Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, we suggest that by promoting chromatin–nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. We show here that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in  S. cerevisiae Mps3, a SUN homologue is involved in the recruitment of heterochromatic sequences such as telomeric repeats to the nuclear envelope (NE), an essential process needed for spindle formation in the course of chromosome segregation [The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, implicated in mechanical coupling between the cytoplasm and nucleoplasm, has been proposed to control chromatin organization and the epigenetic state of chromatin ,2,3,4. Tregation . These sDrosophila larval muscles lacking functional LINC complex. We found that differentiated muscles lacking functional LINC complex exhibited increased epigenetic repression that correlated with enhanced genome binding of Polycomb and HP1a, together with reduced binding of RNA-Pol II to a set of essential muscle genes. The increased chromatin repression observed in the SUN/koi mutant muscle nuclei correlated with increased size of tri-methylated H3K27 chromatin clusters, and with partial translocation of chromatin from the periphery to nuclear center quantified from live 3D imaging. Simulations of the 3D distribution of tri-methylated H3K27 sites indicated that reduced binding of chromatin to the nuclear lamina leads to increased repressive cluster size. These results suggest that the enhanced chromatin repression observed in the LINC mutants was induced by weakening of chromatin binding to the nuclear lamina. Altogether our results imply that by coupling the cytoskeleton and the nucleoskeleton, the LINC complex restrains gene repression in mature muscle fibers.Previously, we demonstrated the functional consequences of disrupted LINC complex on fully differentiated larval muscle fibers, affecting proper larval muscle contractions, and nuclear position along the entire muscle fiber ,21, as w84/Cyo-dfd-eYfpkoi (FBst0025105) described previously [koi-RNAi (FBst0040924), delta KASH , ts/TM2tubP-GAL80 (FBst0007017), GAL4-Mef2.R (FBst0027390), UAS-Spastin , ubi-H2B-mRFP/CyO;Dr/TM6B [tsTub-Gal80; Mef2-Gal4 , UAS-2E12LI-EGFP(III)/TM6B , His2Av-GFP (FBst0005941). Fly lines for Targeted Dam-ID : UAS-LT3-Dm (FBtp0095492), UAS-LT3-Dm-RpII215 (FBtp0095495), UAS-LT3-Dam-Pc, UAS-LT3-Dam-HP1a [The following stocks were used: eviously , koi-RNAFBst00409, delta K;Dr/TM6B , Tub-Galfp FBst00105 descrSUN/koi and ΔKASH mutant larvae were selected by non-Cyo-YFP and confirmed with mis-localization/aggregation phenotype of muscle nuclei. Temporal SUN/koi knockdown (koi-RNAi) and overexpression of spastin was performed using a combination of Mef2Gal4 and tstubGal80 drivers, as follows: embryo collection was performed at 25 °C for 12 h, followed by transfer to a permissive temperature of 18 °C. Larvae were transferred to the restrictive temperature of 29 °C for 3 days (koi-RNAi) or 1 day (spastin) before the late 3rd instar (wandering larvae). tsTub-Gal80; Mef2-Gal4 fly line was used as control for SUN/koi mutant, koi-RNAi and spastin over-expression experiments.All crosses were carried and maintained at 25, 18, or 29 °C and raised on cornmeal agar. Homozygous We drove the expression of Dam-Polycomb , Dam-HP1a and Dam-Pol II (RpII215) and Dam only in control and SUN/koi mutant muscles using Mef2-gal4 driver . Temporal control of Dam expression was achieved with a ubiquitously expressed temperature-sensitive gal80. Each genotype contained 3 independent replicates, with 25 larvae per group. Eggs were laid for 6 h and developed in 18 °C until late larval second instar stage and transferred to 29 °C for 10 h to allow muscle-specific Dam expression. Larvae were then dissected and stored at −80 °C until all triplicates were further processed together following the previously described protocol . Brieflyhttps://github.com/owenjm/damidseq_pipeline (accessed on 6 November 2022). Mean occupancy per gene was then calculated using polii script https://github.com/owenjm/polii.gene.call (accessed on 22 September 2020). The reference genome was taken from flybase https://ftp.flybase.net/releases/FB2016_02/dmel_r6.10/ (accessed on 30 March 2016) and GATC sites from the damid pipeline website. After filtering genes with DamID false discover rate (FDR) < 0.05 (per sample), the three replicates from each group were directed to a clustering algorithm based on pairwise correlation.Each sample was processed separately by the damid pipeline to generate normalized ratios for each pair of samples; Dam fused to protein of interest versus Dam only SUN/koi mutant, relative to control, a regression on principal component analysis was performed with a z-score for each of the points around the regression line. The cut-off criteria for significantly altered binding to a gene was set to FDR < 0.05, z-score > 1.96 (2-tailed), and GATC sites > 1, corresponding to an approximately 95% confidence interval. The full list of identified genes for each group is provided in the https://data.mendeley.com/datasets/czjj5m7btr/1 (accessed on 17 March 2022). Independently, binding profiles  of specific genes were further visualized for control and SUN/koi mutant using the IGV browser [http://metascape.org (accessed on 17 March 2022) [To determine the genes with statistically significant occupancy in  browser . Gene Onch 2022)  using geQuantitative immunofluorescence of epigenetics marks was performed on 3rd instar, wandering larvae that were dissected in phosphate-buffered saline (PBS) and fixed, as previously described . BrieflyDrosophila larvae was designed in our laboratory, to be placed on top of a confocal microscope stage, as previously described [For imaging live nuclei in their intrinsic environment, a minimal constraint device for escribed . For staImmunofluorescence images of epigenetic marks were acquired at 23 °C on a confocal microscope Zeiss LSM 800 with a Zeiss C-Apochromat 40×/1.20 W Korr M27 lens and an Immersol W 2010 immersion medium. The samples were embedded with a coverslip high precision of 1.5 H ± 5 µm  and acquired using Zen 2.3 software (blue edition).Live imaging of H3K27me3-GFP was performed using an inverted Leica SP8 STED3× microscope equipped with internal Hybrid detectors, an acousto-optical tunable filter  and a white light laser excitation laser. Nuclei were imaged with a HC PL APO 86×/1.20 water STED white objective at a scan speed of 400 Hz, a pinhole of 0.8 A.U. and a bit depth of 12. Z-stacks were acquired with 0.308 µm intervals. The acquired images were visualized during experiments using LAS-X software .DNA-FISH imaging was performed using a Dragonfly spinning disk confocal system  connected to an inverted Leica Dmi8 microscope (Leica Microsystems CMS GmbH). The signals were detected simultaneously by two sCMOS Zyla (Andor) 2048 × 2048 cameras, 2 × 2 binning, CF40 pinhole, and 12-bit depth. Images were acquired with a 63×/1.3 glycerol objective and excited with 4 different laser lines (two channels per camera): 405, 488, 561 and 637 nm laser lines.Arivis Vision4D 3.1.2-3.4 was used for image visualization and analysis. Quantitative immunofluorescence analysis was performed with a dedicated pipeline that automatically segmented multiple nuclei per stack in 3D, using denoising and Otsu threshold operation on the independent Hoechst channel. The automated analysis restricts signal quantification only to the nucleus by segmenting the nuclear borders from the full z-stack, therefore eliminating the effects of cytoplasmic background on the mean nuclear fluorescence intensity. Nuclear volumes and total fluorescent intensities of the epigenetic marks and the DNA were calculated and exported to MATLAB R2019b (MathWorks) for further analysis.3 were filtered out. Radial 3D chromatin distribution analysis was performed as previously described [Quantification of live H3K27me3 puncta was performed by first assessing the signal intensity profile of each nucleus and subtracting the nuclear background intensity for each nucleus individually, preserving only the bright repressive puncta. Individual puncta were then automatically segmented in 3D using Otsu operation, and puncta below volume of 0.01 µmescribed  by generDrosophila genome with N = 35,240 beads connected by springs to model each chromosome, where a bead of diameter Drosophila, to a sphere which represents the nucleus. To model the relative lengths of the euchromatin, PCH and H3K27me3 blocks, we analyzed the experimental chip sequence data at 5 Kbps resolution and converted it into beads units [Drosophila genome regions are euchromatic and 30% are identified as PCH [Drosophila genome contains H3K27me3 markers based on its percentage in euchromatin and PCH. Using these values, we generated beads identified as H3K27me3 patches from an exponential distribution with a mean length of 17 beads, which covers 40% of the genome. These H3K27me3 patches were generated randomly along the genome using a Monte Carlo method [Drosophila nuclei [i j where i j can be E (euchromatin) or H (heterochromatin). Heterochromatin beads . Quantitative immunofluorescence parameters from control and  effects . The num effects , in whicSUN/koi mutant muscles in Drosophila third instar larvae. Whereas the two Nesprin-like genes of Drosophila have additional functions in cells, due to non-KASH containing isoforms, SUN/koi, the only SUN representative in Drosophila does not appear to exhibit LINC-unrelated functions [SUN/koi mutants in vivo using the Targeted DamID (TaDa) technique that allows tissue-specific profiling of DNA-binding proteins in the intact living organism [SUN/koi mutated and control muscle fibers of two chromatin factors: (1) Polycomb, the H3K27me3 reader representative of the polycomb-group associated heterochromatin, (2) HP1a, the H3K9me3 reader representative of the HP1a-associated heterochromatin, and in addition, the profile of RNA-Pol II subunit RpII215, representative of actively transcribed euchromatin. Polycomb, the subunit of the Prc1 complex Cbx8, HP1a, and RpII215, the main catalytic subunit of RNA-Po II, fused to the DNA adenine methyltransferase (Dam) were driven to Drosophila larval muscles using Mef2-GAL4 driver in a temporally controlled manner , inducing expression of the Dam-fusion proteins only at the third instar larval stage, when muscles are mature and fully differentiated. The experiment included n = 3 independent replicates of Dam–Polycomb, Dam-HP1a, Dam-RNA Pol II, and Dam only (as reference), for control and for SUN/koi nutant genotypes, with n = 25 larvae in each group. Following 10 h of Dam expression, larvae were dissected, DNA was extracted and digested with DpnI at the Dam methylated GATC sites. Further amplification, processing, and bioinformatics analysis were performed as described [Although the LINC complex has been implicated in the regulation of the epigenetic state, chromatin organization and overall gene expression , it is nunctions ,42. We torganism . Here, wSUN/koi and control muscle fibers, we performed a PCA regression analysis on Dam-Polycomb, Dam-HP1a, and Dam-Pol II, normalized to Dam binding alone  < 0.05, z-score > 1.96, and GATC sites > 1. The gray dots in SUN/koi mutant, and genes above the regression line represent decreased binding in SUN/koi compared to control. Overall, we detected significantly altered binding to 148 genes in the Polycomb group, 173 genes in the HP1a group and 206 genes in the RNA-Pol II group. The full list of identified genes for each group is provided in the https://data.mendeley.com/datasets/czjj5m7btr/1 (accessed on 17 March 2022). Note the robust increase in Polycomb binding observed in the SUN/koi mutant, where 143 out of 148 significant genes showed enhanced Polycomb occupancy. Notably, there was only 0.5 % and 2.4% overlap between the Polycomb-RNA-Pol II and HP1a-RNA-Pol II groups, respectively, and 6.3% overlap between the Polycomb-HP1a groups. SUN/koi mutant muscles for only one chromatin factor (red boxes) with increased