Arabidopsis. In terms of exogenous stress conditions, GCN5 is also involved in the responses to heat stress, cold stress, and nutrient element deficiency by regulating the related gene expression to maintain the homeostasis of some key metabolites  or ions ; in addition, GCN5 is involved in the phytohormone pathways such as ethylene, auxin, and salicylic acid to play various roles during the plant lifecycle. Some of the pathways involved by GCN5 also interwind to regulate specific physiological processes or developmental stages. Here, interactions between various developmental events and stress-resistant pathways mediated by GCN5 are comprehensively addressed and the underlying mechanisms are discussed in the plant. Studies with some interacting factors such as ADA2b provided valuable information for the complicated histone acetylation mechanisms. We also suggest the future focuses for GCN5 functions and mechanisms such as functions in seed development/germination stages, exploration of novel interaction factors, identification of more protein substrates, and application of advanced biotechnology-CRISPR in crop genetic improvement, which would be helpful for the complete illumination of roles and mechanisms of GCN5.Histone modifications are the main epigenetic mechanisms that regulate gene expression, chromatin structure, and plant development, among which histone acetylation is one of the most important and studied epigenetic modifications. Histone acetylation is believed to enhance DNA access and promote transcription. GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5), a well-known enzymatic protein responsible for the lysine acetylation of histone H3 and H4, is a universal and crucial histone acetyltransferase involved in gene transcription and plant development. Many studies have found that GCN5 plays important roles in the different development stages of  Epigenetics play important roles in eukaryotes development, including reversible modifications on DNA and chromatin, post-transcriptional and post-translational embellishment, amongst which histone modifications, DNA and RNA methylations, and the regulation of non-coding RNA are the three pillars in epigenetic studies. At present, the known main modifications of histone include acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ribosylation, etc. , which pGCN5 can lead to increasing or reducing a large number of genes expression . Fr. FrGCN5 GCN5 is very important for the maintenance of the root and shoot stem cell niche and meristem functioning upstream of some key potency factors   , howeverArabidopsis trichomes are unicellular non-glandular trichomes with one stalk and two to four branches developed from epidermal cells . En. EnArabind ADA2b . So, it branches , indicatECERIFERUM3 (CER3), a key candidate gene of stem cuticular wax synthesis, is a target gene of GCN5. Compared with the wild type, the acetylation level of H3K9/K14 in the CER3 promoter region of the gcn5-2 mutant is significantly reduced, which results in the downregulation of its transcript. Additionally, the over-expression of CER3 restored the biosynthesis defect of the cuticular wax of the stem caused by the GCN5 mutation [CER3 transcription is regulated directly by GCN5 through histone acetylation, which is partially involved in the cuticular wax synthesis  biosynthesis and derivatization pathways . ECERIFEmutation . These synthesis . HoweverArabidopsis from juvenile to adult processes, and from vegetative growth to reproductive growth with multifarious downstream factors  has not been reported.In summary, GCN5 plays important roles in the development of  factors . As an ed leaves ,32, whicclv1-1 gcn5-1 double mutant exhibits constitutive ethylene reaction indicating that CLAVATA and GCN5 show an effect in ethylene signaling synergistically and negatively [gcn5-1 mutant, it showed the reduced H3 acetylation in promoters of ERF1 and EBF2, two direct targets of EIN3/EIL1, which was restored to similar to or even higher level in the double mutant clv1-1 gcn5-1 than those in the wild type. Further, the expression levels of ERF1 and EBF2 were slightly increased after ethylene treatment in clv1-1 gcn5-1 [ERF1 and EBF2 loci, and histone acetylation associated with GCN5 is the partial upstream activator of ERF1 and EBF2. Intensive research indicated that the restored H3 acetylation in clv1-1 gcn5-1 could result from the activity of other HATs recruited at the ERF1 and EBF2 loci. More genetic studies showed that CLV/GCN5 requires EIN3 activity to modulate ethylene-induced gene expression, but they do not regulate EIN3 expression. ChIP experiments revealed that some ethylene pathway genes such as ERS1, ERF1, EBF2, and CTR1 are directly regulated in transcription expression by GCN5 through histone acetylation [Ethylene is a type of gaseous plant hormone involved in many developmental processes  and responses to environmental stresses including, biological stress, and abiotic stress tolerance . Both GCgatively  This int1 gcn5-1 ,45,46. Stylation ,45,46. Agcn5 and ada2b mutants exhibit aberrant auxin-related growth phenotypes [gcn5-1, the expression of IAA3, a key negative regulator of auxin signaling is decreased compared with that in the wild-type and clv1-1 mutant plants. Genetic analysis of the clv1-1 gcn5-1 showed that the clv1-1 mutant enhances the gcn5-1 effect on IAA3 expression. In addition, 1-aminocyclopropane-1-carboxylate (ACC) treatment weakened the DR5: GFP signal accumulated in the dark-grown seedlings in all genotypes tested [Furthermore, both enotypes ,29. In gs tested , which igcn5 mutants to the bacterial infection [NahG  [SID mutation  [gcn5 mutant, respectively, eliminating the enhanced resistance to pathogen infection in gcn5 mutant compared to the WT [MYC2, DEFENSE NO DEATH2 (DND2), and WRKY33, involved in the control of SA accumulation are down-regulated in expression and show reduced H3K14Ac levels at their loci in gcn5 mutants [Although GCN5 functions in the enhanced acetylation level of H3K14 in the promoter region of its target gene, whether GCN5 binding is systematically associated with gene activation at a genomic level is not clear. A recent study reported that both GCN5 binding and H3K14Ac level are positively correlated with gene expression through several global methodologies . Further GO enrichment analysis revealed that up-regulated genes by GCN5 are enriched in salicylic acid (SA) reaction-related categories, suggesting that GCN5 may be an SA-mediated immune regulator. The phenotypic analysis evidenced the hyposensitivity of nfection . Additioades SA) , and a Snthesis)  were into the WT . Moreove mutants . These rgcn5 mutant shows a decrease in cellulose content and more severe growth inhibition of root compared with the wild type under herbicide isoxaben treatment. Similar to the ctl1 mutant, gcn5 shows an increase in lignin deposition, cell shape blurring, epidermal cell swelling, and ectopic lignification [CTL1 and regulate the levels of H3K9Ac and H3K14Ac to control the gene transcription  and monocots  and dependent on its histone acetyltransferase activity, which provides evidence that GCN5 may take part in the plant architecture regulation.As sessile organisms, plant cells are surrounded by a cellulose-rich wall that is essential for plant morphogenesis and responses to certain external stimuli. Plants have evolved highly complex signaling pathways to participate in cell wall formation, maintenance, and plasticity ,53,54,55fication . ChIP-qPcription C; constiArabidopsis, the enzymes of the integral membrane fatty acid desaturase (FAD) family include FAD2, FAD3, FAD6, FAD7, and FAD8, of which both FAD2 and FAD3 are localized to the endoplasmic reticulum (ER) and responsible for desaturation [Arabidopsis results in an increase of ALA (C18:3) levels from 19% to approximately 40% of the total seed FAs and a corresponding decrease of LA (C18:2) content in the seeds [FAD3, LACS2, LPP3, and PLAIIIβ have been identified as GCN5 targets by RNA-Seq and ChIP detection. Accordingly, the ALA/LA ratio in gcn5 mutants can be restored to the wild-type level by overexpression of FAD3 [FAD3 in Arabidopsis, indicating that GCN5 regulates FA biosynthesis by affecting FAD3 acetylation level and transcription  ester molecules that accumulate fatty acids (FAs) as the most abundant form of reduced carbon chains ,59. The turation . The ovehe seeds . The mut of FAD3 . It is wcription D. These Arabidopsis, which provides some messages for the mechanisms that GCN5 participates in hormone signaling and secondary metabolisms in other plant species.In sum, GCN5 is involved in many kinds of hormone pathways and secondary metabolism processes. In these pathways, GCN5 generally acts on downstream target genes involved in metabolic pathways to modify the corresponding histone acetylation level positively or negatively. In addition, the interaction between GCN5 and CLV affects not only the growth of inflorescence meristem and stamens but also participates in the regulation of the ethylene pathway in gcn5 compared with the wild type under thermal stress [HSFA3 and UVH6, in turn promoting the acetylation of H3K9/K14 and the activation of HSFA3 and UVH6 under thermal response, indicating that GCN5 plays a key role in maintaining the heat resistance of Arabidopsis by regulating key transcription factors  protein is a key transcriptional activator that binds to the DRE/CRT regulatory element and induces COR (cold-regulated) gene expression to increase plant freezing tolerance [CBFs are up-regulated in both gcn5 and ada2b mutants similar to that in wild-type, but the transcription of COR genes is reduced in both mutants [ada2b-1 (but not gcn5-1) plants show more resistant to freezing than wild-type plants [CBF or COR genes directly or indirectly. Therefore, ADA2b and GCN5 may participate in different pathways to regulate diverse plants’ stress tolerance caused by low or freezing temperatures. These results suggest that it would be very valuable to explore the underlying mechanisms involved by different histone acetylation complexes in response to the low temperature of varying degrees.Low temperature causes great harm to the growth and development of plants; therefore, it is necessary to explore the underlying mechanisms of plants in response to low temperature, especially to uncover the key genes in the process of adaptation to cold stress ,72. GCN5olerance . In resp mutants . Some re mutants . Thus, te plants , indicatGCN5 is increased under salt stress; gcn5 mutant shows shortened root length, severe growth inhibition, and cell wall integrity defects compared with the wild type in response to salt stress. Combined with RNA-seq and ChIP analysis, it was found that GCN5-mediated H3K9 and H3K14 acetylation