Candida albicans responds to iron deprivation by a global transcriptome reconfiguration known to be controlled by the transcriptional regulators Hap43 , Sef1, and the trimeric Hap2-Hap3-Hap5 complex. However, the relative roles of these regulators are not known. To dissect this system, we focused on the FRP1 and ACO1 genes, which are induced and repressed, respectively, under iron deprivation conditions. Chromatin immunoprecipitation assays showed that the trimeric HAP complex and Sef1 are recruited to both FRP1 and ACO1 promoters. While the HAP complex occupancy at the FRP1 promoter was Sef1-dependent, occupancy of Sef1 was not dependent on the HAP complex. Furthermore, iron deprivation elicited histone H3-Lys9 hyperacetylation and Pol II recruitment mediated by the trimeric HAP complex and Sef1 at the FRP1 promoter. In contrast, at the ACO1 promoter, the HAP trimeric complex and Hap43 promoted histone deacetylation and also limited Pol II recruitment under iron deprivation conditions. Mutational analysis showed that the SAGA subunits Gcn5, Spt7, and Spt20 are required for C. albicans growth in iron-deficient medium and for H3-K9 acetylation and transcription from the FRP1 promoter. Thus, the trimeric HAP complex promotes FRP1 transcription by stimulating H3K9Ac and Pol II recruitment and, along with Hap43, functions as a repressor of ACO1 by maintaining a deacetylated promoter under iron-deficient conditions. Thus, a regulatory network involving iron-responsive transcriptional regulators and the SAGA histone modifying complex functions as a molecular switch to fine-tune tight control of iron homeostasis gene expression in C. albicans.The human fungal pathogen  Candida albicans, the most prevalent human fungal pathogen, is an opportunistic pathogen and becomes virulent in immunocompromised host  and to the promoters of HAP2, HAP3, and HAP43 transcriptional regulator genes  of low iron-induced genes including several iron uptake genes  II recruitment, thereby providing tight transcriptional control of iron homeostasis.Transcriptional control is intimately linked to the chromatin state . Acetylaalbicans . SeveralA and B) and SC-LIM media (A and B). The elevated Sef1 and Hap3 protein levels in −Fe medium (A and B) correlated well with the upregulation of the cognate mRNAs  or BPS plus FAS (+Fe). Quantification of the western blot data showed that although Hap2, Hap3, Hap5, and Sef1 are all expressed under both −Fe and +Fe conditions, only Hap3 and Sef1 proteins were upregulated (∼3.5-fold) in the BPS-containing YPD , A and BIM media , A and Be medium , A and Bte mRNAs , 14. Alt induced , 14, ourHAP43 expression is controlled at the level of transcription, we constructed a HAP43-lacZ promoter fusion and analyzed expression in WT, hap43Δ, and sef1Δ mutants. The reporter fusion was activated by approximately 6-fold in WT strain in −Fe medium (A), and this activation was not impaired in the hap43Δ mutant indicating that HAP43 expression is not controlled by Hap43. In the sef1Δ mutant, however, the activation of HAP43-lacZ reporter was only partially reduced (approximately 2-fold) compared with the WT level (A). Therefore, we quantified HAP43 mRNA level in −Fe versus +Fe condition and found that the mRNA is induced ∼6-fold in WT, but the induction was lost in the sef1Δ mutant cells (B). Western blot analysis showed that although Hap43 protein level was also substantially reduced (C and D), the Hap43 protein was still detectable in sef1Δ/Δ mutant cell extracts, indicating Sef1-independent partial expression of HAP43 in iron-starved cells.To determine if e medium A, and thWT level A. Therefnt cells B. Wester reduced , C and Dhap3Δ/Δ, or hap2Δ/Δ strain backgrounds. In each case, we used TAP-tagged subunits to CoIP and probe the western blots with anti-FLAG antibody to identify protein–protein interactions. The data showed that Hap5 strongly pulled down Hap3 from WT cell extracts grown in −Fe medium, but not from +Fe cell extracts , likely due to the diminished expression of the Hap3 protein in +Fe compared with −Fe . The Hap2 protein was efficiently coimmunoprecipitated by Hap5 from cell extracts grown in +Fe or −Fe media . Moreover, the Hap2-Hap5 interaction was greatly impaired by the hap3Δ mutation in −Fe but not in +Fe condition . The CoIP data also showed that Hap5 interacts with Hap3 in both −Fe and +Fe cell extracts that is largely unaffected by hap2Δ/Δ mutation . Together these CoIP data indicated that the induced expression of Hap3 under −Fe condition is essential for Hap5-Hap2 interaction and assembly of Hap3 to form the HAP trimeric complex under iron starved condition. In +Fe condition, however, Hap5 interacted with Hap2 despite the diminished Hap3 protein level, thus indicating Hap3-independent interaction of Hap5 and Hap2.To examine the requirement of the HAP subunits for HAP complex formation, we conducted CoIP assays using epitope-tagged Hap5, Hap2, and Hap3 in WT, in vivo occupancy of each subunit of the HAP complex and the Sef1 regulators. Our previous studies showed that under iron deprivation conditions Hap5 was recruited to FRP1 promoter (−84 to −281 with respect to ATG) and ACO1 promoter (−201 to −340) bearing the CCAAT motif  and at the ACO1 promoter (B) in −Fe condition compared with +Fe condition. We also extended the analyses to other low-iron-induced promoters  as well as to another low-iron-repressed promoter (CYC1) using the same ChIP samples. As expected, occupancy of Hap5 was elevated at all three promoters under −Fe compared with +Fe condition (C). While Hap43 occupancy was undetectable at the FRP1 promoter (A), its occupancy was substantially high at the ACO1 promoter (B) and the CYC1 promoter under Fe-deplete condition (C). The ChIP analysis also showed Sef1 occupancy at FRP1, FTR1, SIT1, and ACO1 promoters but not to the CYC1 promoter (C). Given that Sef1 is a transcriptional activator, it was surprising to detect Sef1 occupancy at the ACO1 promoter under −Fe condition. Indeed, previous ChIP-chip data also seemed to identify Sef1 binding to the promoters of ACO1, ISA1, HEM3, and SEF2 genes whose expression is repressed under iron-deficient condition  was elevated and, surprisingly, to the low-iron-repressed ACO1 promoter but not to the CYC1 promoter. Thus, iron deprivation elicited the induction of HAP trimeric complex occupancy at both iron uptake and iron utilizing gene promoters. Furthermore, iron deprivation also elicited the induction of Sef1 and Hap43 occupancy at the iron uptake and the iron utilizing gene promoters, respectively.Together these ChIP data confirmed that the HAP trimeric complex is recruited to low-iron-induced iron uptake gene promoters as well as to the low-iron-repressed iron utilizing gene promoter under iron deprivation conditions. Hap43, however, showed a robust recruitment only to the low-iron-repressed promoters. Furthermore, Sef1 occupancy to low-iron-induced promoters  and ACO1 promoters (B) in −Fe medium was abolished in the hap2Δ/Δ mutant indicating that Hap2 is required for Hap5 promoter occupancy. Our previous study showed that Hap5 recruitment is also dependent on Hap3 . Furthermore, deletion of hap43Δ/Δ also abolished Hap5 recruitment at ACO1 and FRP1 promoters in Fe-deplete condition (A and B). Together these data demonstrated that the HAP complex recruitment is highly dependent on both Hap43 and Sef1.Next, to address the requirement of Sef1 for recruitment of the HAP complex, we conducted ChIP assays in Δ mutant , A and Bondition , A and Bhap5Δ/Δ and sef1Δ/Δ mutant strain backgrounds. The ChIP data showed that whereas Hap43, as expected, is not recruited to FRP1 (C), its recruitment to ACO1 promoter is completely dependent on the Hap5 subunit of the HAP trimeric complex (D). Next, we investigated the requirement of Sef1 for Hap43 recruitment to the ACO1 promoter. The sef1Δ/Δ mutation did not abolish Hap43 recruitment to the ACO1 promoter but was only reduced by ∼2.5-fold (D) indicating that Sef1 is not essential but required for full potential of Hap43 recruitment to promoter in vivo, consistent with partial dependence of HAP43 mRNA expression on Sef1 (D).To address the requirement of the HAP trimeric complex and Sef1 for Hap43 recruitment, we conducted Hap43 ChIP analysis in  to FRP1 C, its re complex D. Next, 2.5-fold D indicat on Sef1 D.SEF1 in WT and hap3Δ/Δ strain backgrounds. This experiment showed that Sef1 occupancy at the FRP1 promoter, as shown above, was stimulated upon iron deprivation, but interestingly the hap3Δ/Δ mutation did not impair the Sef1 occupancy in Fe-depleted medium (E). Furthermore, the Sef1 occupancy at the ACO1 promoter was also independent of Hap3 (E), indicating that Sef1 is recruited independent of the HAP complex at both FRP1 and ACO1 promoters.To assess if the HAP complex is required for Sef1 recruitment, we carried out ChIP assays using TAP-tagged d medium E. Furthe of Hap3 E, indicaFRP1 promoter since both Sef1 and the HAP complex are recruited to the same location in the FRP1 promoter (A). The FRP1 promoter contains the CCAAT motif at −134 with reference to the FRP1 start codon, and this motif was shown to be critical for Hap5 binding and regulation of FRP1 expression in response to iron deprivation (FRP1 promoter and identified an elaborate sequence 5′CGG(N6)GGC(N18)CCAAT(N10)CCG with two apparent 5′CGG motifs in opposite orientations beginning at position −164 that nested the CCAAT motif (A).To determine how Sef1 and the HAP complex mediate transcriptional activation, we chose the promoter A. The FRrivation . Althougrivation , the exaAT motif A.Figure in vivo, we cloned the FRP1 promoter and mutated the 5’CCAAT sequence to 5’TGCGC by site-directed mutagenesis. As the in vivo requirement of the 5’CGG motif was not known, we introduced mutations in the two 5’CGG motifs and constructed FRP1 knock-in strains bearing either WT as control or those bearing point mutations within the CCAAT and CGG motifs (A) and examined the occupancy of Sef1 and HAP complex. The ChIP analysis showed Hap5 occupancy at the WT FRP1 promoter in the knock-in cells in −Fe medium (B). However, the ChIP data also showe′d that the 5′CCAAT– mutation impaired Hap5 occupancy to FRP1 promoter in vivo (B), consistent with the requirement of the 5′CAAT motif for Hap5 binding to FRP1 promoter in vitro reported previously . But the CGG– mutation abrogated Sef1 occupancy (B), indicating the requirement of this promoter sequence for Sef1 binding to chromatin in vivo. Surprisingly, Hap5 