Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding  Macrophage activation is integral to innate immunity and inflammation, and involves transcriptome remodeling leading to the rapid upregulation of pro- and anti-inflammatory effector genes. Here the authors show that the negative elongation factor (NELF) complex controls the transcription of anti-inflammatory genes through Pol II pause release. The potentially harmful effects of inflammation necessitate the existence of precise regulatory mechanisms to control its magnitude and duration5. Indeed, activation of macrophages, an essential component of inflammatory responses, is subject to exquisitely tight control at multiple levels. For example, during activation, macrophage transcriptome undergoes extensive reprogramming with hundreds of genes rapidly upregulated and downregulated hundreds or even thousands of fold6, pointing at transcription as a critical node of regulatory circuitry in these cells. Macrophage activation is typically triggered by ligation of cell surface or endosomal receptors which initiates intracellular signaling, culminating in recruitment of sequence-specific transcription factors and RNA polymerase (Pol) II to the target gene loci7. Historically, Pol II recruitment and transcription initiation were considered the major rate-limiting steps for gene activation8. However, this view was later challenged by several studies reporting that in resting macrophages, transcription start sites (TSS) of many inflammatory genes such as Tnf are preloaded by Pol II10 raising the possibility that the rate-limiting steps to their activation occur post transcription initiation.Inflammation has evolved as a rapid response to environmental cues and contributes to a series of physiological and pathological processes including host defense, tissue damage, and metabolic alterationsDrosophila and stem cells have described Poll II promoter–proximal pausing, pause-release and entry into productive elongation as equally susceptible to regulation11. Specifically, after formation of the preinitiation complex (PIC), Pol II initiates transcription, synthesizes short (20–60 nt) nascent RNAs and then pauses. Further productive elongation requires signal-dependent pause-release to mobilize Pol II into the gene body regions. Given the importance of Pol II pausing, establishment of pause and its release are highly regulated by a plethora of positive and negative factors, including negative elongation factor (NELF), DRB sensitivity-inducing factor (DSIF), and positive transcription elongation factor-b (P-TEFb)13. In the canonical pause-release model derived from biochemical studies, the four-subunit NELF complex binds and retains Pol II within the promoter–proximal regions14. Pause-release is believed to be triggered by signal-induced phosphorylation of NELF by the heterodimeric P-TEFb complex composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1, which results in dismissal of NELF from promoters. In addition, P-TEFb phosphorylates DSIF converting it from pausing to elongation-promoting factor and serine 2 residues within the heptad repeats in Pol II C-terminal domain , which together is thought to facilitate Pol II entry into gene bodies and productive transcription elongation15.Indeed, numerous recent studies conducted mainly in 20. The contribution of post-initiation mechanisms to immune cell function has not been widely appreciated although several pioneering studies have provided strong evidence for the existence of this type of regulation especially in cells such as macrophages that respond rapidly to environmental cues23. Ligation of TLR4 followed by NF−kB recruitment leads to P-TEFb binding to numerous gene loci25. In fact, studies by us and others have shown how P-TEFb loading and transcription elongation are targeted by negative regulators of inflammation including the glucocorticoid receptor and other transcription repressors26, underscoring the physiological importance of immune gene regulation during early elongation. Nevertheless, these studies mainly focused on specific subsets of genes of interest, whereas the characteristics and a global impact of post-initiation control of transcription to macrophage activation remain to be thoroughly investigated.Post-initiation regulation of transcription is implicated in key biologic processes, including embryogenesis and developmentNelfb in macrophages, we identify a functionally and transcriptionally diverse group of NELF-regulated genes that display aberrant responses to inflammatory signaling, and define a pathway linking paused genes under direct transcriptional control of NELF to their downstream effectors in the immune system. Finally, we describe the consequences of macrophage-specific NELF depletion in vivo thereby establishing a physiological role of NELF in mammalian inflammatory response.Here, by employing genomic, pharmacological, and biochemical approaches, we comprehensively mapped the post-initiation transcriptional landscape during macrophage activation. We describe the surprisingly global and dynamic interactions of the “pausing factor” NELF with chromatin over the course of inflammatory activation of macrophages and the unexpected contribution of the lineage-determining transcription factor PU.1 to this process. Using genetic disruption of 27.To comprehensively define the global Pol II pausing patterns as related to signal-induced transcription in murine primary bone marrow-derived macrophages (BMDM), we performed Pol II chromatin immunoprecipitation followed by high throughput sequencing (chromatin immunoprecipitation (ChIP)-seq) and precision nuclear run-on sequencing (PRO-seq). Out of 10,076 unique genes expressed in BMDM as defined by RNA-seq (referred to as “BMDM transcriptome” hereafter), an overwhelming majority of genes displayed features of promoter–proximal pausing as computationally defined by high pausing index (PI) calculated based on Pol II ChIP-seq signals in the TSS regions versus gene body regions Fig. . Highly − E subunit of the NELF complex in resting BMDM by ChIP-seq. P-TEFb plays a critical role in promoting elongation in a signal-dependent manner28 and, consistently, showed little occupancy in resting BMDM  compared to only 8% of group 3  and group 3 (Il1b) along with their PI are shown in Fig. Widespread Pol II pausing in resting BMDM suggested that pause-release may mediate activation of macrophage transcriptome by inflammatory signals. Following 1 h exposure of BMDM to LPS, a TLR4 agonist, activation of group 1 and 2 genes—approximately two-thirds of LPS-responsive genes—displayed a dramatic increase in both Pol II loading and elongation of paused Pol II into gene bodies indicative of pause-release Fig. . In cont31. Measuring global CDK9 occupancy in LPS-activated BMDM by ChIP-seq , and reloaded to the TSS regions by 1 h approaching levels seen in resting BMDM  inhibitor—but not SB203580, an inhibitor of p38 MAPK less relevant to NF-κB activation—also prevented LPS-induced NELF chromatin dismissal   Fig. . Thus, aNelfb gene . Consistent with previous reports in other cell types33, deletion of Nelfb led to destabilization of the NELF complex and degradation of other subunits including NELF-E  were superinduced in Nelfb KO compared to WT controls pointing at a profound role of NELF in early inducible transcription  that promoted Il10 expression. Among NELF-repressed genes  were in fact AP-1 targets  were generated by crossing Nelfbfl/fl mice to Lyz2Cre mice. Myd88−/− mice were purchased from the Jackson Laboratory. Age and gender matched mice were used for experiments.The laboratory animal facility has been accredited by AAALAC  and the IACUC  of Tsinghua University approved all animal protocols used in this study. All mice were housed in isolated ventilated cages (maxima six mice per cage) barrier facility at Tsinghua University. The mice were maintained on a 12/12-h light/dark cycle, 22–26 °C, 40–70% humidity with sterile pellet food and water ad libitum. 49 and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 10% L929 cell supernatant as conditioned medium providing macrophage colony stimulating factor. Cell culture grade LPS (Escherichia coli 0111:B4) and Pam3CSK4 were purchased from InvivoGen and used at a concentration of 10 ng/ml. SP600125 and SB203580 were purchased from Selleck, Bay 11-7082 was obtained from Sigma-Aldrich and flavopiridol was purchased from Santa Curz Biotechnology. All inhibitors were added 0.5 h prior to LPS addition and were present throughout LPS exposure.Murine BMDM were obtained6 cells per condition were fixed in 1% methanol-free formaldehyde (Thermo Scientific) for 5 min at room temperature followed by quenching with 125 mM glycine for another 5 min. Nuclear extracts were prepared50 and chromatin DNA was sonicated to an average size of 300 bp using a Bioruptor (Diagenode). For immunoprecipitations, antibodies against Pol II , NELF-E , CDK9 , PU.1, c-Fos, and c-Jun were used. After purification, immunoprecipitated DNA was analyzed by RT-qPCR  as indicated in Figure legends. 