Transcriptional enhancers are generally known to regulate gene transcription from afar. Their activation involves a series of changes in chromatin marks and recruitment of protein factors. These enhancers may also occur inside genes, but how many may be active in human cells and their effects on the regulation of the host gene remains unclear.We describe a novel semi-supervised method based on the relative enrichment of chromatin signals between 2 conditions to predict active enhancers. We applied this method to the tumoral K562 and the normal GM12878 cell lines to predict enhancers that are differentially active in one cell type. These predictions show enhancer-like properties according to positional distribution, correlation with gene expression and production of enhancer RNAs. Using this model, we predict 10,365 and 9777 intragenic active enhancers in K562 and GM12878, respectively, and relate the differential activation of these enhancers to expression and splicing differences of the host genes.http://regulatorygenomics.upf.edu/Projects/enhancers.html.We propose that the activation or silencing of intragenic transcriptional enhancers modulate the regulation of the host gene by means of a local change of the chromatin and the recruitment of enhancer-related factors that may interact with the RNA directly or through the interaction with RNA binding proteins. Predicted enhancers are available at The online version of this article (doi:10.1186/s12864-015-1704-0) contains supplementary material, which is available to authorized users. Transcriptional enhancers are characterized by specific chromatin signatures, which differ depending of whether the enhancer is active or not –5. Transin vitro [Although enhancers are typically defined to regulate gene transcription at a distance, about 50 % of potential enhancers predicted by high-throughput methods lie within protein-coding genes  and somein vitro , and thain vitro . In thisth release of GENCODE, human assembly GRCh37 (hg19). ChIP-Seq and RNA-Seq datasets were downloaded from ENCODE [Annotated human enhancers with a mouse homologous enhancer that has been experimentally validated were downloaded from VISTA . The genm ENCODE  for K5626 vs ~2.2⋅106) in both cases the amount of windows with signal was similar (~1.5 million windows), which were then kept for further processing. The relative enrichment of chromatin signals between 2 cell lines was calculated to predict active enhancers in K562 (relative increase of activation marks in K562 with respect to GM12878) and silent enhancers in K562 . Full quantile normalization for counts and GC content was applied using EDASeq [pyicoenrich function (https://bitbucket.org/regulatorygenomicsupf/pyicoteo). A vector of z-scores per region was obtained, which we refer to as attributes, consisting of the 17 enrichment z-scores for the ChIP-Seq and Input datasets. A positive z-score for a region indicates an increased in ChIP-Seq signal in K562 relative to GM12878 in that region, whereas a negative z-score indicates a decreased signal in K562 relative to GM12878; and z-scores close to zero indicate no significant differences between the cell lines. For all datasets, except for the ChIP-Seq with non-specific antibody and for the RNA-Seq datasets, we used replicates. The relative enrichments were calculated with respect to the distribution described by the comparison between replicates. When replicates were not available, these were simulated by pooling the two conditions and dividing them using random sampling [We considered sliding windows of 1500 nt along the entire genome, as suggested by the length distribution of experimentally validated enhancers , 21  [Enhancers were linked to genes by selecting the closest TSS on either direction and by using ChIA-PET data for RNAPII in K562 cells for two replicates from ENCODE . An enhacensus/) .--library-type fr-firststrand --no-effective-length-correction --min-frags-per-transfrag 5 and masking all rRNAs, tRNAs and mitochondrial sequences. The relative changes in transcript abundance were obtained using Cuffdiff with parameters --library-type fr-firststrand --min-reps-for-js-test 1, using the merged GTF file obtained from Cufflinks for GM12878 and K562, along with the bam files of GM12878 and K562 with replicates. This provided 3552 genes (6.68 %) with relative changes in expression between the two cell lines.For every gene in GENCODE (v7) annotation , the moshttps://bitbucket.org/regulatorygenomicsupf/suppa). Only events that do not overlap any other alternative splicing event were kept, giving rise to a total of 5319 events. For exon skipping events, defined by an exon triple E1–E2–E3, the inclusion level (PSI) of the middle exon E2, was calculated as the fraction of reads that include the exon over the total number of reads that include and skip the exon:12, n23 and n13 are the number of reads that span the junctions E1–E2, E2–E3 and E1–E3, respectively. PSI values were calculated using junction reads only, since enhancers can produce RNA as well, so the enhancer-related RNAs may be mistakenly included in the PSI calculation when they overlap an event. Reads at junctions were counted with sjcount [-read1 1 and -read2 0 parameters. For this analysis, RNA-Seq reads were mapped using STAR [--outSJfilterOverhangMin −1 -1 -1 -1 and --sjdbScore 100 in order to use only annotated junctions. A genome index was previously generated with STAR over the Gencode.v7 annotation using the --sjdbOverhang 75 parameter in order to adjust the splice junction database to the length of the RNA-Seq reads. Finally, only events with a total of 20 or more reads mapping at the junctions were kept. This gave a final number of 3227 and 3192 events with PSI values from the nuclear and cytosolic RNA-Seq experiments, respectively.Alternative splicing events from the Gencode v7 annotation  were cal sjcount  from theing STAR  with parWe defined the events to be regulated if they had |delta PSI| > 0.1 in at least one replicate comparison between cell lines. Using two pairings of the replicates, this gave rise to 339 and 293 events (148 in common) with the cytosolic samples, and 367 and 378 (210 in common) for the nuclear samples. Additionally, we defined a set of alternative events that do not change splicing by imposing |delta PSI| < 0.05 between the same replicate comparisons used before. This gave rise to 1722 and 1534 (1328 in common) for the cytosolic samples, and 1627 and 1497 (1278 in common) for the nuclear samples.We built a computational predictive model based on the relative differences in various chromatin marks between two cellular conditions. We applied this model to study the differences between the ENCODE cell lines K562, a leukemia cell line, and GM12878, a blood cell derived cell line. Using windows along the entire genome  in PU.1 Fig.  and RNAPWe additionally investigated whether enhancer-associated RNAs (eRNAs) are found in our predictions. Enhancer activity correlates with the production of polyA+ Fig.  and polyAlthough enhancers can regulate genes from afar, they tend to be enriched upstream of genes . We theWe additionally searched for evidence of direct physical interactions for the enhancer-TSS pairs calculated above by using ChIA-PET data for RNAPII . AlthougActive enhancers regulating the expression of nearby genes have been observed in exons , 15 and In order to predict intragenic active enhancers, we considered 1.5 kb sliding windows inside genes, starting 500 bp downstream of the first TSS and eliminating all windows that overlap with a 1 kb region around every annotated alternative TSS  or active in GM12878 (80.61 %) fall in intronic regions, and 26.02 % in K562 (22.07 % in Gm12878) overlap at least partially with an exon. However, comparing the proportion of exonic and intronic regions covered by enhancers with the actual proportions of these regions in genes, we find no preference for exons or introns  show a significant expression up-regulation in the corresponding cell line . We thenThe change in chromatin state induced by the activation or silencing of an enhancer may affect the processing of the pre-mRNA. There is evidence that localized intragenic chromatin states can produce changes in alternative splicing through various mechanisms –48. MoreWe next decided to evaluate whether there is any association between the presence of enhancers and regulated events in genes. To this end, we compared only genes that have one or more of the 5319 calculated alternative splicing events  and sepaAs an example, we show the case of a regulated exon in the microtubule-actin crosslinking factor 1 gene (MACF1) Fig. . We obseWe have developed a novel semi-supervised method that exploits the relative enrichment and depletion of multiple signals from ChIP-Seq experiments to predict enhancers that are active in one cell line relative to another. Applying this method to ENCODE data we predicted a total of 21,420 enhancers that are active in K562 relative to GM12878 (silent in GM12878 cells) and 21,694 enhancers that are active GM12878 relative to K562 (silent in K562), including intragenic and intergenic enhancers.The number of active enhancers is cell type specific and very much dependent on the method used to detect them . AlthougOur predicted enhancers are H3K27ac dependent and are defined almost entirely by chromatin signals. The relevant predictive features confirm that active enhancers are characterized not only by the presence of H3K4me1, but also by the presence of H3K27ac, H3K4me3 and RNAPII , 12, 13.When we applied the same predictive model to predict intragenic enhancers, we found a similar number of active intragenic enhancers as for intergenic ones. This result suggests that there exist in cells a considerable number of differentially activated intragenic enhancers, which may have a relevant contribution to the mechanisms of cell-specific gene regulation. Since active enhancers are characterized by a local modification of the chromatin state, we hypothesized that our predictions could be linked to relative differences between the same two cell lines in expression and splicing. We observed that intragenic transcriptional enhancers, upon activation or silencing, affect the activity of downstream alternative transcription start sites. Surprisingly, they can also affect the most upstream TSS. This generalizes previous findings indicating that intragenic enhancers can act as internal alternative promoters . We alsoOn the other hand, we found here a strong association of PU.1 (SPI1) to active enhancers in K562 cells and in particular, a significant increase in PU.1 occupancy in 26.8 % of active enhancers. PU.1 has been shown before to be an essential co-factor for enhancer activity  and is kIn summary, there is increasing evidence that changes in the chromatin state can affect the processing of the pre-mRNA , 61–65 a


