The vitamin D receptor (VDR) mediates 1,25-dihydroxyvitamin D3 pleiotropic biological actions through transcription regulation of target genes. The expression levels of this ligand-activated nuclear receptor are regulated by multiple mechanisms both at transcriptional and post-transcriptional levels. Vitamin D3 is the natural VDR activator, but other molecules and signaling pathways have also been reported to regulate VDR expression and activity. In this study, we identify valproic acid (VPA) and natural short-chain fatty acids (SCFAs) as novel transcriptional activators of the human VDR (hVDR) gene. We further report a comprehensive characterization of VPA/SCFA-responsive elements in the 5′ regulatory region of the hVDR gene. Two alternative promoter DNA regions (of 2.4 and 3.8 kb), as well as subsequent deletion fragments, were cloned in pGL4-LUC reporter vector. Transfection of these constructs in HepG2 and human Upcyte hepatocytes followed by reporter assays demonstrated that a region of 107 bp (from −107 to −1) upstream of the transcription start site in exon 1a is responsible for most of the increase in transcriptional activity in response to VPA/SCFAs. This short DNA region is GC-rich, does not contain an apparent TATA box, and includes two bona fide binding sites for the transcription factor Sp1. Our results substantiate the hypothesis that VPA and SCFAs facilitate the activity of Sp1 on novel Sp1 responsive elements in the hVDR gene, thus promoting VDR upregulation and signaling. Elevated hepatic VDR levels have been associated with liver steatosis and, therefore, our results may have clinical relevance in epileptic pediatric patients on VPA therapy. Our results could also be suggestive of VDR upregulation by SCFAs produced by gut microbiota. Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors, which is expressed in multiple normal and transformed cell types. The promoter regions of many nuclear receptor genes have been characterized. They resemble housekeeping genes, and many are embedded in GpC islands ,2. Anoth2D3, calcitriol or VitD3), the hormonal form of cholecalciferol [Specifically, VDR was discovered as a protein activated by 1,25-dihydroxyvitamin D3 lciferol . The genlciferol .VDR is a member of the superfamily of nuclear steroid/thyroid hormone receptors that localizes in the cytoplasm and nucleus of the cells. Following ligand-binding, the VDR forms a heterodimer with retinoid X receptor (RXR) to regulate gene expression via binding to vitamin-D-responsive elements (VDRE) in regulatory regions of target genes. The VitD3/VDR signaling pathway controls calcium and phosphorous homeostasis, immune response, hormonal systems, and cell growth . The groVDR abundance is controlled by transcriptional and post-transcriptional (mRNA stability) regulations, post-translational modifications, and ligand-induced receptor stabilization . At the Regarding VDR gene transcription, several mechanisms are involved. First, homologous upregulation of VDR mRNA by VitD3, the natural ligand for VDR, has been demonstrated both in vitro and in vivo, in a tissue-dependent manner ,17. In aIn previous studies we identified VDR as a pro-lipogenic nuclear receptor, that upon activation, triggers a coordinated gene response causing alterations in glycerolipid and phospholipid metabolism and leading to triglyceride accumulation in hepatocytes ,28. We aVPA has been the first-line, most-frequently prescribed, anti-epileptic drug in children for the past fifty years  and is aHere we identify a novel role of VPA/SCFAs in the induction of VDR expression in human hepatocytes and investigate the transcriptional activation of VDR by VPA/SCFAs through specific promoter DNA elements. For that purpose, two 5′ flanking regions of the VDR gene, previously described as promoter regions, were cloned upstream of the luciferase-coding gene to check functional activity in two different human hepatic cell models. After deletion and segmentation, a short promoter region of 107 bp, which includes two putative Sp1 binding sites, was identified as the principal VPA/SCFA response region in the hVDR gene.2/95% air in Ham’s F-12/Leibovitz L-15  supplemented with 7% fetal bovine serum , 50 U/mL penicillin , and 50 μg/mL streptomycin (Gibco). Cells were used or passaged at 70–80% confluence. For subculturing purposes, cells were detached with 0.25% trypsin/0.02% EDTA in PBS at 37 °C.HepG2 cells were routinely grown in culture grade flasks at 37 °C under a humidified atmosphere 5% CO® hepatocytes  are cells derived from primary human hepatocytes by a novel technology that forces cells to a limited proliferation, without immortalization, while maintaining specific liver functions. Unlike HepG2 cells, they express characteristic hepatic genes [Human Upcyteic genes  and presic genes ,36. UpcyCells were treated with the appropriate compound or with an equal volume of vehicle (PBS) for 24 h. Valproate (Cat. no. P4543), butyrate (Cat. no. B5887), octanoate (Cat. no. C5038), hexanoate (Cat. no. C4026), propionate (Cat. no. P1880), and acetate (Cat. no. S2889) were purchase from Sigma-Aldrich .Total RNA was purified from cultured cells by using a mixed TriZol–column-based protocol. More specifically, following lysis with TriZol and phase separation with chloroform, the aqueous phase was mixed with an equal volume of buffer and processed according to the protocol of RNAeasy kit (Qiagen). The integrity of total RNA was assessed by running 500 ng of total RNA on 1% agarose gel and by a microcapillary electrophoresis , whereas purity was assessed by the A260/A280 ratio obtained after measurement with NanoDrop1000.Gene expression analysis by RT-qPCR were performed essentially as already described . PBGD  and a sequence of 2440 bp upstream and within exon 1c (fragment between −1253 and +1187 of the transcription start site of exon 1c) of the hVDR gene previously described as putative promoters ,11 were Chimeric luciferase reporter constructs with the two fragments of 3836 bp (from −1899 to +1937 around exon 1a transcription start site) and 2440 bp (from −1253 to +1187 around exon 1c transcription start site) of the flanking region of the human VDR gene were obtained by cloning the PCR products (digested with KpnI-HindIII) into the promoterless pGL4.10 vector  previously digested with the same enzymes, which contains the firefly luciferase reporter gene. Lack of mutations in the cloned sequence was assessed by Sanger DNA sequencing and contig assembly.pGL4.10-VDR-3836 was digested with enzymes KpnI-EcoRV, to get a fragment of 1307 bp (−1899 to −592), and EcoRV-HindIII to get a fragment of 2528 bp (−591 to +1937) which were subcloned into pGL4.10.The sequences of 1438 bp (+499 to +1937), 1968 bp (−98 to +1870), and 489 bp (−591 to −102) were obtained by digesting the plasmid pGL4-VDR-2528 with ApaI, SacI, and KpnI-SacI, respectively, and subsequently subcloned in pGL4.10 digested with same enzymes.The sequence of 597 bp (−98 to +499) was obtained by digesting the plasmid pGL4-VDR-3836 with SacI-ApaI, and the resulting fragment was ligated into pGL4.10 digested with same enzymes.Sequences corresponding to 245 bp (−97 to +148) and to 332 bp (+167 to +499), were PCR cloned from pGL4-597-VDR with specific primers . Amplicons were digested with KpnI and XhoI and the resulting fragment was ligated into pGL4.10 digested with same enzymes.Sequences corresponding to 107 bp (−107 to −1), 96 bp (−7 to +89), and 60 bp (+88 to +148) were PCR cloned from pGL4-332-VDR plasmid as previously described with specific primers . Amplicons were digested with SacI and HindIII and the resulting fragment was ligated into pGL4.10 digested with same enzymes.5 cells per well. Cells were incubated at 37 °C overnight, and 1 h before transfection, medium was replaced with fresh antibiotic-free medium. Prior to transfection 200 ng of Renilla-coding pRL-CMV vector  and equimolar amounts of either pGL4.10 empty vector or VDR-promoter constructs were mixed in Opti-MEM media, followed by the addition of the reagent X-tremeGENE HP DNA Transfection Reagent  in a 1:1 DNA:lipid ratio. The mixture was incubated for complex formation at room temperature for 30 min. DNA:lipid complexes were added dropwise to the cells. Next day, medium was changed, and VPA/SCFAs were added to the cells for 24 h to activate reporter constructs. Cell lysates were prepared by applying PLB buffer (Promega). Lysates were cleared by centrifugation at 10,000× g for 1 min. Next, 10 µL of each lysate were pipetted in a white microplate, followed by addition of 30 µL of LAR II reagent, and the luminescence produced by firefly luciferase was recorded. Finally, 30 µL of Stop and Glo reagent (Promega) was added to quench the firefly signal and to detect the luminescence produced by the Renilla luciferase. Each experiment was performed in duplicate and replicated between three and eight times.Transient transfections with VDR-reporter plasmids were performed in HepG2 and Upcyte cells seeded in 24-well plates at a density of 1.4 × 10t-test or ANOVA as appropriate, with Graph Pad Instat Software . Means were considered significantly different if p-values < 0.05 were obtained. Quantitative variables were expressed as mean ± standard error of the mean (SEM), but individual values for each replicate were also depicted.The luciferase assay data were statistically analyzed by In a previous study, we demonstrated that active VitD3/VDR signaling triggers a coordinated gene response leading to triglyceride synthesis and to perturbations in lipid metabolism, specifically in glycerolipids and phospholipids . The aboCells were treated with sub-cytotoxic concentrations (2.5–10 mM) of SCFAs (butyrate and acetate) or VPA and VDR mRNA levels analyzed by RT-qPCR. No significant effect was observed for acetate, whereas butyrate and VPA dose-dependently induced VDR expression both in HepG2 and hepatocytes A,B. A tiPrevious studies ,11 delimAs shown in To precisely delineate the DNA sequence responsible for hVDR activation by VPA, further deletions were obtained and cloned. First, the 3836 bp sequence was fragmented into two regions of 1307 bp and 2528 bp.As shown in Subsequently, we determined whether natural SCFAs also modulate reporter gene activity of the construct pGL.4-VDR-2528. Similar reporter assays were conducted in HepG2 cells treated with acetate, octanoate, hexanoate, butyrate, propionate, and VPA. As shown in To further delimitate the VDR gene sequence responsive to VPA/SCFAs, the fragment of 2528 bp was subsequently divided into shorter regions: 1968 bp (−98 to +1870), 1438 bp (+499 to +1937), and 489 bp (−591 to −102).Promoter functionality of the new constructs was again evaluated with luciferase reporter assays . The 196To gain further insight into the sequence of interest (between −98 and +499), pGL4-VDR-3836 was digested with ApaI and SacI to obtain the sequence of 597 bp (−98 to +499) which was cloned and two subsequent fragment regions of 245 bp (−97 to +148) and 332 bp (+167 to +499) were amplified by PCR from pGL4.10-3836 and subcloned into pGL4.10. Results shown in Results in To our knowledge this is the first study showing that VPA and natural SCFAs are able to transcriptionally upregulate the hVDR gene. To gain insight into this novel mechanism of hVDR regulation, we cloned two 5’ upstream regions previously described as putative promoters ,11 and e2-terminal tails of core histones is central in the specification of a “histone code” that influences the expression of target genes. Recruitment of histone acetyltransferases by transcription factor complexes is associated with a more open DNA conformation that, in general, facilitates gene transcription. Conversely, deacetylation of core histones by HDACs is associated with a closed chromatin conformation and  repression of