Drosophila wing, they demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers.Uyehara et al. show that hormone-induced transcription factors control temporal gene expression by regulating accessibility of DNA regulatory elements. Using the  Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility.Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the  A defining feature of metazoan development is the organization of cells into tissues. The physiological function of a given tissue is determined by the identity of its constituent cells as well as their arrangement within the tissue. As a result, building tissues during development requires precise spatial control of gene expression over extended periods of time. Whereas many of the genes required for the development of different cell and tissue types have been identified, the mechanisms through which spatial information is coordinated with temporal information remain incompletely understood.Spatially, a select and conserved group of transcription factors, sometimes termed “master” transcription factors, often specifies the distinct identities of different cell and tissue types . GeneticOne proposed mechanism to explain the distinctive power of master transcription factors is that they control where other transcription factors bind in the genome by regulating chromatin accessibility . In vivoIf nucleosomes prevent transcription factors from accessing DNA, then how do transcription factors come to occupy their binding sites? Biochemical studies have identified a class of transcription factors termed “pioneer” factors that have the unique ability to bind nucleosomal DNA and subsequently enable binding by other transcription factors . The proDrosophila embryos, an intrinsic cascade of transcription factor expression specifies the distinct temporal identities of neural stem cell progeny . In contrast to stem cell lineages, coordinating the timing of gene expression across fields of cells, such as a tissue, often involves the use of secreted signals. For example, thyroid hormone controls the initiation and progression of metamorphosis in frogs  to show that gene expression is temporally dynamic as wings differentiate and undergo the complex morphogenetic events that create the adult appendage. We then carried out open chromatin profiling and transgenic reporter analysis to show that these changes in gene expression are accompanied by genome-wide changes in the accessibility of temporal-specific transcriptional enhancers. Finally, we used ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) and loss-of-function analyses to show that the ecdysone-induced transcription factor E93 is required to drive the normal sequence of chromatin accessibility changes. Importantly, E93 is required for not only increasing the accessibility of late-acting enhancers but also decreasing the accessibility of early-acting enhancers. Together, these findings demonstrate that E93 specifies temporal identity by directly regulating accessibility of temporal-specific transcriptional enhancers. More broadly, this work helps to explain how hormone signaling can influence tissue-specific gene expression programs to drive development forward in time.Here, we examined the mechanisms controlling temporal gene regulation in Drosophila pupal wing development. By the end of larval development . Gene ontology (GO) analysis showed enrichment for biological processes known to occur at these times , whereas genes increasing between 24 and 44 h include those involved in actin regulation . Conversely, genes decreasing between L3 and 24 h include those involved in DNA replication , whereas genes decreasing between 24 and 44 h include those involved in mitosis  . Thus, the first 2 d of pupal wing development are marked by temporally dynamic changes in gene expression.To examine the mechanisms underlying temporal regulation of gene expression, we focused on the early stages of elopment A, the wielopment . During elopment B,C, cellelopment . This tielopment . Not surelopment A, “L3” aelopment B, “24 h”elopment C, “44 h”elopment . Pairwisse times . For exaSupplemental Fig. S2A). For example, the tenectin gene (tnc), which encodes a constituent of the extracellular matrix that binds α-PS2 integrin . Of the top 7699 FAIRE peaks from each time point , 2154 sites increase and 1333 sites decrease in accessibility between L3 and 24-h wings  . Henceforth, we refer to sites that decrease in accessibility between successive time points as “closing” and sites that increase in accessibility between successive time points as “opening.” We found that the great majority of these temporally dynamic open chromatin sites (78%–89%) is located distal to gene promoters . Finally, plots of the average FAIRE signal in temporally dynamic open chromatin indicate that many temporally dynamic open chromatin sites are used transiently in development. For example, sites closing between L3 and 24 h tend to stay closed, and sites opening between 24 h and 44 h tend to be closed at L3    on L3, 2integrin , exhibitintegrin A. These -h wings A. Similaynthesis , containA levels A and theA levels . At the twofold) B. Similato 24 h) C. Thus, Open chromatin sites are highly correlated with functional DNA regulatory element activity . Our fintnc locus. As mentioned above, tnc encodes an extracellular matrix protein involved in cell adhesion. Consistent with a role for tnc in mediating adhesion between the dorsal and ventral surfaces of the wing pouch, RNAi-mediated knockdown of tnc results in defects in wing morphology . While neither reporter is active in L3 wing discs, there is activity in 24-h wings. We termed these the tncblade (blade) and tncwv (wing vein) enhancers. tncblade is active most strongly in the interveins between the first and second and between the fourth and fifth longitudinal veins and in cells near the proximal posterior margin. It is also active at lower levels in the intervein between the third and fourth longitudinal veins . Thus, tncblade and tncwv are active in complementary domains of the 24-h pupal wing. Since tnc is expressed nearly ubiquitously at this stage of wing development, these open chromatin sites likely correspond to bona fide transcriptional enhancers that interpret different spatial inputs.We first examined two candidate enhancers from the rphology . We clonal veins A. tncwv nubbin (nub) locus, which encodes a transcription factor required for proximal-distal axis and vein development in wings . Immunofluorescence experiments show that a reporter carrying the distal site is not active in L3 wing discs. By 44 h, it shows strong activity in the L3 and L5 wing veins and weaker activity in the L2 and L4 veins , again consistent with defects observed in nub hypomorphic alleles  locus, which encodes a family of transcription factors active in third instar and early prepupal tissues, including the wing . These candidate enhancers were cloned upstream of GAL4 to allow for flexibility in the reporters used. Crossing these GAL4 drivers to flies containing UAS-GFP revealed that both open chromatin sites are transcriptional enhancers active in L3 wing discs. The brdisc enhancer is located ∼40 kb upstream of the br promoter. Similar to Br protein, brdisc is active nearly ubiquitously in wing imaginal disc epithelial cells, with higher levels along the anterior–posterior and dorsal–ventral boundaries in the wing pouch . This enhancer is located ∼30 kb upstream of the br promoter and is active in L3 wing discs in a pattern similar to the adepithelial cells located in the notum of the wing. Like the brdisc enhancer, there is no sign of brade reporter activity in the wing blade by 24 h, consistent with expectations, since the adepithelial cells remain in the notum to form the indirect flight muscles. Notably, br is required for proper differentiation of these cells into adult muscles . Moreover, the timing of each factor's expression coincides with the timing of its requirement in Drosophila development. For example, br is required for the transition from larval to prepupal stages  in temporally dynamic FAIRE peaks relative to temporally static FAIRE peaks. We observed significant enrichment (P < 0.05) for multiple motifs in FAIRE peaks that open or close between successive time points . At 96 h, following the period of E93 expression, E93 mutant wings are dramatically smaller than wild-type wings, with significant defects in vein development  that can be immunoprecipitated with antibodies to GFP. Importantly, the E93GFSTF chromosome complements a deletion encompassing the E93 locus [Df(3R)93FX2], demonstrating that the fusion protein is functional. Supporting this functionality, immunostaining of E93GFSTF flies shows clear fusion protein expression in 24-h and 44-h wings .The above findings reveal that the temporal changes in gene expression that occur during pupal wing development coincide with temporal changes in the accessibility of thousands of open chromatin sites, many of which could be transcriptional enhancers. We next sought to examine the role of E93 in this process. As a first step, we performed ChIP-seq to identify sites in the genome to which E93 binds. We used an  Project . In thisDrosophila transcription factors , demonstrating that the E93 ChIP signal is specific and not simply an indirect consequence of open chromatin. This is further supported by differences in the distribution of E93 ChIP and 24-h FAIRE peak locations across the genome: E93 binds preferentially to promoter-distal sites in the genome, whereas FAIRE peaks overlap proximal promoter regions with greater frequency . We next examined the relationship between E93 binding and temporally dynamic open chromatin in pupal wings. Fifty-one percent of FAIRE peaks that change in accessibility between L3 and 24 h are directly bound by E93 at 24 h. Likewise, 51% of FAIRE peaks that change in accessibility between 24 h and 44 h are directly bound by E93 at 24 h . In contrast, only 4% of sites that change in accessibility during embryogenesis overlap an E93-dependent FAIRE peak  . FAIRE peaks that do not change in accessibility between L3 and 24 h in wild-type wings exhibit no change in accessibility in E93 mutants  is possibly due to a persistent failure in chromatin accessibility from the earlier time point, such as sites that fail to open in E93 mutants at 24 h and stay closed at 44 h .We next asked whether sites that depend on E93 for proper chromatin accessibility correspond to temporally dynamic FAIRE peaks. Indeed, 70% of E93-dependent FAIRE peaks are temporally dynamic between L3 and 24 h in wild-type wings C. This iE93 mutants are due to a failure to make temporally required changes in the accessibility of DNA regulatory elements genome-wide. To directly test this hypothesis, we examined the consequences