CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in local tissues can result in significant exposure of cytotoxic T cells to the lactate metabolite. Lactate has been known to act as an immunosuppressor, at least in part due to its association with tissue acidosis.To dissect the role of the lactate anion, independently of pH, we performed phenotypical and metabolic assays, high-throughput RNA sequencing, and mass spectrometry, on primary cultures of murine or human CD8+ T cells exposed to high doses of pH-neutral sodium lactate.The lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and can displace glucose as a carbon source. Activation in the presence of sodium lactate significantly alters the CD8+ T cell transcriptome, including the expression key effector differentiation markers such as granzyme B and interferon-gamma.Our studies reveal novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells. This allows the regeneration of the NAD+ cofactor, essential for enzyme-catalyzed oxidations . For T cell purification, wild type donor and recipient C57BL/6J (RRID: IMSR_JAX:000664) animals were purchased from Janvier Labs.2.2Sodium Chloride  and Sodium L-Lactate  solutions were prepared by dissolving each compound into RPMI 1640  at a maximum concentration of 500 mM. Solutions were serially diluted, and osmolality measurements were obtained with a micro-osmometer .2.36 (24-well plate) or 5x105 (48-well plate) CD8+ T cells were activated with Dynabeads Mouse T-Activator CD3/CD28  in a 1:1 bead to T cell ratio and cultured in 2 ml (24-well plate) or 1 ml (48-well plate) RPMI 1640  supplemented with 10% Fetal Bovine Serum , 50 µM 2-mercaptoethanol , 100 U/ml penicillin-streptomycin , and 10 U/ml recombinant human IL-2 , and incubated at 37˚ C for 3 days in a humidified CO2 incubator.Spleens were harvested from 8-12 week old C57BL/6J (RRID : IMSR_JAX:000664) mice, mashed over a 40 µm cell strainer , and CD8+ T cells were purified by positive magnetic bead selection  according to manufacturer’s instructions. Purified CD8+ T cells were counted and cell diameter measured using a Moxi Z mini  or a TC20  automated counter. 1x102.45 (48-well plate) or 1x105 (96-well plate) naïve CD8+ T cells were activated with Dynabeads Human T-Activator CD3/CD28  in a 1:1 bead to T cell ratio and cultured in 1 ml (48-well plate) or 200µl (96-well plate) RPMI 1640  supplemented with 10% Fetal Bovine Serum, , 100 U/ml penicillin-streptomycin , and 30 U/ml recombinant human IL-2 , and incubated at 37˚C for 4 days in a humidified CO2 incubator. For long-term expansion after 4 days, Dynabeads were removed using a DynaMag-2 Magnet , washed with PBS, counted, and resuspended in fresh media supplemented with 30 U/ml IL-2 and incubated as described above. Media supplemented with cytokines was added every 2-3 days to maintain cell densities below 2x106/ml. Cells were counted again at day 7 and maintained in culture under the above-described conditions until day 14.Peripheral blood mononuclear cells (PBMCs) were harvested from standard buffy coat preparations of healthy donors, aged 20 to 40 years old, obtained from the Department of Transfusion Medicine at Karolinska University Hospital and processed no later than 4 hours after collection. PBMCs were isolated by gradient centrifugation using Histopaque-1077  and enriched for CD8+ T cells by magnetic bead selection, according to manufacturer’s instructions . Purified CD8+ T cells were counted and 5x102.5Sodium L-Lactate , L-Lactic Acid  and Sodium Chloride  were prepared as 10x concentrated solutions in complete media. Compounds were added to T cells at the point of activation (day 0) minutes before addition of CD3/CD28 Dynabeads. Sodium Chloride and/or plain media was used as control. For long-term expansion, after beads removal cells were cultured in complete media supplemented with cytokines without further addition of compounds.2.6After magnetic bead purification, CD8+ T cells were loaded with 5 µM CellTrace CFSE or CellTrace Violet (CTV) dyes  according to manufacturer’s instructions. CFSE or CTV dilution was determined by flow cytometry.2.7Single-cell suspensions were washed and stained with Fixable Near-IR Dead Cell Stain Kit  followed by staining of extracellular antigens with fluorochrome labelled antibodies. The Fixation/Permeabilization Solution Kit  was used for exposing cytoplasmic antigens. The Transcription Factor Buffer Set  was used for exposing nuclear antigens. Fluorochrome-labelled antibodies against mouse antigens CD44 (clone IM7), CD8 (clone 53- 6.7), CTLA-4 (clone UC10-4F10-11), LAG-3 (clone C9B7W), and PD-1 (clone J43) were purchased from BD Biosciences, CD27 (clone LG.3A10), CD28 (clone 37.51), CD62L (clone MEL-14), and ICOS (clone C398.4A) were purchased from BioLegend, and 4-1BB (clone 17B5), CD127 (clone A7R34), CD25 (clone PC61.5), Eomes (clone Dan11mag), T-bet (clone eBio4B10), and anti-human/mouse Granzyme B (clone GB12) was purchased from Thermo Fisher. Cell counting was performed with CountBright Absolute Counting Beads . Samples were processed in a FACS Canto II flow cytometer (BD Biosciences). Data analysis was performed with FlowJo .2.8ex vivo and cells harvested at 0, 6, 12, 24, 48, and 72 hours post-activation. For each time point, total protein was extracted using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit  and the cytoplasmic fraction used for western blots. Total RNA was extracted using the RNeasy Mini Kit . For each sample, 15 µg of total protein were separated in SDS-PAGE, blotted onto a PVDF membrane and probed with antibodies against MCT1 , MCT4 , GzmB  and PPIB  and detected using infra-red labeled secondary antibodies in an Odyssey imaging system (LICOR). Western blot data were analyzed with the Image Studio Lite software . REVERT Total protein stain  was used for lane normalization. Two micrograms of total RNA were reverse transcribed using iScript cDNA synthesis kit (BioRad) in a total volume of 20 μl. Real-time RT-PCR was used for mRNA quantification . All primers were designed to cover exon-exon boundaries to avoid amplification of genomic DNA. Predesigned qPCR primers used to detect mouse Slc16a1 , Slc16a3 , granzyme B (GzmB) , and Hprt  were purchased from IdtDNA or Sigma. All reactions were performed in 96-well MicroAmp Optical plates in duplicates. The total reaction volume was 15 µl, containing 5 µl sample cDNA, 0.4 mM of each primer forward and SYBR Green PCR Master Mix . All quantification reactions were controlled with a melting curve and primer efficiency was tested with standard curves, and did not differ between the primer pairs. Gene expression data was normalized to Hprt levels.Mouse CD8+ T cells were activated 2.9Mouse CD8+ T cells were activated for 72 hours in the presence of either 40 mM sodium lactate or plain media. At the end of activation cells were washed twice with PBS and cell pellets were snap-frozen in RLT Plus lysis buffer . Total RNA was extracted with the Qiagen’s RNeasy kit according to the manufacturer’s instructions. All samples were quality checked with Agilent Tapestation RNA screen tape. To construct libraries suitable for Illumina sequencing, the Illumina TruSeq Stranded mRNA Sample preparation protocol which includes cDNA synthesis, ligation of adapters, and amplification of indexed libraries was used. The yield and quality of the amplified libraries were analyzed using Qubit by Thermo Fisher and the Agilent Tapestation. The indexed cDNA libraries were normalized and combined, and the pools were sequenced on the Nextseq 550  for a 50-cycle v2.5 sequencing run, generating 71 bp single-end reads (and 2*10 bp index reads). Demultiplexing was performed using bcl2fastq , generating fastq files for further downstream mapping and analysis.2.10Bcl files were converted and demultiplexed to fastq using the bcl2fastq v2.20.0.422 program. STAR 2.7.5b  was used2.116 cells were harvested and washed 3 times with PBS before adding 800 µL of ice-cold extraction mixture consisting of chloroform:MeOH:H2O (1:3:1), containing 500 pg/µL of 2-Deoxy-D-glucose 6-phosphate and 70 pg/µL of myristic acid-13C3 were added to each sample. Metabolites were extracted using a mixer mill set to a frequency 30 Hz for 2 min, with 1 tungsten carbide bead added to each tube. Obtained extracts were centrifuged at 14000 rpm for 10 min. The collected supernatants were divided for GC-MS and Sugar-P analysis. 300 µL of the supernatant was transferred into GC and LC-vial respectively, the supernatants were evaporated until dryness using a SpeedVac.For metabolite analysis, 5x102.12For derivatization, dried samples were dissolved in 20 µl of methoxylamine and incubated on a heat block at 60°C, 30 min. After overnight incubation at room temperature, 12µl of 1- Methylimidazol and 6 µl of propionic acid anhydride were added and heated at 37°C for 30 minutes. The reaction mixture was then evaporated to dryness by N2 gas. Prior to LC-MS analysis, derivatized metabolites were dissolved in 100µl of aqueous 0.1% formic acid. Quantitative analysis was performed by combined ultra-high-performance liquid chromatography electrospray ionization-triple quadrupole-tandem mass spectrometry (UHPLC-ESI-QqQMS/MS) in dynamic multiple-reaction-monitoring (MRM) mode. An Agilent 6495 UHPLC chromatograph equipped with a Waters Acquity BEH 1.7 µm, 2.1 x 100 mm column  coupled to a QqQ-MS/MS  was used. The washing solution, for the autosampler syringe and injection needle, was 90% MeOH with 1% HCOOH. The mobile phase consisted of A, 2% HCOOH and B, MeOH with 2% HCOOH. The gradient was 0% B for 1 min followed by linear gradients from 0.1 to 30% from 1 to 3 min then 30 to 40% B from 3 to 6 min, hold at 40% B from 6 to 10 min, followed by 40 to 70% B from 10 to 12.5 min, hold at 70% B from 12.5 to 15 min, and thereafter 70 to 99% B from 15 to 17.5 min. B was held at 99% for 0.5 min, and thereafter the column was re-equilibrated to 0% B. The flow rate was 0.65 mL min-1 during equilibration and 0.5 mL min-1 during the chromatographic runs. The column was heated to 40°C, and injection volumes were 1 μL. The mass spectrometer was operated in negative ESI mode with gas temperature 230°C; gas flow 12 L min-1; nebulizer pressure 20 psi; sheath gas temperature 400°C; sheath gas flow 12 L min-1; capillary voltage 4000 V (neg); nozzle voltage 500 V; iFunnel high pressure RF 150 V; iFunnel low pressure RF 60 V. Fragmentor voltage 380 V and cell acceleration voltage 5 V. Data were processed using MassHunter Qualitative Analysis and Quantitative Analysis  and Excel  software.2.13The GC-MS samples were spiked with 1050 pg of each GC-MS internal standard before evaporation. Derivatization was performed according to Gullberg et al. . In deta2.1413C6] glucose, ii) 11mM [U13C6] glucose and 40 mM lactate, or iii) 11 mM glucose and 40 mM [U13C3] sodium lactate, and isotopic labelling was performed for 24, 48 and 72 hours. Sodium chloride (40 mM) was added to i) to control for osmolality. Cells harvested at each time point were washed with PBS, and metabolic activity quenched by freezing samples in dry ice and ethanol, and stored at -80°C. Metabolites were extracted by addition of 600 μl ice-cold 1:1 (vol/vol) methanol/water  to the cell pellets, samples were transferred to a chilled microcentrifuge tube containing 300 μl chloroform and 600 μl methanol . Samples were sonicated in a water bath for 8 min at 4°C, and centrifuged (13000 rpm) for 10 min at 4°C. The supernatant containing the extract was transferred to a new tube for evaporation in a speed-vacuum centrifuge, resuspended in 3:3:1 (vol/vol/vol) methanol/water/chloroform  to phase separate polar metabolites (upper aqueous phase) from apolar metabolites (lower organic phase), and centrifuged. The aqueous phase was transferred to a new tube for evaporation in a speed-vacuum centrifuge, washed with 60 μl methanol, dried again, and derivatized by methoximation  and trimethylsilylation trifluoroacetamide + 1% trimethylchlorosilane)  for ≥ 1 h. GC-MS analysis was performed using an Agilent 7890B-5977A system equipped with a 30 m + 10 m × 0.25 mm DB-5MS + DG column (Agilent J&W) connected to an MS operating in electron-impact ionization (EI) mode. One microliter was injected in splitless mode at 270°C, with a helium carrier gas. The GC oven temperature was held at 70°C for 2 min and subsequently increased to 295°C at 12.5°C/min, then to 320°C at 25°C/min (held for 3 min). MassHunter Workstation  was used for metabolite identification by comparison of retention times, mass spectra and responses of known amounts of authentic standards. Metabolite abundance and mass isotopologue distributions (MID) with correction of natural 13C abundance were determined by integrating the appropriate ion fragments using the GC–MS Assignment Validator and Integrator (GAVIN)  11 mM glucose to the labelling of pyruvate and lactate, but not to PEP or ribose  or hypoxic conditions (1% O2), using wild type or HIF-1α null CD8+ T cells  but with low transcript abundance - 64 TPM (present data) and 14 TPM (single-cell). HIF-1α had a motif-enrichment (OR 2.2 p 1.5e-11) and high T cell transcript abundance - 388 TPM (present data) and 92 TPM (single-cell). The transcription factor with the highest affinity score was HIF-1α  together with c-MYC  .All data generated are included in the manuscript and supporting files. The RNA sequencing data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. All experiments and protocols were approved by the regional animal ethics committee of Northern Stockholm .LB and PV contributed equally toward experimental design, performance, and analysis; interpretation of results; drafting and revision of the manuscript. PG contributed to the design, performance, and analysis of carbon tracing experiments. PC conducted experiments. IF contributed to experiment design and discussion. DB contributed to the bioinformatic analysis of RNA-seq data and GEO submission. ER contributed to the transcription factor motif enrichment analysis. HR contributed to the study conceptualization. RJ and HR supervised the work and revised the manuscript. All authors discussed and edited the manuscript. All authors contributed to the article and approved the submitted version.