transcription. Therefore, inhibition of HDACs typically leads to derepression of transcription [Although the mechanisms by which VPA/SCFAs modulate Sp1 and upregulate VDR transcription has not been addressed in this study, we speculate on several hypothesis based on previous knowledge of VPA and SCFA modes of action . The reccription ,42.HDACs are the target of a wide number of structurally diverse compounds known as HDAC inhibitors (HDACi) . AccordiHaving this in mind, one possibility is that the increased levels of hVDR reporter activity described in this work are due to VPA-mediated inhibition of HDACs, through either direct or indirect mechanisms. This possibility is also supported by the fact that HDACs are recruited by a variety of corepressor complexes and transcription factors, such as Sp1. Indeed, it has been shown that HDAC1 can directly interact with Sp1 via its C-terminal domain ,45, but There is also increasing evidence of transcription factors being acetylated at lysine residues, thus modifying their transcriptional or binding activity to the DNA response elements ,48. PrevIn that sense, VPA could inhibit the deacetylation of Sp1 by HDACs—thus disrupting their interaction and reducing the ability to direct HDAC-1 and -2 to target gene promoters. Indeed, a similar mechanism has been described for Npr1 where promoter deletion analysis demonstrated that Sp1 binding sites were essential for HDACi-mediated upregulation of Npr1 promoter activity .From a physiological point of view, VDR activation by SCFAs may lead to different effects. On the one hand, it may have a positive role on cholestatic conditions since it has been shown that active VDR induces CYP3A4 ,58, whicFinally, the role of VDR in cell-growth inhibition and differentiation in many target tissues ,63 and tSCFAs, the main products of bacterial fermentation of dietary fibers, have been identified as mediators of diet-induced crosstalk between the microbiome and the host. SCFAs are important for health as they not only provide energy for the intestinal epithelium, but also have many bioactive roles. They regulate immunity, chemotaxis, phagocytosis, and cell proliferation, and have anti-inflammatory, antitumorigenic, and antimicrobial effects, and alter gut integrity . WhetherAltogether, the identification and cloning of this hVDR promoter responsive to VPA and SCFAs provides the basis for future studies aimed at identifying the molecular mechanism of this regulation. Moreover, given the enormous relevance of the VitD3/VDR pathway in human pathophysiology, supported by a wealth of continuous studies, our mechanistic results provide the backdrop and rationale for future translational clinical studies.


In this study, Mittal et al. report that sequence-specific transcription factors and their tethered cofactors  are generally bound to promoters prior to induction (“poised”), rather than recruited upon induction, whereas induction recruits the preinitiation complex (PIC) to DNA. Their findings suggest that inducible systems, where present, evolved on top of constitutive systems. Genome-wide, little is understood about how proteins organize at inducible promoters before and after induction and to what extent inducible and constitutive architectures depend on cofactors. We report that sequence-specific transcription factors and their tethered cofactors  are generally bound to promoters prior to induction (“poised”), rather than recruited upon induction, whereas induction recruits the preinitiation complex (PIC) to DNA. Through depletion and/or deletion experiments, we show that SAGA does not function at constitutive promoters, although a SAGA-independent Gcn5 acetylates +1 nucleosomes there. When inducible promoters are poised, SAGA catalyzes +1 nucleosome acetylation but not PIC assembly. When induced, SAGA catalyzes acetylation, deubiquitylation, and PIC assembly. Surprisingly, SAGA mediates induction by creating a PIC that allows TFIID (transcription factor II-D) to stably associate, rather than creating a completely TFIID-independent PIC, as generally thought. These findings suggest that inducible systems, where present, are integrated with constitutive systems. Saccharomyces (budding yeast) genome  in essentially any condition that supports growth. They have also evolved inducible systems at specific genes that allow a wide range of expression when faced with distinct environmental conditions . We rece) genome . This prPreviously, we defined 5378 yeast promoters with measurable RNA polymerase (Pol) II preinitiation complexes (PIC) in rich media . A PIC cConstitutive promoters  lack this inducible architecture . They gedep) appears to dominate most but not all promoters (ind), SAGA-dominated promoters (The concept of constitutive and inducible promoters dovetails with the notion of two distinct PIC assembly pathways either through TFIID (transcription tactor II-D) or through SAGA . While tromoters . Althougromoters . Importaromoters . High GTromoters . An alteromoters  but has romoters . Here weWhile constitutive promoters are simple and largely homogenous in factor organization and regulation, inducible promoters are quite diverse . In the ind and TAFdep PICs. In contrast, SAGA is neither present nor appreciably active at constitutive promoters. At constitutive promoters, TAFs are largely left to their own devices to recruit low levels of TBP, and this is assisted by Gcn5-dependent (not SAGA) acetylation of the +1 nucleosome.We found that SAGA's TBP delivery activity is more restricted than even the earliest studies had suggested , being lN = 150 but imputed for all 5378 PIC-containing promoters using multiple criteria . Briefly, RP promoters were selected first, followed by induced promoters, then poised promoters, and finally constitutive promoters . They represent poised promoters that demonstrably can be induced.To provide granularity on inducible versus constitutive regulatory mechanisms on a genomic scale, we established the following five promoter classes representing broad but distinct archetypes of regulation: (1) RP, (2) induced, (3) poised, (4) constitutive, and (5) condition-specific (induced by heat shock). Since the constitutive class is quite distinct from the other four classes, we collectively refer to the latter set as “inducible.” These labels are metaphors for very specific selection criteria used in this study. They are not exclusive of other criteria that could use the same labels. We aimed to test the hypotheses that (1) induced promoters can be characterized simply by relatively high GTF:TAF ratios , (2) poised promoters can be characterized simply by high SAGA occupancy after removing RP and induced promoters, and (3) constitutive promoters can be characterized as having “normal” GTF:TAF ratios after removing those defined as inducible. This then provides a basis for defining precise protein/DNA architectures and interrelated mechanisms of PIC assembly at inducible versus constitutive promoter classes. Details of membership criteria for the five classes and its justification are provided in the Materials and Methods section. Class membership was capped at romoters . It has romoters  but alsoSupplemental Table S2; N = 70, 10 per column) organized across the five promoter classes (rows) and aligned by their most relevant promoter-organizing center: UAS, Sua7, or +1 nucleosome. Sua7 (TFIIB) peak locations were used to provide high-accuracy PIC locations, since yeast transcription start sites (TSSs) are more variable due to Pol II template scanning prior to productive initiation  data sets representing ∼400 different proteins in ∼40 genome-wide meta-assemblages . Both NuA4 and SAGA contain Tra1, which interacts with ssTFs , and this is further confirmed by earlier studies  was slightly enriched at RP core promoters in the kin28as mutant . However, this enrichment was very low compared with the high Mediator enrichment at the equivalently expressed induced promoter class. Mediator at RP promoters was on par with the very low amount detectable at constitutive core promoters. At all promoter classes, Pol II (Rpb3) enrichment in the kin28as mutant relative to WT was high at the core promoter and also 100–200 bp downstream from the PIC. Both regions are where Pol II stalls during stress . The induced class was also enriched with NuA4 and SAGA but to a lesser extent than RP and the poised class. The poised class had ssTFs  that largely differed from those at RP and the induced class  and cofactors  . This ratio was substantially higher at poised (and uninduced condition-specific promoters) compared with induced promoter classes. Thus, while Mediator and other cofactors are tethered to ssTFs at the UAS  the types of ssTFs present, (2) the conformational state of the coalesced cofactors, (3) the level of PIC assembly, and (4) the amount of Pol II transcription. Such toggling has been demonstrated for Tup1 . This exRP, induced, and poised promoter classes had additional characteristics, such as the binding of lesser-studied proteins at the PIC site. For example, induced PICs had particularly high Sub1 occupancy K. Sub1 hThe constitutive class, despite lacking ssTFs and stably bound cofactors , was enriched with well-positioned genic nucleosomes that have an abundance of transcription-linked histone marks , H2A.Z/SWR, and Bdf1/2 than would be expected of their low levels of PIC occupancy and transcription Z. Thus, Supplemental Material, including Supplemental Figures S2 and S3A, and Supplemental Table S4. In addition to examining inducible and constitutive promoter states under normal growth conditions , we examined responses to acute heat shock  to assess the role of SAGA modules and TFIID in rapid genome-wide reprogramming. To this end, membership in the H-series of promoter classes  paralleled the M-series criteria except being defined under acute heat shock. All depletions were conducted for 30 min at 25°C before applying heat shock (6 min at 37°C) or a mock equivalent (6 min at 25°C).To define the functional interplay among factors within inducible and constitutive architectures, we focused on the well-studied but unresolved relationship between SAGA and TFIID during PIC assembly. We examined TFIID and the four activities of SAGA on a genomic scale using deletion and/or depletion mutants and then measured protein–DNA architectures at the five promoter classes, along with impacts on nascent transcription. Strain validation, system controls, and data quality assessments are described in the Supplemental Fig. S4). SAGA was precisely centered on Hsf1-bound sites and UASs at other inducible promoter classes such as RP.Since we examined genome reprogramming by heat shock, we selected Hsf1 as a representative ssTF for functional contribution. As expected, Hsf1 bound the UAS region of heat shock-induced promoters (H02a and H02aNotM02ab) before and after heat shock and was not enriched at other promoter classes . Thus, SAGA occupancy depends on promoter-bound Hsf1, but Hsf1 occupancy does not depend on SAGA and NuA4. NuA4 plays a central role in TFIID-directed PIC assembly (Both Hsf1 and SAGA occupancy increased at induced promoters upon heat shock. We tested whether Hsf1 recruits SAGA by rapidly depleting Hsf1 at 25°C and then measuring SAGA (Sgf73) occupancy after 6 min of heat shock. SAGA became markedly depleted only at promoter classes that were normally enriched with Hsf1 B. In conassembly , possiblassembly . With TFassembly C. Thus, Supplemental Fig. S3A,B). Little or no loss of PICs was observed at constitutive promoters. The same conclusions were drawn when all promoters were examined . This fits with our observed absence of SAGA at constitutive promoters  and TFIID (Taf1) at the five promoter classes. As expected, the induced and constitutive classes had the highest and lowest