The Herpes Simplex Virus (HSV-1) immediate-early protein ICP22 interacts with cellular proteins to inhibit host cell gene expression and promote viral gene expression. ICP22 inhibits phosphorylation of Ser2 of the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) and productive elongation of pol II. Here we show that ICP22 affects elongation of pol II through both the early-elongation checkpoint and the poly(A)-associated elongation checkpoint of a protein-coding gene model. Coimmunoprecipitation assays using tagged ICP22 expressed in human cells and pulldown assays with recombinant ICP22 in vitro coupled with mass spectrometry identify transcription elongation factors, including P-TEFb, additional CTD kinases and the FACT complex as interacting cellular factors. Using a photoreactive amino acid incorporated into ICP22, we found that L191, Y230 and C225 crosslink to both subunits of the FACT complex in cells. Our findings indicate that ICP22 interacts with critical elongation regulators to inhibit transcription elongation of cellular genes, which may be vital for HSV-1 pathogenesis. We also show that the HSV viral activator, VP16, has a region of structural similarity to the ICP22 region that interacts with elongation factors, suggesting a model where VP16 competes with ICP22 to deliver elongation factors to viral genes. Suppression of host gene expression during HSV-1 productive infection may not only dampen host immune responses but also increase the cellular resources available for viral gene expression and requires interaction between viral and host factors. Expression of HSV-1 ICP22, one of five immediate early genes, is initiated by the viral tegument transcription activation protein VP16 in the absence of de novo viral protein synthesis ,4. WhileCCR (residues 193–256) is sufficient for interaction with and inhibition of P-TEFb, a cyclin-dependent kinase complex that phosphorylates the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) [Several studies indicate that ICP22, in particular via the second and third domains, physically interacts or colocalises with host nuclear proteins ,15,16,17in (CTD) . The polin (CTD) ,19. The in (CTD) ,21. The in (CTD) . In addiin (CTD) ,25, add in (CTD) ,27,28. Win (CTD) . Pol II CCR has the same effect on elongation of transcription by pol II through checkpoints at the beginning and end of genes as inhibition of P-TEFb by small molecules like DRB. We also show, through a combination of pulldown-mass spectrometry approaches, that ICP22 interacts with several crucial pol II transcription elongation factors. Using amber suppression and UV crosslinking to ectopically-expressed ICP22 in cells, we show that ICP22 interacts directly with the FACT elongation complex.In order to gain a better understanding of the mechanism of action of ICP22, we have explored the interaction of ICP22 with host cell proteins using in vivo and in vitro approaches. Here we confirm that the ICP22Our findings emphasize that ICP22 acts as a selective viral repressor at the level of transcription elongation of human genes.® CRL-1573™) were maintained in DMEM medium supplemented with 10% foetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine at 37 °C and 5% CO2. Cells were transfected with mammalian expression plasmids using jetPRIME® transfection reagent (Polyplus) as per the manufacturer’s recommendations.HEK293 (ATCCCCR was described previously [CCR and His-IpgC (kindly provided by Susan Lea) were generated using restriction cloning into pET28a, and GST-CDK9FL and GST-Cyclin T11-266 were cloned into pGEX-6P-1.The mammalian expression plasmid encoding Myc-ICP22eviously . The mamp-values were calculated by two-tailed unpaired t-test in which p < 0.05 was considered to be statistically significant.ChIP was performed as described previously , using ag for 15 min at 4 °C and the nuclear pellet was lysed in IP buffer . The lysate was sonicated in a chilled water bath and treated with 25 units of Benzonase  for 1 h at 4 °C. After centrifuging for 15 min at 13,000× g at 4 °C, the soluble supernatant was transferred into a fresh tube. Pre-equilibrated anti-Flag M2 beads  were added into the supernatant and incubated overnight at 4 °C on a wheel. The beads were washed three times with IP buffer.CoIP was performed from a 15 cm dish of HEK293 transfected with mammalian expression plasmids. Cells were harvested in ice-cold PBS supplemented with protease inhibitor cocktail. The cell pellet was lysed in lysis buffer . The suspension was centrifuged at 13,000× ® 800CW Goat anti-Mouse IgG (925-32210), IRDye® 680RD Goat anti-Mouse IgG (925-68070), IRDye® 800CW Goat anti-Rabbit IgG (925-32211), IRDye® 680RD Goat anti-Rabbit IgG (925-68071), IRDye® 800CW Donkey anti-Goat (925-32214), IRDye® 680RD Donkey anti-Goat IgG (925-68074). Blots were visualized on an Odyssey CLx Imaging System , and quantification of the protein intensity was performed with Image Studio Lite software.Proteins in Laemmli sample buffer were boiled for 10 min at 95 °C and electrophoresed in 8% or 12% SDS-PAGE gels to resolve proteins ≥20 kDa. Smaller proteins were resolved by SDS-PAGE with Tris Tricine-SDS running buffer . ProteinEscherichia coli RosettaTM (DE3)  by induction overnight with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18 °C. Lysis buffer  was used to purify His-tagged recombinant proteins and lysis buffer  and protease inhibitor cocktail) was used to purify GST-tagged recombinant proteins. The suspension was centrifuged at 14,000× g for 30 min at 4 °C. His-tagged recombinant proteins were retained on nickel magnetic beads , and GST-tagged recombinant proteins were retained on glutathione magnetic beads . On-bead recombinant proteins were washed with the respective lysis buffer. GST-tagged recombinant proteins were eluted with 10 mM reduced glutathione for pulldown assay with His-tagged recombinant proteins. For pulldown assay with HeLa nuclear extracts (Ipracell), the extracts were diluted 1:1 with IP buffer and precleared by incubating with nickel magnetic beads at 4 °C on a wheel for 2 h. On-bead His-tagged recombinant proteins were added to the extract and incubated overnight. Bound proteins were washed three times with IP buffer.Recombinant proteins were expressed in Sup) and aminoacyl tRNA synthetase, and mutated derivatives of pcDNA3.1-Flag-ICP22 in medium supplemented with p-benzoylphenylalanine (Bpa)  at a final concentration of 2 mM. After 48 h, the medium was replaced with 5 mL PBS. Cells that were still attached to the plates were irradiated at 365 nm UV (4 J/cm2) (UVP 95-0007-06 UV lamp) for 45 min on ice. Cell pellets were resuspended in lysis buffer . The suspension was centrifuged at 13,000× g for 15 min at 4 °C and the supernatant was collected. The nuclear pellet was lysed in stringent IP buffer . After this point, the lysates were prepared for coIP using the coIP protocol detailed above. To prepare mass spectrometry-compatible silver-stained gels, the crosslink samples separated by SDS-PAGE gel were fixed in 50% ethanol, 10% acetic acid and 0.01% formaldehyde for 1 h before washing with 5 μg/mL dithiothreitol for 1 h. After being rinsed in water, gels were stained in freshly prepared 0.1% silver nitrate for 1 h and developed in 3% sodium carbonate and 0.01% formaldehyde. The reaction was terminated by the addition of 50% ethanol and 10% acetic acid. Gel bands of the crosslinked products and equal size bands from untransfected and non-irradiated cells were excised and analysed by mass spectrometry.UV-crosslinking followed the previously described protocol with minor changes . HEK293 Mass spectrometry analysis was performed by the proteomic facilities at the University of Oxford or the Queen Mary University of London, essentially as described . ProteinCCR on Ser2P in a time-course experiment  by Western blot. The level of Ser2P decreases at 8 hpt of Myc-ICP22CCR with little effect on the total pol II level, indicating specific loss of CTD phosphorylation. To understand how ICP22 affects the control of transcription elongation on host genes, we carried out pol II and Ser2P pol II CTD chromatin immunoprecipitation (ChIP)-qPCR on a model cellular gene, KPNB1 (CCR as this time point should provide a snapshot of the repressive action of ICP22 on actively-transcribing pol II. The level of pol II (KPNB1 is significantly elevated at the TSS (TSS+0.9) compared to that of control and mock, suggesting increased pol II pausing near the EEC. In addition, the level of pol II is significantly decreased downstream of the TSS (TSS+5.3), suggesting that the newly-initiated pol II is unable to make the transition to productive elongation. The level of pol II in untreated, mock and transfected cells in the gene body  remains unaffected, suggesting that