Bacillus genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They can be applied as a standard element for synthetic biology. Characterization of the binding motif between UP elements and RNAP may assist with rational and effective engineering. In this study, 11 Bacillus constitutive promoters were screened for strength in Bacillus licheniformis. The motif in UP elements from a strong native promoter, PLan, was characterized. The influence of specific sequences on RNAP binding and expression strength was investigated both in vitro and in vivo. It was found that sequences up to 50 base pairs upstream of the consensus motif significantly contributed to α-CTD  association. Meanwhile, two repeats of a proximal subsite were able to more strongly activate the expression (by 8.2-fold) through strengthening interactions between UP elements and RNAP. Based the above molecular basis, a synthetic UP element, UP5-2P, was constructed and applied to nine wild-type promoters. Fluorescence polarization results demonstrated that it had an apparent effect on promoter–α-CTD interactions, and elevated expression strength was observed for all the engineered promoters. The highest improved core promoter, Pacpp, was more strongly activated by 7.4-fold. This work thus develops a novel strategy for Bacillus promoter engineering. Escherichia coli based on the specific interaction between the bacteriophage-derived, T7-specific RNA polymerase (RNAP) and the strong T7 promoter. Recent research concerning this Gram-negative bacterium has focused more on the engineering of native promoters to create highly regulated gene expression circuits [Bacillus, fewer methods for their rational engineering have been shown to be highly efficient, compared with that of E. coli ones.Well-evaluated promoters of diversified strength are crucial elements for complex in vivo expression in synthetic biology. To this end, they are believed not only to be standardized, but also to exhibit predictable and reliable activity under a variety of different conditions ,2. Stroncircuits ,4,5. AltE. coli [E. coli promoters in a study that employed designing and optimizing UP elements of the promoter, with strengths up to 9.4-fold higher than their wild-type sequences [Structurally, bacterial promoters contain two recognition elements for RNAP and the assembled sigma factor: the classical −35/−10 hexamer and the UP element. The −35 and −10 elements, or the promoter core sequences, recognized by the sigma subunit of RNAP, determine to a great extent the strength of a promoter . A vast equences . This UPBacillus RNAP also contains α2 (dimer), β, β′, and σ subunits [A  binds to promoters to initiate transcription. Kang et al. designed synthetic Bacillus promoters by shuffling the promoter core sequences recognized by different sigma factors [Bacillus promoters, modifying promoter activity through the engineering of UP elements has received limited attention in the field. Here, we seek to engineer a previously verified α-CTD-binding Bacillus redox-sensitive promoter TrxA [Bacillus promoter engineering through simply altering the UP elements alone in a manner that is independent of the core promoter itself.subunits . The hol factors . A set oter TrxA , based oB. licheniformis was developed by using BlR2G as an example. Fluorescent signals from cells loaded with different concentrations were investigated. Results showed that an OD600 range of 0.8–2.5 in a single well of the 96-well microtiter plate guaranteed a linear correlation between cell counts and the fluorescent signals  in P43 at 24 h. More importantly, comparison between different sampling times revealed huge variations in transcription initiation. For example, Pshuttle09 was the most active promoter at both 12 and 24 h, with a relative fluorescence intensity of more than 2.3-fold higher than that of the other promoters. From 24 h to 36 h, however, PLan experienced a great boost in relative fluorescence intensity (by more than 13-fold). Its activity continued to increase by 2-fold from 36 h to 48 h. At the same time, 9 of 11 promoters showed a significantly decreasing trend in activity. When relating the above results to cell growth of the strains, we found that 24 h was near the division point between exponential and stationary, so that these promoters exhibited transcription initiation patterns corresponding to different cell growth stages. Finally, the estimated promoter activity of PLan could be as high as 75-fold higher when the most frequently used promoter P43 was used as a reference. This promoter was then selected for further analysis and engineering.Then, the cell cultures corresponding to different promoters were sampled at 12, 24, 36, and 48 h, respectively, and relative fluorescence intensity (fluorescence intensity (Au)/OD600) was used to demonstrate the promoter activities . The selB. licheniformis, PLan exhibited much higher activity than the other ones tested. This promoter was mined and predicted based on our previous transcriptome data. We wondered why it mediated hyper-expression and if it could be a source of UP elements for promoter engineering. Therefore, its genome loci were retrieved to find the possible answers. The highly expressed transcript RS00925 mediated by PLan is located within a lantibiotic biosynthesis gene cluster and codes for the putative propeptide. The amino acid sequence of this peptide is close to the one reported in B. subtilis and other bacteria [Lan promoter. Inside this cluster, four genes have been annotated as being signal transduction histidine kinase (LanC), transcriptional regulatory protein (LanB), and two hypothetical proteins (LanD and LanA). Subsequent sequence alignment indicated that those sequences were consistent with the agr QS system containing AgrBDCA, which was well characterized in Gram-positive Staphylococcus aureus. This is related to cell density-dependent control of behaviors such as virulence and biofilm formation [Lan is involved in an agr-like QS system. Most QS systems possess the characteristic of autoinducer-dependent transcriptional activation to ensure hyper expression of QS operons. It has also been reported that C-terminal domain of the alpha subunit of RNA Polymerase (α-CTD) plays an important role in this activation by making contact with the promoter regions [B. licneniformis.In bacteria ,23. Howeormation ,25,26. S regions ,28. We tB. licheniformis was expressed by using pET-28(a)/DE3 system in E. coli. After the construct EcCTD was cultivated and induced, cell lysate was subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Compared with the control one, the sample corresponding to EcCTD apparently showed a band of 15 kD, which is consistent with the predicted molecular weight of α-CTD , different shifted bands were observed. It could be inferred that the complex α-CTD-PLan was formed in a protein concentration-dependent manner, confirming that α-CTD specifically bound to the promoter PLan.We therefore performed additional EMSA to investigate whether PB. subtilis, α-CTD can interact with specific UP elements located in a region upstream of the PtrxA core promoter. This UP element is believed to act as an activating sequence for certain promoters and has been consistently shown to increase promoter strength by facilitating RNA polymerase association with DNA. To determine the precise α-CTD binding site or the UP element, DNase I footprinting assays were performed with 5′-6-carboxyfluorescein (6-FAM)-labeled probes containing a sequence upstream of PLan core promoter region. As can be seen in Lan, in which UPL was removed through overlapping PCR. As expected from DNase I footprinting data, no shifted band was obtained with the truncated PLan, from which a positive UP element could be inferred. Compared with previously reported UP elements in PtrxA (terms as UPA here), UPL contained a high-consensus sequence of “AAGAAATAAT” and sheared adenine (A) and thymine (T) dinucleotide repeat tracts. This has been proven to be a crucial element for RNAP recognition and binding in B. subtilis [It is known that in subtilis . HowevertrxA. On this basis, we then selected the truncated UP element fragments with different lengths in flanking base pairs as DNA probes, and evaluated their interactions with α-CTD in vitro and the performance on expression activity. In this effort, we sought to identify a stronger UP element based on the ability to amplify expression of the PtrxA core promoter. For in vitro investigation, Fluorescence polarization (FP) provides a nonradioactive approach for measuring protein–DNA interactions. An increase in molecular volume due to binding of a small fluorescent molecule to a protein impedes the fluorescent molecule’s rotational motion in solution and results in an increase in polarization. According to this principle, the interaction between α-CTD and the engineered promoters with different flanking base pairs in UP elements was indicated by the degree of polarization change (DPC). It showed a clear correlation between the lengths of motif-flanking sequences and the polarization values (p < 0.0001).UP elements as expression activators have been tested in different bacteria . It was n values . As shiftrxA promoter activities mediated by the above different UP elements, cells harboring recombinant vectors were cultivated and collected for promoter strength detection, by using the same fluorescence method described in promoter selection. Wild-type PtrxA promoter was used as the control. As shown in To demonstrate the PtrxA were constructed . Several synthetic promoters harboring 1 to 5 tandem repeats of intact UP5 upstream of Pstructed . In preltrxA-UP5, respectively. After those synthetic promoters were synthesized, they were labeled and incubated with α-CTD. FP results exhibited a wide distribution for DPC. The most repeats (five) of the proximal subsite had the highest DPC of 52%, suggesting a significantly changed interaction. The same repeat number of the distal subsite also had a DPC of 35%. Seven in ten synthetic promoters exhibited polarization values corresponding to stronger binding. In terms of fluorescence intensity, only four in ten synthetic promoters were higher than that of the parent promoter with only a single copy of UP5. Remarkably, the strongest performing sequence was not the most repeats of R5 but R2 (UP5-2P) for the proximal subsite, which more strongly activated PtrxA expression by 