HIF1A locus. We demonstrate that this contrasts with chronic hypoxia, which decreases KDM4A, KDM4B, and KDM4C activity, leading to hypertrimethylation of H3K9 globally and at the HIF1A locus. Altogether, we found that demethylation of histones bound to the HIF1A gene in intermittent hypoxia increases HIF1A mRNA expression, which has the downstream effect of increasing overall HIF-1 activity and expression of HIF target genes. This study highlights how multiple oxygen-sensing pathways can interact to regulate and fine tune the cellular hypoxic response depending on the period and length of hypoxia.The cellular response to hypoxia is regulated through enzymatic oxygen sensors, including the prolyl hydroxylases, which control degradation of the well-known hypoxia inducible factors (HIFs). Other enzymatic oxygen sensors have been recently identified, including members of the KDM histone demethylase family. Little is known about how different oxygen-sensing pathways interact and if this varies depending on the form of hypoxia, such as chronic or intermittent. In this study, we investigated how two proposed cellular oxygen-sensing systems, HIF-1 and KDM4A, KDM4B, and KDM4C, respond in cells exposed to rapid forms of intermittent hypoxia (minutes) and compared to chronic hypoxia (hours). We found that intermittent hypoxia increases HIF-1α protein through a pathway distinct from chronic hypoxia, involving the KDM4A, KDM4B, and KDM4C histone lysine demethylases. Intermittent hypoxia increases the quantity and activity of KDM4A, KDM4B, and KDM4C, resulting in a decrease in histone 3 lysine 9 (H3K9) trimethylation near the  Chronic hypoxia is common in solid tumors and is associated with treatment resistance and aggressive disease . Along w2+-activated calpain proteases (The cellular response to chronic hypoxia is largely coordinated by hypoxia inducible factors (HIFs). HIF is controlled by the prolyl hydroxylase (PHD) oxygen-sensing enzymes, which use oxygen to hydroxylate HIF-α subunits, targeting them for degradation. Factor inhibiting HIF (FIH) also uses oxygen to hydroxylate HIF-α subunits, which blocks binding to the transcriptional coactivators p300/CBP. In the presence of insufficient oxygen, PHD and FIH activity substantially decreases, enabling HIF-α to bind to HIF-β and initiate the transcription of target genes . Similarroteases .Epigenetic modifications of histones are also involved in the response to chronic hypoxia . In the HIF1A and therefore controlling the HIF-1 response in rapid intermittent hypoxia. This pathway is distinct from the canonical oxygen-dependent degradation pathway and does not occur in chronic hypoxia.In this study, we describe a unique role for the KDM4A, KDM4B, and KDM4C histone demethylases in regulating the expression of 2 v/v) or chronic hypoxia (0.5% O2 v/v). Relative to normoxia, HIF-1α protein increased under both chronic and intermittent hypoxia but to a lesser extent in intermittent hypoxia, similar to HCT116 cells (A). We also found that expression of select HIF-1 target genes increased (We have previously shown that HIF-1α protein and HIF-1 target gene expression increases in intermittent hypoxia (5 min/5 min cycles) in HCT116 cells . To exam16 cells  Fig. 1AA2 v/v orncreased . Overallncreased .Figure 1B, all tested periods of intermittent hypoxia . As shown in Figure 1HIF1A gene is believed to be constitutive under most conditions  and MCF7 cells (C) when compared to normoxia. In contrast, when cells are exposed to chronic hypoxia, expression of HIF1A mRNA decreases (C). We also compared the levels of HIF1A mRNA in brain (U251), prostate (PC3), and additional breast (MDA-MB-231) cancer cell lines following exposure to normoxia, chronic, and intermittent hypoxia  expression following intermittent and chronic hypoxia to determine if expression was regulated in a similar manner to HIF1A and found that it was not  significantly increased HIF1A expression when compared to normoxia and chronic hypoxia, except 3 min/3 min, which did not change from normoxia (D). This indicates that expression of HIF1A is controlled differently in chronic hypoxia and intermittent hypoxia and this effect can depend on the period of the intermittent hypoxic cycles. The 5 min/5 min cycles of intermittent hypoxia promoted the largest increase in HIF1A mRNA.To assess how different protocols of intermittent hypoxia affected normoxia D. This iHIF1A mRNA (C), HCT116 cells were treated with cycloheximide (CHX), a protein synthesis inhibitor, followed by either chronic or intermittent hypoxia for 6 h. CHX blocks de novo protein synthesis, therefore HIF-1α levels would reflect protein stabilization and degradation processes. As shown in Figure 1E, CHX reduced HIF-1α protein levels in both chronic and intermittent hypoxia, indicating that HIF-1α accumulation is dependent on continuous protein synthesis in both forms of hypoxia. This also suggests that HIF-1α still undergoes some proteasomal degradation in the presence of either form of hypoxia, though the rate of degradation under individual conditions may vary. Based on Figure 1E, the increase in HIF-1α observed in the absence of CHX for both chronic and intermittent hypoxia was due, at least in part, to new protein synthesis.To determine whether the increase in HIF-1α in intermittent hypoxia is dependent on the increase in F1A mRNA C, HCT116F), HIF-1α accumulated due to blocked oxygen-dependent and oxygen-independent proteasomal degradation (lane 2). In chronic hypoxia, HIF-1α was stabilized (lane 3) and the addition of MG262 slightly increased HIF-1α protein (lane 4), confirming that low levels of proteasomal degradation still occurs in chronic hypoxia. In intermittent hypoxia, there were lower levels of HIF-1α protein (lane 5) when compared to chronic hypoxia (lane 3) but still more than in normoxia . However, when MG262 is added to cells under intermittent hypoxia (lane 6), HIF-1α accumulated well above the observed level in chronic hypoxia, so much so that it was difficult to capture all conditions within the dynamic range of chemiluminescent detection. This indicates that more proteasomal degradation of HIF-1α occurs in intermittent hypoxia than in chronic hypoxia, which explains the lower HIF-1α levels in intermittent hypoxia relative to chronic hypoxia. Taken together with the HIF1A mRNA (C and D) and CHX  data, this result also suggests that the increase in HIF1A mRNA expression in intermittent hypoxia directly contributes to the levels of HIF-1α protein, and when competing degradation processes are inhibited, this leads to a greater increase in HIF-1α .To further explore HIF-1α protein degradation in intermittent and chronic hypoxia, we treated HCT116 cells with MG262, a proteasome inhibitor followed by exposure to normoxia, chronic hypoxia, and intermittent hypoxia for 18 h. The proteasome is the primary degradation pathway for HIF-1α and by using MG262, we can observe how chronic and intermittent hypoxia affect HIF-1α protein expression without competing degradation processes affecting the final protein quantity. When normoxic HCT116 cells were treated with MG262 F, HIF-1αHIF1A mRNA synthesis in intermittent hypoxia (which does not occur in chronic hypoxia) enables a more robust HIF-1 response to a severe hypoxic event following intermittent hypoxia. To test this hypothesis, we exposed MCF7 cells to 18 h of normoxia, chronic hypoxia, or intermittent hypoxia that was immediately followed by an additional acute period of chronic hypoxia (4 h of chronic hypoxia) . As expected, 18 h of normoxia showed no HIF-1α . The addition of 4 h of chronic hypoxia following 18 h of normoxia led to a substantial increase in HIF-1α (lane 2), similar to the amount of HIF-1α present after 18 h of chronic hypoxia (lane 3). Eighteen hours of intermittent hypoxia led to a moderate increase in HIF-1α . However, the largest increase of HIF-1α in any of the conditions occurred when cells were exposed to 18 h of intermittent hypoxia followed by 4 h of chronic hypoxia (lane 5). Similar to the MG262 data, this experiment demonstrates that the increased HIF1A mRNA in intermittent hypoxia is translated into higher levels of HIF-1α protein, though in intermittent hypoxia some of this is likely degraded via oxygen-dependent degradation. When oxygen-dependent degradation is halted following intermittent hypoxia exposure, via a brief exposure to more severe chronic hypoxia, there is potential for a more robust HIF-1α response due to the increased level of HIF1A expression and protein synthesis generated by the preceding intermittent hypoxia.These results led us to hypothesize that an increase in o HIF-1α G, lane 1HIF1A mRNA expression, while chronic hypoxia dampens HIF1A mRNA expression, indicating differential regulation depending on the type of hypoxic exposure. Histone methylation patterns alter the expression of genes and chronic hypoxia is associated with histone hypermethylation (HIF1A gene (H). Surprisingly, and in contrast to chronic hypoxia, we found that intermittent hypoxia decreased levels of H3K9me3 below the levels observed in normoxia (H). H3K9me3 is associated with heterochromatin and gene silencing; therefore a global decrease in H3K9me3 induced by intermittent hypoxia is likely resulting in an increase in expression of associated genes.We identified that intermittent hypoxia increases hylation , 10, 11,F1A gene , therefoF1A gene , we founF1A gene H. Surprinormoxia H. H3K9meHIF1A mRNA. We found that global H3K9me2 levels slightly decreased following intermittent hypoxia . Interestingly, 3 min/3 min cycles did not decrease H3K9me3 (I) and this corresponds with no increase in HIF1A mRNA under the same conditions (D).To assess how varying periods of intermittent hypoxia affect H3K9me3, we measured trimethylation levels in MCF7 cells following varying lengths of alternating normoxia and hypoxia. We found that 5 min/5 min, 8 min/8 min, and 12 min/12 min intermittent hypoxia cycles all decreased global H3K9me3 as compared to chronic hypoxia I. Intere H3K9me3 I and thinditions D.HIF1A gene expression in chronic hypoxia (H) was specifically occurring at the HIF1A gene locus, we carried out a chromatin immunoprecipitation quantitative PCR (qPCR) assay using MCF7 cells. Following exposure to normoxia, chronic hypoxia, and intermittent hypoxia, cells were lysed and H3K9me3 was immunoprecipitated. Coimmunoprecipitated DNA was purified and then subjected to qPCR using primers designed to detect HIF1A introns. For all regions measured, we found an increased quantity of the HIF1A gene in chronic hypoxia compared to normoxia (J). This indicates enrichment of H3K9me3 at the HIF1A gene in chronic hypoxia and supports previous work done in RKO cells (HIF1A in intermittent hypoxia compared to normoxia (J), indicating a loss of H3K9me3 at the HIF1A gene.H3K9me3 is a repressive marker for  hypoxia . To dete hypoxia H was spenormoxia J. This iKO cells . In contnormoxia J, indicaKm