Mycobacterium tuberculosis genome lead to better understanding of transcription units and gene functions, interactions occurring during transcription initiation between RNA polymerase and promoters is yet to be elucidated. Different stages of transcription initiation include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance. We have now analyzed these events with four promoters of M. tuberculosis viz. PgyrB1, PgyrR, PrrnPCL1 and PmetU. The promoters differed from each other in their rates of open complex formation, decay, promoter clearance and abortive transcription. The equilibrium binding and kinetic studies of various steps revealed distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. Surprisingly, the transcription at gyr promoter was enhanced in the presence of initiating nucleotides and decreased in the presence of alarmone, pppGpp, a pattern typically seen with rRNA promoters studied so far. The gyr promoter of M. smegmatis, on the other hand, was not subjected to pppGpp mediated regulation. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. Moreover, the distinct rate limiting steps during transcription initiation of each one of the promoters studied point at variations in their intracellular regulation.Although sequencing of  M. tuberculosis is one of the most formidable pathogens known to mankind. The resurgence of the pathogen, its alliance with HIV infection and emergence of the drug resistant strains has resulted in a global challenge to combat tuberculosis E. coli and other bacteria E. coliviz initiation, elongation and termination . During the initiation, RNA polymerase (RNAP) binds to the promoter, leading to the formation of several intermediates which differ from each other in their kinetic properties rrn, initiator tRNA) achieve higher promoter strength in exponential phase due to the stabilization of open complex by initiating nucleotides (iNTPs). In contrast, during the stationary phase, increase in the concentration of guanosine tetra/penta phosphate ((p)ppGpp), leads to inhibition of transcription from these promoters Transcription constitutes the first stage in gene expression and comprises of multiple steps M. tuberculosis. We have carried out promoter-polymerase interaction studies using a few of the house-keeping promoters to characterize the mechanisms of transcription initiation. The kinetics of RNAP-DNA interactions was measured in different promoter sequence contexts to determine their key rate–limiting steps. Further, the role of iNTPs and pppGpp in regulating transcription initiation was studied. While the M. tuberculosis ribosomal RNA promoter exhibited the characteristics seen with E.coli and other bacteria, the promoters for the gyrase operon showed unusual and hitherto unknown pattern of transcription initiation. Most significantly, the promoters for the same genes from M. smegmatis and M. tuberculosis differed markedly in their kinetic properties and response to the effectors.The present work is the first detailed kinetic analysis of the events during transcription initiation in viz. ribosomal RNA, initiator tRNA  and another house-keeping promoter (PgyrB1 ) were chosen in addition to a weak promoter (PgyrR) . M. tuberculosis has only a single operon for rRNA transcription driven by two promoters rrnPCL1 is the major house-keeping promoter and is stronger than PrrnP1rrnPCL1 is found in the genome of every sequenced species of mycobacteria and appears to be conserved across the genus metU is the only promoter driving the transcription of the single initiator tRNA gene in M. tuberculosisA consensus sequence gyr operon of M. tuberculosis included in the study are illustrated in gyrB1 is the major promoter of the gyr operon that directs the high levels of transcription from the gyrB - gyrA dicistron A dependent promoter consensus sequence. PgyrR is an overlapping and divergently organized promoter to PgyrB1 whose activity is appoximately 13 times weaker than PgyrB1 and hence is a representative weak promoter in this study For comparison of the promoter-RNAP interactions, two stable RNA promoters  (PgyrR) . M. tubein vivo activities of the promoters subjected to the present analysis were determined using the promoter-lacZ transcriptional fusion constructs transformed to 2 155M. smegmatis mc. PmetU showed the highest activity followed by PgyrB1 and PrrnPCL1. The activities were also measured at different times of growth . The promoter strength of PmetU did not vary significantly at different growth phases, while the activities of both PrrnPCL1 and PgyrB1 decreased as the cells entered the stationary phase  suggesting the regulation of these promoters is growth phase dependent. To understand the mechanism of differential regulation of the promoters, equilibrium and kinetic analysis of transcription initiation was carried out.The relative M. tuberculosis promoters with RNAP, binding constant (KB) for closed complex formation was measured. KB is the determinant of the strength of the closed complex, which is obtained by titration of the promoters with the RNAP and plotting the ratios of the bound fraction to total DNA (RPc/RPc+P) against the RNAP concentrations . The affinity of RNAP for PrrnPCL1 and PgyrB1 during closed complex formation was 5 and 3.3 fold higher respectively compared to the other stable RNA promoter, PmetU. Kd which is the determinant of the strength of the open complex, was obtained by plotting the ratio of RPo/RPo+P (fraction DNA bound) against RNAP concentrations . From the rrnPCL1 and PgyrB1 have comparable Kd values (lower than PgyrR and PmetU). Thus, although the affinity of RNAP for both PrrnPCL1 and PgyrB1 was higher than other two promoters studied, (see above) the overall extent of open complex formation was low, which could be either due to slow rate of formation or reduced longevity of open complex. Therefore, next,the rates of formation as well as stability of open complexes were measured for all the promoters. The rates of formation of open complex for the promoters were determined by measuring the rate constants of isomerization (see next paragraph). The stability of the open complexes was determined by monitoring the decay of the complexes with time.For the equilibrium binding analysis of ) it is evident that the rate of open complex formation was highest for the PgyrB1 followed by PrrnPCL1, thus providing an explaination for the higher strength of these two promoters. Notably, the rate of isomerization at PmetU was slow, although the in vivo activity of this promoter was high (see ). PgyrB1 also showed slower dissociation rate indicating another contributing feature for its strength. As expected, like other well studied rRNA promoters, higher dissociation rate was seen at PrrnPCL1, compared to the more stable open complexes at PgyrB1 and PmetU . Biphasic curve obtained in all these cases could be an indication of an initial faster dissociation of closed complex and a slower dissociation of a comparatively more stable open complex.Pseudo first order rate constant (k') which is the measure of isomerization rate was calculated by monitoring the time dependent interaction of RNAP and promoters. From the results of association kinetics, Table 1high see  E, suggerrnPCL1 and also PgyrB1 varied with the growth phase (see ), we next examined the effect of iNTPs on the isomerization and the open complex stability of the PrrnPCL1, PgyrB1 and the other two promoters. The open complex at PrrnPCL1 increased on addition of +1 iNTP, and in presence of both +1 and +2 iNTPs . In contrast to the rRNA promoter, the other stable RNA promoter, PmetU, did not respond in a similar fashion when the +1 iNTP was added. Notably, the isomerization was stimulated at PgyrB1 in the presence of +1 iNTP with the further increase in the presence of both +1 and +2 iNTPs, similar to the pattern seen with PrrnPCL1, which could be attributed to the intrinsic stability of the complexes at these promoters in the absence of initiating nucleotides. In vitro assays carried out in the presence of pppGpp showed that transcription at PrrnPCL1 and PgyrB1 was inhibited . There was no significant effect of pppGpp on transcription at PmetU, revealing that the promoter is not subjected to a similar kind of regulation.rRNA promoters from diverse organisms show enhanced promoter activity in the exponential phase. Since the promoter strength of P. The rate of promoter clearance was faster at two stable RNA promoters in contrast to the gyr operon promoters. The promoter which drives the dicistron transcription (PgyrB1) had 2.5 and 10 times slower clearance rate compared to PmetU and PrrnPCL1 respectively. The lower clearance rate seen with the gyr promoters seems to be resulting out of higher abortive transcription delaying the escape of RNAP from these promoters . When the amount of run-off transcripts synthesized at these promoters were compared in the single and multiple round conditions, fewer run-off transcripts were synthesized at PmetU in the single round transcription compared to the PgyrB1 and PrrnPCL1(data not shown). However, after multiple rounds of transcription, the accumulation of run-off transcripts at PmetU was comparable to that of PgyrB1 and PrrnPCL1.In addition to the efficiency in DNA binding and melting, overall promoter strength also depends on the rate of promoter clearance by RNAP. Thus the extent of abortive initiation during the transition from the initiation to elongation also has an important bearing on transcription initiation , . The in vivo strength is the net result of cumulative effect of all the steps. In the present study with the four promoters of M. tuberculosis, we have dissected the individual steps in the transcription initiation to understand their characteristic rate limiting steps.Kinetics and equilibrium binding studies provide an insight into the strength and the mechanism of transcription initiation at the promoters. In addition to the sequence of promoter elements and overall promoter architecture, the strength of a given promoter is governed by events occurring at various stages of the transcription initiation process and the rrn operons are transcribed by the strongest house-keeping promoters. Very high frequency of initiation at rrn promoters is a characteristic feature that contributes to the abundance in rRNA transcripts rrnPCL1 of M. tuberculosis is no exception to this paradigm. The high strength of the promoter can be attributed to its −10 and −35 elements, which closely resemble to the σA consensus sequence M. smegmatisrrnPCL1 of M. tuberculosis with that of M. smegmatis also revealed interesting differences . PrrnPCL1 of M. smegmatis showed slower promoter