occupancy was also impaired by the 5′CGG– mutation (B) suggesting that a WT 5′CGG motif also promotes HAP complex binding. Interestingly, Sef1 occupancy was not impaired by the 5′CCAAT– mutation (B), suggesting that Sef1 occupancy at FRP1 promoter in vivo occurs independent of the 5′CCAAT motif. Together these data suggest that the HAP complex recruitment is dependent on Sef1 occupancy but recruitment of Sef1 seemed to be independent of the HAP complex occupancy at the FRP1 promoter in vivo.Next, we conducted ChIP assays to examine Sef1 recruitment and found robust Sef1 recruitment to the WT ondition B. But thccupancy B, indicamutation B suggestmutation B, suggesFRP1 promoter by ChIP assay. The data showed that FRP1 promoter was hypoacetylated in Fe-replete medium in the WT strain, but was strongly hyperacetylated in Fe-deplete medium (A). Deletion of SEF1, HAP5, or HAP43 reduced the hyperacetylation in Fe-deplete medium (A). At the ACO1 promoter, the H3-K9Ac level was strongly elevated in Fe-replete medium in the WT strain (B), and this hyperacetylation was dependent on both Sef1 and Hap43, but not on Hap5 (B). In Fe-deplete medium, the ACO1 promoter was not hyperacetylated at the H3-K9, but interestingly, hap43Δ/Δ and hap5Δ/Δ mutations led to hyperacetylation (B). Three conclusions can be drawn based on these ChIP data. First, at the FRP1 promoter, Sef1, Hap5, and Hap43 stimulate H3K9 hyperacetylation under −Fe condition. Second, at the ACO1 promoter, Sef1 and Hap43 promote H3K9 hyperacetylation under +Fe condition, and third, Hap5 and Hap43 repress H3K9 acetylation at the ACO1 promoter under −Fe condition.Next, we assessed the status of nucleosomal histone H3-Lys9 acetylation, a transcriptional activation-linked chromatin mark, at the two iron homeostasis gene promoters. The H3-K9Ac level, normalized to total H3, was determined at the e medium A. Deletie medium A. At theT strain B, and th on Hap5 B. In Fe-tylation B. Three FRP1 and ACO1 promoters, we carried out ChIP analysis in the WT and the hap43Δ, hap5Δ, and sef1Δ mutant strains using an RNA pol II CTD phospho Ser5 antibody. We found that Pol II occupancy at the FRP1 promoter is highly dependent on Sef1 and Hap5 in −Fe medium (C). Interestingly, the hap43Δ mutation led to elevated Pol II levels at the FRP1 promoter compared with that in WT strain in −Fe medium (C). In +Fe medium, however, the sef1Δ, hap5Δ, and hap43Δ mutations did not significantly alter Pol II levels (C) at FRP1 promoter.To examine the recruitment of Pol II to e medium C. Interee medium C. In +FeI levels C at FRP1ACO1 promoter, we observed a high level of Pol II enrichment in +Fe medium in a manner dependent on Sef1 but not Hap5 and Hap43 (D). In contrast, no Pol II enrichment was seen at this promoter in −Fe medium. Interestingly, the hap5Δ and hap43Δ mutations, but not the sef1Δ mutation, led to a significant increase in Pol II occupancy compared with that in WT strain at the ACO1 promoter in −Fe medium (D).At the nd Hap43 D. In cone medium D.FRP1 and ACO1 genes, we measured mRNA levels by qRT-PCR. FRP1 mRNA was highly induced (∼80-fold) in −Fe medium compared with +Fe medium in WT strain (E). The deletion of SEF1, HAP5, or HAP43 genes substantially reduced FRP1 induction in −Fe medium. The ACO1 mRNA was strongly downregulated in the WT strain in −Fe medium compared with +Fe medium (F). Interestingly, deletion of HAP5 or HAP43, but not SEF1, caused derepression of ACO1 expression as the −Fe/+Fe expression ratio was ∼1.0 in the mutants compared with ∼0.04 in the WT strain indicating the repressive activity of HAP5 and HAP43 at the ACO1 gene (F). Moreover, the derepression of ACO1 expression upon HAP5 or HAP43 deletion is consistent with the elevated H3K9Ac level (B) and Pol II occupancy (D). Although hap43Δ/Δ mutation led to elevated Pol II recruitment at both FRP1 and ACO1 promoters under −Fe conditions (C and D), only ACO1 mRNA level is derepressed in −Fe medium suggesting a requirement for further activation of FRP1 promoter postrecruitment of Pol II. Together, the H3-K9Ac and Pol II occupancy data along with the mRNA data indicated that while Sef1 functions as an activator of Pol II recruitment and transcription of FRP1 in −Fe medium and that of ACO1 in +Fe medium, the HAP trimeric complex functions as a dual regulator of Pol II recruitment and transcription, i.e., as an activator at FRP1, and as a repressor at ACO1 promoters.To correlate the H3K9 acetylation and Pol II occupancy to the transcription state of T strain E. The dee medium F. IntereCO1 gene F. MoreovAc level B and Polccupancy D. Althounditions , C and DC. albicans strains bearing deletion mutations in SAGA subunits, namely GCN5 encoding the CaKAT2 histone acetyl transferase and two key SAGA-specific subunits SPT7 and SPT20. The mutants and the control WT strains were tested for growth in −Fe condition (+BPS plates). Compared with the WT parental strain, the gcn5Δ, spt7Δ, and spt20Δ null mutants were impaired for growth in −Fe plate (A). Of the three, the spt20Δ mutant showed the strongest growth defect as compared with the gcn5Δ and spt7Δ mutants. The three mutants also showed slow-growth phenotype in the control SC-LIM plate, and thus the BPS-sensitive growth defect appears to be less pronounced in BPS-containing plate . However, iron supplementation to the BPS plate rescued most of the growth defect of the mutants . Also, reintroduction of a WT copy of GCN5, SPT7, and SPT20 genes in the respective mutants complemented the growth defects in −Fe as well as in the control plate (A) indicating that the three SAGA genes are required for C. albicans response to iron deprivation.The Gcn5-containing SAGA complex is the major histone acetyl transferase that mediates H3K9 acetylation in yeast , 27. TheFe plate A. Of theate −Fe; A. Howevents +Fe; A. Also, ol plate A indicatFRP1 and ACO1. The expression of FRP1 mRNA was induced ∼100-fold in −Fe (+BPS) medium compared with +Fe medium (+FAS) in the WT strain (B). The FRP1 mRNA induction, however, was significantly reduced in all three SAGA mutants, namely gcn5Δ/Δ, spt7Δ/Δ, and spt20Δ/Δ (B), with the reduction between ∼2.0-fold in gcn5Δ/Δ and ∼10.0-fold in the spt20Δ/Δ mutant, suggesting that mutations in the structural components of SAGA had a major impact possibly due to the spt7Δ/Δ and spt20Δ/Δ mutations substantially affecting the integrity of the SAGA complex as reported previously for the budding yeast SAGA (ACO1 mRNA expression had a fold change (plotted as +Fe/−Fe ratio) of ∼15.0, indicating that ACO1 expression is repressed in −Fe medium compared with +Fe medium in the WT strain (B). In the gcn5Δ, spt7Δ, and spt20Δ mutants, unexpectedly, the ACO1 mRNA expression was further elevated in +Fe medium (B) indicative of a repressive function for SAGA at this promoter under Fe-replete conditions. In any case, these data provided evidence that the SAGA complex is a critical effector of iron homeostasis gene regulation in C. albicans.Next, we analyzed if the SAGA subunit genes are required for expression of the iron homeostasis genes T strain B. The FRspt20Δ/Δ B, with tast SAGA , 29, 30.T strain B. In thee medium B indicatgcn5Δ mutant strains. ChIP assays were carried out from chromatin extracts prepared from WT and gcn5Δ mutant strains cultured under iron-deplete or iron-replete conditions, using either histone H3-K9 acetyl antibody or the total H3 antibody, and FRP1 and ACO1 promoters were probed using qPCR. The results depict that histone H3-K9 is hyperacetylated at the FRP1 promoter in −Fe medium compared with +Fe in WT cells (C). However, in the gcn5Δ mutant strain, the hyperacetylation was completely lost indicating that Gcn5 is the critical histone acetyl transferase under iron-deficient conditions (C). At the ACO1 promoter, however, we found hyperacetylation in +Fe consistent with a high level of ACO1 mRNA expression in +Fe compared with −Fe medium (C). The gcn5Δ abrogated H3K9 acetylation at ACO1 promoter in +Fe medium (C), demonstrating that SAGA-mediated acetylation is also critical for ACO1 expression in +Fe medium.To further establish a direct role for acetylation by Gcn5, we assessed nucleosomal histone acetylation at the two iron homeostasis gene promoters in WT and WT cells C. Howevenditions C. At thee medium C. The gce medium C, demonsC. albicans and other fungal pathogens maintain iron homeostasis by modulating their intracellular iron pool through multiple iron acquisition pathways and by conserving iron pools by downregulating iron-requiring proteins. The iron homeostasis gene regulation is controlled by multiple transcriptional regulators in C. albicans and other fungi , indicating that although Hap43 is not recruited to the FRP1 promoter, the HAP trimeric complex recruitment is Hap43-dependent. Given that Hap43 is bound to multiple iron uptake genes that are induced under −Fe conditions, we would not be able to rule out if the Hap43 cross-linking efficiency at the FRP1 promoter is weaker than that at other promoters identified in the Chip-ChIP study. Besides, Hap3 is essential for Hap2-Hap5 interaction in cell extracts (C–F) indicating that the efficient assembly of the trimeric complex is also dependent on intersubunit interactions within the HAP complex. Thus, it is conceivable that the HAP complex assembly in C. albicans is a two-step process similar to that in Aspergillus spp. and mammals  and the presumptive Sef1-binding site (CGG motif) on the FRP1 promoter and found that while the HAP complex recruitment was dependent on Sef1, the recruitment of Sef1 itself was independent of the HAP complex indicating that Sef1 acts upstream to the HAP complex at the FRP1 promoter. As Sef1 has been shown to be recruited to the HAP3 promoter and induce transcription (FRP1 promoter (B). Furthermore, the Sef1 bound at the ACO1 promoter could also potentially promote the recruitment of the HAP trimeric complex and Hap43 to the ACO1 promoter (B).It was previously reported that ∼7% of the iron-responsive gene promoters are bound by both Hap43 and Sef1 indicating a synergy between the two regulators. Therefore, to test the interdependency of Sef1 and the HAP complex for their recruitment to cription , Sef1 lipromoter B. Furthepromoter B.Figure FRP1 promoter under Fe-deplete conditions  indicating a chromatin-mediated control of iron homeostasis. Our results established that the SAGA complex subunits encoded by GCN5, SPT7, and SPT20 are required for cellular growth under Fe-depleted conditions and for optimal regulation