values measuring in a similar range as the PHD oxygen sensors (Trimethyl (me3)  marks can be removed from H3K9 by the KDM4 demethylase subfamily, therefore we focused our attention on this group of enzymes. KDM4 enzymes require oxygen to function and belong to the same 2-OG-dependent family of oxygenases as the PHDs . KDM4A,  sensors , 16, and sensors , 18, 19.KDM4B and KDM4C, as was seen with other HIF-target genes in We hypothesized that KDM4A, KDM4B, and KDM4C would lose activity in chronic hypoxia (increasing H3K9me3), while retaining activity in intermittent hypoxia due to sufficient periodic oxygenation, enabling ongoing demethylation of H3K9 similar to normoxia. Furthermore, if intermittent hypoxia increases expression of the HIF-target genes, A). In contrast, protein levels of KDM4B and KDM4C rose substantially in chronic hypoxia and to an intermediate level in intermittent hypoxia (A). KDM4A mRNA levels decreased in chronic hypoxia and did not change from normoxia in intermittent hypoxia, supporting previous data indicating that KDM4A is not a HIF-target gene or induced by hypoxia (B). Expression of KDM4B and KDM4C mRNA increased in a similar pattern to the results in Fig. 2B). These data indicate that under chronic and intermittent hypoxia, there are increased protein levels of KDM4B and KDM4C enzymes, with stable levels of KDM4A.To test this hypothesis, we exposed MCF7, HCT116, and MDA-MB-231 cells to normoxia, chronic hypoxia, and intermittent hypoxia and assessed the expression of KDM4A, KDM4B, and KDM4C protein and mRNA. We found no changes in the protein levels of KDM4A in cells exposed to normoxia, chronic hypoxia, or intermittent hypoxia A. In con hypoxia A. KDM4A  hypoxia B. ExpresHIF1A siRNA to knock down HIF-1 activity in MCF7 cells. Knockdown with HIF1A siRNA significantly decreased mRNA expression of HIF1A in chronic and intermittent hypoxia, which led to a significant reduction in KDM4B and KDM4C but not KDM4A mRNA levels . While increasing enzyme quantity can lead to an overall increase in enzyme activity, activity also relies on the availability of substrates and cofactors. Molecular oxygen is required for KDM4A, KDM4B, and KDM4C activity, which is limited in chronic hypoxia while present in varying levels in intermittent hypoxia. To compare the effects of chronic and intermittent hypoxia on KDM4A, KDM4B, and KDM4C activity, we measured enzymatic turnover of recombinant KDM4A, KDM4B, or KDM4C under normoxia, chronic hypoxia, and intermittent hypoxia using a KDM4 enzyme assay kit (Abcam). Five hundred nanograms of recombinant KDM4A, KDM4B, and KDM4C were added to microplate wells stably coated with H3K9me3 and exposed to 4 h of normoxia, chronic hypoxia, and intermittent hypoxia. Following exposure, demethylated H3K9me3 was captured using the provided primary antibody, and an horseradish peroxidase–linked secondary antibody was used for absorbance detection. All three enzymes  had decreased demethylase activity in chronic hypoxia as expected. However, in intermittent hypoxia, there was no difference in the amount of demethylated H3K9 product produced when compared to normoxia at equivalent amounts of enzyme (C–E). This indicates that at least for our protocol of intermittent hypoxia, there is sufficient oxygen present for enzyme activity and that our conditions have no apparent difference on demethylation than in normoxia. These results imply that chronic hypoxia limits the amount of oxygen available to KDM4 enzymes, decreasing demethylase activity. In contrast, oxygen is not a limiting factor for KDM4A, KDM4B, or KDM4C activity in intermittent hypoxia.In both intermittent and chronic hypoxia, KDM4B and KDM4C levels increase, while KDM4A levels remain stable A. While C–E), results from Figure 2A indicate that there are higher protein levels of KDM4B and KDM4C in cells exposed to intermittent hypoxia. The quantity of KDM4B and KDM4C also increases in chronic hypoxia but limited oxygen availability appears to hinder their activity (C–E). Given that oxygen is not a limiting factor for KDM4 activity in intermittent hypoxia, we hypothesized that the increase in the quantity of KDM4B and KDM4C in intermittent hypoxia would result in overall greater H3K9me3 demethylation than in normoxia or chronic hypoxia.While KDM4 enzymatic activity is similar in normoxia and intermittent hypoxia when equivalent amounts of enzyme are used , C–E, reactivity , C–E. GiD and E, KDM4B and KDM4C demethylase activity in intermittent hypoxia was the same as normoxia at all comparable concentrations (F and G). In contrast, KDM4B and KDM4C demethylase activity was significantly reduced in chronic hypoxia when compared to the same enzyme concentration in normoxia or intermittent hypoxia.To test this, we measured the demethylase activity of recombinant KDM4B and KDM4C using a range of enzyme concentrations under intermittent hypoxia and compared activity levels to normoxia and chronic hypoxia. Similar to the results from trations , F and GAltogether, these data show that intermittent hypoxia both increases the quantity of KDM4 enzymes  and does not limit substrate availability for KDM4 enzymes  to function optimally. This contrasts with normoxia, which produces the lowest quantity of KDM4 enzymes, and chronic hypoxia, where oxygen availability is limited. The combined effect of an increase in the quantity of KDM4 enzymes and oxygen availability for enzymatic function in intermittent hypoxia is an overall greater increase in KDM4 demethylase activity relative normoxia or chronic hypoxia.HIF1A gene expression in intermittent hypoxia.We showed that intermittent hypoxia increases KDM4B and KDM4C proteins and retains demethylase enzymatic activity of KDM4A, KDM4B, and KDM4C, leading to the highest levels of demethylation in intermittent hypoxia. The next step was to determine whether the increase in KDM4 activity is responsible for the demethylation of H3K9 and subsequent increase in HIF1A expression. Simultaneous knockdown of KDM4A, KDM4B, and KDM4C was highly effective at reducing mRNA and protein levels of each enzyme (A–D). While H3K9me3 levels in chronic hypoxia were already high relative to normoxia, knocking down KDM4A, KDM4B, and KDM4C led to a greater increase in the levels of H3K9me3, indicating some residual demethylase activity in chronic hypoxia (D). Furthermore, where H3K9me3 was nearly undetectable in intermittent hypoxia, the triple knockdown increased H3K9me3 to similar levels to those seen in the chronic hypoxia triple knockdown, confirming that the methylation status of H3K9 in intermittent hypoxia is dependent on KDM4A-C activity (D). As shown in Figure 3E, the triple knockdown had little effect on HIF1A mRNA expression under normoxia and chronic hypoxia. However, in intermittent hypoxia, the triple knockdown significantly reduced HIF1A mRNA. The triple knockdown also reduced HIF-1α protein levels under intermittent hypoxia, while having little effect on HIF-1α in chronic hypoxia (2% oxygen v/v) (D). We also identified that knockdown of KDM4A, KDM4B, and KDM4C reduces the mRNA expression of known HIF-target genes, HK2 and PLOD2 (F and G), and HK2 at the protein level (D). The knockdown data shown in To test this, we used a combined siRNA approach to simultaneously knockdown KDM4A, KDM4B, and KDM4C in MCF7 cells. This was followed by measurement of H3K9me3 levels and h enzyme , A–D. Wh hypoxia D. Furtheactivity D. As shogen v/v) D. We alsnd PLOD2 , F and Gin level D. The knIn vitro tumor spheroids replicate some aspects of in vivo tumor biology, and they contain both oxygenated, proliferating cells on the exterior and chronically hypoxic cells in the center . GFP expression in the chronically hypoxic spheroid cores was blocked with HIF1A siRNA, indicating GFP expression was specific to HIF-1 activity (B). Furthermore, we saw increased H3K9me3 as the spheroid grew in size (B). The level of KDM4B, KDM4C, and glycolytic protein  overexpression and the degree of H3K9me3 correlated with the increase in hypoxia observed via GFP (A).HCT116 cells were transfected with a GFP reporter gene linked to the hypoxia response element (HRE) to which HIF-1 binds. As expected, as spheroids grew larger over time, they developed hypoxic cores with increased HIF-1 activity as indicated by GFP A. GFP exactivity . The chractivity B. Furthe in size B. The le via GFP A.Figure C). The face of the spheroid at the membrane has direct exposure to surrounding oxygen environment, including any fluctuations, which mimics the perfused cells on the tumor exterior. As oxygen must diffuse through the spheroid, this leads to spatiotemporal gradients of fluctuating oxygen until it reaches the chronically hypoxic regions at the spheroid core. We wanted to explore how HIF-1 activity would be affected by these spatiotemporal oxygen changes.Next, we wanted to observe how intermittent hypoxia stemming from the exterior might interact with the chronically hypoxic spheroid core. The 3-day-old HCT116 spheroids stably expressing 5HRE-GFP were placed onto an oxygen-permeable membrane and left to incubate over 24 h to allow cells closest to the membrane to attach C. The faD, Panel 4, top), as the cells are oxygenated. We saw increased GFP expression in the hypoxic core of the normoxic spheroids , as compared to the oxygenated exterior of the spheroid. When spheroids were placed in intermittent hypoxia, GFP expression was much higher in the cells directly attached to the membrane, confirming that HIF-1 activity increases in cells directly exposed to intermittent hypoxia . GFP expression also increased in the core of the intermittent hypoxia spheroids . Interestingly, GFP expression (and therefore HIF-1 activity) was higher in the core of spheroids exposed to intermittent hypoxia than in the spheroids grown in normoxic conditions. This indicates that regions exposed to both chronic and intermittent forms of hypoxia may lead to the greatest HIF-1 activity. These data are supportive of our Western blots in Figure 1G showing the highest levels of HIF-1α are found in cells exposed to intermittent hypoxia, followed by chronic hypoxia. Cells may respond to a range of spatially and temporally distinct forms of hypoxia through dynamic or overlapping signaling pathways that combine to enhance activity of HIF-1.Attached spheroids were exposed to either normoxia or intermittent hypoxia over 18 h to assess HIF-1 activity within a three-dimensional cell culture, which introduces additional oxygen gradients. Immediately after exposure, spheroids were stained with nuclear red live (DNA stain) and confocal images were taken through multiple transverse sections of spheroids. This enabled us to examine GFP expression in cells directly attached to the membrane compared to cells deeper into the spheroid, which are more hypoxic due to chronically limited oxygen diffusion. As expected, in normoxia grown spheroids, there was little to no GFP expression in the cells directly