levels of TBP occupancy, respectively, in accord with their expression level and the transient nature of constitutive PICs . Either romoters A,B. Bothromoters , which wSupplemental Fig. S3A) despite having similar levels of SAGA  and Pol II occupancy . Therefore, SAGA and its recruitment by ssTFs are of central importance for augmented PIC assembly primarily at induced promoters, with a lesser contribution when promoters are poised. Little or no direct contribution is made at constitutive promoters.The poised promoter class showed less SAGA dependency than induced promoters ( of SAGA A. Since dep (loss of TBP when Taf1 was depleted), while the induced (M02a and H02a) class was apparently TAFind . However, additional control experiments described below cast doubt on this generally accepted principle of TAF independence and drove us to reconsider the relationship between SAGA and TFIID in PIC assembly.For purposes of consistency with prior studies , we examy TAFind . Trends When Taf1 was depleted by anchor-away and auxin-induced degradation, we monitored actual Taf1 occupancy loss at each promoter class. As expected, Taf1 was lost at RP, poised (M03a and H03a), and constitutive (M04 and H04) promoter classes . Surprisind) or redundantly with TFIID .We next examined H3K9 acetylation dependency on Gcn5. Gcn5 exists as part of at least two complexes: SAGA/SLIK and ADA . Like otGCN5 (gcn5Δ) or depleting Gcn5 at 25°C resulted in a substantial loss of H3K9ac density, whether assayed under normal or heat shock conditions . This confirms prior genome-wide observations at 25°C . This occurred under normal and heat shock conditions. The extent of H3K9ac loss was similar to that seen upon loss of Gcn5. Little or no loss was observed at constitutive promoters. Thus, SAGA acetylates +1 nucleosomes at all classes of inducible promoters but not at constitutive promoters. Gcn5 apart from SAGA or ADA acetylates constitutive promoters.Deletion or depletion of SAGA-specific Spt20 resulted in loss of H3K9ac density predominantly at inducible promoters   might stall the cycle at any point, resulting in elevated or depressed H2Bub levels. We observed this among the inducible promoter classes. For example, RP promoters showed increased H2Bub, whereas induced promoters showed decreased H2Bub in an spt20Δ strain. At constitutive promoters, loss of SAGA had little or no impact on H2B deubiquitylation under normal or heat shock conditions, reaffirming the lack of SAGA involvement at constitutive promoters.We next examined SAGA's H2B deubiquitylation activity at the five promoter classes. H2Bub predominates at genic nucleosome positions +1 through +4 . As a trind TBP. These properties, along with enrichment of less-studied proteins such as Bye1, characterize the architecture of a constitutive promoter. While constitutive promoters largely have a simple protein architecture that can include sequence-specific insulation from upstream events, a more granular analysis would be needed to determine whether additional architectural subclasses exist.Constitutive and inducible genes have distinct promoter architectures . About 8About 20% of all promoters are inducible. Their function is to sense environmental signals and create a greater dynamic range of expression compared with constitutive promoters. The inducible class tends to have a UAS that anchors ssTFs and multiple cofactors  to the promoter region . ConsequThe ssTFs at poised promoters differ from those at induced promoters. This may reflect ssTFs at induced promoters being activated by existing environmental signals, whereas the ssTFs at poised promoters may be tuned to a different set of signals that are not present. The notion of a poised state makes sense in light of a rapid response to acute changes in environmental conditions. Induced promoters appear much like poised promoters, except their cofactors actively augment PIC assembly, which results in high levels of gene expression.Since we had not demonstrated that the poised promoter class can in fact be induced, we also examined a set of condition-specific heat shock promoters . They are quiescent at 25°C but are rapidly and robustly induced by 6 min of heat shock (37°C). Their induction is defined as having high GTF:TAF (Sua7:Taf2) ratios at 37°C but not at 25°C. Like poised promoters, we found these condition-specific promoters to have the main complement of ssTFs and cofactors at the UAS both before and after induction.ssTFs such as Hsf1 and Gal4 do not require inducing signals to bind their cognate site(s) , althougWith ssTFs/cofactors already present in the NDR, the historical view of gene activation in budding yeast may be expanded. At some promoters, certain ssTFs might invade and evict nucleosomes while recruiting cofactors and the PIC. However, at most other inducible promoters, ssTF and cofactors are generally present, and resident nucleosomes are already depleted. They proceed through a measurable state change that augments the recruitment of PIC components. This is manifested by a locally broadened ChIP-exo pattern for cofactors. ssTFs/cofactors, whether latent or active, generally do not stably engage GTFs, TAFs, or Pol II, as we see little cross-linking at the UAS for these PIC components compared with ssTFs and cofactors. However, transient interactions are not ruled out. Rap1 is an exception in having measurable TAF interactions at the UASs of RP promoters. Reciprocally, we saw little or no cross-linking of ssTFs and cofactors at core promoters like we saw for PIC components. Thus, unlike the main cofactors , PIC components are not stably tethered and poised at the UAS, waiting to be deposited upon induction. Reciprocally, the main cofactors are not stably engaged with the PIC.Our data suggest that yeast promoters generally have a single TFIID-containing PIC position. TFIID may have evolved to be intrinsically inefficient in PIC assembly, which is manifested at constitutive and poised promoters. There, Taf1 may inhibit TBP/DNA interactions and may have weak interactions with acetylated +1 nucleosomes . The weaAt induced promoters, two alternative high-efficiency TAF recruitment mechanisms may be in play in addition to the low-affinity interaction that is characteristic of constitutive promoters. One high-efficiency mechanism is exemplified at RP promoters, although a limited number of other induced promoters that use a similar mechanism is not ruled out. At RP promoters, Rap1 directly engages with TAFs and TFIIA in a high-affinity but dynamic interaction that may eliminate the inefficiencies inherent at constitutive promoters and allows for high PIC assembly . In suppIn the second high-efficiency TAF recruitment mechanism, ssTFs (but not Rap1) and SAGA deliver TBP to the TATA-box at induced promoters to form a PIC. This is a stable interaction of TBP with DNA that supports transcriptional bursting. Consequently, and unlike the other mechanisms, TBP binds DNA first and then engages the TAF complex, possibly through TFIIA . This alHow SAGA contributes to transcriptional regulation has been the subject of much debate. Early studies showed that SAGA contributed to the expression of highly regulated genes, with minor contributions at most genes , which aOur study suggests a unifying principle whereby essentially all Pol II promoters contain a TFIID-anchored PIC. This might allow inducible and constitutive promoters to evolutionarily interconvert through a gain or loss of a UAS, along with adjustments to core promoter sequences such as the TATA-box (and likely other regions). Inducible regulatory mechanisms are outfitted with multiple regulatable molecular switches, including environment-sensing ssTFs, nucleosome remodeling, and PIC assembly. The same switches are well established in multicellular eukaryotes, including humans, and so the intertwined mechanisms of constitutive and inducible PIC assembly described here are likely to be applicable in other eukaryotic systems.Further information and requests for reagents may be directed to and will be fulfilled by B.F.P. (fp265@cornell.edu).S. cerevisiae cultures were grown in 50 mL of YPDA medium to an OD600 of 0.6–0.8 at 25°C. Formaldehyde at a final concentration of 1% (v/v) was used to cross-link cells for 15 min at room temperature, and 125 mM glycine was used to quench for 5 min. For inducing acute heat shock at 37°C, 50 mL of YPDA at 55°C was added to 50 mL of cultures and quickly transferred to a shaker incubator  were constructed in a modified W303 background. Hsf1-FRB and corresponding parental strains were in BY4742 background and were a gift from Dr. David Gross (Louisiana State University).ncubator  for 6 mincubator . Cells wExponentially growing yeast cells were immobilized on microscope slides with agarose pads and imaged on a Leica fluorescent microscope (Leica DMI6000). Images were acquired and recorded with a Hamamatsu ORCA-R2 C10600 camera. Fluorescence and phased-contrast images were captured and processed using developed Matlab software. To deplete D-tagged factors from the nucleus, cells immobilized on agarose pads were incubated with 1 μg/mL  rapamycin for 60 min at room temperature. Changes in fluorescence were subsequently measured and recorded.Exponentially growing D-tagged and corresponding parental strains were serially diluted 10-fold, and 5 μL of cultures was spotted on YPDA agar plates containing 1 mM  auxin or 1μg/mL rapamycin alone or in combination. Plates were incubated for 2–3 d at 30°C.Supplemental Fig. S2A; SPT20, SPT7, GCN5, TRA1, TAF1, and SUA7 genes.Depletion was achieved using a combined anchor-away (AA) and auxin-induced degradation (AID) system in which a D tag was fused to the C terminus of the factor to be removed  along with 1 mL of 0.5-mm acid-washed zirconia beads. Lysis was performed by bead beating for four cycles of 3 min on and 7 min off in a Mini-Beadbeater-96 machine (Biospec). Whole-cell lysates were centrifuged at 14,000 rpm for 3 min at 4°C. Cytoplasmic supernatants were discarded, and nuclear pellets were resuspended in 200 μL of FA lysis buffer supplemented with 0.1% SDS and 75 μL of 0.1-mm zirconia beads. Nuclear samples were sonicated in a Bioruptor (Diagenode) for four cycles of 30 sec on and 30 sec off. Samples were centrifuged at 14,000 rpm for 10 min to collect sonicated chromatin fraction, which was predominantly enriched with DNA fragments in a range of 100–500 bp in size (verified on agarose gels).The following antibodies were used in ChIP-exo: anti H3K120ub , anti-H2B (Abcam 1790), nonspecific IgG (Sigma i5006), anti-TBP (Abcam 61411), anti-Pol II (Santa Cruz Biotechnology 8WG16), anti-H3 (Abcam 1791), anti-H3K9ac (Millipore Sigma 07-352), and anti-H3K14ac (Abcam ab52946). Sera against Hsf1, Taf1, and SAGA subunits  were obtained from David Gross (Louisiana State University), Joe Reese (Pennsylvania State University), and Tony Weil (Vanderbilt School of Medicine), respectively.Chromatin obtained from 50 mL of culture was used for a single ChIP experiment. Appropriate antibodies (3–5 μg per ChIP) were conjugated to protein A/G magnetic Sepharose resin  for 6–8 h at 4°C. Unconjugated antibody was removed, and then chromatin samples were incubated overnight (12–16 h) at 4°C with constant mixing. For TAP-tagged samples, 40 μL of IgG-Dynabead slurry was used per sample. Subsequent steps were performed as described previously .S. cerevisiae genome (sacCer3) using BWA-MEM (v.0.7.9a) . Sequenced reads were aligned to the .0.7.9a) . Aligned.0.7.9a)  to remov.0.7.9a) , except https://www.ncbi.nlm.nih.gov/geo) with accession numbers GSE147927 .https://github.com/CEGRcode/scriptmanager, doi:10.1145/3491418.3535161).Analysis was performed with CLI ScriptManager v.013 . Links to the up-to-date curated and interactive data will be available through the GitHub repo.