of E93 loss-of-function on the activity of the temporally dynamic transcriptional enhancers that we identified above. ChIP-seq shows that the nubvein enhancer is directly bound by E93 at 24 h, and FAIRE-seq in wild-type wings shows that nubvein progressively opens after the L3 stage , which use different transcription factors to determine their identities, share nearly identical open chromatin profiles. Moreover, these shared open chromatin profiles change coordinately over developmental time. There are two possible explanations for these findings. Either (1) different transcription factors produce the same open chromatin profiles in different appendages or (2) transcription factors shared by each appendage control open chromatin profiles instead of the master transcription factors of appendage identity. We favor the second model for several reasons. Since the appendage master transcription factors possess different DNA-binding domains with distinct DNA-binding specificities, it is unlikely for them to bind the same sites in the genome. Supporting this expectation, ChIP for Scalloped and Homothorax, two transcription factors important for appendage identity, shows clear tissue-specific binding in both the wing and eye–antennal imaginal discs  and differentially regulate the activity of these enhancers to control spatial patterns of gene expression, thus shaping the unique identities of individual appendages.E93 mutants, ∼40% of these open chromatin changes fail to occur. Importantly, nearly three-quarters of sites that depend on E93 for accessibility correspond to temporally dynamic sites in wild-type wings. Thus, chromatin accessibility is not grossly defective across the genome; instead, defects occur specifically in sites that change in accessibility over time. This finding, combined with the large fraction of temporally dynamic sites that depend on E93 for accessibility, indicates that E93 controls a genome-wide shift in the availability of temporal-specific transcriptional enhancers. Supporting this hypothesis, we show that temporal-specific enhancers depend on E93 for both accessibility and activity. Since we propose that the response to ecdysone is shared across the appendages, we predict that similar defects occur in appendages besides the wing. It remains to be seen whether other ecdysone-induced transcription factors besides E93 control accessibility of enhancers at different developmental times. It also remains to be seen how the temporal transcription factors work with the appendage master transcription factors to control appendage-specific enhancer activity.Our experiments with E93 provide direct support for this model. In wild-type wings, thousands of changes in open chromatin occur after the large pulse of ecdysone that triggers the end of larval development. In Our findings suggest that E93 controls temporal-specific gene expression through three different modalities that potentially rely on three distinct biochemical activities. The enrichment of E93 motifs and binding of E93 to temporally dynamic sites indicate that it contributes to this regulation directly. We propose that these combined activities drive development forward in time by turning off early-acting enhancers and simultaneously turning on late-acting enhancers.tncblade enhancer, E93 appears to function as a conventional activator. In the absence of E93, tncblade fails to express at high levels, but the accessibility of the enhancer does not measurably change. This suggests that binding of E93 to tncblade is required to recruit an essential coactivator. Importantly, this finding demonstrates that E93 is not solely a regulator of chromatin accessibility. E93 binds many open chromatin sites in the genome without regulating their accessibility and thus may regulate the temporal-specific activity of many other enhancers. In addition, since the tncblade enhancer opens between L3 and 24 h even in the absence of E93 , E93 protein trap , UAS-nls-GFP , UAS-nls-GFP , E934 (gift of Craig Woodard), Df(3R)93Fx2 (gift of Eric Baehrecke), and UAS-destabilized-GFP (gift of Brian McCabe).Flies were grown at 25°C under standard culture conditions. The genotype of the wild-type strain was wn = 717 Rep1; n = 1280 Rep2) from both male and female E93 protein trap flies. Wings were dissected in 1× PBS and kept on ice. Batches of wings from 20 pupae were fixed in 4% paraformaldehyde, 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA for 20 min at room temperature followed by quenching with 125 mM glycine in 1× PBS and 0.01% Triton. Fixed wings were dounce-homogenized in 10 mM HEPES, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton, and 1 mM PMSF. Nuclei were pelleted at 4500g for 20 min and resuspended in 10 mM HEPES, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01% Triton, and 1 mM PMSF. After nutating for 10 min at 4°C, nuclei were pelleted again and resuspended in 140 mM NaCl, 10 mM HEPES, 1 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, and 0.1% SDS followed by sonication on ice with a Branson sonifier until the average chromatin fragment size was 200 base pairs. The soluble chromatin fraction was used for ChIP. Briefly, extracts were precleared with protein A Dynabeads for 2 h at 4°C, and cleared extracts were incubated with 5 µg of rabbit anti-GFP antibody  overnight at 4°C. Bead pull-down was performed for 3 h the following day. Antibody–bead complexes were washed successively and then eluted with 1% SDS, 250 mM NaCl, 10 mM Tris, and 1 mM EDTA. Samples were treated with RNase A and proteinase K and heated overnight at 65°C to reverse cross-links, and purified DNA was recovered by phenol-chloroform/ethanol precipitation. Rubicon Thruplex DNA-seq kit was used for library construction. All samples were sequenced on an Illumina HiSeq 2500 at the University of North Carolina High-Throughput Sequencing Facility. Additional details are available on request.A minimum of 40 wings were dissected from staged female flies in 1× PBS and transferred to ice for subsequent processing. RNA was prepared as described previously , and theZ-scores were calculated using the mean and standard deviation per chromosome arm. To focus on a high-confidence set of peaks, we chose a MACS2 −log10-adjusted P-value of 40 from the 44-h wild-type data set and selected an equivalent number of peaks (n = 7699) from the remaining data sets. edgeR  cloning was used to move candidate enhancers into destination vectors. Injections were performed at BestGene. The brdisc and brade enhancers were cloned into the pϕUGG GAL-4 destination vector . The nubvein and nubmargin enhancers were cloned into a modified pDEST-HemmarG vector (tncwv and tncblade enhancers were cloned into a modified pDEST-HemmarR vector (tnc reporters with existing GFP-marked GAL4 drivers. Each nub and tnc enhancer was integrated into the attP2 site on chromosome 3. Integration of each reporter into its respective attP site was confirmed by PCR. Immunostaining and confocal imaging were performed as described previously (Candidate enhancers were cloned from wild-type n vector  and inteG vector  in whichR vector  in whicheviously . The fol


Plasmodium falciparum intraerythrocytic cycle. In order to elucidate the role of the P. falciparum (Pf)NF-YB transcription factor we searched for target genes in the entire genome. PfNF-YB mRNA is highly expressed in late trophozoite and schizont stages relative to the ring stage. In order to determine the candidate genes bound by PfNF-YB a ChIP-on-chip assay was carried out and 297 genes were identified. Ninety nine percent of PfNF-YB binding was to putative promoter regions of protein coding genes of which only 16% comprise proteins of known function. Interestingly, our data reveal that PfNF-YB binding is not exclusively to a canonical CCAAT box motif. PfNF-YB binds to genes coding for proteins implicated in a range of different biological functions, such as replication protein A large subunit (DNA replication), hypoxanthine phosphoribosyltransferase (nucleic acid metabolism) and multidrug resistance protein 2 (intracellular transport).Little is known about transcription factor regulation during the  Plasmodium falciparum merozoites invade red blood cells (RBC), and over 48 hours develop and multiply through the ring, trophozoite and schizont stages [in vitro. The genome sequence has contributed enormously to our understanding of molecular and cellular aspects of the life cycle [P. falciparum is still poorly understood.t stages , 2. Thisfe cycle , howeverP. falciparum genome and the complex life cycle involving differential gene expression at morphologically distinct stages make it difficult to predict and to characterize transcription factors. Analysing the genome revealed a relative paucity of transcription-associated proteins and specific cis-regulator motifs [Few transcription factors or regulatory motifs have been identified and characterized in malaria parasites -6. Gene r motifs , but ther motifs , 15.P. falciparum, and recently a new set of 129 cis-regulatory elements was reported [Using bioinformatics, including genomic and proteomic tools, some orthologues of general transcription factors such as the TFIIB family  have beereported .P. falciparum and P. chabaudi parasites [3, Ca2+ and cyclic AMP [P. falciparum -26. A variety of regulatory factors are modulated by second messenger signaling in vertebrates [2+ has a role regulating long-term cell adaptation by controlling gene expression .http://www.plasmodb.org), Pf11_0477 (now described as PF3D7_1146600) transcript was predicted to have around 3906 bp according to northern blotting results [P. berghei, has been identified a Pf Nf-YB orthologue named ORP1 (Oocyst Rupture Protein 1) also containing an histone-fold domain (HFD) NF_YB-like. The authors found that mutant parasites in which orp1 gene has been deleted, oocyst rupture, normally occurring in mosquito midgut around day 12 post blood meal, is inhibited but growth of asexual stages was not affected [On PlasmoDB database , B and C (Pf14_0374) [2/M phase of the cell cycle and regulates the expression of topoisomerase IIα, cyclin B1, CDC25C, E2F, CDC2 and thymidine kinase [P. falciparum, the target gene(s) bound by PfNF-YB during the parasite cycle life have not been identified, and the role of this transcription factor is unknown. Our group reported that a cAMP analogue (6-Bnz-cAMP) modulates PfNF-YB transcript and proteins levels, raising the possibility of a role for this protein in parasite signaling pathways [We have demonstrated that melatonin induces posttranslational modification of the PfNF-YB protein by increasing its ubiquitination, allowing PfNF-YB abundance to be controlled by the ubiquitin-proteasome system . In tran and C Pf_0374 [1414_0374) -38. For e kinase -41. In cpathways .In this