COX-2methylation and expression in N-methyl-N-Nitrosurea (MNU)-induced intestinal GCin six Sapajus apella animals. The partial promoter sequence ofS. apella COX-2 gene was obtained and used to identifytranscription factors and cis-regulatory element binding sites. TheCOX-2 methylation pattern was assessed usingMethylation-Specific PCR (MSP), and expression was analyzed byimmunohistochemistry (IHQ). A total of 20 samples were obtained. A 675 bpfragment of the S. apella COX-2 promoter region was obtained,and it was 99.2% and 68.2% similar to H. sapiens and S.boliviensis, respectively. Similar to humans, several transcriptionfactors and cis-regulatory element binding sites were identified in theS. apella sequence. MSP revealed that all samples weremethylated. However, IHQ results demonstrated positive COX-2 expression in allpre-neoplastic and tumoral samples. The results suggest that the analyzedfragment is not crucial in COX-2 regulation of GC in S.apella.Gastric cancer (GC) remains one of the main causes of cancer-related deathworldwide. There are two distinct histological types of GC: diffuse andintestinal. The latter is characterized by the presence of pre-neoplasticlesions. One of the most frequently altered enzymes in intestinal GC is COX-2,an important lesion marker. This work aimed to study  Helicobacter pylori (H. pylori)infection, smoking, dietary habits, and host genetic susceptibility (Gastric cancer (GC) is the fourth most diagnosed cancer and the second highest mortalityrate among all types of cancers worldwide. Its incidence is influenced by severalfactors, including COX-2 may playan important role. Cyclooxygenase-2 (COX-2) is an inducible enzyme that catalyzes theconversion of arachidonic acid to prostaglandins in response to several inflammatorystimuli. The progression from initial gastric lesions to gastric cancer has beencorrelated with COX-2 over-expression, suggesting that its activity may be involved ingastric carcinogenesis onset , Pará State, Brazil, weretreated with oral fresh doses of N-Methyl-N-nitrosourea (MNU) at a dosage of 16 mg/kg body weight, and also received drink water containing MNU inlight-shielded bottles daily. As previously described, this concentration wasresponsible for chemically-induce gastric carcinogenesis  were performed throughout the treatment for monitoring. Biopsy samples weresubjected to histopathological analysis and sent to the Laboratório de BiologiaMolecular at Universidade Federal do Pará (UFPA) for molecular analysis. During theexperiment, five animals that developed pre-neoplastic lesions died from intoxication,showing typical symptoms such as confusion, sleepiness, tremor, hyperthermia, diarrhea,vomiting, urinary retention, cutaneous eruptions, and ulcerative oral lesions. They alsopresented renal, hepatic and respiratory failure and steatosis. Animals suffering andwith presumed terminal illness due to adverse side effects were euthanized byintravenous administration of Ketaral , Dormonid and Methotrimeprazine . The survivinganimal received Canova treatment and was submitted to a surgical removal of the tumor,being clinically monitored for one year after the end of the experiment. During thisperiod it did not show any complications resulting from the treatments (DNA was obtained using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’sinstructions and quantified using a NanoDrop 1000 Spectrophotometer v3.7 (ThermoScientific).S. apella COX-2 promoter region wasobtained from primers designed using conserved regions of the COX-2sequence from Homo sapiens (GenBank access code: NG_028206) andSaimiri boliviensis (GenBank access code: AGCE_01110177). TheCOX-2 primer sequences were as follows: sense5’-GATCACTTCAAAATGAATTCAGGAT-3’ and antisense 5’-GCTACGAAGATAGATTACAGTTATG-3’.The genomic sequence for the 2 and 0.5 U Taq DNApolymerase (Invitrogen). After PCR, the DNA fragments were sequenced using an ABI 3130automated sequencer (Life Technologies). The sequencing reaction was performed using theBigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies).Polymerase chain reaction (PCR) was performed on the normal gastric mucosa samples. Thereaction had a final volume of 25 μL containin: 50 ng of template DNA, 10 pM of eachprimer, 0.20 mM of each dNTP, 2.5 mM MgClS. apella with the human sequence using the Transcriptional Factorsearch program (TFsearch) . Computational analyses revealed the presence of a 127-bp CpG islandcontaining seven CpG sites located between positions 388 and 514 of the sequencedfragment.To analyze the methylation pattern of the of COX-2 promoter methylation pattern was performed by MSPusing selected primers by the MethPrimer software  and 68.2% similar to the S. boliviensis promoter sequence.When we used the normal mucosa samples, we obtained a 675 bp identical fragment (GenBankaccession number KR011346) of the cis-regulatory element binding sites. We identified severaltranscription factor binding sites, primarily CdxA, GATA-1 and GATA-2, andcis-regulatory elements, including p53 Responsive Elements (P53RE),NF-Y and STAT .We then analyzed the promoter fragment to identify potential transcription factor andand STAT . Of all COX-2 promoter, allsamples were methylated, regardless of disease state  .When examining the methylation pattern of the  tissue) .Immunohistochemistry was performed to examine COX-2 protein expression in the samples.The results showed that normal samples did not express COX-2, while samples withpre-neoplastic lesions  or tumorswere positive for COX-2 expression  and4.Animal models are a valuable tool to study the origin and molecular mechanisms of cancerand can be used to develop and test new therapeutic strategies, including gastric cancer,confirming that it was a conserved portion of the promoter region.As the COX-2 promoter region is approximately 1,700 bp long andcontains several binding sites for cis-regulatory elements andtranscription factor binding sites  transcription factor revealed in ouranalysis has a conserved leucine zipper sequence involved in its homo- andheterodimerization and DNA binding. The presence of the C/EBP binding site in theWe can also highlight AP-1, which plays an important role in carcinogenesis promotion. BotharCOX-2 gene expressionand our MSP results revealed a hypermethylated pattern in the S. apellaCOX-2 gene promoter. However, a comparison of these results withimmunohistochemical analysis did not identify a negative correlation that would confirmthe involvement of methylation in COX-2 gene inactivation. Among thevarious hypotheses for this lack of correlation between the methylation profile andimmunohistochemistry, it is possible that the S. apella promoter regionanalyzed here does not represent a key region for COX-2 expression(COX-2 transcriptional regulation necessary.DNA methylation has also been reported to regulate Our immunohistochemical analysis demonstrated the expression of COX-2 protein in allpre-neoplastic lesions and tumor samples and the lack of expression in normal gastrictissue samples which is confirmed by the results of S. apella and humanCOX-2 promoter sequence, suggesting that S. apellais a good animal model for gastric carcinogenesis. Moreover, the lack of correlationbetween promoter methylation and immunohistochemistry results suggest that thisepigenetic mechanism in the analyzed promoter region is not crucial in S. apellaCOX-2 regulation.Our results suggest a similarity between the


CAPN14, a gene with reported esophagus-specific expression. CAPN14 is dynamically up-regulated as a function of EoE disease activity and after exposure of epithelial cells to interleukin-13 (IL-13). Herein, we aimed to explore molecular modulation of CAPN14 expression. We identified three putative binding sites for the IL-13-activated transcription factor STAT6 in the promoter and first intron of CAPN14. Luciferase reporter assays revealed that the two most distal STAT6 elements were required for the ∼10-fold increase in promoter activity subsequent to stimulation with IL-13 or IL-4, and also for the genotype-dependent reduction in IL-13-induced promoter activity. One of the STAT6 elements in the promoter was necessary for IL-13-mediated induction of CAPN14 promoter activity while the other STAT6 promoter element was necessary for full induction. Chromatin immunoprecipitation in IL-13 stimulated esophageal epithelial cells was used to further support STAT6 binding to the promoter of CAPN14 at these STAT6 binding sites. The highest CAPN14 and calpain-14 expression occurred with IL-13 or IL-4 stimulation of esophageal epithelial cells under culture conditions that allow the cells to differentiate into a stratified epithelium. This work corroborates a candidate molecular mechanism for EoE disease etiology in which the risk variant at 2p23 dampens CAPN14 expression in differentiated esophageal epithelial cells following IL-13/STAT6 induction of CAPN14 promoter activity.Eosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease resulting in eosinophilic esophageal inflammation. We recently found that EoE susceptibility is associated with genetic variants in the promoter of  Eosinophilic esophagitis (EoE) is a chronic, allergic disease associated with marked mucosal eosinophil accumulation  belongs to the classical calpain sub-family, a set of calcium activated intracellular regulatory proteases  that is highly associated with EoE risk and located in the promoter of CAPN14 was sufficient to decrease CAPN14 promoter activity in a manner that is consistent with the eQTL seen in individuals with and without EoE  culture   to various levels of confluence (80% or 100%) with relatively low (0.09 mM) or high (1.8 mM) CaCCL26 nanoluciferase reporter construct was engineered using the firefly reporter construct reported previously . For the CAPN14 promoter reporter construct, PCR primers (listed in Table S1) were used to amplify the 1988 base pair region around rs76562819 and subsequently clone it into the pNL1.1 nanoluciferase reporter vector. Site-directed mutagenesis was performed to generate the construct with scrambled STAT6 binding sites and the EoE non-risk allele of rs76562819 using the GeneArt Site-Directed Mutagenesis System . Sequences were confirmed by Sanger sequencing. All mutagenesis primers are provided in Table S1.The 250 ng of nanoluciferase experimental constructs were transiently co-transfected into esophageal epithelial cells with 250 ng of pGL3-control firefly luciferase reporter plasmid. After 24 hr, cells were treated with 100 ng/mL of IL-13 or IL-4 . 