of FRP1 and ACO1 promoters under Fe-deplete and Fe-replete conditions respectively (A and B). Although FRP1 expression was impaired in all three SAGA mutants tested, the spt7Δ and spt20Δ mutations had a greater impact on gene expression as compared with gcn5Δ (B), with spt20Δ being most debilitating. There are compelling previous reports indicating such differential SAGA subunit effects. Proteomics studies of ySAGA  have less effect on mRNA levels than the double deletions (gcn5Δ spt3Δ). Moreover, deletion of subunits of a particular SAGA module showed similar expression profiles, whereas deletion of subunits of different SAGA modules impacted different subset of genes . Furthermore, Sef1 and the HAP complex are also required for Pol II recruitment at the FRP1 promoter under Fe-deplete conditions (C). At the ACO1 promoter, however, Hap5 and Hap43, but not Sef1, are required to maintain this promoter in a hypoacetylated state under Fe-deplete conditions (B), consistent with the repressive function of the Hap43-HAP complex at the ACO1 promoter. Consistent with the transcriptional repressive function of the Hap43-HAP complex at ACO1, Pol II recruitment is greatly stimulated upon hap5Δ/Δ and hap43Δ/Δ deletion under Fe-deplete conditions (D). Furthermore, we are surprised that the hap43Δ/Δ mutation led to a greater than WT level of Pol II recruitment at the FRP1 promoter (C); but the reduced FRP1 mRNA level in the hap43Δ/Δ mutant perhaps suggests that Pol II recruitment is not sufficient to activate the FRP1 promoter under Fe-deplete conditions indicating a postrecruitment control of Pol II activation for FRP1 transcription. We posit that the HAP complex could be minimally sufficient to directly stimulate H3-K9 acetylation upon FRP1 promoter binding, and Sef1 could just mediate HAP complex recruitment. In this context, other transcriptional activators stimulate H3 acetylation by SAGA recruitment through activation domain interactions . Paradoxically, the loss of HAP complex in +Fe led to a hyperacetylated state of ACO1 promoter (B). It is conceivable that the ACO1 hyperacetylation in +Fe is likely due to SAGA activity recruited by Hap43, as the deletion of HAP43 (B) or GCN5 (C) impaired H3K9Ac at ACO1 under +Fe condition.Based on the data presented in this study together with previous studies, a model emerges wherein iron deprivation elicited the transcriptional induction of ke genes  and is bcription . Moreoveetylated A. Paradopromoter B. It is of HAP43 B or GCN5 or GCN5 C impaireFRP1, while the HAP trimeric complex, Hap43, and Sef1 are recruited to the promoter of ACO1 (B). The recruitment of the trimeric HAP complex and Sef1 stimulates SAGA activity and Pol II recruitment at FRP1 leading to transcriptional activation of FRP1 (B). Although Hap43 is not occupied at FRP1, Hap43 is required for HAP3 expression and thereby stimulates HAP recruitment to FRP1. The Sef1 regulator induces the expression of HAP43 as well as HAP3 under −Fe conditions and thus also stimulates HAP complex assembly at the FRP1 and ACO1 promoters (A and B). In contrast, recruitment of trimeric HAP complex and Hap43 to the ACO1 promoter leads to inhibition of SAGA activity and the consequent loss of ACO1 expression in −Fe medium (B). Thus, the Hap2-Hap3-Hap5 trimeric complex and Hap43 have dual roles in regulation of iron homeostasis by transcriptional activation at FRP1 and repression of ACO1  as described previously  or 0.5 (mutants) and grown for 5 h and harvested, and cell pellets were used immediately or snap frozen in liquid nitrogen and stored at −80 °C till use.eviously . Iron-deC. albicans strains and plasmids used in this study are listed in the C. albicans parental strain SN152 and SN95 . The Hap5-TAP strain RPY453 was described previously  bearing Hap5-TAP and Hap3-FLAG were constructed by integration of the His6-FLAG3 cassette from plasmid pSH26-5  and KNC693  deletion cassette from plasmid pHAH1 (GCN5 (ONC601/ONC115 and ONC601/ONC114), SPT7 (ONC602/ONC115 and ONC603/ONC114), or SPT20 (ONC605/ONC115 and ONC606/ONC114) and amplified the respective up-split and down-split fragments, combined and transformed into C. albicans strain SN95 and selected for Arg+ colonies. The correct integrations were confirmed using PCR, and the resultant heterozygous strains KNC641, KNC651, and KNC653 were grown nonselectively in YPD and Arg+ His+ colonies representing homozygous deletion strains KNC642, KNC652, and KNC654 were selected and confirmed by PCR.To construct id pHAH1 , using pGCN5, SPT7, and SPT20 into the homozygous deletion strains KNC642, KNC652, and KNC654, plasmid pNIM1 was sequentially digested with BglII/MluI to obtain nourseothricin resistance marker CaSAT1 while plasmid CIp10 was digested with NotI/XbaI and end filled with Klenow. Then linearized plasmid pCIp10 and insert CaSAT1 was ligated and transformed to electrocompetent bacterial cells DH10B to obtain plasmid pCIp10-SAT1(pPI1). Then, genes were amplified along with 1000 bp upstream and 500 bp downstream of ORF sequences using primer pairs ONC1156-ONC1157 (GCN5), ONC1154-ONC1155 (SPT7), and ONC1152-ONC1153 (SPT20) respectively. The amplified product was digested with BamHI (for SPT7 and SPT20) and SacI (for GCN5) and cloned into plasmid pPI1 digested with the respective enzymes and plasmids pPI2 (GCN5), pPI3 (SPT7), and pPI4 (SPT20) were obtained.For reintegration of gcn5Δ/GCN5), PRI2 (spt7Δ/SPT7), and PRI3 (spt20Δ/SPT20) respectively.The plasmids pPI2, pPI3, and pPI4 were linearized with PmeI, BstEII, and Afl II respectively and transformed into KNC642, KNC652, and KNC654 strains, and positive transformants were confirmed by PCR using ORF specific primer pairs ONC661/ONC662, ONC663/ONC664, ONC708/ONC709 and obtained PRI1  by electroporation to integrate the reporter plasmid at the RPS1 locus and obtained KNC332, KNC340, and KNC348. The correct integration was confirmed by PCR.Strains for β-Gal assays were constructed as follows. The id pAU36  was subcid pAU36  to constFRP1 promoters were constructed in several steps. First, the WT FRP1 coding sequence along with 500 bp upstream region was deleted in strains RPY453 and KNC379 to obtain strains KNC467 and KNC481 respectively. The homozygous frp1Δ deletion was confirmed by PCR. Next, a DNA fragment containing the FRP1 coding sequence flanked by 1kb upstream region and 300 bp downstream region was amplified as a KpnI-SacI fragment using ONC630-ONC631 and cloned into pLitmus28 to obtain pNA51. The WT FRP1 fragment was then subcloned into pSN69  and the CCAAT motif (−134 with reference to ATG) was mutated to TGCGC as reported previously  in the eviously  was muta600 and 0.5 OD600 for WT and the mutant strains (hap43Δ/Δ and sef1Δ/Δ), respectively. For iron-replete conditions , cells were diluted to a starting density of 0.25 OD600 for all the strains and grown for 5 h till ∼1.0 OD. Cells were harvested and cell pellets washed with Z-buffer  at a starting density of 0.3 ODZ-buffer , resuspeZ-buffer  and plotThe cell pellets were resuspended in chilled Winston Buffer  for 4 h at 4 °C, washed with Winston buffer, followed by TEV buffer. The beads were then incubated with AcTEV protease (Invitrogen) overnight at 4 °C. The eluate was collected and 20% of total eluate (IP) and 100 μg whole-cell extracts (Input) were separated on 10% SDS-PAGE gel, transferred onto a Protran nitrocellulose membrane  and probed with anti-FLAG, anti-TAP oranti-Glucose-6-P-dehydrogenase antibodies detected with ECL-Prime Western blot detection reagent  and exposed to X-ray film.C. albicans cells and cDNA was synthesized essentially as described before  formaldehyde, quenched with 125 mM glycine, cells harvested, and chromatin extracts prepared by shearing in Bioruptor . For chromatin immunoprecipitation of TAP-tagged proteins, the sheared chromatin extracts equivalent to ∼25OD600 cells from control and experimental strains were immunoprecipitated with about 25 μl of IgG-Sepharose 6 Fast Flow  BSA) for 3 h at 4 °C. For Hap43 ChIP, 2 μl affinity-purified antibody  and 1:5 (IP DNA), and probed by quantitative real-time PCR for specific regions of interest as well as for the control nonspecific region (ca21chr1_1573500–1574000). For all chromatin immunoprecipitations, enrichment was calculated for target regions and for the control nonspecific region (ca21chr1_1573500–1574000) and specific enrichment calculated with respect to input total chromatin. For the TAP chromatin immunoprecipitations, the fold enrichment was determined by further normalizing the background amount of immunoprecipitation obtained in mock immunoprecipitation conducted using chromatin extracts from untagged strain. Similarly, for Hap43 chromatin immunoprecipitations, the specific enrichment was calculated by further normalizing the background amount of immunoprecipitation obtained in the t-test in Graph Pad Prism 6, and a p-value ≤ 0.05 was considered statistically significant. The p-values are indicated as ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), ∗∗∗ (p ≤ 0.001), ∗∗∗∗ (p ≤ 0.0001), and ns (p > 0.05).The statistical significance of quantitative data was determined by Student's All data reported here are contained within the article or as This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The metastasis suppressor function of NM23 proteins is widely understood. Multiple enzymatic activities of NM23 proteins have also been identified. However, relatively less known interesting aspects are being revealed from recent developments that corroborate the telomeric interactions of NM23 proteins. Telomeres are known to regulate essential physiological events such as metastasis, ageing, and cellular differentiation via inter-connected signalling pathways. Here, we review the literature on the association of NM23 proteins with telomeres or telomere-related factors, and discuss the potential implications of emerging telomeric functions of NM23 proteins. Further understanding of these aspects might be instrumental in better understanding the metastasis suppressor functions of NM23 proteins. The Non-metastasis 23/Nucleoside diphosphate kinase NM23/NDPK protein family contributes to several cellular pathways owing to their interaction with multiple effector proteins and several known enzymatic functions of the NM23 proteins. This multifunctional family of proteins (10 in humans) is broadly divided into two groups based on their identity . NM23-H1 and H2, the cytoplasmic/nuclear localizing proteins, and NM23-H4, reported