clearance and greater amount of abortive transcription in addition to an intrinsically unstable open complex M. smegmatis PrrnPCL1 in comparison to M. tuberculosis . The two promoters also differed significantly in their response to iNTPs and pppGpp (24 and this work); the stimulation and inhibition by the two effectors was much more pronounced at PrrnPCL1 of M. tuberculosis. However, inadequacy of the PrrnPCL1 of M. smegmatis appears to be compensated by the very strong PrrnB, which appears to be one of the strongest promoter in the organism. Moreover, the presence of a second functional rRNA operon also ensures adequate rRNA transcription. All these observations indicate the importance of species specific variations in promoters to meet the cellular requirements. The constitutive high level transcripts synthesized from the single rRNA operon of M. tuberculosis seem to fulfill the need of the metabolic machinery of the cell possibly due to the slow growth characteristics of the organism. As a consequence, the present day M. tuberculosis strains and other closely related pathogens seem to have lost the second rRNA operon (rrnB) found in the fast growing species of the genus rrnPCL1 by iNTPs and inhibition of its transcription initiation by pppGpp point out at the remarkable conservation of rRNA transcription regulation across the diverse bacterial species and this sensing mechanism appears to provide a unified theme for the growth phase dependent regulation of rRNA transcription. However, as summarized in Generally, in every organism as if by a rule, the rrn promoter, the other stable RNA promoter, PmetU, which drives the transcription from a single initiator tRNA gene in M. tuberculosis, does not appear to be influenced by iNTPs and pppGpp. In this context, studies with E.coli tRNA promoters provide interesting parallels. E. coli has two initiator tRNA promoters, PmetY and PmetZ, transcribing the single initiator tRNA and an operon containing three tandemly repeated tRNAf1Met respectively . While the transcription from PmetY is unaffected by pppGpp, the PmetZ is subjected to inhibition rrn operons, regulation of promoters of initiator tRNA is not universally conserved. Also in contrast to the PrrnPCL1, the isomerization step was found to be the rate limiting step at PmetU in M. tuberculosis. The lower amounts of run-off transcripts from the promoter in single round transcription assays, compared to the gyr and rrn promoters could be due to the slower rate of formation of the open complex. However, the high promoter strength of the PmetU under in vivo conditions could be accounted by its higher open complex stability, faster promoter clearance and low levels of abortive transcription. The accumulation of large amounts of transcripts after multiple rounds of transcription at PmetU . Slower isomerization rates, faster promoter clearance, lower abortive initiation are the characteristic features of the transcription initiation at Pgyr from M. smegmatisgyr promoter of M. tuberculosis, the subject of the present analysis, exhibited entirely opposite effects viz. faster rate of open complex formation, slower promoter clearance and higher abortive transcription. Notably, the two promoters exhibit markedly distinct mode of ‘Relaxation Stimulated Transcription’ (RST), a homeostatic control employed by cells to regulate gyrase activity and topological status of the genome gyrB1 could ensure control of the promoter linked to the growth phase. These differences in the regulation of the gyr operons between the two different species may indeed reflect their growth rates, physiology and contrastingly different life-style.Surprisingly, the present studies reveal that the process of transcription initiation at M. tuberculosis promoters studied is subjected to different rate-limiting steps and regulation. While unstable open complexes appear to serve as the sensors of initiating nucleotide concentration in rRNA promoter, distinctly, tRNA promoter is rate-limited at open complex formation and not subjected to growth phase dependent control. The opposing effects of the regulatory effectors, on the principle promoter of the gyr operon of the organism indicate the fine control connecting growth phase to supercoiling homeostasis of the genome, a mechanism probably required for metabolic shut down.To conclude, during initiation of transcription, each of the Table S3. Since promoters of gyr operon are divergent and overlap, the sequences of these promoters were altered such that only one of the two promoters was functional. The sequence of -10 element of PgyrB1 was rearranged from TACAGT to ACTTAG in the fragment containing PgyrR and the sequence corresponding to -10 element of PgyrR was changed from TCTTCT to CTCGTG in fragment containing PgyrB1  x volume of culture (ml) x optical density at 600 nm] M. 2155smegmatis mc transformed with the vector pSD5B in vivo promoter strength in different growth phases, the cultures of M. 2155smegmatis mc transformed with the promoter fusion constructs were grown for 12, 18, 24 30, 48 hours and the β-galactosidase reporter assay was carried out as described before.The cells were grown in MB7H9 (Difco) medium supplemented with 2% glucose (Sigma) and 0.05% Tween80 (Sigma). Promoter strength was measured by β-galactosidase reporter assay and the activity represented in Miller units  ATP and T4 polynucleotide kinase (New England Biolabs) at 37°C for 30 min. The labeled strand was annealed with two molar excess of complementary strand. The binding reactions were carried out in transcription buffer containing 50 mM Tris HCl, (pH-8.0 at 25°C), 3 mM magnesium acetate, 100 µM EDTA, 100 µM DTT, 50 mM KCl, 50 µg ml−1 BSA, 5% glycerol For EMSA, oligonucleotides having the individual promoter sequences were used. The 5' promoter fragments were end labeled at their 5' ends of one of the strands with  challenge for 1 min.The fractions were resolved on 4% native-PAGE at 37°C. The equilibrium dissociation constant for the heparin resistant complexes (Kd) was measured by the equation Y = Ymax[RNAP]/Kd+[RNAP], where Ymax corresponds to binding maximum To study the RNAP-DNA closed complexes, 1 nM of promoter fragments were titrated with varied amounts of RNAP. The incubation was carried out at ice for 20 min and the fractions were resolved on 4% native-PAGE at 4°C. The K−1) followed by immediate loading onto 4% native-PAGE electrophoresed at room temperature to analyze the bound fractions. For dissociation assays, open complexes were formed by incubating promoter fragments and RNAP for 15 min at 37°C and the assay mixtures were subjected to heparin challenge (50 µg ml−1). Aliquots (10 µl) were withdrawn at time intervals ranging from 0 to 48 min followed by loading onto 4% running native-PAGE electrophoresed at room temperature. The first order and dissociation rate constants were calculated by fitting the values as described earlier For determination of association rate constants, closed complexes were pre-formed as described. The aliquots (9 µl) from the assay mixture were withdrawn at different time points (0 to 48 min) and challenged with heparin (50 µg mlgyrR) in the presence of ribonucleotides. The ribonucleotides were added to a final concentration of 100 µM in different combinations . The reactions were incubated to form competitor resistant complex as described above and supplemented with NTP mix (100 µM), 1 µCi (α- P32) UTP and incubated at 37°C for 15 min. The reactions were terminated with 2x stop dye  bromophenol blue, 0.025% (w/v) xylene cyanol, 5 mM EDTA and 0.025% SDS and 8 M urea). The samples were kept at 95°C for 1 min and snap chilled before loading onto 8% urea-PAGE. The effect of iNTPs on open complex was also checked by EMSA. Briefly, the labeled promoter fragments were incubated with RNAP  in the presence of ribonucleotides (100 µM). The reactions were incubated to form competitor resistant complex as described above.Initially promoter DNA was incubated with RNAP  UTP and incubated at 37°C for 15 min and terminated with 2x stop dye  bromophenol blue, 0.025% (w/v) xylene cyanol, 5 mM EDTA and 0.025% SDS and 8 M urea). The samples were heated at 95°C for 1 min and snap chilled before loading onto 8% urea-PAGE. For single round transcription, 50 µg ml−1 heparin was added along with NTP mix (100 µM) and 1 µCi (α- P32) UTP. For promoter clearance analysis, promoter DNA (50 nM) and RNAP (100 nM) were incubated in transcription buffer and the reactions were carried out as described . +2 NTP in the Initial Transcribed Sequence (ITS) of each promoter was used as the labeled nucleotide. (α- P32) ATP was used to label the transcripts in case of PgyrB1, PmetU (α- P32) UTP for PrrnPCl1 and PgyrR. The samples were analyzed in 23% urea-PAGE (10∶1) to resolve abortive transcripts. A 10 µl aliquot from the same assay mix was loaded onto 8% urea – PAGE (19∶1) to resolve the run-off transcripts. The run-off transcripts were quantified as arbitrary units (AU) and plotted against time. The time corresponding to 90% of the maximum transcript formed, at each promoter, was calculated as the promoter clearance rate (PC90%).After RNAP [100 nM] and promoter DNA (50 nM) were incubated at 37°C for 10 min for open complex formation, RNA synthesis was initiated by the addition of NTP mix (100 µM), 1 µCi  was incubated with pppGpp (1 mM) in transcription buffer (with 35 mM potassium glutamate) for 15 min. Figure S1Effect of iNTPs on formation and dissociation of open complex. A iNTPs were incubated with promoter fragments and RNAP as described in Materials and Methods to determine their effect on isomerization. The initial transcribed sequence of each template is shown on the left side of the picture. B The amount of RNAP-promoter complex formed in the presence and absence of iNTPs was quantified (AU) and plotted. Slower moving complex was quantified in case of PrrnPCL1.(TIF)Click here for additional data file.Figure S2Iin vivo promoter activity of PrrnPCL1from M.smegmatis and M.tuberculosis.in vivo activites of PrrnPCL1 from M. smegmatis (M.smeg) and M. tuberculosis (M.tb) was measured by β galactosidase assay and plotted on Y axis as Miller units.(TIF)Click here for additional data file.Table S1Comparison of transcription at rRNA promoters of E. coli, M. smegmatis and M. tuberculosis.(PDF)Click here for additional data file.Table S2Comparison of transcription at gyr promoters of E. coli, M. smegmatis and M. tuberculosis.(PDF)Click here for additional data file.Table S3Strains, plasmids and oligonucleotides used in this study.(PDF)Click here for additional data file.Table S4Sequence of primers used in this study.(PDF)Click here for additional data file.


