Despite the functional importance, little is known about the regulation of rpoA polycistron. In this work, we show that mTERF6 directly associates with a 3′-end sequence of rpoA polycistron in vitro and in vivo, and that absence of mTERF6 promotes read-through transcription at this site, indicating that mTERF6 acts as a factor required for termination of plastid genes’ transcription in vivo. In addition, the transcriptions of some essential ribosome subunits encoded by rpoA polycistron and PEP-dependent plastid genes are reduced in the mterf6 knockout mutant. RpoA, a PEP core subunit, accumulates to about 50% that of the wild type in the mutant, where early chloroplast development is impaired. Overall, our functional analyses of mTERF6 provide evidence that it is more likely a factor required for transcription termination of rpoA polycistron, which is essential for chloroplast gene expression and chloroplast development.The expression of plastid genes is regulated by two types of DNA-dependent RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). The plastid  Arabidopsis)2. The Arabidopsis plastid genome contains 87 potential protein-coding genes, 4 rRNA genes and 37 tRNA genes3. These plastid genes are transcribed into monocistronic and polycistronic mRNAs4. Two types of DNA-dependent RNA polymerases, nuclear-encoded RNA polymerase (NEP) and plastid-encoded RNA polymerase (PEP), are responsible for the transcription of plastid genes6. NEP is a single-subunit enzyme that transcribes housekeeping genes, including rpoA, rpoB, rpoC1 and rpoC28. PEP possesses the major RNA polymerase activity in the chloroplast and transcribes over 80% of the plastid genes7. The four core subunits of PEP  are encoded by the genes rpoA, rpoB, rpoC1 and rpoC2, respectively, and are translated from two polycistrons, L23-L2-S19-L22-S3-L16-L14-S8-L36-S11-rpoA and rpoB-rpoC1-rpoC29.Chloroplasts are semi-autonomous organelles derived by endosymbiosis from a relative of present-day cyanobacteria. The plastid genome is a conserved circular double-stranded DNA of 120 to 160 kbp , are required for regulating the plastid gene expression mediated by both NEP and PEP14. After post-transcriptional modification, the translational events are initiated on the chloroplast ribosome16. Similar to bacteria, the chloroplast has the bacterial-type 70S ribosome, composed of rRNA and protein subunits and assembled into two multi-components of the 50S (large) and 30S  ribosomal subunits18. The 50S ribosomal subunit consists of 23S, 5S and 4.5 rRNA and 33 proteins, of which 24 are nuclear-encoded, whereas the 30S subunit harbors only one 16S rRNA in addition to 24 proteins, of which 12 are nuclear-encoded19. The rest of the chloroplast gene-encoded proteins of the translation machinery can be divided into essential and non-essential components19. The L23-L2-S19-L22-S3-L16-L14-S8-L36-S11-rpoA polycistron encodes 10 ribosome proteins . Only L36 belongs to the non-essential translation machinery in both bacteria (Escherichia coli) and plants19. Despite the functional importance, the precise mechanisms underlying the transcriptional regulation of the rpoA polycistron remain elusive.The primary transcripts produced by NEP and PEP often undergo post-transcriptional modification, including splicing, maturation, trimming of 5′ and 3′ ends and RNA editing before translation21. mTERF2 binds to the mitochondrial DNA (mtDNA) in a non-sequence-specific manner22. mTERF3 and mTERF2 act as positive and negative regulators in mitochondrial DNA transcription, respectively24. mTERF4 regulates the translation of mitochondrial genes by association with rRNA27. Both mterf3 and mterf4 knockout mice cause embryo lethality, and functional studies suggest their pivotal roles in regulating ribosome biogenesis27.The mitochondrial transcription termination factor (mTERF) family was first identified to be responsible for mitochondrial transcription termination at a site adjacent to the mitochondrial 16S rRNA gene28. Arabidopsis contains 35 mTERF proteins, and at least 11 are predicted to be localized in the plastid29. SOLDAT10/mTERF1 is involved in chloroplast retrograde signaling, and absence of SOLDAT10 leads to embryo lethality30. RUG2/BSM (RUGOSA2/BELAYA SMERT)/mTERF4 encodes a chloroplast protein involved in splicing group IIa introns of plastid genes31. Zm-mTERF4, the maize ortholog of the Arabidopsis protein BSM/RUG2, is required for accumulation of plastid ribosomes and splicing of several group II introns in the chloroplast32. In addition to its direct function in regulating plastid gene expression, mTERF4/COE1 is required for regulating un-processed plastid RNA-triggered plastid retrograde signaling33. MDA1/mTERF5 functions in chloroplast development and abiotic stress responses30. The mda1 mutant exhibits a pale pigmentation34. twr-1/mterf9 was identified as a yellow mutant35 and alters chloroplast development and tolerance to abiotic stress36. Although several mTERF members have been characterized in Arabidopsis and were emphasized to play a role in regulating organellar gene expression, whether these members also involve in the transcription termination of plastid genes is unknown.In stark contrast to mammals, in plants, the functions of mTERFs are poorly understoodArabidopsis37. The knockout mutant mterf6 has an albino phenotype and is defective in the maturation of trnI.237. By functional characterization of mTERF6, we found that mTERF6 is required for the transcription termination of plastid genes in vivo. The decreased RpoA protein level and PEP-dependent plastid gene transcription indicated that mTERF6-dependent transcription termination of the rpoA polycistron is necessary for PEP-dependent chloroplast gene expression and chloroplast development.mTERF6 is dual-targeted to both chloroplast and mitochondria in PIGMENT DEFECTIVE 191 (PDE191)38, from the Arabidopsis Biological Resource Center (ABRC). The CS16125 line was renamed “mterf6-5” to distinguish the reported mutant lines of mterf6-1, mterf6-2, mterf6-3 and mterf6-437 alleles. PCR analysis with T-DNA and genome-specific primers indicated that the mutant plants are homozygous for the insertion. The progenies of the selfed heterozygote segregated with a ratio of green plants to albino plants of 233:67 , which suggests that this mutant is inherited as a single recessive allele. In addition, we obtained another mterf6 allele (SALK_116335) from ABRC that was designated “mterf6-6”. The isolation of the genomic sequence flanking the left border of the T-DNA revealed that the T-DNAs are inserted in the first exon of MTERF6 at positions +58-bp and +34-bp relative to the start codon in mterf6-5 and mterf6-6, respectively  and the 731-bp downstream sequences  via Agrobacterium tumefaciens-mediated transformation39. Among the 40 identified transgenic plants, 5 had the mterf6-5/mterf6-5 background. Meanwhile, we also generated FLAG tagged complementation lines , MTERF6 shows alternative splicing to produce three transcripts named AT4G38160.1, AT4G38160.2 and AT4G38160.3  demonstrated that all three genomic sequences .According to 0.3 Fig. . We desi0.3 Fig. . Semiquased Fig. . The expves Fig. . AT4G381ves Fig. . The expPCR Fig. . OverallMTERF6 are a 37.9-kD protein of 333 amino acids, a 41.3-kD protein of 363 amino acids, and a 43.1-kD protein of 378 amino acids. The structures of these proteins are similar to that of members of the mTERFs family reported in metazoans and plants40. SMART analysis revealed that each protein contains 7 mTERF-motif repeats (http://smart.embl-heidelberg.de/) , and ycf2 and accD were chosen as NEP-dependent genes (class III). The genes atpB and atpE were selected as both PEP and NEP-dependent genes (class II)46. The transcriptional levels of both the class I and II genes were downregulated compared with the WT  and some other plastid genes. Since the exact termination sites of the plastid transcripts are not clear, we designed primer sets to characterize the non-transcribed regions of the plastid genome. We used ycf9 as an example to identify the downstream non-transcription region  inside the gene and several primer sets downstream  that cover the gene interval region, were designed  were higher in mterf6-5 than the WT  of rpoA-3′ transcript is far less than the fold change (mterf6/WT) of rpoA-T transcript  assays as described51. We generated a construct of a native promoter driving the MTERF6.1 genomic sequence (including 1005-bp upstream and 999-bp genomic sequences without stop codons) that was fused with a FLAG tag, pMTERF6::MTERF6.1-FLAG (see Methods). This construct could well complement the phenotypic defects of the mterf6-5 mutant. This transgenic line was used for ChIP assay. The mTERF6.1-FLAG protein was immunoprecipitated from a total leaf protein extract of the transgenic plants by using an anti-FLAG antibody coupled with a magnetic bead-conjugated IgG (IP+). The IgG-conjugated magnetic beads were used for mock ChIP (IP−). The amount of the fusion protein in the immunoprecipitate samples was detected by western blot analysis with an anti-FLAG monoclonal antibody  were pulled down with the FLAG antibody and examined by ChIP quantitative PCR (ChIP-qPCR). The result showed a specific enrichment of mTERF6 at the 3′ ends of rpoA and the other two genes, rbcL and ycf5  in vivo.According to previously characterized mTERF homologues in mammals and green alga, their function in transcription termination relies on direct binding to mtDNAody Fig. . A fragmron Fig. , was usecf5 Fig. , which srpoA gene was direct, we performed electrophoretic mobility shift assay (EMSA). The interval region between rpoA and petD contains a sequence (probe B) that is similar to the binding site of the mitochondrial D-loop binding protein (mtDBP) in sea urchin (Paracentrotus lividus)52  5GT sequence in their 3′-end 52 Fig. . A 122 b)52 Fig. . A shift)52 Fig. , with no)52 Fig. . When wetly Fig. , which s B) Fig. . When pr B) Fig. . Moreove B) Fig. , which send Fig. , which s20, mTERF factors have been demonstrated to belong to a multifunctional protein family regulating mtDNA replication, transcription termination and translation55. One of the most important functions described for the