mostly as mitochondrial, are amongst the most studied members of the family of proteins. These proteins have previously been extensively reviewed [One of the lesser studied yet relatively direct links with cancer initiation, progression and metastasis comes from the association of NM23 proteins with chromosome ends or telomeres, and telomere binding proteins (telosome/shelterin complex) . Evidenc6 were incubated with Glutathione S-transferase (GST)-tagged recombinant NM23-H1 or H2. Only NM23-H2 had binding affinity for the telomeric sequence, but not NM23-H1. NM23-H2 was found to bind with the single-stranded, but not the double-stranded telomeric sequence in vitro. The authors further tested the specificity of the binding of NM23-H2 to the telomeric sequence and found that NM23-H2 associated with both (TTAGAG)6 and (TTGGGG)6, but not the (ATAGGG)6 sequence. The first evidence of NM23 proteins interacting with the telomeric sequence came from Nosaka et al., 1998 . Thirty-Danio rerio known as “NM23-B”, discovered based on 87.3% sequence similarity, was also found to interact with the telomeric DNA sequence (TTAGGG)6 [Chondrus crispus—and therefore named NME5-likeCc—was also found to interact with telomeric DNA (TTAGGG)6 in vitro [The NM23-H2 homologue in zebrafish c-MYC purine-binding factor (PuF)—named based on the characteristic G-rich DNA binding by the transcription factor—to be NM23-H2 [c-MYC promoter [c-MYC promoter NHE that formed telomere-like G-quadruplex, along with NM23-H2 association with telomeric DNA (discussed above), supported NM23-H2-G-quadruplex interactions. Disruption of the G-quadruplex by specific base substitutions, or in the presence of intracellular G-quadruplex binding molecules, resulted in altered binding of NM23-H2 to the c-MYC promoter. In addition, in vitro studies using recombinant NM23-H2 showed NM23-H2-G-quadruplex binding [hTERT promoter to be important for the epigenetic regulation of hTERT (vide infra) [c-MYC promoter NHE [In 1993, Postel et al. identified the human promoter . G-quadrpromoter . Similar binding . Recent e infra) . Interesoter NHE .While the findings discussed above demonstrated the interaction of NM23-H2 with telomeric DNA in vitro, evidence supporting NM23-H2 binding to telomeres in vivo was shown in 2012. In both A549 lung carcinoma and HT1080 fibrosarcoma cell lines, NM23-H2 chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed following expression MYC-tagged-NM23-H2. On analysis of the short sequence reads, for every million ChIP-seq about 6000–9000 reads were from the telomeric region comprising three or four telomeric repeats (TTAGGG/CCCTAA) . Here, itelomerase  that synthesizes telomeres) to the telomeres [The shelterin is an essential multi-protein complex that prevents the telomere ends from being detected as DNA double-stranded breaks, thereby preventing the activation of DNA damage signalling . The sheelomeres . Telomerelomeres . The direct association of NM23-H1 and H2 with TRF2 was tested. Here, the authors found co-immunoprecipitation (co-IP) of TRF2 with NM23-H2 from nuclear extracts of HT1080 fibrosarcoma and A549 lung carcinoma cell lines. This was further validated using reverse-IP using pull-down with anti-TRF2 antibody, where interaction with NM23-H2 was observed. Since both NM23-H2 and TRF2 were known to interact with DNA, it was further tested if NM23-H2-TRF2 interaction was dependent on their association with DNA. NM23-H2-TRF2 interaction was found to be independent of DNA or RNA association [35 labelled methionine in vitro to confirm a strong and direct association of NM23-H2 with TRF1. The interaction of NM23-H1 with TRF1 was again found to be insignificant [Earlier work using the yeast two-hybrid method showed the interaction of a truncated TRF1 protein (14-285aa) with NM23-H2; the interaction of TRF1 with NM23-H1, however, was not significant. Further, the authors used recombinant GST-tagged NM23-H1 and H2 and translated full-length TRF1 with Snificant .The ribonucleoprotein reverse transcriptase telomerase binds to the 3’ overhangs of the telomeres and processes telomere elongation . TelomerFurther, it was observed that NM23-H2 over-expression suppressed hTERT enzymatic activity in HT1080 fibrosarcoma cells. This was independent of the nuclease activity of NM23-H2, suggesting that NM23-H2 did not suppress telomerase activity by cleaving the template DNA necessary for telomere elongation by hTERT . As expeWhile these results supported an inverse relationship between NM23-H2 and telomerase, notably, NM23-H2 and telomerase activity when tested in hepatocellular carcinoma (HCC) patient samples showed that NM23-H2 levels positively correlated with telomerase activity . Here, iFEN1 mRNA in melanoma cell lines [Other than TRF2 and hTERT, the nuclear interactome of NM23-H2 (from mass spectrometry data) showed that telomere-associated proteins flap-endonuclease-1 (FEN1) and Lamin A/C/B1 interacted with NM23-H2 ,30,31,32ll lines ,34. FurthTERT by NM23-H2 was reported. From NM23-H2 ChIP in HT1080 fibrosarcoma and HCT116 colorectal cancer cell lines, NM23-H2 binding at the hTERT promoter was found within +40 to -250 bp upstream of the hTERT transcription start site [hTERT gene expression in HT1080 and HCT116 cancer cells, and MRC5 normal primary lung fibroblast cells. NM23-H2 mediated transcriptional repression of hTERT was dependent on its DNA binding activity: the N69H and R34A DNA binding mutants of NM23-H2 could not repress hTERT expression. The authors also observed that NM23-H2 binding was dependent on the hTERT promoter G-quadruplex. In the case of base substitutions that disrupted the hTERT promoter G-quadruplex, NM23-H2 binding was lost from the hTERT core-promoter. This also resulted in the loss of the NM23-H2-mediated suppression of hTERT [In addition to physical interaction with telomerase, direct transcriptional control of the art site . NM23-H2of hTERT .hTERT promoter. While lysine-specific demethylase 1 (LSD1) binding on the hTERT promoter was reported previously, this study demonstrated that LSD1 was recruited onto the hTERT core-promoter by NM23-H2. Based on the findings from the NM23-H2 nuclear interactome , and because LSD1 was known to be a part of the REST/co-REST/LSD1/HDAC1/2 repressor complex, the interaction of NM23-H2 with the remaining members of the multiprotein repressor complex was tested. Co-Immunoprecipitation experiments with anti NM23-H2 antibody showed enrichment for REST, LSD-1, HDAC1 and HDAC2. In the presence of NM23-H2, the repressor complex was shown to remain bound to the hTERT promoter. To further test the effect of NM23-H2 on the repressor complex, NM23-H2 was silenced. This resulted in a gain of H3K4me1, H3K4me2, and H3K9ac activation marks onto the hTERT promoter, as expected [More detailed mechanistic experiments showed that NM23-H2 interacts with the REST/co-REST/LSD1/HDAC1/2 repressor complex, and this retained the non-permissive epigenetic state of the expected . Taken thTERT expression. The effect was further confirmed using hTERT promoter activity. These data highlight the potential of G-quadruplex stabilizing ligands as anti-cancer therapeutic agents that could function by telomerase suppression. In this context, it would be interesting to test the effect of G-quadruplex stabilizing ligands on cancers with hTERT promoter mutations [hTERT promoter G-quadruplex [Further, the authors treated cancer cells with G-quadruplex stabilizing ligands and demonstrated their repressive effect on utations ,36, moreadruplex ,38,39.+ dependent deacetylase, by modulating the acetylation levels of the protein in N1E-115 mouse neuroblastoma cells [NM23-H4  has been reported for its dual functions as a mitochondrial nucleoside diphosphate kinase and a mediator of selective membrane phospholipid transfer . As a mima cells . Howeverma cells ,47.Reports of NDPK family proteins associated with age-related disorders suggest the significance of NDPK in the physiological process of ageing. Based on increased specific activity in brain tissue compared to others during embryonic development, NDPK proteins were deemed to be important for neuronal functions and developmental stages in the late 1990s . More reCytoskeletal modulations play a critical role in cellular and organismal ageing, neural degeneration, and cancer ,52. Vari2+) influx as an essential signal for the reactivation of naïve (in-activated/resting) T cells [T cell activation is an indispensable arm of the adaptive immune response against foreign antigens. Factors and mechanisms governing the activation and amplification of T cells have been studied extensively. T cells derive the name from “Thymus”, where they are generated and programmed specifically for a particular antigen. T cells exit from the Thymus and enter into circulation, where they recognize their specific antigen presented on the surface of antigen-presenting cells (APCs) held by the Major histo-compatibility (MHC) complex. Antigen and T cell receptor (TCR) binding both in CD4+ helper and CD8+ cytotoxic T cells triggers T cell activation ,64,65. A T cells .2+ influx into T cells occurs via Ca2+ release-activated channels (CARC) present in the plasma membrane. CARC channels function by activating K+ channels 4, or KCNN4)). Therefore, CARC channels mediate Ca2+ influx via K+ efflux. Increased cytosolic Ca2+ activates phosphatase calcineurin, which mediates the assembly of Nuclear factor of activated T cell (NFAT) transcriptional complexes [Caomplexes . This in2+ influx, and the proliferation of human CD4 T cells [NDPK-B/NM23-H2 was shown to activate KCa3.1 by directly binding and phosphorylating histidine (358) at its carboxyl terminus. H358 phosphorylation by NDPK-B was found to be essential for KCa3.1 channel activation, Ca T cells . This st T cells . Further T cells ,69. TogehTERT mRNA and telomerase activity were found to sequentially decrease from naïve (TN) to central memory (TCM) to effector memory (TEM) cells. CD4+ cells had higher telomerase in comparison to the corresponding CD8+ subsets [The molecular mechanisms underlying calcium signalling in T cell activation have been known for a long time . Telomer subsets . The rob subsets ,77,78. T subsets ,71,72,73 subsets . More than one mechanism of telomerase re-activation has been implicated to be involved during T cell activation. These include cytokine-mediated (IL-7 and IL-15) upregulation and NFAT-sp1 mediated transcriptional re-activation, and while not tested in T cells, the