Sympathetic target tissue innervation is abnormal in many target tissues in the absence of neuron death, and like NGF, Egr3 also appears to effect target tissue innervation heterogeneously. In some tissues, such as heart, spleen, bowel, kidney, pineal gland and the eye, Egr3 is essential for normal innervation, whereas in other tissues such as lung, stomach, pancreas and liver, Egr3 appears to have little role in innervation. Moreover, in salivary glands and heart, two tissues where Egr3 has an essential role in sympathetic innervation, NGF and NT-3 are expressed normally in the absence of Egr3 indicating that abnormal target tissue innervation is not due to deregulation of these neurotrophins in target tissues. Taken together, these results clearly demonstrate a role for Egr3 in mediating sympathetic target tissue innervation that is independent of neuron survival or neurotrophin deregulation.Nerve Growth Factor (NGF) is a target tissue derived neurotrophin required for normal sympathetic neuron survival and target tissue innervation. NGF signaling regulates gene expression in sympathetic neurons, which in turn mediates critical aspects of neuron survival, axon extension and terminal axon branching during sympathetic nervous system (SNS) development. Egr3 is a transcription factor regulated by NGF signaling in sympathetic neurons that is essential for normal SNS development. Germline Egr3-deficient mice have physiologic dysautonomia characterized by apoptotic sympathetic neuron death and abnormal innervation to many target tissues. The extent to which sympathetic innervation abnormalities in the absence of Egr3 is caused by altered innervation or by neuron death during development is unknown. Using Bax-deficient mice to abrogate apoptotic sympathetic neuron death  The sympathetic nervous system (SNS) is a division of the autonomic nervous system that has essential roles in maintaining tissue and organ homeostasis. A detailed understanding of the mechanisms involved in establishing and maintaining normal sympathetic target tissue innervation is of considerable interest because abnormal development and degeneration of the SNS is a component of many human neurological diseases. A variety of molecules involved in different stages of SNS development have been identified. For example, migration of neural crest cells from the margins of the dorsal neural tube, which give rise to postganglionic sympathetic neurons, is dependent on many diffusible factors and their cognate receptor signaling including neuregulin-1/ErbB NGF has been known for many decades to be essential for sympathetic neuron survival Egr3 is a transcriptional regulator that is induced in sympathetic neurons by NGF, its expression is coupled to Ras/MAPK signaling, and germline Egr3-deficient mice have physiologic dysautonomia characterized by apoptotic sympathetic neuron death and abnormal target tissue innervation −/−; Bax+/+ (3-B+), Egr3+/+; Bax−/− (3+B−), and Egr3−/−; Bax−/− (3-B−) mice using optical dissector neuron counting and unbiased stereology. In this genetic background, 3-B+ mice had approximately 1/3 fewer SCG neurons compared to wild type (3+B+) mice, similar to previously published results Previous studies demonstrated that approximately 1/3 of sympathetic neurons die by apoptosis shortly after birth in the absence of Egr3 and that neuron loss was correlated with decreased sympathetic target tissue innervation and physiologic dysautonomia 2<0.00001). These results indicate that Egr3 mediates at least some trophic responses of sympathetic neurons that are similar to NGF function Target tissue-derived NGF has an essential role in sympathetic neuron survival and it has a trophic role in their differentiation and neurite outgrowth during development. In the absence of Bax, sympathetic neurons do not require NGF for survival, but they continue to respond to NGF by undergoing cellular hypertrophy and extending neurites In the absence of Bax, it was possible to examine whether Egr3 has a role in sympathetic target tissue innervation independent of concurrent neuron death. Egr3-deficient mice have blepharoptosis which is a physiological sign of abnormal sympathetic innervation from the SCG neurons to the eye and a failure to properly elevate upper and lower eyelids Using tyrosine hydroxylase (TH) immunofluorescence and semi-quantitative densitometry, we examined target tissue innervation arising from neurons in several sympathetic ganglia. For example, similar to the eye, sympathetic innervation from the SCG to the pineal gland was reduced by 55% in the absence of Egr3 and neuron death 3-B−;  comparedSympathetic innervation to the small bowel which arises primarily from pre-vertebral mesenteric sympathetic ganglia was also highly disrupted in 3-B− and 3-B+ relative to 3+B+ mice. Using TH immunohistochemistry on intact vascularized small bowel, sympathetic innervation was visualized coursing along the mesenteric vessels , arrow. While many tissues showed significant innervation abnormalities using semi-quantitative TH immunofluorescence and/or terminal axon staining with the DτlZ reporter transgene in 3-B− mice, some tissues such as thymus, lung, stomach, pancreas, liver and brown fat  showed nNGF is expressed by target tissues where it is essential for sympathetic neuron survival and target tissue innervation NGF is produced and released by target tissues where it mediates sympathetic neuron survival and innervation during development, a concept that has been clearly established in mice lacking NGF or its cognate tyrosine kinase receptor TrkA (NTRK1) In similar studies performed using Bax/NGF double knockout mice or sympathetic neurons isolated from Bax-deficient mice devoid of NGF treatment in vitro, terminal axon extension and innervation abnormalities were observed to differing degrees in target tissues Currently, the specific target genes regulated by Egr3 during sympathetic target tissue innervation are not known. One clue related to the mechanisms of Egr3 action in sympathetic neurons may come from the observation that axons prematurely defasciculate from the major mesenteric vessels as they innervate the bowel in the absence of Egr3. Perhaps genes that regulate axon fasciculation and/or axon guidance along blood vessels may be involved. This seems plausible as axon guidance molecules expressed by neurons such as neuropilin-1 (npn1) have a major role in axon fasciculation and target tissue innervation patterning NGF has an essential role in regulating sympathetic neuron target tissue innervation that is in part regulated by Egr3. Although the current results indicate that Egr3 does not regulate NGF or NT-3 in peripheral tissues, it has not been formally demonstrated that Egr3 has its primary function within sympathetic neurons to impact their target tissue innervation. A formal proof that Egr3 has an essential role in regulating target gene expression within sympathetic neurons to mediate target tissue innervation, and potentially to maintain innervation homeostasis, will require more sophisticated genetic studies to demonstrate its sympathetic neuron autonomous function.All experiments with animals reported complied with relevant national and international guidelines. All experimental procedures involving mice complied with a animal studies protocol (#2009-1886) approved by the Northwestern University Institutional Animal Care and Use Committee (IACUC) and PHS animal welfare assurance to Northwestern University (#A3283-01).−/−) and Bax (Bax−/−) were previously described and backcrossed >5 generations into the C57BL/6J isogenic background Mice with a germline deletion of Egr3 (Egr3Genotyping was performed by PCR using genomic DNA isolated from tail biopsy tissue. The sequences of the genotyping primers and PCR conditions are available upon request. Adolescent and adult mice (4–20 weeks old) were used for all analyses with the exception that newborn mice were used to obtain protein lysates from submandibular gland for Western blotting. For most experiments littermate or age-matched wild type mice were used as control. In experiments using Egr3/Bax double knockout (3-B−) mice, littermate mice lacking only Bax expression (3+B−) were used as control to examine the effects of loss of Egr3 on SNS development in the context of inhibited neuron death Anesthetized mice were perfused through the heart with 0.1 M phosphate buffered (pH = 7.2) 4% paraformaldehyde (PFA) and tissues were post-fixed at 4°C for 1–4 hours. Some tissues were cryoprotected overnight at 4°C in graded (15–30%) sucrose, embedded in OCT and 12 µm frozen sections were analyzed. For some experiments, tissues were isolated fresh for protein and/or RNA isolation. For quantitative studies involving neuron counts and morphometry, PFA fixed tissues were embedded in paraffin and serial sectioned at 16 µm.+/+; Bax+/+ (3+B+), Egr3−/−; Bax+/+ (3-B+), Egr3+/+; Bax−/− (3+B−), and Egr3−/−; Bax−/− (3-B−) mice were determined using unbiased stereology and optical dissector methods  on every fifth serial section as previously described SCG neuron numbers from 8–12 week old Egr3Immunofluorescence staining for tyrosine hydroxylase  to identify sympathetic terminal axon innervation and Renin  to identify the juxtaglomerular apparatus in kidney was performed on frozen tissue sections. A secondary antibody conjugated with Cy3 (Jackson Immunoresearch) or Alexa-488 (Invitrogen) was used to localize primary antibody binding. In some experiments tissues were stained intact without sectioning using TH primary antibody and immunoperoxidase detection as previously described +/+ (3+) and Egr3−/− (3-) newborn (P0) submandibular glands.Western blotting was performed as previously described Quantification of target tissue innervation was performed using fluorescence densitometry: Fluorescent images were captured with a Zeiss LS510 confocal microscope using identical aperture and photomultiplier tube voltage settings to ensure accurate comparison between tissues from 3+B- and 3-B- mice. The density of TH-positive terminals was calculated using MetaMorph software as the ratio of total immunofluorescence in a specified area and averaged for 6 separate confocal frames at 200x total magnification. Representative sympathetic target tissues, such as heart (left and right ventricles), distal ileum, trachea, esophagus, stomach, thymus, pineal gland, lung, pancreas, liver, spleen and kidney were analyzed in areas with the highest density of axons identified in control (3+B−) tissues. The “relative innervation” in 3-B− mice was expressed as a mean percent of control (3+B−) from 3–8 animals of each genotype as indicated.Whole mount lacZ enzyme histochemistry of periorbital tissue, SCG, kidney, spleen and distal ileum from 4–8 week old 3+B− and 3-B− mice that also carried the DτlZ sympathetic reporter transgene was performed as previously described GAPDH: , NGF: , NT-3:  and Egr3: .qPCR was performed as previously described in detail


GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a “feed-forward model” of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells. Gtf2i and Gtf2ird1, display a dynamic expression pattern during mouse embryo development TFII-I and BEN, products of paralogous genes Gtf2i or Gtf2ird1 in mouse embryos does not lead to peri-implantation lethality bona fide BEN-binding sites located in the upstream regulatory regions of Hoxc8, Tnn1 Gsc, Scand3, Cfl, Shrm and Ezh2 genes H promoters of IgH genes cis element RBEIII (ACTGCTGA) necessary for transcription of Human Immunodeficiency Virus Type 1 c-Fos as well as the set of estrogen-responsive genes by recognizing the Initiator sequence VEGFR-2 and β-globin genes, for example, occurs through the recruitment of TFII-I to the Initiator and E-box elements, respectively Cfdp1, Sec23a and Nsd1Although the TFII-I family members are expressed very early in development, they are not required for maintenance of pluripotency, because ablation of either in vivo targets of TFII-I factors are poorly defined. In the present work, we report genome-wide promoter mapping in mouse ESCs and embryonic craniofacial tissues. Chromatin immunoprecipitation (ChIP)-coupled DNA microarray analysis (ChIP-chip) revealed multiple TFII-I and BEN binding sites across the upstream regulatory regions of many developmental regulators. In addition to recognizing the previously defined consensus sequences, these proteins associate with the novel cis-regulatory elements. Collectively, our data indicate that chromatin recognition by TFII-I transcription factors is more complicated and diverse than previously considered.Despite these findings, the direct The promoter ChIP-chip analysis was used to identify regions bound by TFII-I and BEN in the mouse ESC lines E14tg2a and Ainv15 and embryonic craniofacial tissues (ETs) derived from E10.5 mouse embryos . The chr5,744 TFII-I and 625 BEN binding peaks (FDR <0.19) were identified within 5,024  and 577 We next investigated the occupancy of TFII-I factors in embryonic craniofacial tissues isolated from E10.5 mouse embryos. This experiment yielded 1,181 TFII-I and 1,520 BEN binding peaks (FDR <0.19) in 970  and 1,41Our next goal was to investigate a distribution of DNA-binding sites with respect to gene structure. CEAS software provides summary statistics including the percentages of peaks that reside in the proximal promoters (1 kb upstream from RefSeq 5′ start), 1st exon, 1st intron, and enhancer regions (>1 kb from RefSeq) Because we cannot rule out a probability that the chromatin recognition by the TFII-I family requires cooperation with additional transcription factors, DNA sequences around TFII-I and BEN bound sites were searched to identify potential protein partners listed in TRANSFAC and JASPAR databases. CEAS identified a number of hits for each motif both within the ChIP-regions and in the whole genome   and S8. cis-regulatory elements The TFII-I and BEN bound sequences were also analyzed for the presence of new GscGsc distal element corresponding to a stereotypical configuration of the two conserved R4 sequences  analysis using the DAVID software We also observed dramatic differences in the composition of genes targeted by TFII-I in mouse ESCs and ETs with regard to their biological functions. The most enriched cellular function among TFII-I bound genes in embryonic tissues is ‘signal transduction’ owing to a large group of olfactory, vomeronasal and taste receptor genes . At the We addressed the question whether the genomic sites occupied by TFII-I exert transcriptional regulatory activity. We analyzed the Affymetrix expression data (GEO database: GDS1616 record) derived from the same E14tg2a mouse ESC line used for ChIP-chip. The default P-value cut-offs (0.04 and 0.06) provide boundaries for defining Present, Marginal, or Absent calls. Absent indicates that the expression level is below the threshold of detection and close to zero. Marginal call indicates the cases of an uncertainty. The distribution of gene targets marked as absent, marginal and present is 49.1%:2.4%:48.5% for BEN and 48.7%:2.6%:48.7% for TFII-I, respectively. This observation clearly indicates that binding of TFII-I factors does not correlate with the transcriptional activity of the corresponding genes. Quantitative mRNA expression analysis from mouse ESCs http://www.informatics.jax.org/phenotypes.shtml). A chi-squared test was performed to estimate the average deviation of actually observed genes from the expected value of random variables  post transfection. Average 9.8-fold decrease in Gtf2i expression (>90% knockdown) and 5.8-fold decrease in Gtf2ird1 expression (∼80% knockdown) was observed 9 h post transfection. This silencing effect persisted through day 3 with the remaining 2.5-fold decrease of Gtf2i and 5-fold decrease of Gtf2ird1 expression, respectively  performed on the same GPL8943 platform (mouse 385 K RefSeq Promoter Array) in B6D2F2 mouse ESCs and Nsd1  is signicis-regulatory sites in ETs. Fourth, BEN predominantly recognizes promoters active in ETs.Our studies revealed that TFII-I and BEN recognize multiple distinct sequence motifs. In ESCs predominantly TFII-I primes the majority of genes associated with signal transduction and cell fate commitment. On a contrary, in embryonic craniofacial tissues BEN binds genes linked to tissue and organ development –11. We dGscThe under-representation of genes targeted by TFII-I in embryonic craniofacial tissues is in a strike contrast to the abundance of genes recognized by BEN –12. In pGsc promoter  and epigenetic modulators (Ezh2 and Nsd1 histone methyltransferase and Hdac4 histone deacetylase).According to this model TFII-I binds to the promoters of bivalent genes with delayed expression except of a few developmental regulators  that together with TFII-I, regulate other genes  . Among T5 ESCs were allowed to aggregate in suspension culture in Petri dishes. E14tg2a cells were grown in a feeder-free environment in KD MEM  containing 15% knockout serum replacement , 1000 U/ml LIF , 0.1 mM non-essential amino acids, penicillin-streptomycin-L-glutamine and 2-mercaptoethanol. The cell envelope-associated alkaline phosphatase (ALP) activity was determined using a commercially available kit ALP-10 . Briefly, a test cell population was washed twice in PBS, lysed in 0.1% Triton X for 5 min prior to addition of the p-nitrophenyl phosphate buffer.Mouse ESCs Ainv15 (SCRC-1029) and E14tg2a (CRL-1821) were obtained from ATCC, VA. Ainv15 was maintained on the feeder cells in DMEM  supplemented with 15% fetal calf serum , 1000 U/ml LIF , non-essential amino acids , penicillin-streptomycin-L-glutamine and 2-mercaptoethanol. The formation of embryoid bodies was initiated in E14tg2a cells by removal of LIF and 2-mercaptoethanol. After depletion of the feeder cells, 5×10Gtf2i or Gtf2ird1 in JoMa1.3 cells was performed using ON-TARGETplus SMARTpool siRNA . Three independent transfections were performed for each gene. To ensure minimal non-specific effects on gene expression we used the control negative siRNA (ON-TARGETplus Non-targeting Pool).Mouse neural crest cell line JoMa1.3 Gtf2ird1 (GenBank accession #AY030288) was amplified by PCR using Herculase II DNA polymerase . The PCR product was cloned into pLox vector and resulting construct plox/Gtf2ird1 was co-electroporated with pSalk-Cre into the genetically modified for this purpose cell line Ainv15 Murine full-length cDNA for 12 primers. Quantitative RT-PCR was performed on Applied Biosystems 7300 or on Bio-Rad MiniOpticon MJ Real-Time PCR system using TaqMan expression assays . The list of assays used in this study is given in Supplemental table S1. PCR reactions were performed in duplicates for each cDNA sample. All transcript levels were normalized to that of Gapdh and the relative expression ratios were calculated using the delta-delta Ct method cDNA was synthesized from 0.1–2 µg of RNA with Omniscript Reverse transcription kit  using oligo dT or random N2 ratio of the IP versus input samples was calculated and the transcription start site mapping was performed using SignalMap software . The promoter recruitment was validated experimentally by independent ChIP.ChIP experiments were performed with mouse ESC lines E14tg2a and Ainv15 or embryonic tissues using ChIP-IT kit . About 120 mouse embryos on gestational day 10.5 (E10.5) were dissected by a tungsten needle to produce craniofacial embryonic material for ChIP. Chromatin samples were sheared using Branson sonifier 150 . Chromatin-binding proteins were reverse cross-linked and digested with Proteinase K . 10 µl of the chromatin supernatant was saved as Input DNA before immunoprecipitation (IP). ChIP experiment was performed with goat anti-BEN polyclonal IgG M-19, goat anti-TFII-I polyclonal IgG V-18, mouse anti-HA monoclonal IgG2b 12CA5  and with rabbit anti-TFII-I polyclonal antibodies 4562  and antibody-chromatin complexes were purified using protein G-coated magnetic beads . After reverse cross-linking and purification on QIAquick spin columns , immunoaffinity-enriched DNA fragments (IP) and the input samples were amplified using whole-genome GenomePlex Complete WGA kit . IP and input samples were labeled in separate reactions with Cy5 and Cy3, respectively, and then were co-hybridized to Mouse 385 K RefSeq Promoter Arrays . The data were extracted using NimbleScan software. Peaks were detected by searching for four or more probes with a signal above a cut-off value using a 500-bp sliding window. A logGtf2i or Gtf2ird1 was performed in mouse ESCs E14tg2a using ON-TARGETplus SMARTpool siRNAs . Anti-H3K27me3 , anti-H3K4me3 , and anti-H3  antibodies were used for ChIP experiment. Quantitative data were obtained by qRT-PCR with SYBR Green and custom-made primers and expressed as a ratio of methylated H3 versus total H3 histone. For co-locaization analysis, H3K4me3 and H3K27me3 ChIP-Seq data were downloaded from NCBI GEO (GSM307618 and GSM307619).For analysis of histone methylation, siRNA-mediated knockdown of http://liulab.dfci.harvard.edu/MACS/). The TFII-I-binding peaks and methylated regions were considered co-localized if they overlapped or the distance between the closer ends of the two genomic coordinates was less than 200 bp apart from each other. The numbers for the co-localization between all different combinations were converted into percent ratios and used Heatmap2 (http://hosho.ees.hokudai.ac.jp/~kubo/Rdoc/library/gplots/html/heatmap.2.html) package in R to produce the heat-map visualization. Heatmap represents the numbers in color code chart with different colors used to represent different z-scores for the data. To compare DNA sequences the EMBOSS Pairwise Alignment Tools were used . Statistical analysis was performed with InStat 3.0b .The putative TFII-I and BEN binding sites were searched as a pattern consensus on both strands using the MacVector 9.5 software . Histone peaks were determined using the peak finding program MACS  from Dana-Farber Cancer Institute and Harvard School of Public Health in Boston, MA  Colocalization of TFII-I binding in mouse ESCs (a) and BEN binding in embryonic craniofacial tissues (b). For the density map, 5744 TFII-I-binding peaks from ESCs (a) and 1520 BEN-binding peaks from embryonic craniofacial tissues (b) were aligned relative to transcription start sites (left panels) and the corresponding BEN-binding peaks (a) or TFII-I-binding peaks (b) are shown in the right panel. Red dashed lines indicate position of the aligned “reference” peaks. (B) The novel consensus sequences recognized by TFII-I factors. (C) The canonical TFII-I and BEN binding motifs.(DOC)Click here for additional data file.Figure S2The gene ontology and KEGG pathway analysis in mouse ESCs. (A) The basic cellular functions. (B) The basic cellular processes. (C) The KEGG pathways. The red and blue bars represent fold enrichment of the TFII-I and BEN bound genes, respectively. The red stars indicate the statistically significant functional categories .(DOC)Click here for additional data file.Figure S3Gene ontology and KEGG pathway analysis of TFII-I target genes in mouse embryonic craniofacial tissues. (A) The basic cellular functions. (B) The basic cellular processes. (C) The developmental categories.(DOC)Click here for additional data file.Figure S4siRNA knockdown efficiency of TFII-I and BEN in JoMa cells.(DOC)Click here for additional data file.Table S1TFII-I binding sites in the ESC promoters.(XLS)Click here for additional data file.Table S2BEN binding sites in the ESC promoters.(XLS)Click here for additional data file.Table S3Promoters in ESCs recognized by TFII-I factors.(XLS)Click here for additional data file.Table S4Promoters in embryonic craniofacial tissues bound by TFII-I.(XLS)Click here for additional data file.Table S5Promoters in embryonic craniofacial tissues bound by BEN.(XLS)Click here for additional data file.Table S6Promoters in ESCs recognized by TFII-I and BEN to the same sequence.(XLS)Click here for additional data file.Table S7Enrichment for transcription factor binding motifs across the TFII-I and BEN bound promoter regions in ESCs.(XLS)Click here for additional data file.Table S8Enrichment for transcription factor binding motifs in the TFII-I and BEN bound promoter regions in embryonic craniofacial tissues.(XLS)Click here for additional data file.Table S9The consensus binding motifs within the TFII-I and BEN bound promoter regions.(DOC)Click here for additional data file.Table S10Pathway analysis in mouse ESCs.(DOC)Click here for additional data file.Table S11Pathway analysis in mouse embryonic craniofacial tissues.(DOC)Click here for additional data file.Table S12TFII-I transcription factors target a large set of developmental regulators.(DOC)Click here for additional data file.


