A correlation between histone acetylation and transcription has been noted for a long time, but little is known about what step(s) in the transcription cycle is influenced by acetylation. We have examined the immediate transcriptional response to histone deacetylase (HDAC) inhibition, and find that release of promoter–proximal paused RNA polymerase II (Pol II) into elongation is stimulated, whereas initiation is not. Although histone acetylation is elevated globally by HDAC inhibition, less than 100 genes respond within 10 min. These genes are highly paused, are strongly associated with the chromatin regulators NURF and Trithorax, display a greater increase in acetylation of the first nucleosomes than other genes, and become transcriptionally activated by HDAC inhibition. Among these rapidly up-regulated genes are HDAC1 (Rpd3) and subunits of HDAC-containing co-repressor complexes, demonstrating feedback regulation upon HDAC inhibition. Our results suggest that histone acetylation stimulates transcription of paused genes by release of Pol II into elongation, and that increased acetylation is not a consequence of their enhanced expression. We propose that HDACs are major regulators of Pol II pausing and that this partly explains the presence of HDACs at active genes. A correlation between histone acetylation and transcription was noted for the first time by Vincent Allfrey in the 1960s , a more To examine the immediate effects of changes in acetylation to transcription, we have used precision run-on sequencing (PRO-seq) to measure transcription globally in response to the histone deacetylase (HDAC) inhibitor Trischostatin A (TSA). HDACs can be divided into four classes based on sequence homology (reviewed in Transcription of mRNA genes involves promoter recognition by RNA Polymerase II (Pol II), followed by initiation, elongation, and termination of transcription (reviewed in Drosophila S2 cells from DGRC (S2-DRSC stock #006) for most of our experiments. These cells were cultured at 25°C in Schneider's Drosophila Medium (Gibco # 21720024), supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin and 100 μg/ml streptomycin. Human HEK 293 cells were maintained in DMEM (Gibco) supplemented with 10% FBS and 100 Units/ml penicillin and 100 μg/ml streptomycin. Drug treatment was performed for 10 or 30 min on cells resuspended in FBS-free media with either the HDAC inhibitor Trichostatin A (TSA) at 400 nM final concentration, with the transcription inhibitors Flavopiridol (FP) at 300 nM or Triptolide (Trp) at 500 nM final concentration, or in combination, and treatment with the solvent dimethyl sulfoxide (DMSO) was used as a control.We used in vitro transcription using the Megascript RNAi kit (Ambion). dsRNA directed against Rpd3 and GFP were 491 and 690 bp respectively. S2 cells (5.2 × 106 cells /treatment) were collected, washed twice and resuspended in 2.5 ml of FBS free medium. 37 nM of dsRNA (Rpd3 or GFP) was added to cells and immediately transferred to a T25 flask and incubated for one hour at 25°C, followed by addition of 2.5 ml medium containing 20% FBS to reach 10% FBS in the total volume. After 2 days the cells were treated again with the same amount of dsRNA in 10 ml of FBS free media and divided into two T75 flasks. An hour later medium containing 20% FBS was added to reach 10% FBS in the total volume and cultured for another 3 days before harvest.Double-stranded RNA (dsRNA) targeting Rpd3 and GFP (control) was generated by PCR amplification from cDNA clones using primers containing the T7 RNA polymerase promoter followed by sequences specific for the target genes shown in . PurifieDrosophila S2 cells. In brief, cell pellets consisting of 10 million cells treated with DMSO or TSA were resuspended in cold PBS before proceeding to nuclei isolation. At this stage, 0.1 million HEK cells (1% spike-in) also in cold PBS were added to each sample just prior to centrifuging at 700g for 5 min. Nuclei isolation was performed by resuspending cells in buffer A , 4 u/ml RNase inhibitor  and immediately dounced 25 strokes with a loose pestle. The nuclei pellet was recovered by spinning at 700g for 5 min and washed twice in the same buffer. Finally, isolated nuclei were resuspended in 100 ul buffer D  for every 10 million cells and stored at −80°C. Nuclear run-on was performed exactly as previously described (Isolation of nuclei and the precision run-on reaction was performed as described previously . Since Hescribed .2 0.1% IGEPAL), followed by tagmentation and library preparation as described earlier  and ChIP B  for 10 min at 4°C followed by resuspension in Sonication buffer . Chromatin was sheared using a Bioruptor (Diagenode) until an average fragment size range of 200–500 bp was achieved. The solubilized chromatin fraction was cleared by centrifugation and used for immunoprecipitation. Chromatin from HEK-293 cells (corresponding to 1%) prepared in similar buffer conditions was added to serve as spike-in control. Immunoprecipitation with 2 μg of either H3 , H3K14ac , H3K27ac  or RNA Pol II ser2P  antibodies was carried out at 4°C overnight. A mix of Protein A and G Dynabeads (Invitrogen) blocked with BSA (1 mg/ml) was used to capture the antibody-chromatin complexes. Chromatin and antibody bead complexes were formed during at least 3 hours followed by 10 minute washes with sonication buffer , WashA , WashB  and TE. Beads were resuspended in 100 ul TE supplemented with 20 μg/ml RNase A and incubated at 50°C for 30 min. Tris pH 8.0 and SDS were added to these tubes to a final concentration of 50 mM and 0.1% respectively and reversal of the cross-links performed at 68°C for at least 4 h, followed by protein digestion by Proteinase K treatment. Finally, DNA was purified using ChIP DNA Clean & Concentrator™ (Zymo research #D5205) and eluted in 60 μl of DNA elution buffer.ChIP experiments were performed as previously described , with miChIP samples were sequenced on Illumina NextSeq platform at BEA core facility, Stockholm. ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB) and single-end (1 × 75 bp) sequenced.For ChIP-qPCR, 2 μl of DNA was used as template and run in duplicates using 300 nM primers and EvaGreen reagents (Solis BioDyne) on a CFX96 Real-Time system (BioRad). Average Cq was calculated for each ChIP sample and compared to input. To normalize for the background of each individual ChIP, two intergenic sites devoid of known histone modifications and chromatin factors was used. ChIP values of histone modifications were further corrected to the total amount of histone H3. All the primers used in this study are listed in Drosophila S2 cells and human HEK293 cells. RNA from the isolated nuclei resuspended in Buffer A and solubilized in TRIzol LS reagent (Ambion # 10296010), was extracted using Direct-zol RNA MicroPrep kit (Zymo research # R2060) following the manufacturer's protocol. RNA (1.5 ug) was digested with DNase I (Sigma # AMPD1-1KT) to eliminate genomic DNA. The RNA without any further purification was converted to cDNA using High-Capacity RNA-to-cDNA Kit (ThermoFisher scientific # 4387406) and random primers. RT-qPCR was performed on a CFX96 Real-Time system (BioRad) using gene-specific PCR primers mixed with EvaGreen reagent (Solis BioDyne) and diluted cDNA (7-fold dilution) as template. The PCR primers (Drosophila) or ActB (human), using the delta-delta Ct method.We employed exactly the same nuclei isolation protocol as in PRO-seq section (described above) for both  primers  used wer2 0.1% IGEPAL). After 5 min incubation on ice the nuclei were pelleted at 700g for 10 min at 4°C. 1× Laemmli buffer was directly added to the pellet and boiled for 10 min at 95°C. HEK293 cells were washed once with ice cold PBS and resuspended in 1x Laemmli buffer and boiled for 10 min at 95°C.Following drug treatment, nuclear proteins were isolated from S2 cells and crude protein extracts were made from HEK293 cells. S2 cells were washed once with ice cold PBS and re-suspended in cold nuclear lysis buffer  and bands were quantified using IMAGE studio software.The samples were electrophoresed on 15% SDS-polyacrylamide gel and transferred on to nitrocellulose membrane. Ponceua S stain was used to confirm transfer before blocking and incubating with primary antibodies diluted in TBST–2% BSA: rabbit monoclonal anti-H3K14ac , rabbit polyclonal anti-H3K27ac , rabbit polyclonal anti-Rpd3  or mouse monoclonal anti-H3  overnight. After washing, the membranes were incubated with fluorophore conjugated secondary antibodies IRDyeDrosophila melanogaster (dm3) genome assembly using BWA with default parameters. To obtain the spike-in reads, we mapped the reads to the human (hg38) genome assembly and counted the reads that only mapped to hg38. We generated PRO-seq coverage tracks, normalized by the spike-ins, with separate strands. Based on PRO-seq reads over gene bodies (500 bp from TSS to 100 bp upstream of TES), we performed differential expression analysis (DE) of TSA 10 min versus DMSO and TSA 30 min versus DMSO using DEseq2 (2 transcript per million (TPM) >3 were defined as expressed genes. A pausing index (PI) for canonical transcripts was defined by promoter (−50 to +100 bp) TPM divided by promoter and body (+500 bp to −100 TES) TPM. We obtained chromatin state data and various chromatin regulators and histone modification datasets in S2 cells from the modENCODE project  genome assembly using Bowtie2 with default parameters after adaptor trimming by Trimmomatic (https://github.com/jsh58/Genrich), which took the replicate consistency into account. As we aim to call differential peaks downstream, we here set a relatively relaxed peak-calling threshold  to obtain candidate peak regions. The peaks that overlapped with a Drosophila blacklist were removed  genome assembly. We mapped ChIP-seq reads to the mmomatic . The uni removed . To perf removed .Drosophila melanogaster (dm3) genome assembly using Bowtie2 with default parameters after adaptor trimming by Trimmomatic. The high quality and uniquely mapped reads (MAPQ ≥ 20) were used for further analysis. We performed peak calling of ATAC-seq accessible regions separately for DMSO, TSA 10 min and TSA 30 min by Genrich peak caller with parameter setting (Genrich -t -o -f -r -j -y -d 100 -q 0.05 -e chrM -v). ATAC-seq differential accessibility analysis between TSA and DMSO on the ATAC-seq peaks was performed with the DEseq2 package. Nucleosome positioning analysis was performed by NucleoATAC  or DMSO as control for 10 or 30 min and