Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope.In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In  Schizosaccharomyces pombe, the chromatin remodeler Fft3 is required to maintain the repressed subtelomeric chromatin. Here we show that Fft3 maintains nucleosome structure of insulator elements at the subtelomeric borders. We also observe that subtelomeres and insulator elements move away from the nuclear envelope in cells lacking Fft3. The nuclear periphery is known to harbor repressive chromatin in many eukaryotes and has been implied in insulator function. Our results suggest that chromatin remodeling through Fft3 is required to maintain proper chromatin structure and nuclear organization of insulator elements.In the genome of eukaryotic cells, domains of active and repressive chromatin alternate along the chromosome arms. Insulator elements are necessary to shield these different environments from each other. In the fission yeast  Nuclear architecture, i.e. non-random positioning of chromosomal loci and nuclear components in three dimensions, is important in organizing genome processes such as transcription, DNA replication and DNA repair –3. One cSchizosaccharomyces pombe, the INM protein Man1 interacts with about a third of the genome, mainly at lowly transcribed genes [In the fission yeast ed genes . Especiaed genes . These sed genes ,12. Manyed genes . The bored genes . MaintenS. cerevisiae, ETL1 in mouse and SMARCAD1 in humans [Fft3 belongs to a highly conserved subfamily of SNF2 remodeling enzymes. Fft3 homologs are present in all eukaryotes examined, including Fun30 in n humans ,16. Fun3n humans ,16–22.Here, we explore the interplay between genome organization and transcriptional regulation. We show that Fft3 maintains chromatin structure and peripheral positioning of subtelomeres by binding to and remodeling nucleosomes at their borders. In addition to the subtelomeres, Fft3 interacts with TFIIIC and Pol III at tRNA genes and affects nucleosome positioning and interaction with the nuclear periphery. We propose that Fft3 maintains nucleosome stability and peripheral positioning of these elements and thereby preserves proper genome organization.fft3Δ cells compared to wild type, allowing for a chromatin structure more permissive to transcription. These increases are significant when compared with the rest of the genome  either at the Fft3 binding sites or in close proximity . Assuminome-wide . To studild type . This suSince Fft3 alters interactions with the nuclear envelope at subtelomeres, we wondered whether LTRs are affected similarly. In wild type cells, LTR elements are enriched for binding of both Man1 and the nucleoporin Nup85  Fig. 4D. Bindingfft3Δ cells compared to wild type, suggesting that loss of Fft3 leads to reduced nucleosome stability in these regions. Based on these observations, we hypothesized that the catalytic activity of Fft3 is essential for its functions in subtelomeric chromatin regulation. We therefore constructed a variant of Fft3 with a point mutation in the ATPase domain  or COC (chromosome-organizing clamps) sites across the genome that are occupied by TFIIIC but not by any other components of the Pol III machinery [ETC/COC sites  and performed a co-immunoprecipitation assay. We observed an enrichment of Sfc4-Myc in the anti-TAP purified material from the double-tagged strain compared to the single-tagged strain . The Tfiewed in . Our resS. pombe, Tf2 transposons cluster into Tf bodies that play a role in genome organization [Drosophila gypsy insulators form specialized insulator bodies at the nuclear periphery, and transposon-derived MAR/SAR sequences can create chromatin loops [S. pombe LTR elements localize close to the nuclear periphery and nuclear pores. It is possible that this anchoring of LTR elements helps to divide loops of chromatin with different properties, such as the subtelomeric chromatin and neighboring euchromatin.It has been suggested that insulator elements function by changing higher-order chromatin structure (reviewed in ). Insulanization . Furtherin loops ,45. In aS. cerevisiae homolog, Fun30, is enriched over tRNA genes and the human homolog, SMARCAD1, purifies together with the TFIIIC complex [S. pombe it is plausible that budding yeast and human Fun30 homologues also directly interact with TFIIIC. Like in S. pombe, tRNA genes function as insulators in other eukaryotes and play a role in genome organization by clustering at specific sites in the nucleus . It is iewed in . It is tAt the nucleosome level, we observed a reduction in nucleosome occupancy at Fft3 targets when the remodeler was missing. A similar decrease has also been observed at genes regulated by Fun30 in budding yeast . This suTrypanosoma brucei and Plasmodium falciparum, the subtelomeres habor genes encoding surface markers that can be varied through recombination [T. brucei, these loci move from the nuclear periphery towards the nuclear interior when activated during differentiation [Beyond yeasts, subtelomeres are important in several other eukaryotic systems. In parasites such as bination  and contntiation . In humantiation . Deletiontiation ,52. TakeIn this study, we provide an example how a chromatin remodeler affects both local chromatin structure and genome-wide nuclear organization. Further studies will be necessary to shed light on this interplay between nuclear architecture and transcriptional regulation that seems to have a major role genome function in eukaryotes.S. pombe cells were grown at 30°C and in YES medium unless stated otherwise. The S. pombe strains used in this study are listed in fft3+ gene through homologous recombination were selected by hygromycin resistance and confirmed by DNA sequencing.DNA was immunoprecipitated as described earlier  using 2μS. pombe Tiling 1.0FR was performed by the Affymetrix core facility at Novum (http://apt.bea.ki.se) according to Affymetrix standard protocols.For microarray hybridization, immunoprecipitated DNA was amplified to 5 μg DNA as described in , with thAll experiments were done as biological duplicates.For real-time quantitative PCR, immunoprecipitated DNA was amplified in the presence of SYBR Green using Applied Biosystems 7500 real-time PCR machine. The primers used are listed in http://apt.bea.ki.se).Genomic DNA was extracted as described previously , with anAll experiments were done as biological duplicates.RNA was extracted using the hot phenol method as described in  and reveAll experiments were done as biological duplicates.Cells were grown to early log phase at 30°C in YES medium, fixed with adding paraformaldehyde to 1,75% for 40 min and processed for IF with mouse monoclonal anti-myc antibodies . For FISH, cells were refixed with 3% paraformaldehyde for 30min at room temperature. Chromosomal DNA was denatured by successive 15 min incubation time in 2SSC, 2SSC 10% formamide, 2SSC 20% formamide and 2SCC 40% formamide at room temperature. Subtelomeric FISH probes were generated using the sequencing cosmid c186  as template. c186 spans 30176bp on chr1R (Range 5524764 to 5554939), from SPNCRNA.283 to SPAC186.09.Images were taken using a Nikon A1+ laser scanning microscope with a 60x Lambda S oil-immersion objective (NA 1.4). Imaging was set up fulfilling the Nyquist criterion in xy and z, with a minimum zoom of 2. For each cell, z-stacks were acquired with 0.2μm spacing and subjected to a blind 3D deconvolution algorithm using the NIS Elements Advanced Research software version 4.12. After decapping (removing the top three and bottom three z-stacks) of each nucleus, distances from the center of each FISH spot to the nuclear periphery were measured. Measurements were then assigned to three concentric equal volume zones . SignifiCells were grown over night at 30°C in PMG liquid medium. For mounting, 35 mm glass bottomed culture dishes (MatTek Corp) were coated with soy lectin (Sigma). A log phase cell suspension was added on top of the coated surface and cells were left to sediment for five minutes before pre-warmed PMG medium was added on top. During imaging, the culture dish was kept at 30°C inside the microscope incubator and cells were imaged for no longer than three hours.Images were taken using an inverted Nikon A1R laser scanning microscope with a 60x Lambda S oil-immersion objective (NA 1.4). Imaging was set up fulfilling the Nyquist criterion in xy and z, with a minimum zoom of 2. For each nucleus, z-stacks were acquired with 0.125μm spacing in a 5 μm range around the center of the nucleus. Deconvolution of each stack was carried out using a 3D non-blind algorithm in NIS Elements . After decapping (removing the top three and bottom three z-stacks of the nuclear volume) of each nucleus, distances from the center of each mCherry spot to the nuclear periphery were measured in the z-plane with the brightest signal. The radius of each nucleus was measured and used to normalize the distance measurement. Normalized distances were then assigned to one of three concentric equal volume zone . SignifiS. pombe genome using Bowtie2[Mononucleosomal DNA fragments were digested and purified as described in . Fragmeng Bowtie2 with stag Bowtie2 was thenProteins were extracted using a FastPrep-24 machine  and separated by SDS PAGE. Immunoblot analysis was carried out using anti-myc  and anti-actin  antibodies.www.hybrigenics.com). The full-length open-reading frame of the fft3 gene (SPAC25A8.01c) was cloned into the pB27 vector as a C-terminal fusion to LexA. The construct was use as bait to screen a S. pombe cDNA library. 