8.2-fold. Likewise, R2 for the distal subsite also exhibited the highest expression intensity. These results indicate that repeats of the specific subsite within an UP element could improve the downstream promoter performance by facilitating promoter–α-CTD interactions. Excessively strong interactions between UP elements and RNAP could decrease gene expression.As α-CTD binds to UP elements at both the proximal subsite and the distal subsite, we hypothesized that each subsite could influence the initiation of transcription individually. It is therefore worth trying to engineer promoters using the respective subsite as an activator. To do so, we designed two sets of synthetic promoters with tandem repeats of “CAAAATAGC”  and “GGCATTTTG” , inserted upstream of UP5 sequences in PtrxA, especially those without a known α-CTD binding site. For this purpose, UP5-2P was inserted upstream of the other nine constitutive promoters screened previously. Just as predicted, as shown in acpp, was able to be more strongly activated by 7.4-fold. Interestingly, all these promoters have not been reported to have an α-CTD binding site other than PtrxA and PspovG. These results suggest that the function of UP5-2P is transferable when it is embedded upstream of multiple core promoters.The above results motivated us to investigate whether the synthetic UP element, UP5-2P, could function independently to improve the performance of promoters other than PBacillus respresents a group of important bacterial hosts for synthetic biology, especially for its excellent performance at an industrial scale [Bacillus expression system lags far behind E. coli or yeast. This can be largely attributed to the lack of strong promoters. The most currently used promoters are wild-type ones and originated in B. subtilis, such as P43 and PHpaII. The strongest constitutive promoter reported in B. subtilis is a hybrid one, PShuttle-09, whose activity was 8 times that of P43. Interestingly, this hybrid promoter was assembled artificially from two B. licheniformis genes: ylyB and luxS [Shuttle-09 was first evaluated in B. licheniformis, and the results also suggested that it was stronger than most reported Bacillus promoters within the first 24 h of cultivation. However, the activities of PspovG and PLan were highly significant after 24 h, and PLan exceeded PShuttle-09 by more than 28-fold in activity after 48 h. These results indicated that in addition to activities, theses promoters exhibited various patterns of transcription initiation corresponding to specific stages of cell growth. Based on the time point with the highest fluorescent intensity, PtrxA and PShuttle-09 can be classified as “exponential active promoters”and PspovG and PLan as “stationary active promoters” in B. licheniformis. It has also been reported that the expression of a series of genes was significantly upregulated during the transition from exponential growth to the stationary stage [Lan in this study was supposed to be involved in an uncharacterized quorum-sensing system. In addition to the obvious time-course in the changes of fluorescent signals, sequence alignment also suggested that the gene cluster transcribed from PLan may serve the functions of recognition and secretion of cell–cell signaling molecules. This is the first time a B. licheniformis quorum-sensing-based promoter has been described; it may be used as a strong expression element for synthetic biology. Unlike the exponential active pattern of most currently used promoters [The genus al scale . Howeverand luxS . In our ry stage . This tyromoters , the celBacillus. As emphasized by Phillips et al., RNAP binding to the UP element occurs independently of the other promoter element [ERNAP [E. coli, the conclusion is consistent with our findings in B. licheniformis. A moderately repeated element, UP5-2P, more strongly activated PtrxA expression by 8.2-fold. Moreover, the wide range of activation or inhibition seen in the tandem proximal/distal subsite constructs suggests that the UP element has a significant effect on promoter function. A standardized UP element can be developed as a plug-and-play regulatory element for genetic circuit designing, in which the expression levels of the target gene could be tuned to an appropriate input level for a certain pathway. A clear advantage of this type of synthetic element is its portable and modular characteristics. We showed that when UP5-2P was placed upstream of nine constitutive promoters, all of them showed increased expression strength. This may be because these promoters were weaker compared with PLan, and they received the greatest benefit upon addition of the UP element. We propose that the inhibition effect of a strong UP element, such as UP5-2P, would also be general and efficient for multiple wild-type promoters.RNAP has two points of physical contact with a promoter: the core promoter sequence (-10 and -35 element) and the UP element. This study confirms that the interaction between the specific DNA sequences in the UP element and α-CTD domain of RNAP can be utilized for rational engineering of promoters in  element . We show element . Perhaps element . Based ot [ERNAP . They suBacillus hosts, as it is now inconvenient to construct complex genetic circuits due to a limited range of promoter activity. As UP elements are also likely to be important components of promoters recognized by secondary sigma factors, it is hypothesized that these UP elements could activate core promoters corresponding to various other sigma factors to a predictable extent.Finally, this study shows that UP element manipulation alone is competitive against other methods of promoter engineering and can be applied to modulate the activity of promoters with different core regions. This is especially beneficial for Bacillus transformation were prepared according to Li [E. coli and Bacillus were grown in terrific broth (TB) based on Xiao’s methods [E. coli. Bacillus transformants were grown with 10 μg/mL erythromycin or 20 μg/mL tetracycline. Cultivation was performed at 37 °C in Luria–Bertani (LB) broth unless otherwise stated.The bacterial strains and plasmids used in this study are listed in ng to Li . E. coli methods . AmpicilBacilli strains and the predicted promoters in this study were cloned into pHY300-PLK, together with the egfp gene. All pHY plasmid constructs are listed in egfp fragment was cloned and inserted between the HindIII and SalI sites of pHY300-PLK. Then, promoters from different origins were assembled upstream of egfp at the HindIII site by using primer pairs of HpaII-F/R ~ R2-F/R. Specifically, PHpaII was cloned using the plasmid pMA5 as the template. PspovG, PydzA, P43, and PTrxA were amplified from genomic DNA of Bacillus subtilis 168. PglvA, PLan, PDS, and Pshuttle-09 were amplified from genomic DNA of B. licheniformis B1391. Finally, Pacpp and Pr2 were synthesized according to the genomic sequence of B. megaterium DSM319 and B. amyloliquefaciens XH7, respectively.For the purpose of strength evaluation, the selected promoters reported from other E. coli JM109.For construction of synthetic promoters with different flanking sequence lengths, UP0~UP5 were introduced by primer pairs UP0-F~UP5-F and GFP-R. These PCR reactions were performed by using the plasmid pTrxAGFP as the template. The amplified fragments were also assembled in plasmid pHY300-PLK and then transformed into E. coli JM109.For the construction of synthetic promoters with a repeated specific subsite in the UP element, UP5-1P~UP5-5P and UP5-1D~UP5-5D were introduced by primer pairs UP5-1P-F~UP5-5P-F, UP5-1D-F~UP5-5D-F, and GFP-R. These PCR reactions were performed by using the plasmid pTrxAGFPUP5 as the template. The amplified fragments were also assembled in plasmid pHY300-PLK and then transformed into E. coli JM109.For the construction of synthetic promoters with the engineered UP element, UP5-2P, upstream of different parent promoters, primer pairs UPHpaII-F ~ UPR2-F and GFP-R were used to incorporate the engineered UP element, by using plasmids pHpaIIGFP~pR2GFP as the templates, respectively. The amplified fragments were also assembled in plasmid pHY300-PLK and then transformed into BamHI and SalI sites of pET-28(a) and transformed into competent E. coli DE3. Standard cloning and E. coli transformations were performed according to Sambrook and Russell [B. licheniformis, a method of electrotransformation was used [The gene encoding α-CTD in the core enzyme of RNA polymerase was amplified using primer pairs CTD-F/R and gnomic DNA of B1391. This was then cloned between  Russell . To tranwas used . PCR reaB. licheniformis involved inoculation in 30 mL of TB medium supplemented with 20 g/L glucose at an inoculation volume of 3%. This was then kept at 37 °C with orbital shaking at 250 rpm. The culture (1 mL) was sampled at different time points. Cells were collected by centrifugation at 9000 g, washed with 0.9% saline, and diluted to OD600 = 0.5–1.0. GFP fluorescence was measured  using a 96-well microtiter plate at an excitation wavelength of 485 nm, emission wavelength of 535 ± 15 nm, and a gain value of 100. Average fluorescence and standard deviation were calculated from the geometric mean fluorescence values of technical triplicates.Overnight culture of the recombinant 600 of the cell culture reached 0.8, IPTG was added at a concentration 0.5 mM. After 12 h of incubation, cells were harvested by centrifugation and washed twice with a phosphate buffer (pH 7.4). They were lysed using a Sonics VCX 750  ultrasonic processor and the crude enzyme was subjected to affinity purification using Mag-Beads His-Tag , following the manufacture’s instruction.The strain EcCTD harboring pCTD was inoculated in 30 mL TB media. When the ODEMSA was carried out following Zhang’s report , with so2, and 0.1 mM DTT) at room temperature for 20 min according to the method of Zhang [DNA probes were amplified by PCR using FAM labeled primers (synthesized by Sangon biotech) and the different promoters as templates. All the primers are shown in of Zhang . Then, tDNase I footprinting experiments were performed as described in a previous study . The DNAp values of less than 0.05 indicated significant results.All experiments were performed at least three times, and the results were expressed as the means ± standard deviations (SDs). Statistical analyses were performed using Student’s t test and ANOVA followed by a Tukey’s post hoc test.