mTERF factors is the termination of mtDNA transcription at specific sites56. Human mTERF, also called mTERF1, is one of the best-studied mTERFs. It can regulate mtDNA transcription termination by binding two sites in mtDNA57. Also, the homologs of mTERF1 are involved in transcription termination in the mitochondria of other metazoans60 and green alga (Chlamydomonas reinhardtii)50. Arabidopsis contains 35 mTERF proteins with approximately 11 mTERF members localized in the chloroplasts29. To date, four members of the mTERF family have been functionally characterized in chloroplasts of Arabidopsis, and their mutants show defective plastid gene expression and chloroplast development61. However, whether these mTERFs are also involved in the transcription termination of plastid genes is unclear, although mTERF6 was reported to localize in the chloroplast and has transcription termination activity in vitro37. Here, we showed that mTERF6 is directly involved in the transcription termination of the plastid gene rpoA . Pol II termination depends on RNA 3′-end processing signals and termination factors66. In Arabidopsis, a nuclear-encoded factor, RHON1, was reported to terminate the transcription of the plastid gene rbcL67. It can bind to both mRNA and single-strand DNA67. Here, we show that mTERF6 binds with higher capacity to single strand versus double strand of 3′-end DNA of rpoA , which suggests that the function of RHON1 in transcription termination is less conserved67. Protein sequence alignment showed that mTERF6 is well conserved among the plant species  and transcription termination factors (DNA/RNA-bound proteins) control transcription termination. In the prokaryote, transcription is terminated by intrinsic termination and Rho-dependent terminationpoA Figs . The postrnI.2 gene directly37. In terms of our data, it seems that mTERF6 binding regions contain the common motif ATT(N)5GT  is also much less than that of its corresponding gene 5 which is important for plastid gene transcription and translation. mTERF6 could directly associate with the rpoA polycistron for transcription termination  in mterf6-5 . These findings and conclusions were reinforced in the mterf6-1 mutant as the molecular function of mTERF6 was further confirmed in seedlings of the mterf6 weak allele (mterf6-1)37  and have yellow leaves, which can later turn green43. The transcription levels of the PEP-dependent plastid genes in mterf6-5 are similar to those of ecb2-2 and ys143  and mutant Arabidopsis plants were grown on Murashige and Skoog (MS) medium containing 0.7% (w/v) agar at 22 °C with a day/night cycle of 16 h/8 h at 120 μmol m−2 s−1. The T-DNA insertion lines CS16125 and SALK_116335 were obtained from the Arabidopsis Biological Resource Center . The mterf6-1 (GABI_152G06) mutant was kindly provided by Dr. Tatjana Kleine.Wild-type  was designed using the left sequence of the T-DNA. The specific primers, containing an LP and an RP, were designed by using the upstream and downstream sequences of the insertion sites, respectively. For CS16125, the primers were for LP: 5′-ACTCTGCACAAGGCATCAACC-3′; and RP: 5′-TGATGCCACATCGCTTTGAGC-3′. For SALK_116335, they were for LP: 5′- ATTGTACCCTTGTCACGCATC-3′; and RP: 5′-CGATGCTGTAGCTGATAAGCC-3′ (Supplementary Table http://www.toyobo.co.jp) and was fused to the pCAMBIA1300 vector. The primer pairs were for MTERF6-Sac-F: 5′-gagctcGTGGGGACGTTAGGAGAGTGACCAG-3′; and MTERF6-Sal-R: 5′ -gtcgacTGATTACCTGCGATTATAGCCATTC-3′ (Supplementary Table MTERF6/mterf6) by Agrobacterium tumefaciens-mediated transformation39. Then, transgenic plants were screened by PNS culture medium with 80 mg L−1 hygromycin and further identified with the genomic-specific primers F: 5′-ACTCTGCACAAGGCATCAACC-3′; and R: 5′-TGATGCCACATCGCTTTGAGC-3′.As for the genetic complementation experiment, the full-length 3229-bp genomic fragment (containing the sequence 1005-bp upstream of the promoter to 2224-bp downstream from the start of transcription) was amplified by KOD plus polymerase  under normal culture conditions. Later, a Hitachi H7500 transmission electron microscope  was used to observe the ultrastructure of chloroplasts73. For generation of transgenic plants, the 2004-bp MTERF6.1 genomic sequence before the stop codon of MTERF6.1 was amplified with the primer set MTERF6-Sac-F/MTERF6.1-Sal-R and cloned into the pCAMBIA1300 vector, following FLAG and 39-Nos sequences. Furthermore, the genomic sequences of MTERF6.2 (2375 bp) and MTERF6.3 (2495 bp) were cloned for construction by using the same method  were screened and identified by using the same method as for the complementation lines mentioned above.For phenotype characterization, plants grown on MS medium were photographed by using an anatomic microscope. The small leaf segments were collected from 7- and 14-day-old Col-0 and 46. The specific primers are in Supplementary Table The procedures for the purification of total RNA, for cDNA synthesis, RT-PCR, and quantitative real-time RT-PCR were described previously74. The anti-RpoA polyclonal antibody was raised against a synthetic peptide of RpoA by the GL Biochem company (http://www.glbiochem.com)47.Total protein from a 7-day-old seedling was extracted as describedin vitro, cDNA of mTERF6 without the region of transit peptide, was cloned into the pMAL-c5X vector in-frame with the maltose-binding protein (MBP) gene. Nde I and BamH I sites were introduced into forward and reverse primers, respectively (see Supplementary Table http://www.thermoscientific.com). Each 20-μL binding reaction, containing 2 μL binding buffer, 5 mM MgCl2, 1 mM EDTA, 2.5% glycerol, 0.05% NP-40, 50 ng/μL poly (dI-dC), 20 fmal probe and 0.5 μg recombinant protein, was performed and incubated at room temperature for 20 min.To obtain the mTERF6 protein pMTERF6::MTERF6.1-FLAG in the mterf6-5/mterf6-5 background) were identified and used as material for ChIP experiments. The ChIP assay was performed as previously described51. Briefly, 3 to 5 g young leaves harvested from 3-week-old plants planted in soil were immersed in ~100 mL of ice-cold TBS  supplemented a final concentration of 0.01% (v/v) Silwet L-77 and 1% (v/v) formaldehyde. Leaves were fixed by vacuum-infiltration and washed with fresh TBS solution containing 0.3 M glycine at 4 °C. Leaves were ground in liquid nitrogen. The powder was resuspended in 3 mL of lysis buffer  and homogenized by vortexing. The resultant slurry was filtered through two layers of Miracloth, then the crude extracts in the filtrate were sonicated on ice with a sonifier Bioruptor . The sample was centrifuged at 15000 g for 20 min at 4 °C. The supernatant was immunoprecipitated using antibody (anti-FLAG)-coupled magnetic protein G beads (Life Technologies) (IP+) and using protein G beads (IP−) as a control. The DNA was isolated from immunoprecipitated products and used for ChIP-qPCR analysis.The complemented T2 transgenic plants (Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: mTERF6 (AT4G38160), RpoA (ATCG00740), PetD (ATCG00730), RbcL (ATCG00490), Ycf5 (ATCG01040), TUB8 (AT5G23860), L23 (ATCG01300), L2 (ATCG00830), S19 (ATCG00820), L22 (ATCG00810), S3 (ATCG00800), L16 (ATCG00790), L14 (ATCG00780), S8 (ATCG00770), L36 (ATCG00760), S11 (ATCG00750), PsaB (ATCG00340), PsbA (ATCG00020), PsbB (ATCG00680), Ycf2 (ATCG00860) and AccD (ATCG00500), AtpB (ATCG00480), AtpE (ATCG00470), Rps14 (ATCG00330), PsbC (ATCG00280), PsbJ (ATCG00550), Ycf9 (ATCG00300), RpoB (ATCG00190), RpoC1 (ATCG00180), RpoC2 (ATCG00170) TrnI.2 (ATCG00930).Sequence data from this study can be found in the Supplementary Information


Streptomyces species are faced with large variations in carbon or nitrogen sources, phosphate, oxygen, iron, sulfur, and other nutrients. These drastic changes in key nutrients result in an unbalanced metabolism that have undesirable consequences for growth, cell differentiation, reproduction, and secondary metabolites biosynthesis. In the last decades evidence has accumulated indicating that mechanisms to correct metabolic unbalances in Streptomyces species take place at the transcriptional level, mediated by different transcriptional factors. For example, the master regulator PhoP and the large SARP-type regulator AfsR bind to overlapping sequences in the afsS promoter and, therefore, compete in the integration of signals of phosphate starvation and S-adenosylmethionine (SAM) concentrations. The cross-talk between phosphate control of metabolism, mediated by the PhoR–PhoP system, and the pleiotropic orphan nitrogen regulator GlnR, is very interesting; PhoP represses GlnR and other nitrogen metabolism genes. The mechanisms of control by GlnR of several promoters of ATP binding cassettes (ABC) sugar transporters and carbon metabolism are highly elaborated. Another important cross-talk that governs nitrogen metabolism involves the competition between GlnR and the transcriptional factor MtrA. GlnR and MtrA exert opposite effects on expression of nitrogen metabolism genes. MtrA, under nitrogen rich conditions, represses expression of nitrogen assimilation and regulatory genes, including GlnR, and competes with GlnR for the GlnR binding sites. Strikingly, these sites also bind to PhoP. Novel examples of interacting transcriptional factors, discovered recently, are discussed to provide a broad view of this interactions. Altogether, these findings indicate that cross-talks between the major transcriptional factors protect the cell metabolic balance. A detailed analysis of the transcriptional factors binding sequences suggests that the transcriptional factors interact with specific regions, either by overlapping the recognition sequence of other factors or by binding to adjacent sites in those regions. Additional interactions on the regulatory backbone are provided by sigma factors, highly phosphorylated nucleotides, cyclic dinucleotides, and small ligands that interact with cognate receptor proteins and with TetR-type transcriptional regulators. We propose to define the signal integration DNA regions  that assemble responses to different stress, nutritional or environmental signals. These integrator sites constitute nodes recognized by two, three, or more transcriptional factors to compensate the unbalances produced