Escherichia coli, to expand the toolkit of genetic parts that can be used to engineer novel synthetic QS-based systems. The library was constructed via polymerase chain reaction with highly constrained degenerate oligonucleotides, designed according to the consensus -35 and -10 sequences of a previously reported constitutive promoter library of graded strength, to maximize the probability of obtaining functional clones. All the promoters have a lux box between the -35 and -10 regions, to implement a LuxR-repressible behaviour. Twelve unique library members of graded strength (about 100-fold activity range) were selected to form the final library and they were characterized in several genetic contexts, such as in different plasmids, via different reporter genes, in presence of a LuxR expression cassette in different positions and in response to different AHL concentrations. The new obtained regulatory parts and corresponding data can be exploited by synthetic biologists to implement an artificial AHL-dependent repression of transcription in genetic circuits. The target transcriptional activity can be selected among the available library members to meet the design specifications of the biological system.The genetic elements regulating the natural quorum sensing (QS) networks of several microorganisms are widely used in synthetic biology to control the behaviour of single cells and engineered bacterial populations via ad-hoc constructed synthetic circuits. A number of novel engineering-inspired biological functions have been implemented and model systems have also been constructed to improve the knowledge on natural QS systems. Synthetic QS-based parts, such as promoters, have been reported in literature, to provide biological components with functions that are not present in nature, like modified induction logic or activation/repression by additional molecules. In this work, a library of promoters that can be repressed by the LuxR protein in presence of the QS autoinducer N-3-oxohexanoyl-L-homoserine lactone (AHL) was reported for  Vibrio fischeri [luxI, encoding for an autoinducer synthase, responsible for the production of the small signalling molecule N-3-oxohexanoyl-L-homoserine lactone (AHL), and luxR, encoding for the LuxR transcription factor that is activated by AHL. The activated complex LuxR-AHL binds a DNA domain called lux box, located upstream of the Plux promoter, thus activating the transcription of downstream genes, involved in bioluminescence, in a concentration-dependent fashion [Quorum sensing (QS) is a cell-to-cell communication mechanism exploited by Gram-negative and Gram-positive bacteria to implement a coordinated and density-dependent control of gene expression –4. The efischeri . Here, t fashion .QS elements have been widely used in synthetic biology to control the behaviour of single cells and bacterial populations via ad-hoc constructed gene networks. Such elements have been exploited to construct exogenously inducible input devices to regulate gene expression in several synthetic devices, such as a lysis device , logic glux and las systems [Synthetic circuits have also been constructed with the aim of improving the knowledge on the behaviour of natural QS systems , 24. Nat systems .In order to improve the knowledge on the quantitative behaviour of QS elements and to expand the list of available parts, novel synthetic components were designed and built up starting from known regulatory elements. Promoters represent key regulatory elements in gene network design. The design of their response logic and the tuning of their activity in a rigorous fashion is necessary in order to meet the specifications derived from circuit design.Constitutive active variants of the LuxR transcription factor were constructed by mutagenesis or truncation to create an AHL-independent LuxR-inducible promoter, functioning as an amplifier that can be re-used in different synthetic networks .QS-based synthetic promoters have been designed to be responsive either to multiple autoinducer molecules  or to adesaR promoter, repressible by the EsaR-AHL complex, to obtain a set of QS-inducible systems with disparate gene expression levels, regulatory range, AHL sensitivity and timing [lsrR genes (starting from the E. coli master QS regulator) able to show novel activation and repression logic [Many efforts have been carried out to create promoters able to sense two or more inputs. Tamsir et al. constructed tandem promoters composed by the sequence of two promoters with the same orientation, behaving as an OR gate with two QS signalling molecules (or other molecules) as inputs , while Bd timing . They ald timing . The twoon logic .lux box, AraC, LacI and TetR operator sites) in the same promoter, in the distal, core or proximal position, obtaining a library of mixed-regulated promoters, responding to one or more stimuli and exhibiting different activation logics [Cox et al. randomly combined different transcription factor binding sites  the E. coli consensus -35 (TTGACA) and -10 (TATAAT) promoter regions. This promoter exhibited an inverted logic, compared to the wild type Plux promoter, being repressed by the LuxR-AHL complex.Egland and Greenberg  reportedThere is no accordance between the findings of Cox et al.  and EglaSerious efforts have been performed in the development of predictive tools to relate promoter strength to the DNA sequence, but no strategy has shown to fully capture this cryptic relation . In factSeveral techniques for libraries generation have been exploited, such as random mutagenesis of existing promoters via error-prone PCR , PCR witIn this work, we report the generation of a library of LuxR-repressible promoters see , inspireThe obtained synthetic promoters, constituting the library, are available in the Registry of Standard Biological Parts (Registry) with BioBrick codes from BBa_J107100 to BBa_J107111 and all the documentation and experience has been shared in this repository .E. coli (Invitrogen) were used for parts cloning and tests. Plasmids were propagated in recombinant strains grown in selective LB medium [TOP10 B medium . TOP10 bApart from the newly synthesized promoters, whose construction is described below, all the parts and plasmids used in this study were assembled from basic or composite parts from the Registry 2009 or 2010 DNA Distribution , accordiDNA-modifying enzymes were purchased from Roche. DNA purification kits were purchased from Macherey-Nagel. All parts were verified through sequencing, performed via the BMR Genomics DNA analysis service . AHL  was dissolved in deionized water to prepare a 2 mM stock, routinely stored at -20°C.The resulting library includes 12 members. lux box) for the binding of the LuxR-AHL activated complex in the core region, between the -35 and -10 sequences [lux box was maintained unaltered.The LuxR-repressible promoters are characterized by the presence of a 20-bp DNA domain  were included in the consensus determination. The resulting consensus sequence is reported in The Anderson constitutive promoter collection  includesreenberg . The Andlux box sequence is present in the ‘core’ position, to provide the AHL-repressible behaviour. However, this part cannot be readily assembled upstream of a gene expression system composed by an RBS-gene-Terminator sequence because the promoter ends with the -10 region. For this reason, the transcription start site (+1) would lie after the beginning of the RBS sequence and the assembly of a spacer between promoter and RBS would be required to properly express the downstream gene. To overcome this problem, a new LuxR-repressible promoter was constructed via oligonucleotide annealing, according to the Anderson library sequence and with the -35 and -10 sequences of BBa_R0061 and BBa_J23119. Two oligonucleotides (sense—J107100_FWD and anti-sense—J107100_REV) were designed according to the structure of the BBa_J23119 promoter  and -10 (TATAAT) regions as the BBa_J23119 promoter of the Anderson collection and the lux box in the core region, overlapping for one nucleotide with both the -35 and -10 sequences  with P_LIB_FWD and P_LUXH_REV primers see , to remoE. coli TOP10 and the grown colonies were selected according to a visual inspection of RFP expression: transformation plates with colonies bearing low-copy plasmids were used to try to select phenotypes only for very strong promoters and not to give a metabolic burden impairing cell growth, while plates with colonies bearing high-copy plasmids should allow the selection of medium to weak promoters. The selected clones were assayed as described in the next section both in the constitutive and regulated conditions. Then, only the clones with detectable fluorescence and which showed repression upon AHL induction were considered. All the promising candidates in the high-copy condition were then transferred in the low-copy plasmid pSB4C5 and all the resulting promoters in the low-copy condition were sequence-verified. Promoters with unique sequences were selected as members of the library. The members of the LuxR-repressible promoter family obtained via the first PCR round with degenerate primers are named BBa_J1071xx, with xx = 01,02,…,10.The obtained parts in both the low- and high-copy plasmids were transformed in The library members were characterized in different contexts. The ‘constitutive’ mode indicates the design that does not include a LuxR expression cassette , while tThe constitutive version of the library, obtained as described above, is shown in E. coli TOP10 cells bearing the RFP-LC library members with the pLuxR-MC plasmid, which is the pSB3K3 plasmid containing the LuxR expression cassette BBa_S03119 as insert. This configuration is named ‘RFP-LC-LUXR’  . Like inGFP was assayed as a different reporter. The BBa_E0240 GFP generator  was XbaI/PstI-digested and inserted in both the SpeI/PstI-digested RFP-MC and RFP-MC-LUXR constructs (thus replacing the RFP expression system with a GFP expression system). These contexts are named GFP-MC and GFP-MC-LUXR, respectively . Here, tLlacO1, PlacIQ and PR promoters, instead of the members of the Anderson library (in this case with the standard DNA scar 'TACTAGAG').The Anderson promoter library members were used to investigate an experimentally observed and previously uncharacterized repressible behaviour of BBa_J23101 by the LuxR-AHL complex. All the sequence-verified Anderson promoters available in the Registry  on the activity of the BBa_J107100 promoter was assayed by constructing redLC (BBa_J107120), redLC-luxRup (BBa_J107121), redLC-luxRdown (BBa_J107122), redLC-luxRMC (BBa_J107120 co-transformed with BBa_S03119), greenMC (BBa_J107123), greenMC-luxRup (BBa_J107124) and greenMC-luxRdown (BBa_J107125) parts. These constructs were submitted to the Registry and are described in 4, 0.1 mM CaCl2, 0.2% casamino acids and 0.4% glycerol), incubated at 37°C, 220 rpm for 16 hours. Cultures were 100-fold diluted in 200 μl of selective M9 in a 96-well microplate. When required, 2 μl of properly diluted AHL were added into the plate wells to reach the desired final inducer concentration. Deionized water was used to prepare the AHL dilutions. No water was added to the wells where AHL-inducible cultures were assayed without inducer. The plate was then incubated in the Infinite F200 microplate reader (Tecan) and the following kinetic cycle, programmed with the i-control software (Tecan), was used