research, we identified several DNA binding sites candidates for PfNF-YB transcription factor at the schizont stage. Our results suggest that PfNF-YB is involved in the control of numerous different biological functions in the parasite, including protein translation folding and modification, intracellular transport, nucleic acid metabolism and cell redox homeostasis.P. falciparum asexual stage we followed its transcript expression throughout the intraerythrocytic developmental cycle using a synchronized parasite population. Figure http://plasmodb.org and published for P. falciparum 3D7 strain [In order to investigate PfNF-YB function during the 7 strain . The higP. falciparum NF-YB is highly expressed in late stages and located at least in part in the nucleus of both trophozoite and schizont forms. During these developmental stages, DNA duplication, followed by nuclear division and cytokinesis to produce new merozoites is essential to allow successful parasite multiplication and invasion of new red blood cells. Therefore, we asked whether PfNF-YB at the schizont stage (44 hpi) can bind to DNA and regulate expression of target genes involved in the parasite cell cycle. To answer this question, a genome-wide PfNF-YB chromatin immunoprecipitation (ChIP-on-chip) assay was performed using a high density whole genome tiling array (Roche NimbleGen Inc) and material purified from schizonts. Chromatin immunoprecipitation was done using PfNF-YB-specific rabbit antibody attached to beads, and, as controls, non-specific rabbit IgG attached to beads and beads alone. Before performing the ChiP-on-chip assay, qPCR with selected genes (PFI1665w and Pf14_0489) was used to test whether an enrichment of promoter regions was obtained using the PfNF-YB antibody. Using this approach, we observed that the putative promoter region of both PFI1665w and Pf14_0489 was enriched in the fraction bound to the PfNF-YB-specific antibody compare to the fraction bound to nonspecific rabbit IgG or beads alone  to confirm the enrichment and to measure the sensitivity and specificity of the detection by qPCR . From 297 targets identified by the ChIP-on-chip experiment, 42% are of unknown function, 40% are proteins with putative function, 16% are of known function, 1% are pseudogenes and 1% are non-coding RNA Figure . With reP. falciparum , and the review , 49, 50. testing , 52 in os Figure . The P.  to date , 52 and Plasmodium may exhibit some sequence diversity  was important to elucidate its binding gene target. ChIP is one of the most powerful tools to elucidate protein-DNA interaction and has been applied to study histone- or transcription factor-DNA interaction in Plasmodium , 56-59. ce CCAAT  and richT motifs . Our resin vivo promoter occupancy by PfNF-YB at schizont form  ont form , we can Protein translation and modification processes are among the most promising areas to target for the development of new anti-malarial drugs. PfNF-YB binds to the genes for several translational proteins, for example translation initiation factor IF-2 (PF13_0069) and peptide release factor 1 (PF14_0265). Moreover, we also identified the eukaryotic translation initiation factor 3 subunit 8 (PFL0310c) and several 40S and 60S ribosomal proteins. The third class of gene that PfNF-YB binds is related to transport. Our results demonstrate that PfNF-YB binds to promoter regions of genes for 15 transporter proteins, among them Sec24b (PFD0250c), Rab GTPase 18 (Pf08_0110) and PfMDR2 (Pf14_0455).in silico transcription factor binding sites we applied bioinformatics approaches, such as WebLogo (http://WebLogo.berkeley.edu/). Logos are commonly used for the representation of transcription factor binding site preferences [P. falciparum NF-YB binds to a variety of motifs. Although the NF-Y transcription factor binds to the cis-element CCAAT motif in most eukaryotic cells, in P. falciparum we showed by in silico analysis that PfNF-YB does not bind exclusively to this cis-element, since 157 genes do not possess the CCAAT binding site in the region immunoprecipitated with PfNF-YB antibody. ChIP-on-chip analysis shows the interaction between protein and DNA, but does not address whether this interaction is directed or not. Therefore the 157 target genes lacking a CCAAT binding site may be recognized by PfNF-YB due to interactions with other transcription factors.Besides the molecular characterization of the transcription factor, it is very important to know the type of motif it binds. In order to infer ferences . It alsoferences , 67. TheArabidopsis thaliana [P. falciparum. Furthermore, the WebLogo analysis identified the cis-element AATAT (313 matches) that was identified by in silico TRANSFAC database search as a TATA-box. The presence of a TATA-box motif corroborates the binding of the PfNF-YB transcription factor to putative promoter regions of target genes. The AATAT motif is also associated with gene signaling in inorganic phosphate starvation in ArabidopsisArabidopsis cis-element sequences in the P. falciparum genome, since the malaria parasite genome is more similar to that of Arabidopsis than other non-Apicomplexan taxa [Our analysis showed the presence of the pentamer motif AAATG  we found that PfAp2-l and PfNF-YB proteins regulate 11 common genes in P. falciparum  and PfNF-YB. This data suggests that P. falciparum transcription factors AP2 and NF-YB regulate DNA/ chromatin genes and host-cell remodeling.Recently several works have shown the importance of AP2 genes in controlling blood stage and sexual developmental cycle of ogenesis , 70. By ogenesis , both folciparum . PfAp2-lBased on target gene function, PfNF-YB can control nucleic acid metabolism, protein translation, transport and regulation of gene expression. However, it is important to highlight the fact that 42% of all target genes identified have no known function. We demonstrate here that PfNF-YB binds to a variety of motifs showing a divergence compared to the situation in most eukaryotic cells, where the CCAAT motif alone is required for NF-YB interaction with a promoter region. In addition, previously we showed that proteasome inhibitor, bortezomibe (BTZ), besides blockage of ring and trophozoite development also decreases PfNF-YB expression , leadingP. falciparum strain 3D7 parasites were cultured in flasks at 37°C and 5% hematocrit in RPMI 1640 medium supplemented with 10% human plasma, and gassed with 90% N2, 5% O2 and 5% CO2 [d 5% CO2 . For synd 5% CO2 .t-test using three independent assays.Samples from parasites were harvested at 10, 15, 25, 30, 44 and 48 hr after invasion for qPCR assay. Parasitized erythrocytes were lysed using Trizol LS (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was performed with 300 ng of random primers and 500 ng of total RNA previously treated with DNAse, using the Superscript II Kit (Invitrogen) as described in the manufacturer’s instructions. Quantitative analysis of the relative levels of specific transcripts in triplicate samples was assessed by SYBER green quantitative real-time PCR (qPCR) in a 7300 Real Time PCR System (ABI) under the following conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 sec; 55°C for 30 sec; 60°C for 30 sec. The efficiency of amplification for each gene was previously determined using dilutions of cDNA or gDNA. Genes outside the 90-110% range were discarded from the analysis. For each reaction 2 to 8 ng of cDNA, oligonucleotides  in Max Mix PCR buffer (ABI) were used. The mRNA was amplified using the following primers. Pf11_0477: 5’-acaagcgaggctagtgacag-3’, 5’-ttcagatattcggttaatggttc-3’; MAL-18S: 5’-aacacaaggaagtttaaggcaacaa-3’, 5’-gcgtgcagcctagttca-3’. The 18S ribosomal RNA gene was amplified as an endogenous control and used to normalize the quantitation results. The reactions were carried out in triplicate and the expression values shown represent the relative amplification from each cDNA sample compared to the control. The significance of the difference between the relative gene expression levels was determined by Student’s P. falciparum cultures and on three independent occasions were used in chromatin immunoprecipitation assays. Parasites were released from red blood cells by saponin treatment  as described above, and crosslinking was performed using 2% formaldehyde at 37°C for 10 minutes. Excess formaldehyde was quenched by addition of glycine, with gentle agitation for 5 minutes at room temperature.. The parasites were then lysed in 5 mM HEPES pH 8.0, 85 mM KCl, 0.5% Triton X-100, plus protease inhibitors for 15 minutes. The nuclear fraction was harvested by centrifugation at 3000 rpm and the pellet was incubated with 500 μl of nuclear lysis buffer . The DNA was sheared into 200 to 1000 bp fragments by sonication (10 times for 10 seconds using a Sonics Vibra cell Model), and the fractionated chromatin was visualized on an agarose gel stained with ethidium bromide. One milliliter of the supernatant was pre-cleared with 100 μl of protein A-Sepharose beads, by incubation overnight at 4°C with agitation, then the mixture was centrifuged and the pellet discarded. The clarified solution containing fragmented chromatin was incubated overnight at 4°C with 20 μg anti-PfNF-YB or a nonspecific rabbit IgG attached to beads, or with control beads (no antibody attached). The mixture was centrifuged at 300 rpm for 3 minutes and the pellet together with one tenth of the input supernatant were retained. The pellet was washed three times with immunoprecipitation buffer  and then 6 times with wash buffer . Bound material was uncoupled from the Sepharose by reversing the crosslinking at 65°C overnight. The DNA was then purified using the phenol extraction method and dissolved in 30 μl of sterile distilled water. PCR analysis of the DNA immunoprecipitated with PfNF-YB antibody or a control rabbit IgG, and in the beads alone and input fractions was performed as described [P. falciparum genome was produced by Roche NimbleGen (USA). The ChIP samples were hybridized with this full P. falciparum genome and the results were analyzed using the Deva program from Roche. The ChIP-on-chip data were normalized using the Tukey bi-weight function to account for differences between the dyes on the array. Roche NimbleGen scaled the ratios in the.gff files by subtracting the bi-weight mean for the probe log2-ratio values for all features on the array from each log2-ratio value.Schizont stage parasites (44 hour post invasion) were isolated from escribed , 75. Theescribed . PCR prohttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE48894).The raw ChIP-on-chip data presented and discussed in this publication were deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE48894  were loaded with samples as recommended by the manufacturer; the micro channels were filled with 9 μl of gel-dye mix in the appropriate wells and the 5μl marker and the samples in 1μl running buffer were loaded. The samples were gently mixed and then the chips were immediately inserted into the Agilent 2100 Bioanalyzer and processed.The validation of the ChIP-on-chip assay was performed by qPCR using specific primers . SamplesP. falciparum genome sequences available in the databases: http://plasmodb.org/plasmo/ and http://genome.ucsc.edu/. Sequences upstream of the ATG translation initiation codon, were evaluated as potential promoter regions by analyzing 500 bp upstream and downstream of the PFNF-YB binding site. The WebLogo program was used to represent the DNA sequence of all target genes from the ChIP-on-chip assay.To analyze the promoter regions of putative candidate genes, we used t test or Monte Carlo methods, as indicated. The Monte Carlo permutation test [The statistical significance of differences between means was analysed by Student’s ion test , 78 was


Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.  •The CFP1/SET1 complex engages in dynamic and stable chromatin-binding events•CFP1 uses multivalent chromatin interactions to select active CpG island promoters•SET1A occupancy at CpG island promoters is predominately defined by CFP1•CFP1 targets SET1 to shape promoter-associated H3K4me3 and gene expression Brown et al. show that the SET1 complex is driven to active CpG island promoters via the CFP1 protein, which engages in multivalent chromatin binding to recognize both non-methylated DNA and H3K4me3. This is necessary for normal H3K4me3 at active gene promoters and appropriate regulation of gene expression. Gene expression is controlled by transcription factors that bind to DNA sequences in gene regulatory elements and control how RNA polymerase engages with transcription start sites . HoweverMethylation of histone H3 on lysine 4 (H3K4me) is evolutionarily conserved from yeast to human and widely associated with gene regulatory elements, where it is thought to support gene activity  through its phosphorylated C-terminal heptapeptide repeat (CTD) . This prGiven the fundamental role that the SET1 complex plays in depositing H3K4me and sustaining normal development, here we have focused on understanding how this complex is targeted to chromatin. By combining live-cell imaging and functional genomics, we discover that the CFP1 component of the SET1 complex preferentially binds to CGIs of actively transcribed genes through multivalent interaction with chromatin, which requires recognition of non-methylated DNA and H3K4me3. We demonstrate that CFP1 is the predominant targeting module for the SET1A complex, whereas co-transcriptional recruitment appears to play only a minor role in SET1A occupancy. Importantly, CFP1 guides H3K4me3 deposition by the SET1A complex and is required for the appropriate expression of a subset of its target genes.1/2 recovery times in the nucleoplasm of ∼1 s  to map CFP1 binding genome-wide A and 2B.To explore the determinants of CFP1 binding, we carried out ChIP-seq using a GFP-specific antibody in lines stably expressing GFP alone, wild-type GFP-CFP1, or GFP-CFP1 with mutations in the CXXC, PHD, CXXC/PHD, or SID G–2I. ImpThe CFP1 CXXC domain is known to bind non-methylated DNA , and the1H,15N heteronuclear single quantum coherence titration experiments. Addition of the H3K4me3 peptide to the 15N-labeled CFP1 PHD domain induced substantial chemical shift perturbations, suggesting a tight interaction of the CFP1 PHD domain with H3K4me3. In contrast, interaction with H3K4me0 was considerably weaker, as evident from small chemical shift perturbations and fast exchange , in which addition of tamoxifen induces Cfp1 deletion  and SET1A enrichment was greatest at CFP1-bound NMI TSSs  and identified significantly misregulated genes  that had changes in expression of at least 1.5-fold. Importantly, following CFP1 deletion, CFP1 target genes predominantly exhibited reduced expression , whereas a smaller subset (584) showed increased expression -associated histone modifications . Given tAlthough our observations provide a potentially simple explanation for how the CFP1/SET1 complex regulates H3K4me3 at actively transcribed CGI-associated genes, it remains less clear how the H3K4me3-predominating state would be initiated in the first place, given that the CFP1/SET1 complex must recognize pre-existing H3K4me3. One possibility is that the CFP1/SET1 complex binds to CGIs where the MLL1/2 complexes have already initiated low-level deposition of H3K4me3. However, removal of MLL1/2 has little effect on the levels of H3K4me3 at actively transcribed genes in ESCs, suggesting that the MLL1/2 and SET1 complexes do not act in a simple linear pathway or that MLL1/2 is not required to maintain elevated H3K4me3 once it has been initiated . Alterna∗ mutant effectively, we used an acquisition rate of 13 fps (1/2) were calculated using a biexponential fit. Briefly, this involved deriving t1/2 values from individual cells  that there was no difference between the wild-type and mutant versions of CFP1.FRAP experiments were performed on an UltraView spinning disk microscope (Perkin Elmer) equipped with an EM-CCD camera (Hamamatsu) using a 60x/1.4NA oil objective. 50 pre-bleach and 1,000 post-bleach images were captured at a rate of 8 frames per second (fps) B and C af 13 fps E and F. f 13 fps . Half real cells F and S1Gransgene H and S1I1H,15N HSQC spectra of 0.1–0.2 mM uniformly 15N-labeled CFP1 PHD finger in 20 mM Tris-HCl buffer, pH 6.8, 100 mM NaCl, 2.5 mM DTT, and 7% D2O were collected on a Varian INOVA 600 MHz spectrometer. Spectra were recorded at 298K using 1,024 × 128 increments, and a spectral width of 8,820 × 1,974 Hz in the 1H and 15N dimensions, respectively. The binding was characterized by monitoring chemical shift changes as histone tail peptides (synthesized by the University of Colorado Denver Peptide Core Facility) were added stepwise. The dissociation constants (Kds) were determined as described in the The Kd values were determined as described in the Spectra were recorded at 25°C on a Fluoromax-3 spectrofluorometer (HORIBA). The samples containing the CFP1 PHD finger in 20 mM Tris-HCl buffer, pH 6.8, 100 mM NaCl, and 2.5 mM DTT and progressively increasing concentrations of the histone peptide were excited at 280 nm. Emission spectra were recorded over a range of wavelengths between 320 and 380 nm, with a 1-nm step size and a 1-s integration time and averaged over 3 scans. The Sequencing libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina and sequenced on either an Illumina HiSeq2500 or NextSeq500.ChIP was performed as described previously , with miCfp1fl/fl cells were spiked with a fixed number of Drosophila SG4 cells  for 20 min and then RNA was isolated by TRIZOL (Thermo Fisher Scientific) extraction. RNA was incubated with Biotin-HPDP and biotinylated RNA was captured with μMACS streptavidin beads (Miltenyi). Biotinylated RNA was depleted of ribosomal RNAs using the Low Input RiboMinus Eukaryote System v2 kit (Thermo Fisher Scientific). cDNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit and subjected to sequencing on the Illumina NextSeq500 platform.Cfp1fl/fl ESCs were cultured for 96 hr in the presence or absence of 4-OHT. For each condition, 4 × 106 cells were spiked with 1 × 106Drosphila SG4 cells. Nuclei were extracted, and an aliquot of nuclei corresponding to 4 × 105 cells was collected for genomic DNA extraction, whereas the remaining nuclei were subject to conventional RNA extraction using TRIZOL (Thermo Fisher Scientific). Nuclear RNA was depleted of ribosomal RNAs using the NEBNext rRNA Depletion kit, and cDNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit. In parallel, genomic DNA (gDNA) was used to prepare “input” DNA libraries using the NEBNext Ultra DNA Library Prep Kit. Nuclear RNA (nucRNA) and gDNA libraries were sequenced on the Illumina NextSeq500 platform.D.A.B. (ORCID ID 0000-0001-7531-8230), V.D.C. (ORCID ID 0000-0002-2920-1274), A.F., J.A., S.I., L.S., T.G.K., H.K., and R.J.K. conceived the project. T.G.K., S.K., T.G.K., H.K., and R.J.K. secured funding. D.A.B., V.C.D., A.F., J.A., S.I., N.P.B., E.D., M.N., M.M., H.K.L. (ORCID ID 0000-0002-5694-0398), A.H.T., and H.W.K. (ORCID ID 0000-0001-5972-8926) generated reagents and performed the experiments. D.A.B., V.D.C., A.F., N.P.B., S.K., L.S., T.G.K., H.K., and R.J.K. wrote and edited the manuscript.


Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of DOI:http://dx.doi.org/10.7554/eLife.21064.001 Polycomb group (PcG) proteins are evolutionarily conserved epigenetic repressors of developmental genes. PcG-mediated gene silencing involves at least two distinct enzymatic activities directed to histone tails: the first mediates Histone H2A mono-ubiquitination at K119 (H2AK119ub1) by the polycomb repressive complexes 1 (PRC1), while the second mediates H3 tri-methylation at K27 (H3K27me3) by the polycomb repressive complex 2 (PRC2) . AccordiThe molecular complexity underlying PcG-mediated gene silencing could be partly explained by the diversity of the PCGF factors (from PCGF1 to PCGF6) that directly associate with RING1A/B proteins. For example, canonical PRC1 (cPRC1) may include PCGF2  or PCGF4 . These cPRC1 complexes can be further classified into specific sub-complexes according to their association with CBX (CBX2/4/6/7/8) or PHC  . CBX proNon-canonical PRC1 (ncPRC1) associates with PCGF1, PCGF3, PCGF5 or PCGF6 and RYBP (RING1 and YY1-binding protein) or YAF2 (YY1 Associated Factor 2), but does not interact with CBX2/4/6/7/8 or PHC1/2/3 . In addiAnother PCGF homolog, PCGF6, was first identified in a multimeric protein complex associated with the E2F6 transcription factor . This coPcgf6 conditional allele, we show that PCGF6 and RING1B common targets are enriched for meiosis- and germ cell-related genes in ESCs, and that such genes are robustly de-repressed in the absence of PCGF6 (Pcgf6-KO). Importantly, silencing of germ cell-related genes by PCGF6 likely plays a role in proliferation and growth of ESCs. We further demonstrate that PCGF6 is involved in pre- and peri-implantation mouse development. Indeed, loss of Pcgf6 leads to pleiotropic defects in vivo, including aberrant axial development and impaired placenta formation. We also reveal a unique recruitment mechanism amongst PRC1 complexes whereby PCGF6-PRC1 utilizes its MGA and MAX components as a heterodimeric DNA binding module to directly recognize and repress expression of germ cell- and meiosis-related genes to support ESC maintenance and embryonic development.In this study, we therefore purified the PCGF6-PRC1 complex and examined the contribution of PCGF6 to ESC maintenance and embryonic development. We demonstrate that PCGF6 mediates repression of target genes by recruiting RING1B and facilitating H2AK119ub1. Taking