MSTN gene regulation. This review discusses the MSTN gene promoter, focusing on its structure in several animal species, both vertebrate and invertebrate. We report the important binding sites considering their degree of phylogenetic conservation and roles they play in the promoter activity. Finally, we discuss recent studies focusing on MSTN gene regulation via promoter manipulation and the potential applications they have both in medicine and agriculture.Myostatin (MSTN) is one of the key factors regulating myogenesis. Because of its role as a negative regulator of muscle mass deposition, much interest has been given to its protein and, in recent years, several studies have analysed The online version of this article (10.1186/s40104-019-0338-5) contains supplementary material, which is available to authorized users. Mstn knockout leads to hyperplasia and hypertrophy of muscle fibers, resulting in a striking increase in skeletal muscle when compared to wildtype animals. This increased musculature is a result of the influence MSTN has on cell cycle control genes, in particular p21, leading myogenic progenitor cells to permanently withdraw from the cell cycle [Myostatin (MSTN) is a member of the TGF-β superfamily of growth and differentiation factors which acts as a negative regulator of skeletal muscle mass deposition . In miceMSTN are associated to the double muscled phenotype in several animals, such as the Belgian Blue and Piedmontese breeds of cattle, dogs and sheep [MSTN gene and presenting increased musculature was also identified [Noticeably, natural mutations in nd sheep –6, indicentified , which fin vivo. For instance, a DNA vaccine was shown to increase body weight and skeletal muscle mass, without altering blood biochemistry or causing apparent side effects [In addition to its role during skeletal muscle formation, MSTN also regulates the homeostasis of this tissue after birth. In fact, higher levels of MSTN protein are observed in the blood stream or muscle fibers of patients suffering from muscle loss or wasting processes, such as cachexia, muscular dystrophies and other muscle disorders –10. Impo effects . A neutr effects . Thus, tMSTN expression is also regulated at different levels. For instance, the microRNA miR-27b is able to attenuate MSTN expression in a posttranscriptional manner, supposedly through a putative recognition sequence in the 3′-untranslated region [Although most of the strategies to block or regulate MSTN activity have focused on its protein or receptor, it has already been reported that d region . This inGene promoters are specific DNA sequences where RNA polymerase and basal transcription factors bind to drive gene expression . PromoteMSTN gene promoter have shown that this regulatory element is conserved among animals and represents a potential target for the development of new strategies to modulate MSTN transcription. In this scenario, in the current review we will discuss the MSTN gene promoter structure and activity in different animal groups, as well as its conservation among them, in order to understand similarities and particularities. Lastly we will explore potential targeting strategies for medical and livestock production purposes.In the past years, analyses of the MSTN promoter activity in human was based on a 3.3-kb segment of the 5′ regulatory region [MSTN gene [The first study detailing the structure and mechanisms involved in controlling y region . This rey region . In factSTN gene , bindingSTN gene . Also ofSTN gene .MSTN gene expression in different contexts. In fact, the 5′ regulatory region was already shown to have higher activity in differentiated myotubes than in proliferating myoblasts [MSTN mRNA [MSTN promoter on gene expression in a myogenic context, which might be explored for medical applications in humans. Nonetheless, the majority of studies about the mechanisms controlling MSTN promoter activity were performed either in vitro or using model organisms, mainly the mouse, as will be discussed below.Deletion analyses of the human 5′ regulatory region revealed that it contains a proximal segment of 327 bp containing essential elements for basal promoter activity, as well as for the responsiveness to glucocorticoids, which were shown to induce promoter activity, and increase endogenous MSTN, in a dose-dependent manner . In addiyoblasts , as was STN mRNA  and endoSTN mRNA . This coin vitro, indicating that these elements are also essential for Mstn expression, as in humans. The first five E-boxes, contained in the proximal 1 kb, were shown to be sufficient for obtaining the highest promoter activity in vitro and E-box number 5 appears to be crucial and capable of inducing maximal promoter activity by itself, being preferentially bound by the MRFs MYOD and MYF5, which were able to cause a 6-fold and 2-fold increase in the promoter activity, respectively [Mstn promoter has been greatly studied under several conditions of human interest, as described below.The study of 2.5 kb of the murine 5′ regulatory region showed the presence of several binding sites, such as for MEF2, C/EBP and CCAAT among others  increases promoter activity most likely by an increase in cytoplasmic calcium [Mstn promoter [Mstn promoter [Mstn transcription during early myogenesis [In the myogenic context, several activators of the  calcium , which l calcium . Upon capromoter . A bindipromoter  and deleogenesis . Furtherogenesis . HoweverMstn promoter are also known. Mstn gene promoter is negatively regulated by the nuclear factor I/X (NFIX), which binds to specific sites and directly represses its activity in mouse myoblasts and in vivo [Mstn promoter activity in mouse myoblasts, being possibly involved in the regulation of fatty acid use in skeletal muscle [Mstn transcription by targeting its promoter.A few antagonists of  in vivo . By doinl muscle . These fin vitro [Mstn promoter. Curiously, these binding sites present complementary effects, since mutation of the first (− 100 bp) almost completely abolished basal promoter activity, but did not alter promoter responsiveness to C/EBP or GR (glucocorticoid receptor), while disruption of the second (− 50 bp) impaired promoter’s responsiveness to C/EBP while increasing basal activity [Mstn promoter activity in pluripotent C3H10T1/2 cells induced to differentiate into adipocytes [Glucocorticoids are known inducers of muscle atrophy and, in contrast to the observed for human and other animals (see below), the mouse promoter was only modestly induced by glucocorticoids in vitro . This inactivity . In anotipocytes , indicatMstn transcription is sensitive to glucocorticoids. When plasmids were transferred into the thigh muscle of mice treated with high doses of dexamethasone, there was an increase of 34% in Mstn promoter activity. This induction seems to be mediated by a GRE (glucocorticoid responsive element) motif, which, when mutated causes a 17% reduction of the promoter activity [Mstn promoter in different manners according to the species, cellular context and through different pathways, both indirect and direct.In vivo assays, however, indicate that activity . TogetheMstn promoter was also shown to be activated by FOXO1, which is a known regulator of genes involved in muscle atrophy, also increased during caloric restriction [The murine triction . Five putriction .Mstn promoter contains several regions responsive to hormones, it was suggested the alteration in promoter activity of hypothyroid rats induced by hormonal changes (in an indirect manner) might be involved in the muscle loss observed in such conditions.MSTN levels are also increased in hypothyroid rats . Since MMstn promoter was shown to be the involved in the development of sarcopenia, a muscle degenerative condition that accompanies hepatic cirrhosis [In vitro experiments showed that treatment of mouse myoblasts with ammonium acetate caused an increase in the binding of the nuclear factor kappa B – subunit 65 (NF-κB-p65) to the Mstn promoter, activating gene transcription. When NF-κB-p65 sites were deleted from the promoter region, as well as when NF-κB gene was silenced, this stimulation was abrogated [irrhosis . It was brogated .Mstn is expressed in adipose, cardiac and liver tissues [Mstn promoter activity is low in 3 T3-L1 mouse preadipocytes and after 4 days of differentiation, it was increased when cotransfected with expression constructs for the adipogenic transcription factor enhancer-binding protein-α (C/EBPα), proliferator-activated receptor-γ (PPARγ) and sterol regulatory element-binding protein 1c (SREBP-1c), indicating an upregulation of Mstn gene during conditions of adipose tissue growth. This indicates that Mstn promoter might have a role in the alterations observed in growth and metabolism of lean and fat tissues observed during obesity [Besides muscle,  tissues , 37, and obesity .Mstn promoter activity was shown to be activated by angiotensin II (ANGII), a key molecule in cardiac remodeling and hypertrophy [Mstn promoter, the increased activity induced by ANGII was abolished. This was also observed by the addition of MEF2 siRNA and a p38 MAP kinase inhibitor, suggesting that ANGII stimulation of Mstn promoter activity depends, at least in part, on p38 MAP kinase and MEF2, and may serve as a negative feedback mechanism to counteract the pathologic hypertrophy effects of ANGII [In rat cardiac myocytes, ertrophy . Howeverof ANGII .The study of 1.6 kb of the bovine upstream region showed it presents 79% of sequence identity with the human 5′ regulatory region and also bears many putative binding sites, including a CAAT site, three TATA boxes, besides a MEF2, which was shown to enhance promoter activity in vitro , 40  were already detected in the bovine 5′ regulatory region of several breeds, such as Belgian Blue, Piedmontese, Limousine, Marchigiana, Black-and-White bulls, Holstein, Hanwoo, Jeju Black Cattle and Qinchuan cattle , 42–45. MSTN gene revealed a high degree of conservation with human (84.4%), mouse (71.6%), bovine (83.7%), sheep (82.2%) and goat (82.5%) [The analysis of 1.2 kb of the 5′ regulatory region of the porcine  (82.5%) , 47. Des (82.5%) , indicat (82.5%) , 48. NonMstn gene [The alignment of these regions revealed the presence of two TATA boxes, which were conserved in all species, except in mouse. Based on mRNA sequences, it was possible to determine that the most distal TATA box (located between nucleotides − 71 and − 67 in pig) is responsible for transcription in pig and human, while the most proximal one is involved in the mouse transcription, resulting in a slightly smaller 5′ UTR in the rodent . Severalstn gene .MSTN 5′ regulatory region and presented high degree of conservation with the ones observed in the bovine promoter [Eleven E-boxes organized in clusters were identified in the porcine promoter  , an upregulation of C/EBPβ binding to the MSTN promoter region as well as an increase in MSTN expression were reported, indicating that C/EBPβ is involved in both the immediate and the long-term effects of maternal dietary protein on offspring MSTN transcription [MSTN promoter , indicating a possible role for these transcription factors in the activation of MSTN upon low protein maternal diet [Maternal nutrition was shown to have effects on us breed . In the cription . Furthernal diet .MSTN promoter region were already reported by several authors [MSTN promoter [SNPs in the porcine  authors , 52, 53, authors , 52 and  authors , a knownpromoter , leading435 -G447 -T879), B (G435 -A447 -T879), C (A435 -A447 -A879) and D (A435 -A447 -T879), in which haplotype D is the most ancestral, haplotypes B and C are derived from haplotype D, probably by artificial selection, and haplotype A originated from haplotype B [MSTN promoter harbouring haplotype A is significantly higher than the others, and its presence is associated with higher values of total meat weight and meat percentage in breeds such as Yorkshire, Landrace, Laiwu and Dapulian [Interestingly, three of these SNPs are organized as four possible haplotypes: A  and the CAAT box (at position 202–207 from the start codon).The analysis of approximately 1.5 kb of the 5′ regulatory region of sheep revealed high degree of conservation with goat (98.1%), bovine (95.8%), porcine (86.9%), human (80.2%) and mouse (67.7%) . Three dpromoter . Using tpromoter  concludeSeveral muscle specific binding sites were also predicted in this region, as indicated in Additional file Within approximately 1.2 kb of the promoter, eight E-boxes were found , 2 , 3  and 4  [The search for SNPs in several horse breeds revealed the presence of two modifications in the promoter region  , 65. Acc, −C156) .MSTN promoter region of Thoroughbred horses at − 146 position [MSTN promoter, the two variants (with and without the SINE) were cloned in a vector upstream to the GFP reporter gene and were transfected in human HeLa cells and horse fibroblast cells. In both cases, the presence of the SINE caused a reduction of reporter gene activity to almost undetectable levels, indicating that this insertion disrupts the MSTN promoter activity [MSTN promoter activity in both undifferentiated and differentiated C2C12 cells [A 227-bp long insertion was identified in the position . Furtherposition , 67. Besposition . A putatposition . Furtherposition . Additioposition . To testactivity . Accordi12 cells . Noticea12 cells . HoweverMSTN gene has not been extensively studied, some authors identified the occurrence of SNPs in this region, as summarized in Table Although the 5′ regulatory region of the rabbit MSTN 5′ regulatory region was studied in birds revealing the presence of several E-boxes, which are conserved in number and location among chicken and quail [MSTN 5′ regulatory region from Wengshang Luhua chicken sites for MEF2 and GRE are found, besides 13 E-boxes [in vitro, but only modifications in E-box 3 depicted reduction in vivo, showing that other regulatory mechanisms present in the organisms and not in the cell culture are involved in the promoter regulation [A 1.2-kb fragment of nd quail   E-boxes . Naturalgulation .MSTN promoter of broiler chicken was shown to be highly polymorphic [MSTN gene were associated with muscle and fat deposition [ymorphic –77 and sposition , corrobomstn genes is much more complex, since this group of animals underwent an additional round of whole genome duplication in comparison to other jawed vertebrates [mstn genes: mstn-1  and mstn-2  and even up to four copies in salmonids, because of an additional duplication in this group [In teleost fishes the study of tebrates . Thus, tis group .Danio rerio), Xu et al. [mstn-1 5′ regulatory region  were identified, as well as several SNPs [mstn-2a and mstn-2b are more closely related than mstn-2c [mstn-2b was shown to be more frequent, followed by mstn-2c, while mstn-2a was rather rare and found only in a heterozygous state [mstn-2a (five extra sites) and mstn-2b (six extra sites). A shorter variant of allele mstn-2a, lacking two E-boxes, was also identified. All three promoter alleles were tested for their activities, through reporter assays using both muscle and non-muscle cells, and were shown to be activated in both, although at a higher rate in the first ones, especially upon differentiation. Interestingly, promoter activity was upregulated upon neural cells (PC12) differentiation, which is in agreement mstn-2 expression in brain. Deletion experiments have shown that the core promoter is contained within the most proximal 127 bp upstream of the ATG site, which include an atypical TATA sequence and a CCAAT box, as well as a site for SP1 and the initiator element (INR). In vivo experiments using zebrafish embryo injection of GFP reporter constructs directed by the different promoter alleles as well as luciferase assays of intramuscular injections confirmed the ability of these sequences to drive gene expression, in muscle and non-muscle tissues. Although all alleles functioned as promoters, allele mstn-2b was shown to be stronger than mstn-2a and mstn-2c in driving gene activity [mstn-2b allele presents a higher activity, it would be interesting to identify exactly the molecular mechanisms responsible for that difference. It is possible that there have been changes in TFBS sequences in this particular allele to allow chances in transcriptional activity.In the marine fish s region . In regaral SNPs . All thr mstn-2c . As for us state . The seaus state  (see Addactivity . KnowingLarimichthys crocea), a fish cultivated in China, has many potential binding sites gathered in mstn-1 and mstn-2 promoters  was almost twice the size of the one analysed in mstn-2 (643 bp). The regulatory regions were also divergent in sequence, since while mstn-1 promoter presented up to 90% of sequence identity when compared to sequences of other teleosts, mstn-2 promoter region was shown to be less conserved in relation to other teleost mstn-2, as expected. Nevertheless, other putative sites were identified in both promoters, as shown in Additional file mstn-1 and mstn-2 promoters that work in a muscle specific manner [The large yellow croaker (promoter . In mstnc manner .Epinephelus coioides) displays a TATA and CAAT boxes at positions − 18 and − 66 bp, respectively, as well as ten potential E-boxes in a fragment of approximately 1.9 kb of the 5′ region of stn-1 [in vitro and in vivo [mstn-1 promoter activity was also investigated using a pEGFP-1 reporter vector which was co-transfected into grouper cells (GF-1), and was shown to be downregulated upon treatment with nodavirus, a member of the Betanodavirdae family, the causative agent of viral nervous necrosis or fish encephalitis. This might be the reason why naturally infected groupers show MSTN protein downregulation. On the other hand, infection with the nervous necrosis virus (NNV) significantly induced mstn promoter activity, indicating a possible role in immune response [The orange-spotted grouper (of stn-1 . Other m in vivo . Promoteresponse .Lateolabrax japonicus) mstn-1 gene has a putative TATA box in its 5′ regulatory region, as well as seven E-boxes [The sea perch Lateolabr E-boxes .Lates calcarifer) promoter region of mstn-1 gene, a putative TATA box is located 28 bp upstream from the transcription start site and its surrounding sequences (± 30 bp) were shown to be conserved across teleost fish [In the barramundi (ost fish . Furtherost fish .Verasper variegatus) contains putative TATA and CAAT boxes, as well as three E-boxes in a 800-kb fragment of mstn-1 upstream region. Some SNPs were identified and possibly have an effect on promoter activity  mstn-2 gene promoter has two putative TATA binding protein sites, one TATA box and two CCAAT boxes within 1.3 kb of its 5′ regulatory region, as well as six E-boxes and several TFBSs  promoter regions of mstn-1a, mstn-1b and mstn-2a contain several TFBSs, some of them common to specific gene orthologs and others more specific , 500 bp upstream of mstn-1 and mstn-2 genes present two TATA boxes, as well as MEF2, E-boxes, and several other TFBSs , and GRE were also found, and in the mstn-1 5′ regulatory region a SF1 site was identified. Despite the similarities in the promoters, these genes are expressed at different tissues of the brook trout [In brook trout (ok trout .Oncorhynchus mykiss), the analysis of 2 kb of the gene promoter from rtMstn-1a and rtMstn-1b revealed the presence of a TATA box, several putative E-boxes  and − 2b (1.5 kb), including seven putative E-boxes, although differences in the TFBSs might explain the different expression patterns observed for the two genes [In the rainbow trout . Within ruminants, large species including cattle formed a separate branch from ovine and caprine [When closely related animals were compared, the multiple alignment of  caprine .MSTN 5′ regulatory region with mammal species  revealed a low level of sequence similarity (30% between chicken and human) [Xenopus tropicalis and zebrafish (mstn-1 and -2), indicating sequence variation between such organisms [mstn-1, a short region of MSTN promoter was found conserved  , which wrganisms . Howeverur group , sites fur group , showingmstn in teleosts fish is the mstn-1 gene [mstn-1 gene would be expected to display higher levels of conservation with other species than mstn-2. This can be observed in Fig. Mstn-1 promoter to mammals or birds. Accordingly, Chen et al. [mstn-1 promoter region from orange-spotted grouper (Epinephelus coioides) and found that it was homologous to genomic sequences of bovine, sheep and zebrafish mstn genes, although the authors do not present information about the size and level of conservation among the promoters of the species compared. However, Funkenstein et al. [mstn-1 of six different fish orders, indicating that the sequences of teleost fish are in general more divergent. In agreement, among the 12 fish mstn-1 promoter sequences analysed in this review, there is just a small conserved block of 140 bp for fish mstn-1, indicating a higher variability of the 5′ regulatory region of fishes. Sequence identity was already shown to be higher among the orders Perciformes and Pleuronectiformes, especially in the ~ 400 bp proximal promoter [mstn-1 promoters from Salmonids (Protacanthopterygii) are more conserved among themselves than to other fishes, so in phylogenetic analyses they are grouped together, as are the members from Ostariophysi and Acanthopterygii groups [According to phylogenetic analysis, the closest ortholog of tetrapod n-1 gene . Thus, tn et al.  analysedn et al.  observedpromoter , where si groups .mstn-2 gene promoter region is less conserved among the fish species included in our sample , but are present in all the other animals, almost every time accompanied by a CAAT box. Another binding site present in nearly all 5′ regions analysed is MEF2, and the combination TATA+CAAT+MEF2 + E-boxes is present in 68% (21/31) of the studied promoters , indicating a highly conserved basic structure. Since the methods used by the different authors varied, it is possible to speculate that this common core is present in all promoters, but some sites were not detected by the conditions/softwares applied. For instance, CAAT boxes were not identified in the articles discussed above in D. rerio mstn-2, O. mykiss mstn-1a and -1b, L. calcarifer mstn-1 and A. japonicus, however further analysis performed by our group were able to localize these sites  but modified in the small ones (mouse and rat): an additional TATA box, a CACCC motif, two A/T rich sequences and a palindrome-like sequence (PAL). Allen and Du  altered MSTN promoter works in association with other regulatory regions to provide the appropriate expression output. This was observed, for instance, in cattle, where two different breeds (Qinchuan and Red Angus) with different muscle phenotypes, present very similar promoters (99% homology) with comparable activity levels, indicating the necessity of other regulatory elements to adjust gene activity [It is also interesting to note that the MSTN gene through variations in TFBSs, some studies have also investigated the role of epigenetics on the MSTN promoter activity. For instance, in the mouse the Mstn promoter was shown to be induced by treatment of Trichostatin A (TSA), a histone deacetylase inhibitor. Such inhibitors facilitate histone acetylation, causing the unwinding of packed chromatin and activation of genes [In addition to the variety of possibilities for transcriptional regulation of the of genes . This prMSTN promoter in the skeletal muscle of pigs at finishing stage [MSTN promoter [MSTN gene promoter in low protein diet piglets [MSTN mRNA levels observed in such piglets. On the other hand, the porcine MSTN promoter activity was shown to be downregulated by sulforaphane, an inhibitor of histone deacetylases, and this hypoacetylation seemed to diminish binding of MYOD to an E-box, thus causing inactivation of Mstn transcription [Moreover, maternal dietary also caused alterations in histone modifications on the ng stage . A materpromoter . Two gen piglets . These acription .mstn promoter of sea perch (Lateolabrax japonicus), Abbas et al. [mstn is expressed in this species, was highly susceptible to digestion, whereas less susceptibility was observed for heart, and even less for kidney and liver tissues, where no mstn expression is observed, suggesting the promoter region/gene are compacted into heterochromatin in such tissues [In contrast, when studying the s et al.  have see tissues .Because of its potential applications both in medicine and livestock, the mechanisms to manipulate MSTN have been extensively studied. The gene promoter itself has been target to some investigation, as described below and summarized in Fig. MSTN gene promoter might be activated by an increase in NF-κB levels caused by Sporadic-inclusion body myositis (s-IBM), a common disease that causes progressive muscle waste and weakness. Since MSTN levels are increased in muscle fibers suffering from s-IBM, and considering the NF-κB binding sites are found in the MSTN promoter region [MSTN activity by binding to NF-kB binding sites in the promoter region under normal physiological conditions, preventing the activation promoted by NF-kB binding. This suggests that restoration of SIRT6 expression may function as a therapeutic strategy to alleviate muscle wasting processes associated with chronic diseases, such as cachexia. Furthermore, pharmacologic inhibition of NF-κB by pyrrolidine dithiocarbamic acid (PDA) abolished MSTN promoter upregulation in response to ammonium acetate and increased myotube diameter in vitro, indicating the important role Mstn promoter has on sarcopenia caused by hyperammonemia in cirrhosis and other hepatic malfunctions [Regarding medical applications, it was suggested by Nogalska et al.  that ther region , 92, ther region  have shounctions .MSTN activity by interfering with its promoter. For instance, MSTN expression can be downregulated up to 50% with the use of siRNAs complementary to a promoter-associated transcript in C2C12 cells in culture [Several methods have already been shown to decrease  culture . FurtherMSTN expression was also shown to be downregulated via decrease in its promoter activity by ursolic acid, a ubiquitous plant compound used in the treatment of muscle wasting conditions [MSTN promoter activity. Furthermore, this compound was able to suppress the MSTN promoter overactivity normally caused by dexamethasone [MSTN promoter, upregulating its activity. Upon ursolic acid treatment, C/EBP-δ levels were decreased, failing to overregulate MSTN promoter activity in the presence of dexamethasone. This allows improvement of overall muscle physiology, making ursolic acid a potential aid in the treatment of cachexia that accompanies several diseases [MSTN promoter activity in cells exposed to dexamethasone was partially blocked by addition of glutamine, a conditionally essential amino acid during catabolic states [nditions . It was ethasone . This sudiseases . Furtherc states . Glutamic states .MSTN in a more efficient manner than zinc finger nucleases targeting MSTN exons in bovine cells [MSTN exons by eGFP and neomycin genes, both driven by the MSTN promoter, and cells were shown to be healthy and could possibly be employed in somatic cell nuclear transfer. The use of this and other genome editing technologies such as CRISPR-Cas9 may allow the modification of specific TFBSs or the number of E-boxes, which are known for their importance in modulating MSTN promoter activity. By targeting MSTN promoter, minor adjustments in the skeletal muscle phenotype of animals will become possible, and will enable to meet specific demands for improvement of animal production.Targeting a breeding perspective, a promoter trap vector was also shown to knockout ne cells . The proMSTN gene promoter and 5′ regulatory region have both conserved and variable regions and that conservation parameters vary depending on the size and regions compared. It is important to notice that different authors have used various fragment sizes and different approaches for their analysis, resulting sometimes in conflicting data. For instance, our group have analysed a 260 bp MSTN promoter and concluded that, between chicken and human, there is 75% of sequence identity [Compiling the data mentioned in this review, it is possible to see that the identity , while Gidentity  found onMSTN promoter is consensus. They work in an additive manner to induce promoter activity under normal conditions, although some are stronger and more important than others for the performance of the promoter. Together with TATA and CAAT boxes, they compose the core of this regulatory region in the majority of animals. MEF2 completes the scenario for all tetrapods, and together, E-boxes+TATA-box+CAAT-box+MEF2 orchestrate MSTN promoter operation in a vast number of species. Another important feature for MSTN promoter, especially in mammals, is the ability to respond to hormones, and this should be taken into consideration when conditions and/or treatments that involve alterations or the use of hormones are involved.The role of E-boxes on MSTN by targeting its gene promoter for both therapeutical and breeding purposes, indicating the great importance of studying the mechanisms underlying MSTN transcription. It is noticeable, however, that most studies aimed at identifying SNPs usually target the identification of markers for animal selection, and do not pursue functional analysis to understand what exactly these alterations cause in the dynamics of the gene promoter, leaving gaps in the whole comprehension of MSTN gene regulation.In several different species, SNPs in the promoter region were shown to be related to body structure, indicating that this is a region of potential interest for analysis and possible interference, especially for breeding purposes. In fact, recent studies have been demonstrating potential manners to interfere with MSTN gene and protein. The role of several transcription factors [Lastly, it is relevant to mention that besides the promoter, several other regulators are also important for the proper functioning of  factors , epigene factors , and pos factors , 109 conAdditional file 1:Table S1. Summarized information on the different MSTN promoters analysed in this review. Information includes accession numbers, number of E-boxes and presence (x) or absence of TFBS in each species. TFBS or sequences identified independently by our group are indicated by a red asterisk (*) [risk (*) . (XLS 23Additional file 2:Table S2. Percent identity matrix of MSTN promoter sequences for all the vertebrate species analysed in this review. Multiple sequence alignment was used to calculate the % id matrix in BioEdit [ BioEdit . (XLS 18


TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the  TBC1D4 gene, encoding an NFκB binding site, can alter transcription factor binding, and use CRISPR-Cas9 to reveal localization of the transcription factor to be the regulator of chromatin accessibility and p65 binding and ultimately TBC1D4 expression.Functional variants have been proposed to alter transcription factor binding. Here, the authors provide direct evidence that functional variants within the  A locus influencing DNA methylation in cis is referred to as a methylation QTL (meQTL)5, while there are also examples of loci influencing cis chromatin states6.The variability in genomic sequences between individuals that is explored for disease associations includes loci that have obvious functional properties. Some of these loci change the coding sequences of genes, but a large proportion exist in the non-coding majority of the genome and are associated with changes in genomic function. The main functional property of the genome that has been explored for association with DNA sequence polymorphism is gene expression. Differences in the level of expression of a gene that depends on the allele present at a locus nearby defines that nearby locus as an expression quantitative trait locus, or eQTL10. By changing TF binding, it is furthermore assumed that gene expression, DNA methylation, and chromatin states are altered as consequences12. Supporting the model of DNA sequence polymorphism influencing TF binding as the primary mechanism for these downstream functional consequences, chromatin immunoprecipitation has shown differences between individuals13 or alleles15 at a locus for TF binding associated with sequence variation at that site. While this is strong evidence for the TF binding model, it remains an association and not a direct test of the hypothesis. As an alternative hypothesis, it is proposed that genetic polymorphism at functional genetic variants influences enhancer RNA structure, altering its ability to act as an aptamer to recruit proteins locally, preventing it from facilitating local looping of chromatin16.When studied at the sequence level, these functional variants can be described to contain binding sites for sequence-specific transcription factors (TFs), with the presence of a sequence variant within that site assumed to alter the binding of the TF at this locus18. These approaches use reagents that have sequence-specificity to target a pre-defined locus, including zinc finger domain proteins19, the TALE binding domain of transcription activator-like effector nucleases (TALENs)22, and, more recently, the CRISPR system with an inactivated nuclease18. With the idea that ‘epigenetic’ regulators are those influencing chromatin states and DNA methylation, a number of enzymes that modify these biochemical properties at a locus have been targeted, with varying efficacies and gene regulatory outcomes39.These observations contrast with the assumptions being made in a second current area of genomic research, the use of ‘epigenetic editing’ to modify regulatory events at specific loci in the genomeThere is, however, some lack of coherence between the ideas being pursued by those studying functional variants and those performing epigenetic editing. The former group would regard TFs as the primary mediators of events that include transcriptional, DNA methylation, and chromatin state changes as consequences. The premise of many ‘epigenetic editing’ approaches is that altering chromatin states and DNA methylation is sufficient to alter transcriptional regulation, ignoring any need for TFs to function locally. It remains to be seen whether the successes or failures of these two independent approaches will reveal insights into physiological transcriptional regulation, for example, whether chromatin modifications by themselves can allow transcription to occur in the absence of local TF binding.Here, we seek to test the model that TF binding mediates functional genomic outcomes at a polymorphic and regulatory locus that we identified in the human genome. We use CRISPR for targeted mutagenesis at a TF binding site overlapping this multifunctional variant, followed by the further use of CRISPR as a targeting system to deliver to the edited locus the TF that could no longer bind to the site—a form of epigenetic editing that assumes a primary role for the TF in regulating the locus. The results reveal insights into both the normal physiology of functional genomic variants as well as guidance for epigenetic editing strategies.40, on which deep (Illumina Platinum genomes) sequencing has been performed41, and for which lymphoblastoid cell lines (LCLs) are available from the Coriell Institute. By studying a family, we could identify effects mediated by variants that are rare in wider populations.Our primary goal was to identify a small set of polymorphic loci with very strong functional effects on gene expression, chromatin accessibility, and DNA methylation. We performed our studies on the CEPH/Utah Pedigree 1463, which includes 17 members in three generations . When samples were harvested as a batch, different combinations of samples were used for biomaterial extraction. All batch information was noted and used as metadata in exploration of the sources of variability in the data analyses. We noted that two samples (GM12877 and GM12893) grew at a slower rate than the other 15. We also checked carefully whether GM12889 was an outlier in any of the functional assays, as we have found this cell line to have a previously unrecognized mosaic copy number neutral loss of heterozygosity of distal chromosome 1143, but did not observe any distinctive results from this sample.To reduce technical variability, substantial care was taken with the assays based on cultured LCLs. A single batch of reagents was used for all cell cultures, and all genome-wide assays were performed on the same flask of cells  assays using our optimized Illumina HiSeq X-based assay44 to profile DNA methylation throughout the genomes of these cell lines.We performed a set of genome-wide functional assays on the 17 samples. Directional RNA-seq was used for gene expression profiling, small RNA-seq to test miRNA expression, and the assay for transposase-accessible chromatin (ATAC-seq) to map nucleosome-free, BHLF1 gene, which is expressed early in the lytic cycle of EBV infection and expressed in subclonal populations of LCLs45. DNA sequence variants found in these assays were compared with those from the Illumina Platinum Genomes annotations41 to confirm sample identities. The influences on our functional assays are summarized in principal component plots in Supplementary Fig. Our analytical studies started by testing for sources of variability of our results. One of the cell lines that were growing more slowly than the others, GM12893, in addition to batch-affected lines GM12880 and 12879 were also outliers in terms of their gene expression  in this family46. This allowed us to compare the target genes affected by eQTLs (eGenes) between our study and the prior analysis of this family. We found 21 eGenes in common between the studies (Supplementary Data p < 1.3 × 10−10) despite the prior study’s older haplotype annotation and their use of an mRNA-seq assay46 as opposed to our RNA-seq assay that was not solely dependent upon oligo-dT priming. We further validated the results from both studies by performing qRT-PCR on several eGenes and found that those discovered by both studies almost universally showed the predicted expression differences among the children (8 out of 9)  to allow sensitive identification of all miRNAs being expressed, pooling reads across all 17 samples for prediction purposes. We show there to be 632 known human miRNAs expressed in these samples, plus 13 from the EBV genome, and we identified 112 candidate novel human miRNAs. We confirmed one of these novel human miRNAs using stem-loop PCR . Analytically, we mapped ATAC-seq reads with the steps of extra stringency to exclude blacklisted regions of the human genomeHaving identified these individual eGenes and caSummits, we then narrowed the focus to define candidate multifunctional variants affecting both gene expression and chromatin accessibility. We identified 62 haplotype blocks containing both an eGene and a caSummit which overlaps a heterozygous SNP. We also filtered to require that there be co-variation of the informative haplotype and SNP. For example, if the peak is associated with one but not the other paternal haplotype, but the SNP variation is carried on the maternal haplotype, the variation in chromatin accessibility cannot be attributed to that particular SNP within the paternal haplotype. As a further filter, with the assumption that one allele being active and the other silenced should be associated with a pattern of intermediate DNA methylation locally, we chose the subset of loci in which CG dinucleotides with DNA methylation values of 20–80% were found within the peak. The overall filtering process is summarized in Fig. GSDME and TBC1D4 genes. We focused on the TBC1D4 multifunctional variant, located in a ~6 Mb haplotype on chromosome 13q  to encode a canonical NFκB binding site52, with the human-specific (HS) allele changing that binding site in 3 positions, alterations strongly predicted to disrupt NFκB binding. We therefore chose this as the locus for genomic editing experiments. TBC1D4 is a gene encoding a Rab-GTPase-activating protein that responds to insulin by dissociating itself from the glucose transporter 4 (GLUT4) protein, allowing the latter to migrate to the plasma membrane where it can act to transport glucose into the cell53. Mutations of TBC1D4 have been associated with type 2 diabetes mellitus55, indicating that the protein has a role to maintain normal glucose homeostasis.The list of high-confidence candidate multifunctional variants included sequences within the 13q Fig. , upper, o49 Fig. , lower. tes Fig. . The Geotes Fig. 50. UsingTBC1D4 but not the adjacent COMMD6 or UCHL3 genes within the same interaction domain  have higher levels of expression of ain Fig. . More deain Fig. . In the ned Fig. .Fig. 4ExAs there are no informative CG dinucleotides immediately adjacent to the multifunctional variant, we are unable to test haplotype-specific DNA methylation patterns. However, we observe that DNA methylation in immediately flanking CGs is lower in individuals with the AA haplotype Fig. .Fig. 8DNcis-regulatory locus show it to be specifically active in LCLs57  strategy for ‘scarless’ editing60. It has recently been highlighted that CRISPR-mediated genomic editing can be associated with mutations that can be large in size and potentially missed if the screen is solely focused on a small, targeted desired mutation61. We, therefore, added long-range PCR of the 4 kb DNA region flanking the targeted locus as well as reverse transcriptase PCR (RT-PCR) of overlapping 1 kb segments of the 6.4 kb TBC1D4 transcript which spans 197.4 kb on chromosome 13. Of the 5 clones found to have edits at the desired site, these PCR-based analyses revealed 2 clones to have large mutational events at the locus by testing DNA and RNA58. We, therefore, focused our analyses on 3 clones, 2 of which had the desired clean replacement of the Ancestral (AA) with the human-specific (HS) allele, and one with a 7 bp deletion spanning the NFκB binding site that appeared by PCR and densitometry to be hemizygous58, an even more deleterious pair of mutations for comparison  to test the effect of replacing the functional, NFκB-binding AA with the inactive HS allele or the cell line with the completely deleted NFκB binding site. We show the results in Fig. nes Fig. . We thennes Fig.  and the nes Fig.  at the eTBC1D4 activity >2-fold relative to a condition in which the same system without the p65 protein was introduced into the LCLs with the HS allele. However, we were concerned that these effects could be partially or wholly due to the increased amount of this NFκB subunit in the cell, binding to other cis-regulatory sites at this gene. We, therefore, employed two additional strategies. We developed a control system to target a known NFκB binding site at the OXTR gene62, validating