Genome editing technology has become one of the hottest research areas in recent years. Among diverse genome editing tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins system (CRISPR/Cas system) has exhibited the obvious advantages of specificity, simplicity, and flexibility over any previous genome editing system. In addition, the emergence of Cas9 mutants, such as dCas9 (dead Cas9), which lost its endonuclease activity but maintains DNA recognition activity with the guide RNA, provides powerful genetic manipulation tools. In particular, combining the dCas9 protein and transcriptional activator to achieve specific regulation of gene expression has made important contributions to biotechnology in medical research as well as agriculture. CRISPR/dCas9 activation (CRISPRa) can increase the transcription of endogenous genes. Overexpression of foreign genes by traditional transgenic technology in plant cells is the routine method to verify gene function by elevating genes transcription. One of the main limitations of the overexpression is the vector capacity constraint that makes it difficult to express multiple genes using the typical Ti plasmid vectors from Agrobacterium. The CRISPRa system can overcome these limitations of the traditional gene overexpression method and achieve multiple gene activation by simply designating several guide RNAs in one vector. This review summarizes the latest progress based on the development of CRISPRa systems, including SunTag, dCas9-VPR, dCas9-TV, scRNA, SAM, and CRISPR-Act and their applications in plants. Furthermore, limitations, challenges of current CRISPRa systems and future prospective applications are also discussed. Gene expression involves multiple processes, including transcription of DNA into messenger RNA (mRNA), splicing of mRNA, translation, and post-translation modification. Accurate regulation of DNA transcription into mRNA is the first step to control the complex process of gene expression. Directional regulation of gene expression will contribute to our understanding of cell physiology, and it is essential for advances in biotechnology.For the regulation of endogenous gene expression, manipulating transcription factors (TFs) to target the specific target gene promoters to activate/inhibit gene transcription is the classical strategy and a successful one . For exaTranscription factors can only bind to fixed sites and lack flexibility. Site-specific nucleases (SSNs) such as TALEN and CRISPR/Cas9 have emerged as multipurpose tools which can greatly enhance molecular biologists’ capability such as knock out, base edit, knock in, knock up and knock down the target gene ,5,6,7. TIn this paper, we summarized the emergence and development of transcriptional activation system based on CRISPR/dCas9 and its application in plant research. To provide a reference for plant researchers to compare the differences of different activation systems and to regulate the activation efficiency of endogenous genes in plants in the future. In addition, the potential applications, existing problems, and challenges for these new technologies were also discussed.Escherichia coli genome, which are near the iap gene sequence and were called Clustered Regularly Interspaced Palindromic Repeats (CRISPR). However, its biological significance was unclear at that time [present in this powerful defense system [The CRISPR/Cas system was first investigated in 1987: scientists discovered a kind of unique DNA sequences from hat time . Subsequhat time . Researce system . Cas proe system .According to the repeat sequence identity of CRISPR and their Cas protein sequence homology, CRISPR/Cas system was classified into two classes and six types  and trans activated crRNAs (tracrRNAs). Then the RNA complex will find and identify a suitable Protospacer Adjacent Motif (PAM), such as the 5‘-NGG-3’ sequence of the target sequence [Cas9 is a specific DNA endonuclease that existed in bacteria species such as sequence . Finallysequence ,26,27.Nicotiana tabacum, Zea mays, and Arabidopsis thaliana (as shown in rpoZ) in E. coli, and this dCas9-ω complex increased the reporter gene transcriptional level up to 2.8-fold [Qi et al. (2013) mutated two conserved endonuclease domains of Cas9 in CRISPR/Cas9 system. The aspartic acid at position 10 of RuvC domain was mutated to alanine (D10A) and histidine at position 840 of HNH domain was mutated to alanine (H840A), so that Cas9 protein lost endonuclease activity and became dead Cas9 (dCas9), which cannot cut DNA but still can bind to specific target DNA sequences with the guide RNA . Therefo2.8-fold . The modGuide RNA (gRNA) of CRISPR/Cas9 system is a specific RNA sequence composed of two elements: crRNA and tracrRNA. The gRNA recognizes the target DNA and directs Cas9 protein to produce double-strand breaks (DSB) in target DNA . Any comTranscription regulatory factors are essentially chimeric proteins, and their DNA binding domains are connected with the functional domains that control the transcription mechanism by promoting the recruitment of key cofactors to regulate transcription . In the On the other hand, transcriptional activators such as herpes simplex Virus Protein 16 (VP16) Transcriptional Activator Domain (TAD) or tetrameric repeat VP64  could elAs mentioned previously, transcriptional activators are essential for the regulation of transcriptional activation through dCas9. Regulation of target gene activation can be achieved by fusing transcriptional activators with dCas9, or by directing gRNA into scaffolds to recruit transcriptional activators. A comparison of different activation systems using these two strategies and evaluating their efficiency is summarized below:Studies showed that the dCas9 protein fused with the transcription factor VP64 can recruit and stabilize the promoter complex to form dCas9-VP64 . HoweverResearchers found that the combination of multiple transcription factors with a single promoter significantly enhanced transcriptional activation of the downstream gene. This principle of signal amplification through protein polymers had been applied in biological system imaging and engineering design . Based oIn the Super Nova Tag (SunTag) system, dCas9 is fused with tandem General Control Nonderepressible 4(GCN4) peptide repeats and each repeat connects a transcription regulator via an anti-GCN4 antibody called Single Chain Fragment Variable (scFv). The domain has highly affinity with relatively short nucleic acid sequences, allowing for protein polymerization on a single RNA template. Compared to the dCas9-VP64, SunTag enables multiple transcription factors such as Ten-Eleven Translocation (TET) and regulatory elements X to be recruited in one system. Therefore, multiple genes can be synergistically activated by different transcriptional regulators in tandem  as shownIn natural gene regulation system in cells, many transcription factor Activation Domains (ADs) can make changes in transcription through a coordinated collection of necessary activators. Chavez et al. (2015) hypothesized that transcriptional activation could be enhanced by combining multiple Ads . A serieCurrently, the CRISPRa system is continually being optimized and improved in animal cells, whereas it is still at an early stage for plant cells. The dCas9-TV system is the first CRISPR/dCas9 activation system applied in plant cells .LUC with a five-fold increase; an improved performance on the dCas9-VP64 (only a two-fold increase). TAD was then introduced as a second step to enhance dCas9-VP128 activity, including plant-specific EDLL and modified ERF2 (ERF2m) and TAD from the herpes simplex virus’ TALE element. Results showed that the combination of VP128 with a tandem ERF2m-EDLL motif (up to four copies) activated LUC transcription up to 12.6-fold compared to the base level, while combination of VP128 and TALE TAD (up to six copies) increased LUC transcription up to 55-fold. The results suggested that dCas9-6TAL-VP128 was a strong transcriptional activator and was called the dCas9-TV system [Li et al. (2017) modified dCas9-VP64 with VP16’s octahedron VP128 instead of VP64 and this system activated V system ,57 as shA common method for studying RNA localization is to insert multiple MS2-binding RNA scaffolds into the target RNA molecule and then tag the RNA molecule with GFP by recruiting MS2-GFP fusion proteins . The inhZalatan et al. (2015) constructed a scaffold RNA (scRNA) system and demonstrated that it can effectively activate gene transcription in yeast and human cells . ScRNA sASCL1 (12%) and MYOD1 (37%) transcription activation [Synergistic Activation Mediator (SAM) continues to be constructed by modifying gRNA loops. Initially, the researchers tried to find the best anchoring position for the activation domain in the Cas9-sgRNA complex. Previously, dCas9-based transcription activators relied on trans activation domains fused to the N or C ends of dCas9 proteins. For an investigation regarding alternative anchorage sites, Konermann et al. (2015) investigated the crystal structure of dCas9 and revealed that the distal gRNA loops did not interact with dCas9 at all. Other research revealed that the gRNA loops could fuse with protein-interacting RNA domains to promote transcription factor recruitment to dCas9 . They cotivation . Some retivation .Os11g35410, Os03g01240 and Os04g39780 in rice protoplasm [The CRISPR-Act 2.0 system is an improved system for multiple transcriptional activation in plants, which is also dependent on MS2. It is capable of recruiting four VP64 proteins to gRNA and the fifth VP64 was carried by the dCas9-VP64 complex. As shown in otoplasm ,64. The Based on the CRISPR-Act 2.0 system, Pan et al. (2021) combined dCas9-VP64, gR 2.0 scaffolds, 10xGCN4 SunTag and a newly developed 2xTAD activator to build a novel CRISPR-activating system, CRISPR-Act3.0 , as showA. thaliana and N. benthamiana for transcription activation test to evaluate the transcriptional activation activity of dcas9-VP64. The data suggested that dCas9-VP64 had weak activation when a single sgRNA was designed in the vector. Then, the adoption of multiple sgRNAs to target the same gene promoter resulted in higher level of gene activation [A. thaliana and O. sativa and rice cells was achieved with a 201-fold and 2745-fold increase, respectively [OSER1 gene up to 4000-fold [N. benthamiana [First, the researchers used tivation ,35,47,66ectively . Xiong e000-fold . Researc000-fold . Selma ethamiana .A. thaliana when SAM or SunTag were fused with the dCas9 [A. thaliana cells with a 1500-fold increase [Arabidopsis (4000-fold) and tomato (240-fold), respectively [Similarly, transcriptional activation of endogenous target genes was also efficiently applied in he dCas9 ,71. Whenincrease . CRISPR-ectively . It is iectively ,66. It iAgrobacterium, Polyethylene Glycol (PEG) or particle bombardment techniques. The donor plants should harbor the T-DNA insertion with CRISPR/dCas9 fragment may be regulated under the biosafety framework for GM plants since they contain transgene element in the genome. This