Polycomb binding to alphaTub84B, increased HP1a binding to Nup62 and decreased RNA-Pol II binding to the Act57B gene. The corresponding Zpca scores are listed for each gene on To compare gene binding profiles between ng alone A. The cuSUN/koi mutant muscles. SUN/koi versus control) fold change occupancy for each chromatin factor, such that red indicates increased occupancy and blue indicates decreased occupancy. Non-significant genes are labeled in gray. Gene clustering was defined by k-means, and the number of clusters was estimated with Gap statistics. Overall, we observe minimal overlap between the group of genes that showed altered binding to the three factors, namely, Polycomb, HP1a, and RNA-Pol II, as the majority of genes colored in each group are non-significant (gray) in the others. Cluster 1 in SUN/koi mutant corresponded to distinct functions. Cluster 2, predicted to include genes that were transcriptionally repressed, included genes associated with muscle function, e.g., genes coding for contractile proteins and for myosin complexes. It also included genes associated with pupal adhesion, predicted to be expressed by salivary gland cells, possibly due to leaky expression of Mef2-GAL4 in the salivary gland. Notably, not all of the genes that increased binding to RNA Pol II are predicted to be transcribed, as RNA Pol II transcription might be paused. Cluster 1, predicted to include genes that were also transcriptionally repressed, contained genes coding for components of mTOR signaling involved in muscle cell growth [SUN/koi mutant muscles. Such transcriptional repression explains the significantly thinner muscle fibers and impaired contraction of the SUN/koi mutant muscles. Interestingly, we also observed decreased binding of RNA-Pol II and increased binding of HP1a to a group of genes involved in nuclear transport, predicting impaired functionality of the muscle fibers as well. In addition, a group of transcriptional regulators showed increased Polycomb binding and decreased RNA-Pol II binding, again predicting impaired function of the muscle fibers, and explaining the weakness of the larval muscles and their thinner phenotype, resulting in slower larval movement.We further paralleled genome-wide changes in Polycomb, HP1a and RNA-Pol II occupancy in the l growth . Figure Taken together, our genome-wide analysis indicates that defective LINC complex function associates with increased chromatin repression, mostly through increased Polycomb binding, and decreased transcriptional activation through reduced RNA-Pol II binding.Drosophila dm6 genome  and counted the number of genes that are in a distance <10,000 bp (expected number of close genes). To match the HP1a-, RNA-Pol II- and Polycomb-upregulated hits distributions over the genome, we picked the same number of genes, corresponding to the upregulated positive hits for each group. The process was repeated Nruns = 1000 times and the p-values were then calculated for the fraction NGE + 1/Nruns + 1, where NGE is the number of simulations with a value greater than or equal to the number of hits for each factor . The number of upregulated gene hits for RNA-Pol II and HP1a was significantly lower; however, a similar tendency for clustering was observed, where nine RNA Pol II upregulated genes were located within 10 kbp compared to a median of five from Monte Carlo simulation  and nine HP1a upregulated genes clustered within 10bp compared to a median of five from Monte Carlo simulation . Similar analysis of the distribution of RNA Pol II downregulated genes did not show such a clustering tendency  and an 82% increase in H3K9me3 (p < 0.01). The overall increased intensity of repressive chromatin marks is in agreement with the increased Polycomb and HP1a binding observed with the targeted DamID. As previously reported, the SUN/koi mutant displays smaller and variable myonuclear size and increased DNA ploidy [SUN/koi group compared to the control . We first compared fitted mixed models for all pulled nuclear volumes and found significant differences between the SUN/koi and control groups for H3K27me3 and H3K9me3 repressive marks (p < 0.01). To assess the contribution of the smaller nuclei, which are present only in the SUN/koi groups, we then fitted mixed models only for the larger nuclei, with overlapping volumes between the SUN/koi and control groups. The larger, overlapping volume nuclei showed no significant difference between the SUN/koi and control models for both repressive marks (p > 0.2), suggesting that the smaller nuclei in the SUN/koi groups are the major contributors to the increased H3K27me3 and H3K9me3 repression. To address whether the increased repressive epigenetic modifications represent muscles lacking LINC function, we characterized larval mutant muscles deficient in both Msp300 and klar using a double mutant combination of klar;Msp300 lacking the KASH domain (ΔKASH) [p < 0.001) and a 25% increase in mean fluorescent H3K9me3 intensity (p < 0.001) signals, and the repressive mark intensity inversely correlated with nuclear volume  . We founr volume .Drosophila muscle nuclei are polyploid, and we showed previously that chromatin volume scales linearly with nuclear volume, maintaining a constant chromatin volume fraction constant [SUN/koi mutant muscles, suggested increased chromatin condensation, which might contribute to the excessive chromatin repression [SUN/koi and control myonuclei. SUN/koi and control groups . A linear mixed model analysis confirms a significant difference between the SUN/koi and control fits (p < 0.01) with a leftward-shift in the SUN/koi total DNA–volume relationship, indicating that for each SUN/koi nucleus, the DNA is condensed in a smaller nuclear volume relative to the control. Interestingly, we did not observe a similar trend of DNA condensation in the ΔKASH mutant muscle fibers  did not cause significant defects in nuclear positioning or sarcomere arrangement, nor did it affect nuclear volume, it did recapitulate the increased levels of H3K27me3 modification with a 45% increase in the mean nuclear fluorescent intensity (p = 0.011). Consistently, the temporal SUN/koi knockdown in differentiated muscles showed no dependance of H3K27me3 levels on nuclear volume  network, normally exerting high compressive forces on the nucleus, was induced by conditional overexpression of spastin, a MT severing protein. This treatment disrupted the MT in muscles for a short time period without affecting sarcomere organization or muscle size . Figure SUN/koi mutant muscle fibers (SUN/koi mutant (p < 0.01). Notably, the active H3K9ac mark showed no correlation with nuclear volume   . OverallSUN/koi mutant muscle nuclei, we performed live 3D imaging of larval muscles utilizing the H3K27me3-GFP mintbody [SUN/koi mutated nuclei is shown in SUN/koi mutant (right) muscle nuclei labeled with H3K27me-GFP mintbody. The strong H3K27me3 signal appears punctuated within the nucleus, with weaker nuclear and cytoplasmic background. Compared to control distribution, the H3K27me3 puncta in the SUN/koi mutant nuclei appeared more spread out throughout the chromatin, resulting in larger repressive H3K27me3 hubs. We quantified the spread of the puncta by measuring the volume  with no significant change in the number of the puncta between the groups. Thus, the live 3D distribution of H3K27me3 puncta demonstrates increased spreading of this repressive mark to neighboring chromatin regions in the SUN/koi mutated muscle nuclei, further supporting the clustering of positive Polycomb binding location across the genome  to the lamina. The Drosophila genome was simulated by beads connected by springs to model each chromosome, where a bead of diameter Drosophila, to a sphere which represented the nucleus. The chromosomal chains consisted of three types of beads: euchromatin beads, pericentromeric heterochromatin (PCH) beads and H3K27me3-modified beads , we identified enrichment for two GO terms functionally associated with TOR signaling, a major signaling pathway regulating muscle growth. Enhanced transcriptional repression of TOR signaling in the  muscles ,46. Sincparation , but alsparation . Our obsPolycomb is a member of polycomb repressive complexes (PRCs) and has been extensively studied in development and differentiating cells. PRC binding to epigenetically modified chromatin leads to a transcriptionally poised state, which then prevents unscheduled differentiation . TherefoA number of studies suggested a link between nuclear mechanical stimulation and chromatin epigenetic repression. For example, long-term stretch of epidermal progenitor cells led to increased H3K27me3 occupancy, resulting in silencing of epidermal differentiation ,53. FurtSUN/koi mutant muscles reveals genes in Cluster 2 with decreased binding to the transcriptional machinery complex. According to GO analysis, this cluster contains a group of genes coding for muscle contractile proteins. Reduced transcription of such genes is expected to result in muscle weakening and failure in larval locomotion, and indeed, both phenotypes were observed in the SUN/koi mutants. Notably, because the DamId analysis was performed at a late stage of third instar larvae, some of the muscle-specific genes undergo transcription reduction due to the transition from larvae to pupal stage (flybase.org). For example, muscle-specific genes, including myosin heavy chain (MHC), sallimus (Drosophila titin-like gene), MSP300 (Nesprin-like gene), alpha actinin (Actn) and others, show a significant reduction in mRNA levels in the late third instar larval stage. In such genes, alterations of RNA-Pol II binding in the mutant muscles might not be detectable. Nevertheless, a group of other muscle genes did show significantly reduced binding to RNA-Pol II, e.g., myofilin (Mf), tropomyosin1 (Tm1), myosin light chain (Mlc), actin 57B (Act57B), troponinC73F (TpnC73F) and others. Consistently, antibody staining indicated reduced TpnC protein levels , predicting their increased repression in the SUN/koi mutant muscles. Lastly, transcription itself might be attenuated due to the increased binding of Polycomb and HP1a to components of the transcription machinery, such as mediator complex proteins med10 and med28. The decreased levels of H3K9 acetylation in the SUN/koi mutant muscles are consistent with the paralleled reduction in RNA-Pol II binding to genes in Cluster 2. Importantly, the lack of overlap between the group of genes that showed increased Polycomb binding (Cluster 1) and the group of genes that exhibited decreased RNA Pol II binding (Cluster 2) suggests that although both processes are regulated by the LINC complex, they might result from distinct effects of this complex on repressed and active chromatin regions.The RNA-Pol II binding profile in n levels . In addiSUN/koi knockdown. The simulation results described in SUN/koi mutant nuclei, implicating weakening of chromatin–nuclear lamina binding. Consistently, recent studies indicated that in LINC mutants, the LEM domain protein Emerin which, together with BAF, promotes chromatin tethering to the lamina, fails to oligomerize at the inner nuclear membrane [Although enhanced DNA condensation and compaction could explain the upregulation of epigenetic repression in the LINC mutant nuclei, we do not favor this explanation, since we found that elevated epigenetic repression is also observed in nuclei of normal size with conditional membrane , and in membrane ; both faIn summary, this study analyzed the contribution of the LINC complex to chromatin epigenetics and the binding of transcriptional regulators in fully differentiated muscle fibers. We demonstrate that in the absence of functional LINC complex, chromatin epigenetic repression is elevated, mainly due to enhanced binding of Polycomb and excessive clustering of repressed chromatin in the nucleoplasm, while Pol II binding to muscle-specific genes decreases. This forms the basis for abrogated muscle structure and function in LINC mutant observed in striated muscles, as well as in LINC-associated cardiomyopathies.