is associated with CTL1, POLYGALACTURONASE INVOLVED IN EXPANSION3 (PGX3), and MYB54 activation under salt stress, and the cellulose gene CTL1 as a direct target of GCN5 plays important roles in cell wall integrity and salt tolerance [Excessive soluble salt in the soil is harmful to the growth and development of plants by producing toxic ions, osmotic stress, and oxidative stress on plant cells ,75. Cellolerance   controlling iron homeostasis are the direct targets of GCN5 [FRD3 mediated by GCN5 determines the dynamic expression of FRD3. Moreover, overexpressing FRD3 saved the iron reserve defect of gcn5 and partially restored plant fertility. These results show that GCN5 plays a key role in FRD3 mediating iron homeostasis to involve plant development.Iron is an important micronutrient and affects root morphogenesis, photosynthesis, nitrogen fixation, respiration, flower color, and fertility, whose homeostasis is essential for plant development ,78. On t of GCN5   AT4 is a direct target of GCN5, and GCN5-mediating acetylation of H3K9/K14 determines the dynamic expression of AT4. In the absence of phosphorus, Arabidopsis gcn5 mutants display attenuated phosphate accumulation between roots and shoots, while the constitutive expression of AT4 in gcn5 mutants can partially restore phosphate redistribution. Further studies evidenced that miR399 and its target PHOSPHATE 2 (PHO2) mRNA level are influenced in the regulation of AT4 by GCN5 [Phosphorus is an essential macronutrient in plant metabolism and plays several important roles in plant growth and development such as energy metabolism, synthesis of nucleic acid, cell membrane formation, photosynthesis, and respiration. Phosphorus deficiency is a factor limiting plant growth and productivity in 40% of the world’s arable soils . Therefo by GCN5 . In summdicer like1 (dcl1), hyponastic leaves1 (hyl1), serrate (se), and argonaute1 (ago1), produce pleiotropic developmental abnormalities [gcn5-2 are elevated. Some miRNAs, such as miR159, miR172, and miR399 are increased more significantly than others  suggesting the preferable role of GCN5 on specific miRNA types, but the underlying mechanism is unclear. The increase of most miRNAs measured is consistent with the corresponding decrease of pri-miRNAs, revealing that the maturation process of these miRNAs may be enhanced in gcn5 mutants. The negative function of GCN5 in miRNA generation may be realized by indirectly inhibiting miRNA mechanism genes, such as DCL1, HYL1, and AGO1 [Arabidopsis GCN5 interferes with miRNA pathways at both transcriptional and post-transcriptional levels, and histone acetylation is an epigenetic mechanism involved in regulating miRNA production. However, the underlying mechanisms of GCN5 interacting with other epigenetic modifications  in miRNA generation is not clear up to now.MiRNAs, encoded by specific genes in the genome, are transcribed as primary transcripts called primary miRNAs (pri-miRNAs). Many miRNAs regulate plant development by delimiting the accumulation of target transcripts that function in development. Mutants with miRNAs accumulation deficiency, such as malities ,86,87,88and AGO1 . ChIP anArabidopsis, the systematic analysis of Arabidopsis promoters (SAP) repertoire was constructed, bringing a total set of 22, 260 in silico amplicons (http://www.psb.ugent.be/SAP/ (accessed on 24 February 2021). Through ChIP-chip assay, an antibody against the GCN5 was used to detect the associated chromatin fragments in wild-type and gcn5 mutants lacking the bromodomain, respectively. The results showed that 40% of the tested promoters were associated with GCN5 and most of which did not require the effective GCN5 bromodomain to bind. However, the bromodomain was necessary for binding to 11% of the promoter regions and related with acetylation of H3K14 in these promoters. Integrated analysis of ChIP-chip and transcriptome implied that binding of GCN5 does not show a strict correlation with gene transcription activation [gcn5-1 and gcn5-2 is decreased significantly at 5′ terminals but increased at 3′ terminals suggesting that GCN5 reversely controls the deposition of the marker at the 5′ and 3′ ends of the gene, which proved that GCN5 has the dual and opposite functions on regulating H3K14Ac level at the 5′ and 3′ ends of its target gene. Additionally, the genome-wide level changes of H3K14Ac in the gcn5-1 mutant are also related to the changes of H3K9Ac at the 5′ and 3′ ends [Given the general effects of GCN5 in plant development, it is worth investigating the genome-wide binding sites and gene expression profiles mediated by GCN5. In tivation ,15,48. EApart from acetylation, eight types of Lysine acylations have recently been identified on histones including propionylation, butyrylation, 2-hydroxyisobutyrylation, succinylation, malonylation, glutarylation, crotonylation, and β-hydroxybutyrylation which affect gene expression and are structurally and functionally different from the histone Lys acetylation . A strucCAB2, RBCS1A, and IAA3 to control the photomorphogenesis; the key factor HY5 play some bridge role in this network, which indicates the key roles of histone acetylation and some hints for the photomorphogenesis in the terrestrial plant, whereas the delicate underlying mechanisms are unclear.GCN5 has been intensely studied to prove that it is a general histone acetylation modifier involved in diverse eukaryote development and response to adverse conditions. But, even so, the emerging evidence is exploring the much more novel functions and mechanisms of GCN5. This review summarizes the updated roles and underlying mechanisms of GCN5 in the development of floral organs, epidermis, organogenesis, and resistance to stresses as well as some hormonal and secondary metabolism pathways in the plant . More inGCN5 transcripts have been reported in humans as well as Brachypodium distachyon  [cis-elements facilitate the locus transcription and gene activation, however, which one is the leading activator is unknown to the specific gene regulation. The application of some advanced technologies such as high-resolution Electron Cryomicroscopy and 3D image capture would be beneficial to explore the bona fide machinery of SAGA complex associated with GCN5 and underlying mechanism. HATs and HDACs are responsible for two types of reaction to balance the histone acetylation, respectively, however, whether the direct interaction between these two kinds of enzymatic complexes occurs in the cell is unclear, and associated research needs to be reinforced. These studies indicate that the investigation of other unknown interacting factors of GCN5 would contribute to the understanding of the GCN5 network in cellular and molecular levels of eukaryotes development.Additionally, alternative splicing in ariants) , in whicariants) . Thus, pariants) . All theArabidopsis, a study revealed that HDA14 is an organellar-localized HDA1 class protein in the chloroplasts and can target and regulate RuBisCO activity through the deacetylation at Lysine 438 of RuBisCO [gcn5 and acetylome analysis, this issue could be partially explained.To date, proteome and acetylome analyses have explored massive non-histone proteins acetylation in mammalian cells. Acetylation on non-histone proteins affects protein functions through regulating protein stability, enzymatic activity, subcellular localization, and crosstalk with other post-translational modifications  In Arabi RuBisCO . These rHAT mechanism increased the promoter activity of the interest gene. Therefore, dCAS9 and HAT fusion (dcas9HAT) combined with directed targeted sgRNAs seems to be the potential to actively regulate the activity of targeted promoters and genes [In recent years, CRISPR-Cas nuclease-directed plant genome editing has become a promising method to change gene function as required and create new plant varieties. Taking advantage of this system and the characteristic of GCN5 activating gene expression by chromatin modification, a CRISPR activation (CRISPRa) system, expressing chimeric dCas9HAT machinery was established, and the associated stable transgenic plants were first generated. The results showed that CRISPRa dCas9nd genes , which pArabidopsis. GCN5 exhibits versatile roles in the plant through interacting with and regulating key factors, some metabolisms, or phytohormone pathways. Moreover, we should extend our focus from the model plant to other crops such as cotton, maize, wheat for protein acetylation study, and investigate more GCN5 homologs in other plant species. More work should focus on the internal connection and interaction factors of GCN5 in the development of different plant organs  to illuminate the complete network of GCN5 in the plant. The further functional characterization of the novel interaction factors, substrates as well as regulators will pave the way for a deeper understanding of molecular mechanisms of GCN5 in eukaryote development.In this paper, we focus more on how GCN5 regulates plant growth and development as well as stress response during the process of acetylation of H3K9/K14 in


The first reference to the “C-value paradox” reported an apparent imbalance between organismal genome size and morphological complexity. Since then, next-generation sequencing has revolutionized genomic research and revealed that eukaryotic transcriptomes contain a large fraction of non-protein-coding components. Eukaryotic genomes are pervasively transcribed and noncoding regions give rise to a plethora of noncoding RNAs with undeniable biological functions. Among them, long noncoding RNAs (lncRNAs) seem to represent a new layer of gene expression regulation, participating in a wide range of molecular mechanisms at the transcriptional and post-transcriptional levels. In addition to their role in epigenetic regulation, plant lncRNAs have been associated with the degradation of complementary RNAs, the regulation of alternative splicing, protein sub-cellular localization, the promotion of translation and protein post-translational modifications. In this review, we report and integrate numerous and complex mechanisms through which long noncoding transcripts regulate post-transcriptional gene expression in plants. Unlike in prokaryotes, genomes in eukaryotes exhibit a large variability in their size ,2, whichcis-NATs) or are complementary to a sense transcript located in a distinct genomic locus (trans-NATs) [LncRNAs form the most diversified group of ncRNAs, exhibiting a large range of sizes varying from 200 bases to over 100 kb in length. They are expressed in various tissues, cell-types, and cell-states, and function in the nucleus or cytoplasm ,12. Givens-NATs) ,13. Circns-NATs) . Alternans-NATs) ,16.It is increasingly clear that lncRNAs participate in virtually every