10–25 × 10BMDM RNA was isolated using Qiagen RNA-easy kit. Total RNA was polyA enriched and Illumina-compatible sequencing libraries were prepared with TruSeq mRNA-Seq sample preparation kit (Illumina). Quality control of RNA and libraries was performed with the BioAnalyzer 2100. Pair-end sequencing was performed at the Weill Cornell Epigenomics Core using HiSeq2500.51. BMDM from WT and Nelfb KO mice were treated with or without LPS for 0.5 h, then BMDM nuclei (40–50 × 106 cells per condition) were isolated by swelling cells for 5 min in ice-cold douncing buffer  on ice, then gently homogenized and pelleted. The nuclei were washed twice in ice-cold douncing buffer and resuspended in storage buffer  at the density of 1 × 107/100 μl, and stored at −80 °C. Nuclear run-on experiments were carried out with 2-biotin labeled NTP. Briefly, preheat 2× nuclear run-on buffer  to 37 °C. Next, 10 × 106 nuclei in 100 μl of storage buffer were mixed with 100 μl of 2× nuclear run-on buffer and incubated at 37 °C for 3 min. Reaction was terminated by TRIzol addition, and RNA was isolated and fragmented by base-hydrolysis with 0.2 N NaOH on ice to an average size of 40–100 nt. Nascent RNA was isolated with magnetic streptavidin-coated beads . The 5′ ends of RNA products were repaired by incubating with RppH  and PNK , followed by another round of biotin-streptavidin affinity purification. RNA was prepared for constructing library with NEBNext Multiplex Small RNA Library Prep Set for Illumina . Briefly, RNA was ligated to 3′ DNA adapter by incubating for 18 h at 16 °C, hybridized with the reverse transcription primer, and ligated to 5′ RNA adapter by incubating at 25°Cfor 2 h then at 16 °C for 18 h. RNA was reverse transcribed and PCR-amplified (15 cycles). Quality control of RNA and libraries was performed with the BioAnalyzer 2100. Pair-end sequencing was performed with Hiseq X-ten.PRO-seq experiments were performed to detect de novo transcriptsTM. Antibody against NELF-B (ab167401) was from Abcam. All the other antibodies were obtained from Cell Signaling Technology. Relative density of blotting bands was quantified using Image J (v1.52a).Whole-cell lysates were prepared by direct lysis in sodium dodecyl sulfate (SDS) loading buffer. For immunoblotting analysis, lysates were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore) for probing with specific antibodies. Antibodies against p38 (sc-535), PU.1 (sc-352) were purchased from Santa Cruz Biotechnology. Antibody against NELF-E (10705-1-AP) was from Proteintech31. Briefly, cells were rinsed twice with ice-cold PBS, scraped into microcentrifuge tubes, and pelleted at 1500×g, 4 °C for 5 min. Cells were resuspended in 5× packed cell volume (PCV) of ice-cold buffer A  for 1 min and centrifuged at 250×g, 4 °C for 2 min. The cell pellets were resuspended in equal volume of Buffer A (with 0.5% NP-40) for 1 min to break down the cell membrane and nuclei pelleted at 2500×g, 4 °C for 2 min. The nuclei were extracted twice by incubating with 1× PCV of low-salt buffer  on ice for 10 min followed by centrifugation at 5000×g, 4 °C for 2 min. The low-salt extracted nuclei (containing chromatin-associated factors) were boiled with 5.6× PCV of 1× SDS-loading buffer to generate chromatin-associated fraction for immunoblotting. Antibodies for NELF-B , NELF-E , Pol II S2P , NF-κB p65  were used to detect chromatin-associated proteins. Actin levels as detected by an anti-actin antibody  served as loading control.Chromatin-associated fraction was prepared using stepwise fractionation protocolGapdh). Primer sequences are listed in the Supplementary Data RNA was extracted from whole cell lysates with a Total RNA Purification Kit (GeneMark) and reverse-transcribed to cDNA with Moloney Murine Leukemia Virus Reverse Transcriptase. qPCR was performed in triplicate with an ABI StepOnePlus thermal cycler. Threshold cycle numbers were normalized to triplicate samples amplified with primers specific for glyceraldehyde-3-phophate dehydrogenase , Mouse IL-6 ELISA Set , Mouse TNF (Mono/Mono) ELISA Set , and Mouse IL-12 (p40) ELISA Set  according to the manufacturers’ instructions.Secreted cytokines were measured after LPS treatment by ELISA. IL-10, IL-6, TNF and IL-12 (p40) were quantified by using the Mouse IL-10 ELISA MAX52. Nine hour post-injection, mice were sacrificed by carbon dioxide exposure, and peritoneal cavities were washed with 5 ml PBS. Numbers of total peritoneal cells were counted using a hemocytometer. For IL-6 rescue experiment, in addition to LPS administration, mice were intraperitoneally injected with 40 ng IL-6/mouse in 500 μl PBS. Nine hour post-injection, mice were sacrificed, and peritoneal cells were isolated and analyzed by FACS.Peritonitis was induced by intraperitoneal injection of 100 ng LPS/mouse in 500 μl PBS53. Briefly, peritoneal exudate cells were washed out with ice cold PBS. After washing once with PBS, peritoneal cells were resuspended in DMEM supplemented with 10% FBS and 1% P/S. Cells were then allowed to adhere for overnight in petri dishes at 37 °C. Non-adherent cells were removed by gently washing three times with warm PBS. The adherent cells were used as peritoneal macrophages.Resident peritoneal macrophages were prepared+CD11b+F4/80+; macrophages in bronchio-alveolar lavage fluid (BALF): CD45+CD11c+SiglecF+; monocytes: CD45+CD11b+Ly6C+; neutrophils: CD45+CD11b+Ly6G+.After removing red blood cells with Ack lysis buffer, cells were stained with the following fluorescence-conjugated antibodies for 30 min at 4 °C: CD45R-APC/Cy7 , CD11b-PE/Cy7 , F4/80-APC , Ly6G-PerCP/Cy5.5 , Ly6C-PE , CD11c-PE/Cy7 , and Siglec F-BV421. Cells were washed three times and analyzed on FACS Fortessa flow cytometer (BD Biosciences). Further analysis was implemented using Flowjo software (Treestar). Cell populations were defined as follows: macrophages in peritoneal cavities and spleen: CD45Cutadapt (V1.14), and the reads that failed to pass the quality control (Q > 10) were discarded. PU.1 ChIP-seq data set was downloaded from NCBI GEO DataSet under the GEO accessions: GSE38379 (GSM940924). SRA files were converted to fastq files using fastq-dump included in SRA toolkit. ChIP-seq and PRO-seq reads in fastq files were aligned to mouse genome (UCSC mm10) using Bowtie (version 1.1.2)54 to generate alignment files of uniquely mapped reads with maximum allowed mismatch of 2 (-m 1 -n 2) for each ChIP-seq data set, and PRO-seq reads aligned with short seed length (-l 10). ChIP-seq reads aligned to genome were extended to 150 bp from their 3′ end for further analysis. ChIP-seq and PRO-seq alignment files were visualized as bedgraph files with normalized reads count (per 10 million reads) at 1 bp resolution, which were generated by using HOMER (v4.7.2)38. Bedgraph files were loaded to IGV , and individual gene tracks were obtained as snapshots from IGV.Pol II ChIP-seq, CDK9 ChIP-seq, NELF-E ChIP-seq and PRO-seq data (the reverse reads from paired-end data) were collected. Adapter sequences were trimmed from the ends of reads by 55 with the parameters -i 70 -g 1–no-novel-indels–coverage-search, and only uniquely mapped reads were preserved.RNA-seq data were collected and pair-end reads were aligned to mouse genome mm10 using TopHat 2.1.0As PRO-seq reads are complementary to in vitro nuclear run-on products, the beginning of PRO-seq reads reflects the actual transcription-active site. Therefore, we shortened PRO-seq reads to 1 bp for quantification, and used the reads aligned to anti-sense strand as the transcription-active signals in sense strand for each gene. To determine ChIP-seq and PRO-seq signals around TSS, we first counted ChIP-seq extended reads (-fragLength 150) and PRO-seq shortened reads (-fragLength 1 -strand -) every 10 bp from TSS to both upstream 1 kb and downstream 2 kb regions for each gene by using annotatePeaks.pl program in HOMER. The output counting matrixes were next used to generate signal heat map around TSS regions by Cluster Treeview 1.1.6. The average signals around TSS regions were calculated as the average reads count per bin (10 bp) per gene.Nelfb KO/WT) ≥ 1.4 for up-regulated genes and fold changes (Nelfb KO/WT) ≤ 0.6 for down-regulated genes. Counting PRO-seq reads for each gene was implemented using annotatePeaks.pl program in HOMER.Upregulated and downregulated genes in PRO-seq were defined as normalized PRO-seq reads (RPKM + 1) fold changes .56. Genes with q-value < 0.05 and (FPKM + 1) fold changes ≥ 1.5 between WT resting and LPS treated (1 h) BMDM were regarded as LPS-inducible (n = 449). Upregulated and downregulated genes in Nelfb KO BMDM were identified as p value < 0.05, (FPKM + 1) fold changes (Nelfb KO/WT) ≥ 1.4 for upregulated genes and (FPKM + 1) fold changes (Nelfb KO/WT) ≤ 0.6 for downregulated genes. LPS-induced (n = 83) and LPS-suppressed (n = 161) genes at 0.5 h were identified with p value < 0.05 and (FPKM + 1) fold change (LPS 0.5 h/0 h) ≥ 1.4 for induced genes and fold change (LPS 0.5 h/0 h) ≤ 0.6 for suppressed genes. “Superinduced” and “more-suppressed” genes in Nelfb KO BMDM were defined as LPS-induced or LPS-suppressed (0.5 h) genes in the WT, which were further upregulated or downregulated in Nelfb KO BMDM.For coverage of mapped RNA-seq reads in transcripts, the expression level of each gene transcript was calculated as the average fragments count of three biological replicates, which was subsequently normalized as the fragments per kilobase of transcript per million mapped reads (FPKM). BMDM-expressed genes were defined as genes with FPKM ≥ 1 in resting BMDM. Both expression level (FPKM) and differential gene expression between experimental conditions was identified by using Cuffdiff program in Cufflinks 2.2.1Refseq gene annotation for mm10 was obtained from UCSC table browser, and genes from mitochondrial and random chromosomes were filtered out. 24030 total unique genes were defined as the total longest variant for each gene with unique gene symbol. To precisely calculate Pol II PI for genes with sufficient gene body length, we chose unique genes longer than 1 kb for further Pol II distribution pattern analysis in Pol II ChIP-seq replicate 1.