Vibrio natriegens is a halophilic bacterium with the fastest generation time of non-pathogenic bacteria reported so far. It therefore has high potential as a production strain for biotechnological production processes or other applications in biotechnology. Culture media for V. natriegens typically contain high sodium chloride concentrations. The corresponding high chloride concentrations can lead to corrosion processes on metal surfaces in bioreactors. Here we report the development of a low-chloride chemically defined medium for V. natriegens. Sodium chloride was completely replaced by the sodium salts disodium hydrogen phosphate, disodium sulfate, and sodium citrate, while keeping the total concentration of sodium ions constant. The use of citrate prevents the occurrence of precipitates, especially of ammonium magnesium phosphate. With this defined medium, high-cell-density fed-batch cultivations in laboratory-scale bioreactors using exponential feeding yielded biomass concentrations of more than 60 g L−1.A defined medium for V. natriegens that only contains traces of chloride was developedCorrosion processes on metal surfaces in industrial bioreactors can thus be preventedHigh yields of biomass can be achieved in fed-batch cultivation with this mediumThe online version contains supplementary material available at 10.1007/s00253-023-12799-4. Vibrio natriegens is a marine, facultatively anaerobic, gram-negative proteobacterium  from the German collection of microorganisms and cell cultures GmbH .The microorganism used in this study was Vibrio natriegens was preserved as 1.5 ml aliquots in a 20 g L−1 glycerol solution in LB medium at −80 °C. These frozen stock cultures were used for inoculum preparation. The bacteria were first cultivated in a preculture with complex medium. For this purpose, 250 μL bacterial suspension containing V. natriegens was taken from a cryo vial and added to 75 ml modified LB medium with increased NaCl concentration  in a 500 ml shake flask with 4 baffles and dissolved oxygen (DO) sensor spots . The inoculated preculture was cultured at 30 °C and a shaking frequency of 200 rpm for 6–7 h in a Multitron shaking incubator  using the Shake Flask Reader SFR from PreSens for online measuring of DO.−1 NaCl. The medium was adapted in preliminary tests and is referred to here as standard medium VN1. It contains the following composition:This preculture was used to inoculate the shake flasks used for the medium development. The standard medium used as the starting medium for media development is a modified M9 medium supplemented with 20 g L−1); glucose (12 g L−1); Na2HPO4 × 2 H2O (8.5 g L−1); KH2PO4 (3 g L−1); NH4Cl (1 g L−1); MgSO4 × 7 H2O (0.74 g L−1); (NH4)2SO4 (5 g L−1); 1% (v v−1) trace element solution.NaCl (20 g L2-EDTA × 2 H2O (6 g L−1); FeSO4 × 7 H2O (1.64 g L−1); CaCl2 × 2 H2O (1.35 g L−1); MnSO4 × H2O (1 g L−1); ZnSO4 × 7 H2O (0.1 g L−1); CuSO4 × 5 H2O (0.03 g L−1); NiCl2 × 6 H2O (0.002 g L−1).The trace element solution was taken from Keilhauer et al.  with EDT2HPO4, Na2SO4, and Na3-citrate. In the first part of the medium optimization, NaCl was replaced by Na2HPO4 and Na2SO4 in different compositions according to Table 2HPO4, Na2SO4, and Na3-citrate in different compositions as shown in Table −1 3-(N-morpholino) propanesulfonic acid (MOPS) to keep the pH as constant as possible. The pH was adjusted to 7.0 by adding 3 M KOH or 2 M H3PO4.During the media optimization described in the “2 concentration. At the end of the experiments, 1 ml samples were taken from each flask, and OD, osmolality, pH, and glucose concentration were measured.For media optimization, the bacteria were cultivated in 500 ml shake flasks (4 baffles) with DO sensor spots in the ShakeFlask Reader SFR. The shake flasks were filled with different defined media according to Tables The optimized media in Table −1); K2HPO4 (60 g L−1); Na3-citrate × 2 H2O (24 g L−1); MgSO4 × 7 H2O (14.8 g L−1); Na2-EDTA × 2 H2O (0.54 g L−1); FeSO4 × 7 H2O (0.148 g L−1); CaCl2 × 2 H2O (0.122 g L−1); MnSO4 × H2O (90 mg L−1); ZnSO4 × 7 H2O (9 mg L−1); CuSO4 × 5 H2O (2.7 mg L−1); NiCl2 × 6 H2O (0.2 mg L−1).Glucose .For the fed-batch cultivations, two parallel benchtop bioreactors were used . The maximum working volume is 1 L. Agitation was achieved through three 6-blade Rushton turbine impellers. The temperature was set to 30 °C and the pH value was controlled at 6.75 (± 0.05) using 25% ammonia water and 2 M phosphoric acid. Antifoam  was fed when required. DO was controlled by a multivariate controller such that the DO value was always above 25% air saturation as described in Biener et al. . The manμS was 0.3 h−1.The bioreactors were filled with 500 ml medium and inoculated using a preculture with the same defined medium to an initial OD600 of 1.7 ± 0.2. Feeding was started after glucose depletion, which was indicated by a sharp increase in DO. The feed solution was fed exponentially according to Eq. cX, 0) was estimated from a previous correlation of OD600 with cell dry weight (CDW) values and the corresponding volume V0 was 0.55 L. The biomass/glucose yield coefficient YX/S was 0.4 grams biomass per gram glucose (gg−1) and the maintenance mS was 0.1 grams biomass per gram glucose and hour (gg−1 h−1). Glucose feed concentration was 540 g L−1. The exponential feed rate was controlled gravimetrically.The biomass concentration at feeding start . Glucose concentration was determined enzymatically using BIOSEN C-line . Osmolality was measured using the freezing point osmometer Osmomat 3000 .For analyses of supernatant components, samples were inactivated at 80 °C for 10 min and centrifuged at 16,000 The main objective of the medium optimization was to replace NaCl in the standard medium with other sodium salts in order to prevent chloride-associated corrosion processes in bioreactors.2HPO4 and Na2SO4 in different compositions according to Table In the first series of experiments, NaCl was replaced by NaV. natriegens grow similar in all media used. The highest OD600 value of 21.6 ± 1.3 was obtained in medium VN4 with Na2HPO4 without the use of Na2SO4. Medium VN2 with NaCl replaced by Na2SO4 had the lowest OD600 value (18.4 ± 2.3). The results show that NaCl can be replaced completely by other sodium salts.Figure −1, which was lower compared to the standard medium VN1 with NaCl (1.08 Osmol kg−1). The final pH values were in the range of 6.1 to 6.8.The osmolalities in the new media at the end of the cultivations were between 0.82 and 0.91 Osmol kgThe fermentation broth was also visually examined for precipitate formation before and at the end of cultivation. Before the start of the cultivations, no precipitates were visible in the media. In some cases, the samples showed precipitations at the end of the cultivations. Precipitates were clearly visible as a white precipitate below the cell pellet in the centrifuged sample see Fig. .Fig. 2PrMicroscopic examination of these deposits indicates precipitation of ammonium magnesium phosphate (AMP) due to the characteristic crystals see Fig. . AMP cry2HPO4) at room temperature of 20 °C was first examined without cells. Since precipitation can also occur several hours after preparation of the solutions 2SO4 to further reduce the chloride content. This medium is referred to here as VN10. The composition of the optimized growth media VN6 and VN10 is summarized in Table In all media, no precipitations were detected before inoculation and at the end of the runs. Precipitation was practically prevented in all media with sodium citrate. Media VN5 through VN8 were stored sterile without cells at room temperature for another 7 days. Slight precipitation occurred in medium VN5 with the lowest citrate content, while all other media remained precipitation-free. A good compromise between high biomass yield and elimination of precipitation is medium VN6. In a final step to optimize the medium VN6, the existing NHSodium citrate was also added to the feed solution as described in section “−1 and therefore higher than that of the other media VN2 to VN10 (between 0.8 and 0.91 Osmol kg−1), because the total concentration of osmotically active particles is higher in the standard medium VN1. Therefore, in the second cultivation (run B), medium VN11 was used, in which the concentrations of Na2HPO4, Na2SO4, and sodium citrate were increased (see Table 4Cl was replaced by (NH4)2SO4.Fed-batch cultivations were performed in the Multifors bioreactor with the optimized low-chloride medium. With the Multifors, it is possible to operate two bioreactors in parallel. In the first batch (run A), medium VN6  are shown. The batch phase lasts approximately 4.2 h for run A and B and 3.5 h for run C and D until glucose was depleted. Biomass concentration increased exponentially while glucose concentration decreased during this phase. The maximum specific growth rate during the batch phase was between 0.9 h−1 in run B and 0.96 h−1 in run D.In Fig. 2-content in aeration for run C and D .Figure see Fig. . During see Fig. . The finDuring the batch phase, the bacteria produced the by-product acetate see Fig. . After gThe ammonium concentration in both reactors increases continuously during the batch phase due to the addition of ammonia water by pH control see Fig. . As expeV. natriegens contain high concentration of NaCl  values, respectively. This could lead to higher space-time yields. Another strategy often used in fed-batch cultivations is to switch to a constant feed rate after the oxygen transfer rate (OTR) reaches its maximum during exponential feeding. However, Thiele et al.


RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes  express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in  The pha2+]i) , 7 and dl muscle , 9, 10.  for MHS . CACNA1St (IVCT) . Most vaificance . To dated T1354S , 21, 22.V1.1-R174W is within the S4 segment of repeat I, T1009 is within the S5-S6 linker of repeat III, R1086 is located in the cytoplasmic loop between repeats III and IV, and T1354 is located in the S5-S6 linker of repeat IV; all highly conserved regions. Functional studies of CaV1.1-R174W and CaV1.1-R1086H expressed in CaV1.1-null myotubes, which lack expression of CaV1.1-WT, have demonstrated increased sensitivity to caffeine-induced Ca2+ release  mouse myotubes, CaV1.1-R174W was shown to result in significantly increased sensitivity to both caffeine and the volatile anesthetic halothane  and homozygous (HOM) Cacna1s-p.R174W mice have normal survival and reproductivity when maintained under home cage conditions. Contrary to expectations, neither HET nor HOM mice displayed adverse outcomes characteristic of a fulminant MH syndrome when exposed to halothane anesthesia, nor are they sensitive to moderate environmental heat stress.To directly address whether CaV1.1-R174W is shown in Figure 1A. Genomic DNA from a mouse HOM for the CaV1.1-R174W mutation was amplified by PCR using primers F1 and R1 (B). The PCR product included the CaV1.1-R174W mutation site and the intron region used for targeting during knock-in cloning. Sequencing of the PCR product was done using primers F1, F2, R1, and R2. CaV1.1-R174W sequencing results aligned with the Cacna1s sequence and confirmed the presence of the mutation and the leftover landing pad containing the LoxP site (B). All other sequence was identical to Cacna1s in the database (B). Quantitative reverse transcription-PCR showed that transcripts for both CaV1.1-WT and CaV1.1-R176W were detected with a ratio of 1:1 in HET skeletal muscle, whereas only the respective transcripts for both CaV1.1-WT and CaV1.1-R176W were detected in WT and HOM samples, respectively (C and D).The targeting vector used to knock-in putative MHS mutation Ca1 and R1 B. The PCoxP site B. All otdatabase B. Quantiectively , C and DA and B). Awake HET and HOM mice survived the heat stress alone protocol lasting 60 min despite a rise in their core body temperature to ∼40.7 °C (A). Upon return to their home cages at 23 to 24 °C, both HET and HOM mice recovered to their initial core body temperatures within 20 min and failed to trigger over the ensuing 24 h when they were sacrificed. Mice subjected to 1.5% halothane maintenance anesthesia maintaining their body temperature of 37 to 38 °C, an environmental temperature empirically determined to maintain their baseline core body temperature during anesthesia, failed to exhibit hyperthermia or advance to skeletal muscle rigidity over the 60 min test period. Once returned to their home cage HET and HOM mice did not exhibit fulminant MH (B). Similar stress conditions have been shown to readily trigger fulminant events in mouse lines expressing verified RYR1 MHS mutations, including RyR1-Y522S  died of heat stress intolerance within 30 min of commencing the heat stress alone protocol, exhibiting a biphasic response of core body temperature with a very steep rise commencing at 20 min and reaching 41 °C at time of death (A). Death was associated with whole-body hypercontraction of skeletal muscles. When subjected to the heat stress + anesthesia protocol HET RyR1-R163C mice (n = 3) exhibited a steep rise in core body temperature that commenced soon after induction and culminated with fulminant MH between 17 and 23 min (B). Four CaV1.1-R174W HET mice surviving their initial exposure to the anesthesia protocol were retested at a bed temperature of 38 to 39 °C and survived without triggering fulminant MH (data not shown).Mice were subjected to an elevated ambient temperature 38 °C) without or with maintenance anesthesia with 1.5% halothane applied through a nose-cone for up to 60 min. Under either of these conditions, heat stress alone or heat stress + anesthesia, were sufficient to trigger fulminant MH in HET  or HOM  mice survived the protocol , A and B∼40.7 °C A. Upon rinant MH B. Simila8 °C withR1-Y522S , and RyRof death A. Death d 23 min B. Four Cp < 0.05, ANOVA; A–D and V1.1 channels present in the transverse tubules of all three genotypes. Robust L-type Ca2+ currents were present in WT fibers  of current density between those of HET and HOM indicate no significant difference (p > 0.05). Taken together, the nearly equivalent charge movement and current amplitudes between WT and HET fibers suggested a preferential t-tubular accumulation of the WT allele in the HET mouse.Similar maximal intramembrane charge movement with no detectable alterations in voltage-dependence was observed in WT, HET, and HOM flexor digitorum brevis (FDB) fibers  compared to 138 ± 9 nM  in HET and 171 ± 17 nM  in CaV1.1-R174W HOM muscles (A). We also found that [Na+]i was elevated in muscle fibers from CaV1.1-R174W (HOM > HET) than WT. The mean [Na+]i in WT muscles was 8 ± 0.1 mM (N = 5) compared to 8.6 ± 0.4 mM  in HET and 9.4 ± 0.8 mM  in HOM CaV1.1-R174W muscles (B). Exposing the animals to 1.5% halothane had no effect on [Ca2+]i in WT  or CaV1.1-R174W HET  skeletal muscles. Despite the fact that it did not trigger an MH episode in CaV1.1-R174W HOM animals, exposure to halothane caused a significant increase in [Ca2+]i from 167 ± 25 to 182 ± 28  (C).The mean Ry maximum binding capacity was not significantly different among genotypes (A–D). However, the same preparations from HET mice indicated a trend to a higher equilibrium dissociation constant  compared to either WT or HOM  for [3H]Ry binding, though the differences did not reach statistical significance .PN200-110 and ryanodine bind with high affinity and specificity to Cacoupling . We therenotypes , A–C. Lienotypes , A–D. HoV1.1, calsequestrin—a high-capacity Ca2+-binding protein within the sarcoplasmic reticulum (SR) lumen and FKBP12 , the major T cell immunophilin. A–D and V1.1 in HET and HOM samples exhibited broader variability among preparations from individual animals compared to the other triadic proteins measured (B).Preparations of skeletal muscle homogenates from individual mice  were compared for their levels of four key triadic proteins by Western blotting; RyR1, Cameasured B.Figure 2+ and Na2+ homeostasis that can be attributed, at least in part, to dysregulation of the transient receptor potential canonical (TRPC) channels, TRPC3 and TRPC6  caffeine concentrations compared with CaV1.1-WT expressed in dysgenic myotubes, likely influenced by enhanced Ca2+entry (V1.1-T1354S in control populations (https://gnomad.broadinstitute.org/variant/1-201020165-T-A?dataset=gnomad_r2_1) is, however, considered too high for it to contribute a major gene effect to MHS , RyR1-R163C(4) RyR1-G2435R(6) and RyR1-T4626I(5) mice. To our knowledge, the CaV1.1-R174W mouse line is the first to show discordance between the MHS halothane or caffeine responses in human IVCT and the ability to elicit a fulminant MH in response to triggering anesthetic or an adverse heat stress response. In this regard, the existing MH susceptible mouse lines that express RyR1 variants pathogenic for MHS in humans have both in vitro and in vivo responses expected of an MH positive phenotype.To date, five Ca086H/C/S , 21. An a2+entry . The pret to MHS . Expresshenotype . HoweverV1.1-R174W mouse model does not reflect the human phenotype, it is worth considering the nature of the clinical reactions in the human index cases. In Eltit et al, 2012  in the T-Tubule and RyR1 residing in the terminal cisternae of SR  are expressed in dysgenic FDB, immobilization-resistant charge movement and Ca2+ current densities are restored to levels indistinguishable from those measured in FDB expressing either WT or CaV1.1-R174W cDNAs alone. Considering that expression of CaV1.1-R174W alone restores immobilization-resistant charge movement in the absence of Ca2+ current in the dysgenic FDB model and supports skeletal muscle EC coupling in mice indicates that CaV1.1-R174W is capable of inserting in the T-tubule membrane and fully supports skeletal type EC coupling in vivo. Moreover, results from quantitative reverse transcription-PCR demonstrate that skeletal muscle from HET mice express a 1:1 ratio of WT and CaV1.1-R174W mRNAs, whereas all three genotypes express indistinguishable levels of CaV1.1 protein when measured by Western botting and [3H]PN200-110 binding analyses. Paradoxically, CaV1.1-R174W is considered a disruptive mutation since the positive charge of arginine is substituted with lipophilic tryptophan in the highly conserved S4 helix of repeat 1, a region critical for sensing membrane potential  and T4826I-RyR1 HET and HOM MH susceptible mouse muscle, which lack CaV1.1 and negative regulation . By contrast, resting [Ca2+]i levels in CaV1.1-R174W HET and HOM muscles are lower than those measured in RYR1-R163C HET or those measured in the double mutant RYR1-R163+dominant negative, both of which trigger with fulminant MH in response to halothane , which both have a high affinity, but a low capacity for Ca2+, pumping Ca2+ either out of the cell (plasma membrane Ca2+ pump) or into the SR, respectively   , 25, 48,rse mode , and an otential , resulticalpains , which cfunction .