performed precision run-on sequencing (PRO-seq). This method captures transcriptionally engaged polymerases at base-pair resolution (2 transcripts per million (TPM) >3 were defined as expressed genes  and increased PRO-seq read densities in the gene bodies  as well as H3K27ac (marking active promoters and enhancers) antibodies. We performed ChIP-seq experiments for both of these histone modifications in TSA-treated S2 cells with human HEK293 cell chromatin as spike-in. Consistent with a global increase in histone acetylation after TSA addition as determined by Western blot Figure , elevateInterestingly, the increase in histone acetylation after 10 min of TSA was greater in the 96 genes that were transcriptionally up-regulated than in unaffected genes Figure  and D. TWe also examined histone acetylation changes at enhancer regions, defined as CBP peaks located more than 100 bp from the TSS . Up-reguRpd3, Apc, AdenoK, CG2911 and sgl after 10 min of TSA-treatment, whereas the change at the non-affected genes Hsp70, ap and CG11268 and some intergenic regions was either smaller or not significant  or Flavopiridol (FP). Trp inhibits TFIIH and thereby prevents transcription initiation, whereas FP is a PTEF-b inhibitor that blocks release from promoter–proximal pausing (reviewed in n Figure . We thenn Figure . To examn Figure . TogetheTo investigate if increased H3K27ac occurs in TSA-treated cells when transcription is inhibited, we treated cells with both FP and TSA for 10 min and performed ChIP-qPCR Figure . IncreasDespite the strong correlation between histone acetylation and transcription, little is known about the mechanisms by which transcription is stimulated by acetylation. We show here that release of paused Pol II into elongation is the predominant mechanism by which acetylation increases transcription. Several studies have suggested that histone acetylation results in open chromatin that is more accessible to DNA-binding proteins . AlthougA recent study showed that Sirt6 restrains release from promoter–proximal pausing by preventing release of the negative elongation factor NELF, and by impeding recruitment of positive elongation factors such as BRD4 and the P-TEFb kinase . HoweverAlthough histone acetylation is increased globally already by 10 min of HDAC inhibition, few genes respond transcriptionally. Interestingly, all of these 96 genes are activated, suggesting that the only direct effect HDACs have on transcription is repression. This indicates that the strong HDAC occupancy that is often observed at active genes functions to restrict gene expression. Similarly, although promoter–proximal pausing restrains Pol II from entering productive elongation, it is positively correlated with gene expression (Interestingly, up-regulated genes gain acetylation to a larger extent than other genes when HDACs are inhibited, presumably because they are highly occupied by HDACs. Consistent with this idea, dMi-2, a subunit of the HDAC-containing NURD complex is more strongly associated with up-regulated than with non-affected genes. Of note, the larger gain in acetylation is mainly confined to the first 2 kb downstream of the TSS. This indicates that HDACs are associated with promoters and downstream positions close to the TSS where they function to restrain Pol II elongation. However, there are also genes that gain acetylation to the same extent without being up-regulated. This shows that histone acetylation is not sufficient for transcription activation at many genes. Perhaps other proteins are being acetylated that cause gene activation. It is also possible that up-regulated genes are associated with features that allow them to respond to increased acetylation. We found that the chromatin remodeler NURF and the Trithorax histone methyltransferase occupy up-regulated genes more strongly than genes that gain acetylation without an increase in transcription. One possibility is that NURF and Trithorax restrict P-TEFb recruitment to these genes making them highly paused, and that increased acetylation results in enhanced P-TEFb recruitment causing release into productive elongation.Whether histone acetylation is a consequence of transcription or causes an increase in transcription is debated . A recenAmong the up-regulated genes are cell cycle regulators such as the p21/p27 CDK inhibitor Dacapo, which is also a major target for HDACs in mammalian cells and is believed to contribute to the cell cycle arrest induced by HDAC inhibitors in tumor cells (reviewed in In conclusion, we have found that HDAC inhibition causes rapid transcriptional up-regulation of highly paused genes by releasing promoter–proximal paused Pol II into productive elongation Figure . Our reshttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141871. UCSC genome browser session: https://genome.ucsc.edu/s/Jeanwen/dm3_TSA.The datasets generated and analysed during the current study are available in the GEO repository, accession GSE141871. gkaa234_Supplemental_FilesClick here for additional data file.


Human T-lymphotrophic virus type 1 (HTLV-1) and Bovine leukemia virus (BLV) belong to the Deltaretrovirus genus. HTLV-1 is the etiologic agent of the highly aggressive and currently incurable cancer adult T-cell leukemia (ATL) and a neurological disease HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). BLV causes neoplastic proliferation of B cells in cattle: enzootic bovine leucosis (EBL). Despite the severity of these conditions, infection by HTLV-1 and BLV appear in most cases clinically asymptomatic. These viruses can undergo latency in their hosts. The silencing of proviral gene expression and maintenance of latency are central for the establishment of persistent infection, as well as for pathogenesis in vivo. In this review, we will present the mechanisms that control proviral activation and retroviral latency in deltaretroviruses, in comparison with other exogenous retroviruses. The 5′ long terminal repeats (5′-LTRs) play a main role in controlling viral gene expression. While the regulation of transcription initiation is a major mechanism of silencing, we discuss topics that include (i) the epigenetic control of the provirus, (ii) the cis-elements present in the LTR, (iii) enhancers with cell-type specific regulatory functions, (iv) the role of virally-encoded transactivator proteins, (v) the role of repressors in transcription and silencing, (vi) the effect of hormonal signaling, (vii) implications of LTR variability on transcription and latency, and (viii) the regulatory role of non-coding RNAs. Finally, we discuss how a better understanding of these mechanisms may allow for the development of more effective treatments against Deltaretroviruses. Deltaretrovirus genus is Human T-lymphotrophic virus type 1 (HTLV-1), the etiologic agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [Bovine leukemia virus (BLV), an oncogenic B-lymphotropic retrovirus that infects cattle and is associated with enzootic bovine leucosis caused by neoplastic proliferation of B cells [The first reported human retrovirus and the representative of the  B cells . HTLV-1  B cells .gag) and envelope proteins (env), as well as key enzymes (pol) and accessory proteins. In deltaretroviruses, accessory proteins are encoded by a sequence located 3′ of the env gene called the pX region. Both in HTLV-1 and BLV, the pX region encodes the Tax and Rex proteins that are involved respectively in transcriptional and posttranscriptional regulation of viral gene expression. Reverse genetics demonstrated that Tax and Rex are essential for viral infectivity in vivo [hbz in HTLV-1) [Human immunodeficiency virus (HIV) lentiviral genome codes for six accessory genes embedded within core gene regions, such as tat, rev, vif, vpr, vpu and nef, and the one putative AntiSense Protein (ASP) from the antisense [Similar to other exogenous retroviruses, the deltaretrovirus genome comprises two identical copies of a single-stranded RNA molecule. Reverse transcriptase is the enzyme responsible for transcription of the genomic RNA into double-stranded proviral DNA. The integrase enzyme is essential for the insertion of virus DNA into the host chromosome, resulting in formation of a stably integrated provirus . Once in in vivo ,6. In ad HTLV-1) ,9,10,11.ntisense ,13,14. Hntisense . The Taxntisense . Tat alsntisense .in vivo. Shortly after infection, a strong immune response controls BLV/HTLV-1 load through destruction of cells expressing viral antigens [Retrovirus-infected cells are able to undergo a productive infection with generation of progeny virions or alternatively can enter a non-productive latent state in which viral gene expression is restricted to miRNAs and antisense transcripts ,19. In Hantigens ,21. Onlyantigens ,23. Duriantigens ,25,26. Iantigens ,28,29. Vantigens . The epiantigens . Intraceantigens ,33,34. Tantigens . These dDevelopment of EBL and ATL requires Tax expression, at least in the early stages of infection. Activation of viral expression from latently-infected cells and escape from the cytotoxic lymphocyte (CTL) response is an essential step in pathogenesis. However, it still remains unclear how viral expression becomes reactivated in latent (low level-expressing) infected leukocytes in vivo. Experimentally, transcriptional activation can be initiated ex vivo by various exogenous stimuli, such as polyclonal activators, T cell receptor (TCR) ligands, antibodies , lectins (PHA), phorbol esters (PMA), ionomycin, prostaglandins, cellular stress , histone deacetylase inhibitors (HDACi) and DNA demethylation agents ,39,40,41cis-regulatory elements, which determine the maximal rate of viral transcription initiation. However, the cell type and its differentiation state with respect to variety of cell activation signals may lead to substantial variations in transcriptional activity of a LTR depending on the chromatin context [LTRs act as promoters for virus gene expression, and thus are key regulatory elements within the provirus. LTR segments are subcategorized into the U3, R, and U5 regions . The 5′- context . All theIn this review, we summarize current knowledge on viral and host processes involved in the regulation of deltaretrovirus transcription and latency, including, (i) epigenetic control of the provirus, (ii) the cis-elements present in the LTR, (iii) enhancers with cell-type specific regulatory functions, (iv) the role virally-encoded transactivator proteins, (v) the role repressors in transcription and silencing processes, (vi) the effect of hormonal signaling, (vii) implications of LTR variability on transcription and latency, and (viii) the regulatory role of non-coding RNAs. Finally, we discuss how these integrated mechanisms contribute towards maintenance or release from viral latency in the context of antiviral therapeutics.Chromatin architecture is important for the control of viral gene expression since transcriptional activation by cellular and/or viral transactivating factors is dependent on accessibility to the promoter DNA. Epigenetic changes, such as DNA methylation and histone modifications , areChromatin modifications related to transcriptional modulation are not exclusively linked to protein components; they can be associated with direct changes in the DNA. Once a retrovirus is integrated into the host genome as a provirus, it can be subject to DNA methylation as an integral part of the host cell genome. Methylation occurs mainly in sequences containing CpG dinucleotides. It has been observed that DNA is less methylated in transcriptionally active regions of the genome compared to inactive regions that are high methylated . MethylaFor HTLV-1, it was shown in vitro that DNA methylation suppresses basal, as well as Tax induced, promoter activity of the LTR ,47,48. Ttax gene expression is associated with unmethylated or weakly methylated 5′-LTRs at seroconversion [Initially, high levels of DNA methylation of the 5′-LTR was considered to be an essential mechanism of HTLV-1 persistence in vivo . Howevernversion . 