to assay the cultures: every 5 min, 15 s linear shaking (3 mm amplitude), 5 s wait, optical density at 600 nm (OD) measurement, fluorescence measurement. The total duration of the kinetic cycle was about 18 hours. RFP detection was carried out with the 535/620 nm filters for excitation/emission and acquisition gain as appropriate, ranging from 50 to 100. GFP-expressing cells were assayed through the same procedure, but using the 485/540 nm filters for fluorescence acquisition. In every experiment, 200 μl of M9 supplemented medium and a non-fluorescent TOP10 culture were also included to measure the background of OD and fluorescence, respectively [Glycerol stocks were streaked on LB agar plates supplemented with the proper antibiotics. Plates were incubated at 37°C for 16 hours. Single colonies were picked and used to inoculate 1 ml of selective M9 supplemented medium  and Relative Promoter Units (RPUs) during the exponential growth phase for each culture. OD, RFP and GFP were background-subtracted as previously described [ln(OD) time series in our experiments. The slope m of this line was computed as the average of the numeric time derivative of ln(OD) in the exponential phase to yield the cell growth rate. Doubling time was computed as ln(2)/m.Raw absorbance and fluorescence time series were processed to obtain doubling time, average RFP and GFP synthesis rate per cell . Relative units, to express the activity of promoters in standardized measurement units, were computed as RPUx = Scell,x/Scell,ref, where x is the culture of interest and ref is the culture bearing the standard reference promoter (BBa_J23101) with RFP or GFP, in the same copy number conditions and with identical measurement system (and DNA junction between promoter and downstream reporter expression system) as the culture of interest, and acquired with the same gain factor.A signal proportional to the RFP or GFP synthesis rate per cell was computed as the numeric time derivative of fluorescence, divided by OD over time. This signal was averaged over the exponential growth phase to obtain the mean M is the switch point of the Hill function, δ+VMAX is the maximum activity in absence of AHL and η is the Hill coefficient. Y is the RPU of the system and 'A' is the AHL concentration in the culture medium.Repression curves were fitted with the Hill equation The coefficient of variation (CV) of the estimated parameters was computed via a Monte Carlo simulation method. In particular, 10,000 synthetic datasets were generated by adding Gaussian noise . Least square optimization was performed using the Matlab Flow cytometric data from single-cell experiments, in FCS format, were exported and processed using an FCS data reader package  via MatScell. Cell-to-cell variability (in terms of CV) was also calculated for every experiment.RPUs were calculated as described above, by using the background-subtracted fluorescence values instead of Although the originally proposed formulas for RPU computation include the growth rate , here itStatistical analysis was performed as reported in : the ANOAll the promoters were initially present in low-copy (LC) or high-copy (HC) vector with an RFP expression cassette downstream, thus enabling quantitative measurements via reporter protein. A total of 90 clones were selected and screened by measuring constitutive RFP expression and repression capability in the regulated mode (promoters 1–90). At the end of this screening, the functional promoters in high-copy were transferred into the pSB4C5 low-copy vector.A total of 28 candidates, all in low-copy plasmid, were obtained and sequenced via the VF2 and VR primers . 10 uniqThe screening procedure for library generation was based on fluorescence measurements in single clones without replicates, in heterogeneous plasmid measurement systems (LC and HC). In order to fully characterize the library performance, the promoters were then tested in different contexts and with a suitable number of biological replicates.All the 11 members of the library of repressible promoters have been assembled in the six contexts represented in The constitutive activity is shown in LtetO1 promoter (according to unpublished experiments of our group). For the promoters with activities that are significantly different among the three measurement systems , CV was computed. Promoters with RPU values greater than 0.1 always have a CV lower than 61%, while CV is much higher for the two weakest promoters, probably because of the high noise levels affecting measurements. Considering library members with RPU>0.1, 7 out of 9 promoters show a statistical difference among the tested conditions. CV entities are consistent with previous studies of our group on context-dependent variability of individual promoters [The strongest promoters have RPU activities comparable to the widely used Promoters , 49, wheromoters , 50, 51.MAX is highly correlated with the constitutive strength of promoters  involving devices with the wild type Plux inducible promoter, where the luxR gene was expressed at high levels by the PLtetO1 promoter (like in this study) in the MG1655 strain [LlacO1 promoter in the MG1655-Z1 strain [MAX values . This might be due to cell overburdening or nonlinear saturation phenomena occurring in the cell, related to copy number increase [MAX values in the regulated low-copy context  did not show a significant context-dependent variation. On the other hand, the use of GFP or RFP as reporter system did not result in important differences in promoter activity.Single-cell measurements have been performed in order to evaluate the library activity with a different measurement system, and to evaluate if fluorescence distributions in cell populations have a unimodal behaviour. Library members have been assayed in constitutive and regulated modes, in the GFP-MC context. Regulated modes were assayed in the full (100 nM), intermediate (5 nM) and no induction conditions. Fluorescence distributions are shown in The 11-member library obtained so far shows an uncovered constitutive activity range between about 0.7 and 2 RPUs see . PromoteIn order to investigate the effects of the presence of flanking sequences on promoter activity and to evaluate their impact in different configurations, seven recombinant strains have been constructed see , by varyThe activity of the LuxR-repressible promoter was measured in absence of AHL (maximum activity) as described in the Methods section and the results were used to perform ANOVA tests. Significant differences in promoter activities have been observed  between the four strains expressing RFP in LC plasmid see , howeverIn the GFP expressing strains, ANOVA test showed that a significant difference is present between promoter activities  and post-hoc comparisons showed a clear separation between the device without the LuxR expression cassette (greenMC) and the ones with the LuxR expression cassette, upstream or downstream of the promoter (greenMC-luxRup and greenMC-luxRdown). In the latter case, promoter activity is reduced by about 2-fold. This result suggests that the presence of an additional expression cassette in MC plasmid can be responsible of the overload of cell machinery, thus causing a diminished promoter activity, even though no effect on the doubling times was observed , PlacIQ  and PR  in order to validate their behaviour in presence of the LuxR-AHL complex.This phenomenon has been further investigated by characterizing all the available promoters from the Anderson library, in LC plasmids in presence of LuxR from the pLuxR-MC plasmid. Other promoters were also included in this analysis: PThe results of a preliminary test are reported in To our knowledge, such phenomena were not reported in literature, and additional efforts should be required to elucidate the underlying mechanism, especially when such promoters are used in complex gene networks where their specific constitutive activity is crucial to enable the correct functioning. This effect has been documented in the Experience section of the BBa_J23101, BBa_J23102 and BBa_J23104 Registry pages.Naturally-occurring cell-to-cell communication mechanisms are widely used as a source of genetic elements to engineer biological systems that show disparate complex functions . In ordeAt the state of the art of synthetic biology, the cryptic relation occurring between promoter nucleotide sequence and strength has not been fully captured, thereby limiting the availability of predictive tools to drive the de-novo design process. The creation of libraries of graded-strength promoters can face such limitations by providing synthetic biologists with a catalogue of parts to choose the one with the desired behaviour.E. coli. The LuxR-repressible behaviour depends on the presence of a lux box  in the core sequence of all the promoters, while the graded strength is obtained by varying the nucleotide sequences of the -35 and -10 regions, according to the consensus determined from an existing constitutive promoter library .lux box in the core sequence was inspired by the work of Egland and Greenberg [The construction of promoters with the reenberg , who valreenberg . Consistin vivo via two different instruments: a microplate reader to perform population-based acquisitions and a flow cytometer to provide single-cell data. Since all the obtained promoters can be used as constitutive or repressible elements, their activity in absence or presence of LuxR was measured, respectively, to provide data on the use of the promoters in both contexts.The availability of catalogues of quantitatively characterized parts can support the rational design of biological systems by enabling the choice of suitable components for the correct functioning of the target system, which can be studied, for instance, via mathematical modelling tools. A central problem in synthetic biology is the context-dependent variability of parts performance when they are used in different circuits or conditions. For this reason, the characterization of parts activity in different contexts and measured via different instruments can strongly support the re-use of genetic parts and could increase the probability of obtaining the expected quantitative behaviour in the designed system. To further support this process, standardized units are also crucial for the re-use of components in different laboratories where different measurement equipment is available. In light of the mentioned requirements, we carried out a multi-faceted characterization of our new library of components by reporting promoter activities in RPU. Two reporter measurement systems (RFP and GFP) were tested downstream of the promoters, in plasmids that are maintained at two different per-cell copy number, and measurements were carried out The final LuxR-repressible promoter library described in this work is composed by 12 members spanning an activity range from about 0.03 to about 3 RPUs and with about 42% mean spacing between the activities of adjacent members. All of them can be tightly repressed by the LuxR-AHL complex, as demonstrated by dose-response experiments.One of the 12 final members (BBa_J107100) did not result from the random screening process, but it was independently designed according to the -35 and -10 sequences of BBa_R0061, a LuxR-repressible promoter present in the Registry, that was designed according to the promoter constructed by Egland and Greenberg . BecausePromoter