GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome “remodeler” rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4′s action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription—one that requires the action of SWI/SNF recruited by the activator, and a slower one that does not—clarifies our understanding of the early events of gene activation, and in particular corrects earlier reports that SWI/SNF plays no role in GAL gene induction. Our finding that chromatin structure is irrelevant for repression as studied here—that is, repression sets in as efficiently whether or not promoter nucleosomes are allowed to reform—contradicts the widely held, but little tested, idea that nucleosomes are required for repression. These findings were made possible by our nucleosome occupancy assay. The assay, we believe, will prove useful in studying other outstanding issues in the field.The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation  and repression (by glucose) of the  In this paper, we examine activation and repression of transcription of a gene in yeast. This gene, like the typical human gene, is wrapped in DNA-protein packets called nucleosomes. It is widely believed that these condensed packets are unwrapped, in a process called nucleosome removal, as transcription begins. Here, we describe a new quantitative nucleosome assay that allows us to measure the time course of nucleosome removal and replacement as the gene is activated and repressed. The yeast activator Gal4, bound to DNA, effects activation of gene transcription in two separate steps. First, it recruits to the gene an enzyme that strips off nucleosomes; and second (as we had shown previously), it recruits the transcriptional machinery. We also show that transcription of the gene can be turned off well before nucleosomes have been returned to the gene. In this case, the activator continues to recruit the nucleosome-remover, but either the transcriptional machinery is not recruited, or if it is, it is soon destroyed A new nucleosome-occupancy technique reveals how the transcriptional activator Gal4 determines chromatin structure as genes are activated and repressed. Saccharomyces cerevisiae. Galactose, added to the growth medium, frees Gal4 of its inhibitor Gal80, and the DNA-bound activator quickly and strongly induces genes required to metabolize the sugar. Two such genes are the divergently transcribed GAL1 and GAL10, between which lie four Gal4 binding sites comprising the so-called upstream activating sequence, galactose (UASg). A wide array of studies shows that Gal4 recruits to nearby yeast genes protein complexes required for transcription [GAL genes in several ways. The strongest direct effect is repression of GAL4 and of GAL2, which encodes the galactose permease. A smaller effect is that the GAL1,10 genes are also directly repressed  would cover DNA that must be available for the transcriptional complex to form. And indeed, several experiments show that these nucleosomes, present on the inactive promoters, are missing when the genes are transcribed [PHO8 gene of yeast: in this case, the nucleosome remodeling complex SWI/SNF, recruited to a gene by the activator, removes promoter nucleosomes in an early step in the process of gene induction [GAL genes [Gal4, like other eukaryotic transcriptional activators, must work despite the fact that DNA is wrapped in nucleosomes. For example, nucleosomes in the nscribed –17. One nscribed . A seconnduction . It has AL genes ,21.GAL gene transcription ceases, promoter nucleosomes rapidly reform at these genes [GAL genes, Gal4 is either inactive  or is depleted by one of the long-term effects of glucose as mentioned above. The possibility that Gal4 might continue to function in the simultaneous presence of galactose and glucose, and if so to what end, has not, to our knowledge, heretofore been considered.Just as removal of promoter nucleosomes is correlated with activation of transcription, so is their reformation typically correlated with the turning off of transcription. For example, when cells are transferred from galactose to glucose, and se genes ,17. WhetHere, we reexamine these matters using a quantitative micrococcal nuclease protection assay that measures, at any given moment, and for any specified DNA fragment, the fraction of the population that is occupied by a nucleosome in vivo. We show that in an early step of gene activation, Gal4 recruits SWI/SNF and quickly removes promoter nucleosomes. In the absence of SWI/SNF, a high level of transcription is also reached and promoters cleared of nucleosomes, but the process proceeds significantly more slowly. We confirm that upon transferring cells from galactose to glucose, transcription quickly diminishes, and nucleosomes rapidly reform at the promoter . In contGAL1 promoter), we can determine the fraction of that DNA segment, in the population, that is occupied by a nucleosome at the moment of cross-linking. This allows us to determine nucleosome positioning and occupancy prior to induction and to follow the time courses of nucleosome removal and reformation upon induction and repression. Consistent with many previous studies using micrococcal nuclease , the, theGAL1k, blue lines in UASg, a matter we return to below.In initial experiments, we found, as expected, that DNA, purified from cells not exposed to the cross-linking reagent (hereafter referred to simply as purified DNA), yielded, in every case, a first-order decay function when digested and analyzed as outlined above. The digestion rates of the segments  was some 200-fold slower than that in the faster digesting portion  DNA segments. The nuclease sensitivities of 40 such segments are represented by the colored bars above the gene schematic  or glucose. The depicted positions of these nucleosomes are consistent with earlier analyses [The repeat length of 160 bp is that expected if nucleosomes are positioned as shown in analyses ,14,16.UASg were protected in essentially every member of the population. Several experiments indicate that this protection is caused by some molecule bound to the UASg and that this molecule is neither Gal4 nor a nucleosome. First, the UASg isolated from cross-linked chromatin (see GAL4 (unpublished data). Third, as noted above, the ChIP experiment of UASg fragment, defined by the distance separating the flanking HS sites, is considerably smaller than that of the repeat length of a nucleosome (130 bp versus 160 bp). Finally, as we shall see  . Our experiments, as well as those of others, indicate a nucleosome disposition in the GAL1 ORF different from that found in the promoter. Thus, ChIP experiments indicate the presence of histones more or less uniformly across the ORF  and recruitment of the transcriptional machinery to the GAL1 promoter . snf2Δ mutants, mRNA production reached levels obtained with the wild-type strain, but only over a much longer time course that paralleled promoter nucleosome loss. These experiments also support the finding alluded to above that the recruited transcriptional machinery, readily detected by ChIP assays, does not protect against micrococcal nuclease digestion. complex ,31, had cription C. FigureSNF2 has no effect on induction of transcription of the GAL genes [snf2Δ strain of Kundu et al., and found that this strain, as well as the snf2Δ strain to which a wild-type SNF2 allele had been added, behaved identically to our corresponding strains in an assay for the rate of synthesis of mRNA upon induction  reformed on a fraction of templates equal to that occupied prior to induction. The other two nucleosomes in UASg over the course of the experiments of GAL genes we observed upon transfer to glucose plus galactose was not due to a nonspecific effect on transcription. Thus, three other genes , which are constitutively active, remained so in the presence of glucose in our experiments (unpublished data).A strikingly different result was obtained if, instead of transferring cells from galactose to glucose, the cells were resuspended in medium containing glucose (2%) plus galactose (2%). UASg over these time courses  and into glucose, promoter nucleosomes rapidly reform. Most of our findings were made possible by our quantitative micrococcal nuclease protection assay that measures fractional nucleosome occupancy in the population for any given DNA site. We now further discuss some of these matters.Our experiments, taken with other work, describe the following scenarios. Prior to induction, nucleosomes positioned so as to compete for binding with the recruited transcriptional machinery can be found on GCN5. Our results also show that in the absence of SWI/SNF, nucleosome removal and transcription were elicited by the action of Gal4, but the reactions were considerably slower than in the presence of SWI/SNF (requiring hours versus minutes). In this case, assuming the recruited machinery competes away the promoter nucleosomes, the reaction may be facilitated by the fact that these nucleosomes spontaneously vacate the promoter relatively frequently (see Promoter Nucleosomes below). Such a scenario might also explain the ability of fusion proteins, bearing a DNA binding domain attached to a component of the transcriptional machinery , to activate transcription to a high level. Consistent with this idea, we have found that activation by such a fusion protein follows a slow time course approximating that triggered by Gal4 in a strain mutant for SWI/SNF . A previous claim, emanating from this laboratory, that SWI/SNF is not involved in induction of the GAL genes [Our finding that none of several components of the transcriptional machinery is required for rapid removal of promoter nucleosomes indicates that the action of SWI/SNF, recruited by Gal4 and perhaps assisted by chaperones , sufficeAL genes , is explAL genes . Our resGAL80, the promoter remained nucleosome-free at this time point even in the absence of galactose. We surmised that Gal4 maintains the promoter nucleosome-free under repressive conditions just as it does when activating transcription, namely, by recruiting SWI/SNF. As predicted by this scenario, in a strain mutated for SWI/SNF, promoter nucleosomes rapidly reformed upon addition of glucose whether or not galactose remained present.We had no reason to anticipate our finding that galactose can continue to signal to Gal80, removing its inhibitory effect on Gal4, even as transcription is repressed by glucose. This was first suggested by the observation that the extent of nucleosome reformation some 30 min following addition of glucose was approximately inversely proportional to the concentration in the medium of galactose. This suggestion was confirmed by our finding that in a strain deleted for GAL4 and GAL2, even if immediate, would have an effect on GAL1,10 expression only as the previously synthesized Gal4 and Gal2 proteins were diluted away. And direct repression of GAL1,10, as measured hours after addition of glucose, is reported to be weak, only some 2–3-fold [GAL1,10 region by the specific DNA binding protein Mig1, and in preliminary experiments, we have found little if any alleviation of early glucose repression of GAL1,10 by deleting MIG1 . As expected from these various considerations, in our experiments, nucleosomes do slowly reform at promoters in the presence of galactose and glucose, presumably a consequence of depletion of Gal4 and Gal2.The ability of Gal4 to work at early times following the addition of glucose, maintaining the promoters nucleosome-free, does not contradict the known mechanisms for glucose repression alluded to in the Introduction. Thus, direct repression of 2–3-fold ,9. This An implication of our findings is that early negative effects of glucose on transcription cannot be ascribed to an elimination of all Gal4 recruiting activities. We do not know whether other recruiting activities of Gal4 remain functional, but if so, it is possible that