+]i in muscle fibers from CaV1.1-R174W (HOM > HET) compared to WT. This is similar to previous observations in MH muscle cells with RyR1 mutations and is consistent with upregulation of a nonspecific cation channels (TRPCs) in the sarcolemma attempting to keep SR [Ca2+] normal which we see here as well. Therefore, it is plausible to suggest that the elevated intracellular [Ca2+] observed in CaV1.1-R174W muscle cells could be the result of an increase in RyR1 leak caused by reduced negative modulation due to the mutation in CaV1.1  in HOM. Furthermore, it did not trigger the expected fulminant MH episode as have been observed in RyR1 MH susceptible mice  at the University of California, Davis. All animals were maintained in a vivarium with constant temperature and humidity with a 12:12 light–dark cycle and provided food and water ad libitum. Mice used in the studies were adults 4 to 8 months of age, including both male and/or female WT (C57BL6/J), HOM Ryr1-p.T4826I (Ryr1-p.R163C (CACNA1S-p.R174W knock-in mouse lines were created for the investigators by INGenious Targeting Laboratory (www.genetargeting.com). The specifics for vector design and confirmation of homologous recombination of the mutation are found in the The methods for establishing the p.T4826I  and Ryr1The 4 to 8-month-old mice were placed in a restrainer (Kent Scientific HLD-MS-T) and a lubricated rectal probe with bead thermistor  inserted rectally for the duration that the mouse was in the restraint. The temperature was monitored continuously with the probe connected to a Single Channel Temperature Controller  and recorded every 2 min. After 5 min at room temperature (RT) (∼24 °C), the mice were placed in a temperature-controlled chamber  for a maximum of 60 min or until the mouse temperature exceeded 41 °C or the animal succumbed to the heat stress. After 60 min if the mouse survived it was removed from the heat chamber and returned to RT and observed for an additional 20 min, with the rectal temperature still being recorded every 2 min. At the end of the experiment, the mice were euthanized by cervical dislocation and decapitation.V1.1-R174W HET (4 mice) surviving their initial testing were subsequently exposed to the anesthesia protocol at a bed temperature of 38 to 39 °C.For induction, mice were exposed to 2% halothane  in a small induction chamber . After 3 to 5 min, the anesthetized mouse, as indicated by a lack of response to gentle rocking, was removed from the small chamber and placed on a warmed platform set to 38 °C as described previously : 145 tetraethylammonium-methanesulfonic acid, 10 CaCl2, 10 Hepes, two MgSO4, 1 4-aminopyridine, 0.1 anthracene-9-carboxylic acid, 0.002 tetrodotoxin, pH 7.4 with tetraethylammonium-OH. 1 mM LaCl3 and 0.5 CdCl2 were added to the external recording solution to block Ca2+ currents during charge movement recordings. N-benzyl-p-toluensulfonamide  was always present in the bath solution to prevent contractions. Linear components of leak and capacitive current were corrected with -P/4 online subtraction protocols. Output filtering was at 2 to 5 kHz and digitization was either at 5 kHz (currents) or 10 kHz (charge movements). Cell capacitance (Cm) was determined by integration of a transient from −80 mV to −70 mV using Clampex 10.3 (Molecular Devices) and was used to normalize charge movement (nC/μF) and current amplitude (pA/pF). To minimize voltage error, the time constant for decay of the whole-cell capacity transient was reduced as much as possible using the analog compensation circuit of the amplifier. QON was then normalized to Cm and plotted as a function of test potential (V) and the resultant QON-V relationship was fitted according to the equation:max is the maximal QON, VQ is the potential causing movement of half the maximal charge, and kQ is a slope parameter. I-V curves were fitted according to the equation:rev is the reversal potential, Gmax is the maximum Ca2+ channel conductance, V1/2 is the half-maximal activation potential and kG is the slope factor.Whole-cell patch-clamp experiments were performed with FDBfibers 1 to 6 h following dissociation as described . Patch p2+]i and [Na+]i measurements were carried out in vivo, on left vastus lateralis fibers in euthermic (37 °C - heating pad) and anesthetized (100 mg/kg ketamine and 5 mg/kg xylazine) WT, CaV1.1-R174W HET, and CaV1.1-R174W HOM mice  .i and [Na+]i was carried out while normal Ringer solution containing (in mM) 135 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, 18 NaHCO3, 1.5 NaH2PO, and 5 glucose, pH 7.3 to 7.4 was used to keep muscle fibers moist. All in vivo experiments were performed at 37 °C.Recording of ryanodine ([3H]Ry) to skeletal muscle homogenate (0.25–0.50 mg/ml) was measured at 36 °C for 3 h with constant shaking in buffer consisting of 1 to 70 nM [3H]Ry , 250 mM KCl, 20 mM Hepes, pH 7.4 and 50 μM free Ca2+ obtained by the addition of EGTA calculated according to the software Bound and Determined (3H]Ry binding was determined in the presence of a 5 μM nonradioactive ryanodine. Bound and free ligand were separated by rapid filtration through Whatman GF/B glass fiber filters using a Brandel cell harvester (Whatman) with three washes with 5 ml of ice-cold buffer . Retained [3H]Ry in filters was quantified by liquid scintillation spectrometry using a scintillation counter (Beckman model 6500). Binding analysis was performed on muscle preparations from individual animals of each genotype  performed in triplicate  as detailed in Figure Legends.The equilibrium binding of [termined . Nonspec3H]PN200-110  were performed at 25 °C for 60 min in the dark, incubated in buffer containing 140 mM NaCl, 15 mM KCl, 50 μM Ca2+, 20 mM Hepes, pH 7.0, and 0.25 to 0.50 mg/ml skeletal muscle homogenate. Nonspecific binding was determined in the presence of 10 μM nifedipine. The bound and free ligand was separated by rapidly washing samples through the filters with 10 X volume of cold binding buffer. Binding analysis was performed on muscle preparations from the same individual animals of each genotype  used for [3H]Ry binding analysis in triplicate . The number of animals used was indicated in Results and figure legends.The equilibrium binding assays of (+)- antibody ab191564); polynoclonal GAPDH 1/1000 to 1/5000 ; Rabbit β-Actin Polyclonal Antibody 1/1000 (Invitrogen REF PA1-16889) in blocking buffer and then washed with Tris-Gastrocnemius muscles from all genotypes were dissected, homogenized, and processed using total protein extraction kit (Millipore). Total protein concentration was determined using the bicinchoninic acid method (Thermo Fisher Scientific). Denatured, SDS-gel separated and membrane immobilized proteins were incubated overnight at 4 °C with primary antibodies: anti-TRPC3, dilution 1:2500 , anti-TRPC6, dilution 1:2500 , anti-actin, dilution of 1:5000 (SC8432), and secondary fluorescent antibodies (Abcam). The resolved bands were detected with a Storm 860 Imaging System . Protein levels were quantified using myImage. Analysis software (Thermo Fisher Scientific) and normalized to -actin.No specific power calculation was conducted prior to experimentation; sample sizes used were based on our previous studies using other MH and non-MH animal models. Randomization methods were not used to assign the animals to be studied, and blinding of the investigators was not used.2+]i and [Na+]i data are presented as mean ± SD. We used histograms and the D'Agostino–Pearson test to assess the distribution of the data . Data were discarded: (i) from the muscle cell whose membrane potential was less negative than minus eighty mV; (ii) when a drift of more than 5 mV between the first and the second microelectrode calibration curves in the range between pCa6–7 for Ca2+ selective-microelectrodes and 1 to 10 mM for the Na+ selective-microelectrode; or (iii) when broken microelectrode tips which did not allow us to carry out the post measurement calibration curves.[Cathe data . Statistt test with p < 0.05 considered significant All the data at 0.05 level were significantly drawn from the normally distributed popluation . Statistical comparisons were made by unpaired 3H]ryanodine and [3H]PN200-110 binding isotherms obtained from individual animals were fitted by nonlinear curve fitting using OriginPro 2018b. Statistical analysis for differences in ryanodine and PN200-110 binding constants (KD and Bmax) among the three genotypes were assessed using Prism GraphPad Software to obtain mean, SD, SEM, and lower/upper 95% confidence interval of mean using one-way ANOVA with Tukey’s multiple mean comparisons test.[p values (GraphPad Prism 9.0). Summary data of F statistic and p values are included in Analysis of Western blots was performed on the same sample membrane preparations measured for radioligand binding indicated above. HET and HOM signals were normalized to GAPDH or -actin, then to the WT signal on the same blot. Normalized band densities in HET and HOM were subsequently normalized to WT bands to generate scatter plots of mean normalized densities ± SD and lower and upper 95% confidence interval. Statistical comparisons among genotypes were performed using a one-way ANOVA with Tukey’s multiple comparisons test applied to obtain adjusted All data reported in this manuscript are available in the figures, Experimental procedures section, and Supporting Information section.This article contains This work was prepared while R. A. B. was employed at the University of Colorado Anschutz Medical Campus. The authors declare that they have no conflicts of interest with the contents of this article.