24 hr after cytokine administration, cells were lysed and nano and firefly luciferase activity was assayed using the Promega Nano-Glo Luciferase Assay System. The nanoluciferase measurement for each well was divided by the firefly luciferase measurement in order to account for cytotoxicity, small differences in cell number, and transfection efficiency. Each well was further normalized to the promoterless pNL1.1 vector to control for baseline activity of the vector.6 cells with 0.8% formaldehyde by adding 1 ml of 10X fixation buffer  to 9 ml of growth medium for 8 min at room temperature with shaking. The reaction was stopped by adding glycine to a final concentration of 125 mM for an additional 5 min. After washing with PBS, pellets were frozen at −80° for at least overnight. Nuclei were prepared by re-suspending pellets in 1 ml of L1 buffer  and incubated at 4° for 10 min. Nuclei were pelleted and re-suspended in 1 ml of L2 buffer  and rotated for 10 min at room temperature. Nuclei were briefly washed with sonication buffer (Tris-EDTA [TE] buffer + 0.1% SDS) and re-suspended in 1 ml of sonication buffer. All buffers were supplemented with complete EDTA-free protease inhibitors . Sonication was performed by a Covaris S220 focused ultrasonicator  at 175W Peak Incident Power, 10% output, 200 bursts for 10 min in 12×12-mm, round-bottom glass tubes. Efficient DNA fragmentation was verified by agarose gel electrophoresis. Anti-histone mark ChIP was performed by SX-8G IP-Star Automated System  in RIPA buffer  following the protocol of the manufacturer with 2–4 μg of indicated antibodies. Libraries were sequenced on the Illumina HiSeq 2500, and ChIP-seq data were visualized and analyzed in Biowardrobe . RNA was reverse-transcribed with the High-Capacity RNA-to-cDNA kit . Gene expression was determined by real-time PCR using a 7500 Real-time PCR system  using Taqman Gene Expression Assays CAPN14- Hs00871882_m1, CCL26- Hs00171146_m1 and GAPDH- Hs02786624_g1. Relative quantification was calculated by the comparative CT method . BrieflyFor the RNA-sequencing study performed to identify the isoforms of CAPN14 expressed in the esophageal biopsies of people with and without EoE , RNA was isolated from distal esophageal biopsy RNA from EoE patients  with active disease and from unaffected controls  as previously described  and Halt phosphatase inhibitor cocktail. Samples were exposed to electrophoresis in NuPAGE Novex 4–12% Bis-Tris Gels and transferred to nitrocellulose using the iBlot system . Primary antibodies: anti-CAPN14  and anti-β-actin . Secondary antibody: IRDye 800CW donkey anti-rabbit . Nitrocellulose membranes were imaged using the Odyssey CLx system (LI-COR) and analyzed with Image Studio (LI-COR). EPC2 cells that constitutively express Statistical analysis and data visualization were done with GraphPad Prism .CAPN14 promoter reporter construct, PCR primers (listed in Table S1) were used to amplify the 1988 base pair region around rs76562819 and subsequently clone it into the pNL1.1 nanoluciferase reporter vector. All mutagenesis primers are provided in Table S1. RNA sequencing data has been deposited in NCBI Geo database as GSE113341. Supplemental material available at Figshare: https://doi.org/10.25387/g3.7565792.Constructs are available upon request. For the CAPN14 expression similar to CCL26, a chemokine whose promoter activity is dependent on the STAT6 transcription factor . Of the two isoforms of CAPN14 that are expressed, the major isoform (ENST00000444918) includes exon 7, while the less highly expressed isoform of CAPN14 (ENST00000398824) does not. Furthermore, the less highly expressed isoform of CAPN14 (ENST00000398824) is predicted to undergo nonsense mediated decay based upon manual annotation from the VEGA/Havana projects (2 > 0.8), but functional genomic and biochemical evidence supported a specific role for rs76562819  rs76562819, have 50% less 76562819 . We founk allele . Thus, tCAPN14 in esophageal epithelium. Unlike CCL26, CAPN14 expression is highly dependent upon the cell culture conditions. Esophageal epithelial differentiation is a physiological process that increases mechanical strength and barrier function to squamous epithelia .We next sought to determine the factors leading to expression of n is low . Followi< 0.0001 . At a pr< 0.0001 . As seenCAPN14. These results are important because calpain-14 represents a candidate for treatment and prevention of EoE. Previous studies supported a model in which IL-13 induction of CAPN14 occurs in a STAT6-dependent manner. We identified the STAT6 binding sites, the necessity of these sites for CAPN14 promoter activity, and the binding of STAT6 to the promoter of CAPN14 in esophageal cells stimulated with IL-13. We demonstrated genotype-dependent expression of CAPN14 and identified rs76562819 as the genetic variant that is sufficient to produce genotype-dependent promoter activity. Indeed, the 40.0% reduction of CAPN14 promoter activity in the genotype-dependent reporter assays mirrors the effect size of the eQTLs measured in subjects with and without EoE , genetic (rs76562819), and epithelial differentiation factors that regulate the expression of CCL11, CCL24, CCL26) through STAT6-dependent mechanisms . It was therefore important to establish that two STAT6 sites in the CAPN14 promoter control IL-13-stimulated expression of CAPN14. IL-13 shares many functional properties with IL-4, stemming from the fact that they share a common receptor subunit, the alpha subunit of the IL-4 receptor . Despite the evidence presented in this study, future experiments in esophageal epithelial cells deficient in STAT6 signaling are needed to fully conclude that STAT6 is necessary in the transcription of CAPN14.IL-13 is a critical cytokine in the pathoetiology of EoE . Indeed,receptor . Both ILreceptor , so it iCAPN14 , other genes have more nuainced regulatory factors affecting expression . In this study, we demonstrated that epithelial differentiation was important; however, we were unable to establish specific tissue-specific molecular pathways that lead to the differences in CCL26 and CAPN14 expression in response to IL-13.Very little is known about the factors regulating the expression, function, and tissue specificity of CAPN14 . A growiCAPN14 transcription. Future studies will assess the post-translational regulation and activity of calpain-14. The structure of calpain-14 strongly suggests that it might dimerize with calpain-S1 (This study focused on the regulation of lpain-S1 . We usedlpain-S1 . CalpainCAPN14 expression. Altogether, this study makes an important advance toward identification of the factors that control the genotype-dependent transcriptional regulation of CAPN14 in esophageal epithelium.The current study identifies critical factors that regulate


In a sharp contrast, if the inactivation rates change with the promoter states, then m(t) may display multiphasic growth patterns. Upon the activation of signals that oscillate periodically, m(t) also oscillates later, almost periodically at the same frequency, but the magnitude decreases with frequency and is almost completely attenuated at high frequencies. This gives a surprising indication that multiple promoter states could filter out the signal oscillation and the noise in the random promoter state selection, as observed in the transcription of a gene activated by p53 in breast carcinoma cells. Our approach may help develop a theoretical framework to integrate coherently the genetic circuit with the promoter states to elucidate the linkage from the activation signal to the temporal profile of transcription outputs.The transcription of inducible genes involves signalling pathways that induce DNA binding of the downstream transcription factors to form functional promoter states. How the transcription dynamics is linked to the temporal variations of activation signals is far from being fully understood. In this work, we develop a mathematical model with multiple promoter states to address this question. Each promoter state has its own activation and inactivation rates and is selected randomly with a probability that may change in time. Under the activation of constant signals, our analysis shows that if only the activation rates differ among the promoter states, then the mean transcription level  In response to these challenges, cells show a remarkable regulatory plasticity to adapt themselves for normal cell functions and physiological homeostasis ,2. Gene γ > 0. Transcripts are produced with rate ν > 0 when the gene is active and are degraded in rate δ > 0 . It was found that q1 decreased with [LR], while q3 increased with [LR]. Interestingly, q2 first increased and then decreased with [LR] and did not surpass 50%.A primary step in the use of our model is to determine the promoter state probabilities in terms of the strength of activation signals. In the following, we show through an example of how the probabilities can be obtained. Sepúlveda et al.  monitorements in , the proactivity . The proq1(t), q2(t) and q3(t) in the following formulae:a1 = 8.8 × 10−3 and a2 = 3.922 × 10−5. As shown in R2) 0.9943, 0.9857 and 0.9936 for q1(t), q2(t) and q3(t), respectively.According to the equilibrium thermodynamic model ,22,25, wt)] can be carried over to the transcription level m(t) of lacZ gene. We consider the case where [LR(t)] increases in t and approaches the steady-state value A in response to rapid external stimuli. Since lambda repressor is much more stable than the corresponding mRNA molecules, we follow the work of Thattai et al.  as in figure 6a. By comparing the signal profile in figure 6a with the growth pattern of m(t) shown in figure 6b and many additional examples of m(t) we examined, including for instance the one shown in figure 6c withAfter a short time period of rapid growth, m(t) starts oscillation that turns out to be periodic later with the same period of the signal. (2) ωThe oscillation magnitude of m(t) decreases in the signal oscillation frequency . (3) As shown in the bottom panels of figure 6b,c, ω ≥ 0.8, for which m(t) behaves as if the transcription is activated by constant signalsthe oscillation magnitude of m(t) becomes very small and the oscillation is almost completely damped when .In the example, we assume that a gene is regulated by two transcription modes sharing the same mRNA production rate on as in  with an ic model ,25. Let m(t) in the bottom panels of figure 6b,c resemble the two typical growth patterns generated by the classical two-state model with a single promoter state and constant kinetic rates: m(t) either increases monotonically for all t > 0 as in figure 6b, or peaks uniquely and decreases thereafter as in figure 6c. The observed resemblance gives a theoretical proof that the cross-talking of multiple pathways, or more generally, multiple promoter states, can potentially filter out not only the oscillation in activation signals but also the randomness in the selection of promoter states. We now use the transcription of C12orf5/TIGAR gene in MCF-7 cells as an example to further testify this interesting finding. In its promoter region, there are two p53 binding sites [q1(t) and q2(t) byν1 = ν2 = 1 h−1; and the decay rates at δ1 = δ2 = 0.18 h−1. We take the activation and inactivation rates, in the same unit h−1, ata), we can determine uniquely the mean transcription level m(t). As shown in figure 7b, the function m(t) determined by our model increases monotonically and fits extremely well with the measured data from [R2 = 0.9395. It shows that the two p53 binding promoter states filter out the periodic oscillation of p53 and the random switching between the promoter states, and offers an experimental validation of our theoretical prediction. It also offers an alternative explanation of the filtration of periodic p53 signals that was previously attributed to the mRNA decay rates in [The two growth curves of ng sites : the lowng sites , we estian cells ,37. By sata from  with therates in ,35.Figu5.Cells in living organisms are constantly exposed to environmental or intracellular challenges. Gene transcription regulation is a central process in cells to adapt themselves for these challenges by up- or downregulating the production of mRNA molecules ,2. Upon m(t): (1) When only the activation rates differ among the promoter states, our theorem 3.1 shows that the presence of multiple promoter states brings on no