MaACS1, MaACO1, MaACO4, MaACO5, and MaACO8, were upregulated in ripening bananas. NAC  transcription factor, MaNAC083, a ripening and ethylene-inhibited gene, was discovered as a potential binding protein to the MaACS1 promoter by yeast one-hybrid screening. Further in vitro and in vivo experiments indicated that MaNAC083 bound directly to promoters of the five ethylene biosynthesis genes, thereby transcriptionally repressing their expression, which was further verified by transient overexpression experiments, where ethylene production was inhibited through MaNAC083-modulated transcriptional repression of ethylene biosynthesis genes in banana fruits. Strikingly, MaMADS1, a ripening-induced MADS  transcription factor, was found to directly repress the expression of MaNAC083, inhibiting trans-repression of MaNAC083 to ethylene biosynthesis genes, thereby attenuating MaNAC083-repressed ethylene production in bananas. These findings collectively illustrated the mechanistic basis of a MaMADS1–MaNAC083–MaACS1/MaACOs regulatory cascade controlling ethylene biosynthesis during banana fruit ripening. These findings increase our knowledge of the transcriptional regulatory mechanisms of ethylene biosynthesis at the transcriptional level and are expected to help develop molecular approaches to control ripening and improve fruit storability.The hormone ethylene is crucial in the regulation of ripening in climacteric fruit, such as bananas. The transcriptional regulation of ethylene biosynthesis throughout banana fruit ripening has received much study, but the cascaded transcriptional machinery of upstream transcriptional regulators implicated in the ethylene biosynthesis pathway is still poorly understood. Here we report that ethylene biosynthesis genes, including  S-Adenosyl-l-methionine (SAM) is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) via catalysis by ACC synthase (ACS) [Ethylene, a versatile phytohormone, modulates numerous physiological processes, spanning seed germination to organ senescence . Due to se (ACS) . The secse (ACS) . Thus, ese (ACS) .cis-acting regions of gene promoter DNA [rin and nor, researchers have steadily been interested in the central TFs' role in regulating fruit ripening. Members of the MADS and NAC families of TFs are encoded by the nor and rin genes, respectively [SlACS2 and SlACO4 [SlACS2 and SlACS4 [Gene expression regulation requires precise transcriptional programs, involving binding of transcription factors (TFs) to oter DNA . With thectively . RIN andectively . Throughd SlACO4 , 10. NORd SlACS4 . Regulatd SlACS4 –15.Musa acuminata) is the most widespread type of fresh fruit in the world, produced mainly in developing countries due to the special climatic conditions needed to grow them, and are a multimillion-dollar export product consumed primarily in wealthier nations [MaACS1  genes and one ACS gene were repressed by ripening  was detected. Previous studies showed that Ma10_g12120 has the closest evolutionary relationship with Arabidopsis ANAC083 [MaACS1 promoter by employing MaNAC083 full-length coding DNA sequences as prey. The MaACS1 promoter did not exhibit basal activity in yeast with AbA, while yeast cells expressing MaNAC083 triggered the AbA resistance gene expression controlled by the MaACS1 promoter and developed well on media containing AbA, showing that MaNAC083 protein physically interacts with the MaACS1 promoter  libraries in search of proteins interacting with their respective promoters. Due to the inability of aureobasidin A (AbA) to inhibit promoter activities of  ANAC083 ; hence, promoter .MaACS1, MaACO1, MaACO4, MaACO5, and MaACO8 showed the presence of the NAC recognition sequence (NACRS) in their promoters  was utilized to determine if MaNAC083 can interact with all promoters directly. The purified recombinant MaNAC083 protein  system  system . To inve) system . MaNAC08) system . Utilizi control . MaNAC08MaACS1, MaACO1, MaACO4, MaACO5, and MaACO8 promoter activities were inhibited after MaNAC083 co-transfection when compared with co-transfection with an empty vector. A lower LUC/REN ratio was reported, showing that MaNAC083 could repress the ethylene biosynthesis gene promoter  hastened fruit ripening and resulted in a faster yellowing phenotype  and MaACO8  exhibit high expression levels during postharvest banana ripening [MaACS1, MaACO1, MaACO4, MaACO5, and MaACO8 generally align with ethylene production during banana ripening  [MaACS1, MaACO1, MaACO4, MaACO5, and MaACO8 genes and represses their expression, which in turn inhibits ethylene biosynthesis and banana ripening  . Howeverripening –4. TheseMaNAC083 to inhibit its expression, which in turn attenuated MaNAC083's repression of the MaACS1, MaACO1, MaACO4, MaACO5, and MaACO8 genes, and eventually promoted ethylene biosynthesis and fruit ripening  at 75–80% maturity were gathered from a plantation near Guangzhou, China. The fruits were then subjected to ripening by incubation with 100 μL L−1 ethylene for 18 h. As Zhu et al. [Pre-climacteric bananas , qRT–PCR reactions were carried out via CFX96 real-time PCR . As Chen et al. [Total banana fruit RNA was isolated utilizing the hot borate technique . Using tn et al.  indicateMaACS1 promoter (−499 bp upstream of ATG) or MaNAC083 promoter (−676 bp upstream of ATG) was cloned into the pAbAi vector to generate the bait plasmid, followed by linearization and transformation into the Y1H Gold strain to produce the bait-specific reporter strain, which was preceded by screening a cDNA library of banana fruits. MaNAC083 or MaMADS1 coding sequences were cloned into the pGADT7 prey vector and introduced into bait-reporter yeast strains for re-transformation. According to the manufacturer's guidelines, DNA–protein interactions were assessed based on the co-transformants' ability to grow on SD/−Leu media with AbA.Y1H screening was done utilizing the Matchmaker™ Gold Yeast One-Hybrid System . The short fragment of the Escherichia coli BM Rosetta (DE3) was purified. Fragments containing NACRS in the promoter of MaACS1, MaACO1, MaACO4, MaACO5, and MaACO8 were manufactured , and labeled with biotin at the 5′ end. As stated in our previously published work [The MaNAC083 coding sequence was inserted in-frame into pGEX-4 T-1 vector with a GST tag. Using glutathione–Sepharose 4B beads, GST-MaNAC083 fusion protein in hed work , EMSA waMaNAC083 or MaMADS1 promoter was extracted and placed in the pGreenII 0800-LUC reporter vector to induce the firefly luciferase reporter (LUC) gene. CaMV35S-REN/MaNAC083pro-LUC or CaMV35S-REN/MaMADS1pro-LUC constructs were transferred into tobacco protoplasts using a PEG-mediated method. Using a dual LUC assay kit (Promega), LUC and Renilla luciferase (REN) activities of transformed protoplasts supplemented with 0 (–ETH) or 10 μL L−1 ethylene (+ETH) were measured.Using a previously described technique , promoteMaNAC083 and MaMADS1 coding sections were transferred into the pBI221-GFP vector to bind with the GFP reporter gene utilizing NLS-mCherry  as a nucMaNAC083 or MaMADS1 coding sequence into the pGreenII 62SK-BD vector as an effector. The double-reporter vector featured an LUC gene controlled by five GAL4-binding element copies. The effector and recombinant reporter constructs were co-transfected into tobacco leaves, as in our prior research [Transcriptional capability was accessed by inserting full-length research . As repoMaNAC083 or MaMADS1 was performed into pGreenII 62-SK vector effector, and cloning of target gene promoters was performed into pGreenII 0800-LUC reporter vector [Agrobacterium tumefaciens strain EHA105 and pSoup vector. The LUC/REN ratio reflected target promoter transcriptional repression. Each pair had six transient assay replicates.To evaluate MaNAC083 or MaMADS1's influence on promoters of target genes, cloning of r vector . TobaccoMaNAC083 or MaMADS1 were cloned into the pCXUN-HA vector, respectively. For vector construction in VIGS, MaNAC083 or MaMADS1 was cloned into the pTRV2 vector. Cells of the A. tumefaciens strain EHA105 carrying recombinant pTRV2 and pTRV1 were combined at a 1:1 ratio. The pulp was injected with A. tumefaciens harboring the constructed plasmids. On day 1 following inoculation, the transformed fruits were exposed to 100 μL L−1 ethylene and kept at 20°C for 5 days. The samples were collected to evaluate ethylene generation, gene expression, and protein accumulation.As previously mentioned, transient overexpression investigation and silencing were done in banana fruit , 30. Thet-test was utilized, as appropriate, to identify statistically significant differences between samples.SPSS  was used for execute statistical analysis. Data were represented as mean ± standard error of three or six independent biological replicates. Analysis of variance (ANOVA) or Student's Supplementary Data Table S2 lists the primers employed for this study.This study was supported by the National Key Research and Development Program of China (grant no. 2022YFD2100103), the National Natural Science Foundation of China (grant no. 32072279), and the China Agriculture Research System of MOF and MARA (grant no. CARS-31).W.S. conceived and designed the study, while W.W. performed experiments alongside W.S. Data analysis was carried out by a team consisting of W.W., Y.Y.Y, C.J.W, J.F.K., W.J.L., and W.S. The manuscript writing and revision were done by J.Y.C., W.S., and W.W.; all authors participated in manuscript discussions.All study data are incorporated in the submitted article.None declared.Web_Material_uhad177Click here for additional data file.