is maybe the major concern for wide application of the current CRISPR/dCas9 mediated transcription activation system in plant. In addition, with the miniaturization of Cas protein and the maturity of virus introduction technology, it will leave the tissue culture stage and accelerate the efficiency of related research. It is anticipated that transcriptional regulation using CRISPR will be further improved by overcoming the technical difficulties mentioned above in the near future.The current CRISPR/dCas9-based transcription system needs to be delivered in plant by This paper reviews the different strategies developed based on CRISPR/dcas9 activation system in recent years. These strategies have been widely employed to study gene transcription regulation in animal cells; however, their application in plants is still at its early stages. In plants, this system is mainly used in the model plant species such as Arabidopsis and rice, but not in other major crop plant species. Nevertheless, there is still a great deal of potential for research in this field. First, the multi gene activation system enables large-scale transcriptional regulation in plants in order to better understand gene regulatory networks. Second, the up-regulation of multiple key genes in the metabolic pathway can generally result in the production of valuable commercial products, and synthetic biology is likely to make a significant breakthrough in the field of agriculture . AdditioA. thaliana [Ongoing efforts are being made to update the transcriptional activation system based on CRISPR/dCas9. For example, Chiarella et al. (2020) improved CRISPR/dCas9-based transcriptional activation using Chemogenetic Epigenetic Modifiers (CEMs). By employing endogenous chromatin activators, this system was able to activate target gene expression without requiring exogenous transcriptional activators, leading to dose-dependent activations of target genes . Gamboa thaliana .As opposed to ribozymes with cutting activity, which often cause uncertain genomic modifications and result in chromosome rearrangements or deletion during multiple point editing, dCas ribozymes offer a superior solution to these problems . HoweverEpigenetic regulation is an important way to regulate gene expression and has certain reversibility. The researchers used CRISPR/Cas9 to knock out epigenetic factors to determine the role of epigenetic factors in the regulation of endogenous genes in plants . In addiFinally, with the wide application of single-cell sequencing technology in plants , it will


POLR3G gene expression and SNAR-A gene features in cancer contexts. Our results support a model in which Pol III identity functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.RNA polymerase III (Pol III) includes two alternate isoforms, defined by mutually exclusive incorporation of subunit POLR3G (RPC7α) or POLR3GL (RPC7β), in mammals. The contributions of POLR3G and POLR3GL to transcription potential has remained poorly defined. Here, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. Particularly sensitive is snaR-A, a small noncoding RNA implicated in cancer proliferation and metastasis. Analysis of Pol III isoform biases and downstream chromatin features identifies loss of POLR3G and snaR-A during differentiation, and conversely, re-establishment of  RNA polymerase III changes its subunit composition during mammalian development. Here the authors report that loss of subunit POLR3G, which re-emerges in cancer, promotes expression of small NF90-associated RNA (snaR-A), a noncoding RNA implicated in cell proliferation and metastasis. Structural and biochemical studies of the Pol III complex, gene promoter architectures, and essential transcriptional regulators have extensively decoded the Pol III transcriptome and layers of transcriptional regulation5. Well-defined classes of small noncoding RNA (ncRNA) synthesized by Pol III include tRNA and 5 S ribosomal RNA (translation), 7SK and U6 small nuclear RNA , RMRP and H1 , and 7SL 15. Additional classes of Pol III-transcribed ncRNA, whose functions remain less well understood, include vault RNA (regulation of apoptosis and autophagy)17, Y RNA 19, BC200 (translation regulation)20, and snaR RNA, which interacts with RNA-binding protein ILF322. BC200 and snaR-A, a specific isoform of snaR ncRNA, are upregulated in a variety of cancer contexts, yet the underlying mechanisms and disease contributions of these ncRNA species remain poorly understood27.The cellular division of transcriptional labor includes the RNA polymerase III (Pol III) apparatus, specially tuned for production of small, noncoding RNAs critical for translation, transcriptional and post-transcriptional regulation, and other fundamental processes28. Pol III recruitment and activity is also controlled through interactions with oncogenic and tumor-suppressor signaling pathways, connecting growth-related synthesis of tRNA and other small RNAs to extracellular growth cues29. Specific pathways converge on the transcription factor complex, TFIIIB, as well as MAF1, a transcriptional repressor that antagonizes Pol III occupancy and activity34. However, MAF1 is a chronic repressor of Pol III activity in both proliferative and differentiated contexts, suggesting additional mechanisms may also contribute to context-specific Pol III transcription regulation at the gene level35. Previous genomic mapping studies of Pol III occupancy suggest some level of tissue-specific activity patterns39, and we and others have reported dynamic loss of Pol III occupancy and transcription at specific tRNA genes during cellular differentiation and exit from proliferation41. Comprehensive profiles of Pol III activity within cellular models of development are needed to better understand the level of dynamic transcription at other classes of Pol III-transcribed genes, and the mechanisms contributing to restricted Pol III activity during differentiation.Three distinct promoter architectures categorically classify Pol III-transcribed genes based on internal  and external (type III) DNA sequence features and complementary transcription factor repertoires42. Unique components include the large subunits, POLR3A (RPC1) and POLR3B (RPC2), which constitute the catalytic DNA-binding core, POLR3D (RPC4) and POLR3E (RPC5), which establish a stably associated subcomplex involved in transcription initiation and termination, as well as POLR3C (RPC3), POLR3F (RPC6), and POLR3G (RPC7α), which together form a heterotrimeric subcomplex required for recruitment of Pol III via direct interactions with transcription factor complex, TFIIIB47. POLR3G is highly expressed during early development and subsequently attenuated during differentiation49. A paralogous subunit, POLR3GL (RPC7β), is expressed at later stages of development and required for long-term survival in vivo50. Replacement of POLR3G and incorporation of POLR3GL into the analogous RPC3-RPC6-RPC7 heterotrimeric subcomplex during development imply that Pol III-transcribed genes are expressed by distinct complex identities depending on the spatiotemporal regulation of POLR3G and POLR3GL genes51.Structurally, Pol III is the largest RNA polymerase protein complex and is comprised of multiple subunits that are either Pol III-specific or otherwise shared with Pol I and/or Pol II52. Whether differences in Pol III composition defined by POLR3G or POLR3GL mechanistically drive unique transcription patterns has not been well established. However, initial mapping of POLR3G and POLR3GL, as well as functional analysis of these subunits in mice, suggests a strong level of redundancy with respect to gene occupancy and contribution to developmental potential, respectively52. Despite evidence of redundancy, ectopic expression of POLR3G, but not POLR3GL, enhances RNA levels for a subset of Pol III-transcribed genes in IMR90 cells, and knockdown of POLR3G in human pluripotent stem cells induces changes in specific Pol III-transcribed ncRNA levels51. Recent Pol III structural analysis suggests that interactions between the conserved C-terminal tail regions of POLR3G and POLR3GL and the active center of Pol III may function as an autoinhibitory mechanism that precludes transcription in the absence of direct interactions with TFIIIB53. While potential differences in POLR3G- and POLR3GL-mediated autoinhibition require further investigation, additional structural insight suggests that subunit POLR3G, but not POLR3GL, may block the site of MAF1 interaction required for repression of Pol III activity54. These findings imply that POLR3G and POLR3GL may not be strictly redundant, and that complex identity may indeed exert some level of context-specific control of Pol III transcription potential.Subunits POLR3G and POLR3GL share 46% amino acid identity and were established by a gene duplication event in the common ancestor of vertebratesSNAR-A gene clusters, specifically, and expansion of Pol III transcription potential, generally. Our results suggest developmental loss of POLR3G restricts the transcription potential of Pol III, supporting a nonredundant model in which subunits POLR3G and POLR3GL contribute to a dynamic transcriptional landscape.To better understand the role of subunit composition as a potential underlying mechanism of Pol III transcription regulation, we established an unprecedented genomic map of Pol III subunit occupancy and transcription in proliferating THP-1 monocytes and traced the effects of POLR3G loss on Pol III activity during differentiation and experimental disruption Fig. . We addihttps://rnacentral.org), a collection of multiple noncoding RNA annotation databases56.We used a combinatorial genomic approach to identify active Pol III–transcribed genes, relying on the extended fragment size and corresponding sequence reads of chromatin immunoprecipitation (ChIP) experiments to confidently identify Pol III complex localization. Measures of Pol III occupancy are subsequently integrated with profiles of nascent and steady–state small RNA abundance. Included in our expansive genome–wide Pol III map are the binding profiles for both large subunits, POLR3A and POLR3B, shared subunit POLR1D, multiple subunits of the RPC3-RPC6-RPC7 heterotrimer subcomplex involved in transcription initiation, including POLR3C, POLR3G, and POLR3GL, and both subunits of the RPC4-RPC5 heterodimer subcomplex involved in transcription initiation and termination, POLR3D and POLR3E Fig. . We addi28. Overall, maps of Pol III subunit occupancy are broadly consistent across individual Pol III subunits, with specific genes featuring high levels of signal intensity across each subunit, and other genes featuring moderate to low signal intensity for all mapped subunits. Rank normalization of Pol III and transcription factor signal intensities illustrate this relationship, with high occupancy genes featuring strong signals for all subunits  induces monocyte-to-macrophage differentiation and entry to a quiescent cellular stateted Fig. . Mirroriged Fig. . These dged Fig. . Whereasged Fig. . The dynBCYRN1), and multiple