Transcriptional reprogramming is an integral part of plant immunity. Tight regulation of the immune transcriptome is essential for a proper response of plants to different types of pathogens. Consequently, transcriptional regulators are proven targets of pathogens to enhance their virulence. The plant immune transcriptome is regulated by many different, interconnected mechanisms that can determine the rate at which genes are transcribed. These include intracellular calcium signaling, modulation of the redox state, post-translational modifications of transcriptional regulators, histone modifications, DNA methylation, modulation of RNA polymerases, alternative transcription inititation, the Mediator complex and regulation by non-coding RNAs. In addition, on their journey from transcription to translation, mRNAs are further modulated through mechanisms such as nuclear RNA retention, storage of mRNA in stress granules and P-bodies, and post-transcriptional gene silencing. In this review, we highlight the latest insights into these mechanisms. Furthermore, we discuss some emerging technologies that promise to greatly enhance our understanding of the regulation of the plant immune transcriptome in the future. However, plants do respond to pathogens by activating their robust yet specialized innate immune system. General pathogen-associated molecular patterns (PAMPs) and specific apoplastic pathogen effectors are perceived by the plants’ surface-localized pattern-recognition receptors (PRRs), leading to activation or prevention of pattern-triggered immunity (PTI), respectively. In addition, specific pathogen effector molecules that are secreted into plant cells are recognized by intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), activating effector-triggered immunity (ETI) . Dependi2+) ,7. The p2+) . Here, wThe timing and efficiency of elicitation of the immune transcriptome is essential for plants to halt pathogens. Of all the cellular components that are involved in transcriptional reprogramming, the role of transcription factors (TFs) in regulating crucial defense responses is best studied. Mutations in TFs including WRKYs, TGAs, NACs, CBP60s/SARD1, ERFs, bZIPs, bHLHs, MYBs, CAMTAs, and TCPs alter plant disease resistance against different pathogens . Some ofXanthomonas oryzae pv. oryzicola (Xoc) were shown to activate expression of OsF3H03g, encoding a 2-oxoglutarate-dependent dioxygenase that negatively regulates SA-related defense and promotes susceptibility against Xoc in Oryza sativa [Xanthomonas sp. induce SWEET sugar transporter genes, resulting in an increased availability of sugar for the pathogen, thereby promoting pathogenesis [Another well-studied example of direct interference with plant immune transcription is that of transcription activator-like effectors (TALEs), which are deployed by many plant-pathogenic xanthomonads. TALEs bind to effector binding elements (EBEs) in the promoters of host susceptibility (S) genes that contribute to disease ,25. Recea sativa . Moreoveogenesis ,28.P. syringae pv. tomato DC3000 (Pto) [The plant immune transcriptome encompasses both activation and repression of genes with diverse molecular functions, ranging from the control of general metabolic processes to highly specific responses that are directed toward a particular organism . The rel00 (Pto) ,35. Furt00 (Pto)  and epig00 (Pto) .2+-dependent protein kinases (CDPKs). These can directly activate TFs, such as the defense-regulating CaM-binding TF family CAMTA and CBP60g, or WRKY28, WRKY33 and WRKY48, which are phosphorylated by CPK5 and CPK6 (2+-activated CAMTA3 (or AtSR1) TF represses expression of the SA regulator NPR1 and the SA biosynthesis gene ICS1 [P. syringae [A rapid influx of calcium and a change in redox status are vital parts of plant immunity and they play intertwined roles in PTI and ETI . Calciumand CPK6 E. This lene ICS1 . Howeverene ICS1 ,51, its ene ICS1 . Moreovesyringae .in vitro only [2-NPR12-TGA32 complex, and possibly also form complexes with other transcription regulators, to regulate the immune transcriptome [The production and signaling of reactive oxygen species (ROS) is tightly connected to that of calcium, as these molecules can (in)directly regulate each other’s cellular concentrations . Both PTtro only . Howevercriptome .Botrytis cinerea by regulating crucial defense-related responses such as camalexin production in Arabidopsis [Post-translational regulation of TFs can alter their activities E. This ibidopsis ,62. The bidopsis , and thebidopsis ,63–66. Mbidopsis . The phobidopsis ,68. GeneArabidopsis against B. cinerea [Phosphorylation has also been shown to be important for the transactivation activity and binding specificity to DNA motifs of the ERF TF ORA59, which is required for defense induction in  cinerea . The hor cinerea . This ca cinerea .Ubiquitination also regulates TF activities via protein turnover. For instance, SA induces TF ORA59 ubiquitination and degradation via the 26S proteasome pathway ,72. The Arabidopsis after infection with an engineered non-pathogenic P. fluorescens strain either expressing the effector AvrRps4 (thus causing both PTI and ETI) or a non-recognized effector mutant (thus causing PTI only), and this was compared with RNA-seq data [Chromatin context is a major determinant for transcriptional activities in all eukaryotic cells. The accessibility of chromatin can influence when and where TFs, other regulators and RNA polymerases find their targets to activate transcription. The chromatin state can be altered through modification of histone tails and deposition of histone variants F. Recentseq data . Over onseq data . In anotseq data . Repositseq data . Notablyseq data ,80, indiIt is still unclear whether the accessibility of the regulatory DNA regions precedes transcription or vice versa. It was found that TF WRKY33 enhances accessibility of genes to reinforce gene transcription. The chromatin remodeling complex SWR1 and the MAPK-WRKY33 module promote deposition of H2A.Z , a variaChromatin remodeling also plays a role during ETI-triggered PCD. During this process chromocenters (regions with heterochromatin) get less dense and different chromatin marks get redistributed, such as the repressive marks H3K9me2 and H3K27me3, leading to altered transcription . StudiesAGO1 results in reduced JA-induced gene expression [Chromatin remodeling can also lead to altered transcription via a non-canonical function of the gene-silencing-related component ARGONAUTE1 (AGO1). AGO1 binds to chromatin around specific genes, likely dependent on its association with specific small RNAs and through interaction with several subunits of the SWI/SNF chromatin remodeling complex . There, pression .DNA methylation is generally associated with suppression of activity of transposable elements and with transcriptional repression G. DNA meDemethylation of DNA occurs either passively during replication or actively by different demethylases under specific conditions. The DNA methylome is altered upon pathogen attack, which modulates the immune response ,86. The dme revealed that DME alters methylation of hundreds of genomic regions and is affected in defense against bacterial and fungal pathogens [dme was silenced in the background of a triple mutant of the DNA methylases ros1, dml2 and dlm3 (rdd) suggest that DME acts redundantly with other demethylases to regulate expression of defense genes via demethylation [Another example is the demethylase DEMETER (DME). Loss-of-function mutants of this enzyme are lethal, but recent studies using plant lines with a weak allele generated by CRISPR-CAS9 or silencing of athogens . Moreovehylation .rdd is impaired in resistance against Pto induced by the immune-stimulating molecular patterns flg22, elf18 and Pep2 [rdd mutant, which is associated with a higher number of differentially methylated promoter regions of defense-related genes and their higher expression level in wild-type compared with mutant. Altogether, the studies discussed here show that DNA demethylation of specific regions is important for a proper immune transcriptome. It has not been explored how the four demethylases change their activity during an immune response, so the spatiotemporal relevance of each enzyme in the regulation of the immune transcriptome remains to be determined.In addition, the demethylation-deficient mutant and Pep2 . The flgPto, demonstrating the essential role of phosphorylation of the CTD of PolII in regulation of immunity against this pathogen [Arabidopsis homologue of CPL1 has previously been associated with miRNA processing [During immune activation PolII is phosphorylated H. For expathogen . Anotherpathogen . Additiopathogen . Howeverocessing  and RdDMocessing .Alternative transcription initiation can expand the regulatory repertoire of the genome, since it may involve alternative promoters that are differentially induced upon different stimuli, or result in different transcripts and proteins I. RecentTFs recruit PolII through interactions with the multi-subunit Mediator complex  Figure J. RecentMED18 and NRPD2a are highly expressed after B. cinerea infection, and mutants and overexpressors of these genes corroborate their importance for a part of B. cinerea-induced gene expression and for resistance against B. cinerea [nrpd2a mutant and possibly also of the med18 mutant. However, this still needs to be investigated.Although Mediator usually connects TFs to PolII, it can also recruit other RNA polymerases that are relevant for defense. The Mediator subunit MED18 interacts with NUCLEAR RNA POLYMERASE D2 (NRPD2a), a subunit of PolIV and PolV . MED18 a cinerea ,101. Pol cinerea , suggestAGO1 and several AS regulators are among its targets, suggesting that apart from its own role in AS, it also indirectly affects gene silencing and AS [cpr5 background [Nuclear im/export of transcriptional components is a selective mechanism to control the plant immune transcriptome K. This wg and AS . In addickground . This waRegulation of messenger RNA (mRNA) largely impacts the formation of proteins. For example, AS of NLR genes and JAZ genes generates isoforms with diverse activities or subcellular localizations, by which plants can control immunity activation  Figure L. TreatmLupinus luteus and Arabidopsis are retained in the nucleus during hypoxia, and released in the cytosol upon reaeration [Moreover, RNA can be (temporarily) stored in the nucleus, or in special aggregations that are involved in temporary storage and/or degradation, which decreases the pool of translating RNAs M–P. For aeration . It is uaeration ,111. Theaeration . It was aeration . This leaeration .Heterodera glycines [Different non-coding RNAs, like small RNAs and long non-coding RNAs (lncRNAs) regulate different steps in gene expression. For a comprehensive overview on non-coding RNAs in plant immunity we refer to a recent review . Small Rglycines . They foELENA1 is induced by treatment with the PAMPs flg22 and elf18 [ELENA1 show that it promotes PR1 and PR2 expression and resistance to Pto. RNA-seq revealed that a subset of elf18-induced defense genes overlaps with ELENA1-induced genes. Upon elf18 treatment, ELENA1 interacts with MED19a in vivo, which promotes binding of MED19a to the promoter of PR1 and possibly other genes [A recent study reported that the lncRNA nd elf18 . Overexper genes   different immune-stimulatory treatments should be compared and (ii) different whole-genome, multi-omics data sets should be combined, (iii) followed by advanced integrated data analysis including the use of mathematical modeling tools . ExampleAt a finer resolution, namely the cell level, other critical questions need attention. Which of the immune responses are cell-type specific? And does that determine whether the initial infection of a certain cell type propagates further to adjacent cells or is halted? Furthermore, how does cell homeostasis, related to different internal and external conditions, such as plant age, time of day, abiotic stress, and spatiotemporal distance from the infection site, influence the plant immune transcriptome? To answer these questions, single-cell methods instead of bulk analyses using the omics assays and molecular tools mentioned in this review would be extremely meaningful (reviewed by ), especiThe plant immune transcriptome is induced upon pathogen perception and required for disease resistance.Many pathogens use effectors to tweak the plant immune transcriptome to their own advantage.Plants regulate their immune transcriptome at multiple scales, e.g. post-transcriptional regulation of TFs, modulation of DNA accessibility, and modulation of mRNAs during their journey from transcription to translation.A combination of multi-omics datasets can provide new insights into immune-related gene regulatory networks.