activity in the constitutive mode, i.e., without a source of LuxR, showed a reasonably contained context-dependent variability among the three biological measurement systems used, with a CV not greater than 31% for promoter members with activity higher than 0.4 RPU. This variability value indicates the expected activity deviation that can be observed when re-using the promoter library members in a new context, such as a gene regulatory network or an expression cassette for a different gene. Although we studied different parts with different measurement systems, the CV among the tested contexts was consistent with our previous works, where promoters with a similar RPU range was investigated , 49. In MAX parameter) which was consistent with their constitutive activity, as expected, but was also dependent on the copy number context. In fact, VMAX in medium-copy (with both RFP and GFP reporter systems) was 2-fold lower than in the low-copy context, measured via RFP. The KM parameter was highly consistent with the one of the wild type Plux promoter when characterized in presence of the same LuxR generator device [For promoters in the regulated mode, i.e., in presence of LuxR, the AHL-RPU data were fitted with a Hill function and the resulting parameters can be used to summarize the quantitative behaviour of the repressible devices. Although some of the promoters showed fitting problems due to their low activity, the overall distribution of the estimated parameters did not have a clear strength- and context-dependent behaviour, except the maximum activity of promoters  in low- (with RFP) and medium- (with GFP) copy contexts with the LuxR expression cassette in different positions: upstream or downstream in the same plasmid, in a different plasmid (only for the low-copy with RFP context), or absent. The results clearly demonstrated that the above observed activity difference was due to the presence of two expression cassettes in a medium-copy plasmid, probably causing a metabolic overload in the host cell, while no significant upstream sequence-dependent effect could be detected when the LuxR expression cassette was present upstream of the reporter expression cassette driven by BBa_J107100.Flow cytometry was used to measure the activity of promoters in one of the above tested contexts  via different measurement equipment. All the tested constructs showed a unimodal fluorescence distribution, whose arithmetic mean was highly consistent with the one obtained via population-based measurements. Single-cell data also gave the occasion to estimate cell-to-cell variability of clones. Excluding promoters with RPU<0.1, an average 55% CV was observed, independently from promoter activity, presence of LuxR expression cassette, and AHL addition. The quantification of cell-to-cell variability has an important impact on the bottom-up design of synthetic networks from basic modules, since it can be used to model and predict interconnection effects that cannot be trivially obtained from population-based measurements . HoweverlacIQ, PLlacO1 and PR promoters. This phenomenon should be taken into account when these parts are used as constitutive promoters in circuits containing the luxR gene and AHL is present, since unpredictable effects may occur, due to a non correct evaluation of their transcriptional strength. Although the maximum repression entity was obtained at 20 μM AHL, which is a very high concentration, it is worth noting that commonly used AHL levels (10 nM to 1 μM) also resulted in a non-null repression.In addition, an undocumented LuxR-dependent repression of three members of the widely used Anderson promoter collection  in presence of AHL was reported in this study. This repression entity was 78%, 28% and 49%, respectively, at 20 μM AHL and cannot be explained by cell growth variation, which is almost unaffected (data not shown). Other 9 tested members of the Anderson collection were unaffected, as well as the three popular PE. coli and other organisms [LtetO1 strong promoter and also with the activity of BBa_J23119 constitutive promoter [Considering the initial 11-member library construction process, the number of obtained members (10 unique clones of graded strength) is consistent with other works in rganisms , 54, altrganisms , 40, 55 rganisms , 39. Ourpromoter , whose -The 'efficiency' of a library generation process is a controversial term, since different works provide it according to different definitions, such as the percentage of final unique members over the initial colonies, or the percentage of colonies with visible fluorescence over the total number of colonies on the transformation plate , 56 and Regulated promoters such as the ones here constructed are widely used in biotechnology and synthetic biology research for several tasks, such as driving the expression of recombinant proteins, optimizing industrially-attractive metabolic pathways and constructing complex circuits with customized behaviour. The graded strength can be exploited to optimize the process yield, in concert with other factors such as plasmid copy number, RBS and codon optimization. The repressible behaviour of our novel components (as well as the inducible behaviour of the currently existing QS-based parts) enables the control of gene expression timing. AHL can be exogenously added or provided via other QS elements  to engineer complex, coordinated and unnatural biological functions. We expect that the herein reported library will be used in different QS-based synthetic circuits or as tunable (or constitutive) promoters in simple gene expression systems. The provided characterization data will strongly support the use of such elements in the circuit of interest according to design specifications, which can also be met with the help of mathematical models where the quantitative features of the reported promoters can be included . The libS1 FigPanels A, B and C show the results in the RFP-LC, RFP-MC and GFP-MC contexts, respectively. The doubling time of TOP10 bearing the BBa_J23101 is also shown in each context. Bars represent the average value, while error bars represent the 95% confidence intervals of the mean value, computed on at least three biological replicates. Results show that no significant promoter activity-dependent trend is present and, for each context, the doubling times are comparable between constitutive and regulated mode and with the strains bearing the BBa_J23101 promoter. We only considered promoters in the regulated mode without AHL, since we found that, given a context, RFP expression never exerted a statistically significant AHL-dependent doubling time increase .(TIFF)Click here for additional data file.S2 FigEach panel shows the curves corresponding to the promoter indicated by the number. Solid lines are the fitted curves, while circles represent the average of three experimental replicates and error bars represent the 95% confidence intervals. Blue indicates the LC-RFP context, red the MC-RFP context and green the MC-GFP context.(TIFF)Click here for additional data file.S3 FigA) Constitutive activity of the 12-member promoter library in the RFP-LC context, showing that promoter 122 (green bar) has successfully filled an activity gap between promoters 2 and 35. B) AHL-dependent Hill repression curve of promoter 122 in the regulated mode. Estimated parameters are reported with their 95% confidence intervals. Bars (panel A) and circles (panel B) represent the average value, while error bars represent the 95% confidence intervals of the mean value, computed on at least three biological replicates. The average doubling time of TOP10 bearing promoter 122 in the RFP-LC context was 89.7 min  and 84.7 min  in the constitutive and regulated (without AHL) modes, respectively. As observed in the other promoters, no significant AHL-dependent variation in doubling time was present in the regulated mode .(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 FigLlacO1, PlacIQ and PR). Promoters were assembled in LC plasmids and were co-transformed with pLuxR-MC (including a LuxR expression cassette in the pSB3K3 vector). Blue bars represent the activity when no AHL was added to the medium, while green bars represent the repressed activity (AHL 20 μM was added to the medium). Only BBa_J23101, BBa_J23102 and BBa_J23104 showed repression and, for them, percent repression is reported. B) Repression curves for the three promoters studied. Differently from the library members, for the used AHL concentrations these promoters do not show a full-repression upon induction.A) Constitutive and repressed activity of all available sequence-verified promoters from the Anderson promoter collection, and three additional popular promoters (P(TIFF)Click here for additional data file.


For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health. Embryos obtained by fertilization develop to adulthood more frequently than those obtained by other methods, such as nuclear transfer , suggestXenopus laevis. The use of spermatids, immediate precursors of sperm, suits such comparisons because (1) spermatids have completed meiosis and have the same DNA content as sperm, and (2) spermatid chromatin structure resembles that of somatic cells  to the cytoplasm of unfertilized eggs (ICSI)  (We first compared the development of embryos obtained by transplanting somatic cell nuclei (SCNT) with the development of sperm-derived embryos. To minimize the experimental difference in the way the embryos were generated, both types of embryos were generated by nuclear injection: cloned embryos were obtained by transplanting nuclei of embryonic cells to enucleated eggs A, and sps (ICSI) C. In tha < 0.05) D,E. This < 0.05) . Spermat < 0.05) B–E, suggIn conclusion, embryos can be generated in the same way from sperm or spermatids, and spermatids show reduced developmental potential compared to sperm. Therefore, the comparison of sperm and spermatids was subsequently used to investigate why sperm support better development than other cell types.Xenopus have been widely used to investigate mechanisms of replication   B, recapi < 0.05) E. Previo < 0.05) . Our resSupplemental Experimental Procedures) and collected at gastrulation, before the onset of developmental defects. Gene expression was then analyzed by RNA-seq, using a set of 34,373 transcripts . Out of 18,340 expressed genes, 255 were differentially expressed in spermatid-derived embryos compared to sperm-derived embryos (FDR < 0.05) . One hundred of these transcripts showed consistent changes in at least six out of seven experiments, and we will refer to them as “misregulated” . The majority (82/100) were up-regulated in spermatid-derived embryos, and they include transcriptional regulators  as well as morphogens  essential for embryonic development. Accordingly, gene enrichment analysis revealed that several development-related terms were significantly enriched in the list of misregulated genes   D.r = −0.17, P-value < 0.05) . Lastly, we have verified that the synthesis of rRNAs is not affected in spermatid-derived embryos. Indeed, in mouse, defect in rRNA synthesis has been proposed to explain the developmental defect of nuclear transfer embryos .The fact that most of the misregulated genes were up-regulated in spermatid-derived embryos raised the possibility that these genes were actively transcribed in spermatids and continued to be transcribed in embryos. Indeed, the