+ TSS regions, and the other 1993 genes are defined as genes with Pol II− TSS regions.We defined TSS region for each gene as −250 bp to +250 bp relative to TSS, and the gene body was defined as the +250 bp from TSS to TTS . Using the findPeaks program in HOMER, we set 500 bp window to search Pol II enriched region in Pol II ChIP-seq replicate 1 data in resting BMDM, and obtained the threshold value of reads count for statistically Pol II enriched TSS regions (FDR < 0.001) as 38. Therefore, in 10076 BMDM-expressed genes (≥1 kb), 8083 genes with more than 38 reads in TSS regions are defined as genes with Pol II+ TSS regions, Pol II PI were calculated as the ratio of Pol II ChIP-seq reads density (reads per 1 kb) in TSS regions to gene body regions in resting and LPS activated BMDM. Genes with PI ≥ 3 were considered as highly paused and genes with 1.5 ≤ PI < 3 were defined as moderately paused. Genes with PI < 1.5 were defined as non-paused along with genes with Pol II− TSS region.For 8083 genes with Pol IISimilar to gene groups categorized by Pol II ChIP-seq defined PI, we used PRO-seq dataset to re-define pausing status in resting BMDM. PRO-seq defined PI were calculated as the ratio of shortened (1 bp) PRO-seq reads density (reads per 1 kb) in TSS regions to that in gene body regions. Using the same method and criteria as Pol II ChIP-seq defined pausing status, among 10076 BMDM-expressed genes (≥1 kb), 8809 genes with more than 13 shortened PRO-seq reads in TSS regions were defined as genes with significantly (FDR < 0.001) transcription-active TSS regions, whereas the other 1267 were defined as genes with transcription-inactive TSS regions. Among 8809 genes with transcription-active TSS regions, 7562 genes with PRO-seq PI ≥ 3 were considered as highly paused and 777 genes with 1.5 ≤ PRO-seq PI < 3 were defined as moderately paused. 470 genes with PRO-seq PI < 1.5 along with 1267 genes with transcription-inactive TSS regions were defined as non-paused.57 to assess differential NELF occupancy at TSS regions. BED files for TSS regions of all genes were used as peak region input, and sorted BAM files for two replicates of NELF ChIP-seq were used as input for reads counting. We used EdgeR mode in DiffBind to identify differential binding, and considered fold change (LPS 0.5 h/0 h) of NELF−E ChIP-seq reads count less than 0.5 and p value < 0.05 as threshold for significant NELF dismissal in TSS regions. (2) Peak calling in ChIP-seq is often utilized to identify differential occupancy. We used findPeaks program in HOMER to call NELF-E ChIP-seq peaks, with LPS 0.5 h condition as background and LPS 0 h condition as enrichment, to identify genes with TSS regions showing significant NELF dismissal upon LPS stimulation.We used two methods to classify NELF occupancy at TSS regions. (1) We used DiffBind (v2.12.0)To identify enriched known transcription regulatory elements in target TSS regions, we used findMotifsGenome.pl program in HOMER to find enriched motifs. Sequences from TSS regions of these genes were used as inputs and randomly extracted sequences from the genome were used as background.t test or other model where indicated. p < 0.05 was taken as statistically significant unless otherwise indicated. Statistical analyses were performed using GraphPad Prism 7 and R 3.3.0.Statistical analysis was performed by using two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data 1Reporting Summary


Oct4 and Sox2 promoter-interacting lncRNA as identified by Chromatin RNA In Situ reverse Transcription sequencing (CRIST-seq). Further understanding of their potential actions will provide a basis for the development of regenerative medicine for clinical application. This work offers comprehensive details and better understanding of the role of lncRNA in determining the fate of stem cells and paves the way for clinical stem cell applications.Pluripotent stem cells have broad applications in regenerative medicine and offer ideal models for understanding the biological process of embryonic development and specific diseases. Studies suggest that the self-renewal and multi-lineage differentiation of stem cells are regulated by a complex network consisting of transcription factors, chromatin regulators, signaling factors, and non-coding RNAs. It is of great interest to identify RNA regulatory factors that determine the fate of stem cells. Long non-coding RNA (lncRNA), a class of non-coding RNAs with more than 200 bp in length, has been shown to act as essential epigenetic regulators of stem cell pluripotency and specific lineage commitment. In this review, we focus on recent research progress related to the function and epigenetic mechanisms of lncRNA in determining the fate of stem cells, particularly pluripotency maintenance and lineage-specific differentiation. We discuss the role of the  Oct4, Nanog, Sox2, and Klf4. The collaboration and synergy of these transcription factors are crucial for maintaining stemness and pluripotency. The cell fate of PSCs is controlled or regulated by a complex process, involving transcription factors, signaling pathways, non-coding RNAs and their collaborative interaction. Hence, understanding the molecular mechanisms underlying determination of PSC fate will be of significance in manipulating their self-renewal and differentiation into specific cell lineages for potential application.Pluripotent stem cells (PSCs), including ESCs and iPSCs, have great potential in regenerative medicine. ESCs are derived from the ICM of a mammalian blastocyst and can differentiate into different cell types to form the endoderm, mesoderm, and ectoderm . On the Long non-coding RNA refers to a family of non-coding RNAs with more than 200 bp in length and is transcribed by RNA polymerase II, usually 5′ capped, spliced and polyadenylated . LncRNA Oct4, Sox2 and Nanog in iPSCs, indicating that they are crucial for maintenance of stem cell pluripotency. In a second study, more than 130 lncRNAs were examined by shRNA knockdown for their function in the of ESC pluripotency maintenance  is suppressed by p53 -contained remodeling complex to the Zbtb3 promoter and activates Zbtb3 expression to induce Nanog expression (lncRNA-ES1 (AK056826), lncRNA-ES2 (EF565083) and lncRNA-ES3 (BC026300) are differentially expressed in hESCs nuclei, and they can bind near the TSS of the OCT4 and NANOG promoters to enhance hESC pluripotency  and SOX2 in hESCs. SOX2 may induce H3K27 methylation during the neuroectoderm lineage commitment, leading to gene silencing at these loci. Furthermore, SOX2 binds lncRNAs to prevent the binding of other core pluripotency-associated transcription factors. LncRNA-ES3 silencing displays a phenotype similar to NANOG knockdown in hESCs  approach to compare promoter DNA binding and chromatin architecture between iPSCs and the “non-iPSCs” that expressed lentiviral reprograming-initiating factors, but failed to complete reprograming to pluripotency. It is interesting to note that the formation of a promoter-enhancer intrachromosomal loop architecture is a critical epigenetic barrier that must be overcome for the successful pluripotency induction . The nonSox2 and Oct4 promoters using this approach. Among the identified lncRNAs, Snhg14 was abundantly expressed in iPSCs and ESCs. Functional assays confirmed that Snhg14 was required to maintain stem cell pluripotency. Oplr16 (Oct4 promoter-interacting LncRNA 16) as another pluripotency-associated chromatin RNA factor. Oplr16 enhanced reprograming epigenetically by coordinating intrachromosomal looping and DNA methylation in the Oct4 promoter. Peblr20 (Pou5F1enhancer binding lncRNA 20) as another reprograming-associated lncRNA. Notably, Peblr20 binds to the Oct4 enhancers, where it recruits TET2 demethylase and activates the enhancer RNA (eRNA) pathway. Thus, Peblr20 functions as a critical pluripotent lncRNA that utilizes a novel trans epigenetic eRNA mechanism to control stem cell fate. Exploration of the genome-wide binding sites of central transcription factors and the lncRNA interactome during cell fate conversion may promote comprehensive understanding of the transcription network in reprograming.Long non-coding RNA consists of essential components of the three-dimensional genome structure. Several previous studies have identified lncRNAs that mediate the formation gene regulatory chromosome loops . FunctioPluripotent stem cells can differentiate and commit to a specific lineage in a given environment. The acquisition of cellular identities is a well-organized, precisely timed, and robustly executed process. The lack of effective differentiation and accurate specification of cell types hindered the widespread use of stem cells. Lineage differentiation of stem cells is regulated by a diverse and complex system that includes lncRNA. We will discuss the role of lncRNA in cell lineage commitment and focus on how lncRNA regulates PSC differentiation into adipocytes, osteogenic, muscle, cardiomyocytes, neurons, skin, and blood cells.Long non-coding RNA is involved in the adipogenic differentiation of stem cells. Understanding the mechanisms underlying adipogenesis regulation by lncRNA may reveal new therapeutic targets to combat obesity and its related diseases .SRA is the first lncRNA identified for functional adipogenesis promotion  is induced by adipogenesis and positively regulates both PPARγ and CEBPA expression by recruiting PA1 (a component of the MLL3/4 complex) to the CEBPA promoter and impacting H3K4me3 and H3K27me3 histone modification  and MIR31HG expression. BDNF-AS is transcribed from the BDNF antisense, while MIR31HG is from chromosome 9 and can be induced by the activated NF-kB. BDNF-AS can inhibit osteogenesis possibly via inverse regulation on BDNF and other osteogenic signaling  to their gene promoters to induce H3K4me, increasing Nanog, Sox2, and Fgf4 activity . Further luciferase and RIP assays