Transposable elements (TEs) are mobile genetic elements that can randomly integrate into other genomic sites. They have successfully replicated and now occupy around 40% of the total DNA sequence in humans. TEs in the genome have a complex relationship with the host cell, being both potentially deleterious and advantageous at the same time. Only a tiny minority of TEs are still capable of transposition, yet their fossilized sequence fragments are thought to be involved in various molecular processes, such as gene transcriptional activity, RNA stability and subcellular localization, and chromosomal architecture. TEs have also been implicated in biological processes, although it is often hard to reveal cause from correlation due to formidable technical issues in analyzing TEs. In this review, we compare and contrast two views of TE activity: one in the pluripotent state, where TEs are broadly beneficial, or at least mechanistically useful, and a second state in human disease, where TEs are uniformly considered harmful. Transposable elements (TEs) are mobile genetic elements that are found in multiple copies in the genome. TEs were first discovered as mutable loci in 1944, in the study of the corn kernel and leaf color variegation in maize . BarbaraThe functionality of TEs has always been under some debate. Back in 1972, Sozumu Ohno termed TEs as “junk” DNA in the Brookhaven Symposium in Biology journal . Indeed,TE abundance varies from several copies up to several thousands of copies of the same element. Although any individual element of the same type is different, due to mutation and truncations, they nonetheless retain some homology. Indeed, even though a TE is inactive due to truncations or mutations, high copy number TEs with similar sequences can provide a sufficient template for recombination and genome rearrangement . Thus, eNewly inserted mobile elements could lead to structural variants including deletions, insertions, duplications, and inversions, which may exist as polymorphisms within the population. Strikingly, there are more than 16,000 polymorphic TEs in the human genome, accounting for ∼24% of all known structural variants; many of these are common variants, with over 6,500 (36%) having a minor allele frequency >0.01 . These iOver the past few decades, scientists have mainly focused on several areas in the study of TEs, particularly the annotation, classification, and evolution of TEs, and less emphasis has been paid to their functions. However, recent studies have increasingly shown that TEs play a vital role in a wide range of biological processes . TEs canIn addition to the direct co-option of protein-coding TE sequences, TE sequence fragments also play subtle and complex roles in other cellular processes. TE sequences can be transcribed and can influence RNA activity when TE-derived sequence fragments are embedded in RNA sequences  Figure . Indeed,TEs have been reported to be expressed in a highly tissue-specific manner . AlthougTo explain the high activity of TEs in early embryo cells and hPSCs, two hypotheses have been proposed. The first suggests that the relatively high activity of the TEs in hPSCs is a consequence of the strategies used by TEs to duplicate themselves across generations . If a TEOnly a handful of TEs (<0.05%) are still capable of transposition in the human genome , the majTE activity is tightly regulated in biological systems. In somatic cells, the regulation of TE activities is controlled by DNA methylation . HoweverTEs play important roles in the regulatory networks of ESC . They coThe transcription of TEs in human pluripotent stem cells (hPSCs) has been extensively investigated. Several studies have reported various proportions of TEs in human transcripts, it is widely agreed that TEs contribute more to lncRNAs than protein-coding transcripts, suggesting they are a major component of lncRNAs . For exaTranscript stability and subcellular localizations are largely controlled by RNA binding proteins . MechaniA similar pattern is seen in hPSCs, and TE-sequences in RNAs are bound by RBPs. Analysis of the RBPs DDX6, ILF2, FUS, and DCP1B in hPSCs revealed that TE-containing transcripts have unique RBP interaction . For exaThe involvement of TEs in harmful mutations, gene dysfunction, DNA double-strand breaks, gene recombination, gene expression dysregulation, and other types of mutations implies that TEs might contribute to human disease. There is growing evidence of a link between TE sequence fragments, PSCs, and cancer. TEs can act as oncogene-specific enhancers, promoters, and exons for pluripotency-specific genes, this drives their expression and converts them into oncogenes . This buTE transposition activity has been suggested to contribute to human genetic diseases, primarily through the transposition of LINE-1, SINE Alu, and SVA (SINE-VNTR-Alu) TEs . StrikinAPC tumor suppressor gene. The insertion included the 3′ part of a LINE-1 and around 180 base pairs of polyadenylated sequence   . In addi (c-Met) . Also, g (c-Met) . In addi (c-Met) , suggest (c-Met) . Taken tIn addition to LINE-1, the expression of ERVs has also been reported in cancer , includicis-regulatory elements to mediate the activation of the autoimmune gene expression in Fanconi anemia and Aicardi-Goutières syndrome  and other neurological diseases . In factTEs are active in different cell types and developmental stages, they are hyperactive in hPSCs where they function in transcriptional regulation, transcript processing such as splicing, RNA stability, and translational processes. Intriguingly, TEs are broadly positive in early embryogenesis, contributing to gene regulation pathways, and acting as a substrate for evolutionary innovation. This is in contrast to the role of TEs in somatic tissues, which tend to be more negative, being associated with the development of human diseases. TEs and their derived peptides or sequence fragments have complex roles in the cell. However, many of these roles remain unclear. Considering the vast number of TEs in the human genome it has, and remains, challenging to study them. However, there is likely much critical information that remains to be discovered concerning both the advantageous and deleterious functions of TEs in both embryogenesis and somatic cells.


The GRIN1, ASCL3, and NOS1 genes are associated with various phenotypes of neuropsychiatric disorders. For instance, these genes contribute to the development of schizophrenia, Alzheimer’s and Parkinson’s diseases, and epilepsy. These genes are also associated with various cancers. For example, ASCL3 is overexpressed in breast cancer, and NOS1, in ovarian cancer cell lines. Based on our findings and literature data, we had previously obtained results suggesting that the single-nucleotide polymorphisms (SNPs) that disrupt erythropoiesis are highly likely to be associated with cognitive and neuropsychiatric disorders in humans. In the present work, using SNP_TATA_Z-tester, we investigated the influence of unannotated SNPs in the TATA boxes of the promoters of the GRIN1, ASCL3, and NOS1 genes (which are involved in neuropsychiatric disorders and cancers) on the interaction of the TATA boxes with the TATA-binding protein (TBP). Double-stranded oligodeoxyribonucleotides identical to the TATA-containing promoter regions of the GRIN1, ASCL3, and NOS1 genes  and recombinant human TBP were employed to study in vitro (by an electrophoretic mobility shift assay) kinetic characteristics of the formationof TBP–TATA complexes and their affinity. It was found, for example, that allele A of rs1402667001 in the GRIN1promoter increases TBP–TATA affinity 1.4-fold, whereas allele C in the TATA box of the ASCL3 promoter decreasesthe affinity 1.4-fold. The lifetime of the complexes in both cases decreased by ~20 % due to changes in the rates ofassociation and dissociation of the complexes . Our experimental results are consistent withthe literature showing GRIN1 underexpression in schizophrenic disorders as well as an increased risk of cervical,bladder, and kidney cancers and lymphoma during ASCL3 underexpression. The effect of allele A of the –27G>ASNP (rs1195040887) in the NOS1 promoter is suggestive of an increased risk of ischemic damage to the brain incarriers. A comparison of experimental TBP–TATA affinity values (KD) of wild-type and minor alleles with predictedones showed that the data correlate well . Previously, using Web service SNP_TATA_Comparator and in vitro experiments, westudied effects of SNPs in TATA boxes within core promotersof human genes to predict potential SNP markers, for example,markers of obesity , autoimmune diseases, Alzheimer’s disease , aggressiveness ,circadian rhythm disorders ,anomalies of female reproductive potential , erythropoiesis disorders , andresistance to anticancer therapy . Then,on the basis of our results and literature data, we made findingssuggesting that SNPs that disrupt erythropoiesis are likely tobe associated with cognitive and neuropsychiatric disordersin humans .The aim of the current work was to search for and to experimentallyverify in vitro the effects of unannotated SNPsin TATA boxes within promoters of genes NOS1, GRIN1, andASCL3 (which are involved in neuropsychiatric disordersand cancers) on the affinity and kinetic characteristics ofTBP–TATA complexes. Identification of causal regulatorymechanisms of diseases is becoming a common practice, butexperimental annotation of variants in target genes, especiallyin regulatory regions, is still a major bottleneck for the useof such genetic data in personalized medicine. Therefore,experimental quantitative methods for annotating SNPs inregulatory regions of specific genes remain important andrelevant. This paper presents predictions of effects of unannotatedSNPs in TATA boxes of genes GRIN1, ASCL3, andNOS1 on the thermodynamic and kinetic characteristics ofTBP–TATA complexes as well as the results of their experimentalverification in vitroThe GRIN1 gene, located in chromosomal region 9q34.3,codes for the GluN1 (NR1) subunit of the N-methyl-Daspartatereceptor (NMDAR) and plays a key role in synapticfunctions . The protein encoded by GRIN1 isa critical subunit of N-methyl-D-aspartate receptors, which aremembers of the superfamily of glutamate receptor channels.The latter are heteromeric protein complexes with multiplesubunits arranged to form a ligand-gated ion channel. These subunits play a key part in synaptic plasticity, which is thoughtto underlie memory and learningThe first meta-analysis and convergence