tRNA species are particularly depleted for Pol III localization following PMA treatment. Visual inspection of individual genes marked by diminished Pol III occupancy clearly affirms the loss of subunit localization after 72-h PMA treatment, including at SNAR-A genes, tRNA genes, and BCYRN1 which, again, exhibit comparatively weak POLR3GL ChIP-seq signal prior to treatment with PMA  cells, CD4 + T cells, CD8 + T cells, and γδ T cells typically express high levels of POLR3GL and limited-to-absent levels of POLR3G mRNA. Analogous gene expression profiling in immune cells isolated from patients with acute myeloid leukemia (AML) identifies co-expression of both POLR3G and POLR3GL mRNA in pre-leukemic hematopoietic stem cells (pHSCs), leukemia stem cells (LSC), and leukemic blast cells (Blast) , a database of clinical and integrated molecular signatures collected from primary human cancer tissues, provides an invaluable resource for exploring both dysregulated gene activity and chromatin features in a multitude of cancer contexts70. Analysis of POLR3G and POLR3GL expression levels in these data uncover significant increases in POLR3G gene expression bias in several primary solid tumors when compared to patient-matched normal tissues, including lung squamous cell carcinoma, lung adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, kidney chromophobe, cholangiocarcinoma, colorectal adenocarcinoma, bladder urothelial carcinoma, and uterine corpus endometrial carcinoma  and conserved regions of an autoinhibitory motif in the C-terminal tail of POLR3G72. ML-60218 exposure causes POLR3G depletion and a switch from POLR3G enrichment towards POLR3GL enrichment in coimmunoprecipitation experiments with other Pol III subunits73. Though the mechanism of POLR3G-sensitivity to ML-60218 requires further investigation, the overlapping effects reported for POLR3G knockdown and ML-60218 exposure help to explain why ML-60218 fails to effectively inhibit Pol III transcription in specific contexts, such as those recently reported during preadipocyte-to-adipocyte differentiation30. Instead, ML-60218 is likely an effective strategy for inhibiting Pol III transcription in early developmental windows and other contexts with high POLR3G abundance.The Pol III-specific inhibitor, ML-60218, was discovered as an analog of small molecule inhibitors of Pol III transcription in We explored the effects of ML-60218 on THP-1 cells as an additional, complementary approach to further dissect the contribution of POLR3G to Pol III transcription potential in our system. THP-1 cells exhibit a narrow range of ML-60218 sensitivity, with limited levels of growth disruption observed at 25–50 uM, and loss of cell viability at 75–100 uM Fig. . ExposurSNAR-A , directed at conserved sequences within the core and tail regions of snaR-A ncRNA, to disrupt snaR-A levels and examined the downstream consequences of snaR-A disruption on cell proliferation in THP-1 monocytes. snaR-A ncRNA levels are measurably diminished by both ASO designs, though most significantly when targeting both the core and tail regions of snaR-A , 10% fetal bovine serum, and 1% penicillin–streptomycin. For differentiation of THP-1 cells, non-adherent cells were diluted to 2 × 105 cells/mL and grown overnight, and a final concentration of 100 nM PMA was added the following morning. THP-1 derived macrophages were collected after 72-h exposure to PMA by aspirating media and any non-adherent cells, and incubating adherent cells with TrypLE (ThermoFisher) for 15 min followed by cell wash in phosphate-buffered saline (PBS) buffer. Drug-disruption of POLR3G/Pol III was performed in THP-1 cells by treatment with RNA polymerase III inhibitor ML-60218  at a final concentration of 25 μM and cells collected at 1, 2, 3, and 4 h post-exposure followed by cell wash in PBS buffer.THP-1 cells were obtained from ATCC (Lot # 62454382) and grown for multiple passages in T-75 flasks between 2-8 × 10m = 2' O-Methyl RNA base; * = Phosphorothioate Bond (Integrated DNA Technologies). THP-1 cells were transfected with ASOs mixed with OPTI-MEM media and Lipofectamine RNAiMAX Transfection Reagent  and added to THP-1 cells absent antibiotic selection at a final concentration of 1 uM. Small RNA was extracted 48-h post transfection using the mirVana PARIS RNA and Native Protein Purification kit  following standard protocols.Knockdown of MYC was performed in THP-1 monocytes in media without penicillin–streptomycin, using Silencer Select predesigned siRNAs , mixed with OPTI-MEM media and Lipofectamine RNAiMAX Transfection Reagent  per manufacturer’s instruction and added to THP-1 at a final concentration of 500 nM. A second knockdown step was carried out 48-h post transfection, and THP-1 lysate, total RNA, and small RNA were extracted on day 4 (96-h) using the mirVana PARIS RNA and Native Protein Purification kit  following standard protocols. Custom Antisense Oligonucleotides were designed against SNAR-A core region , the SNAR-A tail region , and scramble control sequence  5 cells/mL in 12-well plates overnight. A final concentration of 1 uM PMA was added the following morning, and THP-1 cells were allowed to differentiate for 72-h. Non-adherent cells were aspirated following 72-h exposure and replaced with fresh media containing 10 ug/mL polybrene . THP-1 macrophages were transduced with prepackaged lentiviral particles of either [1] copGFP control , [2] control activation , or [3] POLR3G activation . THP-1 macrophages were spun in the presence of 200 uL activation particles / well at 300 × g for 45 min. Polybrene-containing media was replaced with fresh media 8-h post transduction. Confirmation of positive lentiviral transduction of THP-1 macrophages was monitored by GFP visualization and THP-1 macrophages harvested 72-h following transduction. Total and small RNA fractions were purified using the mirVana PARIS RNA and Native Protein Purification kit  following standard protocols.THP-1 monocytes were plated at 2 × 10ACUUUUUUUU; SNAR-A context sequence 2 = UCCAGGGCACGAGUUCGAGGCCAGCCUGGUCCACAUGGGUCGGAAAAAAGGAUUUUUUUU. mRNA and small RNA abundances presented were determined as the relative fold change normalized with two reference genes per RNA type .mRNA changes in THP-1 monocyte and THP-1 macrophages were quantified using the Cells-to-CT 1-Step Taqman Kit , with the exception that total and small RNA fractions were instead extracted with the mirVana PARIS RNA and Native Protein Purification kit , following standard protocols for separate isolation of total and small RNA. Real-time Quantitative PCR was performed with a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) using TaqMan 1-Step qRT-PCR Mix and predesigned TaqMan Gene Expression Assays  for selected genes: Hs02786624_g1 (GAPDH); Hs99999903_m1(ACTB); Hs04978644_g1 (POLR3G); Hs01113209_g1 (POLR3GL); Hs00153408_m1(MYC). Changes in small RNA were similarly profiled using the TaqMan MicroRNA Cells-to-CT Kit  with the exception that small RNA fractions were instead extracted with the mirVana PARIS RNA and Native Protein Purification kit , following standard protocol for separate isolation of total and small RNA. For small RNA, Reverse Transcription (RT) of purified small RNA was performed with a multiplex RT reaction step using an undiluted primer pool consisting of RNU19 , Z30 , SNAR-A context sequence 1 , SNAR-A context sequence 2 . Small RNA changes were measured using RT product via Real-time Quantitative PCR  using TaqMan Master Mix (2×) and predesigned TaqMan MicroRNA Gene Expression Assays  for selected genes: RNU19 , Z30 , SNAR-A (context sequence 1) , SNAR-A (context sequence 2) . SNAR-A context sequence 1 and context sequence 2 were purchased from ThermoFisher Scientific using the Custom TaqMan Small RNA Assay  by submitting sequences corresponding to SNAR-A1 and SNAR-A13 (context sequence 1) and SNAR-A4 (context sequence 2). Context sequences provided by ThermoFisher Scientific: SNAR-A context sequence 1 = CCAGGGCACGAGUUCGAGGCCAGCCUGGUCCACAUGGGUCGGAAAAAAGGTHP-1 viability and proliferation were analyzed using the CyQUANT MTT Cell Viability Assay . 12 mM MTT was prepared as instructed by product protocol, adding 1 mL of PBS into 1 vial of 5 mg MTT. Vial was vortexed until completely dissolved and stored at 4 °C, protected from light. In a transparent 96-well tissue culture plate, 10 uL of 12 mM MTT was aliquoted into each well per data point, with an extra well of MTT for negative control. 100 uL of cell culture suspension was then added to the aliquoted MTT and gently pipetted up and down to mix. Cell culture suspension was well agitated before collecting sample to ensure homogenous suspension sampling. In negative control well, 100 uL of cell culture media was added. 96-well plate was then incubated at 37 °C, 5% CO2, for 4 h, protected from light. After incubation, plate was spun down for 5 min at 300 g. 85 uL of cell culture suspension was carefully removed from each well, without disturbing the sediment at the bottom. 50 uL of DMSO was then added and carefully pipetted up and down to mix, avoiding air bubbles. 96-well plate was then incubated for 5 min in 37 °C and then scanned using an M1000 Infinite Tecan Microplate reader. Absorbance was determined at 540 nm. Assay was repeated across 5 days and data plotted across time.Total steady-state small RNA was purified from equal numbers  of THP-1 monocytes ± ML-60218 exposure and THP-1 macrophages (72-h PMA) using the mirVana miRNA isolation kit according to instructions . Nuclear and cytoplasmic THP-1 fractions were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents  according to instructions, with two additional nuclear washes prior to nuclear extraction. Fractionation purities were assessed by β-tubulin and Lamin B2 contamination  for 1 h at room temperature followed by addition wash in TBST. Proteins were visualized using either SuperSignal West Pico  or SuperSignal West Femto . Antibodies used for immunoblotting include Rabbit Anti-POLR3A  , Rabbit Anti-POLR3B  , Rabbit Anti-POLR3C  , Rabbit Anti-POLR3D  , Rabbit Anti-POLR3E  [1:333], Rabbit Anti-POLR3G  , Rabbit Anti-POLR3GL  [1:150], Rabbit Anti-MYC  , Rabbit Anti-Lamin B2  , and Rabbit Anti-TUBB  . Source Data file includes uncropped immunoblot sections.THP-1 lysates were extracted using the mirVana PARIS RNA and Native Protein Purification kit  following standard protocols, in tandem with total and small RNA isolation experiments where applicable. Total protein concentration was determined using Pierce BCA protein assay kit , and equivalent protein fractions, diluted in cell lysis buffer, were prepared in 4× NuPAGE LDS sample buffer  and 10× NuPAGE reducing agent . Proteins were separated on NuPAGE 4-12% Bis-Tris Protein Gels  using MES SDS Running Buffer  and transferred onto polyvinylidene difluoride membrane 0.2 um . Transfer membranes were blocked with 5% blotting-grade blocker, nonfat milk , followed by incubation with primary antibody at 46 cells/mL and cross-linked with rotation at room temperature in 1% formaldehyde for 10 min. Cross-linking was quenched with the addition of 200 mM glycine and an additional 5 min of rotation at room temperature. Cross-linked cells were then spun down and resuspended in 1× RIPA lysis buffer, followed by chromatin shearing via sonication . Individual ChIP experiments were performed on pre-cleared chromatin using antibody-coupled ChIP grade Protein G magnetic beads . POLR3A antibody was obtained from Abcam (ab96328 lot#GR318563). POLR3B antibody was obtained from Bethyl (A301-855A). POLR1D antibody was obtained from Bethyl (A304-847A). POLR3C antibody was obtained from Bethyl (A303-063A). POLR3G antibody was obtained from Invitrogen (PA5-51120 lot#UG2803044). POLR3GL antibody was obtained from Novus Biologicals (NBP1-79826). POLR3D antibody was obtained from Bethyl (A302-295A). POLR3E antibody was obtained from Bethyl (A303-708A). BRF1 antibody was obtained from Abcam (ab74221). GTF3C1 antibody was obtained from SigmaAldrich (PLA0180). IgG antibody was obtained from ThermoFisher Scientific (NeoMarkers NC-100-P0). 