62.4 million interactions were tested.The yeast-2-hybrid screen and data analysis were performed by Hybrigenics, Paris, France  and lysed in a FastPrep machine (5x30sec at 6.5) with 1 volume of glass beads. The extract was centrifuged for 20 min, 14,000 rpm at 4°C, and the supernatant was incubated with 60μl 50% IgG-bead slurry for 40 min at 4°C. The beads were washed five times with lysis buffer. Bound proteins were eluted with 30μl NuPAGE LDS Sample Buffer (Invitrogen) at 70°C for 10 min and subjected to immunoblot analysis using anti-myc antibody . The membrane was then stripped and incubated with anti-TAP antibody 32P labelled using T4 PNK (USB Affymetrix) and hybridized in 5x Denhardt’s solution, 6xSSC, 10mM EDTA, 0.5% SDS, 0.1mg/ml salmon sperm DNA (Invitrogen) at respective temperatures for 16 hours. The blots were washed 3 times in 2xSSC 0.1% SDS and exposed to Phosphorimager screens (Fuji). Screens were scanned using Molecular Imager FX (BioRad). Band intensities were quantified using the Quantity One software (BioRad). Probe sequences are listed in 2.5ug of total RNA was electrophoresed in 10% polyacrylamide, 8M urea, 0.5x TBE gel and blotted onto Hybond Nx membrane  in 0.3x TBE using a semi dry blotter, followed by UV crosslinking. Oligonucleotide probes were http://www.r-project.org) using the Bioconductor (http://www.bioconductor.org) packages “affy”, “affxparser” and “preprocessCore” with standard parameters. CEL-files were imported and normalized as described in [All analysis was performed in R (ribed in . For visftp://ftp.sanger.ac.uk/pub/yeast/pombe/GFF/pombe_09052011.gff). For averaging of microarray scores across different genomic features, the “feature” column was used for all items with the following exceptions: features matching “LTRTF2”, “Tf2” (together with the feature category “protein_conding_gene”), “origin_of_replication” and “external_name = intron” were annotated as LTRs, Tf2s, replication origins and introns, respectively.To determine probe distributions for the subtelomeres, probes mapping to the following regions were used: 1–98950 bp and 5496300–5579133 bp on chromosome 1, 1–96400 bp and 4437300–4539804 bp on chromosome 2. For probe distributions across tRNA genes, probes were used which overlapped with sequence features marked “tRNA” in the GFF-annotation file downloaded from the Pombase website  were performed using a circular permutation as described in .Microarray data can be accessed at NCBI GEO under the accession number GSE58013.S1 Fig(A-C) Increase in enrichment of RNAP II, H2A.Z and H4K12Ac at subtelomeres was confirmed by qPCR. Data shown as fold difference in enrichment at SPBPB8B6.04c over actin, normalized to input. Error bars represent the standard deviation of duplicate experiments.(PDF)Click here for additional data file.S2 FigData shown as fold difference in enrichment over Dam-only control. Error bars represent the standard deviation of duplicate experiments.(PDF)Click here for additional data file.S3 Fig(B) Subtelomeres move towards the interior in fft3Δ bqt4Δ cells. Percentages of cells in each of the zones in different strains are shown, with number of cells measured in the legend. Significance testing was done using a two-sided Chi-square test.The nuclear envelope was stained with Nup61-myc-Cy3 (red) and the subtelomeric FISH probe was labeled with FITC-anti-DIG (green). Cells were scored as belonging to either of three equal volume zones depending on the distance between the nuclear envelope and the FISH signal. (PDF)Click here for additional data file.S4 Fig(A) Nup85 is significantly enriched at LTR elements. Nup85-Dam enrichment over Dam-only control is shown as boxplot. (B) Nup85 levels decrease at LTRs in fft3Δ cells. Data shown as fold difference in enrichment at LTR elements over Dam-only control. Error bars represent the standard deviation of duplicate experiments. (C) The subtelomeric border element (SBE) LTRs on the left arm of chromosome 2 loose their association with the nuclear envelope in fft3Δ cells. Data shown as fold difference in enrichment at SBE LTRs over Dam-only control. Error bars represent the standard deviation of duplicate experiments. (D) Insertion of an LTR into the ura4 gene is not sufficient to increase Fft3 interaction. Data from anti-myc ChIP qPCR of a strain with wild-type ura4 and a strain with an LTR inserted into the ura4 coding sequence (see schematic). Data is shown as enrichment over control locus (LTR close to SPBPB10D8.04c) with error bars representing the standard deviation of duplicate experiments.(PDF)Click here for additional data file.S5 Fig(A) the right subtelomere on chromosome I, (B) the left subtelomere on chromosome II and (C) the right subtelomere on chromosome II. Genes are shown in light grey, LTRs in dark grey. Asterisks mark positions were nucleosome occupancy is reduced. The left subtelomere border on chromosome II contains four identical copies of the gene SPBPB10D8.04c and an LTR, so the profiles in (B) are shown as an average over all four copies.Nucleosome occupancy at subtelomeric borders is affected by Fft3. Map shows nucleosome occupancy profiles (top panel) and Fft3 enrichment (blue) over the border of (PDF)Click here for additional data file.S6 Fig(A) The Fft3 ATPase mutant is recruited to the central core domain of centromere 1 (cnt1). Data from ChIP-qPCR of non-tagged wildtype, Fft3-myc and Fft3-K418R-myc is shown as fold difference in enrichment at Cnt1 over dg, normalized to input. Error bars represent the standard deviation of duplicate experiments. (B) The Fft3 ATPase mutant is recruited to valine tRNA genes. Data/error bars as in (A). (C) The Fft3 ATPase mutant is recruited to proline tRNA genes. Data/error bars as in (A). (D)Fft3-K418R cells show the same temperature sensitivity as fft3Δ cells. Cell suspensions were diluted into five different concentrations, spotted on YES plates and grown at the indicated temperatures for 3 days.(PDF)Click here for additional data file.S7 Fig(A) RNA Polymerase II levels are increased in fft3Δ and Fft3-K418R-myc. Data from ChIP-qPCR of non-tagged wild-type, fft3Δ and Fft3-K418R-myc is shown as percent of input. Error bars represent the standard deviation of duplicate experiments. (B) H3K9Ac levels are increased in fft3Δ and Fft3-K418R-myc. Data/error bars as in (A), except that data was normalized to H3 occupancy. (C) H2A.Z levels are increased in fft3Δ and Fft3-K418R-myc. Data/error bars as in (A). (D) Man1-interaction is reduced in Fft3-K418R cells compared to wild-type. Data from DamID qPCR is shown as Man1-enrichment over Dam-only control. Error bars represent the standard deviation of duplicate experiments.(PDF)Click here for additional data file.S8 Fig(A) Fft3 binds to various genomic elements, e.g. LTRs, tRNA genes and snRNA genes. Boxplots show averages of Fft3-myc enrichment over anti-myc control for each class. (B) Fft3 is enriched over 5S rRNA genes. Fft3-enrichment over wildtype anti-myc control is shown as boxplot. Fft3-myc is ChIP-chip data from [(C) Nup85 is enriched at 5S rRNA genes in wildtype S. pombe. Nup85-Dam enrichment over Dam-only control is shown as boxplot. Nup85-Dam data is from [(D) The INM protein Man1 is enriched at 5S rRNA genes in wildtype S. pombe. Man1-Dam enrichment over Dam-only control is shown as boxplot. (E) Fft3 is not required for 5S rRNA peripheral association. Man1-Dam enrichment over Dam-only control in fft3Δ cells is shown as boxplot. (F) Nucleosome occupancy at 5S rRNA genes is reduced in fft3Δ cells. The average number of reads mapping to each rRNA is shown for wild type and fft3Δ. P-value was obtained using paired, two-sided Mann-Whitney U test.ata from . (C) Nup is from . (D) The(PDF)Click here for additional data file.S9 FigEnrichment of the condensin subunit Cnd2 over anti-GFP control is shown as boxplot. Scores for probes mapping to 129 of the total 170 tRNA genes are shown in dark grey, scores for all other probes shown in light grey.(PDF)Click here for additional data file.S10 Fig(A) Levels of Proline tRNAs, but not Alanine tRNAs are increased in fft3Δ and fft3-K418R cells, compared to wildtype. Northern blots of total RNA were probed with 32P-labelled oligonucleotide probes, using snoRNA33 as a control for equal loading. (B) Quantification of the tRNA levels in (A). The log2-ratio of fft3Δ / wildtype is shown in dark grey, the log2-ratio of fft3-K418R-myc / fft3-myc is shown in light grey. Error bars represent the standard deviation of duplicate experiments. (C) Expression changes of Proline and Alanine tRNAs verified by qPCR. Total RNA was extracted and reverse transcribed into cDNA. The expression levels of two tRNA classes were measured with RT-qPCR. Error bars represent the standard deviation of duplicate experiments.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Here, we examined the patterning of G9a/GLP-dependent H3K9me2 in HSPCs and the consequences for chromatin structure.The formation of chromatin domains is an important step in lineage commitment. In human hematopoietic stem and progenitor cells (HSPCs), G9a/GLP-dependent H3K9me2 chromatin territories form We profiled chromatin accessibility across the genome of HSPCs treated with either a small molecule inhibitor of G9a/GLP or DMSO. We observed that chromatin accessibility is dramatically altered at the regions of H3K9me2 nucleation. We have characterized the regions of H3K9me2 nucleation, with our analysis revealing that H3K9me2 is nucleated in HSPCs at CpG islands (CGIs) and CGI-like sequences across the genome. Our analysis furthermore revealed a bias of H3K9me2 nucleation towards regions with low rates of C- > T deamination, which typically lack DNA methylation. Lastly, we examined the interaction of H3K9me2 and DNA methylation and determined that chromatin accessibility changes upon loss of H3K9me2 are dependent on the presence of DNA methylation.These results indicate that H3K9me2 nucleation is established at specific sequences that have base composition similar to CGIs. Our results furthermore indicate that H3K9me2 nucleation leads to local changes in chromatin accessibility and that H3K9me2 and DNA methylation work synergistically to regulate chromatin accessibility. During pression .de novo during lineage commitment [in vitro expansion and increased expression of lineage-affiliated genes and certain gene clusters, suggestive of changes in regulation of chromatin structure [We recently observed that in adult human hematopoietic stem and progenitor cells (HSPCs), H3K9me2 chromatin territories are absent in primitive cells and are formed mmitment . In commmmitment . A recenmmitment . We havemmitment . These rmmitment ,9. We futructure .