attached to the membrane . Because antibody penetration can be variable in spheroids even with permeabilization, we used total H3 to account for any bias in antibody penetration. Following confocal microscopy, fluorescence data for both H3K9me3 and total H3 were measured in 20 individual cells from each condition and the ratio of H3K9me3:H3 calculated.To determine how intermittent hypoxia affects H3K9me3 in an HCT116 spheroid, we used similar conditions as in Figure 4E and F, there was a region of single cells grown in monolayer on the membrane surrounding the spheroid. These cells would be exposed to oxygen conditions like those from the monolayers used to produce Western blots in Figure 4B. We found that the ratio of H3K9me3:H3 decreased in monolayer cells exposed to intermittent hypoxia, supporting our findings in Figs. 4G and Figs. 4G and As shown in HIF1A in vivo. We had access to frozen liver tissue from a Sprague–Dawley rat model of OSA. Rats were previously exposed to 8 h of cycling intermittent hypoxia via inhaled gases , which generates fluctuating blood oxyhemoglobin levels and intermittent hypoxia within the liver and other tissues (B and C), supporting our results in HIF1a mRNA increased in the livers of rats exposed to intermittent hypoxia , indicating that systemic intermittent hypoxia can lead to H3K9me3 demethylation and an increase in expression of HIF1a in the liver. We also measured HIF-1α levels and saw a trend toward increased HIF-1α in rats exposed to intermittent hypoxia .We wanted to test whether intermittent hypoxia would decrease H3K9me3 and increase  tissues . This fl tissues . We obtaversus chronic hypoxia (minutes versus hours)  . In agres hours) , namely ple KDMs , 10, 11 ple KDMs , and theHIF1A mRNA expression, though the full effects of this increase are not immediately reflected at the protein level due to competing HIF-1α degradation controlled by the PHDs. However, when degradation of HIF-1α is halted, either by a drug  or through an additional period of chronic hypoxia (G), the surplus of HIF1A mRNAs is converted into protein, with little degradation, resulting in a corresponding large increase in HIF activity. In this unique situation, an abundance of HIF1A mRNA could prove to be beneficial during ischemia by enabling maximum activation of the HIF-1 pathway. While our studies did not use cardiac cells or ischemia models, this is an intriguing hypothesis to be tested in future.Our results are intriguing when viewed in light of studies that focus on the ‘preconditioning effects’ of sleep apnea and cardiovascular disease. Intermittent hypoxia from mild to moderate sleep apnea has been suggested to have a protective effect against myocardial infarction in both humans and animal models , 29, 30.g MG262, F or thro hypoxia G, the suversus chronic) in the different regions (periphery versus core) of the spheroid. Several studies have suggested that fluctuating oxygen levels in tumors lead to greater phenotypic variation and a competitive advantage for tumors  of hypoxia , 1× Glutamax, penicillin (50 IU/ml), and streptomycin (50 μg/ml). HCT116 cells were cultured in McCoys 5A media (Gibco). MCF7, MDA-MB-231, and U251 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) media (Gibco). PC3 cells were cultured in DMEM/F12 media (Gibco). Cell cultures were To overcome the challenges of slow oxygen diffusion through culture media, cells were grown onto oxygen-permeable polydimethylsiloxane (PDMS) membranes to enable cells to access oxygen from the ambient air directly through the membrane. PDMS culture dishes were purchased from Sarstedt, Zell-Kontakt, or Coy Lab Products. Cells were seeded onto PDMS culture dishes in media supplemented with Glutamax and 10% FBS. After overnight incubation, the media was replaced with fresh media with 0.5% FBS. Cells were placed in hypoxia chambers and exposed to different oxygen conditions, as defined later. Construction of hypoxia chambers are described in . Oxygen HIF1A gene , 5 min/5 min, 8 min/8 min, or 12 min/12 min. Altering the periods of normoxia and hypoxia led to different peak and nadir oxygen values in the pericellular media as measured using a PreSens Oxygen Microsensor oxygen electrode (PM-PSt-7 with Oxy-1 ST)  . For theCells were seeded onto PDMS dishes. After overnight incubation, media was removed and replaced with fresh media with 0.5% FBS containing 10 μg/ml CHX  or vehicle control (1% dimethyl sulfoxide). Dishes were then exposed to normoxia, chronic hypoxia, or intermittent hypoxia for 6 h.Cells were seeded onto PDMS dishes. After overnight incubation, media was removed and replaced with fresh media with 0.5% FBS containing 1 mM MG262  or vehicle control (1% dimethyl sulfoxide). Dishes were then exposed to normoxia, chronic hypoxia, or intermittent hypoxia for 18 h.Cells were transfected with siRNA using HiPerfect (Qiagen). We used 20 nM KDM4A FlexiTube GeneSolution siRNA , 5 nM siKDM4B , 5 nM siKDM4C , and 20 nM HIF1A Flexitube GeneSolution siRNA . AllStars Negative control siRNA  was used as a negative control. Cells incubated for 48 h prior to exposure to normoxia, chronic hypoxia, and intermittent hypoxia. Immediately prior to exposure to oxygen conditions, media was replaced with fresh media supplemented with 0.5% FBS.To extract total protein, cells or tissues were lysed in radioimmunoprecipitation assay buffer. To extract nuclear protein, nuclear lysates from cells or tissues were prepared as described previously . TissuesTotal RNA was extracted using the RNeasy Mini Kit (Qiagen). RNA was quantified using a Nanodrop (Molecular Devices). Total RNA (2 μg) was used as a template for complementary DNA (cDNA) synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For quantitative RT-PCR, predesigned TaqMan Gene Expression assays were used with TaqMan Gene Expression Master Mix . Twenty nanograms cDNA was used per reaction. qPCR was performed using a QuantStudio 7 Flex Real-Time PCR System. Relative gene expression was normalized to eukaryotic 18S rRNA (Hs03003631_g1) or ACTB (Hs01060665_g1) and fold change was calculated using the ΔΔCt method. Target gene TaqMan primers (Assay ID) used for qPCR (ThermoFisher) were as follows: SLC2A1 (Hs00892681_m1), HK2 (Hs00606086_m1), LDHA (Hs01378790_g1), PLOD2 (Hs01118190_m1), P4HA1 (Hs00914594_m1), P4HA2 (Hs00990001_m1), EGLN1 (Hs00254392_m1), EGLN3 (Hs00222966_m1), HIF1A (Hs00153153_m1), KDM4A (Hs00206360_m1), KDM4B (Hs00392119_m1), and KDM4C (Hs00909577_m1). Data are expressed as the mean ± SEM from three or more independent experiments.KDM4 demethylase activity was measured using the JMJD2/KDM4 Activity Quantification Assay Kit  according to the manufacturer’s instructions. Recombinant KDM4A , KDM4B , or KDM4C (ab198094) were added to wells coated with H3K9me3 in a range of concentrations (0–800 ng). Reactions took place in the same hypoxic chambers used for cell cultures under normoxia, chronic hypoxia, and intermittent hypoxia over 4 h. Demethylated H3K9me3 were captured using a primary antibody overnight. Wells incubated in a horseradish peroxidase–linked secondary detection antibody for 2 h. Absorbance readings were taken at 450 nm with a reference wavelength of 655 nm using the Infinite M1000 PRO microplate reader (TECAN).http://n2t.net/addgene:46926; RRID:Addgene_46926)   using lie_46926) . SpheroiF) and fluorescence of H3K9me3 and Histone H3 was quantified.Fluorescence images were acquired using a Zeiss Primovert microscope with 5× and 10× objective. Live cell confocal images for GFP expression were acquired using a Leica TCS SP8 confocal laser scanning microscope with 25 × W 0.95 objective. Three-dimensional images were collected and stitched using LASX Navigator module. Confocal imaging of fixed spheroids labeled with H3K9me3 and Histone H3 was performed using a Zeiss LSM 880 microscope with 20× air objective, NA 0.8. For analysis, 60 μm z-stacks (step size 15 μm) were acquired using Zen software . Images were processed and analyzed using Fiji ImageJ . Single Fig. 42, 10 mM KCl, 0.1% NP-40, 1 mM DTT, EDTA-free protease inhibitors). Nuclei were resuspended in sonication buffer  and sonicated at 70% amplitude for 40 min,  at 4 °C (QSonica 800R) to shear DNA to an average fragment size of 200 to 1000 bp. Prior to immunoprecipitation, 10% of each lysate was aliquoted to be used as an input reference sample. The remaining lysates were diluted 1:10 in dilution buffer  and incubated in 10 μg H3K9me3 antibody  overnight at 4 °C. Immune complexes were captured by incubation with Protein G Dynabeads (Thermo) at 4 °C for 2 h. Dynabeads were washed sequentially with a low salt wash buffer , a high salt wash buffer , a LiCl wash buffer , followed by TE buffer , and eluted with 120 μl elution buffer . Crosslinks were reversed by incubation with 0.2 M NaCl and 10 mg/ml RNase A at 65 °C for 4 h. Proteins were digested by incubation with 20 mg/ml proteinase K for 1 h at 60 °C. DNA was purified using the Monarch PCR & DNA Cleanup Kit (NEB), according to the manufacturer’s instructions. Immunoprecipitated DNA was quantified by qPCR relative to the total amount of input DNA. Primer sequences for introns on the HIF1A gene are described in . All experiments were conducted on adult male Sprague–Dawley rats weighing 300 to 500 g, housed three per cage in the Heart Research Institute biological facilities animal holding room. Rats acclimatized in the holding room for at least 7 days before experiments and lived in a 12/12 h light-dark schedule with lights on at 0700 AM. Room temperature was set at 22 ± 2 °C with relative humidity of 40% to 60%. Food and water were available ad libitum.Twelve rats were randomly allocated to either an intermittent hypoxia cohort or a control cohort. The intermittent hypoxia or Sham protocol was started between 8 to 9 AM. Food was removed during the 8 h intermittent hypoxia period but water remained ad libitum.2) within the chamber were continuously monitored  and recorded in Spike2 software (Ver.8 software CED Ltd). Intermittent hypoxia was delivered in 4-phase cycles of varying oxygen and nitrogen flow rates. Oxygen levels were rapidly driven from 21% down to 6% then sustained for 1 min. Oxygen was then rapidly ramped back up to 21% and maintained for 2.5 min. This sequence was repeated for 8 h. The timed sequence and flow rates for the Sham cohort exactly matched those of the intermittent hypoxia sequence but with 21% O2 delivered across all four phases. Following the 8 h protocol, rats were anaesthetized with sodium pentobarbitone  and blood collected via cardiac puncture. Rats were transcardially perfused with ice-cold PBS (400 ml). The right median lobe of the liver was extracted and immediately freeze clamped in liquid nitrogen. The resultant wafer was stored at −80 °C for future use.Rats were individually exposed to 8 h of intermittent hypoxia designed to mimic blood oxygen changes seen in OSA or to a Sham sequence inside a modified home cage. High purity