Nanog+/−;Sall4+/−, Nanog+/−;Utf1+/−, Nanog+/−;Esrrb+/− and Sox2+/−;Sall4+/−). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction.A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e.,   •PSCs with complex haploinsufficiency maintain their developmental potential•Fibroblasts with one allele elimination in two PSC genes show reprogramming delay•Fibroblasts with one allele elimination in two PSC genes display methylation defect•The KI/KO approach to introduce a reporter gene should be used carefully In this article, Buganim and colleagues investigate the effect of complex haploinsufficiency on PSCs. By eliminating single alleles in different combinations of two key pluripotency genes, they show that, although mutant PSCs can contribute equally to chimeras, their derived fibroblasts exhibit reprogramming blockage. Abnormal methylation around vital genes underscores the necessity of maintaining two intact alleles for effective pluripotency induction. Embryonic development and cell fate induction require appropriate gene dosage for the activation of the regulatory circuits that control cellular identity.Oct4 and Sox2  by their fibroblast derivatives, which can only be partially overcome by high levels of exogenous OCT4, SOX2, KLF4, and MYC (OSKM) factors (While a complete knockout (KO) of an important gene may be detrimental to the cell as seen for and Sox2 , a compl factors . In contSox2 [Nanog [Utf1 [Nanog may result in suboptimal reprogramming, producing low-quality iPSCs  approach, leaving only one intact allele of the targeted gene. Such reporter lines  are usety iPSCs .Utf1, Esrrb, Sall4 , NGFP1 , and SGFP1 . Interestingly, while all double heterozygous mutant lines contributed to chimeras similarly to their parental iPSC controls , multiple derivations of fibroblasts from these lines resulted in poor reprogramming efficiency. This reduced reprogramming efficiency was evident in the nuclear transfer (NT) technique as well.To address that, we engineered three secondary systems, NGFP2 (Nanog-GFP#2 ), NGFP1 Sall4 or Nanog along the reprogramming process revealed that only a very small fraction of cells activated these loci, a result that cannot explain the global reprogramming blockage seen in the double heterozygous mutant lines. We then profiled the CpG-rich methylation landscape of fibroblasts derived from SGFP1S2+/−;S4+/− and SGFP1S2+/− control, and noted a clear difference in the methylation levels of multiple developmental and pluripotent loci in the double heterozygous mutant fibroblasts. Accordingly, treating all double heterozygous mutant fibroblasts for 2 days before factor induction with 5-azacytidine rescued the reprogramming blockage and allowed the induction of pluripotency. This study emphasizes the importance of having two intact alleles for proper pluripotency induction and normal embryonic development, and raises a concern regarding the often used KI/KO technique for the purpose of introducing reporters.Tracing the stochastic expression of Considering the vital role of functioning core ESC circuitry to pluripotency, we hypothesized that even a slight decrease in the expression of key pluripotency genes could significantly impact the developmental potential of the cells or the ability of their somatic cell derivatives to undergo reprogramming. We focused our research on secondary iPSC systems -inducible OSKM factor integrations in their genome), as these systems contribute to chimeras and exhibit stable and reproducible reprogramming efficiency by minimizing cell heterogeneity .Nanog , Nanog+/−; Utf1+/−  and Nanog+/−; Sall4+/− . To validate the reduced levels of the targeting genes, we cultured the cells in 2i/L medium (GSK3β and MEK inhibitors and Lif) that recapitulates the ground pluripotent state and facilitates gene expression from both alleles  or 2i/L conditions were analyzed for GFP and tdTomato activity using flow cytometry. In agreement with the western blot analysis, cells grown under S/L conditions exhibited 68% GFP reporter activity (reporter that was introduced in frame and contains polyA) in NGFP2N+/− control and NGFP2N+/−;E+/− iPSC lines, and 55% and 58% in NGFP2N+/−;S+/− and NGFP2N+/−;U+/− iPSC lines, respectively  ESCs (V6.5), grown in either S/L or 2i/L conditions. Pearson correlation heatmap clustered the cells into two main groups based on the culture conditions. Nevertheless, within the S/L group some changes in gene expression were noted in NGFP2N+/−;S+/− and NGFP2N+/−;U+/− compared with NGFP2N+/−;E+/−, parental NGFP2N+/−, and control WT ESCs  validated the Pearson correlation heatmap, separating S/L conditions from 2i/L conditions by PC1 and NGFP2N+/−;S+/− and NGFP2N+/−;U+/− that were grown under S/L conditions from the rest of the samples by PC2  or with additional viruses encoding for OSK and reprogramming was scored. Both Nanog or each of the corresponding factors showed either partial or complete rescue of the reprogramming blockage, while additional OSK further boosted the reprogramming process  F and 2G.N+/− cells, and NGFP2N+/− cells that were infected with empty vector) after 6 days of reprogramming. We chose this time point as it showed a clear reprogramming delay in the double heterozygous mutant plates compared with control plates. NGFP2N+/− MEFs and the parental NGFP2N+/− iPSCs were profiled as well. Hierarchical clustering analysis showed that all the double heterozygous mutant lines clustered together and were different from the control lines  that are upregulated solely in the control groups and 18 genes that are upregulated exclusively in the double heterozygous mutant lines . GO termThy1, Col5a1, Postn, and Des) and EMT regulators , and noticed a comparable downregulation between the control and the double heterozygous mutant lines  that the strong effect seen in NGFP2N+/−;S+/− is a result of approximately a 50% decrease in the transcript levels of Sall4 , the SGFP1S2+/−;S4+/− double heterozygous mutant cells produced approximately 2%–2.5% of iPSCs. This difference can be explained by the levels of the Oct4 transgene that is much higher in SGFP1S2+/− cells compared with the NGFPN+/− cells  cassette upstream to a tdTomato reporter gene and M2rtTA transactivator at the Rosa26 locus  were exposed to tamoxifen (Tam) and the percentage of tdTomato-positive cells was scored by flow cytometry .The fact that additional exogenous expression of OSK factors rescued the phenotype of the double heterozygous mutant cells G suggestOct4” and are associated with “Hippo signaling”  and reprogramming experiments were carried out. In agreement with the RRBS results, treatment of 5′azaDC rescued the reprogramming defect F.N+/− control iPSCs and the double heterozygous mutant iPSC lines   . This ov0.00001) C and S6D0.00001) E and S6FPSCs in 2i/L culture are less affected by differentiation cues due to robust inhibitor-based protection. Conversely, those in S/L conditions are more prone to differentiation signals, resulting in greater transcriptome heterogeneity. In this scenario, any pluripotency gene expression dysregulation can disrupt pluripotency maintenance, potentially affecting somatic cell derivative development.Nanog+/−;Sall4+/−, Nanog+/−;Esrrb+/−, Nanog+/−;Utf1+/−, and Sox2+/−;Sall4+/−) and used different PSC systems to exclude any system-specific effect.Here, by using PSCs as a tested model we aimed to understand how reduced levels of pluripotency genes affects cell’s function. We deleted a single allele from various combinations of two pluripotency genes  to a later blockage at the stabilization step just before the acquisition of pluripotency (NGFP1 and SGFP1 lines).Sall4 or Nanog allele is a frequent event and occurred in most induced cells at early stages of reprogramming. Using tracing systems for Nanog and Sall4 we show that, only a small number of induced cells could activate the targeted alleles following 6 days of factor induction, suggesting that reduced stochastic expression of these genes is not responsible for the global reprogramming delay seen in the double heterozygous mutant cells.Given that the affected genes were shown to play a major role during the stochastic phase of the reprogramming process, we examined the possibility that reduced stochastic expression of the targeted genes hinders the capability of the cells to pass the stochastic phase and to induce pluripotency. To support this hypothesis, one should show that the activation of the Sall4, Nanog, Utf1, Esrrb, and OSK) can either partially or fully rescue the observed blockage; thus, we next hypothesized that epigenetic barrier in the double heterozygous mutant fibroblasts may cause the observed delay. Indeed, CpG-enriched DNA methylation analysis demonstrated a clear difference in the DNA methylation levels in regions within pluripotent and developmental genes between the two fibroblast lines, suggesting that even a 50% reduction in the levels of two pluripotent genes is sufficient to induce aberrant DNA methylation during development. In fact, although Oct4 expression was unaffected in the iPSCs, GO enrichment analysis of the derived MEFs revealed the loss of Oct4’s core pluripotency function. This discrepancy can be attributed to the reduced levels of key pluripotent genes in the iPSCs, including Nanog, Sox2, Sall4, and Esrrb, which are known to regulate the core DNA methylation machinery .Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author, Yosef Buganim  of the Hebrew University and Hadassah Medical Center. The Hebrew University is an AAALAC international accredited institute.Statistical analysis was performed by 2-tailed unpaired t test calculated by GraphPad Prism (8.3.0). All data are presented mean ± SD. p < 0.05 was considered statistically significant. Sufficient sample size was estimated without the use of a power calculation. Data analysis was not blinded.