analysis  of available genome-wide expression studies regardingepileptogenesis in humans and model animals made itpossible to identify several major candidate genes, includingGRIN1. Animal models and postmortem studies on patients’brains have shown that transcription and expression levels ofthe gene of the GluN1 protein in schizophrenia differ fromthose in controls , althoughthe changes varied among different regions of the brain .The achaete-scute complex-like (ASCL) gene family consistsof five members, namely ASCL1, ASCL2, ASCL3, ASCL4,and ASCL5. The ASCL3 gene (SGN1) is located on chromosome11, and its product was originally characterized as atranscription factor specifically localized to cells of salivarygland ducts . Dysregulation of ASCL familygenes has been reported to play a key role in psychiatric andneurological disorders . All ASCLgenes encode the basic helix-loop-helix transcription factorsthat control nervous system development ; thus, they are called proneural genes. Expression ofASCL family genes and the impact of their products on cells arenot limited to the nervous system. For example, ASCL familymembers have been shown to be expressed in progenitor cellsduring muscle and intestinal-cell differentiation .Using bioinformatic analyses, researchers have determinedpotential involvement of several ASCL family members inthe initiation and progression of tumors in various types ofcancer. ASCL3 is overexpressed in breast cancer  but is underexpressed  in kidney, cervical, and bladder cancers as well aslymphoma and melanoma. Analysis of different subtypes ofkidney tumors has revealed that ASCL3 is downregulatedin renal oncocytoma. As has already been mentioned, inlymphoma and cancers of the cervix, bladder, kidney, andepithelium, a decrease in the expression of ASCL3 has beendocumented , suggesting that thisgene is a suitable research object not only in psychiatric andneurological disorders but also in cancers.The NOS1 gene codes for the major isoform of nitric oxidesynthase and is widely expressed in all tissues, and NOS1produces approximately 90 % of nitric oxide in the central nervoussystem . The gene is mapped to chromosomalregion 12q24. Several studies indicate that NOS1variants are associated with such disorders as Alzheimer’sdisease , schizophrenia , and Parkinson’s disease . Using reverse-transcriptionpolymerase chain reaction (RT-PCR), some authors  demonstrated that protein expression ofNOS1 is constitutively high in ovarian cancer cell lines andthat mRNA expression of NOS1 varies among such cell lines.The results of that study mean that NOS1 promotes malignantcharacteristics of ovarian cancer cells, including proliferation,invasion, and chemoresistance, thereby constituting a potentialtherapeutic targetDNA sequences. Unannotated SNPs  were retrieved from the dbSNPdatabase . Promoter sequences within the region relative to a transcription start site wereretrieved from the Eukaryotic Promotor Database (EPD) , and the presence of TATA boxes in these regionswas determined in the same databaseAnalysis of DNA sequences in silico. DNA sequences ofhuman genes GRIN1, ASCL3, and NOS1 between nucleotidepositions –100 and –1 upstream of the transcription startsite, which were taken from the reference genome, wereanalyzed using our Web service SNP_TATA_Z-tester,which is a modified version of SNP_TATA_Comparator.Synthetic double-stranded oligodeoxyribonucleotides(ODNs). For experimental verification, we used 26 bp ODNsidentical to the reference and minor alleles of genes GRIN1,ASCL3, and NOS1; the ODNs were synthesized and thenpurified by polyacrylamide gel electrophoresis by Biosan.Sequences of these double-stranded ODNs – identical tothe promoter regions of genes GRIN1, ASCL3, and NOS1containing TATA-like elements – were as follows :GRIN1 (WT) – 5′-tggagggggACAAAGACAgggtggtg-3′GRIN1 (–34g>a) – 5′-tggaggaggACAAAGACAgggtggtg-3′ASCL3 (WT) – 5′-tcgaaaaaTAAAATAAAAtaaaacat-3′ASCL3 (–45T>c) – 5′-tcgaaaaaTAAAAсAAAAtaaaacat-3′NOS1 (WT) – 5′-tgtttcctGATAGAAAaaaaaaatgg-3′NOS1 (–27G>a) – 5′-tgtttcctGATAaAAAaaaaaaatgg-3′Labeling of the ODNs at 5′ ends with 32Р-γАТР. Toprepare labeled double-stranded ODNs, both their strandswere labeled with 32P-γATP  bymeans of T4 polynucleotide kinase , annealed at 95 °C (at an equimolar ratio), and slowlycooled to room temperature. The annealed duplexes werepurified and analyzed by electrophoresis in a nondenaturing15 % polyacrylamide gel followed by autoradiography ona phosphorimager, Molecular Imager PharosFX Plus . Unlabeled duplexes were prepared in the same way and used without further purification bypolyacrylamide gel electrophoresisIsolation and purification of recombinant TATA-binding(TBP) protein. We used recombinant human TBP expressedin Escherichia coli BL21(DE3) cells via the pAR3038-hTBP plasmid . Expression and purification of TBPwere performed according to ref. , except for theconcentration of isopropyl β-D-1-thiogalactopyranoside (1.0instead of 0.1 mM) and induction time (3 instead of 1.5 h).Determination of kinetic parameters  and equilibriumdissociation constants, KD, of TBP–ODN complexes.Association rate constants (ka) and dissociation rateconstants (kd), which characterize the rates of formation anddisintegration of complexes, were determined by measuringthe kinetics of TBP binding to the TATA-containing doublestrandedODNs identical to either a WT TATA box  or a TATA box carrying an SNP. The experimentswere conducted at several concentrations of the 32P-labeledODNs and a fixed concentration of TBP (0.4 nM unless statedotherwise). TBP–ODN binding experiments were performedat 25 °C in a buffer composed of 20 mM HEPES-KOH(pH 7.6), 5 mM MgCl2, 70 mM KCl, 1 mM dithiothreitol,100 μg/ml bovine serum albumin, 0.01 % of NP-40, and5 % of XYZ, as described in detail before . The electrophoretic mobility shift assay was carriedout in a native 5 % polyacrylamide gel in Tris-glycine buffer(pH 8.3) for 40 min at 10 °C. The gels were dried, and ImagingScreen-K (Kodak) for Molecular Imager PharosFX Plus (Bio-Rad) was exposed to the gels. Each screen was scanned on thephosphorimager, and the autoradiographs were quantified inQuantity One v.4.5.0 software (Bio-Rad).Statistical analysis. The comparison of the predicted andexperimental values of TBP–TATA complexes’ affinity for the“normal” and minor alleles was performed using the Statisticasoftware package .Transcription, as a rule, is the gene expression stage mostsensitive to internal signals and external signals entering thecell and is the main mechanism that controls gene expression.Here, we analyzed its initial stage, i. e., the interaction of TBPwith a promoter: the process that triggers the assembly of thetranscription complexThe Table presents the results of in vitro verification of ourpredictions (made by the SNP_TATA_Z-tester Web service)regarding the effect of substitutions in the TATA boxes ofgenes GRIN1, ASCL3, and NOS1 on TBP–TATA affinity.These data include experimental affinity (KD) values for WTand minor alleles and association and dissociation constants, reflecting the rates of formation anddisintegration of TBP–TATA complexesThe results given in the Table were obtained by theelectrophoretic mobility shift assay. Figure 1 shows electropherogramsfor the GRIN1 gene as an example.As readers can see in the Table, the TBP–TATA affinityfor WT GRIN1 can be described as low-specificity binding (KD = 320 nM). Although the TATA box sequence is AT-rich(ACAAAGACA), the third T, which has the greatest weightin the TATA box, is missing. In addition, conformationalflexibility of the three As, which is clearly not enough togenerate the conformation suitable for TBP binding, is blockedon both sides by high-melting-point base pairs containing Cand G. The substitution (–34G>a) in the sequence flankingthe TATA-like element did not significantly increase theflexibility of this DNA region, but the affinity strengthenedalmost 1.4-fold after introduction of the substitution  as compared to the WT allele (KD = 200 nM). Therate of formation of the TBP–ODN complex increasedtwofold: 2.8 versus 1.4 M–1·s–1 . The rate of complexdissociation was also slightly higher, by 20 %, in the case ofthe minor allele. Overall, these changes somewhat shortenedthe complex’s half-life (from 25 to 20 min), that is, made itless stable. Judging from the sequence of the ODN in question,which is identical to the region of the GRIN1 promoter, thispromoter does not contain the TATA box consensus sequencebut contains a G-rich box, which can bind to transcription factor SP3, which activates transcription of genes in chickenembryonic cortical neurons and represses it in undifferentiatedcells , as demonstrated in a study onthe chicken GRIN1 gene.The observed (in our study) weak affinity of TBP for theTATA-like element of the GRIN1 promoter, which impliesgene underexpression , is in agreementwith findings of low GRIN1 expression in animal models ofschizophrenic disorders. On the basis of our data on enhancedTBP–TATA affinity for minor allele “a” (rs1402667001), wecan theorize that carriers of this allele are at a reduced risk ofschizophrenia as compared to carriers of WT allele G. It shouldbe noted that despite active research, the role of GRIN1 in theetiology of schizophrenia remains uncertain. For instance,in ref. , using Sanger DNA sequencing,researchers conducted a case-control study to investigate theassociation between GRIN1 and the risk of schizophreniain a population of northern China. Distributions of botha genotype and allele of rs117783907 (–1945G/t) significantlydiffered between the case group and control group (p < 0.0083).In that article, genotype frequencies of rs138961287 andrs11146020 are statistically significantly different too(p < 0.05), indicating that rs138961287, rs117783907, andrs11146020 are associated with schizophrenia. In anotherassociation study conducted in a northern Chinese Hanpopulation, allele “c” of rs11146020 was reported to reducethe risk of schizophrenia , although Saadat(2010) found that this allele is a risk factor of schizophrenia inan Italian population. Furthermore, a meta-analysis  suggests that allele “c” of rs11146020 is associatedwith an increased risk of schizophrenia, i. e., the results areinconsistent among studies. It is likely that among differentethnic groups, the influence of environmental and geneticfactors and their interactions may differ in their impact on therisk of mental disorders . The risk ofpsychosis increases with the accumulation of multiple variantscarrying a genetic risk and with