Mechanistic studies on these genes indicate that during gene activation a local increase of phospho-S2 CTD nearby the promoter impairs the phospho-S5 CTD-dependent recruitment of Set1 and the subsequent recruitment of specific HDACs, which leads to nucleosome depletion and efficient transcription. The early increase of phospho-S2 results from the phosphorylation of the CTD S2 kinase Lsk1 by MAP kinase in response to cellular signalling. The artificial tethering of the Lsk1 kinase at the ste11 promoter is sufficient to activate transcription. Therefore, signalling through the CTD code regulates promoter nucleosomes dynamics.The phosphorylation of the RNA polymerase II C-terminal domain (CTD) plays a key role in delineating transcribed regions within chromatin by recruiting histone methylases and deacetylases. Using genome-wide nucleosome mapping, we show that CTD S2 phosphorylation controls nucleosome dynamics in the promoter of a subset of 324 genes, including the regulators of cell differentiation DOI:http://dx.doi.org/10.7554/eLife.09008.001 The process of activating genes—known as gene expression—involves a number of steps. During the first step, the gene's DNA is copied or ‘transcribed’ to produce a molecule of messenger RNA. However, most of the DNA in a cell is wrapped around proteins called histones to make structures known as nucleosomes, and the DNA has to be unpacked to allow the enzymes that make messenger RNA to access it.Cells regulate how the DNA is packed by attaching chemical groups to the histone proteins. Adding acetyl groups to histones usually causes the nucleosomes to unwrap and creates loosely packed DNA that helps with gene expression. On the other hand, the addition of methyl groups has the opposite effect.RNA polymerase II is the enzyme that carries out transcription of messenger RNAs in all eukaryotic cells—that is, the cells of organisms like plants, animals, and fungi. Like all enzymes, RNA polymerase II is made of smaller building blocks called amino acids. One end of the RNA polymerase II enzyme, called the C-terminal domain (or CTD), contains a unique sequence of amino acids that serves as a scaffold to recruit other proteins involved in transcription and histone modifications. Different amino acids in this region of RNA polymerase II can be modified by the addition of phosphate groups. The pattern of these modifications is often thought of as a code and can influence which other proteins get recruited.It remains poorly understood how RNA polymerase II regulates nucleosomes to allow transcription to occur. Materne, Anandhakumar et al. have now addressed this issue by engineering mutant yeast cells in which phosphate groups cannot be added to specific amino acids in the RNA polymerase II enzyme. Most genes were expressed as normal in these yeast cells, but a few hundred genes were not expressed.ste11, which allows transcription to occur.Materne, Anandhakumar et al. then used a technique called MNase-Seq to map the position of nucleosomes across the genome and found that there were more nucleosomes near to start of these down-regulated genes. Further experiments showed that the addition of phosphate groups onto the RNA polymerase II is required to deplete the nucleosomes at the start of a gene called ste11 gene was sufficient to oust nucleosomes and activate transcription by RNA polymerase II.Materne, Anandhakumar et al. also found that artificially tethering the enzyme that adds phosphate groups to the C-terminal domain to the start of the Future work will address if this newly discovered mechanism is implicated in the activation of specific patterns of gene expression during the development of more complex organisms.DOI:http://dx.doi.org/10.7554/eLife.09008.002 The integration of various aspects of the transcription of DNA into mature mRNA relies on combinatorial phosphorylations of the flexible scaffold structure formed by the RNA polymerase II (PolII) subunit Rpb1 C-terminal domain (CTD), which is comprised of repeats of the consensus heptad YSPTSPS . Genes tThe distribution of histone H3K4 and K36 methylation mirrors CTD phosphorylation due to the direct recruitment of the H3 methyltransferases Set1-COMPASS (for H3K4) and Set2 (for H3K36) by the S5P and S2P of PolII, respectively . Set1 isDespite a strong positive correlation between the H3K4 and K36 methylations and active PolII occupancy, their primarily function appears to be to repress histone acetylation and transcription because both serve as a binding platform for recruitment and the activation of histone deacetylase complexes (HDACs) including Set3 complex (SET3C) and Rpd3C(S) . This paKnown methyl-lysine-binding domains include the plant homeodomain (PHD) finger and the chromodomain. Available data support that these domains are responsible for the H3 methylation-dependent recruitment of HDACs. For example, the chromodomain protein Eaf3 is a subunit of Rpd3C(S) deacetylase and binds H3K36me . ComplemHow the balance between acetylation and deacetylation at promoters is regulated during transcription is poorly understood, and a role of the phosphorylated CTD in regulating that process is unknown. Here, we show that S2P affects promoter nucleosome occupancy at a subset of genomic loci. Mechanistic studies reveal that upon gene activation, the Sty1 MAP kinase directly phosphorylates the CTD S2 kinase Lsk1, which results in increased S2P nearby the promoter. We also show that the doubly phosphorylated S2P-S5P CTD has lower affinity for Set1 compared to the S5P CTD. Therefore, the peak of S2P at the promoter counteracts H3K4 methylation and the recruitment of histone deacetylases. Our results indicate that cellular signalling mediated by the PolII CTD directly controls promoter nucleosome dynamics and gene transcription of a subset of genes.rpb1 CTD S2A (S2A) mutants in fission yeast and budding yeast are viable  deletion and the S2A mutant. Although both screens highlighted a genetic link between S2P and RNA maturation, about a third of the shared interactions found fell in chromatin remodelling and modifications , it did not significantly affect the length of the NDR at TTS .Taken together, these analyses reveal an unexpected role of S2P in regulating nucleosomes occupancy and position at the promoter of 324 genes .ste11, we therefore analyzed the occupancy of H3 in the presence or absence of S2P, which revealed an increased level of H3 occurring specifically at the promoter of ste11, while the transcribed region was barely affected. The effect was not observed at the adh1 locus whose transcription does not require S2P  and HDAC on the expression of ste11 locus  and K36R (H3K36R) histone H3 mutants  by Set1, which itself depends on the S5P  pathway to increase histone acetylation at the promoter of f ste11, . It was f ste11, . Howeverhylation . Importa1 mutant .10.7554/Based on these data, we speculated that the burst of S2P at the promoter could interfere with the binding of Set1 to the CTD S5P. In order to test this possibility, we expressed plasmid born NLS-GST-CTD (full-length) fusions harbouring wt, S5A, or S2A repeats in fission yeast. Preliminary experiments revealed that the wild-type version was phosphorylated on both S2 and S5, while the S2A or S5A versions lacked the corresponding S2P or S5P, respectively . When exste11 counteracts the S5P-dependent methylation and deacetylation occurring at the promoter by physically displacing Set1. This model raises the question of the recruitment of Lsk1 in the 5′ region of the transcribed unit.Taken together, the previous data indicate that the timely burst of S2P in the promoter region of lsk1 deletion and shows in vitro kinase activity similar to the wt, which suggests a regulatory rather than catalytic role for the N-terminal extension  of the Ste11 transcription factor  on the total level of PolII (based on the 8WG16 antibody). We next determined the ratio of normalized S2P observed at T1 on T0. This revealed that PolII reached its highest level of S2P close to the promoter during the induction of ste11 and inv1. By contrast, the increase of S2P over the fbp1 locus .Many important insights into transcriptional regulation have come from studies on glucose repression of the  sources . Remarka1 kinase . Moreovepromoter . In orde as inv1  was stro as inv1 , reminisinv1 regulatory region, the absence of S2P resulted in decreased acetylation and increased occupancy of histones, which was associated with a marked decrease of PolII occupancy (set1 deletion mutants fully suppressed the defect resulting from the lack of S2P (inv1 expression (data not shown). A previous genome-wide analysis reported that glucose repression of inv1 requires the Rpd3C(L) (Clr6)-dependent promoter histone deacetylation  complex to the promoter of inv1 and allows transcriptional activation upon glucose starvation and MAP kinase signalling.These data indicate that, similarly to the case of The phosphorylation of the PolII CTD and the methylation of H3K4 are probably the best markers of active transcription, but it is unclear how their interplay is regulated to affect gene expression. Here, we show that cellular signalling through the CTD code directly modulates H3K4 methylation-dependent deacetylation of promoter nucleosomes at some specific loci, which is required for gene activation during cell differentiation and metabolic adaptation.ste11 and inv1, two model genes that we have analyzed in more details. These analyses revealed that the phosphorylation of S2 is critical for gene activation by reversing the effects of histone deacetylation by HDAC at the promoter.Genome-wide MNase-Seq in a strain where S2P is absent revealed that a subset of 324 genes displays specific changes of promoter nucleosome dynamic, defined as position shift, fuzziness change, and occupancy change as defined by the DANPOS bioinformatics pipeline, which leads to a shorter NDR at TSS. These target genes tend to have larger than average NDR and 5′-UTR, which may indicate that they are under more complex regulation. This is clearly the case of Histone deacetylases including Rpd3C(L) and the SET3C complexes localize to promoter regions through their capacity to bind H3K4me, which links them to Set1 and S5P. In the subset of genes we have analyzed, we propose that during transcriptional induction, the local increase of S2P nearby the promoter prevents the efficient association of Set1 with the S5P CTD . Interesste11 and inv1. The Rpd3C(L) has been proposed to regulate transcriptional burst frequency, while the SET3C complex is thought to modulate the burst size , peroxidase–antiperoxidase (PAP) (Sigma), anti-S2P , anti-S5P  antibodies.Fission yeast growth, gene targeting, and mating were performed as described . TAP purescribed . The expPhosphatase  treatment was performed as described. Iodine staining was performed as described .act1 mRNA was used for normalization. Antibodies used in ChIP were PAP (Sigma), anti-Rpb1 S2P (Chromotek), anti-RNA polymerase II (Millipore), anti-HA (Covance), anti-H3 (abcam), anti-H3K14ac (Millipore), anti-H3K36ac (Millipore), anti-H3 K4Me2 (Millipore), anti-H3K4Me3 (Millipore), anti-H3K36me (a kind gift of Jean-Christophe Andrau), anti-Myc (Covance).Chromatin immunoprecipitations were performed using a Bioruptor  and Dynabeads . Note that when a tagged protein is used , the IP/T ChIP ratio is shown in the main figures and the corresponding untagged control strain treated simultaneously is shown in For all ChIP experiments, each column represents the mean percentage immunoprecipitation value ± SEM (n = 2–4). All oligonucleotides used are listed in All the site-directed mutagenesis were performed using the Quickchange kit (Stratagene) following the instructions of the manufacturer.HMG-lsk1-HA, the sequence of the insert was designed by gene synthesis (Integrated DNA Technologies) and the resulting fragment was cloned in the pREP-1 plasmid using the BamHI-SmaI restriction sites.To generate the pREP-1 In order to clone the full-length versions of NLS-GST-CTD, the wt, S2A, and S5A GST-CTD inserts were amplified from pGEX4-T1 vectors  adding tlsk1 mutants were integrated at the lsk1 locus by replacement and 5-FOA selection in a lsk1::ura4 strain. All the mutants were amplified from a plasmid after Quickchange mutagenesis using the Expand TAQ polymerase (Roche).The integration of the histone mutants is described in lsk1::natR deletion strain and the rpb1 S2A-natR strain, both strains were first transformed with a plasmid expressing the ste11 gene, which rescues the sterility. To test for genetic interaction, the query strains were mated to the array