Escherichia coli to achieve a robust adaptation. Here, we demonstrate that an indirect coherent feedforward loop and mutual inhibition designs can experimentally improve the fold change of promoters, by reducing the basal level while keeping the maximum activity high. We applied both designs to six different promoters in E. coli, starting with synthetic inducible promoters as a proof-of-principle. Then, we examined native promoters that are either functionally specific or systemically involved in complex pathways such as oxidative stress and SOS response. Both designs include a cascade having a repressor and a construct of either transcriptional interference or antisense transcription. In all six promoters, an improvement of up to ten times in the fold change activation was observed. Theoretically, our unitless models show that when regulation strength matches promoter basal level, an optimal fold change can be achieved. We expect that this methodology can be applied in various biological systems for biotechnology and therapeutic applications.Computations widely exist in biological systems for functional regulations. Recently, incoherent feedforward loop and integral feedback controller have been implemented into  Gene regulatory networks in cells are often sensitive to the fold-change response of bio-molecular signals and not to their absolute change, e.g. bacterial chemotaxis sensory systems, signaling pathways, and human perception of sound intensity, light intensity, smell and weight . Such a So far, genetic circuits have been implemented in living cells using two major computational paradigms . The firThe basal levels of promoter activity and their FCA play a significant role in determining the behavior of gene circuits in both digital and analog designs. In a digital design, the FCA can be used to tune the threshold of the response function Figure . CircuitSeveral methods have been applied to manage the basal level of promoter activity, such as using antisense transcription ,30–33, fTo this end, we have developed two designs: (i) an indirect coherent feedforward (ICF) circuit Figure , and ii. In thisEscherichia coli was achieved by using repressor and antisense promoters based on two regulatory mechanisms. The first mechanism involves antisense RNA that can bind to the mRNA preventing its translation . The OL circuit includes a part under test (PUT) that is directly connected to a subtraction with a strength of Fs (OUTOL = OUTPUT – Fs). Evidently, enhancing the Fs level decreases the basal and maximum levels of OL circuit by the same amount. According to this simple analysis, we do not expect that an increase in the Fs strength can enhance the FCA in the OL circuit. Furthermore, since in reality signals cannot fall below zero, thereby the FCA level decreases as Fs levels increase  for ICF, and Fs= β for DNF design). Our qualitative analysis of the β – Fs relations for the ICF and DNF circuits shows areas where the MDL and FCA are optimal . n, m and h are Hill coefficients, Fs is the strength of the feedforward/feedback loops, β is basal level and α is a parameter representing the activation strength of X on Z. The simulation results of FCA for the nonlinear molecular ICF network is shown in Figure Fs strength. The MDL of the molecular ICF circuit also rises when the sF increases, compared to the MDL of the linear model which has an optimum. Interestingly, simulation of FCA and MDL for the molecular DNF network  in the ICF and DNF designs with non-linear operations (e.g. Hill-function) \dock Figure  show an 2O2), sodium (meta)arsenite (AsNaO2), hemin, arabinose, isopropyl-β-D-1-thiogalactopyranoside (IPTG), anhydrotetracyclin hydrochloride (aTc) and acyl homoserine lactone 3OC6HSL (AHL) were used as inducers and were obtained from Sigma-Aldrich. For a list of parameters, see Supplementary Information, Table S4.Nalidixic acid (NA), hydrogen peroxide (HEscherichia coli (araD139 D(ara-leu)7697 fhuA lacX74 galK (W80 D(lacZ)M15) mcrA galU recA1 endA1 nupG rpsL (StrR) D(mrr-hsdRMS-mcrBC). The PkatG, PrecA and ParsR promoters were obtained by PCR amplification (Phusion High-Fidelity PCR Kit – New England Biolabs) from the genome of MG1655 E. coli (F− λ− ilvG− rfb-50 rph-1), with primers listed in Supplementary Information, Table S3. For part amplification from the genome, 5 ml of MG1655 strain E. coli were inoculated from frozen glycerol stocks and were grown for 16 h. The next morning, 5 μl from the overgrown culture was mixed with 15 μl of DNase and RNase free water, heated at 96°C for 6 min and incubated at –80°C for 10 min. 2 μl from this solution was added into PCR mixture with total volume of 50 μl. The primers were synthesized by Integrated DNA Technologies . Plasmids for cloning were transformed into chemically competent E. coli 10-beta with a standard heat shock protocol  restriction endonucleases, Thermo Scientific FastDigest Restriction Enzymes, T4 DNA Ligase were used for plasmid construction. All plasmids in this work were built and transformed to NEB 10-beta protocol . BacteriE. coli 10-beta or MG1655 E. coli either through heat-shock protocol  were transformed into competent protocol  or electprotocol .E. coli for cytometry measurements except from PLhrtO and PrecA, which were transformed into MG1655 E. coli wild-type strain. The bacterial cultures were inoculated from colonies on agar plate the previous day and grown in 5 ml of LB with appropriate antibiotics at 37°C and 300 r.p.m. In the morning, the overnight grown bacterial cultures were diluted 1:100 into fresh LB medium  or were diluted 1:50 into fresh M9 minimal medium   for the flow cytometry experiment with appropriate concentrations of antibiotics and incubated for specific time for regrowth and adaptation in fresh media, as described in Supplementary Information, Section 3 for each promoter. Bacterial cultures were transformed into 96-well plates with known concentrations of inducers to total volume of 200 μl, incubated in microplate shaker  for relevant time described in Section 3 for each promoter until they reached optical density of OD600 nm ∼ 0.4–0.7. Then, the fluorescence and scattering of bacterial cultures were analysed through flow cytometry analyzer (CytoFLEX S Flow Cytometer). In all experiments 10000 events have been obtained and the fluorescence and forward and side scattering were taken using CytExpert 2.2 software. The fluorescence distribution data over population data were extracted together with its geometric mean from each well in 96-well plate and plotted using MATLAB. Fluorescence measurement was based on geometric mean of flow cytometry populations from three experiments. The flow cytometry data for one representative experiment for each combination, which was independently repeated for two more times, is provided in Supplementary Information, Section 4. Next, the figures were built in EXCEL, based on geometric mean of flow cytometry populations with error bars representing the standard deviation errors of the geometric mean.Different combinations of plasmids forming different synthetic circuits were transformed into competent NEB 10-beta l-arabinose utilization system  and araO2 (O2) DNA binding sites by rigidly holding the DNA binding domains through its N-terminal arm. This conformation creates a loop in the DNA that prevents the RNA polymerase from binding to initiate transcription. When arabinose is present, the physically closed loop is released and AraC moves to bind araI1 (I1) and araI2 (I2) DNA binding sites. The opened loop allows RNA polymerase to freely access the promoter, and the positioning of a DNA binding domain of AraC at I2 facilitates transcription initiation by RNA polymerase. According to this explanation, a diagram model that describes the PBAD promoter system is shown in Figure BAD promoter, the free AraC represses the PBAD promoter. Since the total amount of AraC is equal to the amount of free AraC and that of the arabinose-AraC complex, in our model, the arabinose participates in two circuit branches. The first is driven by the complex (arabinose-AraC) which directly activates the output. The second branch indirectly activates the output through double inhibitions: the free AraC and what resides from the complex. Motivated by this model, we modified the wild-type PBAD that has I1/I2 and O2 binding sites, by removing O2 binding sites .,47.l-aras Figure . The newr Figure .lux promoter in opposite orientation to promoter under test (PPUT), which inhibits the PPUT activity. We start with the transcriptional interference system  Plux promoter, where both DNA strands are fully transcribed in both directions to produce mRNA and antisense RNA. This special organization allows interference between forward and reverse promoters due to direct interaction between mRNA and antisense RNA. Further analysis of transcriptional interference and antisense transcription is provided in Supplementary Information, Section 2.1 and PUT. In the first two steps, we characterize the behavior of the transcriptional interference unit . In the third step, we implement an inverting switch through TetR repressor  or by varying anhydrotetracycline (aTc) concentration. After selecting an optimal aTc concentration, which gives the highest ON/OFF ratio of LuxR levels represented by mCherry, we can implement the ICF and DNF gene circuits. Then, we will apply ICF and DNF circuits by combining the inverting switch as described in step three with the transcriptional interference unit .We started to implement the ICF and DNF designs in living cells by mimicking a subtraction using a transcriptional interference system and an antisense transcription system. In both systems, we placed the Pe system  that invcription   the basal level decreases to very low values, while the maximum level only slightly decreases  without compromising the MDL. We also experimentally tested PlacO promoter with an isopropyl β-d-1-thiogalactopyranoside (IPTG) inducer using the transcriptional interference model. The OL and ICF circuits showed consistent behaviors with our mathematical models . In the wild-type circuit  and maximum FCA level (∼500) were obtained at the same AHL (∼4nM), while for the DNF, the maximum sensitivity and maximum FCA levels were obtained at different AHL concentrations . The transcriptional regulator, OxyR, is activated by oxidation by H2O2 which in turn activates several genes involved in bacterial defense mechanisms, among them is the KatG gene (katG) allowing its activation with FCA level of 10  in biological systems where chemical inputs directly interact with target promoters by binding transcription factors. However, biological systems often activate multiple pathways in response to chemical signals and more specifically in response to toxic chemicals. For example, cells induce repair systems by activating cascades of regulators, e.g. oxidative stress response  and SOS atG gene . In the el of 10 . We implel of 10 . At a sps Figure . The FCAs Figure . FurtherrecA gene and its transcriptional repressor LexA (recA) and LexA repressor was transformed into bacterial cells. The circuit is induced by Nalidixic Acid toxin and demonstrated a FCA level of 50  exhibits a strong dependency on Nalidixic Acid concentrations  of a target promoter by reducing its basal level while maintaining its maximum activity at high inducer levels. Two circuit designs were implemented, first is an indirect coherent feedforward (ICF) circuit, which is known as a coherent feedforward type 4 system, and second is a mutual inhibition by double negative feedback loop (DNF). Both designs are common motifs in natural regulatory networks. It has been reported that the ICF design can reject