In oleaginous yeast, nitrogen limitation is a critical parameter for lipid synthesis. GATA-family transcriptional factor GAT1, a member of the target of rapamycin (TOR) pathway and nitrogen catabolite repression (NCR), regulates nitrogen uptake and utilization. Therefore, it is significant to study the SpGAT1 regulatory mechanism of lipid metabolism for conversion of biomass to microbial oil in gat1, the lipid yield of OE::gat1 increased markedly in the low carbon and nitrogen ratio (C/N ratio) mediums, while the lipid yield and residual sugar of SpMIG1 expression on the high C/N ratio medium. MIG1 deletion has been found in earlier research to affect glucose metabolic capacity, resulting in a prolonged lag period. Therefore, we speculated that SpGAT1 influenced glucose consumption rate across SpMIG1. Based on yeast one-hybrid assays and qRT-PCR analyses, SpGAT1 regulated the glyoxylate cycle genes ICL1, ICL2, and pyruvate bypass pathway gene ACS, irrespective of the C/N ratio. SpGAT1 also could bind to the ACAT2 promoter in the low C/N medium and induce sterol ester (SE) accumulation.Compared with WT, S.podzolica zwy-2-3, but that its regulatory patterns varied depending on the C/N ratio. When the C/N ratio was high, SpGAT1 interacted with SpMIG1 to affect carbon absorption and utilization. SpGAT1 also stimulated lipid accumulation by regulating essential lipid anabolism genes. Our insights might spur more research into how nitrogen and carbon metabolism interact to regulate lipid metabolism.Our findings indicated that SpGAT1 positively regulated lipid metabolism in The online version contains supplementary material available at 10.1186/s13068-022-02177-z. Because of the shortage of fossil fuels and the rising use of first-generation biofuels, renewable and sustainable alternatives , like mid yields , 4. Whend yields , 5.In recent years, scovered . \documeeveloped –12. Howeo medium . While eChlamydomonasreinhardtii, Trichosporonoleaginosus) , Y2H [pGADT7 +pGBKT7-SpMIG1] and Y2H [pGADT7-SpGAT1+pGBKT7-SpMIG1] were diluted at 0.1, 0.01, and 0.001 for serial dilution and spotted on DDO, TDO, QDO, and QDO+30mM 3-AT plates to detect the interaction of SpGAT1 with SpMIG1, respectively. 3-AT was for suppressing self-activation of prey protein.For yeast two-hybrid assays, refer to the Matchmakermentclass2pt{minimPDC, ICL1, ICL2, ACAT1, ACS and ACAT2 were inserted into the pAbAi vector. Then, linearize pAbAi-promoter vectors with BstBI digestion sites and transform linearized vectors into Y1H, the positive transformants were gradiently diluted at 0.1, 0.01, and 0.001 for serial dilution and spotted on SD/-Ura and SD/-Ura/AbA to detect promoters’ self-activation. Next, the negative control (pGADT7) and the experimental group (pGADT7-SpGAT1) were transformed into Y1H [pAbAi-promoter] competent cells according to the manufacturer’s instructions (Clontech); finally, the positive transformants were gradiently diluted at 0.1, 0.01, and 0.001 for serial dilution and spotted on SD/-Leu and SD/-Leu/AbA to detect whether SpGAT1 bound to the promoter.For yeast one-hybrid assays, refer to the MatchmakerAdditional file 1: Table S1. Strains and plasmids. Table S2. Primers for gene amplification and qRT-PCR. Table S3. cDNA sequence of SpGAT1 and SpMIG1.Additional file 2: Fig. S1. Single cell lipid yield and fatty acid profile of S.podzolica zwy-2-3 cultivated in rapamycin treatment and different C/N ratio mediums. a Single cell lipid yield stained by Nile red. b Fatty acid profiles analyzed by GC-MS. Fig. S2. Results of SpGAT1 mutant identification. a Results of amplifying hygromycin gene, homologous arm, and part of SpGAT1 gene . b Results of amplifying full length of SpGAT1 . c Results of amplifying hygromycin gene by colony PCR . d Detecting SpGAT1 expression level in WT and OE::gat1 by qRT-PCR. Fig. S3. Effects of SpGAT1 on fatty acid profiles and different carbon/nitrogen sources utilization in S.podzolica zwy-2-3. a Fatty acid profiles in WT, Δgat1 and OE::gat1 cultivated in low and high C/N ratio mediums. b Utilization different nitrogen sources in WT, Δgat1, and OE::gat1. c Utilization different carbon sources in WT, Δgat1, and OE::gat1. Fig. S4. Results of subcellular localization observed by fluorescence microscopy. Cyto: transcription factors were cytoplasmic localization, Nuclear: transcription factors were nuclear localization, Nucl-Cyto: transcription factors were located in cytoplasm and nuclear. Fig. S5. Prediction of SpGAT1 interacting protein. a Predicted by GeneMANIA database, b Predicted by String database.


