The Plant Cell, Lilan Hong, Byron Rusnak, Clint S. Ko, and colleagues  with the complete enhancer or the R2 reporter lines. In the full enhancer and R2 reporter lines, the downregulation of the TCPs did not affect the formation of giant cells but displayed a reduced signal. Two putative TCP binding motifs were found in the R2 region. Electrophoretic mobility shift assay (EMSA) confirmed that TCP4 binds to the R2 region. Additionally, mutations of the TCP binding motifs in the complete enhancer or R2 reporter lines caused a near-total loss of the reporter signal. In summary, the TCP protein binding to R2 activates expression driven by the enhancer.To identify trans-factors acting on R1 and R2 to regulate enhancer activity, the authors conducted a yeast 1-hybrid (Y1H) assay with nearly all the transcription factors in Arabidopsis. Of the 150 identified interactors, proteins belonging to the Class II CINCINNATA-like TCP  interacted with R2 and a 100 base pair (bp) junction sequence between R1 and R2. To functionally validate the protein–DNA interactions, the authors crossed the DOF2.2, HCA2, or DOF5.8 reduced the expression of the enhancer in the giant cells and presented altered sepal development. These results demonstrate the role of R1 and Dofs in reducing the expression driven by the enhancer.The authors hypothesized that regulators binding to the R1 region confer expression specificity to the giant cells. In the initial Y1H assays, several DNA-binding one zinc finger (Dof) proteins  interacted with the R1 and the 100 bp junction region which possess three Dof TF binding motifs. The addition of the Dof binding motifs to Region 2 restricted its expression in the giant cells. On the other hand, the deletion of the Dof binding motifs expanded and displayed an epidermal expression pattern. Overexpression of ATML1 and found ectopic giant cells in the sepals, suggesting a role in the activation of enhancer activity. EMSA and mutational analyses of reporter lines verified that ATML1 binds to the HD ZIP binding motif in R2, and the enhancer activity is reduced when the binding is disrupted. The authors generated reporter lines with varying levels of ATML1 expression  and MERISTEM LAYER 1 (ATML1), as regulators of giant cell identity . The autIn summary, this study illustrates how complex mechanisms control gene expression underlying cell-type specification and cell-specific gene expression. In this case, the formation of giant cells depends on the activation by TCP and HD ZIP transcription factors combined with the repression activity of Dof transcription factors in different regions.


MYC, a key member of the Myc-proto-oncogene family, is a universal transcription amplifier that regulates almost every physiological process in a cell including cell cycle, proliferation, metabolism, differentiation, and apoptosis. MYC interacts with several cofactors, chromatin modifiers, and regulators to direct gene expression. MYC levels are tightly regulated, and deregulation of MYC has been associated with numerous diseases including cancer. Understanding the comprehensive biology of MYC under physiological conditions is an utmost necessity to demark biological functions of MYC from its pathological functions. Here we review the recent advances in biological mechanisms, functions, and regulation of MYC. We also emphasize the role of MYC as a global transcription amplifier. The Myc gene was first identified in the early 1980s as a cellular homolog of the retroviral v-Myc oncogene . Its disThe MYC family of proteins consists of three paralogs, MYC (c-MYC), MYCN (N-MYC) and MYCL (L-MYC) . AlthougUpon binding at promoters, the transregulatory domains of MYC and its isoforms, are believed to project its influence onto target genes through patches of amino acids that share high sequence homology among the three MYC isoforms. These patches are referred to as MYC boxes (MBs). From the amino-to carboxyl terminus, there are six conserved MBs: MB0, I, II, IIIa, IIIb, and IV. They are generally unstructured and can adopt specific conformations induced upon partner-protein binding. The degree of plasticity for each MB upon complexing with different partners has not been explored. The inventory and functional roles for MB-interacting partners that have been most intensively investigated are involved in transcription and chromatin process, or control MYC turnover, has recently been reviewed .A sampling of the MYC-interactome shows MB0 interactions with general transcription factor TFIIF . MBI andTranscription activation involves the binding of transcription factors to specific DNA sequences, which recruit the transcriptional machinery, coactivators, and chromatin modifiers to form a transcriptional complex that initiates gene transcription. Transcription factors can recruit coactivators such as CBP/p300, which possess histone acetyltransferase activity and can acetylate histones to promote an open chromatin structure that allows for gene transcription. In addition, transcription factors can recruit chromatin modifiers such as SWI/SNF, which can remodel chromatin to allow access to the transcriptional machinery . Unlike When viewed a transcriptional activator, the expectation and goal were to identify specific, direct MYC target genes to provide insights into the crucial downstream targets and biological processes responsible for mediating the physiological functions and oncogenic pathology of MYC. Numerous studies were undertaken to identify MYC-regulated genes by employing techniques such as microarray or next-generation sequencing to compare RNA expression profiles and genome-wide mapping of MYC-bound chromatin. The notion that MYC and MYC-MAX complexes regulate a limited and well-defined set of target genes for their various roles has been largely challenged . StudiesThe complexity of transcription amplification can be influenced biologically by input signals, cis-elements, other transcription factors, and analytically by the algorithms and pipelines used for analysis. These factors can highlight or obscure the relationship between MYC binding and promoter output in omics studies. To exclude such interfering or biased factors, minimal promoter and the reporter-based assay was designed to interrogate MYC function . Basal rAlthough, it has been suggested that the binding of transcription factors to enhancer elements, super-enhancers, or other regions that drive the recruitment and activity of the transcription machinery plays a critical role in the non-linear mode of transcriptional amplification , the dirin situ hybridization (smFISH) in fixed cells and MS2-tagging of RNA in live cells were used to assess the immediate impact of MYC on transcription bursting. The findings demonstrate that MYC influences the length of time that other core transcriptional factors reside at promoters. Elevated MYC levels uniquely influence the dwell time of various transcriptional machinery complexes. The glucocorticoid receptor (GR) remained unchanged, while SPT5, TBP, and RNAP II exhibited increased dwell time, and MED1, a mediator component, showed decreased dwell time. Elevated MYC enhanced RNA output from its target genes and alterations in burst duration were attributed to changes in the residency of transcriptional machinery and hence altered transcription output. Overall, MYC universally extended the duration of transcriptional bursts , without altering their frequency  or SP1 that recruit corepressor, or changes in chromatin accessibility driven by epigenetic modifications which lead to the displacement of DNA-bound coactivators to ultimately achieve gene repression . FurtherAs discussed above, MYC is an integral part of transcription progression, acting as a global amplifier, it is indispensable for both embryonic development and the maintenance of self-renewing tissues in adults . MYC proSKP2 ubiquitin ligase complex, which direct the phosphorylation, degradation, and proteasome-mediated turnover of p27  and DNA damage . ThroughMYC has been shown to regulate rDNA transcription. MYC interacts with components of the SL1 complex, enhancing the association of TBP and TAF complex with the promoter and recruiting HATs to facilitate RNAP I recruitment and transcription at rDNA promoters. Consequently, the upregulation of UBF expression mediated by MYC positively influences the transcriptional activity of RNAP I, ultimately resulting in enhanced rRNA synthesis . SumoylaMYC proteins are intrinsically disordered proteins (IDPs). They tend to interact with different proteins simultaneously and has been speculated that MYC forms liquid-liquid phase separation when present at high concentration . It has myc mRNA transcripts, while the P2 promoter accounts for 75%–90% of the transcripts . The MYC-TAD harbors a multitude of super-enhancer regions that intricately regulate the expression of the MYC  led to a ∼50% reduced MYC level  regulates the expression of MYC by binding to ∼50 kb downstream of the enhancer   within the IDR region of MYC, thereby reducing the thermal stability of MYC-MAX dimerization and subsequently its function . MYC funMYC protein is a crucial transcription regulator that plays a central role in regulating gene expression in different cellular situations. Its capacity to amplify transcriptional responses contributes to the precise control of cellular processes and the maintenance of a balanced state within cells. It is not known whether MYC exerts its pathological action from an augmentation of its normal transcription amplifier role or whether MYC neopathologic functions are elicited at supraphysiological levels. It is important to understand mechanistically how MYC regulates different kinetic steps of transcription by all three RNAPs. A deeper understanding of the mechanisms through which MYC amplifies transcription, and the factors that influence this process in physiological and pathological conditions will enhance our knowledge of gene regulation and offer valuable insights for developing targeted therapeutic approaches for MYC-related disorders.