disruptions in indirect pathways or create delays in gene expression . The DNFlux) which allows us to easily tune the loop strength with AHL concentration. Experimentally, we could not find any advantage of applying the ICF over DNF. However, we prefer using the ICF design, since it has fewer regulatory elements and is predicted to achieve better performance in wide range of biological contexts.Here, we started with simple linear models, which allowed us to study the behavior of the ICF and DNF circuits when the strength of feedback/feedforward loop is varied. Based on these models, we verified that such designs could improve the FCA level of the circuit/part under test. Next, by replacing the linear operations with non-linear equations such as Hill-function and repression equations, we developed molecular models for the ICF and DNF circuits including three nodes. The molecular models showed that the ICF design has an advantage over the DNF design. While the DNF circuit showed an optimum in the FCA behavior, the FCA of ICF circuit monotonically increases with the loop strength. Finally, we developed genetic models for the ICF and DNF networks using four nodes. These genetic models show that for both designs an optimum can be achieved when the loop strength is varied . These sgfp gene. However, such promoters, known as combinatorial promoters . The first was transcriptional interference, where the reverse promoter is located directly downstream adjacent to the promoter under test and upstream of the reporter gene. The second is antisense transcription, where the reverse promoter is located downstream of the reporter gene. Although the antisense transcription involves possible interruptions between RNAP of forward promoter and RNAP of reverse promoter, here we assumed that the main mechanism for repression is the binding of antisense RNA to the sense mRNA to block translation. Although experimentally we found that both mechanisms have simple constructs and yield similar results in terms of FCA, sensitivity and cell growth (e.g. toxicity), we suggest using antisense transcription because it has been demonstrated to function in a wide range of biological contexts (e.g. cell types). Other biological mechanisms can also be used for implementing a subtraction, for example, by embedding the LuxR binding site inside the target promoter that drives the romoters , requireBADsyn and IPTG/PlacO, as a proof-of-principle. Then we examined native promoters that are either functionally specific or systemically involved in complex pathways in living cells such as oxidative stress and the SOS response. Improved FCA levels were achieved in all six promoters, with improvement varying from three to ten times. Furthermore, using the proposed methodology, we increased the sensitivity of the target promoters. The minimum detection limit for some of these promoters was increased. Notably, the open loop design for the PlacO and PkatG promoters, where the reverse promoter controls GFP only, showed an optimal behavior. This is because the reverse promoter can both reduce the activities of PlacO and PkatG and increase their switching thresholds, in contrast to other promoters where the reverse promoter acts as a pure subtraction and reduce the expression level only. In addition, the highest FCA level of the recA promoter was obtained using the open loop design due to the sensitivity of the PtetO promoter to Nalidixic Acid.In this study, we successfully applied the ICF and DNF designs to six different promoters starting with synthetic promoters, such as arabinose/P1) and mixed it with a strain containing an ICF circuit (N2) in different ratios  by increasing the FCA, i.e. ON/OFF ratio (or the output dynamic range), and sensitivity has long remained an important goal of synthetic biology. For example, it has been shown that FCA can be increased by tuning the ribosome-binding sequences  or fusinThe Flow Cytometry experiments generated during the current study are available at the FlowRepository with Repository ID: FR-FCM-Z3DB.gkab253_Supplemental_FileClick here for additional data file.


Genes spanning long chromosomal domains are coordinately regulated in human genome, which contribute to global gene dysregulation and carcinogenesis in cancer. It has been noticed that epigenetic modification and chromatin architecture may participate in the regulation process. However, the regulation patterns and functional elements of long-range gene regulation are unclear.Based on the clinical transcriptome data from different tumor sets, a novel expressional correlation analysis pipeline was performed to classify the co-regulated regions and subsets of intercorrelated regions. The GLAM2 program was used to predict conserved DNA elements that enriched in regions. Two conserved elements were selected to delete in Ishikawa and HeLa cells by CRISPR-Cas9. SAHA treatment and HDAC knockdown were used to change the histone acetylation status. Using qPCR, MTT, and scratch healing assay, we evaluate the effect on gene expression and cancer cell phenotype. By DNA pull-down and ChIP, the element-binding proteins were testified. 3C and 3D-FISH were performed to depict the alteration in chromatin architecture.In multiple cancer genomes, we classified subsets of coordinately regulated regions (sub-CRRs) that possibly shared the same regulatory mechanisms and exhibited similar expression patterns. A new conserved DNA element (CRE30) was enriched in sub-CRRs and associated with cancer patient survival. CRE30 could restrict gene regulation in sub-CRRs and affect cancer cell phenotypes. DNA pull-down showed that multiple proteins including CTCF were recruited on the CRE30 locus, and ChIP assay confirmed the CTCF-binding signals. Subsequent results uncovered that as an essential element, CRE30 maintained chromatin loops and mediated a compact chromatin architecture. Moreover, we found that blocking global histone deacetylation induced chromatin loop disruption and CTCF dropping in the region containing CRE30, linked to promoted gene regulation. Additionally, similar effects were observed with CRE30 deletion in another locus of chromosome 8.Our research clarified a new functional element that recruits CTCF and collaborates with histone deacetylation to maintain high-order chromatin organizations, linking to long-range gene regulation in cancer genomes. The findings highlight a close relationship among conserved DNA element, epigenetic modification, and chromatin architecture in long-range gene regulation process. In eukaryotic genomes, the co-expression patterns of genes within long chromosomal domains have long been noted , 2. ThesThe regulatory mechanisms of the coordinated gene expression have been explored at both genetic and epigenetic levels. One potential mechanism is the sharing of regulatory elements among genes in these regions, similar to divergent co-expression gene pairs  and paraIn the cancer genome, the connection between long-range gene dysregulation and DNA copy number variants was established very well , but a lIn this study, using a new analysis pipeline, we explored coordinately regulated regions (CRRs) in eight different tumor sets and classified a subset of CRRs (sub-CRRs) that exhibited a high interregional correlation. We calculated the reoccurred DNA elements in sub-CRRs; one novel DNA element was found to be enriched in sub-CRRs of endometrial cancer and was associated with cancer patient survival. The element was further determined to affect regional gene regulation in sub-CRRs. We clarified that the element contributes to the maintaining of chromatin loops and is associated with regional gene regulation restriction, possibly through CTCF recruiting. We also found that histone acetylation destroyed chromatin loops and disrupted CTCF binding, which was linked to alteration of long-range gene expression in sub-CRRs in cancer genomes. The expression matrix of genes and the clinical information were collected from The Cancer Genome Atlas Program (TCGA) database; the hg19 UCSC Gene standard track was used for gene location annotation. In each tumor set, every five consecutive genes on the chromosomes were defined as a unit, and the average of the Pearson correlation coefficient of every two genes in a unit was defined as this unit’s average correlation coefficient.p value was less than 0.05. All CRRs with adjacent start positions were merged into one CRR. This definition was more strict than those in previous studies  to it. These counts showed a bimodal distribution . We used GLAM2 . Primers are listed in CRISPR-Cas9 was performed as described by Ran et al.  with somCells were trypsinized and seeded into 12-well plates with medium containing 10% FBS  or 0.5% FBS (serum-starvation group). After 12 h of incubation, cells were collected. For SAHA treatment, fresh medium was changed with 30 μM SAHA or DMSO and incubated for 24 h before isolation. For SAHA treatment followed by serum starvation, SAHA was added into medium 12 h before starvation; the drug concentration was then maintained for another 12 h during the starvation process. siRNA oligos are listed in Total RNA was extracted with TRIzol  and reverse transcript using the PrimeScript RT-PCR Kit . Real-time PCR was performed by the FastStart Universal SYBR Green Master  on the Real-Time PCR System . Primers for real-time PCR are described in -2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded into 96-well plates; a 12-h culture was proceeded before experiments. MTT was added, and absorbance at 550 nm was measured in time points of 0, 24, 48, and 72 h. At least 6 biological replicates were set for each experiment. For the scratch wound healing assay, cell scratch healing assay was performed as described previously . A 3-mm DNA pull-down was based on ref . Biotin-ChIP assay was performed according to a previous description  with som3C-qPCR experiments were performed according to the Nature Protocol  with somCustomization FISH probes and a hybridization buffer were purchased from Empire Genomics LLC. (USA); a probe mapped to the upstream boundary of sub-CRR95 was labeled with 5-fluorescein and the downstream boundary probe labeled with 5-ROX. The experiments were performed according to the protocol of the manufacturer. Cells were seeded onto a round coverslip; after 24 h of culturing, cells were fixed with 4% PFA, permeabilized with 0.5% Triton X-100, then immerged in 20% glycerine; 3× freeze/thawing cycles were performed in liquid nitrogen. Coverslips were incubated with 0.1 M HCl and then 0.5% Triton X-100 with twice PBS washes before and after each step. Before hybridization, coverslips were immerged in 50% formamide/2xSSC for at least 30 min, then incubated in each of 2xSSC and 70%, 85%, and 100% ethanol sequentially. Probe solutions  were added then sealed with rubber cement. Slides were heated at 74°C for 4 min and incubated at 37°C for 24 h. Coverslips were washed in WS buffer1  at 73°C for 2 min then transferred into WS buffer2  for 1 min at room temperature. Coverslips were dried in the dark and sealed with anti-fade regent containing DAPI . Images of 3D-FISH were captured using a fluorescence confocal microscope , then the 3D distance between red and green loci was measured by ZEN software. 