by metabolic stresses. This interplay mechanism acts as a safety net to prevent major damage to the metabolism under extreme nutritional and environmental conditions.Soil dwelling  The promoter region of the actinorhodin regulatory gene actII-orf4 is recognized by at least nine transcriptional factors of different families, including GlnR, AfsS, AfsQ1, AdpA, AtrA, DasR, DraR, AbsA2, and AbsC  serve to integrate multiple signal cascades that respond to different nutritional and environmental stress signals in Streptomyces , it is likely that each of these transcriptional factors covers at least the major groove or a full turn of DNA (11 nucleotides). Moreover, some of these regulators, e.g., GlnR or PhoP, act as dimers or even oligomers and therefore cover a relatively large stretch of DNA. Mutations affecting these integrator sites alter not only the binding to one transcriptional factor but also to other interacting factors.The interactions between transcriptional factors and regions upstream of some gene clusters are really elaborated and suggests that there is a fine tuning of the expression of important gene clusters by alternative transcriptional factors. There are many reports of putatively interacting transcriptional factors that affect the biosynthesis of secondary metabolites but the molecular evidence supporting those interactions is scarce. In this article we focus on the most relevant and best-known cases of overlapping interactions between transcriptional factors that allow us to get an insight into how the cells integrate inputs from environmental and nutritional stresses. Those studied for which there are experimental evidence of DNA binding and/or footprinting data are summarized in Streptomyces lividans and S. coelicolor  in PhoP, that causes a rearrangement of the protein structure resulting in an activation of the DNA-binding domain in the carboxy-terminal region of this transcriptional regulator. The phosphorylated form of PhoP binds specific sequences named PHO boxes and generally activates, but occasionally represses, the expression of genes of the Pho regulon. The Streptomyces PHO boxes are formed by direct repeat units (DRus) of 11 nucleotides of which seven nucleotides (GTTCACC) are well conserved  changes the charge of this region thus weakening the binding of the mutated PhoP to the DNA backbone. These observations were supported by electrophoretic mobility-shift assay (EMSA) which showed that the mutation to glutamate removed the binding ability of PhoP and, therefore, altered the control of expression of the Pho regulon genes .Since the 1970s, it is known that erythromycin is formed by condensation of 7-three carbon units provided by one propionyl-CoA as starter unit and six methylmalonyl-CoA elongation units . Propionoduction . Interesoduction . This isS. coelicolor is controlled by the AfsR regulator that belongs to the large size SARP family  that controls expression of the act and red gene transcriptional regulators .Expression of actinorhodin and undecylprodigiosin genes in P family . AfsR  concentration level. Methionine is one of the major methyl donors in microbial metabolism. This amino acid regulates positively or negatively the biosynthesis of many antibiotics, including actinorhodin  as carbon source promotes drastically the expression of phoR–phoP, pstS, and other key genes of the Pho regulon. The findings that in S. tsukubaensis glucose stimulates significantly the genes involved in phosphate transport, and the similar effect of fructose in S. lividans indicates that an increase in the concentration of these hexoses in the culture medium favors the uptake of inorganic phosphate to balance the carbon and phosphate metabolism through the glycolysis, pentoses phosphate pathway, and TCA cycle. Reciprocally, inorganic phosphate affects many of the genes of the carbon metabolism and this regulation is mediated by PhoP .The interplay between GlnR and PhoP in pstSCAB operon was expressed as a transcriptional unit in A. mediterranei  and also alters secondary metabolism , member of the MtrAB TCS has an important role in nitrogen regulation . The mtrutamicum . In S. ctabolism , b. ThesamtB–glnK–glnD cluster  and is dependent on the cAMP levels in the cells. Several studies have reported that in S. coelicolor, the carbon catabolite regulation of alternative carbon sources by glucose is not mediated by a CRP-homologous protein. Instead, carbon catabolite regulation is mediated by the glycolytic enzyme glucose kinase  was initially found as a result of the observation that it increases the production of actinorhodin and undecylprodigiosin in S. coelicolor  the pstS gene involved in high affinity phosphate transport; and (3) several bidirectional promoters related to cell differentiation including whiD and bldM   that whe species  and has ptomycin . ExpressdpA gene . Therefo ligands  with theStreptomyces species , whose function is still partially unclear. Sequencing of Streptomyces genomes has revealed that these actinobacteria contain many alternative sigma factors, 69 (66 in the chromosome and 3 in plasmids) in S. coelicolor . The result is that the interaction of the cognate sigma factor with the RNA polymerase is blocked by binding of the corresponding anti-sigma factor. This adds an additional level of control on the interaction of transcriptional factors and sigma factors in the safety network. An additional protein that interacts with the RNA polymerase core enzyme is the ancillary protein RpoZ that in S. coelicolor is controlled by the phosphate regulator PhoP; mutant in rpoZ are impaired in cell differentiation and secondary metabolites biosynthesis ppGpp may influence positively or negatively antibiotic biosynthesis .Another group of molecules that interact with the RNA polymerase are the guanosine hyperphosphorylated molecules, namely guanosine tetra-phosphate (ppGpp) and guanosine pentaphosphate (pppGpp) These compounds initially characterized in ino acid  and conselA/spoT . Based oabolites . Indeed,abolites . Mutantsclusters . Global ynthesis . Among td mutant . Howeverynthesis . The higynthesis . There ielicolor . Bioinfoognition . The S.  culture . Interesin vitro . Howeverand redD . cAMP haS. venezuelae (S. coelicolor. Disruption of genes encoding these c-di-GMP biosynthetic and degradative enzymes impaired actinorhodin and undecylprodigiosin formation but the mechanism involved in this effect is unknown. Cell differentiation and spore pigmentation were also affected in the c-di-GMP-null mutant (B. subtilis and they are also present in actinobacteria including Streptomyces species (Streptomyces genomes. The intracellular c-di-AMP levels affect many aspects of cell metabolism, including cell wall preservation and DNA integrity maintenance, but the major aspect regulated by this nucleotide is the ion homeostasis of the cell, particularly osmotic stress balance (Another modified nucleotide, c-di-GMP, has been studied recently in nezuelae . This nul mutant . The las species . The int balance . The intStreptomyces and related actinobacteria (A further layer of interactions in the protection net is exerted by small ligands that bind to receptor proteins and caused distinct regulatory effects. These ligands are small organic molecules different from normal nutrients that are produced by bacteria and fungi in complex habitats and exert effect in the same species or between different species . Severalbacteria . The disbacteria , and havbacteria , do not bacteria . In concStreptomyces species and related actinobacteria but equivalent protection nets exist in many other bacteria and in lower eukaryotes. However, the molecular mechanisms that protect the cells in response to nutrient starvation and environmental stresses are rather different from those observed in Streptomyces. For example, in E. coli there are only seven alternative sigma factors. The mechanism of control of the Pho regulon in E. coli is somehow different from that observed in Streptomyces species (S. coelicolor (E. coli the phosphorylation of the response regulator PhoB occurs also by acetyl-phosphate. Moreover, the phosphorylation of the PhoB regulator is also performed by histidine kinases of other two-components systems, as is the case of CreC histidine kinase (B. subtilis and again there are differences with respect to the Streptomyces network; for example, the phosphate starvation response is mediated by a phosphorelay mechanism that involves at least three different steps in a cascade through the PhoR–PhoP TCS, the Spo0 system, and the ResD/ResP system (This article focuses on the protection net in  species . In contelicolor , in E. ce kinase . InterplP system . In concStreptomyces that are recognized by interacting transcriptional factors.All available evidence indicates that there are signal integration sites in the genome of atrA promoter region that is bound by PhoP and AdpA. It will be of utmost interest to establish the hierarchy of the integration sites to get a clear idea of the role of these sites on regulation of overall metabolism, particularly to advance our knowledge of the system biology of these actinobacteria.These nucleotide sequences upstream of key genes constitute integrative nodes that serve to compensate the unbalance produced by the lack of a key nutrient or for its excess that would impair the cell metabolism. Probably there are several hundreds of these integrator sites; however, it remains to be confirmed that some of these sites serve to integrate signals for the major transcriptional factors such a PhoP, GlnR, MtrA, and so on, whereas other sites probably integrate two or a few signals as appears to be the case of the Finally, an interesting conclusion is that mutations that initially may be designed to change a specific regulatory phenomenon that affects a single cluster may, however, alter the expression of many different clusters through the interplay of these major transcriptional factors. An intriguing question is whether the signal integration sites in the DNA correspond to open “unprotected DNA region” at difference of other DNA regions that may be protected by histone-like proteins.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.