significant change to the temporal profile of m(t). It either grows for all t > 0 or peaks uniquely as predicted by the classical two-state model with a single promoter state. (2) When the inactivation rates also change with promoter states, m(t) can develop rather complicated dynamics. Our theorem 3.3 proves that m(t) may develop a biphasic growth pattern with two peaks even when there are only two promoter states. This indicates that inactivation rates play a more prominent role than activation rates in shaping the temporal profiles of mRNA level in the rewire of transcriptional programmes. Both cellular signals and regulatory factors can actively control the inactivation rates, which may increase in the concentration of transcription repressor [When the signals inducing the transcription of a gene are stable so that the kinetic rates and the promoter probabilities are all constants, our analysis yields two distinct scenarios for the mean transcription level epressor  or decreepressor . Some trepressor .Escherichia coli provides a rather precise fitting, with the average coefficient of determination R2 > 0.99, to the experimental data of [m(t) is expected to develop a complex dynamics as well [t > 0. Under the activation of a periodic signal with an oscillation frequency ω > 0, our simulation shows that m(t) grows rapidly in a short time period and then oscillates periodically in the same frequency ω. The oscillation magnitude of m(t) decreases in ω and the oscillation is almost completely damped when ω becomes large, for which m(t) behaves as if the transcription is activated by constant signals. The attenuation in the oscillation of m(t) was further testified by the transcription of C12orf5/TIGAR gene in MCF-7 cells [m(t) determined by our model increases monotonically and fits extremely well with the data from [R2 = 0.9395. Our interesting finding indicates that multiple signal transduction pathways, or more generally, multiple promoter states, are capable of filtering out both the oscillation in activation signals and the noise in the random switching between the promoter states and producing simple transcription dynamics as in the case of a single promoter state activated by constant signals.When the gene activation signals change in time, the promoter state probabilities may also change accordingly and a primary step is to determine the probabilities in terms of the signal strength. We followed the approach of the equilibrium thermodynamic model ,22,25, a as well ,6,7,19.  as well . Surpris-7 cells . The funata from  with R2 Our analysis shows that the mean transcription level does not always mirror the dynamics of the activation signals. Counterintuitively, it sometimes displays exactly the opposite: In the presence of multiple promoter states, the transcription level may develop a biphasic growth with two peaks under the activation of constant signals or simply increases all the time under the activation of periodic signals. The set of promoter states behaves like a conversion hub that transforms cellular signals passing through the gene regulatory circuit to the production of mRNA molecules. It maps signalling dynamics to a broader spectrum of temporal profiles for transcription output. The map is not expected to be injective in general, as similar dynamical behaviours may be observed from distinct signalling sources. Our knowledge on the architectures of gene regulatory networks has been largely enriched due to the tremendous efforts in past years ,4,33,37.et al. [There remain many challenges in advancing or extending our theoretical approach. In our hypothesis (H2), the kinetic rates, including the rates of gene activation, inactivation and mRNA production, are assumed to be constants. The temporal dependence of gene transcription on activation signals is attributed to the time dependence of promoter state selection. The constant assumption on the kinetic rates for given promoter states was also made in our previous studies –30,41. Tet al. . More exet al. ,22,25. B


Cacng2 function is dependent upon expression level, but currently, little is known about the molecular mechanisms that control expression of this gene. To address this deficit and investigate disease-related gene variants, we have cloned and characterized the rat Cacng2 promoter and have defined three major features: (i) multiple repressive domains that include an array of RE-1 silencing transcription factor (REST) elements, and a calcium regulatory element-binding factor (CaRF) element, (ii) a (poly-GA) short tandem repeat (STR), and (iii) bidirectional organization with expressed lncRNAs. Functional activity of the promoter was demonstrated in transfected neuronal cell lines (HT22 and PC12), but although selective removal of REST and CaRF domains was shown to enhance promoter-driven transcription, the enhanced Cacng2 promoter constructs were still about fivefold weaker than a comparable rat Synapsin-1 promoter sequence. Direct evidence of REST activity at the Cacng2 promoter was obtained through co-transfection with an established dominant-negative REST (DNR) construct. Investigation of the GA-repeat STR revealed polymorphism across both animal strains and species, and size variation was also observed in absence epilepsy disease model cohorts . These data provide evidence of a genotype (STR)-phenotype correlation that may be unique with respect to proximal gene regulatory sequence in the demonstrated absence of other promoter, or 3′ UTR variants in GAERS rats. However, although transcriptional regulatory activity of the STR was demonstrated in further transfection studies, we did not find a GAERS vs. NEC difference, indicating that this specific STR length variation may only be relevant in the context of other (Cacna1h and Kcnk9) gene variants in this disease model. Additional studies revealed further  complexity at the Cacng2 promoter, and we identified novel, co-regulated, antisense rat lncRNAs that are paired with Cacng2 mRNA. These studies have provided novel insights into the organization of a synaptic protein gene promoter, describing multiple repressive and modulatory domains that can mediate diverse regulatory inputs.CACNG2  is a multi-functional regulator of excitatory neurotransmission and has been implicated in the pathological processes of several brain diseases. The online version of this article (10.1007/s12031-018-1208-x) contains supplementary material, which is available to authorized users. This 323 amino-acid (in human) protein is an auxiliary subunit of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptors, a so-called trans-membrane AMPAR regulatory protein (TARP) that regulates receptor function and distribution at the synapse  CaRF target. In the latter study, a CaRF-associated, CaRE1 element upstream of Cacng2 was also identified  1986 Act of Parliament and Cardiff University ethical review. GAERS and non-epileptic control (NEC) strains were maintained as described . Rat genomic sequence was amplified from initial rat brain RNA extracts which contain a sufficient background of intact genomic DNA. Mouse genomic DNA was amplified from Maxwell kit-purified mouse tissue . Where cDNA was required from either rat brain or cell culture samples (see below), RNA extracts were DNaseI-purified (Promega protocol), and cDNA was generated with the Superscript II protocol  using an Oligo (dT) primer. PCR was conducted using standard procedures with either REDTaq ReadyMix  or Q5 Hot-Start High-Fidelity DNA polymerase . Oligonucleotides used for amplification are listed in Table Se Table S.Cacng2 promoter, genomic sequence of different lengths . For the subsequent analysis of GA-repeat length variation, similar procedures were used to obtain “F11-R10” constructs containing sequence-verified GAERS and NEC rat GA repeats . A previously characterized dominant-negative REST (DNR) expression construct  promoter, widely used as an experimental promoter with relative neuronal specificity  were transfected  with plasmid constructs  and maintained for 24 h prior to either reporter assay, or, in some cases RNA extraction (see above). Plasmids were used at different amounts for 24- and 12-well plates (100 or 150 ng/well respectively), and were co-transfected with the control pRL-TK plasmid . Where used, the DNR construct was transfected at 250 ng. In other experiments, HT22 cell cultures were treated with N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt  or vehicle for 24 h, and then RNA was extracted as described above. Transcription reporter assays were conducted with the Dual-luciferase reporter assay system (Promega), using a Luminometer . Each transfection was replicated either sixfold  or eightfold , as indicated in the “Hippocampal HT22 cells (provided by JD) were maintained in DMEM (Invitrogen/Thermo Fisher) with 10% Fetal Bovine Serum , and PC12 cells  were grown in DMEM with 10% horse serum (Invitrogen) and 5% FBS. Both culture media were supplemented with 1x antibiotic/antimycotic (Invitrogen), and cells were grown on 12- or 24-well Costar CellBIND® plates  at 37 °C, and in 5% COCacng2 gene promoter sequence was predicted from flanking genomic sequence associated with mammalian Cacng2 exons mapped on the UCSC genome browser (genome.ucsc.edu), and also with reference to predicted mouse and human promoters on the Eukaryotic promoter database . Species and strain sequence variants were obtained from the STR catalog viewer . Potential trans-regulatory sites were identified using the Meme suite (meme-suite.org/) and Lasagna (biogrid-lasagna.engr.uconn.edu/lasagna). Potential regulatory sites in the Cacng2 3′ UTR sequence were searched with “AREsite” that detects AU-rich sites . Sequence identity was initially confirmed using BLAST (blast.ncbi.nlm.nih.gov), and sequence comparisons were conducted with Clustal Omega . Statistical analysis was conducted with IBM SPSS Statistics version 20  by applying different tests as indicated in the Results, and accepting p < 0.05 as the significance level.Rat http://genome-euro.ucsc.edu) on 02/10/2017.The Genotype-Tissue Expression Project (GTEx) data used for the analyses described in this manuscript were obtained from the UCSC Genome Browser , and directly analyzed for promoter activity using a transient transfection approach in two cell lines, HT22 and PC12. For comparison with another neuronal promoter sequence that has been extensively characterized for other species, the rat Syn1 promoter was similarly cloned . Subsequent experiments utilized a number of different sized Cacng2 promoter constructs, and all of these constructs were found to be markedly less active than the Syn1 construct, driving only modest levels of transcription even following the selective removal of multiple different sequence blocks .In further experiments, the ector pGL.10, and Cacng2 constructs , F = 37.789, n = 6/group). Our inference of independently acting repressive elements is also generally supported by additional comparisons between different constructs in this analysis , F = 34.707, n = 6/group).The extensive promoter analysis conducted in HT22 cells revealed three repressive regions: a distal, upstream region containing the identified CaRE element, a larger distal region containing the multiple REST elements, and a third, downstream repressive region Fig. . Evidenccts Fig.  revealedsis Fig. . The abssis Fig. ” sectionCacng2 F11R10 1234 bp construct was also selected for analysis of the functional properties of the