exposure to multiple adverseenvironmental factors .Some authors  investigated the expressionof a large group of genes in the brains of patients with majordepressive disorder and controls . Theresults showed elevated expression of most of glutamatergicgenes  tested in the dorsolateral prefrontal cortex (mainlyin women). Based on these findings, it can be assumed that rs1402667001 (studied by us), which enhances the affinityof TBP for the TATA-like element, may be a candidateSNP marker of an increased risk of schizophrenia. Despiteconflicting results, some researchers  believethat the association of GRIN1 with schizophrenia and otherpsychotic disorders is undeniable and that subunit NR1encoded by this gene may be a promising therapeutic targetin schizophreniaAs readers can see in the sequence of the ASCL3 promoterregion, which is identical to the region where a TATA boxis usually located (positions –20 to –70 relative to thetranscription start site), it is enriched in A nucleotides andcontains a T. The latter has the greatest weight in the TATAbox sequence (for the binding to TBP) and can take the thirdposition in our case. Accordingly, we observed strong affinityof the TBP–TATA complex: KD = 12 nM. SNP –45T > C(rs1049743008), replacing T with high-melting-point C, ledto a 1.4-fold weakening of the affinity (KD = 17 nM), althoughthe rate of formation of the TBP–TATA complex increasedslightly (12 %), while the rate of dissociation increaseda little more: by 20 %. As a consequence, the complex’shalf-life with the minor allele is also slightly shorter (11.5versus 14.4 min), i. e., stability decreased. Because changesin the affinity of the TBP–TATA interaction correlate withalterations of gene expression , it canbe hypothesized that carriers of the C allele (with weakenedTBP–TATA affinity and ASCL3 expression) are at a higher riskof a malignant tumor: lymphoma and cancers of the cervix,bladder, epithelium, and kidneys. This notion is confirmed bythe results in ref. , where a databaseanalysis revealed that out of 21 analyzed tumor types, fivecorrelate with the ASCL3 expression that is diminished tovarious degreesThe NOS1 promoter contains a TATA-like element withsequence GATAGAAA, to which TBP binds with 70 nMaffinity. When high-melting-point G was replaced in ourstudy by A, the affinity strengthened, albeit slightly: by 14 %(KD = 59 ± 9 nM). In this context, the rate of TBP–TATAcomplex formation (ka) increased by a factor of 1.7, while thedissociation rate of the complex (kd) accelerated by a factorof 1.4, and the lifetime of the complex diminished ~1.4-fold.Based on the results in ref.  indicatingthat NOS1 inhibitors can effectively reduce the severity ofischemic brain damage, it can be theorized that the A allele – with enhanced TBP–TATA affinity and gene expression – may bea candidate marker of an elevated riskof the ischemic brain injury associatedwith cerebral palsy. The associationof NOS1 with various diseases pointsto a pleiotropic role of NOS1 in manyphysiological processes and potentiallyto a pathogenesis that is shared amongthese diseases.Our comparison of the experimentalaffinity values (KD) of TBP–TATAcomplexes of reference (WT) allelesand minor alleles with those predictedby the SNP_TATA_Z-tester Web service indicatesthat the data correlate well .Thus, we determined the affinity andkinetic characteristics of the interactionof TBP with TATA boxes containingunannotated SNPs. We found that theseSNPs may be functionally significantand correlate with an increased riskof such neuropsychiatric diseases asschizophrenia and ischemic braindamage as well as the risk of malignant tumors:lymphoma and cancers of the cervix,epithelium, bladder, and kidneys.The results show effects of the analyzedSNPs  in the TATA boxeswithin promoters of genes GRIN1,ASCL3, and NOS1 on affinity and the rates of formation and disintegration of TBP–TATA complexes .Our experimental data suggest that the identified candidate SNP markers inneuronal genes can contribute to the development of not only neuropsychiatric butalso oncological diseases, in agreement with the results obtained by other authors.Our findings about the influence of SNPs on TBP–TATA affinity and therefore onthe expression of the genes in question point to their possible contribution to ahigher risk of the diseases associated with these genes. 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Drosophila. dCas9-VPR was targeted to the TAHRE telomeric element, an example of constitutive heterochromatin, and to promoters and enhancers of the HOX Ultrabithorax (Ubx) and Sex Combs Reduced (Scr) genes in the context of facultative heterochromatin. dCas9-VPR robustly activated TAHRE transcription, showing that although this element is heterochromatic, dCas9-VPR was sufficient to activate its expression. In the case of HOX gene promoters, although Polycomb complexes epigenetically silence these genes, both were ectopically activated. When the artificial TF was directed to enhancers, we found that the expression pattern was different compared to the effect on the promoters. In the case of the Scr upstream enhancer, dCas9-VPR activated the gene ectopically but with less expressivity; however, ectopic activation also occurred in different cells. In the case of the bxI enhancer located in the third intron of Ubx, the presence of dCas9-VPR is capable of increasing transcription initiation while simultaneously blocking transcription elongation, generating a lack of functional phenotype. Our results show that CRISPRa system is able to activate transcription in any type of heterochromatin; nevertheless, its effect on transcription is subject to the intrinsic characteristics of each gene or regulatory element.Transcription factors (TFs) activate gene expression by binding to elements close to promoters or enhancers. Some TFs can bind to heterochromatic regions to initiate gene activation, suggesting that if a TF is able to bind to any type of heterochromatin, it can activate transcription. To investigate this possibility, we used the CRISPRa system based on dCas9-VPR as an artificial TF in  In some cases, such as the transcriptional activation mediated by nuclear hormone receptors, the GAGA factor and the heat shock factor, the transcriptional activator binding site could be located near the promoter5. However, in metazoans, most of the developmentally regulated genes, as well as genes that respond to different signal transduction pathways, are activated by the action of enhancers7. These cis-regulatory elements (CREs) are recognized by multiple transcription factors (TFs) and are able to operate at large distances from the promoter by the formation of chromatin loops through the interaction between transcription factors and components of PIC10. In general, the interaction of the TF with coactivators and/or with elements of the PIC occurs through a specific region known as the activation domain11. Transcriptional activators recruit protein complexes that modify and/or remodel chromatin to maintain transcriptional permissive regions known as euchromatin14.Gene expression mediated by RNA polymerase II (RNPII) in eukaryotic cells is modulated by the action of activators that recruit the basal transcription machinery to the promoter to form the preinitiation complex (PIC)15. In general, two types of heterochromatin have been identified based on the degree of compaction, which is linked to specific histone modifications. Facultative heterochromatin is present in regions that include silenced genes in specific cell types, and its promoters are enriched with the repressive mark tri-methylation of lysine 27 of histone 3 (H3K27me3)17. This histone modification is introduced by components of the Polycomb group of genes in Drosophila, which include Polycomb Repression complexes 1 and 2  through the action of the methyltransferase Enhancer of Zeste [E(z)]18. The action of PRC1 and PRC2 ensures the epigenetic silencing of genes during development19. In contrast, constitutive heterochromatin, considered to have a higher state of compaction of chromatin, is mostly present in telomeric and centromeric sequences, and it is characterized by enrichment of the trimethylation of lysine 9 of the histone 3 (H3K9me3) mark20.On the other hand, in nontranscribed regions, the chromatin conformation, called heterochromatin, is highly compacted into a “closed stage”14. It seems that this is the first event in transcription activation, suggesting that sending a transcriptional activator to any region in the chromatin could potentially cause its transcriptional activation.It has been established that the conformation of chromatin is a determinant in the activation of gene expression. However, many TFs, mostly pioneers, recognize specific DNA elements in compact chromatin and are able to alter the structure of the nucleosome and recruit factors to activate transcription and open the chromatin21. To induce the transcription of specific genes, a dead Cas9 (dCas9) has been fused to the activation domains of several TFs, with dCas9-VPR being one of the most widely used22. VPR is comprised of three activation domains derived from VP6, p65 and Rta TFs, and it has been used to activate transcription in a variety of models23. The dCas9 fused to activator domains is usually directed upstream of the transcription start site (TSS) of a specific gene, and recently, it has been used to activate CREs such as enhancers25.The CRISPR/Cas9 system has been modified either to activate (CRISPRa) or to repress (CRISPRi) transcriptionDrosophila as a model organism since the combination of the GAL4-UAS system with CRISPRa-dCas9-VPR allows precise genetic manipulation in a complete animal26. To test this system in constitutive heterochromatin, we analyzed whether the telomeric TAHRE element, which is maintained as silenced constitutive heterochromatin, can be activated by dCas9-VPR. For the activation of facultative heterochromatin, we selected the HOX genes Ultrabithorax (Ubx) and Sex combs reduced (Scr). These genes are highly regulated during fly development, and their regulated silencing is maintained by PRC1 and PRC227.Based on this information, our main question was whether the dCas9-VPR system could activate transcription in any type of heterochromatin in a complete organism. For this purpose, we used bxI enhancer located in a Ubx intron increases transcription initiation, but at the same time, it seems to block the passage of RNPII, generating a loss-of-function Ubx phenotype. These results show that the activation