ZBTB7A, in particular, is reported to be implicated in a wide variety of physiological and cancer-related cell events. These physiological processes include regulation of erythrocyte maturation, B/T cell differentiation, adipogenesis, and thymic insulin expression affecting consequently insulin self-tolerance. In cancer, LRF/ZBTB7A has been reported to act either as oncogenic or as oncosuppressive factor by affecting specific cell processes  in opposed ways, depending on cancer type and molecular interactions. The molecular mechanisms via which LRF/ZBTB7A is known to exert either physiological or cancer-related cellular effects include chromatin organization and remodeling, regulation of the Notch signaling axis, cellular response to DNA damage stimulus, epigenetic-dependent regulation of transcription, regulation of the expression and activity of NF-κB and p53, and regulation of aerobic glycolysis and oxidative phosphorylation (Warburg effect). It is a pleiotropic TF, and thus, alterations to its expression status become detrimental for cell survival. This review summarizes its implication in different cellular activities and the commonly invoked molecular mechanisms triggered by LRF/ZBTB7A’s orchestrated action.Transcription factors (TFs) consisting of zinc fingers combined with BTB  domain (ZBTB) are a highly conserved protein family that comprises a multifunctional and heterogeneous group of TFs, mainly modulating cell developmental events and cell fate. LRF/ ZBTB7A) appears in the literature with several synonyms: POK erythroid myeloid ontogenic factor or POZ and Krüppel-erythroid myeloid ontogenic factor (Pokemon), factor binding IST protein-1 (FBI-1), HIV-1 inducer of short transcripts binding protein, TTF-I-interacting peptide 21, DKFZp547O146, etc. The lymphoma/leukemia-related factor (LRF) preferentially refers to the protein product encoded by the ZBTB7A gene.Zinc finger and BTB domain containing 7A  BTB domain, capable for the formation of homo- or hetero-dimers. These features provide LRF/ZBTB7A with collaboration abilities and highly contextual activities towards cellular function, such as transcription co-repressor activity, protein and histone acetyltransferase binding, proximal promoter sequence-specific DNA-binding, and DNA-binding with consecutive attraction and modulation of other TFs activities.Characteristic domains of the LRF/ZBTB7A exerts its action within the nucleus compartment; herein, cell responses may differ in different tissues and local cell microenvironments. Cell processes disturbed by the aberrant expression of LRF/ZBTB7A TF are discussed in the present manuscript in an effort to give prominence to its both widespread gene regulatory effects and components of cellular life-cycle affected. Its basic ability to cooperate and attract various complexes to the broader “targeted” regulated genome area determines not only its pleiotropic and sometimes conflicting action, but also its potential to act on a case-by-case basis, depending on the epigenetic profile of the genome, due to its preferred binding sites within rich CG regions  targeted genes in early human preadipocyte differentiation [Zbtb7a, it was evident that PPARγ and aP2 mRNAs were both significantly upregulated [Zbtb7a showed evidence for accelerated adipogenesis with earlier induction of differentiation markers and enhanced lipid accumulation, suggesting that this TF contributes significantly in the differentiation process due to downregulation of E2F-4 [ZBTB7A protein levels peak at the end of mitotic clonal expansion, it has been hypothesized that this TF facilitates the cells’ terminal differentiation during adipogenesis. Indeed, 3T3-L1 cells stably overexpressing Lrf/Zbtb7a showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, Lrf/Zbtb7a reduced the transcriptional activity of the cyclin A promoter [ZBTB7A reduces the activity of cyclin A promoter indirectly by inhibiting Sp1 while represses the activity of E2F-4 promoter directly [ZBTB7A is induced during the early phases of human and murine preadipocyte differentiation where it contributes to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation by exhibiting thus a dual function of promoting both growth arrest and terminal adipocyte differentiation.Ιn human adipose tissue, LRF/ntiation , 116. Coegulated , and thiof E2F-4 , a transof E2F-4 . Since tpromoter . Similardirectly  Fig. 3)ZBTB7A mRZBTB7A, AP-1 (JUN), and EWS RNA binding protein 1 (EWSR1), to the human insulin gene promoter alone or joined to a class I or class III VNTR allele, located 596 bp upstream of the insulin gene [Type 1 diabetes (T1D) is a complex disease with a strong genetic background, and there are more than 40 loci identified to correlate with increased T1D susceptibility , 121. Inlin gene –126. Thelin gene , 128. Thlin gene . The AIRlin gene , 131 andlin gene .ZBTB7A, AP-1, and EWSR1) could induce insulin expression in transfected HEK-293 cells, but LRF/ZBTB7A provided the most robust results especially in the presence of the AIRE, with an additional 11-fold increase of the insulin mRNA levels from a co-transfected reporter driven by the class III VNTR allele. Consequently, LRF/ZBTB7A was identified as a strong candidate for the regulation of thymic insulin expression [Zbtb7A promoter activity and enhances endogenous Zbtb7A mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the PI3K/AKT cascade and transcription factor Sp1, known to recognize and bind methylated regions in genes’ promoter(s) [Zbtb7a expression in HepG2 cells [All three proteins (LRF/pression . On the moter(s) , have beG2 cells .ZBTB7A presents pleiotropic actions and is involved in the regulation of many fundamental physiological cell processes as follows: the terminal erythrocyte and adipocyte differentiation, the lineage cell fate decisions of pre-mature and mature B and T cells, the insulin self-tolerance, and also, pathophysiological conditions such as numerous cancer types.LRF/ZBTB7A is rising to a central player controlling proper cell proliferation and differentiation by participating in several molecular mechanisms and interacting with an impressive variety of binding partners in a tissue- and context-dependent manner to exert either oncogenic or oncosuppressive functions. Altered expression profile of LRF/ZBTB7A has been detected in several types of cancer, supporting the idea that it potentially bridges discrete downstream cell signaling pathways as well as intrinsic/extrinsic apoptotic pathways via regulation of mutual participants. To this end, LRF/ZBTB7A functions mainly as a co-operator with other TFs rather than a master or “pioneering” TF and does not directly activate or repress gene promoters as has been established for NF-κB or the Hypoxia Inducible Factor (HIF), but alternatively influences the accessibility of other TFs and DNA-binding proteins.Collectively, LRF/ZBTB7A feature is its penchant for binding CG reach DNA sequences, located within regulatory gene regions and its evident ability to from homo- or hetero-dimmers capable to recruit chromatin remodeling complexes such as HDACs and NuRD. It remains to be clarified whether LRF/ZBTB7A belongs to the recently introduced generation of TFs, with the capability to sense epigenetic events and orchestrate reciprocal action as a response, which further transduces chromatin context and triggers changes in gene expression.A critical LRF/


HOXA9 (Homeobox A9) is a homeotic transcription factor known for more than two decades to be associated with leukemia. The expression of HOXA9 homeoprotein is associated with anterior–posterior patterning during embryonic development, and its expression is then abolished in most adult cells, with the exception of hematopoietic progenitor cells. The oncogenic function of HOXA9 was first assessed in human acute myeloid leukemia (AML), particularly in the mixed-phenotype associated lineage leukemia (MPAL) subtype. HOXA9 expression in AML is associated with aggressiveness and a poor prognosis. Since then, HOXA9 has been involved in other hematopoietic malignancies and an increasing number of solid tumors. Despite this, HOXA9 was for a long time not targeted to treat cancer, mainly since, as a transcription factor, it belongs to a class of protein long considered to be an “undruggable” target; however, things have now evolved. The aim of the present review is to focus on the different aspects of HOXA9 targeting that could be achieved through multiple ways: (1) indirectly, through the inhibition of its expression, a strategy acting principally at the epigenetic level; or (2) directly, through the inhibition of its transcription factor function by acting at either the protein/protein interaction or the protein/DNA interaction interfaces. Antennapedia and Bithorax ancestors, the HOX-Like subgroup corresponds to the HOX cluster genes and is the only group of HD proteins conventionally named “HOX genes.” Organized into four paralog clusters in animals, the number and identity of HOX genes varies depending on the species. In humans, 39 HOX proteins are organized from 1 to 13  in the four paralog clusters A to D. After their spatiotemporal expression, critical for patterning of the anterior–posterior axis in embryos, HOX gene expression is largely repressed in adults but controlled reactivation allows dynamic expression in adults to lead or reactivate some cellular processes, including hematopoiesis [Transcription factors represent a large class of proteins with more than 1500 members, of which 15%–20% are considered oncogenes ,4. Derivopoiesis ,7,8, wouopoiesis , vasculaopoiesis ,13,14,15opoiesis ,18,19,20opoiesis .A large proportion of HOX cluster genes are considered oncogenes due to their implication in translocations, mutations or improper expression dynamics in some tissues. They are associated with cancer at the initiation, development or metastasis stages and are over-expressed in cancer cells relative to normal tissue , are frequently over-expressed in BXH-2 murine leukemic cells . It was In human leukemia, the first implication of HOXA9 was highlighted by the discovery of the NUP98-HOXA9 fusion protein resulting from t translocation ,32, a raBesides expressed as an oncogenic fused protein, the implication of structurally unmodified HOXA9 as a