of single deletion strains (Bioneer deletion library) on SPA plates. All steps were performed manually using V&P Scientific pin replicators allowing either 96 or 384 wells format. Immediately following the transfer of cells onto the mating plate. Cells were then allowed to sporulate at 26°C for 3 days, and mating plates were subsequently transferred to 42°C for 3 days in order to eliminate unmated/unsporulated cells, thereby enriching for spores. Following heat treatment, spores were transferred on YES plates and allowed to germinate for 2 days at 32°C. To select for recombinant double-mutant haploids, cells were arrayed on YES plates containing G418 and nourseothricin and allowed to grow for a further 2 days.Due to the sterility of the 2 5 mM, CaCl2 1 mM, NP-40 0.75%) supplemented with 7.6 ml NP buffer + 0.5 µl β mercaptoethanol + 400 µl spermidine 10 mM) and split into 2 Falcon tubes . Add 50 µl MNase (32 units) to one tube and incubate 20 min at 37°C without agitation. Add 500 µl Stop buffer, 200 µl RNase A (0.4 mg/ml), and 225 µl proteinase K (20 mg/ml) and incubate at 65°C overnight. Potassium acetate was added (1.25 ml of a 3 M solution) and the mix was incubated 5 min on ice. After phenol extraction, 200 µl NaCl 5 M, 1.7 µl Glycogene (20 mg/ml), and 3.5 ml of isopropanol were added. After precipitation and ethanol wash, the pellet was resuspended in 200 µl of TE buffer. The samples were run on agarose gel (1.5%) and the bands corresponding to the mononucleosomes were cut and purified with Qiagen. Q-PCR with the primer pairs of the set of overlapping amplicons was performed. Nucleosomal DNA enrichment calculated as the ratio between the amounts of PCR product obtained from DNA samples generated from the mononucleosomal gel purification to that of the input  DNA.A culture of 500 ml of fission yeast cells was grown to OD 0.5 at 32°C and crosslinked with 7 ml of formaldehyde 37% for 20 min at 25°C, 60 rpm. The crosslink was stopped by the addition of 27 ml of Glycine and cells were pelleted. The pellet was resuspended in preincubation solution  supplemented with 100 µl β mercaptoethanol/50 ml and incubated 10 min at 30°C. The cells were centrifugated and resuspended in 10 ml of Sorbitol 1 M/Tris pH 7.4 50 mM buffer containing 200 µl Zymolase (0.01 g/200 µl water) and incubated 20 min at 30°C . After centrifugation, the pellet was resuspended in 7.5 ml NP buffer .Total RNA was prepared as described  and puriinv1, Northern blots were preferred to Q-RT-PCR because the act1 mRNA showed variations during the course of the experiment.Note that while studying the induction of The preparation of mononucleosomal DNA and the sequencing of mononucleosomal DNA were previously described in details . The nuchttps://code.google.com/p/danpos/). Clonal reads  were removed from the reads previously mapped on the Schizosaccharomyces pombe genome with BWA (http://bio-bwa.sourceforge.net). Variation in size resulting from MNase treatment was compensated by shifting each read toward the 3′ direction for half of the estimated fragment size. Nucleosome occupancy was then calculated as the quantile-normalized count of adjusted reads covering each base pair in the genome. After this processing, DANPOS calculates the differential signal at single nucleotide position based on a Poisson test. Dynamic nucleosomes are then identified by peak calling on these signals. TSS and TTS-associated NDR length were quantified as the length of the longest DNA segment whose proximal border is located closer than 65 bp from the TSS (or TES) with nucleosome occupancy levels lower than an arbitrary threshold (mean(occupancygenome) − standard_deviation(occupancygenome)) at any point.MNase-seq data were processed and dynamic changes were detected using DANPOS . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and three peer reviewers, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work cannot be considered further for publication in eLife.Thank you for choosing to send your work entitled “Promoter nucleosome dynamics regulated by signaling through the CTD code” for consideration at Although we recognize the potential novelty of the work describing a function of Ser-2 phosphorylation at the promoter, we believe that the phenomenon is likely not general, but most importantly in the absence of mechanism it only remains as an observation.Reviewer #1:S. pombe. Further analysis revealed that nucleosome dynamics and occupancy at promoters was altered in CTD S2A mutants in a subset of genes, including the genes ste11 and inv1. Molecular and genetic experiments identified altered histone acetylation as a possible reason for increased histone occupancy at promoters.The authors identified chromatin remodellers and chromatin modifiers in a synthetic screen analyzing mutants of Pol II CTD serine 2 (S2A) and the CTD S2 kinase lsk in ste11 expression after artificial tethering of LSK 1 at the ste11 promoter. While the association of HDACs with promoters is CTD S5-P dependent the authors suggest that the combined phosphorylation of S5 and S2 can abolish this association, leading to hyperacetylation of chromatin and gene activation.Histone deacetylation at promoters is under the control of H3K4 methylation and the H3K4 methylation dependent recruitment of HDACs SET3C and RPD3 to promoters. The authors show that CTD S2 phosphorylation can block the recruitment of HDACs to promoters and thereby contribute to gene activation. Further, phosphorylation of kinase Lsk and its recruitment to promoters is growth regulated and under the control of MAP kinases. Mutation of MAP kinase specific phosphorylation sites in the N-terminal domain of Lsk prevents its recruitment to promoters, phosphorylation of CTD S2, and thereby the dissociation of HDACs from promoters. In line with these observations the authors can activate S. pombe could be a more general mechanism is supported by supplementary data in paper. Inactivation of Ctk1, the S. cerevisiae CTD S2 kinase, abrogates promoter histone acetylation and results in low gene expression.The assumption the CTD S2-P dependent mechanism of gene activation described here for All together this is a very detailed and carefully performed analysis and all conclusions are supported by data.How CTD S2-P marks mechanistically can abrogate the association of HDACs with promoters remains open but this question is beyond this paper.Reviewer #2:Schizosaccharomyces pombe. The authors provide a large number of experimental data ranging from genome-wide synthetic genetics arrays (SGAs) for growth phenotypes to locus-specific chromatin modifications. However, the presented results fail to provide compelling evidence for their thesis that 'promoter nucleosome dynamics is regulated by the CTD code'. Most importantly, the provided data do not discriminate between cause and effect or even bystander effects.The manuscript by Hermand and coworkers investigates a functional link between CTD phosphorylation at serine-2 and histone dynamics in the yeast Many results and conclusions of this study have multiple interpretation issues.rpb1 CTD-S2A mutant allele. As expected the identified genes display a significant overlap, but it is troublesome that only single subunits of chromatin regulatory complexes were identified. Also, there is little evidence of a systematic enrichment of ATP-dependent chromatin remodellers, which regulate nucleosome dynamics. Surprisingly, the authors did not investigate the identified Rsc1 and Swr1 genetic interactions in any detail. Next, the nucleosome density is determined in the rpb1 CTD-S2A, but not in the lsk1 deletion. Compared to wild type yeast the authors observe small differences in global MNase sensitivity and they make conclusions on nucleosome dynamics. This is highly surprising. Nucleosome dynamics is defined as nucleosome turnover or half-lives at specific genomic loci. Therefore, the conclusion that 'these analyses reveal an unexpected role of S2P in regulation nucleosome dynamic' is flawed.First, the authors present the combined results from two genome-wide SGAs using a CTD-S2 kinase deletion and an ste11 and inv1 genes for locus-specific effects. The rationale for this is sketchy and not linked to the results from the SGA screens as the H3K4 and H3K36 (de)methylation or H3K14 (de)acetylation machineries were not identified in these screens. Also, the reason to analyze involvement of the PAF complex (via the LEO1 gene) in CTD-S2 phosphorylation dependent regulation of ste11 is not clear. The conclusion that 'defects in initiation or elongation cannot explain the increased level of histone H3 observed at the ste11 promoter in the absence of S2P' is not substantiated, as the authors did not analyze RNA polymerase II occupancy at ste11 in the CTD-S2A strain.Next, the authors focus on the histone modifications at the Reviewer #3:S. pombe. A synthetic lethal screen found many interactions between the S2A mutant or Lsk1/Cdk12 (S2 kinase) and chromatin-related factors. Nucleosome mapping revealed a subset of genes with altered nucleosome positions near promoters in these mutants. The rest of the paper focuses on one of these genes (ste11), with a few other experiments done on the inv1 gene. The story then gets a little confusing. The model proposed is that these promoters are repressed by H3K4 methylation recruiting either SET3C or Rpd3L HDACs. To activate the genes, the MAPK Sty1 phosphorylates Lsk1, which in turn phosphorylates S2 near the ste11 promoter to block H3K4 methylation and subsequent HDAC recruitment. It's a complicated model, and the many experiments needed to look at each of these steps makes the paper difficult to follow.This paper follows up earlier results showing that a CTD serine 2 to alanine mutant has gene-specific, rather than general, effects in Major issues:1) A big gap in the paper is understanding why only a small number of promoters are affected, since CTD phosphorylation and H3K4 methylation are seen at all active genes. At several points in the paper, the text suggests this is a general mechanism, but the data don't support that. What specifies which promoters are repressed, and is Sty1 recruited to these promoters specifically? The change in the NDR suggests remodelers may be involved—maybe these promoters are more dependent on SWI/SNF? Are there overlapping ncRNAs causing methylation over the promoter regions?2) A critical element of the model is that double S2P+S5P no longer binds Set1 complex (and/or HDACs). This should be tested physically, maybe by peptide binding.