glucose somehow causes destruction of any transcription complex that might be recruited to the promoter. This notion would be consistent with previous suggestions that certain mutations in that complex can diminish the negative effects of glucose –36.UASg). Thus, for example, where we find DNA locations that yield biphasic digestion curves, indicating two subpopulations, we have identified the slow digesting fraction as occupied by a nucleosome, the fast digesting portions as naked, i.e., simply lacking nucleosomes. And, we have argued, that the progressive increase in the fraction naked, as induction proceeds, reflects nucleosome loss. A more complicated description for the naked fraction could be imagined. Thus, for example, perhaps prior to induction, the naked regions bear nucleosomes in some aberrant configuration that would expose a segment of DNA so as to render it “naked” in our experiments, and according to this notion, as induction proceeds, instead of falling off, the nucleosomes increasingly adopt that aberrant configuration. It is difficult to completely exclude such a scenario. However, our ChIP analysis, which probed for histone H2B  obtained from EUROSCARF . Additional strains used were CY1069 (snf2Δ) and its corresponding wild type [SNF2 expression plasmid, pM4724 [snf2Δ derivative and to CY1069. For all experiments reported, cells were grown exponentially for at least 16 h at 30 °C prior to harvesting in synthetic complete medium (SC) or, in the case of snf2Δ and its wild-type control, in yeast extract peptone medium (YP). All sugars were added at a final concentration of 2% unless otherwise indicated. For the galactose induction experiments, prewarmed and aerated galactose was added directly to the media. In cases in which harvested time points where less then 5 min apart, medium containing 4% galactose was added at a one-to-one ratio to the growing cells. For the glucose repression experiments, exponentially growing cells were precipitated, washed with the original medium, and then added to prewarmed and aerated glucose containing medium under conditions in which the original medium was diluted greater then 100-fold. For all experiments, cells were harvested at an optical density at 600 nm (OD600) between 0.5–0.9 by fixing the cells with freshly diluted formaldehyde at a final concentration at 0.5% for 1 to 5 min. The fixing reaction was stopped by addition of glycine at a final concentration of 0.125 M.Strains, both wild type and deletions (except gged H2B ), were dild type . We also, pM4724 , to both2, 100 mM KCl, 400 μM dNTPs, 4% DMSO, 2× SYBR Green I (Molecular Probes), 0.01% Tween 20, 0.01% NP40, 1–4 ng/μl of each oligo primer, and 0.025–0.1 U/μl of Taq polymerase (Roche) was mixed with an equal volume of DNA being quantitated, resulting in a total reaction volume of 5 μl. The time that the oligo primers are mixed with the 2× reaction buffer is limited to less then 10 min before it is mixed with the DNA sample to limit primer dimer formation. A typical real-time PCR reaction measured 80 unknown samples plus 16 known samples (two standard curves) in quadruplicate, i.e., 96 samples transferred into four different positions of a 384-well plate. Seven of the eight samples within a standard curve consisted of 3.33-fold dilution series of yeast chromosomal DNA. The final sample of the standard curve contained no DNA. All real-time PCR reactions were performed on the Light Cycle 480/384 from Roche. Reactions were run for 40 to 50 cycles (depending on the primer pair) at 95 °C for 4 s, 59 °C for 26 s, and 72 °C for 4 s with the florescence of the SYBR Green being read at the 72 °C step. Since the specific activity of Taq polymerase varied considerable from lot to lot, care was taken to test by titration each batch of Taq to find its optimal concentration.DNA and cDNA were quantitated by real-time quantitative polymerase chain reaction . A 2× reaction buffer: 20 mM Tris-HCl (pH 8.3), 13 mM MgClOutliers of quadruplicate measurements were eliminated if dropping one of the four measurement reduced the standard deviation by greater then 2-fold and the original standard deviation was above the 50th percentile for the plate. An average quadruplicate measurement was eliminated if it was not greater then 2-fold above the measured value of the no DNA control. The DNA concentration was then determined by comparing real-time PCR measured values to a linear fit of the known chromosomal concentrations.GAL1, GAL10, GAL7, GAL3, and GAL2 and for controls HHF1, ACT1, and RBP11  of cells by a modified version of the hot acidic phenol technique  in whichChIP assays were performed as described .2O was added at a range of concentrations from 4 U to 0.000488 U in a 2-fold dilution series; two tubes had no nuclease. The reaction was started by adding 5.6 μl of 2 mM CaCl2 to each tube. The reactions were incubated for 1.5 h at 37 °C and stopped by the addition of 8.8 μl of 0.5 M EDTA each. Ten microliters of a solution containing 200 mM Tris (pH 7.4), 4 M NaCl, and 0.2 μl of Protease K  were added to each tube, followed by incubation at 42 °C for 1 h and at 65 °C for at least 4 h. The DNA was purified using the QIAquick 96 PCR Purification Kit (Qiagen). DNA was typically quantitated at 16 or more positions  and eight loci near the control genes TUB2 and PHO5.Reaction conditions: 100–200 ml of a yeast cell culture were spun down and resuspended in 500 μl of FA lysis buffer without EDTA: 50 mM Hepes-KOH (pH 7.5), 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate. The resuspended cells were sonicated twice for 10 s using a Branson Sonifier 250 equipped with a micro tip with the output set at 4. Cell debris was then spun down and the chromatin supernatant transferred to a new tube. A total of 26 μl was then distributed to 16 separate tubes, and 120 μl of FA lysis buffer without EDTA was added. To each tube, 10 μl of a micrococcal nuclease solution in HFor purposes of discussion, the DNA from each level of micrococcal digestion will be referred to as a sample. All 16 samples from the same harvest digested at the range of micrococcal nuclease concentrations will be referred to as a group. The real-time PCR-measured value for each sample at each position (locus) will be referred to as the sample-locus value, and the group of values at each locus as group-locus values.UASg was fit to the one-state decay function ekMN)− divided by its calculated one-step curve fit value. The normalized rescaled values for each group-locus is then fit to the two-state decay function (1 − fr2) ek1−(MN) + fr2 ek2−(MN), where MN is the concentration of micrococcal nuclease, k1 is the adjustable parameter representing the rate of digestion of the unprotected DNA, k2 is the adjustable parameter representing the rate of digestion of the protected DNA, and fr2 is the adjustable parameter representing the fraction of the DNA that is protected. The curve is fit by adjusting all three parameters to minimize the sum of the squares of the differences between the two-state decay function and the normalized rescaled group-locus values, where k1 is at least 50-fold greater than k2, and k1 is no less than a cutoff value set for each group . Sample-locus outliers from this fit were eliminated if the absolute difference of the normalized rescaled sample-locus value compared to its respective curve was greater then five times the average absolute difference for the entire group. The curves were then fit again to the remaining data points as described above. The error for each adjustable parameter was calculated by incrementally adjusting the parameter away from its best fit while allowing the other two parameters to adjust to their minimum until the sum of the squares of the differences increased by greater than 10%.For each group-locus, the undigested DNA concentration was rescaled to one by dividing each sample-locus value of a group-locus by the average of the undigested sample-locus values. To compensate for the varying yields of DNA for each sample within a group , the rescaled values for each group-locus measured at the 2 values was seen at the control loci (the variation was less then 15%). To correct for this, the average value for each control was assumed to be its true value. For each group, the measured fr2 values at the control loci were plotted against their average values, and a curve fit was then performed on this plot. The curve used was the line segments defined by the points , ) and , ), where the x-axis is the measured fr2 control values and the y-axis is the average control values. A least-squares fit was performed by adjusting x and y under conditions in which the slope of either line segment was between 0.5 and 2. The fit curve was then used to rescale all fr2 measurements from the group.A slight systematic variation in frFigure S1(A) This redrawing of the data of (B) The same as for (A) except that 20 min prior to harvesting the cells, galactose (2%) was added to cells growing in raffinose. This figure shows that after induction, despite the presence of the transcriptional machinery as revealed by ChIP analysis (see (300 KB PDF)Click here for additional data file.Figure S2UASg for cells growing in raffinose. These data were generated as a “high-resolution” ChIP. That is, after cross-linking and sonication, the chromatin was treated lightly with micrococcal nuclease before immunoprecipitation. The experiment that generated the data represented by the thin dotted line, in contrast, omitted this nuclease step. Sonication alone generates fragments of about 500 bp, and the addition of the nuclease step evidently decreases this fragment length sufficiently to dramatically improve resolution.The thick line is identical to that of (228 KB PDF)Click here for additional data file.Figure S3UASg (A) and one from the GAL1 ORF (B) (blue lines). Also shown are digestions of these segments as found in cross-linked chromatin (magenta lines). The curves in (B) are identical to those shown in UASg (A) confers less protection than does a nucleosome (B).Shown are nuclease digestions of two purified DNA segments, one from the (306 KB PDF)Click here for additional data file.Figure S4GAL1,10 promoter (excluding the UASg) or at one or another position in the GAL1, PHO5, orTUB2 ORFs. Cells were grown in the absence of galactose and the presence of phosphate, and so GAL1 and PHO5 are off. The highest points of occupancy in the GAL1,10 promoter correspond to the centers of the positioned nucleosomes flanking the UASg. The nucleosomes in the ORFs are not well positioned. The figure shows that, at many positions in the ORFs, the average protection is higher than in the GAL1,10 promoter. Note the values below 20% found in the promoter region—these are HS sites, and none are found in the ORFs.Each dot represents an approximately 60-bp fragment found in the (255 KB PDF)Click here for additional data file.Figure S5SNF2-deleted strains . Each strain was transformed with a plasmid expressing SNF2 (pM4724) to produce two pairs of putative isogenic SNF2+/− strains. The figure shows that for each pair, the strain expressing SNF2 induced significantly more quickly, as assayed by production of GAL1 mRNA, than did the strain lacking SNF2.The depicted experiment used two putative (354 KB PDF)Click here for additional data file.Figure S6Two ChIP experiments are shown using an antibody to Gal4.(A) Cells were pregrown in galactose and raffinose, and at time zero transferred to glucose.(B) Cells were pregrown in galactose and raffinose, and at time zero transferred to medium containing three sugars: glucose, galactose, and raffinose Click here for additional data file.Table S1The table lists each primer pair used for this paper with its: name, size, midpoint (relative to the ATG for promoter and ORF primers and relative to the stop site for terminator primers), and the sequence of each oligo.(217 KB PDF)Click here for additional data file.


Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the “core”, which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5′ core with a novel 3′ 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors. Chromatin insulator elements are boundary elements that separate active transcriptional domains in the genome to allow differential regulation of genes, and prevent the spread of heterochromatin towards active transcriptional units Chromatin insulator elements can confer an important safety feature to these randomly integrating vectors. A DNase hypersensitive fragment upstream of the chicken β-globin gene locus, the chicken hypersensitive site-4 (cHS4) is a prototypic insulator that has been extensively characterized. It has been shown to have enhancer-blocking activity Two distinct and separable insulator activities of cHS4 are confined to the 5′ 250 bp termed the “core” C), two tandem repeats of the core (sBG2C), 5′ 400 bp (sBG400), 5′ 800 bp (sBG800) or the full-length 1.2 Kb cHS4 insulator (sBG-I). All vectors carried the human (h) β-globin gene and promoter and the locus control region enhancer. The different insulator fragments were cloned in the forward orientation into the U3 region of 3′ LTR, so that upon reverse transcription, integrated provirus in target cells has the insulated 3′ LTR copied to the 5′LTR, and flanks the hβ-globin expression cassette at both ends. To assess whether elements outside the 5′ 250 bp core merely provided a spatial scaffold, vectors with inert DNA spacers downstream of the core, sBG400S and sBG800S, were also tested. All vectors were compared to the uninsulated control, sBG . Although there was no apparent integration bias between uninsulated and insulated single copy clones tested, the possibility that insulated vectors land in regions of the genome which are either more active or less prone to epigenetic silencing cannot be formally excluded. The presence of the cHS4 core (sBGC), or extended sequences of the insulator downstream to the core, up to 800 bp, did not increase the % hβ+ cells further; neither did tandem repeats of the core sequence, even though the latter has been shown to confer enhancer blocking effect in plasmid-based systems Consistent with our previous results 400S and sBG800S, showing that the reduction in CV was specific to the insulator core, and in contrast to the data on % of hβ+ cells, which required the full-length insulator to be present.Another phenomenon seen with transgene expression is clonal variegation, defined as varying levels of expression in daughter cells with the same integration site. A quantitative way to determine clonal variegation is by FACS analysis of transduced clones and calculation of the coefficient of variation (CV) of expression of the transgene around the average expression of the transgene in the clone. The CV is a unit-less measure of variability calculated as ratio between sample standard deviation (SD) and the sample average. We observed a high CV in the uninsulated sBG clones  MEL clones having both copies of the core deleted from both LTRs. We did not observe deletion of the insulator sequences in clones from all other vectors. Southern blot analysis of sBG2C MEL pools confirmed deletion of one/both copies of the core in the majority of cells It was notable that PCR for insulator sequences showed absence of the insulator sequences only in sBGC, sBG2C, sBG400 and sBG-I groups of mice  and (2) TER-119 positive erythroblasts showed no difference in the percentage of TER-119+ cells between different vector groups (not shown). However, significantly higher % of hβ+ cells were only present in secondary CFU-S with the sBG-I vector. Again, the CV was significantly lower in CFU-S transduced with all the vectors carrying the core, compared to uninsulated sBG transduced CFU-S (Supplementary figure S2). However, expression from sBGC, sBG2C and sBG400 transduced CFU-S was not significantly different from that of sBG CFU-S. Taken together, these data suggest that the 5′ 250 bp core sequences in sBGC, sBG400, sBG400S, sBG800 and sBG800S specifically reduced the clonal variegation of hβ-globin expression. However, the full-length cHS4 element was required for improved probability of expression from different integration events.The chromatin position effects were next confirmed in single copy secondary CFU-S. The secondary colony forming units-spleen (CFU-S) assay is considered the most stringent assay that is a ‘gold-standard’ for studying epigenetic effects of chromatin insulator elements in cells derived from hematopoietic stem cells. Notably, we did not observe any transduced CFU-S that was positive by PCR for vector-specific sequences that did not express hβ-globin by FACS, consistent with our results reported on lack of transgene silencing with erythroid-specific SIN lentivirus vectors C, sBG400 and sBG800, three vectors that carried the “core”.Next we determined the epigenetic modifications that accompany the specific effects seen with the various insulator regions by comparing the relative levels of active histone marks acH3, acH4 and H3K4me2 and repressive histone marksH3K9me3 and H3K27me3 between different proviruses in MEL clones. ChIP analysis was performed on the cHS4 core in three representative clones that were pooled together for each vector  and all other vectors, which carried the “core”, to assess whether differences in histone patterns over the transgene promoter in vectors may have contributed to the reduced clonal variegation. There was a small but significant reduction in repressive chromatin patterns H3K27me3 with sBGThese data show that the “core” sequences and extension of the core upto the 5′ 800 bp of cHS4 reduced activation marks over the transgene promoter to a small extent. However, a major reduction in repressed histone modifications over cHS4 and the transgene promoter region only occurred when the distal 3′ 400 bp sequences of cHS4 were present in addition.The anemia, reticulocytosis and other RBC indices were improved even with the sBG vector (2mα2) in sBG-I mice as compared to 19±6% in the sBG mice, while that in sBGC, sBG400 and sBG2C group of mice was not significantly different from control , derived MEL clones and transplanted mice with sBG3′400-transduced LSK cells. Note that unlike vectors described earlier, this vector does not contain the 5′250 bp “core” sequences (3′400 vector had no effect on % of hβ+ cells in MEL clones or the % hβ+ RBC in mice (C). However, like all vectors carrying the 5′ core, sBG3′400 significantly reduced the CV of hβ-globin expression in MEL clones and in RBC , but was at least 2-fold lower than that seen in the sBG-I mice  (The amount of hβ-globin protein in the sBG P<0.01) Figure 3C vector , and was similar to sBG-I clones. Likewise, the CV of the sBG650 clones was comparable to sBG-I clones , but was at least 2-fold higher than that seen in the sBG mice . Five months after transplant, secondary transplants were performed to generate CFU-S, which confirmed that the sBG650 vector restored insulator activity similar to that seen with sBG-I vector (650 proviruses (When we combined the 5′ 250 bp core and the 3′ 400 bp sequences of cHS4 insulator (sBG and RBC  was sign and RBC  was sign and RBC  was sign and RBC  was sign and RBC  was sign3′400) when present alone (sBG3′400), or when in combination with the 5′ core (in sBG650 and sBG-I)  Figure 8C, sBG3′400, sBG650 and sBG-I proviruses from MEL clones. ChIP analysis was performed using semi-quantitative PCR and qPCR. When primers to the core region were used to amplify ChIP products, CTCF and USF-1 recruitment to the 5′ core region was evident  likely occurs via USF-1.The 3′ 400 bp region, however, has no known CTCF or USF-1 motifs, that have been shown to impart enhancer blocking and barrier activity, respectively, to cHS4. It is conceivable; however that CTCF and/or USF-1 may perhaps be recruited to the 3′400 region. Using antibodies to USF-1 and CTCF, we immunoprecipitated chromatin from sBG650 had very reasonable titers that were only 2.5±0.9 fold lower than sBG, in contrast to 10.4±2 fold lower titers of sBG-I (n = 3). Therefore, this optimized insulator can be used for the design of safer gene therapy vectors which would provide uniform and therefore higher expression and be scalable to large-scale production.The 1.2 Kb cHS4 remarkably lowers titers of SIN-lentivirus vectors, limiting large-scale virus production for human trials. We have recently shown that the mechanism of reduction in titers is specifically due to the length of the insert in the 3′LTR 2C proviruses recombine The full-length cHS4 insulator has been previously shown by us C) or the extended 150bp sequences beyond the core (sBG400), although the increase was not statistically significant. In their study, an uninsulated and 400 bp cHS4 insulated self-inactivating lentivirus vector was compared in MEL clones, but no comparisons were made to a lentivirus vector with full-length cHS4, which in our hands, shows the highest hβ-globin expression. We confirmed using stringent assays in vivo by extending the core to 400 and 800 bp, with no significant improvement in the probability of expression from different integration sites.Emery and colleagues have shown that extending the 5′ core by 150bp provides good insulator activity C, sBG400 and sBG800 vectors, where no significant differences in these epigenetic marks were observed. Recently, Emery and coworkers have shown a 10-fold increased GFP expression from a 1.2Kb cHS4 insulated gamma-retroviral vector, which correlated with hyper-acetylation near the core and the promoter region Regions surrounding the cHS4 insulator and β-globin promoter have been shown to constitutively higher marks of active chromatin in the native location C, sBG400, sBG800 and sBG3′400 vectors. This effect required the vector carrying the full length cHS4 , thus increasing active chromatin marks. However, we observed no such increase in acH3, acH4 and H3K4me2 over the core or the 3′ 400bp when they flanked the transgene in the sBGChIP analysis over the hβ-globin promoter showed that compared to an uninsulated vector, the core alone reduced repressive chromatin marks over the promoter to some extent  supplemented with 10% heat-inactivated fetal bovine serum  and differentiated as described th3/+ thalassemia mice, a kind gift of Dr. Kaarin Gaensler , were used for transplants. All animal studies were done using protocols approved by the Institutional Animal Use and Care Committee. Enrichment of lineage−Sca-1+c-kit+ (LSK) hematopoietic stem/progenitor cells was performed on single cell suspension of bone marrow by immunomagnetic separation and FACS sorting  were dissected at day 12 after transplant of bone marrow cells from primary mice 24wk after transplant, as described earlier HbbComplete blood counts were performed on a Hemavet . Reticulocyte count was analyzed by staining 1µl of whole blood with 200µl of Retic-COUNT reagent  and enumerated on the FACSCalibur (BD). Quantitative analysis of hβ-globin protein in RBC was performed on hemolysates of blood by high performance liquid chromatography (HPLC), as previously described Table S1) in triplicate, and data analyzed as previously described [Ct (Input) − Ct (ChIP)] divided with [dilution rate (ChIP)/dilution rate (Input)]. Ct values of all PCR products were determined by the SDS 1.2 software (Applied Biosystems). Mean and SEM values were determined for the fold difference, and two-tailed paired t tests to determine statistical significance (p<0.05).ChIP analysis was performed on MEL clones as described Ligation-mediated (LM) polymerase chain reaction was performed as described by Modlich et al Vectors were compared to the sBG vector Student's ‘t” test (unpaired and two tailed). ANOVA (Dunnett multiple comparison test) was also performed between groups for multiple comparisons. Data was expressed as mean ± SEM. P<0.05 was considered significant.Figure S1Representative histograms (FACS) showing hβ expressing cells in mock, sBG, sBGC, sBG2C, sBG400 and sBG-I single copy CFU-S. The % of hβ+ cells are indicated within the histogram.(0.99 MB EPS)Click here for additional data file.Figure S2Human β-globin messenger RNA (mRNA) expression in single copy secondary CFU-S of sBG, sBGC, sBG2C, sBG400 and sBG-I by qPCR. Murine α-globin expression served as the internal control against which hβ-globin expression was normalized. P values are shown in the figure. ** indicates P<0.01.(0.87 MB EPS)Click here for additional data file.Table S1The primers and probes used in chromatin immunoprecipitation (ChIP) is shown. ‘F’ represents forward primer and ‘R’ represents reverse primer.(1.40 MB EPS)Click here for additional data file.Table S2http://genome.ucsc.edu..Insertional site analysis on single copy MEL clones from uninsulated sBG and insulated sBG-I vector with gene hits according to (0.37 MB PDF)Click here for additional data file.Materials and Methods S1(0.03 MB DOC)Click here for additional data file.