INO1 gene. Here, loss of CDK8 suppressed the reduced mRNA expression and RNAPII occupancy levels of CTD truncation mutants.The C-terminal domain (CTD) of RNA polymerase II (RNAPII) is composed of heptapeptide repeats, which play a key regulatory role in gene expression. Using genetic interaction, chromatin immunoprecipitation followed by microarrays (ChIP-on-chip) and mRNA expression analysis, we found that truncating the CTD resulted in distinct changes to cellular function. Truncating the CTD altered RNAPII occupancy, leading to not only decreases, but also increases in mRNA levels. The latter were largely mediated by promoter elements and in part were linked to the transcription factor Rpn4. The mediator subunit Cdk8 was enriched at promoters of these genes, and its removal not only restored normal mRNA and RNAPII occupancy levels, but also reduced the abnormally high cellular amounts of Rpn4. This suggested a positive role of Cdk8 in relationship to RNAPII, which contrasted with the observed negative role at the activated  SRB10/CDK8 gene. Here, using gene expression microarrays, we determined the set of genes most sensitive to alternations in CTD function and uncovered unexpected links between RNAPII-CTD and Cdk8.RNA Polymerase II (RNAPII) is the enzyme responsible for the transcription of all protein-coding genes. It has a unique extended domain called the C-terminal domain (CTD). This domain is highly conserved across species and is composed of repeats of a seven amino acid sequence. The CTD functions as a recruiting platform for regulatory and RNA processing factors, making the CTD a master orchestrator of transcription. Previous work revealed a critical role for CTD length in the transcription of induced genes. However, how CTD length is generally required for transcription is currently unclear, as is the mechanism underlying the observed suppression of CTD truncation phenotypes by loss of the  Conversely, our data also revealed that deletion of CDK8 suppressed the activation defects of CTD truncation mutants at the INO1 locus thus indicating that Cdk8 also functioned to repress transcription and RNAPII association in CTD truncation mutants.The work here highlighted the functional circuitry between the RNAPII-CTD and Mediator in the regulation of cellular homeostasis, gene expression, and the transcription factor Rpn4. Our data uncovered a length-dependent requirement of the CTD for genetic interactions and mRNA levels of genes expressed under normal growth conditions. Truncating the CTD primarily resulted in increased expression and RNAPII association at a subset of genes, in part mediated by changes to transcription initiation. These genes had preferential association of Cdk8 at their promoters and were regulated by the transcription factor Rpn4. The expression and RNAPII binding defects of the majority of this subset of genes were suppressed by deleting rpb1-CTD11, rpb1-CTD12, rpb1-CTD13 and rpb1-CTD20 respectively) against a library of 1532 different mutants involved principally in aspects of chromatin biology and RNA processing RPB1 locus by addition of a TAG stop codon followed by a NAT resistance marker. As a control for the genetic integration strategy we also generated RPB1-CTDWT, which contained a NAT resistance marker following the endogenous stop codon. While the minimal CTD length for viability is 8 repeats, we focused on strains starting at 11 repeats as mutants bearing shorter CTDs were significantly unstable in our hands, consistent with previous findings rpb1-CTD11, rpb1-CTD12 and rpb1-CTD13 and the second consisting of rpb1-CTD20 and RPB1-CTDWT  suggesting that shortening the CTD results in genetic interaction patterns most similar to mutants affecting transcription initiation  to generate genetic interaction profiles of CTD truncation mutants containing 11, 12, 13 or 20 heptapeptide repeats  using an antibody specific to the RNAPII subunit Rpb3. Despite the use of different platforms, antibodies and normalization methods, the obtained genome-wide Rpb3 occupancy profiles obtained in wild type cells were highly correlated with those previously published by several groups (rpb1-CTD11 and wild type cells (Spearman's rho 0.85), agreeing with the limited transcriptional differences detected by the expression analysis. Nonetheless, our Rpb3 occupancy plots showed clear RNAPII occupancy differences along genes that were identified as either having increased or decreased mRNA levels in the rpb1-CTD11 mutant , thus suggesting a direct effect of truncating the CTD on RNAPII levels and mRNA synthesis at specific loci . Specifically, we measured RNAPII in l groups  [35]– and pRS314 [rpn4 S214/220A] were obtained from Dr. Youming Xie (Wayne State University School of Medicine). Reporter plasmids were generated by cloning 450 bp of the desired promoter into the Sal1 BamH1 sites of pLG669-Z Strains and plasmids are listed in Supplementary materials. Partial, complete gene deletions or integration of a 3XFLAG tag was achieved via the one-step gene replacement method E-MAP screens were performed and normalized as described previously rpb1-CTD11 cdk8Δ mutant. The Yeast Promoter Atlas database was used for transcription factor enrichment by performing a Hypergeometric test with Bonferroni correction  Microarrays were performed in duplicate as previously described Yeast cultures were grown in media containing 200 µM of inositol (uninduced) and switched to media lacking inositol for 4 hrs (induced) ChIP-on-chip cultures were grown overnight in YPD, diluted to 0.15 OD600 and grown to 0.5–0.6OD600 units. Cross-linking and chromatin isolation were performed as above. 5 µl of anti-Rpb3 (Neoclone), 4.2 µl of anti-FLAG (Sigma) or 4 µl of anti-H3K36me3 (Abcam ab9050) were coupled to 60 µl of protein A magnetic beads (Invitrogen). DNA was amplified using a double T7 RNA polymerase method, labeled and hybridized as previously described et al 1998. Average gene profiles were generated by averaging all probes that mapped to genes of interest. For averaging, probes corresponding to ORFs were split into 40 bins while probes corresponding to UTRs were split into 20 bins.CHROMATRA plots were generated as described previously rpb1-CTD11 mutants and assayed as previously described Reporter plasmids were transformed into wild type and Overnight cultures grown on YPD or –TRP media were diluted to 0.5 OD600, 10-fold serially diluted and spotted onto YPD or –TRP plates with or without the indicated amounts of hydroxyurea (Sigma), formamide (Sigma), or on plates lacking inositol. Plates were incubated at the indicated temperatures for 2–4 days.Whole cell extracts were prepared from logarithmic growing cells by glass bead lysis in the presence of trichloroacetic acid. Immunoblotting was carried out with 3E10, 3E8, 4E12, 8WG16 (Millipore), YN-18 (Santa Cruz), Rpb3 (Neoclone), HA-Peroxidase (Roche) and Pgk1 (Molecular Probes) antibodies INO1 mRNA levels were normalized to ACT1 mRNA RNA was extracted and purified using the Qiagen RNeasy Mini Kit. cDNA was generated using the Qiagen QuantiTect Reverse Transcription Kit. cDNA was analyzed by qPCR as described above. Overnight cultures were diluted to 0.3 OD600 and grown to 1.0 OD600. 10 OD600 units were collected to constitute time 0 and a final concentration of 100 ug/ml of cycloheximide (Sigma) was added to the remaining culture. 10 OD600 units were collected at the indicated time points. Proteins were extracted using trichloroacetic acid.Figure S1Sample genetic interaction network of CTD truncations mutants revealed CTD length-dependent genetic interactions. Subsets of genetic interaction profiles depicting genes involved in transcription and how they interacted with the CTD as it was progressively shortened. Blue and yellow represent aggravating and alleviating genetic interactions respectively. Gray boxes represent missing values.(PDF)Click here for additional data file.Figure S2et al 1998. (B) Chromosome plot of a 55-kilobase pair region on chromosome 5 .Comparison of previously published Rpb3 genome-wide association profiles. (A) CHROMATRA plots of RNAPII occupancy (PDF)Click here for additional data file.Figure S3et al 1998.Truncation of the RNAPII CTD leads to changes in the genome-wide association of transcription association factors.  CHROMATRA plots of relative occupancy of transcriptional associated factors (PDF)Click here for additional data file.Figure S4CDK8 suppressed CTD-associated growth phenotypes. (A) The sensitivity of CTD truncation mutants containing 11 or 12 repeats to known and novel growth conditions was suppressed by deleting CDK8. Ten-fold serial dilutions of strains containing the indicated CTD truncations with and without deletion of CDK8 were plated and incubated on YPD media at 16, 30 and 37°C and YPD media containing the indicated concentrations of hydroxyurea or formamide. (B) Immunoblots of whole cell extracts with CTD phosphorylation specific antibodies. YN-18 detects the N-terminus of Rpb1 and was used as a control for Rpb1 protein levels. Rpb3 was used as a loading control.Deletion of (PDF)Click here for additional data file.Figure S5Genome-wide Cdk8 occupancy plots agreed with previous reports. Average Cdk8 occupancy at all genes separated by transcriptional frequency revealed a preference of Cdk8 for binding to the promoter of highly transcribed genes (left) and confirmed that Cdk8 binding at coding regions was independent of transcriptional frequency (right).(PDF)Click here for additional data file.Figure S6GCN4 was not involved in the suppression of rpb1-CTD11 phenotypes by loss of CDK8. The sensitivity of rpb1-CTD11, cdk8Δ and gcn4Δ single, double and triple mutants in the W303 background was tested by plating ten-fold serial dilutions on YPD media at 16, 30 and 37°C and YPD media containing the indicated concentrations of hydroxyurea or formamide.(PDF)Click here for additional data file.Figure S7rpb1-CTD11 defects by loss of CDK8. The sensitivity of rpb1-CTD11, cdk8Δ, rpn4Δ single, double and triple mutants carrying an empty vector, or a plasmid containing either RPN4 or RPN4 S214/220A was tested by plating ten-fold serial dilutions on YPD media at 16, 30 and 37°C and YPD media containing the indicated concentrations of hydroxyurea or formamide.Phosphorylation of Rpn4 at S214/220 is not involved in the suppression of (PDF)Click here for additional data file.Table S1rpb1-CTD11, 12, 13, 20 and full length mutants.E-MAP profiles of (XLSX)Click here for additional data file.Table S2CDK8 and strains containing 13 or 20 repeats or full length CTD (see attached excel file). M value is the log2 of the ratio between the two samples per gene.Gene expression profile of strains containing 11 or 12 heptapeptide repeats with or without deletion of (XLSX)Click here for additional data file.Table S3rpb1-CTD11 mutant.Biological process gene ontology terms enriched in genes with increased or decreased mRNA levels in the (XLS)Click here for additional data file.Table S4CDK8 in rpb1-CTD11 mutants.Biological Process gene ontology terms enriched in the subset of genes with increased or decreased mRNA levels that were suppressed by loss of (XLS)Click here for additional data file.Table S5Strains used in this study.(XLS)Click here for additional data file.Table S6Plasmids used in this study.(XLS)Click here for additional data file.Table S7Primers used in this study.(XLS)Click here for additional data file.