[Editors’ note: further revisions were requested before acceptance.]eLife. Your submission has been considered by James Manley and the Reviewing Editor who oversaw the initial review.Thank you for choosing to send your work entitled “Promoter nucleosome dynamics regulated by signaling through the CTD code” for consideration at We remain unconvinced of the general significance of the observations and are still concerned regarding the limited mechanistic insight. However, if you can: 1) show that the phenomena observed (ser-2 phosphorylation) occurs at a significant number of promoters in yeast; and 2) provide more solid insights into the mechanism, then we would be willing to consider a new manuscript on this topic. eLife.Thank you very much for reviewing our manuscript entitled “Promoter nucleosome dynamics regulated by signaling through the CTD code”. I am aware of the fact that this manuscript is somehow challenging the current view of role of CTD S2P during transcription, which is precisely why we submitted it to Reviewer #2:The manuscript by Hermand and coworkers investigates a functional link between CTD phosphorylation at serine-2 and histone dynamics in the yeast Schizosaccharomyces pombe. The authors provide a large number of experimental data ranging from genome-wide synthetic genetics arrays (SGAs) for growth phenotypes to locus-specific chromatin modifications. However, the presented results fail to provide compelling evidence for their thesis that 'promoter nucleosome dynamics is regulated by the CTD code'. Most importantly, the provided data do not discriminate between cause and effect or even bystander effects.This is a comment with which I strongly disagree. The core of our conclusion is twofold:a) The S2 kinase Lsk1 (Cdk12) is regulated by MAP kinase dependent phosphorylation, which influences its chromatin distribution;b) CTD S2P directly affects the occupancy of promoter nucleosomes, which leads to transcription activation.ste11 and inv1), we also engineered a chimeric S2 kinase that we can artificially tether to the ste11 promoter, which results in transcriptional activation and bypasses the MAP kinase regulation methylation or H3K14 (de)acetylation machineries were not identified in these screens. Also, the reason to analyze involvement of the PAF complex (via the LEO1 gene) in CTD-S2 phosphorylation dependent regulation of ste11 is not clear.The SGA screens look for synthetic lethality. Why would the referee expect us to find H3K4 and K36 demethylases or HDAC? Based on our model, their deletion should rather result in synthetic improvement and rescue  A big gap in the paper is understanding why only a small number of promoters are affected, since CTD phosphorylation and H3K4 methylation are seen at all active genes. At several points in the paper, the text suggests this is a general mechanism, but the data don't support that. What specifies which promoters are repressed, and is Sty1 recruited to these promoters specifically? The change in the NDR suggests remodelers may be involved—maybe these promoters are more dependent on SWI/SNF? Are there overlapping ncRNAs causing methylation over the promoter regions?We really don’t suggest that the mechanism we describe is general, as it is repeatedly stated that it affects only a subset of genes. And indeed, as suggested by the referee, it is the Sty1 MAP kinase that gives the specificity. At most loci, Sty1 is not present and therefore a classical CTD S2P pattern is observed.2) A critical element of the model is that double S2P+S5P no longer binds Set1 complex (and/or HDACs). This should be tested physically, maybe by peptide binding.We now have an experiment using GST-CTD, GST-CTD S5A and GST-CTD S2A in vivo that clearly indicates that Set1 association with the CTD is only favoured by the S5 phosphorylated CTD in the absence of S2P. This experiment would nicely address the issue and complement the mechanistic aspect of the paper by showing that the early S2P phosphorylation interferes with the binding of Set1 to the CTD.[Editors’ note: further revisions were requested before acceptance.]Thank you for choosing to send your work entitled “Promoter nucleosome dynamics regulated by signaling through the CTD code” for consideration at eLife. Your submission has been considered by James Manley and the Reviewing Editor who oversaw the initial review.We remain unconvinced of the general significance of the observations and are still concerned regarding the limited mechanistic insight. However, if you can: 1) show that the phenomena observed (ser-2 phosphorylation) occurs at a significant number of promoters in yeast; and 2) provide more solid insights into the mechanism, then we would be willing to consider a new manuscript on this topic.rpb1 CTD S2A mutant. This list is highly enriched in genes we have previously identified as having an early CTD S2P profile , and in genes downregulated in the CTD S2A mutant . These data are presented in the subsection headed “CTD S2P regulates promoter nucleosome dynamics at a subset of genes” of the manuscript. They establish that the phenomena we describe occur at a significant number of promoters. We propose that the gene specificity is established by MAP kinase signaling, which is essential to recruit the CTD S2 kinase nearby the promoters of the ste11 and inv1 genes that we have analysed in more details. In addition, the number of gene affected may be underestimated by the fact that the large-scale analyses were performed in a single growth condition. Regarding the mechanism, we provide a completely new figure (I am happy to say that we have addressed these issues in a new version of the manuscript. We report a list of 324 genes that show dynamic changes at the -1 nucleosome in the w figure  where th


Enterobacteriaceae. Genes may be positively or negatively regulated depending on the presence or absence of each aromatic amino acid, all three of which function as cofactors for TyrR. In this report we detail the transcriptional control of two divergently transcribed genes, akr and ipdC, by TyrR, elucidated by promoter fusion expression assays and electrophoretic mobility shift assays to assess protein-DNA interactions. Expression of both genes was shown to be controlled by TyrR via interactions with two TyrR boxes located within the akr-ipdC intergenic region. Expression of ipdC required TyrR bound to the proximal strong box, and is strongly induced by phenylalanine, and to a lesser extent by tryptophan and tyrosine. Down-regulation of akr was reliant on interactions with the weak box, and may also require a second, as yet unidentified protein for further repression. Tyrosine enhanced repression of akr. Electrophoretic mobility shift assays demonstrated that TyrR interacts with both the strong and weak boxes, and that binding of the weak box in vitro requires an intact adjacent strong box. While the strong box shows a high degree of conservation with the TyrR binding site consensus sequence, the weak box has atypical spacing of the two half sites comprising the palindromic arms. Site-directed mutagenesis demonstrated sequence-specific interaction between TyrR and the weak box. This is the first report of TyrR-controlled expression of two divergent protein-coding genes, transcribed from independent promoters. Moreover, the identification of a predicted aldo-keto reductase as a member of the TyrR regulon further extends the function of the TyrR regulon.The TyrR transcription factor regulates genes involved in the uptake and biosynthesis of aromatic amino acids in  Escherichia coli K12, the TyrR regulon consists of eight operons and nine genes [6-TTTACA) are defined as strong, while those with fewer then ten matches are referred to as weak boxes [in vivo, whereas weak boxes require an adjacent strong box to bind TyrR [The TyrR transcription factor controls the expression of genes involved in the uptake and metabolism of aromatic amino acids. In ak boxes . An invaak boxes . Strong ind TyrR . The thrind TyrR , allowinind TyrR .E. coli tyrP gene in the presence of tyrosine [aroG in the presence of phenylalanine and tryptophan [tyrR gene by binding upstream of the −35 sequence [tyrB in the presence of tyrosine [aroP gene from its primary promoter P1 is inhibited when TyrR, bound to two sites downstream of the aroP transcription start site, recruits RNA polymerase to an alternate promoter P3 which overlaps the P1 promoter but is located on the opposite DNA strand. This recruitment prevents open complex formation at P1, thereby inhibiting transcription [mtr promoter by TyrR is also dependent on DNA structure; negative supercoiling stabilized by the DNA binding proteins IHF and HU is necessary for repression with tryptophan [TyrR typically represses transcription by preventing access of the RNA holoenzyme to the core promoter, or inhibiting the later stages of open complex formation and elongation. A strong TyrR box overlapping the −35 element represses expression of the tyrosine , 9. A siyptophan . TyrR ausequence , 12. Tyrcription –15. Repryptophan .mtr gene of E. coli contains a pair of adjacent TyrR boxes, one strong  and one weak , upstream of the −35 sequence, and separated from each other by eight base pairs [mtr. In the presence of phenylalanine, TyrR binds to both sites and further enhances mtr transcription above the level of induction by tyrosine [folA is activated via hexameric TyrR in response to tyrosine. Binding of IHF within the folA promoter induces DNA bending and facilitates TyrR interaction with RNA polymerase to enhance transcription [While the primary mechanism of TyrR transcriptional control is repression, the expression of several genes is enhanced by TyrR. The se pairs , 18. In tyrosine , 19, 20.cription .E. coli, experimental evidence has shown that the TyrR regulon is restricted to genes required for aromatic amino acid uptake and biosynthesis. However, bioinformatic and functional analysis suggests that TyrR homologues in other bacteria have expanded or different regulons. Microarray assays indicated that the TyrR homologue in pseudomonads, PhhR, is a much more global regulator, controlling 21 genes which, in addition to proteins for aromatic amino acid uptake and biosynthesis, encode enzymes for catabolism of phenylalanine and other aromatic compounds, ABC transporters, a LysR type transcription factor, and several proteins of unknown function [Shewanella spp., the orthologous TyrR was predicted to control a regulon that comprises 35 genes spanning 16 operons [Shewanella TyrR orthologue has undergone regulon expansion, and is proposed to regulate expression of genes for branched chain and aromatic amino acid degradation and components of the glyoxylate shunt of the tricarboxylic acid cycle [In function , 23. Usi operons . The Sheid cycle .Enterobacter cloacae UW5, the TyrR regulon includes the ipdC gene encoding the enzyme indolepyruvate decarboxylase which converts indole-3-pyruvate to indole-3-acetaldehyde, a key step in the indole-3-acetic acid (IAA) biosynthetic pathway [ipdC expression and IAA production. This strong TyrR box is centered directly between ipdC and a divergently transcribed gene, akr, encoding a putative aldo-keto reductase , we characterize the roles of the strong and weak TyrR boxes in the activation and repression of ipdC and akr, respectively. Genes that are co-regulated are often functionally related and understanding how akr is controlled by TyrR is a first step towards elucidating its possible role in production of IAA, an important signaling molecule in bacterial-plant interactions [Previously, we showed that in the rhizobacterium  pathway . Bindingeductase . In addisequence . This seractions .E. coli and E. cloacae were routinely grown at 37°C and 30°C, respectively, in either Luria Bertani (LB) broth or M9 minimal media (Difco). Media was supplemented with antibiotics when required at final concentrations of 100 μg/mL ampicillin, 25 μg/mL kanamycin, 20 μg/mL gentamicin or 5 μg/mL tetracycline. L-Aromatic amino acid supplements to the growth media were supplied at a final concentration of 1 mM when indicated.The bacterial strains and plasmids used in this study are listed in akr and ipdC. Overnight cultures of E. cloacae UW5 were inoculated 1:100 into fresh M9 media with or without 1 mM of each aromatic amino acid . Cultures were grown to mid-logarithmic  and stationary  phase, at which point 1 mL of culture was transferred to a microfuge tube containing 0.5 mL RNAprotect (Qiagen), and vortexed for ~30 s. Cells were then centrifuged, supernatant discarded and the pellet stored at −20°C. Total bacterial RNA was isolated using the RNeasy Mini Kit (Qiagen). RNA quality was assessed by running a sample on a 2% agarose gel, and RNA quantity and purity was measured spectrophotometrically at 260 nm using a SpectraMax M5 plate reader (Molecular Devices).5’-Rapid amplification of cDNA ends (RACE) was used to identify the transcription start sites of akr and