leukemic driver was then evidenced as part of its over-expression in leukemic cells. Indeed, the relevance of HOXA9 expression in global survival of human leukemia patients was first demonstrated on gene expression signatures relating to patient outcome: HOXA9 was evidenced as the protein presenting the highest correlation with poor prognosis in a series of nearly 7000 genes . This asThe most described HOXA9-associated leukemias are: (1) acute leukemia (either myeloid or lymphoid) bearing MLL  fusions ,45,46,47The AML subtype MPAL preferentially affects infants or is developed as a therapy-induced leukemia. MPAL is associated with poor prognosis with a five-year survival rate of less than 40% in infants compared to ~90% for non-MPAL . The genIn mice, grafting of bone marrow cells with retroviral transduction of MLL fusion proteins deregulated the expression of Hox genes . All MPAThe NPM1 mutations are generally present in de novo adult AML with normal karyotype and were evidenced to be correlated with the over-expression of HOXA, MEIS1, and FLT3 genes ,74. UndeIn parallel, Brunetti et al. demonstrated that HOXA (and HOXB) genes are not only direct downstream targets of NPM1c+ protein but also that the interaction between exportin-1 (XPO-1), a nuclear pore exporter, and NPM1c+ protein, maintains mutated-NPM1 in the cytoplasmic compartment as an important point explaining AML occurrence . MoreoveThe over-expression of HOXA9 is also found in many NUP98 fusions containing AML samples, accounting for a total of 1%–3% of AML . Some exTrisomy 8 (+8) represents ~10% of all AML and also correlates with a high level of HOXA9 expression . Alone, In many other well-defined cytogenetic or molecular alterations associated with AML, HOXA9 can be frequently over-expressed but not in all patients of the same sub-group, suggesting that further analysis of additional alteration would be required. For instance, EVI1 over-expressing leukemia results from t associated with poor survival in 8%–10% AML and presents over-expression of the HOXA9 gene with a large spread of HOXA9 expression from positive to negative. In total, the proportion of HOXA9-over-expressed AML is ~70%. This high proportion highlights HOXA9 as an interesting potential target to treat such AML. The oncogenic function of HOXA9 in AML is associated with cell proliferation, differentiation blockade, increased malignancy of leukemic cells, and progenitor self-renewal maintenance . InvalidIn order to understand HOXA9-mediated leukemogenesis, the key point would be to identify the network underlying its transcriptional activity using ChIP-sequencing. However, due to the lack of ChIP and ChIP-seq grade antibodies directed against HOXA9 and other HOX proteins to identify endogenous targets for each HOX protein on the chromatin, most global ChIP analyses have used exogenous expression of tagged proteins. This is notably the case of a HA-tagged Hoxa9 protein in a Hoxa9- and Meis1-transformed murine bone marrow cell model to identify thousands of genomic binding regions of the murine Hoxa9 transcription factor, being associated or not with Meis. These studies identified several pro-leukemic Hoxa9 target genes such as Erg (ETS-related gene), Flt3, Lmo2 (LIM domain only 2), and c-Myb (myeloblastosis) . HoweverAlternatively, microarray or RNA-seq analyzes have been successfully performed. Gene expression analyses in models over-expressing or interfering  with HOXA9 expression showed significant transcriptomic modulations in which HOXA9 could act as an activator or a repressor, depending on target gene and cell context . Most HOIf HOXA9 could act alone to trigger leukemia, it requires cofactors to increase its propensity to induce leukemia. For HOXA9, the leukemogenic activity of MEIS1 was discovered through in vivo experiments in BXH-2 mice, a pro-viral insertion model in which 15% of induced AMLs are caused by pro-viral insertion into the Meis1 gene locus . Like HOinhibition of its expression;blockade of the specific protein/protein interaction crucial for its mechanism of action; or, more specifically as part of a transcription factor, the blockade of the interaction with its cognate sequence on the DNA.As HOXA9 is associated with a large proportion of AML, its inhibition is an interesting strategy against AML that could be achieved by different ways:DOT1L, which interacts with a large number of fused partners of MLL, themselves present in the non-oncogenic MLL complex; menin/LEDGF complex, which interacts with the N-terminal domain of MLL; WDR5, which interacts with the C-terminal part of MLL and binds to me-H3K4 marks during the transcription elongation process. Binding of WDR5 in complex with RbBP5 and ASH2L proteins with a WDR5 interacting motif (WIN) changes the conformation of the SET-domain of MLL to activate epigenetic function of the wild-type MLL complex;pTEFb complex, which makes contact with both MLL and DOT1L proteins and traps BRD4 to a large MLL-fusion complex.As presented above, it is well established that HOXA9, together with posterior HOXA genes, is regulated in MPAL at the expression level by epigenetic control driven by the oncogenic fused MLL proteins within a large epigenetic complex associating  the following proteins :DOT1L, MOF, which binds to the SET domain in the C-terminal part of MLL protein and acetylates H4K16 position at transcriptional initiation stage ;UBE2O, which promotes wild-type MLL degradation in response to IL1 treatment and subsequent IRAK4 (interleukin-1 receptor-associated kinase 4) activation pathway  in orderHowever, it will lose interaction to other factors that bind to the C-terminal part of the wild-type MLL protein that is removed in MLL chimeras:In a more global view of the proteins interacting with MLL chimera complexes, also involved are the epigenetic regulators LSD1 (KDM1A) , HDAC 66,67, KDMAll these proteins and/or their protein/protein interaction interfaces represent interesting opportunities to inhibit the oncogenic processes of MLL fusion proteins on HOXA9 expression.Since MLL chimeras interact with multiple protein partners, different strategies were developed with the aim of decreasing HOXA +/- MEIS1 gene expression, restoring differentiation and, hopefully, consequently treating AML.The DOT1L histone methyltransferase (HMT) methylates lysine 79 of histone H3 (H3K79) and is aberrantly recruited at the MLL complex through interaction with the MLL fusion partner moieties, such as AF9 , AF4 5858, AF6 , AF10 1, or ENL Interestingly, DOT1L inhibitor treatments of AML with MLL-PTD , mutateda; Other DOT1L inhibitors a;  were als9e, that interacts with DOT1L at the µM range and reduces HOXA9 and MEIS1 expression [the pyrimidyl-aminoquinoline derivative pression ;3 and 9, identified by high throughput screening [compounds creening ; DC_L115, which binds DOT1L at sub-micromolar concentration ;12 and aza-benzimidazole derivative 13, as two orally bioavailable DOT1L inhibitors [2-chloro benzothiophene derivative hibitors ; the phenoxyacetamide derivatives L01, L03, L04, and L05, identified by hierarchical docking-based virtual screening and molecular dynamic simulation ; and massonianoside B, identified by use of a pharmacophore-based in silico screening .This is, for instance, also the case for:b; The menin/LEDGF complex interaction with MLL is also crucial for MPAL and controls the expression of HOXA transcription factors. Development of menin/MLL inhibitors is another promising therapeutic strategy  , but at the micromolar range in non-MLL leukemic models. In animal models, KO-539 was given by the oral route and evidenced weak toxicity in mice and potent leukemic cells differentiation (CD11b-expressing cells in bone marrow of treated mice) and anti-leukemic effects  in both the MV4-11 MPAL model and patient-derived xenografts from the NPM1c+/FLT3-mut AML subtype . Among oc; Interestingly, DOT1L collaborates also with the bromodomain protein BRD4 , an acetd; The interaction of WDR5 with MLL protein is the third approach presented here d; . Inhibithoxa9 and hoxa10 gene expression level or genes associated with G1/S phase arrest and apoptosis. Cell cycle arrest, cell death and expression of the myeloid surface marker CD11b were also detected in the human MV4-11 cell model but not in the K562 cell line [The macrocyclic MM-401 and MM-589 peptidomimetics are also high affinity WDR5 binding compounds. MM-401 induces modification of gene expression profile with common features with that of the depletion of MLL-fusion in the murine MLL-AF9 cell model, among which are the ell line . MM-589 ell line .In addition to peptidomimetics mostly having poor cellular permeability, small molecules were designed as antagonists. Among these is WDR5-0102, which binds the arginine pocket of WDR5 , and alse; The MLL-fusion proteins also interact with pTEFb, a transcription elongation factor complex that phosphorylates serine 2 residues located in the heptad repeats “YSPTSPS” from the carboxyl-terminal domain of the largest subunit of RNA-PolII, thus activating the transition from the initiation to the elongation step of the transcription process. Therefore, targeting pTEFb was evidenced as a strategy to treat MPAL e; . pTEFb iOther CDK9 or pan-CDK inhibitors are in developmental stage, such as LS-007/CDKI-73 , LY28577f; Recently, the inhibition of the degradation by the proteasome of the wild-type MLL was proposed as an alternative strategy against MPAL with the use of IRAK (interleukin-1 receptor-associated kinase) inhibitors f; . In respg; In MPAL, MLL translocations lead to the loss of methyltransferase function, resulting in the recruitment of other antagonist epigenetic modulators such as the histone lysine-specific demethylase 1 (LSD1/ KDM1A) g; . LSD1 deThe most promising LSD1 inhibitors in AML appear to be (i) the tranylcypromine-based compounds such as tranylcypromine (TCP) trentinoin  against relapsed/refractory AMLs, (ii) GSK2879552 (GlaxoSmithKline), evaluated in small cell lung cancer and AML, and (iii) ORY-1001 , which entered phase I clinical trials in AML ,188. Tho32 active at the nanomolar range on LSD1 as evidenced by surface plasmon resonance [In addition to those compounds that have entered clinic trials, others are also being evaluated in pre-clinical models. The