demonstrate that lncRNA-1604 can interact and sponge miR-200c to suppress the expression of the core transcription factors ZEB1/2 and the subsequently neural differentiation  and POU3F3  is exclusively expressed in the heart and can promote cardiovascular lineage commitment of mESCs by activating functional upstream genes of the MesP1, a permissive regulator of multipotent cardiovascular progenitor . Therefore, CARMEN may initiate a cardiogenic differentiation program after damage, and it may be a potentially attractive therapeutic target for future regenerative and cell-based therapies  and vascular development related genes. PUNISHER deficiency downregulates histone H3 phosphorylation, and impairs human vessel maturation  locus and expressed in parallel with T during hESC cardiac commitment. Mechanically, yylncT worked as an essential activator of T by inhibiting DNMT3B activity to maintain the hypomethylation at the T/yylncT locus (Recently, cT locus . CollectANCR deficiency reduces keratinocyte differentiation marker expression, including transcription factors that promote differentiation. Mechanistically, ANCR can recruit the PRC2 complex to the epidermal differentiation related MAF and MAFB promoters although ANCR can also promote osteoblast differentiation by up-regulating Runx2 expression (TINCR is one of the most highly induced lncRNAs in the cytoplasm of keratinocyte progenitors during keratinocyte differentiation. TINCR deficiency decreases differentiation-related gene expression and affects the formation of lipid barrier and keratohyalin granules in keratinocytes. PMA and RIA-seq demonstrate that TINCR can bind to mRNA decay protein STAU1, and interact with and stabilize MAF and MAFB mRNAs, as well as CALML5 protein to promote keratinocyte differentiation (HULC can promote ADSCs differentiation into epithelial and smooth-muscle-like cells. Its overexpression increases the expression of Uroplakin-II, AE1/AE3, α-SMA, SM-MHC, Calponin, and SM-22α. Mechanistically, HULC positively regulates BMP9 expression and the Wnt/β-catenin activation, but it inactivates the Notch signaling during ADSC differentiation (Anti-differentiation ncRNA is preferably expressed in progenitor keratinocytes, but significantly down-regulated in terminally differentiated cells . ANCR depression . Transcrntiation . HULC cantiation .EGO is crucial for an eosinophil development (EGO silencing impairs the expression of basic protein and neurotoxin that are essential for eosinophil development, suggesting that EGO can positively regulate eosinophil lineage differentiation (HOTAIRM1 is a human HoxA cluster transcribed antisense lncRNA that is highly expressed in myeloid progenitor cells upon granulocytic differentiation. HOTAIRM1 silencing inhibits HoxA1, HoxA4, CD11b and CD18 expression, impairing myeloid cell differentiation (HoxBlinc is a specific marker for Flk1 + mesoderm that can promote hematopoietic differentiation. Mesoderm-derived Flk1 + cells can be induced to be cardiogenic and hemangiogenic progenitors. HoxBlinc knockout downregulates the expression of HoxB gene and other cardiac/hematopoietic differentiation related genes and the components of the Notch signaling. In mechanism, HoxBlinc can bind to the HoxB genes and activated HoxB gene expression through recruiting the Setd1a/MLL1 complex and mediating long-range chromatin interactions. HoxBlinc can also regulate the Wnt/Notch signaling and Hox pathways (Hematopoietic stem cells can differentiate into different types of blood cells. Besides transcription factors and miRNAs, recent studies reveal that lncRNA participates in hematopoiesis, particularly in the myeloid lineage development. The lncRNA elopment . EGO silntiation . LncRNA ntiation . LncRNA pathways .MEG3 is a positive regulator of both osteogenesis (Oct4 and Sox2 promoters and affect reprograming through epigenetic pathways, including coordination of intrachromosomal loops, alteration of the methylation levels, and activation of the eRNA pathway for stemness genes (As a major part of mammal genome, lncRNAs play essential roles in determining stem cell fates via different mechanisms. Some lncRNAs may be involved in different biological processes. For example, lncROR may maintain stem cell pluripotency  and promogenesis  and neurogenesis , while iogenesis . LncRNA ogenesis . They foss genes . It is iThe current understanding of lncRNA is more comprehensive due to improved technologies for lncRNA functionality, structure and interacting partners. LncRNA is important player in stem cell pluripotency maintenance, differentiation and their dysfunction-related human diseases through different mechanisms. The recently discovered lncRNAs that maintain stem cell pluripotency and determine their lineage differentiation are summarized in JC, YW, and CW collected the data. JC wrote the manuscript. J-FH and WL provided guidelines and edited the manuscript. All authors read and approved the final manuscript.The 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.


Epstein-Barr virus (EBV) infection is associated with a variety of malignancies including Burkitt’s lymphoma (BL), Hodgkin’s disease, T cell lymphoma, nasopharyngeal carcinoma (NPC), and ∼10% of cases of gastric cancer (EBVaGC). Disruption of epigenetic regulation in the expression of tumor suppressor genes or oncogenes has been considered as one of the important mechanisms for carcinogenesis. Global hypermethylation is a distinct feature in NPC and EBVaGC, whereas global reduction of H3K27me3 is more prevalent in EBVaGC and EBV-transformed lymphoblastoid cells. In BL, EBV may even usurp the host factors to epigenetically regulate its own viral gene expression to restrict latency and lytic switch, resulting in evasion of immunosurveillance. Furthermore, in BL and EBVaGC, the interaction between the EBV episome and the host genome is evident with respectively unique epigenetic features. While the interaction is associated with suppression of gene expression in BL, the corresponding activity in EBVaGC is linked to activation of gene expression. As EBV establishes a unique latency program in these cancer types, it is possible that EBV utilizes different latency proteins to hijack the epigenetic modulators in the host cells for pathogenesis. Since epigenetic regulation of gene expression is reversible, understanding the precise mechanisms about how EBV dysregulates the epigenetic mechanisms enables us to identify the potential targets for epigenetic therapies. This review summarizes the currently available epigenetic profiles of several well-studied EBV-associated cancers and the relevant distinct mechanisms leading to aberrant epigenetic signatures due to EBV. A wide  latency , 3. ThisThe EBV latency proteins include six EBV nuclear antigens , and three latent membrane proteins  . StudiesBRCA1/2, p14, p16, and hMLH  to the cytosine nucleotide, becoming 5-methylcytosine (5mC). This process is catalyzed by a family of DNA methyltransferase (DNMT) enzymes , utilizing S-adenosylmethionine (SAM) as the substrate for the methylation  and suppresses its expression , resulting in down-regulation of AQP3 , EBVaGC is characterized by a higher prevalence of DNA methylation than for other GC cancers. This is commonly known as the EBV-CpG island methylator phenotype (EBV-CIMP) . When EBRASSFI , CDKN2A   and LMP promoters (LMPp) to maintain latency I in BL cells . The samAberrant histone modification is another epigenetic feature associated with cancer development , 78. Thede novo methylated loci identified from NPC remarkably overlap with the bivalent marks derived from human ESCs , but loss of these marks makes them susceptible to DNA methylation  study showed that the EBV genome, in particular the oriP region, preferentially associated with human chromosomes 2, 3, 4, 6, 7, and 13, and these EBV-interacting regions usually exhibited higher A-T content, reduced H3K9me3, as well as higher H3K27ac or H3K4me1 signals   and H4 , increased H3K27me, and DNA methylation (BIM promoter region (BIM may be due to the inhibition of phosphorylation of Pol II by the presence of EBNA3A/C (BIM promoters (CDKN2A), was highlighted by Lenka and colleagues as well (CDKN2A promoter was previously reported in LCLs (The global level of H3K27me3 in LCLs is different from NPC, but similar to EBVaGC, showing significant reduction . The gloectively . Howeverectively . By analectively . Some onectively . In factectively . The epihylation . Subsequhylation . This str region . The dowEBNA3A/C . Also, Eromoters  (Figureromoters , 114. Th as well . They ob as well . As DNA  in LCLs , the met in LCLs . Althoug in LCLs .via EBNA1 binding results in repressive of gene expression and is associated with H3K9me3 (The EBV genome was further shown to interact with the host genome enriched with EBNA1, EBF1, and RBP-jK binding sites . Another H3K9me3 , 117. FTo summarize, it is evident that EBV infection results in hypermethylation in NPC and EBVaGC, driven by LMP1 and LMP2A, respectively, and is associated with down-regulation of TSGs. Although promoter methylation of a few TGSs in EBV-infected B cells is observable, it is unlikely that EBV contributes to global hypermethylation in B cells. With regard to the histone modification status, while H3K27me3 is globally prevalent in NPC, a critical elevation level of H3K27me3 is not obvious in EBVaGC and EBV-infected B cells. The distinct methylation and histone modification status in the relevant cancer types is summarized in MML: conceptualized and wrote the manuscript. MLL: guidelines and proofreading. All authors contributed to the article and approved the submitted version.The 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.