+ HSPCs treated with UNC0638 or dimethyl sulfoxide (DMSO) control  have been hypothesized to have a more “open” chromatin structure that might help maintain a multipotent state by, for example, allowing transcriptional priming of lineage-affiliated genes -12. One +CD90+CD38loCD45RA– cells [+ HSPCs (composed mainly of committed progenitors), a nucleation stage ensues in which H3K9me2 marks appear at discrete loci across the genome [cis from sites of nucleation, to form characteristic patterns in mono-lineage cells such as CD41+CD61+ committed megakaryocytes or CD3+ T-cells [+ HSPCs revealed mutual exclusivity in signals for H3K9me2 and chromatin accessibility  in chromatin accessibility  in CD348 Figure D. To evaOur previous results indicated that a large percentage of the H3K9me2 nucleation sites (~50%) were at CpG islands , but it We next stratified CGIs based on whether they are promoter associated or “orphan” sites, as defined by Illingworth et al.  and anal+ HSPCs at CpG islands that do not necessarily meet thresholds based on sequence content to be considered CGIs. The initial characterization of CpG islands was based on the experimental observation that there is an enrichment of unmethylated CpG dinucleotides in the mouse genome [The GC and CpG content of H3K9me2 nucleation sites suggest that H3K9me2 is established in CD34e genome . Recentle genome . Examinide novo. To our knowledge, this is the first observation of coordination between H3K9me2 patterning, promoter and orphan CGIs, DNA methylation, and chromatin structure.In summary, we find that H3K9me2 patterning regulates chromatin structure at promoter and orphan CGIs and other sites of H3K9me2 nucleation, specifically promoting “closed” chromatin states. These results support the notion that G9a/GLP-H3K9me2 participates in global changes in chromatin structure in addition to histone patterning during HSC lineage formation. However, the biological significance of this patterning remains a question. One possibility is that H3K9me2 patterning helps reinforce chromatin states at sites of transcription during lineage specification, which may need to be reset in certain lineages and re-formed + cells from healthy adults were purchased from the Fred Hutchinson Cancer Research Center Cell Processing Shared Resource, as described previously [+ cells were treated with 2 μM of UNC0638 or 0.02% DMSO for 48 h, as described previously [Human CD34eviously . UnfractFAIRE was performed as previously described . Paired-P value threshold of 1 × 10-10. This analysis resulted in 43,159 peaks.Aligned bam files for H3K9me2 ChIP-seq data were obtained from . Wiggle + HSCs and CD34+ HSPCs  HOXA9 and (B) MLLT11 loci. Primer coordinates for qPCR are indicated by green bars. (C) Corresponding qPCR results. Relative values represent qPCR values normalized to GAPDH.Click here for fileFAIRE fold change for H3K9me2 nucleation sites as compared to the whole genome. The increase in chromatin accessibility as measured by the log of the fold change of the FAIRE signal for UNC0638 over DMSO for all H3K9me2 nucleation sites (H3K9me2) as compared to the log of the fold change for FAIRE (UNC0638/DMSO) for all 1 kb windows across the genome tiled in 50 bp increments (Background).Click here for fileThe FAIRE signals for UNC0638 and DMSO for three classes of CGIs: (green) those with low rates of C- > T deamination that are predicted to be mostly unmethylated, (red) those that display rapid G/C gain that are predicted to be constitutively methylated, and (blue) those under selection.Click here for file+ DMSO and (B) CD90+ cells.DNA methylation distributions for probes in (A) CD34Click here for file


CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.DOI:http://dx.doi.org/10.7554/eLife.06535.001 Cancers are often caused by mutations in genes that allow cells to proliferate uncontrollably. One gene that is frequently mutated in many cancers encodes a protein called MYC. The activity of this gene is normally tightly controlled, but the mutations found in human cancer cells mean that this gene is constantly switched on, and so too much MYC protein is produced.Previous studies have shown that a protein complex called ‘positive transcription elongation factor b’ (or P-TEFb for short) is essential to control the expression of the gene for MYC. P-TEFb works with an enzyme called RNA polymerase II to copy the instructions contained in protein-coding genes into long molecules called messenger RNAs. This process is called transcription and it involves a number of stages. P-TEFb is needed to start of one these stages, which is known as the ‘elongation’ step.P-TEFb consists of two protein subunits; one of which—an enzyme called CDK9—is the catalytic subunit. Most of the P-TEFb complexes in a cell are held in an inactive form, in which the activity of the CDK9 subunit is suppressed. If CDK9 is active when it should not be, certain proteins—including the MYC protein—can be produced in abnormally high amounts. This means that inhibiting CDK9 has been investigated as one way to reduce the production of the MYC protein. While some CDK9 inhibitors already exist, these compounds have the undesirable effect of also inhibiting other related enzymes and thus killing normal cells. Hence, new and more selective inhibitors of CDK9 are urgently needed.Lu, Xue et al. have now developed a new inhibitor of CDK9, called i-CDK9. The experiments show that i-CDK9 can potently inhibit CDK9 activity; and in human cells, very low levels of i-CDK9 prevented RNA polymerase II carrying out elongation for many genes. Unexpectedly, Lu, Xue et al. observed that more messenger RNA molecules that encode MYC were produced after cells were treated with low levels of i-CDK9. Further investigation revealed that this unexpected result occurred because the P-TEFb complexes were released from the inactive form and brought to the MYC gene by another protein called BRD4. This stimulated production of the MYC messenger RNAs. When P-TEFb was bound by BRD4, the CDK9 activity was also protected against inhibition by i-CDK9. Moreover, the reason that the MYC expression was induced by i-CDK9 is because the cells can compensate for the loss of CDK9 by using MYC to maintain the production of messenger RNAs of many key genes; these genes include the gene for MYC itself. These results suggest that CDK9 and MYC must be simultaneously inhibited in order to effectively treat cancers.DOI:http://dx.doi.org/10.7554/eLife.06535.002 Drosophila to mammals have shown that promoter-proximal pausing of Pol II is a prevalent feature of many genes and that the regulated release of Pol II is essential for synchrony and robustness of their induction  II is employed to execute a series of interconnected stages that collectively constitute the transcription cycle. In the past, the early stages of this cycle involving the recruitment of Pol II to gene promoters and assembly of active pre-initiation complexes were considered the primary step where transcription is controlled . Howevernduction .During transcription, the extensive and dynamic modifications of the Pol II C-terminal domain (CTD) have been linked to specific stages of the transcription cycle and mRNA processing. Among these, the CTD Serine-2 phosphorylation, which is a hallmark of productive elongation and RNA processing, is catalyzed by the positive transcription elongation factor b (P-TEFb), which is composed of CDK9 and its cyclin partner T1 (CycT1) or the minor forms T2a and T2b. Additionally, P-TEFb also phosphorylates the SPT5 subunit of DSIF and the NelfE subunit of NELF, which antagonizes the inhibitory actions of these two negative elongation factors and promotes the release of paused Pol II and transition into productive elongation .The importance of P-TEFb in transcriptional elongation requires that its activity be tightly controlled in the cell. Indeed, under normal growth conditions, the majority of P-TEFb is sequestered in the inactive 7SK snRNP, in which the CDK9 kinase activity is suppressed by HEXIM1 or 2 in a 7SK snRNA-dependent manner . The remA number of reagents and conditions that can globally impact growth and/or induce stress response have been shown to cause the release of P-TEFb from 7SK snRNP and formation of the BRD4-P-TEFb complex for stimulation of transcriptional elongation . In HIV-MYC occupies a central position downstream of many growth-promoting signal transduction pathways. As an immediate early response gene activated by many membrane-associated ligand–receptor complexes, MYC links growth factor stimulation to regulated cellular proliferation and cell cycle progression under normal conditions . i-CDK9 has a N2′-(trans-4-aminocyclohexyl)-5′-chloro-N6-(3-fluorobenzyl)-2,4′-bipyridine-2′,6-diamine scaffold that is structurally distinct from flavopiridol and all the other known non-selective CDK inhibitors . It occu50 value below the detection limit of 0.0004 µM -based kinase assay, i-CDK9 was shown to potently inhibit the CDK9-CycT1 catalytic activity with an IC.0004 µM . In the DK8-CycC . In contDK8-CycC .As there are no commercial or in-house kinase assays available for CDK3, CDK5, CDK11 and CDK13, the inhibitory abilities of i-CDK9 toward these CDKs were thus evaluated in the DiscoveRx KINOMEscan assay, which is based on a proprietary active site-directed competition-binding platform . At 1 µM50 of ∼2 nM under the current experimental conditions, no obvious inhibition of CDK12-F was detected even at 80 nM of the inhibitor . While Ser2 phosphorylation by CDK9-CycT1 was mostly inhibited by 80 nM i-CDK9, CDK12-CycK was not significantly inhibited until 640-1200 nM i-CDK9 was added into the reactions . Thus, bhttp://www.discoverx.com/ReferenceTreeImages/Flavopiridol.htm), some of which  had affinities that are similar or even higher than that for CDK9.Finally, the selectivity profile of i-CDK9 toward other non-CDK kinases was also assessed using the KINOMEscan platform. Among the >400 kinases evaluated, only 9  in the DSIF subunit SPT5 in a dose-dependent manner in HeLa cells . InhibitTo investigate the global impact of CDK9 inhibition on transcriptional elongation by Pol II, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was performed to examine the genome-wide occupancy of Pol II before and after HeLa cells were treated with i-CDK9. Similar to the situation reported in other cell types , among tTo investigate how the changes in TR value may correlate with changes in expression of the genes, we conducted DNA microarray studies to determine the effect of selective CDK9 inhibition on global gene transcription. A comparison between the two data sets indicates that TR values for those 138 genes whose expression was up-regulated at least twofold by i-CDK9 were significantly less affected compared to the rest of the genes . FurtherAmong the small group of genes that showed at least twofold increase in expression after 8 hr of i-CDK9 treatment, the proto-oncogene MYC has caught our special attention because of its extreme importance in cell growth control and oncogenic transformation and also the fact that its expression has been shown to depend on CDK9 . MicroarMYC amplification states (http://www.broadinstitute.org/ccle/home). Notably, the extent of the initial decrease and then subsequent induction of MYC mRNA production varied among the cell lines. For example, after 2 hr of i-CDK9 (0.5 µM) treatment, the MYC mRNA levels in U87MG (MYC copy number [CN] = 2) and HeLa (CN = 4) cells showed only a slight decrease compared to the DMSO control, whereas they were significantly reduced in A375 (CN = 2), NCIH441 (CN = 8), and A2058 (CN = 4) cells. Furthermore, the prolonged treatment for 8–16 hr caused the MYC mRNA to reach levels much higher than those in the control cells in all cell lines except NCIH441 . As the initial reduction in MYC mRNA level was not prominent in HeLa cells, we focused our attention on the major up-regulation of MYC expression detected upon 8–16 hr of treatment