Reduction in oxygen levels below normal concentrations plays important roles in different normal and pathological conditions, such as development, tumorigenesis, chronic kidney disease and stroke. Organisms exposed to hypoxia trigger changes at both cellular and systemic levels to recover oxygen homeostasis. Most of these processes are mediated by Hypoxia Inducible Factors, HIFs, a family of transcription factors that directly induce the expression of several hundred genes in mammalian cells. Although different aspects of HIF regulation are well known, it is still unclear by which precise mechanism HIFs activate transcription of their target genes. Concomitantly, hypoxia provokes a dramatic decrease of general transcription that seems to rely in part on epigenetic changes through a poorly understood mechanism. In this review we discuss the current knowledge on chromatin changes involved in HIF dependent gene activation, as well as on other epigenetic changes, not necessarily linked to HIF that take place under hypoxic conditions. Metazoans organisms utilize molecular oxygen during cellular respiration to produce sufficient amounts of ATP, as continuous and proper oxygen supply is an absolute requirement for survival. In response to hypoxia, defined as the reduction of oxygen levels below normal values, complex mechanisms are triggered to allow adaptation to this environmental condition. These adaptive responses comprise changes at the systemic, tissue and cellular levels that tend to reduce oxygen consumption, and improve oxygen supply. Cardinal adaptations to hypoxia include angiogenesis, erythropoiesis, a switch from oxidative to glycolytic metabolism, and a partial shut-down of major energy consuming cellular processes –3.Remarkably, hypoxic conditions take place during normal physiological processes, being a necessary stimulus for embryonic development and stem cell maintenance –7. DurinThe cellular response to hypoxia is largely dependent on changes in gene expression, which are mainly commanded by a unique family of transcription factors named HIFs, for Hypoxia Inducible Factors –13. HIFsEukaryotic DNA is complexed with different histone and non-histone proteins to form the chromatin –24. HistBesides the posttranslational modifications of the histone tails, chromatin can also be modified by various ATP-dependent chromatin remodeling complexes, which change the accessibility of the transcription machinery to particular loci, by promoting nucleosome sliding, nucleosome exchange or DNA exposure –34.Alteration of the gene expression profile is a common cellular response to different types of stress. Changes in gene expression under stress are intimately associated with alterations in chromatin structure, mediated by histone modifying and chromatin remodeling complexes . In thisIn order for transcription to take place, an initiation complex conformed by the RNA Polymerase II and general transcription factors must overcome the physical barrier posed by the chromatin. It has been demonstrated that HIF recruits co-activators that modify the chromatin structure thereby facilitating access of the transcriptional machinery to the DNA. These so called HIF co-activators include the histone acetyltransferases (HAT) p300 and CREB-binding protein (CBP) –40, someHistone acetylation, a process strongly associated with transcriptional activation, takes place on lysine residues localized in histone N-terminal tails . Histonep300 and CBP are two paralogues with strong HAT activity, highly conserved in most multicellular organisms . In addiBoth HIF-1α and HIF-2α have been shown to interact with p300/CBP through two distinct transactivation domains termed C-TAD –51 and NInteraction between p300/CBP CH1 and HIF C-TAD is regulated by oxygen, mainly through the hydroxylation of a single asparagine residue within the HIF C-TAD . This hyin vitro. These authors demonstrated that simultaneous deletion of the CH1 domain of both p300 and CBP provoked very diverse effects on the induction of 40 different HIF target genes. This implies that the requirement of this domain is gene-specific, being essential for some genes and dispensable for others. In agreement with this, a recent study has shown that individual knock down of p300 or CBP does not reduce hypoxic induction of the HIF target genes LDH-A and PGK, suggesting that p300 and CBP are either redundant or dispensable in the expression of these and possibly other HIF targets [In addition to FIH-1 dependent regulation, several other factors and pathways have been shown to modulate the interaction between p300/CBP and HIF-α subunits. These additional regulatory factors include positive regulators, such as casein kinase II and mitogen-activated protein kinase (MAPK), as well as negative regulators, including CITED2 andCITED4 ,57. Sirt targets .Why does deletion of the CH1 domain of the two HATs provoke only a moderate effect on the expression of certain HIF target genes? In principle, one explanation could be that p300/CBP are recruited to specific promoters through their intact CH3 domains. Nevertheless, this is apparently not the case, since CH1 deletion severely reduces the recruitment of p300/CBP to HIF target promoters, without significantly affecting gene expression. Thus, it seems likely that p300/CBP are dispensable for HIF dependent induction of a subset of HIF target genes .Histone acetylation is controlled by the opposing activities of HATs and HDACs . In mammRECK is downregulated through a mechanism that involves recruitment of HDAC1 to its promoter [By targeting histones or non-histone proteins, HDACs generally have a negative effect on transcription , but intpromoter . It was promoter  , and that this recruitment is required for full EPO induction under hypoxia [Drosophila cells aimed to detect novel HIF activators. Several components of the SWI/SNF complex  have emerged as hits of the screen, suggesting that the requirement of the SWI/SNF complex in the transcriptional response to hypoxia is a conserved feature in animal evolution [It has been demonstrated that components of the SWI/SNF complex are recruited to the  hypoxia   or arginine (R) residues, which can appear as mono-, di-, or tri-methylated forms. Methylated histone residues induce alterations in compaction of the chromatin, and also provide binding sites for different proteins that regulate gene expression, resulting in the inhibition or enhancement of transcription –87. Alth2), Fe(II) and 2-oxoglutarate to remove methyl groups from specific histone residues through an hydroxylation reaction. Several lines of evidence have linked JHMD function to hypoxia [The Jumonji C (JmjC)-domain containing histone demethylases (JHDM) constitute the largest family of lysine demethylases. JHDM family members utilize molecular oxygen  reduce their overall rate of transcription to about 50% of their usual normoxic levels. This sharp reduction in mRNA synthesis correlates with several histone modifications, that are not only associated with transcriptional repression, but also with transcriptional induction. Again, as a particular histone mark can promote different, even opposite biological effects, it cannot be ruled out that these “positive” histone marks globally induced under oxygen deprivation might have negative effects in the particular physiological and epigenetic contexts that take place under this condition.It has been shown that hypoxia provokes general reduction of gene transcription . Howeveret al. [2 the activity of the methyltransferase G9a increases, supporting in part the accumulation of methylated species. In addition, they have shown that demethylation of H3K9me2 is reduced under hypoxia, although the identity of the particular demethylases whose activity diminishes is unknown. As has been mentioned above, JDHM activity is partially inhibited at 0.2% O2, so one possibility is that reduction of H3K9me2 demethylation is a consequence of partial loss of activity of one or more JHDM.An increase in di- and tri-methylation of H3K9, two typically repressive marks, are among the best characterized global modifications induced under hypoxia –68,94,97et al.  have demRemarkably, deacetylation of H3K9, another chromatin modification associated with transcriptional repression, globally increases under oxygen deprivation conditions –67,89,94et al. [In hypoxia, global induction of H3K4 di- or tri- methylation, both of them modifications usually associated with transcriptional activation, have also been well documented –68,89,94et al.  suggest In summary, oxygen deprivation induces a widespread combination of histone modifications that are usually associated with either transcriptional repression or transcriptional activation. The field is now beginning to define more precisely these modifications, although more research is required to fully understand their biological roles, as well as to identify the enzymes and signaling pathways involved. It will be highly relevant to assess the impact of preventing the global epigenetic changes that occur under hypoxia, over general transcriptional repression and specific gene induction.In order to activate gene transcription, HIF recruits a range of gene-specific co-factors that acetylate/ormethylate histones, or change chromatin structure. It would be very interesting to define the mechanisms that recruit co-factors to specific hypoxia-inducible genes. On the other hand, the hypoxic stimulus induces a general repression of transcription, presumably supported by global chromatin changes. Therefore, it seems that epigenetic mechanisms play a dual role in gene regulation under hypoxia, controlling HIF target gene induction and the downregulation of general transcription.


Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in  in vivo is a poorly understood factor in the regulation of eukaryotic gene expression. Topological changes are constantly generated by DNA tracking processes and may influence gene expression if not constantly removed by DNA topoisomerases. For decades it has been generally accepted that these enzymes regulate transcription by removing excess topological strain generated during tracking of the RNA polymerase, but we still lack a more holistic view of how these enzymes influence gene transcription in their native environment. Here, we examine both global and gene-specific changes in transcription following lack of DNA topoisomerases in budding yeast. Taken together, our findings show that topoisomerases play a profound role during transcriptional activation of genes with a repressible/inducible mode of regulation. For the PHO5 gene, which is investigated in more detail, we demonstrate that topoisomerases are required for binding of a transcription factor, which is crucial for promoter opening during PHO5 activation. Our data thus suggest that inducible gene promoters are highly sensitive to changes in DNA superhelicity.Gene expression is controlled at many different levels to assure appropriate responses to internal and environmental changes. The effect of topological changes in the DNA double helix on gene transcription  Saccharomyces cerevisiae, DNA superhelicity is influenced by topoisomerases I and II (Top1p and Top2p), encoded by the TOP1 and TOP2 genes, respectively top1Δtop2ts mutants grown under restrictive conditions display a decreased rate of both rRNA and mRNA synthesis Early studies of transcription have demonstrated that DNA topoisomerases are important in the transcription process in vitro and in vivo studies Transcription and DNA supercoiling are linked by a cause-effect relationship that operates in both directions. The transcriptional effect on supercoiling is explained by the Twin-Supercoiled-Domain-Model, which predicts that two domains of DNA supercoiling are generated during transcription elongation, provided that the RNA polymerase cannot rotate freely around the template, and that DNA rotation is hindered in vivo, where chromatin structure adds another layer of complexity. Dissociation and re-association of nucleosomes will release and absorb negative superhelicity, respectively, with a potential impact on transcription in vivo and facilitate dissipation of topological strain The crosstalk between DNA supercoiling and transcription still remains elusive Several studies have suggested that the individual topoisomerases play a role during transcription initiation. Thus, human topoisomerase I has been demonstrated to affect transcription initiation from TATA-containing promoters, functioning as a repressor of basal transcription but as an enhancer of activated transcription S. pombe, Top1p was suggested to be directly responsible for nucleosome disassembly in gene promoters prior to transcription S. cerevisiae, Top1p and Top2p were suggested to act redundantly to allow recruitment of RNA polymerase II to nucleosome-free promoters rather than to act in nucleosome removal per seRecent studies of transcription using genome-wide approaches have further substantiated a role of topoisomerases during transcription initiation. In a study performed in S. cerevisiae strains lacking either one or both DNA topoisomerases to unravel the implications of these enzymes on transcription. Although we demonstrate that the requirement for topoisomerases generally correlates with transcriptional activity we find that DNA topoisomerases have a major impact on transcription of a subset of genes, which are not unified by being highly transcribed per se. Rather, the most affected genes are characterized by features associated with highly regulated transcription initiation. Studies of several genes from this subgroup demonstrate that topoisomerases indeed are required for adequate and timely transcriptional induction. Finally, in case of the inducible PHO5 gene, we demonstrate that topoisomerase-mediated relaxation is required for binding of the Pho4p transcription factor, whereas constitutive PHO5 transcription is unaffected by topoisomerase deficiency.In the present study, we have combined microarray gene expression analyses and single-gene studies using S. cerevisiae polyadenylated transcriptome by microarray analysis in top1Δ, top2ts, top1Δtop2ts and the isogenic wild-type strain. To bypass genome-wide effects of topological challenges caused by replication To investigate the impact of DNA topoisomerases I and II  on genome-wide transcription, we examined the top1Δtop2ts, reflecting an absolute drop in mRNA abundance at the cellular level of ∼30% . In contrast to the double mutant, the single mutants show no correlation between transcript changes and wild-type mRNA abundance, consistent with the redundant nature of the two enzymes in genome-wide transcription.As shown in Relative measures of transcriptional activity for every gene were obtained from measures of mRNA abundance by taking gene specific values of polyA mRNA breakdown into account as described by Schreiber and co-workers top1Δtop2ts transcript changes to a genome-wide survey of transcript lengths top1Δtop2ts gene expression changes . In conclusion, the data demonstrate that the requirement for topoisomerases during global gene transcription increases with increasing transcriptional activity, but is independent of transcript length.We next related the top1Δtop2ts , galactose- , glucose-  and inositol-regulated (INO1) promoters. In order to study the induction capabilities, wild-type and top1Δtop2ts cells were cultured under repressive conditions and transferred to the respective inducible conditions , where the wild-type cells are fully repressed . This enzyme only relaxes negative supercoiling and has earlier been used to alter DNA superhelicity on a global scale PHO5 transcript levels upon transfer to inducing conditions, strongly suggesting that PHO5 activation is supercoiling sensitive.To further investigate this we took advantage of the PHO5 activation in top1Δtop2ts cells, we next studied the impact of topoisomerase deficiency on regulation of crucial steps upstream of transcription initiation. A previous study has shown an absolute requirement for nucleosome removal in trans from the PHO5 promoter region for transcription initiation to occur, which is dependent on binding of the Pho4p transcription factor to the PHO5 promoter PHO5 with a nucleosome map, illustrating Pho4p binding sites and four highly positioned nucleosomes covering the promoter region.To examine the underlying cause of the perturbed top1Δtop2ts we used Chromatin immunoprecipitation (ChIP) with an antibody against histone H3 to measure nucleosome occupancy in the PHO5 promoter in wild-type and top1Δtop2ts cells during PHO5 induction. As shown in top1Δtop2ts. These results suggest that topoisomerases are either required directly for the removal of repressive nucleosomes from the PHO5 promoter or for a step prior to this activity. We reason that this step cannot be de-regulation of a chromatin remodeling factor as strains disrupted for such factors have been shown to merely give rise to a kinetic delay in PHO5 activation and not an absolute inhibition To investigate if nucleosome removal is affected in PHO5 induction in top1Δtop2ts is indirectly caused by disruption of the physiological stimulus leading to transcriptional activation, we analyzed the cellular localization of GFP-tagged Pho4p in wild-type and top1Δtop2ts cells. As expected, both strains exhibit nuclear accumulation of Pho4-GFP after 90 and 180 min under phosphate-free conditions at the restrictive temperature (To eliminate the possibility that defective perature . The resPHO5 promoter (the low affinity UAS1 site and the high affinity UAS2 site)   is pertunditions . Even afi) highly transcribed genes are generally more dependent on topoisomerase activity than poorly transcribed genes, and (ii) genes strongly dependent on topoisomerases are characterized as highly regulated and chromatin-dependent with a repressible/inducible mode of regulation. In support of the genome-wide findings our single-gene studies suggest that topoisomerases play a previously unidentified and important role in transcription of highly regulated, inducible genes. Finally, the in-depth analysis of the inducible PHO5 gene has revealed that topoisomerase activity is required for binding of the Pho4p transcription factor upstream of promoter nucleosome removal. The findings provide novel insight into the role of DNA topoisomerases for in vivo transcription.The global gene transcription analyses of budding yeast cells deficient for topoisomerases I and II reveal two major effects: ? For the majority of these genes, rich media will neither give complete repression nor complete activation of transcription. Rather, a subpopulation of cells will have a given gene in an active form, while the remaining cells will have the gene in a repressed form. We interpret a drop in transcript levels between wild-type cells and top1Δtop2ts cells . As we have used G1 arrested cells to exclude replication-associated effects in top1Δtop2ts rather than exponentially growing cells, it remains possible that different supercoiling levels exist in different phases of the cell cycle, which may underlie the discrepancy between ours and earlier studies concerning the dependency of GAL1 transcription on topoisomerases.In further support of a role of topoisomerases for regulated gene transcription we show that the enzymes stimulate transcriptional activation of twelve different inducible genes, including the and INO1 . AttentiPHO5 induction, as arrested cells have been demonstrated to accumulate polyphosphate, a vacuolar Pi reserve, which will influence the rate of PHO5 induction PHO5 induction observed in the top1Δtop2ts cells, as these cells, due to their decreased metabolic activity, would be expected to need more time to consume the Pi reserve relative to wild-type cells. To exclude this possibility we deleted VTC1 and VTC4 in wild-type and top1Δtop2ts, as these genes are the two main genes required for polyphosphate synthesis. As seen in top1Δtop2ts cells, induction of PHO5 still does not take place in vtc1Δtop1Δtop2ts and vtc4Δtop1Δtop2ts cells, confirming that it is the lack of topoisomerases and not excessive Pi reserves, which causes inhibition of PHO5 induction. This was further demonstrated by studying PHO5 induction using exponentially growing cells rather than G1 arrested cells , we find it unlikely that either one of these enzymes play a more specific role during transcription initiation of PHO5. Rather, our data suggest that it is lack of their redundant DNA relaxation activity that influences PHO5 transcription. In support of this, we find that E. coli TopA-mediated changes in global supercoiling levels in wild-type cells result in altered transcriptional output from PHO5 . Furthermore, we expect most of these effects to result in delayed PHO5 induction kinetics, as seen for the topoisomerase single mutants, rather than a total inhibition We reason that indirect effects caused by a potential transcriptional de-regulation of co-factors in the  nucleus , and onltop1Δtop2ts, may have profound effects on chromatin-regulated promoters. Topoisomerase deficiency may have pleiotropic effects affecting polymerase recruitment PHO5, where binding of a transcription factor is inhibited. The different scenarios are not mutually exclusive, and DNA topoisomerases most likely function at numerous