90–120 loci were measured for each sample. Methods) was used to calculate the expression correlation of adjacent genes in genomes; the coordinately regulated regions (CRRs) were identified as regions in which the absolute value of the Pearson correlation coefficient between any pair of genes was greater than 0.4 (adjusted p < 0.05). The characteristics of CRRs in all eight tumor datasets are shown in To explore the regulation patterns and functional elements of long-range gene regulation in cancer genomes, we first assessed coordinate expression patterns in eight cancers from the TCGA database Table 1.Methods), and these suggested a randomly occurred correlation and were excluded from subsequent analysis. The right-side peak consisted of CRRs that have more correlated partners than random. This result shows that the correlations between some CRRs were significantly higher (p < 2e-16) than those in random cases. These CRRs were defined as sub-CRRs and were obtained using a clustering method based on the Gaussian mixture model  with it, and the distributions were represented by density curves (We further observed that many genes in sub-CRRs were closely related to cancer characteristics. Gene Ontology (GO) analysis of genes contained in sub-CRRs revealed functions related to the cell cycle and proliferation . Seventy conserved DNA sequences with a high frequency in sub-CRRs were obtained in endometrial cancer. We found that the No. 30 DNA sequence was significantly enriched in the sub-CRRs compared with whole genome , and we named this sequence as core regulation element 30 (CRE30). CRE30 contains mostly G and C bases and exhibits an extraordinarily high GC content  and PCR (right boundary)  on CRE30in situ hybridization (3D-FISH) to examine whether loss of CTCF binding that is induced by CRE30 deletion could affect the chromatin architecture around sub-CRR95. Green and red fluorescence probes were designed on the two boundaries of sub-CRR95 , we performed chromosome conformation capture (3C) experiments. A bait primer was designed in the Hind Ш restrict fragment which contains the CRE30 locus, and restriction fragments that cover CTCF-binding peaks were designed as the detection targets , 39, we Together, these results indicated that through recruiting CTCF and maintaining chromatin loops in sub-CRR95, CRE30 acts as an essential element for holding compact chromatin architecture. CRE30 deletion led to loop disruption and chromatin decondensation, which further linked to changes in gene regulation patterns within the region. We further wondered if CRE30-mediated chromatin loops were modulated in cancer genomes. Previous studies suggested that direct epigenetic modifications, such as histone acetylation on CTCF-binding sites and chromatin loop boundaries, impact the maintenance of chromatin loops –43. We uTo assess the effects of histone acetylation on chromatin architecture, we performed 3D-FISH experiments on Ishikawa and HeLa cells, and the results showed a significant increase of the 3D distance between sub-CRR95 boundaries after SAHA treatment compared with controls  for each gene among different samples. Genes were divided into four groups: genes in sub-CRRs with CRE30, genes in sub-CRRs without CRE30, genes in CRRs (sub-CRR genes excluded), and genes in the whole genome (CRR genes excluded) 6K left,Based on above findings, we developed a long-range gene regulation model based on the functional DNA element in cancer genomes Figure 7Research on mechanisms for epigenetic remodeling including chromatin architecture and direct epigenetic modifications has confirmed their regulatory roles on long-range gene expression in specific regions of the cancer genome , 8, 28. Co-regulated regions have been reported in some different cancer genomes identified by different calculating methods , 8, 9. IIn this study, the conserved DNA element CRE30 was found to recruit CTCF in different sub-CRR loci. The relationship between CTCF binding and chromatin architecture–induced gene regulation has been well established , 44. To Previous studies suggested a weak influence of high-order chromatin structure on gene regulation in some loci in the absence of stress condition , 47. IbrLong-range epigenetic remolding was found to associated with high-order chromatin alteration and gene regulation , 48. RecIn our study, cancer case classification using gene expression diversity in sub-CRRs showed significant differences in patient survival time, which indicated that the regions that we identified might have practical values for clinical pathological feature prediction. Similar characteristics were also reported in co-expression regions in other studies , 9. MorePrevious studies showed that the histone deacetylase inhibitor leads to epigenetic remodeling and chromatin architecture alteration of genomes . SAHA, aIn conclusion, our research developed a novel expressional correlation analysis pipeline to classified groups of co-regulation regions (defined as sub-CRRs here) exhibiting similar regulation patterns. A conserved DNA element (CRE30) was identified to enrich in sub-CRRs and proved to affect gene regulation and cancer cell proliferation and migration. CRE30 acts as an essential element to recruit CTCF and maintain chromatin loops, which restricts gene regulation in compact chromatin regions. Meanwhile, the regulatory restriction is relieved when histone acetylation disrupts CTCF binding and chromatin loops. Our results provide new findings into the collaboration among conserved DNA elements, epigenetic modification, and chromatin architecture on the long-range gene regulation process.The original contributions presented in the study are included in the article/YuL and RT designed the research. RT performed the experiments. YiL and XL analyzed the data. FH analyzed the data and drew the figures and tables. ZL performed experiments on sub-CRR96. HS and XZ performed some 3D FISH experiments and supplementary data. HN and QJ performed the writing and reviewing of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the project from the National Natural Science Foundation of China , Heilongjiang Touyan Team (HITTY-20190034), and Heilongjiang Postdoctoral Science Foundation funded project (LBH-Z20064). 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.


Escherichia coli RpoE, which is the best characterized ECF and whose structural and functional studies have provided key insights into the transcription initiation by ECF-RNAP holoenzymes, and the ECF15-type EcfG, the master regulator of the general stress response in Alphaproteobacteria.The ability of bacterial core RNA polymerase (RNAP) to interact with different σ factors, thereby forming a variety of holoenzymes with different specificities, represents a powerful tool to coordinately reprogram gene expression. Extracytoplasmic function σ factors (ECFs), which are the largest and most diverse family of alternative σ factors, frequently participate in stress responses. The classification of ECFs in 157 different groups according to their phylogenetic relationships and genomic context has revealed their diversity. Here, we have clustered 55 ECF groups with experimentally studied representatives into two broad classes of stress responses. The remaining 102 groups still lack any mechanistic or functional insight, representing a myriad of systems yet to explore. In this work, we review the main features of ECFs and discuss the different mechanisms controlling their production and activity, and how they lead to a functional stress response. Finally, we focus in more detail on two well-characterized ECFs, for which the mechanisms to detect and respond to stress are complex and completely different:  Bacteria, like all other living organisms, have adapted and evolved to inhabit a specific environment, tuning their physiology and metabolism to the pervading conditions provided by their habitat. However, bacteria also need to be prepared to face constant changes in their environment. These fluctuations may lead to important deviations from the optimal growth conditions for a species, threatening bacterial survival, and are known as stress conditions. Stress may be generated by environmental changes and by the biochemical processes performed in the cell, although it may also appear as nutrient starvation or interactions with a host. Furthermore, a stress-inducing condition is subjective and specific for each species, so that different bacterial representatives may differ in their response to the same stimulus.Eukarya [When the conditions shift to a less favorable situation, bacteria fight for survival by activating a number of mechanisms and regulatory circuits whose main effect is the mitigation, or removal, of the stress. The gene products synthesized to overcome stress conditions are variable depending on the stress. For example, temperature shifts may lead to the production of chaperones that aid in protein folding and proteases to remove misfolded proteins . These sEukarya . This ocEukarya  and cont70 factor, which ensure the bulk of the transcriptional control of house-keeping genes, alternative σ factors control the expression of a number of specific genes (regulons) with a defined collective function, which may go beyond the stress response. In the early 1990s, certain representatives of this group caught the attention of molecular microbiologists  holoenzyme essential for transcription initiation, due to their role in guiding the catalytic core of the RNAP in promoter recognition. Moreover, their specific helicase activity on the -10 box facilitates the formation of the transcription bubble through formation of the open complex. Apart from the vegetative σiewed in ) becauseiewed in .Since their discovery, much effort has been focused on unraveling all biological aspects of ECFs. These include their role in bacterial physiology, their evolution and classification, the regulation of their production and activity, the stimuli that activate them and their target genes, their structural features and their transcription initiation mechanisms. Our knowledge on ECFs is continually growing, and excellent recent reviews have covered the findings of this broad ranging research field. Nevertheless, it is of interest for the scientific community to place our knowledge on ECFs into a broader context by discussing the various aspects of their biology, thus regarding their overall role in cellular physiology from a holistic viewpoint. For this reason, in this review, we comprehensively cover the most prominent aspects of the biology of ECFs, such as their role in the response to stress, how they are regulated, their classification, their mechanism of action and the future perspectives we consider to be important in future research on these regulators.70 family, and the other much smaller group includes the RpoN family , [70 family members melt the DNA near the TSS in an ATP-independent manner and without the aid of additional regulatory proteins [2 domain is able to melt the DNA at the -10 box once the RNAP holoenzyme is correctly positioned thanks to the DNA-binding activity of σ4 and σ3 domains to other promoter regions, such as the -35 box and the extended -10 region, respectively [1.1), form the basis of the typical structure feature of the primary σ70 factor, ascribed to Group I  except for the absence of the region σ1.1 [70 homologs. Members of Group III, such as E. coli RpoH and FliA, essentially conserve domains σ2, σ3 and σ4 [3 and σ4 bind different promoter regions, they are considered redundant, since the RNAP holoenzyme can bind and orientate efficiently in the absence of one of these domains or one of their DNA-binding determinants [2 and σ4 separated by a linker shorter than 50 residues. Actually, “Group IV” would be a more appropriate term to describe these σ factors, since it is meanwhile clear that some ECFs respond to intracellular stimuli [When considered from a structural point of view, σ factors can be divided in two main, structurally unrelated families: one large group includes the primary σ or σN), ,14. The proteins . The σ2 ectively . These s Group