Given that induction of slo gene expression plays an important role in the acquisition of tolerance to sedating drugs, we investigated the molecular mechanism of gene induction. Using chromatin immunoprecipitation followed by real-time PCR, we show that a single brief sedation with the anesthetic benzyl alcohol generates a spatiotemporal pattern of histone H4 acetylation across the slo promoter region. Inducing histone acetylation with a histone deacetylase inhibitor yields a similar pattern of changes in histone acetylation, up-regulates slo expression, and phenocopies tolerance in a slo-dependent manner. The cAMP response element binding protein (CREB) is an important transcription factor mediating experience-based neuroadaptations. The slo promoter region contains putative binding sites for the CREB transcription factor. Chromatin immunoprecipitation assays show that benzyl alcohol sedation enhances CREB binding within the slo promoter region. Furthermore, activation of a CREB dominant-negative transgene blocks benzyl alcohol–induced changes in histone acetylation within the slo promoter region, slo induction, and behavioral tolerance caused by benzyl alcohol sedation. These findings provide unique evidence that links molecular epigenetic histone modifications and transcriptional induction of an ion channel gene with a single behavioral event.Tolerance to drugs that affect neural activity is mediated, in part, by adaptive mechanisms that attempt to restore normal neural excitability. Changes in the expression of ion channel genes are thought to play an important role in these neural adaptations. The  Drosophila melanogaster as a model system to determine how the nervous system becomes tolerant to the sedative effects of organic solvents. Sedating Drosophila with an organic solvent  increases the expression of the slo K+ channel gene, which accelerates recovery from sedation. To elucidate the molecular mechanics underlying these phenomena, we documented dynamic changes in a chemical modification  that occurs within the slo regulatory region after sedation. These changes were mediated by a transcription factor and are linked to both slo induction and behavioral tolerance. Increased expression of slo channels is predicted to alter the signaling properties of neurons. This alteration, we propose, directly speeds the recovery from sedation.A startlingly large number of adolescents abuse organic solvent inhalants, common components of glues, paints, and cleaning solutions. Our focus is on the molecular basis of tolerance—reduced response to a drug over time—which promotes increased drug consumption and accelerates the process of addiction. We use the fruit fly  Drosophila--is due to epigenetic modification of theslo potassium channel gene.Tolerance to solvents of abuse--at least in Drosophila melanogaster to study the neuronal basis of tolerance to organic solvent sedation.Drug tolerance can be defined as reduced responsiveness to an effect of a drug caused by prior exposure to the drug . With reDrosophila acquire rapid tolerance to subsequent solvent sedation. Rapid tolerance is defined as reduced drug responsiveness caused by a single prior exposure to the drug. In flies, this manifests itself as a reduction in the duration of sedation. Changes in the expression of the slo Ca2+-activated K+ channel gene have been linked to the production of rapid tolerance. It has been shown that sedation by a variety of methods induces slo gene expression in the nervous system, that slo mutations block the acquisition of behavioral tolerance, and finally, that transgenic induction of slo phenocopies the tolerant phenotype. Thus, the transcriptional regulation of the slo gene appears to be of general importance for the production of tolerance to sedation by various organic solvents [slo transcriptional control region. We continue to use BA as a model organic solvent anesthetic in this endeavor because it is well tolerated by flies and can be easily administered.When exposed to a small sedating dose of an organic solvent, such as benzyl alcohol (BA) or ethanol, solvents . We wishOne of the first steps in transcriptional activation is commonly thought to be the alteration of chromatin structure. Specific amino acids in the N-termini of core histones can be modified by phosphorylation, acetylation, methylation, or ubiquitylation . In partRecent studies indicate that histone acetylation contributes to the regulation of neural excitability and synaptic plasticity. It has been shown to have roles in learning and memory, in the production of circadian rhythms, and in the response to seizure, and has been identified as an important component underlying cocaine-induced neural plasticity –16. MoreDrosophila slo channel gene is very complex. It has a 7-kb regulatory region that includes at least five transcriptional promoters that mediate developmental and tissue-specific gene expression. The two upstream promoters are neural specific. Neural expression is requisite for the acquisition of tolerance to both the anesthetic BA and to ethanol [slo transcriptional control region, there exist multiple putative CREB DNA binding sites. The history of CREB makes it an attractive candidate for the sedation-mediated activation of the slo gene and the production of tolerance.Transcriptional regulation of the  ethanol ,4,19,20.cis-regulatory DNA element cAMP-response element (CRE) to induce target gene expression. In mammals, CREB has been implicated in the production of neuronal changes associated with drug tolerance and addiction. Exposure to addictive drugs, including opiates, cocaine, nicotine, and ethanol, induces CREB phosphorylation and CRE-mediated gene expression in the mouse nucleus accumbens (NAc), a major brain reward region [CREB is a key transcription factor involved in regulating neural plasticity in response to various environmental stimuli . Activatd region –27. The d region . Althougd region –34. Howed region .Drosophila, the cAMP-PKA signaling pathway, a pathway activating CREB, has been implicated in the development of drug tolerance. Drosophila carrying a mutation in amnesiac, a gene encoding a neuropeptide that activates the cAMP pathway, show increased sensitivity to alcohol [One way that CREB can stimulate transcription is by recruiting CBP, which is a HAT . By indu alcohol ,38. Conv alcohol .slo promoter region and that these changes are linked to increased slo expression, which produces behavioral tolerance to the sedative effect of BA. Furthermore, we show that the CREB transcription factor has an essential role in the production of acetylation changes, slo induction, and behavioral tolerance to organic solvent sedation.Here, we show that a sedating dose of BA changes the chromatin acetylation state of the slo transcriptional control region, following BA sedation. The antibody used in ChromIP recognizes histone H4 acetylated at lysines K5, K8, K12, and K16 in Drosophila and mammals [A strong link exists between gene induction and the acetylation of amino-terminal lysines in histone H4 in promoter regions ,40,41. W mammals ,42.slo transcriptional control region , the two neural promoters (C0 and C1), and the muscle promoter (C2)  A. As a cslo expression is not yet apparent . dCREB-A is weakly expressed in the adult brain [dCREB2 gene is most similar to mammalian CREB and CREM genes and is considered to be the Drosophila homolog of these genes [In lt brain  and has se genes .dCREB2 is expressed in the adult brain, and has been implicated in the formation of learning, memory, and circadian behavior [Drosophila, it has been shown that the KID domain of most dCREB2 proteins exists in a phosphorylated active state. As a result, in Drosophila, CREB activity has been postulated to be controlled at the level of DNA binding to its DNA elements [slo control region by sequence analysis. Furthermore, the highly conserved 55b element contains an AP-1 binding site motif. Both AP-1 and CREB belong to the b-ZIP family of transcription factors which share a similar DNA binding motif, the leucine zipper. The AP-1 response element (TGACTCA) is quite similar to the CREB response element, with only one base difference (CRE sequence: TGACGTCA). Both CREB and AP-1 can induce local histone acetylation by recruiting CBP HAT [behavior –54. As welements . Two put CBP HAT .Drosophila dCREB2 DNA binding domain . We determined the relative occupancy of dCREB2 protein within the slo promoter region both before and after drug sedation. The data indicate a clear drug-induced increase in CREB binding at and around the 55b region  and that elevated protein levels persist up to 24 h [As in mammals, the dCREB2 transcript is alternatively spliced. The dCREB2a splice variant acts as a transcriptional activator, whereas the dCREB2b isoform has repressor activity . A domin to 24 h .dCREB2b transgene was induced, and histone acetylation levels were measured after BA sedation. These acetylation levels were compared to those from flies of the same genotype in which the transgene had not been activated. We observed that the induction of dominant-negative CREB by a brief heat pulse 1 h prior to BA sedation eliminated the histone acetylation peak around 55b . We observed that induction of the dominant-negative CREB transgene 1 h prior to BA sedation suppressed slo induction . This may mean that increased accessibility of 6b is required to maintain slo induction. We propose that this dynamic pattern of hyperacetylation represents an unfolding regulatory program that leads to a transient and self-limiting boost in slo channel expression.The acetylation spike at 55b occurs immediately prior to the induction of cription . Finallyslo gene expression and whether it could also produce a tolerance-like phenotype. We observed that sodium butyrate consumption induced slo expression and phenocopied the BA sedation–induced tolerance phenotype. In various preparations, sodium butyrate has been shown to enhance histone acetylation and to induce expression of a small fraction of the genes in the eukaryotic genome (estimates range from 1% to 7%) [slo, and demonstrate other similarities to the effects of BA sedation. Using slo null mutations, we were able to show that the capacity of sodium butyrate to phenocopy tolerance is dependent on slo expression in the nervous system, as is rapid tolerance induced by BA or ethanol sedation [slo transcription control region. Sodium butyrate also caused hyperacetylation at the conserved 6b sequence, but did not detectably change acetylation at C1, cre1, or 55b sites. This mimics the acetylation pattern observed 24 h after BA sedation. Since sodium butyrate inhibits HDAC activity, the enhancement of 6b acetylation by sodium butyrate suggests that it has inhibited an HDAC that is chronically positioned near or at the 6b site. The simplest interpretation is that enhanced availability of 6b permits slo induction.We used the HDAC inhibitor sodium butyrate to artificially induce histone acetylation in the genome. We asked whether a simple change in acetylation would induce % to 7%) ,61,62. Tsedation . In addiDrosophila species [slo gene. Drosophila species.Both 55b and 6b are non–promoter-containing DNA sequences that