5 ug of antibody per ChIP was coupled to 18 uL of beads and rotated overnight with sheared chromatin at 4°C. Beads were then washed 5× in ChIP wash buffer (Santa Cruz), 1× in TE, and chromatin eluted in TE + 1% SDS. Cross-linking was then reversed by incubation at 65°C overnight, followed by digestion of RNA (30 min RNase incubation at 37°C) and digestion of protein (30 min proteinase K incubation at 45°C). ChIP DNA was then purified on a minElute column (Qiagen), followed by DNA library preparation (NEBNext Ultra II DNA Library Prep Kit for Illumina) and size selection of 350-550 bp fragments via gel extraction (Qiagen).Equal numbers of THP-1 monocytes, THP-1 derived macrophages, and ML-60218 treated monocytes were collected (~10 million cells per ChIP experiment) and resuspended in growth media at 1 × 10https://egg2.wustl.edu/roadmap/web_portal/ /processed_data.html#RNAseq_uni_proc. The Cancer Genome Atlas (TCGA) primary solid tumor Pol III subunit expression levels were extracted across all disease cohorts from the Broad Institute TCGA Genome Data Analysis Center (GDAC) Firehose mRNASeq Level 3 RSEM gene normalized data files at https://gdac.broadinstitute.org. Primary solid tumor ATAC-seq alignment bam files were retrieved from the Genomic Data Commons Data Portal . Clinical patient survival outcomes and Kaplan–Meier survival probability data were retrieved from the Kaplan–Meier Plotter Pan-cancer RNA-seq analysis platform 92. High POLR3G, POLR3GL, or MYC expression were defined using a uniform upper tertile patient split across all cancer types, compared to the remaining cohort as shown. KMplot data visuals were generated using R packages surviplot (https://github.com/aroneklund/surviplot) and survival . Model cell-type illustrations were created using BioRender.com.Previously reported THP-1 RNA-seq, ATAC-seq, and nascent small RNA sequencing files were obtained from Gene Expression Omnibus (GEO) series GSE96800. ATAC-seq and gene expression data derived from primary immune progenitor and primary differentiated immune cell populations were obtained from GEO series GSE74912 and GSE118119, respectively. NIH Roadmap Epigenomics gene expression profiles related to immortalized cell line (ENCODE) and multi-tissue Pol III subunit levels  prior to downstream sequence alignment and analyses. Trimmed paired-end ChIP sequencing reads were mapped to the GRCh38 genome using bowtie version 2.2.4 with settings “bowtie2 -t –sensitive -x”93 and processed with samtools version 1.3.194. Read counts were extracted over a comprehensive RNAcentral database annotation of noncoding RNAs56 using bedtools version 2.23.095. Gene annotation set and corresponding GRCh38 coordinates were downloaded from the contemporaneous RNAcentral database (release version13):ftp://ftp.ebi.ac.uk/pub/databases/RNAcentral/releases/13.0/genome_coordinates/bed/homo_sapiens.GRCh38.bed.gz. RNAcentral coordinate IDs were mapped to Ensembl, Gencode, GtRNAdb, HGNC, Lncbase, Lncbook, Lncipedia, Lndcrnadb, miRbase, NONCODE, Refseq, and Rfam. To remove duplicate coordinate gene entries derived from multiple databases, genes with start and end coordinates within 50 bp of each other were merged into singular coordinate entries. Gene-specific raw ChIP-seq signal counts were extracted and include the flanking 150 bp for each RNAcentral gene coordinate entry. ChIP-seq data were used to compare Pol III subunit binding patterns  were sequenced together on an Illumina HiSeq4000 . Sequencing was performed by the Genome Sequencing Service Center by Stanford Center for Genomics and Personalized Medicine. Sequencing reads were trimmed using Trim Galore v.0.4.0 : ftp://ftp.ebi.ac.uk/pub/databases/RNAcentral/releases/13.0/genome_coordinates/bed/homo_sapiens.GRCh38.bed.gz. RNAcentral coordinate IDs were mapped to Ensembl, Gencode, GtRNAdb, HGNC, Lncbase, Lncbook, Lncipedia, Lndcrnadb, miRbase, noncode, Refseq, and Rfam. To remove duplicate coordinate gene entries derived from multiple databases, genes with start and end coordinates within 50 bp of each other were merged into singular coordinate entries. Gene-specific raw small RNA signal counts were extracted and include the flanking 25 bp for each RNAcentral gene coordinate entry. Differential count statistics corresponding to small RNA changes were derived from the exactTest function of the edgeR package for differential expression analysis97 over the entire RNAcentral annotation set. Downstream analyses and visualizations subset on canonical Pol III-transcribed genes and, specifically, the 350 genes confidently identified as Pol III-occupied in our system by ChIP-seq analysis.For analysis of Pol III-transcribed small RNA abundance, we mapped nascent and steady-state small RNA reads to the entire genome space (GRCh38) to avoid false-positive signal arising from sequence reads unrelated to Pol III-transcribed genes that might otherwise occur using a limited reference setPrimary Immune cell populations: Simultaneous ATAC-seq and gene expression data derived from primary immune progenitor and primary differentiated immune cell populations were obtained from GEO series GSE74246, GSE74912, GSE118165, and GSE11811965. Pol III subunit expression profiles were extracted from previously processed gene abundance analysis files (GSE74246_RNAseq_All_counts.txt and GSE118165_RNA_gene _abundance.txt). For the purpose of Pol III identity and gene accessibility correlations, the log2 ratio of POLR3G and POLR3GL abundance levels within individual samples was considered. Raw ATAC-seq data were downloaded and mapped to the GRCh38 genome using bowtie version 2.2.4 with settings “bowtie2 -t –sensitive -x”93. Read counts were extracted over a comprehensive RNAcentral database annotation of noncoding RNAs56. Gene annotation set and corresponding GRCh38 coordinates were downloaded from the contemporaneous RNAcentral database (release version13): ftp://ftp.ebi.ac.uk/pub/databases/RNAcentral/releases/13.0/genome_coordinates/bed/homo_sapiens.GRCh38.bed.gz. Gene-specific raw ATAC-seq signal counts were extracted and include the flanking 150 bp for each RNAcentral gene coordinate entry. Gene accessibility signal counts were rank normalized across 248 ATAC-seq experiments, including unstimulated and stimulated immune cell experiments65. In total, 188 simultaneous ATAC-seq and RNA-seq profiles were matched by sample ID, and the Pearson correlation determined for the log2(POLR3G/POLR3GL) ratio and normalized gene accessibility profile in individual samples : Pol III subunit expression levels were extracted across all TCGA disease cohorts from the Broad Institute TCGA Genome Data Analysis Center (GDAC) Firehose mRNASeq Level 3 RSEM gene normalized data files at https://gdac.broadinstitute.org. For analysis of Pol III subunit expression changes in individual disease cohorts, patient-matched normal tissue (11) and primary solid tumor (01) expression profiles were merged by participant ID. Disease cohorts with ≥5 patient-matched samples were considered . TCGA POLR3G/POLR3GL expression ratios were derived as the intra-sample ratio of RSEM gene normalized POLR3G expression counts/POLR3GL expression counts, an internally controlled comparative measure of the relationship between POLR3G and POLR3GL in either normal or tumor samples. Primary solid tumor ATAC-seq alignment bam files were retrieved from the Genomic Data Commons Data Portal . Read counts were extracted over a comprehensive RNAcentral database annotation of noncoding RNAs56. Gene annotation set and corresponding GRCh38 coordinates were downloaded from the contemporaneous RNAcentral database (release version13): ftp://ftp.ebi.ac.uk/pub/databases/RNAcentral/releases/13.0/genome_coordinates/bed/ homo_sapiens.GRCh38.bed.gz. Gene-specific raw ATAC-seq signal counts were extracted and include the flanking 150 bp for each RNAcentral gene coordinate entry. Gene accessibility signal counts were rank normalized across 410 TCGA ATAC-seq experiments. In total, 388 ATAC-seq and RNA-seq profiles were matched by primary solid sample ID, and the Pearson correlation determined for the log2(POLR3G/POLR3GL) ratio and normalized gene accessibility profile in individual samples  are considered. Maximum enrichment scores , that are shared between 2 or more independent experiments  at a given cutoff level wherein the number of top genes (res Fig.  represenFurther information on research design is available in the Supplementary InformationReporting Summary