Arabidopsis tissues at different developmental stages and have identified a number of promoters that are well expressed in both transient (Nicotiana benthamiana) and stable (Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target sites in a subset of the screened promoters, converting them into NOR logic gates. The work here establishes a pipeline to screen for additional constitutive promoters and can form the basis of constructing more complex information processing circuits in the future.To support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. We have developed a pipeline to identify genes that have stable expression across a wide range of  Several projects have produced measurable gains in photosynthetic efficiency  promoter, and promoters from members of the ubiquitin and actin families  transient agroinfiltration assays and identified 16 promoters that had expressions that were significantly different from the negative control  with the intensity of RUBY colouring quantified in Fiji .To determine whether the promoters showed constitutive expression in N. benthamiana doesnot perfectly predict expression in Arabidopsis, we included two promoters  that did not show expression in tobacco infiltration in our Arabidopsis stable transformation experiment. Interestingly, AT1G54080 displayed RUBY expression in roots and pollen. AT5G37830 had visible expression in pollen, siliques, stems, and roots. AT3G08530 had the most ubiquitous expression and had visible expression in the flowers, pollen, siliques, stems, and roots. A visual summary of the Arabidopsis and N. benthamiana experiments can be found in Supplementary Figure S5.Given that expression in Arabidopsis (Supplementary Table S7). This discrepancy could be attributed to the RUBY reporter or potential limitations in identifying additional transcriptional regulators  and two without (AT1G64550 and AT2G29080). The two lines without visible RUBY expression both had qPCR expression level between the two lines with visible RUBY expression in the roots. This result suggests that RUBY was not a reliable reporter for these low-expressing promoters and that the promoters were indeed functional in bidopsis . To furtbidopsis  for morebidopsis . The restors see .Arabidopsis genome. A constitutive promoter with two unique gRNA target sites can function as a NOR gate (a two-input logic gate where the output is only ON when neither of the inputs is present) in the presence of a dCas9-guided repressor. The inputs for such a gate are the gRNAs. When either or both of the gRNAs are present, the dCas9-guided repressor should be able to keep the promoter OFF (Supplementary Table S8).To make the promoters screened in this experiment more versatile, we next introduced two gRNA target sites into six of the promoters screened with target site sequences not found in the oter OFF . Only whSupplementary Figure S6). The repressibility of the modified promoters was tested in N. benthamiana with each infiltration containing all constructs required for repression .Working with native promoters also provided an opportunity to learn more about the biology of promoters themselves. Yamamoto and colleagues suggested that plant promoters can be grouped into a few core promoter categories based on the presence or absence of certain location-sensitive motifs . Minimum intron length was set to 60, and maximum intron length was set to 6000  were normalised using the Median Ratios method from the DESeq2 package in R  and 3′UTR+terminators  of the remaining genes were extracted using the Araport11 genome annotation and the ‘3,000 bp upstream and downstream’ sequence files from the TAIR website. The extracted sequences were screened for BbsI and BsaI restriction enzyme cut sites and only those without were kept. Any genes with their promoter+5′UTR and 3′UTR+terminator overlapping annotations from neighbouring genes in the Araport11 annotation were also removed.Promoter+54.4.The promoter sequences of the remaining genes were uploaded onto PlantRegMap using the ‘Binding Site Prediction’ function  and promoter and terminator sequences were extracted as described above. The promoter core type was annotated by Tokizawa et al. . A list 4.6.′UTR and 3′UTR+terminator for each candidate genes as defined above were cloned with PCR from extracted genomic Col-0 DNA into their respective MoClo level zero acceptors   through Gibson assembly by replacing the original sequence , 100 uM acetosyringone). The OD of the cultures were measured and about 1~2 mL volume mixture with 5.0 OD for construct to be tested and 5.0 OD for P19 were prepared. The infiltration mix was rotated to mix at room temperature for 3 hours before injecting into fully emerged N. benthamiana leaves with a 1mL syringe. The injections were always injected as triplicates on three separate leaves on three separate tobacco plants. Each leaf is also always injected with a pUBQ10:mTURQ control.In total, 5 mL cultures of 4.9.At 3 days post infiltration, the leaves were clipped off and visualised in the Azure C600 Western Blot imaging system with exposure times Cy5 = 0 sec, Cy3 = 15 sec, Cy2 = 5 sec. Two hole punches were taken out of representative regions of each injection, and damaged regions with high background fluorescence were avoided. The leaf discs were placed in a 96-well plate on top of 200 uL of water, and the plates were read with a TECAN SPARK plate reader with YFP: excitation 506(15) and emission 541(15), Gain 100. mTurq: excitation 430(15) and emission 480(15), Gain 50. mScarlet: excitation 565(15) and emission 600(15), Gain 100. Settings: Multiple Reads Per Well; Circle (Filled) 4 × 4 with border 800 uM. Each leaf disc was read 12 times giving a total of 24 datapoints per injection per leaf. The output data was read into a custom R file for annotation, clean-up and visualisation. Multiple biological replicates were assayed for each promoter being tested, and the three replicates closest to the median of all replicates were kept for visualisation and statistical analysis. Each injection’s YFP value is subtracted by the median YFP value of the pUBQ10:mTURQ negative control on the same leaf. The YFP value is then divided by the mTURQ value for each injection to normalise across results. A Dunnett Test, a post hoc pairwise multiple comparison test from the DescTools package, was used to determine whether the injections were significantly different from the negative control  with gRNA target-sites driving NLS-YFP and pUBQ10:NLS-mTURQ internal control as the reporter. The mPromoters (5OD) were co-injected with P19 (1OD), TPL repressor (1OD), self-cleaving gRNA_1 (1OD), and self-cleaving gRNA_2 (1OD). To test the mPromoters’ NOR gate functionality, two gRNA inputs were required. In cases where only one input is present, the other self-cleaving gRNA will be a non-matching guide to the mPromoter. When neither inputs are present, sometimes two non-matching gRNAs (1OD each) were co-injected and sometimes only one (2OD), but the final total OD were always consistent. The exact injection combinations can be found in the R script. The TPL repressor construct contains pUBQ1:tdTomato-pUBQ10:dCas9_TPL(N188) and is modified from Khakhar et al. . Self-cl4.11.Constructs with the candidate promoters driving RUBY were transformed into Col-0 through floral dipping method  and the result was read using the Bio-Rad CFX Maestro software and analysed using standard methods . 1 μg of extracted RNA was then used with the iScript cDNA synthesis kit (BIO-RAD). qPCR was performed using the iQ SYBR Green Supermix (BIO-RAD). PP2AA3 was used as a reference gene and the primers for PP2AA3 and RUBY can be found in  Pfaffl, .Yang and Nemhauser supplementary materialYang and Nemhauser supplementary materialYang and Nemhauser supplementary materialYang and Nemhauser supplementary materialYang and Nemhauser supplementary materialYang and Nemhauser supplementary material 13 October 2022Dr. Olivier HamantEditor-in-ChiefQuantitative Plant BiologyDear Dr. Hamant:Attached please find our manuscript entitled “Expanding the synthetic biology toolbox with a library of constitutive and repressible promoters”, which we are submitting for consideration as an article. None of the material has been published or is under consideration elsewhere. We have submitted a preprint: MS ID#: BIORXIV/2022/511673.For the ambitious engineering projects currently under development in plants, one of the major bottlenecks is the lack of well-characterized, constitutive promoters. Reusing promoter parts in large genetic constructs makes cloning challenging and increases the likelihood of transgene silencing, among other concerns. In our manuscript, we describe the development and implementation of a pipeline to identify broadly expressed genes. We then cloned out presumptive promoters