were originally identified because of their conservation between two  species ,63. ConsTwo putative CREB binding sites are located close to 55b. The cre1 site is TGACGAA and cre2 is TGACGTAA. The cre1 site matches the canonical CRE motif, whereas cre2 differs in a single nucleotide. Both include the first five bases of the consensus CRE sites (TGACG), which are sufficient for CREB-mediated transcription . FurtherThe AP-1 and CREB transcription factors have been implicated in the neural response to abused drugs. AP-1 transcription factor complexes are dimers formed from the Jun and Fos family of transcription factors. Acute administration of certain abused drugs causes complex changes in the pattern of the Fos family members ,66–68. Tslo promoter region (unpublished data). However, ChromIP did show that CREB bound in this region and that drug sedation enhanced CREB occupancy. We further implicated CREB, in drug-mediated slo expression and behavioral tolerance, by showing that a dominant-negative CREB transcription factor could block hyperacetylation surrounding 55b, the induction of slo, and behavioral tolerance caused by BA sedation.Both AP-1 and CREB can stimulate gene expression by recruiting cofactor CBP, which helps to position the basal transcriptional machinery ,72. CBP slo in response to BA sedation. That is; drug sedation activates the CREB signaling pathway. CREB binds the CRE sites flanking 55b and perhaps within 55b itself, and then recruits the CBP HAT to acetylate histones in the neighborhood of 55b. This makes sequences at 55b available for binding by factors that lead to further modifications and result in the inhibition of the HDAC positioned at 6b. Inhibition of this HDAC augments 6b acetylation, making it available for binding by a factor , slo4, ash218/slo4, and the hs-dCREB2b. All fly stocks were raised on standard cornmeal agar medium (12/12-h light/dark cycle). Newly eclosed flies collected over a 1- to 2-d interval were studied 4–5 d after eclosion. The hs-dCREB2b transgene expresses a dCREB2b cDNA from a hsp70 promoter [promoter .https://www.fishersci.com) in food was fed to 1- to 2-d-old flies for 3 d. Controls were fed unadulterated food.Age-matched female Canton S flies were divided into six groups of 15. Three groups were sedated with 0.4% BA, and three were mock sedated as previously described . Sodium Tolerance was measured as described . In the About 1,500 wild-type flies were either BA sedated or mock sedated for 5 min and then were allowed to recover in a BA-free environment . Thirty http://www.upstate.com) with minor modifications. One milliliter of soluble chromatin (1 mg/ml) was adjusted to RIPA buffer and then precleared with 50-μl salmon sperm DNA/protein A agarose slurry for 1 h at 4 °C to reduce nonspecific binding. Ten percent of the preimmunoprecipitation lysate (100 μl) was held back and used to determine the input of DNA. The input-level control was processed with the eluted immunoprecipitations (IPs), beginning with the cross-linking reversal step. The polyclonal antibody  against acetylated H4 at K5, K8, K12, and K16, and anti-CREB antibody  were used at 1:200 dilution. Five microliters of antibody were added to each sample in 1-ml RIPA buffer and incubated overnight at 4 °C with gentle mixing. Immunocomplexes were recovered by adding 80 μl of the salmon sperm DNA/protein A agarose beads, incubating for 3 h at 4 °C with rotation. The beads were sequentially washed three times in RIPA , twice in high-salt buffer, once in LiCl buffer, and twice in TE buffer, 10 min each. The cross-linking between histones and DNA was reversed by incubating at 65 °C overnight, and DNA fragments were purified with phenol-chloroform extraction followed by acid ethanol precipitation. ChromIP assays were performed more than three times with independent tissue samples.The ChromIP assay was performed as described . Within the slo transcriptional control region experiment/2 ^ (CTinput – CTIP)control. The entire protocol was repeated in triplicate a minimum of three times, and the mean and SEM were calculated. Significance was determined by one-way ANOVA.All amplicons have differences in standard-curve amplification slopes of less than 0.1. Melting curves were used to detect nonspecific amplification. Amplifications were run in triplicate, and the changes on histone H4 acetylation were calculated by the ΔΔCT method. Fold enrichment over control equals 2 ^ (CTinput − CtIP). The entire protocol has been repeated in triplicate, and the mean and SEM calculated. Significance was determined with the two-way ANOVA.For ChromIP to measure CREB binding, all real-time PCR measurement values were normalized to input DNA in both the BA-sedated and mock-sedated control. The amount of DNA recovered in the IP was expressed as the ratio of input DNA with the equation: IP/input = 2 ^ (Cthttp://www.nanodrop.com). Global histone H4 acetylation levels were expressed as the ratio of the amount of DNA associated with the acetylated chromatin to input DNA. The entire protocol has been repeated four times, and the mean and SEM calculated. Significance was determined with the Student t-test.Chromatin from cross-linked fly heads was sonicated and immunoprecipitated with anti-H4 antibody as described above. DNA coimmunoprecipitated with acetylated histone H4 and input DNA were purified by reverse cross-linking followed by phenol-chloroform extraction. DNA was then quantified in a NanoDrop spectophotometer .Heat-induction protocol for the HS-dCREB2b transgene dCREB repressor (dCREB2b) transgene was induced at 37 °C for 30 min in a humidified incubator as described . Immediahttp://www.ncbi.nlm.nih.gov) accession numbers for genes mentioned in this paper are (32595), dCREB-A (39682), dCREB2 (32817), Gpdh (33824), and slo (42940).The National Center for Biotechnology Information (NCBI;


Cyanobacteria are photosynthesizing microorganisms that can be used as a model for analyzing gene expression. The expression of genes involves transcription and translation. Transcription is performed by the RNA polymerase (RNAP) holoenzyme, comprising a core enzyme and a sigma (σ) factor which confers promoter selectivity. The unique structure, expression, and function of cyanobacterial σ factors (and RNAP core subunits) are summarized here based on studies, reported previously. The types of promoter recognized by the σ factors are also discussed with regard to transcriptional regulation. Life depends on organic matter and oxygen produced by photosynthesizing organisms. Cyanobacteria, photosynthesizing organisms capable of producing oxygen, came into being about three billions years ago. It is generally accepted that the ancestor of cyanobacteria gave rise to plant plastids via endosymbiotic events, conferring the photosynthetic ability. Cyanobacteria are Gram-negative prokaryotes that perform oxygenic photosynthesis, found almost all over the earth. There are many kinds of cyanobacteria, including unicellular, filamentous, freshwater, marine, thermostable, and drought-resistant species. Some species are capable of fixing nitrogen, can produce hydrogen, and are possible sources of food. In addition, cells have become fossil fuels. Meanwhile, cyanobacteria have been recognized as a source of numerous natural products2 (RpoA × 2), β (RpoB), and β′ (RpoC), plays a central role. The core enzyme functions in RNA polymerization and requires the σ subunit for specific transcription initiated at the promoter.Gene expression involves regulation in four steps; transcription, post-transcription, translation, and post-translation. In transcription, the eubacterial RNAP holoenzyme, composed of a σ subunit and a core enzyme containing the major subunits αN and σEscherichia coli.N covers transcription from nitrogen-related genes in E. coli. Although cyanobacteria possess a system for nitrogen metabolism, no σN-types exist in cyanobacteria. Also in cyanobacteria, the σThe σ factors can be divided into two families, σ1.1–1.2ββ′(RpoA × 2/RpoB/RpoC). We present a schematic outline for the RpoA (41 kDa) and RpoB (131 kDa) subunits of the cyanobacterium Synechocystis sp. PCC 6803 (2ββ′γ (2ββ′β″). In order to avoid confusion, it needs to be noted that plant β′ and β″ are encoded by rpoC1 and rpoC2, respectively. The eubacterial β′ (RpoC) subunit, for example of E. coli, possesses eight highly conserved domains, A to H,E. coli RpoC. The insertion is suggested to be a DNA-binding domain as in the jaw module of the largest subunit of RNA polymerase II, RPB1.in vivo and in vitro.E. coli)-type core enzymes are essentially the same in molecular structure and function, however, differences in promoter recognition have been observed between them even with the same promoterE. coli or Thermus aquatics.As mentioned in the Introduction, most eubacterial RNAP holoenzymes consist of a core enzyme with subunits of αPCC 6803 . The Rpo3 (2ββ′γ . This arand RpoB 1 kDa sub1–2.1–2–1.Synechocystis sp. PCC 6803 (hereafter PCC 6803) (http://bacteria.kazusa.or.jp/cyanobase/) have been completely determined by the neighbor-joining method.E. coli RpoD (group 1 σ factor) which has generally been used as a standard σ factor.Anabaena sp. PCC 7120 (hereafter PCC 7120), which is a filamentous and heterocyst-forming cyanobacterium. These multiple proteins except for Alr3800 (SigB2) are encoded on extra-chromosomal plasmids. Therefore, we subdivided the B cluster into Bc (encoded into a chromosome) and Bp (encoded on plasmids). The C- and D-type σ factors have a single group 2 σ factor in each cluster. In the E clade, this type of σ factor is not found in the freshwater unicellular cyanobacterium Synechococcus elongatus PCC 7942 (hereafter PCC 7942), the unicellular thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (hereafter BP-1) and marine cyanobacteria  and have been classified into groups 1, 2 and 3, based on phylogenetic analyses.CC 6803) . This tibacteria .30 In adbacteria , 30 implbacteria . Therefo1–2–2.As mentioned in the Introduction, σ factors have evolutionally conserved regions critical for promoter recognition and forming a pre-initiation complex. Next, we summarize the composition and structural features of group 1–3 σ factors of PCC 6803. SigA (type-A) has regions 1 (1.1 and 1.2), 2, 3, and 4. In composition, group 2 σ factors  are similar to SigA but lack region 1.1, which has been identified upstream of region 1.22.In this section, we mainly summarize the research on group 1–3 σ factors in PCC 6803, the most intensively investigated cyanobacterium.2–1.Microcystis aeruginosa K-81 (hereafter K-81).sigA transcription. Concomitantly, the SigA protein level was slightly decreased in heat-shocked cells.Eubacterial group 1 σ factors are also known as principal σ factors, and this type σ factor comprehensively contributes to transcription in a cell. The group 1 σ factors are thought to be indispensable for the expression of housekeeping genes, since it is impossible to disrupt their genes. Consistent with this, the levels of the transcript and protein for the PCC 6803 group 1 σ factor, SigA, are constant despite growth phases under normal physiological conditions.In vitro transcription systems with reconstituted RNAP are a powerful tool for the biological characterization of the promoter selectivity of σ factors. So far, such systems have been established in PCC 6803, PCC7942, PCC7120, and BP-1.E. coli core enzyme and group 1 σ factors, have been tried for analyses of promoter selectivity in the cases of PCC 6803 and K-81.in vitro  approaches, group 1 σ factors specifically and efficiently recognize the E. coli RpoD consensus type promoters (−35 and −10 elements as TTGACA and TATAAT). The in vitro transcripts synthesized by RNAP-SigA (RNAP core enzyme + SigA) of PCC 6803 were undetectable when the mutated −35 element of the psbA2 promoter (mutated −35 and −10) was used as a template,In BP-1, the functional characterization of region 1.1 of SigA has been reported.rpoD1 (a group 1 σ factor gene) Promoter 2 facilitated transcription in K-81.psbA2 transcription involving RNAP with the group 1 σ factor.50Moreover, it is known that the tertiary architecture of DNA influences gene expression, and sequence-directed  or protein-induced DNA bends refer to changes in the DNA double helix. Previous studies presented evidence that a right-handed superhelical curvature upstream of 2–2. 