SNR6. The gene is essential, transcribed by the enzyme RNA polymerase III, and codes for the U6snRNA required for mRNA splicing. A translationally positioned nucleosome on the gene body enables the assembly factor TFIIIC binding by juxtaposing its otherwise widely separated binding sites, boxes A and B. We found histone depletion results in the loss of U6 snRNA production. Changing the rotational phase of the boxes and the linear distance between them with deletions in 5 bp steps displayed a helical periodicity in transcription, which gradually reduced with incremental deletions up to 40 bp but increased on further deletions enclosing the pseudoA boxes. Nhp6 influences the transcription in a dose-dependent manner, which is modulated by its previously reported co-operator, an upstream stretch of seven T residues centered between the TATA box and transcription start site. Nhp6 occupancy on the gene in vivo goes up at least 2-fold under the repression conditions. Nhp6 absence, T7 disruption, or shorter A–B box distance all cause the downstream initiation of transcription. The right +1 site is selected with the correct placement of TFIIIC before the transcription initiation factor TFIIIB. Thus, the T7 sequence and Nhp6 help the assembly and placement of the transcription complex at the right position. Apart from the chromatin remodelers, the relative rotational orientation of the promoter elements in nucleosomal DNA, and Nhp6 regulate the transcription of the SNR6 gene with precision.A small non-histone protein of budding yeast, Nhp6 has been reported to specifically influence the transcription of a yeast gene,  Nucleosomal arrays are non-randomly punctuated by the nucleosome-free regions (NFRs), which are generally hotspots of high transcription activity, promoter and enhancer elements, replication origins, fragile genomic sites, etc. The U6 snRNA gene is one of the few examples where positioned nucleosomes have been shown to cause its transcriptional activation  gene has an unusual organization  were derived from the plasmid pCS6 . The chromatin was assembled using the well-established plasmids . The in n method . All traSNR6 gene was shown to enable the binding of TFIIIC and high transcriptional activation in vitro (δ element (YLRCdelta5)  is activated  . The PICnd box A . We had nditions . We founctivated , confirmSNR6 and some tRNA genes in vivo  could  reduces transcription from 4) could . In agre4) could , with th4) could . Moreove4) could . In the 4) could . Surpris4) could . The afoin vitro chromatin transcription of a tRNA gene  activation in the presence of TFIIIC (p = 0.0082) activation for dT7 -TFIIIC resulted in repression  of transcription in the absence of Nhp6 , indicating a reduced TFIIIC binding to the dT7 chromatin. Consistent with an earlier report (7 element to manifest its influence fully on the transcription of SNR6.The Nhp6 effects on the TFIIIC-dependent dT of Nhp6 , whereasr report , the res7 sequence regulates the dose-dependent effects of Nhp6 on the TFIIIC-dependent chromatin transcription of the SNR6 gene. Taken together, this study has demonstrated that reducing the distance by short 5–40 bp deletions between the terminator and box B does not improve transcription; a longer deletion including extragenic pseudoA boxes does. Chromatin transcription from yeast SNR6 is activated at lower and repressed at higher Nhp6 levels. Nhp6 increases transcription fidelity by abolishing non-canonical initiations in favor of +1 transcription. This transcriptional activation depends on TFIIIC and the cis promoter element T7 stretch. Occupancy of specifically the Nhp6A isoform on the gene goes up under repression, attributing a repressive role to Nhp6 in keeping the highly active SNR6 gene expression under check in vivo.The aforementioned results show that the TSNR6 gene sequence directs the assembly of nucleosomes with unique rotational settings on the whole gene (Reducing the distance between the terminator and box B may constrain the TFIIIC binding whenever the A–B boxes do not fall in phase. For a particular DNA sequence wound over the nucleosome surface, rotational positioning decides the DNA phase accessible for a DNA-binding factor , while pole gene . The nucole gene . Consideole gene . Therefoin vivo (Further deletions may either include the A/B boxes in the core DNA wound over the nucleosome making TFIIIC/B binding non-productive, or the TFIIIC binding may exclude the nucleosome, alleviating the chromatin repression. The increase in TFIIIC-dependent ND transcription on d55, d60, and d65 plasmids may be explained by the absence of the interfering pseudoA boxes, whereas the opposite results on the chromatin may be the outcome of two effects. First, the TFIIIC binding may lead to nucleosome exclusion but a steric obstruction of the gene body may reduce transcription. Alternatively, in the TFIIIC absence, the nucleosome may be found only downstream of the +85 bp position, as seen earlier in vivo . This woSNR6 in a strain with deletion of 42 bp between boxes A and B, was found similar to that in a strain with a lethal point mutation on box B (SNR6 is reported to be an inefficient step during transcription complex assembly in vitro (SNR6 transcription and synthetic lethality of Nhp6 with a 42 bp deletion between the terminator and box B, reducing the distance between boxes A and B to the near subnucleosomal size (SNR6 TATA box mutations (nhp6ΔΔ condition (SNR6in vitro (in vivo chromatin structure altered around the TATA box region of SNR6 in the nhp6ΔΔ cells was taken as an indication of altered TFIIIB binding, which could be a reason for the transcriptional repression of SNR6 (SNR6. This may be the reason that earlier a deletion of 42 bp between the terminator and box B showed synthetic lethality with several other promoter mutations in SNR6 (The chromatin footprint on on box B . The recin vitro  and Nhp6in vitro . A positmal size , synthetutations , and nhpondition , all couin vitro . The in  of SNR6 . Therefo in SNR6 .7 mutations do not abolish the TFIIIB footprint but show lethality in the absence of Nhp6 (7 stretch are found near the exit point of DNA in the US nucleosome, box A sits close to the DNA entry spot in the A–B box nucleosome. Nhp6 is generally found in the NFR near the entry/exit points of nucleosomal DNA (7 stretch, just upstream of TSS and stabilize the TFIIIC interaction with box A in turn. This is consistent with the highest association of Nhp6 with TFIIIC, out of all the components of the pol III transcription complex (7 may interfere with the encroachment of NFR by the US regulatory nucleosome on SNR6 (The T of Nhp6 . While tomal DNA  and Nhp6omal DNA . Togethe complex . The inh on SNR6 .SNR6 ND was seen even up to 500-ng Nhp6 addition (in vitro. At lower levels, it caused even activation by enhancing the +1 transcription initiation. The requirement of both the upstream T7 stretch and TFIIIC for transcription activation by Nhp6 implies a balancing role for the T7 element in the dose-dependent effects of Nhp6. As Nhp6A is an abundant, non-sequence-specific DNA-binding protein, its effects may easily be dose-dependent in vivo. Increased Nhp6A occupancy on the SNR6 gene under repression is consistent with a role for Nhp6 in further establishing the repressed chromatin state of SNR6.Transcription was found refractory to ∼300 ng Nhp6, whereas after saturation at ∼100 ng, higher Nhp6 additions inhibited chromatin transcription . No tranaddition , whereasSNR6 is regulated by its unique chromatin organization and targeted by a plethora of epigenetic regulatory complexes (SNR6 transcription. The effects are individually small but subtle and significant when together. Enhancing transcription activation on the SNR6 chromatin by Nhp6 is the outcome of a combined influence of TFIIIC and the T7 element on chromatin transcription while T7 stretch also affects TFIIIC binding. It appears that every part of the SNR6 gene sequence has evolved with a unique role in fine-tuning its chromatin expression levels, making SNR6 a specific target for Nhp6.Yeast omplexes . This st


IgH) gene, is responsible for the expression of all the different antibody isotypes. Transcriptional regulation of the IgH gene is complex and involves several regulatory elements including a large element at the 3’ end of the IgH gene locus (3’RR). Animal models have demonstrated an essential role of the 3’RR in the ability of B cells to express high affinity antibodies and to express different antibody classes. Additionally, environmental chemicals such as aryl hydrocarbon receptor (AhR) ligands modulate mouse 3’RR activity that mirrors the effects of these chemicals on antibody production and immunocompetence in mouse models. Although first discovered as a mediator of the toxicity induced by the high affinity ligand 2,3,7,8-tetracholordibenzo-p-dioxin (dioxin), understanding of the AhR has expanded to a physiological role in preserving homeostasis and maintaining immunocompetence. We posit that the AhR also plays a role in human antibody production and that the 3’RR is not only an IgH regulatory node but also an environmental sensor receiving signals through intrinsic and extrinsic pathways, including the AhR. This review will 1) highlight the emerging role of the AhR as a key transducer between environmental signals and altered immune function; 2) examine the current state of knowledge regarding IgH gene regulation and the role of the AhR in modulation of Ig production; 3) describe the evolution of the IgH gene that resulted in species and population differences; and 4) explore the evidence supporting the environmental sensing capacity of the 3’RR and the AhR as a transducer of these cues. This review will also underscore the need for studies focused on human models due to the premise that understanding genetic differences in the human population and the signaling pathways that converge at the 3’RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions, including emerging infections such as SARS-CoV-2.One gene, the immunoglobulin heavy chain ( IgH) gene is responsible for the expression of all Ig classes/isotypes, i.e. IgM, IgD, IgG, IgE, and IgA. These isotypes have different effector functions in mediating immunity  production significantly impacts human health by weakening the ability to maintain immunocompetence and survive infectious diseases –4. Conveimmunity –11. Therfunction . Howeverfunction , 14. Stufunction . Severalfunction . In animss clear Table 1.sitivity , 71. Thisitivity –75. Furtsitivity –79. Howe defined  as well d humans . These dThe AhR signaling pathway was first characterized in the upregulation of Phase I and II metabolic enzymes upon exposure to halogenated aromatic hydrocarbons (HAH), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). The AhR is a ligand-activated nuclear receptor that when activated by ligand translocates from the cytosol to the nucleus where it binds the AhR nuclear translocator (ARNT). The AhR/ARNT transcription factor complex binds to dioxin response elements (DRE) and through interactions between the AhR transactivation domain and co-activators or co-repressors modulates transcription of sensitive genes  Figure, 83. HowC. elegans (platelmint) AhR homolog does not bind dioxins and has low amino acid identity with mouse and human in the ligand binding domain, suggesting either binding to unknown endogenous ligands or ligand-independent effects , which is also a nuclear receptor, can bind a wide range of ligands. An increasing number of chemicals in the environment, including natural and industrial chemicals, as well as dietary and pharmaceutical chemicals have been shown to bind the AhR with varying affinity , 103. Inb]carbazole (FICZ) is more potent at inducing AhR activity in a rat hepatic cell line as compared to one from human  to bind bacterial ligands and efficiently fine tune a more complex immune response to commensal and pathogenic bacteria. There is a growing body of literature supporting a role of the AhR in most aspects of the immune system  region and a constant (C) region. The V regions encode for the antigen-binding pocket and have high sequence variability, whereas the C regions have very little sequence variability. The C region of the heavy chain portion of the Ig defines the five Ig classes  and consequently determines the antibody properties and effector functions. The main function of antibodies is to bind or opsonize (i.e. coat) the antigen to either neutralize it by preventing it from binding its cellular target, or to flag it for destruction and/or clearance by soluble immune mediators (e.g. complement proteins) and