levels to influence DNA superhelicity for maintenance of transcriptional competency.Taken together, our investigations suggest that DNA topoisomerases are required to maintain the genome in a state competent for transcription initiation. Top1p and Top2p seem to exert this role by a mutually redundant relaxation of DNA supercoils, thus influencing highly transcribed genes and highly regulated, chromatin-dependent genes. Any imbalance in net DNA superhelicity, which likely appears in S. cerevisiae strains are derivatives of W303a, and the associated manipulations for obtaining derivate strains are according to standard genetic techniques. For microarray analysis, yeast strains were grown to exponential phase at 25°C in YPD and further grown for 90 min at 25°C in YPD with α-factor  to synchronize cells in G1. Cultures were then placed at 37°C for another 90 min for conditional inhibition of Top2p, where more α-factor was added to keep cells in G1. Cultures were adjusted, so that an equal number of cells could be used for all yeast strains (6×107 cells). Finally, cells were harvested by centrifugation at 37°C. For each sample, aliquots were collected for fluorescence-activated cell sorting analysis as previously described PHO5 activation experiments, cells were prepared as for the microarray analysis, but instead of YPD they were cultured in high phosphate medium . Glucose was added to 2%, amino acids were added to standard concentrations, and KH2PO4 was added to a concentration of 15 mM. After cell cycle arrest in G1 and conditional inhibition of Top2p, cells were shifted to phosphate-free medium  for induction of PHO5. The fold increase observed in PHO5 mRNA levels, when cells are kept in phosphate-free medium for 180 minutes varies from experiment to experiment in the range from 50 fold to 350 fold. For this reason we show PHO5 inductions as percentages of maximum transcript accumulated in the wild-type strain. For transcriptional repression of PHO5, cells were first cultured in phosphate-free medium at 25°C for 4 h to obtain high PHO5 transcription, where α-factor was added after 2,5 h. The temperature was then increased to 37°C to inhibit Top2p, and after 15 min at 37°C, cells were transferred to high phosphate conditions (15 mM KH2PO4) for PHO5 repression. See All For the microarray experiments total RNA was initially prepared by acid phenol extraction. Immediately prior to RNA extraction, external spike-in Poly-A RNA's  were added to an equal number of cells from all four yeast strains to enable external normalization. High-quality RNA was obtained by further purification of phenol-extracted RNA on RNeasy columns  according to manufacturer's directions. RNA quality was assessed by gel electrophoresis and spectrophotometry . Gene expression profiling was performed using Affymetrix Yeast Genome 2.0 GeneChip oligonucleotide arrays essentially according to Affymetrix protocols. Normalization procedure and data processing can be viewed in detail in http://www-genome.stanford.edu/turnover/), as described by Schreiber and colleagues To estimate global mRNA changes we calculated and compared the total intensity from all detectable probe sets on the mutant and wild-type arrays. For correlation to wild-type transcription levels, all mRNA abundances were averaged across biological triplicates . Arbitrary transcriptional activities were calculated by dividing average expression levels in the triplicate wild-type arrays by genome-wide mRNA half-life data http://www.nature.com/ng/journal/v41/n4/suppinfo/ng.319_S1.html). Analysis of nucleosome occupancy was performed with the use of a recent map of nucleosome positions in S. cerevisiaehttp://chemogenomics.stanford.edu/supplements/03nuc/). The data on nucleosome positions aligned according to transcription start site were used.Measures of transcriptional plasticity for every gene were obtained from Barkai and co-workers 8 cells) were taken at the indicated time points. RNA was purified as for the microarray analysis followed by DNase I treatment, and cDNA was made by SuperScript II RT-PCR  using oligo dT primer. Real-time PCR was performed with DYNAmo SyBR Green qPCR kit  and used to quantify mRNA levels, using a Stratagene MX3000 . For each yeast strain, Ct-values from triplicate qPCR amplifications were averaged across three independent measurements. ChIP was performed on 2.5×108 cells as described previously YOL151W) not affected by topoisomerase activity as assessed by qPCR (data not shown), and the 0 min time point was set to 1. Normalizing to a telomeric locus (TEL06R) gave similar results. Pho4-13xcMyc ChIP was calculated in the same way, but using the GAL1/10 promoter region as control region. Primer sequences are listed in For analysis of transcription levels, cells were grown as described above, and samples  plotted against average RNA polymerase II (RNA pol II) occupancy in open reading frames as a 200 gene moving average. Data on RNA polymerase II occupancy were gathered from Topoisomerase dependency correlates with RNA polymerase II binding in ORF's. (EPS)Click here for additional data file.Figure S2top1Δtop2ts microarray data set (% genes) were calculated. De-regulated genes were defined as being up- or down-regulated with +0.5 and −1 cutoffs in the signal log2 ratio between mutant and wild-type, respectively. Upper histogram, the average mRNA abundance for genes in each of the functional gene groups (groups 1–15) was normalized to the genome-wide average (arbitrarily set to 1). Lower histogram, the fraction of de-regulated genes in top1Δtop2ts is shown for each functional gene group. The MIPS functional gene groups and their number of genes are listed to the right. The functional gene groups with highest and lowest expression are indicated by orange and blue colors, respectively. For comparison, the TATA-less and TATA-containing gene groups Association between gene expression level and topoisomerase dependency for functionally classified gene groups. Gene groups based on the most general and overall functional classifications were retrieved from the MIPS FunCat database (EPS)Click here for additional data file.Figure S3top1Δtop2ts gene expression changes as a 200 gene moving average.Transcriptional plasticity correlates with topoisomerase dependency independent of gene expression levels. The analysis presented in (EPS)Click here for additional data file.Figure S4top1Δtop2ts correlate with expression changes obtained from yeast strains with perturbation of different chromatin factors. Gene expression changes in top1Δtop2ts  are plotted as a function of gene expression changes  generated from perturbation of different chromatin regulators. (A) spt6tsspt16tstaf1-2ts spt3(E240K)gcn5(KQL)paf1Δtop1Δtop2ts + TopA vs. top1Δ  R denotes the Pearson correlation coefficient, and the associated correlation P-value (P) was calculated by permutation testing. Genes in the lower 0.05 and upper 0.95 percentiles for expression changes were specified as the most de-regulated genes in each dataset, and oP denotes the P-value of the overlap between de-regulated gene sets from the chromatin regulators and top1Δtop2ts, using a hypergeometric test.Expression changes in (TIF)Click here for additional data file.Figure S5top1Δtop2ts. Error bars represent 95% confidence intervals.Confidence intervals for nucleosome occupancy in promoter regions of genes from different gene groups. Profiles of the average nucleosome-binding pattern (EPS)Click here for additional data file.Figure S6top1Δtop2ts cells. Time course experiments of control gene expression in wild-type and top1Δtop2ts cells under inducible conditions, where the experimental setup was as shown in ESC1, ACT1 and GAPDH) were quantified by qPCR at the indicated time points after transfer of cells to either phosphate- (upper panel), galactose- (middle panel), or glucose-inducible conditions (lower panel). mRNA levels were normalized to the mRNA level obtained in the wild-type at the 0 min time point (set to 100%). Averages from two individual experiments are shown with error bars representing ± one standard deviation.Transcription of non-inducible genes under inducible conditions in wild-type and (EPS)Click here for additional data file.Figure S7PHO5 induction kinetics. Time-course experiments of PHO5 transcription in wild-type and Pho4-13xcMyc cells. The experimental setup was as described for PHO5 mRNA levels were normalized to the wild-type mRNA level at the 180 min time point (set to 100%). Averages from two individual experiments are shown with error bars representing ± one standard deviation. Number indicates the mean fold increase in PHO5 mRNA levels at the latest time point.Pho4-13xcMyc cells display wild-type (EPS)Click here for additional data file.Figure S8PHO5 induction in topoisomerase deficient cells is not caused by internal polyphosphate storages. Time-course experiment of PHO5 transcription in vtc1Δ and vtc1Δtop1Δtop2ts cells (left panel), vtc4Δ and vtc4Δtop1Δtop2ts cells (middle panel) or wild-type, vtc1Δ and vtc4Δ cells (right panel) after transfer from high phosphate to phosphate-free conditions, where the quantified PHO5 mRNA levels were normalized to the vtc1Δ level at the 180 min time point (set to 100%), the vtc4Δ level at the 180 min time point (set to 100%), or the wild-type level at the 180 min time point (set to 100%), respectively. Averages from two individual experiments are shown with error bars representing ± one standard deviation. Numbers indicate the mean fold increase in PHO5 mRNA levels in the indicated strains at the latest time point. The experimental setup was as described for vtc1Δ and vtc4Δ cells (right panel) demonstrates that vtc1Δ and vtc4Δ cells show more rapid and higher maximum activation of PHO5 relative to wild-type cells in agreement with previous studies Lack of (EPS)Click here for additional data file.Figure S9PHO5 induction in topoisomerase deficient cells is not caused by G1 cell cycle arrest. Upper panel, experimental setup, where +Pi and −Pi indicate high and no phosphate, respectively. Lower panel, time-course experiment of PHO5 transcription in asynchronous growing wild-type and top1Δtop2ts cells after transfer from high phosphate to phosphate-free conditions. The quantified PHO5 mRNA level was normalized to the wild-type level at the 180 min time point (set to 100%). Average from three individual experiments is shown with error bars representing ± one standard deviation. Number indicates the mean fold increase in PHO5 mRNA levels in wild-type cells at the latest time point.Lack of (EPS)Click here for additional data file.Table S1top1Δtop2ts. Using Funspec software Gene Ontology analysis of genes affected 2-fold or more in (EPS)Click here for additional data file.Table S2top1Δtop2ts. aGene sets were tested for overlap with genes regulated by the stress-related SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex P). bGene sets were tested for overlap with TATA-box containing genes P). cA measure for responsiveness to environmental changes was derived for every gene using the Gasch dataset P values for higher responsiveness to environmental changes are reported. dDistance to the closest telomere was compared to the rest of the genes by the t-test.Characterization of genes de-regulated 2-fold or more in (EPS)Click here for additional data file.Table S3S. cerevisiae strains used in this study.(EPS)Click here for additional data file.Table S4Primers used in qPCR for quantification of ChIP levels and gene expression levels.(EPS)Click here for additional data file.Text S1Supplementary Materials and Methods.(DOCX)Click here for additional data file.