aspect of gene expression. In plants, although the functional characterization of ncRNAs is still in its early stages, several lncRNAs have been described as regulators of gene transcription, capable of conditioning the epigenetic environment of their genomic targets and of modulating the activity of transcriptional complexes . In addicis-NATs generated from a single locus showing sequence complementarity with their corresponding sense transcript or trans-NATs that are transcribed from different distant loci and typically display imperfect complementarities with their target endogenous RNA [Arabidopsis thaliana [Natural antisense transcripts (NATs) constitute an important class of lncRNAs, exerting a wide variety of molecular functions in eukaryotes ,19. Theynous RNA ,21  mRNA in A. thaliana female gametophytes [asHSFB2a in transgenic plants leads to the absence of HSFB2a RNA while the overexpression of HSFB2a results in a complete loss of asHSFB2a expression, suggesting that HSFB2a and asHSFB2a are mutually repressive [DELAY OF GERMINATION 1 (asDOG1) was found to be a negative regulator of DOG1 expression, a gene involved in the control of germination [asDOG1 transcription increases the levels of DOG1 sense mRNAs, enhancing seed dormancy [A. thaliana, a screening of lncRNAs using a custom-made array led to the identification of the circadian-regulated lncRNA CDF5 LONG NONCODING RNA (FLORE), a NAT of the CYCLING DOF FACTOR 5 (CDF5) transcript, which likely connects the circadian clock to the photoperiodic flowering pathway [FLORE is specifically expressed in the vasculature and regulates CDF5 in cis as well as CDF1 and CDF3 in trans. Interestingly, FLORE and CDF5 show a mutual inhibition behavior, suggesting that the CDF5/FLORE NAT pair constitutes a circadian regulatory module, which buffers its own circadian oscillation and photoperiodic flowering [TWISTED LEAF (TL) is transcribed from the opposite strand of the OsMYB60 locus encoding an R2R3 MYB transcription factor [TL by RNA interference leads to a significant increase in OsMYB60 expression levels and twisted leaf blades. It was suggested that TL may play a cis-regulatory role on OsMYB60 by affecting H3K27me3, H3K36me2, and H3K36me3 histone mark deposition [NATs can affect positively (concordant regulation) or negatively (discordant regulation) the expression of sense transcripts. An example of discordant regulation is provided by the NAT-lncRNA tophytes . The ovepressive . Similarmination . Down-redormancy . As a la pathway . FLORE ilowering . In ricen factor . Downregposition . A. thaliana [SRO5 mRNA allows the production of 24 nucleotides (nt) siRNAs from the region overlapping with Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) transcripts. Subsequently, P5CDH transcripts are cleaved to generate 21nt siRNAs [CesA6 transcript levels leads to the production of 21 and 24nt siRNAs that correlates with the down-regulation of CesA6 gene and several loci in trans involved in the regulation of cellulose rates and in the modulation of cell wall biosynthesis [Petunia hybrida, the Sho gene involved in the production of cytokinin phytohormones contains an antisense ORF partially overlapping with the ORF of the Sho sense transcript that encodes the SHO protein [cis-NAT SHO leads to the association of Sho sense and antisense transcripts in a double-stranded RNA likely targeted by a DICER complex for degradation into 24nt siRNAs [A. thaliana. The NAT-lncRNAs NAT398b and NAT398c are cis-NATs of the MIRNA genes MIR398b and MIR398c, respectively. Knock-down of NAT398b/c promotes the accumulation of MIR398b and MIR398c, while the overexpression of NAT398b and NAT398c represses the processing of miR398. Notably, the overexpression of siRNAs derived from NAT398 overlapping transcripts, so-called nat-siR398, reduces the levels of pri-miR398b and pri-miR398c [In addition, various NAT-lncRNAs have been reported to cause post-transcriptional silencing through the production of regulatory small interfering (si) RNAs derived from NAT pairs. This mechanism was first described in the regulation of salt tolerance in thaliana . Under st siRNAs . Similart siRNAs ,34. In bynthesis . Similar protein . The tist siRNAs . Another-miR398c .A. thaliana revealed that antisense transcription is associated with micro (mi) RNA-targeted mRNAs [INHIBITOR of WAX1 (Iw1) contains an inverted repeat showing more than 80% identity to the WAX1-COE gene, encoding a carboxylesterase-like protein that controls glaucousness [Iw1 transcript is able to form a miRNA precursor-like long hairpin which produces small RNAs, including the 21nt-miRNA miRW1. The accumulation of miRW1 is linked to the down-regulation of W1-COE and its paralog W2-COE, the cleavage of W1-COE transcripts and glaucous repression [Interestingly, computational analyses performed for ed mRNAs . In wheacousness . The Iw1pression .In addition to capping and polyadenylation, the production of mature mRNAs from pre-mRNAs relies on the prior removal of introns and the ligation of the majority of exons in the order in which they appear in a gene, a process known as RNA splicing. Under certain circumstances, some exons can be skipped, generating various isoforms of mature mRNAs from a single pre-mRNA. This process, called alternative splicing (AS), is mediated by the spliceosome and involves a subclass of small nuclear RNAs referred to as nuclear uridine-rich RNAs. They function in collaboration with core small nuclear ribonucleoprotein complex subunits (snRNPs) and non-snRNPs splicing factors (SFs) whose interaction with lncRNAs likely condition their stability and sub-cellular localization ,41,42,43ALTERNATIVE SPLICING COMPETITOR (ASCO) interacts in vivo with the plant-specific SFs NUCLEAR SPECKLE RNA-BINDING PROTEINS (NSRa and b), which localize in nuclear speckles and are involved in splicing [nsra/b exhibited over 2200 genes differentially regulated in comparison to wild-type plants, as well as a reduced number of lateral roots suggesting a decreased sensitivity to auxin. Interestingly, the identification of RNA processing events in the nsra/b mutant revealed an important number of intron retention events and differential 5′ start or 3′ ends in a subset of genes, including a high number of auxin-related genes that behave accordingly in the ASCO overexpressing lines [ASCO competes with mRNA-targets for the binding to NSRs, suggesting that ASCO regulates the AS of pre-mRNAs in response to auxin by hijacking NSRs [A. thaliana revealed that lncRNAs are overrepresented among NSRa targets [ASCO led to the deregulation of expression and splicing of a large number of genes related to biotic stress and flagellin response in A. thaliana [ASCO plants and the double mutant nsra/b were found to exhibit a different response to flagellin, suggesting that ASCO also modulates AS in an NSR-independent manner. Consistently, an ASCO-directed chromatin isolation by RNA purification (ChIRP) coupled to mass spectrometry and RIP assays allowed the identification of other putative ASCO protein partners, including the pre-mRNA-processing-splicing factor 8A (PRP8a) and the spliceosome-core component SmD1b [ASCO overexpression also competes for PRP8a binding to particular mRNA targets [In plants, AS plays crucial functions in the control of gene expression, boosting the protein-coding capacity and contributing to developmental plasticity ,45. In Asplicing . NSRs pang lines ,47. Remaing NSRs . More re targets . As NSRa targets . In agrethaliana . Remarkant SmD1b ,50,51. Ant SmD1b , ASCO ov targets .A. thaliana [SEPALLATA 3 (SEP3) gene increases the abundance of the naturally occurring exon-skipped AS variant SEP3.3, which lacks exon 6 [SEP3 splicing gives rise to floral homeotic phenotypes [SEP3 exon 6 circRNA can directly interact with its cognate DNA locus, forming an RNA:DNA hybrid (R-loop), which results in transcriptional pausing and correlates with the recruitment of splicing factors and AS. This mechanism suggests that circRNAs expressed from distant loci may increase the splicing efficiency of their cognate exon-skipped messenger RNAs and that chromatin conformation and R-loop formation are critical modulators of splicing patterns [An additional mechanism of AS regulation involving circular non-coding RNAs (circRNAs) was reported in thaliana . The oves exon 6 . SEP3 isenotypes ,53. Remapatterns .A. thaliana revealed a large proportion of convergently overlapping pairs (COPs) with the potential to form double-stranded RNAs [trans-NATs in Arabidopsis led to the identification of 1320 putative trans-NAT pairs [The analysis of transcription data for overlapping gene pairs in ded RNAs . Interesded RNAs . ConsistAT pairs . Most ofAT pairs . Taken tEARLY NODULIN 40 (ENOD40) genes known to participate in root symbiotic nodule organogenesis, contain only sORFs whose transcripts may encode short peptides [GmENOD40 encodes two oligopeptides of 12 and 24 aa residues that may have a transport function and specifically bind to sucrose synthase subunit nodulin 100 to control the use of sucrose in nitrogen-fixing nodules [M. truncatula, MtENOD40 is rapidly induced by symbiotic rhizobial bacteria in the root pericycle and is also detected in the differentiating cells of the nodule primordia [MtENOD40 has been described as highly structured and not associated to polysomes [MtENOD40 RNA directly interacts with the constitutive RNA Binding Protein 1 (MtRBP1), a close homolog of lncRNA-interacting AtNSRs [MtENOD40 to cytoplasmic granules. Hence, while ENOD40-encoded peptides are likely involved in sugar metabolism, the highly structured ENOD40 RNA contributes to nucleocytoplasmic trafficking [Short open reading frame (sORF) mRNAs are atypical mRNAs that contain only sORFs (shorter than 100 amino acids) and accumulate in the cytoplasm where they can be translated into oligopeptides acting as signal molecules ,55. Remapeptides ,57,58. Irimordia ,57. MtENolysomes ,59. Yeasg AtNSRs , locatedg AtNSRs . During fficking   gene is involved in the export of phosphate to the apoplastic space of xylem vessels [PHO1;2 encodes the associated cis-NAT PHO1;2. Both genes are controlled by promoters active in the vascular cylinders of roots and leaves, but while PHO1;2 promoter is unresponsive to phosphate, cis-NAT PHO1;2 promoter is strongly activated under phosphate deficiency [cis-NAT PHO1;2 transcripts and PHO1;2 protein amount increase, although PHO1;2 mRNA levels remain unchanged. In addition, the downregulation or constitutive overexpression of cis-NAT PHO1;2 leads, respectively, to a decrease or strong increase in PHO1;2 protein levels, whereas the level of expression and nuclear export of PHO1;2 mRNA are not affected. Notably, the expression of cis-NAT PHO1;2 is associated with the shuttle of the PHO1;2–cis-NAT