LSD1 antagonist SP2509 was active in the MPAL cell model MOLM-13, but also in the NPM1c+ cell model OCI-AML3, inducing cell differentiation, but the effect on HOXA9 expression was not evaluated. SP2509 evidenced promising results used in combination with the HDAC inhibitor panobinostat in OCI-AML3 and patient-derived xenograft mice models . NCD38, esonance .N1-(2-((2-chlorophenyl)thio)benzyl)-N1-methylethane -1,2-diamine compound  [h), and SD70 [i; 28d was able to reduce HOXA9 and MEIS1 expression in MPAL models [The epigenetic writer H4R3 methyltransferase PRMT1 and the eraser Jumonji domain-containing H3K9 demethylase KDM4C (JMJD2C/GASC1) are two proteins that also associate with the oncogenic MLL complex, as evidenced in MLL-AF9 and MLL-GAS7 translocation models . Those tR049_12)  against and SD70  against L models .j; The H4K16 histone acetyl-transferase (HAT) MOF is another epigenetic writer associated with MLL-AF9- and NUP98-HOXA9-driven leukemias. The MOF inhibitor MG149 evidenced anti-leukemic activities of MLL-fused leukemia and other AML cell models that are associated with HOXA9 over-expression, such as the CALM-AF10 fusion model U937, but also the K562 cell model (BCR-ABL fusion) that does not express HOXA9 , suggestTargeting epigenetic modifiers could also prevent HOXA9 transcription in other AML sub-types. The activity of these epigenetic enzymes is modulated, for instance, through metabolites produced by mitochondrial metabolism.k; The dihydroorotate dehydrogenase enzyme (DHODH) is involved in nucleic acid synthesis and the cell cycle, as well as in the post-translational glycosylation of important protein targets such as epigenetic enzymes . The inhl; Both IDH1 and IDH2 proteins are glycolytic enzymes that catalyzes the conversion of isocitrate into α-ketoglutarate. IDH1/2 mutations are found in ~15%–20% of secondary and de novo AML and globally impact the normal catalytic activity by leading to the production of 2-hydroxyglutarate (D-2-HG). This oncometabolite is a competitive inhibitor of the epigenetic TET2 enzyme, thus leading to the decrease of 5hmc marks ,205. It m; At the level of epigenetically modified DNA, different mutations of the DNA methyl transferase protein DNMT3A are associated with modifications of the DNA methylation status, leading to a leukemia associated with HOXA9/MEIS1 expression ,210,211.®, Sanofi Aventis, Gentilly, France), vorinostat , the cinnamic hydroxamic acid analogue panobinostat , romidepsin , belinostat , the phenylbutyrate-derivative AR-42 [n; At the histone level, HDAC inhibitors such as valproic acid  is a nucleolar phosphoprotein that preferentially binds to quadruplex DNA, a function and localization that is lost in NPM1c+; inhibitors of NPM1 have been developed . The retAs presented in the previous section, indirect inhibition of HOXA9 leading to the repression of its expression could be achieved through a broad diversity of transcription regulators used as targets, and related drug candidates.Direct targeting of HOXA9 protein is undoubtedly an interesting strategy to treat a large proportion of AML. As a transcription factor, HOXA9 does not belong to receptor or steroid families that could be targeted by ligand mimetics through the binding to a binding pocket. Two other strategies are currently being developed: targeting HOXA9 at the transcription factor/cofactor protein/protein interface and targeting HOXA9 at the DNA binding level.Targeting the protein/protein interface is one strategy that has already been developed to target the HOX protein though its interaction with specific co-factors , since tYPWM) and a repeat of nine arginine residues to facilitate cell delivery: WYPWMKKHHRRRRRRRRR. HXR9 disrupts the interaction of the hexapeptide sequence from multiple HOX proteins, which is used to interact with different PBX partners. HXR9 binds to the pocket in PBX, which otherwise would be recognized by HOX proteins. Such interaction shuts down the expression of various genes involved in apoptosis [50 around 10 µM), associated with an increase in apoptotic cell death, as evidenced by flow cytometry after propidium iodide/annexin V double staining. Of interest, HXR9 is also active in the HOXA9-negative cell line K562, an erythroleukemia cell line derived from a chronic myeloid leukemia patient in blast crisis and associated with BCR-ABL fusion kinase that may express other HOX proteins.The peptide inhibitor HXR9 , point ppoptosis ,237,238.poptosis ,239, renpoptosis , ovarianpoptosis ,242), brpoptosis , prostatpoptosis , asbestopoptosis , and orapoptosis ,247. HXRpoptosis ,248,249,KWMKKAARRRRRRRRR; underlined are the modified amino acids relative to HXR9) also effective against numerous cell lines and human tumors, and is also licensed by HOX Therapeutics Ltd. . Among them are the PC3 prostate and MDA-MB-231 breast cancer models against which the peptide inhibitor HTL-001 presented higher efficacy that the parental HXR9 peptide [The HXR9 derivative peptide HTL-001 , point p peptide . Given tMore recently, HOX Therapeutics Ltd. developed a small molecule, HTL-002 , point pThe PBX and MEIS proteins are not the only HOXA9-interacting proteins. For instance, SMAD4 (Mothers against decapentaplegic homolog 4), a TGF-β and bone morphogenic protein (BMP) signaling pathways associated factor, interacts with HOXA9 , point qThe histone H3 lysine 9 methyltransferase G9a (KMT1C/EHMT2) is also known to physically interact with HOXA9 in AML as determined by co-immunoprecipitation  Figure , point rProtein/DNA is another interface that could be targeted to inhibit a transcription factor. This could be achieved through binding to the DNA binding domain when a binding pocket is present or identified or, more commonly, through binding to their DNA cognate sequence ,258. InhThe alternative to target transcription factor/DNA binding is to interfere at the level of DNA interaction. Such interference was already achieved using well-known alkylating drugs with either trapping of the protein/adduct or inhibition of the transcription factor/DNA interaction  ,268,269.Recently, we selected a series of heterocyclic diamidines as minor groove DNA ligands on the HOXA9 cognate sequence , point sOverall, these data demonstrate for the first time the propensity of sequence-selective DNA ligands to inhibit HOXA9/DNA binding both in vitro and in a murine Hoxa9-dependent leukemic cell model . EvaluatIn summary, the different approaches presented in this manuscript are gathered in We showed in this review that the physical (indirect) and functional (direct) inhibition of HOXA9 in AML could be achieved through multiple strategies, some being used/evaluated in clinic, and many others being in preclinical stage. For instance, indirect targeting strategies for epigenetic complex actors or cofactors are currently under development and in clinical trials, as illustrated by DOT1L inhibitors. MLL abnormalities, which are found in about ~5% of AML, are associated with a poor patient prognosis, justifying the strong need for new therapies against MPAL ,295. HowMoreover, besides being an attractive strategy to control HOXA9 expression at the epigenetic level, it is worth noting that most, if not all, of the epigenetic drivers presented here have an effect on the expression of many other genes. Consequently, such approaches that change the epigenetic marks may not exclusively be active on the leukemic cells of AML patients, but may also present side-effects and off-target effects by altering normal cells ,301,302.STRK located in a conserved region of its homeodomain. In particular, phosphorylation of the S204 residue by PKC strongly decreased HOXA9/DNA and HOXA9/PBX1/DNA complex formation in vitro, and PKC inhibition by bisindolyl-maleamide 1 decreases TPA-induced differentiation in a HOXA9-transformed murine cell line [Based on actual knowledge, new therapeutic opportunities may also come from other aspects of the regulation of HOXA protein expression  or activity . Indeed, the binding of CCCTC-binding factor (CTCF) at chromatin loop bases around the HOX locus is associated with the silencing of HOX cluster expression with (CTCF) binding sites located at the loop bases, subsequently stabilizing the polycomb repressive complex 2 (PRC2) binding to DNA, and resulting in H3K27me3 repressive epigenetic mark trimethylated lysine 27 of histone H3 to lock the HOXA cluster ,304. In ell line   share a closely related HOX/PBX interaction interface, and some similarities in the DNA binding sequence and a better knowledge of their respective mechanisms of action would be determinant.Identifying and targeting new HOXA9/protein interactions is another alternative research area of interest. At present, two non-PBX/MEIS proteins were identified as potential protein partners: SMAD4 and G9a. Identification of the precise protein/protein interfaces and subsequent drug screening may identify new potential drugs to treat AML.Finally, there is also still some limitation in the targeting of the HOXA9/DNA interface, but a better knowledge of HOXA9 target genes and the way HOXA9 binds to the genome from comparison with the other 38 HOX proteins would help develop new strategies. Indeed, for the DNA binding inhibition strategy, the precise knowledge of the HOX specificity on the genome of a cancer cell is not fully addressed. This is mainly due to the absence of ChIP-seq analyses for each HOX protein expressed in a specific cell . AML is a nice model to compare HOXA regulated genes , binding sequences (using ChIP-seq as examples) and protein partners (using immunoprecipitation-coupled mass spectrometry of endogenous protein (RIME) or bimolecular fluorescence complementation (BiFC), for instance). However, the ChIP-seq and RIME approaches may be properly performed only by use of highly specific and efficient antibodies, validated against each HOX protein, and those antibodies are unfortunately not yet available. Such kinds of global approaches, compiled with other high throughput and next-generation sequencing analyses  analyses, methylome, ATAC-seq, and binding energy topography by sequencing (BET-seq), etc.) would also provide opportunities to identify critical downstream factors as new potential targets in AML and other cancers associated with HOXA9 over-expression.