5′-LTR nversion . The LTRs of HTLV-1 (i.e. HTLV-1a) and BLV (L267 cells) contain 47 and 23 CpG sites, respectively . In contMechanistically, viral proteins can affect DNA methylation. In BLV, Tax downregulates DNA methyltransferase (DNMT) activity, which in turn leads to a lower level of DNA methylation in the CRE site of the promoter . This meIn addition to direct DNA methylation, lysine (Lys) methylation of histones can play divergent roles in transcriptional regulation. Retroviral promoters are subject to silencing through the formation of facultative heterochromatin, which is typically created by the Polycomb repressor complex 2 (PCR2) . The PRCSaccharomyces cerevisiae, fully activated promoters are rich in methylated H3K4, while H3K9 methylation is associated with gene silencing [Lys methylation of the N-terminal ends of histones H3  and H4 (K20) affect transcriptional regulation and the chromosome organization . In Saccilencing . BLV sililencing . Concomiilencing . In HIV, the PRC2 repressive complex modulates latency. Chromatin immunoprecipitation assays using latently HIV-infected Jurkat T cell lines showed that the catalytic subunit of the PRC2 complex, HMT Enhancer of Zeste Homolog 2 (EZH2), is present at high levels at the LTRs of silenced HIV proviruses and is rapidly displaced following proviral reactivation . PRC2-meHistone acetylation is among the best characterized modifications of integrated retroviral genomes (reviewed in ). HistonSo far, most studies on histone modification of HTLV-1 proviruses were conducted using in vitro cell culture methods, where Tax expression is the primary mechanism of 5′-LTR activation through a positive feedback loop. Tax is known to interact with the chromatin remodeling complex, change nucleosome positioning, and induce transcriptional activation from the 5′-LTR . Tax, toHTLV-1 sense-strand transcription is associated with a significant increase in H3K4me3 and H3K9ac in HTLV-1-infected PBMC cultures ,75. In tThese clinical trials on HTLV-1 were supported by evidence obtained in BLV. Administration of valproate to BLV-infected sheep induced BLV expression and cured leukemia . The moscis-active elements critical for the modulation of promoter activity. The cellular RNAPII initiates transcription at the U3-R boundary of the 5′ LTR. The LTR promoter contains a TATA box that provides a binding site for RNAPII, determines the site of initiation and affects the efficiency of the initiation [The protein-binding signatures within retroviral LTRs are essential docking sites for initiation of transcription factor (TF) complexes. The U3 region plays a crucial role in the induction of retroviral transcription since it contains the Tax-responsive itiation . Besidesitiation ,84. OtheAvian Leucosis Virus (ALV) to the numerous elements in case of HTLV-1/BLV group and HIV-1 retroviruses [Mouse Mammary Tumor Virus (MMTV), Murine Leukemia Virus (MLV) and Simian Foamy Virus (SFV).Enhancer sequences consist of binding motifs for sequence-specific transcription factors. Notably, the complexity of cis-acting elements within the basal LTR promoters of retroviruses varies from relatively few in the case of oviruses . A precioviruses . Figure The main TFs involved in deltaretrovirus transcription are: cAMP-responsive element-binding protein/activating transcription factor (CREB/ATF), activating enhancer binding protein (AP), erythroblast transformation specific family members (Ets), nuclear factor kappa B (NF-κB), specificity protein (Sp) and myeloblastosis proto-oncogene protein (c-Myb). Cell type-specific TF for deltaretroviruses include: PU.1 and Spi-1 , CREB and ATF1 (abundantly expressed in T- and B-cells), and c-Myb (primarily expressed in hematopoietic cells). Unlike HIV, deltaretroviruses do not contain binding sites for nuclear factor of activated T cells (NF-AT), T-cell specific/lymphoid enhancer binding factors (TCF/LEF-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF).Enhancer sequences are usually 10-15 base pairs in length and may be located within the first ≈200 base pairs upstream of the cap site . An examAs for HTLV-1, the major regulatory elements in BLV are three repeated 21-bp TREs that include imperfectly conserved CREs matching the consensus sequence 5′-TGACGTCA-3′ . These CAdditional elements of transcription control have been identified within R and U5 sequences of 5′ LTR. For HTLV-1, the downstream regulatory element (DRE) in the R region includes the YB-1 binding site that is required for basal transcription . The U5 Despite basic similarities among retroviruses, there are several distinctive features with respect to their transcriptional regulatory elements located in 5′-LTR that determine the level of viral gene expression, as well as control of cellular tropism of retrovirus replication. + T lymphocytes [+ T cells, B cells, monocytes, dendritic cells and microglial cells in vivo, although the role of these cell populations in latency and infection remains poorly understood [+ T-cells, much higher levels of transcription from the HTLV-1 LTR has been observed in primary CD4+ T-cells, suggesting that cellular factors within CD8+ T-cells may be insufficient to support efficient functioning of the HTLV-1 promoter [HTLV-1 gene expression is dependent on several cellular transcription factors and their different expression profiles within a given infected cell. The main cellular targets of HTLV-1 in infected individuals are CD4phocytes ,106. HTLderstood ,108,109.derstood . The higderstood . Once Taderstood . HTLV-1 derstood . Phosphoderstood . Elevatederstood . Alternaderstood . Comparepromoter ,116. + T cells, CD8+ T cells and γ/δ T cells, the major cellular target of BLV is the B lymphocyte [cis-regulatory elements for two Ets members PU.1 and Spi-B proteins [Although BLV may be present in monocytes, granulocytes, CD4mphocyte ,120,121.mphocyte . In addiproteins . During proteins ,123,124.proteins ,126. TheHTLV and BLV gene expression in monocytes and DCs is mediated primarily through the enhanced DNA-binding activity of AP-1 to TRE-I. In HTLV-1 infected cells of the monocyte/macrophage lineage, stimuli that trigger monocytic differentiation or activate protein kinase C (PKC) pathway enhance the expression and DNA-binding activity of AP-1 transcription factor (Fos/Jun) ,127,128.BLV has also been detected in other tissues including endothelial and mammary epithelial cells ,131,132.Basal transcription from the LTR is insufficient to promote productive virus replication. Delta- and Lenti-retroviruses are able to increase the rate of gene expression through virus-encoded transcriptional activator proteins (transactivators). Although the effect is very similar in BLV/HTLV-1/HIV, the mechanisms are quite different ,139,140.Deltaretroviruses induce a polyclonal expansion of lymphocytes that evolves into an aggressive monoclonal leukemia in approximately 5% of infected individuals after a variable but frequently long period of latency ,146. AltSense HTLV-1 proviral expression can also be inhibited by the antisense protein HBZ, which counteracts Tax by competing for binding to CREB and CBP/p300  and othetax gene  [tax gene at positions 7464, 7469, 8045 were identified in HTLV-1 carriers and patients with ATL. Multiple deletions and/or insertions in the tax gene in ATL patients is a mechanism that abrogates transactivation activity and promotes escape from the immune system [Since Tax is an immunodominant target antigen for cytotoxic T-lymphocytes (CTLs) in vivo, the proviral clones might escape from the host immune system by suppressing Tax expression. Therefore, immortalized cells that induce weaker CTL responses are favored during the development of adult T-cell leukemia (ATL) . During pitopes) ,150,151.e system ,149. More system .The U3 region of the BLV LTR also contains three TREs , which are important for Tax-mediated activation of the LTR. A NF-κB-binding site is located between TRE2 and TRE3 of the U3 region . This sitax gene expression also occur in BLV. Naturally occurring single amino acid substitution E303K impairs Tax function and induces the silent BLV phenotype in BLV-induced B-cell tumor [As in HTLV-1, mutations that affect ll tumor . Reversell tumor . The D24ll tumor . Amino all tumor . In BLV-infected cattle, different Tax variants can be associated with changes in white blood cell numbers . A majorCollectively, these data reveal that Tax is an essential regulator of viral transcription upon interaction with the TRE elements and CREB/ATF transcription factors. Tax nevertheless becomes counter-selected by a series of mechanisms including proviral mutations. LTRs also interact with repressors of transcription . These iBox 1C2H2-type zinc finger proteins (ZNFs) representlarge family of the transcription factors characterized by multiple tandemlyrepeated zinc fingers, which allow high flexibility in target recognition [ognition . Most ofognition .The bioognition . Interesognition . In addiognition . Interesognition .In addiognition . HUB1 haognition .Howeverognition .Collectognition . One mai    Another C2H2 zinc finger transcription factor,COUP-TF-interacting protein 2 (CTIP2), was found to repress HIV-1transcription via a direct association with the LTR-bound transcriptionfactor Sp1 and chicken ovalbumin upstream promoter transcription factor(COUP-TF).  