Aspergillus fumigatus is a key step in development of this problematic clinical phenotype. We and others have previously described a C2H2-containing transcription factor called FfmA that is required for normal levels of voriconazole susceptibility. Null alleles of ffmA exhibit a strongly compromised growth rate even in the absence of any external stress. Here, we employ an acutely repressible doxycycline-off form of ffmA to rapidly deplete FfmA protein from the cell. Using this approach, we carried out RNA-seq analyses to probe the transcriptome cells acutely deprived of FfmA. A total of 2,000 genes were differentially expressed upon acute depletion of FfmA, illustrating the broad transcriptomic effect of this factor. Interestingly, the transcriptome changes observed upon this acute depletion of FfmA expression only shared limited overlap with those found in an ffmAΔ null strain analyzed by others. Chromatin immunoprecipitation coupled with high throughput DNA sequencing analysis (ChIP-seq) identified 530 genes that were bound by FfmA. More than 300 of these genes were also bound by AtrR, a transcription factor important in azole drug resistance, demonstrating striking regulatory overlap with FfmA. However, while AtrR is an upstream activation protein with known specificity, our data suggest that FfmA is a chromatin-associated factor that binds DNA in a manner dependent on other factors. We provide evidence that AtrR and FfmA interact in the cell and show reciprocal expression modulation. Interaction of AtrR and FfmA is required for normal gene expression in A. fumigatus.Transcriptional regulation of azole resistance in the filamentous fungus  Aspergillus fumigatus FfmA is a C2H2 transcription factor that acts globally to control expression of nearly 20% of the genome. Paul et al. show using ChIP-seq, western blot and co-immunoprecipitation analyses that FfmA extensively interacts with the transcription factor AtrR to control azole drug resistance. The authors’ data argue that FfmA is a chromatin-associated protein that is recruited to DNA via interaction with AtrR and other factors. Aspergillus fumigatus . Each of these changes was capable of decreasing the susceptibility of the fungus to azole drugs, and their combination led to an enhanced level of resistance  present in the 34-bp repeat . The Dox-Off-ffmA_Ble (SPF182) and the Dox-Off-FLAG-ffmA_Ble (SPF185) used in this study have been described in A. fumigatus strains were typically grown at 37°C in a rich medium . Selection of transformants used a minimal medium ; trace elements, vitamins, and nitrate salts are as described in the appendix of All strains used in this study were derived from AfS35 strain  were used to replace hasB with the hygromycin resistance marker cassette amplified from the plasmid pSP62  supplemented with 0.01% Tween 20 (1 × PBST). The spore suspension was counted using a hemocytometer to determine the spore concentration. Spores were then appropriately diluted in 1 × PBST. Approximately 100 spores (in 4 ml) were spotted on a minimal medium in the presence or absence of 25-mg/ml doxycycline. The plates were incubated at 37°C and scanned after 3 days.Fresh spores of Western blotting and immunoprecipitation were done as described in https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped to the A. fumigatus Af293 genome using HISAT2 in paired-end read mode (https://www.htslib.org). Mapped reads were assembled onto annotated Af293 genes with Stringtie using the -eB parameter  cutoff of 0.05 and a maximum allowable gap between peaks of 100 bp (The reads were mapped to the To generate coverage plots with the Recoup R package  mapped rS. cerevisiae. The 2 C2H2 domains of FfmA are located in the center of the factor (Modulator of transcription) as a sequence-specific DNA-binding protein involved in regulation of a wide range of different genes including ones involved in mating pheromone response  experiment to profile the transcriptomes produced  or down-regulated (259). Since growth of the DO-ffmA strain under this condition was closer to that of the wild-type than in the case of the ffmAΔ null strain, we anticipated that the transcriptome would also be less disturbed. Using FungiFun2  were repressed . These repressed genes could be broadly separated into loci involved in either transport or metabolism . Compared to our previous analysis of the ChIP-seq profile of AtrR, we noticed many more peaks for FfmA inside or downstream of the coding sequence (∼40%) compared to those for AtrR (∼22%) .AFUA_8g02440 (erg25A) gene in which AtrR binds upstream of the transcription start site (TSS) but the shared FfmA peaks are located within the transcription unit. The transcription factor-encoding zfpA gene contains a number of AtrR peaks in its promoter region, and these are closely paralleled by the appearance of FfmA binding density. Note the strong induction of zfpA transcription upon addition to doxycycline to the DO-ffmA gene. Finally, the abcG1 gene is shown as the effect of FfmA loss of this factor caused a reduction in AbcG1 expression by western blot analysis .The top panel illustrated data from the ffmA cells treated with doxycycline with the 530 genes showing binding of FfmA using the 2 different immunological reagents to perform ChIP-seq. Only around 20% of the 530 genes exhibited altered gene expression under these conditions and of these, only 6 genes were seen to be significantly enriched in the list of genes induced in the presence of doxycycline in DO-ffmA cells. These genes were all membrane transporter proteins and an amine oxidase protein  or with anti-FLAG antibody. A secondary anti-mouse antibody bound to beads was used to recover immunoprecipitates. These were washed and then resolved on SDS-PAGE. Recovered proteins were detected by western blotting  of these data sets led to the detection of an element very similar to that for the ATRE. This sequence motif was also detected in a minority of FfmA ChIP-seq peaks (45/600) and was not centrally located in these peaks. This behavior is more consistent with an element peripherally associated with the binding of FfmA as an authentic binding site would be expected to be centrally located in MEME-ChIP analysis as this involves inspecting 250 bp on either side of the detected ChIP-seq peak and deriving a consensus . We were able to detect general DNA binding but no sequence specific site recognition. Conversely, this was relatively straightforward for AtrR  that shaFinally, the Recoup plotting of the ChIP-seq peaks is the most striking indication that the action of FfmA and AtrR are different. This algorithm plots the average sequence coverage across all genes by the factor of interest. If the Recoup treatment of AtrR ChIP-seq data is inspected, a clear enrichment of binding can be seen on the 5′ end of all genes . Note thThe chromatin-bound FfmA protein functions to control the expression of a wide range of genes. We found roughly 2,000 genes that were significantly changed by at least 2-fold in cells that were depleted of FfmA in an acute manner . This laA. fumigatus AFUA_1g14750 gene is detected by ChIP-seq using either the FLAG-tagged or the wild-type FfmA protein and showed significant induction upon doxycycline-mediated loss of FfmA. This gene encodes the A. fumigatus homolog of the S. cerevisiae Sfp1 protein that, when overproduced, led to the induction of ribosomal protein gene transcription . While these genes all contain peaks for FfmA binding by ChIP-seq, it is possible that these cannot be recognized by FfmA in vitro. Given that only a fraction of FfmA can interact with AtrR in the cell, binding of this C2H2 protein might require additional interaction partners.The interaction of FfmA with DNA is not likely to be as relatively simple as for AtrR. Our efforts to detect direct DNA-binding of recombinant FfmA centered on use of the promoter regions from the Drosophila melanogaster called GAGA factor or GAF has a single C2H2 domain and can bind DNA in vitro but exhibits more complex interactions to determine its DNA-binding pattern in vivo including cooperative binding and recruitment of chromatin remodelers (While there are many examples of C2H2 domain-containing proteins functioning as upstream acting sequence specific binding proteins, other functions of this domain are known. A factor from atrR gene have modest phenotypes unless challenged with azole drugs in vitro (ffmAΔ null mutants that are severely compromised in growth. Second, doxycycline-mediated depletion of FfmA using the DO-ffmA allele caused a dramatic induction of the has regulon. Increased expression of these proteins has been shown to cause increased production of the hexadehydroastechrome iron chelator (has regulon expression. Control of the has regulon also demonstrated that, while clear AtrR DNA-binding could be seen in the intergenic hasA/B promoter, no significant FfmA DNA-binding was observed (abcG1. An important future goal is to probe the overall chromatin structure of strains with lowered FfmA levels to test this idea.The model that FfmA may act to globally impact gene expression is supported by at least 2 different observations. First, cells lacking the observed . We suggobserved . This cljkad173_Supplementary_DataClick here for additional data file.