ipdC, respectively. cDNA was purified using Qiagen’s PCR purification kit and end tailed with dCTP using terminal transferase (New England Biolabs) in a reaction containing 1X TdT buffer, 2.5 mM CoCl2 and 10 mM dCTP. Reactions were incubated at 95°C for two min and cooled to room temperature before addition of 20 U terminal transferase and further incubation at 37°C for 20 min. Tailing reactions were stopped by heating to 65°C for 10 min.A total of 2 μg RNA was treated with amplification grade DNase I (Invitrogen) to remove any contaminating DNA. cDNA synthesis was performed using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s recommendations with primers IPIF and IPDC-GSP1  for akr akr and ipdC, respectively. Touchdown PCR cycling conditions were as follows: initial denaturation at 94°C for 2 min; 20 cycles of 94°C for 30 s, 58°C −0.5°C per cycle for 30 s, 72°C for 30 s; 25 cycles of 94°C for 30 s, 48°C for 30 s, 72°C for 30 s; final elongation at 72°C for 7 min. Ten microliters of PCR products were used as templates in a second round of PCR performed with a common AUAP primer (Ten microliters of tailed cDNA were then used in a 50 μL touchdown PCR with an abridged anchored primer (AAP) and gene specific primers, AKR-GSP2 and IPDC-GSP2  for akr P primer  and geneakr and ipdC tailed cDNA fragments were ligated into pGEM-T Easy (Promega), generating the plasmids pGEM-IP1 and pGEM-AP1 (E. coli JM109 cells (Promega). Inserts were then sequenced using standard T7 and SP6 primers  to identify the transcription start site.Both pGEM-AP1 , respectakr and ipdC was examined using plasmid-based reporter gene fusions. The suicide plasmids pJQSIPG and pJQSIAPG contain the ipdC and akr promoters, respectively, fused to the uidA reporter gene encoding β-glucurionidase [ipdC::uidA and Pakr::uidA fusions were excised as ApaI-XbaI fragments from pJQSIAPG and pJQSIAPG, respectively, and inserted into a similarly digested derivative of the pBBR1MCS-2 broad host range plasmid [lacZ promoter (pBBRL). The lacZ promoter was excised from pBBR1MCS-2 using the restriction enzymes KpnI and NsiI. Remaining 5’ overhangs were removed by Klenow digestion followed by re-circularisation by blunt end ligation with T4 DNA ligase (NEB) to generate pBBRL. Following insertion of the PipdC::uidA and Pakr::uidA fusions, the resulting plasmids pBBRLI and pBBRLA, respectively, (E. coli JM109 (Promega), purified and introduced into E cloacae UW5 and J35 by electroporation.The impact of the TyrR transcription factor on expression of ionidase . The Pip plasmid  lacking ctively,  were tra2, 20 mM Tris-CL, pH 8.0, 5 mM DTT) to remove non-mutated template DNA. The reaction was stopped with 30 μL H-buffer  before SLIM-PCR hybridisation at 99°C for 3 min; followed by two cycles of 65°C for 5 min; 30°C for 40 min. Plasmid DNA was purified and transformed into CaCl2 competent E. coli S17-1 (λpir) cells. Successful mutagenesis was confirmed by sequencing the plasmids (Robarts Research Institute). Following sequence confirmation, plasmids were introduced into wild-type E cloacae UW5 and tyrR null mutant E. cloacae J35 by electroporation. The resulting plasmids are listed in The involvement of two predicted TyrR binding sites was examined by introducing point mutations into the binding sites using site-directed ligase-independent (SLIM) PCR mutagenesis  with theipdC and akr promoter-driven reporter gene expression was quantified using the method of Cowie et al. (2006) [E. cloacae UW5 and tyrR null mutant E. cloacae J35 cells containing the reporter gene constructs described above were grown in triplicate from independent colonies overnight in LB broth with appropriate antibiotics, pelleted, and washed twice in 1X M9 salts. Cultures were then diluted 100-fold into 1 mL M9 minimal media in 2.2 mL 96-deep well plates , with or without each of the aromatic amino acids. Cultures were grown to mid-logarithmic  and stationary  phase, at which time they were assayed for β-glucuronidase activity. Briefly, 20 μL of cell culture were withdrawn to wells of a 96-well plate  containing 80 μL of reaction buffer  and 0.44 mg/mL p-nitrophenyl β-D-glucuronide (PNPG), and incubated at room temperature until development of a strong yellow precipitate, at which time the reaction was stopped with the addition of 100 μL 1 M Na2CO3. Absorbance of the reaction products was measured at 405 nm using a SpectraMax M5 plate reader (Molecular Devices) and used to calculate specific promoter activity in Miller units (1000 x OD405nm)/(time x OD600nm x culture volume in reaction). Wild-type E. cloacae UW5 and uninoculated medium were included as controls to assess background levels of absorbance at 405 nm.. (2006) . Wild-ty6 tag [E. coli M15 carrying plasmids pREP4, which constitutively produces high levels of lac repressor protein, and pQEtyrR were grown at 37°C in LB to mid logarithmic phase  before induction with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and allowed to incubate for an additional four hours. His6-TyrR was purified under native conditions using the QIAexpressionist Ni-NTA metal affinity chromatography kit (Qiagen). The native purification protocol was followed according to the manufacturer’s instructions with the exception of a modified wash step and elution buffer. Wash buffer 1 contained 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole; wash buffer 2 was as described for wash buffer 1 with 10% glycerol; the elution buffer contained 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 8% glycerol. Purified protein was quantified using the Quick Start Bradford protein assay (Bio-Rad), and purity assessed on a 12% SDS-PAGE gel stained with Coomassie brilliant blue G-250.The previously constructed expression vector pQEtyrR  was used6 tag . E. coliakr and ipdC were amplified with Phusion High Fidelity DNA polymerase (NEB) using the wild-type and mutant promoter fusion plasmids described above as templates. Primers CF1 and DIG-CR1  with 1 μg poly d(I-C), 0.1 mM ATP and 0.1 mM of either L-tryptophan, L-phenylalanine or L-tyrosine in binding buffer  for 5 min at room temperature. Following incubation, 50 ng of specific DIG-labeled DNA was added and incubated at room temperature for a further 30 min. Competition assays were performed by incubating His6-TyrR first with 100-fold molar excess unlabelled wild-type DNA (500 ng) for 5 min in reaction buffer at room temperature, followed by addition of 50 ng wild-type DIG-labeled DNA. Binding reactions were stopped with 5 μL loading buffer , and loaded onto a 6% native-PAGE gel . Protein-DNA complexes were resolved at 80 volts for 2–3 h at 4°C.Protein-DNA binding reactions were carried out as described previously . PurifieDNA was transferred to a positively charged nylon membrane (Roche) using a Trans-Blot SD Semi-Dry cell (Bio-Rad) and visualised using the anti-DIG-Detection system according to the manufacturer’s instructions (Roche). Developed membranes were imaged with a Bio-Rad ChemiDoc MP (Bio-Rad).p values of <0.05. Unless stated otherwise, all main effects and interactions were significant.Statistically significant differences in gene expression from each promoter during logarithmic or stationary growth phases were detected by two-way analysis of variance (ANOVA) with post-hoc comparisons by TukeyHSD test using JMP 7 . The genakr and ipdC, the location of the promoters of both genes relative to the predicted TyrR boxes was first determined. 5’-RACE was used to identify the transcription start sites from which the core promoter sequences of the two genes were inferred. The transcription start site for ipdC was found to be a purine base located 26 nucleotides upstream of the ATG start codon  the akr coding sequence is conserved among closely related bacteria with annotated genomes, including the start codon, and (ii) the identified adenine was located at the end of five contiguous adenines, and reverse transcriptase is known to pause or prematurely terminate activity when it encounters a poly-adenine tract [Salmonella enterica sv. typhimurium SL1344 were identified by RNA sequencing, including those for the homologous ipdC and akr genes [S. enterica SL1344 and E. cloacae UW5 akr-ipdC regions suggested that the E. cloacae UW5 akr transcription start site is a cytosine nucleotide 25 base pairs upstream of the akr start codon  is consistent with the E. cloacae UW5 ipdC transcription start site identified in this study. Visual inspection of the sequence upstream of the predicted E. cloacae UW5 akr transcription start site revealed a possible −10 element that matches four out of six nucleotides of the sigma-70 consensus sequence [akr. No definitive −35 hexamer was identified upstream of the −10, although the lack of a −35 element does not preclude RNA polymerase interaction [akr.5’-RACE identified a transcription start site for the ne tract . The genkr genes . Alignmert codon . The S. sequence . This poeraction . A candiakr and ipdC genes, uidA transcriptional fusions were generated using a derivative of the low copy number plasmid pBBR1MCS-2 [lacZ promoter. Consistent with previous results [E. cloacae UW5 cells containing the ipdC::uidA fusions were grown and assayed for β-glucuronidase activity in stationary phase, expression from PipdC was 3.5-fold greater than that in the tyrR null mutant E. cloacae J35 strain which lacks a functional TyrR  compared to expression levels in wild-type E. cloacae UW5, indicating that TyrR normally represses akr. Inclusion of the aromatic amino acids tryptophan and phenylalanine did not result in significant down-regulation of akr, however, the inclusion of tyrosine in the medium resulted in a 1.5-fold decrease in akr expression relative to M9 medium alone during log phase. Transcription from the akr promoter remained at relatively constant levels during both logarithmic and stationary phases of growth when assayed in wild-type E. cloacae UW5.In contrast to the positive regulatory effects of TyrR on the  by TyrR . When exipdC, four promoter variants were constructed via SLIM-PCR mutagenesis . The high identity of the weak box palindromic arms to the TyrR consensus sequence, coupled with the optimal spacing would ensure that TyrR is bound with high affinity to both sites [To examine the effect of TyrR binding at the weak box on the th sites . The resakr, the weak TyrR box is situated to overlap the predicted −10 sequence of the akr promoter, suggesting that binding of TyrR to the weak box competes with RNA polymerase for access to the promoter. Mutation of the weak box (pBBRLAW) resulted in a 3.5- and 5.3-fold higher level of expression from the akr promoter compared to the wild-type promoter (pBBRLA) in logarithmic and stationary phases, respectively  had no effect on akr expression in either growth phase suggesting that the observed repression is mediated by the weak box  yielded the same level of repression as from the wild-type promoter in wild-type E. cloacae UW5; however, in the tyrR null mutant E. cloacae J35, transcription increased 3.8- and 9.3-fold in log and stationary phases, respectively. The effect of the TyrR box mutations on akr expression was similar in M9 medium with and without aromatic amino acid supplements , compared to the DNA fragment alone , and prn of IAA , 39. In n of IAA . E. cloaelopment . Plant gelopment . Identifelopment ).ipdC expression in a TyrR dependent manner, with phenylalanine as the strongest inducer. The center of the strong TyrR box is located 33 base pairs upstream from the center of the predicted ipdC −35 element. This would position a TyrR dimer bound at the strong box on the same face of the DNA as RNA polymerase bound to the ipdC promoter, and in an optimal location for interactions with the RNA polymerase α-CTD [ipdC is due to