role of c-myc in telomerase re-activation has also been discussed, along with the post-transcriptional stabilization of telomerase transcripts   and mainKar et al. demonstrated the nuclear interaction of NM23-H2 with hTERT that was found to be independent of DNA binding . HoweverhTERT is also known to activate the transcription of several gene targets ,91. The Another important aspect of NM23-H2 is its association with TRF1/TRF2. These interactions might be crucial in genome-wide telomere length-dependent transcriptome changes. This is because, recently, the extra-telomeric association of TRF2 was shown to extensively impact genome-wide gene expression changes that were dependent on telomere length .Additionally, similar to NM23-H2, TRF2 is also shown to bind to G-quadruplexes and gene promoters carrying potential G-quadruplex forming sequences ,94,95. TWe further highlight the indications from the association of NM23-H2 with lamins and FEN1 . TRF2 isFinally, multiple pieces of evidence of the mitochondrial NM23 protein suggests an association with ageing. Simultaneously, observations on the cytoskeleton, NM23 family proteins, telomeres, and ageing are connected. These suggest potential roles of NM23 proteins that remain to be addressed. Further work will be required to illuminate these molecular interactions and the implications of such cross-talk, if any.NM23 proteins have multiple activities and functions that assist their metastasis suppressor role. While some of these are well understood, others continue to be explored. Here, we discussed the interactions and regulatory effects of NM23 proteins related to telomeres and telomere-binding proteins . The emeThe dual regulation of telomerase, both at the transcriptional and post-translational levels, by NM23-H2 emphasizes its regulatory effect on telomeres and telomere-associated functions, including metastasis and ageing. While based on current findings it is difficult to say whether NM23 proteins have any role in these aspects, it would be interesting to investigate the reasons and implications of NM23 association with telomeres and telomere binding proteins.Additionally, the role of NM23 proteins needs to be further explored with reference to ageing-associated pathologies. From the association of NM23 proteins with the initiation/progression of diseases such as Alzheimer’s, progeria and cancer, it is possible that NM23 family members might influence pathological contexts. Direct exploration of these questions would be important.


Long non-coding RNAs (lncRNAs) have emerged during the post-genomic era as significant epigenetic regulators. Viral-like 30 elements (VL30s) are a family of mouse retrotransposons that are transcribed into functional lncRNAs. Recent data suggest that VL30 RNAs are efficiently packaged in small extracellular vesicles (SEVs) through an SEV enrichment sequence. We analysed VL30 elements for the presence of the distinct 26 nt SEV enrichment motif and found that SEV enrichment is an inherent hallmark of the VL30 family, contained in 36 full-length elements, with a widespread chromosomal distribution. Among them, 25 elements represent active, present-day integrations and contain an abundance of regulatory sequences. Phylogenetic analysis revealed a recent spread of SEV-VL30s from 4.4 million years ago till today. Importantly, 39 elements contain an SFPQ-binding motif, associated with the transcriptional induction of oncogenes. Most SEV-VL30s reside in transcriptionally active regions, as characterised by their distribution adjacent to candidate cis-regulatory elements (cCREs). Network analysis of SEV-VL30-associated genes suggests a distinct transcriptional footprint associated with embryonal abnormalities and neoplasia. Given the established role of VL30s in oncogenesis, we conclude that their potential to spread through SEVs represents a novel mechanism for non-coding RNA biology with numerous implications for cellular homeostasis and disease. Based on the latest Gencode reference annotation, the mouse genome contains >13,000 long non-coding RNAs (lncRNAs) . Given tOne of the known functional lncRNAs in the mouse genome is the RNA transcript from viral-like 30 elements (VL30s) that was characterised as early as 40 years ago . VL30 RNVL30 lncRNAs may affect cellular physiology by at least two distinct mechanisms. First, the vast majority of VL30 RNAs contain sequences that render them retrotransposition-competent . VL30 reSmall extracellular vesicles (SEVs) are created by cells as a delivery mechanism of proteins, lipids or nucleic acids and as a medium of extracellular communication. Upon their discovery, SEVs were considered as a waste disposal mechanism. Today, SEVs have emerged as major players in several cellular processes, including immunoregulation, CNS development and homeostasis, tissue regeneration, inflammation and coagulation . A recenThe aim of the current report was to find the origins and functions of VL30s that are packaged in SEVs and to establish whether they are a distinct group within the VL30 family. Towards this aim, we performed the analysis of phylogenetic and genomic distribution and enriched gene pathways and networks data. Our results support that the SEV enrichment motif occurred 4.4 million years ago (MYA) and spread through retrotransposition to at least 40 elements. Network analysis suggest a stress-associated transcriptional footprint. We conclude that enrichment into SEVs leads to the horizontal transfer of oncogenic VL30 lncRNAs with apparent implications in cellular pathophysiology.http://genome.ucsc.edu/ accessed on 14 September 2021) was used for finding SEV enrichment motifs in the latest assembly (mm39) of the mouse genome [https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 14 September 2021) was used to query for the presence of SEV enrichment motifs in the genomes of the Mus genus. The chromosomal distribution of SEV-VL30s was depicted in the Ensembl Genome Browser (https://ensembl.org/ accessed on 14 September 2021) [https://great.stanford.edu/great/ accessed on 5 October 2021) was used to annotate SEV-VL30s in relation to transcription start sites (TSSs) of mouse genes. GREAT associates genomic regions with their nearby genes and applies the gene annotations to the regions, based on binomial and hypergeometric statistical tests, over regions and genes, respectively. Association is two-step. Every gene is assigned a regulatory domain, and then, each region is tested whether it overlaps with gene regulatory domains.Blast Like Alignment tool (BLAT) web interface in the UCSC Genome Browser  ,30. The er 2021)  was perfer 2021)  web implhttps://www.ebi.ac.uk/Tools/msa/muscle/ accessed on 5 October 2021). The phylogenetic tree was constructed following evolutionary history inference using the Neighbour-Joining method [Mus genus was generated using TimeTree [http://www.timetree.org/ accessed on 5 October 2021).Multiple sequence alignment was conducted using the MUSCLE algorithm , as implg method . The evog method  and are g method . The phyTimeTree  web intehttps://genemania.org/ accessed on 6 October 2021), using Gene Ontology-based weighting methods. Enriched pathway analysis was performed in Enrichr [https://maayanlab.cloud/Enrichr/ accessed on 6 October 2021), using default settings. Briefly, the gene set of interest was compared with preset gene set libraries in the Enrichr server, for significant correlations (p < 0.05) based on the Fisher exact test. The results were viewed using Appyters [Network analysis was performed in Genemania, a binary classification algorithm for network construction ,39, in t Enrichr ,41  is associated with the presence of a 26-nt-long preserved SEV enrichment motif . We analIn a later step, we systematically annotated the structural properties of SEV-VL30s. As found in the previous section, 25/36 full-length elements contain identical 5′ and 3′ LTRs, consensus sequences that are prerequisites for retrotransposition. LTRs, primer binding sites (PBS) and the polypurine tract (PPT) are the required sequence hallmarks for retrotransposition competence. Importantly, we found that all full-length elements contain both PBS and PPT sequences . A totalSFPQ binding of VL30 RNA is mediated by two distinct sequence motifs that are hallmarks of VL30 family members . ImportaThe results of structural and functional analysis reveal that: (1) SEV-VL30s are potent for retrotransposition, since they contain the hallmark sequences of LTR, PBS and PPT, and (2) SFPQ binding, since they contain at least one SFPQ binding motif. Collectively, the horizontal transfer of SEV-VL30s on target cells may lead to genetic and/or epigenetic disequilibrium that is associated with oncogenesis.In order to identify the evolutionary history of SEV binding in VL30s within the mouse germline, we performed phylogenetic analysis for SEV-VL30s. Using the MUSCLE algorithm, 5′ LTRs of SEV-VL30s were aligned, and a phylogenetic tree was drawn using the Neighbour-Joining method . The timMus genus contains a number of highly similar species that diverged from a common ancestor about 10 MYA. To gain further insights into the origins of SEV enrichment features, we explored the existence of the SEV motif in the 30 known species that belong to the Mus genus and found that the conserved 26-nt consensus SEV motif is present only in the genomes of Mus musculus and Mus spretus  integrates high-throughput epigenetic data on DNAseI digestion sites, the histone modifications of H3K4me3 and H3K27ac and CTCF ChIP-seq data produced by the ENCODE and Roadmap Epigenomics Consortia. The existence of cCREs is considered a strong indication for transcriptional activity . We screUsing the GREAT tool, which uses a combined statistical analysis to associated gene regulatory domains with genomic regions, we calculated the distance between cCRE-related SEV-VL30s and transcription start sites (TSSs) of the nearest mouse genes. The 25 elements reside in the vicinity of TSSs from 47 mouse genes, in distances as near as 4429 bp . To furtTo further evaluate possible shared functions of SEV-VL30 genes, we performed enrichment analysis in Enrichr. Based on data from MGI Mammalian Phenotype Level 4 2021, we found that our dataset is enriched, among others, in the following datasets : embryonCollectively, most SEV-VL30s reside in transcriptionally active regions, as characterised by cCREs. Nearby genes are transcribed into proteins that physically interact and their expression