PHO1;2 sense-antisense pair towards the polysomes, supporting a role for cis-NAT PHO1;2 in the promotion of PHO1;2 translation through polysomal recruitment, to regulate phosphate homeostasis [In rice, the  vessels ,72,73. Tficiency . In phoseostasis .A. thaliana led to the identification of novel lncRNAs controlling cognate mRNA translation [cis-NATs showed an induction correlated with the enhanced translation of their cognate sense transcripts, including two ATP BINDING CASSETTE SUBFAMILY G transporters (ABCG2 and ABCG20) and a POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 7 (PRK7) family member, associated with nutrient uptake, lateral root formation, and root cell elongation, respectively [trans-NATs showed a positive correlation between their expression and their target mRNA levels, and the expression of four trans-NATs was found to correlate with a change in target mRNA polysome association under low phosphate conditions [More recently, global analyses of polysome-associated RNAs and ribosome footprints in nslation ,75. Undeectively . In addiMiRNAs are ncRNAs of 20–22 nucleotides that play key regulatory roles in various biological processes in plants . They arA. thaliana, the lncRNA INDUCED BY PHOSPHATE STARVATION 1 (IPS1) is a functional endogenous target mimic (eTM) of miR399 involved in inorganic phosphate (Pi) homeostasis [AtPHO2 (Phosphate 2), encoding an E2 ubiquitin conjugase-related protein, which negatively regulates Pi remobilization and Pi content in shoots. The sequences of the mRNA AtPHO2 and lncRNA IPS1 contain a similar motif of 23 nucleotides complementary to AtmiR399. However, in contrast to AtPHO2, IPS1 pairing with AtmiR399 is interrupted by a mismatched loop in the expected AtmiR399 cleavage site, which prevents its degradation. When IPS1 sequesters AtmiR399, the authentic AtmiR399-target AtPHO2 is accumulated, leading to a decrease in shoot Pi content [PI-DEFICIENCY-INDUCED LONG NONCODING RNA 1 (PILNCR1) functions as an eTM for ZmmiR399, thwarting the ZmmiR399-guided posttranscriptional repression of ZmPHO2 and favoring maize adaptation to Pi deficiency [Medicago truncatula, the PI-DEFICIENCY-INDUCED LNCRNA 1 (PDIL1) was reported to regulate Pi transport by inhibiting the degradation of MtPHO2, also acting as an eTM for MtmiR399 [lncRNA23468 involved in the resistance to Phytophthora infestans [lncRNA23468 induces a significant decrease in miR482b accumulation and an increase in the miR482b target genes NBS-LRR (nucleotide binding sites-leucine-rich repeat) expression. It was thus proposed that lncRNA23468 may decoy miR482b for targeted cleavage, thereby increasing the expression levels of NBS-LRRs genes, enhancing tomato resistance to P. infestans [In eostasis . The Pi  content . More reficiency . AdditioMtmiR399 . Anothernfestans . Overexpnfestans . Arabidopsis thaliana and rice [slylnc0195 and slylnc1077 involved in the tomato yellow leaf curl virus response were predicted to be eTMs of miR166 and miR399, respectively, and the functionality of slylnc0195 was also validated using a transient agro-infiltration assay [A. thaliana to identify lncRNAs involved in blue light-directed plant photomorphogenesis and acting as ceRNAs. The lncRNA BLUE LIGHT-INDUCED LNCRNA 1 (BLIL1) was found to inhibit hypocotyl elongation under blue light and in response to mannitol stress by serving as a ceRNA to sequester miR167 [Computational analyses also led to the identification of putative eTMs originating from intergenic or noncoding genes for 20 highly conserved miRNAs in and rice . The ideand rice . In tomaon assay . Recentlr miR167 .A. thaliana, a collection of transgenic plants expressing aTMs predicted to reduce the activity of most of the miRNA families was generated, leading to morphological abnormalities in the aerial part for ~20% of the miRNAs targeted [Interestingly, the mechanism of target mimicry can be engineered and exploited to inhibit specific miRNAs via artificial miRNA TMs (aTMs) in order to establish their functionality. In targeted . MIKKI (“decoy” in Korean) was identified as an eTM for miR171, known to target mRNAs encoding SCARECROW-Like (SCL) transcription factors for cleavage [MIKKI is a TE-derived locus including Osr29 Long Terminal Repeat (LTR) retrotransposon, and its mature transcript contains an imperfect binding site for miR171, generated by a splicing event and likely attenuating the cleavage activity of miR171. In roots, MIKKI transcripts bind to miR171, stabilizing SCL mRNAs, which play an important role in root development [Finally, transposable element (TE)-derived transcripts that contain binding sites for miRNAs can also function as eTMs. In rice, the retrotransposon-derived transcript cleavage . MIKKI ielopment . In mammals, several examples illustrate the action of lncRNAs in protein post-translational modifications. By bringing together target proteins and specific kinases, phosphatases, or ubiquitin-ligases, lncRNAs can regulate post-translational modifications that will modulate the activity of enzymes ,92, the Polycomb Group (PcG) proteins are critical regulators of gene expression, essential for development in many organisms. They form complexes that modify post-translationally histones tails of target genes. In plants, the histone H3K27 trimethyltransferase CURLY LEAF (CLF) functions as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) complex ,96,97. HA. thaliana, the FLOWERING LOCUS C (FLC) gene encodes a MADS-box-containing transcription factor (TF) that acts as a critical repressor of flowering [FLC transcription is antagonistically regulated not only by the active histone modifications H3K4me3 and H3K36me3 but also by the repressive histone modification H3K27me3 [FLC expression [FLC transcriptional regulation depends on cis-acting lncRNAs, including COOLAIR, COLD ASSISTED INTRONIC NONCODING RNA (COLDAIR), and COLDWRAP [COOLAIR is a set of antisense transcripts physically associated with the FLC locus, linked to the synchronized replacement of H3K36 methylation with H3K27me3 during the early stages of vernalization, independent of Polycomb complexes [COOLAIR directly binds to the RNA binding protein FLOWERING CONTROL LOCUS A (FCA), which further interacts with the PRC2 component CLF. This allows the recruitment of CLF at FLC for H3K27me3 deposition [COLDAIR is transcribed from the first intron of FLC and cooperates with COLDWRAP, derived from FLC proximal promoter, to facilitate the establishment of H3K27me3 during the late stage of vernalization, through the formation of a repressive intragenic chromatin loop that retains CLF at the FLC promoter [COLDAIR suppresses H3K27me3 and induces H3K4me3 at the FLC locus depending on the recruitment of ATX1 and removal of CLF, leading to enhanced FLC expression [MADS AFFECTING FLOWERING4 (MAS), transcribed from the MADS AFFECTING FLOWERING 4 (MAF4) locus, is also involved in the regulation of flowering [MAS is induced by cold and activates MAF4, encoding a MADS-box containing TF, by interacting with WDR5a, a structural core component of a COMPASS-like H3K4 histone methylation complex. MAS mediates the recruitment of WDR5a to MAF4 for H3K4me3 deposition and activation of MAF4 [LRK Antisense Intergenic RNA (LAIR) is transcribed from the antisense strand of its neighboring LRK (leucine-rich repeat receptor kinase) gene cluster and can interact with OsWDR5 as well as with the histone H4K16 acetyltransferase OsMOF [LAIR overexpression is associated with higher H3K4me3 and H4K16ac levels at the LRK1 chromatin region and with the upregulation of RLK1, leading to increased grain yield [AG second intron encodes several ncRNAs, including AGAMOUS INTRONIC RNA 4 (AG-incRNA4), which recruits CLF and represses AG transcription likely through the deposition of H3K27me3 [In lowering . FLC traH3K27me3 . Upon trpression . RemarkaCOLDWRAP ,105,106.omplexes . COOLAIRposition . COLDAIRpromoter ,105. Strpression . Remarkapression . The NATlowering . MAS is  of MAF4 . In ricese OsMOF . LAIR ovin yield . AGAMOUSin yield ,113,114.H3K27me3 .A. thaliana, the PRC1 protein LHP1 recognizes H3K27me3 deposition and ensures the spreading of this repressive mark, controlling global genome topology [AUXIN REGULATED PROMOTER LOOP (APOLO) is transcribed from the promoter region of its neighboring gene PINOID, a key regulator of polar auxin transport, and interacts with LHP1 in vivo [APOLO recognizes a subset of auxin-related genes in trans, through sequence complementarity and DNA–RNA hybrid formation (R-loops). Remarkably, overexpression of APOLO leads to the decoy of LHP1 from common target genes over the genome, and is associated with a decrease in H3K27me3 deposition as well as with modifications of chromatin conformation [MARNERAL SILENCING (MARS) is transcribed in response to abscisic acid (ABA) from the marneral cluster, which includes the marneral synthase MRN1 gene and the two P450 cytochrome-encoding genes CYP705A12 and CYP71A16 [MARS over-accumulation is associated with the decoy of LHP1 and a decrease in H3K27me3 distribution throughout the marneral cluster. Loss of H3K27me3 likely allows the formation of a chromatin loop bringing together an enhancer element enriched in ABA-related TF binding sites and MRN1 proximal promoter, resulting in the transcriptional activation of MRN1 and a delay in seed germination [In topology . In resp in vivo . APOLO rormation . SimilarCYP71A16 . MARS ovmination .SVALKA [Compelling evidence supports the involvement of lncRNAs in diverse and numerous aspects of post-transcriptional gene regulations in plants. Future research will likely shed light on the basis governing lncRNA interaction with diverse molecular partners, including DNA, proteins, or transcripts. The noncoding transcriptome has been shown to differ across ecotypes of the same species, notably in response to the environment . This obSVALKA , suggestSVALKA . SimilarSVALKA . The advSVALKA , will li


Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is correlated with differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network driven by cell-type-specific enhancers.During  The pioneer transcription factor Zelda is essential for the reprogramming of germ cells to totipotent cells of the early Drosophila embryo. Here the authors examine the function of Zelda later in development in the larval brain to show that Zelda promotes the undifferentiated stem-cell fate. They further identify factors that limit the reprogramming capacity of this pioneer factor. These features of reprogramming pioneer factors allow them to gain access to silenced regions of the genome and drive new gene expression profiles to change cell fate. Indeed, misexpression of pioneer factors within an organism leads to dramatic gene expression changes that can cause disease10. Despite the ability of these factors to engage silenced portions of the genome, there are barriers to pioneer-factor binding and efficient reprogramming17. Many studies have leveraged the advantages of cell culture systems to identify impediments to pioneer-factor binding and reprogramming. However, many fewer studies have identified limitations to pioneer-factor-driven cell-fate changes within the context of an entire, developing organism.During development, the genome is differentially interpreted to give rise to thousands of distinctive cell types. Once terminally differentiated, cells within an organism are generally incapable of transitioning to a less differentiated fate. By contrast, in cell culture the addition of a cocktail of transcription factors can reprogram differentiated cells back to a pluripotent state. Many of these reprogramming factors, including Oct4, Sox2, and Klf4, function as pioneer-transcription factors: a specialized set of transcription factors that can bind DNA within the context of nucleosomes, facilitate chromatin accessibility and subsequent binding by additional transcription factors21. This maternal-to-zygotic transition (MZT) is necessary for development and is orchestrated, in part, by factors that reprogram the zygotic genome for transcriptional activation20. Factors that activate the zygotic genome have been identified in many species and all share essential features of pioneer factors. Perhaps the best characterized of these transcriptional regulators is Zelda (Zld), which we and others have shown functions as a pioneer factor to reprogram the early embryonic genome in Drosophila melanogaster28.Immediately following fertilization, the specified germ cells must be reprogrammed to form the totipotent cells that can ultimately differentiate to generate a new organism. During this time, the zygotic genome is transcriptionally silent, and maternally deposited mRNAs and proteins control early embryonic development22. This binding is distinctive even for pioneer-transcription factors31. For example, the extensively studied pioneer factor FOXA2 only binds ~10% of its motifs in a variety of different cell types12. The chromatin environment in the early embryo is naïve as compared to later in development32, and this may contribute to the widespread occupancy of CAGGTAG motifs by Zld at this stage of development. In the Drosophila embryo, similar to other organisms, there are relatively few post-translational modifications to the histone proteins, and the genome is packaged by a unique linker histone dBigH1, which is essential for proper development34. In addition, early development is characterized by a series of 13, rapid, semi-synchronous nuclear divisions that each occur over approximately 10 minutes and are comprised of only synthesis (S) and division (M) phases32. While it is possible that the reprogramming function of Zld requires these distinctive properties of early development, Zld is also necessary for development after the MZT26. It remains unclear whether Zld defines cis-regulatory regions in tissues outside the early embryo and if so, how this activity is regulated by the cell-type-specific chromatin established during development.In the early embryo, Zld binding is driven strongly by sequence with between 40–65% of the canonical Zld-binding motifs (CAGGTAG) bound by Zld during the MZT37. Both types of neuroblasts undergo asymmetric division to self-renew and to generate a descendant that exits the multi-potent state and begins to differentiate. While type I neuroblasts directly contribute to neurogenesis, type II neuroblasts divide asymmetrically to self-renew and to generate a sibling cell that commits to an intermediate neural progenitor (INP) identity and functions as a transit-amplifying cell. One of the newly born neuroblast progeny, marked by the absence of Deadpan (Dpn) expression, first transitions into a non-Asense-expressing (Dpn−Ase−) immature INP, and then, after three to four hours, to an Asense-expressing (Dpn−Ase+) immature INP. Dpn is re-expressed in mature INPs (Dpn+Ase+) that undergo 6–8 rounds of asymmetric division to exclusively generate differentiated cell types  in the larval brain provide an excellent in vivo system for investigating temporal regulation of enhancer activity. In the larval brain lobe, there are predominantly two types of neural stem-cell populations: type I and type IIpes Fig. . Thus, d38. However, despite reactivation of Notch signaling in the mature INPs, these cells do not revert back to stem cells. Downregulation of Notch signaling in newly born INPs allows the transcriptional repressors Earmuff (Erm) and Hamlet (Ham) to become sequentially activated during INP commitment40. Erm and Ham function through histone deacetylase 3 (Hdac3) to silence expression of tailless (tll), which encodes a master regulator of type II neuroblast fate41. Suppressor of Hairless (Su(H)), the DNA-binding partner of Notch, binds the tll locus in larval brain neuroblasts, suggesting that tll is a Notch-target42. Thus, in the INPs Erm- and Ham-mediated silencing of tll prevents reactivation of Notch signaling from inducing tll expression and driving their reversion to neuroblasts. Consistent with this model, INPs in erm or ham-mutant brains spontaneously revert to type II neuroblasts and knocking down Notch function can suppress the supernumerary neuroblast formation in these mutant brains43. These results support a model whereby sequential silencing by Erm and Ham during INP commitment renders the tll locus refractory to aberrant activation in INPs, but the precise mechanisms and the enhancers upon which these repressors act remains unclear.As in many stem-cell populations, Notch signaling is essential for maintaining type II neuroblasts in an undifferentiated state: loss of Notch signaling causes premature commitment of neural stem cells to INP fate, and constitutive activation leads to extra neuroblasts44. Thus, the type II neuroblast lineage provides a powerful system to investigate the function of this pioneer factor in a tissue apart from the early embryo. We demonstrate that Zld functions with Notch signaling to maintain type II neuroblasts in an undifferentiated state. Exogenous expression of Zld during INP commitment can promote INP reversion into neuroblasts. However, Erm and Ham expression limit this reprogramming capacity. We show that in type II neuroblasts chromatin occupancy by Zld is reorganized as compared to the embryo and that this reorganization is correlated with changes in chromatin accessibility. Nonetheless, target genes such as dpn and tll are shared between developmental stages. We propose that Zld drives key developmental transitions in the neuroblast lineage and the early embryo through a shared gene-regulatory network by binding to cell-type-specific enhancers.Zld has been previously shown to be expressed in type II neuroblasts, but not in their progenyzld function had no detectable effect on the neuroblast, but instead resulted in defects in INP proliferation44. This discrepancy between the Zld expression pattern and the phenotype induced by RNAi-mediated zld knockdown prompted us to re-evaluate the role of zld in the larval brain  neuroblasts using well-defined cell-fate markers. zld-mutant clones contained a single neuroblast and 15.4 ± 7 INPs, indistinguishable from wild-type clones (16.1 ± 6.2 INPs) . These data indicate that Zld is either dispensable in the type II neuroblast lineage or redundant pathways compensate for the absence of Zld.Despite Zld expression being limited to the type II neuroblast, a previous study reported that knocking down ain Fig. . We ther47. We therefore tested whether Zld might act synergistically with Notch signaling to regulate type II neuroblasts. For this purpose, we assessed mosaic clones in a sensitized genetic background in which Notch function was reduced by RNAi. 22.2% of Notch-RNAi type II neuroblast clones lacked identifiable neuroblasts, and 33.3% of the clones contain neuroblasts with reduced cell diameter and Ase expression; two characteristics indicative of differentiation , allowing us to limit expression to the larval stages. While wild-type third instar larval brain lobes invariably contained 8 type II neuroblasts, 72 h of Zld overexpression starting in the L1 larval stage resulted in 61.1 ± 13 type II neuroblasts per lobe  or all four zinc fingers of the DNA-binding domain mutated (ZnF6). Overexpressing either protein resulted in supernumerary type II neuroblast formation , but expression of either mutant protein generated significantly fewer supernumerary neuroblasts than overexpression of wild-type Zld  to expression only in INPs  -Gal4 resulted in 25.4 ± 8.7 type II neuroblasts per lobe (n = 10 brains). While fewer supernumerary type II neuroblasts  were identified when Zld misexpression was limited to late immature INPs and mature INPs (Erm(III)-Gal4), these data demonstrate that Zld misexpression can revert partially differentiated neuroblast progeny back to an undifferentiated stem-cell fate. Unlike Zld misexpression in immature INPs, expression of Zld in mature INPs driven by Opa-Gal4 was not sufficient to induce supernumerary neuroblast formation . Thus, the ability of misexpression of Zld to promote the undifferentiated state is limited along the neuroblast lineage. This stands in contrast to aberrant Notch activation in mature INPs that can result in supernumerary neuroblasts. Thus, Zld promotes an undifferentiated state by functioning in parallel to, and not downstream of, Notch.Our data showed a genetic interaction between Zld and Notch, supporting a role for Zld in promoting an undifferentiated state in type II neuroblasts by functioning in parallel to or downstream of Notch. Aberrant activation of Notch signaling in either immature or mature INPs drives supernumerary neuroblast formationl4) Fig. . Zld ove50. However, Dpn remains expressed in type II neuroblasts even in the absence of Notch signaling, suggesting that additional activators can drive expression49. Zld binds to the cis-regulatory regions of dpn in the early embryo, and embryonic dpn expression depends on maternally encoded Zld -binding motifs, or both Zld and Su(H)-binding motifs mutated. Similar to the endogenous locus, expression of the reporter depended on Zld binding for expression in the embryo23 -Gal4  was strongly suppressed by loss of a single copy of dpn -Gal4 is active in both immature INPs and mature INPs, this observed suppression is not due to reducing Zld-induced dpn expression in mature INPs, where Notch signaling is reactivated. This is because dpn overexpression in mature INPs cannot induce a supernumerary type II neuroblast phenotype. Thus, Zld functions in parallel to Notch to maintain type