variable GA-repeat sequence. For this purpose, two additional F11R10 constructs were synthesized containing the longer 58 bp (NEC-representative) and 60 bp (GEARS-representative) GA-repeat length sequences. A three-way comparison between the GA-50 (Sprague-Dawley), GA-58, and GA-60 constructs in further transfection experiments revealed a modest but significant increase in activity of the GA-58 and GA-60 constructs compared with GA-50 construct, but no apparent difference in activity between the latter two constructs , F = 31.592, n = 8/group). The GA-repeat sequence is therefore identified as having regulatory function in this promoter context. Additional analysis of this repeat sequence on the UCSC genome browser revealed a high level of conservation  HT22 cells, and compared with that of Cacng2 mRNA . Interestingly, however, the control lanes used for the end-point PCR analysis , an observation that requires investigation in further studies .Following the demonstration of brain-specific expression of mRNA Fig. in whichCacng2 promoter with associated lncRNA transcripts, further RT-PCR and transfection studies were performed. Initially, RT-PCRs were designed to identify more 5′ lncRNA sequence. This analysis was based on the genomic location of a (+ strand) rat EST (AI535545.1), and a similar mouse AK043153 RNA that are both initiated upstream of the rat lncRNA transcripts identified above, therefore indicating proximity to the Cacng2 promoter. Using primers RlncF2 and RlncR3 . Therefore, the lncRNA(s) are initiated (at most) just 594 bp from the Cacng2 initiator sequence, and, given the location of AI535545, possibly as little as 555 bp. Taken together with our Cacng2 promoter analysis, this sequencing confirms the predictions of current genome annotation (see above), namely that the (reverse-oriented) Cacng2 and lncRNA transcripts are initiated proximally, and may therefore share a bidirectional promoter. To test this hypothesis, further transfection experiments were conducted using an additional (reverse orientation) construct based on the 801 bp Cacng2 promoter construct that includes the initial 25 bp of the 5′ lncRNA sequence identified above. Using both HT22 and PC12 cells, it was shown that the 801 bp promoter sequence significantly enhanced transcription in both orientations, providing strong, functional evidence for the presence of a bidirectional promoter in this region  = 16.781, p < 0.05, Dunnett T3 test; PC12: F (2.17) = 30.298, p < 0.05, Dunnett T3 test; n = 6/group). Interestingly, the reverse orientation construct was relatively more active in PC12 cells, driving significantly more transcription than the (forward (Cacng2)-oriented) construct in this cellular context . Further studies are required to identify the possible physiological significance of this finding, but clearly, the promoter can be relatively more active in this orientation, which is interesting given the high levels of brain lncRNA (relative to Cacng2) transcripts identified in our previous RT-PCR analysis  contributory genetic factor in this polygenic animal model  was also identified, but was not found to correlate with ns Table . FurtherCacng2 GA-50 and Cacng2 GA-58/60 is unknown, but may involve TF binding. In Drosophila sp. two zinc finger proteins, GAF (GAGA factor) and CLAMP (Chromatin-linked adaptor for MSL proteins) bind GA repeat sequences and regulate gene expression, for example, at a bidirectional histone promoter  shows that conserved Cacng2 REST sites in other species are associated with additional repressive associations, including Sin3A and HDAC, providing a basis for powerful, co-repressor-dependent silencing of this neuronal gene , a common observation for these non-coding RNAs ESM 2(XLSX 34 kb)


Drosophila melanogaster embryos that low-affinity Ultrabithorax (Ubx)-responsive shavenbaby (svb) enhancers drive expression using localized transcriptional environments and that active svb enhancers on different chromosomes tended to colocalize . Here, we test the hypothesis that these multi-enhancer ‘hubs’ improve phenotypic resilience to stress by buffering against decreases in transcription factor concentrations and transcriptional output. Deleting a redundant enhancer from the svb locus led to reduced trichome numbers in embryos raised at elevated temperatures. Using high-resolution fluorescence microscopy, we observed lower Ubx concentration and transcriptional output in this deletion allele. Transcription sites of the full svb cis-regulatory region inserted into a different chromosome colocalized with the svb locus, increasing Ubx concentration, the transcriptional output of svb, and partially rescuing the phenotype. Thus, multiple enhancers could reinforce a local transcriptional hub to buffer against environmental stresses and genetic perturbations, providing a mechanism for phenotypical robustness.We previously showed in  Svb is ax (Ubx) . Thus, aDrosophila melanogaster embryos that Ubx transiently but repeatedly explores the same physical locations in a nucleus, which are likely clusters of binding sites  are Ubx-responsive and preferentially appear near or overlap spatially with transcription sites of the endogenous svb gene, despite being on different chromosomes  on a different chromosome. We observed that Ubx concentration around active transcription sites of the DG3-deletion allele and their transcriptional output increased when the svbBAC is physically nearby. Moreover, we found that trichome formation was partially rescued at high temperature. As a result, our findings support the hypothesis that shared microenvironments provide a mechanism for phenotypic robustness.To understand the mechanistic implications of having multiple enhancers in a shared microenvironment, here we examined the ability of the svb enhancers DG3, E3 and 7 . In wild-type embryos, the DG3 reporter gene was expressed ventrally in stripes along segments A1-A7, in addition to narrow thoracic stripes in T1-T3  and can induce ectopic expressions when overexpressed. These data are consistent with our previous observation of the localization of DG3-driven transcription sites within Ubx microenvironments svb) led to reduced phenotypic robustness of svb under non-optimal temperatures, with reduced numbers of trichomes produced svb) allele (108Df(X)svb flies to other lines . Also, we observed defects in trichome formation in the dorsal edges of the stripe pattern, which are exclusively covered by DG3 svb allele developed at 32°C produced fewer trichomes compared to wild-type larvae  allele , which wn of DG3 . Howevere larvae . We alsoe larvae . These re larvae . However108Df(X)svb deletion allele, we imaged Ubx distributions and the transcriptional output of the svb gene in fixed Drosophila melanogaster embryos using high-resolution confocal microscopy. We reasoned that the defect could be due to changes in the transcription factor concentration around the enhancers (input) and/or the transcriptional output of the gene (output). As Ubx is specifically needed to drive DG3 and svb expression on the ventral surface of the A1 segment, we used it as a metric for the transcription factor distributions around svb transcription sites. The samples were stained with immunofluorescence (IF) for Ubx and RNA fluorescence in situ hybridization (FISH) for svb transcription sites as previously described svb allele, raised at either 25°C or 32°C. For all imaging experiments involving the svb allele with the deletion, we selected only homozygous embryos for imaging . Because DG3 expression in the A1 segment showed a clear link to changes in Ubx level, we focused most of our subsequent imaging quantifications in this segment.To address molecular sources that may lead to the reduced number of ventral trichomes we observed for the escribed . We imagsvb transcription intensity’ in materials and methods). In nuclei from the A1 segment, Ubx intensities around svb transcription sites with the wild-type allele did not significantly change between 25°C and 32°C svb) allele had a local Ubx concentration that is slightly lower than wild-type at 25°C svb allele at 25°C showed reduced levels of transcriptional output slightly lower than the wild-type under heat-shock. At 32°C, the transcriptional output further decreased in the mutant. When the various conditions were plotted by their svb transcriptional output and Ubx intensity  centered on the transcription site svb allele  flies  (svbBAC-dsRed and 108Df(X)svb flies, suggesting that the co-localization of related regulatory regions could occur under stressed conditions. This effect was not observed for the unrelated regulatory region of diachete driving the expression of gfp, which was inserted on a BAC in the same chromosomal location as svbBAC.To test the rescue, embryos or larvae with a 8) flies . Similar3 and 7) . This obsvb regulatory region was able to rescue both molecular and functional defects observed from the loss of the region containing DG3. Both svb transcriptional output . The pairs of transcription sites under and above this threshold clustered in two groups distinguishable also by Ubx and svb transcription levels (1118w) x svbBAC-dsRed embryos and larvae were similar to wild-type in both trichome number and Ubx levels around svb transcription sites , Ubx concentration increases after a certain point were not clearly coupled to increases in svb transcriptional output. Additional regulatory mechanisms could be at play beyond transcription factor retention that determines the final transcriptional output of the locus. Determining the complete response function would be complicated, due to reasons such as the enhancers each having clusters of factor binding sites and the overlap of expression patterns from related enhancers. As svb also accepts inputs from many additional transcription factors  at the svb locus instead of the DG3 enhancer on the svbBAC driving svb expression in trans. Interactions in trans may serve an auxiliary role through influencing the properties of the local transcriptional environments.We were able to partially rescue the n allele . This suproteins  or otherproteins  could ovproteins . Interessvb transcriptional output and the number of cells fated to become trichomes is sigmoidal  would not lead to significant changes in trichome production and would appear to be wild-type in phenotype. The cis-regulatory region of wild-type svb under ideal conditions likely already saturates the system, as evidenced by the lack of change in Ubx concentration and trichome numbers even with the addition of svbBAC  it is in and how the gene loci positioned themselves into these locations. Future works to visualize and track genes, regardless of their transcriptional state, in fixed and living embryos would answer key questions on how they find, form and interact with transcriptional microenvironments.Summarizing our observations, we hypothesize that the relationship between f svbBAC . AlthougDG3-deletion allele of svb, we have shown that transcriptional microenvironments could span multiple enhancers that share similar transcription factor binding sites. These microenvironments of transcription factors could form the protein core of transcription factor ‘hubs’ that have been proposed to form through phase-separation mediated through protein-protein interactions between disordered domains svb svX)svb108 ; svbBAC-AC-dsRed ; diBAC-gvering D . Unless D. melanogaster strains were maintained under standard laboratory conditions, reared at 25 °C, unless otherwise specified. For heat-shock experiments, these conditions were followed: for staining with fluorescent antibodies, flies were allowed to lay eggs on apple-juice agar plates for 5 hr at 25 °C and then kept in an incubator at 32 °C for 7 hr before fixation; for cuticle preps, dechorionated embryos were kept at 32 °C until they emerged as larvae. 