In this single-molecule study on bacterial Rho factor-dependent terminators, we confirm that the three Rho-dependent termination routes operate compatibly together in a single terminator, and discover that their termination efficiencies depend on the terminational pauses in unexpected ways. Evidently, the most abundant route is that Rho binds nascent RNA first and catches up with paused RNA polymerase (RNAP) and this catch-up Rho mediates simultaneous releases of transcript RNA and template DNA from RNAP. The fastest route is that the catch-up Rho effects RNA-only release and leads to 1D recycling of RNAP on DNA. The slowest route is that the RNAP-prebound stand-by Rho facilitates only the simultaneous rather than sequential releases. Among the three routes, only the stand-by Rho's termination efficiency positively correlates with pause duration, contrary to a long-standing speculation, invariably in the absence or presence of NusA/NusG factors, competitor RNAs or a crowding agent. Accordingly, the essential terminational pause does not need to be long for the catch-up Rho's terminations, and long pauses benefit only the stand-by Rho's terminations. Furthermore, the Rho-dependent termination of   Transcriptional pauses play critical roles for gene regulation in all stages of transcription. In bacteria and archaea, for example, the pauses provide time windows for RNA polymerase (RNAP) to bind transcription factors ,2 and coSpecifically, transcriptional pause is considered essential for efficient termination, as it provides a time for stable elongation complexes (ECs) to undergo the conformational changes necessary for releasing transcript RNA at termination. The eukaryotic RNAP II pausing beyond polyadenylation sites facilitates terminational breaking up with left-over RNAs . In bactrut (Rho utilization) site and moves down along the RNA using ATP hydrolysis energy to catch up with the RNAP that is pausing at a downstream termination site. In this catch-up termination, a.k.a. tracking  and the individual route termination efficiencies (TEs) of several ρ-dependent terminators using the single-molecule assays that we lately developed  using the EZTM Total RNA Miniprep Kit purchased from Enzynomics.e enzyme  and RpoDyme  and biotinylated PEG (Laysan Bio Korea). Consequently, they were immobilized on both surfaces in a scattered way via biotin-streptavidin-biotin conjugation , 10 μM ATP , 10 μM CTP  and 340 nM RNAP holoenzyme with σjugation .2, 150 mM KCl and 1 mM dithiothreitol, while the buffer was supplemented with 5 mM protocatechuate acid and 100 nM protocatechuate-3,4-dioxygenase to reduce photobleaching of the fluorophores (E. coli and 5% PEG-8000 (New England Biolabs) were added, and the MgCl2 and ribocil-C (MedChemExpress) concentrations were varied as indicated. After washing, transcription was resumed by adding an NTP mixture (200 μM each) to the chamber using a syringe pump Fusion 100 (Chemyx).Fluorescence images were obtained from the samples immobilized on the quartz slide rather than the cover slip using a home-build prism-type total internal reflection fluorescence microscope with a 1-s exposure time in the alternating laser excitation mode  so the trophores  and withrophores . When neSingle-molecule images were taken while both Cy3 and Cy5 were alternately excited by a 532-nm green laser Excelsior-532-50-CDRH and a 640-nm red laser Excelsior-640c-60-CDRH (Spectra-Physics), respectively. The fluorescence signals were collected by a water-immersion objective lens UPlanSApo 603 (Olympus) and filtered through a 532-nm notch filter NF03-532 E-25 and a 632-nm notch filter NF03-633 E-25 (Semrock) to reject scattered laser lines. They were then separated with a dichroic mirror 635dcxr (Chroma) before imaged onto an electron-multiplying charge-coupled device camera iXon DU-897 (Andor Technology). Software IDL 7.0 (ITT), MATLAB R2018a (MathWorks), Excel 2016 (Microsoft) and Origin 8.5 (OriginLab) were used for data analyses.r was estimated for linear correlation between tp and ρ-dependent TE using Origin 8.5. Tested additionally was a null hypothesis that the linear fitting slope is zero, and calculated was probability P of the null hypothesis using the formula below.The Pearson's tN denotes the Student's t-distribution whose degree of freedom is N, b1 denotes the fitting value of the slope, and SE denotes the standard error of fitting value. All the reported P values are one-sided and statistical significance is defined as P ≤ 0.05.where E. coli holoenzyme (30 nM) and a template DNA (15 nM) with the wild-type or a mutant of mgtA were incubated with or without E. coli ρ hexamer (100 nM) in a transcription buffer of 40 mM Tris–HCl, pH 8.0, 200 μM ATP, 200 μM GTP, 200 μM UTP, 25 μM CTP, 0.75 μCi of [α-32P]CTP (3000 Ci/mmol), 10 mM MgCl2, 150 mM KCl and 2 mM dithiothreitol at 37°C for varying reaction time. The reaction was quenched by addition of a 2× urea-denaturing gel loading buffer and analyzed on a 6% polyacrylamide–7 M urea gel.E. coli RNAP  when the 5' Cy3-end of RNA is still proximal to RNAP at the stalling site, as the protein binding restricts the cyanine dye's photoisomerization from fluorescent rescence . The Cy3The Cy5 also displays PIFE when its photoisomerization is hindered by the RNAP contacting the downstream Cy5-end of DNA in two instances. One is when transcription readthrough takes place at the termination site and the continuously transcribing RNAP comes to the end for runoff transcription. It is here called readthrough runoff. The other instance is when termination occurs with RNA-only release and the 1D recycling RNAP diffuses on DNA to the end, i.e. after the recycling termination. No PIFE is shown if RNAP with or without RNA falls off DNA before arriving at the end.In the stand-by ρ assays Figure , we pre-P279S  tabulated in Transcriptional pause at the termination sites is required for both catch-up and stand-by terminations like all other types of termination and here called terminational pause. Additionally, it has been presumed that RNAP pauses at the termination sites long enough to be caught by the catch-up ρ, whereas the stand-by ρ already pre-bound to RNAP does not need such extra length of pause for its action. It has been recently found, however, that the stand-by terminations usually occur later than the catch-up terminations . Thereuptp with ρTEs of the three routes measured in the two ρ assays. Firstly employed in this study were the same DNA templates with the mgtA, rho, ribB, trp-t’ or tR1 ρ-dependent terminator as we have previously confirmed their termination proficiencies and characteristics using both single-molecule and bulk transcription assays (n = 34 741) than the previous one (n = 12 838).The terminational pause durations cannot be directly measured but can be approximated in our experimental settings. We then examined the correlations of n assays , but ovetp values, we measured the ρ-free elongation timespans trun that transcribing RNAP takes to run from the stalling site to the downstream Cy5-end  and 20 s (tR1) with the five terminator templates  to 263 s (mgtA) and are an order of magnitude larger than the tinc values, indicating that the elongation on each template is much retarded by lengthy or abundant pausing. Then, tp is diverse from 93 (tR1) to 250 s (mgtA), or 1.6 to 4.2 min, which is a range of 2.7-fold difference.The NTP each . When its Figure . Contrastp is the sum total of all the pause durations. However, the contribution of non-terminational pauses to tp estimation appears negligible on the mgtA template and its eight mutants described below, according to the time-course bulk transcription assays  and little significant for the catch-up decomposing  but negatively linear for the catch-up recycling .The mutants' n Figure . They ar5.3–8.4% . All thenal ones . The tp-n Figure  is positmgtA , while only 5.9 and 0.9 pp rise in the catch-up decomposing and recycling ρTEs, respectively -sensing riboswitch. With abundant FMN, the ribB rut site is open to allow for efficient termination to attenuate the downstream ribB expression and riboflavin synthesis. With scarce FMN, the rut site is closed in a hairpin structure to impede the termination and facilitate the ribB transcription. Because the greenish-yellow fluorescence of FMN interferes with the single-molecule imaging of Cy3, we used non-fluorescent ribocil-C in place of FMN as a riboswitch ligand  in order to see if the addendum modifies the tp-ρTE correlations.All the above experiments were performed with a minimal set of transcription complex components including core RNAP, σ and ρ but without any other associating factors. We then carried out three auxiliary sets of experiments each with an addendum that may affect transcriptional pause and termination. Firstly, NusA and NusG factors are known to bind RNAP and affect its pause, termination or both in one way or another ,46. Thusrut sites but also nonspecifically at other sites. The nonspecific binding could produce a sponge effect to reduce the effective concentration of ρ for the specific binding , little significant for the catch-up decomposing  and less positive for the catch-up recycling  as shown in Figure r = 0.84, P = 0.038, m = 0.11%/s) but barely significant for the catch-up decomposing  or recycling . Similarly, with PEG-8000, the positive linear correlation  but not for the catch-up decomposing  or recycling .With NusA/G, the linear n Figure  is signitp and ρTE are distinctly altered in individual terminators by the inclusion of NusA/G, competitor RNAs or a crowder. After all, with either of them, the linear tp-ρTE correlations remain qualitatively the same for the slow decomposing termination routes; still much positive for the stand-by decomposing route and poor for the catch-up decomposing route. On the other hand, the correlation is poor or only slightly positive for the fastest, catch-up recycling termination route.Thus, both tp. Meanwhile, the catch-up ρ’s minor route for recycling outcome, the fastest route, is either unaffected or affected positively or negatively by pause elongation depending on the situation.We have recently unveiled the three coexisting but kinetically different routes of ρ-dependent termination . Here, wrut site and catches up with the paused RNAP, so the pause has to be long enough for the catching-up and has been deemed sensitive to the pause duration. However, the catch-up decomposing ρTE turns out to be little affected by pause extension or reduction in a range of 1.6 to 4.2 min. The rut sites are mostly only 10–20 nt away from the 3' end of RNA and rarely farther than 100 nt (rut site and move along RNA by the distance but not much longer. Conclusively, a pause is imperative but does not need to be long for efficient termination via the catch-up routes, and the long pauses observed with many ρ-dependent terminators may have a role for something else.These findings depicted in Figure n 100 nt . As ρ trn 100 nt , ρ wouldrut site, so the stand-by ρ has been presumed ready for execution and not to need long pauses. However, pause shrinkages to a minute or so hamper the stand-by route. How so? We hypothesize that the RNAP-prebound ρ is inactive or only partially active and takes time to get fully activated. This hypothesis could be consistent with that the RNAP-bound ρ requires conformational changes to capture a rut site (E. coli ρ hexamer ring, one protomer designated ρ6 is bound to another protomer ρ5 and NusA factor, but the rut-site binding is stabilized only after the ρ6 moves by ∼4.5 nm towards another protomer ρ1 and RNAP β’ subunit, shifting the ring opening from ρ6·ρ1 to ρ5·ρ6 junction. Furthermore, a series of additional conformation changes occur in the ρ·RNAP pre-terminational complex (Second, following the stand-by route, which is generally less frequent than the two catch-up routes combined , ρ pre-brut site ,46. In t complex .rut recognition in the stand-by termination route? Riboswitch RNA folding can be regulated thermodynamically or kinetically. In the kinetic controls, ligand recognition occurs fast as a co-transcriptional process before a thermodynamic equilibrium is reached. In the thermodynamic controls, by contrast, ligand recognition occurs after an equilibrium is established. Looking forward to obtaining more experimental proofs, we postulate that the mgtA and ribB riboswitch terminators are thermodynamically regulated primarily by utilizing the stand-by ρ’s slow rut recognition and long pauses.What can be an advantage of the slow activation for Initially in this study, we used a minimal transcription system comprising only linear DNA template, RNAP holoenzyme and the general termination factor ρ. In order to emulate the intracellular environment as much as practically possible, we performed the three In this study, we have shown that the three compatible routes of ρ-dependent termination are not only kinetically different but also play distinct regulatory roles. Including the ρ-free decomposing background termination, it is the four termination routes that operate compatibly together in any ρ-dependent terminator. We and others have previously shown that two termination routes are compatible in bacterial intrinsic terminators, and that the hairpin recycling route is much more frequent than the hairpin decomposing route ,54. MultIn summary, the three ρ-dependent termination routes dissimilarly depend on transcriptional pauses. The pause duration-termination efficiency correlation is invariably and highly positive only for the stand-by decomposing termination among the three routes, regardless whether NusA/G factors, competitor RNAs or a macromolecular crowder is added or not. Moreover, the two ρ-dependent terminator-associated riboswitches largely regulate the stand-by ρ’s slow termination. These results could provide unprecedented insights about the roles of transcriptional pauses in bacterial ρ-dependent terminations and possibly the general transcription termination mechanism.All data are available from the corresponding authors upon reasonable request.gkad051_Supplemental_FileClick here for additional data file.