CTIP2 also facilitated HIV Tat protein relocation totranscriptionally inactive regions of chromatin .CTIP2 rSpecificity protein (Sp) is a well-known family oftranscription factors that includes Sp1, Sp2, Sp3 and Sp4, which areimplicated in cell growth, differentiation, apoptosis and carcinogenicity [genicity . Sp1 progenicity .cis-regulatoryelements [    In the HTLV-1 LTR region six Sp1 binding sites wereidentified: two Sp1 binding sites located in U3 5′-LTR region that arecrucial for basal and Tax-transactivated promoter expression, two Sp1 bindingsites located in U5RE sequence of LTR region that acts as repressor forHTLV-1 gene expression in absence of Tax, and two new functional Sp1 siteslocated within the R region that acts as negative elements . Howeverelements ,165,172.Basic helix-loop-helix-zipper (bHLH-ZIP) familyincludes transcription factors Activating enhancer binding protein 4 (AP-4)and E protein (E47). They contain a basic domain, responsible for DNA bindingon the E-box motif (CAGCTG), and HLH and ZIP domains, which are implicated inprotein oligomerization [rization . For therization . Overexrization ,176,177.    In addition, another bHLH factor, named E47 has shownsilencing activity of the HTLV-1 promoter, both in the presence and absenceof Tax. It is possible that E47 binds to the putative E box in the promoter28 bp upstream of the TATA box and negatively modulates the binding of HATp300/CBP to the CRE-binding protein site within 21-bp Tax-responsive elements,178. The2) or progesterone (P4) [Modulation of virus replication by hormones is physiologically relevant, and crucial to our understanding of sex-differences in virulence and immunity to retroviruses ,180. Manone (P4) .The current knowledge of the endocrine system control of HTLV-1 viral gene expression is very limited . No hormIn contrast, BLV infection has been correlated with breast cancer development, although these observations are still controversial ,191,192.Immunocytochemical studies of mammary epithelial cells infected in vivo with BLV suggest that these cells are in a state of latency and after administration of glucocorticoids and prolactin together, may stimulate the production of viral particles . HoweverRepression of viral expression is a major strategy to evade developed by Deltaretroviruses immune response. Considering that (i) several stressor factors are associated with animal handling in production facilities, (ii) animals exposed to stress stimuli present higher levels of glucocorticoids and (iii) the role of glucocorticoids as strong immunosuppressive agent has been extensively demonstrated, it would be possible under certain physiological circumstances  that glucocorticoids might favor BLV replication and spread in vivo. Opposite to what was observed from in vitro studies, mutations in the GRE region of BLV did not affect virus infectivity and replication using an in vivo ovine model of BLV infection . In agreRetroviruses, similar to other RNA viruses, exhibit generally a high mutation rate because of polymerization errors occurring during DNA synthesis by reverse transcriptase (RT), which lacks proofreading activity. Cytidine deamination of RT products through APOBEC3G activity also contributes towards hypermutation of proviral DNA . ProviruIn vitro, the BLV RT is 10-fold more faithful than other retroviral polymerases. The error rate in BLV is 4.8 × 10−6 and in HTLV-1 is 7 × 10−6 nucleotides, as compared to 2.5–5.9 × 10−4 for purified HIV-1 RT and 3.4 × 10−5 measured during single-cycle HIV-1 infection [LTR sequence alteration can affect the modulation of viral expression and latency ,206. Unlnfection ,210,211.nfection ,213. Altnfection . Although most of the deltaretroviral genome remains stable, variations are noted in the promoter region of these retroviruses. Among these, a G(232)A transition in the CREB-binding site in the TRE-1 of the HTLV-1 LTR is commonly present in HTLV-1 asymptomatic carriers. This nucleotide variation correlates with increased viral proliferation and proviral load . FurtherIn BLV, few studies have evaluated proviral LTR sequences for the presence of single nucleotide polymorphisms (SNP), insertions and deletions ,153,216.The U3R region of the BLV 5′ LTR is involved in the control of both basal and Tax-dependent transcription of the BLV genome though its interaction with several cellular transcription factors. Therefore, mutations affecting promoter nucleotide sequence might drive potentials differences in the biological properties of diverse BLV strains. In support of this, the spontaneous substitution T175C within the TATA box is associated with virus productivity in vitro, which was closely related with BLV transmissibility ,219. The functionality of these mutations can be investigated by reverse genetic experiments. In this context, it is noteworthy that high level of promoter activity does not necessarily correlate with efficient replication. Indeed, the substitution of the suboptimal CRE elements in the 3 TREs increases promoter activity but reduces proviral loads . This suNon coding RNAs of cellular or viral origin can modulate LTR-directed transcription, replication and pathogenesis . Long non-coding RNAs (lncRNAs) are defined as RNA transcripts larger than 200 bp without any coding potential . They ap+T lymphocytes can act as a negative regulator of transcription factor NFAT. Since NFAT promotes HIV-1 expression by binding to the downstream sequence of TAR, NRON is able to suppress HIV transcription [In HIV, noncoding repressor of NFAT (NRON) which is a highly expressed lncRNA in resting CD4cription . NRON alcription . Anothercription . lncRNA cription . In HTLV-1, the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL) cooperates with EZH2 to enhance the proliferation of HTLV-1 infected cells .+ T cells, while being almost undetectable in resting CD4+ T cells [In HIV, antisense transcripts of ncRNAs suppress HIV gene expression by recruitment of a repressive complex containing DNMT3a, HDAC-1 and EZH2 ,229,230. T cells ,232. HBZ RNA is expressed mainly in ATL cells, and primarily localizes to the nucleus, where it functions as an lncRNA to induce T cell proliferation [HBZ RNA functions to transforms T cells [HTLV-1 and BLV also transcribe antisense RNAs, respectively HBZ and AS1/2 ,233. Altferation ,235. How T cells . HBZ RNA of HTLV-1, the transcripts are retained in the nucleus, indicating that the main functions of AS1-S/L and AS2 are exerted by their lncRNA form. These antisense RNAs may play role in regulation of BLV, silencing of the 5′-LTR and/or leukemogenesis [BLV provirus also constitutively expresses antisense transcripts in all leukemic and asymptomatic samples. Similarly to the ogenesis .+ T cells as compared to activated CD4+ T cells, interact directly with the 3′ ends of HIV-1 mRNA and decrease replication [Several host microRNAs (miRNAs) were demonstrated to have direct or indirect effects on retroviral replication. In HIV-1, a series of miRNAs  are enriched in resting CD4lication . The mirlication . As Cycllication . HTLV-1 Tax downregulates host miRNAs to promote continuous viral transcription . As discpol-derived miR-H3 targets the 5′-LTR TATA box and activates transcription of the viral promoter [Viruses also express miRNAs that control the viral life cycle, cell survival and immune evasion. Many viral miRNAs downregulate the expression of host factors harboring antiviral activity . In HIV,promoter ,242. In promoter . BLV transcribes a cluster of viral miRNA through RNA Pol III dependent promoters. BLV miRNAs are abundantly expressed in both bovine B-cell tumors and in experimentally-infected bovine calves . The miRThe existence of HTLV-1 and BLV antisense transcripts implies that the LTR promoter is bidirectional. The abundance of sense and antisense transcripts fluctuates in transient bursts via a still unexplained mechanism ,247. DurRetroviruses having a DNA intermediate in their replication cycle can undergo transcriptional silencing and hide from host immunity. Understanding the molecular mechanisms underlying silencing are necessary to clear the viral reservoir from an infected host .Transcriptional and posttranscriptional mechanisms participate in initiation and/or maintenance of latency. In this review, we outlined a wide range of mechanisms underlying deltaretrovirus transcriptional regulation and latency .Activation of LTR-driven sense transcription may have clinical outcomes due to its therapeutic potential in a shock-and-kill strategies. This clinical approach consists of three-steps: (1) the reactivation of viral transcription from latently infected cells, followed by (2) the killing of cells expressing viral antigens, and (3) anti-retroviral therapy for protection of uninfected cells. Transcription can be targeted by HDACs inhibitors like valproic acid (VPA) or methylation inhibitors such as 5-azacytidine (5-AzaC) ,78. HDACThese therapeutic approaches are not practical for BLV-infected cattle in agricultural settings, mainly due to animal management restrictions and economic costs. However, BLV infection in cattle could be used as an in vivo model for HTLV viral infection in humans. For BLV, applying test-and-elimination strategy led to the successful eradication of the EBL in some European countries. Since this strategy is economically untenable in countries with high levels of BLV prevalence, test-and-segregation programs have been recommended but are rarely implemented. Therefore, a better understanding of modes and mechanisms of transmission of BLV, might aid the development of novel control strategies for BLV. We hypothesize that external stimuli  might trigger virus reactivation from latency, contributing to higher viral loads and virus transmission. In this scenario, reducing stressors as well as separating high provirus load herds could prevent BLV transmission and reduce overall prevalence.Moving forward, an improved understanding of the fundamental regulatory mechanisms controlling deltaretroviral transcription will assist in the development of novel and more efficient anti-retroviral prophylactic strategies and therapeutic agents.