spermatid, as opposed to the sperm, is a transcriptionally active cell, and this difference might explain why genes are up-regulated in embryos originating from spermatids rather than sperm. To test this possibility, we compared the expression level of genes in spermatids and in spermatid-derived embryos. We did not observe any correlation between the expression of misregulated genes in spermatid-derived embryos and their expression in spermatids ( < 0.05) E. This s < 0.05) . All emb embryos  and coul embryos . We obseWe conclude that the developmental failure of spermatid-derived embryos is not associated with carried over spermatid RNAs or with defects in rRNA expression. Rather, we observe a correlation between developmental defects and misexpression of a set of developmentally important genes in spermatid-derived embryos. We hypothesized that differences in gene expression between sperm- and spermatid-derived embryos might result from epigenetic differences of sperm/spermatid chromatin.Supplemental Tables S2, S3; Supplemental Fig. S5; Xenopus, the transition from spermatid to sperm is characterized by histone H3 and H4 retention and partial loss of H2A and H2B . Secondly, we analyzed DNA methylation profiles in sperm and spermatids by MBD-seq. DNA methylation was higher around sperm TSSs than spermatid TSSs . A lower level of nucleosome occupancy and DNA methylation in spermatids compared to sperm could therefore explain the up-regulation of genes in spermatid- compared to sperm-derived embryos.To investigate potential links between the epigenetic marking of paternal chromatin and embryonic gene expression, we carried out epigenetic profiling of mononucleosomal chromatin from sperm and spermatid using an extensive MNase digestion protocol applied by others to probe for histones stably associated with chromatin in mature sperm in mouse and human  as well as for the overall methylation levels  around TSSs for each of these modifications. When compared to all genes, the set of misregulated genes was significantly enriched for H3K27me3 in both sperm and spermatids  or repression  of transcription. We looked for peaks  showed an enrichment at promoters of misregulated genes in spermatids but not in sperm C,D, provSupplemental Fig. S9). Therefore, the difference between sperm- and spermatid-derived embryos is best explained by the fact that, in contrast to sperm, where H3K27me3 is overrepresented at genes differentially expressed in haploid embryos, in spermatids H3K27me3 coexists with H3K4me2/3 on these genes, thereby contributing to their up-regulation in spermatid-derived embryos.The epigenetic features analyzed  can individually provide a possible explanation for the observed differences in expression in sperm- and spermatid-derived embryos. However, complex interactions involving more than one epigenetic feature might better explain this differential embryonic gene expression. In order to identify such interactions, we have performed a partial correlation analysis. In this analysis, all the measured parameters are assessed simultaneously to produce maps indicating the way epigenetic features associate with each other and with gene expression. The aim of such analysis is to extract more general principles describing the paternal epigenetic program underlying gene expression and identify which epigenetic features are likely to have the strongest contribution to embryonic gene expression. We applied the partial correlation analysis to identify links between the measured epigenetic features in sperm and the expression of the misregulated genes in sperm-derived embryos A, or betXenopus sperm was a conserved feature across species. For that purpose, we investigated how these histone marks were distributed in human sperm . mRNAs encoding histone demethylases or control mRNAs were first injected into immature oocytes. After allowing 24 h for the enzymes to be expressed, the oocytes were in vitro–matured into eggs (IVM) and injected with sperm or spermatids (ICSI). The resulting embryos were collected at the gastrulation stage for RNA-seq analysis. In this protocol, histones from both maternal and paternal chromatin are demethylated when the embryo is generated. By comparing embryos produced with different paternal chromatin (sperm or spermatid), we can evaluate the effect of paternal epigenetic mark removal on embryonic gene expression.To test the function of epigenetic marks from sperm or spermatid chromatin on the regulation of embryonic transcription, we experimentally removed these marks in embryos . Importantly, genes down-regulated in sperm-derived embryos showed only limited overlap with those down-regulated in spermatid-derived embryos  and 80% (1392 genes) of all differentially expressed (FDR < 0.05) genes were down-regulated in sperm- and spermatid-derived embryos, respectively . Again, there was only a partial overlap between genes affected by KDM6B in sperm- and spermatid-derived embryos . These These analyses allowed us to conclude that sperm is not merely a carrier of DNA, but that it also contributes epigenetic information required for proper embryonic gene expression. We then focused our analysis on the sperm chromatin as it represents the most likely vector of such epigenetic information.Xenopus laevis, core histones H3 and H4 are retained, whereas ∼90% of core histones H2A and H2B are lost   , lower tse (∼1%) , and simse (∼1%) . Nucleosturation I. We shoan sperm C, 5C. SoXenopus and mouse  using forceps and paper tissues. It is crucial to clean the testes well from any nontesticular tissues, as otherwise the cells released from the tissues may negatively affect the final purity of isolated cells. Subsequently, testes were torn into small pieces with forceps and homogenized with 2–3 strokes of a Dounce homogenizer (tissue from one testis at a time). The cell suspension was then filtered to remove tissue debris and cell clumps  and spun down at 800 rcf, 4°C, for 20 min. Supernatant was discarded and the cell pellet was resuspended in 12 mL of 1 × MMR. If any red blood cells were visible at the bottom of the pellet , only the uncontaminated part of the pellet was recovered, taking extreme care not to disturb the red blood cells. Subsequently, step gradients of iodixanol  in 1×MMR final were manually prepared in prechilled 14 mL ultraclear centrifuge tubes  in the following order from the bottom to the top of the tube: 4 mL of 30% iodixanol, 1 mL of 20% iodixanol, 5 mL of 12% iodixanol , and 2 mL of cell suspension in 1 × MMR on top. Gradients were spun down in a prechilled SW40Ti rotor at 7500 rpm , 4°C, for 15 min, deceleration without brake . The top interface fraction (between 1 × MMR and 12% iodixanol), containing spermatids, and the pelleted fraction, containing mature sperm, were collected. Collected fractions were diluted six times with 1 × MMR and collected by spinning first at 805 rcf, 4°C, for 20 min and respinning at 3220 rcf, 4°C, for 20 min to pellet remaining cells. Pelleted cells were subjected to nuclei preparation (see below).For each round of sperm and spermatid purification, testes from six adult 1/2–11 and processed for gene expression analysis .Sperm and spermatid nuclei were permeabilized as described before  and stor2, 0.2 mM DTT, 5 mM HEPES, pH 7.5) supplemented with protease inhibitors (PI) , transferred into centrifugation tubes , supplemented with 1 mL of EB buffer with PI and 100 µg/mL of cytochalasin B , and placed on ice for 10 min. Subsequently, eggs were spun briefly at 350g for 1 min at 4°C , and excess buffer was discarded. Eggs were then spun at 18,000g for 10 min at 1°C, the extract was collected with a needle, transferred to a fresh, prechilled tube, supplemented with PI and 10 µg/mL of cytochalasin B, and respun using the same conditions. Extract was collected with a needle and used fresh for the replication assay (see below).Eggs were collected in 1 × MMR, de-jellied with 0.2 × MBS, 2% cysteine (pH 7.8–7.9)  and washed with 0.2 × MMR. Subsequently, eggs were activated for 3 min at room temperature (RT) with 0.2 × MMR supplemented with 0.2 µg/mL calcium ionophore . Eggs were rinsed with 0.2 × MMR, and subsequently all abnormal or not activated eggs were removed. Eggs were washed with 50 mL of ice-cold extraction buffer (EB) . Permeabilized cells were added to a final concentration of 200 nuclei/µL of extract and incubated at RT for 2 h (tapping every 10 min). The reaction was stopped by adding 10 volumes of ice-cold 1 × PBS , and cells were spun down at 1000g, 4°C, for 7 min. Cells were resuspended in 50 µL of 1 × PBS and mixed immediately with 50 µL of melted (at 65°C) 2% low melting point agarose  in 1 × PBS. After solidification, the agarose plug was incubated overnight (O/N) at 50°C with 1 mL 0.5M EDTA, pH 8, 100 µL 10% sarkosyl , 1 mg/mL Proteinase K , followed by three washes in TE pH 6.5. Subsequently, the plug was incubated twice in TE supplemented with 0.1 mM PMSF  for 30 min at 50°C and washed four times with 1 mL of 50 mM MES , pH 6.35, 1 mM EDTA (1 h at RT each wash). Then, the solution was removed; the plug was melted in 400 µL of MES pH 6.35, 1 mM EDTA at 68°C for 20 min, and the agarose was digested with 2 units of β-agarase  O/N at 42°C.Replication on single DNA fibers was performed as described before  with sliSilanized coverslips were prepared as described before . Thirty 2O and used to generate cDNA sequencing libraries using an Illumina TruSeq Kit (#RS-122-2001), according to the manufacturer's protocol.Spermatid (1 million) or a pool of five stage 10.5–11.5 embryos were collected and frozen at −80°C. RNA extractions were performed using a Qiagen RNeasy Mini Kit according to the manufacturer's protocol. RNA was eluted in 50 µL of DEPC H2O). Embryos were cultured at 18°C and collected for Western blot analysis at stage 21 (Mouse KDM6B (aa1025–1642) or KDM5B (aa1–770) were cloned using the p-Entry cloning system  into a pCS2+ plasmid with a C-terminal HA-tag and NLS-tag. mRNA was synthesized in vitro using a MEGAscript SP6 Kit  following the manufacturer's instructions. Eggs were in vitro–fertilized and de-jellied using a 2% cysteine solution in 0.1 × MMR. Injections into one-cell stage embryos were performed in injection solution  using a stage 21 . WesternSperm and spermatids were separated as described above. Chromatin fractionation and chromatin immunoprecipitation (ChIP) were performed as described before  with sliSperm and spermatid chromatin were separated as described above, and 200 ng of digested genomic DNA were used to purify methylated DNA using the Methyl Collector TM Ultra Kit . The purification was carried out according to the manufacturer's instructions, using the low salt buffer to wash the bead-methyl DNA complexes. The purified methylated DNA and the input DNA were then subjected to library preparation with the TruSeq DNA Kit .Supplemental Material.Details of the sequencing data analysis methods used in this study are described in the http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE75164.All ChIP-seq, RNA-seq, MBD-seq, and MNase-seq data sets from this study have been submitted to the NCBI Gene Expression Omnibus (GEO;