It is now definitively established that a large part of the human genome is transcribed. However, only a scarce percentage of the transcriptome (about 1.2%) consists of RNAs that are translated into proteins, while the large majority of transcripts include a variety of RNA families with different dimensions and functions. Within this heterogeneous RNA world, a significant fraction consists of sequences with a length of more than 200 bases that form the so-called long non-coding RNA family. The functions of long non-coding RNAs range from the regulation of gene transcription to the changes in DNA topology and nucleosome modification and structural organization, to paraspeckle formation and cellular organelles maturation. This review is focused on the role of long non-coding RNAs as regulators of cyclin-dependent kinase inhibitors’ (CDKIs) levels and activities. Cyclin-dependent kinases are enzymes necessary for the tuned progression of the cell division cycle. The control of their activity takes place at various levels. Among these, interaction with CDKIs is a vital mechanism. Through CDKI modulation, long non-coding RNAs implement control over cellular physiology and are associated with numerous pathologies. However, although there are robust data in the literature, the role of long non-coding RNAs in the modulation of CDKIs appears to still be underestimated, as well as their importance in cell proliferation control. An unanticipated and surprising breakthrough of the last two decades is the discovery that a large part of the genome is transcribed and what was mistakenly called junk DNA is actually recognized to play crucial roles in determining cell phenotypes and in human diseases. As a matter of fact, the ENCODE (ENCyclopedia Of DNA Element) project has clearly and definitely demonstrated that genes codifying for untranslated transcripts correspond to 60–75% of the human genome ,2. This Generally, the DNA regions encoding lncRNAs are located in distinct genomic loci including, for the most part, intergenic regions that represent a majority of the human genome. LncRNAs could also result from the expression of sense or antisense transcripts that overlap with specific sequences of protein-coding genes. Usually, RNA polymerase II (Pol II) is responsible for the transcription of lncRNAs, although other RNA polymerases might be employed ,10. The A number of lncRNAs might result from the transcription, often in an antisense direction, of a region placed near the promoter sequence of a protein-encoding gene ,14 usualThe function of PROMPT is in large part unclear, although some hypotheses have associated these RNAs with the transcriptional modulation under stress conditions . It is tA further class of lncRNAs includes eRNAs (enhancer RNAs) that are significantly longer than PROMPTs, extending for about 2000 bps ,23. TheyA further class of lncRNAs is the so-called lincRNAs (acronym of long intervening noncoding RNAs) ,35,36. TIn addition to this categorization, a number of alternative classifications have been reported. For example, lncRNAs have been distinguished into intronic lncRNAs, antisense lncRNAs, sense lncRNAs, intergenic lncRNAs, bidirectional lncRNAs, and others. in cis or in trans. LncRNAs also modulate the maturation of primary messenger RNAs, their turn-over  and their translation efficacy. Finally, lncRNAs are able to interact with proteins and, even, favor or hamper organelle formation. Following, we will briefly discuss some of these mechanisms.LncRNAs may regulate a number of cellular processes and particularly gene transcription. It is particularly fascinating that not only the transcript sequence but, also, the lncRNA gene itself  is able to interact directly with other DNA regions thus affecting their topology and, in turn, their activation/repression. The effect might be on neighboring genes or might involve very distant DNA regions. Additionally, lncRNA transcript/DNA interactions could occur and might have effects Sphingosine Kinase 1 (probably due to the interaction of eRNA with Sphingosine Kinase 1 enhancer) is an example of triple helix formation ) . Other lle, PVT1 . Finallyer cells . In FiguCDKN2A, CDKN2B, and CDKN1C. Intriguingly, these genes mapped in loci  with a high density of lncRNA-codifying genes and where some well-known lncRNA genes are localized, namely H19, KCNQ1OT1, ANRIL, and LincRNA-p21. These lncRNAs are conserved in mammalian species, a finding that indirectly argues for their functional significance. Although a large amount of solid data on lncRNAs has been published, several aspects of these molecules need to be clarified. These include, in particular, their exact relevance to cell phenotype. It is indeed known that a large number of lncRNAs are present in scarce amounts that could not be sufficient for affecting their target activities. The development of animal models ablated for the lncRNA genes  might help in clarifying this vital aspect. On the other hand, the possibility of enhancers underlying the lncRNA-codifying sequence should be taken into consideration. Particularly, a detailed genetic (transcriptome) and protein (proteome) analysis might shed light on the role of specific lncRNAs. On the other hand, as always happens in all fields of biological and clinical sciences, lncRNA research can still be full of unexpected, exciting, and powerful discoveries. Undoubtedly, the members of the INK4 and CIP/Kip families are major protagonists in determining a specific cell phenotype. Additionally, the importance of their alterations in human chronic and acute diseases is largely established. Although all the components of these two families show remarkable structural homologies (relatively to the respective family) and a certain level of functional redundancy has been observed, the strict specificity of some of their roles has been clearly demonstrated in that each of them can be only partially replaced by others. Additionally, the ablation of a gene encoding each specific CDKI results in a peculiar phenotype. Numerous mechanisms regulate the level and activity of CDKIs, including transcriptional and translational control, rate of degradation, post-synthetic modifications, subcellular localization, and binding with a variety of interactors. In all instances, the discovery of a novel regulatory mechanism has contributed to clarifying a physiological process or has been associated with important human diseases. An additional layer of modulation has emerged in recent years and is related to the ability of numerous lncRNAs to control the expression of different CDKIs, namely


Up to seven members of the histone H1 family may contribute to chromatin compaction and its regulation in human somatic cells. In breast cancer cells, knock-down of multiple H1 variants deregulates many genes, promotes the appearance of genome-wide accessibility sites and triggers an interferon response via activation of heterochromatic repeats. However, how these changes in the expression profile relate to the re-distribution of H1 variants as well as to genome conformational changes have not been yet studied. Here, we combined ChIP-seq of five endogenous H1 variants with Chromosome Conformation Capture analysis in wild-type and H1.2/H1.4 knock-down T47D cells. The results indicate that H1 variants coexist in the genome in two large groups depending on the local GC content and that their distribution is robust with respect to H1 depletion. Despite the small changes in H1 variants distribution, knock-down of H1 translated into more isolated but de-compacted chromatin structures at the scale of topologically associating domains (TADs). Such changes in TAD structure correlated with a coordinated gene expression response of their resident genes. This is the first report describing simultaneous profiling of five endogenous H1 variants and giving functional evidence of genome topology alterations upon H1 depletion in human cancer cells. DNA is packaged within the nucleus to efficiently regulate nuclear processes. Chromatin packing involves several hierarchical levels of organization that have been mostly described by chromosome conformation capture techniques, among others. First, at megabases scale, the genome can be segregated into the so-called A and B compartments. The A compartment represents active, accessible chromatin with a tendency to occupy a more central position in the nucleus. The B compartment corresponds to heterochromatin and gene deserts enriched at the nuclear periphery . Second,Several studies support the idea that H1 variants are not redundant and that functional specificity may exist with H1 variants non-randomly distributed in the genome and interacting with different protein partners . For exaTo study the consequences H1 depletion in human cells, we have previously generated a derivative T47D cell line containing a short-hairpin-RNA that affects the expression of several H1 genes as well as the protein levels of mainly H1.2 and H1.4 . In such2 in RPMI 1640 medium, supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, as described previously , transferred to a PVDF membrane, blocked with Odyssey blocking buffer (LI-COR Biosciences) for 1 h, and incubated with primary antibodies overnight at 4°C as well as with secondary antibodies conjugated to fluorescence  for 1 h at room temperature. Bands were visualized in an Odyssey Infrared Imaging System (Li-Cor). Coomassie staining or histone H3 immunoblotting were used as loading controls. ImageJ software was used for immunoblot quantification.Chromatin immunoprecipitation was performed according to the Upstate (Millipore) standard protocol. Briefly, cells were fixed using 1% formaldehyde for 10 min at 37ºC, chromatin was extracted and sonicated to generate fragments between 200 and 500 bp. Next, 30 μg of sheared chromatin was immunoprecipitated overnight with the indicated antibody. Immunocomplexes were recovered using 20 μl of protein A magnetic beads, washed and eluted. Cross-linking was reversed at 65ºC overnight and immunoprecipitated DNA was recovered using the IPure Kit (Diagenode). Genomic regions of interest were identified by real-time PCR (qPCR) using SYBR Green Master Mix (Invitrogen) and specific oligonucleotides in a Roche 480 Light Cycler machine. Each value was corrected by the corresponding input chromatin sample. Oligonucleotide sequences are detailed in previous studies .Qualified ChIP and Input samples were subjected to end-repair and then 3’ adenylated. Adaptors were ligated to the ends of these 3’ adenylated fragments. Fragments were PCR-amplified and PCR products were purified and selected with the Agencourt AMPure XP-Medium kit. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library and then quality-checked. The library was amplified to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated in the way of sequenced by combinatorial Probe-Anchor Synthesis (cPAS).genomecov -ibam -bga -scale). MACS2 (v2.1.2) (bdgcmp -m subtract).Single-end reads were quality-checked via FastQC (v0.11.9) and aligned to the human GRCh37/hg19 reference genome using Bowtie2 (v2.3.5.1)  with def(v2.1.2)  was usedChIP-Seq data on histone H1 variants and H3K9me3 epigenetic modification from T47D multiH1 shRNA cells treated or not with Dox has been deposited in NCBI’s Gene Expression Omnibus and is accessible through GEO Series accession number GSE156036. ChIP-Seq data on histone H1 variants from WT T47D cells is at GSE166645.Specific antibodies recognizing human H1 variants used for ChIP/ChIP-seq were: anti-H1.0/H5 clone 3H9 , anti-H1.2 , anti-H1.4 , anti-H1.5  and anti-H1X . ChIP-seq of H3K9me3 was performed using anti-H3K9me3 . Other antibodies used were: anti-H1.0 , anti-H1.3 , anti-H1.5 , anti-H3  and anti-Lamin A .Hi-C libraries were generated from T47D multiH1 shRNA cells treated or not with Dox, as single replica (r1) or duplicate (r2 and r3), as previously described ,28. In bTADbit  with a radius of 100 nm. Density measures a proxy for local DNA compactness as the ratio of DNA base pairs and the distances between two consecutive particles in the models – the higher the density, the more compact the DNA. Walking angle measures the angle between triplets of consecutive particles—the higher the value, the straighter the models— and can be used as a proxy for the stiffness of the chromatin fiber. Finally, radius of gyration measures 3D structure compactness as the root mean square distance of the all particles in a model from its center of mass.it model  was usedit model ,36. For it model . ConsistBAMPE mode. Filtered BAM files were also used to compute the ATAC-Seq genome coverage, which was normalized to reads per million .ATAC-Seq data identified by the accession number GSE100762 was reprocessed as previously described  with sliGenome-wide GC content, G bands coordinates at 850 bands per haploid sequence (bphs) resolution and chromosomes coordinates were obtained from the UCSC human genome database ,39. G baCDK2 (active) and NANOG (inactive) genes  or termination sites (TTS) and around coding genes was calculated using CEAS software and is shown in Furthermore, differential genomic distribution of H1 variants in T47D cells established by ChIP-seq here is compatible with immunofluorescence imaging of H1 variants within the nuclei . H1.2 shIn summary, ChIP-seq data in T47D cells demonstrated that H1 variants are differentially distributed through the genome in two profiles: H1.2, H1.5 and H1.0 enriched towards low GC regions and H1X and H1.4 more abundant at high GC regions. Still, all H1 variants are abundant within heterochromatin or inactive regions of the genome. Upon H1.2 and H1.4 depletion, H1.2, H1.0 and H1.5 did not significantly change their genomic distribution, whereas H1X increased at high GC regions, where H1.4 was selectively depleted. H1.0, whose expression and protein levels increased, was homogeneously incorporated throughout the genome.Chromosome conformation capture techniques such as Hi-C allows to detect local and distal contacts within the genome and to establish the position of borders flanking the so-called topologically associating domains (TADs). Hi-C experiments also allow to establish a division of the genome into two compartments, active (A) and inactive (B). To address the consequences of histone H1 depletion on genome architecture, we prepared nuclear DNA from untreated and 6-days Doxycycline-treated multiH1 shRNA cells, in two independent experiments with a total of 3 replicates, and performed the Hi-C protocol . After aThe segmentation of the genome into the A and B compartments remained largely unchanged upon H1 KD  or decreased (D-score< 0) interactions . In other words, those regions of the genome with high H1.2 overlap are likely to result in increased local interactions once H1 is depleted while regions with high H1X are likely to decrease interactions.The observed increased border strength was associated to an increase in intra-TAD  both within A and B compartments and a decrease of inter-TAD interactions  within the A and between A and B compartments Figure . The incs Figure . Next, wNext, to identify if there was a correlation between the observed changes in H1 variants upon H1 KD within the spatial genome and the underlying chromatin state, we further classified TADs by their content in H1.2 and H1X variants , TADs with a bidirectional transcriptional response , and TADs with no annotated genes . Models were built based on our Hi-C data at 10 kb resolution using TADbit as previously described (h1’ group) as well as TADs with no genes (‘wi’ group) overall resulted in more different structural properties. Specifically, both h1 and wi TADs are more compact (lower radius of gyration) compared to the rest of the groups  models compared to the wi, which are the least dense of all. Other groups have similar density values and between these two extremes. It is important to note that there is an apparent discrepancy in TAD structural features and ATAC-seq data for some TAD groups such as h1. These TADs result in models that tend to be dense/compact while highly accessible in the ATAC experiment. Nevertheless, at the level of resolution of the 3D models (that is 10 kb) it is impossible to assess whether the apparent discrepancy is due to the data or the modeling exercise. The reason is that the measures are averaged over entire TADs and the comparison of both datasets cannot be done directly as ATAC-seq data is ∼100 base pair resolution and our models are 10 kb resolution. The high gene expression rate and GC content in h1 TADs group explains the high ATAC accessibility at low resolution. However, at 10 kb resolution the ‘density’ measure says that at the TAD level the DNA fiber is more compacted.To further characterize architecturally changes within TADs with coordinated transcriptional response to H1 KD, we next generated 3D models of genomic regions harboring TADs that contained at least 90% of genes down or up-regulated , while the changes were more evident in the h1 group and also in the ud group independently of the direction of the changes in gene expression. Indeed, although without significance, changes in the TADs with a coordinated transcriptional response to H1 KD  have the same trends, indicating that TADs that were coordinately up- or down-regulated were similarly structurally altered upon H1 KD. Our 3D models indicate that all TADs are altered in a similar way due to H1 KD, with different consequences in gene expression deregulation that might depend on local features or distinct H1 abundance.Finally, the models indicate that upon H1 KD and across all types of TAD groups there is a significant increase of the walking angle measure indicating a change of stiffness of the chromatin Figure . In genes Figure . In geneIn this study, we have analyzed the genomic distribution of five endogenous H1 variants within T47D breast cancer cells by ChIP-seq using specific antibodies. This is almost the whole somatic H1 complement of this cell line with the exception of H1.1, which is not expressed in these cells, and H1.3 that was not profiled due to the lack of ChIP-grade antibodies. This is, to our knowledge, the first time that most of endogenous variants have been profiled in a mammalian cell. Antibodies for H1.2, H1X and H1.0 were used before on ChIP-seq experiments ,20; H1.4In previous studies, we mapped endogenous H1.2 and H1X, demonstrating that they have different distributions across the genome ,17,20. Obona fide heterochromatin marker.In T47D cells, the H1 content was estimated to be 9% for H1.0, 23% for H1.2, 13% for H1.3, 24% for H1.4 and 31% for H1.5 . Our disTo study whether alteration of the total H1 content and relative abundance of the different variants affected the genomic localization of remaining histones, we performed ChIP-seq in T47D cells knocked-down for H1.2 and H1.4 with an inducible system, previously characterized . InteresImmunofluorescence analysis of H1 location within the nuclei confirmed the expression changes described upon H1 KD, but any redistribution within chromatin states is difficult to pick up with this technique. Still, it was possible to confirm that H1.4 and H1X localization differs from H1.2, H1.5 and H1.0. Further studies at super resolution fluorescence microscopy might help to characterize, in the future, the differential localization of H1 variants and their role in chromatin organization and genomic functions.In this work, we have shown that H1 KD caused changes in chromatin accessibility and H3K9me3 distribution, shifts in A/B compartments and TAD borders, and changes in the 3D architecture of TADs Figure . Some ofWe reported before that multiH1 KD (H1.2 + H1.4) effects were more drastic than the simple addition of H1.2 or H1.4 KD effects, e.g. in the number of genes being deregulated, or in causing the induction of the interferon response due to de-repression of heterochromatic repeats  22). Th. Th22). Previously, we and others have shown that epigenetic states and H1 distribution are more homogeneous within a TAD, suggesting that TAD borders prevent the spreading of these features ,20. In oSevere H1 depletion causes cell cycle arrest and transcription-replication conflicts ,53,54. OWe have shown here and previously that H1 variants selective depletion results in changes in expression of hundreds of genes, including de-repression of intergenic and intronic RNAs, as well as heterochromatic repeats and ERVs, which leads to the induction of the interferon response . MoreoveOverall, our results indicate that the non-random genomic distribution of H1 variants, their re-location upon variant depletion, and the subsequent genome structural changes have a read-out in their direct  change of the gene expression program.https://genome.ucsc.edu/s/NSP/GSE156036_GSE166645.Hi-C and ChIP-seq data on T47D breast cancer cells reported here and deposited in NCBI’s Gene Expression Omnibus are accessible through GEO Series accession number GSE172618 and GSE156036/GSE166645, respectively. A link to a UCSC genome browser session displaying the uploaded ChIP-seq tracks is provided: gkac226_Supplemental_FileClick here for additional data file.