pol II that passed the early elongation checkpoint before or during early accumulation of Myc-ICP22CCR is able to continue elongation of this 36 kb-long gene. The level of pol II is significantly reduced downstream of the poly(A) site , suggesting that Myc-ICP22CCR affects the level of transcribing pol II at the end of KPNB1. ChIP analysis of Ser2P  site at the end of KPNB1 gene, in the same way as the CDK9 inhibitors KM05382 and DRB [We have investigated the ability of the CCR of ICP22 to induce loss of pol II CTD Ser2 phosphorylation, a mark indicative of transcription elongation, by monitoring the effect of ectopic expression of Myc-ICP22periment A. Myc-ICe, KPNB1 B at 7 hpf pol II C; Pol IIof Ser2P C; Ser2P CCR , the FACT complex (SPT16 and SSRP1), the PAF1 complex (PAF1 and CDC73), P-TEFb substrates SPT5 and NELF-E and a component of the P-TEFb inactive complex (MEPCE).To identify any additional interacting proteins, we carried out a pulldown assay using bacterially-expressed recombinant His-ICP22 control . Importayclin T1 . ICP22CCtrometry F. AdditiCoimmunoprecipitation of endogenous protein complexes and the combined approaches of pulldown-mass spectrometry performed in this study demonstrate that ICP22 interacts with the host transcription elongation apparatus. On the basis of these results, we propose that ICP22 acts not only through interaction with and inhibition of CTD kinases but also through association with other elongation factors, including the chromatin-specific transcription elongation FACT complex.CCR, several residues between 193–256 and three residues just outside this region  were replaced by an amber stop codon (TAG) in N-terminal Flag-tagged ICP22 (Sup) and aminoacyl tRNA synthetase specific for both the tRNASup and the unnatural photoreactive amino acid p-benzoylphenylalanine (Bpa). Expression of ICP22Bpa was optimized to produce photo-cross-linkable ICP22 analysed by anti-Flag Western blot. Only truncated ICP22 is produced in the absence of Bpa, whereas full-length ICP22 that migrates with a molecular weight 60–75 kDa is successfully expressed for a range of mutants when Bpa is added to the culture medium  was strongly detected upon UV irradiation of ICP22Bpa L191 and Y230 compared with other mutants and Flag-ICP22WT , UV-irradiated samples were silver-stained, and mass spectrometry analysis was performed from a gel slice containing proteins with a molecular mass of approximately 250 kDa with controls of untransfected cells or samples not subjected to UV-irradiation .Interestingly, both FACT subunits, SPT16 and SSRP1, are specifically detected after crosslinking. Coimmunoprecipitation of ICP22rosslink D. Residund SSRP1 E, indicaCCR may provide insight into how ICP22 inhibits crucial host factors involved in host transcription elongation. However, structural information is lacking at present. Sequence alignment and homology modelling suggest that the ICP22CCR could adopt a helix-turn-helix conformation that closely resembles the concave surface of the VP16 core region . Alternatively, host cell proteins that interact with VP16 may be needed to reverse inhibition of pol II CTD kinases. The core region of VP16 may therefore perform a critical role by competing P-TEFb away from ICP22 to allow delivery to viral genes for activation.Thus, VP16 can overcome the effect of ICP22 on pol II CTD kinases, but VP16The HSV-1 viral genes are arranged in the same way as host-cell pol II-transcribed genes, with TATA box-containing promoters, introns and polyadenylation signals ,39,40. HCCR, we identified CDK9 and other transcriptional kinases, CDK12, CDK11B and CDK13, that interact with and may be inhibited by ICP22. CDK12 is of particular interest as it is another elongation-phase kinase, which has been reported as being sensitive to DRB [A time course of transient expression of ICP22 in HEK293 revealed that ICP22 could cause an identical pattern of inhibition of transcribing pol II to treatment of cells with small molecule CDK9 inhibitors, like DRB [e to DRB ,47. Moste to DRB ,48. Becae to DRB ,47, we pe to DRB ,35, we fe to DRB .The interaction of ICP22 and P-TEFb-associated factors, including the FACT complex, further validates the role of ICP22 in regulating pol II-mediated transcription elongation of host-cell genes. FACT is a histone chaperone that facilitates transcription by disruption of the DNA-histone interface, which then enables the passage of elongating pol II through nucleosomes . This fuCCR may therefore be positioned close to the dimerization domain of SPT16 and SSRP1. It is also possible that ICP22 crosslinks to the C-terminus of histone-free FACT complex, thus impairing its essential role as a histone chaperone. The sequence alignment of Cyclin T1 and N-terminal regions of SPT16 and SSRP1 reveal that residues within these ICP22-interacting proteins are poorly conserved (We show that multiple residues within the core conserved region of ICP22 crosslink to FACT in living cells, which implies that the association is stable, and both FACT complex subunits were associated with ICP22 after purification under stringent conditions. Both the SPT16 and SSRP1 subunits of FACT share general structural features such as a dimerization domain nearby the N-terminal region, a middle domain and a short, highly acidic C-terminal region that are responsible for flexible interaction with histones and nucleosomal DNA (onserved , suggestSurprisingly, our amber suppression-mediated photo-crosslinking experiments did not detect a stable association between ICP22 and P-TEFb. However, this approach does not necessarily reflect the inability of ICP22 to interact with host proteins like P-TEFb, and selection of other residues to modify or alternative photo-crosslinkers such as arylazide- or diazirine-containing amino acids may be necessary to detect this interaction .CORE has potential structural homology to the ICP22CCR and can inhibit phosphorylation of Ser2 pol II CTD, the VP16CORE may be able to compete with ICP22 for P-TEFb to bring it to the viral genes. On the basis of these new and previous findings, we propose a working model where ICP22 represses the activity of the Ser2 pol II CTD kinases P-TEFb and/or CDK12 and interacts with the FACT complex, resulting in promoter-proximal stalling and premature termination of elongating pol II on cellular genes (HSV-1 VP16 is widely studied as a potent transactivator in the early phase of infection. VP16 is a type IIB activator that strongly stimulates initiation, as well as elongation of transcription and phenylalanines within its C-terminal activation domain, which are key for VP16 activity ,60. ICP2ar genes B. VP16 iCollectively, our results suggest that ICP22 selectively inhibits transcription elongation, at least in part by association with Ser2 pol II CTD kinases P-TEFb and/or CDK12 and the FACT elongation complex. Exactly how ICP22 orchestrates distinct interactions with the individual components of transcription elongation complexes leading to downregulation of host gene transcription and activation of viral genes by VP16 at a particular stage of HSV-1 infection in the context of a viral infection remain to be determined. Our work provides a foundation for future studies using live HSV ICP22 mutant that will further validate its role as a selective viral repressor protein.


Nuclear endosperm development is a common mechanism among Angiosperms, including Arabidopsis. During nuclear development, the endosperm nuclei divide rapidly after fertilization without cytokinesis to enter the syncytial phase, which is then followed by the cellularized phase. The endosperm can be divided into three spatial domains with distinct functions: the micropylar, peripheral, and chalazal domains. Previously, we identified two putative small invertase inhibitors, InvINH1 and InvINH2, that are specifically expressed in the micropylar region of the syncytial endosperm. In addition, ectopically expressing InvINH1 in the cellularized endosperm led to a reduction in embryo growth rate. However, it is not clear what are the upstream regulators responsible for the specific expression of InvINHs in the syncytial endosperm.in planta, we further demonstrated that this enhancer region is required for InvINH1 expression in the syncytial endosperm. One of the MADS box genes, AGL62, is a key transcription factor required for syncytial endosperm development. Using promoter-GFP reporter assay, we demonstrated that InvINH1 and InvINH2 are not expressed in agl62 mutant seeds. Collectively, our data supports the role of AGL62 and other type I MADS box genes as the upstream activators of InvINHs expression in the syncytial endosperm.Using protoplast transient expression system, we discovered that a group of type I MADS box