Therefore, Synechocystis has a much simpler carotenoid pool than Arabidopsis for identifying the functions of exogenous enzymes  driven by the Ampr promoter (Ampr::Kanr) from pGBK‐T7 as a selection marker. Carotenoid metabolic enzyme genes driven by the promoter of RbcL from Synechocystis to ensure a high‐level expression were cloned downstream of Ampr::Kanr. When two enzyme genes were to be expressed together, a ribosome‐binding sequence  was used to separate their open reading frames, so that the RbcL promoter could drive their expression simultaneously  was introduced, the transgenic ZEP+ strain accumulated violaxanthin instead of zeaxanthin. This demonstrated that Synechocystis is suitable to support the function of ZEP.s Figure c. We fure Figure d. This fet al., Solanum lycopersicum (SlNSY) together with AtZEP. No neoxanthin was found in transgenic NSY+ cells. This is most probably because the function of NSY in higher plants needs two additional proteins, ABA4 and NXD1, which we could not find homologs from the Synechocystis genome. However, compared with ZEP+, the NSY+ cells had a lower level of violaxanthin and a higher level of β‐carotene, supporting a primitive function of NSY in catalysing the β‐cyclization of lycopene, as previously reported  gene from Arabidopsis (AtLut2) and observed the production of α‐carotene in the corresponding Lut2+ strain. This confirms that AtLUT2 collaborates with endogenous CruA to catalyse the ε‐ and β‐cyclizations, respectively, of the two open ends of lycopene. We further incorporated the P450‐type carotene ε‐hydroxylase gene from Arabidopsis (AtLut1), together with AtLut2, into the cassette. The transgenic LUT1/2+ cells accumulated a significant level of lutein, indicating a collaboration of endogenous carotene β‐hydroxylase (CrtR) and exogenous AtLUT1 to hydroxylate both ends of α‐carotene. This indicated a successful construction of the β,ε‐ branch of carotenoid metabolism in Synechocystis and demonstrated the feasibility of utilizing this cyanobacterial platform to identify enzymes that catalyse the metabolism of lutein. It is interesting that, compared with the LUT2 strain, the LUT1/2+ cells accumulated much lower amounts of β‐carotene and zeaxanthin. This revealed that the expression of additional carotene ε‐hydroxylase resulted in a decreased metabolic flux towards the biosynthesis of carotenoids with two β‐rings in their structures.Carotenoids with ε‐rings are rarely synthesized in cyanobacteria. To test whether Haematococcus pluvialis (HpBKT) and the bacterium Paracoccus haeundaensis (PhCrtW), both of which produce astaxanthin, and expressed each of these genes in Synechocystis. Instead of astaxanthin, canthaxanthin and its isomers were accumulated in transgenic cells of both strains. Echinenone was also found in PhCrtW‐transformed cell. This showed that both HpBKT and PhCrtW preferentially use β‐carotene as their substrate, and the endogenous CrtR is unable to hydroxylate canthaxanthin to produce astaxanthin, as it was recently demonstrated in rice endosperm  with an additional CrtZ‐type carotene 3,3′‐hydroxylase that uses canthaxanthin as a substrate  cyclizes both ends (Cunningham and Gantt, Synechocystis as a new platform for the functional characterization of enzymes involved in carotenoid metabolism and for the screening of unknown factors that interfere with the activities of enzymes such as SlNSY and LsLCYE. Although it is difficult to compare the carotenoid production in Synechocystis with that in either E. coli or Arabidopsis, Synechocystis has the advantage of being able to grow both autotrophically and heterotrophically. This means that silencing of its genes for carotenoid biosynthesis is not as crucial as in higher plants for the growth, acclimation, and propagation, and its carotenoid productivity is also released from the restriction of the photosynthetic capability (Fiore et al., Synechocystis platform can also be improved as a candidate chassis for the biosynthesis of different carotenoid compounds.Taken together, we demonstrated the feasibility of engineering TJC, LEY and SL designed the experiment. TJC, LJW, XQH and YYD performed the experiments. LEY and SL wrote the manuscript.


Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) β2 is known to mediate the negative regulation of the prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene suggest that there is a tethering mechanism, whereby liganded TRβ2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSHβ gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TRβ2 and GATA2 in cultured neuronal cells. Here, we confirmed the expression of the GATA2 protein in the TRH neuron of the rat PVN using immunohistochemistry with an anti-GATA2 antibody. According to an experimental study from transgenic mice, a region of the rat prepro-TRH promoter from nt. -547 to nt. +84 was able to mediate its expression in the PVN. We constructed a chloramphenicol acetyltransferase (CAT) reporter gene containing this promoter sequence (rTRH(547)-CAT) and showed that GATA2 activated the promoter in monkey kidney-derived CV1 cells. Deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. Unexpectedly, T3-dependent negative regulation was maintained after mutation of the reported negative T3-responsive element, site 4. T3 also inhibited the GATA2-dependent transcription enhanced by cAMP agonist, 8-bromo-cAMP. A rat thyroid medullary carcinoma cell line, CA77, is known to express the preproTRH mRNA. Using a chromatin immunoprecipitation assay with this cell line where GATA2 expression plasmid was transfected, we observed the recognition of the GATA-RE by GATA2. We also confirmed GATA2 binding using gel shift assay with the probe for the GATA-RE. In CA77 cells, the activity of rTRH(547)-CAT was potentiated by overexpression of GATA2, and it was inhibited in a T3-dependent manner. These results suggest that GATA2 transactivates the rat prepro-TRH gene and that liganded TRβ2 interferes with this activation via a tethering mechanism as in the case of the TSHβ gene. Prepro-TRH expression is negatively regulated by the liganded T3-receptor (TR) at the transcriptional level  co-transfected with TRs in addition to GATA2 and Pit1 [26], two transcription factors that determine thyrotroph differentiation and activate the TSH� promoter. We modified the following sentence at the lines 151-154 in the 1st submission as follows. In solution and on the GATA-responsive elements (GATA-REs) of the TSHß promoter DNA, we detected the protein-protein interaction of TRß2 with GATA2 [29]. We deleted the sentences at the lines 158-160 and 447-452 in the 1st submission. With regard to the molecular characteristics of Sim1 and Arnt2---We added a paragraph at the line 416 in the 1st submission Sim1 and Arnt2 are two basic helix-loop-helix/PAS (Per-Arnt-Sim) transcription factors, which form heterodimers on the central nervous system midline enhancer (CME) to control the differentiation of neuroendocrine lineages in the hypothalamus [30]. Interestingly, SIM1-null mice show hypocellularity in PVN and supraoptic nucleus (SON) and absence of the expression of TRH, corticotropin-releasing hormone, oxytocin, vasopressin and somatostatin in these two nuclei and the anterior periventricular nucleus. Using Tet-On system in Neuro-2a cells, where Sim1 of which C-terminal domain was replaced with VP16 activation domain can be induced together with Arnt2, Liu et al. identified 268 potential target genes for Sim1/Arnt2 heterodimer [31]. As mentioned above, the GATA2 gene is included in addition to TRß2 gene. According to Liu et al., there is a putative CME in the TRß2 gene but not in the mouse preproTRH promoter [31]. Although the presence of CME in the GATA2 gene should be investigated in future, it should be noted that the regulatory mechanism of GATA2 gene in PVN may be different from that in thyrotroph because production of TSH was comparable to that of control pituitaries even in the absence of Sim1 [30]. With regard to the mechanism of the regulation of the rat preproTRH promoter-----We modified the following sentences in the line 164-165 in the 1st submission. As mentioned above, the rat prepro-TRH gene is known to be activated by the signals from multiple membrane receptors via cognate transcription factors including CREB, KLF10/TIEG1, STAT3, SP-1 and AP-1 [1-4]. On the other hand, two the transcription factors, Sim1 and Arnt2, are reported to be essential for the differentiation of the PVN in the hypothalamus [30]. We also modified the sentence at the lines 476-500 in the 1st submission as follows. As shown in Fig. 2A and 8, the GATA2-dependent transactivation of the prepro-TRH gene may be potentiated by the multiple transcription factors [3]. For example, catecholamine and αMSH signals induce phosphorylation of CREB via PKA pathway while leptin signal activates STAT3 via Janus kinase 2 pathway [1-4]. KLF10/TIEG1, which is expressed in CA77 cells and various parts of the adult rat brain, stimulates the rat prepro-TRH promoter via a KLF binding site  [54]. In addition, prepro-TRH gene expression is also stimulated by protein kinase C (PKC) signaling via the AP-1 site [76] and, presumably, GATA-RE (dr-GATA) [10]. Although site 4 appears to be dispensable for T3-dependent inhibition, this sequence may be physiologically relevant [23] because its mutation (site 4m) decreases the basal promoter activity . Thus, while the profiles of T3-dependent repression of them are very similar [56], the basal transcription level of the prepro-TRH gene is different from that of the TSHß gene. This may explain the observation from 152,261 human subjects where the relationship between serum TSH and free T4 is not a simple log-linear curve [58], but is instead an overlap of two negative sigmoidal curves [73]. With regard to the superiority of rats to mice as experimental animals in physiological studies---Because there are differences in the genomic structure of this gene among species, it is important to describe why we chose the preproTRH promoter of rats but not mice. We modified the sentence at the lines 521-523 in the 1st submission as follows. In this study, we chose the rat, but not mouse, preproTRH gene because (i) the anatomical and physiological findings of hypothalamus have been accumulated in this species, (ii) in vivo mapping of the rat preproTRH promoter was reported [47] and (iii) the transcription factors that regulate the rat preproTRH gene were characterized in detail . On the other hand, the screening of GATA-RE(s) in the prepro-TRH genes of the various species using expression plasmids for wild-type and mutant GATA2  may provide insights into the evolution of the H-P-T axis [16].With regard to uf-GATG in Figures---We added it in Figure 4 and 6. With regard to “mg” protein of CV1 whole cell extract---We described the amount (mg) of whole cell extract of CV1 cell transfected with TRß2 expression plasmid and that of CA77 cells at the lines 317 and 635 in the1st submission. We also added “mg” protein of CV1 whole cell extract in the section of Materials and Methods and Fig. 7A. With regard to the grammatical errors and unusual description---We corrected all the words and the sentences that you kindly pointed. With regard to mutant GATA2 (C349A) ---We modified the sentences in the line 356-359 as follows.