Despite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed.We examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1.Our study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study.The online version contains supplementary material available at 10.1186/s13100-023-00308-3. Transposable elements litter the genomes of eukaryotes, constituting over 50% of the human genome . These tIn mammals, nearly all characterized examples of transposon-derived regulatory elements are derived from endogenous retroviruses (ERVs). Over time, most ERVs in the human genome have lost their protein coding regions through truncation and recombination, leaving behind the Long Terminal Repeat (LTR) elements. Retroviral LTRs are often rich with transcription factor binding sequences that originally evolved to hijack the host transcriptional machinery to promote proviral transcription , 10, 11.Long Interspersed Line Elements (LINEs) are another subclass of transposable elements, and are the only class capable of autonomous transposition in the human genome . Given tIn this study, we investigated the epigenetic status of LINEs in the context of immune gene regulation by analyzing publicly available chromatin profiling data from human primary blood cells  and a lyWe chose one such instance to study more closely: a LINE insertion of the L1M2a family within the first intron of the Interferon Alpha/Beta Receptor 1 (IFNAR1) gene. The 5’ end of this element appears to be intact, but the 3’ end is truncated, rendering it transpositionally inactive. It is nevertheless silenced by the HUSH complex , and yetTo investigate the epigenomic features of LINE elements, we examined publicly available chromatin immunoprecipitation (ChIP) sequencing datasets from primary human immune cells . We confInterestingly, we identified a subset of LINE insertions that showed both repressive and activating marks. A fraction  of LINE-1s overlapped both repressive H3K9me3 peaks and enhancer associated peaks , but not those marked by H3K9me3 only, were enriched near immune genes in immune cells, suggesting a possible regulatory role  gene that is marked by HUSH-dependent H3K9me3 , but exhWe also found evidence that IFNAR1.L1M2a shows inducible enhancer activity upon type I IFN signaling. ATAC signal increases markedly upon treatment of cells with interferon alpha (IFNɑ), and antisense PRO-Seq signal increases upon treatment with interferon beta (IFNβ) in GM12878 cells . Because IFNAR1.L1M2a is nested within the first intron of IFNAR1, we predicted IFNAR1 to be the most likely regulatory target of IFNAR1.L1M2a. Three nearby genes were also considered as potential target genes based on their proximity to the element, with transcription start sites (TSS) within about 100 kb of the LINE: Interleukin 10 Receptor Subunit Beta (IL10RB), Interferon Alpha/Beta Receptor 2 (IFNAR2), and Interferon Gamma Receptor 2 (IFNGR2)  to compare gene expression profiles in wildtype and knockout cells over the 0, 4, 12 and 24 h treatment time course. Both IFNAR1 and IL10RB show significantly different expression profiles in knockout cells, but IFNAR2 and IFNGR2 do not , 2'-5'-Oligoadenylate Synthetase Like (OASL), and Interferon Stimulated Gene 15 (ISG15)   was used2, 0.1% IGEPAL, 0.1% Tween-20, 0.01% Digitonin), and incubated for 5 min on ice, then 1 ml of wash buffer  was added and mixed. Cells were then pelleted again for 10 min at 4 °C and the supernatant aspirated. Pellets were resuspended in 50ul of transposition buffer , then incubated for 30 min at 37 °C. Samples were purified using the Zymo DNA Clean and Concentrator Kit . Fragments were amplified with a 5 cycle PCR, then an additional cycle with Nextera DNA CD Indices , then re-purified with the DNA Clean and Concentrator Kit. Fragments were filtered using a BluePippin (Sage Science) for fragments smaller than 100 base pairs. Libraries were quantified using Qubit (Invitrogen) and pooled, then bead-cleaned using 1.5 volumes of AMPure XP beads . Libraries were sequenced using a NextSeq 500 at a depth of ~ 20 million reads per sample.Cells were treated with either 100 U/ml human IFNα2  or 0.001% DMSO for 1 h. Libraries were prepared using a protocol modified from previously published protocols . Cells wRaw datasets were processed through the following pipeline. Raw fastq files were quality checked using FastQC v0.11.5 . Adapter2.GM12878 lymphoblastoid cells were obtained from the Coreill Institute . Cells were cultured in Roswell Park Memorial Institute (RPMI) media  with 15% Fetal Bovine Serum  by volume and 1% Penicillin/Streptomycin , according to the recommendations of the Coriell Institure and were grown at 37 °C in 5% COGuides for CRISPR/Cas9 knockout of IFNAR1.L1M2a.enh were designed using the UCSC genome browser track “CRISPR/Cas9-NGG Targets, whole genome .” Three CRISPR/Cas9 knockout was performed using IDT’s Alt-R system and the Neon Transfection System . Guide RNAs were purchased from IDT and combined with Alt-R tracrRNA  in nuclease free IDTE , heated to 95 °C for 5 min and then cooled to room temperature. Alt-R S.P. HiFi Cas9 Nuclease V3  was diluted in Neon Resuspension Buffer R  and added to the RNA mixture for, incubated 10–20 min at room temperature. Alt-R Cas9 Electroporation Enhancer  was added. Electroporation was performed according to Neon Transfection System recommended protocols, and GM12878 cells were electroporated using three 10 ms pulses at 1200 V.Single cell dilutions were plated to 92-well plates in order to isolate clonal cell lines. Once expanded, clonal lines were screened for homozygous knockout of IFNAR1.L1M2a.enh. Genomic DNA was isolated from cells using Quick Extract DNA Extraction Solution . Initial screening for knockouts was performed by PCR amplification of the locus. Primer sequences are listed in Additional file Cells were either untreated or cultured in media containing 10 U/ml human IFNβ  for 4 h. CUT&Tag was performed using a protocol modified from Steven Henikoff’s lab . Brieflyhttps://github.com/crmnbttlr/IFNAR1.L1M2a_2023.Raw sequencing datasets were processed through the following pipeline. Raw fastq files were quality checked using FastQC v0.11.8 , adapterCells were either untreated or were cultured in media containing 10 U/ml human IFNβ  for either 4, 12, or 24 h. Wildtype GM12878 cells, clonal wildtype cells  and clonal knockout cells  were each collected in duplicate for each of the four treatment conditions. RNA was extracted from cells using the Zymo Quick RNA Miniprep Plus Kit  according to manufacturer protocols. RNA-Seq libraries were prepared using the KAPA mRNA Capture Kit , using their recommended protocol. Libraries were sequenced at a depth of ~ 15 million reads per sample by the University of Colorado Anschutz Cancer Center Genomic Core using NovaSeq6000.https://github.com/crmnbttlr/IFNAR1.L1M2a_2023.Raw RNA-Seq datasets were processed through the following pipeline. Files were quality checked using FastQC v0.11.8 , reads wCells were either untreated or cultured in media containing 10 U/ml human IFNβ  for 24 h. Cells were then pelleted by centrifugation at 500 rpm for 5 min. Media was aspirated. Cells were resuspended and spun down again, twice in serum-free RPMI media, then added dropwise to clean, sterile glass coverslips treated with human fibronectin  to allow them to settle onto the glass. Media was aspirated, cells were washed once with phosphate buffered saline (PBS), then fixed by treating for 25 min with 4% paraformaldehyde . Coverslips were washed twice with Hank’s Balanced Salt Solution (HBSS), then stained with 5 ug/ml Wheat Germ Agglutinin Alexa Fluor 488  for 10 min at room temperature, protected from light. Coverslips were then washed once with PBS, then cells were permeabilized by treatment with 0.2% Triton × 100  for 10 min at room temperature. Coverslips were washed again with PBS, then stained with DAPI , diluted 1:1000 in PBS, for 5 min at room temperature. They were washed again with PBS, then blocked for 30 min in a solution of 10% bovine serum albumin , dissolved in PBS. Primary antibody staining was conducted for 1 h at room temperature. IFNAR1 was labeled using a rabbit anti-IFN-alpha/beta R1 antibody . IFITM1 was labeled using a rabbit anti-IFITM1 Alexa Fluor 488 antibody . Both were diluted 1:200 in a solution of 6% BSA in PBS. After primary staining, coverslips were washed with PBS, then stained with a secondary antibody, a goat anti-Rabbit IgG H&L Alexa Fluor 647 , diluted 1:200 in a solution of 6% BSA in PBS for 1 h at room temperature. Coverslips were then washed three times for 10 min in a wash solution of 0.2% BSA and 0.05% Triton × 100 in PBS. Coverslips were then mounted to glass slides in a mounting medium of Fluoromount G , and sealed around the edges with clear nail polish. Imaging was performed using a Nikon Ti-E Spinning Disc Confocal Microscope, with 488 nm (20%), 405 nm (20%) and 640 nm (25%) lasers, a 40 × air objective (Nikon), and an EMCCD camera (Andor iXon Ultra 888) at 300 ms exposure and 10 MHz EM Gain.2, with circularity between 0 and 1, excluding edges and including holes. Generated ROIs were manually inspected, and incorrectly segmented ROIs that either included multiple cells or partial cells were removed from the ROI manager. Measurements of area, mean, standard deviation, and integrated density were collected for all ROIs remaining in the manager. Integrated density per cell was used to approximate protein abundance. A pipeline in ImageJ is available at https://github.com/crmnbttlr/IFNAR1.L1M2a_2023.Immunofluorescence images were analyzed using ImageJ  to mask Cells were either untreated or were cultured in media containing 1000 U/ml human IFNɣ  for 4 h before RNA extraction. Duplicates of two wildtype clones (clones 2D3 and 2D9) and two knockout clones (clones L3.A4 and MN8.C12) were treated. RNA was extracted using the Zymo Quick RNA Miniprep Plus Kit  according to manufacturer protocols. RT-qPCR was performed in a 384-well plate using the Luna Universal One-Step RT-qPCR Kit  on a Bio-Rad CFX Opus 384 Real-Time PCR System. Normalized expression was calculated as 2 to the power of the difference between the Cq value of the gene of interest and the Cq value of CTCF. CTCF was quantified using primers ACCTGTTCCTGTGACTGTACC and ATGGGTTCACTTTCCGCAAGG. IFNAR1 was quantified using primers CGCCTGTGATCCAGGATTATCC and TGGTGTGTGCTCTGGCTTTCAC. IFITM1 was quantified using primers GGCTTCATAGCATTCGCCTACTC and AGATGTTCAGGCACTTGGCGGT.RNA-Seq from all wildtype vs all knockout cells was used as input for Stringtie  to assemAdditional file 1: Supplemental Figure 1. Enrichment of histone marks among transposon families. Giggle [Supplemental Figure 2. Overlap of repressive and active histone marks at LINEs in naïve B cells. Heatmaps and metaplots of individual LINE-1s (A) and LINE-2s (B) that overlap repressive H3K9me3 signal only (navy), active marks H3K4me1 or H3K27ac (yellow), both H3K9me3 and active marks (cyan), or which overlap none of these three histone marks (green). Supplemental Figure 3. Gene function enrichment near epigenetically marked LINE-1s in naïve B cells. GREAT [Supplemental Figure 4. The IFNAR1 gene locus. (A) The IFNAR1.L1M2a element (light blue) lies within the first intron of the IFNAR1 gene, and includes a predicted distal enhancer [Supplemental Figure 5. Sequencing validation of CRISPR knockout of IFNAR1.L1M2a.enh. Four clonal lymphoblastoid cell lines  were isolated with homozygous CRISPR knockout of IFNAR1.L1M2a.enh. The knockout regions were validated by sanger sequencing, and the alleles are displayed here, with the deleted regions marked with dotted lines. Supplemental Figure 6. Chromatin profiling of the IFNAR1.L1M2a locus upon IFNβ signaling. CUT&Tag [Supplemental Figure 7. Quantification of IFNAR1 expression by RT-qPCR. RT-qPCR was used in parallel with RNA-Seq to assess changes in the transcription of IFNAR1 upon IFNAR1.L1M2a.enh knockout. Significantly lower expression of IFNAR1 was observed in knockout cells (purple) compared with wildtype (gray) at all timepoints, using a student’s t-test. * indicates p<0.05. ** indicates p<0.01. *** indicates p<0.001. Supplemental Figure 8. RNA-Seq and transcript assembly at the IFNAR1 locus. (A) Transcript assembly using Stringtie [Supplemental Figure 9. Transcriptional Response of wildtype and knockout cells to IFNɣ. Quantitative PCR measuring expression, normalized to CTCF, of IFNAR1 (A) and representative IFN stimulated gene IFITM1 (B), in wildtype compared with IFNAR1.L1M2a.enh knockout cells, under untreated and IFNɣ treated conditions. Two clonal cell lines of each genotype were used, each in duplicate. There was no significant difference between wildtype and knockout cells under the same treatment conditions. Supplemental Figure 10. Immunofluorescence of IFITM1. Immunofluorescence was used to approximate quantify IFITM1 protein abundance at the single cell level. (A) Representative images of IFITM1 labeling in wildtype and IFNAR1.L1M2a.enh knockout cells, under untreated and IFNβ-treated conditions. Scale bar is 25 microns. (B) Quantification of IFITM1 staining intensity per cell. Both wildtype and knockout cells show induction of IFITM1, though in knockout cells the induction appears less robust, in agreement with RNA-seq data .. Giggle  was useds. GREAT , 86 was enhancer , 65 (B) enhancer . (C) Traenhancer . (D) ChIenhancer  shows thenhancer  which sh CUT&Tag  was usedtringtie  shows noAdditional file 2: Supplemental Table 1. Epigenetically Marked LINE-1s in Primary Naive B Cells. Lists of LINE-1s in the human genome which overlap the epigenetic marks of interest in naive B cells [ B cells .Additional file 3: Supplemental Table 2. Epigenetically Marked LINE-1s in Lymphoblastoid GM12878 Cell Line. Lists of LINE-1s in the human genome which overlap the epigenetic marks of interest in lymphoblastoid GM12878 cells [78 cells .Additional file 4: Supplemental Table 3. Genes Proximal to Epigenetically Marked Genes in Primary Naive B Cells. Lists of genes with potential cis interaction with epigenetically marked LINE-1s in naive B cells [ B cells , accordi B cells , 86.Additional file 5: Supplemental Table 4. Transcription Factor Binding in the IFNAR1.L1M2a.enh region according to ReMap. List of transcription factors found to bind within the IFNAR1.L1M2a.enh region in various cell types, according to ReMap 2022 [Map 2022 .Additional file 6: Supplemental Table 5. CRISPR Guides and Primers. Sequences of guides used to conduct CRISPR/Cas9 knockout of IFNAR1.L1M2a.enh, PCR primers used to validate knockouts, and sequences of knockout regions in four clonal knockout cell lines: L3.A4, L3.E4, MN6.G11, and MN8.C12.Additional file 7: Supplemental Table 6. DESeq Differential Expression Analysis Outputs. Tabular outputs of differential sequencing analysis using DESeq2 [g DESeq2 , includiAdditional file 8: Supplemental Table 7. Immunofluorescence of IFNAR1. Tables of IFNAR1 immunofluorescence intensity, as well as area-normalized mean intensity, for segmented individual cells. Datasets were randomly subset to normalize n values.Additional file 9: Supplemental Table 8. Immunofluorescence of IFITM1. Tables of IFITM1 immunofluorescence intensity, as well as area-normalized mean intensity, for segmented individual cells. Datasets were randomly subset to normalize n values.