advantage of a Previous proteomic approaches have repeatedly identified PCGF6 as a component of multimeric protein complexes designated as PCGF6-PRC1 that included MAX, MGA, E2F6, TFDP1, RING1B, RING1A, CBX3, RYBP, L3MBTL2, YAF2 and WDR5 in human cell lines . To addrPcgf6-deficient (Pcgf6-KO) ESCs (We went on to confirm these results by immunoprecipitation followed by immunoblotting (IP-IB). For this purpose, we stably expressed FLAG-tagged PCGF6 in KO) ESCs  or FLAG-KO) ESCs . We obseKO) ESCs . We indeRing1A/B leads to widespread disruption of PRC1-dependent gene repression (Pcgf6-KO ESCs. Our results considerably overlapped with those reported recently (Tex19.1 (testis-expressed protein 19A) and Tdrkh (Tudor and KH domain containing)  and ncPRC1 is mediated by RING1A and RING1B . Consistpression . To deterecently  (Yang etrecently . This antaining) . Indeed,Hoxd (homeobox protein Hox-D) gene cluster, were not bound by PCGF6 -bound genes. As CBX7 is a key component of cPRC1 , exclusiKdm2b-KO ESCs  ESCs (Pcgf6 (Pcgf6-KO)  ESCs . To gainKO) ESCs . This ancgf6-KO) , left. W-KO ESCs , left. W-KO ESCs  in the PdKO ESCs , right, dKO ESCs . TogetheTo gain insight into the physiological role of PCGF6-PRC1-mediated gene regulation in ESCs, we performed gene ontology (GO) analyses, which revealed that PCGF6-PRC1 target genes are enriched in meiosis- and germ cell-related genes as suggested previously , unlike Pcgf6, revealing a role for PCGF6 in mediating H2AK119ub1 deposition  and Mael (maelstrom spermatogenic transposon silencer) was indeed considerably depleted in the absence of Pcgf6  and Pcgf4 (Bmi1), both of which are essential components of the cPRC1 complex, led to reduction of RING1B binding of only cPRC1 target genes (PCGF6-CBX7+RING1B+)  in a conserved residue critical for interaction with RING1B . We expr and MAX . ChIP-qP and MAX  and, as  and MAX .L3mbtl2-KO ESCs  ESCs  ESCs . We did  in ESCs .Eed-KO ESCs (deficient for H3K27me3), and compared PCGF6-binding levels at target genes. We did not observe any significant differences in FLAG-PCGF6 binding between WT and Eed-KO cells  showed significant up-regulation in Max-KD or Mga-KD ESCs (Ddx4 [DEAD (Asp-Glu-Ala-Asp) box polypeptide 4], Tdrd1 (Tudor domain containing 1), Stag3  and Mael loci, and confirmed our RNA-seq results  in FLAG-PCGF6 expressing ESCs. The e siRNAs , top. Wenchanged , bottom.-KD ESCs . We perf results .In contrast, genes bound by PCGF6 and RING1B, but not MAX (MAX-PCGF6+RING1B+), were barely up-regulated . IntriguMax-KD or Mga-KD ESCs revealed that PCGF6 binding to target loci depends on MAX and MGA  led to slower ESC proliferation and cell differentiation  domain [hereafter referred to as MAX(VD) or MAX(Δb)] ] , left, fMAX(Δb)] , right. Max-KO ESCs  and fetal bovine serum as suggested previously , which already express such germ cell-related genes. We induced the PGCLCs via epiblast-like cells (epiLCs), using an in vitro culture system that we have previously developed  mice were viable and fertile, but were not born at the normal Mendelian ratio (Pcgf6−/− homozygous embryos could be observed as early as the blastocyst stage [3.5 days post coitum (dpc)]. Furthermore, embryonic death in the Pcgf6−/− line could be continuously detected during post-implantation development. Consistently, we found that about one third of the surviving Pcgf6−/− embryos at 10.5 dpc exhibited growth retardation . We mated homozygous or heterozygous males with heterozygous females, and cultured the blastocysts in ESC inducing conditions until ESC-like colonies appeared. From 73 blastocysts collected, we could establish 22 ESC-like lines. Remarkably, among these 22 lines, not a single one was homozygous Pcgf6−/−  specification of the axial skeleton is well characterized . Given tese mice . To furtese mice . We furtvertebra . These sox genes . We indePcgf6−/− mice  and Tpbpa , respectively, was dramatically decreased. These findings demonstrate further roles of PCGF6 in peri-implantation development, including A-P specification and regulation of placental development.In addition to the skeletal phenotypes, we also noted significant reduction of placental size, manifested by hypoplasticity and malformation of the labyrinth layer, in the −/− mice . In partIn the present study, we show that PCGF6 constitutes a non-canonical PRC1 complex, PCGF6-PRC1, that includes the core PRC1 component RING1B. PCGF6 physically interacts with RING1B, and plays a role in recruitment of RING1B to target genes, followed by deposition of H2AK119ub1 and transcriptional silencing. We have previously reported that the PCGF1-associated ncPRC1 (PCGF1-PRC1) recruits PRC2 to target genes . Here, wLike PCGF1-PRC1, PCGF6-PRC1-mediated targeting of PRC2 also takes place in endogenous genomic loci, in particular at promoters of a certain group of genes that are enriched for germ cell-specific and meiosis-specific functions . HoweverBy surveying DNA sequences of the PCGF6-PRC1 target genes, we find that they are enriched for the binding motif of the bHLHZ  transcription factor MAX . PreviouPcgf6-KO ESCs   . Such ab)  that was modified to express an N-terminal Flag and 2XStrep2 tag, an N-terminal 3xFlag tag, or an N-terminal HA tag. All PCR generated constructs were verified by sequencing. Mutations of the PCGF6 RING-finger domain (H155Y) and the MAX basic domain (L31V and E32D) were introduced into the wild type Pcgf6 and Max constructs, respectively, using the PrimeSTAR Mutagenesis Basal Kit .The full length mouse AB_262044), HA-tag , RYBP , L3MBTL2 , MAX , MGA , PCGF1 , PCGF2 , PCGF6 , EZH2 , SUZ12 , SET1 , trimethylated Histone H3 lysine 27 (H3K27me3) , dimethylated histone H3 lysine 9 (H3K9me2) , H3K27ac , monoubiquitinated histone H2A lysine 119 (H2AK119ub1) , mouse IgM , histone H3 , and LAMIN B .The RING1B mouse monoclonal antibody has been described previously . A rabbiPcgf6 (Rosa26::CreERT2 (ERT2-Cre) transgenic mice purchased from Artemis Pharmaceuticals (Germany). ESCs we(Germany). Male ES KO ESCs  were geng/+ ESCs , Pcgf2/4dKO ESCs , Kdm2b-K-KO ESCs , and Max KO ESCs  were des KO ESCs . Prior tMax and Mga were selected according to a previous report , 1 mM EDTA, 140 mM NaCl, 0.4% NP-40, and 0.5 mM PMSF] and sonicated for several seconds. After centrifugation, the supernatant was collected, and then incubated with anti-FLAG antibody  for 120 min at 4°C. The immune complexes were captured by incubation with protein A/G magnetic Dynabeads  for 60 min at 4°C. The bead-bound proteins were washed with IP buffer, eluted in SDS sample buffer under reducing condition, separated on SDS-PAGE gels, and subjected to western blot analysis. Tandem mass spectrometry (LC–MS/MS) was performed as described previously . MateriaGapdh as housekeeping gene. For RNA-seq studies, RNA integrity was assessed on a BioAnalyzer; all samples had a RNA Integrity Number (RIN) ≥9.5 (Agilent Laboratories). Sequencing libraries were generated according to Illumina’s instructions and sequenced on the Illumina HiSeq platform as described previously  with the RNase-Free DNase Set (Qiagen) according to the manufacturer's instructions. Genomic DNA-free RNA samples were further purified using the RNeasy kit RNA cleanup protocol. For subsequent RT-PCR analysis, cDNA was synthesized with the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). Quantitative real-time PCR was performed by using the Brilliant III SYBR Green QPCR Master Mix , using eviously .6 cells. Antibody bound proteins were isolated on protein A/G magnetic Dynabeads (Invitrogen), washed extensively, eluted, cross-links were reversed, and then the samples were sequentially treated with RNase and proteinase K before being purified using phenol-chloroform extraction. Real-time qPCR was performed using the Brilliant III SYBR Green QPCR Master Mix (Agilent). For H2AK119u1-ChIP using E6C5 (Millipore #05–678), pre-cleared chromatin from 2–3 × 106 cells was incubated with 40 ul of E6C5 antibody (Millipore #05–678) , and 30 ul of original Protein A dynabead slurry (Invitrogen) was incubated with 3 ug (=3 ul) of rabbit anti-mouse IgM antibody (Millipore #12–488) . Next day, the chromatin-1st antibody complexes were immunoprecipitated with second antibody - preconjugated protein A dynabeads, and subjected to the conventional ChIP experiments as described above.Chromatin immunoprecipitation (ChIP) was performed as previously described , with miKdm2b-KO ESCs were also obtained from NCBI GEO (Series GSE55698).ChIP-seq libraries were prepared according to Illumina’s instructions accompanying the NEBNext ChIP-Seq Library set  and quantified by the KAPA Library Quantification Kit , and their sizes were confirmed by Bioanalyzer (Agilent). Libraries were sequenced using Illumina  HiSeq system as described previously . The peahttp://meme-suite.org/). Briefly, 500 bp sequences around the centers of ChIP-seq peaks were collected from the mouse genome and enriched motifs were calculated using the above program with default parameters. Graphical representation of the sequence logo, where letter height was determined by Shannon entropy, was generated using our in-house program.Binding motifs of PCGF6, RING1B, CBX7, MAX and MYC were detected using a computer program DREME included in the motif analysis suite MEME version 4.9.1  and GSE87484 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=sbebmckgtlyhlat&acc=GSE87484).Sequencing and microarray data can be accessed via the geo accession GSE84480 .Thank you for submitting your article "PCGF6-PRC1 suppresses premature differentiation of embryonic stem cells by silencing germ cell-related genes" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.This study presents an interesting investigation into the role of the PRC1 cofactor PCGF6 in PRC1-mediated gene silencing. Following up on the recent characterization of non-canonical PRC1 sub-complexes the authors investigate the function of one such sub-complex containing the PCGF6 protein. They present data suggesting that PCGF6 is involved in the recruitment of non-canonical PRC1 to a subset of genes that encode germ cell regulators. Mechanistically, they propose that recruitment of PCGF6-PRC1 is mediated by MAX/MGA transcription factors.Although the subject of the study is important in the field of Polycomb biology, the data presented lack clear evidence for a role of MAX in recruitment. It remains mostly correlative and does not provide direct evidence that MAX/MGA transcription factors recruit PCGF6-PRC1. There are a number of other concerns, controls and clarifications that need to be addressed in a revision before the study can be considered for publication.1) A major concern of the work is that the evidence for a role of MAX in specifying PCGF6-PRC1 recruitment is overstated. Motifs enriched within PCGF6 binding sites are not exclusive