 6–8), 96 species of osmolyte (PM plate 9) and 96 different pH conditions (PM plate 10) (E. coli BW25113 and JW0872 (lrp single-gene deletion mutant of BW25113). The time-course of cell growth was monitored by measuring the cell density-dependent increase in respiration .After Genomic SELEX screening, we recognized a sudden and marked increase in the list of regulation targets of Lrp, indicating that Lrp plays as-yet unidentified regulatory roles in overall transcription of the late 10) . We meash curves . Growth lrp mutant strain exhibited slower growth under a total of 59 conditions, of which 50 were in the presence of specific nitrogen sources, four in the presence of specific carbon sources, four in the presence of nutrient supplement and one at specific pH . Likewise, transcription factors of the genes for amino acid metabolism, including AdiY and CysB (regulator of Cys synthesis), LeuO (regulator of Leu synthesis), and TdcA and TdcR (regulator of Thr synthesis), are all under the direct control of Lrp  of LysRS]. After the Genomic SELEX screening, Lrp was found to bind the promoter region of at least eight aminoacyl-tRNA synthetase genes , implying the involvement of Lrp in transcription regulation of these genes.Results of the SELEX-chip screening supported the concept that Lrp is a global transcription regulator for the set of genes involved in transport, synthesis and degradation of amino acids. The results of the PM assay and metabolome analyses are both consistent with this concept. In addition, Lrp was found to be involved in regulation of the genes for the utilization of amino acids in the pathway of translation, such as tRNA, tRNA aminoacylation, rRNA and ribosomal proteins . E. coliin vivo of these aminoacyl-tRNA synthetase genes by Lrp, we performed Northern blot analysis for detection of mRNA from these genes. RNA samples were prepared from both E. coli WT BW25113 and the lrp mutant JW0872, and subjected to Northern blot analysis (lysU, the known target of Lrp, was virtually undetectable in the WT strain under the culture conditions employed, but a high level of lysU mRNA was detected in the lrp mutant strain, indicating strong repression of the lysU gene by Lrp. Next, we analysed the level of mRNAs for seven other aminoacyl-tRNA genes. The levels of serS, tyrS and thrS were low in WT cells, but increased in the lrp mutant, as in the case of lysU. mRNAs of other aminoacyl-tRNA genes were detected even in WT cells, but alaS mRNAs increased, albeit at low levels, in mutant cells. Thus, we concluded that Lrp participates in transcription regulation of at least eight aminoacyl-tRNA synthetase genes, of which expression of five aminoacyl-tRNA synthetase, including AlaRS, LysRS, SerRS, ThrRS and TyrRS, is repressed by Lrp. So far only minimal Lrp-dependent changes have been observed in the microarray analysis (In order to examine regulation analysis . mRNA ofAfter SELEX-chip screening, at least 296 regulation targets were identified for Lrp, resulting in an increase of ∼2.3-fold. One group of the novel targets includes the genes for utilization of amino acids such as the genes encoding tRNA, aminoacyl-tRNA synthetase, rRNA and ribosomal proteins. Here, a total of eight aminoacyl-tRNA synthetase genes were identified to be under the direct control of Lrp, but this number increases by setting the cut-off level of SELEX pattern < 10 . In the E. coli contains as many as 300 species of transcription factors, each monitoring a specific factor or condition in the environment (E. coli transcription factors belong to the one-component signal transduction system, in which a single polypeptide contains both an effector-binding sensory domain and a DNA-binding domain. The activity of this group of transcription factors is controlled by a single species of the effector ligand, i.e. inducer or co-repressor. In some cases, the involvement of two effectors has been identified: allantoin and glyoxalate for AllR (ironment . The majfor AllR , glyoxylfor AllR , and urafor AllR , and Sdifor AllR . The nexlrp gene itself (In the collection of a total of 296 Lrp targets selected by SELEX-chip screening, a set of 21 transcription factor genes was identified, including the e itself . InteresE. coli, such as GadW for acid response, YedW for copper and peroxide response, EnvR for response to drugs, QseB for quorum sensing, BluR for biofilm formation, and MatA and SlyA for planktonic growth. Thus, the life style of E. coli under these transcription factors is also under the control of Lrp, which monitors the nutritional conditions in the environment. Lrp is located upstream of this hierarchic network including these two groups of transcription factors, thereby regulating a large number of genes indirectly besides the total of ∼300 direct targets.In addition to these amino acid-related transcription factors, Lrp was found to regulate a total of 15 transcription factors (