transcription factors can form dimers to activate InvINH1 promoter. Promoter deletion assays carried out in the protoplast system revealed the presence of an enhancer region in InvINH1 promoter, which contains several consensus cis-elements for the MADS box proteins. Using promoter deletion assay Our findings revealed several type I MADS box genes that are responsible for activating InvINH1 in the syncytial endosperm, which in turn regulates embryo growth rate during early stage of seed development.The online version contains supplementary material available at 10.1186/s12870-021-03399-3. The seeds of angiosperms are made up of three distinct tissues, the seed coat, the embryo, and the endosperm. The embryo and the endosperm are both products of double fertilization , 2. Besifis mutants such as mea, fis2, fie, and msi1, the endosperm fails to cellularize, which in turn leads to embryo abortion [agl62 mutants, the endosperm enters cellularization prematurely [Several regulators have been reported to control the developmental transition from syncytial endosperm to cellularized endosperm. The syncytial program is negatively regulated by a chromatin repressive complex, the FIS-PRC2 (Polycomb Repressive Complex 2) complex. In abortion –11. The abortion –15. Besiabortion –19, indiabortion . In agl6maturely . Therefo6GG called the CArG-box [Even though regulators specifically expressed in the micropylar or the chalazal endosperm have been reported , 22–24, CArG-box , 29. TheCArG-box , 31. MotCArG-box .fis2 mutant, InvINH1 mRNA level is dramatically up-regulated [Prior work in our lab has identified a putative invertase inhibitor, InvINH1, as a suppressor of embryo growth . InvINH1egulated . Our labegulated . The ovefis2 [Our prior work has shown that invertase inhibitor 1 (InvINH1) is expressed in the syncytial endosperm and is up-regulated in PRC2 mutants such as fis2 . To idenfis2 , 26. MorThe type I MADS-box family is further classified into Mɑ, Mβ, Mγ, and Mδ subfamilies . The majSince InvINH1 expression is higher in the micropylar region and absent in the chalazal region of the syncytial endosperm , we nextin planta that are responsible for the micropylar-preferred expression pattern of InvINH1.Even though some AGL monomers also activated the InvINH1 promoter at low levels  by deleting a 100-200 bp fragment at a time from the 5′ end of the InvINH1 full-length promoter 7G) were identified at locations -5 bp (CAAAAAATG), − 185 bp (CATTAAATG), − 269 bp (GCAAATATTGC), − 333 bp (CAATTTTG), − 374 bp (CTTAAAATG), − 426 bp (CTAAATTTG), and -966 bp (CAATAAAAG). Interestingly, four of these seven sites  are located within the − 453 to -265 bp region, which was renamed as enhancer 1 6GG , 35–37. To determine whether enhancer 1 alone is sufficient to recruit the AGL dimer to activate transcription, we cloned the 189 bp enhancer 1 sequence in front of the 35S minimal promoter. Promoter activity was then analyzed with protoplast transient assays in the presence of the AGL40-AGL90 dimer. The 35S minimal promoter alone displayed roughly 1% of the full-length InvINH1 promoter activity (data not shown). The addition of enhancer 1 led to 3.83 ± 1.39 fold increase in promoter activity over the 35S minimal promoter , there are three promoter deletions  that resulted in an increase in promoter activity  to suppress embryo growth during the syncytial phase. In this study, we discovered that a group of type I MADS-box transcription factors form dimers to activate InvINH1 promoter. Even though type I MADS-box genes are already known to form dimers in yeast two-hybrid system, our study is the first one to demonstrate that the dimers formed by type I MADS-box genes are capable of activating transcription in plant cells. Some of the type I MADS-box genes identified in this study are the known targets of the FIS-PRC2 complex, which is a chromatin remodeling complex required for endosperm cellularization. Collectively, our findings revealed a regulatory pathway that is responsible for activating InvINH1 in the syncytial endosperm, and for down-regulating InvINH1 after endosperm cellularization. This specific expression pattern of InvINH1 is likely responsible for the observed difference in embryo growth rate before and after endosperm cellularization in Arabidopsis.Arabidopsis thaliana plants (ecotype Col-0) and fis2–8 mutant [agl62–1 (SALK_137707) and agl62–2 (SALK_022148) mutants [− 2·s− 1 light intensity. The rest of the plants were grown under long-day condition (16-h light/8-h dark) with 180–200 μmol·m− 2·s− 1 light intensity.Seeds for wild type 8 mutant  were obt2 SALK_0248 mutant mutants . In brieThe constructs for overexpressing AGL (pUC19-d35Stev-AGL) in the protoplast transient expression assay were created by cloning the respective AGL coding region downstream of a double 35S promoter and a translational enhancer from tobacco etch virus in a pUC19 backbone . Since tD1-GUS to pUC-pInvINH1D8-GUS), fragments of the InvINH1 promoter were amplified from pUC-pInvINH1-GUS with the iProof high-fidelity DNA polymerase (Fisher Scientific) using the primers listed in Additional file To construct the full-length InvINH1 promoter-GUS reporter (pUC-pInvINH1-GUS) used in the protoplast transient expression assay, the 1172 bp 5′ flanking region of InvINH1, including the entire 5′ intergenic region and the coding region for the first seven amino acids, was subcloned from pBN-pInvINH1-GFP  into pBID5d1-GUS to pUC-pInvINH1D5d8-GUS), or to delete the CArG sites from the InvINH1 promoter D5 fragment . The internal deletions were generated by amplifying pUC-pInvINH1D5-GUS through inverse PCR with non-overlapping primers followed by ligation. All mutagenesis reactions  were carried out following the manufacturer’s instructions. All the primers used in mutagenesis PCR are listed in Additional file A Q5 mutagenesis kit (New England Biolabs) was used to generate internal deletions in the InvINH1 promoter D5 fragment in 20 bp increments (pUC-pInvINH1D5-GUS and cloned into pUC-35S(− 90)-TEV-GUS between the HindIII and XbaI sites. To construct the enhancer 1 trimer, the enhancer 1 region (wildtype or 𝝙CArG) was first PCR-amplified from the pUC-pInvINH1D5-GUS or pUC-pInvINH1D5𝝙CArG1,2,3,4-GUS as separate HindIII-XbaI, XbaI-BamHI, and BamHI-PstI fragments, then digested and ligated to form the enhancer 1 trimer, which was cloned into pUC-35S(− 90)-TEV-GUS between HindIII and PstI site. The resulting trimers had 6 bp linkers between each enhancer 1 repeat. To include the entire CArG4 sequence, the enhancer 1 region (− 453 to -256 bp) used in these constructs was expanded from -265 bp to -256 bp. The primers used for all PCR reactions are listed in Additional file To clone the enhancer 1 upstream of the 35S minimal promoter, we first generated the construct pUC-35S(− 90)-TEV-GUS. Briefly, a region including the CaMV 35S minimal promoter (− 90 bp) and the TEV translational enhancer was PCR amplified from pUC19-d35Stev-GFP, then cloned into pUC-pInvINH1-GUS between XbaI and BamHI site, replacing the InvINH1 promoter. The enhancer 1 monomer was then PCR amplified from pUC-pInvINH1Constructs used for plant transformation were generated by cloning fragments of the InvINH1 promoter into vector pBN-GFP . The Inv2, 0.1% BSA, and 1 mM β-Mercaptoethanol (14.3 M), then vacuum-infiltrated for 30 min to remove residual air bubbles. The petri dish was then incubated for 2.5 h at 21 °C in the dark. Afterwards, the protoplasts were released by gentle shaking. The protoplast suspension was filtered with a 40-μm nylon cell strainer (Corning). After two washes with ice-cold W5 solution , protoplast density was determined using a hemocytometer, then adjusted to 2 × 105 protoplasts / mL in MMg solution .Arabidopsis mesophyll protoplasts were isolated from the rosette leaves of four to five-week-old Col-0 plants using modified versions of two published protocols , 47. In 5 protoplasts / mL) and 10uL mixed plasmid DNA solution (14.4 μg). The plasmid DNA mixture was made of 0.4 μg pUC19-35S-LUC [2), mixing by gently inverting the tubes four times and incubating at room temperature for 10 min. Reactions were stopped by adding 880 μL W5. The protoplasts were spun down and resuspended in 200 μL W5. Protoplasts were added to 6-well plates coated with 5% calf-serum containing 800 μL W5 solution and incubated at 21 °C in the dark for 19 h. After incubation, protoplasts were pelleted at low speed and frozen in liquid nitrogen. Samples were stored at − 80 °C until further analysis. Two biological replicates were run sequentially at a time. The total number of biological replicates used to calculate each data point was described in figure legends.Each protoplast transfection reaction contained 100 μL protoplast suspension  and incubated