Although reduced, sympathetic innervation in 2 distinct DCM models invariably exhibited transmural  gradients, with the endocardium being devoid of sympathetic nerve fibers versus controls. Further, the severity of transmural innervation gradients was correlated with VAs. Transmural innervation gradients were also identified in human left ventricular free wall samples from DCM versus controls. We investigated mechanisms underlying this relationship by in silico studies in 1D, 2D, and 3D models of failing and normal human hearts, finding that arrhythmogenesis increased as heterogeneity in sympathetic innervation worsened. Specifically, both DCM-induced myocyte electrical remodeling and spatially inhomogeneous innervation gradients synergistically worsened arrhythmogenesis. Thus, heterogeneous innervation gradients in DCM promoted arrhythmogenesis. Restoration of homogeneous sympathetic innervation in the failing heart may reduce VAs.Ventricular arrhythmias (VAs) in heart failure are enhanced by sympathoexcitation. However, radiotracer studies of catecholamine uptake in failing human hearts demonstrate a proclivity for VAs in patients with reduced cardiac sympathetic innervation. We hypothesized that this counterintuitive finding is explained by heterogeneous loss of sympathetic nerves in the failing heart. In a murine model of dilated cardiomyopathy (DCM), delayed PET imaging of sympathetic nerve density using the catecholamine analog [ Ventricular arrhythmias (VAs) remain a leading cause of death in nonischemic dilated cardiomyopathy (DCM) . The autNgf) and increased production of the neurorepellant semaphorin 3A (Sema3a) by cardiomyocytes in the failing heart (123I]-mIBG). In this study, reduced sympathetic innervation, identified by lower heart/mediastinal uptake of [123I]-mIBG, was associated with higher risk of severe VAs, heart failure hospitalization, and mortality.Paradoxically, while congestive heart failure (CHF), whether ischemic or DCM, is characterized by chronic sympathetic excess, it is associated with reduced cardiac sympathetic innervation in humans  and in cng heart –13. The ng heart , using dHowever, the mechanisms by which loss of sympathetic innervation in chronic CHF enhances arrhythmogenesis remain poorly understood. We hypothesized that 1) the loss of sympathetic nerves in the failing heart is heterogeneous and 2) this heterogeneity permits the emergence of dynamic substrates that permit reentry and the triggers that initiate them during sympathetic activation. The goal of the present study was to test this hypothesis in an established mouse model of nonischemic DCM (DCM Tg9) –17. ThisWe demonstrate that progression of DCM increases susceptibility to ventricular arrhythmogenesis. Importantly, we correlate this susceptibility to an increased transmural gradient of sympathetic innervation. Finally, we show how the heterogeneity in sympathetic innervation gradients promotes arrhythmogenesis using in silico models of normal and failing human myocardium and heart. This study provides what we believe are novel insights into how neural remodeling in nonischemic heart failure promotes ventricular arrhythmogenesis.First, we confirmed that the DCM mouse model exhibits features of human heart failure, particularly differential changes in functional sympathetic innervation as assessed by delayed PET imaging. DCM mice exhibited cardiomegaly compared with controls , progres11C]meta-Hydroxyephedrine ([11C]-mHED) PET-CT was performed to quantify functional sympathetic cardiac innervation on delayed imaging in DCM and control mice. To validate the synthesis of [11C]-mHED and its specificity for sympathetic nerve endings, delayed PET-CT imaging was performed following depletion of cardiac sympathetic nerves using 6-hydroxydopamine (6-OHDA). As shown in https://doi.org/10.1172/jci.insight.157956DS1), mice treated with 6-OHDA demonstrated little to no sympathetic innervation on immunohistochemistry. Consistent with this, PET-CT imaging using [11C]-mHED demonstrated severely reduced uptake in the heart but not hind leg or mediastinum, where sympathetic innervation is lower. When performed in late-stage DCM versus age-matched control littermates, noncardiac tissues, including liver and hind leg muscle, were not significantly different in radioactivity level. However, the base, apex, anterior wall, and posterior wall of the heart showed significantly less radioactivity in DCM mice compared with control mice  across the entire left ventricle in short axis  and long axis  . As expeAcsl1+), an established model of lipotoxicity, LV dysfunction, and HF  compared with control animals  at the b, and HF . In thisradients , A–C.To verify the presence of a transmural gradient in human cases of nonischemic HF, blinded analysis was conducted on human samples of healthy controls and nonischemic HF to examine sympathetic nerves with immunoreactivity to tyrosine hydroxylase (TH) . GreaterGiven that denervation in human HF and canine and rodent models of HF have implicated altered expression of neurotrophin/neurorepellant in cardiomyocytes, we sought to determine whether there are transmural differences in the neurotrophin NGF and the neurorepellant Sema3a in failing hearts versus WT. Transmural sections rapidly frozen and separated into epicardial and endocardial segments demonstrated greater mRNA expression of Sema3a in the endocardium of DCM compared with control hearts, while epicardial levels of Sema3a and endocardial or epicardial levels of NGF were not significantly different between DCM and WT hearts .To explore possible arrhythmic consequences of the increased innervation gradients, continuous ECG recordings were performed in DCM and control mice  under liCaL) throughout the sympathetic surge ramp protocol. This resulted in several types of behaviors as shown in top = 1.0, βbot = 0.0), PVCs that emerged and propagated from the gradient region , and whole cable EAD alternans in the case of homogeneous innervation (βtop = βbot = 1.0).To mechanistically investigate the arrhythmogenic effects of sympathetic innervation gradients, we performed computer modeling simulations of cardiac tissue . First, CaL conductance during a sympathetic surge  as well as reduced the sympathetic innervation level for the βbot while maintaining βtop = 1.0, which created a gradient of innervation in the cable. The resulting phase diagram shown in Ca,max (4 μm/s with these parameters) required for PVCs to occur. Second, once this minimum level is met, a larger denervation gradient  generally results in more PVCs , we observed only normal sinus rhythm at a PCa,max of 5 μm/s. However, in Ca,max to 6 μm/s , we now observed PVCs and T-wave repolarization abnormalities . Finally, in Ca,max = 6 μm/s, βendo,top = 1.0, βendo,bot = 0.2), we now saw a clear VT to VF reentrant arrhythmia. The asymmetry in innervation gradients allowed for a PVC to originate from one of the endo-epi gradients but not the other, leading to a reentrant arrhythmia forming, which eventually underwent wave break into VF.These 2D simulations exhibited several behaviors depending on the denervation pattern. Ca,max and the sympathetic innervation level to elicit different behaviors. This time, however, as we had 2 regions of different innervation in the endocardium, we had to vary both βendo,top and βendo,bot, resulting in a 3D phase diagram. endo,top plotted against βendo,bot for 3 values of PCa,max = 4, 5, 6 μm/s, representing a low, medium, or high sympathetic surge, respectively. The identity diagonal along the plot represents a homogeneous epi-endo gradient across the tissue. The off-diagonal elements represent varying degrees of heterogeneity of epi-endo gradients between the top and bottom of the tissue, as defined by βendo,top and βendo,bot. With PCa,max = 4 μm/s, only sinus rhythm was observed at all combinations of gradients. As the sympathetic surge was stronger with PCa,max = 5 μm/s, we began to see PVCs or VF appear with certain combinations of endocardial innervation heterogeneity. Note that the severe arrhythmias only manifested with large heterogeneity of gradients, the top left and bottom right corners of the plot. At an even stronger sympathetic surge with PCa,max = 6 μm/s, all combinations of gradients exhibited some arrhythmia, but the diagonal representing homogeneous gradients exhibited only benign PVCs, while the large-heterogeneity off-diagonal regions again exhibited more severe VT and VF arrhythmias.As in the previous cable simulations, we simultaneously varied the Pepi = 1.0 and βendo,base and βendo,apex varying between the base and apex regions of the endocardium. Three different scenarios were simulated: homogeneous high denervation , homogeneous mild denervation , and heterogeneous mixed denervation . Finally, to verify these results with a more realistic physiological geometry, we then performed simulations using a 3D anatomical ventricle geometry. The simulation setup is shown in endo,base = 0.8, though again favoring the LV (see voltage snapshot 4). The apex region this time did not excite ectopically because of the heterogeneity of denervation where βendo,apex = 0.2, and thus the PVC was able to propagate asymmetrically from base to apex. A focal polymorphic VT developed, and this asymmetry eventually resulted in a competing focus developing, causing wave break, which degenerated into VF.functional sympathetic ventricular denervation, as demonstrated by [11C]-mHED PET-CT, which was mirrored by an increased epicardial-to-endocardial sympathetic innervation gradient in the left ventricular myocardium on IHC. Moreover, we directly translate these findings to the clinical setting, as similar innervation patterns were observed in human HF tissue. Last, we correlate these changes in innervation gradients to increased arrhythmogenesis through in vivo ECG recordings with a NE challenge and through computer models that assess the arrhythmogenic consequences of the observed innervation gradients.This study demonstrates that 1) heterogeneity in sympathetic innervation is characteristic of DCM in mice and humans and 2) this heterogeneity seemingly increases susceptibility to VAs. We show that DCM mice experience To our knowledge, this is the first study characterizing spatial dispersion of sympathetic innervation in HF and directly correlating these patterns to increased arrhythmogenesis. This