is associated with embryonic abnormalities and neoplasia. The genomic distribution of SEV-VL30s is distinct to the remaining non-SEV elements. We conclude that SEV-VL30s are related to specific cellular functions, distinct from non-SEV VL30s.In this study, we established the origins of VL30s that are packaged in SEVs and proved that they formed a distinct functional group of 40 elements within the VL30 family. SEV enrichment evolved ~4.4 MYA and expanded as an inherent hallmark of the VL30 family along with recent VL30 expansions through retrotransposition. Most current SEV-VL30 integrations contain hallmarks of active transcription, retrotransposition competency and are associated with developmental abnormalities and neoplasia. In conclusion, the potential of VL30 lncRNAs through SEVs suggests a significant pathophysiological role in target cells. The SEV-VL30 lncRNAs are associated with cancer with at least two mechanisms. First, high-frequency retrotransposition as a result of stress is associated with epithelial-to-mesenchymal transition and cancer stem cell formation . Second,Mus germline ~17 MYA and the largest wave of expansion is calculated at <1 MYA, resulting in 86 full-length elements in today’s mouse genome [VL30 elements appeared in the e genome . We sugge genome . TherefoThe transcriptional profile of individual VL30s is known to be cell-type-specific  and induOur results agree with the conceptual framework that retrotransposon induction is associated with the orchestration of regulatory networks, such as the ones observed during early human development that assist stem cell pluripotency . The bal


Eukaryotic genomes undergo pervasive transcription, leading to the production of many types of stable and unstable RNAs. Transcription is not restricted to regions with annotated gene features but includes almost any genomic context. Currently, the source and function of most RNAs originating from intergenic regions in the human genome remain unclear.We hypothesize that many intergenic RNAs can be ascribed to the presence of as-yet unannotated genes or the “fuzzy” transcription of known genes that extends beyond the annotated boundaries. To elucidate the contributions of these two sources, we assemble a dataset of more than 2.5 billion publicly available RNA-seq reads across 5 human cell lines and multiple cellular compartments to annotate transcriptional units in the human genome. About 80% of transcripts from unannotated intergenic regions can be attributed to the fuzzy transcription of existing genes; the remaining transcripts originate mainly from putative long non-coding RNA loci that are rarely spliced. We validate the transcriptional activity of these intergenic RNAs using independent measurements, including transcriptional start sites, chromatin signatures, and genomic occupancies of RNA polymerase II in various phosphorylation states. We also analyze the nuclear localization and sensitivities of intergenic transcripts to nucleases to illustrate that they tend to be rapidly degraded either on-chromatin by XRN2 or off-chromatin by the exosome.We provide a curated atlas of intergenic RNAs that distinguishes between alternative processing of well-annotated genes from independent transcriptional units based on the combined analysis of chromatin signatures, nuclear RNA localization, and degradation pathways. Studies estimate that up to 85% of the human genome is pervasively transcribed by RNA polymerase II (Pol II), resulting in a plethora of RNA products –4. Many Xist [7G cap and poly(A) tail) with mRNAs [Many well-characterized lncRNAs, such as the X-inactive-specific transcript Xist , share-pth mRNAs  and haveth mRNAs –16. In sth mRNAs , 17–20, th mRNAs . For exath mRNAs –25. Howeth mRNAs –29.In addition to mRNAs and lncRNAs described above, downstream of gene transcripts (DoGs) arises when Pol II terminates far downstream of the ends of genes . These rIn a recent study , we perfTo gain a comprehensive overview of the transcriptional landscape, we identified 38 publicly available datasets containing chromatin and nuclear fractionated RNA-seq samples. These cover 5 human cell lines  and four subcellular fractions . The initial processing and mapping to the human genome yielded > 2.5 billion uniquely mapped reads Fig. ” sectionWe classified TUs into two broad groups based on their genomic location relative to existing gene annotations Fig. a, b. (i)Assessing the expression levels in HeLa cells, it is apparent that the relative abundance of TUs is higher in the nucleus (4.28% of mapped reads) compared with the cytoplasm  and 571 independent TUs. As controls, we paired gene-associated TUs with their corresponding protein-coding genes, and we identified 3462 lncRNA genes in a similar size range to independent TUs.Next, we sought to understand the transcriptional origins of gene-associated TUs. Here, we focus on data from HeLa cells unless stated otherwise, as it is the cell type with the largest variety of measurements.Figure Since the novel TUs are transcribed by RNA Pol II, we asked if the unannotated TSSs initiating UoG transcription have previously been detected through cap analysis of gene expression (CAGE). To this end, we used annotated CAGE peaks derived from a large collection of cell lines and tissues , 41. UoGFigure Threonine-4 (T4P) phosphorylation is a hallmark of terminating Pol II and causes a characteristic NET-seq signal near transcription end sites (TESs) of protein-coding genes . Among pFinally, we examined the ChIP-seq profiles for four histone marks associated with transcriptional activity, as well as the histone acetyltransferase EP300 , mRNAs with processing defects , 52, andSince we observed a predominant chromatin retention Fig. a and no + RNA-seq technologies. Despite their study proving valuable to better characterize intergenic fragments associated with transcription of annotated genes, the methods used limited their detection potential. Indeed, the authors were able to identify a large number of novel alternative exons, although the functional implications of the intergenic transcripts they defined remained elusive [In 2010, van Bakel and colleagues explored transcribed unannotated transcripts by using tiling arrays and poly(A) elusive . Here, wTo date, transcription upstream of canonical genes has been reported as a consequence of bidirectional transcription from neighboring promoters or enhancers, with the transcript being generated in the antisense direction. In contrast to these transcripts, the UoGs identified here originate from the same strand as the associated downstream genes, thus limiting the possibility that these are products of enhancer- or promoter-derived divergent transcription. The presence of CAGE peaks on opposite strands around the beginning of these transcripts suggests that a minor fraction could instead originate from convergent transcription . Either Studies have recently highlighted the presence of widespread transcription of intergenic regions downstream of protein-coding genes in mice and humans in response to heat shock, osmotic stress, or oxidative stress , 31. AltAlthough intergenic transcription has been commonly considered a consequence of pervasive transcription and, therefore, having no apparent functional role, accumulating evidence indicates that such processes can have major repercussions on the activities of neighboring genes . Indeed,These mechanisms of transcription-associated chromatin regulation are not necessarily confined to intergenic regions linked to previously annotated genes but might be broadened to independent TUs. However, since these regions are usually found in gene-poor portions of the genome, their products are more likely to exert their functional role in trans. This and their similarities in terms of transcriptional activity, chromatin state, and degradation patterns to lncRNAs support the hypothesis that independent TUs could be novel lncRNA loci.In summary, we assembled publicly available RNA-seq data to identify and classify intergenic transcripts based on their expression and location relative to annotated genes. We showed that gene-associated and independent RNAs have characteristic patterns of transcription and that they are highly sensitive to nuclear degradation processes. Our data are consistent with recently reported chromatin remodeling and gene expression regulatory mechanisms associated with transcription. Collectively, the results expand the current categories in gene annotation and provide the tools to further investigate the underappreciated role of intergenic transcription as a function of gene expression and regulation.--twopassMode Basic --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --outFilterMultimapNmax 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --outFilterType BySJout --outSAMattributes All --outSAMtype BAM SortedByCoordinate, and specific options for gapped  and ungapped  alignments. PCR duplicates were removed using Picard MarkDuplicates (v2.18.3) with default parameters. Quantification of expression was performed using QoRTs (v1.3.0) [Sequencing quality checks were performed on all experiments using FastQC . Adaptor(v1.3.0)  and the (v1.3.0) .