in the dark for 1 h at 37 °C. The reaction was stopped with 100 μL 0.2 M Na2CO3 and analyzed on a Synergy™ H1MD plate reader (BioTek) with the following settings: Excitation, 360 nm; Emission, 460 nm; Optics, Top; Gain, 50; Light Source, Xenon Flash; Lamp Energy, High. Each cell lysate was used in two duplicate reactions to act as technical replicates. Two technical replicates were also carried out for the no-cell-lysate control reaction to account for background noise.For the GUS fluorescence assay, 2 μL protoplast lysate was mixed with 25 μL MUG substrate solution  / LUCexperiment, since the value of LUCcontrol was close to zero. For graphing purposes, relative promoter activity was reported as a percentage of the reference promoter activity. The data point used as the reference point was described in figure legends. The average and standard deviation for each data point were calculated using Excel.Promoter activity was calculated using the formula  for 7–10 days, or until the green seedlings were large enough to be transferred to soil. After transplantation, seedlings were kept in the dark overnight before being transferred to standard growing conditions.T1 seeds were sterilized using 70% EtOH and bleach, then suspended in 0.1% low melting-point agarose and plated on 0.5x MS media containing 35 μg/mL kanamycin and 50 μg/mL cefotaxime. Plates were kept at 4 °C in the dark for 3 days, then incubated in a growth chamber  at 22 °C under long-day conditions (16-h light/8-h dark with 80–100 μmol·mGenomic DNA was extracted from leaf tissue using glass bead maceration, according to published protocols . To summDeveloping seeds were dissected from the siliques at globular or early torpedo stage as described previously , 55. BriAdditional file 1.Additional file 2.


GATA2 is a transcription factor that is critical for the generation and survival of hematopoietic stem cells (HSCs). It also plays an important role in the regulation of myeloid differentiation. Accordingly, GATA2 expression is restricted to HSCs and hematopoietic progenitors as well as early erythroid cells and megakaryocytic cells. Here we identified aberrant GATA2 expression in B-cell acute lymphoblastic leukemia (B-ALL) by analyzing transcriptome sequencing data obtained from St. Jude Cloud. Differentially expressed genes upon GATA2 activation showed significantly myeloid-like transcription signature. Further analysis identified several tumor-associated genes as targets of GATA2 activation including BAG3 and EPOR. In addition, the correlation between KMT2A-USP2 fusion and GATA2 activation not only indicates a potential trans-activating mechanism of GATA2 but also suggests that GATA2 is a target of KMT2A-USP2. Furthermore, by integrating whole-genome and transcriptome sequencing data, we showed that GATA2 is also cis activated. A somatic focal deletion located in the GATA2 neighborhood that disrupts the boundaries of topologically associating domains was identified in one B-ALL patient with GATA2 activation. These evidences support the hypothesis that GATA2 could be involved in leukemogenesis of B-ALL and can be transcriptionally activated through multiple mechanisms. The findings of aberrant activation of GATA2 and its molecular function extend our understanding of transcriptional factor dysregulation in B-ALL. BCR-ABL1, BCR-ABL1 like, and KMT2A (MLL) rearranged have a high risk of treatment failure and poor prognosis (Pediatric acute lymphoblastic leukemia (ALL) is the most common malignant disease in children . Althougrognosis –7.DUX4, PAX5, IKZF1, and TCF3, among others  and maintains the HSC pool  predisposition gene  datasets of 629 B-ALL patients obtained from St. Jude Cloud  from the LNCaP cell line was used to calculate the distance between GATA2 peaks and the transcription start site (TSS) of the nearest gene to determine the genomic range for motif analysis. According to the results, the 200 bp sequences centered on the TSS of protein coding genes across the whole genome were analyzed with FIMO  for the GATA2 was calculated with in-house R script.The heterozygous markers and RNA-seq BAM file for SJALL043839_D1 were obtained from previously published data . AllelicThe protein structure of the in-frame KMT2A-USP2 fusion was visualized with ProteinPaint . The topGATA2 and potential target genes was calculated with Pearson correlation. All analysis were performed in R (version 3.5.3).The DEG analysis was performed with DESeq2. GATA family member expression between different lineages of leukemia was compared using Wilcoxon test. The correlation in expression between GATA1, GATA2, and GATA3) in a total of 1,644 pediatric leukemia patients with AML (n = 297), T-ALL (n = 99), and B-ALL  using RNA-seq data available at St. Jude Cloud  and T-ALL , and GATA3 showed the highest level of transcription in T-ALL . On the other hand, transcription levels of all three GATA genes were low in B-ALL. In particular, we found that GATA2 was not transcribed in the majority of B-ALL patients, with FPKM values <1 in 79.4%  cases (median FPKM = 0.297). The lack of GATA2 transcription in B-ALL was in accordance with its role in common lymphoid progenitor cells during normal hemopoiesis (GATA2 was actively transcribed in a subset of B-ALL patients (n = 257 with FPKM > 1).We examined the RNA expression of three hematopoietic GATA transcription factors . Secondly, aberrantly high GATA2 transcription was observed in a subset of B-ALL patients across different subtypes. We next identified the cases with outlier GATA2 transcription defined as GATA2 transcription levels greater than three standard deviations of the mean in each subtype. Aberrant transcription was analyzed within each subtype to minimize the variation across different subtypes. According to this criterion, a total of 13 B-ALL cases with aberrant activation of GATA2 were identified from 7 subtypes (KMT2A rearrangements (MLL), two with ETV, and one each with DUX4 rearrangement (DUX4), low hypodiploid (HYPO), BCR-ABL1 (PH), and BCR-ABL1-like (PHL) (GATA2 transcription was identified for the following four subtypes: TCF3-PBX1 (E2A), MEF2D rearrangements (MEF2D), PAX5 alterations or mutations (PAX5), and ZNF384 rearrangements (ZNF384)  as the GATA2-normal group.We next asked if subtypes , accountke (PHL) . In addi(ZNF384) . To furtGATA2 transcription on B-ALL, we performed differential expression analysis between the GATA2-outlier group and the GATA2-normal group using DESeq2 with B-ALL subtypes as a covariate. Out of 10,505 expressed protein coding genes (FPKM >1 in >30% patients), we identified 1,150 differentially expressed genes (DEGs), including 699 up-regulated and 451 down-regulated, in GATA2-outlier cases  (p < 0.001)  , while n< 0.001)  and none< 0.001) . Consistgulation  from whi process , implyinGATA2 activation that are potentially involved in B-ALL, we combined genome-wide computational prediction of GATA family transcription factor binding motifs and the differential expression analysis described above. We first analyzed GATA2 chromatin immunoprecipitation sequencing (ChIP-seq) profiles of LNCaP cancer cell line published previously . Notably, we found that both patients carried the same KMT2A-USP2 fusion. Further checking confirmed that these patients were the only two carrying this fusion in the cohort analyzed (GATA2 could be trans-activated by KMT2A-USP2. KMT2A-USP2 is a relatively rare type of KMT2A rearrangement and was recently reported as a recurrent fusion in pediatric B-ALL (KMT2A (NM_005933) and intron 2 of USP2 (NM_004205), which resulted in an in-frame fusion consisting of exons 1–21 of KMT2A and exons 3–13 of USP2  data for both the B-ALL cell line Nalm6 and neuroblastoma cell line Kelly  and SJBALL021549_D1 was a 9-year-old child, the expression levels of GATA2 in these two cases were comparable, indicating that trans-activation of GATA2 by KMT2A-USP2 is not influenced by age but rather by the function of this fusion. Different from other common KMT2A rearrangements with breakpoints located in the major breakpoint cluster region (BCR), the KMT2A-USP2 breakpoint appeared to be inside the minor BCR and joined the plant homeodomain (PHD) domain of KMT2A with the conserved ubiquitin carboxyl-terminal hydrolase (UCH) domain of USP2  with  of USP2 . The bioproteins , 44. Howcription , indicatHOX loci  and thatHOX loci , 47, it s fusion . Our resGATA2 by the KMT2A-USP2 fusion, a cis-activating mechanism is also involved in GATA2 dysregulation. ASE and high outlier expression of GATA2 were found in one B-ALL patient with a somatic focal deletion detected downstream of GATA2 (GATA2 in B-ALL. This mechanism is similar to the activation of other oncogenes including TAL1 and LMO2 by disruption of TADs (KMT2A-MLLT3). As the KMT2A rearrangement was an unfavorable prognostic factor . The GATA2 ChIP-seq data used in this study can be accessed from the gene expression omnibus under accession nos. GSM1600544, GSM935347, GSM935589, and GSM935373 for LNCaP, HUVEC, SHSY5Y, and K562 cells, respectively, with the called peaks (in BED format) available upon request. Whole-genome sequencing and RNA-seq data for SJALL043839_D1 are available in St. Jude Cloud GenomePaint (https://genomepaint.stjude.cloud/). The Hi-C data of Nalm6 and Kelly cell lines are also available from St. Jude Cloud GenomePaint.The RNA-seq data for the St. Jude leukemia patients  were downloaded from St. Jude cloud  and Shanghai Key Laboratory of Clinical Molecular Diagnostics for Pediatrics (20dz2260900 to YL).