An allelic variant of the transcription factor GAME9 reduces accumulation of toxic steroidal glycoalkaloids in ripe tomato fruit via altered binding to the promoter of a steroidal glycoalkaloid biosynthesis gene. GLYCOALKALOID METABOLISM 9 (GAME9) is responsible for the variation in SGA content in tomato (Solanum lycopersicum). During a sequential analysis we found a 1 bp substitution in the AP2/ERF binding domain of GAME9. The 1 bp substitution in GAME9 was significantly associated with high SGA content and determined the binding capacity of GAME9 with the promoter of GAME17, a core SGA biosynthesis gene. The high-SGA GAME9 allele is mainly present in S. pimpinellifolium and S. lycopersicum var. cerasiforme populations and encodes a protein that can bind the GAME17 promoter. In contrast, the low-SGA GAME9 allele is mainly present in the big-fruited varieties of S. lycopersicum and encodes a protein that shows weak binding to the GAME17 promoter. Our findings provide new insight into the regulation of SGA biosynthesis and the factors that affect the accumulation of SGA in tomato.Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules found in the family Solanaceae. SGA content varies among different plant species and varieties. However, the genetic mechanisms regulating SGA content remain unclear. Here, we demonstrate that genetic variation in  Solanum alkaloids, are secondary metabolites primarily found in the Solanum family , commonly known as −1 tomatine inhibits the growth of Tribolium castaneum larvae. A total SGA level that exceeds 200 mg kg−1 FW of edible tuber is considered unsafe for humans  was the first gene reported to encode an SGA biosynthetic enzyme   transcription factor ORCA2 regulates terpenoid idole alkaloid biosynthesis in GAME9. The high-SGA GAME9 allele is mainly represented in Solanum pimpinellifolium (PIM) and S. lycopersicum var. cerasiforme (CER) and encodes a protein that can bind a cis-element in the promoter of GAME17—a core gene for SGA biosynthesis. In contrast, the low-SGA allele is mainly present in the big-fruited varieties of S. lycopersicum (BIG) and encodes a protein that shows weak binding to the GAME17 promoter. These findings provide insight into new strategies for regulating toxic compounds and the genetic improvement of tomato. They also provide information useful for engineering reduced levels of anti-nutritional compounds in other plants.In tomato, GAME9 serves as a master transcriptional regulator of SGA biosynthesis by binding GCC box elements in the promoters of target genes  and signiﬁcance thresholds  of all the SNPs were defined at a uniform threshold. VCFtools was used to perform local linkage disequilibrium (LD) analysis. The 83.9–84.2 Mb regions of SNPs (P<2.4×10–7) centered on the lead SNP (ch01:84016748) were selected to calculate the r2 of LD. The physical locations of the SNPs were identified based on tomato genomic sequence version SL2.50. All tomato plants were self-pollinated. Tomato fruit was hand-harvested at physiological maturity.A total of 2 678 533 single nucleotide polymorphisms  for 442 accessions were used in a GWAS, which included 31 PIM accessions, 124 CER accessions, and 287 BIG accessions that were from the same populations as used previously  were harvested uniformly at their mature green (MG), breaker, and red ripe (RR) stages. The freeze-dried samples (100 mg) were extracted for 8 h at 4 °C with 1.0 ml 80% methanol: water (V:V) containing 0.1 mg ln=3), the actin gene (BT013524) was used as an internal control. Primer sequences are list in JXB online.Total RNA was isolated using the Trizol reagent (Sigma-Aldrich). cDNAs were synthesized form DNaseI-treated total RNA using a Hiscript II 1st Strand cDNA Synthesis Kit . Expression analysis of SGA-related genes in the fruits (peel and flesh tissues without the seeds and gel) of wild-type and transgenic lines was performed using three biological replicates (GAME17 were amplified from TS-303 genomic DNA with specific primers (see Supplementary GAME9135A and GAME9135V with specific primers (Saccharomyces cerevisiae) genome and cultured on SD/−Ura at 30 °C for 3 d. The bait-reporter strain was transformed with the prey vector. After 3 d, the positive yeast strains were picked and diluted with sterile water. These suspensions were spotted on an SD/−Leu medium that contained from 0 to 100 ng ml−1 aureobasidin A. The plates were incubated for 3–4 d at 30 °C. The negative (pGADT7+N-AbAi) controls were prepared as described above. The DNA–protein interactions were analysed based on the growth of the yeast strains relative to the negative controls on media with different ABA concentrations.Promoter fragments from  primers  were ampGAME9135A and GAME9135V were amplified from the genomic DNA of TS-303 and AC, respectively, with specific primers (see Supplementary Agrobacterium tumefaciens (GV2260)-mediated transformation was used to transiently express both reporters and effectors in Nicotiana benthamiana leaves. The empty pGreen II 62-SK was used as the negative control. Three days after the transfection, both fLUC and rLUC activities were measured using the dual luciferase assay reagents  and an Infinite M200 plate reader . To generate β-glucuronidase (GUS) reporter proteins for observation, the two alleles GAME9135A and GAME9135V were amplified from the genomic DNA of TS-303 and AC, respectively, and fused to pHellsgate8 vector, a CaMV 35S promoter-driven effector, and the fragment of the promoter was cloned into pMV2 vector (derived from the pHellsgate8 vector with a deleted CaMV 35S promoter) harboring the GUS reporter gene. The empty pHellsgate8 was used as the negative control. Three days after the transfection, both the negative control and the fusion proteins were incubated at 37 °C for 12 h in staining buffer 6, 0.5 mM K4Fe(CN)6, and 1mg ml−1 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid), followed by washing with 70% (v/v) ethanol. The relative expression of GUS reporter gene and negative control was determined with the GUS primers, and the actin gene (BT013524) was used as an internal control. Primers sequences are list in The two alleles GAME9135A and GAME9135V were amplified from TS-303 and AC, respectively, and then inserted into pET15d. The recombinant fusion protein with a MBP-tag was expressed in Escherichia coli DE3 (BL21) cells  and affinity purified using magnetic agarose  according to the manufacturer’s instructions. To label the probes, FAM was attached to the sense oligonucleotides at their 5′ end. Sense, antisense, and mutated oligonucleotides  glycerol, 50 ng μl−1 poly (dI-dC)). After 30 min, the reaction products were separated by electrophoresis on 6% non-denaturing polyacrylamide gels. After migration, the FAM-labeled probe on the non-denaturing polyacrylamide gel was visualized using an Amersham Imager 600.The whole coding sequences of GAME9 open reading frame (ORF) was amplified from TS-303. The GAME9-silenced fragment was amplified from AC cDNA using gene-specific primers with 5′-attB1 and 3′-attB2 extensions on the forward and reverse primers. The full-length GAME9 ORF was cloned into overexpression vector pMV2 (derived from the pHellsgate8 vector) using a CaMV 35S promoter. The GAME9-silenced fragment was recombined into the RNAi vector pHellsgate 2 using the Clonase BP reaction (Invitrogen). All vectors were introduced into the A. tumefaciens strain C58 for tomato transformation. TS-286 was transformed with the overexpression vector driven by a CaMV 35S promoter and the silencing vector. The genomic DNA from these transgenic plants was analysed with PCR-based genotyping experiments. The pertinent primers are listed in The full-length ERF1 (Solyc01g090300), ERF2 (Solyc01g090310), ERF3 (Solyc01g090320), ERF5 (Solyc01g090370), GAME1 (Solyc07g043490), GAME2 (Solyc07g043410), GAME4 (Solyc12g006460), GAME6 (Solyc07g043460), GAME7 (Solyc07g062520), GAME9 (Solyc01g090340), GAME9 homolog from potato (Sotub01g029510), GAME11 (Solyc07g043420), GAME12 (Solyc12g006470), GAME17 (Solyc07g043480), GAME18 (Solyc07g043500), and MYC2 (Solyc08g076930).Sequence data from this article can be found in the Sol Genomics Network databases with the following accession numbers: P=2.25×10–14), hydroxytomatidenol , hydroxytomatidine , dehydrofilotomatine , neorickiioside A , lycoperoside H  and lycoperoside A  (P<2.4×10–7) fell into a 122.7 kb region of 83.9–84.2 Mb; there were a total of 11 genes in this region (Solyc01g090330), and an AP2/ERF (Solyc01g090340) named GAME9, which has been reported to regulate the biosynthesis of steroidal alkaloids  that showed a strong association with the levels of hydrotomatidine   (Zhu et 6×10–12) . All thes region . A haplos region , and mans region . Haplobls region , an unknmatidine .GAME9 locus, we analysed the nucleotide sequence of GAME9 in 374 tomato accessions with diverse SGA content. The resequencing of the GAME9 coding region indicated that a polymorphism  that causes an alanine to valine substitution at position 135 (A135V) in the N-terminus of AP2/ERF binding domain explained 19.87% and 23.5% of the variation in the level of hydrotomatidine and the seven SGAs in tomato fruit, respectively  between the levels of the seven SGAs in the two alleles of GAME9 , and the ‘high-SGA’ content phenotype as GAME9135A, which is mainly present in the PIM (100%) and CER (71.2%) populations. Further, we randomly selected 14 accessions of high SGA and 14 accessions of low SGA and measured expression of GAME9 in red ripe fruit by quantitative RT-PCR. The expression of GAME9 showed no significant difference in red ripe fruit between high- and low-SGA accessions  assay indicated that GAME9135A can bind to the N1 motif (−898 to −378 bp) of the GAME17 promoter and that GAME9135A cannot bind the N2 motif (−666 to −378 bp) of the GAME17 promoter  leaves. We found that the P1 sequence  was essential to activate a luciferase (LUC) reporter gene. In contrast, the P2 sequence  was not necessary (GAME9135A protein can bind the core sequence between −898 and −671 bp in the GAME17 promoter. We tested