--normalizeUsing CPM --binSize 20 --smoothLength 60 options, and --filterRNAstrand for the selection of forward and reverse strands.Deduplicated unique alignments were converted to stranded normalized coverage bigWig files using deeptools (v3.0.2)  with --n-f 0.2 -g 100 -j 3 -t. The individual annotation GTFs from all wild-type RNA-seq datasets (no treatment condition was used to annotate the intergenic regions) were then used as input for StringTie with the --merge option to generate a non-redundant set of predicted transcripts. The output, which consists of a GTF file with merged gene models, was filtered using the gffcompare utility [-C option to discard predicted transcripts that were fully contained within larger annotated regions.Deduplicated uniquely mapped reads were assembled into a de novo annotation GTF using StringTie (v1.3.4c)  with the utility  with thesetdiff function from the GenomicFeatures R package [Upstream of gene (UoG): the unit is located upstream of the associated gene.Downstream of gene (DoG): the unit is located downstream of the associated gene.Linker of genes (LoG): the unit is located between two genes and transcriptionally associated with them.A custom pipeline written in R was used to process the GTF file generated as described above. The pipeline performs several steps, the first of which is the discrimination of the purely intergenic regions  from the known features . This operation is performed by the  package  on the gcountOverlapsUnion function. Features are considered co-transcribed if expressed (TPM ≥ 1) in the same cell line and in at least two datasets. At this level, the categorization is also re-evaluated and, if necessary, TUs can be re-assigned to the proper sub-group .To confirm the co-occurrence of the annotated gene(s) and gene-associated features, their expression is re-assessed across the RNA-seq datasets within R using a customized intersect to calculate the individual overlaps with annotated features retrieved from NONCODE [-s (stranded) and -f (to test the fractions of overlaps from 0.1 to 1). The number of independent TUs not annotated in any of the databases was obtained by performing sequential intersections (adding the options -wa -v).Expressed independent TUs were used as input for BEDtools  NONCODE , CHESS [ NONCODE , and Ref NONCODE  databaseview, and gapped alignments  were extracted based on their CIGAR information . Reads were then assigned to “long ncRNA” or “independent TU” features using the countOverlapsUnion function from the GenomicFeatures R package [Deduplicated unique alignments were parsed using samtools  view, an package . For eacSince the large majority of data available for validation derived from HeLa cells, we decided to focus our analysis of intergenic features only to those expressed in this cell line. Therefore, we generated a set of annotated genes and gene-associated and independent TUs where each feature had an average expression of ≥1 TPM across the HeLa RNA-seq datasets. In addition, we required the gene-associated features to be connected to annotated protein-coding genes, thus reducing the chance to include poorly annotated genes for which start and end genomic coordinates are not reliable . We retained only the independent TUs located ≥10 kb from any annotated feature on the same strand orientation, to ensure that their transcription is not directly linked to known genes. Finally, we discarded features with length < 5 kb to avoid signal overlaps between the start and end positions in metadata profiles.ScoreMatrixBin function from the genomation R package [The metadata profiles were generated using the CPM-normalized coverage bigWig files see “” section package . The wraWe collected the “fold change over control” and merged replicates ChIP-seq bigWig files from ENCODE. The list of epigenetic modifications and associated accession numbers can be found in Additional file We retrieved the hg38 CAGE reprocessed data  from theGenomicAlignments R package [countOverlapsUnion function. The estimateSizeFactorForMatrix function from the DESeq2 R package [ggpubr R package was used to visualize the results and perform the statistical tests .We collected data from cells in wild-type/untreated conditions and after the knockdown of several proteins from different sources (Additional file  package , and the package  was usedAdditional file 1. Integrated supplementary Figures and Tables. Contains figures from S1 to S5 and tables from S1 to S4.Additional file 2. Review history.


SNA is one of the essential EMT transcriptional factors capable of suppressing epithelial maker while upregulating mesenchymal markers. However, the mechanisms for SNA to transactivate mesenchymal markers was not well elucidated. Recently, we demonstrated that SNA collaborates with EGR1 and SP1 to directly upregulate MMP9 and ZEB1. Remarkably, a SNA-binding motif (TCACA) upstream of EGR/SP1 overlapping region on promoters was identified. Herein, we examined whether four other mesenchymal markers, lymphoid enhancer-binding factor (LEF), fibronectin (FN), cyclooxygenase 2 (COX2), and collagen type alpha I (COL1A1) are upregulated by SNA in a similar fashion. Expectedly, SNA is essential for expression of these mesenchymal genes. By deletion mapping and site directed mutagenesis coupled with dual luciferase promoter assay, SNA-binding motif and EGR1/SP1 overlapping region are required for TPA-induced transcription of LEF, FN, COX2 and COL1A1. Consistently, TPA induced binding of SNA and EGR1/SP1 on relevant promoter regions of these mesenchymal genes using ChIP and EMSA. Thus far, we found six of the mesenchymal genes are transcriptionally upregulated by SNA in the same fashion. Moreover, comprehensive screening revealed similar sequence architectures on promoter regions of other SNA-upregulated mesenchymal markers, suggesting that a general model for SNA-upregulated mesenchymal genes can be established. The poor prognosis of hepatocellular carcinoma (HCC) is due to frequent metastasis occurring via complicated processes, including epithelial mesenchymal transition (EMT), migration, and invasion of primary tumor . Among tThe cultured conditions for HCC340 and HepG2 cells were the same as described in our previous report . HCC340 Tetradecanoyl phorbol acetate (TPA) was purchased from Sigma-Aldrich . The monoclonal Snail (C15D3) antibody; and the antibodies against EGR1, SP1, MMP9, ZEB1, LEF1, Fibronectin, COX2, COL1A1, Histone H3, and GAPDH were purchased from Cell Signaling  and Santa Cruz Biotechnology , respectively. The snail expression plasmid, p-Snail, driven by CMV promoter within the p-cDNA3 vector, was a gift from Dr. Cheng K.K., Tzu Chi University.The promoter regions in the full-length promoter plasmids of LEF, FN, COX2, and COL1A1 were amplified from the genomic sequence of each gene. The PCR products were ligated into pGL3 vector . The promoter plasmids of deletion constructs were derived from each full-length promoter by double digestion with various restriction enzymes, followed by filling in the restriction site overhangs by Klenow enzyme. Subsequently, the digested DNA fragments were ligated into pGL3 vector.The full-length promoter plasmids of LEF, FN, COX2, and COL1A1 were used as templates for site-directed mutagenesis using a GeneEditor in vitro site-directed mutagenesis system  to obtain various mutant promoters according to the manufacturer’s protocol. The bases changed in the site-directed mutagenesis for the proposed Snail binding motif (TCACA), and the EGR1/SP1 overlapping binding region were the same as those described in our previous report ,18.Transwell and wound healing migration assays were performed as described in previous report ,24.Dual-Luciferase promoter assay was performed according to the protocol of manufacture .ChIP assay was performed as described in our previous Reports ,18. SuppRT-PCR was performed as described in our previous report ; the priEMSA was performed as described in our previous report . NuclearWestern Blot was performed as described in our previous Reports ,18.shRNA Technology was performed as described in our previous Reports . The seqt-test in Excel. All the quantitative studies were performed at least in triplicate, as appropriate. Statistical significance between groups were indicated by * p < 0.05 and ** p < 0.001.Data were analyzed using Student’s Initially, by RT-PCR, we found TPA elevated mRNA of SNA by 3.7-fold at 1 h and declined until 6 h, whereas it significantly increased mRNA of FN, LEF, and COX2 by 1.5–2-fold within 1–2 h, which was further increased to 2.5–4.8-fold until 6 h in HepG2, one of the conventional HCC cell lines a. Also, We further examined whether TPA can induce promoter activation of FN, LEF, COX2, and COL1A1 in a SNA-dependent manner. To this end, full-length promoter plasmid of FN (FNpro1150), LEF (LEFpro1595), COX2 (COX2pro979), and COL1A1 (COL1A1pro1090) were constructed by inserting the promoter fragment containing 1150 bp and 1595 bp, 979 bp and 1090 bp, respectively, upstream of the translation start site of each promoter into PGL3 vector. As shown in Further, we sought to identify the TPA-responsive element on promoters of FN and LEF, COX2, and COL1A1 and examined whether it contained the proposed SNA binding region “TCACA” and putative EGR1/SP1 overlapping motif, as described previously . AccordiTo prove whether the candidate SNA binding motifs are required for transcription of the aforementioned mesenchymal genes, site-directed mutagenesis coupled with promoter analysis were performed. For FN, promoter mutants with the altered sequence (TCACA→TTGTA) at indicated SNA motifs (PM 1–3) were produced for analysis in both HepG2 . In addiThus far, it appears that SNA coupled with EGR1/SP1 activate transcription of the aforementioned mesenchymal genes, whether the relevant transcriptional factors can bind to the indicated putative regions was examined within cells using ChIP assay. As shown in We further confirmed the binding of SNA on the aforementioned promoters by EMSA. Nuclear extract of HCC340 treated with TPA at different time points were incubated with biotin-labeled probe containing the SNA binding motif on FN, LEF, COX2, and COL1A1 promoter . As shown in In the past decades, it appeared that the mechanisms by which SNA upregulates the expression of mesenchymal genes were more complicated than it downregulates epithelial markers. As demonstrated in this and our previous  reports,Previously, the role of SNA in upregulating transcription of FN, LEF, COX2, and COL1A1 has been reported in different contexts. SNA can mediate TGFβ-triggered LEF expression for EMT in MDCKII cells . OverexpThus far, we have demonstrated six of the mesenchymal genes, FN, LEF, MMP9, ZEB1, COX2, and COL1A1, are transcriptionally upregulated by SNA in the same manner, i.e., by direct binding of SNA coupled with EGR1/SP1 on their putative regions. To predict whether this could be a general model, we screened the mesenchymal genes that can be upregulated by SNA from PubMed and examined whether they contain the aforementioned SNA binding motif (TCACA) coupled with a downstream EGR1/SP1 overlapping region in the gene bank. In addition to what we have investigated, five of the well-known mesenchymal genes such as vimentin, vitronectin, α-SMA, N-cadherin, and TWIST1 were identified . All of In conclusion, we proposed a general model for SNA to upregulate transcription of mesenchymal genes via collaboration with EGR1/SP1 in HCC. Remarkably, a specific putative SNA binding motif “TCACA” close to the EGR1/SP1 overlapping region, required for activating their promoters, was identified. In the future, this model will be validated after more of the candidate mesenchymal genes are investigated in HCC and other cancers.