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chromatin organizes nuclear genome in the restricted space of the nucleus and contributes to all nuclear processes which occur in the absence of internal membranes. Chromatin structure is highly dynamic allowing the unconstrained but controlled reprogramming of nuclear processes, including gene expression, in response to internal and external cues. This is particularly important in plants that, as sessile organisms, constantly need to modify their development and growth . Recent progress in decoding the functions of histone H3 lysine 9 di-methylation (H3K9me2) in the model plant Arabidopsis thaliana (Arabidopsis) are discussed by Xu and Jiang. In their review, Xu and Jiang, give an overview of the methyltransferases involved in the methylation of H3K9 and how this modification is properly deposited at its target genomic regions. Current knowledge on the readers and functional outcomes of H3K9 methylation are also highlighted (Xu and Jiang). In an original research article, Demidov et al. shed light on the functions of the phosphorylation of the centromere-specific histone 3 (CENH3), a variant that replaces the canonical histone H3 in centromeric regions. Using a modification-specific antibody, Demidov et al. showed that Arabidopsis CENH3 is phosphorylated at serine 65 (CENH3 pS65) in vivo. CENH3 pS65 may have a role in reproductive development as suggested by its enrichment in floral buds and the defects in reproductive tissues, and plant growth and development, caused by perturbations in this modification. The authors also provide evidence that the kinase Aurora3 may be involved in the phosphorylation of CENH3 S65 .As key structural components of the chromatin, histones are the main target of regulatory complexes and are subjected to an array of posttranslational modifications. One of the best studied histone modifications is methylation, from which histone H3 lysine 9 methylation has been shown to be associated to the silencing of genomic parasites and repetitive sequences in plants and most eukaryotes , the system employs the pH sensor protein SEpHluorinA227D targeted to different cellular membranes (including plasma membrane or inner nuclear membrane) or to specific chromatin loci tagged by Tet or Lac operator (Tet/Lac-O) sequences. This is combined with Tet/Lac-O-targeted fluorescent proteins that allow monitoring chromatin dynamics at these loci. Using the system, the authors show that addition of eATP can lead to reduction of pH at sites of chromatin-bound proteins, which correlates with changes in dynamics of chromatin-bound proteins . In a different article of this Research Topic, the groups of Holger Puchta and Andreas Houben use MS2 or PP7 aptamers, short RNA oligos that can be recognized by RNA-binding proteins fused to a reporter, that are incorporated into sgRNA to amplify the GFP signal for in vivo labeling of plant telomeric sequences using the CRISPR/deactivated Cas9 (dCas9) system. The system improved the detection possibilities and signal/noise ratio of telomeres in transiently transformed Nicotiana benthamiana . Unfortunately, it proved not functional in stably transformed plants, including N. benthamiana, Arabidopsis or Daucus carota (carrot), perhaps, as the authors speculate, due to the interference of stable CRISPR/dCas9 RNP binding to telomeres with plant growth and development . Nevertheless, the use of aptamers within sgRNA is a promising strategy for signal amplification and sensitivity during in vivo imaging of plant chromatin.Grasser has reviewed our current knowledge of one of these complexes, the heterodimeric histone chaperone FACT that is well-conserved among eukaryotic organisms and controls nucleosome assembly/disassembly linked to some of the most important DNA-related processes . In addition, the impact of FACT on plant developmental switches through the regulation of the expression of key developmental genes, such as FLOWERING LOCUS C (FLC), master repressor of flowering, and DELAY OF GERMINATION 1 (DOG1), key repressor of germination, is highlighted. However, how this histone chaperone complex is recruited to the chromatin or what poses specific genes to be more dependent on FACT-mediated regulation are still open questions that will require further investigation (Grasser). The laboratories of José A. Jarrillo and Manuel Piñeiro have contributed to our understanding of the activities of the NuA4 complex in plant chromatin remodeling. In this Research Topic they provide an overview of the Arabidopsis putative NuA4 complex during flowering and also describe the essential role of this complex in other cellular processes  . Furthermore, the complex scenario of the interactions of NuA4 with accessory proteins to form different complex variants in other organisms is profusely covered and used to make an elegant comparison of the situation in Arabidopsis .Protein-protein interactions of nuclear components to form different type of multimeric complexes play a key role in the regulation of chromatin dynamics. Meng et al. reports that in maize, ZmCHB101 is necessary for proper physiological responses and gene expression changes upon nitrate treatment. ZmCHB101 is a close homolog to Arabidopsis SWI3D, one of the subunits of the SWI/SNF remodeling complex . The authors identified two ZmCHB101 target genes involved in nitrate transport, and observed that nucleosome occupancy and selected histone modifications at these loci were affected in ZmCHB101 RNAi lines. Interestingly, the presence of nitrate seems to negatively affect ZmCHB101 binding to these targets, which in turn may facilitate the binding of another nitrate-associated regulator, ZmNLP3.1 . How the occurrence of nitrate in the cell is exactly translated into observed downstream effects is an intriguing question that needs further investigation. The connection of metabolism with epigenetics is reviewed in this Research Topic by Leung and Gaudin. The review discusses current knowledge about how metabolites modulate chromatin-modifying machineries. Specially, it focuses on acetylation and methylation and the key substrates for these modifications, acetyl-coenzyme A and S-adenosylmethionine, which provide most of the current experimental data. However, as the authors emphasize, DNA and histones  have been shown to be subjected to a myriad of different chemical modifications (Leung and Gaudin). Collectively, they may form a crucial link between the metabolic status of the cell, largely dependent on environmental conditions, and the epigenetic information with its output on gene expression. Plants, with their lifestyle and ability to produce vast amounts of secondary metabolites are especially interesting organisms to study these phenomena.Chromatin-based mechanisms are involved in transcriptional regulation of virtually all major developmental and growth processes, including key metabolic pathways. The research article by Moreno-Romero et al.). We envisage that these and future advances in the chromatin and epigenetic field will become essential for understanding the fundaments of chromatin dynamics and, very importantly, to bridge the gap between this knowledge and its implementation for the epigenetic control of plant traits.The diverse scope of themes covered by the articles in this Research Topic finely reflects the dynamic nature of the plant chromatin research field. Some of the most recent advances in this area presented during the 2019 European Workshop on Plant Chromatin (EWPC) have been collected and summarized in a review article in this Research Topic (All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila) species with small genome sizes.Core promoters have a substantial influence on various steps of transcription, including initiation, elongation, termination, polyadenylation, and finally, translation. The characterization of core promoters is crucial for exploring the regulatory code of transcription initiation. However, the current understanding of