2–2–1.2–2–1–1.sigB transcripts were also expressed upon the exposure of cells to heat-shock,sigB knockout strain, indicating that responses to heat-shock involve the autoregulation of sigB in PCC 6803.hspA encoding a small heat-shock protein was first identified to be a target of the heat-shock induced σ factor SigB.hspA promoter consistently observed in vitro was by RNAP-SigB among the group 1 and 2 σ factors.hspA transcript remained even in the sigB knockout strain (approximately 40% of the amount in the wild-type strain). No or almost no effect on hspA expression by other group 2 type σ factors, except for SigB, was observed under heat-shock conditions. Therefore, the remaining hspA transcript in the sigB knockout strain may depend on the promoter recognition by SigA. Consistent with this idea, transcription from the hspA promoter by reconstituted RNAP-SigA was observed in vitro.groESL1, groEL2, dnaK2, and grpA was not altered in the sigB mutant.Bacillus subtilis.groES, groEL1, and groEL2 but not hspA) were negatively regulated by HrcA, which acts as a repressor and prevents the expression of heat-shock proteins by binding directly to CIRCE (Controlling Inverted Repeat of Chaperone Expression).htpG, hspA, dnaK, groEL1, and groES was less induced in the sigB knockout strain after temperature upshift .sigB knockout strain may be due to different experimental conditions. However, SigB plays a central role cooperating with SigA/HrcA in the transcription of heat-shock inducible genes. Physiological studies indicated that the sigB knockout strain showed retarded growth at 43 °C compared with the parental wild-type strain.sigB, compared to 20% for wild-type cells.Under heat-shock  at the mid-exponential phase in PCC 6803, the protein level of SigB significantly increased 12.7-fold compared to that in the control experiment (30 °C). By contrast, the levels of other group 1 and 2 σ factors decreased only slightly or remained constant, and group 3 σ factors were not detectable under heat-shock conditions.2–2–1–2.b6 f complex), suggested that the oxidative state of the components downstream of the PQ induces SigB synthesis. In darkness, SigB is involved in the regulation of gene expression. The level of dark-induced lrtA transcription was not increased in the sigB knockout strain.in vitro transcription from the lrtA promoter.sigB knockout strain relative to the wild type,The SigB protein level also significantly increased 2-fold after a shift from continuous light to darkness. In contrast, the protein level decreased upon illumination to approximately 25% of that in dark-adapted cells.2–2–1–3.glnB (PglnB-54/-53) located at -54/-53 (+1 is the initiation codon), which is regulated by NtcA, a transcriptional activator of the CRP family, is due to specific recognition by SigC in the stationary  growth phase;glnB promoter in the logarithmic  growth phase. In the process of identifying the σ factor using group 2 σ factor knockout strains, it was found that SigB specifically contributed to transcription of glnB under nitrogen deprivation in the logarithmic phase, but other group 2 σ factors did not. The glnB gene expression was also slightly affected in the sigB knockout strain in the stationary phase, indicating that SigB is involved in the regulation of glnB expression in both phases.glnA, sigE, amt1, and glnN. Consistent with such observations, the SigB protein level was increased approximately 2-fold in response to nitrogen deprivation in both growth phases. Furthermore, transcripts synthesized from the NtcA-dependent glnB, glnA, amt1, and glnN in vitro were detected by RNAP-SigB and their levels were significantly increased when NtcA and 2-oxoglutarate  were added to the reaction mixture.Synechococcus sp. PCC 7002 (hereafter PCC 7002) sigB, corresponding to the PCC 6803 sigB (sigB-type σ factors in its genome (sigB2) and plasmids   . Among t2–2–1–4.sigB is induced in the cell upon exposure to osmotic, salt, or oxidative stress.sigB expression is regulated in response to a common signal, as mentioned below. SigB is an autoregulated heat-shock σ factor, which can specifically recognize the hspA promoter.sigB (about a 5 to 10-fold increase) and hspA (about a 3 to 90-fold increase) transcripts are remarkably accumulated upon the exposure of cells to salt- or osmotic-stress, which also leads to inactivation of PS II activity.sigB knockout strain after heat treatment  than in the wild type.sigB transcript was controlled by a two-component regulatory system comprising Hik2  and Rre1 , and also indicated that an osmotic responsive hspA transcript completely disappeared in the rre1 knockout strain.sigB transcript was reduced to less than half at 60 min after heat treatment  in the wild type, however, it remained high in the hik34 knockout strain, suggesting that Hik34 (Slr1285) contributes to the repression of sigB expression after the transient induction of sigB expression. Therefore, it is conceivable that sigB expression is controlled by a specific two-component regulatory system, which senses some signal (one candidate is the redox status of the photosynthesis electron transfer chain), and then SigB attempts to adapt to environmental conditions through the up- and down-regulation of gene expression.It has been also reported that transcription of rpoD2 resulted in a low amplitude but still rhythmic expression of psbAI. On the other hand, another study indicated that inactivation of rpoD2 resulted in an elevated level of psbAI expression.kaiB  and purF (encoding an amidophosphoribosyltransferase) were increased by about 1 hour in the rpoD2 mutant, whereas amplitude was similar to that of the wild-type strain. Overproduction of RpoD2 increased the period length of psbAI by about 2 hours, indicating that RpoD2 is involved in the rhythmic expression in a promoter-dependent manner. They also showed that the expression of rpoD2 itself peaked at subjective dusk like most genes in PCC 7942, and that RpoD2 negatively regulates its own transcription.rpoD2 mutant, the promoter activity of psbAI was elevated by inactivation of rpoD5/sigC.psbAI was prolonged by 2 hours in the rpoD5/sigC mutant. As for purF and kaiB expression, both periods were unaffected in the mutant, showing that separate timing circuits with different periods coexist in a cell.psbAI expression by about 2 hours as RpoD2 was overproduced. rpoD5/sigC itself showed a rhythmic peak in expression at subjective dusk as rpoD2 did, but the amplitude of its promoter activity was higher than that of rpoD2.rpoD3, its deletion resulted in about a 50% decrease in the amplitude of psbAI expression and a shift in phase by ~4 hours.purF expression was prolonged by about 3 hours and its amplitude was decreased in the mutant. On the other hand, the expression of kaiB was not significantly affected in the rpoD3 mutant. Therefore, the rpoD3 mutation affected promoter activities in three different ways. Interestingly, although overproduction of RpoD3 resulted in arrhythmic expression of psbAI,kaiB was not affected. The expression of rpoD3 rhythmically peaked at subjective dusk as for rpoD2 and rpoD5/sigC.The regulatory mechanism for the circadian clock has most extensively been studied in PCC 7942 among the cyanobacterial radiation.2–2–2.2–2–2–1.sigC knockout cells was slightly reduced and viable cell counts on BG11 plates were significantly lower (3.2 × 106 ml) in the stationary phase, compared with the value for wild-type cells (7.8 × 107 ml). The reduced cell viability in the sigC mutant at the stationary phase was also confirmed by trypan blue staining. Furthermore, they also confirmed that the number of lysed cells was larger in the mutant than wild-type culture by transmission electron microscopy. It was recently reported that differences in cell density between the wild-type and sigC knockout strain after prolonged heat treatment can be partially ascribed to differences in the stationary phase between these strains.sigC is a group 2 type σ factor, which is effective for cell viability in the stationary phase.In the process of investigating the function of SigC, Asayama et al.2–2–2–2.sigC knockout strain in the stationary phase under white-light illumination revealed that the signal corresponding to a nitrogen regulatory gene (glnB) product, PII, was significantly reduced in intensity in the sigC mutantsigC knockout cells under nitrogen depletion at the stationary phase, but not logarithmic phase of growth. Moreover, results obtained by transcriptome using high density gene filters  and quantitative real time (QRT)-PCR analyses revealed a decrease in the glnB transcript in the sigC knockout strain at the stationary phase, but not logarithmic phase. These results showed that glnB transcription specifically depends on SigC at the stationary phase. Further high-resolution analysis of the contribution of SigC to glnB expression from the two transcription start sites (PglnB-54/-53 and -33)sigC mutant at a mid-log phase of growth was equal to that in the wild type, whereas a significant induction in response to nitrogen deprivation was not observed in the sigC knockout strain. The inactivation of sigC did not affect the transcription from the PglnB-33 promoter.glnA, amt1, and sigE were decreased in the sigC knockout strain under nitrogen deprivation during the stationary phase, but not affected during exponential growth. Consistent with these findings, in vitro transcription analyses using reconstituted RNAP-SigC demonstrated that SigC specifically and effectively recognized NtcA-dependent promoters in the presence of NtcA and 2-OG.glnN promoter was observed in vivo and in vitro.glnA and glnN are essentially different.glnN, and pointed out possible regulation by a palindromic inverted repeat sequence in the region upstream of the NtcA-binding motif of glnN . The lack of recognition of the glnN promoter by SigC may be caused by the unique structure in addition to the low similarity of NtcA-binding motifs of glnN among the NtcA-dependent promoters.sigE, corresponding to PCC 6803 sigC .Primer extension analyses revealed that the sigC, implying that PCC 7120 SigC contributes to nitrogen metabolism in connection with iron homeostasis.gfp transcript to investigate the temporal and spatial pattern of expression of sigC (PsigC-gfp) revealed a rapid increase of GFP fluorescence in the sigC reporter strain 4 hours after a nitrogen step down (from a nitrate-plus medium to nitrate-free medium),E. coli σN-type σ factor groESL and hspA transcripts were not altered in the sigC knockout strain compared with the wild-type strain. Thus, the inability of the sigC knockout strain to grow at high temperature in normal air with CO2 is partly dependent on the poor availability of inorganic carbon at high temperature and partly on another, yet unknown, factor.Recently, Tuominen et al.2–2–3.2–2–3–1.