FLOWERING LOCUS C (FLC) in Arabidopsis thaliana is a useful system to address these questions. FLC is transcriptionally repressed during exposure to cold temperatures, allowing studies of how environmental conditions alter expression states at the chromatin level. FLC repression is also epigenetically maintained during subsequent development in warm conditions, so that exposure to cold may be remembered. This memory depends on molecular complexes that are highly conserved among eukaryotes, making FLC not only interesting as a paradigm for understanding biological decision-making in plants, but also an important system for elucidating chromatin-based gene regulation more generally. In this review, we summarize our understanding of how cold temperature induces a switch in the FLC chromatin state, and how this state is epigenetically remembered. We also discuss how the epigenetic state of FLC is reprogrammed in the seed to ensure a requirement for cold exposure in the next generation.Chromatin plays a central role in orchestrating gene regulation at the transcriptional level. However, our understanding of how chromatin states are altered in response to environmental and developmental cues, and then maintained epigenetically over many cell divisions, remains poor. The floral repressor gene FLOWERING LOCUS C (FLC) regulation provides a paradigm for understanding how chromatin can be modulated to determine gene expression in a developmental context. This review describes our current mechanistic understanding of how FLC expression is genetically specified and epigenetically regulated throughout the plant life cycle, and how this determines plant life-history strategy. Arabidopsis thaliana is regulation of the floral repressor locus FLOWERING LOCUS C (FLC) , temperature signals are noisy. To be capable of inferring seasonal information, plants must have systems that are capable of averaging fluctuating temperature, and also ‘remembering’ previous temperature exposure. Our understanding of how seasonal changes in temperature influence plant development is most advanced for the process of vernalization: the acceleration of flowering through exposure to prolonged cold. A requirement for vernalization ensures that plants over-winter vegetatively and flower in the following spring. Central to this process in ng Figurea , how this cold exposure is maintained during subsequent growth in warm conditions (epigenetic memory), and how FLC is reprogrammed during embryo development, with opposing functions of the autonomous and FRIGIDA pathways setting the FLC expression level and determining reproductive strategy.In this review, we describe the key regulators of FLC expression was shown to progressively decrease with increasing weeks of cold exposure .A key feature of vernalization is its quantitative nature: flowering is progressively accelerated as plants are subjected to increasing cold exposure (weeks and months). This was elegantly explained when te Figurea. When met al., FLC expression change, each cell would have to ‘remember’ a quantitative FLC expression level and pass this on through mitosis to ensure stability of epigenetic repression. Digital repression is an elegant mechanism by which plants may respond quantitatively to cold exposure without the need for individual cells to store complex quantitative information.The decision to flower therefore seems to be distributed across many cells of the plant, with each cell responding independently , which is expressed in the phloem companion cells in the vasculature, and subsequently moves from the leaves to the shoot apex to induce flowering  as well as histone acetylation and histone H2B ubiquitination (H2Bub1) are commonly associated with actively transcribed genes in species from yeast to mammals   to a repressed state (H3K27me3). The second line of evidence is genetic: Proteins responsible for placing activating histone marks such as H3K4me3 (ATX1 and SDG25)  family   among eukaryotes suggests that they play important conserved roles in gene regulation. However, it has been difficult to prove conclusively that particular histone modifications are absolutely required for mediating the activating or repressive effects of Trithorax or Polycomb. The main reason for this is that model organisms with a functional Trithorax/Polycomb system also have many copies of histone H3 genes. Thus, it is difficult create mutations to confirm that specific histone residues are the relevant physiological substrates of these complexes. Progress was made relatively recently through a set of experiments in Drosophila,Caenorhabditis elegans and mammals have been reviewed in detail elsewhere . This is the enzymatic subunit that catalyses H3K27me3 through its SET domain , which has been shown to specifically recognize H3K27me3. In the context of PRC2, H3K27me3 binding by EED results in allosteric activation of PRC2 H3K27me3 methyltransferase activity  transcribed from FLC intron 1  appears to be required during cold exposure to ensure removal of activating chromatin marks and to mediate FLC transcriptional down-regulation. This raises the possibility of the existence of an H3K36me3 demethylase , whose targeting to FLC depends on antisense COOLAIR transcripts  to ensure that they are maintained indefinitely at that locus despite the noisy processes of nucleosome turnover and H3K27me3 demethylation. The concept of histone modifications as carriers of epigenetic information has become so embedded in current thinking that histone modifications are commonly referred to as ‘epigenetic marks’, implying that a region of such marks has the intrinsic capacity to instruct its own maintenance and inheritance in daughter cells. The debate over whether histone modifications are the cause or consequence of epigenetically heritable transcriptional states is on-going , U (unmodified) and A (activating)  that nucleosomes are inherited semi-conservatively as DNA is replicated  may also be passed on to daughter chromosomes in the absence of PRC2  or non-coding RNA produced at the FLC locus could feed into a bistable trans-regulatory network. A recent study used two distinguishable fluorescent reporters of FLC expression in the same cells to investigate the cis-memory storage capability of FLC chromatin  being sufficient for full epigenetic silencing. The remaining three vernalized slowly, with a longer period of cold exposure (10–12 weeks) required for silencing  . However, the increase in FLC expression normally observed during resetting was much lower in elf6-5. This reduced expression and early flowering was inherited over subsequent generations, thus the elf6-5 mutation caused partial trans generational inheritance of vernalization-induced FLC repression , is needed in Arabis alpina for perennial flowering  pathway (activating) and the autonomous pathway (repressive)  or through genotype, and is then switched back to the ON state by reprogramming (reversing the switch). Both the maintenance of states and switching between states involves an intimate connection between chromatin regulators and sense/antisense RNA transcription and processing – a mechanism that has yet to be fully elaborated. Nuclear organization is an additional layer of regulation.The many regulatory inputs make FLC haplotypes with characteristic responses to cold exposure. This may be because many of the regulators of FLC (such as Polycomb/Trithorax and the RNA 3′ processing machinery) are not specific to FLC regulation but instead perform more general tasks all over the genome. FLC is unlikely to be exceptional with respect to gene regulation; it has just been studied in more detail than most genes. Whenever an adaptive trait depends so closely on quantitative gene expression levels, subtle changes in regulation have strong consequences on fitness. In these cases, we may expect a similar level of complexity in gene regulation. As such, FLC continues to provide a valuable paradigm for studies of chromatin-based gene regulation, environmental perception and decision making.Cell-autonomous epigenetic switching performs the biological function of registering and remembering unpredictable and noisy temperature signals. An emerging theme is that the relative stabilities of activated and repressed epigenetic states appear to have been subtly modulated in natural accessions by cis sequence variation to generate a range of


Rb depleted small intestines of mice and performed ChIP-exo-seq (chromatin immunoprecipitation combined with lambda exonuclease digestion followed by high-throughput sequencing). The genome-wide chromatin occupancy of E2F3 and MYC was determined by mapping sequence reads to the genome and predicting preferred binding sites (peaks). Binding sites could be accurately identified within small regions of only 24 bp-28 bp long, highlighting the precision to which binding peaks can be identified by ChIP-exo-seq. Forty randomly selected E2F3- and MYC-specific binding sites were validated by ChIP-PCR. In addition, we also presented gene expression data sets from wild type, Rb-, E2f3- and Myc-depleted crypts and villi within this manuscript. These represent comprehensive and validated datasets that can be integrated to identify putative direct targets of E2F3 and MYC involved in the control of cellular proliferation in normal and Rb-deficient small intestines.E2F3 and MYC are transcription factors that control cellular proliferation. To study their mechanism of action in the context of a regenerating tissue, we isolated both proliferating (crypts) and non-dividing (villi) cells from wild-type and  Among the E2F members, E2F3 is known as a major transcriptional activator that can be inhibited when in complex with RB. During late G1 phase of the cell cycle, CDK mediated phosphorylation of RB leads to the release of E2F3 from RB-E2F3 complexes and the activation of the G1–S transcriptional program, resulting in entry of cells into S phase1. MYC is another important transcriptional regulator of the cell cycle2. It’s an oncogene with a broad functional spectrum, which includes the control of cellular proliferation, cell survival, cell growth and metabolism3. Despite significant functional overlap between E2F and MYC in the control of cell cycle regulation, the mechanisms linking these two critical transcriptional programs are poorly understood.E2F is a family of transcription factors that links up-stream proliferation signals with the timely expression of many downstream cell cycle regulated genes4. At the base of crypts, pluripotent stem cells asymmetrically divide to generate a new stem cell and a transient amplifying progenitor cell. Progenitor cells undergo multiple rounds of cell division to form a transient amplifying zone. As progenitor cells migrate towards the finger-like protrusions called villi, they exit the cell cycle and differentiate4. Thus, proliferating progenitor cells and differentiated enterocytes are located in distinct spatial regions of the intestinal track as depicted in in vivo.The small intestine has been utilized as a model system to study normal cellular proliferation and cancer. A unique characteristic of the small intestine is that the continuous regeneration of the entire intestinal track is neatly organized in a defined pouch-like invagination called the crypt6. Recently, an improved technique to better define chromatin binding regions was developed, ChIP-exo-seq8. The key innovation of this technique is that after ChIP, the lambda exonuclease is used to digest DNA fragments starting from the exposed 5′ end and stopping at the protein-DNA boundary. After library preparation and deep sequencing, the 5′ end of the reads obtained are highly concentrated