insect core promoters is focused on those of Diptera  in the migratory locust, In summary, our results provide novel insights into the effects of genome size expansion on distant transcription initiation.The online version contains supplementary material available at 10.1186/s12915-021-01004-5. The core promoter, which is located towards the 5′ region of a gene on the sense strand, is an upstream regulatory region facilitating the transcription initiation of a protein-coding gene. Core promoters contain necessary sequence features to recruit RNA polymerase II to form transcription initiation complexes and initiate the transcription of protein-coding genes . Core prThe precise annotation of core promoters not only is necessary for understanding the cis-regulatory elements controlling protein-coding gene transcription but also is crucial for genome annotation. Despite the rapid generation of genome sequences of diverse insect taxa, the current official insect gene sets are mostly derived from RNA sequencing (RNA-seq) assemblies. Owing to the inherent technological limitations RNA-seq, the RNA-seq read coverage is strongly biases towards the 3′ landscape of the transcriptome, and the 5′ ends of transcript models are generally inaccurate . The knoThe intron position is one of the critical factors in the regulation of transcription initiation . AlthougLocusta migratoria, is a global species representing a model system with remarkable phenotypic plasticity regulated by gene expression  initiator dinucleotides revealed a severe overrepresentation of pyrimidine–purine (PyPu) dinucleotides in the non-1-bp-wide TSCs located in the 5′-UTRs, coding DNA sequences (CDSs), and intronic and intergenic regions but not in those located in the 3′-UTRs . To identify the genic TSCs for which promoter activities are provided by transposable elements (TEs), we searched for TE-containing TSCs that drive the expression of annotated protein-coding genes. We identified a considerable proportion  of reliable TSCs derived from 36 multiple families of TEs that drive the expression of 2190 annotated protein-coding genes. This observation was more prevalent among TSCs located in intergenic regions. Although all three major classes of locust TEs were present , the percentage of genic TSCs whose expression was driven by TEs was not directly proportional to the composition of TEs in the locust genome  of the reliable TSCs to annotated protein-coding genes, and the remaining 52.0% represented potential initiation sites of unannotated nonprotein-coding genes. The comparison of biological replicates of the tissue and organ data confirmed the ability to quantify promoter expression using the oligo-capping method with excellent quantification reproducibility  and fruit flies , respectively. The percentages of PyPu dinucleotide in the locust (85.38% in the genic TSCs and 6.87% in the random sequences) and fruit fly (86.49% in the genic TSCs and 23.75% in the random sequences) core promoters are statistically different  from those in the random sequences. The statistical differences  remain unchanged for the TATA-box elements in both locusts (18.19% in the genic TSCs and 10.45% in the random sequences) and fruit flies (17.61% in the genic TSCs and 10.47% in the random sequences).To identify the well-accepted Like in fruit flies, we observed an increase in AT contents in the 20 to 40 bp regions upstream of the PyPu dinucleotide in locusts; these are typical regions in which TATA-box elements are located . Therefore, both sharp and broad core promoters in locusts can drive the transcription of protein-coding genes with restricted expression.Transcription can be initiated at precise genomic regions or dispersed genomic regions, a distinction referred to as promoter shape. Distinct promoter classes are defined based on the shape of the TSS distribution: sharp core promoters or broad core promoters , 28. Theτ scores , the τ sP < 0.05, Wilcoxon rank-sum test). Furthermore, we assessed the OTSS diversity of genic TSCs using the Shannon index (H), which is a diversity index that takes into account not only the OTSS number but also the evenness of the relative usage of different OTSSs [H values of locusts were significantly higher than those of fruit flies , suggesting increased OTSS diversity of genic TSCs in locusts. To exclude the potential influences of the different TSS profiling methods applied and unequal sequencing depths in the locust and fruit fly data, we performed down-sampling analyses to examine the robustness of the above results. The P value of the Wilcoxon rank-sum test remained significant in the down-sampled data . To describe the mean relationship between TSC expression and OTSS diversity using a partitioning method, we grouped the genic TSCs into 10 bins based on their expression quantile ranges. The binscatter plot shows that the TSC expression in both locusts and fruit flies is significantly positively  correlated with OTSS diversity, suggesting that increases in TSC expression are generally achieved by the activation of transcription initiation from expanding OTSSs  in insects. When the genic TSCs were grouped into three categories based on PSS values, we found that the H values of the nonsharp core promoters in locusts were significantly higher than those in fruit flies . However, a similar observation was not made for the sharp core promoters. Overall, the increased OTSS diversity of genic TSCs indicates a lower precision of transcription initiation of the broad and intermediate core promoters in locusts compared with fruit flies.In both restricted and ubiquitously expressed TSCs, the locusts showed a greater proportion of PSS values towards the left tail Fig. e than frnt OTSSs . In geneGenomic regions flanking TSSs are enriched in functionally important regulatory elements, which show depletion of single-nucleotide polymorphisms (SNPs) due to evolutionary conservation . To deteBased on the number of assigned core promoters, the protein-coding genes in locusts are divided into two categories: single-core-promoter genes and multicore-promoter genes. The multicore-promoter genes with two or more core promoters included 38.90% of the assigned protein-coding genes in locusts. The proportion of multicore-promoter genes in locusts was similar to that in fruit flies (38.71%). Compared with the fly genome, in which 37.78% of the genome consists of intergenic regions, the locust genome is greatly expanded, and a total of 73.67% of the locust genome consists of intergenic regions. The average length of the intergenic sequences in locusts was much longer than that in the fruit fly genome (217.63 kb in locusts and 4.42 kb in fruit flies). Therefore, despite the remarkable discrepancy in the sizes of intergenic regions between the species, the proportions of multicore-promoter genes are similar between locusts and fruit flies.Ds value) was determined by calculating changes in the OTSS distribution between core promoters for each multicore-promoter gene. The distribution of Ds values approximately followed a normal distribution . Thus, a considerable portion of protein-coding genes contain introns located between the coding and 5′-untranslated first exons, considering that the mean length of mRNA leaders is much shorter than 3 kb.The density distribution of the distances from the annotated start codons to the farthest upstream genic core promoters showed a distinctive bimodal log distribution with a valley at 3 kb in locusts  than those in fruit flies  and fruit flies , the mean intron length in MLTI was significantly longer than that in MLOI. The median length of the mRNA leaders in locusts was 133 bp (25% quantile = 70 bp and 75% quantile = 230 bp), which was similar  to that in fruit flies . Significant increases  in the length of mRNA leaders were accompanied by increases in intron numbers in both locusts and fruit flies  than those in fruit flies in both MLOI and MLTI. However, no significant length difference in mRNA leaders between locusts and fruit flies was observed in the mRNA leaders without introns (MLIPs). With respect to the exon-level comparison of mRNA leaders, it was observed only in fruit flies that significant increases in exon length  were accompanied by increases in intron numbers  than those in fruit flies. In addition, a comparison of the standard deviation showed that the exon lengths (mean = 118.5 bp) of MLTI presented less variance than those of either MLIP or MLOI in locusts  and fruit flies 99.16%), indicating the possibility of strong selection constraints against presenting many introns in mRNA leaders. In mRNA leaders, the intron lengths in locusts  were significantly longer  and multicore promoters  genes of locusts exhibited a significantly greater number