with i-CDK9.First, the induction of MYC protein level was confirmed in HeLa cells treated with i-CDK9. Upon exposure to just 0.1 µM i-CDK9, the MYC level increased 3.6-fold whereas the global pSer2 level was only decreased by 23% . It tookMYC induction.Notably, a very similar MYC induction pattern was also produced by the pan-CDK inhibitor flavopiridol. Just like i-CDK9, flavopiridol also significantly elevated the MYC protein level and this effect required a shorter exposure time  and lower drug concentration  than those needed to fully reduce the global pSer2 level . The neaMYC expression by i-CDK9 requires continuous protein synthesis, we treated cells with or without the protein synthesis inhibitor cycloheximide (CHX) prior to incubation with i-CDK9. Without the pre-treatment, i-CDK9 significantly increased the MYC protein level as expected  from a stably transfected, doxycycline-inducible expression construct driven by the CMV promoter  was performed with a specific shRNA (shBRD4), whose expression was induced by Cre recombinase . The losMYC expression, we tested whether the BET bromodomain inhibitor JQ1 could block the induction. JQ1 is known to competitively bind to the acetyl-lysine recognition pocket within BRD4's bromodomains, leading to the dissociation of the BRD4-P-TEFb complex from acetylated chromatin  of BRD4  kinase activity of the BRD4-bound P-TEFb was also required. This point is illustrated by the demonstration that the overexpressed kinase-inactive CDK9 mutant D167N, which binds to BRD4-like WT CDK9  and actsous CDK9 , preventMYC induction was further demonstrated by the observation that the initial MYC induction by a low level  of i-CDK9 was subsequently shut off by a higher i-CDK9 concentration  to phosphorylate a synthetic Pol II CTD peptide  was measured in the presence of either WT BRD4 or a BRD4 mutant lacking the C-terminal P-TEFb-interacting domain (∆PID). The BRD4 proteins were affinity-purified from transfected HEK293T cells under highly stringent conditions to strip away their associated factors . While W50 = 4.4 ± 0.7 nM). However, the addition of WT BRD4 into the reaction protected CDK9 against i-CDK9 and increased IC50 to 14.7 ± 2.1 nM. BRD4∆PID, on the other hand, displayed a much weaker effect by changing the IC50 value to 7.2 ± 0.6 nM  and H4 (Ac-H4) in whole cell extracts remained unchanged upon exposure to i-CDK9 , they weTaken together, the data presented thus far indicate that the i-CDK9-induced MYC expression is likely caused by a combination of events: (1) the induced transfer of P-TEFb from 7SK snRNP to BRD4; (2) BRD4's promotion of CDK9's kinase activity and resistance to inhibition; and (3) favorable chromatin changes at an early stage of the i-CDK9 treatment that lead to subsequent increase in the occupancy of the BRD4-P-TEFb complex at the MYC locus, promotion of Ser2 phosphorylation on the Pol II CTD, and finally productive transcriptional elongation.MYC is a classic example of the so-called PRGs, which are a set of genes that can be induced in response to both extracellular and intracellular signals without the requirement for de novo protein synthesis . Given tAnother interesting observation about the 23 BDPR genes that had detectable Pol II ChIP-seq signals in HeLa cells . In contHaving identified the mechanism of MYC induction by i-CDK9, we also wanted to determine the biological significance of this phenomenon. Given that MYC induction was triggered by inhibition of cellular CDK9 kinase, we hypothesized that MYC may normally facilitate CDK9's function and that the elevated MYC expression in i-CDK9-treated cells is therefore a cellular attempt to compensate for the loss of CDK9. To test this hypothesis, we first determined whether the shRNA-mediated MYC KD would affect the global pSer2 levels before and after the treatment with i-CDK9. Indeed, even though the KD was incomplete, it markedly reduced pSer2 levels in both untreated cells as well as in cells exposed to a range of i-CDK9 concentrations , revealiNot only did the KD of MYC expression negatively affect the ability of CDK9 to phosphorylate Ser2, the treatment of cells with either JQ1, the BET bromodomain inhibitor that suppressed the BRD4-dependent MYC expression , or 10,0The observation that MYC can bind and recruit P-TEFb to numerous MYC-target genes to activate transcription  provides50) of i-CDK9 and JQ1 as a single agent or in combination on both HeLa and H1792, a non-small cell lung cancer cell line with significant MYC amplification (CN = 8). Our data clearly show that the combination of i-CDK9 and JQ1 synergistically inhibited growth of both cell lines , raise the possibility that some of the effects attributed to this compound may not be caused by the inhibition of CDK9. Because simultaneous inhibition of both CDK9 and cell cycle CDKs makes the data interpretation difficult and confusing, a highly selective CDK9 inhibitor that can specifically block the P-TEFb-dependent elongation phase of Pol II transcription is thus very desirable and urgently needed. In this regard, the current development and characterization of i-CDK9, which demonstrated far superior selectivity against CDK9 than did flavopiridol, is both timely and valuable for our efforts to further investigate CDK9 biology as well as the therapeutic potential of P-TEFb inhibition.The aberrant activation of cyclin-dependent kinases (CDKs) and dysregulation of cell cycle progression is a hallmark of many human diseases that include cancer, cardiac myopathies, and inflammatory processes. Because of this, manipulation of cell cycle progression by means of small molecule inhibitors has long been suggested as a therapeutic avenue to cure these diseases especially cancer. There are now more than 50 pharmacological CDK inhibitors that have been described . Among tOne key finding from the present study is that long-term inhibition of P-TEFb by i-CDK9 potently up-regulated the expression of a number of PRGs that include MYC, FOS, JUNB, ERG1, and others. Notably, a subset of the same PRGs  has also been shown to be potently down-regulated before they were significantly up-regulated following the treatment with the pan-CDK inhibitor flavopiridol . As for In addition to serving as an epigenetic reader and recruitment agent to deliver active P-TEFb released from 7SK snRNP to acetylated chromatin at the MYC locus, BRD4 is also shown in the present study to use its C-terminal P-TEFb-interaction domain (PID) to directly increase the catalytic activity of CDK9 despite the fact that itself failed to display any kinase activity toward the Pol II CTD. This result is consistent with the observations reported recently by the Geyer laboratory, which employed both the synthetic CTD peptides as well as the full-length human CTD containing all 52 repeats in the kinase assays . Likely At this moment, the precise mechanism used by BRD4 PID to promote CDK9's kinase activity and protect against i-CDK9 is yet to be determined. It is interesting to note that the HIV-1 Tat protein appears to possess a similar ability to activate CDK9 upon binding to the isolated, recombinant P-TEFb . While TThe data presented in the present study support the model that the i-CDK9-induced MYC expression and interaction with P-TEFb is important to compensate for the loss of P-TEFb activity in treated cells. Given the fact that P-TEFb is a general transcription factor  and thatFor example, when investigating the MYC-induced global amplification of transcription in lymphocytes and ES cells, the Levens group noticed that the activation of genes that contain the E-box, which are traditionally defined as the MYC-target genes, makes up only a modest fraction of the total number of genes activated in these cells . ConsistWhile the inhibition of CDK9 leads to elevated MYC expression and binding to P-TEFb in order to mitigate the impact of loss of cellular CDK9 activity, previous reports have shown that treating cells with JQ1, which blocks the BRD4-dependent MYC expression, causes the release and activation of P-TEFb from 7SK snRNP . These oN2′-(trans-4-aminocyclohexyl)-5′-chloro-N6-(3-fluorobenzyl)-2,4′-bipyridine-2′,6-diamine, nicknamed i-CDK9 in the current study, was synthesized at Novartis Institutes for BioMedical Research .The antibodies used in immunoblots and cell-based assays were either purchased or raised as follows: pSer2 RNA Pol II , total RNA Pol II (F-12) , Mcl-1 , PARP , SPT5 , β-Actin , MYC , Europium-labeled anti-rabbit antibody . Polyclonal anti-phospho-SPT5 antibodies (pThr775) were raised by immunizing rabbits with a phosphorylated peptide (CGSQT(p)PMYGSGSRT(p)PMY) followed by negative affinity purification against the non-phosphorylated peptide described above, and subsequently by positive purification against the phosphorylated peptide.For ChIP experiments, antibodies against pSer2 Pol II , total Pol II , acetyl-histone H3 , acetyl-histone H4  were purchased commercially. Antibodies against CDK9 and BRD4 were generated in our own laboratory and have been described previously .All cell lines were obtained from American Type Culture Collection  and grown in the media recommended by ATCC.The inhibitory activity of i-CDK9 for CDK1-CycB (Upstate/Millipore), CDK2-CycA (Invitrogen), CDK4-CycD1 (Novartis), CDK7-CycH-MAT1 (Invitrogen), and CDK9-CycT1 (Invitrogen) was assessed in the AlphaScreen -based kinase assay developed at Novartis. Briefly, each CDK–cyclin pair was pre-incubated with i-CDK9 for 15 min, followed by addition of kinase substrates . The mixture was incubated at room temperature for additional 4–5 hr. The reaction was stopped and read on Envision . Selectivity against a large panel of over 400 kinases was evaluated using the KINOMEscan platform  employing a proprietary active site-directed competition binding assay . The ass6 cells were incubated overnight with 3 μg the indicated antibodies per reaction and followed by an 1-hr incubation with Dynabeads Protein A (Life Technologies). Immunoprecipitated DNA was purified by PCR Purification Kit (QIAGEN) and analyzed by qPCR. The sequences of the PCR primers used for amplification of the various MYC regions are listed below:A-Forward: AAGGCCTGGAGGCAGGAGTAATTTA-Reverse: AGTTTGCAGCTCAGCGTTCAAGTGB-Forward: ACGTTTGCGGGTTACATACAGTGCB-Reverse: GAGAGGAGTATTACTTCCGTGCCTC-Forward: GCGCGCCCATTAATACCCTTCTTTC-Reverse: ATAAATCATCGCAGGCGGAACAGCD-Forward: TACTCACAGGACAAGGATGCGGTTD-Reverse: TGAATTAACTACGCGCGCCTACCAE-Forward: TAGTAATTCCAGCGAGAGGCAGAGE-Reverse: TATGGGCAAAGTTTCGTGGATGCGF-Forward: ACTGGAACTTACAACACCCGAGCAF-Reverse: TGGACTTCGGTGCTTACCTGGTTTG-Forward: GGTTCACTAAGTGCGTCTCCGAGATAG-Reverse: AGCGGGAGGCAGTCTTGAGTTAAAH-Forward: TAAGGGTGGCTGGCTAGATTGGTTH-Reverse: AAATTAGCCTGGCATGGTGGTGTGI-Forward: ACAGAAATGTCCTGAGCAATCACCI-Reverse: GCCCAAAGTCCAATTTGAGGCAGTJ-Forward: TTCCTCTGTTGAAATGGGTCTGGGJ-Reverse: ACCTGCCTTCTGCCATTCCTTCTAK-Forward: TCCTGTCCATGGGTTATCTCGCAAK-Reverse: AACAGAATGGGTCCAGATTGCTGCThe assay was performed as described  with somAll qPCR signals were normalized to the input DNA, and signals generated by nonspecific IgG in control immunoprecipitations were subtracted from the signals obtained with the specific antibodies.All Co-immunoprecipitation experiments were performed in NEs prepared from F1C2 cells that stably express CDK9-F or the parental HeLa cells transfected with the indicated cDNAs. The procedures have been described previously .6) were washed twice with 1× phosphate buffered saline (PBS) and fixed in cold (−20°C) 70% ethanol overnight. To measure the sub-G1 population, cells were then stained with propidium iodide (50 μg/ml) for 1 hr at 37°C and analyzed by a Beckman Coulter Epics XL flow cytometer.After incubation with the indicated compound(s), HeLa cells  substrate. The final reaction mixture contained 2 nM CDK9/cycT1, 50 nM CDK7tide, 1 mM ATP, 2.5% DMSO, and various concentrations of i-CDK9 plus or minus BRD4 as indicated in the figures. The reactions were stopped by adding 60 mM EDTA plus 0.01% Tween 20. After the addition of AlphaScreen beads (PerkinElmer) and detection antibody  followed by overnight incubation at room temperature, the plates were read on an Envision 2101 (PerkinElmer).CDK9/CycT1 (Invitrogen) and purified, recombinant full-length or truncated BRD4 were pre-incubated for 45 min in a buffer containing 20 mM HEPES, pH 7.3, 10 mM MgClHeLa or H1792 cells were plated into 96-well microplates. i-CDK9 was serially diluted in the presence of seven different concentrations of JQ1. i-CDK9 or JQ1 was also serially diluted alone. The compounds were added to cells in 96-well dishes and incubated at 37°C for 24 hr for apoptosis assays or 72 hr for cell proliferation assays. Each treatment condition was done in triplicate. Apoptosis was detected by measuring Caspase 3/7 activation using Promega's Caspase-Glo 3/7 assay. Cell proliferation was measured using Promega's Celltiter-Glo assay. The concentrations of each drug in the combinations required to produce 50% inhibition were plotted to generate the isoboles. Isobologram analysis of drug interaction was performed essentially as described .http://bioconductor.org) with RMA method and HGU133Plus2_Hs_ENTREZG custom CDF for target gene definition , which was used to extract total RNA. RNA concentrations were measured on NanoDrop 2000 Spectrophotometer (Thermo Scientific) and RNA integrity determined by the Agilent RNA 6000 Nano Kit on Bioanalyzer (Agilent Technologies). 400 ng of total RNA from each sample that displayed good quality (RIN or RNA Integrity Number > 7) was submitted to the Novartis microarray core facility and analyzed on Affymetrix HGU133Plus2 microarray platform according to the manufacturer's instructions. Normalization of the raw signals was done using bioconductor affy package , and fragmented using Covaris-S2 sonicator (Covaris Inc.). Sonicated lysates were incubated with 5 μg Pol II antibody  overnight. After extensive washes, the isolated chromatin fraction was processed for sequencing library preparation according to the manufacture's instructions (Illumina Inc.). Sequencing was conducted by the Novartis sequencing core facility.Raw reads were aligned using Bowtie v2.1.0  and ChIPGSEA analysis was carried out as specified by Broad GSEA package using default parameters . We used review process). 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  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.After extensive consultation, the reviewers recommend that this paper be revised with new experiments, and the following is a summary prepared by the Reviewing Editor. It should be noted that the very positive Reviewer 1 was least knowledgeable about the literature and relevant issues and was convinced by the other reviewers of the weaknesses of the paper. These weaknesses include both experimental issues and also the relative lack of mechanism . As such, the reviewers are all in accord with this decision.The authors should address all the comments. In particular, experiments that should be done include:1) Specificity of the inhibitor against CDK12.2) A key point to address is a direct test of whether transcription induced by i-CDK9 is dependent on the catalytic activity of CDK9. This would require looking at higher concentrations of the inhibitor and to achieve induction first and then try to shut off with the higher dose.3) Analysis at earlier time-points. The question is whether there is anything different in the chromatin of a transiently repressed gene (MYC) versus a “permanently” repressed one. ChIPs of histone acetylation and BRD4 at 2 hr at minimum .4) The Keskin paper and the paper about DYRK1a mentioned in review  need to be cited. There is some literature on related effects of some genes being activated upon inhibition of CDK9Reviewer #1:eLife. The authors describe a selective inhibitor of CDK9, and the evidence for selectivity is excellent . While, as expected, CDK9 inhibition generally inhibits Pol II release from the pause, they show that MYC and some other genes behave in the opposite fashion. They convincingly show that CDK9 inhibition leads to release of CDK9 from the 7SK snRNA, increased association with BRD4 leading to enhanced CDK9 kinase activity and resistance to inhibition. In addition, they show that increased MYC expression compensates for reduced CDK9 function and that simultaneous inactivation of MYC and CDK9 synergistically induces growth arrest. Taken together, this paper describes a new and potentially therapeutically useful inhibitor of CDK9, as well as a novel mechanistic and biological connection between MYC and CDK9. The paper is very well written.This is a superb paper that should be published in Reviewer #2:The authors describe a new inhibitor of cyclin-dependent kinase 9 (CDK9), with improved potency and selectivity compared to drugs previously available. At the majority of genes, treatment of cells with this compound, i-CDK9, has the predicted effects on Pol II distribution-increased promoter-proximal pausing and decreased occupancy over gene bodies-and on steady-state transcript levels, which are decreased. At a subset of genes, however, CDK9 inhibition had little effect on pausing (measured as the “traveling ratio” or TR), and caused expression levels to increase. Among the genes that responded paradoxically was MYC, a key growth-promoting transcription factor. After transient repression by i-CDK9, levels of MYC transcription recover and then surpass levels in DMSO-treated cells. The authors show that i-CDK9 treatment causes 1) release of CDK9 from inhibitory 7SK RNA/HEXIM1 complexes; 2) increased association with the stimulatory/targeting partner BRD4; 3) enhanced occupancy of Pol II, BRD4 and CDK9 at the MYC gene; and 4) increased phosphorylation at Ser2 of the Pol II CTD on MYC. Paradoxically, increased expression of MYC induced by i-CDK9 appears to depend on CDK9 activity.eLife. My specific concerns are listed below:The experiments are mostly well executed and interpreted. CDK9/cyclin T1, a.k.a. P-TEFb, is a central regulator of gene expression and, possibly, a cancer drug target. A truly selective inhibitor would be of great interest to investigators in the field. The demonstration that MYC can evade repression by i-CDK9 is interesting, but not entirely novel; differential sensitivity of specific genes to inhibitors of “general” transcriptional functions has many precedents. This is MYC, so the mechanism of this evasion will be of significant interest to a wide audience, but the results presented here do not provide that mechanistic understanding. Why certain subpopulations of the released CDK9 would be resistant to such a potent inhibitor, or why certain loci might have an advantage in recruiting transcription machinery, including CDK9, under those conditions, is left unexplained. Extensive additional experiments would be needed to raise the work to the level at which I could recommend it for publication in 1) As stated above, no mechanism is provided to explain the most interesting effects of i-CDK9 on gene expression. I think the authors make a tactical error by choosing to focus nearly exclusively on the late (8-hr) time point when MYC transcription is maximally induced. The real clues to how MYC is being “re-activated” might be found by asking why the initial repression seen at 2 hr does not persist (in comparison with genes that are durably repressed by i-CDK9). Are there differences in the occupancy of transcription machinery at those genes? In chromatin modification patterns? For example, increased histone acetylation on MYC at later time points is not surprising, because transcription is elevated , but ana2) The ability of “transcriptional stress” (including treatments with drugs that target CDK9) to promote the release of CDK9 from 7SK/HEXIM1 is well-known. The authors' analysis of this phenomenon is more detailed than most, but does not illuminate the mechanism, i.e., how lowered CDK9 activity feeds back on CDK9-regulatory pathways. It also raises another question: for all the apparent superiority of i-CDK9 to older drugs , most of the results presented here could have (or have) been obtained with flavopiridol at similar doses. This extends to effects seen at cellular (PARP cleavage), genomic (increased TR) and biochemical levels (decreased Ser2 and Spt5 phosphorylation).3) A paper just published in Molecular Cell , proposes a novel function for DYRK1A-a secondary target of i-CDK9 with an IC50 ∼50 nM-as a Ser2- and Ser5-specific CTD kinase. I do not expect the authors to respond to this paper, but they should acknowledge that it exists and could explain some of the effects of i-CDK9 on Ser2 phosphorylation. It is quite likely, in my opinion, that no single kinase is responsible for any specific phosphorylation on Pol II in human cells, so whenever one sees disappearance of pSer2 (e.g. at i-CDK9 doses > 300 nM in 4) The ChIP data in Reviewer #3:The authors report a new chemical inhibitor of Cdk9, which is more potent and selective than flavopiridol, but has similar effects on the cell. They show that these compounds have effects on cells that are not seen upon knockdown of Cdk9, namely increased expression of MYC, FOS, JUN and other Brd4-sensitive genes. The findings suggest a positive feedforward loop in which cells respond to low levels of Cdk9 by increasing Brd4-binding and activation of P-TEFb.1) Throughout the paper, global RNA pol II Ser2 phosphorylation is used as a marker for Cdk9 kinase activity. However, it has been reported