I . σ factoion σ1.1 , and the3 and σ4 . Althougrminants . ECFs co stimuli .70 family members has relied on the presence of various well-conserved protein domains and subdomains  is composed of several domains and subdomains [1.1, σ2 , σ3 , σ3.2 and σ4  . The σNCR (non-conserved region) subdomain is an adjacent region to the core σ2 domain whose interaction with the core RNAP, in a manner which is antagonistic to the core RNAP–σ2 interaction, facilitates promoter escape allowing the first stages of elongation to occur [3.2 is a conserved linker between the σ3 and σ4 regions that inserts into the RNAP active site and occupies the RNA exit channel. Part of this region, the σ finger, interacts with the DNA template strand pre-organizing it into a helical conformation for accommodating it into the RNAP active site, thus enabling the binding of initiating NTPs to the template strand. For promoter escape and transcription elongation to occur, the DNA–σ3.2 interactions must be disrupted and the σ finger has to be displaced from the RNA exit channel, which may explain from a mechanistic perspective the close relationship between this region and abortive transcription events [Taken as a paradigmatic example, bdomains ,17,38,39.1, 4.2) . Once thactivity a. Upon p complex . Separatlex [1.2 ,41, consto occur . σ3.2 isn events ,29,43,442 and σ4 DNA-binding domains and a non-conserved σ2–σ4 linker of less than 50 residues, which is highly divergent in length and sequence among ECFs. Most of the knowledge about the mechanisms of transcription initiation by ECFs comes from the well-studied E. coli RpoE (σE). Crystal structures of RpoE σ4 complexed with the −35 element [2 complexed with the −10 element [As mentioned above, Group IV σ factors consist of only the σ element , σ2 comp element  and the  element ,37 have 70 factors, divergent mechanisms for the recognition of the conserved -35 sequence motif have also been described [4 domain and the -35 box, for example, using the Thermus aquaticus σ70 factor as a model for structural studies [4 domain of the ECFs RpoE (E. coli) and SigW (B. subtilis), with target -35 boxes GGAACTT and ATTGAAACCTTT, respectively, numerous additional interactions also occurred, except with the central short A-tract [4 domain.Apart from the differences in the interaction with the -10 box between ECFs and primary σescribed ,47. In G studies . When de A-tract ,47. InteMycobacterium tuberculosis σH, σL and chimeric σH/E factors revealed that the σ2-σ4 linker, similarly to the aforementioned σ finger in the primary σ70 factor [3.2 region of the σ70 factor is also suggested by analysis of mutant versions of this region in different ECFs, which show, to different extents, an inability to interact with the core RNAP and isomerize the holoenzyme–DNA closed complex to an open complex. This also affects transcription initiation and the production of abortive transcription complexes (thus indicating a role in promoter escape). However, unlike the σ3.2 region of the primary σ70 factor, the amino acid sequence of the σ2–σ4 linker is not conserved. Rather, ECFs share a similar secondary structure at the ends of this linker, which may indicate, together with the results obtained from structural studies, that these regions of the linker are important for its activity within the RNAP active site and the exit channel [The recent structural studies of transcription initiation complexes using the 0 factor , inserts0 factor ,36,37. B channel ,36,37.70 factor drives the majority of bacterial transcription, alternative σ factors, including ECFs, target small groups of operons. This implies that σ70 must have a broader promoter specificity, whereas alternative σ factors must stringently promote the transcription of their regulon alone [70 factors melt the promoter mainly at the AT-rich -10 sequence of the non-template strand through a set of invariant aromatic residues involving regions σ2.3–σ2.4, opening promoters by flipping out two bases, A-11 and T-7, at the -10 box in E. coli [1.2 is also able to flip out the G-6, thus increasing the melting capacity of the primary σ70 factor [2 domain that interact with each of the nucleotides in the -10 box and the discriminator element are detailed in 70 holoenzyme to induce the DNA melting from three different positions allows a certain divergence in these nucleotides with respect to the consensus A-11, T-7 and G-6. This ensures that, even if a mismatch in any of those positions has a lower potential to be unstacked and buried by the σ70 factor, the open complex can still be formed thanks to the ability to flip out the other nucleotides. A distinctive feature of Group IV σ factors is that the regions σ2.3–σ2.4 are highly divergent from primary σ factors, with region σ2.3 lacking crucial melting residues [2.3 (loop L3), which is essential for specific recognition of only one flipped-out nucleotide at the −10 element . This is due to the presence of a pocket in which the flipped-out nucleotide is accommodated, where specific interactions are stablished with all the functional groups of the respective nucleotide [E. coli RpoE requires a cytosine in the -10 position . Later on, with the analysis of nine newly sequenced Planctomycetes genomes [2 and σ4 domains separated by a linker of less than 50 residues; (iii) they are co-transcribed with a cognate anti-σ factor; and iv) they are positively autoregulated  [2 and σ4 domains as promoter-binding determinants and key contact regions with the core RNAP. These contacts are mainly those of the surface between the σ2.2 region and the β’ subunit, but also those of the surface between the σ4 domain and the β subunit with a weaker intensity [Although ECFs show sufficient sequence and structural similarity to be clustered as homologs in Group IV, their diversity is sufficient to allow their classification into distinct sub-groups based on functional similarities, and this facilitates their further study. The first attempts at their formal classification was published in 2009 by Anna Staroń and collaborators . At that genomes  and more genomes , the cla genomes . At thiske ECFs) . After tntensity ,54,55. Bntensity .The advent of bacterial genome sequencing and annotation projects has led to a great increase in the number of available sequences in the databases, which has helped mitigate the former bias toward highly represented phyla. In its latest version , the claDespite the classification being based on sequence similarity and genomic context of ECFs, nevertheless, due to their degree of conservation, target promoter motifs and potential functions can be inferred for each distinct group with a high level of accuracy based on the few experimentally studied representatives. Even in groups with no experimentally analyzed representatives, regulatory mechanisms and target motifs may be predicted based on the genomic context and the amino acid composition of the DNA-binding domains compared to other ECFs . RegardiFrom the perspective of the regulation of their activity, the functional control of ECFs can be grouped into five general mechanisms, although each type of ECF may present distinctive regulatory features . These iE. coli and B. subtilis, respectively [Anti-σ factors are usually encoded by genes located in the vicinity of the gene encoding the target ECF, or even within the same transcriptional unit. They prevent the ECF from functioning by titration, while it is not needed. To release ECFs from inhibition, anti-σ factors may undergo proteolysis, the most frequent mechanism for membrane-bound anti-σ factors , or theyectively ,63, whicectively .S. melonis and Cupriavidus metallidurans, respectively [Streptomyces venezuelae [4 domain to the -35 box and by sequestering BldN to the membrane, as the anti-σ is a membrane-anchored protein. By comparing the structure of ASDIs and ASDIIs, RsbN served to define the ASDIII anti-σ factor class for the first time.Within ASDIIs, structural studies have reported that in the EcfG-NepR and CnrH-CnrY pairs from ectively ,64, the nezuelae  shows thBacteria compared to their major regulatory roles in Eukarya. However, the presence of coding genes for STPKs in bacterial genomes revealed through the numerous genome annotation projects developed in recent years has attracted the attention of microbiologists. In particular, ECFs classified into groups ECF43, ECF59 and ECF60  is essential for the activation of its cognate ECF (EcfK), which regulates the Type VI Secretion System of this strain [Vibrio parahaemolyticus [2 domain of σ factors and the positively charged helices located in the β’-clamp of the core RNAP is essential for the formation of the holoenzyme [STPKs were initially thought to have a minor role in signal transduction in ent ones ) are encs strain . Recent olyticus , which cloenzyme ,67. In Sloenzyme .2 and σ4 alone, which together minimally define a σ factor. The gene encoding some of these ECFs does not appear near any gene encoding a putative anti-σ factor or accessory regulatory protein that might control function. Instead, they exhibit extensions in their open reading frames, mainly at their C-terminus, but some also have an N-terminal extension . These etail in ,59. Thesrculosis ; (ii) thmpestris ,70; and mpestris , in part members . However members .Planctomycetes genomes [Finally, ECFs with N-terminal extensions have been recently discovered after the analysis of more than one hundred  genomes  and clas genomes . In the  genomes .Pantoea stewartii subsp. stewartia [hrpS. This regulator is able to fully activate the transcription of the ECF HrpL coding gene, which eventually drives the transcription of the hrp and wts virulence factors.In the case of orphan ECFs, with no regulatory partner associated (either anti-σ factor or regulatory extension), the control of their transcription, frequently by TCSs, may stand as the only regulatory mechanism controlling their function . Such istewartia . In thisSphingopyxis granuli TFA [Expression of most of the ECF-containing operons is induced in response to a specific signal. In most instances, with a few exceptions such as the FeclR-like systems and some others , they arnuli TFA . Here, uSinorhizobium meliloti, the ECF RpoE2 is regulated by the anti-σ factors RsiA1 and RsiA2, with RsiA2 being encoded in the same operon as RpoE2 and RsiA1 being encoded at a different position on the chromosome [Rhizobium etli, there are two EcfG σ factors that control its general stress response, with ecfG1 located on the chromosome and ecfG2 located on a plasmid [In spite of these basic regulatory mechanisms that govern the activity of most of the ECFs, there are still cases in which a possible regulatory mode is not clear or cannot be easily predicted. Traditionally, the regulatory partner of an ECF has been its cognate anti-σ factor, which is usually encoded in the same locus. This is also true for STPK- and many TCS-regulated ECFs, according to analysis of conservation of the genomic context used for their classification. However, some ECFs are encoded by a monocistronic operon with no context conservation, or if the genomic context is conserved, unknown elements or elements from which a possible regulatory activity cannot be inferred easily, are found in the vicinity. In the case of ECFs with regulatory extensions, even if they are encoded by isolated genes, a regulatory mechanism can still be predicted. Nevertheless, questions remain about the regulation of ECFs encoded without an obvious cognate anti-σ factor or another putative regulator nearby. There is the possibility that a cognate anti-σ factor actually exists, although it would have to be encoded elsewhere in the genome. For instance, in