108Df(X)svb embryos/larvae with svbBAC-dsRed are readily discernable by the loss of svb and trichomes in the T1 segment (see ment see .108Df(X)svb allele, only larvae lacking trichomes in the T1 segment were imaged  or the first intron and second exon (16 kb) of svb. See Standard protocols were used for embryo fixation and staining . SecondaDrosophila embryos was done in ProLong Gold + DAPI mounting media . Fixed embryos were imaged on a Zeiss LSM 880 confocal microscope with FastAiryscan . Excitation lasers with wavelengths of 405, 488, 561 and 633 nm were used as appropriate for the specific fluorescent dyes. For imaging in embryos carrying 108Df(X)svb, only embryos without svb mRNA expression in the T1 segment were imaged, following the same reason described in the section on ‘Cuticle preparations and trichome counting’. Unless otherwise stated, all images were processed with Fiji/ImageJ  region of interest (ROI) centered on the transcription site is then created. The integrated fluorescent intensity inside the ROI from the Ubx IF channel and the RNA FISH channel are then reported as the local Ubx concentration and the transcriptional output, respectively. The intensity presented in the figures is the per-pixel average intensity with the maximum readout of the sensor normalized to 255.Inside nuclei with svb and dsRed/gfp transcription sites, the centers of the transcription site were identified using the find maximum function of Fiji/ImageJ. The distance between the transcription sites were then computed using the coordinates of the transcription sites. Two sites are considered colocalized when they are within 360 nm of each other.Inside nuclei with Drosophila melanogaster embryos between stages 10 and 12.The ChIP profile for Ubx around the svb cis-regulatory region is from  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article is being evaluated by three peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor.Thank you for sending your article entitled "Multi-enhancer transcriptional hubs confer phenotypic robustness" for peer review at Given the list of essential revisions, including new experiments, the editors and reviewers invite you to respond within the next two weeks with an action plan and timetable for the completion of the additional work. We plan to share your responses with the reviewers and then issue a binding recommendation.Essential points to address are the following:svb that do not correlate with the TF concentration. Thus, it is unclear whether the experiments described here support or reject the hypothesis laid out in the "multi-enhancer "hubs" improve robustness by increasing transcription factor retention near transcription sites."1) The connection between enhancers, Ubx protein concentration, transcriptional output, and phenotype. Although different concentrations of Ubx are measured at the deleted vs. wild-type locus, an important point that the three reviewers agree upon is that gene expression levels and Ubx concentrations are not well correlated. There appear to be other unexplained factors that are influencing the expression of svb transcription within each condition. A positive correlation would support their hypothesis.The authors must consider further whether the global measurements shown in Figure 3 are the most effective measure of this proposed correlation, or whether a more fine-grained analysis might support the idea that boosted Ubx levels from complexed enhancers are driving transcription. As noted below, the authors might show scatter plots of Ubx concentration vs. The trichome phenotype is also not directly correlated to the transcriptional outputs and Ubx concentrations, and as mentioned in the Discussion, further layers of regulation are likely in play. This idea needs to be more fully fleshed out.svb background, only in a deletion background. Does this finding indicate that such hub formation is saturable? And if so, does this indicate that the wild-type svb locus forms a local hub with enhancers in the immediate vicinity, and trans-complementation is not a normal feature of svb function?A finding related to "enhancer hubs boosting local concentration" is the finding that an ectopic BAC appears not to influence Ubx concentration in a wild-type DG3 enhancer drives gene expression in ventral abdominal stripes, but does not rescue Ubx concentrations from a trans setting. A larger deletion that includes DG3 as well as additional Ubx binding regions  impacts transcription, trichome development, and robustness. A yet larger cis-regulatory domain on a BAC rescues some aspects of gene expression and Ubx concentration. The interpretation conflates DG3 with the function of the deleted region; reviewers noted that a more careful interpretation would differentiate results from each of these different cis elements. For instance, the lack of trans-rescue by the DG3 enhancer alone may be due to the inability of a short segment to transvect effectively. The interpretation should explicitly take into account known properties of transvecting regulatory loci in Drosophila.2) A second point raised concerned the exact cis regulatory regions in play. A minimal 3) Several aspects can be addressed by better justification and explanation of methods and data presentation, including– Why sometimes either A1 or A2 trichomes are quantitatively assessed, depending on the figure;– The use of one-tailed (vs. two-tailed) T-tests for statistical relevance;– Recommendations for inclusion of both 25C and 32C phenotypes and data uniformly, and;DG3 and Ubx roles are difficult to know based on the single images shown;– Clarification of exact role of Ubx in T1-T3 regulation, as the conclusions drawn about – The number of data points in Figures 3 and 4 are limited; is there a technical limitation to more extensive sampling of the transcriptional readouts and Ubx intensities?4) In some cases, the actual experimental approach was unclear to the reviewers:DG3 deletion mutants were homozygous, so there would be no wild-type copy of the gene in these embryos – is that indeed the case?– there was some confusion about whether the svb, but not dsRed mRNA expression; is that correct? Imaging of dsRed only used to score a svb locus as "overlapped" vs. "not overlapped"?– Figure 4 shows – Choice of pixel size for ROI.– Signal to noise for Ubx across the nucleus, as well as variation in average Ubx from sample to sample.Reviewer #1:This paper addresses a timely topic, namely how multiple enhancers may coordinate to regulate gene expression. This is being actively investigated in a number of systems using both case studies and genome wide methods, and the field is fueled by new capabilities in high resolution microscopy. Thus, interest in the topic is high. However, I have substantial concerns about how this work contributes to this topic.svb; this increased level of transcription allows svb expression to be robust to high temperature variation. However, the evidence presented in this paper does not consistently support this hypothesis.The central hypothesis of this work is that increased levels of Ubx, provided by micro-environments created through coalescing multiple Ubx-responsive enhancers, increases the level of transcription of shavenbaby, svb transcription.Let's first consider the set of experiments correlating the presence of the enhancer deletion (via a deficiency), the concentration of Ubx and the level of svb enhancer (DG3) decreases resilience to high temperature stress, resulting in smaller numbers of trichomes in A1.– Deleting an svb enhancers localize to a microenvironment where Ubx concentrations are high compared to background.– In the A1 segment, svb enhancer decreases the concentration of Ubx in these microenvironments moderately at high temperature, but has no effect at low temperature.– Deleting an svb decreases at both WT and deficiency alleles.– At high temperatures, transcription of svb decreases under low temperature stress for the deficiency allele.– At low temperatures, transcription of svb transcription is not consistently correlated with Ubx levels in the microenvironment.These results do not support the hypothesis because svb locus is integrated onto another chromosome. This partially rescues the trichome phenotype of the deficiency allele. However, the Ubx levels in microenvironments were unchanged from WT. If the extra svb regulatory region associates with the endogenous svb transcription site, this should either result in above WT levels of Ubx, or there is some additional layer of regulation. This was not discussed.A second concern is the interpretation of the rescue experiment, where a BAC containing the full Reviewer #2:svb.In this paper, Tsai et al. studied the general problem of how low affinity binding sites, which promote enhancer specificity, also permit robust activation. They focus on ventral "shadow" enhancers for shavenbaby/DG3 enhancer, which was less studied before, and responds to Ubx in A1. Indeed, ChIP-seq data shows Ubx binding in a DG3 element that drive reporter gene expression in trichome forming territories, and this reporter responds to perturbations of Ubx.The authors start by focusing on the 108Df(x)svb, removes DG3, as well as several other Ubx-bound regions, and causes trichome formation defects only upon heat stress, and primarily in the region of DG3 activity.A deletion, svb transcription and Ubx protein distribution show a reduction of Ubx concentration in the vicinity of active svb loci, specifically in 108Df(X)svbmutant cells under heat stress, and a reduction of transcriptional output upon heat stress, even in wild-type embryos.State-of-the-art imaging of svb cis-regulatory DNA could rescue the formation of a Ubx-rich "hub" containing the endogenous svb locus, and svb transcription, in 108Df(X)svb mutants, but only when the two loci colocalized.Remarkably, exogenous svb expression and trichome formation. While the specifics are rather specialized, the problem of transcriptional hub formation and robust gene expression is a general problem of importance.Taken together, the data presented is consistent with the notion that multiple enhancers form a Ubx-rich microenvironment necessary for robust Nevertheless, there are still specific issues with the main conclusions based on the work presented, which seems somewhat preliminary.108Df(X)svbremoves more than DG3, including elements that clearly bind Ubx as per Figure 1—figure supplement 1, so it is not clear if the observed defects can be attributed to loss of DG3. Ideally, CRISPR/Cas9 should be used to remove Ubx-bound regions outside DG3 (e.g. Ubx-bound DG2) and to remove DG3 specifically, in order to support the conclusions that the observed effects of 108Df(X)svb are due to a loss of DG3.According to Figure 2, DG3 remains active in the most dorso-lateral cells in Ubx mutant , in a domain that seems to be specifically marked by DG3 reporters . This domain is portrayed as primarily affected by reduction of trichome formation in 108Df(X)svb mutants, but then it is not clear that this defect is due to a lack of Ubx activity on DG3 (both