targeting by testing the Cas9-induced mutation rate at this locus in LCLs  drove a need to identify ‘epigenetic’ marks such as DNase hypersensitivity, or ChIP-seq mapping of active chromatin regulators, demonstrating enrichment for overlap of these candidate regulatory loci with the lead SNPs identified in the GWAS, finding even greater enrichment for other SNPs in linkage disequilibrium65. To progress from these relatively large areas of the genome, containing many candidate functional variants, to the specific variants mediating the disease, the approach has generally been to identify functional variants within these regions. Typically these variants are defined by associating co-variation of transcription levels with sequence polymorphism in cis to that gene, but there are now studies that use DNA methylation66 or chromatin characteristics67 to help identify loci with transcriptional regulatory properties. The locus that we studied within the TBC1D4 gene was selected for its effects on local gene expression, chromatin accessibility and local changes in DNA methylation, as well as its strong predicted effect on the binding of the NFκB TF based on sequence characteristics. This confirms that it should be possible to use different types of functional genomic assays to arrive at the same functional variants, and that the use of multiple functional assays simultaneously67 may be especially valuable in prioritizing certain loci as being more likely to have a regulatory function. A functional variant, once identified, can be linked to variable gene expression as an eQTL, with the target eGene representing a potential insight into the mechanism of the disease, based on the properties of the protein encoded by that gene.The interest in identifying functional variants has arisen from associations implicating haplotypes in disease heritability68, especially apparent from the systematic studies of the GTEx Consortium69. The cis-regulatory landscape of different cell types is well known to be distinctive70, implying sequence-specific mechanisms for targeting post-translational modifications of histones, DNA methylation, and nucleosomal remodeling in different cell types. Candidates for mediating this sequence-specificity should include TFs , but not the enzymatic mediators of chromatin states or DNA modifications, which inherently lack the ability to recognize specific sequences. The model of a primary recognition of specific sequences by different TFs as part of the process of driving lineage commitment is well-established. The fact that some TF binding sites (TFBSs) are variable between individuals, therefore, leads to the idea that the same canonical cell type from individuals differing in their DNA sequences at TFBSs can confer distinctive properties on that differentiated cell between individuals, differences that could be reflected by altered or disease phenotypes.One concern has been that the post-GWAS surveys for sequence variants with regulatory functions may have been performed in a cell type irrelevant to the disease process being studied, prompting the search for functional variants in the cell type(s) assumed to mediate the phenotype of interest. That eQTLs can have a cell type-specific activity is increasingly apparent71 and for DNA methylation influencing the genomic binding of at least one TF, NRF172. There is clear evidence from in vitro studies for DNA methylation influencing TF binding, both negatively and positively73. The question therefore arises whether our targeting of the p65 transactivation domains to a specific locus, an example of epigenetic editing, is exerting its positive effects on TBC1D4 transcription by means of replicating the function of the TF no longer capable of binding to the allele lacking the NFκB motif, or whether it is instead creating a local environment conducive to TF binding in general. As the targeting of the CRISPR complex is mediated through a guide RNA that forms an RNA:DNA hybrid74, loci which we have shown to be mostly inactive in the human genome75, and prior studies have revealed NFκB to bind strongly preferentially to double-stranded DNA76, we believe that, if anything, the targeting of the locus by CRISPR would have an inhibitory effect on local TF binding. While formally unproven by our studies, the primary mechanism for activation of the locus would appear to be more likely to be by the restoration of p65 than by non-specific effects on local chromatin organization. The question, therefore, arises about epigenetic editing more broadly, whether targeting of a locus to induce what may be secondary consequences of local TF binding  is the optimal approach. If the TF (or group of TFs) that would normally bind locally and recruit the mediators of these activation marks is not expressed in that cell, will transcription still be promoted? The different viewpoints inherent to the epigenetic editing and functional genetic communities could end up, even inadvertently, prompting some valuable insights into transcriptional regulation.This train of logic implies a primary role for TFs, with secondary effects on chromatin states and DNA modifications. Such a viewpoint raises a challenge to the idea that epigenetic regulation of the genome, defined in terms of chromatin patterns and DNA methylation, acts instead in a primary role to define where TFs can bind within the genome. There is support for both possibilities, mouse embryonic stem cell studies showing an instructive role for TFs on DNA methylationTBC1D4 gene from a promoter located ~182 kb telomerically.In summary, this study follows up on a wealth of excellent association data indicating a primary role for TFs in mediating effects of sequence variability at functional genetic loci. We harnessed the genetic and epigenetic editing capabilities of the CRISPR-Cas9 system to provide a direct test of this hypothesis, by engineering a locus to fail to bind the cognate TF, then restoring its targeting locally by guiding the transactivation domains of the TF back to the locus using CRISPR, increasing the transcription of the 2 incubator and maintained between 2 × 105 and 8 × 105 cells/ml.Lymphoblastoid cells (LCLs) derived from the 17-member CEPH Pedigree 1463 were purchased from the Coriell Institute and cultured in RPMI 1640 medium, supplemented with 15% fetal bovine serum , 100 IU/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). Cells were kept in suspension in tissue culture flasks  at 37 °C in a 5% CO42. For each cell line, we collected 5 × 105 cells from two different cultures, harvested during the exponential growth phase. The cells were pelleted at 500×g at 4 °C for 5 min and washed with 50 μl of cold PBS. Samples were then centrifuged again at 500×g at 4 °C for 5 min. Cells were lysed in ice-cold lysis buffer  and immediately spun at 500×g at 4 °C for 5 min. The pellet was then resuspended in 50 μl of the transposase reaction mixture (TD buffer supplemented with 100 nM transposase). Following a 30-min incubation at 37 °C, the samples were purified using the DNA Clean and Concentrator-5 purification kit (Zymo). After purification, the libraries were amplified and indexed by combining: 10 µl transposed DNA, 5 µl PCR Primer Cocktail , 15 µl Nextera PCR Master Mix , 5 µl each of Nextera i5 and i7 indexed amplification primers , and 10 µl of nuclease-free water. The PCR reaction was carried out using the following conditions: 98 °C, 30 s; a total of 10 cycles of 98 °C, 10 s; 63 °C, 30 s; 72 °C, 1 min; 72 °C, 5 min. Subsequently, the libraries were purified using Agencourt AMPure XP beads; large fragments were filtered by using 0.6× magnetic bead volume (relative to the PCR mixture volume) and then keeping the supernatant. Primer-dimer and short fragments were removed by collecting bead-associated DNA in a 1:1 (bead solution volume:mixture volume) mixture. The libraries were quantified by Qubit HS DNA kit  and their quality assessed by 2% agarose gel electrophoresis and Bioanalyzer High-Sensitivity DNA Assay. Libraries were sequenced on an Illumina HiSeq 2500 to obtain 100 bp paired-end reads.The assay for transposase-accessible chromatin (ATAC)-seq libraries were prepared similarly to Buenrostro et al.44. A total of 2 × 106 cells was harvested, pelleted at 2000×g for 10 min and frozen at −80 °C until extraction of genomic DNA. Thawed cells were resuspended in 10 mM Tris pH 8.0, 150 mM EDTA solution before the addition of SDS to a final concentration of 1% in a 250 μl volume. Protein was digested overnight at 50 °C with 25 μg of Proteinase K , followed by an hour incubation at 37 °C with 5 μg of RNAse A (Sigma). Extraction of genomic DNA was then achieved through two consecutive phenol:chloroform steps, selecting the aqueous phase. The DNA was concentrated and purified via ethanol precipitation. Libraries were then prepared using 100 ng of genomic DNA and an additional spike-in of 0.5 ng of unmethylated lambda DNA as previously described44. Libraries were 250 bp paired-end sequenced on the Illumina HiSeq X system.Whole-genome bisulphite sequencing (WGBS) libraries for the Illumina HiSeq X (XWGBS) were prepared from each cell line similar to the method that we have previously describedCell pellets were treated with QIAzol lysis reagent (Qiagen) and total RNA was isolated using the miRNAeasy kit (Qiagen) combined with on-column DNase (Qiagen) treatment according to the manufacturer’s instructions. All dirRNA-seq libraries were prepared by removing rRNA from 1 μg of total RNA and generating strand-sensitive reads with a KAPA Stranded RNA-Seq Kit with RiboErase (HMR), following manufacturer’s protocol (Kapa Biosystems). Total RNA was depleted of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase. RNA was then fragmented using heat and magnesium and submitted to first strand cDNA synthesis using random priming. After second strand synthesis and incorporation of dUTP, the ds-cDNA was A-tailed, ligated with adapters, and the library was amplified with primers carrying appropriate adapter sequences. The libraries were 100 bp paired-end sequenced.Libraries were constructed using NEBNext Small RNA Library Prep Kit (NEB). Input total RNA was ligated with 3′ adapters, and the excess of 3′ adapter was hybridized with a reverse transcription primer prior to the ligation of the 5′ adapter. Ligated RNA was converted into cDNA with a first strand reverse transcriptase, followed by PCR amplification. Library size was then selected by loading the PCR product into a 3% agarose dye-free gel of a Pippin Prep system (Sage Science). The libraries were 100 bp single-end sequenced. The metrics for all of the library sequencing performed in this project are shown in Supplementary Data 20 and random hexamers as primers 20 