romosome . Further plasmid . However plasmid ,77,78, t plasmid , may repE. coli homolog of RpoE, since it has been profoundly characterized at the molecular level with regard to its response to cell-envelope stress. Additionally, we will dedicate a section to the ECF15-type EcfG, the master regulator of the GSR in Alphaproteobacteria, an ECF that activates a global response whose activity is regulated by a unique partner-switching mechanism.Since their discovery, a wide variety of ECFs has been experimentally studied without even knowing that they might be evolutionarily related. In this section, we will summarize the main characteristics of two important examples of ECFs involved in stress responses, and whose mechanisms regulating their function are complex and differ dramatically. Firstly, we will focus on the ECF02-type RpoE, one of the first representatives of these proteins to be discovered that is regulated by regulated proteolysis of its membrane-anchored anti-σ factor RseA. We will mainly discuss the role of the E. coli due to its ability to initiate transcription from the third promoter of rpoH (rpoH P3) at lethal temperatures (50 °C) [H factor, responsible for the transcription of heat-shock response genes, encoding products consisting primarily of chaperones and proteases that bind to cytoplasmic proteins and assist them in folding. Apart from rpoH, the second gene known to be transcribed by RpoE was htrA . htrA encodes a 51 kDa precursor protein that is processed to the 48 kDa periplasmic endopeptidase DegP, required for growth and survival at high temperatures (>42 °C) [RpoE was initially identified in  (50 °C) . Transcr(>42 °C) ,80. At t(>42 °C) . In 1993(>42 °C)  found th(>42 °C) ,83.E. coli, in contrast to other ECFs.In general, any kind of environmental stress that causes accumulation of misfolded proteins in the periplasm, such as heat-shock, the presence of ethanol, pH changes, oxidative stress or osmotic stress, results in the activation of the RpoE pathway. In addition, RpoE activity is induced by entry into stationary phase  and partE. coli. Their targets not only include major porins, resulting in inhibition of synthesis of all major OMPs , but also non-OMP targets without envelope-related functions are regulated. MicA and RybB also converge in some messenger RNA (mRNA) targets such as lamB, ompA, ompW and tsx [phoP mRNA. The PhoPQ TCS regulates genes involved in Mg2+ transport, resistance to antimicrobial peptides, acid pH resistance, virulence and also LPS modification under certain stress conditions. Therefore, MicA links these cellular processes to RpoE [micL . MicL represses the translation of Lpp, an OM lipoprotein that forms covalent linkages between the OM and the peptidoglycan cell wall and is the most abundant protein produced by E. coli. However, Lpp has no role in OMP or LPS biogenesis itself, so it has been proposed that reducing the Lpp levels could be a way to help the biogenesis machinery, such as the Lol pathway, to deliver other lipoproteins that do have essential functions in OMP and LPS biosynthesis [Part of its regulon also includes regulatory small RNAs (sRNAs), such as MicA and RybB, through which RpoE downregulates all major OMP synthesis pathways and controls the remodeling of LPS composition . Moreove and tsx . They se and tsx . MicA in to RpoE ,92. Simi to RpoE . The miccL (slrA ) transcrynthesis . Neverthynthesis .rpoE transcription (rpoE P1 and rpoE P2), being rpoE P1 a distal promoter region of unknown regulation and rpoE P2 positively regulated by RpoE itself in response to OMPs misfolding and LPS defects [rpoE have clarified the whole rpoE promoter organization and identified a total of six promoters , -218, -153 and -75, respectively). This organization sustains the rpoE transcription in response to different signals by recruiting different σ factors, TCS response regulators and global regulators that function on individual promoters [rpoE P6 would correspond to the originally named rpoE P2, and the current rpoE P4 would approximately correspond to the original P1. This complex organization and regulation places RpoE as a pivotal regulatory element, where a variety of stresses may converge allowing a rapid response.Two promoters were initially reported to direct the  defects . Furtherromoters . Among trpoE P2 promoter shares the same transcription start site as rpoE P3 and is regulated by RpoN using QseF as an enhancer-binding protein. QseF is a response regulator of the QseEF TCS with homology to the NtrC family of transcriptional activators. The RpoD-dependent rpoE P3 promoter is noteworthy, as it is able to respond to specific LPS defects related to LPS biosynthesis, and this induction is dependent on the Rcs signaling pathway. The RcsBCDF phosphorelay senses damage at the LPS layer particularly in the RcsF OM lipoprotein and transmits it to the RcsB cytoplasmic response regulator, which then binds rpoE P3. The rpoE P2 promoter also responds to LPS defects but to a much lower extent than the rpoE P3 promoter. The rpoE P4 promoter behaves in vivo as a typical RpoS-dependent promoter, in the sense that the rpoE P4 promoter could be involved in inducing the RpoE pathway under stationary phase and general stress conditions [rpoE P6 responds to availability of free RpoE in a positive feedback loop as a result of its transcriptional and post-transcriptional activation (explained below).Briefly, according to , the rponditions . The autThe factors that regulate the activity of RpoE include the RseA anti-σ factor and RseB as negative regulators, and DegS and RseP, two inner-membrane proteases that together with the cytoplasmic proteases (the ClpXP complex), sequentially inactivate RseA by regulated intramembrane proteolysis (RIP). The circuit generated by these proteins provides information on the integrity/health of the cell envelope, ultimately determining RpoE activity, and many of the molecular details have been described ,96 Figu.RseA is an integral membrane protein and its N-terminal cytoplasmic domain functions as an anti-σ, titrating RpoE, keeping it in an inactive conformation. The co-crystal structure of this cytoplasmic domain complexed to RpoE has revealed that RseA inhibits RpoE activity by sterically blocking its association to the RNA polymerase, as explained above. Biochemical studies have shown that RpoE has higher affinity for the RseA cytoplasmic domain than to the core RNAP . Only thBacteria and Eukarya, which also negatively regulate their protease activity [rpoE operon, binds to the periplasmic domain of RseA. RseA cleavage by DegS requires binding disruption of RseB [DegS (site-1 protease), as a member of the HtrA family, harbors a catalytic serine protease domain and a periplasmic C-terminal PDZ domain. PDZ domains are protein modules that mediate specific protein–protein interactions and bind preferentially to the C-terminal 3–4 residues of the target protein. They are found in signaling proteins of activity . The peractivity ,99. RseB of RseB . The Rse of RseB . Therefo of RseB . The sub of RseB .It has been argued that phosphorylation of RseA could be another mechanism regulating its interaction with RpoE . In thatrpoH P3, as reporters, has shown that the RpoE activity is regulated in a growth phase-dependent manner, increasing its activity as the growth rate decreases . In this condition, ppGpp levels are known to increase. In vivo experiments using mutant strains that cannot produce ppGpp (relA and spoT mutants), a rseA mutant background, and combinations of these, have shown the requirement of ppGpp for this response, and that it is distinct from the stress signaling pathway controlled by RseA. In addition, further in vitro experiments have shown a positive regulation of some RpoE-dependent promoters by ppGpp and DksA in multi-round transcription assays from the rpoH P3 promoter, pointing out that this σ factor may respond to other signals apart from those indicating damage of the outer membrane or compromising the integrity of the cell envelope. Such regulation would serve to adjust RpoE levels and activity to meet cellular needs independently of RsaA [Another layer of control over the expression of RpoE is mediated by the alarmone guanosine 3,5-bispyrophosphate (ppGpp) and DksA under nutrient limitation . This en of RsaA .cis/trans isomerases , their enzymatic contribution and their substrates in E. coli, a strain lacking all six cytoplasmic PPIases (Δ6ppi), was constructed and analyzed. This cis/trans proline isomerization is often a rate-limiting step in protein folding, hence the importance of these folding catalysts in accelerating this reaction. A Δ6ppi mutant strain, among other pleiotropic phenotypes, is not viable when growing on rich medium, at a fast growth rate, and presents severe defects in protein folding. This has allowed the identification of proteins significantly affected by this catalysis, which suffered aggregation in the mutant. Pull-down experiments where four PPIases and their derivatives, either affected in their predicted active site or substrate-binding domains, were individually overexpressed in the Δ6ppi mutant and purified have identified RpoE and RseA as targets of the PPI FkpB, which implies another level of post-transcriptional regulation.As part of a recent study carried out by Gracjana Klein and collaborators , and whi6ppi strain led to the identification of the transcription factor DksA (among other factors). An overexpression of DksA in the Δ6ppi mutant background under non-permissive growth conditions is able restore growth to the strain. However, this phenotype seems to require the correct levels (wild-type levels) of GroL/S and RpoE. Intriguingly, purified DksA also has PPIase activity comparable to FkpB in vitro, which may justify its ability to restore the growth defects in the Δ6ppi background [Interestingly, using a multicopy suppressor approach to identify factors that are limiting in the Δckground .In Alphaproteobacteria, the Group IV σ factor EcfG, belonging to the ECF15 subgroup, plays a central role in the regulation of the so-called general stress response. This is a protective response elicited by a wide variety of unrelated stresses . It may In this pathway, three main elements are needed to transduce the signal and activate the response: the EcfG σ factor, which drives the transcription of the GSR regulon, its cognate NepR anti-σ factor, which prevents the EcfG function under non-adverse conditions, and the PhyR response regulator, which mimics EcfG structure, acting as an anti-anti-σ factor. Apart from this, stress-responsive histidine kinases and/or phosphatases regulate the activation of the GSR by modifying the phosphorylation state of PhyR  Figure .Methylobacterium extorquens [P). This phosphotransfer triggers a conformational change in PhyR through which an N-terminal σ-like domain is exposed. This domain, mimicking the true σ factor EcfG, is able to sequester NepR in a more stable complex, thereby releasing EcfG to activate its regulon. This mechanism is possible due to the differences in the interaction kinetics between the pairs NepR-EcfG, NepR-PhyR and NepR-PhyRP [Although the partner-switching regulation was initially identified in the methylotrophic bacterium torquens ,108,109,pR-PhyRP . Once thpR-PhyRP ,111.nepR and ecfG co-transcribed in the same operon and phyR transcribed divergently in another transcriptional unit. The expression of both transcriptional units is driven by GSR-dependent promoters, thus amplifying the GSR once it is activated. Additionally, a sensor or signal transducing histidine kinase (HK) coding gene (which would contain a characteristic HRXXN motif) is found in the vicinity. This