whether the GC-rich element (5′-CGCCCCCC-3′) in this core sequence could serve as a GAME9135A binding site using an EMSA. We compared the ability of GAME9135A to bind probes containing this GC-rich sequence with probes containing mutations in this GC-rich sequence (GAME9135A fusion protein can bind the wild-type cis-element (5′-CGCCCCCC-3′) but not the mutant cis-element  in the tobacco ME genes . Thus, wpromoter . To indeecessary . Thus, wsequence . We foun-element . These dGAME9135V effector (pGreen II 62-SK) to perform transient transactivation and promoter binding assays. The infiltrated plants revealed that the P1 sequence in the GAME17 promoter could support the transactivation activity of both GAME9135V and GAME9135A. However, the transient overexpression of GAME9135A could activate transcription of the luciferase reporter gene (3.8-fold) more than could the transient overexpression of GAME9135V (1.6-fold)  that affects the N-terminal AP2/ERF domain of GAME9, we used a CaMV 35S promoter-driven .6-fold) , B. This control , D. Addi strains , F. Finaragments , H. TakeGAME genes, the transactivation of GAME7 , GAME11 , GAME6 , GAME4 , GAME12 , GAME1 , GAME18 , and GAME2  by GAME9135A and GAME9135V was tested in tobacco leaves. We found that GAME9135A and GAME9135V are capable of transactivating the GAME7 promoter (GAME7 in that GAME9135A was a more potent activator than GAME9135V (GAME17 in red ripe fruit by quantitative RT-PCR. The expression of GAME17 showed a significant difference in red ripe fruit between high- and low-SGA accessions (see GAME9135A can bind the GC-rich element (5′-GCCNNCC-3′) in the promoters of GAME17 and GAME7, and relative to GAME9135A, GAME9135V weakly binds and activates the transcription of genes that contribute to SGA biosynthesis. Taken together, it is clear that the binding activity of GAME9 depends on allelic variation that influences the N-terminal AP2/ERF domain.To test whether this regulatory strategy is used for other promoter . More imAME9135V , 3. In aions see . So thesSolanum tuberosum), and eggplant (Solanum melongena) , to up-regulate GAME4 and GAME7 expression in a different scenario  transcription factors CrMYC2 and NtMYC2, respectively, which potentially function as activators of gene expression by binding G-boxes in the ORCA3 and ERF189 promoters  in GAME9 that can be used to develop a CAPS maker that will be useful for breeding low alkaloid varieties of tomato with better nutritional value.In general, a high concentration of glycoalkaloids and their byproducts adversely affects the nutritional value of tomato fruit and human health . Thus, bJXB online.Supplementary data are available at Fig. S1. Manhattan plots for GWAS on six SGA contents.In vivo function of GAME9.Fig. S2. GAME9-RNAi lines.Fig. S3. SGA pathway-related gene expression at the mature green fruit stage in 135A binds the GAME17 promoter and activates its expression.Fig. S4. GAME9GAME17 in red ripe tomato fruit.Fig. S5. The relative expression of Fig. S6. Alignment of GAME9 amino acid sequences.Table S1. Primers used in this study.Table S2. Genes within 122.7 kb of the SNP (ch01:84016748) most highly associated with fruit hydrotomatidine contentTable S3. The SNP and their P-value (associated with fruit hydrotomatidine content) in the 86 haploblocks.GAME4, GAME7, and GAME17.Table S4. The GC-rich motifs found in the promoters of eraa014_suppl_supplementary_figures_S1_S6_tables_S1_S4Click here for additional data file.eraa014_suppl_supplementary_table_S2Click here for additional data file.eraa014_suppl_supplementary_table_S3Click here for additional data file.


Meiosis is a specialized cell division that gives raise to four haploid gametes from a single diploid cell. During meiosis, homologous recombination is crucial to ensure genetic diversity and guarantee accurate chromosome segregation. Both the formation of programmed meiotic DNA double-strand breaks (DSBs) and their repair using homologous chromosomes are essential and highly regulated pathways. Similar to other processes that take place in the context of chromatin, histone posttranslational modifications (PTMs) constitute one of the major mechanisms to regulate meiotic recombination. In this review, we focus on specific PTMs occurring in histone tails as driving forces of different molecular events, including meiotic recombination and transcription. In particular, we concentrate on the influence of H3K4me3, H2BK123ub, and their corresponding molecular machineries that write, read, and erase these histone marks. The Spp1 subunit within the Complex of Proteins Associated with Set1 (COMPASS) is a critical regulator of H3K4me3-dependent meiotic DSB formation. On the other hand, the PAF1c (RNA polymerase II associated factor 1 complex) drives the ubiquitination of H2BK123 by Rad6-Bre1. We also discuss emerging evidence obtained by cryo-electron microscopy (EM) structure determination that has provided new insights into how the “cross-talk” between these two marks is accomplished. Saccharomyces cerevisiae has a strong dependence on the presence of nutrients in its surroundings. As long as there is enough nutrient availability, cells proceed to mitosis, allowing them to proliferate by budding [The budding yeast  budding . Provide budding . This sp budding . Meiosis budding .The ability to sporulate, which is unique to diploid cells, requires the expression of both MATa and MATα alleles. The products of this locus give rise to an a1/α2 heterodimer, without which sporulation is unable to take place [IME1. This promoter is much larger than that of the majority of yeast genes and contains regulatory elements for all the different factors that could potentially affect IME1 expression. These regulatory elements include binding sites for transcription factors responding to mating type, nitrogen deprivation, and others [IRT1, which nearly encompasses the totality of the IME1 promoter. This lncRNA acts as a cis-element, inhibiting the binding of transcription factors to this region [Most of these elements are able to control the transcription of the master regulator Ime1. The regulation of the expression of this factor is mediated by the Rme1 repressor , which id others . In hapls region .IME1 [IME4 RNA (known as IME4-AS or RME2), whereas in diploids, its anti-sense transcription is impeded by the aforementioned a1/α2 heterodimer. As a result, the Ime4 protein is produced, activating IME1 by the N6-adenosine methylation of its RNA [Furthermore, not only does mating affect the Rme1-dependent pathway, but it modulates the expression of the RNA-methyltransferase Ime4, needed for correctly inducing IME1 . What ma its RNA ,11,12.URS1 site [cis-element is the target of the Ume6 protein, which after binding to the URS1 site displays the capacity of repressing the transcription of these genes during growth, by the recruitment of the Rpd3/HDAC complex and the chromatin remodeler Isw2 [Once Ime1 is activated, a first transcriptional wave is triggered, consisting of the so-called “early” genes, which exhibit in their promoters a common regulatory element: the RS1 site ,14. Thisler Isw2 ,15,16,17URS1 site to which Ume6 is bound, and whose expression is indispensable for the entrance into the premeiotic S phase [IME2, a key regulator in meiosis by mediating Ime1 stability; INO1, which is involved in lipid biosynthesis; or SPO13, which is linked to sister chromatid cohesion [The Ume6 protein has the ability to act as a repressor, but also as an activator. The conversion of Ume6 from repressor to activator was initially thought to be due to impeded interaction with the Rpd3/HDAC complex by the presence of Ime1 . However S phase . Among tcohesion ,23,25.The segregation of homologous chromosomes occurs during the first meiotic division, while the second division is comprised of the separation of sister chromatids. The physical location where homologous chromosomes have exchanged their information is termed the chiasma. It is in this place where the spindle microtubules attach to kinetochores, generating tension. This tension is required for proper segregation at meiosis I . It is iThe recombination characterising meiosis consists of four consecutive steps A; (i) thSaccharomyces cerevisiae [As stated, programmed DSBs are formed during the first meiotic division, a reaction catalyzed by the meiosis-specific, topoisomerase-like DNA transesterase Spo11 ,33 FiguA. Apart revisiae ,35,36. Trevisiae . Among trevisiae .After these steps, crossover recombination and homologous pairing are linked by the synaptonemal complex (SC), which implicates the multimeric assembly of coiled-coil proteins between aligned homologous chromosomes . In spitOnce the recombination process has ended, the expression of Ndt80 implies entering into the middle phase . The nexIn conclusion, DSB formation and repair are precisely regulated to ensure that this step is performed in a coordinated manner during meiotic recombination.S. cerevisiae does not occur at random; it is rather localized into specific regions, called recombination hotspots, where DSBs preferentially form. These short regions are, at the same time, contained within larger regions known as DSB-hot domains—these are opposed to DSB-cold domains, which mainly devoid of DSBs [Nonetheless, the distribution of DSBs in  of DSBs . Among t of DSBs . To corr of DSBs . Of inte of DSBs .As explained above, DSBs occur in DNA. In eukaryote cells, DNA is found wrapped around specialized proteins called histones, making up nucleosomes, the fundamental unit of chromatin . The corAmong others, H3K9ac has been reported in DSB hotspots, and H4K16ac is an important checkpoint for meiotic recombination, which is also facilitated by H4K44ac ,57,58. HFocusing on DSB hotspots, they are characterized by a nucleosome-depleted region (NDR), featuring a noticeable histone mark arrangement directing their activity where H3K9ac is flanking them. After that, H3K4me3 centres on the +1, +2, and +3 nucleosomes. In contrast, H3K4me1/2, H3K36me3, H3K79me2, and H3R2me are found next to the 3’ ends of genes  Figure .S. cerevisiae, despite being a PTM normally linked to several features of the process of transcription [Due to our research interests, we will discuss in the next subsections (i) the trimethylation of histone H3 on the lysine 4 (H3K4me3), which has been the one most correlated with meiotic DSBs in cription ,66; and In humans and higher eukaryotes, H3K4me3 is deposited by a family of histone methyltransferases, known as SET1/MLL. It is made up of six different members: SETD1A, SETD1B, and MLL1, 2, 3 and 4 . These cIn budding yeast, all H3K4 methylation is exclusively mediated by the lysine methyltransferase Set1, by being part of a complex called the Complex of Proteins Associated with Set1 (COMPASS) ,69 or SeThe COMPASS complex consists in yeast of eight subunits, being the catalytic subunit Set1 3–9, Enhancer-of-zeste, and Trithorax), as well as seven additional components: Swd1 (Cps50) and Swd3 (Cps30) , Swd2 (Cps35), Sdc1 (Cps25), Bre2 (Cps60) (required for di- and trimethylation), Spp1 (Cps40) (required solely for trimethylation), and a little subunit, Shg1, of which little information is available ,70.Two different studies published in 2018 shed light into its structure and mechanisms ,74. AccoIn the opposite part of the complex, Bre2 and Sdc1 form a trimeric subcomplex, with one copy of the first and two of the latter. This dimerization of Sdc1 is necessary for the correct stabilization of Bre2. Spp1, Swd2, and Shg1, on the other hand, can be found interacting with the N-terminal SET domain and the RNA-recognition motif (RRM) of Set1 ,76. Sincset1∆ strains display a general reduction in most DSBs, which has been seen to correlate with H3K4me3 levels [The COMPASS plays an important role in recombination initiation during meiosis and DSB generation. Though originally described as a complex involved in transcription, the identification of Spp1 in these events opened up this new function for COMPASS. Spp1 is a subunit of COMPASS that contains a plant homeodomain (PHD) finger and is able to physically interact with H3K4me3/2, as well as with the Mer2 protein (from the RMM subcomplex) . The int3 levels ,83. This3 levels . Moreove3 levels . These rA conserved proof that there is a relationship between H3K4me3 and meiotic-driven DSBs can be found in mammals, where the histone methylase Prdm9 leads DSBs to occur in DNA motifs recognized by its zinc finger domain .Monoubiquitination of histone H2B on its lysine 123  is controlled by a coordinated action of the E2-conjugating enzyme Rad6, the E3 ligase Bre1, and the regulatory cofactor Lge1 ,88. MeanHR6B, has been related to defects in male fertility when it is depleted in mice, due to errors during spermatogenesis. The function of Rad6 is thus thought to be involved with both the synaptonemal complex and recombination in meiosis, highly implying a role in chromatin remodelling [lge1∆ and bre1∆ cells initiate meiotic DNA replication in the S-phase, later than the wild-type, and take much longer to complete it, presenting a reduced DSB formation as well [In addition to its role in gene expression (see later), H2B ubiquitination has also been related to the formation of DSBs . The ubiodelling . Likewis as well ,98.Interestingly, not only does the PAF1c play a crucial role during transcription, but also its component Rtf1 has been described as participating in the meiotic process . Rtf1 isIME1 [Furthermore, Ubp8 from SAGA DUBm is involved in H2B and Cse4 deubiquitination . Cse4 isIME1 .PHO84), leading to the expression of antisense transcription at the 3’ ends of coding regions. In this study, the authors found that this antisense transcription is promoted by H3K4me3, but is not fully dependent on the mark.As noted earlier, H3K4 methylation has been well studied in the context of transcription. H3K4me3 is strongly correlated to transcription activation and active genes, with a prominent peak around the transcription start sites (TSSs) being a feature of its distribution. In addition, this peak has been linked in magnitude to the amount of mRNA for a given gene ,105. It The actual functions for H3K4me in the regulation of transcription, as well as chromatin organization, are not thoroughly known, though it is known that Set1 is mobilized to TSSs by interacting with the RNA polymerase II (RNAPII) when it is phosphorylated at serine 5 of its Rpb1 C-terminal domain (CTD-Ser5P), or via elongation factors ,109,110.The little impact of this modification on gene expression at the genomic level highlights that the role of methylation on transcription is more complicated than anticipated ,112. AltThe role of COMPASS in transcription can be further understood in light of several recent studies, which show that this complex is able to bind to mRNAs both in vitro and in vivo ,115. NotH2Bub has been described to have several roles in gene transcription regulation, since it has been seen to correlate to more RNAPII processivity, being enriched in promoter regions. It has been proposed that, upon H2B monoubiquitination, inter-nucleosomal interactions are disrupted . This isH2B ubiquitination also requires the presence of another complex in yeast: PAF1c. This complex is comprised of five different subunits: Rtf1 , Cdc73, Paf1, Ctr9, and Leo1 . PAF1c irad6∆ cells are devoid of methylation [swd1 deletion completely abolishes any kind of H3K4 methylation by COMPASS [Notably, H3K4me needs H2Bub in order to occur, since hylation  and bothhylation ,123. Thihylation . This mohylation . Howeverhylation . Once ubhylation ,126,127.The main inhibitory region of the COMPASS CM is concentrated on an arginine-rich motif (ARM) that immediately precedes the SET domain of Set1  Figure B. The ARIn summary, these new results help us better understand the molecular mechanism behind the coordination between H2Bub and H3K4me. Further investigations to assess whether this mechanism occurs both in transcription and meiotic recombination and at different genomic positions are required to fully understand the H2Bub/H3K4me connection.Even if it has been stated several times through this work, a well-defined trait characterizing all aforementioned mechanisms is the fact that they are extensively conserved across evolution. Actually, most of the mentioned factors present well-described orthologues in higher eukaryotes that have been involved in numerous disorders, mainly due to the crucial nature of the processes they regulate. A very short summary of some of the diseases associated to mutations in these factors are given below, and are recapitulated in Set1/MLL has been reported to participate in cancer and ageing , being aC. elegans and in yeast [As stated before, Set1/MLL is implicated in lifespan and ageing as well. High levels of H3K4 methylation are undesirable for an extended longevity, where reactive oxygen species (ROS) play a vital role ,148. An in yeast .Furthermore, Set1 is required for proliferation of ESCs, iPSCs, and neuronal stem cells. Besides participating in leukemia, MLL1/MLL2 are indispensable in embryogenesis . A Set1/As described here, recombination hotspots are triggered by PRDM9-mediated H3K4me3. Deficiencies in this gene are associated with defective synapses and male infertility ,141. CXXAmong others, in the case of H2B ubiquitination, Rad6  defects result in male infertility ,134, andAll chromatin-dependent functions are regulated by epigenetic modifications. Among them, histone modifications constitute one of the best-studied regulatory elements that orchestrate specific patterns that will modulate from gene expression to meiosis. The in-depth study of these modifications has provided an extremely interesting view of how different histone modification patterns could lead to the renovation of chromatin structures that allow the signalling required to trigger a specific process. Notably, most of these modifications are executed by multi-subunit complexes that can accomplish different functions depending on their partners and the environment. Our knowledge of how specific histone modifications—for instance, the focus of this review: H3K4me and H2Bub—occur upon a precise signal is far from being complete.Many questions remain unanswered—for instance, (i) how can the histone writers and erasers differentiate between genomic loci? (ii) How can the histone readers discriminate between the distinct chromatin-based processes? (iii) Which is the interconnection between specific histone modifications that regulates different biological processes? (iv) Are the molecular mechanisms behind each modification shared between molecular events? (v) Is there a set of specific factors that regulate the writers and the erasers only during meiotic recombination? An interesting possibility is that the capacity of different subunits to bind to different partners enables them for specific roles. As a matter of fact, the presence of Spp1 in both processes might indicate its implications in both of them and how they are linked, given that it is localized to COMPASS, allows the deposition of the H3K4me3 mark, and when forming a subcomplex with Mer2, is able to read the mark and conduct DSB formation ,81,82. Ijhd2∆ cells show a precocious gametogenesis and stress-sensitive spores [S. pombe the control of a master regulator of cell fate decision, Ste11, depends on different activities of the SAGA complex for the switch from mitotic growth to sexual differentiation [Much effort has been focussed on addressing the role of the histone H3K4me and H2Bub writers ; meanwhile less is known about their erasers . Jhd2 has been better studied, indicating that it is a general regulator, being essential for delaying transcriptional quiescence during sporulation. In fact, e spores ,158. Adde spores ,81. In ce spores ,160,161.e spores , which ie spores . Notablyntiation . Is Ubp8Everything that has been described here brings our attention to the profound relationship between H3K4me and H2Bub, and the need for understanding more deeply how the machineries that write, read, and erase them are able to discern between the different molecular events occurring in chromatin .