“As shown in Fig. 2B, rTRH(547)-CAT was strongly activated by wild type mouse GATA2. We previously reported that a mutant GATA2, C349A, in which cysteine at the codon 349 in its DNA binding domain was substituted to alanine , failed to activate the TSHß promoter . As expected, this mutant GATA2 again failed to potentiate the preproTRH promoter, suggesting the presence of a functional GATA-RE.”In addition, we deleted the sentence “in which cysteine at the codon 349 in its DNA binding domain was substituted to alanine” at the line 589-590 in the 1st submission. With regard to Lezouaic'h in the line 420 in the 1st submission---We corrected it as Lezoualc'h. Reviewer #2: With regard to double-staining of TRH neurons using fluorescent antibody----We have attempted to do double-staining of them using fluorescent antibody for more than 8 months. However, we failed to detect signals with anti-GATA2  although we were able to detect the signal of immune-fluorescent stain using cultured cells . To our knowledge, there is no report of the immuno-fluorescent stain of tissue using this anti-body. Thus, we speculate that this phenomenon is the characteristics of this antibody. Although we attempted again immuno-fluorescent stain of PVN using another anti-GATA2 antibody  three times for the revised paper, we again failed. However, we believe the specificity of our anti-GATA2 antibody  and the co-localization of GATA2 protein at least in some population of TRH neurons . With regard to co-immunoprecipitation or GST pull down assay between GATA2 and TRß2---https://academic.oup.com/mend/article/21/4/865/2738390). In addition, we modified the following sentence at the lines 151-154 in the 1st submission as follows. As described in the line 154-155 in the 1st submission, we already reported both co-immunoprecipitation and detailed GST pull down assay between GATA2 and TRß2. Please see S10 Fig.pdf and S11 Fig.pdf in the “Supporting information3 (a ZIP file)” which were published from our laboratory :865-84, In solution and on the GATA-responsive elements (GATA-REs) of the TSHß promoter DNA, we detected the protein-protein interaction of TRß2 with GATA2 [29]. With regard to the reason why we used CAT but not luciferase assay in this manuscript---https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028916). We attached these data as S12 FigLucCAT1 and S13 FigLucCAT2 in the “Supporting Information3 (a ZIP file)”. Although the artifact with FFL cDNA may be unexpected, it has profoundly impeded the studies on the T3-dependent negative regulation, in particular, of the preproTRH gene. Please see also the description in the line 452-475 and references in the 1st submission.As mentioned in the line 227-228 in the 1st submission  and reference [41], we previously evaluated the enhancer activity of firefly luciferase (FFL) cDNA using CAT-based assay and found that some DNA sequence of it may function as an enhancer that can be stimulated by PKC and repressed by T3-bound TR. (PLoS One. 2012 (1):e28916, AttachmentResponse to Reviewers.docSubmitted filename: Click here for additional data file. 2 Nov 2020GATA2 mediates the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded T3 receptor β2 in the rat hypothalamic paraventricular nucleusPONE-D-20-24793R1Dear Dr. Sasaki,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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Cyclin-Dependent Kinases (CDKs) are well-known reliable targets for cancer treatment being often deregulated. Among them, since the transcription-associated CDK9 represents the sentry of cell transcriptional homeostasis, it can be a valuable target for managing cancers in which the transcriptional machinery is dysregulated by tumor-driver oncogenes. Here we give an overview of some natural compounds identified as CDK inhibitors with reported activity also against CDK9, that were taken as a model for the development of highly active synthetic anti-CDK9 agents. After, we summarize the data on CDK9 inhibition in a group of rare pediatric solid tumors such as rhabdomyosarcoma, Ewing’s sarcoma, synovial sarcoma and malignant rhabdoid tumors (soft tissue sarcomas), highlighting the more recent results in this field. Finally, we discuss the perspective and challenge of CDK9 modulation in cancer. The cyclin-dependent kinases (CDKs) family includes two main subgroups of kinases, those that mainly regulate cell cycle progression  and those that control transcriptional processes  . For somThe CDKs activity depends on specific regulatory cyclins subunits (29 cyclins in humans) with which CDKs form heterodimers . While lConversely, cyclins partnered with transcription-associated CDKs are modulated in a cell cycle-independent manner . Since twww.clinicaltrials.gov). Most of the CDKs inhibitors developed in the pre-clinical and clinical settings are against the cell cycle-associated subgroup that controls the crucial processes of tumor cell survival and growth. In contrast, the development of small molecules selectively targeting CDKs regulating transcriptional programs has just been started.Overall, abnormalities in the expression and/or activation of a number of CDKs are considered a hallmark of cancer development and progression . Over thAmong the transcription-associated CDKs, CDK9 plays a key role in controlling basal gene transcription thus ensuring cell transcriptional homeostasis . This crA group of soft tissue sarcomas (STSs) of childhood express chimeric transcription factors from which they are dependent for survival or have mutations or loss of key components of transcriptional complexes . STSs inIn this review, we briefly describe the roles of CDK9 in transcriptional processes and summarize the last insights on natural products as CDK9 inhibitors in cancer. We also give an overview of the results of CDK9 inhibition in a subgroup of transcriptionally-driven pediatric STSs. Perspectives and challenge of potential clinical applications of CDK9 inhibitors are also discussed.CDK9 consists of two isoforms encoded by the same gene but transcribed from two different promoters . The shoCDK9 binds CyclinT1 or CyclinT2  with which it forms the core component of Positive Transcription Elongation Factor Complex (P-TEFb), modulating gene transcription by phosphorylating key substrates at serine and threonine residues . Gene tr2+ signaling promotes the release of CDK9 from the 7SK snRNP complex  has been identified as the major active ingredient of the traditional Chinese medicine formulation known as Danggui Longhui Wan, and has been used for many years to treat chronic myelogenous leukemia (CML) in China   . Its marent CDK7 . Later, ent CDK7 .Unfortunately, despite its remarkable pre-clinical effects, it does not demonstrate significant activity in clinical studies on patients with cancers with the exception for those affected by hematological malignancies and, in particular by chronic lymphocytic leukemia (CLL) . One of Raphanus sativus), is a CDK inhibitor (R) enantiomer of a 2,6,9-trisubstituted purine . The main anti-cancer mechanism of wogonin and other related flavones such as apigenin, chrysin, and luteolin, has been ascribed to their ability to selectively inhibit CDK9 activity by binding the ATP-binding pocket  targeting CDK9 by the recruitment of the ubiquitin E3 ligase cereblon (CRBN) Figure 2Pediatric soft tissue sarcomas (STSs) are rare malignancies of children and adolescents that accounts for 8% of all pediatric cancers . They arcis-encoded gene  as well as transcription-associated factors have yielded important results in preclinical research on STSs in the last years. In particular, STSs whose cells are dependent from fusion proteins (translocations positive) working as oncogenic TFs or co-activator of oncogenic transcription, such as rhabdomyosarcoma (RMS), Ewing’s sarcoma (ES), and synovial sarcoma (SS), and or from mutated components of transcriptional complexes such as malignant rhabdoid tumors (MRT), have shown promising response to modulations of the gene transcription machinery. Core regulatory complexes of oncogenic TFs drive transcriptional programs in these tumors by binding to enhancer regions . Enhanceded gene . In thisPAX3-FOXO1 is a chimeric oncogenic TF expressed by high risk RMSs characterized by t chromosomal translocation (~ 20% of cases) . Fusion-in vitro the p300 and PCAF acetyltransferases and the SWI/SNF ATP-dependent chromatin remodeling complex, which includes BAF complex families, together with CDK9/CycT2 on promoters/enhancers regions to activate high level transcription of muscle-specific genes  of CDK9 facilitates rather than inhibits the MyoD-dependent transcription of MYOG and muscle genes in differentiating murine myoblasts  and its KD restrained cell number expansion  regulates myoblasts-to-myotubes transition fostering the activity of MYOD also by promoting CycT2 binding to DNA with consequent CDK9/CycT2 complex activation . To do syoblasts . In thisxpansion . In agrexpansion . OverallALK, we showed that while BRD4 inhibitors clearly displayed selectivity for SE-driven luciferase compared to constitutively active promoter-driven luciferase, the CDK9 inhibitor flavopiridol had a general transcriptional inhibitory effect. Further experiments could clarify both the enhancer inhibition selectivity of flavopiridol and the specific role of CDK9 in SE activation in the context of fusion-positive RMS.Recently, we demonstrated that PAX3-FOXO1, whose expression is strictly needed for tumor cell survival , forms aEWSR1 and the FLI1 gene of ETS family  as the only cytogenetic abnormality, which encodes for the fusion oncoprotein SS18/SSX1 considered the driver of malignancy . Other fin vitro. Indeed, CDK9 was reported highly expressed in SS cell lines and in samples from patients with high expression associated with poor prognosis  and dose-limiting toxicities (DLTs) in patients with advanced metastatic or progressive solid tumors who are refractory to established therapy ; Italian Ministry of Health Ricerca Finalizzata (Project # PE-2013-02355271) to RR and AM; Italian Ministry of Health Ricerca Corrente 2020 to RR; PRIN 2016 (prot.20152TE5PK) to AM, and Progetto di Ateneo Sapienza 2017 no. RM11715C7CA6CE53 to DR. SP is a recipient of a Fondazione Veronesi 2020 fellowship.The 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.