−2 s−1), and first identified a σ factor, SigD, whose protein was significantly accumulated in response to the change in light intensity−2 s−1) or darkness was also examined by Western blot analyses.sigD, Hihara et al.−2 s−1 to 300 μmol photons m−2 s−1) in a genome-wide analysis with a DNA microarray. On the other hand, light-induced SigD synthesis was observed in the presence of rifampicin (inhibitor of transcription) but not chloramphenicol (inhibitor of translation),rpoD3, corresponding to PCC 6803 sigD (−2 s−1) at the level of the transcript and protein. This accumulation is specific to high-light, since rpoD3 expression was not significantly altered in cells exposed to high salt (0.5 M NaCl), high osmolarity (0.5 M sorbitol), darkness, high temperature (42 °C), or low temperature (22 °C).rpoD3 deletion mutant rapidly lost viability under high-light conditions. Therefore, the PCC 6803 SigD-type σ factor is evolutionally conserved in cyanobacteria and may function as a key protein for light- and high-light-responsive gene expression in response to changes in light.In 2003, Imamura et al. examined fluctuations in the protein levels of all σ factors under high-light conditions in PCC 6803 , heat-shock (transition from 30 °C to 45 °C), salt stress (0.5 M NaCl), high osmolarity (0.5 M sorbitol), and hydrogen peroxide (0.25 mM H2O2).sigD transcript did not accumulate under heat-shock (transition from 32 °C to 42 °C), low temperature (transition from 32 °C to 16 °C), and high salt (0.5 M NaCl). The different results might be due to different experimental conditions and detection methods. In the normal physiological growth phase, sigD transcripts starting from three putative transcription start TSPs  were detected at almost constant levels, but the protein level of SigD was higher in the exponential phase than stationary phase.24DNA microarray analyses showed that transcription of PCC 6803 sigD transcription were identified by Murata’s group. They showed that the mutation of hik33, encoding a well-known sensory histidine kinase involved in multiple stress responses, led to reduced sigD induction by osmolarity stress or cold-shock.sigD transcript was also reduced by knockout of rre31, a response regulator gene, indicating that the Hik33-Rre31 two-component system transduces osmotic signals for sigD transcription.rpoD3 (as the sigD type of PCC 6803) transcription by binding to the HLR1 (high light-responsive element 1) motif, located just downstream of the TSP of rpoD3, under normal growth conditions, and the dephosphorylation of RpaB induced by high-light stress results in transcriptional derepression of rpoD3. However, the mechanism by which light-sensing signal transduction, involving changes in the redox state of the electron transport chain in photosynthesis, is linked to the light-responsive transcription with σ factors has been unclear. In PCC 7942, results from a recent study indicated that the SasA (as an ortholog of Hik8 in PCC 6803) –RpaA (Rre31 in PCC 6803) two-component regulatory system functions as a major circadian timing mediator.sigD, and Rre26 is implicated in the regulation of sigD transcription under high-light as in PCC 7942. Further studies are needed to clarify the regulatory mechanism of sigD expression in response to environmental stress.Some regulatory factors for 2–2–3–2.psbA2 and psbA3, and contributed to their light-induced transcription.cpcBADC, petBD, and psaAB. This demonstrates a universal function of SigD for light-induced transcription in cyanobacteriasigD knockdown strain was conducted.sigD mutant observed by Imamura et al. and Yoshimura et al. (see above) were not detected as genes with reduced levels in the microarray analysis. As they discussed, the difference may be due to the dynamic range of their system, in which the detection of an ~1.5-fold reduction  is difficult.In 2003, it was reported that the light-induced σ factor PCC 6803 SigD specifically recognized the promoters of photosynthetic genes, 2–2–4.2–2–4–1.8TACN21TAN3T, bound by NtcA (a nitrogen controlled activator), and identified it approximately 270 nucleotides upstream from an initiation codon  of sigE (rpoD2-V) in the PCC 6803 genome. Northern blot analysis indicated that transcription of sigE was indeed induced in the nitrogen-deprived cells. Further 5′-end mapping of the sigE transcript by primer extension showed that the transcription from PsigE-202 was constitutive, whereas the transcription from PsigE-264 increased with nitrogen deprivation. This suggested that the transcription from PsigE-264 was controlled by NtcA. Actually, a gel-shift analysis confirmed that purified NtcA bound to the sigE promoter.sigE expression at the level of the transcript and protein was increased by nitrogen depletion in a NtcA-dependent manner.sigE expression through the induction of transcription from PsigE-264 in response to nitrogen deprivation. On the other hand, the transcription from PsigE-202 is thought to be controlled by the group 1 σ factor, SigA, since the transcript was not affected by disruption of each group 2 σ factor gene.sigC  were found as the first case of σ factor genes in a cyanobacterium.sigE expression exhibits a circadian rhythm at the transcript level.sigE expression at the level of the transcript and protein is regulated by the circadian system.In 1997, light-responsive transcripts of K-81 2–2–4–2.sigE was identified as encoding a nitrogen responsive σ factor, the transcript levels of nitrogen-regulated genes, amt1, glnB, and glnN, in the sigE knockout strain were examined.amt1 or glnB was not significantly altered in the mutant, however, activation of the transcription of glnN was impaired in the mutants; a decrease of about 30% was observed after 4 hours of nitrogen starvation, and recently, the same results were also obtained by Asayama’s group.pfkA (encoding one of two phosphofructokinases), gap1 , pyk1 (one of two pyruvate kinases)], the oxidative pentose phosphate (OPP) pathway , or glycogen breakdown [glgX (one of two glycogen isoamylases) and glgP (one of two glycogen phosphorylases)]. Consistent with the analysis of the transcripts, the sigE knockout strain showed a reduced rate of glucose uptake and an increased intracellular level of glycogen. Moreover, the mutant was unable to proliferate under light-activated heterotrophic growth conditions. These results clearly indicate that SigE functions in the transcriptional activation of sugar catabolic pathway-related genes in PCC 6803  and micropeptin (mip) have been identified and characterized in the freshwater unicellular cyanobacterium Microcystis aeruginosa K-139.mcyA and psm (peptide synthetase of Microcystis) 3A/3I were revealed and their possible promoters exhibited the consensus sequences recognized by the E. coli RpoD-type σ factor. Do σ factors such as the SigA-, SigD-, and SigE-types contribute to light-induced gene expression? This is an interesting question because nonribosomally synthesized peptides produced by some bacterial and fungal species belong to a diverse family of natural products that include antibiotics, immunosuppressants, plant and animal toxins, and enzyme inhibitors for which mechanisms of biosynthesis and light-responsive gene regulation have yet to be elucidated.For light-responsive gene expression, nonribosomal peptide synthetase genes responsible for the biosynthesis of 2–2–4–3.For photosynthesizing organisms, the carbon (stored as starch) produced by photosynthetic reactions is utilized as a source of energy through catabolic pathways in the dark (night). Actually, the expression of genes involved in the catabolism of sugar showed a circadian rhythm, which peaked during darkness .2–3.2–3–1.sigF knockout strain and found a pleiotropic phenotype. Most notably, the knockout strain lost phototactic movement with a concomitant loss of pili, which are abundant on the surface of wild-type cells , from the promoter PpilA1-54, in wild-type cells. The pilA transcript from PpilA1-54 completely disappeared in the sigF knockout strain, but not on the knockout of group 2 and group 3 σ factors. Further in vitro transcription analysis using each reconstituted RNAP with one of the nine σ factors clearly demonstrated that only SigF is capable of recognizing the pilA promoter. The transcription of sigF from PsigF-22/-21sigF knockout strain.sigF transcription was specifically driven in vitro by reconstituted RNAP-SigF, indicating that sigF expression is autoregulated.pilA1 promoter in PCC 6803. Furthermore, in vitro transcription analyses using scanning mutagenized template DNAs of PpilA1-54 identified the region from −39 to −7 including an AG-rich stretch and a core promoter with TAGGC (−32 region) and GGTAA (−12 region) as important for transcription. The core promoter of PCC 6803 pilA1 is identical to that of PCC 6803 sigF  and sll0041 , were experimentally verified to be targets of SigF, their promoter sequences being well similar to the identified SigF recognition core promoter -treated cells by in vivo labeling of proteins with [35S]methionine and subsequent electrophoretic separation of soluble proteins from cell lysates. Results from one- and two-dimensional electrophoresis indicated that the sigF knockout strain exhibited markedly decreased levels of salt-induced de novo protein synthesis. The reduction was not observed under the other conditions, but one (approximately 16-kDa protein) of the proteins was also decreased in the high-light-treated cells. The tolerance of the sigF knockout strain for 684 or 767 mM NaCl was similar to that of the wild type, whereas 859 mM NaCl was lethal to the mutant. It was also found that exposure of cells to high-light  was lethal to the sigF knockout strain. However, the mechanism by which SigF is involved in salt- and high-light-related gene expression is totally unclear.The physiological characterization of the PCC 6803 sigF knockout strain showed much slower growth than the wild type when the cells grown at 38 °C were exposed to 15 °C.sigF mutant had the same growth rate as the wild type, suggesting that sigF is required for the cells to grow at 15 °C but not 22 °C. However, the mechanism has remained obscure.In PCC 7002, the So far, endogenous PCC 6803 SigF protein has not been detected under normal physiological conditions or various stressful conditions by Western blotting using an antibody which specifically recognizes recombinant SigF protein;2–3–2.sigG gene is essential for cell growth, since a completely