nitrogen and oxygen (Coregas) was mixed at the desired percentages by a GSM-4 Gas Mixer (CWE Inc). Oxygen levels (% Ot test was used to compare siCONTROL with siHIF1A or siKDM4. Two-way ANOVA was used for KDM4B and KDM4C enzyme assays at varying concentrations (see Statistics were conducted using GraphPad Prism (GraphPad Software Inc). One-way ANOVA was used when comparing results between normoxia, chronic hypoxia, and intermittent hypoxia. Paired two-tailed ions see  and for ions see . Mann–WhAll data are contained in the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The OC hempseed had higher total protein and lower nonprotein nitrogen content. Protein extracts showed bimodal particle size distributions, with OC showing the smallest and CC1 the largest mean particle diameter (d(0.5)), i.e., 89.0 and 120.0 µm, respectively. Chromatographic analysis showed similar protein profiles for all three protein extracts. The protein extracts were subjected to in vitro simulated gastrointestinal digestion (SGID). Degree of hydrolysis (DH) measurement showed that the highest extent of digestion upon SGID was associated with CC1 (11.0 ± 1.5%), which also had the lowest in vitro antioxidant activity. Only the OC and OC digested samples had lipase inhibitory activity. The results indicate that the cultivation method impacted the composition, physicochemical, digestibility, and biofunctional properties of hempseed proteins.The proteins from two conventionally (CC1 and CC2) and one organically cultivated (OC) hempseed samples were extracted  and compared in terms of their physicochemical, digestibility and  Cannabis sativa L.) is an annual herbaceous plant that has been widely used due to its industrial  iin vitro bioactive properties.Hemp grows well in a variety of climates and soil types and has an excellent ability to grow without fungicides, herbicides and pesticides . Hemp cap > 0.05)) compared to CC1 (21.93 ± 0.14%) and CC2 (21.18 ± 0.39%). The total protein content in the commercial HPI was 41.20 ± 1.25%. The protein content in hempseeds from 10 Canadian cultivars ranged between 22% and 26% [The composition of the hempseed samples and the commercial HPI is summarized in o CC1 21.3 ± 0.14% and 26% , the pro and 26% , this wa and 26% . In addip < 0.05) higher than in OC (2.61 ± 0.19%) and CC2 (2.84 ± 0.07%). Potin, Lubbers, Husson, and Saurel [The NPN content in CC1 (3.28 ± 0.07%) and HPI (3.16 ± 0.14%) was significantly (d Saurel  previousp < 0.05) higher than that of the other samples, i.e., 6.37 ± 0.21 and 6.37 ± 26% for CC1 and CC2, respectively. All the hempseed samples had a higher moisture content than the HPI (4.45 ± 0.05%) sample. The moisture values for the hempseed samples (range 6.37–7.30%) were comparable with the reported average value (6.2%) in hempseed from 10 Canadian cultivars [The moisture content (7.30 ± 0.24%) of the OC hempseed was significantly  lower than that of the HPI sample (8.38 ± 0.16%). The ash content in all three cultivated hempseed samples herein was similar to the mean values of ash content (5.2%) in hempseed from 10 Canadian cultivars [The ash content for all the hempseed protein extracts was similar (ranging from 4.81% to 5.10%) and was significantly  than for the two other samples . All three hempseed samples used herein had significantly (p < 0.05) higher lipid content than HPI (10.13 ± 0.36%). The lipid content of the CC2 sample was similar to that previously reported for the Fasamo cultivar (30.5 ± 0.7%) grown in Canada [The lipid content in CC2 (31.32 ± 1.56%) was higher  was significantly lower than CC1 (26.8 ± 0.6%) and CC2 (26.5 ± 0.6%). The dietary fiber of Finola variety hempseed was previously reported to be 27.6% . The diep < 0.05) and the lowest (p < 0.05) dietary fiber content. CC1 had the highest (p < 0.05) NPN and CC2 the highest (p < 0.05) lipid content. No significant difference (p > 0.05) between the protein content of the OC and CC samples was observed.Overall, the OC had the highest moisture  solubility at pH 12 being equal to 82.2 ± 4.7% herein. The results showed that the solubility of the commercial HPI under neutral pH conditions was low. The protein solubility of HPI was minimum at pH 3–6, while it increased gradually above pH 6  was determined in order to select a suitable pH for protein extraction from OC, CC1 and CC2. The results showed that protein solubility at alkaline pH was higher than under acidic conditions, with the highest  higher protein than the two other samples. The results showed that the spray-dried OC, CC1 and CC2 extracts contained 65.54 ± 1.24, 62.58 ± 1.61, and 70.03 ± 0.94% protein, respectively. The protein profiles of the spray-dried samples were analyzed using RP-UPLC (The spray-dried CC2 protein extract powder contained significantly ( RP-UPLC . The prop > 0.05); however, the CC1 sample had the widest distribution of particle sizes (d(0.9) − d(0.1) = 590.0 µm) (p > 0.05).The results of the particle size analysis of the aqueous reconstituted hempseed protein extracts showed that the smallest particles were associated with the OC sample while the largest was associated with the CC1 sample. The particle sizes for the CC2 sample were in between these two samples. Nevertheless, these values were lower than the mean particle diameter of commercially available HPI with 50% protein, which were recorded to be 200 µm . The uniformity of the protein extract samples was not significantly different (90.0 µm) . Bimodal90.0 µm) . This map > 0.05) different. The results showed that the highest (p < 0.05) DH obtained for the samples subjected to gastric followed by intestinal digestion was associated with the CC1 sample (11.0 ± 1.5%) followed by CC2 (8.7 ± 0.4%) and OC (8.1 ± 0.9%).The DH of the digested OC, CC1 and CC2 samples subjected to the gastric and gastric followed by intestinal digestion was determined . The DHsThe incubation of HPI preheated at 75, 80 and 90 °C with trypsin for 10 min was previously reported to result in DHs of 2.3%, 4.5%, and 6.7%, respectively . In anotThe two main polypeptide chains, the 33 kDa acidic subunit and 18–20 kDa basic subunits of edestin, were observed on the gel of undigested hempseed proteins  appeared to be lower than that for the CC1 (lane 6) and CC2 (lane 9) protein extracts. This was in accordance with the DH results . At the Molecular mass distribution analysis showed that hempseed proteins having MW > 10 kDa were hydrolyzed to smaller components during SGID . The mosin vitro 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS●) scavenging activity analysis showed that at a concentration of 1 mg/mL, the hempseed protein extracts had low antioxidant activity. Subjection of the hempseed protein extracts to SGID increased their antioxidant activity. However, there was no significant difference (p > 0.05) between the antioxidant activity of the gastric digested and gastrointestinal digested sample from the different hempseed protein extracts. Among the intact proteins, the OC sample had the highest (p < 0.05) ABTS● scavenging activity (16.9 ± 0.9%), while no significant difference (p > 0.05) was observed between the antioxidant activity of CC1 and CC2. Among the digested samples, the highest ABTS● scavenging activity (86.9 ± 1.3%) was associated with the OC gastric digested samples, while the lowest (82.4 ± 0.6% at a concentration of 1 mg/mL on a protein basis) was associated with the gastric digested CC1 sample. Therefore, SGID treatment released peptides with ABTS● scavenging activity.The ● scavenging activity of 20–60%, while their subsequently fractionated peptides with sequences corresponding to GSH, WVYY and PSLPA at 0.5 mg/mL had radical scavenging activity of 80%, 70%, and 60%, respectively [● scavenging activity compared to the acid soluble samples [Previously, hempseed protein hydrolysates at a concentration of 0.5 mg/mL were reported to have a DPPHectively . The alk samples . It shou samples . For exa samples .Proteins and peptides with pancreatic lipase inhibitory activity are potentially promising compounds with antiobesity properties. The results herein showed that the intact and digested proteins derived from the two conventionally cultivated hempseeds (CC1 and CC2) had no lipase-inhibitory activity. In contrast, CC1 and CC2 protein extracts and also the digests of CC1 and CC2 at a protein concentration of 1 mg/mL had lipase-inducing activity up to 35–42%. Interestingly, the intact and digested proteins derived from the organically cultivated hempseed (OC) at a protein concentration of 1 mg/mL inhibited ~22% of the lipase activity. Differences between the OC and CC samples may be associated with compositional differences . Limited2SO4), boric acid, trifluoroacetic acid (TFA), Trizma® base, dimethyl sulfoxide (DMSO), 2-mercaptoethanol, methanol, protein markers (6.5–200 kDa), Sudan III, 2,4,6-trinitrobenzenesulfonic acid (TNBS), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), porcine pepsin (2500 U/mg), porcine pancreatic lipase (50 mU/mL), 4-methylumbelliferyl oleate (4-MUO) and Orlistat® were from Sigma-Aldrich . Hexane, MS-grade water and acetonitrile (ACN) were from Honeywell International Inc. . Comassie R, Laemmli buffer and Mini-Protean TGX 4–20% pre-cast polyacrylamide gels were from Bio-Rad Laboratories Inc. . Sodium dodecyl sulfate (SDS) was from National Diagnostics . Corolase PP® (4.4 U/mg) was from AB Enzymes GmbH . Cellulose acetate filters were from Millipore  and polytetrafluoroethylene syringe filters were from VWR .Sodium hydroxide (NaOH), hydrochloric acid (HCl), trichloroacetic acid (TCA) and acetic acid were from Fisher Scientific . Kjeldahl tablets, sulfuric acid ) was grown under license from the Irish Department of Health . The hemp was sown between 25 and 27 May 2019 and CC2 and CC1 were harvested between 11 and 12 October 2019, while the OC crop was harvested on 22 October 2019. The samples were cleaned by physical separation of the whole seeds from any other materials.Two conventionally grown hempseed (CC1 and CC2) and one organically grown hempseed (OC) samples were provided by Hemptech Ireland Ltd., Wicklow, Ireland. The hemp In addition, a commercial hempseed protein isolate  was purchased from a local health-food store.During moisture content analysis, the hempseeds were firstly ground to fine particles using a KG49 Delonghi coffee grinder . Empty sample pans were weighed (M1) and an aliquot (1.5 g) of each hempseed sample and the commercial HPI was then added to each sample pan and the total weight was recorded (M2). Samples were dried  in a hotbox oven with fan size 2  and weighed again once dried (M3). The moisture content was calculated using Equation (1):For ash content measurement, the oven-dried crucibles were cooled and weighed (W1) prior to adding 250 mg of each ground sample and the commercial HPI (n = 3). The total weight was obtained (W2). The samples were dried in a furnace  at 550 °C for 8.5 h and weighed again (W3) after