that Cdk9 knockdown does not affect the global level of Ser2 phosphorylation, and instead that Cdk12 (yeast Ctk1) is the CTD Ser2-kinase (2) The very related question is whether i-Cdk9 inhibits Cdk12? The authors show that i-Cdk9 has fewer off-target effects than flavopiridol, but this paper ignores whether or not i-Cdk9 affects Cdk12 kinase activity. Flavopiridol is a significant inhibitor of Cdk12, and if i-Cdk9 also blocks Cdk12, it would change the conclusions of the paper, and certainly invalidate using Ser2 phosphorylation as a marker for Cdk9 because Cdk12 is critical for cancer cell survival, the 'anti-cancer' effects of flavopiridol, and possibly also i-Cdk9, could have more to do with inhibition of Cdk12 than Cdk9.3) The CTD Ser2 phosphorylation increases in the body of the MYC gene upon treatment with i-Cdk9, consistent with increased transcription of the gene. However, eventually the MYC transcription and CTD Ser2-P should be inhibited by i-Cdk9, at extended times or with higher levels of the drug? To establish the correlation with Brd4, it needs to be shown that CTD Ser2 levels go down under the same conditions at non-Brd-dependent genes.eLife. Your revised article has been evaluated by James Manley (Senior editor), a Reviewing editor, and two reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Thank you for resubmitting your work entitled “Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism” for further consideration at Reviewer 1 strongly suggested a low dose-high dose experiment, but is willing to have the paper published provided if it is acknowledged in the text the drawbacks/pitfalls of both the knockdown and dominant-negative approaches, leaving open the possibility of other interpretations. If this change in the text is done appropriately, the paper will be accepted.Reviewer #1:This is a revised version of a manuscript I previously reviewed. It is improved, most significantly by the demonstration that changes in chromatin modifications and BRD4 recruitment/ retention precede transcriptional induction of MYC and other genes that respond paradoxically to the CDK9 inhibitor . The aut1) I know that the D167N mutant acts dominant-negatively, but that is the problem. Its ability to bind CDK9 partners, ATP and i-CDK9 means that overexpressing it is likely to change the response to the drug, without necessarily showing that CDK9 activity is required for MYC transcriptional induction .2) The same problem applies in reverse to the RNAi experiment: silencing CDK9 expression depletes the primary target of i-CDK9, before the drug is added. If induction of MYC expression by i-CDK9 is through binding to CDK9, it is hard to see how losing this effect when CDK9 is depleted says anything about a catalytic requirement for CDK9 in induced MYC transcription.3) Finally, the fact that high-dose i-CDK9 represses MYC when added at time 0 does not say that the same treatment would shut off transcription induced by the lower dose. The confusion is evident in the sentence “The dependence on catalytically active P-TEFb […] higher i-CDK9 concentration”, which uses the language of a time course  to describe a simple dose response (subsection “The kinase-active P-TEFb and its interaction with BRD4 are required for i-CDK9 to induce MYC expression”). I want to see the actual time course, in cells that are first treated with the lower dose to induce MYC expression and then raised to the higher dose to try to shut it off. From that result I would conclude that MYC transcription induced by i-CDK9 still depended on CDK9 activity. 1) Specificity of the inhibitor against CDK12.To determine how i-CDK9 may affect CDK12 kinase activity, we compared the abilities of CDK12 and CDK9 to phosphorylate GST-CTD in the presence of increasing amounts of i-CDK9. Phosphorylation of Pol II CTD on Ser2 was detected by Western blotting with the pSer2-specific antibody. Two different sources of materials were tested in separate experiments in order to ensure the consistency of the observations. First, Flag-tagged CDK12 (CDK12-F) and CDK9 (CDK9-F) affinity-purified from transfected HeLa cells were tested in kinase reactions shown in new It’s interesting to note that a similar finding was recently made by Dr. Geyer and colleagues, who showed that compared with CDK9, CDK12 was about one order of magnitude less sensitive to inhibition by the pan-CDK inhibitor flavopiridol .2) A key point to address is a direct test of whether transcription induced by i-CDK9 is dependent on the catalytic activity of CDK9. This would require looking at higher concentrations of the inhibitor and to achieve induction first and then try to shut off with the higher dose.The i-CDK9-induced MYC expression is indeed dependent on the catalytic activity of CDK9. This point has already been clearly demonstrated in 3) Analysis at earlier time-points. The question is whether there is anything different in the chromatin of a transiently repressed gene (MYC) versus a “permanently” repressed one. ChIPs of histone acetylation and BRD4 at 2 hr at minimum .Following the reviewers’ suggestion, we performed ChIP analyses of H3 and H4 acetylation and BRD4 binding at the promoters of MYC, a transiently repressed gene, and HEXIM1, an example of a “permanently” repressed gene see , at earlThus, at early time-points of i-CDK9 treatment before MYC transcription is induced, the acetylation state of the MYC promoter is already different from that of the HEXIM1 promoter. It is possible that the simultaneous increase in both Ac-H3 and Ac-H4 levels detected at the MYC promoter is a good prediction of eventual “re-activation” of the gene. In contrast, a strong reduction in Ac-H3 level that cannot be offset by a weak Ac-H4 increase as observed at the HEXIM1 promoter could be an indicator of a permanently repressed state caused by i-CDK9.4) The Keskin paper and the paper about DYRK1a mentioned in review  need to be cited. There is some literature on related effects of some genes being activated upon inhibition of CDK9We thank the reviewers for pointing out the two papers, which have now been discussed in the revised manuscript. For the Keskin et al. paper, we discussed (in the second paragraph of the Discussion section) that just like what we are showing with i-CDK9 in the present study, a subset of the same primary response genes that were examined in our For the DYRK1a paper by Di Vona et al., we discussed (in the last paragraph of the subsection headed “i-CDK9 is a potent and selective CDK9 inhibitor”) that despite a more than 100-fold greater selectivity of i-CDK9 for CDK9 than for DYRK1A , the facReviewer #2:1) As stated above, no mechanism is provided to explain the most interesting effects of i-CDK9 on gene expression. I think the authors make a tactical error by choosing to focus nearly exclusively on the late (8-hr) time point when MYC transcription is maximally induced. The real clues to how MYC is being “re-activated” might be found by asking why the initial repression seen at 2 hr does not persist (in comparison with genes that are durably repressed by i-CDK9). Are there differences in the occupancy of transcription machinery at those genes? In chromatin modification patterns? For example, increased histone acetylation on MYC at later time points is not surprising, because transcription is elevated , but analyzing such marks at 2 hr might be more informative as to the mechanism of activation.Please see our response above to common concern #3 shared by all reviewers.2) The ability of “transcriptional stress” (including treatments with drugs that target CDK9) to promote the release of CDK9 from 7SK/HEXIM1 is well-known. The authors' analysis of this phenomenon is more detailed than most, but does not illuminate the mechanism, i.e., how lowered CDK9 activity feeds back on CDK9-regulatory pathways. It also raises another question: for all the apparent superiority of i-CDK9 to older drugs , most of the results presented here could have (or have) been obtained with flavopiridol at similar doses. This extends to effects seen at cellular (PARP cleavage), genomic (increased TR) and biochemical levels (decreased Ser2 and Spt5 phosphorylation).We are not quite sure about what the reviewer wants us to improve here. With regard to the mechanism and signaling pathway that regulate the release of P-TEFb from 7SK snRNP in response to “transcriptional stress”, they indeed remain mostly unknown at this point, but are not the focus of the current study. There have been several reports on how other types of stress signals (e.g. UV and HMBA) cause the release of P-TEFb, and it is possible that similar mechanisms and signaling molecules may play a role in allowing the “transcriptional stress” to do the same thing.3) A paper just published in Molecular Cell , proposes a novel function for DYRK1A-a secondary target of i-CDK9 with an IC50 ∼50 nM-as a Ser2- and Ser5-specific CTD kinase. I do not expect the authors to respond to this paper, but they should acknowledge that it exists and could explain some of the effects of i-CDK9 on Ser2 phosphorylation. It is quite likely, in my opinion, that no single kinase is responsible for any specific phosphorylation on Pol II in human cells, so whenever one sees disappearance of pSer2 (e.g. at i-CDK9 doses > 300 nM in), it is likely to signify inhibition of multiple kinases. The flip side is that effects seen at lower doses are potentially due to incomplete inhibition of the intended target. This rather than the modest effects of BRD4-binding on CDK9 activity-the ∼2-fold stimulation of kinase  and ∼3-fold increase in IC50 -is probably why MYC expression can be induced by i-CDK9 even though it depends on CDK9 activity. I also disagree with the assertion  that MYC induction is happening “before” CDK9 inactivation, because it rests on a false assumption: that decay of phospho-Ser2 and -Spt5 signals provides an instantaneous measurement of kinase activity in vivo. That is certainly not the case; even if no other kinase can phosphorylate these sites, the loss of phosphates will depend on activity of the phosphatases that remove them. To clarify the situation, I would ask a simple question: can MYC expression induced by 100-500 nM i-CDK9 be shut off by subsequent addition of the same drug at a higher dose?The recent MC paper by Di Vona et al. has been discussed in the last paragraph of the subsection headed “i-CDK9 is a potent and selective CDK9 inhibitor” of the revised manuscript . Regarding the suggested experiment to examine MYC expression in the presence of high concentrations of i-CDK9, it has already been done in 4) The ChIP data inare consistent with the elevated MYC expression at the 8-hr time point. BRD4, CDK9 and Pol II are all increased, as