XBP1 mRNA during the unfolded protein response (UPR) in the cytoplasm. We recently reported that the tRNA-LC coevolved with PYROXD1, an essential oxidoreductase that protects the catalytic cysteine of RTCB, the catalytic subunit of the tRNA-LC, against aerobic oxidation. In this study, we show that the oxidoreductase Thioredoxin (TRX) preserves the enzymatic activity of RTCB under otherwise inhibiting concentrations of oxidants. TRX physically interacts with oxidized RTCB, and reduces and reactivates RTCB through the action of its redox-active cysteine pair. We further show that TRX interacts with RTCB at late stages of UPR. Since the interaction requires oxidative conditions, our findings suggest that prolonged UPR generates reactive oxygen species. Thus, our results support a functional role for TRX in securing and repairing the active site of the tRNA-LC, thereby allowing pre-tRNA splicing and UPR to occur when cells encounter mild, but still inhibitory levels of reactive oxygen species.The mammalian tRNA ligase complex (tRNA-LC) catalyzes the splicing of intron-containing pre-tRNAs in the nucleus and the splicing of  RNA molecules are mostly synthesized as precursors and only become functional upon a series of modifications and processing events such as the removal of intronic sequences during a process called RNA splicing. The so-called “canonical splicing” entails the removal of introns from precursor mRNAs (pre-mRNAs) . In contPre-tRNA splicing occurs in two steps: cleavage of the pre-tRNA by a tRNA splicing endonuclease  and ligaXBP1u mRNA (u for unspliced) upon activation (XBP1 mRNA exons are joined by the tRNA-LC to generate XBP1s mRNA (s for spliced) , a signaling cascade that is triggered when unfolded proteins accumulate in the ER . This pativation . In metaspliced) . Translaspliced) . Besidesmusculus .We have recently reported that the mammalian tRNA-LC is reversibly inhibited by oxidative stress . StructuXBP1 mRNA splicing. We demonstrate that TRX reactivates the tRNA-LC after alleviation of the oxidative stress as well as upon extended UPR through direct interaction with RTCB.In this study, we show that the absence of TRX enhances the sensitivity of the tRNA-LC toward oxidative stress, with an impact on pre-tRNA and TXN mRNA encoding TRX (shTRX cells) . We monitored tRNA-LC activity through the interstrand ligation of a double-stranded, radiolabeled RNA substrate bearing a 3′phosphate (3′P) on one strand and a 5′hydroxyl group (5′OH) on the complementary strand , with IC50 values of 76 µM for shCtrl cells and 37 µM for shTRX cells. RTCB levels remained unchanged in both treatments . Of note, we observed an increase in the activity of the tRNA-LC irrespective of the presence of TRX at 5 and 10 µM menadione, but not in the presence of H2O2. This effect may relate to other conserved cysteine residues in the subunits of the tRNA-LC which are potentially modified during oxidative stress. Yet, the absence of TRX has a clear impact on the sensitivity of the tRNA-LC in the presence of menadione and H2O2.To test whether TRX preserves the enzymatic activity of the human tRNA-LC during oxidative stress, we generated a HeLa cell line expressing a Doxycycline (Dox) inducible short hairpin RNA (shRNA) targeting the X cells) . Both TRy strand . Under sSupplemental Fig. 2A,B). To note, mutating the active site of TRX affected the recognition of FLAG-TRX by the anti-TRX antibody but not by the anti-FLAG antibody, indicating a similar level of overexpression of the constructs. Overexpression of FLAG-TRX CxxC prevented the inactivation of the tRNA-LC by menadione  and FLAG-TRX SxxS (mutant)—in shTRX cells . Inhibition with H2O2 was achieved at 250 µM in siCtrl cells, while 100 µM substantially reduced RNA ligation activity in siTRX cells . To monitor the reactivation of the tRNA-LC after oxidative stress, we treated cells with 30 µM menadione, while further titrations with H2O2 led us to an optimal concentration of 125 µM H2O2. The treatment was done for 30 min and cells were allowed to recover over a time course of 3 h in medium supplemented with cycloheximide (CHX) to inhibit de novo protein synthesis. In siCtrl cells treated with menadione, the tRNA-LC fully recovered after 3 h . These results indicate that catalytically active TRX is required to reactivate the tRNA-LC once oxidative stress levels have decreased.We next investigated the potential role of TRX in the previously described reversibility of the tRNA-LC inhibition upon recovery from oxidative stress , and in 2O2 for 3 min, and carried out elution with FLAG-peptide in nonreducing conditions. We found RTCB enriched in the eluate in a H2O2 concentration-dependent manner, indicating that RTCB is a target of TRX  and simultaneous oxidative stress conditions. The splicing efficiency, determined by the ratio of XBP1s to XBP1u mRNA, decreased in shCtrl and shTRX cells after menadione treatment when measured by RT-qPCR .We next monitored  RT-qPCR C. Howevee RT-PCR D. We alse RT-PCR E. The reXBP1 mRNA splicing reporter in a HEK293 FITR cell line. The reporter encodes a Dox-inducible, GFP-XBP1intron-mCherry transcript that generates a GFP-mCherry fusion protein upon splicing, similar to previously described XBP1 splicing reporters . When TRX was depleted using siRNAs (siTRX cells) and UPR was induced simultaneously with oxidative stress, the levels of GFP-mCherry protein diminished at lower menadione concentrations in comparison to siCtrl cells . Thus, potentially higher levels of ROS generated at late stages of UPR could irreversibly inhibit the tRNA-LC, down-regulate XBP1 mRNA splicing and signal to induce apoptosis.The tRNA-LC is able to catalyze the splicing of e of TRX C–F; howeThis study demonstrates that the redox sensitivity of the tRNA-LC is a tightly regulated process that involves several factors and impacts pre-tRNA splicing, UPR and ultimately, protein biosynthesis. PYROXD1 has been shown to protect RTCB from oxidation  and, rec2. Cells were regularly tested for mycoplasma contamination.HeLa Kyoto, HeLa RIEP  and HEK22 plates and transfected at 90% confluency with 20 µg plasmid DNA and 10 µg helper plasmid , using CalPhos Mammalian Transfection Kit , according to the manufacturer's instructions. The transfection medium was replaced to complete DMEM after 16 h. Virus-containing supernatants were harvested at 36, 48, and 60 h post-transfection and added dropwise onto HeLa RIEP cells for transduction. Cells expressing the shRNA constructs were selected using 1 mg/mL Neomycin . To induce expression of shRNAs, cells were treated for 3 d with 2 µg/mL Dox.For generation of Doxycycline-inducible shRNA cell lines, HeLa RIEP cells were transduced with ecotropically packaged shRNA expression vectors  or rescue vectors , as described before . For retXBP1 splicing reporter was designed similar to previously described studies , flanked by restriction sites and cloned into a pcDNA5-FRT vector containing EGFP and mCherry sequences. For generation of stable HEK293 FITR cells expressing the GFP-XBP1intron-mCherry reporter, cells were seeded into a six-well plate and transfected at 50% confluency. An amount of 1.5 µg of plasmid DNA and 1.5 µg pOG44 (encoding the Flp recombinase) were mixed and transfected using CalPhos Mammalian Transfection Kit . The transfection medium was replaced by complete DMEM after 24 h, and after a further 24 h, the selection of positive clones was started using 0.5 mg/mL Hygromycin B. Colonies appeared after 14 d of selection. For UPR-related experiments, silencing of TRX or RTCB and Archease was done as described below. Expression of the GFP-XBP1intron-mCherry was induced for 5 h; 1 h later, menadione and Tm were added to the cells. Cells were harvested by trypsinization, pellets were resuspended in FACS buffer (PBS + 2% FCS) and analyzed by flow cytometry (BD LSRFortessa) and FlowJo software.The  studies . A 120-mTXN (siTRX) or scrambled control (siCtrl) consisted of siRNA pools of 30 siRNAs and were purchased from siTOOLs Biotech. HeLa cells were transfected at 30%–40% confluency using Lipofectamine RNAiMax , according to the manufacturer's instructions. Subsequent experiments were performed after 3 d of silencing.For depletion of proteins using RNAi, cells were seeded 1 d before transfection. Small interfering RNAs (siRNA) targeting human For treatment with inhibitors or oxidants, HeLa Kyoto cells were seeded into six-well plates and treated at 90% confluency. A list of compounds and inhibitors used in this work can be found in TXN mRNA were designed using the online siRNA prediction tool “SplashRNA” (http://splashrna.mskcc.org)  . The seqkcc.org)  were ampThe sequence of human TRX cDNA was adapted to be nontargetable by shTRX #6. The cDNA of WT (CxxC), C35S (CxxS), and the double mutant C32S/C35S (SxxS) was cloned into pDONR221 (Invitrogen) by Gateway recombination. Sequences were subsequently shuttled from the pDONR221 construct into pBMN-FLAG-IRES-GFP  that was used to generate stable cell lines. The primer sequences for Gateway recombination are shown in DNA constructs were mixed with 50 µL of chemically competent DH5α cells, and incubated at 4°C for 30 min. The bacteria were heat shocked at 42°C for 45 sec and incubated at 4°C for 2 min. An amount of 450 µL of LB medium was added and incubated at 37°C for 1 h while shaking. The bacteria were plated onto agar plates supplemented with the appropriate antibiotic and incubated overnight at 37°C.260nm using a DeNovix DS-11 spectrophotometer.For plasmid isolation, one colony of transformed bacteria was inoculated into 5 mL (Miniprep) or 250 mL (Maxiprep) LB medium, supplemented with the appropriate antibiotic, and incubated overnight at 37°C while shaking at 200 rpm. Isolation of plasmids was done using QIAprep