cooling. The ash content of each powder was calculated using Equation (2):The lipid content of samples was determined according to AOAC (2000) . An aliqThe level of nitrogen in each sample was determined (n = 3) using a KjelDigester K-446  according to the Kjeldahl protein nitrogen determination method . The protein was calculated using a nitrogen-to-protein conversion factor of 5.32 .2O 1:20 (w/v)) and the sample was allowed to stand for 180 min in order to precipitate the proteinaceous compounds. The sample was then centrifuged at 4 °C and 3500× g for 15 min using a 320R Hettich centrifuge . The nitrogen in the supernatant and the pellet were separately analyzed as described above. The nitrogen content in the supernatant indicated the NPN content.The NPN was determined using the TCA precipitation method . Brieflyw/v) in dH2O. The HPI suspension was stirred for 10 min and the pH was then adjusted to pH 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 using HCl (0.5 M) or NaOH (0.5 M) followed by stirring for 30 min at room temperature. An aliquot of the suspension (7 mL) was centrifuged (3000× g at 4 °C for 15 min). The nitrogen content in the supernatant obtained was analyzed (n = 3) using the Kjeldahl procedure. The pH of the supernatant with the highest protein content  was subsequently selected for protein extraction from the hempseed samples.The nitrogen solubility of the commercial HPI at different pH values was firstly assessed in order to select the optimum pH for extraction of the proteins from the hempseeds. For this, the HPI sample was suspended 1:20 ) and stirred at room temperature for 10 min. The pH was then adjusted to that of maximum protein solubility (pH 12). Samples were stirred for 30 min followed by centrifugation  using a 320R Hettich centrifuge . The pH of the supernatant was then adjusted to pH 4, i.e., the pH corresponding to the isoelectric point of hempseed protein [g, 4 °C, 15 min) was resuspended in dH2O (1:10 (w/v)). The suspension was then dried using a B-290 mini spray dryer  at an inlet temperature of 150 °C and an outlet temperature of 85 °C. The protein content of each spray-dried powder sample was determined using the Kjeldahl method as outlined earlier.Protein extraction from the hempseed samples was carried out according to the pH selected from the previous step that indicated the maximum solubility. First, the hempseed samples were de-oiled using a stainless steel laboratory automatic oil press . The de-oiled samples were subsequently ground using a coffee grinder. As described previously, the ground samples were then suspended in dH protein  using 0.v/v) TFA and mobile phase B was 80% (v:v) ACN and 0.1% (v/v) TFA. The samples were reconstituted in dH2O to reach a concentration of 0.5% (w/v). The samples were filtered through 0.2 µm cellulose acetate filters. An aliquot of 10 μL was injected into the Acquity UPLC BEH C18, 130 Å column (2.1 mm × 50 mm × 1.7 mm) equipped with an Acquity BEH C18 (1.7 mm) vanguard precolumn. The flow rate was set on 0.3 mL/min over 51 min with the gradient program as previously detailed by Cermeño et al. [The protein extracted from the OC and CC hempseeds was analyzed using an Acquity UPLC system . Mobile phase A was 0.1% ) protein extract adjusted at pH 7.0 was pipetted into a Mastersizer 2000  equipped with a Hydro 2000S sample dispersion system (set at a stirring speed of 1000 rpm) interfaced with Mastersizer 2000 software . The volume weighted mean , the surface weighed mean , the absolute deviation from the median (uniformity) as well as the average particle size (d(0.5)), the sizes of particles below which 10% and 90% of the sample lie (d(0.1) and d(0.9), respectively) and the specific surface area (SSA) were recorded. The particle refractive index and the continuous phase refractive index used were 1.52 and 1.33, respectively. The beam length was 2.35 mm and the minimum detection limit was 0.02 µm. Measurement integration time was 12,000 ms. The analysis was performed in triplicate with three measurements during each run.An aliquot (1 mL) of reconstituted (5% (w/v) protein) were incubated in a water bath . The pH was adjusted to pH 2 using 1 M HCl. Following addition of pepsin (2.5% (w/v) protein), gastric digestion took place for 90 min before the sample was heated to 90 °C for 20 min, deactivating the enzyme. The pH of the sample was then adjusted to pH 7.5 using 1 M NaOH. Intestinal digestion occurred with the addition of Corolase PP (1% (w/v) protein) and incubation for 150 min at 37 °C before deactivation. The pH values were kept constant throughout each digestion phase using a pH stat . The samples containing digested protein were then freeze-dried . Therefore, three samples were obtained from each hempseed protein extract: a sample prior to digestion, a sample that had undergone simulated gastric digestion and a sample that had undergone simulated gastric and intestinal digestion. These were subsequently characterized for their degree of hydrolysis (DH%), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel-permeation high-performance liquid chromatography (GP-HPLC) profiles, in vitro antioxidant activity, and lipase-inhibitory activity.SGID was carried out on the hempseed protein samples according to Walsh et al., . The samw/v) protein were diluted in 1% (w/v) SDS to reach a concentration of 0.1% (w/v). Samples were heated for 30 min at 50 °C. The 5% (w/v) TNBS stock solution was diluted to 0.05% (w/v) using a 1:1 solution of dH2O and sodium phosphate buffer . An aliquot of 10 μL from each sample was then added to a 96-well microplate  along with 160 μL of TNBS in each well. The absorbance was measured at 50 °C every 5 min during 1 h at λ = 350 nm using a microplate reader . Leucine at various concentrations  SDS) was used as a standard.The DH was determined according to the method described by Nongonierma et al., , where sSDS-PAGE analysis was used to obtain the profiles of each hempseed protein sample. Samples (~4 mg/mL on a protein basis) were mixed 1:1 with a mixture of 2-mercaptoethanol and Laemmli buffer (1:19) in Eppendorf tubes. The tubes were shaken for 5 min at 200 rpm and incubated at 95 °C using a DX-100/DX-100R Dry Block Thermo-Shaker . An aliquot of 5 µL of a protein marker standard and 10 µL hempseed protein samples (~20 µg protein) was pipetted into each lane. The electrophoresis apparatus  was set at 150 V and 15–50 mA for 90 min.w/v) hempseed protein dissolved in mobile phase  were prepared for GP-HPLC analysis. Samples were then filtered into Eppendorf tubes using 0.2 μm polytetrafluoroethylene syringe filters and 200 μL of each sample was transferred into HPLC sample vials. Separation was performed by isocratic elution on a TSK G2000 SW separating column (600 mm length × 7.5 mm ID) fitted with a TSKGEL SW guard column  over 51 min. The absorbance of the eluent was monitored at 214 nm and separation was carried out at a flow rate of 1 mL/min.Protein suspensions containing 0.25%  of this was added to 10 μL of samples. PBS was used as a blank. All samples including the blank were applied to a microplate and the absorbance recorded over 6 min, with shaking at 30 °C using a BioTek Synergy HT microplate reader. The scavenging by the samples of the ABTS● was calculated according to Re et al. [Hempseed proteins and their digested samples (21 mg on a protein basis) were diluted to 1 mL with phosphate-buffered saline  and centrifuged at 3500× e et al. .2. Porcine pancreatic lipase was dissolved in assay buffer (1 mg/mL). The substrate was a solution of 4-methylumbelliferyl oleate (4-MUO) dissolved in DMSO to reach a concentration of 1 mM. Samples (1 mg/mL on a protein basis) were dissolved in DMSO. The assay was performed by addition of preheated (30 °C) lipase  to 50 µL of substrate, 25 µL test sample and 100 µL assay buffer in a microplate followed by shake-incubation at 30 °C for 30 min. Afterwards, the fluorescence was measured at excitation and emission wavelengths of 360 and 460 nm, respectively, using a BioTek Synergy HT microplate reader. The positive control was Orlistat® which was prepared similarly to the test samples. The percentage of lipase inhibition of samples at a concentration of 1 mg/mL (on a protein basis) was reported.The lipase-inhibitory activity assay was performed according to Amigo-Benavent et al. . The ass® Release 15 for Windows, and p < 0.05 was considered statistically significant.Data values are presented as the mean of three measurements ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by the Tukey post hoc comparison test was carried out to test for significant differences using Minitabp < 0.05) in vitro antioxidant and lipase inhibitory activity than CC samples. This study showed that the cultivation conditions had a considerable impact on the physicochemical and biofunctional properties of hempseed proteins. It should be mentioned that these are preliminary results from a limited number of samples and further studies would be required to better understand the impact of cultivation on the nutritional quality of hempseed proteins. More investigations on the possible applications of organically cultivated hempseed proteins as a sustainable alternative to animal-origin protein in food products are warranted.This study focused on the extraction and characterization of the proteins from two conventionally (CC1 and CC2) hempseed samples and one organically cultivated (OC) hempseed sample. The hempseeds contained 20–25% protein that consisted of edestin (65–75%) along with albumin molecules. Minor variations were observed in the protein profiles of the samples. The OC and CC1 hempseeds had the lowest NPN. The particles in suspension of the OC protein extract were more uniform and had a lower mean diameter than the CC samples. Subjection of the protein extracts to SGID resulted in differences in molecular mass distribution of the digested samples, with the OC samples showing lower MWs compared to CC protein extracts. In addition, the digested OC protein extracts had higher (


Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational mechanisms. We have recently shown that the small, noncoding vault RNA1–1 negatively riboregulates p62 oligomerization in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1–1 inhibits p62 oligomerization are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1–122) are necessary and sufficient for specific vault RNA1–1 binding, and we identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1–1 further reveals a central flexible loop within vault RNA1–1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein–protein interactions critical to the activation of specific autophagy. Macroautophagy ensures the clearance of intracellular substrates ranging from single ubiquitinated proteins to large proteotoxic aggregates and defective organelles. The human autophagy receptor p62  guides selective macroautophagy of intracellular cargo to maintain homeostasis in situations of proteotoxic stress or starvation . FollowiIn this process, p62 recognizes ubiquitinated cargo via its carboxy-terminal ubiquitin-associated (UBA) domain  and N-arSince the affinity of p62 monomers for specific cargo and the ATG8-like proteins is rather modest, protein oligomerization is essential for achieving high avidity protein interactions while maintaining selectivity . OligomeSupplemental Fig. 3; We recently discovered that the small noncoding vault RNA1–1 directly binds to p62 and regulates p62 oligomerization and hence function in autophagy . Vault RTo identify the region of p62 that is required for RNA binding in a systematic manner, we generated a series of FLAG-HA tagged p62 truncation constructs A, and as1–122; aa 1–122) significantly increases normalized RNA binding above the level of wild-type p62, suggesting a negative modulatory function of the ZZ domain on RNA binding. However, the PB1 domain alone (aa 1–102) or the ZZ domain with the linker (aa 102–172) display little if any RNA-binding capacity  may play a regulatory role  protein C,D. Addicapacity C,D. Thes1–122 displays specific and maximal vault RNA1–1 binding compared to full-length p62 . In contrast, p62 association with the other vault RNA paralogs is not significantly changed. These findings reflect the prime role of vault RNA1–1 in p62 riboregulation as previously observed  conditions, we performed native immunoprecipitation followed by RT-qPCR of copurified RNA (RIP). In this assay, p62observed  and hint1–122—the PB1 oligomerization domain with its adjacent carboxy-terminal linker—accounts for specific vault RNA1–1 binding to p62. This result is unexpected in light of the previously identified ZZ domain mutant R139A/K141A, which showed reduced RNA binding and implicated the ZZ domain as a critical region for the RNA interaction . Yet, their RNA-binding capacity is not significantly changed . In addition to the in cellulo experiments, we used maltose-binding protein (MBP)-tagging to generate recombinant p62 with low oligomerization potential .We next generated FLAG-HA tagged mutant constructs of p62 to (i) assess, whether RNA binding depends on oligomerization, and (ii) to narrow down the RNA-binding interface in the context of full-length p62. To prohibit PB1-dependent oligomerization, we mutated the negatively charged OPCA motif A introduotential  and test1H, 15N-HSQC NMR spectroscopy with the minimal construct p621–122 which we forced into a monomeric form by the before-characterized triple mutation (D69A/D71A/D73A). We recorded spectra with and without equimolar amounts of in vitro transcribed full-length vault RNA1–1 to assess binding , confirming the direct involvement of these residues in RNA binding. In contrast, the RNA-binding capacity of the linker mutant MBP-p62 R106A/R107A . These results favor the interpretation that mutations targeting the linker and ZZ domain foster multimeric forms of p62 in cellulo that exclude RNA from binding to the autophagic receptor.In contrast, R22 and R96 appear to extend the RNA-binding interface without making a strong contribution to multimerization in the absence of RNA binding. Mutations in the linker region or ZZ domain of p62 both resulted in reduced RNA binding and increased presence of multimers D,E. To dSupplemental Fig. 4; The tertiary structure of vault RNA1–1 is presently unknown. Previous secondary structure analyses using RNase H probing were useful but limited in resolution . We applSupplemental Fig. 3). Overall, our data implicate nucleotides within the central loop of vault RNA1–1 as specific p62 contact points.Next, we conducted RNase A footprinting to identify vault RNA1–1 residues that are protected by p62 binding . When coSupplemental Fig. 5). We compared reintegrated wild-type vault RNA1–1 with two mutants that alter either the central loop triplet U36/U37/U38 alone (M1) or in combination with residues A46/C47 . As expected, the central domain mutants M1 and M2 tend to show reduced p62 binding in cellulo compared to the wild-type vault RNA1–1 . This conclusion is further supported in vitro by EMSAs, where the mutations in the central loop of vault RNA1–1 compete less well for p62 binding than the wt counterpart . This assay also shows that the central loop region of vault RNA1–1 alone is not sufficient for p62 binding in vitro  and reveals the importance of the 3D structure of vault RNA1–1 for p62 binding.To validate the functional importance of the central flexible loop region of vault RNA1–1 for riboregulation of p62, we generated a HuH-7 Flp-IN vault RNA1–1 KO cell line with a single FRT integration site. This KO cell line thus allowed the stable and isogenic reintegration of vault RNA1–1 variants expressed from the endogenous promoter  are both necessary and sufficient for maximal and specific vault RNA1–1 binding. Mutational analyses pinpoint to K7 and R21 as critical amino acids that are necessary for RNA binding  was shown to exert negative auto-regulation through direct interaction with the ZZ domain  that showed strongly diminished RNA binding and increased p62 oligomerization D,E. InteZ domain . In lighZ domain , we hypoZ domain . The excZ domain .Supplemental Fig. 6B; Supplemental Fig. 6B).As proposed before, our new data further corroborate that vault RNA1–1 primarily inhibits p62 oligomerization. Our new data also suggest that vault RNA1–1 may not be able to disrupt p62 oligomers that are formed when cargo binds to the ZZ domain. Integrating all available experimental evidence, we now suggest a refined mechanistic model for riboregulation of p62 by vault RNA1–1: under physiological, nutrient-replete conditions, vault RNA1–1 inhibits p62 oligomerization by binding the critical hinge points K7 and R21. When starvation reduces cellular vault RNA1–1 levels, p62 oligomerization via K7 and R21 is facilitated, stimulating autophagic flux  of vault RNA1–1, we identified nucleotides required for p62 binding , 4. We iThe p62/vault RNA1–1 binding interface may well be targeted by regulatory modifications that potentially influence riboregulation. For example, K7 has previously been found to be ubiquitinated by TRIM21 . Like vaOur data also demonstrate that autophagic clearance can be modulated by experimental changes of intracellular vault RNA1–1 levels . ModulatIn addition to its function as an autophagy receptor, p62 provides a platform for key cellular signaling pathways . While wWith hundreds of newly identified RNA-binding proteins , riboregThis work provides a structure-function analysis to identify critical residues of p62 and vault RNA1–1, respectively, for the specific interaction between the two partners and their function in the riboregulation of p62 in mammalian autophagy. As such, it answers what distinguishes vault RNA1–1 from the other three human vtRNAs, and how vault RNA1–1 achieves control of p62 oligomerization via K7 and R21. In the future, it will be important to examine this interaction at atomic resolution using state of the art structural technologies.Since our experiments have been conducted with cultured human cells, it will also be interesting to examine in vivo models  expressing the mutant RNAs and forms of p62, respectively, which we have defined in this work. The investigation of animal models and clinical samples in the context of viral infections, cancer or neurodegenerative disorders will advance our understanding of the functional importance of p62 riboregulation in organismal function and disease.Supplemental Table 1.Antibodies and reagents used in this study are listed in l-glutamine , and 100 U/mL penicillin/streptavidin  at 37°C and 5% CO2. The cells were routinely passaged 2–3 times per week. For this purpose, the cells were dissociated through the addition of Trypsin-EDTA . HuH-7 Flp-In cell line stocks and their derivatives were cultured in medium containing zeocin  or Hygromycin B Gold  depending on expressed marker genes. The parental hepatocellular carcinoma HuH-7 Flp-In cell line  was first established in HuH-7 cells (derived from a hepatocellular carcinoma of a human aged 57 yr) and their derivatives were cultured in DMEM containing 1 g/L glucose supplemented with 10% heat-inactivated FCS , 2 mM Restriction-free cloning was performed according to Supplemental Table 2; http://crispor.tefor.net; Version May 2017), the highest-scoring candidates ordered from Sigma-Aldrich, annealed, and ligated into BbsI linearized pSpCas9(BB)-2A-GFP/RFP/Cer constructs . Combinations of the generated plasmids were nucleofected into HuH-7 Flp-In cells using the SF Cell Line 4D-Nucleofector X Kit according to the manufacturer's guidelines, with 1Mio cells and 1 µg of total plasmid DNA in a 100 µL setup running program FF137. As a negative control, a mixture of all parental plasmids was used. A fluorescence-activated single-cell sort of double/triple-positive cells was performed 48 h after nucleofection. Upon expansion, the clones were tested for vault RNA1–1 deletion by PCR from genomic DNA using locus spanning primers . Cell lines lost the transiently transfected plasmids after outgrowth as checked via FACS.CRISPR/Cas9 genome editing was based on Transfections were performed using Lipofectamine 3000 according to the manufacturer's guidelines. For cell-based assays, XIE62-1004-A , the protein concentration determined by Bradford assay and the input material adjusted accordingly (1–2 mg/IP). Per IP, 25 µL anti-HA magnetic bead slurry was washed twice with PBS prior to addition to the samples. The IPs were performed for 1–2 h at 4°C with constant rotation. Following, the samples were washed six times with 1 mL of Trit-Lysis buffer and the reaction tubes exchanged after every second wash. For pH elution, 50 µL of 0.1 M glycine pH 2 were added per condition and incubated for 5 min at room temperature. The eluates were transferred into a new reaction tube and neutralized through addition of 7.5 µL of 1 M Tris-HCl pH 8.5.For IPs, one confluent ∅ 15 cm dish per IP served as starting material. The cells were washed twice with ice-cold PBS. The buffer was aspirated completely and the cells lysed on ice through the addition of 750 µL Trit-Lysis buffer  supplemented with cOmplete Protease Inhibitor Tablets  and 5 µg/mL RNasin . The cells were collected through scraping and homogenized by pipetting. Lysates were cleared by centrifugation for 10 min at 13,0002O.For protein analysis, 1% of input material and 15%–20% of elution were analyzed by western blotting. For RNA analysis, 5% of input material and 70% of elution were processed by the addition of 400 µL RNA lysis buffer (Zymo) and RNA extraction using the Zymo Quick-RNA MicroPrep RNA Extraction Kit (Zymo), following the manufacturer's guidelines, including the DNase I on-column DNA digest. Elution was performed with 15 µL RNase-free H2. Subsequently, the cells were lysed on ice with 0.75–1 mL PNK lysis buffer , and transferred into 1.5 mL reaction tubes. The lysates