Escherichia coli by either activating or repressing transcription. While the interaction of IscR with its DNA sites has been studied, less is known about the mechanism of IscR regulation of transcription. Here, we show that IscR recruits RNA polymerase to an activated promoter and that IscR binding compensates for the lack of an optimal RNA polymerase σ70 −35 promoter element. We also find that the position of the −35 promoter element within the IscR DNA site impacts whether IscR activates or represses transcription. RNA polymerase binding at a distally positioned −35 element within the IscR site results in IscR activation. Molecular modeling suggests that this position of the −35 element allows IscR and RNA polymerase to bind to the promoter from opposite faces of the helix. Shifting the −35 element 1 nucleotide upstream within the IscR binding site results in IscR repression and a steric clash of IscR and RNA polymerase binding in the models. We propose that the sequence similarity of the IscR binding site with the −35 element is an important feature in allowing plasticity in the mechanism of IscR regulation.IscR is a global transcription factor that regulates Fe-S cluster homeostasis and other functions in  Escherichia coli, the housekeeping sigma factor, σ70, recognizes promoters containing two conserved hexamers that are positioned around bp −10 (−12TATAAT−7) and bp −35 (−35TTGACA−30) relative to the transcription start site and are separated by an optimal spacing of 17 bp , which activates the suf operon, encoding the alternate Fe-S cluster biogenesis pathway (–suf and isc operons in the presence of O2 increases the capacity of Fe-S biogenesis pathways to repair or replace O2 or reactive oxygen species (ROS)-damaged Fe-S clusters and maintain Fe-S cluster homeostasis.The iron-sulfur cluster regulator (IscR) is a conserved global transcription factor that regulates essential functions for Fe-S cluster biogenesis in many bacteria. Under anaerobic conditions, when the demand for Fe-S cluster biogenesis is low, IscR ligates a [2Fe-2S] cluster (holo-IscR) and represses the  as IscR , 19. Und pathway –23. Thisisc and suf operons, IscR regulates 40 other genes in E. coli (isc operon) are regulated by IscR primarily under anaerobic conditions (suf operon) can be bound by either apo- or holo-IscR, but promoters containing these sites are regulated primarily under aerobic conditions, in part due to the higher levels of apo-IscR present under these conditions (hyaA) promoter showed that the DNA recognition helix of the winged helix-turn-helix motif inserts into a major groove making base-specific contacts with the IscR half-site and that the wing interacts with the adjacent AT-rich minor groove  and mutant promoters. These experiments show that small differences in the position of the −35 promoter element within the IscR binding site play an important role in RNA polymerase recruitment by IscR as well as in determining positive or negative regulation of the promoter by IscR.To understand how promoter architecture affects IscR-dependent transcription regulation, we made mutations in the promoter region that drives the transcription of  of life . While tfication , 28. ThiydiU, 4 of the 6 nucleotides of the −10 hexamer recognized by σ70 are conserved (TACACT [matches to the consensus are in boldface type]), but two matches to two possible suboptimal −35 hexamers, TGTTTG and TGTTTA, were identified . Thu. Thu70 rand K593 . One of and K593 . Thus, tydiU gene by recruiting RNA polymerase to overcome a weak −35 promoter element, then improving the −35 hexamer should bypass the need for IscR to activate PydiU. To test this hypothesis, we replaced the native −35 hexamer with a near-consensus −35 sequence, choosing nucleotides that would not disrupt IscR binding  and with the −35 hexamer of PsufA (B). Shown are gel images following electrophoretic separation of in vitro transcription products from plasmids containing the region from bp −200 to +40 relative to the transcription start site of wild-type PydiU and PydiU variants in the presence of 50 nM RNA polymerase and 0, 100, 200, or 400 nM IscR. The ydiU and control RNA-1 transcripts are indicated. Download FIG S1, PDF file, 0.9 MB.Replicates of Copyright © 2021 Chowdhury et al.2021Chowdhury et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro transcription assays for the improved promoter. The IscR protection pattern of the improved promoter was nearly identical to that observed for the native promoter, except for changes to the hypersensitive sites at position −36 and −38, indicating minor alterations of IscR interaction with its site. Nevertheless, when both proteins were present, protection by both IscR and RNA polymerase was observed, indicating that both proteins can bind simultaneously to the promoter. Altogether, the in vitro transcription and footprinting assays show that IscR compensates for a suboptimal −35 hexamer in activating PydiU and that IscR is no longer required when the suboptimal −35 hexamer is replaced with a near-consensus sequence.RNA polymerase binding to the improved promoter was assayed by DNase I footprinting . ConsistydiU with that of PsufA , matching the consensus hexamer. The basal level of transcript produced from the PydiU mutant with the PsufA −35 hexamer positioned 1 nucleotide closer to the −10 element was 3-fold higher than that of the native ydiU promoter, presumably a result of the stronger −35 hexamer. IscR was also able to activate the promoter albeit to a lesser extent than the native PydiU promoter  Representative image following electrophoretic separation of in vitro transcription products from plasmid templates containing the region from bp −200 to +40 relative to the transcription start site of WT PydiU and PydiU mutants with 2-bp deletions in the presence of 50 nM RNA polymerase and 0, 100, 200, or 400 nM IscR. The ydiU and control RNA-1 transcripts are indicated. (C) Replicates of in vitro transcription assays of PydiU mutants with 1- or 2-bp deletions. The assay was performed as described above for panel B. Download FIG S3, PDF file, 1.2 MB.(A) Sequence comparison of the WT and PCopyright © 2021 Chowdhury et al.2021Chowdhury et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the hyaA promoter, the mutant promoter was converted from one that was activated by IscR to one that was repressed, with a 5-fold decrease in the transcript level at the highest level of IscR tested , which is shifted 1 nucleotide upstream compared to the native −35 hexamer, 5′-TGTTTA, of the native promoter. The length of the RNA polymerase-protected region by DNase I footprinting  gene, which is involved in tRNA hydroxylation, are also activated by IscR from a type II IscR binding site, and the position of the −35 promoter element within the binding site of these promoters is the same as that in PydiU or PsufA . Thin PsufA  36). A . A ydiU ruginosa  37). Th. ThydiU 70 subunits of RNA polymerase to activate transcription  using the plasmid pPK6842, which contains the region between bp −200 and bp +40 relative to the transcription start site of PhyaA ; 20 μM UTP; and 500 μM (each) ATP, GTP, and CTP for 20 min at 37°C in a solution containing 40 mM Tris (pH 7.9), 50 mM KCl, 10 mM MgCl2, 100 μg/ml bovine serum albumin (BSA), and 0.1 mM dithiothreitol (DTT). E. coli RNA polymerase with σ70 (New England BioLabs [NEB]) was added to the assay mixtures at a 50 nM final concentration, and the reactions were terminated after 5 min by adding stop solution (USB). The samples were heated to 90°C for 60 s before being electrophoresed on a 7 M urea–8% polyacrylamide gel in 0.5× Tris-borate-EDTA (TBE) buffer. The transcripts were visualized using a Typhoon FLA-9000 gel imaging scanner and quantified using AzureSpot analysis software (Azure Biosystems).Plasmids were purified using a QIAfilter maxi kit (Qiagen). A total of 2 nM supercoiled plasmid DNA was incubated with 100, 200, or 400 nM IscR; 0.25 μCi of dGTP (PerkinElmer) using Klenow fragment. The radiolabeled fragment was isolated from a 5% acrylamide gel, purified using a QIAquick gel extraction kit, and incubated with either 100 nM E. coli RNA polymerase with σ70 (NEB), 500 nM purified WT IscR, or both for 30 min at 37°C in a solution containing 40 mM Tris (pH 7.9), 30 mM KCl, 100 μg/ml BSA, and 1 mM DTT. A total of 2 μg/ml of DNase I (Worthington) was added to the reaction mix for 30 s, followed by the addition of sodium acetate and EDTA to final concentrations of 300 and 20 mM, respectively, to terminate the reaction. The reaction mix was ethanol precipitated, resuspended in urea loading dye, heated for 60 s at 90°C, and loaded onto a 7 M urea–8% polyacrylamide gel in 0.5× TBE buffer. An A+G ladder was made by formic acid modification of the radiolabeled DNA, followed by piperidine cleavage (DNA fragments containing Pcleavage . The gelWT IscR was purified from strain PK8581 as previously described . The prohyaA (PDB accession number 4HF1) determined by X-ray crystallography was modeled onto the cryo-electron microscopy (cryo-EM) structure of E. coli σ70 bound to the rpsT P2 promoter (PDB accession number 6PSQ) by aligning the −35 hexamer of PhyaA to that of the rpsT P2 promoter (hyaA was transformed into the IscR binding site of PydiU using the “mutate” function in Sybyl. The resulting model of IscR bound to its target site in PydiU was refined using PHENIX.refine, using the PDB accession number 4HF1 electron density for the model base pairs (ydiU was modeled by aligning the −35 promoter element of PydiU to that of the rpsT P2 promoter.The structure of IscR bound to its DNA site from Ppromoter , 31, usise pairs . All modin vitro transcription or DNase I footprinting reactions was done on three separate days, with proteins isolated on three different days. The products were visualized using a Typhoon 9000 FLA scanner, and the images were viewed using ImageJ or AzureSpot software. For in vitro transcription reaction quantification, the intensity of each transcript band was quantified using AzureSpot software, and automatic background correction was done using the rolling-ball method. The WT and mutant ydiU and hyaA transcript levels were normalized to the corresponding RNA-1 levels. The normalized transcript levels from the triplicate experiments were averaged and plotted as a scatter graph. Error bars represent the standard errors of the means.Each set of All data are available within the manuscript and the supplemental material.