other immune cells (e.g. phagocytes) .Antigen recognition by antibodies is highly specific and the immune system has the capacity to produce different Igs specific for different antigens, which is remarkable considering that only two genes, the heavy and light chain Ig genes, encode for antibodies. This diversity is possible due to a process called random or somatic V(D)J recombination, which only occurs in developing B lymphocytes and produces an incredible amount of diversity starting from just a single pair of alleles for both the heavy chain gene and the two light chain genes (i.e. λ and κ) in each individual genome Figure 2Initial activation of B lymphocytes by a particular antigen results in production and secretion of IgM, which is pentameric. Since IgM affinity for antigen is generally low during a primary or initial immune response, a pentameric structure with 10 antigen binding pockets enhances the ability of IgM to interact with the antigen . As actiIgH gene are contributing factors. We will first review studies supporting altered Ig production by chemicals that have been shown to bind the AhR.Because of the central role of Ig in humoral immunity, any impairment in Ig production can lead to increased infections and higher morbidity –3. Primain vivo and in vitro rodent models (+ B cells expressing a high level of lymphocyte-specific protein tyrosine kinase (LCK)  , 41, 42.pulation . Furthero dioxin . Additio+ B cells contrast with mouse studies, which support decreased Ig secretion due to an AhR-mediated transcriptional inhibition of Ig genes  of the gene that encodes AID (i.e. aicda). These results suggest that the AhR, independent of exogenous ligand, is a physiological negative regulator of AID (aicda mRNA and AID protein (H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester]) on stimulation-induced aicda expression (AICDA gene did not identify AhR binding motifs (data not shown). However, this does not preclude the possibility of the AhR binding to DNA through a non-canonical signaling pathway  – was identified as a direct target of the AhR . In thesr of AID . Additio protein . In contpression , 49. Howvia species-specific pathways  and increased hypersensitivity (i.e. non-specific increase in IgE) as supported by animal models and epidemiology studies Table 1.IgH gene is governed through a complex interaction of several regulatory elements, whose activity is B-lymphocyte specific and depends on the cellular maturation state. The most 5’ regulatory element is the variable heavy chain (VH) promoter, which lies immediately upstream of the V region and contributes to B-lymphocyte specific activity of the IgH locus (IgH remodeling via DNA recombination are the 5′ μ enhancer (Eμ) and the 3′ IgH regulatory region (3′RR), an enhancer complex mapped downstream of the cluster of C region genes   . Despiteebrates) –169. Howd by CSR , 170. Adocations , 170.IgH and AhR genes in cartilaginous fish. Unlike the multiple IgH genes that are not conserved beyond cartilaginous fish, multiple AhR genes appear to have been maintained in many species; however, xenopus, mouse, gorilla, and human appear to be limited to having only one AhR gene  demonstrates an increase in C-region genes as well as duplications within specific heavy chain classes  and the Catarrhini Parvorder  because of a large duplication of a portion of the IgH region. This divergence allowed for the emergence of additional antibody subclasses  in the Catarrhini Parvorder  to promote IgH expression and CSR  also exhibited no correlation between allele *2 and high Ig titers . Serone IgH gene has been largely defined in rodents. The mouse 3′RR consists of a set of enhancers, either rodent-specific  or conserved in vertebrates   Figure 5proaches , 241–244proaches , 246. Deproaches , 156–159romoters , 247. Th isotype , 249. Re isotype . Germlin isotype , 251–253 isotype , 254. Tama cells .IgH expression and antibody secretion  is expected to be a factor on the transcription factor milieu and the effects of environmental factors. Multiple transcription factors such as the AhR, NFκB/Rel, POU/Oct, Pax5 have been identified as positive and/or negative regulators of the mouse 3′RR, which may be dependent on the maturation stage of the B cell and the specific enhancer. Co-activation of β-adrenergic receptors  and of CD86 (antigen stimulation) induced binding of OCA-B (Oct-1-associated coactivator)/Oct-2 dimers to octomer motifs within the mouse hs1.2 and hs4 enhancers  involved in antibody production. An additional consideration is the genetic polymorphisms within the human hs1.2 enhancer, which, as discussed in the previous section, has been associated with Ig expression levels and several diseases , these studies suggest a strong cooperative activation when all of the human 3′RR enhancers  are linked together . Howeverelements –199, 201diseases , 222–224In vitro analysis of human hs1.2 enhancer activity using reporter plasmids demonstrated that the hs1.2 alleles harboring a higher copy number of the 40 bp monomer have increased transcriptional activity  only identified ER consensus sites outside of the monomer repeat  in human evolved to adapt to internal stimuli and changing environmental conditions, and that xenobiotics, such as chemicals in pollution and forest fires, and disease conditions significantly disrupt 3′RR activity by high jacking its environmental sensing function and tipping the balance towards a pathological response. Understanding genetic differences and the signaling pathways that converge at the 3′RR will provide valuable insight into individual sensitivities to environmental factors and antibody-mediated disease conditions.Snyder and coworkers  suggesteCS and DF contributed to conception and design of the review. CS and DF wrote the first draft of the manuscript. PD performed the in silico analysis. CS and PA contributed figures and figure legends. CS finalized the manuscript for submission. All authors contributed to the article and approved the submitted version.


Ewing sarcoma (EwS), a type of bone and soft tissue tumor, is mainly driven by the expression of the fusion protein EWSR1-FLI1. Upon binding to chromatin, EWSR1-FLI1 reprograms the epigenetic state, alters gene expression, and thus leads to tumorigenesis. Considerable studies have investigated the epigenomic and transcriptomic profiling of EwS. Nevertheless, a comprehensive view of therapeutic targets is still lacking. This review discusses the epigenetic and transcriptional alterations reported in EwS. Specifically, we discuss the binding characteristics of EWSR1-FLI1 on chromatin, the mechanisms of EWSR1-FLI1 in reprograming epigenome, and EWSR1-FLI1-induced transcriptional alterations. Moreover, we summarize the chemical, RNAi, and CRISPR-cas9 high throughput screens conducted in EwS with the goal of assisting in the development of novel therapies to treat this aggressive disease. In the last decade, next generation sequencing (NGS) technologies have improved our understanding of cancer mechanisms at a higher molecular level . For exaEwing sarcoma (EwS) is the second most common primary malignant bone tumor in pediatric patients . It accoAt the cellular level, EwS has a poorly differentiated stem cell-like phenotype with some degree of neurogenic features ,4,5. HisSince EWSR1-FLI1 was identified in the 1990s . MoreoveActing as an aberrant oncogenic transcription factor, EWSR1-FLI1 binds to DNA via the FLI1′s ETS domain and induces gene expression via the EWSR1 amino terminal TAD domain . MountinIn eukaryotes, DNA is packaged into chromatin through interacting with histones ,28. ChroMounting evidence has shown that dysregulated epigenetic modifications are as significant as genetic mutations and can act as oncogenic driver lesions causing an autonomous growth of cancer cells . A betteAs described above, EWSR1-FLI1 not only binds to chromatin but also changes the chromatin state to rewire cells for malignancy. Studies have sought to identify EWSR1-FLI1 binding sites on chromatin and map the epigenetic state associated with EWSR1-FLI1. To this end, it has been revealed that EWSR1-FLI1 binds not only to promoters in the vicinity to the transcription start sites (TSSs) but also to enhancers at a long distance ,22. At tSince 2008, several research groups have demonstrated that EWSR1-FLI1 uses GGAA-containing elements (microsatellites) as specific response elements to up-regulate genes dispersed throughout the human genome ,20,34,35The binding of EWSR1-FLI1 at canonical ETS binding sites (where ETS transcription factors bind) has also been described in both non-malignant endothelial cells and malignant EwS cells ,21. At tTo map chromatin context that are EWSR1-FLI1, Riggi et al., compared the EWSR1-FLI1 binding sites with four histone modifications H3K4me1, H3K4me2, H3K4me3, and H3K27ac using ChIP-seq . They foTo investigate the underlying regulatory mechanism of how EWSR1-FLI1 alters epigenetic states, Tomazou et al., mapped the epigenome in a cellular model that is dependent on EWSR1-FLI1 and compared the epigenome states of EWSR1-FLI1-high and EWSR1-FLI1-low EwS cells . In thisEWSR1-FLI1 can alter chromatin states, although it does not have histone modification activity. For example, the expression of EWSR1-FLI1 in primary pediatric mesenchymal stem cells (MSCs) increased H3K4me1 and H3K27ac modification levels at EWSR1-FLI1 binding sites. On the contrary, the knockdown of EWSR1-FLI1 in EwS cells reduced H3K27ac levels at EWSR1-FLI1 binding sites. To understand how EWSR1-FLI1 alters histone modifications, several studies have demonstrated that EWSR1-FLI1 acts as a pioneer factor that recruits histone methyltransferases and acetyltransferases on chromatin to modify histones b.In 2014, Riggi et al., showed that 75% of activated EWSR1-FLI1 binding sites were enriched with acetyltransferase p300, a chromatin regulator which acetylates H3K27  Figure b. FurtheAlso known as SWI/SNF (switch/sucrose non-fermentable) complex, BAF is another complex which can be recruited by EWSR1-FLI1 onto chromatin  Figure b. The BALSD1  is another histone modifier that interacts with EWSR1-FLI1  Figure b. As theAnother well-studied EWSR1-FLI1 interacting chromatin modifier is RING1B ,53. ActiAs described above, EWSR1-FLI1 not only binds to promoters but also enhancers. STAG2 has been identified as critical for the binding of EWSR1-FLI1 at the super enhancer regions and EWSR1-FLI1-mediated transcription ,57. STAGBy removing acetylation from proteins including histones, HDACs increase chromatin condensation to repress gene expression. It has been observed that EWSR1-FLI1 could recruit HDACs to gene promoter regions to conduct its gene repression function ,63. ConsAs introduced above, the expression of EWSR1-FLI1 in EwS results in a potent chimeric oncoprotein with novel biological properties and a unique transcriptional signature essential for oncogenesis. Mechanistically, EWSR1-FLI1 could act as both a transcription activator and a transcription repressor; while both functions are essential for