because DG3-LacZ seems to remain active and because the region removed by 108Df(X)svbbinds Ubx outside DG3).108Df(X)svbembryo under stress, and the impact of heat stress on transcription even in wild-type embryos further indicates that changes in Ubx accumulation is only part of the explanation for both the 108Df(X)svb phenotype and the response to heat stress.In Figure 3, the discrepancy between robust accumulation of Ubx, except in 108Df(X)svbchromosome, and then help place the svb locus in a Ubx-rich microenvironment. This is implied but not explicitly stated, or clearly demonstrated by the data.The rescue presented in Figure 4 is a strong result but also difficult to interpret: for instance, it seems likely that the "hub" forms independently of Ubx, which presumably cannot accumulate on the DG3 and Ubx govern the formation of the svb containing micro-environment and svb transcription. As acknowledged also in the Discussion, robustness of trichome formation also involves mechanisms downstream of svb transcription, making the link between Ubx hubs and robust trichome formation even more complex.For instance, Figure 5 adds to the notion that regulatory inputs other than svb locus explains the phenotypes described for 108Df(X)svb. Rescue experiments more directly combining loss of DG3 function with gain of Ubx function should help resolve these issues.Taken together, a few of the criticisms above question whether the accumulation of Ubx, or the lack thereof, in microenvironments containing the Reviewer #3:svb) loci preferentially colocalized with regions of elevated concentration for the transcription factor Ubx, a known regulator of svb activity. Moreover, they observed that other Ubx target genes exhibited a tendency to colocalize to these same Ubx-enriched "microenvironments", even when located on different chromosomes. Based on these findings, they hypothesized that the spatial overlap between multiple distal enhancers and transcription factor microenvironments could result in increased transcriptional output and could be a mechanism for redundancy in the face of environmental stresses. In the present work, Tsai, et al. test this hypothesis directly by deleting the DG3 enhancer from the svb cis-regulatory region and assessing the effects on microenvironment formation, svb transcription, and trichome number (a phenotype tied to svb expression) under normal (25C) and heat-stress (32C) conditions. This manuscript constitutes an intriguing extension of the findings presented in the authors' earlier work on Ubx microenvironments in the vicinity of svb loci. They find that the deletion mutant results in a significant depletion in Ubx concentration around the mutated svb locus at 32C, and that this depletion coincides with a reduction both in svb transcription and trichome number. Furthermore, they find that the addition of the full cis-regulatory region of svb on a different chromosome leads to preferential colocalization between the added region and the original mutated svb locus. The authors report that local concentration of Ubx in the vicinity of these colocalized enhancer regions recovers to wild-type levels under heat shock. They argue that, consistent with their initial hypothesis, the coincident partial recovery of svb transcription and trichome number indicates that this colocalization of multiple Ubx targets to a single microenvironment is indeed functioning to counteract the embryo's mutation-induced sensitivity to heat shock conditions.In their previous work, Tsai, et al. observed that, during fruit fly embryogenesis, actively transcribing shavenbaby  change across conditions, but also with respect to one another.Perhaps most fundamentally, the authors' conclusions hinge upon the assumption that the Ubx enrichment signal they observe at active loci play a causal role in modulating transcriptional activity, yet they present no data to directly support this assumption. Indeed, the box plots in Figure 3 C and D seem to show that, while both Ubx concentration and transcription decrease in response to heat, Drosophila development also necessitates rapid, precise transcriptional responses to changes in transcription factor concentrations. Thus, naively at least, if robustness leads to desensitization of the response of an enhancer to changes in the concentrations of inputs transcription factors, one might expect locus colocalization to actually be deleterious in the case of genes that must respond rapidly to time-varying transcription factor inputs. Augmenting the existing commentary to address this point would help clarify the implications of the present work. Does this trade-off make predictions regarding where and when this kind of clustering might manifest over the course of development (and for what genes)?On a more conceptual note, the authors' proposal that the colocalization of enhancers in transcription factor microenvironments could serve as an added layer of redundancy (akin to multiple TF binding sites and multiple enhancers) to increase the transcriptional robustness is thought-provoking. The concept of robustness is used throughout biology with different meanings, and further discussion of the implications and nuances of this proposal would also help establish what exactly the authors mean by it. For instance, what the authors cast as "robustness" could equally well be termed "desensitization". While robustness to environmental changes could be desirable, the fast pace of General Comments:svb loci per embryo at this point in development. If the full set of svb loci was not used for the analyses presented, can the authors comment on how the analysis subset was selected. Also, given that many of the effects presented are relatively subtle, the addition of data points (to the extent that it is feasible) would greatly enhance the robustness of the analysis and might lead to the discovery of additional trends within the data not evident in such small sample sizes. Given that the authors get multiple nuclei per embryos, is there a fundamental limitation to how much data they can present such as, for example, their data analysis pipeline?Can the authors comment on why the box plots in Figure 3 and Figure 4 contain so few data points? All contain 52 or fewer, yet one would expect there to be many more active As mentioned above, this work leans heavily upon the presumed causal relationship between local Ubx concentration and transcriptional output. The work would be greatly strengthened by the addition of bivariate analyses that rigorously test the relationship between Ubx concentration and transcription within each of the four conditions shown in Figure 3 C and D.svb site is colocalized with the svbBAC more or less recovers Ubx concentration at the endogenous (unperturbed) svb locus, yet the authors do not address whether the resulting signal is less than, equal to, or greater than the sum of the signal at each locus (Df(X)svb and svbBAC) when they are not colocalized. Showing how the enrichment signal at colocalized loci compares to the signal at each separately would indicate whether and to what degree some sort of synergy is at play, or whether the increased signal is merely a result of having more Ubx binding sites in region.The boxplots in Figure 4C show that Ubx concentration at loci where the mutated svb locus vary with the radial separation between loci? What about the Ubx intensity?While the trends in Ubx intensity, radial separation, and transcriptional activity shown in Figure 4C, E, and F are interesting taken individually, much more could be learned from the data by making bivariate plots. How does transcriptional output at the mutated  [Editors' note: the authors’ plan for revisions was approved and the authors made a formal revised submission.]Essential points to address are the following:1) The connection between enhancers, Ubx protein concentration, transcriptional output, and phenotype. Although different concentrations of Ubx are measured at the deleted vs. wild-type locus, an important point that the three reviewers agree upon is that gene expression levels and Ubx concentrations are not well correlated. There appear to be other unexplained factors that are influencing the expression of svb that do not correlate with the TF concentration. Thus, it is unclear whether the experiments described here support or reject the hypothesis laid out in the "multi-enhancer "hubs" improve robustness by increasing transcription factor retention near transcription sites."svb could have a positive but complicated relationship to Ubx concentrations and would depend on more than Ubx alone. We now state this in the second paragraph of the Discussion. However, Ubx is a crucial driver of svb and, specifically, also DG3 expression in the ventral region of the A1 segment  and Figure 1 of this manuscript. We specifically focused most of our analyses and quantifications in A1. We now state this in the Results section, “Transcription sites from the DG3‐ deletion allele have weaker Ubx microenvironment and lower transcriptional output”.The reviewers are correct to point out that there are multiple inputs that control the response of the svb locus . Therefore the response function of svb transcription sites would be a reasonable metric of how local transcription factor concentrations changed when we perturbed the system through mutations and elevated temperature. We now state this in the Results section “Transcription sites from the DG3‐deletion allele have weaker Ubx microenvironment and lower transcriptional output”. With these caveats in mind, we did observe increased Ubx concentrations and around the wildtype svb transcription sites  compared to the deletion allele, and increased phenotype resilience for the wildtype allele at 32 °C. In the svbBAC rescue, we also observed that colocalized svbBAC and DG3‐deleted svb allele had both higher Ubx concentrations and svb transcriptional outputs . We, therefore, believe that our results support the idea that multi enhancer hubs help transcriptional factor retention and can ultimately lead to a more robust phenotype. We believe the analysis that the reviewers suggested below also supports our hypothesis of improved transcriptional factor retention with complexed enhancers.Within this specific body segment, Ubx intensity near The authors must consider further whether the global measurements shown in Figure 3 are the most effective measure of this proposed correlation, or whether a more fine-grained analysis might support the idea that boosted Ubx levels from complexed enhancers are driving transcription. As noted below, the authors might show scatter plots of Ubx concentration vs. svb transcription within each condition. A positive correlation would support their hypothesis.svb transcription vs. Ubx intensity. There is initially a positive correlation between svb transcriptional output and Ubx intensity, in line with the reviewers’ proposal. This trend dissipates at higher Ubx and svb intensities, indicating that the response of svb output to Ubx concentration is not a simple relationship. Capturing the exact dependence of transcriptional output on Ubx concentration is difficult for the reasons we stated in the second paragraph of the Discussion . To fully address this would require future live imaging experiments that can track the gene locus regardless of its transcriptional state, in addition to reporting on its transcriptional activity and transcription factors around it. Numerous