of transcription in different types of heterochromatin by the dCas9-VPR system generates distinct transcriptional responses that depend on their location and the roles they may have in the activation of transcription.Intriguingly, dCas9-VPR was able to transcribe both types of heterochromatic elements during development. In the telomeric element, transcription was efficiently activated, showing that dCas9-VPR can act as a pioneer TF. In the case of HOX genes, dCas9-VPR was able to activate transcription; however, the effect on the activation of transcription was different between enhancers and promoters. In particular, the occupation of the synthetic transcriptional activator in the Drosophila, telomeres are maintained by the transposition of non-long terminal repeat (non-LTR) retrotransposons. These retro-transposable elements (rTE), also referred as the HTT array, are known as HeT-A, TART and TAHRE28. These elements are silenced via the Piwi-piRNA pathway, which requires the transcription of segments of the rTE during oogenesis to induce the formation of heterochromatin29. We chose this region to test the transcription of constitutive heterochromatin using the CRISPRa system. We designed double single guides RNAs (gRNAs) directed upstream of the 5´ region of the TAHRE element, and one of the designed gRNA is also directed to the 3’ region of this rTE . We observed that in adult flies with the CRISPRa genotype, the expression of TAHRE was induced in both testis and somatic tissue compared with the control flies, where we did not detect any transcript  and the anterior part of abdominal segment 1 (A1)35. During larval stages, Ubx is expressed in the haltere and T3 metathoracic leg (T3 leg disc) imaginal discs. In cells where Ubx should not be expressed37, it is found as facultative heterochromatin enriched with the mark H3K27me338. Ubx is transcribed from a single promoter, and its regulatory elements are extended by approximately 100 Kb . For instance, the mutant phenotype in adult wings is observed predominantly in the second (2P) and third (3P) posterior regions as well as in cubital (L4) and distal (L5) veins of the wing  and MS1096  drivers. As shown in Sup. Fig. 1, the effect on Ubx expression was similar to that observed using the actin driver. When the tubulin driver was used, which is stronger than Act5C, the transformation of antennae to legs was more dramatic . Intriguingly, these results showed that there was no detectable expression of Ubx in other tissues, despite that the Act5C driver is ubiquitously expressed in all cells, suggesting that only in some specific tissues the Ubx ectopic expression may be conducted by the dCas9-VPR. Furthermore, similar results were obtained using the αTub and wor (neuroblasts specific) driver to identify if Ubx could be ectopically expressed in the nervous system in embryos . Nevertheless, not evident ectopic expression was found , supporting the idea that only in some particular cells the Ubx ectopic expression may be conducted by the dCas9-VPR system (see discussion).Since the expression of dCas9-VPR was directed by a ubiquitous driver, we wondered if the ectopic expression of Ubx promoter, can induce its variegated ectopic expression in some but not all imaginal discs, generating changes in the cell identity that are manifested in adult organs. In addition, ectopic Ubx expression in the wing imaginal disc preferentially occurs in specific regions, indicating that within the disc, there are cells that seem to be more permissive to derepress Ubx than others. These results indicate that the presence of an artificial transcriptional activator is sufficient to activate Ubx despite its normal silenced state in some cells of the wing and eye-antenna discs.In summary, the dCas9-VPR system, when directed to the Sex comb reduced (Scr) gene. Scr is a HOX gene involved in the differentiation of the labial and prothoracic segments43. Its ectopic phenotypes are identified in male adults by the presence of extra sex combs in the mesothoracic (T2) and metathoracic (T3) legs, similar to those present in the prothoracic leg (T1) in wild-type males44. Similar to Ubx, Scr is highly regulated during development, with specific enhancers localized upstream and inside the second intron, directing its expression at different locations and developmental stages43. The Scr gene encodes two isoforms that are transcribed from two overlapping promoters and, therefore, from two TSSs44. Two gRNAs were designed to bind to a region between the Polycomb Response Elements (PREs) 4/9, which are binding sites for PCR1 and PRC2 that cover the two TSSs45  and bithorax (bx) regions contain several CREs that are in the third intron of Ubx, approximately 30 kb downstream of the TSS 56. We performed RT–qPCR of the three regions around the sites that recognize the gRNAs . These results indicate that the occupancy of dCas9-VPR in this CRE enhances its effect on Ubx transcription initiation only in the haltere disc. However, since this enhancer is within the body of the gene, we propose that the generation of the loss-of-function phenotype is because it blocks RNPII elongation in some cells.To answer this question, we quantified nascent pre-TSS Fig. E. IntrigUbx have shown that its effect on the induction of transcription by dCas9-VPR may not be the same when it is targeted to a promoter as it is when targeted to an enhancer. Thus, we tested the transcriptional effect of the dCas9-VPR system when directed to a nonintronic enhancer that is located far away from the gene it acted on, as is the case of the Scr upstream enhancer, ScrE43. This enhancer is at -33 Kb with respect to the Scr TSS of the two promoters, between the 3´ end of fushi tarazu (ftz) and the 3’ end of Antennapedia (Antp) . We found that these flies have ectopic sex combs in the T2 and T3 pairs of legs in the first tarsomere, indicating derepression of Scr in these discs  Fig. A. ScrE iscs Fig. B, similaype Fig. B–D. Intrype Fig. B–D. Addiype Fig. C,D. In aype Fig. C,D, sugg T1 Fig. C,D.FigurScr expression through Scr immunostaining of leg discs and found that it was ectopically expressed in T2 and T3 discs. In agreement with the extra sex comb phenotype observed in adult male legs of this genotype, there was a lower number of cells in discs that expressed Scr than in discs from flies where dCas9-VPR was directed to the promoter , the RNPII is kept paused even when these genes are not expressed64. Therefore, it is possible that only in certain cells the RNPII is already present in these promoters and therefore the ectopic activation by the dCas9-VPR is facilitated. This aspect has to be taken into account when it is desired to use the CRISPRa system to ectopically express a gene. In any case, in some cells, the expression of Ubx was ectopically activated, bypassing the Ubx silencing exerted by the Pc group genes in these discs. On the other hand, ectopic Scr activation was more robust , comparable to typical phenotypes in animals lacking PcG function. As mentioned above, Scr has two TSSs, and it is possible that the transcriptional activator would activate both promoters, although this has not been determined. In any case, dCas9-VPR  activated  Scr expression in tissues in which it should be silenced by the PcG genes.Regarding the induction of ectopic transcriptional activation of the HOX Ubx or Scr enhancers were different from those where it was sent to the promoters. When dCas9-VPR was in the Scr upstream enhancer, Scr ectopic transcription was activated in T2 and T3 leg discs. However, its expression varied, with low penetrance and expressivity. Noticeably, Scr expression was extended toward the center of the disc, mainly in T1 and T2, causing the appearance of sexual combs in tarsomeres 2 and 3 of legs derived from these T1 and T2 discs.The phenotypes of animals where dCas9-VPR was sent to the Scr enhancer can activate transcription in the T2 and T3 discs suggests that the enhancer has almost all of the requirements necessary to activate the expression of Scr in these tissues and that the addition of a new transcription factor was sufficient to activate CRE. In support of this hypothesis, it has recently been reported in several organisms that the interaction between the enhancer and its target promoter occurs even though the enhancer is not active, both in the tissue where the target gene is expressed and in tissues where it is not67. This observation is relevant from an evolutionary point of view since mutations in Selector genes are known to be the cause of morphological differences between different species. For example, in other Drosophila species, the expression of Scr, unlike in D. melanogaster, causes the presence of sexual combs in tarsomeres 2 and 370, similar to what we have observed when sending the dCas9-VPR to the Scr upstream enhancer.The fact that the occupation of the synthetic activator in the Ubx bxI enhancer, it was able to increase Ubx transcription in the haltere disc, as determined by measuring the nascent Ubx transcript. However, a loss-of-function Ubx phenotype is observed in these flies. How is it possible that the effect of locating dCas9-VPR at the bxI enhancer is giving results at the same time of both Ubx transcriptional activation and repression? This seems to indicate that, as has been previously reported, the occupation of the dCas9-VPR downstream of the TSS, in this case at the intronic bxI enhancer, inhibits the passage of RNPII, causing a net effect of Ubx silencing. On the other hand, when dCas9-VPR occupies the bxI enhancer, it favors its interaction with the Ubx promoter, increasing the levels of transcriptional initiation. However, when RNPII reaches the downstream site where dCas9-VPR is positioned, it is stalled and aborted. Nevertheless, we do not know whether these two processes are occurring simultaneously or if the barrier action of dCas9-VPR on RNPII only occurs when the enhancer does not interact with the promoter. In support of this possibility, it has been reported that as transcription increases due to the action of activators in an enhancer, the distance between the promoter and the enhancer increases71. This could also explain why we observed an Ubx variegated expression pattern in haltere discs.When dCas9-VPR was sent to the TAHRE element) and facultative (the HOX genes) heterochromatin regardless of their silenced state. Likewise, the effect on transcriptional activation by sending this artificial activator to a promoter or to an enhancer of the same gene can