EWSR1-FLI1 in mediating oncogenesis. By altering gene expression at the molecular level, EWSR1-FLI1 plays roles in a variety of cellular processes including cell cycle, apoptosis, metabolism, angiogenesis, chromosome segregation, and cell migration. Identification of dependencies incurred by EWSR1-FLI1 expression would offer a compelling avenue for the development of effective targeted therapies. Previous reports have described that rather than inducing specific signaling pathways, EWSR1-FLI1 activates a larger set of genes which are involved in a variety of cellular processes. Here, we summarize the studies that characterized genes whose expressions are directly regulated by EWSR1-FLI1.Studies have found that EWSR1-FLI1 can regulate gene transcription by interacting with other transcription effectors on chromatin. For example, it has been reported that EWSR1-FLI1 uses transcription factors E2Fs to facilitate its target gene transcription . There aAnother protein which helps EWSR1-FLI1-mediated transcription is NR0B1  ,74,75,76EWSR1-FLI1 also directly interacts with RHA, a member of the DEXH box helicase family of proteins, to regulate transcription and RNA splicing . RHA, alAs described in 3.1.2., while functioning as one of the EWSR1-FLI1-interacting proteins, NR0B1 was identified as one of the highest up-regulated EWSR1-FLI1 targets ,74,75,79Acting as a direct downstream target of EWSR1-FLI1, EZH2 maintains the stemness feature of EwS cells ,12,85,86Demonstrated by Stephen Lessnick’s group, NKX2.2 is another gene that is highly up-regulated by EWSR1-FLI1 ,63,92. AKDM3A  has also been identified as one of the critical mediators for EWSR1-FLI1-drived EwS growth ,102,103.Another critical factor for EWSR1-FLI1-mediated tumor development is GLI1 ,107,108.Due to the binding of EWSR1-FLI1 at super enhancers, EWSR1-FLI1 target genes are enriched with super-enhancer transcripts. For example, MEIS1 has been identified as one of the super-enhancer transcripts that is transcriptionally induced by EWSR1-FLI1 in EwS . MEIS1 iTo identify regulators which are essential for metastasis, Zhang et al., utilized proteomic approaches and revealed a significant up-regulation of IL1RAP in cells resistant to anoikis (detachment-induced death) . From thIn addition, it has also been documented that EWSR1-FLI1 activates platelet-derived growth factor (PDGF)  and cMYCThe first gene that was identified to be suppressed by EWSR1-FLI1 is TGFBR2 , which eIn 2004, Prieur et al., found that IGFBP3 was up-regulated when EWSR1-FLI1 was inactivated by siRNA, suggesting that the expression of IGFBP3 is suppressed by EWSR1-FI1 in EwS cells . The dirBy comparing EWSR1-FLI1 transcriptional profile data and genome-wide EWSR1-FLI1 binding sites on chromatin, Stephan Lessnick’s group found that there is a large set of genes that are repressed by EWSR1-FLI1 and characterized the transcriptional repressive function of EWSR1-FLI1 . For exaThe enrichment of forkhead box (FOX) recognition motifs was observed in the EWSR1-FLI1-repressed gene set . ConsequOther EWSR1-FLI1-repressed genes also include the tumor suppressor SPRY1  and p21 Studies using either gain-of-function or loss-of-function strategies are essential to decipher roles of the specific epigenetic and transcriptional alterations for cancer growth and survival. In recent years, high throughput biochemical and genetic screens enable us to systematically identify functional mechanisms and novel vulnerabilities in cancers in an unbiased manner. Here, we summarize the high throughput chemical screens, RNA interference (RNAi) screens, and CRISPR-Cas9 screens conducted in EwS .In 2007, Stegmaier et al., performed a small molecular library screen to identify modulators of EWSR1-FLI1 activity in EwS . In thisAs an extension of the Stegmaier et al., study, Boro et al., screened a small molecule compound library enriched for FDA-approved drugs to identify the one with the capacity to modulate the expression of EWSR1-FLI1 target genes . This stRecently, Wang et al., conducted a high throughput screen of 1912 compounds in combination with the FAK inhibitor, PF-562271, to identify new combination treatment approaches with FAK inhibition . From thMore recently, Zhang et al., screened 8000 compounds to identify inhibitors for the treatment of EwS cells harboring mutant STAG2 . From thIn 2010, Arora et al., identified essential kinases that regulate the growth and survival of EwS cells using an siRNA library targeting 572 human kinases . To thisIn 2016, Grohar et al., conducted a genome-wide RNAi screen to identify therapeutic vulnerabilities in EWSR1-FLI1 activity . They foIn 2017, He et al., performed a siRNA-based high throughput screen  to identify genes whose function is critical for EWSR1-FLI1 driven cell viability . This stIn 2018, Stolte et al., used a genome-scale CRISPR-Cas9 screen to discover targets specific for TP53 wild type EwS . They foIn 2021, Seong et al., performed a genome-scale CRISPR-Cas9 screen to identify regulators which control EWSR1-FLI1 protein stability . From thMore recently, another genome-scale CRISPR-cas9 screen was conducted to identify genes whose knockout conferred resistance to the LSD1 inhibitor SP-2509 with the aim of developing effective combination strategies . This stThe development and implementation of genome-wide approaches enables us to characterize the epigenetic and transcriptional regulation mediated by EWSR1-FLI1 in a comprehensive manner. To this end, it has been revealed that EWSR1-FLI1 binds to both GGAA microsatellites and the canonical ETS binding sites on chromatin ,20. UponAn improved understanding of the molecular biology of EwS is essential for developing novel therapies. Considerable work has gone into the identification of the transcriptional targets of EWSR1-FLI1. A larger set of genes have been identified as important for EWSR1-FLI1-mediated tumorigenesis. However, despite the effort, very limited targets have been clinically demonstrated to be of prognostic or therapeutic significance. It has been shown that EwS cells are of significant heterogeneity and EwS cells switch between functionally distinct cell states dependent on EWSR1-FLI1 fluctuations. Cells with high EWSR1-FLI1 expression levels proliferate exponentially, whereas cells with low EWSR1-FLI1 expression tend to be migratory and invasive . The hetSince 2007, several studies have conducted high throughput chemical, RNAi, and CRISPR-Cas9 screens to identify therapeutic targets in an unbiased manner ,142,143.Taken together, studies on EWSR1-FLI1-induced transcriptional and epigenetic changes have shed light on the initiation and progression of EwS. Furthermore, the implementation of high through chemical/genetic screens highlighted exciting opportunities for future EwS treatments. We also foresee the adaptation of state-of-the-art technologies such as the CRISPR gene tiling scan and single-cell CRISPR screens  in the E


Antigenic variation is a successful strategy employed by numerous pathogens to evade the host immune system. It describes the frequent exchange of antigens, which prevents detection and elimination by the host immune system. Strikingly, pathogens from diverse phyla undergo antigenic variation, thereby underlining the efficiency of this strategy for pathogen survival . While ttrans—splicing of a common spliced leader RNA to the 5´end of each mRNA. Due to the unregulated nature of transcription, transcript levels are mainly regulated on the post-transcriptional level in the parasite.African trypanosomes are commonly used model organisms to study antigenic variation due to their large antigen repertoire of more than 2000 antigen-coding genes  as well Trans-splicing ensures maturation of the polycistronic pre-mRNA and mature antigen mRNA is additionally stabilized by m6A RNA modifications in the poly-A tail  each trypanosome selectively expresses one antigen and only few different antigens are expressed in the population at any time; (2) the expressed antigen is frequently exchanged. Notably, antigen expression is governed in a specific manner which differs from other protein-coding genes in the parasite: An antigen-coding gene can only be expressed when located in one of fifteen polycistronic expression sites (ESs), of which only one is active in each cell. To ensure stable levels of antigen mRNA, the active ES is expressed by highly processive RNA polymerase I (Pol I) within a nuclear body referred to as expression site body (ESB) . Trans-sy-A tail . Taken tvia a process referred to as liquid-liquid phase separation, and thereby create subcellular environments with specific functions  . More reubstrate , as wellubstrate . Surprisubstrate . Taken tThe specific function of each condensate in this assembly remains to be experimentally characterized. However, the molecular composition of two condensate groups allows speculation about their function: (1) the ESB and the highly SUMOylated condensate contain factors involved in antigen transcription  ; (2) theIn contrast to the transient transcription and splicing condensates that form co-transcriptionally in other eukaryotes, the multi-condensate assembly in trypanosomes seems rather stable according to imaging data from fixed cells . Therefo6A modification, are coordinated within the multi-condensate assembly.Collectively, the comparison of antigen transcription and splicing condensates in trypanosomes to the respective condensates from other eukaryotes allows a careful first interpretation: the stable association of several nuclear condensates with specific functions at the active antigen-coding gene might spatially coordinate the multi-step-process of antigen expression and confer robustness to it. The ESB Pol I reservoir and the highly SUMOylated body most probably mediate high and robust antigen transcription rates. Further, the three splicing condensates, SLAB, NUFIP and Cajal body, fulfill the high demand for efficient antigen mRNA processing, which is required for successful antigen expression. It remains to be shown which specific function each condensate possesses and which additional processes, such as e.g. mSeeding of nuclear condensates is thought to be mediated by spatially restrained chromatin elements such as DNA, histones or RNA, which recruit freely diffusible proteins to a given locus . RepetitInterestingly, several repetitive DNA sequences are implicated in antigen expression in trypanosomes, indicating a potential role as seed structures . The roltrans-splicing, are required for intact multi-condensate formation. Inhibition of Pol I transcription caused VEX1 and VEX2, two proteins that are part of the multi-condensate assembly, to distribute within the nucleus  awarded to Prof. Nicolai Siegel.