and terminators of these loci, and validated their expression in both transient (Nicotiana benthamiana) and stable  transformation assays. To further increase the functionality for building complex circuits, we engineered a subset of these promoters with unique gRNA target sites and successfully generated orthogonally repressible NOR gates. The constitutive promoters screened in this study can help meet the need for additional promoter parts, and by converting them into NOR gates, they form the basis of constructing more complex logic gates in the future.Based on the reception of this work by our colleagues in workshops and meetings, we believe it will appeal to a diverse range of scientists interested in transcriptional regulation, networks and synthetic biology. It would be fantastic if the debut of this toolset could be in an open-access journal like Quantitative Plant Biology.Some potential reviewers for this work include:Jenn Brophy, Stanford, jbrophy@stanford.edu, expertise: plant synthetic biology, esp. logic gatesKevin Cox, Danforth Center, KCox@danforthcenter.org, expertise: transcriptional regulationDavid Ehrhardt, Carnegie Institute, dehrhardt@carnegiescience.edu, expertise: vector and tool developmentNaomi Nakayama, Imperial College London, n.nakayama@imperial.ac.uk, expertise: plant synthetic biology; transcriptional regulation, esp. promoters and terminatorsDiego Orsáez, IBMCP, CSIC Valencia, dorzaez@ibmcp.upv.es, expertise: plant synthetic biology; dCas9-based tool developmentNicola Patron, Earlham Institute, nicola.patron@earlham.ac.uk, expertise: plant synthetic biology; transcriptional regulation, esp. promotersRoss Sozzani, NCSU, rsozzan@ncsu.edu, expertise: vector and tool development; bioinformaticsYoshiharu Y. Yamamoto, Gifu University, yyy@gifu-u.ac.jp, expertise: transcriptional regulation, esp. promotersThank you for your time and consideration.Sincerely,Dr. Jennifer Nemhauser, jn7@uw.eduDepartment of BiologyUniversity of Washington Your manuscript has been now assessed by three reviewers, please apologize the delays. I share with them the opinion that your manuscript “Expanding the synthetic biology toolbox with a library of constitutive and repressible promoters“ is very interesting, timely and potentially relevant for the community. However, several valid concerns have been raised. Among other points, and in particular, the reviewers identified that the choice of RUBY as a reporter is not ideal, including the limitations shown in terms of sensitivity and that the dynamic range is relatively low. In addition, the experimental data doesn´t seem to support the claim of constitutive activation for the promoter. Further comparisons and in silico studies are suggested.I would therefore be glad to consider publication of your article provided that it undergoes a revision tackling the issues raised by the reviewers No accompanying comment. 21 May 2023Dr. Olivier HamantEditor-in-ChiefQuantitative Plant BiologyDear Dr. Hamant:Attached please find our revised manuscript entitled “Building a pipeline to identify and engineer constitutive and repressible promoters”. We thank the editor and reviewers for their careful reading of our work. We have made a number of changes described below in response to the reviewer comments (including modifying the title), as well as adding new experimental results. We believe that these changes improve the manuscript and increase the accessibility of our findings. We hope that you agree.Thank you for your time and consideration.Sincerely,Dr. Jennifer NemhauserDepartment of BiologyUniversity of Washingtonjn7@uw.eduDetailed response to reviews:Reviewer 1 (R1)Major Comments:1) Title is too big to express the contents. Development of an Expression and Repression System for Transgenic Plants, for example, would be more appropriate.Our response: Inspired by the reviewer’s suggestion, we changed the title to: Building a pipeline to identify and engineer constitutive and repressible promoters. We hope that this change captures the spirit of the suggested revision.(R1) 2) Function of gene repression would be the key of the report. However, the demonstrated repression in Fig. 4D is much less effective than expected. This is not like repression but small modulation. The authors should try to develop ten fold repression or more. Otherwise, I don’t think of beneficial applications of the system.Our response: While we understand (and share) the reviewer’s frustration with the relatively subtle impact of the repression we observe, it is worth noting that these parts have not been optimized , and that the overall low level of expression makes strong repression quite difficult to achieve. We have added text throughout the manuscript to emphasize the proof-of-concept nature of this study, and its shortcomings. Despite these issues, we strongly believe that these results will be of use to the plant synthetic biology community, as well as those interested in transcriptional regulation more generally.(R1) Minor Comments:1) Fig. 2 and 3. A positive control (e.g. 35S promoter - NOS terminator) should be included to see expression levels of the developed promoter-terminator pairs.Our response: We can appreciate the reviewer’s desire to have a familiar promoter to serve as a point of comparison; however, we think that the levels of a 35S promoter are far too high to serve as a meaningful control for these endogenous, low-expressing promoters.(R1) 2) Fig. 4D. Degree of repression should be shown. This is important information, and some researchers are really interested in it.Our response: Thank you for this suggestion. We have modified Figure 4D accordingly.(R2) Reviewer: 2Major comments:Although these promoters appear to have constitutive activity based on their expression patterns from transcriptomics data, it is not clear from the data shown in this paper that the 2 kb of upstream sequence (promoter) is capable of conferring constitutive activity in transgenic plants. For this reason, I suggest that the authors remove any statements that these promoters are constitutive, unless experiments are done in transgenic plants where a different (perhaps more sensitive) reporter gene is used  and gene expression is assessed in different tissues throughout the plant, at different developmental stages. Alternatively, RT-qPCR could be done on mRNA from individual tissues from the transgenic plants, at different developmental stages, to show constitutive activity.Our response. We thank the reviewer for improving the precision of our language. We have now changed language throughout the text to differentiate between the objective of our screen versus what we have observed with our promoters. Additional qPCR had also been performed as per reviewer 2 and reviewer 3’s suggestion to confirm the expression of RUBY in the various tissues collected for two of the promoters. .(R2) (Given the large variations in Tobacco data)... It is not clear that these NOR logic gates would function robustly in stable transgenic plants. I suggest this limitation be discussed.Our response: We have added text to the Discussion to incorporate this suggestion as follows:“While N. benthamiana serves as a great prototyping platform, the performance of the gates would also need to be evaluated in stable Arabidopsis lines to validate their viability.”(R2) Minor suggestions:(R2) Line 90: in the legend for Figure 1, define SRA as “Sequence Read Archive”.Our response: We thank the reviewer for the suggestion, and have made this edit.(R2) Lines 144-146: Twelve promoters were selected to transform Arabidopsis, yet one of those did not show activity in N. benthamiana? Why was this one promoter tested in Arabidopsis? This is not clear.Our response: We have added a clarifying sentence regarding the promoter in question: “Given that expression in N. benthamiana doesn’t perfectly predict expression in Arabidopsis, we included two promoters  that did not show expression in tobacco infiltration in our Arabidopsis stable transformation experiment. Interestingly, AT1G54080 displayed RUBY expression in roots and pollen.”(R2) Line 173: I could not find any instances of panels A and C from Figure 4 being mentioned in the text.Our response: We thank the reviewer for the suggestion, and have added callouts for these panels.(R2) Line 190: provide full gene number – AT1G64550Our response: We have made this correction.(R2) Line 199: fluorescenceOur response: We have made this correction.(R2) Line 217: As in my comments above, the reference to these promoters being constitutive should be removed.Our response: We thank the reviewer for the suggestion, and have made this edit.(R2) Lines 323-324: I don’t believe this sentence is referencing the correct Supplementary file numbers.Our response: We have made this correction.