on the protein-DNA boundary, providing better positional resolution and accuracy of the predicted protein binding sites8.ChIP-seq is a widely used technique to profile chromatin occupancy of transcription factors on a genome wide scale. Briefly, chromatin is released from lysed cells and sheared to smaller fragments and specific antibodies are used to immunoprecipitate protein-DNA complexes. Then, the enriched DNA fragments are purified, tagged, amplified and sequenced using next-generation sequencing technology. Finally, genomic regions with enriched read alignments are detected as predicted protein binding sites, which usually span hundreds of base pairsRb deficient small intestines. An average of 70 million to 120 million reads were generated for each ChIP-exo-seq experiment . Experimental manipulation of animals was approved by Institutional Animal Care and Use Committee at the Ohio State University. Mouse strains were bred and maintained in a mixed genetic background (C57BL/6×129×FVB/N). Both genders of mice were utilized in the studies. To induce the expression of Cre recombinase and deletion of alleles flanked by loxP sequences, β-naphthoflavone  dissolved in corn oil  was intraperitoneally injected into 2-month old mice at the dosage of 80 mg kg−1 body weight10. Five injections were performed within 30 h (9am-3pm-9am on first day and 9am-3pm on second day).The description of the mouse strains used and how they were maintained is described in detail in the related research manuscriptImmediately after euthanizing mice, the small intestine was dissected and mesentery and adipose tissues were removed. The intestine was then cut open along their cephalocaudal axis and gently washed in phosphate buffered saline (PBS) to remove undigested food chow. The tissue used for RNA isolation was collected from a 10 cm portion of the intestine proximal to the stomach. For ChIP, the intestine was divided into three parts of equal length and the first 1/3 section proximal to the stomach was used. After cleaning with PBS, the tissue was incubated in 25 ml PBS containing 0.5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT). After incubation for 30 min at room temperature, villus fractions were collected by multiple rounds of gently shaking the tissue, decanting tissue materials in suspension into multiple tubes containing 10 ml ice-cold PBS with 1 mM DTT. After most villi were collected, the remaining tissue was incubated in 25 ml PBS with 0.9 mM EDTA and 1 mM DTT. The crypt fractions were collected by shaking the tissue in multiple tubes containing 10 ml ice-cold PBS with 1 mM DTT until complete separation of epithelial cells from mesenchymal tissues. Crypt-enriched fractions were filtered using 70 μm cell strainer  to remove potential contamination of broken pieces of villi with larger sizes. After centrifuge at 335×g for 5 min at 4 °C, the resulting villus-enriched or crypt-enriched tissue pellets were combined and washed in ice-cold PBS. The tissues were then precipitated by centrifuge at 335×g for 5 min at 4 °C. For RNA isolation and ChIP assays, the cell pellets were processed as described below.http://cancer.osu.edu/research-and-education/shared-resources/genomics/services).Total RNA from purified villi/crypts was isolated using TRIzol reagent following manufacturer’s protocol and further cleaned up with RNeasy Mini Kit . RNA integrity was assessed using Agilent 2100 Bioanalyzer Instruments. Global RNA expression levels were profiled using Affymetrix GeneChip Mouse Genome 430 2.0 Array at the Ohio State University Shared Resources (3 (900 rpm vortex) to retrieve the immunoprecipitated chromatin. The chromatin was then reverse-crosslinked by incubation with 0.5M NaCl and 0.1 mg/ml RNase A overnight at 65 °C (including non-immunoprecipitated inputs), followed by 0.1 mg/ml proteinase K treatment at 50 °C for 1 h. The DNA was purified with Qiagen kits . Quantitative PCR was performed using SYBR Green master mix  with Applied Biosystems StepOnePlus Real-Time PCR System.Freshly isolated crypts/villi were crosslinked in PBS containing 1% formaldehyde at 37 °C for 15 min on a rotator. The crosslinking reaction was terminated by incubation with 0.125 M glycine at 37 °C for 5 min on a rotator. The cell pellets were then washed in ice-cold PBS, followed by cytosolic lysis and nuclear lysis steps (cytosolic lysis buffer: 5 mM PIPES (pH 8.0), 85 mM KCl, 0.5% NP-40; nuclear lysis buffer: 50 mM Tris (pH 8.0), 10 mM EDTA, 1% SDS). The released chromatin was then subject to sonication to generate DNA fragments mainly with 100-300 bp sizes. The fragmented chromatin was diluted with 9 volumes of IP dilution buffer (16.7 mM Tris (pH 8.0), 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100). To minimize non-specific pull-down, the chromatin was incubated with Protein G Plus/Protein A beads  at 4 °C for 1 h on a rotator prior to adding antibodies. Antibodies used in the study were E2F3 , MYC  and normal rabbit IgG . The chromatin was incubated with antibodies overnight at 4 °C on a rotator, followed by incubation with Protein G Plus/Protein A beads  at 4 °C for 2 h on a rotator. The chromatin-antibody-bead complex was then washed twice with each of the following buffers: low-salt buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 150 mM NaCl), high-salt buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 500 mM NaCl), LiCl buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 250 mM LiCl, 1% NP-40 and 1% deoxycholic acid) and Tris-EDTA buffer (pH 8.0). Note that all the steps described above were performed in the presence of proteinase inhibitors. The Protein G Plus/Protein A beads were incubated with 1% SDS plus 0.1M NaHCORb KO mice and villi were collected from 7 wild type mice and 7 Rb KO mice. The subsequent steps, including crosslinking, cytosolic and nuclear lysis, sonication, dilution with IP dilution buffer, pre-clean and immunoprecipitation (incubation with antibodies and beads) were performed as described above in the ChIP section, except Pierce Protein A/G Magnetic Beads  were used instead of Protein G Plus/Protein A beads. Prior to adding antibodies, the pooled chromatin derived from each genetic/tissue compartment was divided into two for E2F3- and MYC-ChIP, respectively. After generating the chromatin-antibody-bead complex, the library construction steps including on-bead enzymatic reactions , elution and reverse-crosslinking, primer extension and P5 exo-adaptor ligation were performed as described11. The resulting DNA was enriched by 13 cycles of PCR using NEBNext High-Fidelity PCR Master Mix . The DNA concentration and DNA size distribution of libraries were profiled using Agilent High Sensitivity DNA Kit. Cluster generation on Illumina cBot and single-end high throughput sequencing on Illumina HiSeq 2500 platform were performed at the Ohio State University Shared Resources (http://cancer.osu.edu/research-and-education/shared-resources/genomics/services).For ChIP-exo-seq experiments, crypts were collected and pooled from 32 wild type mice and 27 12 were utilized. The ‘justRMA’ function was used to read the raw CEL files, perform background correction and normalization. The ‘exprs’ function was used to obtain the log2 transformed expression value. All parameters were set as default.For microarray analysis, functions from R package AffyAfter base calling, adapter trimming, and barcode demultiplexing using the sequencer manufacturer’s software, raw sequences with quality scores (Sanger/Illumin1.9) were stored in FASTQ format. The name of each sample includes the mouse genotype, tissue type and the specific antibody used. Each sample was sequenced in two lanes of an Illumina flow cell and the resulting data was combined into one FASTQ file before mapping onto the genome.7: FDR<0.01; MACS2 GEM (version 2.4.1)homer v4.6IGV Version 2.3.32https://dx.doi.org/10.6084/m9.figshare.2059239)All the scripts used to run tools mentioned before or process intermediate files are uploaded onto figshare (17 (NCBI) under accession number GSE56008 (Data Citation 1). For each sample in GSE56008, a processed data file listing all the predicted binding sites is provided. It is a tab-separated text file in BED format with the following columns: Chromosome Name, Start Coordinate, End Coordinate, Binding Site ID, and Binding Site Score . Links to the corresponding NCBI Sequence Read Archive (SRA) accession, which deposits the raw sequencing data (Data Citation 2), are also provided.ChIP-exo-seq data from 8 samples (4 from crypts and 4 from villi) were deposited in Gene Expression OmnibusMicroarray Dataset 1 in Rb deleted villi. In all ChIP-exo-seq experiments the quality score was determined to be above 30 . above 30 . Over 90above 30 . Thus, bReads aligned to the positive and negative strands formed independent single peak summits for both E2F3 and MYC ChIP experiments. Sequences between the two summits, which were 24 bp–28 bp in length, represent the E2F3 and MYC binding sites . The numE2f3-deficient and Myc-deficient tissues, respectively. All selected E2F3- and MYC-specific peaks were highly enriched in wild type tissues when compared to E2f3- and Myc-deficient tissues . In addition, control IgG ChIP-PCR assays showed low background binding to these selected E2F3- and MYC binding sites. Together, these control experiments highlight the highly specific nature of E2F3- and MYC chromatin profiling by ChIP-exo-seq.To validate the predicted transcription binding sites, forty randomly selected E2F3- and MYC-specific peaks were assessed by ChIP-PCR assays in Rb deficient tissues. Interestingly, canonical MYC binding motifs were only enriched in Rb deficient crypts, highlighting the impact of Rb loss on MYC global chromatin binding. Detailed information of motif sequences and distribution for each experiment is listed in Supplementary Fig. 6b of the reference manuscript9.We then identified transcription factor binding motifs imbedded within 100 bp region of the identified binding sites. As expected, canonical E2F binding motifs were significantly enriched in crypts and villi of both wild type and 2 transformed expression values were used to perform Multidimensional Scaling (MDS) analysis. As shown in et al.,9). Thus, we conclude that the overall reproducibility of microarray gene expression data sets is very good.Microarray gene expression data sets were first background corrected and normalized, and then the loghttps://dx.doi.org/10.6084/m9.figshare.2059239). These scripts were run on a linux system. To deploy the pipeline, please download and decompress the script archives, install required software and configure them in the ‘software.conf’ file. Then copy your raw FASTQ files into the ‘rawdata’ folder and configure them in the ‘samples.conf’ file. Finally, the ‘bash run_ChIP-exo_pipeline.sh’ command is used to execute the analysis pipeline. The predicted binding regions, corresponding target genes, and enriched sequence motifs of each ChIP-exo-seq experiment will be automatically generated. Intermediate files, such as alignments in BAM format and read coverage along the genome, will also be generated and could be visualized using IGV18. The parameters can be adjusted by revising the corresponding scripts. This package may be modified to accommodate other species.Scripts for running Bowtie2, GEM, MACS2, HOMER and scripts for processing other intermediate files have been uploaded to figshare .