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pseudomonas aeruginosa. The transcription of its many Zn transporters is therefore tightly regulated by a known set of transcription factors involved in either the import or the export of Zn. In this work, we show that the Zur protein, a well-known repressor of Zn import, plays a dual role and functions in both import and export processes. In a situation of Zn excess, Zur represses Zn entry, but also activates the transcription of czcR, a positive regulator of the Zn export system. To achieve this, Zur binds at two sites, located by DNA footprinting in the region downstream the czcR transcription start site. In agreement with this regulation, a delay in induction of the efflux system is observed in the absence of Zur and Zn resistance is reduced. The discovery of this regulation highlights a new role of Zur as global regulator of Zn homeostasis in P. aeruginosa disclosing an important link between Zur and zinc export.The control of cellular zinc (Zn) concentrations by dedicated import and export systems is essential for the survival and virulence of The online version contains supplementary material available at 10.1186/s12866-022-02750-4. Among all trace elements, zinc (Zn) is of particular importance because it functions as a cofactor of many essential enzymes and is, more generally, needed to sustain protein structure –3. Zn ister iron .Pseudomonas aeruginosa, several systems are involved in Zn homeostasis [P. aeruginosa, for example, is able to grow and thrive in Zn-poor environments such as the bloodstream, where trace metals are chelated to prevent microbial growth [In the Gram-negative bacterium eostasis , 8. Zn ieostasis , 9. Conteostasis  or a plaeostasis . P. aerul growth . Conversl growth .P. aeruginosa. The major regulator of Zn import is the Zinc Uptake Regulator, hereafter Zur, protein , a Fur ly Np20, . In the ly Np20, , 13. Zn ly Np20, , and Czcly Np20, . Until nP. aeruginosa switches from a low-Zn environment to an excess-Zn situation. We show that Zur accelerates czcR transcription, and thus indirectly activates the transcription of Zn efflux systems by binding to two DNA sites in a region upstream the czcR translation start site (ATG). This new function makes Zur the keystone of Zn homeostasis in P. aeruginosa, acting not only as a repressor of Zn import but also as activator of Zn export.In this study, we discovered that the Zur protein is needed for the full expression of the Zn efflux system when P. aeruginosa genome, we found, as expected, Zur boxes in the promoter regions of genes involved in the import of Zn, but also, intriguingly, upstream of the czcRS genes [czcR upstream region. A clear DNA shift was observed in the presence of Zur  confirmed that a region longer than 292 bps upstream of the ATG is required to sustain czcRS transcription in the presence of Zn , box 2 (mut2), or both (mut1 + 2) Zur boxes were mutated at key positions  was overexpressed in trans by the addition of 0.2 mM IPTG , showedant Fig. . AltogetcR Fig. S.Fig. 5ExP. aeruginosa, eight zinc import and four zinc export systems have been described [P. aeruginosa, being involved not only in the repression of the import of Zn, but also in the early and fast induction of the CzcCBA Zn export system. Zur is therefore a keystone of Zn homeostasis in P. aeruginosa, integrating both the import and export of the ion.Zinc is an essential element whose cellular concentration is tightly regulated . It is vescribed . In caseXanthomonas campestris, for instance, Zur binds a GC-rich sequence overlapping the -35 to -10 region of the XC2976 gene, encoding a CzcD homolog, and directly activates its transcription [Caulobacter crescentus a Zur box, located just upstream the -35 element of a Zn export system operon allows its expression in the presence of Zn [Streptomyces coelicolor, where Zur binds to a Zur box located just upstream the -35 region of the zitB gene, a czcD homolog. Interestingly, at low Zn concentrations, Zur binds as a dimer to this region, weakly activating zitB transcription. As Zn concentration increases, Zur multimerizes on the promoter and increases zitB transcription [Yersinia pseudotuberculosis by binding to the -90 DNA region [Streptomyces avermitilis where Zur, by binding to the transcription start site, activates the transcription of the aveR gene, inducing the synthesis of the Avermectin secondary metabolites [P. aeruginosa, we found that the Zur protein binds in a Zn-dependent manner to two DNA regions (Zur box1 and 2) located 200 bp after the czcR TSS, in the 5’-UTR DNA region. This binding has a positive effect on czcR transcription and, in general, on Zur-mediated Zn resistance.In some other bacteria, Zur also acts as an activator of zinc efflux genes. In cription . In Caulce of Zn . Anothercription . Zur alsA region . This seA region . Other pabolites . In the czcR transcription? Recently numerous two-compoent systems response regulators (TCS-RR) binding sites have been detected in the intergenic region between czcR and czcC [Pseudomonas species analyzed for the presence and location of Zur boxes , only P. aeruginosa possesses boxes upstream of the czcR gene [P. aeruginosa, Zur appears to be the central cog in Zn homeostasis since it controls both the import and export of this metal. CzcR is also known to be capable of controlling the expression of the gene coding for the OprD porin, involved in imipenem resistance, or the lasI gene involved in quorum sensing [czcR expression highlights a novel and important role of Zur in the interaction between Zn homeostasis, virulence factor expression and antibiotic resistance in P. aeruginosa. Deciphering this novel and important regulation occurring by Zur at the czcR gene vicinity is therefore of prime interest.What is the molecular action of Zur for the activation of and czcC . Zur couand czcC , 29. ThezcR gene . This ch sensing , 30. TheP. aeruginosa or 100 μg/mL ampicillin  and 15 μg/mL Tc or Gm for E. coli.The bacterial strains are listed in supplementary table SE. coli DH5α by heat shock, verified by sequencing before being electroporated into the P. aeruginosa wild type or Δzur strains [gfp-fusion assays, the two czcR promoter regions  as indicated in Figs. gfp fusions #1 or #2, overnight cultures were diluted to an OD600 of 0.1 and cultured for 2.5 h in the presence of 30 μM TPEN before adding ZnCl2. Absorbance at 600 nm and fluorescence at 528 nm were then monitored every 15 min. Arbitrary units indicated in the Fig. To monitor the growth of the wt and the mutant strains, overnight cultures were diluted in M-LB medium to an OD600 of 0.05, supplemented with N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine  or ZnClzur:6His on the pMMB66EH or the empty plasmid. 1 ml was collected and centrifuged immediately prior to ZnCl2 addition (t0) and after several time points, as specified in Fig. 600 of 1 gives 0.175 mg/ml of protein) to obtain a final concentration of 2 mg/ml and loaded onto SDS PAGE, using 4–12% precast gels (Thermofisher Scientific). Transfers were performed with iBlot2 transfer stacks (Invitrogen) and nitrocellulose membranes were incubated with the anti-CzcR [Immunoblot analyses were performed as previously described . Brieflynti-CzcR , anti-penti-CzcR  antibodiczcR, was determined by electrophoretic mobility shift assays (EMSA). The znuB gene, the 5’-UTR of czcR, wt or containing the mutated zur box-es , were amplified by PCR from either the P. aeruginosa wt genomic DNA or from the in vitro synthetized mutated DNA  primer as indicated in supplementary table S czcRS plasmid, determined with the same FAM-labeled primers and the Thermo Sequenase Dye kit (Thermofisher Scientific) was used to align the peaks. All fragments were analyzed by capillary sequencing  using ILS600 as a size standard, then peaks were assessed using PeakScanner2 software (Thermofisher Scientific).The P. aeruginosa wt or mutant strains were diluted to an OD600 of 0.1 in M-LB containing 30 μM TPEN and cultured for 2.5 h before being induced with 2 mM ZnCl2. For RT-qPCR, 0.5 ml of culture was mixed with 1 ml of RNA protect (Qiagen) immediately prior to metal addition (t0) and after several time points as indicated in the figures. The P. aeruginosa wt culture intended for the 5’RACE experiment was collected after 5 h of 2 mM ZnCl2 induction. Total RNA extractions were performed as mentioned in previous publications [Overnight cultures of the ications . BrieflyoprF.Quantitative RT-PCR were performed as previously described . BrieflyczcR and czcC genes, were determined using the Rapid amplification of cDNA-5’ends (5’RACE) kit (Roche). Briefly, 5 μg of total RNA from the P. aeruginosa wt were reverse transcribed using the specific primers sp1R for czcR mRNA or sp1C for czcC mRNA. Resulting cDNAs were purified with the Wizard SV Gel and PCR Clean-Up System (Promega) and tailing with dATP and TdT. Tailed-cDNA strands were then amplified with the dT-Anchor primer (Roche) and the specific primer sp2R or sp2C (for czcR cDNA and czcC cDNA respectively). A 20-fold dilution of the first PCR was used as template for a second PCR with primers Anchor (Roche) and sp3R for czcR cDNA or sp2C for czcC cDNA. PCR products were then purified by agarose gel electrophoresis and subcloned into a pCR2.1 vector with the TA Cloning kit (Life Technologies) according to the supplier’s instructions. Plasmids isolated from E. coli clones were analyzed by sequencing. The transcription starts of czcR or czcC indicated in Fig. The transcription starts of the t-test and significance p-values were set at p ≤ 0.05(*) or p ≤ 0.001 (***). For other figures, data points represent the mean of triplicate values.Regarding the graph representations, the mean values of at least three independent experiments are indicated in the figures, along with the corresponding standard deviations. When specified, statistical analyses were performed according to the Student’s Additional file 1:Figure S1. A) DNAse I footprinting of czcR promoter (template strand) in the absence or presence or of 1 µM Zur protein. DNA fragments were analyzed by capillary electrophoresis. The two Zur boxes are indicated by the numbers 1 and 2 in red. The sequencing reaction is visible below the figure (seq1) and the sequence with the two boxes indicated in red is shown below the figure. B) Transcription start site (+1) of czcR and czcC determined by 5’RACE. The CzcR box, according to (1) is indicated in the box and the -35, -10 sequences were determined in silico using BPROM program (2).Additional file 2:Figure S2. Growth curves of the wt PAO1 strain, the Δzur mutant and the ΔzurΔznuB double mutant without or in presence of 2 mM ZnCl2 (Zn). Standard deviations of the triplicates are indicated.Additional file 3:Figure S3. GFP promoter fusions containing 542 bp upstream of the czcC DNA regionincluding either the wt or the mutated Zur boxes (mut1+2). Fusions were transformed into the wt PAO1 strain. Cultures were incubated at 37°C with shaking in a microplate reader and fluorescence measurement was performed every 15 min after the addition of 2mM ZnCl2, or without the addition of Zn, as indicated. The fluorescence values are normalized with cell density. Standard deviations of the triplicates are indicated.Additional file 4:Table S1. Strains and plasmids used in this studyAdditional file 5:Table S2. Primers used in this study