(R2) Line 329: …into regions that do not disrupt…Our response: We have made this correction.(R2) Line 345: Azure C600 Western BlotOur response: We have made this correction.(R2) Lines 387-388: Were these seeds plated on media without selection? T2 plants should still be segregating for the transgenes, so why were these grown without selection? How do you know that a plant not expressing RUBY is not because it doesn’t contain the transgene?Our response: The seedlings were screened without selection to minimize stress on the seedlings. To ensure that at least some proportion of our seedlings did contain the transgene, we examined at least 19 T2 individuals per T1-line (independent insertion event).Reviewer: 3 (R3)Main considerations(R3) The endogenous nature of the selected sequences prompts a question about their conservation, and thus transferability, to other species. The authors do address this when they test the promoters in transient tobacco agroinfiltration, however there is potential information being missed by such an approach. I wonder whether this could be addressed with an in silico approach, at least for the 5-6 promoters which are validated experimentally: for example can synteny of conserved transcription binding sites be compared across Arabidopsis accessions and other brassica species (for which the genome sequence is available). Similarly, the position where to insert gRNA targeted-sequences could be inspected for variability (with the assumption that high variability implies negligible regulatory capacity).Our response: We thank the reviewer for this suggestion. We are also interested in exploring this cross-species promoter analysis. However, we believe the question is best addressed using a broader bioinformatic approach including many additional species, which would be a significant amount of additional work and is outside the scope of the current manuscript.(R3) Ruby usefulness is questioned, based on the results showed in fig 3. However, the authors do not consider potential post-transcriptional regulation that the UTRs included in their design might entail. A comparison between qPCR and protein levels (if any antibody is available to test it) would be very informative.Our response: We thank the reviewer for this suggestion and have added the aspect of post-transcriptional regulation in the discussion section:“Evaluating the promoters using RUBY revealed that the novel reporter had limited sensitivity when driven by weaker promoters. We were able to detect RUBY expression in seedlings and adult tissues without visible coloration using qPCR, a more sensitive assay. However, it is important to note that detecting transcripts doesn’t always imply comparable levels of protein production due to post-transcriptional and post-translational regulation. In our design, we attempted to capture the effects of any post-transcriptional regulation by including the UTRs, but other potential transcriptional regulators could be missed. The lower-than-expected RUBY mRNA levels detected could be due to such regulators. Promoter-proximal introns after the translation start codon, for example, would not be captured in the cloning pipeline though it is known to contribute to gene expression . Distally located regulatory regions would also not be captured, but they should be rare in the compact genome of Arabidopsis .”In regards to protein level analysis, we appreciate the suggestion though we are unaware of an antibody for RUBY.(R3) Figure 2 shows that the selected promoters/terminators regulate genes with a range of expression levels. How does this compare between the endogenous genes and transgenes ?Our response: We thank the reviewer for suggesting this analysis. We have added a brief discussion of this as well as an additional supplementary table addressing the comparison between endogenous gene and transgene levels.“The expression level of RUBY mRNA detected through qPCR is weaker than expected from the RNAseq dataset. While the predicted expression level of all four of the genes in the qPCR experiment are higher than the reference gene PP2AA3, the measured result showed the opposite (Supplementary Table S6). This discrepancy could be attributed to the RUBY reporter or potential limitations in identifying additional transcriptional regulators (see discussion).”(R3) Finally, the constitutive nature of the promoters is only evaluated at the spatial level , but not their resilience to alteration by environmental stimuli (e.g. light/temperature). This could be tested in the T2 lines.Our response: We thank the reviewer for their suggestion. Our main design specification was to identify broadly-expressed promoters, and our selection criteria do not include stress-resistance, therefore we have only evaluated these promoters on their expression pattern. We did, however, include a metric for stress-resistance in our Supplementary_Table_S2 that was determined from RNAseq data.Minor considerations:(R3) The second paragraph sets out the goal of this study and provides a definition for constitutive promoters. The term ubiquitous could be presented as well, as a tissue specific promoter could still be constitutively active at most/all developmental stages, but I appreciate that there is ambiguity in the literature about these terms.Our response: We appreciate the reviewer noting the ambiguity in the use of the term “constitutive.” We have modified our text to state explicitly our definition: “Constitutive promoters are defined here as expressed in all tissues at all times”(R3) The third paragraphs describes the previous approaches to generate constitutive promoters. By the authors own definition of ‘constitutive’, promoters with different expression patterns could not be defined as such. Similarly, the use of exogenous ZF, TALEN or Cas binding sites does not seem to address the problem, as their effectiveness would depend on the abundance of the trans-acting factor, moving the issue of regulation one level up.Our response: We have modified the text to reflect this suggestion: “To expand the number of promoters available, several groups have recently used distinct strategies to engineer both constitutively and conditionally expressed promoters”Results(R3) Can the authors justify why limiting the analysis of transcription factor binding sites to 500 bp? Is regulation potential expected to decrease with distance from TSS?Our response: We have added a few references to clarify this point: “While there are no specific guidelines on optimal placement for gRNA target-sites in plants , studies in other eukaryotes have pointed to -50 to +300bp from TSS in mammalian cells for CRISPRi, and within -200bp from TSS in yeast ”(R3) Figure 2 legend: from how many leaves does the beeswarm plot of datapoint collected from the platereader?Our response: We have added text to the figure legend and methods section to clarify this point.“For each construct, the three replicates with median fluorescence levels closest to the median of the group were selected for visualization and statistical analysis. Each biological replicate is represented by a beeswarm plot of 24 datapoints  collected from the plate reader as well as a single summarizing datapoint representing the median. The boxplots represent all biological replicates.”(R3) Figure 3: the text (l155) mentions expression in the pollen but the resolution of the photo does not allow to see that . More in general Ruby production is assessed locally, but it is unclear which tissue was used for RNA extraction before the qPCR analysis. I believe it would be interesting to repeat these qPCR in (at least some) different tissues, separately, and to compare transgene expression with the Ruby phenotype. It would be interesting to compare this information with transgenic lines where Ruby is driven by traditional constitutive promoters, if such transgenic plants are available.Our response: We have made a clarification in the figure legends that the qPCR data was collected from T2 whole seedlings. Additional qPCR had also been performed as per the reviewer 2 and reviewer 3’s suggestion to confirm the expression of RUBY in the various tissues collected for two of the promoters. .