Double-stranded RNA-binding proteins (dsRBPs) are major players in the regulation of gene expression patterns. Among them, Nuclear Factor 90 (NF90) has a plethora of well-known functions in viral infection, transcription, and translation as well as RNA stability and degradation. In addition, NF90 has been identified as a regulator of microRNA (miRNA) maturation by competing with Microprocessor for the binding of pri-miRNAs in the nucleus. NF90 was recently shown to control the biogenesis of a subset of human miRNAs, which ultimately influences, not only the abundance, but also the expression of the host gene and the fate of the mRNA target repertoire. Moreover, recent evidence suggests that NF90 is also involved in RNA-Induced Silencing Complex (RISC)-mediated silencing by binding to target mRNAs and controlling their translation and degradation. Here, we review the many, and growing, functions of NF90 in RNA biology, with a focus on the miRNA pathway and RISC-mediated gene silencing. Appropriate and controlled gene expression is essential for every living organism. Deregulation of this process can have profound effects and often be responsible for diseases such as cancer and genetic disorder ,2,3. WhiILF3) gene [ILF3 is localized on human chromosome 19 and contains 21 exons, giving rise to at least five distinct transcripts that are generated by alternative splicing [ILF3 gene are NF90 and Nuclear Factor 110 (NF110), which are ubiquitous and generally abundant proteins with an apparent molecular mass of 90 kDa and 110 kDa, respectively [RNA-binding proteins (RBPs) are known to contribute to posttranscriptional gene regulation, mediating mRNA splicing, translation, and turnover ,5. AmongF3) gene . ILF3 issplicing . The mosectively ,9,10,11.ILF2 gene. Heterodimerization of NF90/NF110 with NF45 is important for the stabilization and function of the complex. In particular, it was shown that the binding of NF45 to NF90 leads to thermodynamic stabilization and improves the RNA-binding ability of NF90, enhancing its affinity for RNA substrates [The main protein partner of NF90 and NF110 is Nuclear Factor 45 (NF45), which is transcribed from the bstrates . In mostbstrates ,14. Howebstrates  and as abstrates ,17,18,19bstrates . NF90/NFbstrates , splicinbstrates , post-trbstrates ,24,25. Tbstrates ,26,27. HILF3 gene, localized in human chromosome 19p13 , a nuclear localization signal (NLS), two double stranded RNA-binding motifs (dsRBMs), and a RGG motif . The RGGThe DZF domain is also conserved in NF45 and is responsible for dimerization and consequent stabilization of the heterodimer . However647, leading to translocation of NF90 from the nucleus to the cytoplasm [382, inducing nuclear export of NF90 [188 and Thr315, leading to their nuclear export [482 increase the stability of NF90 [NF90 and NF110 contain multiple residues that can be phosphorylated in both the common region and in the isoform-specific regions . Phosphoytoplasm . In addi of NF90 . The intr export . Moreove of NF90 . Aside f of NF90 .Regarding the regulation of NF90 expression, it was shown that long non-coding RNA (lncRNA) Low Expression in Tumor (lncRNA-LET) destabilizes NF90 by changing its conformation and exposing ubiquitination sites that promote its proteolytic degradation . Recentl10-A motif in the minor groove could contribute to the interaction of NF90 to target dsRNA, further stabilizing it [NF90 appears to recognize target RNAs based on both structure and sequence. In particular, NF90 binding to the adenovirus-expressed Viral Associated 1 (VA1) RNA did not show any specificity for nucleotide sequence, upon extensive mutational analysis. Instead, the structure of the RNA determined NF90 binding, with a requirement for a minihelix-like structure ,27. Furtizing it . In addiizing it . HoweverNF90 is a polyvalent factor that, since its discovery, has been linked to a variety of functions such as transcriptional and translational regulation, viral replication, and miRNA biogenesis . MoreoveIn recent years, NF90 has been found to be differentially expressed in several types of cancers such as ovarian, breast, cervical, hepatocellular and nasopharyngeal carcinomas, leukemia, and bladder cancer ,42,43,44IL2 promoter and enhances its transcription [Although NF90 does not contain a known DNA-binding motif, evidence strongly suggests that it could have a similar function to canonical DNA-binding proteins. NF90/NF45 was originally described as a DNA-binding complex, acting as a transcription factor for the cytokine, interleukin 2 (IL2), during T-cell activation. In particular, it was shown that NF90/NF45 binds the antigen recognition response element 2 (ARRE-2) present in the cription . It was cription ,47.In keeping with its role in viral replication and T-cell activation, NF90 was also shown to regulate the transcription of another cytokine, Interleukin 13 (IL13), by binding to a DNase I hypersensitive site (DHS) . On the c-FOS transcription upon serum induction. NF90/NF45 was shown to bind to the c-FOS enhancer/promoter region while cooperating with general co-activator factors [More recently, NF90/NF45 was reported to be involved in the upregulation of  factors . Similar factors . TherefoThe role of NF90 in the regulation of miRNA biogenesis was recently described. The first evidence for the involvement of NF90/NF45 in the maturation of miRNAs was shown by Sakamoto and colleagues . In thisSince this finding, different miRNAs have been shown to be modulated by NF90/NF45. The complex was found to downregulate myogenic miRNAs such as miR-133a, leading to significant loss and maturation of skeletal muscle and atrophy in NF90/NF45 double-transgenic mice . More reIt was recently demonstrated that NF90/NF45 is able to inhibit the maturation of miR-3173, a miRNA embedded in the first intron of Dicer pre-mRNA, by preventing binding of Microprocessor to pri-miR-3173. Furthermore, in the absence of NF90, the level of pre-miR-3173 increases while Dicer pre-mRNA exhibits splicing defects that lead to its downregulation. Increased progression and metastasis were observed in ovarian cancer cells. Therefore, it was established that NF90 can act as a tumor suppressor in ovarian cancer models. Interestingly, the mature form of miR-3173 is able to target NF90 mRNA by binding to its 3′ UTR, leading to translational repression, mediating a feedback amplification loop that controls Dicer expression and ovarian carcinoma progression .More recently, the extent of the effect of NF90 on miRNA maturation was uncovered using genome-wide approaches . Data inInterestingly, transcriptomic analysis showed that NF90 association with pri-miRNAs may modulate the expression of their host genes, as described previously . For insOn the other hand, NF90 can also bind and stabilize oncogenic pri-miRNAs such as pri-miR-548k, which is implicated in esophageal squamous cell carcinoma (ESCC), promoting cancer progression, metastasis, and poor overall survival ,57. InteIt is not known whether NF45 is also implicated in the regulation of pri-miRNA processing, together with NF90. The stable interaction between these two factors might suggest that NF45 is likely to be involved, although it has not yet been formally demonstrated. In addition, most studies suggest that NF90 regulates pri-miRNA processing via competition with Microprocessor for binding to pri-miRNA structures. However, a more direct functional role of NF90 in inhibiting pri-miRNA processing has not been formally excluded.To conclude, the effect of NF90 on cancer development and progression is likely multifactorial, being dictated not only by the effect of the miRNAs it modulates, but also by their mRNA targets, and the gene that hosts the pri-miRNA. Therefore, the outcome of differential NF90 expression in cancer depends on complex mechanisms that warrant further investigation.In addition to controlling mRNA fate by binding and modulating the processing of miRNAs, NF90 can directly regulate mRNAs’ translation, stability, and decay ,58. AU-rWhile NF90 has been shown to modulate the translation of bound mRNAs, its effect can be either positive or negative, depending on the RNA target. NF90 can inhibit translation by affecting the initiation step, or by retaining target mRNAs in the nucleus . For insVEGF mRNA, promoting its loading onto polysomes and increasing its stability [NF90 has also been shown to activate the translation of a limited subset of mRNAs, such as Vascular Endothelial Growth Factor (VEGF) and cyclin T1 mRNA ,61. Undetability . After Htability .IL2 mRNA, which prevents its degradation [PARP1) mRNA, and therefore promote tumor development [NF90 is also able to increase or decrease the stability of its target mRNAs. For instance, upon co-stimulation of CD28 following T-cell activation, NF90 is phosphorylated by AKT and translocates to the cytoplasm where it can bind the 3′ UTR of radation ,63. Simiradation ,61. In Helopment . On the elopment .BAFF) mRNA by recruiting miR-15a to BAFF 3′ UTR. Moreover, a variant of BAFF mRNA (BAFF-var), which lacks the NF90 binding site, was found to have enhanced translation and is associated with an elevated risk of the development of autoimmune diseases. Interestingly, it was shown that the binding of NF90 and miR-15 to the mRNA correlates with higher Ago2 binding, which suggests that translational inhibition of BAFF mRNA might be mediated by RISC [More recently, several studies have suggested the existence of signaling pathways or regulatory axes involving NF90, and often, miRNAs that impact the development of pathologies, especially cancer progression and metastasis ,37,42,44 by RISC .Interestingly, NF90 has been identified as a subunit of P-bodies, and was recently shown to interact with RBPs involved in RISC-mediated silencing, such as Moloney leukemia virus 10 (MOV10) and Argonaute 2 (AGO2), in an RNA-dependent manner ,64. NF90Besides acting as a cellular mRNA binding factor that controls RNA metabolism and translation, NF90 also binds viral RNA or DNA . The conDespite the numerous and diverse examples of NF90 activity during the response to viral infection, the exact mechanism underlying its role is yet to be fully elucidated. However, it is possible that the complicated contribution of NF90 to antiviral immunity might occur through different mechanisms depending on the type of virus.The most recent described role of NF90 involves circular RNA (circRNA) biology. In fact, it was found that NF90 is a key factor for the biogenesis of circRNAs in the nucleus while their mature form might act as a molecular reservoir of NF90 in the cytoplasm for prompt immune response following viral infection . In partIt was recently shown that circular Actin Alpha 2 (circACTA2) RNA can compete with CDK4 mRNA for the binding of NF90 in vascular smooth muscle cells (VSMC). In particular, increased circACTA2 expression, induced by angiotensin II (Ang II) stimulation in VSMC, reduced the association of NF90 with CDK4 mRNA, leading to Ang II mediated senescence in VSMC . AlthougSince its identification, NF90 has been studied with increasing interest, especially in the last 15 years. Recently, a growing number of functions of NF90 have been described that allow us to define it as a polyvalent and versatile factor in RNA biology and homeostasis. In fact, NF90 has been shown to be implicated in RNA metabolism at all stages, from transcription to degradation. Moreover, its role in miRNA biogenesis, RNA stability, and translation during cancer progression and viral infection highlights its ability to orchestrate cellular responses in different cellular compartments and upon different stimuli. Given the widespread impact of the binding of NF90 to RNAs during homeostasis, viral infection, and cancer progression, it would be tempting to envision the application of this knowledge for the development of therapeutics designed in order to inhibit or promote NF90 binding to selected RNAs. Many NF90-modulated pri-miRNAs are associated with cancer development and progression. Therefore, the modulation of NF90 binding to these pri-miRNAs would affect their abundance, having an effect not only on their mature form but also on the expression of their tumor-related mRNA targets. Moreover, the effect of the competition between Microprocessor and NF90 would in turn also affect the expression of the mRNAs hosting the pri-miRNA, which can be tumor suppressors such as Dicer or oncogenes such as TIAM2. NF90 was recently shown to compete with SMD and RISC activities, leading to increased stability of NF90-bound mRNAs that can be involved in mitosis, viral infection, and hypoxia. A better understanding of how NF90 is implicated in RISC and SMD-mediated silencing might clarify its role in mRNA translation and degradation during viral infection or cancer-induced hypoxia, and potentially open possibilities for targeted therapies. Interfering with NF90 binding to pri-miRNAs or mRNAs using nanotherapeutics could be a novel approach to control the abundance of specific targets in cancer.