generate different transcriptional responses in terms of the strength of an ectopic phenotype, as was the case for Scr. Surprisingly, the presence of dCas9-VPR in an enhancer within the body of Ubx can simultaneously increase the degree of transcription initiation and block the passage of RNPII in the same gene when located 30 kb from the TSS. All these findings show that since transcription is a stochastic and plastic mechanism, synthetic transcriptional activators in complex organisms are incorporated in a specific manner into each gene or regulatory region.In conclusion, by using the synthetic transcription activator dCas9-VPR, we have shown that it is capable of activating transcription by RNPII, both in regions classified as constitutive  as previously reported72. For Gibson Assembly protocol, we used Gibson Assembly Master Mix de New England BioLabs. All guide sequences were designed using the Benchling platform73 and Breaking-Cas tool74. Guide sequences are available in Supplementary Table 1.Double guides, directed to the different targets of the The double gRNA-plasmids in pCFD4 were integrated into the fly genome at the attP40 integration site on the second chromosome with the phiC31 transformation method by the company BestGene.gRNA/CyO;MKRS,Sb/TM6B,Tb,Hu with each gRNA sequences directed to TAHRE, Ubx or Scr or their corresponding enhancers, were crossed with flies W;If/CyO,UAS:dCas9-VPR/TM6B,Tb,Hu (donated by Dr. Norbert Perrimon) in order to found the families with genotype sgRNA/CyO;UAS:dCas9-VPR/TM6B,Tb,Hu. Finally, we crossed these flies with the corresponding GAL4 driver, accordingly to the experiment. We used Act5C-GAL4 driver  and Tub-GAL4  for ubiquitous expression in TAHRE, Scr and Ubx lines respectively, and specially with Ubx promoter lines: MS1096-GAL4/y, and ap > GFP-GAL4/CyO,Tb; +/+ in order to drive expression to the haltere imaginal disc. Fly crosses were conducted at 25 °C, unless for bxI line, which were performed at 28/18 °C each 12 h.Flies with genotype All structures dissected, including antennae, halteres, legs, and wings, were collected from adult fly which were boiled with KOH solution (10% v/v) in a water bath for 5 min. Then, were again boiled in sterile distilled water to remove KOH during 5 min. The structures were dissected in ethanol and mounted in glycerol 50%.sgRNA/Act5C-GAL4;UAS:dCas9-VPR/+ with the g-RNA directed to Ubx and Scr promoters and enhancers as it may apply. As control phenotype we used the corresponding sister lines product of the cross with Act5C-GAL4 driver, which genotypes were sgRNA/CyO;UAS:dCas9-VPR/+ or Act5C-GAL4/CyO;UAS:dCAs9-VPR/+.All cuticles dissected had the genotype: All cuticles were documented in a Nikon Eclipse E600 upright microscope with a digital camera with Aptina CMOS sensor 5.1 MP, KPA.Wandering-stage third instar larval were dissected in cold PBS 1X and fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. Subsequently, larvae were washed 3 times with PBST (1X PBS and 0.2% Triton) during 15 min each wash. Then, larvae were blocked with BBT  at 4 °C for 1 h. Afterwards, the sample was incubated with the primary antibody overnight. After that the larvae were washed again 3 times for 20 min with PBST, the secondary antibody and DAPI was added, and incubated for 2 h at room temperature, and washed 3 times with PBST for 20 min. Finally, the PBST was removed to add the mounting medium and thus extract the imaginal discs for later observation.Ubx promoter experiments, we analyzed flies with genotype Ubx-gRNA/Act5C-GAL4;UAS:dCas9-VPR/+ and lines Ubx-gRNA/CyO;UAS:dCas9-VPR/+ or Act5C-GAL4/CyO;UAS:dCAs9-VPR/+ as siblings lines control. In the case of bxI enhancer experiments, we used the haltere imaginal discs with the genotype bxI-gRNA/CyO;UAS:dCas9-VPR/αTub-GAL4 for CRISPRa fenotype and bxI-gRNA/CyO;UAS:dCas9-VPR/TM6B,Tb,Hu as the control. The change to αTub driver corresponds to the use TM6B, Tb balancer for an easier identification of each genotype during third larval stage and as substitute for another ubiquitous driver similar to Act5C.. Finally, for both Scr experiments, promotor (Scr-gRNA) and enhancer (ScrE-gRNA), we performed the immunohistochemistry of leg imaginal discs with genotype gRNA/Act5C-GAL4;UAS:dCas9-VPR/+ and lines gRNA/CyO;UAS:dCas9-VPR/+  or Act5C-GAL4/CyO;UAS:dCAs9-VPR/+ as siblings lines control.In the case of haltere, wing, and eye-antennae imaginal discs for Antibodies used were mouse monoclonal antibody Ubx , mouse monoclonal antibody Scr . As secondary antibody, we used Goat anti-mouse IgG Alexa Fluor 568  at 1:200 and DAPI concentration 1 ng/μL.Samples were imaged on Olympus FV1000 Multi-photonic Inverted or Olympus FV1000 Confocal Upright microscopes, equipped with a UPLSAPO objective 20X NA: 0.75 AND 60X NA: 1.1. Images were processed using ImageJ (Fiji) and Inkscape vector graphics editor software.gRNA/Act5C-GAL4;UAS:dCas9-VPR/+ expressing gRNAs for TAHRE as CRISPRa lines and the resulting siblings lines from the cross as control were used. Testis and the corresponding carcasses of males without testis (somatic tissue) were analized. Haltere and wing imaginal discs for bxI fly lines with the genotype bxI-gRNA/CyO;UAS:dCas9-VPR/αTub-GAL4 were used. The corresponding siblings lines obtained from the cross were used as controls.Adult specimens with genotype rp49  to use it as a reference gene.The RNA was extracted and purified with TRIzol Reagent following the manufacturer’s protocol. The isolated RNA was used for the synthesis of cDNA, following the protocol for the M-MLV Reverse Transcriptase enzyme (Invitrogen). End-point PCR amplification of the cDNA was carried out to determine the presence of TAHRE transcripts using Taq polymerase. RP49 was used as a reference gene. For the stand-specific experiments, specific primers to sense and antisense TAHRE transcript were used for the cDNA synthesis, as well as the reverse primer of TAHRE experiments and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) for the bxI enhancer experiments in a Roche Real Time PCR LightCycler 1.5. The threshold cycle (CT or 2−∆∆CT) method75 was used to calculate the fold-change for transcript relative quantification. At least three independent biological replicates were analyzed in each case. Statistical analyzes (student t and One-way ANOVA tests) were performed using the GraphPad Prism 8 software. The primers used for RT-qPCR experiments for each target are listed in suppl. Table 2.The RT-qPCR experiments were conducted using LightCycler FastStart DNA Master PLUS SYBR Green I (Roche) for Drosophila melanogaster haltere were previously published49. Raw data was downloaded from the GEO database (GSE166714). Fastq files were aligned to the dm6 genome using Bowtie2 software and peak calling was performed using MACS3. BigWig files were processed using Deeptools library and finally were visualized using the IGV software.ATAC-seq data from Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


These studies reveal novel mechanisms of transformation and provide insights into potential therapeutic targets for intervention in this disease.T Lymphocyte Acute Lymphoblastic Leukemia (ALL) is an aggressive disease arising from transformation of T lymphocytes during their development. The mutation spectrum of T-ALL has revealed critical regulators of the growth and differentiation of normal and leukemic T lymphocytes. Approximately, 60% of T-ALLs show aberrant expression of the hematopoietic stem cell-associated helix-loop-helix transcription factors TAL1 and LYL1. TAL1 and LYL1 function in multiprotein complexes that regulate gene expression in T-ALL but they also antagonize the function of the E protein homodimers that are critical regulators of T cell development. Mice lacking E2A, or ectopically expressing TAL1, LYL1, or other inhibitors of E protein function in T cell progenitors, also succumb to an aggressive T-ALL-like disease highlighting that E proteins promote T cell development and suppress leukemogenesis. In this review, we discuss the role of E2A in T cell development and how alterations in E protein function underlie leukemogenesis. We focus on the role of TAL1 and LYL1 and the genes that are dysregulated in  Consistan T-ALL . Approxian T-ALL . The higan T-ALL . Unfortuan T-ALL . Nonethean T-ALL . A betteNotch1 gene and a requirement for Notch signaling for their survival (E2a-deficieint mice.TAL1 and LYL1 are basic helix-loop-helix (bHLH) proteins that bind DNA in association with the E protein transcription factors . The E psurvival –22. In tTcf3 (E2a) gene encodes 2 bHLH proteins (E12 and E47) through alternative splicing of exons encoding the bHLH domain (TCF12 (HEB) and TCF4 (E2-2), that each code for two E box binding proteins through alternative transcription start sites, resulting in proteins with differing activation domains but identical bHLH domains  and a failure to initiate expression of multiple lymphoid genes  is regulated by NOTCH1 and plays a major role in T cell lineage specification . The STIL regulatory elements are constitutively active in thymocytes, resulting in ectopic TAL1 expression. These TAL1 upstream deletions are specific for T-ALL cells and are most likely caused by erroneous V(D)J recombinase activity  in a region 8kb upstream of TAL1 that create a de novo MYB binding site that results in strong enhancer activity in these leukemias (TAL1 transcription in leukemic cells (TAL1 was identified as a gene involved in the t(1:14) and t chromosomal translocations in T-ALL, which place ectively –63. LYL1mosome 7 . These t of TAL1 . These dactivity , 66. Alteukemias . Chromatic cells . Taken t-/-E2a mice , LIM domain binding (LDB1), and GATA protein families –78. Indepression . Interespression . Experimpression . Using spression . Intereseukemias –85. Of neukemias .Lyl1 significantly increases leukemia latency in LMO2 transgenic mice suggesting that LYL1 supports transformation. Microarray analysis revealed that LMO2 expressing thymocytes have higher Lyl1 expression compared to wild type thymocytes indicating a potential feed-forward mechanism reminiscent of the mechanism seen in TAL1 expressing leukemias. Consistent with this idea, the LYL1 promoter contains ETS and GATA binding sites, which promote the expression of LYL1 in HSCs and both ETS1 and GATA3 are implicated in T-ALL  translocation that placed the 3’ end of the genitors . This tr  and the Janet Rowley Fund (University of Chicago Comprehensive Cancer Center).Our studies on The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.