new reagents  are being developed in the lab to address this.We have modified the analysis in Figure 3C to show The trichome phenotype is also not directly correlated to the transcriptional outputs and Ubx concentrations, and as mentioned in the Discussion, further layers of regulation are likely in play. This idea needs to be more fully fleshed out.svb transcriptional output is positive but complex, likely involving additional factors (paragraph two) and 2) the relationship between svb transcriptional output and phenotype is sigmoidal (paragraph four) and the wild‐type system operates in a saturated regime under ideal conditions (paragraph five). The processes leading to the response functions in 1 and the phenotypical tolerance to a range of svb out in 2 remain to be investigated (paragraphs two and five), specifically with live imaging approaches. As mentioned in the previous paragraph, work is currently ongoing in the lab to address this.We now have a new figure  summarizing our hypothesis and referred to this figure throughout the Discussion to clarify our proposed mechanism. In short, we propose in our revised Discussion that 1) the relationship between Ubx concentration and A finding related to "enhancer hubs boosting local concentration" is the finding that an ectopic BAC appears not to influence Ubx concentration in a wild-type svb background, only in a deletion background. Does this finding indicate that such hub formation is saturable? And if so, does this indicate that the wild-type svb locus forms a local hub with enhancers in the immediate vicinity, and trans-complementation is not a normal feature of svb function?cis‐regulatory region of the wildtype svb is sufficiently in saturation to deal with environmental challenges as seen in trichome numbers. We now state this in the Discussion (paragraph five). Our data, however, does not provide a direct answer as to if trans‐ chromosomal interactions are a normal feature of svb or other genes. Ongoing efforts in the lab to characterize and map these long‐range interactions through imaging and genomics approaches should shed light on their functional impact during development in the future.We agree with the reviewers that hub formation is saturable, based on this observation. We also believe that local hub formation with the 2) A second point raised concerned the exact cis regulatory regions in play. A minimal DG3 enhancer drives gene expression in ventral abdominal stripes, but does not rescue Ubx concentrations from a trans setting. A larger deletion that includes DG3 as well as additional Ubx binding regions  impacts transcription, trichome development, and robustness. A yet larger cis-regulatory domain on a BAC rescues some aspects of gene expression and Ubx concentration. The interpretation conflates DG3 with the function of the deleted region; reviewers noted that a more careful interpretation would differentiate results from each of these different cis elements. For instance, the lack of trans-rescue by the DG3 enhancer alone may be due to the inability of a short segment to transvect effectively. The interpretation should explicitly take into account known properties of transvecting regulatory loci in Drosophila.svb locus as an important factor in if phenotype rescue takes place. We now state that DG3 alone as the rescue locus might have failed due to its inability to pair with the svb locus in the Discussion (paragraph four). We also now compare and contrast the svbBAC‐svb interactions that we observed with transvection and hypothesize that the addition of other topological elements such as insulator to DG3 could overcome this problem (paragraph four). We further state that the svbBAC did not rescue trichomes in regions where DG3 provided exclusive coverage, suggesting that the rescue BAC rescued the phenotype by overdriving the other ventral enhancers E3 and 7 rather than directly restoring DG3 function.We agree that we should take into account the efficiency of the rescue locus in finding the 3) Several aspects can be addressed by better justification and explanation of methods and data presentation, including– Why sometimes either A1 or A2 trichomes are quantitatively assessed, depending on the figure;DG3 in the A2 segment responds to additional inputs beyond Ubx, as explained in the Results section “The DG3 enhancer responds specifically to Ubx in the A1 segment”.This issue was an oversight on our part, and we have updated all the main figures where we counted trichomes to be from the A1 segment for consistency. We moved data from the A2 segment to figure supplements as they also show a similar but weaker trend as in A1. We speculate that this is due to additional factors at work as – The use of one-tailed (vs. two-tailed) T-tests for statistical relevance;We have changed our test to two‐tailed T‐tests throughout, as is the standard. This did not change of our findings.– Recommendations for inclusion of both 25C and 32C phenotypes and data uniformly, and;svbBAC rescue experiments at 25 C as neither the wildtype nor the deletion allele displayed reduced trichome numbers (phenotype output), and the difference between the Ubx intensities (molecular input) around transcription sites of both genotypes was small at this temperature.We added trichome images from 32 C to Figure 2 and for the data analysis involving Ubx intensity and svb transcriptional output in Figure 3. We did not conduct – Clarification of exact role of Ubx in T1-T3 regulation, as the conclusions drawn about DG3 and Ubx roles are difficult to know based on the single images shown,svb, and DG3 specifically, on the ventral surface in segments outside of A1 is more complicated . DG3 expression in the thoracic segments T1‐T3 overlaps with other ventral svb enhancers and responds to changes in Ubx level less that in the A1 segment. T2 and T3 also show only low levels of expression from DG3 on the ventral surface with wild‐type Ubx expression. This issue potentially introduces many confounding factors for quantitation. We have thus confined ourselves to qualitative descriptions of DG3 properties outside of A1 and A2. We now state this concern at several places in the Results and Discussion.As the reviewers noticed, the role of Ubx in regulating – The number of data points in Figures 3 and 4 are limited; is there a technical limitation to more extensive sampling of the transcriptional readouts and Ubx intensities?In the process of doing the additional analysis suggested by the review, we added more data points, and the number of transcription sites and embryos quantified is comparable to the original publication this manuscript is linked to . As in the previous publication, colocalization is a relatively rare event, so we observed fewer transcription sites. The number observed for colocalized sites is also similar to our previous publication.4) In some cases, the actual experimental approach was unclear to the reviewers:– There was some confusion about whether the DG3 deletion mutants were homozygous, so there would be no wild-type copy of the gene in these embryos – is that indeed the case?108Df(X)svballele based on the following phenotype in the T1 segment: the lack of trichomes or the lack of svb mRNA expression, respectively. This is a homozygous marker for the deletion allele as the wild‐type svb allele expresses in this segment. This was mentioned in “Deletion of a region including DG3 enhancer causes defects in ventral trichome formation specifically at elevated temperatures” in Results, “Preparing Drosophila embryos for staining and cuticle preps” in Materials and methods and the legends for Figure 2C in our original manuscript . We have now made it explicit in the Results that we use this to select for animals homozygous for the deletion for both counting trichome number and for confocal imaging. It is now also described in both “Cuticle preparations and trichome counting” and “Imaging fixed embryos” in the Materials and methods section.Yes, we selected larvae or embryos homozygous for the – Figure 4 shows svb, but not dsRed mRNA expression; is that correct? Imaging of dsRed only used to score a svb locus as "overlapped" vs. "not overlapped"?svb mRNA output. Imaging of dsRed transcription site is only used to score colocalization with svb. We displayed image panels for individual nuclei that show a dsRed signal for illustrative purposes. We explicitly stated that we are displaying svb transcription in both the Figure 4E and the legends.Yes, the data analysis in Figure 4 only used only – Choice of pixel size for ROI.DG3‐deletion allele have weaker Ubx microenvironment and lower transcriptional output” in the Results and “Analysis of microenvironment and svb transcription intensity” in the Materials and methods.The 40‐pixel ROI is too large and a mistake in our part. The actual ROI size in the previous manuscript was a 4‐pixel square ROI; this has been changed to be a circle with a diameter of 4 pixels (170 nm). We chose this size because it is the resolution limit of the AiryScan images we acquired. We have corrected this and stated our rationale in the revision when we describe the image analysis in “Transcription sites from the – Signal to noise for Ubx across the nucleus, as well as variation in average Ubx from sample to sample.svb transcription there is a significant difference  from randomly sampled locations in the nucleus. This has also been added to the Results “Transcription sites from the DG3‐deletion allele have weaker Ubx microenvironment and lower transcriptional output”We used exactly the same antibodies and methodology as reported in  to stain for Ubx and the quality of the images acquired were the same. In the previous publication we explored the Ubx cluster characteristics. We now include a new supplemental figure  with Ubx variation across the nucleus within embryos, and from sample to sample (there is no significant difference). In contrast, at the sites of active