Miniprep or Maxiprep kits (Qiagen) according to the manufacturer's instructions. Purified plasmids were resuspended in nuclease-free water, and DNA concentration and purity were measured at A2, 100 mM KCl, 1% Nonidet P-40, 0.1 mM AEBSF, 1 mM TCEP pH 7.0). Whole-cell extracts were cleared by centrifugation at 14,8000 rpm, 10 min, 4°C, the supernatant was transferred into a tube, and protein concentration was measured by Bradford assay.For tRNA-LC activity assays, cells were scraped with cold PBS and transferred into a tube. After spinning at 1000 rpm, 5 min, 4°C, the supernatant was removed and the cell pellet resuspended in HeLa lysis buffer , and diluted with water to obtain 1× SDS-loading buffer. Samples were boiled for 10 min at 95°C and were frozen at −20°C or subjected to protein analysis by SDS–PAGE and western blot.For measurement of protein concentration of whole-cell extracts, Bradford Protein Assay  was mixed 1:5 in water. Concentration standards were prepared with bovine serum albumin  by adding 0, 1, 2, 4, 8, or 16 µL of BSA to 1 mL of Bradford reagent, and a standard curve was prepared by measuring the absorbance at 595 nm. A total of 1 µL of cleared lysate was diluted in 1 mL Bradford reagent, and concentrations were measured at 595 nm based on the standard curve using a DeNovix DS-11 spectrophotometer.A total of 25–30 µg of a sample was separated by SDS–PAGE and transferred onto methanol-activated Immuno-Blot PVDF membranes in 1× Turbo Trans-Blot buffer . Blotting of two membranes was performed at 400 mA for 1 h. Membranes were blocked for 1 h with 5% milk in PBS with 0.05% Tween-20 (PBS-T) or 3% BSA in PBS-T. Blocked membranes were probed with primary antibodies diluted in 5% milk PBS-T or 3% BSA in PBS-T overnight at 4°C while shaking. Membranes were washed three times in PBS-T for 5–10 min and incubated with the appropriate secondary antibody in 5% milk in PBS-T for 1 h at room temperature, before washing three times for 5–10 min in PBS-T. A list of primary and secondary antibodies and their dilutions used in this work can be found in 260/280 nm using a DeNovix DS-11 spectrophotometer. Up to 2 µg RNA was treated with DNase to remove genomic DNA. cDNA synthesis from isolated RNA was prepared using Maxima First Strand cDNA Synthesis Kit for RT-qPCR , according to the manufacturer's instructions. Obtained cDNA was diluted 1:10 in nuclease-free water before downstream analysis.Isolation of RNA from cells was performed using TRIzol reagent  according to the manufacturer's instructions. The obtained RNA pellet was air-dried and resuspended in 20 µL nuclease-free water. Concentration and purity of the sample were measured at AFor a quantitative measurement of mRNA levels using RT-qPCR, a master mix was prepared for each set of primers: for one well of a 384-well plate, 5 µL of 2× GoTaq , 0.4 µL forward primer (0.5 µM), 0.4 µL reverse primer (0.5 µM), and 3.2 µL nuclease-free water were mixed. A total of 1 µL of cDNA was pipetted into the 384-well plate, and 9 µL master mix was added per well. Primers used in this work can be found in XBP1u and XBP1s mRNA levels by semiquantitative reverse transcription PCR (RT-PCR), 1 µL of diluted cDNA was mixed with 12.5 µL ReqTaq Ready Mix PCR reaction mix , 0.5 µL forward primer (0.2 µM), 0.5 µL reverse primer (0.2 µM), and filled up to 25 µL with nuclease-free water. The sequences for XBP1 and ACTB forward and reverse primer pairs can be found in For examination of 2HPO4 pH 7.2, 7% SDS, and 0.1 mg/mL sonicated salmon sperm DNA ) at 50°C for 1 h, and hybridization was done at 50°C (DNA probe) or 80°C (LNA probe) overnight in hybridization buffer supplemented with 100 pmol of [5′-32P] labeled DNA or LNA probes. To obtain the radioactive probe, 1 µL of the respective probe (100 µM) was labeled using 1 µL 10× PNK buffer, 2 µL [γ-32P]ATP, 1 µL PNK, and 14 µL nuclease-free water. The reaction was incubated for 1 h at 37°C. The labeled probe was separated from unincorporated nucleotides using MicroSpin G-25 columns , according to the manufacturer's instructions. The DNA/LNA probes used for northern blots can be found in For northern blot analysis of RNA samples, 5 µg of total RNA was mixed with equal volumes of 2× FA buffer  and boiled for 5 min at 98°C. Samples were resolved using a 10% denaturing urea polyacrylamide gel . After the gel run, the RNA was blotted onto Hybond N+ membranes  for 3 h at 180 mA and cross-linked by UV. Prehybridization was carried out in hybridization buffer  was ligated to 50 pmol RNA oligonucleotide “20.25”  with 1 µL T4 RNA ligase 1  for 1 h at 16°C in 15% (v/v) DMSO, 50 mM Tris-HCl pH 7.6, 10 mM MgCl2, 10 mM β-mercaptoethanol, 200 µM ATP, and 0.1 mg/mL BSA in a total reaction volume of 20 µL. Labeling reactions were stopped with equal volumes of 2× FA buffer , boiled for 5 min at 98°C, and separated on 10% denaturing urea polyacrylamide gel . Labeled RNA was visualized by autoradiography, cut from the gel and passively eluted in 300 mM NaCl, while shaking overnight at 4°C. RNA was precipitated by the addition of three volumes of ice-cold ethanol and recovered by centrifugation. The RNA pellet was resuspended in 200 µL nuclease-free water.A total of 1.11 MBq guanosine-5′-triphosphate  per reaction. The body-labeled pre-tRNA was resolved on a 10% denaturing urea polyacrylamide gel, visualized by autoradiography and passively eluted from gel slices overnight in 300 mM NaCl. RNA was precipitated by the addition of three volumes of ice-cold ethanol and dissolved to 0.1 μM in buffer containing 30 mM HEPES-KOH pH 7.3, 2 mM MgCl2, 100 mM KCl. To assess pre-tRNA splicing, one volume of 0.1 μM body-labeled S. cerevisiae pre-tRNAPhe was preheated at 95°C for 60 sec and incubated for 20 min at room temperature, and subsequently mixed with four volumes of reaction buffer . Equal volumes of this reaction mixture and cell extracts were mixed and incubated at 30°C. At given time points, 5 μL of the mix was deproteinized with proteinase K , followed by phenol/chloroform extraction and ethanol precipitation. Reaction products were separated on a 10% urea-denaturing polyacrylamide gel, and mature tRNA and tRNA exon formation were monitored by phosphorimaging. Quantification of band intensities was performed using ImageQuant software .A PCR was performed using 2 plates, and silencing of endogenous TRX by shRNA was induced with 2 µg/mL Dox for 3 d. Cells were left untreated, stressed for 3 min with H2O2, 15 min with menadione, for 4 or 8 h with 1.5 µg/mL Tm or 300 nM Tm. Cells were washed once and scraped in PBS supplemented with 20 mM NEM. After incubation of the cells in PBS with 20 mM NEM for 7 min, cells were pelleted for 5′ at 1000 rpm, and washed with PBS. The cell pellets were lysed in buffer 1  supplemented with cOmplete EDTA-free Protease Inhibitor  and 20 mM NEM, and incubated for 30 min under slight rotation at 4°C. The lysates were cleared by centrifugation at 14,800 rpm for 10 min, 4°C; protein concentrations were measured by Bradford assay and adjusted to 1–3 mg/mL in 800 µL total volume. A total of 50 µL M2 FLAG-beads  per 15 cm2 plate were washed three times in buffer 1 with 20 mM NEM, and added to the diluted cell lysate. Pulldown of FLAG-TRX variants with trapped proteins was performed for 3 h at 4°C while rotating. Subsequently, beads were washed four times in wash buffer with increasing salt concentrations  and four times in reverse order. Bound complexes were eluted in 50 µL 1 mg/mL FLAG-peptide dissolved in buffer 1 for 30 min, 4°C, while shaking.Trapping of TRX targets using the FLAG-TRX overexpression construct was done as described before . HeLa RIt-test assuming unequal variances, with (*) P < 0.05; (**) P < 0.01; (***) P < 0.005, using GraphPad Prism software.To assess statistical significance in interstrand ligation assay quantifications, mRNA expression levels (RT-qPCR) or FACS analysis, we used unpaired or paired Student's 50 values, quantification data from interstrand ligation assays were normalized to the untreated sample of shCtrl cells. IC50 values were calculated by nonlinear regression and sigmoidal concentration–response curve fit using GraphPad Prism software.For the calculation of IC


Although KCCs transport K+ and Cl− in a 1:1 stoichiometry, two Cl− coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl− in Cl1 and Cl2. Individual mutations of residues in Cl1 and Cl2 reduce or abolish KCC2WT function, indicating a crucial role of both Cl− coordination sites for KCC2 function. Structural changes in the extracellular loop 2 by inserting a 3xHA tag switches the K+ coordination site to another position. To investigate, whether the extension of the extracellular loop 2 with the 3xHA tag also affects the coordination of the two Cl− coordination sites, we carried out the analogous experiments for both Cl− coordinating sites in the KCC2HA construct. These analyses showed that most of the individual mutation of residues in Cl1 and Cl2 in the KCC2HA construct reduces or abolishes KCC2 function, indicating that the coordination of Cl− remains at the same position. However, the coupling of K+ and Cl− in Cl1 is still apparent in the KCC2HA construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr569 in Cl2 shifted in KCC2HA. Thus, conformational changes in the extracellular domain affect K+ and Cl−-binding sites. However, the effect on the Cl−-binding sites is subtler.The K Gene duein CIP1 , 7. NKCC and Cl− . Both usgradient . CCC fameostasis , 9, 10.via post-translational modifications , flanked by the intracellular N and C terminus, as well as a large extracellular domain (ECD) , 12, 13.ications , 21, 22.ications , 25, 26.ications , 35, 36.ications , 39, 40.