Epigenetic regulation is referred to as changes in gene function that do not involve changes in the DNA sequence, it is usually accomplished by DNA methylation, histone modifications , and chromatin remodeling . In plants, the shoot apex produces different lateral organs during development to give rise to distinguishable phases of a juvenile, an adult and a reproductive phase after embryogenesis. The juvenile-to-adult transition is a key developmental event in plant life cycle, and it is regulated by a decrease in the expression of a conserved microRNA-miR156/157, and a corresponding increase in the expression of its target genes encoding a set of plant specific SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) proteins. Recent work has revealed that the miR156/157-SPL pathway is the master regulator of juvenile-to-adult transition in plants, and genes in this pathway are subjected to epigenetic regulation, such as DNA methylation, histone modifications, and chromatin remodeling. In this review, we summarized the recent progress in understanding the epigenetic regulation of the miR156/157-SPL pathway during juvenile-to-adult transition and bring forward some perspectives of future research in this field. Unlike mammals, in which organ formation is completed during embryonic development, plants produce new organs from self-sustaining stem cell populations known as meristems in different developmental processes. In plants, post-embryonic development can be divided into a juvenile vegetative phase, an adult vegetative phase and a reproductive phase, and each developmental phase is marked by changes in a series of distinct phase-specific traits . The traArabidopsis, the juvenile-to-adult transition is characterized by the formation of leaf abaxial trichomes, an increase in leaf length/width ratio and serration, and a decrease in cell size (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes act sequentially with miR172, another miRNA that targets a class of AP2-like transcription factors (TFs), to regulate juvenile-to-adult transition in plants  and four miR157 genes (MIR156A∼D), and those genes function redundantly. The mir156a mir156c double mutant exhibited a similar phenotype to the 35S::MIMICRY156 transgenic plants with significantly reduced levels of miR156, which indicates that MIR156A and MIR156C are the two main loci contributing to the level of miR156 and have dominant roles in vegetative phase change within the miR156 family in Arabidopsis . Here, we review our current understanding of epigenetic regulation of the miR156/157-SPL pathway and the roles of corresponding players in juvenile-to-adult transition in plants.As the master regulator of the juvenile-to-adult transition, miR156/157-SPL pathway has been shown to be subjected to transcriptional and post-transcriptional regulation. Those include the transcriptional regulation of DNA methylation [5-Methylcytosine (5mC)] is a hallmark of epigenetic gene silencing in both plants and mammals . DNA metSpm transposable elements (Mu) element  or demethylation enzymes , as well as bisulfite sequencing of MIR156/157 and SPLs loci, will facilitate to uncover the role of DNA methylation in regulation of miR156/157-SPL pathway and juvenile-to-adult transition in plants.The first indication of DNA methylation plays a role in phases of shoot development comes from the work done by Brink. In the 1950s, Brink noticed the similarity between phase change in plants and changes in cell states in non-plant organisms, he proposed that phases of shoot development might be regulated by reversible changes in chromatin based on his research on paramutation in maize . Subsequelements  and the  element  suggest  element , and an  element . In Arabhenotype , indicate mutant , and 25-e mutant  indicateHistone modification at specific lysine sites functions as transcription repressive marks such as H3K9me, H3K27me, H2Aub, etc., or active marks such as H3K4me, H3K36me, H3Ac, etc., this modification is catalyzed by Polycomb group (PcG) protein complexes and Trithorax group (TrxG) protein complexes, respectively . PcG comMEDEA (MEA), SWINGER (SWN), and CURLY LEAF (CLF) are orthologs of the Drosophila E(z) gene, which function as a histone methyltransferase subunit in the PRC2 complex. MEA appears to function in embryogenesis specifically, and CLF and SWN are broadly expressed and partially redundant in vegetative and reproductive development  through the E(z) SET domain . In the elopment .Arabidopsis uncovered 1000s of gene loci carrying the H3K27me3 mark catalyzed by the PRC2 complex, indicating that H3K27me3 is a major epigenetic silencing mechanism in plants , also carry H3K27me3 mark. However, except for SPL4 and SPL6 which play no obvious roles in juvenile-to-adult transition, miR156/157-targeted SPL genes are largely devoid of the H3K27me3 mark. These results imply that the PRC2 complex promotes SPL gene transcription indirectly by repressing the transcription of MIR156/157 loci   ,c. Loss-henotype ,c. H3K27ansition . TherefoH3K27me3 ,c.Arabidopsis and animals, but it has related functions. In Arabidopsis, the function of PRC1 can be histone 2A mono-ubiquitination (H2Aub) dependent or independent. H2Aub dependent group requires the E3 ubiquitin ligase activity of Arabidopsis B lymphoma Moloney murine leukemia virus insertion region1 homolog 1A (AtBMI1A)/B/C or AtRING1A/B, while H2Aub independent group requires the activity of the EMBRYONIC FLOWER 1 (EMF1)  . BMI1-PR1 (EMF1) .Arabidopsis. BMI1-PRC1 maintains the repression of miR156 and accelerates juvenile-to-adult transition  analyses indicated that ATXR7 binds to a region adjacent to the TSS of MIR156A and deposits the H3K4me3 mark to activate MIR156A transcription , ATX2, and ATXR7 . ATX1 anbidopsis . atxr7-1cription .Histone acetylation is generally considered as an active epigenetic mark, which is a balanced process regulated by histone acetyltransferases (HAG1) and histone deacetylases . Spt–Ada–Gcn5–acetyltransferase-like histone acetyltransferase complex (SAGA-like complex) is conserved in mammals, plants, files and yeast, and General Control Non-repressed 5 (GCN5) functions as the catalytic component for this complex .Arabidopsis, loss-of-function mutants in HAG1 (the Arabidopsis homolog of GCN5), hag1-6 and hag1-7, exhibited a significantly delayed juvenile-to-adult transition phenotype  and H2A.Z coding genes  exhibited a similar pleiotropic phenotype, which indicates that the primary function of SWR1-C is to deposit H2A.Z (ATP-dependent SWR1 chromatin remodeling complex (SWR1-C) functions in exchanging the histone H2A-H2B dimer with the H2A.Z-H2B dimer, and then produces nucleosome variant . In Arabit H2A.Z . Howeverit H2A.Z .arp6 and hat9/hat11 mutants, MIR156A/MIR156C transcripts were reduced and juvenile-to-adult transition was accelerated  is the ATPase subunit of the most widely studied SWI2/SNF2 chromatin remodeling protein complex. It uses the energy derived from ATP hydrolysis to change the histone octamer-DNA interaction . BRM reg MIR156A . BRM als is a CHD3 ATP-dependent nucleosome remodeling protein, which is physically associated with the nucleosome remodeling and deacetylation complex . PKL is d region .Figure 2.Although the miR156/157-SPL pathway has been shown to be the master regulator of juvenile-to-adult transition in plants, yet little is known about the upstream regulator of this pathway, especially for miR157. Recent studies have revealed that DNA methylation, histone modification, chromatin remodeling play important roles in regulating the expression of some components in the miR156/157-SPL pathway. However, there are still some critical questions remain to be solved as illustrated in MIR156/157 and/or SPL loci are subjected to epigenetic regulation to modulate juvenile-to-adult transition in plants. However, these epigenetic regulators, by their own, have no DNA binding specificity. Therefore, a central question is how these epigenetic regulators are recruited to their target genes.Arabidopsis showed that PRC2 components bind to specific DNA motifs called Polycomb response elements (PREs) by interacting with specific TFs . FLC is silenced by depositing H3K27me3 mark under winter cold treatment, and the silenced state was maintained in the mature pollen grains and the egg cells , a seed-specific pioneer TF (FLC re-activation.A similar example of Off-Resetting pattern in plant lifecycle is the regulation of gg cells . In pro-oneer TF , and EARoneer TF , were shMIR156/157, it is still unknown where and when the de novo re-activation occurs. Moreover, whether the resetting of miR156/157 depends on a reversible epigenetic regulation still remains elusive. Further study of when, where and how miR156/157 Off-Reset pattern is initiated during plant life cycle will be an important future task.As for YX and LZ wrote the article, and GW revised it.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.


In eukaryotes, genetic material is packaged into a dynamic but stable nucleoprotein structure called chromatin. Post-translational modification of chromatin domains affects the expression of underlying genes and subsequently the identity of cells by conveying epigenetic information from mother to daughter cells. SWI/SNF chromatin remodelers are ATP-dependent complexes that modulate core histone protein polypeptides, incorporate variant histone species and modify nucleotides in DNA strands within the nucleosome. The present review discusses the SWI/SNF chromatin remodeler family, its classification and recent advancements. We also address the involvement of SWI/SNF remodelers in regulating vital plant growth and development processes such as meristem establishment and maintenance, cell differentiation, organ initiation, flower morphogenesis and flowering time regulation. Moreover, the role of chromatin remodelers in key phytohormone signaling pathways is also reviewed. The information provided in this review may prompt further debate and investigations aimed at understanding plant-specific epigenetic regulation mediated by chromatin remodeling under continuously varying plant growth conditions and global climate change. The packaging of genetic material into nucleosomes is a distinctive evolutionary feature of eukaryotic cells. Nucleosomes are repetitive units consisting of 147 bp DNA strands tightly wrapped around a central octamer comprising one histone heterotetramer (H3/H4) and two histone heterodimers (H2A/H2B). Histones are highly conserved proteins found in all eukaryotes as core nucleosome units . TogetheGene regulatory mechanisms in eukaryotic cells  act upon chromatin structure as a substrate. These activities induce cellular changes and regulate gene expression in a number of biological processes, including genome stability, recombination, developmental reprogramming and response to extracellular signals . The regThere are two main players regulating chromatin dynamics: (1) chromatin remodelers that alter DNA-histone interactions by energy harnessed through ATP hydrolysis and (2) nucleosome-modifying enzymes that modulate DNA and histone residues by specifically adding or removing covalent modifications . OrganisPlant development can be divided into the embryonic and postembryonic phases. The embryonic development phase includes the establishment of the seedling apical-basal axis, the shoot apical meristem (SAM) and the root apical meristem (RAM). The subsequent establishment of the leaf, stem and flower meristems occurs in the postembryonic phase. In this review, we discuss recent investigations underpinning the involvement of SWI/SNF chromatin remodeling ATPases in regulating key plant growth and development processes, including meristem establishment and maintenance, cell differentiation, organ initiation, flower morphogenesis and flowering time regulation. We also discuss the role of chromatin remodelers in the plant response to key phytohormone signals. This overview may be useful for framing the current knowledge gaps, thereby stimulating further debate and research aimed at a comprehensive understanding of epigenetic regulatory mechanisms in plant development. This in turn may guide molecular-based plant improvement techniques for desirable agronomic traits.Chromatin remodeling complexes are evolutionarily conserved multi-unit protein complexes that regulate chromatin structure by altering nucleosome composition and