II neuroblasts in an undifferentiated state by activating dpn expression, and misexpression of Zld in the partially differentiated neuroblast progeny can revert them to an undifferentiated state by activating dpn expression.Because of the ability of Zld and Notch to both activate ive Fig. . The supbrain tumor (brat11/Df(2L)Exel8040). These brat-mutant brains contain thousands of type II neuroblasts at the expense of other cell types and are a well characterized model for studying the transition from an undifferentiated stem-cell state to a committed INP identity51. Supporting the relevance of this tissue for these assays, we demonstrated that Zld levels within a single neuroblast are equivalent between wild-type and brat-mutant brains  52  to identify Zld-binding sites in the type II neuroblasts. These experiments required enriching for type II neuroblasts, as wild-type larval brains only contain eight type II neuroblasts per lobe. For this purpose, we performed ChIP-seq on third instar larval brains dissected from larvae that are mutant for )52 Fig. . Identif)52 Fig. . Supportays Fig. . De novo 56 Fig. .Fig. 3Zl22. Zld is a pioneer factor that can bind to nucleosomal DNA57, and this capacity likely contributes to the unique binding profile in the early embryo. However, the naïve chromatin environment of the early embryo may also contribute to the unique binding profile of Zld at this stage. To determine the relative contributions of the pioneering activity of Zld and the naïve chromatin environment to the binding profile of Zld, we compared the binding of Zld in the type II neuroblasts to that of the early embryo. We realigned previously published ChIP-seq data for Zld from stage 5 embryos22 to the dm6 genome release, using the same parameters for aligning, filtering and peak calling as was used for the neuroblast ChIP-seq data  bound by Zld in the very early embryoion Fig. . Contrarion Fig. . These uion Fig. . Becauseion Fig. 24 and usp-value < 2.2e-16) or randomized regions of the genome   Fig. . Indeed,16) Fig. . Similar 22 Fig. . Unexpec 22 Fig. . Instead 22 Fig. . These ftif Fig. . As had tif Fig. . Togethe58 les Fig. . This cooth Fig. . Regionsoth Fig. . Similaroth Fig. .Fig. 5Ce60. Thus, the correlation between Zld binding and open chromatin might be due to Zld-mediated chromatin accessibility. However, chromatin accessibility also influences transcription-factor binding, such that accessibility may guide Zld binding to some loci. Therefore, we analyzed chromatin accessibility at regions containing the canonical Zld-binding CAGGTA motif. CAGGTA-containing regions bound in the type II neuroblasts and embryo are accessible in both cell types , drives expression in a pattern identical to wild-type, but with reduced levels in the posterior62. Thus, the region bound by Zld specifically in the type II neuroblast is not necessary for expression in the early embryo, suggesting it may define a neuroblast-specific cis-regulatory module.To begin characterizing changes that allow Zld binding to non-canonical sites, we focused on the gene int Fig. . To deteion Fig. 62. Priortll regulatory region. R31F04, which contains sequence corresponding to the region extending from −4.9 to −1.8 kilobase upstream of the tll transcriptional start site, drove expression in the type II neuroblasts  and assayed for GFP in the type II neuroblasts . Consistent with tll expression rapidly diminishing in immature INPs, the region with the most dramatic loss in accessibility over this time course was the Zld-bound, type II neuroblast-specific tll enhancer . To identify factors that could be driving the decrease in accessibility in these clusters, we performed motif searches at the Zld-bound sites and identified cluster 6 as having the largest percentage of sites containing the canonical Zld-binding motif  failed to induce supernumerary type II neuroblast formation, suggesting cell-type-specific features limit the ability of Zld to activate tll expression -Gal4) of brains heterozygous for null mutations in either erm or ham. While loss of single copies of either erm or ham does not result in supernumerary type II neuroblasts41, loss of a single copy of either of these genes enhanced the weak supernumerary phenotype caused by misexpression of Zld . This effect was enhanced when copies of both erm and ham were removed   Fig. . To moreund Fig. . Togethe71. Despite the ability of Zld to promote the undifferentiated stem-cell fate, this capacity is limited as cells differentiate to INPs. We showed that the ability of Zld to drive gene expression is limited by the repressors Erm and Ham. Because of the ability of pioneer factor misexpression to cause disease, understanding these cell-type-specific constraints on pioneer factors has important implications for our understanding of development and disease.Our results demonstrate that Zld, an essential transcriptional activator of the zygotic genome, promotes the undifferentiated state in the neural stem-cell lineage of the larva and can revert partially differentiated cells to a stem cell. Other pioneer factors are known to have similar functions when misexpressed, and this can lead to disease. For example, expression of DUX4, an activator of the zygotic genome in humans, in muscle cells leads to Facioscapulohumeral muscular dystrophy (FSHD), and OCT4 and Nanog are overexpressed in undifferentiated tumors and their expression is associated with poor clinical outcomeszld in the newly generated INPs results in supernumerary type II neuroblasts44. Thus, Zld levels must be precisely controlled to allow differentiation following asymmetric division of the type II neuroblasts. Zld promotes the undifferentiated state, at least in part, through the ability to drive expression of Dpn, a key transcription factor for driving type II neuroblast self-renewal72. dpn is a target of the Notch pathway in type II neuroblasts and constitutively activated Notch signaling drives dpn expression49. However, loss of Notch signaling does not completely abrogate expression of known target genes, including dpn, suggesting that additional activators can drive expression in the absence of Notch49. Indeed, we show that Zld functions as such a factor in driving dpn expression, and loss of a single copy of dpn can suppress the ability of Zld to promote the reversion of immature INPs to type II neuroblasts. We propose that this redundancy with Notch is not limited to regulating dpn expression and that Zld and Notch may function together to regulate a number of genes required for type II neuroblast maintenance. Supporting this, Zld is bound to 49% of the identified direct Notch-target genes in neuroblasts , which has similar genomic occupancy in the embryo and larval imaginal discs76. Thus, unlike for Grh, Zld binding is cell-type-specific and likely governed by changes to the chromatin structure along with the expression of cell-type-specific transcription factors Exel8040/CyO, ActGFP (BDSC#7847)81 and screened for GFP negative larvae. Imaging experiments took advantage of a line with endogenous Zld tagged with superfolder GFP24. For ChIP-seq experiments using the anti-GFP antibody and GFP-Zld quantification, the sfGFP-tagged Zld alleles were combined with brat11 and bratDf(2L)Exel8040 and crossed to obtain brat-mutant brains. Imaging of Six4::GFP (BDSC#67733) and FlyLight GMR31F04-Gal4 (BDSC#46187) and GMR31D09-Gal4 (BDSC#49676) was done with previously published lines.Flies were raised on standard fly food at 25 °C (unless otherwise noted). To obtain the 25 and a gBlock containing ZnF mutations in the DBD of Zld . The mutated C2H2 ZnFs convert cysteines to serines. These transgenes were integrated at ZH-86Fb site on chromosome 3 using φC31-mediated integration82 . The following drivers were used to drive ectopic expression of Zld in the larval type II neuroblast lineage: Worniu-Gal4; Asense-Gal8083 combines the Worniu-Gal4 that expresses in type I and II neuroblasts with the Gal4 inhibitor Gal80 under the Asense promoter to obtain a line that drives expression specifically in type II neuroblasts. Worniu-Gal4; Tub-Gal80ts80 is temperature sensitive thus upon shift from 18 °C to 33 °C, Gal80 repression of Gal4 is relieved and expression is driven. The temperature shift was performed from 24 to 96 h after egg laying, corresponding to larval stages L1-L3. Erm(II)-Gal447 drives expression in the Ase- immature INPs through Ase+ immature INPs and mature INPs. Erm(III)-Gal484 drives expression in the Ase+ and mature INPs. Opa-Gal4  drives expression in the mature INPs.For ectopic Zld expression experiments, the open reading frame for Zld-RB was cloned into the pUASt-attB vector using standard PCR, restriction digest and ligation procedures. Similarly, transgenic UAS driven Zld ZnF mutants were cloned using previously generated plasmidsdpn transgenes we ordered gBlocks  with mutated Zld and Su(H) binding sites in the 5 kb dpn cis-regulatory and used Gateway LR Clonase  to clone the region into the VanGlow vector without the DSCP (Addgene#83342)50. The various transgenic reporters were integrated into the VK37 site on chromosome 2 using φC31-mediated integration82 . L3 brains from reporter fly lines were dissected, fixed and stained to visualize GFP reporter expression in the type II NBs. Embryos from these lines were collected on molasses plates with yeast paste from flies reared in cages for luciferase assays. The embryos were ground in an eppendorf tube with a pestle in 1x passive lysis buffer and luciferase signal was measured using the Dual Luciferase assay from Promega . tll transgenes were similarly cloned with Gateway LR Clonase  to insert PCR-amplified genomic regions into the VanGlow vector with the DSCP (Addgene#83338). The various transgenic reporters were integrated into the VK31 site on chromosome 3 using φC31-mediated integration82 .To create the dpn172, hamDf(2L)Exel7071 (BDSC#7843)41, and erml(2)513843 mutant experiments were done using previously published lines. zld294 26 and Notch-RNAi (BDSC#33611) clones were generated using previously published methods85. Wor-Gal4 was used to drive a GFP reporter to mark neuroblast clones. Briefly, clones were induced by heat shock at 37 °C for 90 min at 24 h after larval hatching. Brains were dissected for clone analysis at 72 h after clone induction.4 containing 4% formaldehyde for 23 min. Fixed brain samples were washed with PBST containing PBS and 0.3% Triton X-100. After removing fix solution, samples were incubated with primary antibodies for 3 h at room temperature. Samples were washed with PBST and incubated with secondary antibodies overnight at 4 °C. The next day samples were washed with PBST and then equilibrated in ProLong Gold antifade mountant . The confocal images were acquired on a