(R3) Figure 4. The legend states that colours represent datapoints from a single leaf (if I understand correctly). It seems to be a lot of datapoints from a single leaf, while in the analysis showed in figure 2 comes from leaves which were infiltrated in only 4 spots.Our response: We have added text to the figure legend and methods section to clarify this point.Discussion:(R3) In the introduction, the authors correctly indicate the need to identify new minimal promoters, to design new full promoters that respond to specific stimuli. They could propose that such an analysis should be undertaken in the future on the promoters they identified in this study.Our response: We thank the reviewer for their suggestion and have modified our discussion section accordingly“If a minimal promoter sequence can be identified from these native promoters, they can also serve as the foundation of additional synthetic promoters where the expression pattern and strength can be freely modified by adding cis-elements or synthetic transcription factor binding sites.”Materials and methods:(R3) A description of how the gRNA target sequences where introduced in the cloned promoters is missing.Our response: We have added text to the methods section to clarify this point. “gRNA target-sites were cloned into regions that does not disrupt any predicted TF binding sites through Gibson assembly by replacing the original sequence ” Your revised manuscript has now been evaluated by the two of the three previously involved reviewers. I apologise for the delays. Thanks for your efforts preparing a new version which is indeed improved. You have tackled most of the scientific issues. I am pleased to accept the manuscript for publication provided that you tackle the minor comments of Rev#1 on Figs 2 and 3. I´m glad to be able to bring you encouraging news and I look forward to receiving the revised manuscript. No accompanying comment. 23 July 2023Dr. Olivier Hamant, Editor-in-ChiefDr. Matias Zurbriggen, Associate Editor:Quantitative Plant BiologyDear Dr. Hamant and Dr. Zurbriggen:Thank you for the wonderful news about the acceptance of our manuscript. We have made addressed the remaining reviewer concerns to the best of our ability, described in detail below.Detailed response to reviews:Reviewer 1 (R1)1) I could not understand why the authors want to show “a pipeline” instead of successfully developed tools, which are functional promoter cassettes. After publication of the article, do the authors expect for other researchers to follow this scheme to get additional constitutive promoters ???Our response: We hope the reviewer will agree that we have successfully used our pipeline to identify promoters that function in tobacco and Arabidopsis. The engineering constraints we applied in this manuscript are almost certainly not universally applicable to all potential uses. We expect that others may find that they can make use of our pipeline, likely with their own set of constraints, for identifying promoters with particular patterns of expression . In addition, the pipeline can be readily adapted to other organisms, an approach we ourselves have taken in another publication currently under review.2) Fig. 2 and 3: It is better to show fold change over negative control levels. Presented figures are difficult to guess strength of the signals obtained from positive samples.Our response: We thank the reviewer for this suggestion and acknowledge that there are other ways to process and represent the data. We maintain that our choice is the clearest for readers, but understand that there is room for disagreement. To facilitate alternative analyses and provide full transparency, all data and scripts  are now linked to Zenodo DOI: 10.5281/zenodo.8170303. The data files include individual outputs generated by the plate reader that are consolidated and annotated using R-script before normalized for plotting, and this pre-normalized consolidated dataset is also available for download.3) Fig. 3A: Signal of RUBY is not easy to see. Pictures need improvements.Our response: We put a significant amount of effort into photographing the subtle RUBY expression found in our transgenic lines, and, in fact, did a number of ‘blinded’ tests to verify that one plant part was indeed redder than the control. To provide more quantitative measurements of the RUBY signal, we have now added an unbiased analysis of the photographs in the manuscript, where the level of “red” within a region of interest was determined by using the a* axis in the CIELAB color space .4) Fig. 4: Degree of suppression are all around 0.5 fold or more, which, for me, indicates failure of the suppression system. These examples do not look like a practical system.Our response: We agree that the repression system can be further optimized, and the practical application of the system would depend on the dynamic range desired.5) I could not find proof of constitutive function of the developed promoters in the figures. This information is necessary.Our response: For two of the transgenic Arabidopsis lines, we performed qPCR on flower, leaf, root, and seedlings. In all cases, we were able to detect reporter mRNA.Thank you for again your time and consideration of our work.Sincerely,Dr. Jennifer NemhauserDepartment of BiologyUniversity of Washingtonjn7@uw.edu Dear authors, thanks for having tackled the last remaining issues and delivered such a good quality article. Please apologise for the long time needed. I’m glad to inform you that your manuscript has been accepted for publication. No accompanying comment.


MADS-domain transcription factors are involved in the control of a multitude of processes in eukaryotes, and in plants, they play particularly important roles during reproductive development. Among the members of this large family of regulatory proteins are the floral organ identity factors, which specify the identities of the different types of floral organs in a combinatorial manner. Much has been learned over the past three decades about the function of these master regulators. For example, it has been shown that they have similar DNA-binding activities and that their genome-wide binding patterns exhibit large overlaps. At the same time, it appears that only a minority of binding events lead to changes in gene expression and that the different floral organ identity factors have distinct sets of target genes. Thus, binding of these transcription factors to the promoters of target genes alone may not be sufficient for their regulation. How these master regulators achieve specificity in a developmental context is currently not well understood. Here, we review what is known about their activities and highlight open questions that need to be addressed to gain more detailed insights into the molecular mechanisms underlying their functions. We discuss evidence for the involvement of cofactors as well as the results from studies on transcription factors in animals that may be instructive for a better understanding of how the floral organ identity factors achieve regulatory specificity. Flowers contain the reproductive organs of angiosperms, the largest group of land plants. They produce much of the food humans and their livestock consume and are therefore pivotal for agriculture and the economy. Early genetic analyses of flower development led to the formulation of the ABC model of floral organ identity specification ,2. This MCM1 from the yeast Saccharomyces cerevisiae, AGAMOUS from the plant Arabidopsis thaliana, DEFICIENS from the plant Antirrhinum majus, and SERUM RESPONSE FACTOR from the human Homo sapiens). The MADS-domain protein family is much enlarged in land plants relative to other eukaryotes  and combidopsis .Transgenic and mutant studies have shown that the transcription factors encoded by the floral homeotic genes are necessary and sufficient to define the identity of lateral organs as specific floral organs . Furt6-GG-3′) and that their preferences for and affinities to individual CArG-box motifs vary depending on the composition of a given dimer [It has been shown that homo- and heterodimers of MADS-domain transcription factors bind to so-called CArG-box sequences (consensus: 5′-CC-(A/T)en dimer . The seqen dimer ,23,24. BChromatin immunoprecipitation assays coupled to next-generation sequencing (ChIP-seq) revealed that several thousand sites are bound by each of the floral organ identity factors in the Arabidopsis genome ,26,27,28In classic experiments, in which the amino-terminal half of the MADS-domain of different floral organ identity factors was replaced by the corresponding sequences of human MADS-domain proteins, it was shown that the expression of the resulting chimeric proteins led to gain-of-function phenotypes similar to those observed in lines overexpressing the unmodified floral organ identity factors . BecauseIn summary, there is evidence that differences in the DNA-binding preferences of individual MADS-domain transcription factors (or MADS-domain protein dimers) account, at least in part, for their functional specificities. At the same time, the closely-related floral organ identity factors show considerable overlaps in their genome-wide binding patterns, raising the question of whether their DNA-binding activities alone are sufficient to determine their clearly separable functions.To understand whether all the binding events that were detected by ChIP-seq were functional, transcriptomics experiments were carried