Translational regulation of mRNAs is critically important for proper gene expression in germ cells, gametes, and embryos. The ability of the nucleus to control gene expression in these systems may be limited due to spatial or temporal constraints, as well as the breadth of gene products they express to prepare for the rapid animal development that follows. During development germ granules are hubs of post-transcriptional regulation of mRNAs. They assemble and remodel messenger ribonucleoprotein (mRNP) complexes for translational repression or activation. Recently, mRNPs have been appreciated as discrete regulatory units, whose function is dictated by the many positive and negative acting factors within the complex. Repressed mRNPs must be activated for translation on ribosomes to introduce novel proteins into germ cells. The binding of eIF4E to interacting proteins (4EIPs) that sequester it represents a node that controls many aspects of mRNP fate including localization, stability, poly(A) elongation, deadenylation, and translational activation/repression. Furthermore, plants and animals have evolved to express multiple functionally distinct eIF4E and 4EIP variants within germ cells, giving rise to different modes of translational regulation. Post-transcriptional regulation of mRNAs is frequently used to modulate gene expression throughout development in all plants and animals . ParticuTranslational repression of mRNAs by RBPs has been studied extensively; most bind to sequence specific elements within 5′ or 3′-UTRs to interfere with ribosome recruitment by blocking the translation initiation machinery or to recruit the Ccr4-Not1 deadenylase complex causing shortening of the poly(A) tail . The latDrosophila embryogenesis, transposon-derived piRNAs bind imperfectly to nos mRNA in an mRNP involving Aubergine, Smaug, and eIF4E complexes to initiate Ccr4-Not1 mediated deadenylation  play a fundamental role in the activation step via their ability to recruit the ribosome to repressed or stored mRNPs. Recent studies have begun to shed light on their unique roles in RNA-regulatory networks across diverse phyla. A model has emerged in which eIF4 factors reside in such mRNPs in both their repressed and active states . Data frper se -binding protein (PABP), while also bridging eIF4 to the ribosome and recruiting eIF4A, the DEAD-box RNA helicase used to unwind secondary structure . Additioper se . These fper se . Becauseper se . It is nper se . Furtherper se . Other lper se . Internaper se .4-Leu-Φ: where X is variable and Φ is hydrophobic)  recognize the lateral surface of eIF4E, displacing eIF4G in preformed eIF4E:eIF4G complexes . Whether or not certain 4EIPs are TOR regulated is largely unknown, yet remains an interesting question.The balance between CD and CI synthesis at the eIF4E:eIF4G node is regulated by nutrients in somatic tissues by target of rapamycin (TOR) kinase via phosphorylation and inhibition of canonical eIF4E-binding proteins (4EBPs), which were among the first 4EIPs discovered but have a much narrower definition, and will not be the primary focus of this review . Regulatophobic) . Additioomplexes . Thus, eomplexes . WhetherC. elegans, multiple eIF4E isoforms (IFE-1 – IFE-5) are expressed in a tissue specific manner and each has unique developmental functions  and mRNAs involved in spermatocyte cytokinesis , as observed by polysome profiling .To add to the complexity to the broad CD vs. CI translational control outlined above, gene duplication events over evolutionary time have given rise to multiple isoforms of eIF4E and 4EIP in both plant and animal species . The evounctions . The twounctions . By measokinesis . In facthenotype . IFE-3 orofiling . Importarofiling . Importaenotypes . Interesng mRNPs . Whether tissues . Each of tissues . Fly eIF tissues . However tissues . This deC. elegans appear distinct from P bodies, whereas in mouse spermatogonia evidence points to the structures being more closely related , and maintains their translational repression, which is critical for body axis patterning during embryogenesis (discussed below). It seems clear that all animal germ cells contain multiple unique eIF4E:4EIP complexes. Regulating the transit and activation of these complexes contributes to differential mRNA translational control used to drive physiological germ cell processes in divergent animal systems.One of the clearest examples of the link between granule dynamics, translational control, and germ cell differentiation occurs in the latter stages of ytoplasm . There Iytoplasm . Importate 4EIPs . Recent  differentiation . LikewisDrosophila oocyte development and early embryogenesis. oskar mRNA (osk) becomes enriched in the posterior pole (germ plasm) during mid-oogenesis and nanos mRNA (nos) becomes enriched in the same region during late oogenesis (gurken (grk) and bicoid (bcd) also exhibit unique and dynamic localization during the maternal-zygotic transition  that marks mature mRNAs at those ligation points as they exit the nucleus   . In the nt (TCE) . This incr4-Not1 . Thus, n oocytes .grk). During egg development grk mRNA also relies on Cup for proper localization, but in contrast to osk and nos mRNAs, it localizes to the dorsal anterior portion of the egg -regulation are used in oocytes and embryos to control the local translation of messages that are required to establish the germ plasm. Similar examples can be found in nematodes, frogs and mice, indicating the prevalence of poly(A)-mediated mRNA regulation throughout germ cell biology -tail. With the induction of meiotic maturation, CPEB becomes phosphorylated on multiple residues, in response to progesterone signaling (nos mRNA repression in Drosophila oocytes and in humans as outlined above (cad mRNA during fly embryogenesis (fog-1 and fem-3) in worms  biology . Polyade biology . Poly(A) biology . Enhanci biology . Multime biology . This dy biology . Dynamic biology . This pr oocytes . There a oocytes . Not all oocytes . The mos oocytes . The seq oocytes . During ng mRNAs  -tails, which reduces translational efficiency or enhances degradation. Thus, germ cell systems institute translational repression early, then maintain that repression until intrinsic or extrinsic signals drastically change cell fate . The signaling cascades activate translation by stimulating the eIF4 factors already resident in the mRNP and thereby disrupt the repressive effects of 4EIPs and RBPs, and activate GLD-2 to elongate the poly(A)-tails of their bound mRNAs.Clearly, the mechanisms that control translational repression/activation switches in maturing C. elegans. Here stem cell proliferation, meiotic entry, and differentiation are all controlled by a remarkably small number of regionally expressed RBPs that bind 3′-UTRs of a subset of mRNAs to repress their translation  mRNA-repressive structures because many of their components have prescribed roles in translational repression or mRNA decay . Howevernslation . Yet thinslation .C. elegans caused very specific translational de-repression of certain mRNAs involved in germline sex-determination (cad mRNA during fly development (H. sapiens, Hoxb4 translational repression relies on 4EHP (The regulated activation of mRNPs by the translational initiation machinery has been especially understudied in germ cells. Remarkably, studies from nematodes, flies, and frogs indicates that the eIF4 group factors are playing very specific developmental roles. Interestingly, a theme emerges from all animals and plants investigated, that multiple isoforms of each factor are expressed during development, providing the opportunity for each isoform to evolve a specialized function over time . Indeed,mination . Likewiselopment . Similar on 4EHP , and oth on 4EHP . WhetherPolyadenylation/deadenylation machinery and localization factors also rely on eIF4E:4EIP complexes to appropriately regulate their target mRNAs by repression/activation and localization . Future HH wrote and revised the manuscript and created the figures. BK assisted in writing and revising the manuscript.The 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.


Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter –10 element, while not affecting interactions with –35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of –10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with –10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials. Bacterial multi-subunit DNA-dependent RNA polymerase (RNAP) is a proven target for a number of antibacterial compounds. About a half of these, including the only two compounds used clinically (fidaxomicin and compounds of the rifamycin class), specifically target transcription initiation. During initiation of transcription, the RNAP holoenzyme (core RNAP joined by initiation factor σ) binds to promoter DNA to form a ‘closed promoter complex’ (RPc) in which the RNAP is bound to double stranded promoter DNA. For RPc to form, conserved substructures of σ in the context of holoenzyme must interact with promoter DNA. σ subunit regions 2 (σR2) and 4.2 (σR4) recognize the core –10 and –35 promoter elements, respectively , while cSpecific inhibitors of initiation of transcription include Fidaxomicin (Fdx), Ripostatin (Rip), Myxopyronin (Myx), Corallopyronin (Cor), GE23077, rifamycins (Rifs), and ureidothiophene (Urd). Fdx blocks RPo (and probably correct RPc) formation by locking the RNAP clamp in an open conformation by binding to the switch region 2 of β′ subunit of RNAP (β′ switch-2), the molecular hinge that facilitates clamp movement . Fdx doeStaphylococcus aureus RNAP holoenzyme in vitro. Urd was shown to be highly active against S. aureus RNAP in vitro, with an IC50 of ∼1 μM  and purified by agarose gel electrophoresis (Qiagen). lacUV5-promoter fragment was produced by PCR with the primers 5′-CTCACTCATTAGGCACCCCAGGC-3′ and 5′-CCAGGCGGTGAAGGGCAATCAGC-3′ from template CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGATTGCCCTTCACCGCC. E. coli RNAP subunits were expressed in T7 express cells (New England Biolabs) transformed with pGEMABC  and pACYCDuet-1_Ec_rpoZ (encoding rpoZ) (Core ng rpoZ) . ExpressS. aureus SH1000 was purified in the same way except the cells were grown in Brain-Heart Infusion liquid medium.Cellular RNAP core from M. smegmatis RNAP σA holoenzyme was purified as described previously  was followed by ion exchange chromatography on Resource Q column , where σA holoenzyme was collected. Identity of σA was confirmed by mass spectrometry.Cellular His-tagged eviously . M. smegT. aquaticus was purified as described previously (Recombinant eviously . SDS-gelS. epidermidis wild-type and E105Q σA RNAP holoenzymes were purified from respective strains of wild-type S. epidermidis ATCC 12228 and S. epidermidis ATCC 12228 harboring an E105Q mutation in σA (see ‘Isolation of ureidothiophene resistant S. epidermidis’ below). RNAPs were purified as described above for S. aureus, apart from the polyethylenimine and ammonium sulphate precipitation steps were omitted, and holoenzymes were collected during ion exchange chromatography.Cellular Escherichia coli σ70, S. aureus σA and T. aquaticus σA were expressed in T7 express cells (New England Biolabs) transformed with pET28 expression vector encoding N-terminally 6xHis-tagged σ subunits. Expression was induced by addition of 0.4 mM IPTG to exponentially growing cells and cells incubated on an orbital shaker (150 rpm) at room temperature overnight. Cells were then harvested by centrifugation and resuspended in grinding buffer. Cells were lysed by sonication and debris cleared by centrifugation. σ subunits were then purified on HisTrap HP column . Urd resistant mutations were introduced in the expression vector by site-directed mutagenesis using QuikChange II kit (Stratagene) and mutant proteins purified as above.2). 1 pmol of E. coli RNAP core with 3 pmol of σ70 or 1 pmol of T. aquaticus RNAP core with 3 pmol of T. aquaticus σA or 1 pmol of S. aureus RNAP core with 3 pmol of S. aureus σA or 1 pmol of M. smegmatis or S. epidermidis RNAP holoenzymes were incubated in TB with 1 μl of DMSO containing or not containing Urd at 37°C (or 60°C in case of T. aquaticus RNAP) for 5 min. Transcription was initiated by the addition of 2 μl mixture of nucleotides and promoter DNA in TB, containing : 10 nM promoter DNA, 25 μM CpA  or 100 μM ApA (for lacUV promoter), 0.2 μl α-[32P]UTP (10mCi/ml) , 10 μM UTP with (run off transcription) or without (abortive transcription) 100 μM ATP, CTP and GTP. Reactions were stopped after 10 min incubation at 37°C (or 60°C in case of T. aquaticus RNAP) for run off transcription or 5 min for abortive transcription by the addition of equal volume of formamide-containing loading buffer. Products were resolved in denaturing polyacrylamide gels, revealed by PhosphorImaging , and analyzed using ImageQuant software Transcription from promoter DNA fragments was performed essentially as described . BrieflyIn vitro transcription on single-stranded M13ori minimal promoter was performed as described in  were then added to the reaction and incubated at 37°C for 10 min. Transcription was initiated by the addition of 1 mM ATP, CTP and UTP, 100 μM GTP and 0.2 μl α-[32P]GTP (10mCi/ml) . Reactions were stopped after 30-min incubation at 37°C by the addition of formamide-containing loading buffer. Products were analyzed as above.ribed in . Briefly32P]-ATP prior to complex assembly, as described . 2 pmol of RNAP core and 6 pmol of σ70 were incubated in EMSA buffer. 2 μl of DMSO containing or not containing Urd were added to the reaction and incubated at 37°C for 5 min. Reactions were supplemented with 0.2 pmol of promoter DNA radiolabelled as above and incubated for a further 5 min at 37°C. Next, heparin  was or was not added, and samples incubated for a further 2 min at 37°C. Complexes were resolved in 4.5% non-denaturing polyacrylamide gel, which was dried and analyzed as above.Reactions were performed in 20 μl final volume of EMSA Buffer . Individual wells contained 1 ml of solid LB agar supplemented with 2.5% pluronic F68 (Thermofisher). Prior to agar setting, DMSO with or without Urd was added to the individual well to a final concertation of 5%. S. epidermidis ATCC12228 was streaked onto LB agar and grown at 37°C overnight. A single colony was picked and grown in liquid LB to 1 × 106 CFU/ml. 10 μl of 106 CFU/ml S. epidermidis inoculant were dotted onto each well and the plate incubated at 37°C overnight. MIC was deduced as the concentration in which no visible cell growth was observed (3.125 μg/ml). Secondly, S. epidermidis ATCC12228 was streaked onto standard LB agar and grown at 37°C overnight. A single colony was picked and grown in liquid LB until ∼1 × 109 CFU/ml. 100 μl of 109 CFU/ml S. epidermidis ATCC12228 was streaked onto an LB agar plate supplemented with 2.5% pluronic F68, containing 4× MIC of Urd (12.5 μg/ml). Urd resistant strain was confirmed by re-streaking on the same media, and sent for full Illumina genome sequencing (MicrobesNG). Genomes were assembled and SNPs identified by CLC Genomics Workbench software (Qiagen)Firstly, Urd MIC of in vitro transcription by the wild-type E. coli RNAP, the most extensively characterized bacterial RNAP. When added to RNAP before DNA, Urd inhibited transcription on a linear DNA template, containing lacUV5 promoter (IC50 ∼15.1 ± 8.1μg/ml)   μg/ml. In contrast, T. thermophilus and M. smegmatis RNAPs were much less sensitive to Urd  Figure . A decre) Figure . We furtt Figure , E. We at Figure . In thisd Figure . These oE. coli RNAP core enzyme. Elongation complexes were assembled with fully complementary template and non-template strands and 5′-radiolabeled RNA  and then incubated with radiolabeled lacUV5 promoter DNA. As can be seen from Figure Urd may altogether block interactions of RNAP with DNA or prevent crucial interactions of RNAP with promoter DNA that are required for either RPc formation, or melting of promoter DNA. In order to distinguish between these possibilities, we first analyzed RNAP-promoter complex formation by electrophoretic mobility shift assay (EMSA). lacUV5 with 5′-radiolabelled non-template strand). The results indicate that Urd does not cause a significant changes in protection in the upstream promoter regions from positions –39 to –25  added before mixing RNAP and the promoter DNA  spontaneous Urd resistant mutant conferring ≥4× resistance to the MIC of Urd (3.125 μg/ml). The mutant strain was resistant to Urd at >100 μg/ml. Genome sequencing revealed a single nucleotide substitution in the rpoD gene encoding the primary sigma factor σA, Substitution resulted in E105Q mutation in σR1.2 of σA   and assessed the effect of the mutation on E. coli holoenzyme sensitivity to Urd. Mutant holoenzyme was ∼6-fold more resistant to Urd than the wild-type RNAP  Figure . This reP Figure . As σR1.The principle finding of this study is a new mode of inhibition of bacterial transcription. We show that Urd inhibits interaction of RNAP with –10 promoter element and other parts of promoter DNA downstream of –35 element during RPc formation. We also show that the action of Urd is manifested through essential regulatory region of σ subunit, σR1.2.DNA footprinting data suggests that, in the presence of Urd, σ cannot stably engage with –10 element and downstream part of promoter DNA, i.e. form a correct closed promoter complex. Urd, thus, acts at earlier stages of transcription initiation than do Rip, Cor and Myx, which target the β′ switch-2, and inhibit transformation of closed promoter complex into RPo ,14. Urd E. coli RPo and holoenzyme, σR1.2 E104 interacts with residue I290 of the β′ coiled-coil domain, while the latter interacts with residue E381 of σR2. This may create a mechanical linkage between σR1.2 and σR2 (Figure The dependence of Urd inhibition on σR1.2 integrity suggests the compound mediates inhibition of transcription by interacting with this particular σ factor sub-region or disrupt conformational change governed by this σ region, which is required for interaction of holoenzyme with downstream promoter DNA. σR1.2 has previously been shown to play a role in formation of stable open promoter complexes, in particular it is implicated in allosteric control of –10 promoter element recognition by σR2 . The ami2 Figure , and thu2 Figure . Furthergkaa591_Supplemental_FileClick here for additional data file.