The main aim of this study was to investigate the molecular and expression characteristics of lncFAM200B along with its crucial genetic variations. Our results showed that bovine lncFAM200B was a 472 nucleotide (nt) non-coding RNA containing two exons. The transcription factor binding site prediction analysis found that lncFAM200B promoter region was enriched with SP1 transcription factor, which promotes the binding of myogenic regulatory factor MyoD and DNA sequence. The mRNA expression analysis showed that lncFAM200B was differentially expressed in embryonic, neonatal, adult bovine muscle tissues, and the lncFAM200B expression trend positively correlated with that of MyoG and Myf5 in myoblast proliferation and differential stages. To identify the promoter active region of lncFAM200B, we constructed promoter luciferase reporter gene vector pGL3-Basic plasmids containing lncFAM200B promoter sequences and transfected them into 293T, C2C12, and 3T3-L1 cells. Our results suggested that lncFAM200B promoter active region was from −403 to −139 (264 nt) of its transcription start site, covering 6 SP1 potential binding sites. Furthermore, we found a novel C-T variation, named as SNP2 (ERZ990081 in European Variation Archive) in the promoter active region, which was linked to the nearby SNP1 (rs456951291 in Ensembl database). The genotypes of SNP1 and combined genotypes of SNP1 and SNP2 were significantly associated with Jinnan cattle hip height. The luciferase activity analysis found that the SNP1-SNP2 haplotype CC had the highest luciferase activity, which was consistent with the association analysis result that the combined genotype CC-CC carriers had the highest hip height in Jinnan cattle. In conclusion, our data showed that lncFAM200B is a positive regulator of muscle development and that SNP1 and SNP2 could be used as genetic markers for marker-assisted selection (MAS) breeding of beef cattle.Skeletal muscle is one of the three major muscle types in an organism and has key roles in the motor system, metabolism, and homeostasis. RNA-Seq analysis showed that novel lncRNA,  Long non-coding RNA (lncRNA) is an important class of non-coding RNAs (ncRNAs), which are involved in a variety of biological processes. LncRNAs are usually greater than 200 nucleotide (nt) in length, mostly were transcribed by RNA polymerase II, and some were transcribed by RNA polymerase III. Similar to mRNAs, the expression of lncRNAs have obviously temporal (the same tissue on different development stages) as well as the spatial (different tissues) specificity. LncRNA gene has its own promoter, which can be recognized by specific transcription factors. In the last decade, lncRNAs have been showed to have multiple functions in many developmental processes, such as regulating gene expression by transcriptional, post-transcriptional, or epigenetic regulation . BesideslncMD, lncYYW, and lnc133b in bovine muscle development have been identified, the roles of numerous lncRNAs are still mysteries to be explored  is a rapid and efficient breeding method, which is based on crucial genetic variation markers . Thus, flncMD and some new lncRNAs (Bos taurus FAM200B gene (NCBI Reference Sequence: AC_000163.1), thus we aptly renamed it as lncFAM200B. In this study, we focused on lncFAM200B as it was differentially expressed in bovine embryonic, neonatal, and adult skeletal muscle . Based on the RNA-Seq results, we speculated that lncFAM200B probably plays an important role in the development of bovine skeletal muscle. lncRNAs . In theslncFAM200B and further, we identified the functional genetic variations in lncFAM200B gene. These results would lay the foundation for the function research of lncFAM200B and provide scientific data for beef cattle breeding.Therefore, in this study, we investigated the sequence and expression characteristics of bovine All experiments in this study were approved by the Faculty Animal Policy and Welfare Committee of Northwest A&F University (no.NWAFAC1008). The care and use of experimental animals is in full compliance with local animal welfare laws, guidelines, and policies.lncFAM200B, multiple tissue samples from Qinchuan steers at three different developmental stages: embryos of about 3 months old, newborns within 1 week, and adults of about 24 months old were collected from Shaanxi Kingbull Livestock Co., Ltd. . For sampling at each of the developmental stages, three individuals were used. For each neonatal and adult individual, seven types of tissue samples were collected . For embryonic stage, only six kinds of tissue samples were collected (without fat). All samples were frozen immediately in liquid nitrogen and stored at −80°C.To explore the expression profile of lncFAM200B.Total RNA was isolated from samples using TRIzol reagent . The quality of total RNA was evaluated by 1% agarose gel electrophoresis and NanoDrop 2000 spectrophotometer . Then PrimeScript™ RT reagent Kit with gDNA Eraser  was used to synthesize complementary DNA (cDNA), which was used as template for quantitative reverse-transcription PCR (qRT-PCR) or full-length amplification of lncFAM200B using bovine fetus skeletal muscle cDNA as template. The 3′RACE was done using PrimeScript™ RT reagent Kit  and 3′ RACE universal primers QT, QO, and QI as described in lncFAM200B were designed based on the sequence obtained from RNA-Seq (lncFAM200B was obtained through sequences assembly based on the results of 3′ and 5′ RACE.Rapid amplification of cDNA ends (RACE) experiments were carried out to identify the full-length of bovine  RNA-Seq Table 1.C/EBPα) and lncRNA H19 imprinted maternally expressed transcript (H19) were also calculated as control. NCBI-Open Reading Frame Finder (ORF Finder) was used to analyze the open reading frame (ORF) of lncFAM200B. The prokaryotic expression system was used to detect the protein coding ability of lncFAM200B. The full length of bovine lncFAM200B and enhanced green fluorescent protein (EGFP) were cloned into vitro prokaryotic expression system pET-28a vector using XhoI and HindIII restriction enzymes and In-Fusion® HD Cloning Kit  (http://www.mirdb.org/) was used to predict the interacting miRNAs, and AliBaba2.1  was used to predict the transcription factors that may bind to the promoter region of lncFAM200B.The coding potential was predicted on Coding Potential Calculator (CPC) website . The kno, China) . The miRlncFAM200B in tissues. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. The primers for qRT-PCR were listed in −∆∆Ct method (t-test (The qRT-PCR was performed to detect the expression of t method . The cor (t-test .2. The proliferation medium for myoblast contains 80% Dulbecco’s Modified Eagle Medium (DMEM), 20% fetal bovine serum (FBS), penicillin (10 U/ml), and streptomycin (10 mg/ml). When myoblast start to fuse, the proliferation medium was replaced by differential medium, which contains 2% horse serum, penicillin (10 U/ml), streptomycin (10 mg/ml), and DMEM. The RNA of the myoblast was collected using TRIzol reagent  at proliferation and differential stages. Mouse C2C12 myoblast cells, mouse 3T3-L1 embryo fibroblast, and human embryonic kidney 293T cells were used to uncover the active region of lncFAM200B promoter or single nucleotide polymorphisms (SNPs) effects on promoter activity. They were grown in 10% FBS, 90% DMEM, penicillin (10 U/ml), and streptomycin (10 mg/ml) medium.The procedure for separating bovine myoblast from skeletal muscle was the same as the previous study of our lab . Then celncFAM200B gene promoter, six fragments of the lncFAM200B promoter region were amplified and cloned into the pGL3-Basic vector  using SacI and SmaI restriction enzymes , pGL3-CT (SNP1-C and SNP2-T), pGL3-AC (SNP1-A and SNP2-C), and pGL3-AT (SNP1-A and SNP2-T) were constructed using overlap PCR to detect the effects of haplotype on promoter activity . Before transfection, cells were seeded into 96-well plate. When cells covered 80% of the culture plate bottom, the plasmids were transient transfected according to the manufacturer’s protocol. To normalize the transfection efficiency, the pRL-TK was transfected with constructed plasmids, and the transfection ratio of constructed plasmids and pRL-TK was 50:1 . All tralncFAM200B promoter region. The samples of Qinchuan cattle (n = 139), Jinnan cattle (n = 121), Nanyang cattle (n = 67), and Ji’an cattle (n = 25) were randomly collected from Shaanxi, Shanxi, He’nan, and Jiangxi provinces, respectively. The detailed information and records of body measurement traits for the cattle were the same as the published papers . After genotyping, the genotypic and allelic frequencies, population genetic diversity indexes  were calculated according to the methods described as http://www.msrcall.com/)  and haplotypes analyses were performed using SHEsis online platform   . Then th groups) .lncFAM200B. The 3′ and 5′ RACE obtained 174 bp and 323 bp sequences, respectively  was obtained by RNA-Seq , the 5′ ectively Figure 1very low . To ensulncFAM200B. Among these miRNAs, 5 miRNA scores were above 60, so we further predicted the target genes of these 5 miRNAs. As a result, some cell proliferation associated genes were uncovered, such as insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2)  was close to lncFAM200B. Thus, lncFAM200B might interact with FGFBP1 and affect cell proliferation and differentiation , and 43 SP1 transcription factor binding sites. lncFAM200B.The miRNA prediction analysis uncovered that 8 miRNAs might interact with ntiation . The tralncFAM200B, we investigated the expression profiles in bovine embryonic, neonatal, and adult tissues. In various bovine tissues, lncFAM200B was widely expressed in three developmental stages  and Myf5   and a novel genetic variant SNP2   Figure 7P > 0.05) in the four populations , Jinnan , and Ji’an  cattle populations showed these two loci were linked in cattle. The r2 reflects the extent of the linkage disequilibrium and r2 > 0.33 indicated that there was a sufficiently strong linkage between the two loci. When different genotypes are evenly distributed in the population, the D’ > 0.33 can also be used to judge that there was a linkage disequilibrium . At SNP2 locus, only CC and CT were identified in the four detected cattle breeds Figure 7ilibrium .P = 0.012). The hip height of the CC genotype carriers was 131.7 ± 6.7 cm, which was evidently higher than that of CA (128.6 ± 6.5 cm) and AA (127.1 ± 5.4 cm) genotype carriers, but we did not observe any significant difference between CA and AA genotype carriers (P = 0.033). The hip height of the CC-CC carriers  was markedly higher than that of CA-CT , CA-CC , and AA-CC  genotype carriers . But no difference was found among the other haplotypes  of SNP1 and SNP2 haplotypes were constructed and transfected into commonly used 293T cells to detect the luciferase activity. The luciferase activity of positive control (Control) was significantly higher compared to that of the negative control (empty Basic) and we found that the relative luciferase activity of pGL3-CC was the highest among the four haplotypes in 293T cells. The luciferase activities of pGL3-CC and pGL3-AT were significantly higher than that of the pGL3-CT haplotypes  to further prove that lncFAM200B was a novel lncRNA.With the rapid development of high-throughput sequencing technology, an increasing number of lncRNAs have been discovered in many animal species. Structurally, the lncRNA resembled protein-coding gene with its own promoter, exons, and introns. The lncFAM200B positively correlated with the expression of MyoG (P = 0.003) and Myf5 (P = 0.035). MyoG, a muscle-specific transcription factor, positively regulated the skeletal muscle fiber development, myoblast differentiation, and fusion, and was found to be indispensable for myogenic differentiation , one novel SNP  was discovered which linked with the nearby SNP1 (rs456951291). The genotypes of the SNP1 and the combined genotypes of SNP1 and SNP2 were significantly associated with the hip height in Jinnan cattle. Interestingly, haplotype CC had the highest luciferase activity and the highest hip height. Our results established that lncFAM200B is a positive regulator of muscle development and we believe that our studies will help in advancing the beef cattle MAS breeding program.The lncRNA The detailed information of SNP2 can be found in the European Variation Archive database after 2019/12/31. Project: PRJEB33081; Analyses: ERZ990081.The animal study was reviewed and approved by Faculty Animal Policy and Welfare Committee of Northwest A&F University (no. NWAFAC1008).SZ and XL conceived and designed the experiments. SZ, ZK, and CP performed the experiments. XS provided the RNA-Seq data. XC, RD, CL, HC, and XL collected the DNA samples. SZ and XL analyzed the data. XL contributed reagents, materials, and analysis tools. XL and SZ wrote the paper.This work was funded by the National Natural and Science Foundation of China (No. 31672400).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.