evidence suggests that the DCM-induced changes in sympathetic innervation patterns play a fundamental role in promoting pro-arrhythmic conditions.11C]-mHED PET-CT data, which was additionally replicated in another murine model of HF in which HF was induced by an overexpression of Acsl1.Prior animal and human studies have demonstrated that HF is associated with cardiac autonomic denervation. For example, Machado et al. showed that patients with DCM associated with Chagas disease experienced cardiac sympathetic denervation . SimilarCardiac sympathetic innervation is established during embryonic development, when sympathetic nerves reach the heart and traverse the myocardial wall from the epicardium to the endocardium , 23. CorP75NTR-null mice bears resemblance to ours and clearly indicates a possible role for NGF/P75NTR signaling in DCM.At the tissue level in our study, cholinergic innervation was localized primarily to the epicardium as shown in IHC staining for vesicular acetylcholine transporter (VAChT) and did not differ between DCM mice and controls . In contAdditionally, Chen et al. recently showed that in patients with nonischemic cardiomyopathies, global sympathetic denervation correlates with the extent of ventricular fibrosis reflected in mean unipolar voltage, which is related to an increased risk for arrhythmogenesis . Prior sCaL was required for PVC formation. If too much denervation is present, there is no longer enough sympathetic induced activation of ICaL for the PVC to occur. Therefore, while in general larger innervation gradients were more conducive to PVCs, there exists an intermediate range where PVCs can form.In this study, we used computer modeling to explore the mechanistic arrhythmia implications of our experimental results, namely how HF can result in heterogeneous denervation of sympathetic fibers. Our simulations showed that gradients in sympathetic denervation can generate PVCs during a sympathetic surge through our previously described R-from-T mechanism. However, some minimal threshold of sympathetic innervation was still required for PVC formation. This is consistent with our previous studies , 35, 36,In addition to isolated innervation gradients, we investigated the effects of gradient heterogeneity, i.e., neighboring regions with different epicardial-endocardial innervation gradients as seen in our experimental results. Both our 2D and 3D anatomical ventricle simulations demonstrated that while PVCs could form with homogeneous innervation gradients, heterogeneity in innervation gradients was required for more severe reentrant and complex arrhythmias such as VT and VF. This arises from the fact that since the main gradient is from the endocardium to the epicardium, any PVC that forms from that gradient will be largely planar, traveling a short distance transmurally until hitting the surface boundary. This geometry of ectopic propagation will always be relatively benign, as the entire PVC will be extinguished quickly without any symmetry breaking. Conversely, with heterogeneous innervation gradients, the spontaneous PVC is now able to propagate longitudinally with much more room and asymmetry for reentry and/or wave break to occur.Crucially, as these arrhythmias follow the R-from-T mechanism, both the sympathetic surge as well as QT prolongation must be present. Therefore, the electrophysiological changes of HF were a necessary component, along with the heterogeneous innervation gradients, for these arrhythmias to occur. Gradient heterogeneity in the setting of normal healthy electrophysiology was not able to produce arrhythmias.The arrhythmia mechanisms demonstrated by computer modeling appear to be consistent with our experimental arrhythmia results as seen in Figure 6. Just as in the simulations, the DCM mice with increased epicardial-endocardial innervation gradients demonstrated more arrhythmias, mainly PVCs. Sustained VT or VF arrhythmias were rare in the mouse experiments, which may be due to the small size of the mouse heart, smaller than the tissue wavelength required for reentry . RegardlThese modeling results suggest that while innervation gradients from HF alone can produce PVCs, more severe arrhythmias may require additional conditions including a heterogeneity of innervation gradients. This may play a role in how early mortality from less severe HF (NYHA II-III) is primarily from sudden cardiac death (SCD), while mortality in severe HF (NYHA IV) is primarily from CHF , though This study was partially performed in a murine model of DCM. Murine pathophysiology and electrophysiology might incompletely translate to humans. However, sympathetic innervation gradients were also observed in human HF tissue. Further, using a computational model of human HF, the arrhythmogenic effects of this gradient were also shown. It should also be noted that this study demonstrates an association and not a causal link between myocardial sympathetic innervation gradients and arrhythmogenesis.CaL without any other effects, focusing on the initial phase of sympathetic simulation. While this should be sufficient for arrhythmia initiation, the next dominant current that β-adrenergic stimulation affects is slow inward rectifying potassium current, which has a slower time course than ICaL and may play a role in arrhythmia termination  were assessed to assess left ventricular size and function.11C]-mHED. Functional imaging of sympathetic innervation was assessed using PET-CT following [11C]-mHED injection. Mice were anesthetized with isoflurane mixed in oxygen , secured to an imaging chamber on the Inveon PET scanner  or G8 PET/CT Scanner (PerkinElmer), and catheterized via tail vein. A 60-minute list-mode PET acquisition was started followed immediately by a bolus injection of [11C]-mHED via the catheter . Mice imaged with the Inveon PET scanner underwent CT imaging on the CrumpCAT microCT (UCLA) for coregistration (Exitron 12000 was used as CT contrast). PET data were plotted as histograms into 8 × 5-minute frames (starting at 2.5 minutes after injection), corrected for isotope decay and detector normalization (and photon attenuation for the G8 scanner), reconstructed using OSEM-MAP or MLEM for G8 scanner, and normalized to %ID/g. Images were quantified by drawing regions of interest (14 mm3) on select tissues using Amide software (SourceForge).See Ventricular arrhythmogenesis was quantified in mice anesthetized under inhaled isoflurane (1%–2%). Recordings were made through subcutaneous needle electrodes in the right forearm and left hind leg to acquire lead II electrocardiograms . LabChart 8 was used to analyze ECG recordings for arrhythmias (AD Instruments). In short, 5-minute baseline recordings were obtained, after which NE (2 mg/kg) was injected intraperitoneally. After injection, ECG recordings were continued for over 30 minutes, or until death.An arrhythmogenicity index was created to denote the severity of arrhythmogenesis based on ECG recordings from in vivo studies in mice, as has been done before in pigs . ArrhythAll IHC studies were performed on hearts following perfusion fixation. Briefly, following cervical dislocation, heparinized saline (50 U/mL) was injected into the left ventricular apex, followed by 4% paraformaldehyde (PFA) for 5 minutes. The heart was isolated and collected in a 0.01 M PBS solution, after which it was suspended in 4% PFA solution and stored at 4°C overnight.Z-stack. All images were exported as.CZI files and converted to maximum intensity projections (MIPs) for subsequent quantitative analysis.Hearts were embedded in approximately 2% agarose solution in an upright position, and transmural sections of 100 μm thickness were obtained. Sections were stained for TH and NPY, or VAChT, using appropriate primary and fluorescent secondary antibodies  to explore the morphology and distribution of sympathetic innervation. Intensity-based, 3D modeling was performed on heart sections stained with TH and NPY. The Surfaces tool was used on individual.CZI files of confocal images taken at original magnification, 20×, with Mouse hearts were rapidly excised and placed on a metal tray on a slab of dry ice, cut in short axis, and rapidly separated into endo and epi regions under a large magnifying glass. Separated epicardial and endocardial tissues were then stored at –80°C. Tissues were homogenized with Trizol, and RNA was subsequently isolated and quantified through RT-qPCR using a NanoDrop device .Whole, transmural mouse heart sections were stained with Masson’s trichrome as described before  and wereConfocal images of mouse heart sections stained with TH and NPY were converted to MIP format. The left ventricle was divided into 8 segments separated by lines on the program Zen Black (Carl Zeiss), and the epicardium and endocardium were separated at the midpoint of the myocardium. The 16-bit, full-resolution images were analyzed on ImageJ software . After binary conversion of the files, thresholding was used to calculate innervation area and tissue area of the epicardium and endocardium for all 8 segments. An epicardium-over-endocardium ratio was made by dividing the relative innervation area in the epicardium over the relative innervation area in the endocardium. Values quantifying the transmural gradient were further analyzed for heterogeneity by comparing a ratio of adjacent regions in the short-axis orientation and another ratio in the long-axis between identical regions in base-mid and midapex segments. Ratios from individual regions were scored to identify the severity of long-axis heterogeneity .Details of modeling studies are provided in P < 0.05 was considered statistically significant. Data in all dot plots are shown as as mean ± SEM.Statistical analysis was performed using GraphPad Prism 9.2.0. All described studies were in accordance with the University of California Institutional Animal Care and Use Committee guidelines. All mice were housed in dedicated pathogen-free facilities at the UCLA. The UCLA Institutional Review Board approved human tissue studies.AHJD, MBL, JTL, RS, ZQ, and OAA designed research studies. AHD, LC, JC, MBL, MDP, EHS, CMW, MAO, JTL, MP, and RS conducted experiments/acquired data. AHD, LC, TJD, JC, MBL, MP, DBH, JTL, RS, ZQ, and OAA analyzed data. AJ, CVR, and PYJ provided reagents/samples. AHD, MBL, DBH, RS, and OAA drafted the manuscript. All authors approved the manuscript.