Members of the NR4A subfamily of orphan nuclear receptors regulate cell fate decisions via both genomic and non-genomic mechanisms in a cell and tissue selective manner. NR4As play a key role in maintenance of hematopoietic stem cell homeostasis and are critical tumor suppressors of acute myeloid leukemia (AML). Expression of NR4As is broadly silenced in leukemia initiating cell enriched populations from human patients relative to normal hematopoietic stem/progenitor cells. Rescue of NR4A expression in human AML cells inhibits proliferation and reprograms AML gene signatures via transcriptional mechanisms that remain to be elucidated. By intersecting an acutely regulated NR4A1 dependent transcriptional profile with genome wide NR4A binding distribution, we now identify an NR4A targetome of 685 genes that are directly regulated by NR4A1. We show that NR4As regulate gene transcription primarily through interaction with distal enhancers that are co-enriched for NR4A1 and ETS transcription factor motifs. Using a subset of NR4A activated genes, we demonstrate that the ETS factors ERG and FLI-1 are required for activation of NR4A bound enhancers and NR4A target gene induction. NR4A1 dependent recruitment of ERG and FLI-1 promotes binding of p300 histone acetyltransferase to epigenetically activate NR4A bound enhancers via acetylation at histone H3K27. These findings disclose novel epigenetic mechanisms by which NR4As and ETS factors cooperate to drive NR4A dependent gene transcription in human AML cells. NR4A1, NR4A2, NR4A3) are products of immediate early genes whose expression is induced by a variety of extracellular signals such as inflammatory cytokines, genotoxic stress, and apoptotic and mitogenic stimuli [AAAGGTCA) or through NR4A homo- and heterodimeric interaction with a NurRE (TGATATTTX6AAAGTCCA). NR4A2 has also been reported to interact indirectly with DNA via tethering to NFkB which results in suppression of NFkB target genes [The NR4A subfamily of orphan nuclear receptors  to identify genome wide NR4A binding sites, and we have integrated these data with gene expression signatures that are acutely regulated upon rescue of NR4A expression in human AML cells to identify direct genomic targets of NR4A1 and investigate its global transcriptional mechanisms of action. Using this integrated unbiased approach, we reveal novel cooperative interactions between NR4As and members of the ETS family of transcription factors to promote transcription of NR4A target genes. We show that NR4As can reprogram ETS factor binding to NR4A bound enhancers to promote p300 histone acetyl transferase (HAT) recruitment and epigenetic enhancer activation via p300 dependent acetylation of histone H3 at lysine 27 (H3K27Ac).In-vitro transcribed RNA (IVT-RNA) transfection system have been previously described [In-vitro transcription (IVT) was performed per manufacturer’s instructions on linearized plasmid containing the NR4A1 coding sequence using the mMESSAGE mMACHINE® T7 Kit (Applied Biosystems). RNA polyadenylation was performed with a Poly(A) Tailing Kit, and resulting IVT-RNA was purified with a MEGA Clearance Kit (Applied Biosystems) according to manufacturer instructions. For electroporation, cells were suspended to a final concentration of 1 million cells per 100uL DPBS. 200uL of cell solution was transferred to 0.4 cm cuvettes (USA Scientific), mixed by pipetting with IVT-RNA at a final concentration of 100 nM, and immediately electroporated at 330V for 5 milliseconds with the GenePulser Xcell system (Bio-Rad).Kasumi-1 cells were purchased from ATCC. Cells were maintained in 1640 RPMI with 20% FBS. The plasmids and escribed . In-vitrβ2m levels.Total RNA was extracted using RNeasy Mini Kit (Qiagen), and RNA was reverse transcribed into cDNA using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems). cDNA was diluted 5 fold with nuclease-free water and quantitated via TaqMan Gene Expression Assays and Master Mix . TaqMan pre-designed primers and probes (Applied Biosystems) were used for target gene qPCR. PCR amplifications were performed using the ABI Step One Plus Sequence Detection System (Applied Biosystems) under standard conditions. Transcript levels were determined by standard curve and normalized to corresponding Total RNA was extracted 6h after IVT transfection using an RNeasy Mini kit (Qiagen). Quality control and processing of human genome U133 Plus 2.0  chips were performed by the Baylor College of Medicine Genomic and RNA Profiling Core. Protocols from the Affymetrix GeneChip Expression Analysis Technical Manual were used for preparation and fragmentation of biotin labeled cRNA. The Affymetrix GeneChip Fluidics Station 400 was used to perform array hybridization, washing, and staining with streptavidin-phycoerythrin. Probe fluorescence values were normalized by Robust Multiarray Analysis (RMA) using RMA Express. Differentially expressed probes were identified by Rank Product Analysis in the TM4 Microarray Software Suite ,20 and c4hr after NR4A1 IVT transfection, duplicate samples of 30 million Kasumi-1 cells were fixed with 1% formaldehyde for 15 minutes at room temperature and then quenched with 0.125M glycine for 5 minutes. Cells were washed twice with cold DPBS and frozen at -80°C. ChIP-sequencing was performed by Active Motif, Inc. Cells were lysed by Dounce homogenization and chromatin was sheared to an average length of 300–500bp. 30ug aliquots of lysate were pre-cleared with Protein A agarose beads and incubated with 2ug NR4A antibody  overnight at 4°C. Immune complexes were captured with Protein A agarose beads for 3 hours and washed with low salt, high salt, LiCl, and TE buffers. Immune complexes were eluted with SDS buffer, and subjected to RNase treatment and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP DNA was amplified by following the Illumina ChIP-Seq DNA Sample Prep Kit protocol. DNA libraries were quantified and sequenced on a Genome Analyzer II using 36nt single end reads.Sequences were aligned to the human genome (NCBI37/hg19) using the BWA algorithm. Alignments were extended in silico at their 3’ ends to a length of 150bp and assigned to 32nt bins. Peak locations were determined using the MACS  algorithm with a p-value cutoff of 1E-10 , and werChromatin immunoprecipitations were performed 4h after IVT electroporation. Cells were crosslinked with 0.75% formaldehyde for 10min at room temperature and quenched with 0.125M glycine for 5min. Cells were washed twice with cold DPBS, and cell pellets containing 5 million cells were re-suspended in 200ul FA Lysis Buffer . Cell lysate was sonicated for 60min using a BioRuptor  with a 30sec on, 30sec off cycle in wet ice to achieve chromatin fragments of 100–300bp. For NR4A1, FLI-1, and p300 ChIPs, 50ug pre-cleared lysate was diluted 10-fold in ChIP dilution buffer  and incubated with 4ug of appropriate antibody overnight. For histone modification ChIPs, diluted lysate was incubated with 2ug of appropriate antibody overnight at 4°C. Immune complexes were captured with Protein A/G agarose beads (Santa Cruz Biotechnology) and washed successively for 5min each at 4°C with Low Salt, High Salt, and LiCl wash buffers followed by three TE buffer washes. Input and immunoprecipitated DNA crosslinks were reversed overnight at 65°C and DNA was purified with QiaQuick PCR Purification Kit (Qiagen). ChIP DNA was quantitated with Power SYBR Green Master Mix (Life Technologies) and normalized to input DNA. Antibodies used for ChIP were anti-NR4A (sc-990), anti-p300 (sc-585), anti-FLI1 (sc-356), and rabbit IgG (sc-2027) (Santa Cruz Biotechnology); anti-histone H3 (ab1791), anti-H3K9Ac (ab10812), anti-H3K27ac (ab4729), anti-H3K4me1 (ab8895), and anti-H3K4me3 (ab8580) (Abcam); and anti-H3K27me3 . ChIP-qPCR primer sequences are listed in Kasumi-1 cells were electroporated with 200nM of each appropriate siRNA or with equal amounts of non-targeting control siRNA (Dharmacon) 48h before electroporation with in vitro transcribed NR4A1 mRNA. RNA was extracted 4 hours or 6 hours after electroporation with NR4A1 IVT for ChIP and gene expression analysis, respectively, and processed as above.We first sought to identify direct NR4A1 target genes through integration of NR4A1-dependent gene expression signatures with NR4A1 genome wide binding sites in Kasumi-1 AML cells. Since Kasumi-1 cells do not express endogenous NR4A1, NR4A2, or NR4A3 proteins, and we have previously demonstrated that NR4A1 and NR4A3 regulate redundant transcriptional programs, we introduced NR4A1 mRNAs via electroporation as a model of NR4A1 activity and used a well validated anti-NR4A antibody  for ChIP-seq analysis of genome wide NR4A1 binding to chromatin. We have previously shown that this antibody is monospecific for reexpressed NR4A1 in this system . Using tWT) and NR4A1 with C284A and E285A mutations of a zinc finger within the DNA binding domain (NR4A1CEAA) that are essential for NR4A DNA binding [WT activates 547 genes and represses 551 genes within 6 hours of expression in Kasumi-1 cells , suggesting that classical DNA binding and transcriptional regulation is required for cell growth inhibition by NR4As . To addr-1 cells . Gene seR4A1CEAA . Gene oned genes . Interese system , consistWT. This analysis revealed that a minority of genome-wide NR4A1 binding corresponded with NR4A1-dependent gene regulation in Kasumi-1 cells since only 10% of the 6982 unique genes bound by NR4A1 were transcriptionally regulated by NR4A1  can be used to identify regions of chromatin accessibility , and motand SDPR , and NR4d CLEC5A .Given the high degree of ETS motif coenrichment within NR4A1 binding sites, we next asked whether NR4A1 and ETS factors cooperate at a genome wide level to regulate NR4A1 target genes. We queried published genome-wide binding data for the ETS factors ERG and FLI-1, two of the most highly expressed ETS factors in human AML-ETO positive AML cells , to deteIL7R, FLT3, and BCL6  groups of induced and repressed direct target genes. Enriched GO annotations of induced genes included lymphocyte differentiation, negative regulation of transcription, protein import into the nucleus, and hematopoiesis. The most enriched biological process, lymphocyte differentiation , includeand BCL6 , and we L6  in contrast to H3K4me3 which reflects a common mark of active promoter elements ,40. The S region . The p15 H3K27me3, was userol loci , and thirol loci . However at BCL6 . Consistenhancer . Taken tH3K27 is a key substrate for acetylation by the highly homologous histone acetyl transferases (HATS), p300 and CBP ,44,45. TENO3  or through homo- or heterodimeric intractions with a Nur response element (NurRE) composed of palindromic repeats of a core AGGTCA motif [MYC, require a functional DNA binding domain of NR4A1 and NR4A3 [CEAA does not regulate genes enriched for NFkB target genes, we did not observe enrichment of NFkB motifs within NR4A1-occupied regions of NR4A1 repressed genes despite constitutive NFkB activity in AML [Analysis of gene specific mechanisms of transcription by NR4As has revealed that they regulate gene expression via direct binding as monomers to an extended nuclear receptor half-site Click here for additional data file.S2 FigA) Heatmap of differentially expressed genes 6hr after GFP, NR4A1WT or NR4A1CEAA IVT-mRNA transfection. (B) Gene set enrichment analysis (GSEA) of differentially expressed genes. (C) Gene ontology annotations generated by DAVID functional annotation of genes induced or repressed by NR4A1WT. (D) Gene ontology annotations of genes induced or repressed by NR4A1CEAA.