type of HK specifically starts the phosphosignaling of the alphaproteobacterial GSR, and although one of them is usually encoded nearby the rest of the regulators, there might be other HRXXN HK-coding genes distributed throughout the genome [PhyR, NepR and EcfG are generally encoded at the same locus, with e genome ,112,113.e genome . Howevere genome ,112,114,e genome , thus fiThe presence of EcfG paralogs has been studied in a few organisms ,116,117,Rhizobium etli genome encodes two EcfG proteins , and although both σ factors share some target genes, they show a functional specialization controlling different sets of genes with distinct promoter sequences [ecfG coding gene presents different phenotypes in R. etli. For example, an ecfG1 mutant shows increased heat sensitivity, while deletion of ecfG2 accounts for reduced oxidative stress resistance [Caulobacter crescentus, two EcfG paralogs are present (SigT and SigU), where sigU is a target of the main regulator SigT and controls the expression of a small subset of genes belonging to the regulon of the SigT, indicating some kind of redundancy [M. extorquens, up to six of these σ factors, with unclear functional relationship, have been described, although one main regulator (EcfG1) has been identified [M. extorquens (490 genes) is transcribed by an additive contribution of different combinations of the six EcfG paralogs encoded in this species (EcfG1-6). In S. granuli TFA, with EcfG1 and EcfG2, differential functions between the two paralogs have been detected by in vitro transcription assays, showing that although they seem to share the same target genes, EcfG2 produces higher levels of transcription than EcfG1, regardless of the promoter, and seems more important to induce GSR [ecfG1 transcription is strongly induced by EcfG2 under stress conditions, reaching sufficient protein levels to activate target genes by itself. The relationships between the different EcfG paralogs regarding their contribution to the activation of the GSR regulon in the aforementioned species are represented in S. meliloti, which presents two PhyR (RsiB1/B2) and two NepR (RsiA1/A2) coding genes, with RsiA1 being essential [For instance, the equences . This orequences . The mutsistance . In Cauldundancy . In M. eentified . Additioduce GSR . Howeverssential . In thesAmong the mechanisms to respond to stress displayed by bacteria, ECFs have a significant representation, due to their diversity and abundance. Their simple, yet efficient, mode of action enables them to exert a quick and specific transcriptional regulation. Additionally, their characteristic functional regulation and target promoters seem to be conserved within each of the distinct ECF groups as is clear based on their classification, their phylogenetic relationships and genomic context conservation. Although not all ECFs respond to stress, a prominent proportion of them appear to be involved in protecting against damage coming from the inside or the outside of the cell. Some of these responses to a particular stress are narrow, involving a limited number of operons, but other responses are very broad , implying the induction of many genes presumably involved in coping with different stresses that may appear together in natural conditions. A well-characterized example of these multi-stress conditions is the stationary phase of growth, which is known to activate the GSR . EspeciaS. granuli, only 37% of the genes have an annotated function [The extensive use of transcriptomics and bioinformatics have allowed the definition of many ECF regulons related to stress responses. Among their target genes, many well-known elements that protect against specific stresses are found. A plethora of examples can be found in the literature, such as catalases and peroxiredoxins to protect against oxidative stress ,4, chapefunction ). These function , such asThe wide variability of the ECFs as a protein family is reflected in the recent expansion of their classification. On the base of updated protein and genome databases, the reclassification built by Casas-Pastor et al. accurately shows the variety of potential modes of regulation, target promoters and phylogenetic relationships of this group of proteins . FurtherRegarding their biological roles in bacterial gene regulation, putting the focus on the function of their target genes, we have attempted to ascribe a broad function to each of the ECF groups, based on the knowledge about experimentally studied representatives. Basically, we have differentiated between those ECFs involved in protecting the cell against internal or external threats and maintaining its integrity (45 groups) and those implicated in nutritional, metabolic and biosynthetic processes (five groups), with some groups comprising representatives that could fit in both of these classes (five groups). The classification has revealed not only that the way of regulation and the target operons within each group seems to be homogeneous, but also the importance of ECFs in the protection of the cell, since most of the documented ECF systems belong to the first class. Moreover, and most intriguing, the majority of the ECF groups defined in the latest classification do not have any experimentally studied representative (102 groups), and thus, their putative biological role is still unaddressed. This gives an idea of the vast universe of potential information  that is yet to be explored, and that will challenge molecular microbiologists in the future, even beyond the stress responses.M. tuberculosis, whose activity is controlled by two Cys residues that form a disulphide bond depending on the presence of oxidants [Conventional transcriptional regulators are, in general terms, DNA-binding proteins that control (positively or negatively) transcription after providing a certain conformation to the promoter region and/or facilitating or impeding the anchoring of the RNAP and the transcriptional initiation . Unlike oxidants . Neverthoxidants , and basoxidants .2 and the σ4 domains), which makes them, together with their target promoters, amenable to be more easily engineered. That race has already started and, for instance, different ECF-promoter partners from different bacterial species and belonging to distinct ECF groups within the classification have been optimized, even forming regulatory cascades, in heterologous systems for E. coli and B. subtilis [E. coli [2 and σ4 domains and their cognate -10 and -35 boxes. A similar rationale was followed to find highly orthogonal ECF-anti-σ pairs, and although specific pairs could be found, the cross-reactivity levels appeared higher than in the ECF-promoter pairs .2 and σ4 domains of ECFs to their most likely cognate nucleotides in the corresponding -10 and -35 boxes has allowed the de novo prediction of the target promoter for nearly 70% of ECFs. Promisingly, this opens the door to design and engineer ECF-promoter systems à la carte in the future.The current methods for a priori target promoter determination of ECFs is limited, since it is based on previously known ECF-promoter pairs and predictions on autoregulated ECFs, comprising only a part of the ECF protein group. Due to this, experimentally studying the ECF-promoter specificity requires high-throughput approaches such as that followed by Rhodius et al. . However


Bombyx mori ERR (BmERR) and confirmed the promoter activity of this region. Interestingly, we found that 10 and 50 nM 20-hydroxyecdysone (20E) up-regulated the transcriptional activity of BmERR promoter. In addition, eight putative ecdysone response elements (EcREs) were predicted in the upstream sequence of BmERR. Based on their positions, the upstream sequence of BmERR was truncated into different fragments. Finally, an EcRE-like sequence (5′-AGTGCAGTAAACTGT-3′) was identified. Electrophoretic mobility shift assay (EMSA) and cell transfection experiments confirmed that this motif specifically binds to the complex formed between ecdysone receptor (BmEcR) and the ultraspiracle (BmUSP), a key complex in the 20E signaling pathway. Interference of BmERR or BmEcR mRNA in the embryonic cells of Bombyx mori significantly affected the expression of BmEcR and BmUSP. Overall, these results suggested that an EcRE element was identified from BmERR, and this will help understanding the detailed regulatory mechanism of ERR in insects.Estrogen-related receptor (ERR) is an orphan nuclear receptor that was first discovered in animals, and play an important role in metabolism, development, and reproduction. Despite extensive research on the function of ERR, its transcriptional regulation mechanism remains unclear. In this study, we obtained the upstream region of  Nuclear receptors are a large family of eukaryotic transcription factors (TFs) with important functions such as regulation of ligand-mediated gene expression and signaling pathways . EstrogeIn mammals, there are three subtypes of ERRs, ERRα, ERRβ, and ERRγ, which play important roles in physiological and pathological functions . In partDrosophila, ERR controls energy metabolism by regulating genes expression in the glycolytic pathway. ERR-knockout in Drosophila strains resulted in glycogen accumulation, and the glycolysis process was blocked, leading to death of the flies  has become a widely used model insect for lepidopteran research, and a general model organism for life sciences, toxicology, and fungal infections (Bombyx mori ERR (BmERR) is involved in the growth and development of silkworm embryos and larvae by regulating the expression of glucose metabolism-related genes (via the 20-hydroxyecdysone (20E) -EcR pathway in silkworm (BmERR including the transcription start site (TSS) using 5′-rapid amplification of cDNA ends (5′RACE), analyzed its transcriptional activity and relationship with 20E, and explored the molecular mechanism of BmERR response to 20E.Since the completion of the genome sequence, the silkworm , BmEcR (GenBank: L35266), and BmUSP (GenBank: U06073.1) using the SYBR Premix Ex TaqTM  and an ABI StepOne v2.1 Sequence Detection System . The relative mRNA expression levels of target genes were calculated with the 2–ΔΔCT method and the silkworm translation initiation factor 4A  was used as an endogenous control. The primers used for PCR were listed in Quantitative real time-PCR was performed to evaluate the expression levels of BmERR 5′-UTR sequence and the transcription start site (TSS), RACE was employed to clone the 5′-untranslated region of BmERR. The universal primer mix (UPM) from the 5′-Full RACE Kit  was used as the forward primer, and the specific reverse primer BmERR-R (BmERR 5′-UTR sequence in our previous fat body transcriptome sequencing results (date not shown). The 5′-UTR was amplified by nested PCR using the synthesized cDNA as a template under the following program: 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 30 s. The PCR products were cloned into the pMD19-T simple clone vector  and then sequenced.To obtain the complete  BmERR-R  was desiBmERR were obtained by 5′RACE-PCR and transcriptome sequencing of silkworm fat body. The cis-acting regulatory elements (CREs) in the upstream sequences of BmERR were predicted using JASPAR.2The upstream sequences of BmERR promoter fragments were cloned from the silkworm genomic DNA with different primers  restriction sites.Using the high-fidelity DNA polymerase , different lengths of shown in  under thB. mori cells. Using the ligation free cloning kit , the open reading frame (ORF) of BmEcR and BmUSP were amplified from the cDNA and cloned into psl 1180-HR3-A4 SV40 vector restricted with BamHI and NotI in the manner of homologous recombination. The primers were given in The psl 1180-HR3-A4-DsRed-SV40  