As sessile organisms, plants have evolved unique patterns of growth and development, elaborate metabolism and special perception and signaling mechanisms to environmental cues. Likewise, plants have complex and highly special programs for transcriptional control of gene expression. A case study for the special transcription control in plants is the expansion of general transcription factors, particularly the family of Transcription Factor IIB (TFIIB)-like factors with 15 members in Arabidopsis. For more than a decade, molecular and genetic analysis has revealed important functions of these TFIIB-like factors in specific biological processes including gametogenesis, pollen tube growth guidance, embryogenesis, endosperm development, and plant-microbe interactions. The redundant, specialized, and diversified roles of these TFIIB-like factors challenge the traditional definition of general transcription factors established in other eukaryotes. In this review, we discuss general transcription factors in plants with a focus on the expansion and functional analysis of plant TFIIB-like proteins to highlight unique aspects of plant transcription programs that can be highly valuable for understanding the molecular basis of plant growth, development and responses to stress conditions. The set of genes that are expressed defines the cell. Transcription is the most important step in gene expression . In eukacis-acting DNA elements of their target genes and recruiting cofactors and Pol II to the target genes. Transcription cofactors are proteins or protein complexes that contribute to activation (coactivators) and repression (corepressors) but contain no DNA-binding activity [Transcriptional regulation of genes is a vital process that allows the cell or an organism to define its identity during growth, development and response to intra- and extra-cellular signals . Transcractivity . These cactivity ,5. The sactivity ,7. Adenoactivity . Gene-spactivity . These mactivity . Chromatactivity .There are thousands of gene-specific transcription factors in a typical eukaryotic organism that determine the tissue- and cell-specificity of gene expression . By contIn eukaryotes, transcription of protein-coding genes is performed by Pol II, which utilizes up to seven different general transcription factors  for promoter recognition and transcription initiation ,2. TFIIBUnlike gene-specific transcription factors, many of which are encoded by large gene families, the families of general transcription factors are usually very small with 2–4 members . In non-Comprehensive search and analysis of TFIIB-like proteins from different eukaryotic genomes including metazoan, fungi and algae species have confirmed that there are in general three or more TFB-like proteins in each genome . HoweverThe expansion of TFIIB-like proteins in plants is intriguing but not completely surprising given their structural, functional and evolutionary relationship with bacterial σ factors and archaeal transcription factor B (TFB). Multi-subunit DNA-dependent RNA polymerase proteins for gene transcription are conserved in bacteria, archaea, and eukaryotes. Bacterial RNA polymerase holoenzyme consists of the RNA polymerase core enzyme and a σ factor ,12. On bHalobacterium salinarum, a halophilic  member of the Archaea that grows in concentrations of NaCl near or at saturation, there are at least seven TFBs that direct environment-specific gene expression programs [H. salinarum NRC-1 to their fitness landscapes, functional hierarchies, and genetic interactions across 2488 experiments, covering combinatorial variations in salt, pH, temperature, and Cu stress, an elegant scheme was revealed in which completely novel fitness landscapes are generated by the introduction of subtle changes to the regulation or physical interactions of duplicated TFBs through gene conversion events [In programs . By corrn events . These in events . These rEven in eukaryotes where the functions of general transcription factors have been discussed almost exclusively in the context of basal transcription, their possible role in the regulation of physiology may have been under-appreciated. In yeast, ethanol production could be enhanced through the mutagenesis of TFIIB, suggesting that altering the function of a general transcription factor can have significant phenotypic consequences . FurtherAlthough expansion of TFIIB-like factors occurs in all plants, functional analysis of the large number of these transcription factors has been reported only in Arabidopsis. Significantly, a majority of these reported studies have revealed critical roles of Arabidopsis TFIIB-like factors in reproductive processes including male and female gametogenesis pollen tube germination and growth guidance, endosperm development, and embryogenesis .AtTFIIB1 is expressed in many tissues including vegetative nuclei and generative cells of pollen grains and pollen tubes, endosperm, and embryos [AtTFIIB2 expression is not found in the endosperm and vegetative nucleus of mature pollen [AtFTIIB1 have been isolated and analyzed for the biological functions of the TFIIB-like proteins. These mutants were drastically reduced in the genetic transmission of the attfiib1 mutations through male gametes but did not affect female gametophytic function [attfiib1 mutant plants transformed with AtTFIIB2 driven by the AtTFIIB1 promoter were restored in pollen tube growth, guidance, and reception completely, but only partially recovered in the seed development [Arabidopsis AtTFIIB1 and AtTFIIB2 are structurally highly similar with 86% similarity in amino acid sequence and both belong to the TFIIB clade in the phylogenetic tree of TFIIB-like proteins . AtTFIIB embryos . On the e pollen . In totafunction . The mutfunction . The mutfunction . The attelopment . These relopment   encoded by a ubiquitously expressed gene and is the founding member of the BRP protein family . Surpris process . Thus, A process . These r process .Cyanidioschyzon merolae and Arabidopsis [It has been proposed that plant BRP1 is a general transcription factor for Pol I but not for Pol II based on chromatin immunoprecipitation (ChIP) assays of BRP1 from red algae bidopsis . ChIP anbidopsis . By contbidopsis . Furtherbidopsis . The Cmpbidopsis . Likewisbidopsis . CmpBRP1bidopsis . Thus, tbidopsis  , a cell cycle-related gene encoding a subunit of the origin recognition complex [AtBRP4 knockdown plants [ORC6 knockdown transgenic plants also displayed the male gametophyte defect as observed in BRP4 knockdown plants, suggesting that ORC6 acts downstream of AtBRP4 and is required for male gametophyte cell-cycle progression [The protein sequence of Arabidopsis AtBRP4 is highly similar to those of AtPBRP2, AtTFIIB1 and 2, but the N-terminal zinc finger and the linker sequence are significantly longer than the three other TFIIB-like factors . AtBRP4 released . AtBRP4 henotype . On the  pollens . These f complex ,33, was n plants . ORC6 kngression . Taken tgression   due to its expression in developing pollen grains [AtBRP5 is expressed in many tissues in inflorescence, developing pollen grains and embryos [AtBRP5 was expressed primarily in developing pollen grains and the tissues with active cell division and differentiation.n grains . Analysi embryos . Express embryos . In the  embryos . No expr embryos . TherefoAtBRP5 with a T-DNA insertion in the 5′ untranslated region of AtBRP5 has been identified but no homozygous mutant plants could be obtained [atbrp5 mutation was drastically reduced through the male gametophyte and that the mutation only had a slight impact on female gametophytic function [A mutant for obtained . Geneticfunction . Furtherfunction . Pollen-function . AtBRP5 function . AtBRP5 function   is zygotically lethal, indicating an essential role of AtMEE12 in plant growth and development [AtMEE2 did not affect pollen development or formation of female gametophytes but disrupted micropylar pollen tube guidance [AtMEE12 in the central cell alone is sufficient to restore the normal pollen tube guidance phenotype of the mutant [AtMEE12 is a TFIIB-like factor highly similar to yeast Rrn7 and human TAF1B TFIIB-related Pol I general transcription factors. Intriguing, genetic and molecular analysis has indicated that AtMEE12 functions in the Pol II machinery to regulate pollen tube guidance . RT-qPCRnd stems . During lization . AtMEE12etophyte . A Ds trelopment . Analysiguidance . Expresse mutant . These rCBP1 is expressed in seedlings, leaves, inflorescences, and flowers [CBP1 expression either through a Ds element insertion in the 3” untranslated region or expression of artificial microRNA results in defective ovules in pollen tube attraction [Subsequent study has shown that AtMEE12 interacts with a novel plant-specific protein called CCG BINDING PROTEIN1 (CBP1) . CBP1 al flowers . It is a flowers . Knockdotraction . Expresstraction . CRPs artraction ,37,38,39traction   are unlikely to act as TFIIB-like factors due to the lack of the conserved N-terminal zinc finger and the two cyclin fold repeats. Among the remaining 13 TFIIB-like factors, 10 have been characterized by reported studies to various extent through molecular and genetic approaches . GeneticDespite these progresses, our understanding of the expanded family of plant TFIIB-like factors is still very limited. First, even though the expansion of the TFIIB-like proteins occurs in all plants, their functional analysis has so far been restricted to Arabidopsis. It is, therefore, unclear about the structural and functional conservation and diversification of the expanded TFIIB-like factors among different plants, which are diverse in a wide range of traits in growth, development and stress responses. Second, even in Arabidopsis, our knowledge about those functionally analyzed TFIIB-like factors is restricted to their structures, expression patterns and mutant phenotypes. We are still unclear about the molecular basis for the established roles of these TFIIB-like factors. In addition to the three major RNA polymerases, plants contain Pol IV and V involved in siRNA-mediated gene silencing. Some of Arabidopsis TFIIB-like factors have been demonstrated to be associated with specific RNA polymerases, but important questions remain. For example, AtMEE12 has been shown to be a transcription factor associated with Pol II in Arabidopsis despite the fact that it is structurally most closely related to human and yeast Rrn7/TAF1B factors, which act as general transcription factors for Pol I. BRP1 has been shown to be a general transcription factor for Pol I in red algae and Arabidopsis but AtBRP1 also plays a key role in the transcription of protein-coding genes regulated by the VirE3 effector from Agrobacterium. Third, understanding of the biological roles of these TFIIB-like factors will require a better knowledge about the specific transcription program directed by the TFIIB-like factors including their direct target genes. Identification of direct target genes and establishment of plant TFIIB-like factors as special transcription factors of a specific Pol will challenge the paradigm of GTFs and can potentially transform our understanding of transcriptional regulation in plants and other eukaryotes. Establishment of plant TFIIB-like factors as master regulators of important plant traits will be a breakthrough, not only for understanding transcriptional regulation of plant growth and development but also for developing novel strategies of engineering to improve crop plants. Elucidating the regulation of plant TFIIB-like factors will help understand signaling pathways for transcription reprograming in plants.