were used). All qPCR primers were designed using the NCBI Primer-BLAST web interface77  using both oligo(dT)Stem-loop primers , decoy version 1 for GRCh38 (hs38d1), and the Epstein Barr Virus genome, was used throughout the analysis79. Uniquely mapped reads were retained using samtools v0.1.19, followed by removal of mitochondrial reads by picard-tools v1.9280. Finally, duplicate reads were removed using both picard-tools (MarkDuplicates.jar) and samtools (samtools rmdup). Read1 reads were shifted using bedtools2 v2.26.0, as previously performed42. Leveraging the cell type similarity among the samples, all of the shifted reads were concatenated for each of the two replicate batches. Peaks were called for each replicate using MACS2 v2.1.0 81. Irreproducible discovery rates (IDRs) were found for the overlapping peaks using the method previously described82, and peaks with an IDR of less than 0.05 were retained and subsequently filtered by consensus blacklisted and mitochondria homologous regions, creating a ‘master’ peak list Kallisto v0.42.5 85. Estimated counts for genes were determined by Sleuth86. Genes on the sex and EBV chromosomes were removed and filtered for those with an average count of at least 10 among the children. Genes whose expression levels were associated with parental haplotype allele inheritance were discovered as aforementioned for caSummits except with additional regressors for batch extraction date and quartiled mean EBV expression. eGenes were defined by an FDR of less than 0.4. The statistical significance of the overlap between our eGene set and the previous study’s set was assessed using GeneOverlap v. 1.16.0.Sequenced reads were pseudo-aligned to GENCODE v24 long noncoding RNAs and protein-coding genes, as well as EBV genes, using 87 using the STAR aligner v. 2.5.2b with several parameter modifications specific to miRNAs . The aligner soft clips the 5′ ends of reads in order to find proper alignments. As this could lead to erroneous alignments, soft clipped reads were removed using samtools as well as the secondary alignments of reads. The primary alignments of all family members were concatenated and passed through miRDeep* for novel miRNA detection88. Only 221 putatively novel miRNAs with more than 50 supporting reads and a miRDeep* score greater than 10 were retained. A final set of 112 novel miRNAs were obtained by further filtering against the following miRNA databases: Rfam database v.1289, GENCODE v2478, fRNAdb90, NONCODE 201691, and FANTOM92.Small RNA sequence reads were aligned to the custom hg38 reference genome and a miRBase v21 human miRNAs gene transcript filesubRead v 1.5.0 featureCounts on all reads uniquely mapped by STAR and a miRBase v21 gtf also containing the 221 putative novel miRNAs93. Only miRNAs with at least 5 counts in 4 children were retained and were subsequently normalized using the trimmed mean of M values method94. These expression levels were regressed by haplotypic inheritance as previously mentioned to find eQTL-miRNAs (e-miRNAs).Quantification of miRNAs was achieved by using 95.To test if e-miRNA expression was mediating the effect of genetic variation on associated eGenes, the expression levels of eGenes were regressed by shared haplotype block miRNA expression levels, as well as by extraction date and quartile of EBV expression. e-miRNAs whose expression levels significantly (FDR < 0.05) predicted the expression of eGenes were not found to target their respective associated genes by testing validated targets stored in the multiMiR database44. Only CpGs with at least ten reads defining its methylation status on either strand were retained. Cytosines with coverage in the 99.9th percentile were removed to mitigate potential PCR artifacts. Finally, only the CpGs for which at least three children had intermediate methylation levels (25–75%) were used for discovering functional variants.To define CpGs with intermediate methylation levels, sequence reads were aligned to the custom hg38 reference genome and methylated and unmethylated counts for CpG were calculated as we have previously performed46 were explored for their potential to disrupt TF motif using HaploReg51. Rare variants were those with a minor allele frequency less than 0.05 in all 1000 Genomes superpopulations96.As shown in Fig. 56 was downloaded from the NCBI GEO database accession GSE55105. The fastq files were aligned using the Burrows-Wheeler Aligner version 0.7.13 (bwa mem –M –t < N > hg38.idxbase < R1 FASTQ >)79 to our custom hg38 genome.The ChIP-seq dataset from Zhao et al.6 cells was resuspended RPMI 1640 medium with 1% formaldehyde and incubated at room temperature under shaking for 10 min. The cross-linking was quenched with 0.125 M glycine, and the suspension was pelleted by centrifuging at 800×g for 5 min at 4 °C, then washed with 4 ml of ice-cold PBS and pelleted again. The pellets were then resuspended in Farnham lysis buffer and spun down at 800×g for 5 min at 4 °C. Subsequently, the samples were processed in a Biorupter using the setting of ON/OFF (30 s/30 s) cycles for 10 min. An average shearing size of 200 bp was obtained for each sample. The sonicated mixture was then spun at 10,000×g for 15 min at 4 °C, and the supernatant was collected. Protein G Dynabeads (ThermoFisher) were added to a 2 ml tube containing 1 mL PBS/BSA, the magnetic beads were washed 3 times to a final volume of 200 μl of PBS/BSA, and 20 μg of anti-p65 antibody  was added to each tube. The antibody was coupled to the beads for 3 h at 4 °C. Antibody-coupled beads were then washed again 3 times with PBS/BSA and then added to the 1 ml chromatin preparation and incubated at 4 °C overnight. The beads containing the immuno-bound chromatin were collected using a magnet, followed by washing 5 times in cold LiCl wash buffer and then washing with 1 ml TE buffer. The supernatant was then discarded and the bead pellet was resuspended in 200 μl IP elution buffer and incubated at 65 °C for 1 h, with vortexing every 15 min to elute the immuno-bound chromatin from the beads. The samples were then spun at 20,000×g for 3 min at room temperature. The supernatant containing the immunoprecipitated DNA was incubated at 65 °C overnight to complete the reversal of the formaldehyde cross-linking and then purified using the DNA Clean and Concentrator-5 kit (Zymo Research). The purified DNA was used as a template for quantitative PCR performed with the Light Cycler 480 Syber Green Master mix (Roche), according to the manufacturer’s instructions. Enrichment of DNA at the TBC1D4 cis-regulatory site was compared to a negative control region located within a gene desert of chromosome 13  where GM12878 ChIP-seq showed no H3K4me1, H3K27ac or NFκB enrichment. Enrichment at the promoter of IκBα, a p65 binding site validated by EMD Millipore, was used to assess the quality of the immunoprecipitation.For each tested cell line , a total of 20 × 1097, a total of 5 × 104 cells were pelleted by centrifugation at 500×g for 5 min at 4 °C and resuspended in 50 μl of ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin and kept on ice for 3 min. The suspension was washed with RSB devoid of NP40 or digitonin, and nuclei were pelleted by centrifugation at 500×g for 10 min at 4 °C. Nuclei were resuspended in 50 μl of transposition mixture . The reaction mixture was incubated 37 °C for 30 min with 3 g orbital shaking. Subsequently, the sample was purified with the DNA Clean and Concentrator-5 kit (Zymo) and submitted to qPCR as described for quantitative ChIP. A locus within a gene desert of chromosome 13  was used as a negative control. The enrichment of accessible chromatin at the TBC1D4 locus was normalized to the promoter region of GIN1, which was found to be highly accessible yet demonstrated invariable signal strength among the children.As performed by to Corces et al.98 was double digested, then purified on agarose gel to obtain a linearized fragment containing both the sgRNA expression and the dCas9 sequences. P2A Reverse complement oligonucleotides were annealed and extended, while the EGFP sequence was amplified by PCR from plasmid pBI-MCS-EGFP (Addgene plasmid #16542)99 using the nucleotides and primers described in Supplementary Data 63 with sequence-specific primers . Plasmid pCAG-eCas9-GFP-U6-gRNA was a gift from Jizhong Zou (Addgene plasmid #79145) and was used in combination with an ssODN template  cells were transfected with 33.3 µg of CRISPR-Cas9 plasmids in addition to 0.4 nmol of ssODN template for editing. The same number of cells was transfected with 2 μg of GFP plasmid to provide GFP control cells, while negative control cells received transfection reagents only. Transfections were conducted with the Cell Line Nucleofactor Kit V (Lonza) according to the manufacturer’s instructions. After transfection, cells were suspended in medium and incubated overnight under cell culture conditions, then replaced with fresh medium. GFP-positive cells were sorted after 48 h following the transfection. Cells were pelleted, washed twice, and suspended in sorting buffer . Cell suspensions were submitted to cell analysis and sorting in a FACSAria II cytometer (BD Biosciences). FACS data were analyzed using FACSDiva software (Becton Dickinson) with gating of single cells using FSC/W and SSC/W, and gating of GFP-positive cells. GFP-positive cells were identified by determining the GFP gating cutoff for control GFP-negative cells, further details are included in Johnston et al.58.LCLs were passaged at 3.5 × 10g, and the supernatant was filtered through a 0.3 micron sterile filter prior to use. When subsequent T7EI assays or qRT-PCR analyses were to be performed, cells were sorted directly into QuickExtract DNA extraction solution or RNAlater, respectively.Single GFP-positive cells were sorted 48 h after transfection into individual wells of a 96-well plate, containing a mixture of fresh and conditioned medium (1:1), in which the FBS concentration was increased to 20%. The 96-well plate was incubated for two weeks under cell culture conditions, then the content of each well was transferred to a new 96-well plate with additional conditioned medium. Conditioned medium was obtained from GM12881 cells, cultured in 20% FBS RPMI 1640 for 24 h. The medium was removed without disturbing cells at the bottom of the flask, centrifuged at 792×TBC1D4 or the OXTR loci, genomic DNA was isolated from transfected and control cell pellets using QuickExtract DNA Extraction Solution (Epicentre) according to the manufacturer’s instructions. DNA was then concentrated by ethanol precipitation. The 1 kb region containing the gRNA targeted region was amplified with forward and reverse primers (Supplementary Data To verify indel efficiencies of gRNAs targeting CRISPR-Cas9 to the Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Reporting SummarySource Data