expected. Why does the increase in CDK9 appear proportionately greater than that of BRD4, however, if the latter is needed to recruit the former? The authors also make the point that the pSer2 distribution is shifted towards the 3' end relative to that of CDK9, but this is consistent with many published results. What looks more noteworthy is the apparent shift of pSer2 towards the 5' end in the i-CDK9-treated cells relative to the DMSO-treated ones. To be sure that this is real, they would need to plot the pSer2:total Pol II ratio (which they should do in any case).Because CDK9 and BRD4 were recognized by their own specific antibodies in the ChIP assay, the somewhat bigger increase of the CDK9 signal compared to that of BRD4 may simply reflect the different dynamic ranges of recognition or sensitivities of the two antibodies. Based on the reviewer’s suggestion, we have plotted the pSer2/total Pol II ratios across the MYC locus under the DMSO and i-CDK9 conditions and the result is shown in Reviewer #3:1) Throughout the paper, global RNA pol II Ser2 phosphorylation is used as a marker for Cdk9 kinase activity. However, it has been reported that Cdk9 knockdown does not affect the global level of Ser2 phosphorylation, and instead that Cdk12 (yeast Ctk1) is the CTD Ser2-kinase . So the authors should show that the knockdown of Cdk9 causes a loss of Ser2 phosphorylation in their hands. Otherwise, the effect of i-Cdk9 on Ser2 phosphorylation could be due to inhibiting a different kinase.We thank the reviewer for giving us an opportunity to explain this perplexing controversy. Indeed, in our own hands, knockdown of both CDK9 and CycT1 (∼10% CDK9 and 50% CycT1 remained in KD cells) caused only a partial reduction of the global pSer2 level  Panel B,2) The very related question is whether i-Cdk9 inhibits Cdk12? The authors show that i-Cdk9 has fewer off-target effects than flavopiridol, but this paper ignores whether or not i-Cdk9 affects Cdk12 kinase activity. Flavopiridol is a significant inhibitor of Cdk12, and if i-Cdk9 also blocks Cdk12, it would change the conclusions of the paper, and certainly invalidate using Ser2 phosphorylation as a marker for Cdk9 because Cdk12 is critical for cancer cell survival, the 'anti-cancer' effects of flavopiridol, and possibly also i-Cdk9, could have more to do with inhibition of Cdk12 than Cdk9.Please see our response above to common concern #1 shared by all reviewers.3) The CTD Ser2 phosphorylation increases in the body of the MYC gene upon treatment with i-Cdk9, consistent with increased transcription of the gene. However, eventually the MYC transcription and CTD Ser2-P should be inhibited by i-Cdk9, at extended times or with higher levels of the drug? To establish the correlation with Brd4, it needs to be shown that CTD Ser2 levels go down under the same conditions at non-Brd-dependent genes.Please see our response above to common concern #2 shared by all reviewers. Indeed, as shown in Reviewer 1 strongly suggested a low dose-high dose experiment, but is willing to have the paper published provided if it is acknowledged in the text the drawbacks/pitfalls of both the knockdown and dominant-negative approaches, leaving open the possibility of other interpretations. If this change in the text is done appropriately, the paper will be accepted.Reviewer #1:This is a revised version of a manuscript I previously reviewed. It is improved, most significantly by the demonstration that changes in chromatin modifications and BRD4 recruitment/ retention precede transcriptional induction of MYC and other genes that respond paradoxically to the CDK9 inhibitor . The authors also demonstrate that CDK12 is inhibited only very weakly by i-CDK9. The remaining major concern is the decision not to do the experiment I suggested: first to induce MYC expression by low-dose (e.g. 300 nM) i-CDK9 treatment for 8 hr, and then try to shut it off with a higher dose (2 µM). The response to this suggestion, both in the revised manuscript (in the last paragraph of the subsection headed “The kinase-active P-TEFb and its interaction with BRD4 are required for i-CDK9 to induce MYC expression”) and in the rebuttal letter, is to restate results that were in the original version, which I understood but found unsatisfactory. Here's why:1) I know that the D167N mutant acts dominant-negatively, but that is the problem. Its ability to bind CDK9 partners, ATP and i-CDK9 means that overexpressing it is likely to change the response to the drug, without necessarily showing that CDK9 activity is required for MYC transcriptional induction .2) The same problem applies in reverse to the RNAi experiment: silencing CDK9 expression depletes the primary target of i-CDK9, before the drug is added. If induction of MYC expression by i-CDK9 is through binding to CDK9, it is hard to see how losing this effect when CDK9 is depleted says anything about a catalytic requirement for CDK9 in induced MYC transcription.3) Finally, the fact that high-dose i-CDK9 represses MYC when added at time 0 does not say that the same treatment would shut off transcription induced by the lower dose. The confusion is evident in the sentence “The dependence on catalytically active P-TEFb […] higher i-CDK9 concentration”, which uses the language of a time course  to describe a simple dose response (subsection “The kinase-active P-TEFb and its interaction with BRD4 are required for i-CDK9 to induce MYC expression”). I want to see the actual time course, in cells that are first treated with the lower dose to induce MYC expression and then raised to the higher dose to try to shut it off. From that result I would conclude that MYC transcription induced by i-CDK9 still depended on CDK9 activity.We have performed the so-called low dose (0.3 μM)-high dose (2 μM) experiment as suggested by Reviewer #1, and the result is the same as what was shown in


Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. Estrogen receptor (ER) α and β are ligand-dependent transcription factors ,2. ERs aImmediately after synthesis, ERα dimerizes and translocates primarily to the nucleus independent of E2 . E2 bindERS as other transcription factors are modular proteins ,2. The sWe therefore envision that the genetic conjugation of repression domains (RDs) from other transcription regulators into the EBM could generate potent monotransrepressors that alter the expression of endogenous ERE-bearing genes and cellular proliferation in opposite directions to those observed with E2-ERα or monotransactivators. To examine this prediction, we generated monotransrepressors containing the RD of the Krüppel associated box (KRAB) of KOX-1 protein  and/or oEBD and CDCEBD were described previously , TGFB2  and UAP1 (UDP-N-acteylglucosamine pyrophosphorylase 1) genes (data not shown). These findings suggest that the regulation of this subset of genes by SK, like E2-ERα and PV, requires ERE interactions and indicate that a transregulator with activator or repressor domains can mediate the expression of the same gene with polar directions within a chromatin context.To examine the effects of monotransregulators on gene expressions, we selected a subset of estrogen responsive genes regulated through ERE-dependent or ERE-independent pathways, which we previously determined using a microarray approach ,10. To aD) genes  as well r) genes  togetherTIFF1  RARA  or TGFA  expression is regulated by E2-ERα through the ERE-dependent signaling pathway  or HAS2  gene expression (data not shown). Interestingly, SK enhanced, the others repressed, the expression of MMP1, suggesting a distinct mechanism in gene regulation.We previously showed that E2-ERα and PV as well as the ERE-binding defective counterparts modulate the expression of the 2) genes , implyines [MMP1 , MMP3 (MCCND1 (Cyclin D1), JAK1 (Janus kinase 1) or IRS1 (insulin receptor substrate 1) gene, which was used as control (data not shown).Constructs had no effect on the transcription of the −9 M E2. Cells were then subjected to cell cycle analysis. Results revealed, as we showed previously [EBD at any concentration (shown at 500 MOI), increased the cell number accumulated in S and G2 phases with a corresponding decrease in G1 phase  or opposite direction (PDLM1) to that of E2-ERα, are consistent with the importance of receptor-specific features on the expression of these genes. On the other hand, all constructs modulated the expression of MMP1, MMP3 or SERPINB2 gene. Although implying a generality of action, the ability of SK to modulate the expression of, for example, MMP1 and that of E2-ERαEBD to regulate PDLM1, also suggest a distinct mode of transcriptional regulation.The underlying mechanisms of transcriptional modulation of estrogen responsive genes through the ERE-independent pathway are unclear. Studies suggest that functional E2-ERα interactions through ADs at both termini with transcription factors bound to their cognate regulatory elements are critical for the ability of the complex to modulate responsive gene expressions in a ligand-, promoter- and cell-context–dependent manner ,53. We p pathway . The inaRegardless of mechanisms, it is apparent that the regulation of cellular proliferation in opposite directions by SK with RDs and PV with ADs as bi-potential transcription factors is dependent upon estrogen responsive genes mediated by the ERE-dependent signaling pathway. Targeted gene expressions and/or genome modifications have critical importance in biological research and in clinical setting. Zinc Finger Proteins (ZFPs), Transcription Activator-Like Effectors (TALEs) and Clustered Regulatory Interspaced Short Palindromic Repeat/Cas9 based RNA-guided DNA endonucleases (CRISPR/Cas9) are the basis of powerful engineering technologies with a wide usage for targeted modulation of gene expressions as well as genome and epigenome manipulations –57. TheyS1 FigA) Synthesis of monotransregulators as a result of different concentrations of recombinant adenoviruses. Cells were infected with recombinant adenoviruses bearing none (Ad5) or a cDNA at indicated MOIs. In all infections, the total MOI was adjusted to 1500, which is the highest concentration of adenovirus used in the experimental series, by supplementing with Ad5. Extracts (10 μg) of infected cells at 48h were subjected to WB using the HRP-Flag M2 antibody. Molecular mases in kDa is indicated. NS denotes non-specific protein detection. (B) Cell extracts (10 μg) at 48h post-infection were also subjected to electrophoretic mobility shift assay (EMSA) without (-) or with (+) the Flag-M2 antibody (Flag-M2). ERE specifies the unbound and P-ERE denotes the protein-bound radiolabeled ERE. DNA indicates the radiolabeled ERE lane only.((TIF)Click here for additional data file.