to MAX. The effects of MAX knockdown on gene expression and observations based on the DNA-binding mutant of MAX do not rule out an indirect mode of action. There is no demonstration direct MAX-mediated recruitment of PCGF6-PRC1 with the enforced tethering system used in the study. The mass spectrometry data uncover an interaction with MGA, but not MAX. Hence, one of the major conclusions of the study is not adequately demonstrated by the data presented in the manuscript. The authors must demonstrate direct MAX-mediated recruitment of PCGF6-PRC1 with the enforced tethering system or alternative approach. If a Tet-MAX can recruit the PCGF6 ncPRC1 complex this would support their conclusions.2) In 3) A suggestion to improve the paper would be to move the 4) In the subsection “PCGF6 forms complexes with PRC1 components”, the authors purify the PCGF6-PRC1 complex and report copurification of CBX1 and CBX3 (HP1) proteins. This is an intriguing result that could link this complex to H3K9me3 and heterochromatin. Later, they confirm the presence of L3MBTL2 at GF6-PRC1 target genes. In the second paragraph of the subsection “PCGF6 recruits RING1B to target genes and facilitates downstream H2K119ub1 deposition” and  […] Although the subject of the study is important in the field of Polycomb biology, the data presented lack clear evidence for a role of MAX in recruitment. It remains mostly correlative and does not provide direct evidence that MAX/MGA transcription factors recruit PCGF6-PRC1. There are a number of other concerns, controls and clarifications that need to be addressed in a revision before the study can be considered for publication.1) A major concern of the work is that the evidence for a role of MAX in specifying PCGF6-PRC1 recruitment is overstated. Motifs enriched within PCGF6 binding sites are not exclusive to MAX. The effects of MAX knockdown on gene expression and observations based on the DNA-binding mutant of MAX do not rule out an indirect mode of action. There is no demonstration direct MAX-mediated recruitment of PCGF6-PRC1 with the enforced tethering system used in the study. The mass spectrometry data uncover an interaction with MGA, but not MAX. Hence, one of the major conclusions of the study is not adequately demonstrated by the data presented in the manuscript. The authors must demonstrate direct MAX-mediated recruitment of PCGF6-PRC1 with the enforced tethering system or alternative approach. If a Tet-MAX can recruit the PCGF6 ncPRC1 complex this would support their conclusions.We thank you for these critical comments and suggestions. Although we failed to detect MAX in our mass spec analysis, we found a considerable association of MAX with FLAG-PCGF6 by IP-immunoblotting with several negative controls as shown in the revised Upon the request, we further examined whether the enforced tethering of MAX could recruit PCGF6-PRC1. We expressed MAX/TETR and ectopically tethered this fusion protein to a pre-integrated TetO array in ESCs . ChIP-qP2) In Thank you for this suggestion. We included a control Flag ChIP-seq data (obtained from a Flag-empty vector cell line) alongside the Flag-PCGF6 data in 3) A suggestion to improve the paper would be to move the  in vivo are much starker than the in vitro differentiation defects currently featured in We appreciate your suggestion and moved these figures to 4) In the subsection “PCGF6 forms complexes with PRC1 components”, the authors purify the PCGF6-PRC1 complex and report copurification of CBX1 and CBX3 (HP1) proteins. This is an intriguing result that could link this complex to H3K9me3 and heterochromatin. Later, they confirm the presence of L3MBTL2 at GF6-PRC1 target genes. In the second paragraph of the subsection “PCGF6 recruits RING1B to target genes and facilitates downstream H2K119ub1 deposition” and Thank you for your important suggestion. We performed ChIP-seq for H3K9me2 and the summary of data is included in We also performed RNA-seq analysis of Cbx1/3-double knockout (dKO) ESCs to investigate the role of CBX1/3 for the regulation of PCGF6-PRC1 targets . We did We revised the main text  accordingly.


RORC gene, plays a critical role in the development of naive CD4+ cells into fully differentiated Th17 lymphocytes. Th17 lymphocytes are part of the host defense against numerous pathogens and are also involved in the pathogenesis of inflammatory diseases, including autoimmune disorders. In this study, we functionally examined four naturally occurring polymorphisms located within one of the previously identified GC-boxes in the promoter region of the gene. The single nucleotide polymorphisms (SNPs) rs774872314, rs116171003 and rs201107751 negatively influenced the activity of the RORγT promoter in a gene reporter system and eliminated or reduced Sp1 and Sp2 transcription factor binding, as evidenced by the electrophoretic mobility shift assay (EMSA) technique. Furthermore, we investigated the frequency of these SNPs in the Polish population and observed the presence of rs116171003 at a frequency of 3.42%. Thus, our results suggest that polymorphisms within the RORγT promoter occurring at significant rates in populations affect promoter activity. This might have phenotypic effects in immune systems, which is potentially significant for implicating pathogenetic mechanisms under certain pathological conditions, such as autoimmune diseases and/or primary immunodeficiencies  syndrome).RAR-related orphan receptor gamma RORγT, a tissue-specific isoform of the  RORC gene by a selection of alternative promoters [IL17A and IL17F. Th17 cells protect against numerous pathogens  [RORγ and RORγT proteins are DNA-binding transcription factors transcribed from the same romoters ,2,3. Botromoters . RORγT inthracis , Staphyls aureus , and Canlbicans) , rheumatlbicans) , psoriaslbicans) , or Gravlbicans)  suggest RORγT expression is strictly limited to the subset of activated CD4+ cells, while RORγ is more broadly expressed. Processes that are responsible for the observed pattern of expression include epigenetic DNA modifications [RORγT and identified several elements important for its expression, including four E-boxes capable of binding upstream stimulatory factor(USF) transcription factors [RORγT gene. This prompted us to investigate their functional relevance in human lymphocytes using a luciferase reporter gene assay and an electrophoresis mobility shift assay, and to confirm the distribution of the identified polymorphisms in the Polish population. ications  and dire factors  and two  factors . Both id factors . After a factors , we foun2. A Jurkat (human T cell lymphoblast-like) cell line was purchased from ATCC  and maintained under standard conditions in Roswell Park Memorial Institute (RPMI)-1640  containing 10% fetal bovine serum  at 37 °C in an atmosphere of 5% CORORγT gene were described in our previous works [® 200 PRO . The culture medium was transferred to 96-well transparent plates, for secreted embryonic alkaline phosphatase (SEAP) activity measurements, and lysis buffer was added to the cells before they were frozen at −70°C. After thawing, the luciferase activity was measured. Alkaline phosphatase control activity was determined spectrophotometrically at 405 nm and was used as a transfection efficiency control . Luciferase values were normalized per corresponding SEAP activity.All promoter constructs for the us works ,13, withRORγT promoter insert using the polymerase chain reaction (PCR)-based method. Following the reaction, the restriction enzyme DpnI  was added to remove the plasmid template. All mutations were identified by restriction enzyme analysis and further verified by sequencing. The mutants were then cloned into the pGL3-Basic vector using the restriction enzymes Acc65I and HindIII . The following primer pairs were used for mutagenesis: 5′-TGGGGCCACCTGGGAGCGGGGGAGCCTGGACCCT-3′ (p1f) and 5′-AGGGTCCAGGCTCCCCCGCTCCCAGGTGGCCCCA-3′ (p1r) (both for the rs774872314 SNP); 5′-TGGGGCCACCTGGGGTCGGGGGAGCCTGGACCCT-3′ (p2f) and 5′-AGGGTCCAGGCTCCCCCGACCCCAGGTGGCCCCA-3′ (p2r) (both for the rs116171003 SNP); 5′-TGGGGCCACCTGGGGGTGGGGGAGCCTGGACCCT-3′ (p3f) and 5′-AGGGTCCAGGCTCCCCCACCCCCAGGTGGCCCCA-3′ (p3r) (both for the rs200231898 SNP); and 5′-TGGGGCCACCTGGGGGCAGGGGAGCCTGGACCCT-3′ (p4f) and 5′-AGGGTCCAGGCTCCCCTGCCCCCAGGTGGCCCCA-3′ (p4r) (both for the rs201107751 SNP).Mutagenesis was performed directly on the pUC18 plasmid carrying −180/+78 sequence of the An electrophoretic mobility shift assay (EMSA) was performed using infrared dye-labeled (IRD-labeled) DNA probes. Nuclear extracts were prepared from Jurkat cells using a Nuclear Extract Kit . The following DNA probes were used:5′-TGGGGCCACCTGGGGGCGGGGGAGCCTGGACCCT-3′ ) and 5′-AGGGTCCAGGCTCCCCCGCCCCCAGGTGGCCCCA-3′ ) (both for the wild type sequence) 5′-TGGGGCCACCTGGGAGCGGGGGAGCCTGGACCCT-3′ (p1f) and 5′-AGGGTCCAGGCTCCCCCGCTCCCAGGTGGCCCCA-3′ (p1r) (both for the rs774872314 SNP); 5′-TGGGGCCACCTGGGGTCGGGGGAGCCTGGACCCT-3′ (p2f) and 5′-AGGGTCCAGGCTCCCCCGACCCCAGGTGGCCCCA-3′ (p2r) (both for the rs116171003 SNP); 5′-TGGGGCCACCTGGGGGTGGGGGAGCCTGGACCCT-3′ (p3f) and 5′-AGGGTCCAGGCTCCCCCACCCCCAGGTGGCCCCA-3′ (p3r) (both for the rs200231898 SNP); and 5′-TGGGGCCACCTGGGGGCAGGGGAGCCTGGACCCT-3′ (p4f) and 5′-AGGGTCCAGGCTCCCCTGCCCCCAGGTGGCCCCA-3′ (p4r) (both for the rs201107751 SNP). The DNA probes were incubated on ice with 2.5 µg of nuclear extract in binding buffer containing 10 mM Tris–HCl (pH = 8.0), 50 mMKCl, 18.5 mMNaCl 1 mM Dithiothreitol (DTT), 0.1% IGEPAL, 5% glycerol, and 100 ng of salmon testis DNA (to prevent the nonspecific binding of proteins to the probes). For the competition assay, 50- and 200-fold molar excesses of unlabeled oligonucleotides (wt) were added to the reaction mixture. The DNA-protein complexes were then fractionated on 5% non-denaturing polyacrylamide gels and analyzed on an Odyssey  infrared fluorescence scanner.The participants were recruited from 2010–2012 for the TESTOPLEK research project and registered as a POPULOUS collection in the BioBank Lab of The Department of Molecular Biophysics of the University of Lodz . Each suThe saliva samples were stored at room temperature until initial processing. DNA was manually isolated from 500 µL of saliva according to the manufacturer’s procedure . The elution volume was 50 µL. DNA was quantified using the broad-range Quant-iT™ dsDNA Broad Range Assay Kit . All DNA samples underwent quality control testing using PCR to determine sex, utilizing melting profile analysis and obtaining specific fragments of DNA from samples of human biological material (Patent number PL406569-A1) . Afterwa® Automated Workstation . The mixture was composed of 2×GoTaq® Colorless Master Mix , 10×LC Green Plus® dye , 0.5 µL of 10 µM primers , and 3 µL of DNA (200 pg/µL), and filled with 0.5 µL of water to the final volume. The