out after altering floral homeotic gene activities. These experiments revealed that: (i) only a subset of the genes bound by the floral homeotic factors respond to a perturbation of their activities; and (ii) despite their similar genome-wide binding patterns (see above), the different floral organ identity factors regulate markedly distinct sets of target genes ,10,25,34SEEDSTICK [In support of such a scenario, several lines of evidence suggest that the floral organ identity factors are part of larger transcriptional complexes: (i) Gel filtration experiments showed that protein complexes containing SEP3 have a molecular weight of around 670 kDa, which is considerably larger than MADS-domain protein tetramers (~120 kDa) . (ii) ThEEDSTICK . DespiteIn addition to transcription factors, the above-mentioned experimental approaches also yielded epigenetic regulators as candidate interactors . For exaIn summary, several independent lines of evidence support the idea that the floral organ identity factors are part of large regulatory complexes that contain cofactors and epigenetic regulators . The exaThere is ample evidence for the role of post-translational modifications (PTMs) in the regulation of transcription factor activities . Common One published example of a PTM of a floral organ identity factor comes from a study that demonstrated farnesylation (a form of prenylation) of AP1 . It has FLM transcripts undergo alternative splicing and that different ambient temperatures lead to the predominant accumulation of one or another splice form [Another mechanism that is well known to affect transcription factor function is alternative splicing, where certain protein domains are either retained or eliminated based on the composition of the mature mRNA after splicing of the corresponding pre-mRNAs. While alternative splicing is not specific to transcription factor-coding genes and likely affects over 60% of intron-containing genes in higher plants . Specifiice form ,64. The Hox genes is that they are typically expressed only in the cells, tissues, or organs they help to specify [Hox genes are expressed often overlap, so that in some cells combinations of Hox proteins are active [Hox genes do not encode MADS-domain but homeodomain transcription factors [Hox and floral organ identity genes, any comparison between the regulation of the corresponding transcription factors has to be approached with caution; however, given the striking similarities in their functions and the fact that evolutionary mechanisms are often conserved between plants and animals [As described above, the activities of transcription factors are known to be controlled by a variety of mechanisms. To better understand which of these mechanisms contribute to the regulation of the floral organ identity factors, it may be instructive to review what is known about functionally analogous transcription factors in animals. We believe that Hox proteins are particularly interesting in this context because they play roles similar to those of the floral organ identity factors, in that they control the specification of different organ and cell types along an axis . Their i specify . Furthere active , just as factors ,67,68. T animals , it may  animals ,67,68. D animals ,67,68. B animals ,67,68. F animals ,71.cis-regulatory modules bound by the Hox transcription factors but that may not bind DNA cooperatively with them [In addition to these variations in binding specificities, the regulation of Hox target genes appears to be dependent in some cases on the presence of cofactors. Arguably the best studied of the Hox cofactors are members of the Three Amino Acid Loop Extension (TALE) class of homeodomain-containing proteins ,67,68. Tith them ,67,68. Tith them , with thIn this review, we have summarized some of the current knowledge about the function and regulation of the floral organ identity factors. While insights into the role of dimerization and tetramerization as well as differences in DNA-binding preferences have been obtained, other regulatory mechanisms that could be at play have not yet been studied in detail. In particular, it seems important to better characterize the composition and dynamics of the regulatory complexes that contain the floral organ identity factors and to study the functions of the different complex components. The identification of several candidates for interactors in recent years has provided a good starting point for this work. Based on what has been found for the TALE proteins interacting with Hox transcription factors in animals (see above), we think it likely that cofactors of the floral MADS-domain proteins also have other developmental functions and may not even be specifically expressed in flowers. This, and the high level of functional redundancy often found among plant genes, could explain why such cofactors have not been identified in the many genetic screens that were conducted in Arabidopsis. It is also possible that there are many cofactors from different families that each control only a small subset of target genes. Thus, there may not be one general mechanism underlying floral organ identity factor function but rather a combination of different and possibly interacting mechanisms that may be specific for each regulatory complex and that might vary depending on the genes they target. With the ever-improving experimental approaches plant biologists have at their disposal, now seems to be a good time to reach to the bottom of how the floral organ identity factors achieve regulatory specificity.


Telomerase activity and telomere elongation are essential conditions for the unlimited proliferation of neoplastic cells. Point mutations in the core promoter region of the telomerase reverse transcriptase (TERT) gene have been found to occur at high frequencies in several tumour types and considered a primary cause of telomerase reactivation in cancer cells. These mutations promote TERT gene expression by multiple mechanisms, including the generation of novel binding sites for nuclear transcription factors, displacement of negative regulators from DNA G-quadruplexes, recruitment of epigenetic activators and disruption of long-range interactions between TERT locus and telomeres. Furthermore, TERT promoter mutations cooperate with TPP1 promoter nucleotide changes to lengthen telomeres and with mutated BRAF and FGFR3 oncoproteins to enhance oncogenic signalling in cancer cells. TERT promoter mutations have been recognized as an early marker of tumour development or a major indicator of poor outcome and reduced patients survival in several cancer types. In this review, we summarize recent findings on the role of TERT promoter mutations, telomerase expression and telomeres elongation in cancer development, their clinical significance and therapeutic opportunities. Cancer cells acquire replicative immortality through a multistep process involving progressive accumulation of genetic and epigenetic changes, escape from replicative senescence and neoplastic transformation . TelomerTelomerase activity is upregulated in neoplastic cells through multiple mechanisms including chromosomal rearrangements, amplification of TERT gene, generation of TERT structural variants, disruption of telomere position effect and activating mutations in TERT promoter . EpigeneThe TERT gene is located approximately one megabase from the end of the short arm of chromosome 5, and its core promoter is delimited by nucleotides located at positions −330 upstream and +37 downstream of the TERT ATG start site . TERT gede novo binding motifs for E-twenty-six transformation-specific (ETS) transcription factors  provided relevant information regarding the distribution of TERT promoter variations in diverse tumour types and histological subtypes . The datde novo 5′-GGAA-3′ binding motifs that are recognized by transcription factors of the E-twenty-six (ETS) family, including the GA-binding proteins alpha and beta (GABPA and GABPB), ETS1 and ETS Variant Transcription Factor 1 (ETV1) (TERT promoter mutations have been shown to activate telomerase expression through the creation of 1 (ETV1) . In part1 (ETV1) , (Bell e1 (ETV1) . TERT pr1 (ETV1) .Three-dimensional (3D) organization of the genome represent an important mechanism by which regulatory elements control epigenetic modifications and gene expression . The binDuplication of GABP binding sites in TERT promoter have been shown to cause the increase of telomerase expression similarly to the hot spot nucleotide changes . DuplicaMoreover, the complex formed by ETS1 with p52 NF-κB, whose expression is specifically controlled by non-canonical NF-κB signalling, has been shown to bind selectively the mutant TERT promoter −146A thus promoting telomerase expression in glioblastoma and melanoma . The ETSRecent studies