Pseudomonas aeruginosa is an opportunistic pathogen that causes a multitude of infections. These infections can occur at almost any site in the body and are usually associated with a breach of the innate immune system. One of the prominent sites where P. aeruginosa causes chronic infections is within the lungs of cystic fibrosis patients. P. aeruginosa uses two-component systems that sense environmental changes to differentially express virulence factors that cause both acute and chronic infections. The P. aeruginosa AlgZR two component system is one of its global regulatory systems that affects the organism's fitness in a broad manner. This two-component system is absolutely required for two P. aeruginosa phenotypes: twitching motility and alginate production, indicating its importance in both chronic and acute infections. Additionally, global transcriptome analyses indicate that it regulates the expression of many different genes, including those associated with quorum sensing, type IV pili, type III secretion system, anaerobic metabolism, cyanide and rhamnolipid production. This review examines the complex AlgZR regulatory network, what is known about the structure and function of each protein, and how it relates to the organism's ability to cause infections. Pseudomonas aeruginosa is a ubiquitous, metabolically versatile, environmental organism with the ability to cause opportunistic infections in humans. Its >6 Mbp genome contains at least 5500 open reading frames (ORFs), encoding hundreds of virulence determinants  patients, where colonization by P. aeruginosa in the CF lung is linked to a worsening disease prognosis  sigma/anti-sigma factor system, encoded by the algU/TmucABCD operon (PA0762–0766)  envelope stress response system in Escherchia coli  Figure . The algP. aeruginosa, the MucA anti-sigma factor, encoded by the mucA gene, sequesters the stress-responsive AlgU/T sigma factor to the inner-membrane  gene  system and the 7,8-dihydro-8-oxo-deoxyguanine repair (GO) system are two levels of genomic repair that are commonly impaired during chronic infection. Mutations in these systems result in highly mutable strains , and at the genetic level from mutations in the chromosome. At the post-translational level, alginate biosynthesis is activated by the regulated proteolytic degradation of MucA. Several excellent reviews describing the details of post-translational regulation and biosynthesis of alginate overproduction are currently available  is the first ORF in an operon (PA3540–3551) comprised of 12 genes  encoding proteins involved in alginate biosynthesis, as well as its modification and export , encoding a phosphomannomutase, is located distally to the algD locus, yet is still part of the alginate pathway  is rather complex  AlgR (see below); (b) KinB/AlgB, a two component system where the AlgB (NtrC-family) response regulator positively regulates expression in a RpoN (σ54) dependent manner   is located −45 bp upstream of the transcriptional start site in the opposite orientation  than toward the algD-RB3 (dK = 3.7 × 10−7)  compared to its consensus sequence . AlgC is a phosphohexomutase enzyme that has dual phosphomannomutase/phosphoglucomutase activity  and algC-ABS3 (5′-CCGTGCGTC-3′) are higher affinity ABSs, algC-ABS2 (5 ′-CCGTTGTTC-3′) has two base pair changes as compared to the consensus sequence, and algC-ABS2 displays lower affinity to AlgR.The utilization of a cosmid library in conjunction with a chemically mutagenized strain library led to the discovery of D Figure . The algalgC-ABS1 acts like a eukaryotic enhancer element . Yet, there was a reduction to 0.5% activity when algC-ABS1 was deleted. Additionally, all three binding sites were required for maximal algC::lacZ expression  as compared to non-mucoid backgrounds      restores mucoidy , nitrogen-fixing, soil-borne organism, which has an alginate-encapsulated cyst as part of its lifecycle. Alginate serves to protect the cyst from desiccation, as well as to protect its nitrogenases from excess exogenous oxygen , which is required for catalyzing the phosphorylation of Asp54  an N-terminal CheY-like receiver (REC) domain  using the last 139 a.a. of AlgR (DNA-binding region) predicts that AlgR carboxyl terminus has at least 9 beta-pleated sheets, and demonstrates a 100% precision  with the AgrA C-terminus domain  regulates the agr (accessory gene regulator) locus in S. aureus. The agr locus encodes a set of regulatory RNAs (RNAIII) that control S. aureus virulence factors in a temporal, cell-density (quorum) dependent manner  and will give additional insight into the structure of this family of regulators.Though the crystal structure of AlgR has not been solved, secondary structure prediction based on the Phyre2 protein fold recognition server     and a CA domain   that AlgZ was considered to be part of this class of HKs. Part of the difficulty in categorizing AlgZ was due to the lack of sequence identity within the carboxyl-terminus. While the H box, having the sequence RPHFLFNSL , shows reasonable identity among this group of proteins, the N box, LQPLLENALIYG  only retains 5 of the 12 amino acids found in the canonical sequence. The obvious similarities between AlgZ and canonical histidine kinases are limited to these two boxes, as AlgZ lacks identifiable G1, F, and G2 boxes that make up the ATP-coordinating flexible lid. Even among its most similar orthologs , AlgZ is unique as LytS contains both G boxes, and YehU contains one of the two. It is possible that AlgZ may bind and coordinate ATP in an atypical manner, or bind a related molecule like GTP, as recently identified in the Bacillus anthracis histidine kinase (BA2291)  now suggest that the cytoplasmic domain of AlgZ (amino acids 151–358) contains the kinase, but it was only after the characterization of its orthologs  in the mucoid strain FRD1 does not affect alginate production  that incorporate into the type IVa pili, and a calcium-dependent retraction protein (PilY1). The pilY2 ORF is located in this operon but is proposed to be a pseudogene  activated fimU transcription, while algZ deletion abrogated fimU transcription , as well as fimU-ABS2 (5′-CCCTCGGGC-3′)  . As these genes are under the control of the Rhl quorum-sensing system, it was surmized that this regulation was through the rhlI gene, encoding the C4-homoserine lactone (HSL) autoinducer synthase. AlgR repressed rhlI transcription and bound the rhlI promoter in vitro directly at the rhlI-ABS (5′-CCGTTCATC-3′), located −28 bp upstream of the transcriptional start site , located −702 bp upstream of the rhlA transcriptional start site  was correlated with increased rhlA and rhlB transcription, where biofilm dispersion would occur  was unable to express rhamnolipids, while a phospho-mimetic isoform  was able to express rhamnolipids. In support of this, rhamnolipid-dependent swarming motility was affected by the algRD54N allele, and complemented with a strain expressing the rhlAB genes  are dependent on AlgR  and mucoid (PA17) strains, P. aeruginosa, type IVa pili mediated twitching motility, and the Rhl quorum sensing and rhamnolipid production in non-mucoid cells, AlgR likely has an even larger role in the organism's physiology. By comparing an algR deletion strain to its parental wild type PAO1 strain, it was demonstrated that AlgR is involved with regulating hundreds of genes in logarithmic, stationary, and biofilm modes of growth  regulate denitrification, and genes required for fermentation of arginine and pyruvate , is a ortholog of  Sawers, . Anr is arcA (PA5171), part of the arcDABC operon encoding anaerobic arginine deiminase enzymes, which catabolizes arginine to generate ATP through substrate level phosphorylation under anaerobic conditions , which encodes an oxygen-independent copropophyrinogen III oxidase important for heme biosynthesis under anaerobiosis , ccoO2 (PA1556), and ccoN2 (PA1557), ccb3-2 type cytochrome oxidase components expressed at low oxygen concentrations , which is an outer-membrane diffusion-driven specific transporter of hydrophobic molecules whose expression increases under anaerobic conditions in the presence of iron , a gene encoding hydrogen cyanide synthase responsible for the production of HCN, and a possible mediator of anaerobic growth   genes are the best studied. Hydrogen cyanide (HCN) is a volatile, highly reactive molecule that inhibits respiratory cytochromes and metalloenzymes. HCN is produced primarily at microaerobic conditions at high-cell density    is located −160 bp upstream of T3 . Interestingly, AlgR regulation of the hcnA gene is strain-dependent. Several P. aeruginosa strains contain chromosomal deletions of a region that would otherwise encode an AlgR binding site within the hcnA promoter region. Some strains  as well as several CF isolates have regions flanking exoY deleted (in the ORFs of PA2192 and PA2190)  is a needle-like structure that injects effector proteins  into host cells. Bacterial surface-contact, and low calcium concentrations in laboratory settings induce the expression of the T3SS in non-mucoid mucA) background  and expression of T3SS effector protein ExoS as readouts, P. aeruginosa carrying a mucA22 allele (conferring mucoidy) exhibits suppressed T3SS expression as compared to non-mucoid organisms. Meanwhile, a mucA22ΔalgR double-deletion restored both exsA and exoS expression to wild type (in strain PAK) levels, and plasmid-expression of algR reduced exsA and exoS expression, indicating that AlgR represses these T3SS genes  is a cAMP-binding transcriptional regulator  increased transcriptional activity from a Vfr-responsive promoter, while algZR expression from a plasmid decreased transcriptional activity from this promoter . RsmA (CsrA) proteins can also activate gene expression by altering mRNA secondary structure, mRNA stability, and/or ribosomal recruitment  . However, recent work showed that exsCEBA expression (encoding regulatory proteins for T3SS activation) was activated either directly or indirectly by free-RsmA. The authors propose that small changes in RsmA availability could explain the target gene's different responses to RsmA/RsmY/RsmZ  was more susceptible to hypochlorite treatment, but more resistant to both hydrogen peroxide and myeloperoxidase compared to its isogenic PAO1 parental strain. The algR mutant was more resistant to killing by murine macrophages and primary human neutrophils, indicating that AlgR may normally repress the genes that are necessary to survive exposure to primary host response cells  results in increased persistence (persistence in this study was defined by the ability to grow in 5 ug/mL of oflaxicin)  was tested in acute septicemia and acute pneumonia models of infection in C57BL/6j mice. Mice infected by intraperitoneal injection with PAO700 show increased survival as compared to mice infected with the wild type PAO1 strain. The same algR inactivated strain was cleared more quickly from murine lungs after acute infection. Co-infection with PAO700 and PAO1 resulted in decreased recovery of PAO700 as compared to wild type from the mice, suggesting an algR requirement for maximal virulence in these models. Interestingly, constitutive expression of algR decreased virulence as well suggesting that AlgR protein levels in P. aeruginosa need to be balanced for pathogenicity of the organism  to which AlgZ and ultimately AlgR respond? How is AlgR able to activate transcription regardless of its phosphorylation state? Current studies are underway to answer these questions.In all, AlgR appears to be a global regulator of n Figure . SeveralThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A mutant transcription factor that has been linked to congenital heart disease has wider effects than previously thought. Related research article Bouveret R, Waardenberg AJ, Schonrock N, Ramialison M, Doan T, de Jong D, Bondue A, Kaur G, Mohamed S, Fonoudi H, Chen CM, Wouters M, Bhattacharya S, Plachta N, Dunwoodie SL, Chapman G, Blanpain C, Harvey RP. 2015. NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets. eLife4:e06942. doi: 10.7554/eLife.06942Image Mutant forms of the NKX2-5 transcription factor are still able to influence how many genes are expressedCongenital heart disease affects at least 1 in every 100 people worldwide, making these developmental defects and their consequences a significant clinical burden . While tProteins called transcription factors bind to specific DNA sequences, such as promoters and enhancers, to control gene expression. Cardiogenic transcription factors, such as NKX2-5, TBX5 and GATA4, play critical roles in ensuring that the embryonic heart forms properly, and dominant mutations in the genes that encode these transcription factors have been implicated in the development of congenital heart defects. A commonly-held assumption is that disease-associated mutations in the DNA binding region of transcription factors act primarily to decrease the functionality of the protein, thus altering the expression levels of their target genes. Now, in eLife, researchers at the Victor Chang Cardiac Research Institute, the University of New South Wales, the Université Libre de Bruxelles and other institutions in Australia, Belgium, Iran and the UK – including Romaric Bouveret as corresponding author and Richard Harvey as last author – challenge this assumption for the cardiogenic transcription factor NKX2-5 .Drosophila where it was named tinman, as without it, the heart fails to form  and more broadly expressed co-factors  in the regulation of gene expression. Furthermore, it suggests that the specific control of cardiac regulatory genes is embedded within a broader and more ancient network that regulates ubiquitous cellular processes.