of distant core promoters than those of fruit flies. Furthermore, the multicore-promoter genes presented a significantly greater number of distant core promoters than the single-core-promoter genes did in both locusts and fruit flies . One distinctive difference between the two types of core promoters in the two species was their transcriptional abundance. As expected, the majority of the adjacent core promoters in fruit flies showed higher transcriptional abundance than the distant core promoters. The transcriptional activities of distant core promoters were significantly weaker than those of adjacent core promoters in fruit flies. However, the scatter plot of TSC expression quantiles indicated that a substantial portion of the distant core promoters in locusts showed high transcriptional activity  between the distances from the annotated start codons to the upstream core promoters and TSC expression (TPM) levels in distant core promoters in locusts. However, no similar positive correlation was observed for either the adjacent core promoters in locusts or the adjacent/distant core promoters in fruit flies , suggesting that both ubiquitous and restricted expression patterns are present in the distant core promoters of locusts and fruit flies. Considering all of these results together, the detection of broad core promoters with restricted expression patterns in locusts reflects only distinct fundamental aspects of gene regulation in locusts and fruit flies, suggesting the lower precision of transcription initiation of locust genic TSCs with restricted expression patterns.To determine whether the overrepresentation of distant broad core promoters in locusts reflects TSC expression specificity, we used Considering TSC expression in relation to the shape dynamics of core promoters, we performed a sliding window analysis using PSS values and TSC transcriptional abundances to determine the relationship between the core promoter shape and TSC expression. By plotting the PSS values and the expression quantiles of each sliding window, we found that the increases in TSC expression were generally accompanied by decreases in PSS values Fig. d. EnhancTribolium castaneum , Bombus terrestris , Helicoverpa armigera , Laodelphax striatellus , Acyrthosiphon pisum , and Aedes aegypti . We also included a chelicerate species, Tetranychus urticae , as it has the smallest genome size among those of the sequenced arthropod species. The same TSC identification and false TSC removal approaches that were used in the locust data were applied in these arthropod data , the genome sizes of which are much smaller than the locust genome  of SE than adjacent core promoters, suggesting a high variation in TFBSs in distant core promoters  of the protein-coding genes in which the mRNA leaders contain introns in locusts is greater than that (24.97%) in fruit flies, despite a better annotation of mRNA leaders in fruit flies. This result implies a greater tolerance of introns in mRNA leaders in locusts. Some specific introns in the mRNA leaders have regulatory impact on promoting the transcription and nuclear export of the corresponding host genes , 48. In Podisma pedestris, has the largest genome size identified to date among insects , which is approximately 170-fold larger than the smallest insect genome studied (0.1 pg), indicating that genome sizes vary greatly among insects [We found that a total of 45.97% of core promoters in locusts are located in the more than 2-kb region upstream of the start codon ATG site. Compared with locusts (~ 6.5 Gb), fruit flies 180 Mb) have a dramatically smaller genome size. Even in the small-sized genome of the fruit fly, a considerable portion (19.41%) of the core promoters are located in the more than 2 kb upstream region of the start codon ATG site, implying a positive correlation between genome size and the distance from the core promoter to the start codon. Because no TSS studies have previously been conducted beyond Diptera, the current catalog of insect core promoters includes only data from this order 0 Mb have. The les insects . These rThe increased OTSS diversity within core promoters suggests a less precise transcription initiation of the broad and intermediate core promoters in locusts than in fruit flies. A large majority of the human genome is pervasively transcribed, and protein-coding genes account for only a small proportion of the total transcriptional output . ConversCis-regulatory elements are composed of motifs that bind to TFs that determine regulatory activity. Thus, the gains and sequence divergence of TFBSs have the potential to modify the regulatory activity of cis-regulatory elements. These results imply that as the genome size has expanded, more sophisticated regulatory mechanisms have appeared to drive distant transcription initiation. Taken together, all these results suggest that distant transcription initiation in locusts is not the byproduct of transcriptional noise of lowly expressed long noncoding RNAs in intergenic regions.In the comparison of core promoters across multiple species, the significant positive correlation between the genome size and the number ratio of distant core promoters to adjacent core promoters suggests that the genome size expansion results in the emergence of distant core promoters to initiate distant transcription. However, based on the comparison between locusts and fruit flies, the mRNA leader lengths in the large-sized genome were significantly shorter than those in the small-sized genome in genes having at least one intron in their mRNA leaders. These results suggest that in the large-sized genome, the emergence of distant core promoters was accompanied by increases in the intron size located in mRNA leaders. A large portion of the intergenic region, which is accessible to the transcription machinery, can initiate transcriptional noise at inappropriate positions within intergenic regions . TherefoDrosophila taxonomic representation and revealing distinct genomic features to deepen the understanding of transcription initiation in insects. After conservative computational correction steps, we identified a total of 38,136 reliable TSCs, including 18,305 genic TSCs, in the locust genome. The availability of the locust TSC atlas offers an unprecedented opportunity for the comparative analysis of insect core promoters. The comparison of locust and fruit fly data showed a number of distinct features: the nucleotide composition of the PyPu dinucleotide; the strength and motifs of TATA-box signals; the distribution of the CpG o/e content; the effects of promoter shape on TSC expression specificity, transcription initiation accuracy; and SNP density patterns around core-promoter regions. Furthermore, we revealed a distinctive bimodal log distribution of the distance from the annotated start codons to the core promoters of locust protein-coding genes and defined the adjacent and distant core promoters using a threshold of 3 kb. We found stricter constraints on the exon length of mRNA leaders and widespread higher TSC expression activities of the distant core promoters in locusts compared with fruit flies, implying an important role of distant transcription initiation in locusts. We further compared core promoters in the seven arthropod species across a broad range of genome sizes to reinforce our results on the emergence of distant core promoters in large-sized genomes, and we also revealed the changes in abundance and divergence of TFBSs of distant transcription initiation in the context of genome size.In this study, we generated high-resolution transcription initiation datasets to define a comprehensive atlas of TSCs in locusts, contributing to the expansion of non-Locusta migratoria) were reared in large, well-ventilated cages (40 cm × 40 cm × 40 cm) at a density of 500–1000 insects per container. These locust colonies were reared under a 14:10 light/dark photoperiod at 30 °C and were fed fresh wheat seedlings and wheat bran.The migratory locusts  according to the manufacturer’s instructions. Genomic DNA was removed using TURBO DNase (Invitrogen), and poly-A RNA was enriched twice using a Dynabeads Oligo (dT) 25 kit (Thermo Fisher Scientific). A total of 500 ng of poly-A RNA was treated with calf intestinal alkaline phosphatase (CIP) at 37 °C for 1 h. The reaction mixture was purified using 2.2X Agincourt RNAClean XP beads (Beckman Coulter). The resulting CIP-treated RNA was treated with Tobacco Acid Pyrophosphatase (TAP) at 37 °C for 1 h and purified with 2.2X Agincourt RNAclean XP beads. A 5′ RNA adapter (5′-AGGCACGGGCUAUGAG-3′) was ligated to the CIP/TAP-treated RNA using T4 RNA ligase at 25 °C for 4 h. First-strand cDNA synthesis was carried out using SuperScript IV Reverse Transcriptase (Invitrogen) with 3′ specific random hexamer primers (5′-GATGGAGCGTGTTAGCGNNNNNN-3′). The cDNA was purified with double size selection using 0.6× followed by 0.9× of AMPure XP beads (Beckman Coulter) and was amplified by PCR via Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) in conjunction with 5A (5′-AGGCACGGGCTATGAG-3′) and 3A (5′-GATGGAGCGTGTTAGCG-3′) primers. Finally, sequencing libraries were constructed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following the manufacturer’s instructions. A total of 14 oligo-capping libraries were generated and sequenced using the Illumina NovaSeq 6000 System. To generate the oligo-capping data for the seven arthropod species, total RNA was extracted from whole bodies and used for library construction using the protocols as described above.The raw sequencing reads were preprocessed to remove Illumina adapter sequences and low-quality reads using the Trimmomatic program version 0.36 . The detq-value of less than 1e−10) of the TGAG motif and their 1-bp-substitution variants were filtered for further analysis. The significant enrichment motifs were identified using the locust data. The false TSCs derived from internal truncated sites were also filtered for further analyses. The proximal and distal core promoters were defined by comparing two core promoters based on the distances to the annotated start codon, respectively. The core promoter located closer to the downstream start codon of the linked protein-coding gene is considered proximal, whereas the other is considered distal. The adjacent (less than 3 kb) and distant (greater than 3 kb) core promoters were defined based on the distances to the annotated start codon using a threshold of 3 kb, respectively. In intron-level comparison, we did not adopt the rebuilt transcript models that were assembled by the oligo-capping paired-end sequencing reads and the official gene set due to the potential assembly errors in isoform reconstruction [Although the TSC is not the same as the promoter, these two terms are used interchangeably in TSS studies because of the strong association between TSCs and promoters , 35. Thutruction . For exptruction . RepeatMtruction .τ , which is the best metric to measure tissue specificity [As a measure of tissue specificity of TSC expression, cificity , was calτ vary from 0 to 1, where 0 < τ < 0.1 indicates ubiquitous expression and 0.9 < τ < 1 indicates restricted expression.The values of The biological process enrichment was determined using the clusterProfiler and GO.db packages of the R version 3.5.0 program . The resThe promoter shape was determined using the promoter shape score (PSS), which quantifies the promoter shape based on the promoter width and distribution of oligo-capping reads within a promoter . The PSSpi is the probability of observing an OTSS at base position i within a promoter. L is the set of base positions that have the normalized TSS expression at a threshold of 3 TPM, and w is the promoter width, which is defined as the interval distance between the 10th and 90th quantiles. The PSS values are positively correlated to the width of core promoters, and a PSS value of 0 indicates that all transcription of a core promoter is initiated in a narrow genomic region. The core promoters can be classified into three categories: sharp (PSS > − 10) core promoters, intermediate (PSS ≤ − 10 and PSS > − 20) core promoters, and broad (PSS ≤ − 20) core promoters [where romoters .Ds value), which calculates changes in the OTSS distribution between core promoters for each multicore-promoter gene. The Ds value was defined by the following equation:The promoter shifting was assessed using the degree of shift  of the proximal and distal core promoters in the tested tissue and Pc and Dc are the expression abundance values of the proximal and distal core promoters in the control. A Ds value = 0 indicates the absence of promoter shifting. A Ds value > 1 and a Ds value < −1 indicate promoter shifting towards the proximal and distal core promoters, respectively. To account for multiple comparisons, the raw P values from the chi-squared tests were adjusted using the Benjamini–Hochberg method to control the false discovery rate (FDR).where The resequencing data of locusts and fruit flies were retrieved from the NCBI Sequence Read Archive (SRA) database under BioProject accessions: PRJNA256231 and PRJNA433455 , 38. TheH) of OTSS diversity for a genic TSC was defined by the following equation:The Shannon index  of multiple sequence alignment was defined to evaluate the TFBS divergence by the following equation:Pi is the fraction of nucleotide bases of nucleotide base type i, M is the number of nucleotide base types, and L is the length of the identified TFBS.HOMER version 4.9 was used to perform a de novo motif analysis using the findMotifs.pl tool . Positio < 1e− 5 . In TFBSAdditional file 1: Figure S1. Nucleotide composition of OTSSs and nucleotide distribution of sequencing reads. Figure S2. Distribution of identified OTSSs in different genomic regions. Figure S3. Correlation of the number of tissues involved and the identified OTSSs. Figure S4. Distribution of the distance between the identified OTSSs and start codon. Figure S5. Width distribution of transcription start site clusters (TSCs) in different genomic regions. Figure S6. Consensus 25-bp sequences surrounding the dominant TSSs in different genomic regions. Figure S7. A significant enrichment of the TGAG motif and its 1-bp-substitution variants in the 1-bp-wide TSCs. Figure S8. Mis-hybridization of the 5′ oligo-capping adaptors and internal RNA sites results in overrepresentation of the TGAG motif. Figure S9. False TSCs derived from internal signals in the possible truncated mRNAs. Figure S10. Density histogram of the 3′ end of insert fragments along the mRNA transcript with lognormal fit. Figure S11. Percentage of removed TSCs by the 3′ end distribution of insert fragments. Figure S12. Quantification reproducibility for individual TSCs for two biological replicates. Figure S13. Number of identified TSCs per annotated protein-coding gene in the migratory locust and fruit fly. Figure S14. Summary of Drosophila core promoter elements in the core promoters of locusts and fruit flies. Figure S15. CpG distribution in the 4-kb flanking region of transcription start sites. Figure S16. Normalized CpG contents of locusts and fruit flies. Figure S17. Mean AT contents in the 10 to 50 bp regions upstream of dominant OTSSs of core promoters in locusts and fruit flies. Figure S18. Distribution of the tissue-specificity index (tau) of genic TSCs in locusts. Figure S19. Scatterplot of enriched GO terms of ubiquitously (Tau = 0) and restricted (tau = 1) TSC expression of locust core promoters. Figure S20. Correlation between TSC expression and OTSS diversity via binscatter estimation. Figure S21. The alternative usage of core promoters (promoter shifting) in the ovary sample when compared to the muscle sample as a control. Figure S22. The alternative usage of core promoters (promoter shifting) of protein-coding genes in different tissue or organ samples when compared to the muscle samples as controls in locusts. Figure S23. Distant transcription initiation in locusts and fruit flies. Figure S4. Mean intron length in mRNA leaders of locusts and fruit flies. Figure S25. Consensus sequences of the 25 bps surrounding the dominant TSSs. Figure S26. Meta-profile of TSCs over the gene body of protein-coding genes in the official gene sets. Figure S27. The density distribution of distances from the annotated start codon of protein-coding genes to the upstream genic core promoters. Figure S28. The abundance distribution of the distances from the TFBSs to the dominant transcription starting site (TSS) in protein-coding genes. Table S1. Sequencing data generated in this study for locusts. Table S2. Overrepresented motifs of TGAG and its variants in the 1-bp-wide TSCs located in the intergenic region. Table S3. Over-represented motifs of TGAG and its variants in the non-1-bp-wide TSCs located in the intergenic region. Table S4. Overrepresented motifs of TGAG and its variants in the 1-bp-wide TSCs located in the intronic region. Table S5. Over-represented motifs of TGAG and its variants in the non-1-bp-wide TSCs located in the intronic region. Table S6. Over-represented motifs of TGAG and its variants in the 1-bp-wide TSCs located in the coding sequence (CDS) region. Table S7. Overrepresented motifs of TGAG and its variants in the non-1-bp-wide TSCs located in the coding sequence (CDS) region. Table S8. Consensus sequences of Drosophila core promoter elements. Table S9. Sequencing data generated in this study for the arthropod species. Table S10. The identified TSCs in the arthropod species.