− extrusion transporter in most adult inhibitory neurons of the central nervous system where it lowers [Cl−]i , die shortly after birth because of motor deficits CCs (1) harbors conserved residues mainly in TM1  and was revealed in cryo-EM structures of NKCC1, NCC, KCC1, KCC3, and KCC4 (1 site in KCC2 (− coordination site (Cl2) is composed of main-chain interaction residues from TM6  and Tyr569 in TM10 (A) (2) reduces or abolishes the transport activity of KCC1, KCC3, KCC4, NKCC1, and NCC indicating that the Cl2 site is important for their function CCs  CCs , 29, 58.and KCC4 , 58. Mol in TM10 A  , 35, 58.function , 29, 35.−-binding sites have been functionally investigated in KCC2, and more importantly, it is still not known whether both sites are required for transport. Therefore, we performed an extensive functional analysis of residues coordinating Cl− in Cl1 and Cl2 sites. Our analyses revealed that both Cl− coordinating sites are required for KCC2 function.None of these ClHA) results in subtle conformational changes of the ECD and affects K+ ion binding  . H. HHA) re+ and two highly conserved Cl−-binding sites (A) (6M23)   . Cl2 is 7.4 Å away from the coordination center of the K+ ion (A). This raises questions about the functional role of both Cl− sites. To address this important question, residues of both Cl−-binding sites were mutated into residues of similar size but different chemical properties. This resulted in the following seven murine KCC2b mutations: rnKCC2bG113A, rnKCC2bV114T, rnKCC2bI115E, rnKCC2bG413A, rnKCC2bI414E, rnKCC2bM415Q, and rnKCC2bY569F. All mutants showed transfection rates similar to rnKCC2bWT in human embryonic kidney 293 (HEK-293) cells (B and 4B). HEK-293 cells transiently expressing rnKCC2bWT displayed a significantly higher KCC2 transport activity (100%) than mock-transfected control cells . The transport activity has been measured by Cl−-dependent uptake of Tl+ in HEK-293 cells as described previously , rnKCC2bV114T , and rnKCC2bI115E  compared with rnKCC2bWT showed diminished KCC2 activity  , 29, 35.) (6M23) ; howevere: 7D99) . Cl1 is  in TM10 A  , 29. Theese ions A. Cl2 ise K+ ion A. This r3) cells B and 4B.eviously , 60. Aftlization  and S2. Fig. 3A. . These r2-binding sites for KCC2 function, we used the mutants rnKCC2bG413A, rnKCC2bI414E, and rnKCC2bM415Q in TM6 and rnKCC2bY569F in TM10. The mutation KCC2bG413A  abolished KCC2 transport activity. The mutants rnKCC2bI414E , Met415 to glutamine , and rnKCC2bY569F  compared with rnKCC2bWT showed reduced KCC2 transport activity, revealing a crucial role in KCC2 function , and Tyr446 (in TM10)  . To inveagenesis .mmKCC2bHA (B). HEK-293 cells transiently expressing mmKCC2bHA displayed a significantly higher KCC2 transport activity (100% ± 11%) than mock-transfected control cells . The Cl1 mutation mmKCC2bHA-G113A  and mmKCC2bHA-I115E  abolished and mmKCC2bHA-V114T  diminished KCC2 transport activity  and mmKCC2bHA-I414E  abolished KCC2 transport activity . These results are in line with the analyzed Cl−-binding sites in Cl2 for KCC2WT (see aforementioned). Contrary to rnKCC2bY569F, the mutant mmKCC2bHA-Y569F  did not alter KCC2 transport activity. Thus, the extension of the EL2 with the 3xHA tag affects side-chain interaction of Tyr but not main-chain interactions of Gly, Ile, and Met with Cl− in Cl2.For ClFig. 6A, , and submmKCC2bHA construct, we previously hypothesized a switch from Asn110, Ile111 (in TM1), Tyr195 (in TM3), Pro409, and Thr412 (in TM6) K+-binding sites to the predicted alternative K+-binding site consisting of Pro409, Ser410 (in TM6), and Tyr446 (in TM10) (B) . HEK-293 cells transiently expressing KCC2bHA displayed a significantly higher KCC2 transport activity (100%) than mock-transfected control cells . The constructs mmKCC2bHA-P409H , mmKCC2bHA-S410A , and mmKCC2bHA-Y446F  abolished KCC2 transport activity  B 59). T. TmmKCC2mKCC2bHA B. HEK-29activity . These drnKCC2b affect its transport activity, we generated the mutations: rnKCC2bP409H, rnKCC2bS410A, and rnKCC2bY446F in rnKCC2bWT. All mutants showed similar transfection rates in HEK-293 compared with rnKCC2bWT (B). HEK-293 cells transiently expressing KCC2b displayed a significantly higher KCC2 transport activity  than mock-transfected control cells  and rnKCC2bS410A  showed diminished transport activities, whereas rnKCC2bY446F  enhanced KCC2 activity  or Cl2  significantly reduced or abolished KCC2 activity. In addition, Tyr195 in TM3 has a bifunctional role in stabilizing K+ and Cl− in Cl1 , KCC3 (Tyr283), or KCC4 (Tyr216) support this hypothesis  . In this scenario, the Cl2 site has an “allosteric” effect in stabilizing the coordination of the K+-binding site , they can change transport direction to load cells with K+ and Cl−   may then induce a conformation that facilitates ion translocation. In our present analyses, we investigated the inward-mediated transport of Tl+ (congener of K+) and Cl−.KCCs mainly serve as K and Cl− . It is hobserved , 61. In observed , 61, likl− → K+) , 61. Her− sites, we used a modified KCC2 construct, in which the EL2 is extended by a 37-amino acid-long HA tag. This modification, which importantly does not compromise transport activity, results in subtle conformational changes of ECD, which is transduced to TM1 and TM6 , and Thr412 (TM3) (A and B) . Mutation of these alternative residues in the KCC2HA variant abolished KCC2 activity, which corroborates the presence of an alternative K+-binding site in KCC2HA.To take a closer look to the function of both Cl and TM6 . These cn KCC2HA . Because12 (TM3) , A and BA and B) . K+ theron sites B. Mutati− coordination sites, we carried out the analogous experiments for both Cl− coordinating sites in the KCC2HA construct.In order to find out whether the extension of the EL2 with the 3xHA tag also affects the coordination of the two Cl− in the Cl1 site are in close proximity to the K+-binding site. Thus, both ions are coupled with each other with a 3.4 Å distance between them (A). The extension of EL2 with the 3xHA tag shifts the K+ coordination site from Tyr195 in TM3 in KCC2WT to Tyr446 in TM7 in KCC2HA . Since K+ and the alternative K+ site are both in close proximity to Cl− in Cl1, coupling of both ions is present in both KCC2WT and KCC2HA. Interestingly, in the K+-independent NCC, mutation of Tyr386 to alanine  abolishes its function  has been performed with Coot  and mouse KCC2b (NM_020333.2) with an HA tag in the extracellular loop 2 3xHA tag (IFYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHAAA) was performed according to the QuikChange mutagenesis system (Stratagene) , 64, 65.+–Cl− cotransporter activity, HEK-293 cells were transiently transfected with the respective constructs, using Turbofect (Fermentas). Cells were seeded in a 6-well plate 24 h prior to transfection. The Dulbecco's modified Eagle's medium was replaced 4 h before transfection. About 150 μl Opti-MEM (Invitrogen), 6 μl Turbofect, and the corresponding DNA amount were thoroughly mixed and incubated for 20 min at room temperature (RT). The mixture was applied to the cells that were then shaken at 300 rpm for 10 min at RT.For immunocytochemistry and measurement of K+–Cl− cotransporter activity measurements, transfected HEK-293 cells were plated at a concentration of 1 × 105 cells/well in a 0.1 mg/ml poly-l-lysine-coated black 96-well culture dish  was applied for 30 min. Primary antibody solution  was added in carrier solution  and incubated for 1 h. After washing three times with PBS, the secondary antibody, which was conjugated to a fluorescent probe , was added to the carrier solution and incubated for 1 h. Again, the cells were washed three times with PBS and completely dried. The dried coverslips were mounted onto glass slides with Mowiol (Roth) and 4′,6-diamidine-2-phenylindole . Photomicrographs were taken using an Olympus fluorescence microscope (Olympus BX63).For immunocytochemistry, all steps were performed at RT. HEK-293 cells grown on poly-−-dependent uptake of Tl+ in HEK-293 cells as described previously  with 2 μM FluoZin-2 AM dye (Invitrogen) plus 0.2% (w/v) Pluronic F-127 (Invitrogen) and incubated for 48 min at RT. Afterward, cells were washed three times with 80 μl preincubation buffer and incubated for 15 min with 80 ml preincubation buffer including 0.1 mM ouabain to block the activity of the Na+/K+ ATPase. Then, the 96-well plate was placed into a fluorometer (Fluoroskan FL), and each well was injected with 40 μl 5× Thallium stimulation buffer . The fluorescence was measured in a kinetic-dependent manner  across the entire cell population in a single well. By using linear regression of the initial values of the slope of Tl+-stimulated fluorescence increase, the transport activity was calculated. The absolute values are normalized by setting the slope of the KCC2WT or KCC2HA construct as 100% and calculating the percentage of the activity of the mutants respectively. Normalization is needed to subtract naturally occurring fluctuations of the fluorescence increase. A comparison of the absolute versus normalized values for exemplar mutations can be seen in Transport activity of KCC2 was determined by Cleviously , 59, 66.rnKCC2bWT, mmKCC2bHA, and mock), using a two-sample t test after Student’s t test for similar variances between samples. In exceptional cases, where the standard deviation differs by more than a factor of 2, Welch's t test was used (p values were corrected using the Benjamini–Hochberg method (p values <0.01. A Tukey honestly significant difference test after Tukey and Kramer was used for multiple comparison for the analyses in The normalized transport activities of the respective mutant were tested against control samples (was used . To avoig method . The chaAll relevant data are available from the corresponding author upon reasonable request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.