were homogenized by sonication on ice  and cleared by centrifugation at 16,000g for 10 min, 4°C. The protein concentration was measured by Bradford assay and the input material adjusted accordingly (1–2 mg/condition). Following, the lysates were treated with 5 ng/µL RNase A and 2 U/mL Turbo DNase for 15 min at 37°C and 1100 rpm. Per condition, 25 µL anti-HA magnetic bead slurry was washed twice with PBS prior to addition to the samples. The IPs incubated for 1.5–2 h at 4°C under constant rotation. Subsequently, the IPs were washed three times with PNK lysis buffer and three times with PNK wash buffer . Radioactive labeling of retained RNA was performed on beads in PNK wash buffer containing 0.1 µCi/µL [γ-32P] ATP, 1 U/µL T4 PNK, and 1 mM DTT for 15 min at 37°C and 850 rpm. After another four washes with PNK wash buffer, the proteins were eluted by addition of 50 µL 0.1 M glycin pH 2 for 5 min and eluates neutralized with 7.5 µL 1 M Tris-HCl pH 8.5. The samples were complemented with 4× sample buffer containing 200 mM DTT, heated to 70°C for 3 min, resolved by SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were rinsed, dried, and a radioactive signal was detected with a phosphorimaging screen for 3–72 h. Following, the membrane was used for western blot analysis. Image analysis was performed with Fiji Image J 2.0.0 (https://imagej.net/Fiji). The relative PNK signal was calculated and normalized to the relative western blot signal to account for variation in IP efficiency.For the PNKs, one 80%–90% confluent ∅10 cm or ∅15 cm dish per condition served as starting material. The cells were washed twice with ice-cold PBS, the PBS was aspirated completely, and the cells were UV-crosslinked at 150 mJ/cmFor standard protein analysis, cells were washed twice with ice-cold PBS, the buffer aspirated completely and cells lysed through the addition of RIPA lysis buffer . Following, the cells were collected by scraping and protein concentration measured by Bradford assay. The lysates were mixed with 4× NuPage LDS Sample buffer supplemented with 200 mM DTT before denaturation at 70°C for 3 min. Typically, 5–15 µg total protein were resolved by SDS-PAGE on 4%–15% TGX Precast gels using 1× Laemmli running buffer . The proteins were transferred onto PVDF or nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad) and transfer efficiency assessed by Ponceau Red staining. The membranes were blocked in PBS-T 5% milk  for 1 h at room temperature. Primary antibodies were diluted in PBS-T 5% milk and added to the membrane overnight at 4°C under constant shaking. After three PBS-T  washes, each for 5 min, the membrane was incubated with HRP-conjugated secondary antibody in PBS-T 5% milk for 1 h at room temperature. Following three PBS-T washes, the western blots were developed using ECL on a Bio-Rad ChemiDoc MP Imaging System with auto-capture function.+ membrane with 0.8 mA/cm2 for 2.5 h in 0.5× TBE. Following, RNA and membrane were crosslinked by UV exposure at 150 mJ/cm2.Typically, 15–20 µg of total RNA were mixed with 2× RNA gel loading dye , denatured for 5 min at 95°C, loaded onto a denaturing 8% polyacrylamide gel , and separated for 1–2 h at 350 V in 0.5× TBE. A semidry blotting apparatus was used to transfer the RNA onto a Hybond N32P-labeled DNA antisense oligonucleotide probe for overnight incubation at 50°C . The DNA probe was labeled with PNK and purified over a Chroma Spin chromatography column  according to the manufacturer's guidelines. Finally, the membrane was washed three times with a high stringency buffer  and three times with a low stringency buffer , before visualizing the radioactive signal by 4–16 h exposure to a phosphorimaging screen.The membrane was prehybridized  for 1 h at 50°C before the addition of a radioactively Protein expression was performed according to 15NH4Cl as the sole nitrogen source at 22°C O/N upon induction with 0.5 mM IPTG. The cells were lysed in a buffer containing 50 mM Tris pH 8, 750 mM NaCl, 10% glycerol, 20 mM imidazole, and cOmplete, EDTA-free Protease Inhibitor Cocktail using a microfluidizer . Subsequently, the protein was purified from the cleared lysate by Nickel affinity chromatography  in the same buffer. The affinity tag was removed by 3C protease cleavage , dialysis, and an additional passage over the HisTrap column. As a final purification step, oligomers were removed by gel filtration on a Superdex S75 10/300 in a buffer containing 20 mM MES pH 6.5, 100 mM NaCl, 0.2 mM TCEP, and 0.05% NaN3.The oligomerization deficient p62 truncation p62 was cloned into pCoofy4 (Ref: PMID: 23410102) using restriction-free cloning , transfo3 were titrated with full-length vault RNA1–1 in the same buffer. At each titration step, an apodization-weighted sampled HSQC was collected  according to the manufacturer's guidelines. RNA was gel purified and phenol-chloroform extracted, dissolved in water and its concentration measured by QuBit assay (Thermo).For biochemical assays: pUC57-T7-vaultRNA1–1 plasmids were linearized and used for in vitro transcription of RNA using the MEGAshortscript Kit  with 2, 2 mM Spermidine-HCl, 40 mM DTT, 0.1 mg/mL BSA, 7.5 mM each NTP, 800 units/mL RNasin, 10 units/mL IPP, 25 µg/mL template DNA, and 10,000 units/mL T7 RNA polymerase and incubated 8 h at 37°C. The RNA was gel purified, extracted by electrophoresis, precipitated, and its concentration measured by NanoDrop.For NMR: pUC57-T7-vaultRNA1–1 plasmids were linearized and used for in vitro transcription of RNA in a large-scale reaction containing 100 mM HEPES-KOH pH 7.5, 10 mM MgCl2. The EMSA reactions typically contained 10 nM–2 mM protein, 10 nM radioactively labeled RNA , 150 nM nonlabeled bacterial tRNA, 1 mg/mL of BSA, 10 mg/mL RNasin, 5 mM DTT, 0.5 mM PMSF, 2.5 mM MgCl2, 100 mM KCl; 20 mM HEPES pH7.9; 0.2 mM EDTA, and 20% glycerol. In the case of competitive EMSAs, the binding reaction was competed with nonlabeled RNA at a typical range of 0.1 to 2 µM that was added to the protein together with the labeled probe. Reactions were incubated for 10 min at room temperature, loaded onto a native 5% acrylamide gel, and run overnight at 70 V in 0.5× TBE. Subsequently, the gel was dried for 1 h at 80°C and signal visualized by exposure to a phosphorimaging screen.Before the reaction, RNA was denatured for 2 min at 95°C and subsequently refolded in the presence of 2.5 mM MgCl32P-γUTP (Hartmann) and purified over a Chroma Spin chromatography column. Prior to RNase footprinting, the RNA was denatured for 10 min at 65°C and allowed to refold by ramping down 1°C/30 sec to room temperature. Per condition, 8 µL containing 20 µM 3′-end 32P-labeled RNA (∼1600 cps) and 6 µM protein were assembled in binding buffer , and incubated for 10 min at room temperature. RNase A (5 ng/µL) was prediluted  in H2O and 2 µL dilution added per reaction. The digest was incubated for 15 min at room temperature. Following, the RNA was extracted by Phenol-Chloroform extraction using TRI Reagent and dissolved in 2× RNA gel loading dye. For the alkaline ladder, 2 µL containing 20 µM 3′-end 32P-labeled RNA (∼400 cps) were mixed with 8 µL 50 mM NaHCO3 pH 9.2, incubated at 95°C for 5 min and supplemented with RNA gel loading dye. The samples and ladder were loaded onto a denaturing 8% polyacrylamide gel  and separated at 42 W (50°C–55°C) in 0.5× TBE. The gel was fixed for 10 min in a bath of 6% acetic acid and 10% EtOH, dried on Whatman filter paper in a vacuum gel dryer at 80°C for 40 min, and the radioactive signal visualized by exposure to a phosphorimaging screen.In vitro transcribed RNA was dephosphorylated with Fast Alkaline Phosphatase (FastAP) according to the manufacturer's guidelines and purified over a Zymo Quick-RNA Miniprep column. Subsequently, the RNA was radioactively labeled in a T4 PNK reaction with 2O, denatured at 95°C for 2 min and directly transferred on ice. The RNA was allowed to fold for 20 min at 37°C upon addition of 12 µL 2× folding buffer . Subsequently, 12 µL protein (15 µM) in 1× folding buffer was added to the reaction and incubated for 10 min at 37°C before splitting the reaction into 4 × 8 µL each. For the modification reactions, 92 µL Mod. buffer A for DMS  or Mod. buffer B for CMCT  were added. For DMS treatments 1 µL of 16×, 4×, or 2× DMS diluted in EtOH was added to the reactions, immediately mixed and incubated for 15 min at 37°C. For CMCT treatments, 5, 10, or 15 µL of CMCT solution (42 mg/mL in H2O) were added to the reactions, immediately mixed and incubated for 15 min at 37°C. The reactions were stopped on ice, RNA extracted with TRI reagent according to the manufacturer's guidelines and EtOH precipitated. Modified residues were detected by primer extension reaction using a radiolabeled DNA oligonucleotide (T4 PNK reaction and purification over a Chroma Spin 10 chromatography column) complementary to the 3′-end of the RNA. For primer extension, 1 µL of 10 µM radiolabeled primer was annealed to 2 µL of extracted RNA and used in a 5 µL reaction with AMV reverse transcriptase according to the manufacturer's guidelines. For the sequencing lanes, in vitro transcribed RNA was used in primer extension reactions that contained respective ddNTPs. The reactions were stopped with 20 µL Stop buffer  and the cDNA EtOH-precipitated and separated on a denaturing 8% polyacrylamide gel  at 42W in 0.5× TBE. Subsequently, the gel was fixed for 10 min in a bath of 6% acetic acid and 10% EtOH, dried on Whatman filter paper in a vacuum gel dryer at 80°C for 40 min, and the radioactive signal visualized through exposure to a phosphorimaging screen.Per compound, four probing reactions were performed with one untreated control and three treated samples of varying concentrations. Therefore, 20 pmol in vitro transcribed RNA were diluted in 12 µL HSupplemental Table 2. The analysis was performed on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR system in the default standard two-step cycling run. Relative expressions were calculated with the ΔΔCT method. The mean Ct value was used when normalizing to multiple housekeeping genes.Reverse transcription was performed with 500 ng of total RNA or 7 µL of purified RNA from IPs in a 10 µL reaction using the Maxima First Strand Synthesis kit  according to the manufacturer's guidelines. Control reactions that did not include the reverse transcriptase (-RT) were included for each sample. The obtained cDNA was diluted 1:20–1:50 in nuclease-free water and assembled in 10 µL qPCR reactions with 5 µL cDNA, 4.6 µL Fast SB SYBR Green Master Mix, 0.2 µL each primer (10 µM). Primer sequences can be found in www.graphpad.com). Details for each experiment can be found in the corresponding figure legend. Image analysis was performed with Fiji Image J 2.0.0 (https://imagej.net/Fiji). Structure representations were generated using PyMOL Molecular Graphics System 2.0.7 (https://pymol.org). NMR analysis was performed with NMRPipe (https://www.ibbr.umd.edu/nmrpipe/) and NMRFAM SPARKY (https://nmrfam.wisc.edu/nmrfam-sparky-distribution/).Statistical analysis was performed with GraphPad Prism 8.3.1 (279; Supplemental material is available for this article.