vector wB. mori embryonic cell line (BmE) was cultured in a 24-wells plate at 27°C in Grace  insect cell culture medium supplemented with 10% fetal bovine serum (FBS) . After 12 h, every well was transfected with a mixture of 1 μg recombinant plasmid, 0.1 μg internal control plasmid pRL-78ML  were dissolved in DMSO  at a stock concentration of 5 mg/mL and stored at −20°C. After 6 h of cell transfection, different concentrations of 20E were added to the 24-well cell culture plate, and the equal amount of DMSO was added as a control. The cells were harvested after 48 h of transfection and assayed with the Dual-Luciferase Reporter System .BmERR promoter were synthesized as probes for electrophoretic mobility shift assay (EMSA) . The single-stranded sequences were labeled with biotin at the 3′-end and annealed to produce a double-stranded probe. To evaluate interactions between the regulatory elements and prokaryotic expressed proteins BmEcR and BmUSP (Oligonucleotide sequences of four EcRE-like motifs 1/2/3/8 (EcRE-like 1/2/3/8) predicted at positions −1,325 to −1,311 (5′-AGTGCAGTAAACTGT-3′), −1,258 to −1,244 (5′-ACCTTTATTAAATT-3′), −1,199 to −1,185 (5′-TAGTGGTACTTTAGA-3′), and −28 to −14 (5′-GCGTTAGCTGATATT-3′) in the nd BmUSP , EMSA waBmERR, BmECR, and BmUSP. The 584- and 504-bp fragments of BmERR and BmECR were, respectively, selected to synthesize double-stranded RNA (dsRNA) (A double-stranded RNA interference (dsRNAi) approach was performed to evaluate the relationships among  (dsRNA) . The frat-tests, and *P < 0.05, **P < 0.01, and ***P < 0.001 were accepted as statistically significant.The results are presented as the mean ± SD from three independent experiments. Statistical analyses were performed using Microsoft excel . Differences between groups were analyzed with Student’s BmERR promoter, we analyzed the upstream region of BmERR from the silkworm genome database.3 The TSS and complete (5′-UTR) sequence were successfully identified through 5′-RACE PCR  20E. After 48 h, by 10 nM  and 50 nby 10 nM . In addincreased .BmERR promoter, and designated EcRE-like 1–8, respectively. We constructed six vectors for cell transfection with different lengths of BmERR promoter fragments as the position of these elements on the promoter. The effects of EcRE-like elements on promoter activity were evaluated by overexpression of BmEcR and BmUSP and Red fluorescent protein (RFP) was transfected as a control in BmE cells. The luciferase activity assay showed that BmERR promoter activity was significantly increased compared with that of the control when BmEcR and BmUSP proteins were overexpressed. However, compared with the control, when the promoter fragments containing EcRE-like 1/2/3 were removed, there was no significant change in BmERR promoter activity, indicating that these elements may play a positive regulatory role. After the promoter fragment containing the EcRE-like elements 4/5/6/7 were truncated, respectively, the promoter activity did not show significant difference under the case of overexpression of BmEcR and BmUSP. But compared with the control, the promoter still showed obviously higher transcriptional activity  into a basal vector pGL3- VgP78ML , which dBmERR, the relationship between BmERR, BmEcR, and BmUSP was evaluated by RNA interference in BmE cells. qRT-PCR showed that the expression of BmEcR was significantly decreased after the dsBmEcR fragment was transfected into BmE cells (BmERR and BmUSP were significantly reduced compared with the control (BmERR caused a significant reduction of BmERR expression (BmEcR and BmUSP were significantly increased (In order to further explore the mechanism of 20E regulating the transcription activity of mE cells , and the control . RNAi ofpression . Contrasncreased . These rDrosophila, and the expression of genes related to carbohydrate metabolism were also significantly reduced (BmERR promoter and confirmed that BmERR promoter can respond to 20E. EMSA showed BmEcR and BmUSP likely bind to the EcRE-like 1 motif on the BmERR promoter. After mutation of EcRE-like 1 motif, the basal promoter will not respond to 20E. This indicated that 20E up-regulated the transcriptional activity by activating the BmEcR and BmUSP complex to bind to the EcRE motif on the BmERR promoter.Twenty-hydroxyecdysone is an important hormone that regulates the growth, development, metabolism, and apoptosis of insects . High 20 reduced . Here, wBombyx mori estrogen-related receptor and BmEcR have functional cross-talking in the BmE cells. Decreasing the expression of BmEcR by dsRNAi reduced the mRNA levels of both BmERR and BmUSP, whereas the expression of BmEcR and BmUSP were increased when the expression of BmERR was down-regulated. This is similar to the research on other insects. In the Teleogryllus emma, ERR and EcR both affected the development of testes. The expression of TeEcR and TeERR are regulated by each other (ch other . In Drosch other . These iTwenty-hydroxyecdysone regulated the expression of glycolysis-related genes in the fat body of the silkworm through the ecdysone receptor EcR-USP . In AnthERR in Drosophila larvae, but there was no significant difference in S2 cells treated with 0.3 μM 20E (ERR was very sensitive to the concentration of 20E. Our research found that 10 and 50 nM 20E could up-regulate the activity of BmERR promoter. Although eight EcRE motifs were predicted on the 1,333 bp BmERR promoter region, there was only one EcRE motif response to 20E. In addition, the 1,333 bp BmERR promoter region had significant transcriptional activity, but it might not contain all hormone response elements completely. It was reported that the distal sequence of the promoter also contained the motifs which respond to 20E (BmERR was not only very sensitive to the dose of 20E, but also depended on length of the BmERR promoter region. In summary, our study analyzed how 20E regulate the expression of ERR and provided a perspective in the regulation of ERR expression in insects.So far, the research on ERR mainly focused on the function and the role in the 20E signaling pathway in insects. Previous report showed that 1 μM 20E inhibited the expression of 3 μM 20E . It showd to 20E . These rThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.JW and GS contributed to the conception and design of the study. JW performed the statistical analysis and wrote the first draft of the manuscript. All authors contributed to experiment, manuscript revision, read, and approved the submitted version.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.


Cancer cells accumulate epigenomic aberrations that contribute to cancer initiation and progression by altering both the genomic stability and the expression of genes. The awareness of such alterations could improve our understanding of cancer dynamics and the identification of new therapeutic strategies and biomarkers to refine tumor classification and treatment. Formalin fixation and paraffin embedding (FFPE) is the gold standard to preserve both tissue integrity and organization, and, in the last decades, a huge number of biological samples have been archived all over the world following this procedure. Recently, new chromatin immunoprecipitation (ChIP) techniques have been developed to allow the analysis of histone post-translational modifications (PTMs) and transcription factor (TF) distribution in FFPE tissues. The application of ChIP to genome-wide chromatin studies using real archival samples represents an unprecedented opportunity to conduct retrospective clinical studies thanks to the possibility of accessing large cohorts of samples and their associated diagnostic records. However, although recent attempts to standardize have been made, fixation and storage conditions of clinical specimens are still extremely variable and can affect the success of chromatin studies. The procedures introduced in the last few years dealt with this problem proponing successful strategies to obtain high-resolution ChIP profiles from FFPE archival samples. In this review, we compare the different FFPE-ChIP techniques, highlighting their strengths, limitations, common features, and peculiarities, as well as pitfalls and caveats related to ChIP studies in FFPE samples, in order to facilitate their application. Chromatin in eukaryotes is a finely organized nuclear complex of genomic DNA, histones, and non-histone proteins. The lowest level of chromatin organization is represented by nucleosomes, which consist of 147 base pairs of DNA wrapped ~1.7 turns around a histone octamer core. The octamer is composed of four heterodimers of the core histone proteins H2A, H2B, H3, and H4, while a fifth histone, histone H1, binds to internucleosomal DNA to stabilize higher-order structures. Chromatin function is mainly regulated by histone post-translational modifications (PTMs), which consist of enzyme-mediated chemical modifications of specific histone residues, among which those targeting the N-terminal tail of histones seem to play a major regulatory role. Different ATP-dependent chromatin remodelers are guided by these modifications to control the chromatin state and regulate the gene expression by making chromatin accessible or not to transcriptional regulatory complexes. Chromatin structure indeed has a crucial role in various processes including activating or repressing transcription to control functions such as the cell cycle, DNA damage repair, and cell fate .,29.22,29In this procedure, which we called pathology tissue chromatin immunoprecipitation (PAT-ChIP), chromatin isolation from FFPE samples is achieved by both physical extraction, using a probe sonicator at high amplitude, and controlled micrococcal nuclease (MNase) digestion. We demonstrated that FFPE-isolated chromatin can be immunoselected, applying the procedure to both genome-wide (coupled with NGS) and to locus-specific  studies using different tissues such as seminoma, breast cancer, and lung carcinomas ,30. In 2Notably, the last 2 years have seen the birth of three other procedures. In 2019, Zhong and colleagues proposed chromatin extraction from FFPE tissues (Chrom-EX PE), a technique that exploits heat (16 h at 65 °C) to extract high-quality chromatin through a focused ultrasonicator . UnfortuA further procedure, named RCRA ChIP-seq, was published a few months ago by Kaneko and colleagues  27]; ag; ag27]; A few months ago, a further technique that combines a novel fusion protein of hyperactive Tn5 transposase and protein A (pA−Tn5) transposition and T7 in vitro transcription to perform chromatin studies in FFPE tissues was proposed  24]. Th. Th24]. In the following sections, we aim to describe in detail and compare the different experimental strategies used to achieve chromatin profiling in FFPE samples, from the preliminary evaluation of the specimens to chromatin extraction and immunoselection, including the final quality tests that might precede the NGS sequencing.Tumor samples are commonly characterized by the presence of variable amounts of normal tissue that hinders the correct identification of the cellular population contributing to a specific phenomenon and decreases the reliability of final data. Thus, a histological analysis by hematoxylin and eosin staining is strongly recommended, while also considering its compatibility, already demonstrated, with chromatin extraction and immunoselection . When thAs already stated, genome-wide studies from FFPE archival samples may be hindered by the low efficiency of chromatin isolation, often due to extensive tissue fixation introduced during routine pathological processing. A solution containing 3.7–4% of formaldehyde (FA) is routinely used as fixative reagent with a length of