reaction was performed on a 384-well microplate using the CFX384™ real-time PCR system  . The reaction conditions were as follows: initial denaturation at 95 °C for 3 min, 50 amplification cycles of denaturation at 95 °C for 20 s, and annealing at 60 °C for 30 s. The plate was read after each cycle. Directly afterwards, the melting curve was determined by incubating the plate at 90 °C for 60 s and 40 °C for 60 s, and increasing from 65 °C to 95 °C (in 0.2 °C increments) for 10 s while reading the plate. The obtained data were analyzed with the Bio-Rad Precision Melt Analysis Software, version 1.2 (Bio-Rad Laboratories Inc.).The single standard reaction mixture (10 µL) was prepared using the JanusGenetic variation observed in HRM melting curve analysis was verified by a direct sequencing method for at least three samples representing each cluster. Preparation of samples for sequencing was conducted using specific primers  according to the previously described protocol . AnalysiFor the detected polymorphisms, the parameters obtained from GenBank were assigned dbSNP IDs (rs numbers) and coding DNA nucleotide positions, followed by reference sequence NM_001001523.1. These data were used below for the variant nomenclature.RORγT gene promoter for potential transcriptional factor binding sites was performed using MatInspector software [p-value of 0.05 or lower was considered statistically significant.Analysis of the software . Single software . StatistRORγT promoter variants in human lymphocytes. Because the rs774872314, rs116171003, rs200231898, and rs201107751 polymorphisms are all located within one of the identified GC-boxes (GC-box 2)  led to an almost complete loss of activity of the RORγT promoter  see  that is oter see .RORγT promoter were determined using EMSA. Based on results from the reporter gene assay, we hypothesized that SNPs could affect protein binding to GC-box 2. To address this hypothesis, we conducted EMSA with probes containing wild-type GC-box 2 and polymorphic residues. The specificity of the observed bands was confirmed using the competition assay with excess unlabeled wild-type probe  and GC-box 2 (+28/+36), which are crucial for the activity of the promoter. Furthermore, site-directed mutagenesis suggested that promoter function is dependent on the cooperative interactions of these two boxes [RORγT promoter in a reporter system.Previously, we showed that the 5′-flanking regions of the wo boxes . AnalyziRORγT promoter activity. Thus, polymorphisms demonstrating the strongest inhibitory effect on promoter activity  see . EMSA pevity see  have alsvity see , which iphocytes . On the phocytes , and thaproteins ,28,29,30IL17A and IL17F by binding to ROR-response elements (ROREs) present in their promoters [Bacillus anthracis [Staphylococcus aureus [Candida albicans [RORγT expression. Very low expression of RORγT is observed in carriers of mutations in the STAT3 gene. This deficiency of RORγT results in the lack of Th17 cells in the bloodstream [Staphylococcus aureus and Candidia albicans. Other SNPs, namely, rs774872314 and rs116171003, also have significant potential to impair the expression of RORγT, which leads to changes in the phenotype of immune cells. Thus, the rs774872314 and rs116171003 SNPs can reduce the activity of this promoter and, in turn, decrease the expression of RORγT. This might be considered a potential protective factor that decreases the chances for the development of autoimmunological disorders that depend on Th17 cells. This would be similar to the rs3811046 and rs3811047/rs2723186 polymorphisms in the IL37 gene [RORγT is considered to be a signature transcription factor for Th17 lymphocytes, as its ectopic expression in CD4+ cells is sufficient to develop the Th17-like phenotype . RORγT iromoters ,33. Th17romoters , rheumatromoters , psoriasromoters , and Graromoters . Howevernthracis , Staphyls aureus , and Canalbicans . Genome albicans ,36,37. Iodstream ,39 and tL37 gene . This isL37 gene ,42, a siRORγT promoter are associated with significant differences in the functionality of this promoter. These observations justify further studies to elucidate the potential effects of the rs774872314, rs116171003, and rs201107751 polymorphisms on the susceptibility of their carriers to certain immunological disorders.In conclusion, our results clearly show that naturally occurring genetic polymorphisms in Sp-binding motifs of the


In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation.Numerous  It haDNA footprinting studies have been instrumental to our understanding of promoter mechanics. This classic approach utilizes the protection from nuclease digestion provided by proteins bound to DNA to produce a highly precise map of the binding site . This mein vitro studies performed on model promoters  have yielded three classes of Crp promoters depending upon the location of the consensus motif sequence(s) relative to the transcription start site (TSS), the number of motif sequences, and the presence of additional transcription factors [in vitro conditions with a focus on characterizing early events during transcription initiation.DNA footprinting has also played a significant role in our current understanding of transcription activation by Crp. Detailed  factors –32. Howein vivo. These techniques have been paramount to studying transcriptional regulators and to construct regulons and transcriptional regulatory networks. However, the information ascertained by application of these methods predominantly provides a binary (present/absent) representation of binding events. Integrating with gene expression analysis allows for expansion of these binary calls to provide conditional activation/repression calls. However, the resolution of ChIP-chip (on the order of kilobases) and ChIP-seq (on the order of hundreds of base pairs) does not enable research to precisely determine the location of the binding event. One of the challenges facing biology is to be able to predict promoter activity. One potential approach to achieve this is by obtaining high-resolution mechanistic information of individual promoters and to convert that mechanistic information into a model of promoter dynamics.Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (chip) or next-generation sequencing have provided genome-scale information on DNA/protein interactions Escherichia coli to elucidate the Fur transcriptional regulon, which predominantly is found to act as a repressor [An enhanced form of ChIP-seq called ChIP-exonuclease (ChIP-exo)  generateepressor .in vitro under in vivo conditions and extend those observations to the genome-scale. Crp provides an ideal entry point for such a study because of the mechanistic and structural information borne out through decades of detailed work on individual promoters [70, with respect to published data on RNAP holoenzyme footprinting data. We then compared the protection pattern provided by Crp to σ70 and surprisingly found tremendous overlap in their DNA footprinting pattern. However, there was very little observed protection of the Crp motif sequence. This phenomenon was then explored in a repressor, ArcA, and the Crp familial protein, Fnr. Lastly, genetic perturbations to Crp/RNAP interactions were introduced and the affects of these mutations were characterized using ChIP-exo.The study of bacterial transcription activation using high-resolution ChIP-exo data could affirm the transcription initiation processes elucidated romoters ,2,36–39.70 ChIP-exo peak distribution provides the bounds of protected DNA regions on the template and nontemplate strand. ChIP-exo profiles across all binding sites were calculated for both the template and nontemplate strand by first calculating the density of the 5’ end of tags for each individual peak region spanning 400 bp centered and oriented relative to the TSS (transcription start site). The median position of the σ70 peak center is 5 bp downstream of the TSS therefore the peak center is found to be an accurate approximation for the TSS  formation, the RPO, the initial transcribing complex (ITC) and the transition to the ternary elongation complex (TEC). However, the closed promoter complex (RPC) does not have an advanced footprint extending to the +20 position.The activity of lambda exonuclease is 5’ to 3’  and, as C complex formation is being captured by ChIP.Unlike the template strand, the ChIP-exo 5’ tag distribution for the nontemplate strand is multimodal. This distribution marks the upstream boundary relative to the TSS. The dominant mode found between -18 and -1 accounts for 28% of the 5’ tag density. Therefore, promoters that belong to this mode have partial to complete protection of the discriminator sequence, the -10 promoter element, and the TGn extended -10 element but little to no protection of the -35 promoter element or any upstream promoter elements . The -35 promoter element is partially protected by the mode farthest upstream which accounts for 9% of the 5’ tag density profile and spans -34 to -23 with a maximum located at -28. The upstream boundary, -3, is located in the center of the -35 element. The downstream mode accounts for 8% of the 5’ tag density and is located downstream of the TSS. The boundaries of this mode are between +4 and +12 with a local maximum at +6. Like the template strand, the DNA protected regions of the different modes on the nontemplate strand provide little to no support that recruitment and RP70 is being capturing in complex with RNAP when assessed in the context of in vitro footprinting studies performed on model promoter constructs. Furthermore, both the template and nontemplate strands provide evidence that σ70 ChIP studies identify stable intermediates during transcription initiation that occur after the recruitment of RNAP and RPC formation. The template strand protected regions in the σ70 ChIP-exo profiles provide the strongest evidence that post-recruitment stable intermediates are being captured. The template strand distribution protection extends to +20 relative to the TSS, which agrees with numerous in vitro studies that have demonstrated a clear transition in the downstream protected boundary from -5 to +5 in RPC to +20 for RPO, ITC, and early TEC complexes (9–12). Hydroxyl radical footprinting studies on RPC formation in the T7A1 promoter showed that the short-lived RPC complex protects DNA to approximately -5 bp (13–15). Similar results were observed in DNase footprinting of the T7A3 promoter, lacUV5, and rrnBP1 . Furthermore, an RNAP mutant with deficient open complex formation was found to have DNase footprints that extend to just +1 at the λPR promoter (18). However, the RPC complex was only observed when the temperature was dropped in most of these studies. The temperature dependent capture of early closed complexes has