segregated sigG mutant has not been obtained.sigG knockout strain showed a decreased growth rate in spite of its incomplete segregation.sigG knockout strain to about 1,500 μmol photons m−2 s−1 for 2–3 hours at 29 °C was lethal. A primer extension analysis revealed that transcripts of sigG were synthesized with three putative transcription start sites  and increased dependent of the growth phase.E. coli promoter recognized by RpoE , are cotranscribed.sigG and sapG gene products interact when produced in yeast cells. The sigG or sapG mutant strain grew as well as the wild-type strain at 38 °C, indicating that these genes are not required for cell growth under optimal conditions. In contrast, the sigG mutant could not grow continuously at 22 °C, and could not grow at all at 15 °C. The sapG mutant showed a similar growth phenotype, suggesting that SapG is a regulatory protein for SigG involved in the same pathway for acclimation to low temperature. Interestingly, the structure of sigG-sapG in the cyanobacterial genome is conserved in PCC 6803, PCC 7942, PCC 7120, BP-1, and NIES-843 (CyanoBase). It is thus conceivable that the action of SapG toward SigG is conserved in cyanobacteria.The necessity of sigG occurred between 9 and 13 hours after a nitrogen step-down according to PsigG-gfp reporter analyses,In PCC 7120, the expression of 2–3–3.sigH gene was performed by primer extension analysis, and two putative transcription start points were identified (PsigH-73 and -36/-35).E. coli RpoE . At this time, however, the transiently activated expression of the heat-shock gene groEL was already reduced nearly to the control level. Thus, it is unlikely that SigH is responsible for the quick heat-shock-responsive transcription. But there is no information available regarding genes controlled by SigH-type σ factors in cyanobacteria.The 5′-end mapping of transcripts for the li RpoE σ and B. s2–3–4.sigI possesses two putative transcription start points (PsigI-100 and -50) that contain the B. subtilis σB recognition promoter, and the transcript levels increased with cell growth.sigI gene is not essential for cell growth, since a completely segregated sigI mutant has been obtained.105Information is still limited about SigI-type σ factors. In PCC 6803, a primer extension analysis revealed that sigI promoter region (206 bp upstream of the sigI coding region) was active in vegetative cells and heterocysts.82In PCC 7120, the PsigI-gfp reporter strain showed that the 2–4.rpoD4 (M1-type) and rpoD6 (M2-type)  mutant.purF expression, the period was lengthened by about 3 hours and the amplitude was decreased. In the mutant, however, the amplitude of kaiB expression was not significantly affected. Thus, disruption of rpoD4 had a similar effect on circadian expression as that of rpoD3. The double mutant of rpoD3 and rpoD4 showed a shortened period of kaiB expression by ~1.5 hours, indicating that the kaiB promoter was redundantly recognized by RpoD3 and RpoD4.PMT2246 (M1-type) and PMT0346 (M2-type) of Prochlorococcus marinus MIT9313 and PMM1697 (M1-type) and PMM1289 (M2-type) of Prochlorococcus marinus MED4 was up-regulated by nitrogen depletion.PMT2246,sigE (see section 2–2–4–1).As mentioned above, the result of a phylogenetic analysis indicated that some cyanobacterial group 2 σ factors compose independent clusters besides SigB-, SigC-, SigD, and SigE-types (see section 1–2–1). Some of their σ factors are classified into the M-type which is divided into four subtypes, M1 to M4,M2-type) , Table 1Anabaena variabilis ATCC29413_Ava3085) formed another cluster of SigF.Nostoc HK-01 sigJ gene (ortholog of PCC 7120 sigJ) was significantly induced in the mid-stage of dehydration, and was upregulated before that of other σ factor genes. Subsequently, they characterized PCC 7120 SigJ under dehydration because a transformation method for cells of Nostoc HK-01 had not been established. It was observed that a higher-expressing transformant of the sigJ gene acquired desiccation tolerance. Furthermore, a genome-wide analysis with a DNA microarray showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the transformant. In accordance with the data from the microarray analysis, the amount of extracellular polysaccharide released into the culture medium was as much as 3.2-fold that released by the control cells. Thus, PCC 7120 SigJ is a key regulator of desiccation tolerance and regulates the synthesis of extracellular polysaccharide. Accommodating dehydration is important for cell survival, since drying causes cell lysis and damage to nucleic acids, proteins, and membranes.sigJ-type gene is not conserved in most cyanobacteria, as shown in Nostoc HK-01, the expression of group 2 and 3 σ factor genes except for sigJ was induced in the late stages of dehydration. Therefore, it seems plausible that some other type of σ factor is also involved in gene expression for adapting to dehydration.There is a unique cluster of group 3 σ-factors, the SigJ-type, adjoining the SigF-type . In PCC How is there universality of gene expression and functions for respective group 2 and 3 σ factors, involving the M- and SigJ-types, among different cyanobacteria? Comprehensive and comparative (across) analyses of type σ factors among different cyanobacteria are awaited.2–4.de novo synthesized RNAs under light. Thus, subsequently, the SigB protein level was examined in group 2 σ factor knockout strains. Surprisingly, a large amount of SigB remained in the sigC knockout strain even 3 hours after exposure to light. Consistent with this, sigB transcript levels were dramatically elevated in the sigC knockout strain, being approximately 11 times higher than in the wild type at 0.5 hours after the shift to light. The mechanism by which SigC represses sigB expression remains unknown, though these results clearly indicated a regulatory network among group 2 σ factors. After that report, some groups revealed a regulatory network among group1, 2, and 3 σ factors in PCC 6803.In 2003, it was reported that a regulatory network exists among σ factors in cyanobacteria, which was discovered during an analysis of SigB expression.in vitro transcription analysis is a powerful tool for demonstrating direct recognition by an σ factor of its target gene(s). How can we obtain substantial evidence for σ factor’s target genes in vivo without using the standard genetic approach? Chromatin immunoprecipitation (ChIP) is widely used for quantifying protein-DNA interactions in living cells.rpoD3 and hliA promoter regions in PCC 7942.in vitro transcription and/or ChIP analyses as well as the conventional genetic approach will be useful for the characterization of σ factors in cyanobacteria.As the expression of group 1, 2, and 3 σ factor genes is controlled by each other, 3.As described in section 2, a unique feature of cyanobacteria is the presence of many group 2 type σ factors. The diversity of group 2 type σ factors impacts on promoter recognition and interference with the expression of group 1 σ factors. The novel ability of a group 3 σ factor, SigF, to recognize promoters has been known in PCC 6803. Promoter types of PCC 6803 connected with RNAP and σ factors are summarized in this section . Also di3–1.psbA genes, encoding the photosystem II reaction center protein D1, has been characterized in PCC 6803.psbA promoters for basal transcription, as the psbA transcripts were still observed on knockout of the group 2 σ factors.psbA under light or high-light conditions.psbA, cpcBACD, petBD, and psaAB.119Type 1 promoters possess both −35 (TAGACA) and −10 (TATAAT) hexamers as the σ3–2.glnB P2 promoter (PglnB-54/-53) has only the −10 hexamer and a GTA-N8-TAC motif for binding by NtcA. This motif confers an enhancement of transcription.in vitro the PglnB-54/-53 type promoters, PglnA-48/-47, Pamt1–142, and PglnN-32, in the presence of NtcA, but exhibit little or no activity without NtcA.lrtA is assigned to a subclass of type 2 having only the −10 hexamer with extended TGTGn and GC sequences,E. coli RpoD and RpoS, respectively. The selectivity of SigB for the lrtA promoter was directly confirmed in vitro by the redundancy of other group 1 and 2 σ factors.psbA promoter (PpsbAmt), whose sequence possesses only the −10 hexamer as an artificial type 2 promoter, has been reported for SigA and SigD.in vitro but SigA can not, indicating that the −35 hexamer is essential for RNAP-SigA in psbA transcription. Therefore, it seems that the transcription is driven by RNAP-SigA on the type 2 promoter. In other words, the group 2 σ factors may change with race, population, and/or activity with (or without) regulators under stress, and contribute to the transcription from the type 2 promoter in the presence of SigA. How do the group 2 σ factors exercise promoter recognition for transcription from type 1 and 2 promoters in replacement of the group 1 σ factor? As mentioned in section 2, there are several possibilities for the replacement of group 1 with group 2 σ-factors: (i) the group 2 σ factors are induced and increase in population under stress, (ii) co-activators and/or substances (e.g. ppGpp) are expressed under stress and enhance the binding of the group 2 σ factor to the core RNAP enzyme, (iii) the group 2 σ factors are modified at the post-translational level and are activated, and (iv) the group 1 σ factors are inactivated by anti-σ-factors (reported as RSD protein in E. coli)Type 2 promoters possess only the −10 hexamer or plus enhancer-motif sequences associated with transcriptional activator proteins that may compensate for the lack of function of the −35 hexamer. Because samples for type 2 promoters are limited , we disc3–3.pilA1, pixJ1, sll0837, and sigF) are distinct from those of the type 1 and type 2 promoters. Transcription from type 3 promoters may also be independent of group 1 and group 2 σ factors, as in the case of the pilA1 promoter recognized by SigF. Actually, in vitro transcription analyses demonstrated that the group 3 σ factors could not recognize the type 1 promoters.in vitro transcription systems together with knockout strains may also be useful for the identification and characterization of novel promoters recognized by group 3 σ factors.Stringent promoter recognition by PCC 6803 SigF was reported recently.4.Transcription in cyanobacteria is driven by the RNAP core enzyme with heterogeneous σ factors which are assigned to groups 1, 2, and 3. The evolutional diversity in terms of the population and function of cyanobacterial group 2 σ factors is unique. The network for gene expression of σ factors influences each other. Coordination among group 1 and 2 σ factors seems to contribute to the sensing of environmental changes or fine tuning for transcription from type 1 or 2 promoters. On the other hand, a stringent control for promoter recognition has been reported for the type 3 promoter. Functional analyses of structure and amino acid sequence, conferring differences in promoter selectivity, are required. Moreover, the characterization of unique structures in core enzyme subunits in RNAP and identification of transcription factors (e. g. response regulators in two-component systems) interacting with RNAP are also awaited for insights into cyanobacterial transcription.