have identified activating mutations in the promoter region of the ACD gene co-occurring with TERT promoter mutations in 5% of cutaneous melanoma . The ACDIn papillary thyroid cancers the ETS variant 5 (ETV5) is abundantly expressed and able to activate mutant TERT promoter in GABP-negative thyroid cancer cells . In addiIn bladder cancer, GABPA is involved in the recruitment of two transcriptional co-regulators of telomerase expression, namely, TRIM28 (tripartite motif containing 28) and TRIM24, to the mutant TERT promoter −124A . The traProteins of the shelterin complex, besides protecting telomeres from unwanted DNA repair processes, have a major role in the formation of loops and regulation of telomerase expression. Indeed, telomeric factors such as TRF1, TRF2 and RAP1 have the ability to interact with non-telomeric binding sites, such as TERT promoter sequences . Recent Somatic mutations in the TERT promoter have been identified with variable frequencies in numerous cancer types . HighestIn glioblastoma, TERT promoter mutations are considered early genetic events of tumour development. in situ . The frequency of TERT promoter mutations in intraepithelial neoplasia grade 1-3 (CIN1-3) and squamous cell carcinoma of the conjunctiva was significantly higher in HIV-positive (between 32% and 46%) compared to HIV-negative patients (between 13% and 22%) . These fVariable frequencies of TERT promoter mutations have been detected in diverse tumour types of the lower genital tract, associated or not with human papillomavirus infection. Two independent studies performed in India and in Italy showed that 20% of cervical squamous cell carcinoma harboured TERT promoter mutations independently from the HPV status . The conSquamous cell carcinoma of the vulva originates from two different etiopathogenic pathways, one associated with human papillomavirus (HPV) infection and the other in the setting of longstanding dermatosis, such as lichen sclerosus . OverallSimilarly to vulvar cancer, penile squamous cell carcinoma either develop through an HPV associated pathway or through other oncogenic mechanisms independent from the HPV infection . The seaSquamous cell carcinoma of head and neck comprises a heterogeneous group of tumours mainly localized in the oral cavity, larynx, oropharynx, hypopharynx and nasopharynx. According to a recent systematic review and meta-analysis including 1830 patients, the overall prevalence of TERT promoter mutations in head and neck cancer was 21% with significant differences according to cancer site . Indeed,Selective reactivation of the telomerase in the large majority of tumours, while remaining inhibited in normal cells, provides the opportunity to develop therapeutic approaches inhibiting telomerase activity . SeveralImmunotherapy targeting telomerase also showed promising results in all cancer types with high telomerase activity . Specifi+ cytotoxic T lymphocytes (CTL), include the peptide I540 , that is the first TERT immunogenic peptide identified as an HLA-A*0201-restricted T-cell epitope and has been evaluated in phase III clinical trials for melanoma treatment  and induce specific CD8reatment . In addireatment . Peptidelomerase . TERT 57patients . UV1 peppatients . UV1 peppatients .The crucial role of telomerase activity and telomere maintenance in cell transformation and development of cancers has known since decades. Mechanisms of telomerase reactivation in cancer cells include complex molecular changes such as chromosomal translocations, TERT gene amplifications, TERT structural variants, transcription binding factors, TERT promoter mutations and spatial chromatin arrangements. Most of these genetic and epigenetic alterations drive strong telomerase activity in a manner that is specific to cancer histotypes. Paradigmatic is the observation that a subgroup of high-risk neuroblastoma patients having mostly unfavourable outcome were characterized by TERT rearrangements or by MYCN amplified tumours, both inducing massive transcriptional upregulation of the TERT gene . SimilarKnowing the temporal occurrence of genetic and epigenetic changes in TERT locus during cancer growth is very important for the development of diagnostic and prognostic markers in diverse tumour types. The cancer driver effect of TERT promoter mutations in tumour development has been shown by a proof-of-concept study showing that correction of the −124A mutation to the wild type −124G blocked the binding of ETS transcription factors to the TERT promoter, reduced TERT gene transcription and telomerase expression, and induced senescence and proliferative arrest of glioblastoma cells . AssessmIn normal cells, TERT promoter is tightly controlled by numerous transcription factors, which bind to regulatory elements and control the silencing in somatic cells and the reactivation in stem cells. These factors include among others activators of TERT transcription, such as the oncogene c-MYC, Sp1, NF-κB, STAT family of proteins, AP-2, GSC, as well as repressors that downregulate TERT transcription, such as MAD1/2, p53, WT1, CTCF, and MZF-2 . The freMutations in the core promoter of the TERT gene are exceptionally high in many cancer types and may represent specific targets for new therapies. Such mutations abrogate the repressive function of G4-structures in the TERT promoter by inducing conformational changes . Recentl


MYC which is under the control of numerous super-enhancers gained in cancer compared to normal tissues. This Review will cover the expanding cell-intrinsic and cell-extrinsic etiology of these super-enhancer changes in cancer, including somatic mutations, copy number variation, fusion events, extrachromosomal DNA, and 3D chromatin architecture, as well as those activated by inflammation, extra-cellular signaling, and the tumor microenvironment.Super-enhancers are large, densely concentrated swaths of enhancers that regulate genes critical for cell identity. Tumorigenesis is accompanied by changes in the super-enhancer landscape. These aberrant super-enhancers commonly form to activate proto-oncogenes, or other genes upon which cancer cells depend, that initiate tumorigenesis, promote tumor proliferation, and increase the fitness of cancer cells to survive in the tumor microenvironment. These include well-recognized master regulators of proliferation in the setting of cancer, such as the transcription factor  Super-enhancers are estimated to contain tenfold more distinct protein factors than typical enhancers and are algorithmically defined using the Rank Ordering of Super-Enhancers (ROSE) script –3. UsingMYC [. Transcriptional regulation by SEs is often critical for downstream gene expression, as CRISPR deletion or interference of the distal SE significantly reduces expression of its target gene. In the case MYC, deletion of its SE in mice results in complete loss of MYC expression in hematopoietic lineages [SEs were first discovered in transgenic mice in and termed locus control regions in 1987. Further characterized in murine embryonic stem cells (mESCs), super-enhancers adopted their moniker in 2013 and were observed near transcription factors required for pluripotency, suggesting they may enrich at genes critical for cell identity in the setting of healthy and diseased states , 5. Sevelineages .The etiologies of enhancer changes described in cancer are almost invariably attributed to cell-intrinsic genomic alterations, including (1) activation of oncogenic signaling and, (2) de novo formation of transcription factor (TF) binding sites, as well as (3) focal amplification of non-coding active regulatory regions –10. HereVHL that encodes the E3 ligase for the HIF transcription factor, directly results in the formation of numerous aberrant SEs due to HIF accumulation [APC that encodes a member of the β-catenin destruction complex [. Accumulated β-catenin subsequently translocates to the nucleus to bind the TCF/LEF family of transcription factors and activate transcription of Wnt target genes including the previously mentioned and known target MYC  in lung cancer results in a global reduction in SE acetylation levels due to inhibition of H3K4 methylation at promoters, including at the transcription factor PER2 which negatively regulates glycolytic genes [PER2-dependent glycolytic genes nevertheless sufficiently reprograms the SE landscape of these genes to sustain a metabolic dependency in this subset of lung cancer with increased sensitivity to glycolysis inhibiton [Inactivating mutations in proteins that directly modify histones can also reprogram SEs, especially proteins governing histone methylation, which are mutated in a variety of cancers . Loss ofic genes . Yet, dinhibiton . Furthernhibiton .In several cancers, whole genome and whole exome sequencing approaches uncovered high frequency mutations