E. urophylla genome and that they drove the expression of the gus gene. There was a noticeable difference in the intensity of color between the adventitious buds on the same callus block, as well as the intensity of color within the same adventitious bud. According to the established two-factor model of blue value, there was a greater difference between the levels of the genotype factor than the promoter factor in eucalyptus leaf discs. Further, the basal and inducible transcriptional levels of the three improved promoters were investigated by qPCR. With the basal transcriptional level of the GWSF promoter normalized to one, the relative basal levels of VGWSF, GWVSF, and GWSFV were 1.40, 1.45, and 4.15, respectively. The qPCR results were consistent with the staining results of GUS histochemical staining. The three improved promoters all had the properties of being induced by salicylic acid, Ralstonia solanacearum, and Phytophthora capsici. The three improved promoters demonstrated a significantly higher TMV induction activity: their induction activity from high to low was GWSFV > GWVSF > VGWSF. The findings will be beneficial to the construction and optimization of artificial promoters for transgenic plants.The transcriptional properties of artificial promoters are closely related to the type and arrangement position of cis-elements. GWSF (374-bp) was an effective SPIP with four cis-element dimers. There were four pathogen-inducible cis-elements in the GWSF promoter  and a minimal cauliflower mosaic virus 35S promoter. V-element dimers were inserted into the upstream (VGWSF), midstream (GWVSF), and downstream (GWSFV) regions of the original GWSF promoter sequence to examine their affect on the position. The expression activity of promoters was analyzed and estimated using the histochemical staining of leaf discs of eucalyptus with transient expression, an image digitization method to extract the color features, and the induction treatment by a plant pathogenic microorganism/inducer and qPCR assays. The histochemical staining results of the adventitious buds indicated that the promoters had been successfully integrated into the  Agrobacterium have the advantages of simplicity, rapidity, and a high transformation efficiency [In tropical and subtropical regions, eucalyptus is the most widely grown hardwood crop due to its high economic benefits, high-stress resistance, and quick growth . Eucalypficiency , which cficiency .Promoters are particular DNA sequences located upstream of the 5′ end of the coding regions, which initiate gene transcription by combining with RNA polymerase and regulate the expression mode and level of downstream structural genes . Hence, In plants, synthetic promoters introduced either stably or instantly are valuable for identifying functional regulatory elements and the transcription factors that govern the amplitude, spatial distribution, and temporal pattern of gene expression . The seqGWSF 374-bp), named for its four cis-element dimers, was a highly efficient SPIP (synthetic pathogen-inducible promoter) capable of causing rapid responses to a wide range of pathogens 4-bp, nam. We refeCaMVmini35S promoter [Some studies on the activity of promoters revealed that promoters containing several types of cis-elements generally exhibit better expression characteristics than promoters containing only one type of element . The strpromoter  will leapromoter .Agrobacterium. We also investigated the positional effect of newly added V-elements on the transcription properties of the original promoter. The results of this study will serve as a reference for the design of artificial promoters.This study aimed to evaluate the gene expression characteristics of different promoters and to find plant pathogen-inducible promoters with a low basal expression and high inducibility by salicylic acid, bacteria, fungi, and viruses. We designed VGWSF, GWVSF, and GWSFV promoters in this work, which are located in the upstream, midstream, and downstream regions of the GWSF promoter sequence inserted by a V-element dimer with virus-inducing activity . An analE. urophylla S.T. Blake were used for establishing the transformation method. Tissue-cultured seedlings of E. urophylla (EU6), E. urophylla × E. grandis (Guanglin 9), and E. urophylla × E. tereticornis (Guangzhou 1) were used for promoter selection, which were provided by the South China Experiment Nursery located in Suixi County, Guangdong Province. The one-year-old seedlings of three genotypes of eucalyptus were grown with vermiculite and cultivated in an artificial climate chamber at 28 ± 2 °C, 500 mmol/m2/s of light intensity, and photoperiods of 14 h light/10 h dark per day.Seeds of CaMV35S minimal promoter is 30 bp in GWSFV, 188 bp in GWVSF, and 330 bp in VGWSF, respectively. Two restriction sites  and cloned into the pUC19 plasmid. The improved promoters were used to replace the wild-type CaMV35S promoter in the plasmid pBI121 by restriction endonuclease digestion and ligase ligation in order to regulate the expression of the β-glucuronidase (gus) gene. As described in the literature [Agrobacterium were conducted.The V-element sequence was 5′-TTGGGAAGGAATTTCCTACT-3′ , and a 6on sites a,b, Hindterature , the conAgrobacterium GV3101 was identified by PCR allied with restriction endonuclease digestion and sequencing. The primers for PCR verification were as follows:The success of the vector construction and transformed VGWSF forward/reverse: 5′-GCAAGCTTTTGGGAAGGAAT-3′/5′-TGGTGGCT GGATTATCTTCA-3′, the length of the amplified fragment was 215 bp; GWVSF forward/reverse: 5′-TGAAGATAATCCAGCCACCA-3′/5′-AGCGTGTCCTCTCCAAAT GA-3′, the length of the amplified fragment was 243 bp; GWSFV forward/reverse: 5′-CCAGAA TACTAGACAGCCACCA-3′/5′-GGAAGGGTCTTGCGGATTAT-3′, the length of the amplified fragment was 231 bp; and GUS forward/reverse: 5′-ACACCGATA CCATCAGG-3′/ 5′-TCACCGAAGTTCATGCCAGT-3′, the length of the amplified fragment was 480 bp.E. urophylla were provided by the Research Institute of Fast-growing Trees, Chinese Academy of Forestry. After surface sterilization, seeds germinated on a half Murashige and Skoog (MS) agar medium for 8 days under darkness at 25 ± 1 °C.Viable seeds of 2/s for 1 d. Seedling hypocotyls were cut from the 9-day-old seedlings and inoculated on an SPCa medium [A. tumefaciens GV3101 solution for 30 min at −0.05 MPa, then were inoculated on the original medium to co-culture for 3 days in darkness. Subsequently, the explants were swirled in sterile water to remove A. tumefaciens. The explants were inoculated on the same medium with 200 mg/L of cefotaxime (Cef) for one week to decontaminate residual A. tumefaciens, followed by the CBIM with 200 mg/L of Cef and 50 mg/L of Kanamycin (Kan). The explants were sub-cultured every 2 weeks on a fresh CBIM medium until the adventitious bud regenerated from the callus, using 50 mg/L of Kanamycin and cefotaxime, gradually decreasing to 100 mg/L, under a 16 h photoperiod with a light density of approximately 20 μmol /m2/s emitted from cool fluorescent light tubes at 25 ± 1 °C. As described above, PCR and sequencing were used to identify the positive calli and positive buds.The seedlings were illuminated at a photon flux density of approximately 20 μmol /ma medium  supplemeAgrobacterium solution with an OD600 of 0.6~0.8, vacuumed at −0.05 MPa for 10 min, and infected for 30 min after they had completely sunk into the liquid. The leaf discs were taken out and placed on sterile filter paper saturated with a 1/2 MS liquid medium (containing 100 mg/L of vitamin C) and cultured in the dark at 28 °C for 48 h. The GUS histochemical assay was performed according to the method of Jefferson et al. [The mature leaves over the fourth internode of the eucalyptus stem were selected as the materials. The lower epidermis was damaged by sandpaper friction and the leaf discs with a diameter of ten millimeters on both sides of the main leaf veins were taken with a punch, soaked in 0.2% Triton X-100 for 30 min, rinsed with sterile water, and then dried with sterile filter paper. The leaf discs were immersed in the n et al. .Different treatment combinations of leaf discs were acquired with a Sony digital camera . A photographic device was used to fix the camera directly above a vertical eucalyptus leaf disc, positioned 30 cm away from the height of the leaf disc. The photographs were taken using the same camera settings .Phytophthora capsici and Ralstonia solanacearum were purified and preserved in our laboratory. The TMV particles were prepared as described previously by Huang et al. [6 CFU (Colony-Forming Units) for Ralstonia solanacearum or Phytophthora, at 50 μL 2 mmol/L salicylic acid solution, or 50 μL extracts containing TMV particles. The leaf discs were cultured in the dark at 28 °C for 24 h, then ground into fine powder in liquid nitrogen, and the total RNA was extracted with Fruit Mate and an RNAiso Plus reagent . The total RNA quality was evaluated with a NanoDrop 2000C spectrophotometer . The total RNA was used as the template to complete reverse transcription to obtain the cDNA following the manufacturer’s instructions .Pathogen strains of g et al. . After tgus gene were evaluated by qPCR using a CFX 96  and SYBR Premix Ex TaqTM II  with the eucons08 gene [gus F/R, 5′-CTGATAG CGCGTGACAAAAA-3′/ 5′-GGCACAGCACATCAAAGAGA-3′; eucons 08 F/R, 5′-TCCAATCCGAGTCGCTGTCATTGT-3′/5′-TGATGAGCCTCTCTGGTTTGACCT-3′. The thermal cycling conditions were as follows: initial denaturation step at 95°C for 30 s, 40 cycles of 9 s at 94 °C, 9 s at 55 °C, and 15 s at 72 °C. The specificity of the primer pairs was verified by melting curve analysis from 65 °C to 90 °C. The quantification of the relative changes in gene expression was performed using the 2−ΔΔCt method as previously described by reference [The transcriptional levels of the s08 gene  as an ineference  and Bio-Statistical analysis was carried out using SPSS 20.0  and a plot was produced using OriginPro . All measurements were repeated three times.CaMV35S promoter in the plasmid pBI121. The transcriptional activity of the improved promoters could be inferred by detecting the activity of glucuronidase by GUS histochemical staining or by quantitative assays [E. urophylla genome and that they drove the expression of the gus gene to produce glucuronidase , E. urophylla × E. grandis (Guanglin 9), and E. urophylla × E. tereticornis (Guangzhou 1), the different promoters showed obvious differences in coloration  has a much smaller p-value than the A factor (promoter), indicating a greater difference between the levels of the B factor than the A factor. Two factors significantly influenced the B values (the GUS gene expression activity), and their interaction also significantly affected the B values.Using Sigmascan pro5.0 software , the color characteristic values of the leaf image were extracted to obtain the RGB values, namely red (R), green (G), and blue (B). As only the blue (B) value varies among the combinations, this study utilized an average B value per unit area in order to evaluate the GUS gene expression. The established two-factor model shows the predicted B values of different treatment combinations in As a result, we believe that the B values were influenced by the genotype and promoter type, and the combination of the GWSFV promoter in GZ1 represented the highest GUS gene expression activity in eucalyptus leaf discs.GUS (β-glucuronidase) is particularly popular as a reporter gene for assaying endogenous promoter activity in plant tissues via histochemical staining, but this can only approximate the transcriptional level of the promoter, whereas qPCR can directly quantify the relative transcriptional activities of the promoters.E. urophylla, with significantly differing GUS histochemical staining between different promoters, was used as the experimental material, and we further investigated the basal and inducible transcriptional levels of the three improved promoters by qPCR. In E. urophylla leaf discs, the basal level of the GWSF promoter, i.e., the CK in the GWSF group, had been normalized to 1  was significantly affected by the genotype and promoter factors by color feature extraction and analysis of GUS staining. Using color feature extraction and the analysis of leaf discs, genotype and promoter factors were significantly associated with GUS gene expression activity.CaMV35S minimal promoter, the higher the virus-inducible activity.The different promoters of transgenic plants were treated by plant pathogenic induction, and the results of qPCR assays showed that: the basal level of the improved promoters increased significantly; the improved promoters had inducible responses to more inducers; the insertion element had an obvious position effect on the transcriptional properties of the original promoter; and the closer the V-element was to the