been shown to be a result greater RPO abundance at physiological temperatures . Conversely, the advanced downstream boundaries centered on +20 has been observed in studies performed on the intermediates leading to RPO and the RPO complex for the T7A1 promoter (13–15), the T7A3 promoter (17), the rrnBP1 promoter , the λPR promoter , and the lacUV5 promoter . Furthermore, the ITC and the transition to the TEC also have a downstream footprint boundary of +20–25. DNase footprinting of T7A1, tac, and lacUV5 promoters showed that the ITC has a slightly advanced footprint at +25 compared with +20 for RPO and the early TEC had a footprint at +30 (25–27).Collectively, the ChIP-exo distribution of the mean 5’ tag density on both strands indicates that σ70 on the nontemplate strand show a multimodal distribution with regions that provide protection to different components of σ70 promoter elements. These were found to form three modes spanning -34 to -23, -18 to -1, and +4 to +12 with the region spanning -18 to -1 accounting for the largest fraction of the 5’ tag density profile. While periodic patterns of DNA protected regions at the upstream boundary are common , the location of the boundaries is supportive of post-recruitment intermediates (27–29). A detailed study on the lacUV5 promoter using DNase I, methylation protection, and exonuclease III protection across transcription intermediates showed that transitions from RPO to ITC undergoing abortive initiation retained strong protection of a region between -24 and -6 to exonuclease III digestion that was reduced to protection of the region downstream of -6 after escaping the abortive transcription phase to produce longer transcripts (27). This is further corroborated by a recent study that showed the lacUV5 promoter has an upstream footprint boundary at -23 in the presence of σ70 compared with -13/-14 for the σ70 lacking transcribing complex (29). A study of the T7A1 promoter using exonuclease III showed a drastic movement in the upstream-protected region from -43 to -3 in the transition from RPO to early transcribing complexes (ITC or TEC) (28). Furthermore, the width of the protected region agrees with studies examining RPO, ITC, and TEC. Early TECs have been found to have footprint regions spanning ~30 bp whereas RPO and ITC have longer footprint seen to be 50+ bp in length .ChIP-exo mean 5’ tag density profiles for σC formation. Genome-scale characterization studies of bacterial transcription have shown the rate-limiting step in transcription predominantly occurs post-recruitment of RNAP . For example, the λPR promoter is limited at the opening of the transcription bubble marked by a slow transition from the closed intermediate to the open intermediate . Furthermore, the promoter λPR’ encodes a promoter-proximal pause site induced by a -10 like sequence downstream of the TSS (36). A similar pause occurs in the lac promoter  and, in fact, it is estimated that the occurrence of promoter-proximal pausing is upwards of 20% in E. coli (38–40). However, numerous additional processes along the trajectory of RPO to TEC formation have been found to be rate determining including scrunching (41), and promoter escape . Therefore, ascertaining the potential bottlenecks in transcription initiation at genome-scale and under in vivo conditions would be of value to genome-scale models of promoter kinetics.Kinetic studies also support the notion that the ChIP-exo data presented here is reflective of stable intermediates occurring post-recruitment of RNAP and RP70 groups . Furthermore, the -10 promoter element has near perfect consensus at the critical -11A position and a partial TGn motif characteristic of the extended -10 promoter element. Group II resembles the motifs found in Group I but with lower sequence conservation in both the -10 and -35 promoter elements. Conversely, Group III has the most divergent -35 motif from consensus and no appreciable motif for the extended -10 promoter element.It is known that promoter sequence elements involved with RNAP holoenzyme recruitment contribute to the post-recruitment kinetics of transcription initiation –24. Thus0 groups  as deter0 groups . σ70-likE. coli Crp. ChIP-exo data showed strong consistency with previously determined Crp binding sites (see deoC promoter is a Class III promoter with two Crp binding sites flanking a CytR regulatory site that represses the activating action of Crp [in vitro [tnaC) and Class II (gatY) promoters that often have no exonuclease protection to the Crp binding site, but instead, have strong protection of the region surrounding the TSS. In fact, these regions correspond greatly with the ChIP-exo profiles generated for σ70 under the same condition but no observed σ70 ChIP-exo peak was detected for the repressed deoC promoter.Transcription factor binding was further studied with ChIP-exo in ites see . ChIP-exites see . The deon of Crp . The ChIin vitro . However70, all Crp ChIP-exo binding profiles were aligned and strand-oriented relative to the TSS. The same was done with the peak center position and the predicted Crp motif sequence  prevents transcription elongation beyond a length of 2–3 nt  and, in E. coli [70 with a strong unimodal distribution on the template strand at +20 and a less defined modal distribution on the nontemplate strand  with restriction sites at the 5’ and 3’ end of the gene. The gene was digested from GeneArt plasmids and ligated into the pKD3 plasmid directly upstream of the chloramphenicol (Cm) resistance gene. Resulting plasmids have the Ar mutant-crp gene, followed by the FRT flanked Cm resistance cassette as in pKD3 plasmid. Linear PCR products were amplified from resulting modified pKD3 plasmids using primers with 5’ overhangs with homology directly upstream of the start codon and downstream of the stop codon of crp gene to direct the insertion. This PCR product was transformed into electrocompetent Δcrp E. coli K12 carrying the pKD46 plasmid, and selected by Cm resistance, correct insertions were verified by Sanger sequencing. The Cm resistance gene was then removed from confirmed mutant strains by FLP recombinase excision transforming with pCP20 plasmid as previously described [structed . The Δcrescribed . The Δcrescribed . The ΔArescribed ,58. The 70, Crp, Δcrp, ΔAr1, ΔAr2, and ΔAr1ΔAr2 experiments, cultures were grown aerobically in shake flasks. Rifampicin conditions were incubated in the presence of rifampicin  for 20 min prior to crosslinking as previously described [M9 minimal media was used for all cultures with 2 g/L of glucose, fructose, or glycerol. For σescribed . Fnr and70 (Neoclone WP004), and anti-Myc (Santa Cruz Biotechnology sc-40). Immunocomplexes were captured using Pan Mouse IgG Dynabeads (Life Technologies). The following library preparation steps were sequentially performed while the protein/DNA/antibody complexes were bound to the magnetic beads: end repair (NEB End Repair Module), dA tailing (NEB dA-Tailing Module), adaptor 2 ligation (NEB Quick Ligase), nick repair (NEB PreCR Repair Mix), lambda exonuclease treatment (NEB), and RecJf exonuclease treatment (NEB). A series of step-down washes were done between all steps using buffers previously described [The ChIP-exo protocol was adapted from Rhee and et al. for the Illumina platforms with the following modifications . DNA croescribed . Strand escribed  with defescribed  with theGene expression analysis was performed using a strand-specific, paired-end RNA-seq protocol using the dUTP method . Total RS1 Fig70 grown on fructose minimal media for the template (dashed black trace) and the nontemplate strand (solid black trace). Also shown are the peak center positions relative to the TSS (blue bars). (B) The mean 5’ tag density profile is shown for σ70 grown on glucose minimal media for the template (dashed black trace) and the nontemplate strand (solid black trace). Also shown are the peak center positions relative to the TSS (blue bars).(A) The mean 5’ tag density profile is shown for σ(EPS)Click here for additional data file.S2 Fig(A) The mean 5’ tag density profile is shown for Crp grown on fructose minimal media (a Crp activating condition) for the template (dashed black trace) and the nontemplate strand (solid black trace). Also shown are the peak center positions (blue bars) and predicted Crp binding sites (gray bars) relative to the TSS. This profile closely resembles the Crp ChIP-exo profile generated on glycerol minimal media. (B) The mean 5’ tag density profile is shown for Crp grown on glucose minimal media (a Crp repressing condition) for the template (dashed black trace) and the nontemplate strand (solid black trace). Also shown are the peak center positions (blue bars) and predicted Crp binding sites (gray bars) relative to the TSS. The peak regions detected do not align well to the TSS, a stark divergence from profiles observed on activating carbon sources.(EPS)Click here for additional data file.S3 Figcrp ChIP-exo profiles generated using the anti-crp antibody shows poor correlation between biological replicates.A whole genome correlation plot is shown for the Δ(EPS)Click here for additional data file.S4 FigCrp protected footprints were examined by adding rif to the culture medium prior to harvest capture RNAP holoenzyme at stable intermediates prior to and including the ITC. Crp ChIP-exo distributions are shown for shared peak regions between rif-treated and wild type cultures grown on glycerol. The protected footprint regions mirror those found in the non-rif treated samples.(EPS)Click here for additional data file.S5 FigThe mean 5’ tag density profiles are shown for wild type Crp, ΔAr1, ΔAr2, and ΔAr1ΔAr2 mutant strains from top to bottom. This plot shows the systematic los off TSS-centered peak regions with successive perturbation of RNAP holoenzyme/Crp interactions. Ultimately, the ΔAr1ΔAr2 profile does not resemble the profiles obtained under activating conditions that indicated protection of transcription initiation at post-recruitment stable intermediates.(EPS)Click here for additional data file.S6 Figin vitro footprinting studies are not observed under physiological conditions studied performed using ChIP-exo. Instead, the longer-lived Crp/RNAP holoenzyme complex associated with post recruitment stable intermediates (RPO shown) is observed leaving the motif sequence available for nuclease digestion.Shown is an illustration of Class I and Class II promoter models for Crp binding events. Initial recruitment involves a relatively short-lived complex consisting of the motif sequence(s), Crp, and RNAP holoenzyme. This complex observed using (EPS)Click here for additional data file.S1 TableThis table contains the identity of the 699 promoters which make up the trimodal distribution in (CSV)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.