in the SWI/SNF family of chromatin modifying enzymes. The SWI/SNF complex features ATP-ase activity capable of de-stabilizing histone–DNA interactions and thereby regulating chromatin accessibility for transcription factor binding. Thus, mutations in the SWI/SNF family, collectively observed in ~ 20% of all human cancers, can impact the enhancer landscape to varying degrees [PBRM1, which encodes the BAF180 subunit of SWI/SNF, is frequently lost in renal cell carcinoma (RCC). However, PBRM1 silenced RCC cell lines exhibit little change in open chromatin and H3K27ac landscapes including at SEs [SMARCB1, which encodes the subunit SNF5. SMARCB1 loss in this context decreases SWI/SNF occupancy at typical enhancers (TEs) while maintaining occupancy at SEs, an observation that was reproduced in other cancer cell lines as well [For instance, g at SEs . Similar as well –24.ARID1A, mutated in endometrial carcinoma, preferentially affects SEs over TEs. Wilson et al. observed ARID1A occupancy at SEs over TEs, which exhibited increased H3K27ac signal and open chromatin accessibility following ARID1A deletion resulting in activation of invasion genes [ARID1A deletion is attenuated with either genetic or pharmacologic inhibition of P300, suggesting it is required in the setting of ARID1A loss [ARID1A deletion did not change significantly change P300 genome-wide occupancy [ARID1A and the repressive histone variant H3.3, which became depleted following ARID1A loss [Conversely, a separate study showed loss of on genes . These son genes . EpistasD1A loss . The exaccupancy . A folloD1A loss .The proximity of constituent enhancers, the density of protein factors, and the level of transcriptional cooperativity led to the hypothesis that SEs exist as membrane-less, phase separated condensates, which have recently emerged as an important driver of protein–protein interactions, especially given the intrinsically disordered domains present among transcription factors, Mediator, and BRD4 Fig.  3, 32–3–332–39. de novo liquid–liquid phase separated puncta with subsequent oncogenic SE formation detected by ChIP-seq [Oncogenic fusion proteins that create aberrant transcription factors can create de novo SEs to maintain transcriptional dependencies. One recurrently detected fusion in leukemia, NUP98–HOXA9, is enriched for intrinsically disordered domains to form a ChIP-seq , 45. NotChIP-seq . ImportaChIP-seq . Other oChIP-seq –30. SEs ChIP-seq .de novo binding site for the transcription factor MYB that resulted in the formation of a SE at this locus  for somatic hypermutation and class switch recombination , 47. Howferation –49.BCL6, BCL2, and CXCR4 [BCL6 SE, a recurrent mutation prevents binding by the transcriptional repressor BLIMP1 and confers increased fitness in DLBCL cells. When these mutations were corrected back to the WT allele, dropout of DLBCL cells was observed compared to isogenic cells retaining the BCL6 SE mutation [In contrast to the Mansour et al. study in T-ALL in which a mutation created a binding site for a transcription factor to form a SE, non-coding mutations in DLBCL occur in existing SEs and alter the binding sequences of transcriptional repressors. This disinhibition results in even greater expression of SE associated oncogenes including nd CXCR4 . At the mutation . These dLMO1 that associated with neuroblastoma susceptibility in GWAS [. The G is the reference and risk allele, critical for the GATAA binding motif for the GATA transcription factor within the SE, while the protective alternative allele T breaks this sequence, resulting in decreased GATA occupancy at the LMO1 SE and gene expression [LMO1, suggested to originate from an APOBEC-like cytidine deaminase mutational signature, that created a MYB transcription factor binding site which increased expression and dependency on LMO1 in T-ALL [Inborn polymorphisms that affect transcription factor binding also occur in SEs. Oldridge et al. report a G > T polymorphism at the SE of the transcriptional co-regulator and oncogene  in GWAS . The G ipression . In a Eupression . A similin T-ALL . Howeverin T-ALL . These eMYCN which is the strongest correlate for high-risk disease and poor prognosis [MYCN results in widespread promiscuous occupation of an ubiquitous CANNTG motif (where N can represent any base) [Amplification is one of the most established etiologies of SE reprogramming in cancer alongside oncogenic signaling. Approximately 25% of neuroblastomas exhibit amplification of the oncogene rognosis . While nny base) . The resny base) . Dysreguny base) .MYC, KLF5, USP12, and PARD6B  ranging from 100 kb to several megabases, a process linked to p53 mutations –64. ThesEGFR as an example, Morton et al. show enhancers potentiating EGFR expression are co-amplified on the same amplicon as the oncogene on ecDNA, confirmed using DNA fluorescence in situ hybridization (FISH) [MYCN in neuroblastoma and Wilms tumor [cis on ecDNA [Using n (FISH) , 66. Thims tumor . DNA brems tumor . Remarkams tumor , 70. Comon ecDNA .MYC [. Hung et al. propose a model, where enhancer activation of the MYC promoter can occur in cis, from an enhancer within the same ecDNA, or in trans, from an enhancer on another ecDNA molecule within the same hub [FGFR2 ecDNA and MYC ecDNA are in a hub, interference of FGFR2 enhancers decreased MYC expression, but interference of MYC enhancers did not affect FGFR2 expression [MYC on chromosome 8 and FGFR2 on chromosome 10, can activate genes in trans through ecDNAs.SE-gene interactions are not restricted to just ecDNAs. One study found that SEs on ecDNA can interact with and enhance expression of genes on chromosomal DNA, thus acting as “mobile enhancers” . ecDNA cMYC , 73. HunA technical difficulty of ecDNA studies have been trying to distinguish between chromosomal versus extrachromosomal reads in sequencing experiments. Recently, Hung et al. from the laboratory of Howard Chang developed CRISPR–CATCH, a novel technique for the isolation of megabase-scale ecDNA that allows targeted investigation of the epigenome within these amplicons . Indeed,SEs are often flanked by CTCF sites with exceptionally strong boundaries believed to concentrate transcriptional activating machinery at a focal locus to potentiate gene expression . These sDNA between two CTCF sites are extruded to form a loop, compartmentalizing enhancers and gene bodies into “neighborhoods” or topologically associating domains (TADs) thereby restricting gene–enhancer interactions to mainly within the loop. However, complex, long-distance SE interactions have also been described, including three-way interactions between multiple SE-containing TADs on the same chromosome . PerturbIDH [Flavahan et al. showed, in two studies, that such disruption can unleash SEs to activate nearby oncogenes. One such mechanism of CTCF perturbation is methylation, a feature of several cancer especially those with mutations in isocitrate dehydrogenase, IDH –83. ThesIDH .PDGFRA amplifications and mutations are common in glioblastoma but appear to be somewhat mutually exclusive with IDH mutant tumors, which nonetheless highly express PDGFRA [. Flavanhan et al. initially found IDH mutant gliomas exhibited a disrupted CTCF boundary that allowed an aberrant interaction between a typical enhancer and the glioma oncogene PDGFRA, each originally insulated from each other, as an alternative method of oncogene activation  silenced tumors [SDH silenced gastrointestinal stromal tumors (GISTs) lose ~ 5% of their CTCF binding sites due to SDH-deficient hypermethylation [FGF3 and FGF4 in the neighboring TAD, while another CTCF site isolates a SE from the GIST oncogene KIT [. CTCF binding loss subsequently leads to aberrant interactions and subsequent oncogene addiction. Aside from methylation, deletions may also inactivate CTCF insulators. Non-coding micro-deletions of TAD boundaries have been described in T-ALL, which merge TAL1 into an adjacent neighborhood, where it is subsequently activated by enhancers [The same phenomenon occurs in succinate dehydrogenase  as well as inflamed non-dysplastic colon (purely cell-extrinsic) [Kras mutations to accelerate lesion formation [Thus, our wealth of ChIP-seq and ATAC-seq data on primary tumors actually reflect three, distinct super-imposed chromatin landscapes: cell-intrinsic, cell-extrinsic, and a unique third subset that arises from the interplay between the latter two . Indeed,trinsic) . In partormation .Cell-extrinsic enhancer reprogramming may also provide insight towards targeting these oncogenic epigenetic changes. Given the role of inflammation towards their induction, future studies should elucidate whether tamping down local inflammation, using FDA-approved TNF-α inhibitors or PROTAC-degraders of STAT3, could attenuate such reprogramming with therapeutic benefit . While m