Leica SP5 scanning confocal microscope . More than 10 brains per genotype were used to obtain data in each experiment. Primary antibodies include Rabbit anti-Ase Antibody (1:400 for IF)43, Rat anti-Dpn Antibody 86, Rat anti-Wor Antibody 86, Chicken anti-GFP Antibody , and Rhodamin phalloidin . Secondary antibodies include Alexa Fluor® 647 AffiniPure Goat Anti-Rat IgG (H + L) , Alexa Fluor® 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H + L) , Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 .Third instar larval brains were dissected in PBS and fixed in 100 mM Pipes (pH 6.9), 1 mM EGTA, 0.3% Triton X-100 and 1 mM MgSO87. We modified the protocol of in situ HCR v3.0 to combine immunofluorescent staining of the larval brain. Third instar larval brains were dissected in PBS and fixed in 100 mM Pipes (pH 6.9), 1 mM EGTA, 0.3% Triton X-100 and 1 mM MgSO4 containing 4% formaldehyde for 23 minutes. Fixed brain samples were washed with PBST containing PBS and 0.3% Triton X-100. After removing fix solution, samples were pre-hybridized with hybridization buffer  at 37 °C for 1 h. Pre-hybridized samples were mixed with 5 nM Sp-1 mRNA HCR probe  and incubated at 37 °C overnight. After hybridization, samples were washed with washing buffer  and then incubated with amplification buffer  at 25 °C for 30 min. During washing period, imager hairpins  were denatured at 95 °C for 2 min. Once samples were equilibrated in amplification buffer, samples were mixed with 3 μM of denatured imager hairpins and incubated at 25 °C for overnight. The next day, samples were washed with PBST and then refixed in 100 mM Pipes (pH 6.9), 1 mM EGTA, 0.3% Triton X-100 and 1 mM MgSO4 containing 4% formaldehyde for 15 min to initiate immunofluorescent staining procedures.mRNA signals in the larval brain were developed by performing in situ HCR v3.0Dpn or Wor were used to identify the type II neuroblast or INP nucleus. The pixel intensities of the reference proteins were measured in nucleus of cells of interest by using Image J software (version 1.53 m) and the pixel intensities of GFP reporter proteins in the identical area were measured. The intensities of GFP signal were corrected based on the difference of intensities of the reference protein in each sample and then the average of GFP intensities for each experiments were calculated. All biological replicates were independently collected and processed.brat11/Df(2L)Exel8040 larvae aged for 5–6 days at 25 °C (L3 stage). The dissected brains were fixed in 1.8% formaldehyde for 20 min, which was stopped by incubation with 0.25 M glycine at room temperature for 4 min and on ice for 10 min. The samples were washed 3 times with wash buffer , flash frozen in liquid nitrogen and stored at −80 °C until all the brains had been collected. Brains were thawed on ice and combined for homogenization in SDS lysis buffer  containing protease inhibitors  to obtain nuclear extracts. The nuclear extracts were disrupted using a Covaris sonicator (S220 High Performance Ultrasonicator) . 7% of the sonicated sample was stored as input and the rest was diluted with 1× volume of dilution buffer  and incubated overnight with antibodies ). Protein A beads  were added and samples were incubated at 4 °C for 4 h. Beads were recovered and washed. , 1× high salt wash buffer , 1× LiCl wash buffer , and 2× with TE buffer .) Washed beads were incubated at room temperature for 15 min with elution buffer  to elute the chromatin. The samples and corresponding input were incubated at 55 °C overnight with PK solution ). Samples were incubated with 0.5 µl 20 mg/ml RNase  at 37 °C for 30 min and moved to 65 °C for 6 h to reverse the crosslinking. Samples were cleaned up by phenol:chloroform extraction, precipitated, and resuspended in 20 µl TE buffer. Sequencing libraries were made using the NEB Next Ultra II DNA library kit  and were sequenced on the Illumina Hi-Seq4000 using 50 bp single-end reads at the Northwestern Sequencing Core (NUCore).Five hundred brains (in 45 min time windows) were dissected in Schneider’s medium  from 88. Adapters and low-quality bases were removed using Trimmomatic-0.39.89. Reads were mapped to the dm6 genome assembly90 using Bowtie 291. Unmapped, multiply aligning, mitochondrial, and scaffold reads were removed. Throughout SAMtools was used to filter and convert file formats92. MACS version 293 was used with default parameters to identify bound regions of chromatin in samples (IP vs INPUT) for both replicates of Zld antibody ChIP in neuroblasts. The Zld antibody ChIP in the embryo did not have a corresponding INPUT for the single IP, so peaks were called without reference to a control. The GFP antibody ChIP in the neuroblasts was called with the parameters above with the exception of lowering the m-fold value to -m 3 50 due to low IP efficiency. Peak summits were extended by 100 bps on either side. High-confidence Zld-bound regions in the neuroblasts were called as 200 bp peak regions with 50% overlap in both replicates using the BEDtools (version 2.29.0) intersect function94. Regions belonging to contigs and unmapped chromosomes were removed. High-confidence regions used for analysis with the GFP antibody ChIP in neuroblasts were called as being bound with 50% overlap in both Zld antibody replicates and the GFP antibody replicate. Comparison of Zld-binding sites between the embryo and neuroblasts were performed by intersecting high-confidence bound regions from the Zld antibody ChIP in the embryo and Zld antibody ChIP in the neuroblasts. Shared regions had 10 bp overlap between the embryo and neuroblasts, and unique regions had less than 10 bp overlap  from merged replicate read counts in 10 bp bins across the entire genome and dividing by the standard deviation.Read quality was checked using FASTQC (version 0.11.9)99 with z score-normalized bigWig files. Average signal line plots were generated using seqplots from z score-normalized bigWig files at 10 base-pair resolution100. Genomic annotations were performed with the Bioconductor R package ChIPseeker  using default settings and the BDGP dm6 genome through TxDb.Dmelanogaster.UCSC.dm6.ensGene package . TSS regions were redefined as −500 to +150 bps. Peak to nearest gene assignments were done using default settings of the annotatePeak function.Heatmaps were generated using deepTools2101 and Hypergeometric Optimization of Motif Enrichment 102. These programs identified motifs enriched in the input relative to shuffled or randomized regions of the genome. The de novo motifs were matched to known motifs from the JASPAR (http://jaspar.genereg.net) and DMMPMM (http://autosome.ru/DMMPMM) databases by the programs. Motifs are given a p-value or E-value  indicating the confidence of the enrichment relative to the control sequences. Additional motif searches were done using the Biostrings package in R (version 2.50.2). The vcountPattern function was used to tally the number of regions containing at least one occurrence of a motif and the vmatchPattern was used to locate regions containing a motif.To test for enrichment of motifs, de novo motif searches were done using the MEME-suite (version 5.1.1)103. Log2(counts) overlapping each peak was plotted and the Pearson correlation calculated for each pair of replicates.To assess ChIP-seq and ATAC-seq replicate reproducibility, the number of reads overlapping each peak was quantified using featureCounts (version 1.6.4)104. brat11/Df(2L)Exel8040 larvae were aged for 5–6 days at 25 °C prior to dissecting brains in Schneider’s medium . 5 dissected brains were transferred to the detached cap of a microcentrifuge tube containing 10 µL cold lysis buffer . Under a dissecting microscope, brains were homogenized with the blunted tip of a pasteur pipette. The cap was transferred to a microcentrifuge tube containing 40 µL of additional lysis buffer. Tubes were spun down for 10 min at 500 × g at 4 °C. Supernatant was removed under a dissecting microscope. This nuclear pellet was used to prepare ATAC-seq libraries using the Nextera DNA Library Preparation Kit . The pellet was suspended in 5 µL buffer TD before adding 2.5 µL water and 2.5 µL Tn5 transposase . Samples were incubated at 37 °C for 30 min. Tagmented DNA was purified using the Minelute Cleanup Kit . DNA was amplified with 12 cycles of PCR using the NebNext Hi-Fi 2× PCR Master Mix . Following PCR, DNA was purified using a 1.2× ratio of Axyprep magnetic beads . Library quality and tagmentation were assessed on an Agilent Bioanalyzer before pooling and submitting for 150 bp paired-end sequencing on a Illumina HiSeq 4000 at NovoGene.The protocol for ATAC-seq on larval brains was adapted from a protocol previously used for ATAC-seq on single embryosChromatin accessibility during the transition from type II neuroblast to INP was characterized using a temperature-sensitive system for synchronous type II neuroblast differentiation . Brains were collected at 0, 6, 12, 18, or 24 h following temperature shift, capturing intermediate stages during the transition from type II neuroblasts to INP. Following temperature shift, brains were collected and ATAC-seq libraries prepared as described above.105. Reads were aligned to the D. melanogaster genome (version dm6) using Bowtie2 using the following parameters: --very-sensitive, --no-mixed, --no-discordant, -X 5000, -k 291. Aligned reads were filtered to include only reads with a mapping quality score >30. Reads aligning to scaffolds or the mitochondrial genome were discarded. To identify fragments that likely originated from nucleosome-free regions, fragments were filtered to include only those <100 bp, as previously described106. All downstream analysis and visualization was performed using these accessible fragments. To call peaks on accessible fragments, accessible fragments from both replicates were merged and MACS2 was used with the following parameters: -f BAMPE, --keep-dup all, -g 1.2e8, --call-summits. Deeptools was used to calculate genome coverage and generate bigWig files used for genome browser tracks and metaplots.Adapter sequences were trimmed from raw sequence reads using NGmerge (version 0.3)22, neuroblast ATAC and stage 5 embryo ATAC59. DESeq2 was used to compare ChIP-seq or ATAC-seq profiles between the embryo and neuroblast and identify differentially bound or differentially accessible regions between the two cell types107. Log2 fold-change values calculated by DESeq were used to correlate differences in binding with differences in accessibility between the two cell types.To compare Zld binding to chromatin accessibility in the embryo and neuroblasts, neuroblast-specific, embryo-specific and shared Zld peaks were merged to create a set of all Zld peaks detected in either cell type. The number of ChIP-seq or ATAC-seq reads overlapping