interactions . A commoArabidopsis SWI/SNF complex subunits have been identified based on sequence similarity with metazoan subunits. These include four SWI2/SNF2 ATPases  (A number of 2/CHR23) ; four SW2/CHR23) . Eukaryo2/CHR23) .Saccharomyces cerevisiae is probably the most comprehensively studied. SWI/SNF subfamily CRCs have a well-established role in gene expression regulation. The SWI/SNF subfamily remodelers contain 8–14 subunits, and the catalytic ATPases of most SWI/SNF subfamily remodelers are composed of a helicase-SANT domain, a post-HSA domain and a C-terminal bromodomain. While the fungal SWI/SNF subfamily ATPases consist of a pair of actin-related proteins (ARPs)  , higher s (ARPs) . The mods (ARPs) . It was ing RNAs .Drosophila melanogaster. They include the nucleosome remodeling factor (dNURF), chromatin accessibility complex (dCHRAC) and ATP-utilizing chromatin assembly and remodeling factor (dACF)  . SANT-lir (dACF) .Xenopus laevis and comprise 1–10 subunits. The N-terminus of their catalytic subunit consists of two chromodomains arranged tandemly, with the composition varying from monomeric in lower eukaryotes to multimeric in vertebrates ; in mammals, it is represented by three complexes, INO80, Snf2-related CBP activator protein (SRCAP) and p400. D. melanogaster has INO80 and p400 complexes  region in the ATPase subunit, as deletion of this region leads to dissociation of a number of subunits from the complex, including the RuvB-like Rvb1 and Rvb2 subunits    are alsod CHR23) .WUSCHEL (WUS) and activating WUS expression in the organizing center (OC) region of the SAM . Consistent with this function, Arabidopsis syd mutant plants exhibit reduced WUS transcript levels accompanied by abnormal SAM development  pathway to maintain the root stem cell niche by altering the expression of PIN-FORMED (PIN) genes responsible for auxin distribution in the RAM, as illustrated in Figure 1. brm mutants exhibit faulty root stem cell niche maintenance, reduced meristem activity and retarded root growth  suggests that AN3 recruits SWI/SNF complexes to promote cell division during leaf development (Arabidopsis indicated that BRM and SWINGER (SWN) (a key component of Polycomb Group Repressive Complex 2 in plants) antagonistically control vegetative phase change through the temporal expression of miR156 at the nucleosome level. Specifically, the accelerated vegetative phase change of brm mutants was accompanied by reduced miR156 expression and increased levels of H3K27me3 at the MIR156A locus (ANGUSTIFOLIA3 (AN3)/GRF-INTERACTING FACTOR1 (GIF1), a member of the GIF family of transcriptional coactivators, plays a key role in elopment , cotyledelopment  and leafelopment . Transcrelopment . BRM is elopment . A forwa6A locus .Arabidopsis SWI/SNF CRCs function in various plant development pathways including the regulation of leaf and flower development   . RNA intelopment .A study of the chromatin remodeler protein ZmCHB101, which is the core subunit of maize SWI3, revealed its key roles in normal maize growth and development. ZmCHB101 controls the transcriptional reprogramming of a set of genes involved in gene expression regulation, photosynthesis, metabolic regulation and stress response. The RNAi maize lines generated in the study exhibited improper tassel and cob development and abaxially curling leaves caused by increased bulliform cell numbers .Arabidopsis . When exposed to light, dark-grown brm plants exhibit higher greening rates, reduced protochlorophyllide accumulation and lower levels of reactive oxygen species (ROS) compared to wild-type plants; there is also increased expression of NADPH:protochlorophyllide oxidoreductase A (PORA), PORB and PORC, enzymes which accelerate a key step in chlorophyll biosynthesis  was shown to regulate chlorophyll biosynthesis in ynthesis .The importance of chromatin remodelers for proper plant growth progression and development is exemplified by their involvement in asymmetric cell differentiation and in initiating organ founder cells. Proper plant growth also involves precise developmental phase transitions, and the antagonism between SWI/SNF ATPases and PcG repressive complex components is crucial for these shifts.The precise regulation of the transition from vegetative growth to flowering is paramount for plant reproductive success. This process is characterized by the transition of the vegetative SAM into an inflorescence meristem (IM) and the initiation of floral meristems (FMs) . While SArabidopsis, a repressor complex that consists of the two MADS box transcription factors MADS AFFECTING FLOWERING 4/5 (MAF4/5), FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) serves as a negative regulator of flowering time. The components directly repress the expression of the floral pathway integrators FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) (FRIGIDA (FRI) promotes higher FLC levels that inhibit flowering (FLC locus (FRI-mediated activation of FLC expression and thereby enables flowering (In 1 (SOC1) . FRIGIDAlowering  through LC locus . Vernalilowering .Arabidopsis SWR1 chromatin remodeling complex (including PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 (PIE1), ACTIN RELATED PROTEIN 6 (ARP6) and SWR1 COMPLEX 6 (SWC6)/SERRATED LEAVES AND EARLY FLOWERING (SEF)) have been shown to play important roles in regulating the proper growth and development of most plant organs. Importantly, SWR1 controls plant development by generating a balance between microRNAs and target mRNAs at the transcriptional level . One line of evidence is that functional disruption of SWR1 prevents H2A.Z deposition at FLC chromatin, suppressing its expression and leading to early flowering  and expression regulation of FLC and MAF4/5 by facilitating the enrichment of H2A.Z at their ends. Independent Atino80-5 and Atino80-6 mutant alleles display similar pleiotropic phenotypes of small plant size, reduced organ size and late flowering. These observations provide a link between plant responses to environmental and developmental signals and epigenetic mechanisms involved in plant chromatin stability  subunit of the LC locus . SWP73A LC locus .SVP, another key flowering repressor, is highly expressed during the vegetative phase to promote growth . One study proposed that BRM controls flowering time in Arabidopsis by directly activating SVP expression  under long days to induce flowering is the FT-encoded florigen protein (FT) (LEAFY (LFY) and APETALA1 (AP1). This activation leads to the formation of the floral primordium, which sequentially generates three types of lateral floral organs   by removing H3K27me3 marks on its second intron   . FT movestamens) . LFY recigure 3) . The SWRy Pol II .PICKLE (PKL) have highlighted its role in plant reproductive development by promoting crosstalk between the sporophytic and gametophytic generations. Loss of PKL in the maternal sporophyte leads to improper development of the Arabidopsis female gametophyte, integument and pollen tube, accompanied by delayed ovule and embryo development . Moreover, the phosphomimetic BRM mutant shows hypersensitivity to ABA (Abscisic acid (ABA) is an important phytohormone that promotes seed dormancy and arrests growth in post-germination embryos under water stress conditions. In the absence of stress stimuli, BRM was initially implicated in repressing the activity of the ABA pathway, with adult olerance . It was y to ABA .GA3ox1 promoter, thereby activating the gene . Arabidopsis brm null mutants exhibit a significant decrease in active GA levels . SWP73B negatively regulates the CK biosynthesis genes ADENOSINE PHOSPHATE-ISOPENTENYLTRANSFERASE (IPT3) and (IPT7) and the CK-regulated cell cycle inhibitor KIP-RELATED PROTEIN7 (KRP7) by hindering the deposition of active histone marks on their gene bodies (Excess cytokinin (CK) production inhibits primary root elongation and weakens lateral root development . SWP73B e bodies .In aggregate, these findings indicate that both phytohormone biosynthesis and degradation in response to extracellular and intracellular cues partly depend on SWI/SNF chromatin remodeling ATPases. The findings also suggest the role of SWI/SNF ATPases as a hub in plant perception and response to phytohormone signaling as well as hormone crosstalk through direct physical interactions with hormone signaling pathway components. The outcomes of these interactions influence the plant response to abiotic stress, enabling plant adaptability under changing climates.Arabidopsis. Phylogenetic analysis suggests that the chromatin remodeling machinery and their modes of operation are evolutionarily conserved across eukaryotes. However, SWI/SNF subunit loss-of-function studies have uncovered varied organism-specific phenotypes. These phenotypes also depend on the strength of a given mutation, leading to phenotypic differences among single and double mutants of SWI/SNF subunit genes. The functional redundancy of some SWI/SNF complex subunits also necessitates further in-depth analyses of their interaction patterns in different plant development pathways.Chromatin remodeling has been extensively studied in the context of various regulatory and developmental processes in a number of eukaryotic organisms, including humans, mice, yeast, fruit fly and Arabidopsis and more recently in maize. Plant-specific molecular and genetic approaches should also characterize regulatory differences between developmental stages and under different growth conditions for a more comprehensive understanding of the involvement of SWI/SNF chromatin modifiers in plant growth and development.As discussed above, a number of plant regulatory and developmental transitions are controlled by chromatin modifiers through complex pathways in response to both endogenous and environmental factors. These precisely orchestrated mechanisms are heritable, underscoring their importance for plant survival in changing habitats. Moreover, the dynamics of these epigenetic controls go hand in hand with changes in plant growth conditions. Positive attributes that could potentially be integrated into plant breeding schemes are of interest to many plant scientists and breeders insofar as they may yield varieties that can withstand adverse weather conditions, early flowering or various biotic stresses and maximize nutrient utilization for improved yield. High-throughput technologies together with the vast genomic data presently available in public repositories should facilitate comparative and functional studies aimed at plant improvement. Future studies in other species of interest  will likely build upon the findings on ATP-dependent CRCs in The following research questions and topics are of particular interest in terms of how CRCs may be involved in various processes either directly or indirectly related to plant development.(1)Medicago truncatula, whose flowering is prompted by winter cold and long-day photoperiods despite the absence of an FLC gene.Involvement of SWI/SNF ATPases in flowering regulation in the temperate legume model plant (2)The role played by chromatin remodelers in oxidative stress remediation processes resulting from various metabolic activities in plant cells other than DNA double-strand break repair.(3)Besides their known function in the phosphate starvation response, there is a need to ascertain other roles of SWI/SNF remodelers in mediating the uptake of essential micro- and macronutrients from the soil in conjunction with nutrient transporter gene families and rhizosphere microbial activities.(4)The mechanism by which the chromatin remodeling complex SWR1 mediates chromatin structural dynamics by replacing the core H2A-H2B histone dimer with the H2A.Z-H2B dimer in octamers is well characterized. However, the molecular mechanisms underlying the reverse process remain elusive.(5)Arabidopsis SWI/SNF CRCs in regulating plant development are only beginning to be understood. The latest example is SWP73 involvement in the cell cycle, leaf development, flowering time and embryogenesis. Much remains to be uncovered in this regard.The specific roles of accessory subunits of SO and SC conceived the idea, wrote the manuscript, and drafted the figures. SP designed and organized figures and revised the manuscript. WL, MY, MA, and HZ revised the manuscript and organized the figures. YQ conceived the idea and revised and organized the manuscript.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.