activation by TyrR complexed with aromatic amino acids, which increases the affinity of TyrR for the DNA and leads to strong activation in promoters with similarly positioned TyrR boxes [mtr gene in E. coli has a similarly orientated strong TyrR box centered 42 base pairs upstream of the −35 element. In the presence of phenylalanine, a TyrR dimer bound to this strong box interacts with the RNA polymerase α-CTD enhancing expression 2.8-fold [E. coli mtr promoter and the E. cloacae UW5 ipdC promoter reveals very similar trends. The mtr gene is activated 2-fold (tryptophan), 2.8-fold  and 1.7-fold (tyrosine); while ipdC is activated 2-fold (tryptophan), 3.7-fold  and 1.4-fold (tyrosine) [Inclusion of any of the aromatic amino acids in the growth media significantly increased se α-CTD , 38. It rR boxes , 42. The2.8-fold , 21. A cyrosine) .akr, identified in this study, was shown to be down-regulated by TyrR. Little is known regarding the function of the protein encoded by the akr gene. From the amino acid sequence it is predicted to be a member of the aldo-keto reductase (AKR) superfamily of proteins, a diverse group of enzymes found in all domains of life that share a common (α/β)8 barrel fold and utilize the nicotinamide adenine dinucleotide (NAD(P)H) cofactor to reduce carbonyl compounds [E. cloacae UW5 AKR groups it with E. coli YghZ (data not shown), which is homologous to the AKR6 family that comprises the β-subunit of voltage gated potassium channels [yghZ gene encodes a methylglyoxal reductase, which has been shown to confer protection against the toxic electrophile methylglyoxal by reducing it to acetol [E. cloacae UW5 AKR is unknown, the sequence and organization of the open reading frame is conserved among closely related bacteria that possess the ipdC gene: when ipdC is present in a sequenced enterobacterial genome, it is found adjacent to, and divergently transcribed from, a homologous akr  and may have abolished the binding site for a second regulator. In wild-type E. cloacae UW5 with a double strong box, the increased affinity of TyrR for the weak box maintained repression of akr at wild-type levels.Down-regulation of akr was down-regulated by TyrR, but not completely repressed may reflect the weak interactions between TyrR and the DNA binding site overlapping the −10 element. A TyrR dimer or hexamer may associate transiently with the weak box allowing RNA polymerase access to the promoter and a low level of expression. This is proposed to be the case with the aroG gene of E. coli, where the TyrR box overlaps the −10 element and competition between the two proteins for DNA binding results in a low but constant level of gene expression [mtr [akr-ipdC intergenic region, increasing its affinity for the adjacent weak box in the presence of tyrosine. This is supported by the requirement for the wild-type strong box for strong TyrR binding to the intergenic region in EMSAs. The observation that mutations in the strong box do not affect akr expression in the reporter gene assays may support the involvement of a second regulatory protein that stabilizes TyrR binding to the weak box in vivo.The observation that ion [mtr  or the fion [mtr . The resion [mtr . TyrR maakr in vivo and by its binding to TyrR in vitro as shown by EMSAs, was one of the most interesting findings of this study. In E. coli, TyrR binds the consensus sequence TGTAAA-N6-TTTACA and requires the conserved G-N14-C pair [akr promoter and the double tandem 5’ arms suggests two possibilities for the spacing of the G-C pair: G-N10-C or G-N16-C. There is little information regarding protein binding to DNA sequences with atypical spacing between palindromic half-sites. The consensus binding sequence for PrrA, a global transcription factor controlling ~25% of the genome in Rhodobacter sphaeroides [The functionality of the weak TyrR box, demonstrated by its necessary role in repression of aeroides , comprisaeroides . Site diaeroides .E. coli demonstrated its interaction in vivo with several promoter regions lacking a discernible LexA DNA binding consensus sequence, while these sites were bound extremely poorly when studied in vitro [E. coli revealed that the protein can recognize and bind to two types of binding sites: one comprised of two 11 base pair inverted repeats separated by a single nucleotide and the other contains only a single 11 base pair arm, with no discernible second inverted arm [akr expression.Genome wide studies of transcription factor interactions with DNA have revealed that interactions with non-canonical sites, which were previously considered artifacts, are more common then previously thought and reflect true protein-DNA interactions. Chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) performed with the LexA regulator of in vitro . ChIP-chrted arm . While irted arm , 53, we S1 FigipdC promoter mutants in M9 minimal medium without amino acid supplements (A) and in the presence of phenylalanine (B), or tyrosine (C) in wild-type E. cloacae UW5 (grey) and tyrR null mutant E. cloacae J35 (black). Cells were assayed for β-glucuronidase activity in both logarithmic and stationary phases of growth. Error bars represent the standard error of the means of three independent replicates. Statistically significant differences of p < 0.05 are indicated by lowercase letters in logarithmic phase, and uppercase letters in stationary phase.Expression from (TIF)Click here for additional data file.S2 Figakr promoter mutants in M9 minimal medium without amino acid supplements (A) and in the presence of phenylalanine (B), or tyrosine (C) in wild-type E. cloacae UW5 (grey) and tyrR null mutant E. cloacae J35 (black). Cells were assayed for β-glucuronidase activity in both logarithmic and stationary phases of growth. Error bars represent the standard error of the means of three independent replicates. Statistically significant differences of p < 0.05 are indicated by lowercase letters in logarithmic phase, and uppercase letters in stationary phase.Expression from (TIF)Click here for additional data file.S3 Fig6  in the absence of aromatic amino acids (A), 1 mM tryptophan (B), phenylalanine (C) or tyrosine (D). Competition assays were performed with 10-fold molar excess of unlabeled-probe over DIG-labelled probe; rightmost lane. Positions of free DNA (F) and the two resolved TyrR-His6-DNA complexes  are indicated.Wild-type DIG-labelled DNA probe was incubated with increasing amounts of TyrR-His(TIF)Click here for additional data file.


FMR1 gene. In contrast to Fragile X syndrome, in which the FMR1 gene harbors over 200 CGG repeats but is transcriptionally silent, the clinical features of FXTAS arise from a toxic gain of function of the elevated levels of FMR1 transcript containing the long CGG tract. However, how this RNA leads to neuronal cell dysfunction is unknown. Here, we discuss the latest advances in the current understanding of the possible molecular basis of FXTAS.Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset inherited neurodegenerative disorder characterized by intentional tremor, gait ataxia, autonomic dysfunction, and cognitive decline. FXTAS is caused by the presence of a long CGG repeat tract in the 5′ UTR of the  Fragile X Mental Retardation 1 (FMR1) gene . Of. OfFMR1  repeats . WhetherFMR4, originates upstream of the FMR1 start site, and covers 2.4 kb of sequence [FMR4 is widely expressed in fetal and adult human tissues, and throughout human and macaque brain regions. Expression of FMR4, like that of ASFMR1 and FMR1, is increased in brain tissue of premutation individuals and is silenced in individuals with the full mutation [FMR4 overexpression was shown to increase cell proliferation, whereas FMR4 downregulation induced apoptosis in vitro [cis-acting effect was observed upon expression of the FMR1 gene. Therefore, it was hypothesized that FMR4 influences proliferation pathways in trans, by targeting distal genomic loci. Current work is focused on defining a role for this transcript, as it has been found to affect the chromatin state and transcription of several genes involved in neuronal differentiation, axon guidance, and synaptic signaling, as well as cell cycle regulators .Another antisense transcript, sequence . FMR4 ismutation . Importain vitro . AdditioFMR1 locus, FMR5 and FMR6, were recently identified [FMR5 is a sense-oriented lncRNA transcribed from approximately 1 kb upstream of the FMR1 transcription start site (TSS). FMR5 is not differentially expressed in human brain from unaffected individuals compared with full mutation and premutation patients, suggesting that its transcription is independent of CGG repeat expansion. Furthermore, the TSS of FMR5 appears not to be affected by the chromatin silencing that occurs within full mutation alleles, or by the open chromatin hypothesized to increase transcription of FMR1 premutation alleles. FMR6 is a spliced long antisense transcript, 600 nucleotides i, whose sequence is entirely complementary to the 3′ region of FMR1[FMR1, and uses the same splice junctions as FMR1. An unexpected finding was that FMR6 is reduced in premutation carriers, suggesting that abnormal transcription and/or chromatin remodeling occurs toward the distal end of the locus. However, the chromatin marks associated with the 3′ end of FMR1 in premutation carriers have yet to be described. The function of FMR6 remains to be identified, but its complementarity to the 3′ region of FMR1 presents several interesting possibilities. FMR6 may bind to FMR1 mRNA, thereby regulating the stability, splicing, subcellular localization, or translational efficiency of FMR1, as has been described for other lncRNAs [FMR6 overlaps miR-19a and miR-19b binding sites in the FMR1 3′ UTR [FMR6 may modulate the stability or translational efficiency of FMR1 by interfering with microRNA binding. Overall, these results highlight the importance of non-coding transcription of the FMR1 locus. However, much work remains to fully understand the relevance of these transcripts to the pathology observed in premutation carriers.Two new transcripts arising from the entified . FMR5 isn of FMR1. It begi lncRNAs . Notably1 3′ UTR , suggestFMR1 transcripts. Further investigation is needed to evaluate the pathological consequences of expression of ASFMR1 or other long non-coding RNA mapping within the FMR1 gene. Finally, although decreased expression of FMRP is probably not the principal cause of FXTAS, it cannot be excluded that a reduction in FMRP plays a role in modulating some of FXTAS features. In that context, the level of FMRP depletion in brain samples from a larger cohort of patients with FXTAS needs to be measured.The restriction of FXTAS clinical features to unmethylated, transcriptionally active alleles with large CGG repeat numbers suggests that the expression of a mutant RNA is pathogenic to neuronal cells . This hyFMR1 mRNA production, protein titration, non-AUG translation, antisense transcription, and decreased expression of FMRP are a number of non-exclusive mechanisms that may all contribute to FXTAS pathology. It is possible that contributions to pathology from more than one mechanism may help to explain the great variability in clinical presentation of premutation individuals, aspects of which have heretofore not been accounted for by CGG expansion size, mosaicism, methylation, alternative spliced isoforms, additional genomic changes, or other known factors. Thus, more work is needed to determine the relative contribution of these processes to disease pathology in this multifaceted disorder (Figure In conclusion, in addition to increased r Figure .The authors declare that they have no competing interests.CS, KU, CP, VJP, FT and NCB wrote the paper. All authors read and approved the final manuscript.