K-mers in Mark-Associated Nucleotides), an approach to infer variation in transcription factor (TF) activity across samples through unsupervised analysis of the variation in DNA sequences associated with an epigenomic mark.Variation in chromatin organization across single cells can help shed important light on the mechanisms controlling gene expression, but scale, noise, and sparsity pose significant challenges for interpretation of single cell chromatin data. Here, we develop BROCKMAN  contains supplementary material, which is available to authorized users. Understanding how the dynamic interaction of transcription factors (TFs) and chromatin governs cell types, differentiation, and responses is a fundamental challenge. TFs recognize and bind to specific DNA sequences and can potentially affect chromatin structure and gene expression through various means, including recruiting histone modifiers, chromatin remodelers, and the mediator complex. In particular, “pioneer” TFs may be able to open chromatin and, in so doing, allow other factors to bind to the now-accessible DNA . MeasureEpigenomic data has recently increased dramatically in scale and complexity, with studies profiling either large numbers of individuals e.g. –7), or u, or u7])However, single cell epigenomics data is inherently sparse, since every locus is present at only two copies per diploid cell , such thk-mers) has been used extensively in the past , and, andk-mell types , 17. Thiark Fig.  - top. Tk-mer words associated with the epigenomic mark, using matrix factorization and dimensionality reduction to: (1) analyze variation in k-mer occupancy across single cells as a basis for distinguishing different cell types, states, and treatments; (2) identify differentially active TFs; and (3) decipher TF-TF interactions. Applying BROCKMAN to scATAC-seq profiles, we show that cell-cell variation in k-mers associated with open chromatin provides a robust and information-rich representation that can readily distinguish different cell types, drug treatments, biological artifacts, and cycling cells without any knowledge of TFs and without requiring peak calling on bulk or pooled single cell profiles. Leveraging known TF specificities, we demonstrate that the individual components of our reduced-dimensionality k-mer space correspond to individual TFs or groups of TFs that tend to be more lowly expressed, consistent with transcriptional bursting causing noisy TF expression. The TFs that co-vary within a k-mer component are more likely to physically interact, consistent with biochemical cooperativity between TFs, which we show is expected to be especially variable. BROCKMAN thus provides a highly effective tool for exploratory data analysis for high-dimensional or single cell epigenomics.Here, we present BROCKMAN, a method for representing epigenomic data by the k-mers) associated with the chromatin mark, we can infer the activity of the motif’s cognate TF . In the context of chromatin accessibility  in different chromatin regions should allow us to uncover such dependencies. In particular, because a TF may recognize multiple related k-mers, these related k-mers should co-vary with each other, reflecting on the (hidden) activity of their joint, cognate TF.Since some TFs can modify chromatin where they bind, the differential activity of TFs should be reflected in differential chromatin states at locations containing the TF’s binding motif. For example, if the levels of a given active TF in a cell are too low for it to bind its motif and modify chromatin, then the chromatin modification will be not be associated with this TF’s motifs. As the level of an active TF rises, it will bind its motif in the DNA and modify chromatin, leaving signature motifs next to the chromatin modification it elicited. Thus, by capturing a motif  at loci associated with a chromatin mark of interest, yielding a matrix of k-mer frequencies by samples. It then (3) decomposes this matrix of k-mer frequencies to identify groups of k-mers that co-vary across the samples and reduces the dimensionality of the data. Finally, (4) we can explore the relationships between cells/samples in this reduced-dimension space, and identify the k-mers (and associated TFs) that underlie differences between cells or samples.To capture these dependencies in k-mers within 50 bp of each transposon integration site , and applied t-stochastic neighborhood embedding (t-SNE) to the resulting significant principal components ), human embryonic stem cells (H1ESC), fibroblasts (BJ), erythroblasts (TF-1), and promyeloblasts (HL-60), sometimes including multiple replicates  Fig. 2a2a. We scnts PCs;  to facilk-mer loadings , and is appropriate for our relatively non-sparse data (most 8-mers (our maximum k) are observed at least 9 times per cell in our analysis). Indeed, performing PCA on a subset of cells yields similar PCs to the entire set and projecting held-out cells onto the learned PCs, results in co-clustering of related cells (data not shown). Factorization by Independent Component Analysis and Sparse Minibatch PCA yielded similar results (data not shown).Note that while there are many factorization approaches, PCA proved highly appropriate because it has been repeatedly successful at capturing biological signals in diverse datasets, allows projection of new samples onto learned components, yields interpretable Cells from the different cell types readily partitioned into distinct clusters, as did cells of the same type (K562) between treatments Fig. . We alsoOne grouping Fig. , “Mixed”Current analyses are typically performed for only a sub-set of reads, often those that reside within peaks and can be uniquely mapped. However, this could lead to loss of key biological information. For example, although reads outside of ATAC-peaks may reflect assay noise, they could also include cell-specific chromatin signatures, especially from regions open only in rare cell types, which may not be evident from bulk ATAC-seq or even from aggregate scATAC-seq data, and would be excluded if only reads within peaks are considered. In another example, although repeat regions may be important loci of gene regulation , challenk-nearest neighbors  , using the sample label for treatment and replicate as the “ground truth”. First, as a local measure, we assessed the number of cells from the same sample among each cell’s ce) Fig. -d; Seconce) Fig. -h.Fig. 3Surprisingly, including only reads outside of peak regions improved cell grouping. To show this, we partitioned reads into two groups, and performed BROCKMAN on each set separately: reads within 250 bp of any of the 46,145 called peaks, and reads outside this window. , indicated that gapped k-mers only improved cell grouping globally  when using gapped k-mers, indicating that gaps may allow for more complex relationships to be captured in fewer PCs.Using the same approach to assess the impact of gapped lly Fig. , but notlly Fig. . Althougk-mers amongst all cells, we found 131 significant PCs, suggesting variation in the activities of individual or combinations of TFs between or within cell types. Specifically, we hypothesized that each PC may represent the differential activity of one or more correlated TFs or sets of TFs, captured by the relevant k-mers . Next, we relate the different PCs to differential TF activity by classifying each k-mer into “cognate” and “non-cognate” for each TF using both the in vitro preference of each TF to individual 8-mers as measured by Protein Binding Microarrays (PBMs) and position weight matrix (PWM) motifs derived from these same experiments and others  ) and the dissociation constant (Kdx) of its binding site in the following relationship, which represents 1 minus the probability the binding site will not be bound:As described previously , a TF’s x can also bind with a partner y, occupancy of x depends on x binding in isolation, as before, but also binding with y as a xy heterodimer, depending on the concentration [xy] and the Kdxy of the heterodimer. At equilibrium, [xy] = [x][y]Kaxy, where Kaxy is the association constant of x and y. Thus, for x binding to a single binding site with or without cooperative binding of y, we have:If TF y] is constant since the same logic holds if x and y are interchanged and for arbitrary [y]. Thus, Kaxy[y] is a constant corresponding to the fraction of x that is in xy form. Assuming Kdxy < Kdx , as [x] changes, this cooperative occupancy is always at least as steep as without cooperativity at concentrations yielding intermediate occupancy, regardless of choice of parameters, resulting in saturation of binding over a shorter range of [x] with cooperative binding. Intuitively, this is because increasing [x] increases cooperative and non-cooperative binding equally when Kdxy = Kdx, but when Kdxy < Kdx cooperative binding increases more rapidly until saturation. Additional file x is in xy form, and Kdxy is 100× lower than Kdx.For simplicity, we can assume that [Additional file 1:Figure S1. BROCKMAN computational pipeline. A bash pipeline and other computational resources are available on GitHub (https://carldeboer.github.io/brockman.html). Tools/functions used for each step are indicated in brackets. (PDF 545 kb)Additional file 2:Figure S2. PCs that distinguish replicates. Shown are the Bonferroni-corrected P-values (y axis) and AUROC values (x axis) for how well each PC separates each untreated K562 replicate from the other two replicates. Colors indicate the replicate being compared to the other two. Red horizontal line: P-value cutoff (0.1) below which PCs were considered to separate batches.) (PDF 185 kb)Additional file 3:Figure S3. The TFs enriched in PCs have lower expression. A, B) CDF of the mean (population) expression  or mean-corrected CV  The relationship between the mean expression (x axis) and CV (y axis) for all genes in WT K562 data (dots). Names of TFs with the highest mean-corrected CV are labeled and AP-1 factors are bolded. Pink, blue: TFs with least and most significant PC enrichment. (PDF 200 kb)Additional file 4:Table S1. Summary of TFs associated with the different untreated K562 cell-variable PCs. TFs are listed in decreasing order of enrichment significance, with TFs filtered for redundancy between motifs as described in the Additional file 5:Figure S4. Cooperativity between TFs results in steeper binding curves. The predicted fractional TF occupancy (y axis) for a given concentration of the TF (x axis), when the concentration of the cooperatively-interacting TF is constant. The two binding curves are aligned at 50% occupancy to emphasize the differences in the slopes. Modeling was done as described in