((PDF)Click here for additional data file.S3 FigA) Genes included in lymphocyte differentiation annotation derived from NR4A1-induced genes. (B) NR4A1 ChIP-seq binding profiles at validated induced genes in Kasumi-1 cells. Arrows denote binding regions validated by ChIP-qPCR. The Refseq transcript for each gene is shown below each locus. The y axis represents cumulative tag counts at each region.((PDF)Click here for additional data file.S4 FigA) Genes included in the cell proliferation annotation derived from NR4A1-repressed genes. (B) NR4A1 ChIP-seq binding profiles in Kausmi-1 cells at validated repressed genes. Arrows denote binding regions validated by ChIP-qPCR. The Refseq transcript for each gene is shown below each locus. The y axis represents cumulative tag counts at each region.((PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Transcriptional regulation of inflammatory gene expression has been at the forefront of studies of innate immunity and is coordinately regulated by transcription factors, including NF-κB, and chromatin modifiers. The growing evidence for involvement of chromatin in the regulation of gene expression in innate immune cells, has uncovered an evolutionarily conserved role of microbial sensing and chromatin remodeling. Toll-like receptors and RIG-I-like receptors trigger these signaling pathways leading to transcriptional expression of a set of genes involved in inflammation. Tightly regulated control of this gene expression is a paramount, and often foremost, goal of most biological endeavors. In this review, we will discuss the recent progress about the molecular mechanisms governing control of pro-inflammatory gene expression by an evolutionarily conserved novel nuclear protein Akirin2 in macrophages and its emergence as an essential link between NF-κB and chromatin remodelers for transcriptional regulation. First, with the exception of methylation, histone modifications can change the net charge of nucleosomes, resulting in a loosening of DNA-histone interactions. This idea is in fact supported by the observation that acetylated histones are easier to displace from DNA both in vivo ,32 and iin vitro ,34.A number of histone acetylases have been identified during the last decade. They are quite diverse in terms of their enzymatic activity and regulation. p300/CBP coactivators were the first major known histone acetylases discovered and initially implicated in transcription, and later shown to enzymatically modify histones ,36,37. TRecent studies have suggested the involvement of chromatin to actively contribute to the selectivity of the response to a stimulus via cell type specific or stimulus specific manner ,42,43,44Albeit different stimuli can activate NF-κB in macrophages, each stimulus activates only a subset of the NF-κB target genes that are competent for activation ,47. SomeThis stimulus specific transcriptional response can be broadly categorized into the primary response and the secondary response, although exceptions do exist ,50,51. PDrosophila melanogaster, and evolutionary conservation of “Toll” signaling molecules  between mammals and Drosophila, led us to investigate the new molecules involved in the NF-κB-dependent transcription. We have previously identified CG8580 in an RNAi-mediated knockdown study and found that the knockdown of this gene led to impaired immune deficiency (Imd) pathway signaling and enhanced sensitivity of flies to Gram-negative bacterial infection [Drosophila, mice, teleosts, and humans. The function of the Akirin protein has remained enigmatic; despite its high degree of conservation, it remains almost completely devoid of known functional domains, catalytic activity, or demonstrable DNA binding activity. Akirin has been reported to be crucial for regulating gene expression in a wide range of contexts [Akirin family members (Akirin1 and Akirin2) [Akirin2 is required for embryonic development and the mice lacking this gene die by embryonic day 9.5. Fibroblast cells derived from Akirin2-deficient mouse embryos showed selective defects in NF-κB-dependent gene expression following stimulation through pathways involving the Toll-like receptor, interleukin-1 receptor or TNF receptor. All of these pathways converge on the activation of NF-κB. Furthermore, neither Akirin1-deficient mice nor cells derived from these animals have any obvious unusual characteristics [The availability of powerful genetic tools to study nfection . We renacontexts ,54,55. Icontexts . Humans Akirin2) . Gene taeristics . HoweverAkirin2 possesses two conserved regions at their N- and C-termini, the former containing a nuclear localization signal. It contains two conserved helical regions, one located in the central region (residues 75–95) and one in the C-terminus (residues 143–193), separated from each other and from the N-terminus by regions predicted to be intrinsically disordered. Neither of the conserved helical regions have significant sequence similarity to proteins of known function, but conserved intrinsically disordered regions are often important for mediating protein-protein interactions . As AkirIl6 and Il12b promoter region in response to lipopolysaccharide (LPS) in mouse macrophages as well as in HeLa cells. The acetylation of H3K9 as well as trimethylation of H3K4 on the Il6 and Il12b, but not Cxcl1 and Tnf promoters was induced in response to LPS in an Akirin2-dependent manner. These data suggested the involvement of promoter specific or stimulus specific responses in the chromatin remodeling which require Akirin2 [Moreover, Akirin2 is also found to be critical for regulating histone modifications on its target gene promoters, such as  Akirin2 .et al. provided a comprehensive view of Akirin function in NF-κB transcriptional selectivity during the innate immune response in Drosophila [Drosophila, the Brahma complex associate with Osa forming the Osa-containing-SWI/SNF-like Brahma (BAP) complex, whereas an association with both Polybromo and BAP170 defines the PBAP complex. Each complex targets a mutually exclusive subset of Brahma-dependent genes [in vivo for efficient antimicrobial peptide synthesis and for the survival of flies following Gram-negative bacterial infection. Upon immune challenge, Akirin is able to bind Relish, forming a link between this transcription factor and the BAP complex on the promoter of a subset of NF-κB target genes. Relish-dependent genes thus fall into two groups, either relying on Akirin and the BAP complex ), or expressing most of the negative regulators of the IMD pathway and AMPs independently of Akirin.One mechanism whereby Akirin might function as a general cofactor for gene expression would be through interactions with chromatin remodeling complexes. In an independent study, Bonnay osophila . In an ant genes ,58. TheySMARCD1, SMARCD2 and SMARCD3 genes, respectively [Il6 gene expression in response to LPS in the macrophage cell line, though BAF60b and BAF60c, but not BAF60a, were important for Il6 gene expression in response to IL-1β in HeLa cells [in vivo.Mammals have a family of BAP60 homologues, comprised of BAF60a, BAF60b and BAF60c, which are encoded by the ectively . Akirin2La cells . AlthougLa cells . BesidesLa cells . InsulinLa cells . In thisIl6 and Il12b, but not Tnf, mRNA expression to TLR stimulation requires an IκB-like molecule called IκB-ζ [The hypothesis that Akirin2 acts in concert with NF-κB family members by physically associating with them, led to an important observation that ed IκB-ζ ,65. Anoted IκB-ζ . Our groed IκB-ζ , suggested IκB-ζ . In conted IκB-ζ .Il6 and Il12b promoters in macrophages in response to LPS. Reciprocally, Akirin2 is also required for the recruitment of IκB-ζ to the same promoters in response to LPS. The interaction between endogenous IκB-ζ and Akirin2 was also observed in resting macrophages. In addition, IκB-ζ was found to form a complex with BAF60 proteins via Akirin2 [Il6 and Il12b promoters.Emergence of IκB-ζ as an Akirin2 binding protein via the C-terminal region of Akirin2 proved to be the missing link in this puzzle. Recent study also reported that IκB-ζ was required for the recruitment of Akirin2 as well as Brg1 to the  Akirin2 . These d Akirin2 ,70, and in vivo.Collectively, the IκB-ζ-Akirin2-BAF60 axis is critical to bridge the NF-κB and SWI/SNF complexes in innate immune cell activation. It is intriguing to speculate that IκB-ζ functions via Akirin2-mediated chromatin remodeling in various immune cell types. Further studies with conditional Akirin2-deficient mice will be required to uncover the broad functional roles of Akirin2 in various cells types Mammalian SWI/SNF complexes contain either of two catalytic subunits, Brg1 or Brm, which are associated with several accessory proteins, known as BAFs (Brg1-associated factors) ,71,72,73Cmpk2, Akap12 and Parp14 was impaired in Brg1 knockdown cells, indicating that Akirin2 target genes require SWI/SNF complex for their expression irrespective of the presence of CpG islands on their promoter regions [This SWI/SNF based selectivity was recently suggested to be dependent on the differential CpG island context of NF-κB target gene promoters, although there are contradictory studies. Most of the SWI/SNF-independent genes were characterized by CpG island promoters, whereas almost all SWI/SNF-dependent genes contained promoters with a low content of CpG dinucleotides ,47. Geno regions . This reDrosophila is unmethylated and lacks classical CpG islands [Drosophila does not display CpG islands or methylation [Drosophila would establish regions of nucleosomal instability precluding any need of Akirin and the SWI/SNF complex for the control of gene transcription.An interesting feature of this evolutionary conservation stems from the observation that the genome of  islands . Even thhylation , bioinfohylation . HoweverDrosophila Akirin has been shown to be required for Twist-mediated gene transcription by controlling chromatin remodeling during embryogenesis [In addition to the control of innate immune responses, ogenesis . Akirin Myc and Ccnd2 promoter (involved in various aspects of cell growth and proliferation) in response to CD40 stimulation in control B cells. In contrast, Akrin2 deficiency abrogated the recruitment of Brg1 to the Myc and Ccnd2 promoter, indicating that Akirin2 is critical for the recruitment of the SWI/SNF complex to the Myc and Ccnd2 promoter [Moreover, a recent study published using B cell specific Akirin2 mice, further emphasize the role of this enigmatic protein in various different cell types . Having promoter .Il6 and Il12b in response to stimulation with TLR ligands, Akirin2 was also found to be important for the induction of type I IFNs and a set of IFN-inducible genes in response to RNA virus infection [In addition to the regulation of nfection . Genome-nfection . Furthernfection . Since Infection ,81, it snfection , it is rRecent technological advances and the growing number of ChIP-Seq, and transcriptome profiling studies have enabled a great and rapid expansion of the knowledge of mechanisms controlling transcriptional responses to inflammatory stimuli. Availability of genome-scale expression data suggested that dynamic changes in gene transcription in innate immune cells are pivotal for evoking inflammation in response to pathogen infection. Gene expression is, in turn, controlled by elaborate and complex mechanisms.Akirin2 represents a new class of transcription cofactor that bridges the transcriptional activation with those of chromatin remodeling complexes to influence gene expression. This mechanism is evolutionarily conserved and plays a pivotal role in regulating gene expression not only in mammals but also in insects . Targeti