Traumatic temporomandibular joint (TMJ) ankylosis can be classified into fibrous, fibro-osseous and bony ankylosis. It is still a huge challenge for oral and maxillofacial surgeons due to the technical difficulty and high incidence of recurrence. The poor outcome of disease may be partially attributed to the limited understanding of its pathogenesis. The purpose of this article was to comprehensively review the literature and summarise results from both human and animal studies related to the genesis of TMJ ankylosis. Temporomandibular joint (TMJ) ankylosis is often described as either fibrous or bony, and, in traditional opinion, fibrous ankylosis can progress into bony ankylosis. The mosIn this review, focusing on bony ankylosis, we will describe the current understanding of the clinical, imaging and pathological features of the disease. Then, we will discuss the underlying condition of the disease based on evidence from both animal and human studies. The hypotheses regarding its pathogenesis will be exhaustively summarised and critically evaluated. We will also introduce the advances of cellular and molecular mechanisms of new bone formation in bony ankylosis, and provide new perspectives to prevent the disease.The onset of disease usually occurs in children under 10 years with a rAccording to the literature and our careful observations, the computerized tomographic features of bony ankylosis can be summarised as follows: ① bony fusion is mostly located in the lateral part of the joint, whereas the atrophic condylar head and rudimentary joint space can often be seen in the medial part of the joint-15 , are the only bone-forming cells. In addition, increasing evidences support the hypothesis that similar signaling molecules and pathways, for example BMP and Wnt signalling, may be employed in both physiological and pathological bone formation,69. TherFor the first question, Xiao et al. providedWnts are secreted glycoproteins highly conserved between species, and there are at least 19 Wnt ligands. The WntBMPs are members of the transforming growth factor β (TGF-β) family well known for their osteogenic potential. As the main regulator of chondrocyte proliferation, survival and differentiation, BMP signalling has a remarkable ability to induce endochondral bone formation. BMP2 isThe potential roles of BMP/Wnt signalling in traumatic TMJ bony ankylosis were recently studied using animal models or human specimens,62,65,66Based on a reliable animal model, Yan et al.,62 demonAccording to the hypotheses of hypertrophic non-union, once TMJ trauma establishes the microenvironment for the bone healing of the 2 articular surfaces, the development of ankylosis is unavoidable. In this situation, whether the outcome is fibrous or bony ankylosis depends on the severity of the primary TMJ trauma. Relatively milder injury of TMJ leads to fibrous ankylosis, whereas serious TMJ trauma results in bony ankylosis. TherefoBesides increasing the mechanical instability between the 2 injured articular surfaces through mouth-opening exercises, other methods for inhibiting bone formation or fracture healing, such as non-steroidal anti-inflammatory drugs, low-dosIt is well known that MSCs, possessing the property of pluripotency, play important roles during the course of bone healing. Changing the cell lineage determination of MSCs by manipulating specific transcription factors may be another suitable prophylactic method for bony ankylosis. We believe that FGF21, a key mediator of Peroxisome proliferator-activated receptor-γ (PPARγ) might be such a promising drug. FGF21 cThe true traumatic microenvironment leading to TMJ ankylosis has not been identified. Although animal models and clinical observations have provided new evidence about the pathogenesis of traumatic TMJ bony ankylosis, the biological events and molecular mechanisms are far from being comprehensively understood. The hypotheses of hypertrophic non-union and its supplement seem to grasp the nature of bony ankylosis and explain the development of the disease. A series of recent clinical and experimental studies have preliminarily verified the hypotheses at the cellular and molecular levels. Current data suggest that targeting pathways such as BMPs and Wnt signalling is likely to convert bony ankylosis into fibrous ankylosis. Alternatively, promoting MSCs of the radiolucent zone into adipocyte differentiation using FGF21 may be a promising strategy to prohibit the onset of bony ankylosis, and even avoid the occurrence of fibrous ankylosis.ank: Progressive ankylosis; ANKH: Progressive ankylosis gene; BMP: Bone morphogenetic protein; EHPVO: Extrahepatic portal venous obstruction; FGF21: Fibroblast growth factor-21; GNAI3: Guanine nucleotide-binding protein (G protein), α inhibiting activity polypeptide 3; MSCs: Mesenchymal stem cells; PLCB4: Phospholipase C, β4; PPARγ: Peroxisome proliferator-activated receptor-γ; Shox2: Short stature homeobox 2; TGF-β: Transforming growth factor β; TMJ: Temporomandibular joint; Wnt: Wingless-related MMTV integration site.The authors declare that they have no competing interests.YBY drafted the manuscript and wrote the text. SXL drafted the manuscript and helped with writing the text. JS and JCZ drafted the manuscript and reviewed it critically. YZ revised the final version of the manuscript. All authors read and approved the final manuscript.Yan YB is an Associate Clinical Professor at the Department of Oral and Maxillofacial Surgery of Tianjin Stomatological Hospital. Liang SX is an Associate Clinical Professor at the Department of Operative Dentistry and Endodontics of Tianjin Stomatological Hospital. Shen J is a Professor at the Department of Oral and Maxillofacial Surgery of Tianjin Stomatological Hospital. Zhang JC is a Clinical Professor at the Department of Oral and Maxillofacial Surgery of Tianjin Stomatological Hospital. Zhang Y is a Head Professor at the Department of Oral and Maxillofacial Surgery of Peking University School and Hospital of Stomatology.


The present study highlights the transcriptional and post-transcriptional regulation of mitochondrial transcription factor A (Tfam) during myogenesis and the relationship this has with the increase in mitochondrial content during differentiation. c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes.The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2–3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2–3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome  The fusion of myoblasts into myotubes is vital for skeletal muscle development during embryogenesis and for the maintenance of muscle fibres throughout the lifespan . ProgresThe induction of nuclear-encoded mitochondrial proteins such as Tfam often occurs via the activation of multiple serine-threonine kinases in skeletal muscle. AMP-activated protein kinase (AMPK) is activated in muscle by increases in the AMP:ATP ratio. This kinase specifically enhances the transcription and activity of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), an important regulator of mitochondrial biogenesis –10. AMPK2C12 myoblasts were cultured on 100 mM gelatin-coated plastic culture plates containing Dulbecco's modified Eagle's medium . Cells were incubated in conditions of 5% CO2 at 37°C. Once reaching 90% confluence, differentiation was induced by switching to DMEM supplemented with 5% heat-inactivated horse serum and 1% P/S. For experiments utilizing 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), cells were treated for 24 h with either water or AICAR (1 mM), to activate AMP kinase [C2C12 myoblasts were washed and scraped with ice-cold Dulbecco's PBS . After centrifugation at 3000 g for 5 min, pellets were resuspended in 100 mM Na-KPO4/2 mM EDTA (pH 7.2), sonicated on ice (3×3s), frozen in liquid N2 and then thawed. Samples were then centrifuged at 16000 g for 5 min at 4°C and the supernatants were mixed with test solutions containing reduced cytochrome c. Enzyme activity was determined by measuring the maximal rate of oxidation of reduced cytochrome c using the change in absorbance at 550 nm on a Bio-Tek Synergy HT microplate reader.C2C12 myoblasts were washed with PBS then scraped with a passive lysis buffer supplemented with protease inhibitors to generate whole cell lysates. Cytoplasmic and mitochondrial fractions were also isolated from cultured cells at 0, 2 and 4 days of differentiation and were prepared using the Mitochondria or the Nuclear Isolation Kits (Pierce). Equal amounts of total protein (35–70 μg) were mixed with loading buffer and separated on SDS/PAGE . Gels were then transferred on to nitrocellulose membranes and blocked in for 1 h in 5% skim milk-1× TBST . Blots were incubated overnight at 4°C with antibodies (diluted in blocking buffer) specific for Tfam , cytochrome c oxidase (COX) IV , cytochrome c , Sp1 , p65 , p-AMPK , human antigen R (HuR) , CUG triplet repeat RNA-binding protein 1 (CuGBP1) , voltage-dependent anion channel (VDAC), GAPDH  and KH type splicing-regulatory protein (KSRP) . After incubation, the blots were washed (3×5 min) in TBST and probed with the appropriate anti-mouse , anti-rabbit  or horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. α-Tubulin was used as a loading control for cell lysate and cytoplasmic fractions. The membrane was then subjected to enhanced chemiluminescence (Amersham Pharmacia Biotech) and exposed to film. Signals were quantified with Sigma Scan Pro.C2C12 myoblasts were harvested and total RNA was extracted using TRIZOL (TRIzol® trademarked product). Total RNA was resuspended in diethyl pyrocarbonate-treated sterile H2O. RNA purity and concentrations were determined spectrophotometrically at 260- and 280-nm. mRNA levels were measured using reverse transcription (RT)-PCR for Tfam at each time point. RT was performed using Superscript II Reverse Transcriptase (Invitrogen) according to the instructions from the manufacturer. Forward and reverse primers  were used. PCR products were analysed on Ethidium Bromide-stained agarose gels and expected size products were quantified using SigmaScan Pro (version 5) software. The quantification of all target genes was corrected using the internal control β-actin.CA 2-kb fragment of the mouse Tfam promoter was generated by PCR using genomic DNA . The mouse Tfam promoter was sequenced, matched to the GenBank sequence (Accession number AC153509) and used for PCR primer design  to produ2C12 myotubes was extracted with TRIZOL reagent (Invitrogen) and RT was performed using Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions DNA fragments of 746 and 600 bp corresponding to the 3′-UTR of Tfam mRNA were generated by PCR from the reverse transcribed cDNA. These amplified 3′-UTR of Tfam fragments were gel extracted, digested with EcoRV and BamHI and then ligated into the HpaI and BamHI sites to replace the SV40 3′-UTR at the 3′-end of the Renilla Luciferase gene in the pRL (Renilla luciferase)-CMV  vector.Total RNA from C2C12 myoblasts were transfected and analysed as previously described [Cescribed . Cells w2C12 myoblast were transfected with 3.6 μg each of the control plasmids (pRL-CMV) or with 3.6 μg of Renilla luciferase/3′-UTR chimeras pTfam 746 or pTfam 600, as described above. Transfection efficiency was corrected using the 2051 kb Tfam promoter firefly luciferase construct (0.4 μg/well) and normalized to day 0 of Tfam promoter luciferase activity. Second, fully differentiated C2C12 myotubes were treated with either 10 μg/ml actinomycin D to inhibit transcription or methanol as a vehicle-matched control, for 