fixation that is influenced by different factors  . DespiteIn addition, although preventing hydrolysis during prolonged storage minimizes protein degradation, FFPE tissues deteriorate over time . Thus, tGiven this evidence, it is suggested not to enroll, when possible, over-fixed samples  and samples stored for a long time to maximize the chances of extracting large quantities of high-quality chromatin.Before starting to analyze how the different techniques deal with chromatin extraction from FFPE specimens, some additional considerations must be taken into account. Independently from formalin fixation and paraffin embedding, the type of tissue is per se an important source of variation. Cellular density and structural features of each tissue may indeed impact chromatin extraction efficiency. Thus, it is strongly suggested to adjust sonication conditions in function of the tissue processed and to test different conditions before starting a new study.For example, chromatin fragmentation should be checked by running decrosslinked and purified DNA on an agarose gel or through automated capillary electrophoresis prior to immunoselection. Best results are obtained when most of the fragments range between 200 and 1000 bp, since lower-size fragments normally indicate high degradation of the sample , while higher-size fragments are normally lost during library preparation and could reduce the final resolution of the assay. Many of the FFPE-ChIP procedures herein described agree to evaluate the fragmentation of the extracted chromatin before proceeding with the immunoselection ,24,25,27The amount of starting material can also influence chromatin extraction by limiting the effect of sonication. Thus, chromatin should be extracted starting from equal amounts of starting material to reduce possible bias deriving from extraction-dependent chromatin selection.With the intent of deciphering which procedural parts related to chromatin extraction from FFPE are most relevant, we conducted a comparative analysis of the different procedures proposed in these years. The thickness of the sections processed varies from 5 to 20 µm, while the number of sections required is normally quite low, ranging from 1 to 4, with FiT-seq being the only one suggesting a higher number  or limonene-based agents (PAT-ChIP and EPAT-ChIP). Rehydration is also quite standardized, with minor variations in passages in decreasing alcohol concentrations, from 100% to 0% . The onlConcerning the issue of an extensive fixation of the tissue, introduced during routine pathological processing , the FFPHowever, while DNA digestion is quite easy to control through a simple run on an agarose gel, protein digestion may be “dangerous” considering that a protein is just the target of the assay. The failure in the application of the FiT-seq by our research group and the statement by the authors themselves that FiT-seq fails to work with H3K27ac may indicate the necessity of a tricky fine-tuning of the technique .The main step forward in chromatin extraction from FFPE tissues is probably the introduction of a heating step to partially revert the crosslinking of FFPE tissues. The first technique to develop such strategy was EPAT-ChIP in late 2018  24], fo, fo24], A final and important point to discuss regards the conditions of sonication. In addition to the different types and concentrations of detergents used to achieve extraction and solubilization of chromatin, the main distinction found is between the usage of probe sonicators or focused ultrasonicators. PAT-ChIP and EPAT-ChIP, together with the procedure described for the commercial kit ChIP-IT FFPE Chromatin Preparation II from Active Motif, are the only procedures that involve the use of a “canonical” probe sonicator  22,24].,24.22,24Once the objective of getting high amounts of good-quality chromatin is achieved, the criticisms related to chromatin immunoselection are largely comparable to those of standard ChIP techniques. Incubation conditions are influenced by the ratio between the amount of chromatin and antibody, the volume of incubation, the presence of detergents and salts, and the time and the temperature of incubation. All these aspects, together with the system used to capture the chromatin–antibody complexes and the washes used to eliminate the not specific binding of chromatin, must be carefully gauged. Each ChIP technique, regardless of the starting material, makes use of its own strategy to reach the final goal, which is to maximize the binding of the target epitopes, while reducing as much as possible the noise caused by unwanted nonspecific binding of chromatin.Regarding the chromatin required for each immunoselection (input), many of the techniques examined suggest to decrosslink, purify, and quantify the DNA from an aliquot of the extracted chromatin to precisely estimate its amount ,25,26. TInterestingly, FiTAc-seq makes use of a carrier-assisted ChIP method developed by other authors  that entAlthough the composition of the incubation mixture can vary a lot, almost all the procedures perform the incubation with the antibody for 16 h at +4 °C, with the intent of optimizing the interaction between the antibody and its target epitopes . HoweverChromatin–antibody complexes are normally captured using protein G and/or A beads that can be agarose or sepharose beads and magnetic or not magnetic . Column As for all ChIP protocols, the quality of the antibody is fundamental for the feasibility of the entire procedure. For this reason, it is strongly suggested to get, when available, ChIP- or ChIP-seq-grade antibodies. However, based on our direct experience, the single lots of antibodies may not be tested by suppliers and the observation of high lot-to-lot variation is not to be considered a remote event. Thus, the preliminary test of different lots of antibodies is fundamental to ensure a homogeneous experimental condition. In addition, coupling this test with the real-time qPCR analysis of regions known to be enriched or not enriched by the histone PTM under study can provide an idea of the enrichment that can be reached with a specific immunoglobulin (see next section for details). The best condition will be the one showing the higher level of specificity of the immunoselection , while getting sufficient final DNA to produce a high-quality library . Details on the strategies to evaluate the specificity of the immunoselection are described in the next section.Once final DNA (bound) is obtained, it is strongly suggested to perform a few quality checks before proceeding with library preparation and sequencing. DNA is normally fluorometrically quantified, and the enrichment of total DNA, with respect to the input, can be estimated. DNA quantitation is important to ensure library preparation under controlled and comparable conditions, especially if several samples must be compared, limiting library and sequencing bias that can negatively impact the final data. As already stated before, some authors indicate that an evaluation of the specificity of the immunoselection should be performed prior to library preparation ,25. To rPrimer pairs for the analysis of histone PTMs such as H3K4me3, H3K27me3, H3K27ac, H3ac, H4ac, RNA polymerase II, and CTCF in human and murine models are already available in literature ,24,29,30To ensure control over the reproducibility of the assays performed at different times, a technical control of immunoselection might also be considered. For example, chromatin extracted from a cell line routinely used in the lab and already immunoselected with the same antibody can be used to this end. Lastly, negative controls, such as no antibody (mock) or an unrelated antibody , can also be added to get an idea of the nonspecific binding of chromatin. However, it should be remembered that both these controls have limitations that can give a wrong idea of the specificity of the immunoselection; thus, the evaluation of enrichment over background described above should be preferred.Recently, enzyme-tethering strategies, such as CUT&RUN  and CUT&A few months ago, a further technique exploiting an enzyme-tethering strategy for chromatin profiling in FFPE samples was published . This teBefore setting their procedure, the authors demonstrated that the standard CUT&Tag protocol is not suitable for FFPE samples, suggesting that fragmentation of DNA induced during crosslinking reversal of archival FFPE chromatin may be the cause of such failure. Thus, they thought to overcome this problem using pA-Tn5 loaded with an adapter containing the T7 promoter sequence. They hypothesized that, even if there are DNA breaks in the middle of DNA fragments, they could still decode the insertion sites of pA–Tn5 transposase by transferring broken DNA fragments into RNA molecules with IVT. The IVT RNA is then reverse-transcribed in cDNA, purified, and converted to double-stranded cDNA; then, the library is prepared using, again, Tn5 tagmentation .Through their work, the authors reached the objective of being the first to develop a technique based on enzyme-tethering strategies, which allows the epigenomic profiling of a low number of nuclei (about 4000) from FFPE tissues. Unfortunately, unlike the standard CUT&Tag procedure, the protocol is time-consuming  and is based on several steps requiring enzymatic activities that may increase the experimental variability, with the need of a complicated fine-tuning of the technique. As declared by the same authors, the technique must be adapted as a function of the histone modification investigated  . The FAChttp://www.roadmapepigenomics.org (accessed on 14 November 2021)) has generated detailed and accurate descriptions and classifications of the functional states of regulatory elements in human stem cells and primary ex vivo tissues [The chromatin regulatory landscape combines histone modifications, TF binding, and the function of genomic elements to determine the biological function of chromatin. The large-scale epigenomic studies from the Roadmap Epigenomics Mapping Consortium ( tissues ,60,61,62Chromatin is profoundly rearranged in cancer inducing the activation of oncogenes, as well as the inactivation of tumor-suppressor genes, which play a role in cancer progression and invasion. FFPE samples represent the gold standard for storage of pathology samples. After our first proposal of a ChIP technique applicable to FFPE tissues, several other procedures have been published in the last 5 years. Recent protocols share the introduction of a heat-mediated limited reversal of crosslinking that facilitates the subsequent extraction of chromatin by sonication, suggesting it as a useful way to overcome the problem related to FFPE sample fixation.However, in some cases, clinical biopsies may have very limited material, leading to significant sensitivity problems. Enzyme-tethering strategies working in situ to produce high-quality chromatin profiles are currently available for fresh/frozen cells and tissues. A first attempt to apply such techniques to FFPE archival samples was recently developed, although there is, in our opinion, considerable scope for improvement.Single-cell technology is another important frontier in chromatin studies. Tissues are complex mixtures of cells and, although strategies to enrich the content of a tissue component such as macro- and microdissection can be useful, the presence of several cellular components may normally impact the final resolution of the assay and gives an incomplete picture of the epigenomic cellular landscape. scRNAseq and scATACseq techniques are currently available, and a few ChIP techniques allowing single-cell analysis have also been introduced in recent years ,49. We b