each peak was quantified using featureCounts for the following datasets: neuroblast Zld ChIP, nuclear cycle 14 embryo Zld ChIPTo identify regions with dynamic chromatin accessibility during the transition from type II neuroblast to INP, DEseq was used to perform differential accessibility analysis across data from the 5 ATAC-seq time points. The likelihood ratio test was used to identify 11,127 regions with differential accessibility across any of the time points. K-means clustering was performed to separate sites into groups with different patterns of chromatin accessibility over the time course. We initially tested values of k from 2 to 15 and found that 6 clusters were sufficient to capture the following patterns of change in the dataset: early increase, late increase, transient increase, early loss, late loss, transient loss. Clusters obtained using values of k larger than 6 did not identify additional patterns of change. K-means clustering was performed in R with the following parameters: nstart = 25, max.inter = 1000. For analysis of Zld motifs at differentially accessible sites, regions were considered to contain a Zld motif based on presence of the CAGGTA motif within 200 bp of the peak summit (Supplementary Data Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32, and both phages encode a protein similar to bacterial RNA polymerase promoter specificity σ subunit. Here, we investigated the temporal pattern of 7-11 gene expression during infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays, we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11 σ subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus, GTAAtg-(16)-aCTA, and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzymes formed by the phi32_gp36 σ factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by the RNA polymerase holoenzyme containing the host primary σ70 factor. Unlike the case of phiEco32, no shut-off of σ70-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme. Salmonella enterica serovar Newport phage 7-11 is a podovirus with a distinctive, strongly elongated virion head ) ultracompetent cells (Stratagene) were used for molecular cloning UTP. Reactions proceeded for 10 more minutes at 37 °C and were terminated by the addition of an equal volume of formamide loading buffer. Primers used to generate templates for transcription are listed in Transcription reactions were performed in transcription buffer -5′-end-labeled DNA fragment was used and transcription buffer did not contain DTT. After preincubation for 15 min at 37 °C, the reactions were probed with 2 mM KmnO4 for 20 s at 37 °C. Reactions were terminated by the addition of 0.5 volume of stop solution containing 600 mM β-mercaptoethanol, followed by ethanol precipitation and 10% piperidine treatment at 95 °C for 15 min. Samples were chloroform-extracted and ethanol-precipitated. Pellets were dissolved in formamide loading buffer and reaction products were analyzed on sequencing gels.Promoter complexes were formed as described in the in vitro transcription section with the following modifications: 100 nM of (γ-70 protein labeled at position 517 with fluorescein (σ70*) was obtained as previously described [70*, 1 or 3 nM RNAP core complexed with Rifampicin (Rif) and 12 nM gp47 (if indicated). The RNAP core–Rif complex was prepared by incubation of RNAP core (1 or 3 nM) and 1 uM Rif for 10 min at 25 °C [Fluorescence measurements were carried out at 25 °C using a QuantaMaster QM4 spectrofluorometer (PTI) in transcription buffer containing 0.02% Tween 20 . The σ70at 25 °C . The fluE. coli phage phiEco32 was determined in 2008 [E. coli (APEC) phages NJ01 (NC_018835), ECB2 (NC_018859), SU10 (KM044272) and 172-1 (KP308307) [E. coli phages  and Cronobacter sakazakii phage vB_CsaP_GAP52 (GAP52) (NC_019402) are also related to phiEco32-like phages. Comparison of phiEco32 and 7-11 genomes is schematically presented in Kuravirus phages, we consider phages 7-11 and GAP52 as members of a distinct “7-11 subgroup” among the phiEco32-like bacteriophages.Transcription of phiEco32 genome was studied by macroarray and primer extension analysis and conventional early, middle and late expressed classes of phage genes were revealed . Since pAll phiEco32-like phages encode a σ factor . Previously, we identified six phi32_gp36 promoters located upstream of middle and late genes of the phage [The putative σ factor of 7-11, SaPh711_gp47, is a distant homologue (15% identity) of phi32_gp36 and other phiEco32 subgroup σ factors, which likely explains our inability to find 7-11 promoters using the phiEco32 middle and late promoter consensus motifs. We have searched all 7-11 intergenic regions (that were defined as DNA sequences between nucleotides positions “−100” and “+200” relative to predicted start codons) for the presence of overrepresented DNA motifs by MEME suite  and the 70-dependent hosts and early phage phiEco32 transcription but activates phi32_gp36-dependent transcription in vitro [Transcription factor phi32_gp79 inhibits σ70 factor and middle and/or late promoters dependent on the holoenzyme containing SaPh711_gp47. Since we could not predict SaPh711_gp47 promoters bioinformatically, we decided to proceed from the fact that out of six phiEco32 genes  located downstream of phi32_gp36-dependent promoters  two types of promoters in the 7-11 genome: early promoters dependent on the host RNAP holoenzyme containing the primary σnfection A. Since The products of primer extension shown in 12, 22, 28 and 69  from DNA fragments that contained identified 7-11 late promoter sequences. Efficient in vitro RNA synthesis was observed from each late promoter detected in vivo . Conversely, the SaPh711_gp47-holoenzyme did not transcribe from a DNA template containing a strong σ70-promoter T7 A1 [70 RNAP holoenzyme did not lead to the appearance of bands corresponding to late phage promoter transcripts, indicating that the two σ factors compete for the same RNAP core binding site. Surprisingly, the dual transcription regulator of phi32_gp79 that inhibits σ70 transcription in vitro and activates phi32_gp36-dependent transcription [As expected, the RNAP holoenzyme containing the σer T7 A1 . The add4) probing. Since thymines in single-stranded, but not double-stranded, DNA are sensitive to oxidation by KMnO4, this reagent allows one to determine the size of the transcription bubble in the promoter complex. As can be seen, the region of localized DNA melting included the TSS and the -10 promoter element  pro70 subunit fluorescently labeled at amino acid 517 (σ70*)  .70* is adjacent to the binding site of rifampicin (Rif), an inhibitor of transcription elongation that interacts with RNAP core [70* decreases, due to quenching of the σ70* fluorophore by Rif via the FRET mechanism (black curve) [70 (red curve). When RNAP core bound to rifampicin was preincubated with SaPh711_gp47, followed by the addition of fluorescently labeled σ70*, no quenching was observed, even after a 1 h incubation with σ70* (blue curve), presumably because the σ70 binding site was occupied by SaPh711_gp47. When σ70 and SaPh711_gp47 were added to the Rif-bound RNAP core simultaneously, fluorescence quenching (green curve) was only moderately less than that observed in the same ratio of RNAP core/σ70 in the absence of SaPh711_gp47 (red curve). Since this experiment was performed under conditions when the concentration of SaPh711_gp47 was eight times greater than that of σ70, it follows that the formation of the holoenzyme with σ70 is faster than the formation of the SaPh711_gp47 holoenzyme. However, if the complex with SaPh711_gp47 is already formed, σ70 is unable to displace bound SaPh711_gp47.In the context of the holoenzyme, the labeled amino acid of σNAP core . Upon fok curve) . The deg70-depedent promoters were predicted in the phage 7-11 genome 2-8  are commonly found, predominantly in intergenic regions, of many phages [32 (DNA polymerase) transcript [Robust production of SaPh711_gp47 in amounts necessary to compete with σCGG loop C. An idey phages . They ary phages . The knoanscript ,29. The anscript . We prop70, is alone able to efficiently compete for the binding such that enough SaPh711_gp47 holoenzyme to serve the needs of the virus is formed. The lack of host transcription shut-off and the absence of the middle expression class of phage genes may be due to the absence of a homologue of phi32_gp79, a middle gene product that inhibits σ70 transcription and activates phi32_gp36-dependent transcription [There does not appear to be specific shut-off of either early phage or host transcription during 7-11 development. Presumably, SaPh711_gp47, which binds RNAP core better than σPromoters recognized by SaPh711_gp47 consist of two consensus elements: GTAAtg –(16)- aCTA. Promoters recognized by phi32_gp36 consist of a single consensus element tAATGTAtA. For both cases, transcription start sites are located 6–9 bp upstream relative to the promoter sequence. The absence of the -35 element in phi32_gp36 promoter can be explained by the absence of homology with region 4 of other sigma factors (region 4 is responsible for the recognition of -35 promoter elements) in phi32_gp36. Conversely, the amino acid sequence in the C-terminal part of SaPh711_gp47 is homologous to region 4 of known σ factors and should be therefore capable of sequence-specific recognition of DNA.24 from Winogradskyella sp. PG-2 (WP_045474069.1), a σ70 family factor from Aeribacillus pallidus CIC9 (WP_094245485.1) and a hypothetical protein from Desulfuromonadales bacterium (HIJ97285.1). Promoter specificity for RNAP holoenzymes containing these most closely related to SaPh711_gp47 proteins is unknown. Given the evolutionary distance between phiEco32 and 7-11 σ factors, it is clear that the ancestors of these phages must have acquired their σ factor genes independently and from different sources. Yet, the locations (but not the sequences) of promoters recognized by phi32_gp36 and SaPh711_gp47 holoenzymes in their respective genomes are conserved, providing an interesting evolutionary example of how functional constraints on the expression strategy of phage genes lead to common but independent solutions. Given that organized transcription of nonearly phage genes in phi32 and 7-11 group phages depends on independently acquired σ factors recognizing multiple distinct promoters, we predict that further analysis of related phages shall either reveal interesting hybrid variants or, conversely, additional phages with other, yet to be described, pairs of σ -factor genes and promoters they recognize.The 7-11 σ factor, SaPh711_gp47, is a distant homologue of phi32_gp36 and other phiEco32-like phages’ σ factors (~15% similarity). The closest homologues of SaPh711_gp47 (~50% similarity) are σ