2, 4 or 6 h. Total RNA was extracted with TRIZOL reagent (Invitrogen) according to manufacturer's instructions. The mRNA expression of mitochondrial transcription factor A (Tfam) over days of differentiation was quantified using 7300 StepOnePlus™ Real-time PCR System (Applied Biosystems Inc.) and SYBR® Green chemistry . First-strand cDNA synthesis from 2 μg of total RNA was performed with primers using SuperscriptIII transcriptase (Invitrogen) according to manufacturer's directions. Forward and reverse primers  were designed based on sequences available in GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) using the MIT Primer 3 designer software (http://wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and were confirmed for specificity using the basic local alignment search tool (www.ncbi.nlm.nih.gov/BLAST/). β-2 Microglobulin was used as a control housekeeping gene, the expression of which did not differ between actinomycin D or vehicle treatment groups. All samples were run in duplicate simultaneously with negative controls that contained no cDNA. Melting point dissociation curves generated by the instrument were used to confirm the specificity of the amplified product. Primer efficiency curves were generated for each set to ensure 100±2% efficiency. Linear regression analysis of Tfam mRNA availability in actinomycin D-treated day 1 compared with day 4 differentiated C2C12 cells were plotted. The decay rates were determined by comparing the slopes of day 1 compared with day 4 of Tfam mRNA availability.Two methods were used to assess Tfam mRNA stability during differentiation. First, mRNA stability was assessed using the transfection of Tfam 3′-UTR constructs ligated to a Renilla luciferase reporter. CmtDNA and nuclear DNA (nDNA) were isolated with a Mammalian Genomic DNA Miniprep kit (Sigma–Aldrich). Forward and reverse primers for mtDNA : Forward 5′-CCTATCACCCTTGCCATCAT-3′/Reverse 5′-GAGGCTGTTGCTTGTGTGAC-3′); and nDNA  were used. PCRs were conducted and the amplified products were analysed on Ethidium Bromide-stained agarose gels and expected size products were quantified using SigmaScan Pro (version 5) software. The quantification of all target genes was corrected using the internal control S12.ttest was used to analyse the mtDNA and COX I mRNA data. All other experiments were analysed using a one-way ANOVA to examine the effect of differentiation. Data are reported significant if P<0.05.Data are expressed as means±S.E.M. The luciferase activity graphs utilizing various Tfam promoter lengths for AICAR-treated cells and non-treated cells and Renilla luciferase/Tfam 3′-UTR chimera constructs were analysed using two-way ANOVAs followed by Bonferroni post-hoc tests. Students' unpaired 2C12 cells to assess changes in mitochondrial content during differentiation. COX IV (P<0.05) and cytochrome c (P<0.05) protein levels were significantly elevated by 3- and 2-fold respectively, on day 4 of differentiation compared with day 0. To further confirm that mitochondrial biogenesis occurred in C2C12 cells with differentiation, a functional marker of mitochondrial content was measured. Differentiation resulted in a significant 3-fold increase in COX activity by day 2 of differentiation, when compared with day 0 (P<0.05).Nuclear-encoded mitochondrial proteins were measured in C. COX IV A, P<0.05chrome c B, P<0.05th day 0 C, P<0.05P<0.05). This occurred in concordance with an approximate 2-fold increase in Tfam mRNA levels (P<0.05) on day 4 of differentiation. Additionally, differentiation produced an approximate 2-fold increase in Tfam protein level (P<0.05) on day 4 of differentiation relative to myoblasts on day 0, which paralleled the induction of Tfam mRNA. Thus, significant increases in differentiation-induced transcriptional activation on day 2 preceded elevations in Tfam mRNA and protein expression evident by day 4.Tfam transcription, mRNA and protein levels were analysed in relation to overall changes in mitochondrial content during differentiation. Tfam transcriptional activity increased by approximately 13-fold on day 2 of differentiation compared with day 0 and remained elevated by 30-fold on day 4 when compared with day 0 A, P<0.05A levels B, P<0.05in level C, P<0.05p<0.05). At days 2 and 4, Tfam accumulated within the mitochondria and was reduced in the cytosol. This indicates the progressive translocation of a greater abundance of Tfam into the mitochondrial fraction as differentiation proceeds.In order to localize Tfam during differentiation, mitochondrial and cytosolic fractions were isolated. At day 0, Tfam protein level was significantly higher in the cytosolic fraction , p<0.05.To further investigate the differentiation-induced changes in Tfam expression, we analysed possible changes in Tfam mRNA stability. Knowing that the 3′-UTR of mRNAs is primarily responsible for the stability of mature mRNA, we examined this region of the Tfam mRNA. Analyses revealed the identification of several potential binding sites for either stabilizing (HuR) or destabilizing  RNA-binding proteins A, suggesP<0.05) on day 4 of differentiation when compared with day 0. Furthermore, mtDNA copy number increased by 1.5-fold (P<0.05) in fully formed myotubes on day 4 in contrast with the day 0 myoblasts.To assess the functional nature of the increase in Tfam protein during differentiation, we measured downstream targets of Tfam transcription. COX I, an mtDNA-encoded protein subunit of complex IV, was elevated by approximately 2-fold A, P<0.051.5-fold B, P<0.05A thorough examination of the mouse Tfam promoter sequence up to −2051 bp upstream of the transcription start site was conducted to investigate the location of putative binding sites for transcription factors involved in regulating Tfam transcription during differentiation. The promoter sequence was retrieved from Transcriptional Regulatory Element Database (Accession Number 48715) and the sequence was subjected to analysis for the presence of consensus transcription factor-binding sites that were 6–8 nts long using PATCH software. A number of binding sites of known transcription factors involved in mitochondrial biogenesis were identified .2C12 cells to determine transcriptional activity. The data indicate that Tfam transcription increased with each day of differentiation for all promoter truncations investigated. The activity of the 1706 construct was 4.5-fold higher, on average, than the full-length 2051 bp construct from days 2 to 4 of differentiation (P<0.05), indicating the presence of a major repressor element(s) between 1706 and 2051 bp upstream of the start site. Further truncation of the promoter produced progressive decreases in promoter transcriptional activity, such that the activity of the 78 bp construct reporter activity was 50% lower (P<0.05) than the 1706 mTfam construct during successive days of differentiation.To investigate possible differences in the functions of the regions within the 2 kb Tfam promoter, 5′-deletion constructs were transfected into Cntiation , P<0.05,0% lower , P<0.05 2C12 cells. Following treatment with AICAR, AMPK activation via phosphorylation on Thr172 was increased by 2.5-fold (P<0.05). In response to AMPK activation, Tfam promoter activity was increased by approximately 2-fold when transfected with the −393 and −78 kb Tfam promoter constructs (P<0.05).AICAR was utilized to investigate the possible role of AMPK in the transcriptional control of Tfam expression in C2.5-fold B, P<0.05nstructs C, P<0.05P<0.05). In contrast, Sp1 (P<0.05), a transcriptional regulator of a variety of mitochondrial proteins, remained unchanged during differentiation.Of the multitude of putative binding sites  within tast, Sp1 B, P<0.05The differentiation of myoblasts into myotubes is accompanied by a pronounced rise in mitochondrial content , which iWe began to investigate this by establishing how differentiation affects the expression of Tfam. Our results demonstrate enhanced transcriptional activity of the Tfam promoter on day 2 of differentiation, with subsequent increases in Tfam mRNA levels observed on day 4. Changes in the transcriptional activity of the Tfam promoter exceeded the increase in steady-state mRNA levels. This suggested the likelihood of rapid Tfam mRNA decay at the onset of the differentiation period to counterbalance this large increase in transcription. Indeed, Tfam mRNA degradation was much more rapid at the onset of differentiation (day 1), as compared with day 4. In contrast with this complex interaction between transcription (synthesis) and degradation leading to steady state Tfam mRNA levels, the progressive elevation in Tfam protein levels observed over the differentiation period paralleled that of the mRNA levels. This suggests a simpler model for the regulated expression of Tfam protein, which is controlled by the level of mRNA, with little regulation evident at the level of translation.2C12 myotubes in proportion to each other. These data suggest that the observed increase in mitochondrial Tfam protein during differentiation is functional and that following translocation into the mitochondria, its predominant influence is on the rate of mtDNA replication, rather than transcription. Thus, adaptations in the Tfam expression pathway are a normal component of the adaptive responses of the organelle during mitochondrial biogenesis induced by differentiation. Our previous results in mature adult skeletal muscle also showed that contractile activity-induced elevations in Tfam protein levels permitted greater Tfam binding within the D-loop of mtDNA and that this corresponded to greater expression of mitochondrial-encoded COX subunit III transcript [The specific targeting sequence within the N-terminal of the Tfam dictates the import of the protein into the mitochondria, where it can carry out its functional role on mtDNA . Cellulaanscript . This inOther studies have shown that the transcriptional rate of the Tfam gene appears to be regulated, in part, by nuclear respiratory factors and Sp1 . Motif mSeveral studies have established that the expression of a variety of mitochondrial genes is increased via the activation of multiple serine-threonine kinases in skeletal muscle. This often occurs through alterations in the energy status of the cell that activate AMPK as a result of increased AMP:ATP ratios . ActivatDiffering models of mitochondrial biogenesis produce similar adaptive responses in mitochondrial proteins  and tranIn summary, we have investigated the regulation Tfam expression in muscle cells during differentiation, which is accompanied by a marked increase in mitochondrial biogenesis. Our results suggest that the increase in total cellular Tfam protein is regulated by a combination of transcriptional activation, as well as by changes in mRNA stability, with little regulation at the level of translation. A negative regulatory element within the distal promoter region probably attenuates transcription and therefore overall protein expression, within muscle cells. In turn, this transcriptional activation is probably influenced by the energy state of the cell during differentiation, which affects the activity of AMPK. The import of Tfam to mitochondria may represent an additional site of Tfam activity regulation, but this remains to be specifically determined. However, it is evident that as it becomes progressively localized to the organelle matrix, Tfam becomes active in the replication of mtDNA and the generation of mtDNA gene products which contribute to the co-ordinated regulation of organelle biogenesis.