FLO1 gene was to regulate transcription elongation. Here, we discuss possible mechanisms by which histone acetylation might regulate RNA polymerase II processivity, and comment on the contribution to transcription of chromatin remodelling at gene coding regions and promoters.Recently, we reported that a major function of histone acetylation at the yeast  DNA in eukaryotic cells exists as a nucleoprotein complex called chromatin. The fundamental subunit of chromatin is the nucleosome which comprises two copies each of the core histones, H3, H4, H2A and H2B, around which is wrapped ∼147 bp of DNA . Indeed,The histone modifications most commonly associated with active gene transcription are histone acetylation and histone methylation . NumerouArguably the most commonly studied aspect of chromatin remodelling has been the analysis of chromatin at gene promoters in response to gene activation and repression . The genFLO1 gene [FLO1 encodes a lectin-like cell wall protein which mediates a cell aggregation stress-response known as flocculation, whereby yeast cells within the ‘floc’ are protected against the outside environment [We recently investigated the role of histone acetylation in the regulation of transcription of the yeast LO1 gene . FLO1 enironment . This phFLO1 transcription is regulated via the antagonistic action of the Tup1-Cyc8(Ssn6) co-repressor and Swi-Snf co-activator complexes which function to repress or activate transcription, respectively [FLO1 gene is associated with a Tup1-Cyc8 dependent array of strongly positioned, hypoacetylated nucleosomes which span the promoter and upstream region. Conversely, FLO1 de-repression correlates with increased promoter histone acetylation, and the Swi-Snf dependent disruption of this nucleosome array. In a mutant deficient for Swi-Snf activity, nucleosome remodelling and FLO1 transcription are abolished suggesting transcription of this gene is Swi-Snf dependent [We had previously shown that ectively ,12  which histone acetyltransferase (HAT) enzyme was required for transcription, (ii) which histone lysine (K) residues were involved, and (iii) to determine the mechanism of action of histone acetylation in FLO1 transcription. We hypothesised that histone acetylation would drive Swi-Snf recruitment and nucleosome remodelling at the FLO1 promoter, which would enable the initiation of transcription. Surprisingly, this proved not to be the case. Instead, our results suggested the dominant role for histone acetylation at FLO1 was in the open reading frame (ORF) to enable Pol II elongation  was the critical target for acetylation by these HATs that was required for FLO1 transcription.Specifically, we confirmed FLO1 gene following de-repression. We then examined H3 acetylation, Swi-Snf recruitment, histone depletion, and Pol II occupancy at the promoter and ORF following gene activation in the presence and absence of Gcn5 and Sas3-dependent histone H3 acetylation.To investigate the role of these HATs in regulating transcription, we used the anchor-away technique to conditionally and rapidly deplete the Tup1 repressor from the nucleus so as to monitor the time-course of transcription-coupled chromatin events at the FLO1 transcription and expression of the flocculation phenotype. When the experiment was repeated in a strain deficient for both Gcn5 and Sas3 activities, although H3K14 acetylation (H3K14ac) and FLO1 transcription were abolished, Swi-Snf recruitment, histone eviction and Pol II occupancy at the promoter persisted. However, we could not detect Pol II in the ORF. Together, this suggested that Swi-Snf recruitment was not dependent on Gcn5 and Sas3-dependent histone H3K14ac, and that histone eviction at the promoter was insufficient to enable FLO1 transcription. Instead, these data were consistent with histone H3K14ac in the ORF being a critical requirement for transcription elongation.Our data showed that following removal of the co-repressor from the promoter, there was a rapid acetylation and depletion of H3 which was accompanied by Swi-Snf recruitment to the promoter and Pol II enrichment in the promoter and ORF . This coFLO1 ORF following the removal of the Tup1-Cyc8 co-repressor when Sas3 and Gcn5 dependent H3K14ac was abolished could be due to either (i) a failure to release Pol II from the promoter, or (ii) prevention of Pol II from entering into the ORF. We favour the latter possibility based on evidence of a role for histone acetylation in the eviction of ORF nucleosomes during transcription elongation. Indeed, it has been shown that NuA3 occupancy can be detected globally at gene coding regions [GAL1 ORF is decreased, and the Pol II occupancy profile across the ORF is altered, indicating Pol II fails to fully traverse the coding region [S. pombe have also shown that Gcn5 is required for ORF nucleosome eviction and Pol II progression at stress responsive genes [esa1 mutant shows defects in Pol II processivity consistent with a failure of Pol II reach the end of the active genes tested. Most strikingly, Pol II has a slower elongation rate in the esa1 mutant [The absence of Pol II in the  regions . Furtherve genes . A similve genes . This co1 mutant . PresumaFLO1 gene coding region which is required to drive histone eviction to allow Pol II progression across the ORF. If this hypothesis is correct, we would predict that following FLO1 activation we would see increased H3 occupancy in the FLO1 ORF in an H3K14ac deficient mutant. It would also be pertinent to confirm in the histone acetylation-deficient background if the Pol II at the promoter is initiation competent, and to determine if Pol II enters the ORF, and if so, to measure how far it travels.We therefore hypothesise that a major role of Sas3 and Gcn5 is to acetylate H3K14 within the FLO1 ORF in the context of the NuA3b complex, whereas Gcn5 is functioning in the context of the SAGA complex acting from its primary location in the promoter to acetylate histones in the 5’ ORF. SAGA(Gcn5) might also be present in the ORF at greater levels than our chromatin immunoprecipitation (ChIP) analysis suggests, perhaps due to it being difficult to detect in this region using this technique.The Sas3 protein is the catalytic subunit of the yeast NuA3 histone acetyltransferase complex which predominantly acetylates histone H3 K14 and K23 . Gcn5 isFLO1 promoter is consistent with the occupancy of Gcn5 seen at almost all active Pol II-dependent genes where SAGA can be recruited by transcription factors to upstream activator sequences [FLO1 have not been identified.It has been proposed that promiscuous HAT and histone deacetylase (HDAC) activity across the genome maintains a steady state level of global chromatin acetylation . Localisequences ,26. HoweFLO1 ORF, and the high occupancy we see of Sas3. Indeed, the observed occupancy of Gcn5 at the 5’ ORF is consistent with its recruitment by the Sgf29 sub unit of SAGA which binds to H3K4 methylated (me) chromatin which we would expect to be enriched in this region of the gene when active [HAT occupancy within ORFs could result from tethering of the HATs to Pol II at the promoter, which then enter into the coding region. Alternatively, HATs could be recruited directly to actively transcribed gene coding regions via chromatin which has been post-translationally modified in some way. The latter mechanism may be more applicable to both the low occupancy we see of Gcn5 in the active n active .In vitro evidence shows that the YEATS domain of Taf14 can bind to acetylated H3K9, whereas in vitro and in vivo evidence shows that the Phd finger of Yng1 and the PWWP domain of Pdp3 can respectively target Sas3 to H3K4 and H3K36 methylated chromatin within gene coding regions [FLO1 gene is 4.1 kb long and is marked by H3K9ac and H3K36me when active , it would fulfil the criteria for a Sas3-enriched gene. Thus, we would propose that at FLO1, Gcn5 and Sas3 might be recruited to the actively transcribed gene coding region by the transcription-coupled deposition of H3K4me and H3K36me expected to be found at the 5’ and 3’ ORF regions. Histone acetylation in the 5’ ORF might also be a consequence of Gcn5 acting at a distance from its location in the promoter.With regards Sas3 occupancy, the Yng1 and Taf14 subunits common to NuA3 complexes contain a PhD finger, and YEATS domain respectively, whilst the unique Pdp3 sub-unit of NuA3b contains a PWWP domain.  regions . Interes regions . ConsideIt has been proposed that histone acetylation contributes to the recruitment of ATP-dependent chromatin remodelling complexes which bring about nucleosome repositioning, disassembly or eviction. For example, the Snf2 subunit of the Swi-Snf complex contains a bromodomain which has an affinity for H3 acetylated chromatin which has been shown to determine its association with active gene promoters and DNA double-strand break sites ,29. The in vivo nucleosome eviction activity of such chromatin remodelling complexes has been shown to require histone chaperones to tether the evicted histones [HO promoter and PHO5/PHO8 promoters, respectively [in vivo evidence to show that the Fun30Fft3 ATP-dependent chromatin remodelling complex cooperates with the FACT histone chaperone to promote nucleosome eviction within ORFs to enable Pol II elongation [FLO1 promoter, which ATP-dependent remodellers or histone chaperones are functional at the FLO1 ORF is currently unknown.The histones . One of histones ,34. Indeectively ,36. Moreongation . Thus, wFLO1 gene coding region via the deposition of transcription-coupled histone methylation marks (FLO1 transcription may also be required for gene activation.We have proposed a model in which Sas3, and to a lesser extent Gcn5, are recruited to the active on marks . We furtFLO1 gene. We have shown that in a strain deficient for Sas3 and Gcn5 activities, Pol II could not be detected in the ORF even though Swi-Snf recruitment, histone eviction and Pol II occupancy still occurred at the promoter. Thus, Swi-Snf recruitment and activity at the promoter does not require Sas3 and Gcn5-dependent H3K14ac, and FLO1 transcription is not solely dependent upon promoter histone eviction. We propose that promoter and ORF chromatin remodelling are independent events which are critical for FLO1 transcription initiation and elongation, respectively. A challenge for the future will be to determine the interplay between the histone PTMs, chromatin remodelling complexes and histone chaperones that correlate with transcription elongation, whilst taking into account the concomitant nucleosome eviction and reassembly taking place.Our data indicate that Sas3 and Gcn5-dependent H3K14ac in the gene coding region is required to enable transcription elongation of the Swi-Snf dependent