Defective autophagy and lipotoxicity are the hallmarks of nonalcoholic fatty liver disease. However, the precise molecular mechanism for the defective autophagy in lipotoxic conditions is not fully known. In the current study, we elucidated that activation of the mammalian target of rapamycin complex 1 (mTORC1)–G9a–H3K9me2 axis in fatty acid–induced lipotoxicity blocks autophagy by repressing key autophagy genes. The fatty acid–treated cells show mTORC1 activation, increased histone methyltransferase G9a levels, and suppressed autophagy as indicated by increased accumulation of the key autophagic cargo SQSTM1/p62 and decreased levels of autophagy-related proteins LC3II, Beclin1, and Atg7. Our chromatin immunoprecipitation analysis showed that decrease in autophagy was associated with increased levels of the G9a-mediated repressive H3K9me2 mark and decreased RNA polymerase II occupancy at the promoter regions of Beclin1 and Atg7 in fatty acid–treated cells. Inhibition of mTORC1 in fatty acid–treated cells decreased G9a-mediated H3K9me2 occupancy and increased polymerase II occupancy at Beclin1 and Atg7 promoters. Furthermore, mTORC1 inhibition increased the expression of Beclin1 and Atg7 in fatty acid–treated cells and decreased the accumulation of SQSTM1/p62. Interestingly, the pharmacological inhibition of G9a alone in fatty acid–treated cells decreased the H3K9me2 mark at Atg7 and Beclin1 promoters and restored the expression of Atg7 and Beclin1. Taken together, our findings have identified the mTORC1–G9a–H3K9me2 axis as a negative regulator of the autophagy pathway in hepatocellular lipotoxicity and suggest that the G9a-mediated epigenetic repression is mechanistically a key step during the repression of autophagy in lipotoxic conditions. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved lysosomal degradative pathway important for survival, differentiation, development, and maintaining cellular homeostasis , 2, 3. INonalcoholic fatty liver disease (NAFLD), also called as metabolic-associated fatty liver disease, is a common disorder and refers to a group of conditions ranging from simple fatty liver to steatohepatitis (fatty liver plus inflammation), fibrosis, cirrhosis, and hepatocellular carcinoma , 16, 17.In the present study, we report that mTORC1–G9a–H3K9me2 axis negatively regulates autophagy in lipotoxic conditions. Furthermore, we identified histone methyltransferase G9a as a key epigenetic regulator of autophagy and suggested that G9a could act as a potential therapeutic target in NAFLD.A). We also evaluated the cytotoxicity induced by free fatty acids in immortalized mouse hepatocyte AML 12 cells. AML 12 cells showed relatively more sensitivity as compared with HepG2 cells with 18 to 25% cytotoxicity at 0.25 mM palmitate and 35 to 40% at 0.5 mM . In order to understand the mechanism of lipotoxicity, we decided to proceed with 0.5 mM palmitate only. Next, we treated the HepG2 cells with palmitate and measured the mRNA levels of three important lipogenic genes: sterol regulatory element–binding protein 1c, fatty acid synthase, and stearoyl CoA desaturase 1. All the genes show increased expression in palmitate-treated cells as compared with untreated control cells . We also measured the triglyceride levels, and as expected, triglyceride levels were also increased in palmitate-treated cells . Increased lipogenesis and defective autophagy are the hallmarks of lipotoxicity in NAFLD. To look at the status of autophagy in palmitate-treated cells, we observed decreased LC3II/β-actin ratio (D and G) and increased levels of SQSTM1/p62 (D and H), a protein known to be degraded exclusively by autophagy. We also measured the protein and mRNA levels of autophagy-related genes (ARGs) in palmitate-treated cells. The mRNA as well as the protein levels of Beclin1, mammalian ortholog of Atg6 which play a central role in autophagy, and Atg7, an enzyme for the ubiquitin-like autophagy proteins, were significantly decreased in palmitate-treated cells . The immortalized mouse hepatocyte AML 12 cells were also treated with 0.5 mM palmitate for 24 h. Protein levels of Atg7 and Beclin1 were significantly decreased, while as the autophagic cargo SQSTM1/p62 was increased in palmitate-treated cells as compared with untreated control cells (J–M). Taken together, increased lipogenesis and lipotoxicity validated our hepatocellular model of lipotoxicity, while as the decreased levels of LC3II, Beclin1, and Atg7, and the accumulation of SQSTM1/p62 in palmitate-treated cells suggests the impairment of autophagic machinery in lipotoxic conditions.Previous studies have reported that long-term exposure to free fatty acids increases lipogenesis and induces lipotoxicity in various cellular models of lipotoxicity . We testFig. 1in vivo and for translocation of the mTORC1 to the lysosome, an important step required for mTOR activation (A and B). Both mTORC1 activation and histone methylation have been shown to put brakes on autophagy; therefore, we tested whether there is any association between mTORC1 activation and expression of histone methyltransferase G9a, an enzyme that methylates histone H3 at lysine 9, a mark associated with gene repression. Interestingly, the activation of mTORC1–P70S6 kinase axis in palmitate-treated HepG2 cells was associated with significant increase in the expression of histone methyltransferase G9a (A and C). As mentioned previously, both mRNA and protein levels of Beclin1 and Atg7 were decreased in palmitate-treated cells (D and E). AML 12 cells also showed increase in H3K9me2 occupancy at the promoter regions of Atg7 and Beclin1 in palmitate-treated as compared with untreated control cells (K and L). Collectively, these results suggest a possible connection between mTORC1 activation and G9a-mediated repressive H3K9me2 mark in lipotoxicity.mTORC1 is considered as the major gateway to autophagy. Moreover, SQSTM1/p62–mTORC1–autophagy connection has been well established, and it has been consistently reported that SQSTM1/p62 accumulates in the cells with hyperactive mTORC1 , 27. Furtivation . Therefotivation , A and Brase G9a , A and Ced cells ; therefoed cells , D and Eol cells , K and LA–C). Activation of AMPK by metformin in lipotoxic cells increased the phosphorylation of AMPK-Thr172 and blocked the palmitate-induced activation of mTORC1–P70S6K axis . ChIP experiments using H3K9me2 antibody show decreased occupancy of G9a-mediated repressive H3K9me2 mark at the promoter regions of Beclin1 and Atg7 in palmitate- + rapamycin-treated cells as compared with palmitate-treated cells (E and F), thereby suggesting that increased G9a-mediated repressive H3K9me2 mark at the promoter regions of Beclin1 and Atg7 in palmitate-treated cells could be the result of mTORC1-mediated increased G9a expression and that targeting of this mTORC1–G9a axis could restore the expression of Atg7 and Beclin1 and hence autophagic machinery. Furthermore, RNA pol II occupancy, a readout of active transcription, increased at the promoter regions of Beclin1 and Atg7 in palmitate- + rapamycin-treated cells as compared with palmitate-treated cells only (G and H), which again testify the importance of mTORC1–G9a axis in the regulation of autophagy in lipotoxic cells.We used rapamycin (100 nM) to inhibit the mTORC1 hyperactivity in palmitate-treated cells. Rapamycin effectively downregulated the mTORC1 activity in palmitate-treated cells as evident by the decreased phosphorylation of its downstream targets: P70S6 kinase and 4E-BP1. This inhibition of mTORC1 activity was associated with decreased G9a protein levels in palmitate-treated cells , A–D. Ched cells , E and Flls only , G and HA–D). We also checked the mRNA level of Atg7 and Beclin1 using quantitative RT–PCR (qRT–PCR) and observed that mTOR inhibition in palmitate-treated cells restored the mRNA levels of Beclin1 and Atg7 (E and F). In order to understand that the aforeobserved phenomenon is not HepG2 specific, we tested whether inhibition of mTORC1 will lead to restoration of Atg7 and Beclin1 in immortalized mouse hepatocytes and murine lipotoxic cell model AML 12 cells. The results are consistent with HepG2 data. Our results with AML 12 cells indicate that mTORC1 activation upon palmitate treatment downregulated the expression of autophagy genes Atg7 and Beclin1 and increased the accumulation of autophagic cargo SQSTM1/p62, thereby suggesting the autophagic blockade in lipotoxic AML 12 cells. Inhibition of mTORC1 in lipotoxic mouse hepatocytes restored the levels of Atg7 and Beclin1, which was further associated with decreased accumulation of SQSTM1/p62 (G–K).Since inhibition of mTORC1 activity in palmitate-treated cells resulted in the decreased expression of histone methyltransferase G9a and G9a-mediated repressive H3K9me2 mark at Atg7 and Beclin1 promoters, we tested whether downregulation of G9a is associated with restoration of autophagy in palmitate-treated HepG2 cells. Western blot results showed that mTORC1 inhibition in palmitate-treated cells restored the protein levels of Beclin1 and Atg7, which was further associated with decreased levels of SQSTM1/p62, an autophagic cargo, thereby suggesting restoration of autophagy by mTORC1 inhibition in palmitate-treated cells , A–D. Weand Atg7 , E and FSTM1/p62 , G–K.FigA–E). Furthermore, inhibition of G9a decreased the repressive H3K9me2 mark at Atg7 and Beclin1 promoter regions (F and G). To test whether this mechanism is conserved, we used murine lipotoxic cellular model AML 12 cells. Our results show increased G9a-mediated H3K9me2 marks at promoter regions of Atg7 and Beclin1 in palmitate-treated cells. Inhibition of G9a using G9a-specific inhibitor UNC-0642 (10 μM) in lipotoxic AML 12 cells restored the expression of Atg7 and Beclin1 protein levels (H–J) and reduced H3K9me2 levels at the promoter regions of Atg7 and Beclin1 (K and L). Taken together, these results suggest that mTORC1–G9a–H3K9me2 axis negatively regulates autophagic process and that targeting this axis either by inhibiting mTORC1 or G9a restores the expression of genes involved in autophagosome formation and the process of autophagy  increased the LC3II/β-actin ratio and restored the expression levels of Atg7 and Beclin1 to that of control cells , A–E. Fu regions , F and Gn levels , H–J and Beclin1 , K and Lutophagy .Figure 7The adaptive and pathological changes occur at several levels within the cellular system when exposed chronically to free fatty acids. These changes serve to fine tune the complex cellular machinery controlling the cellular metabolism to a new fate. The evolution of these changes and their crosstalk is the determining factor in response to the changes. To understand the nature of these changes, we studied human hepatoma HepG2 cells and mouse hepatocyte AML 12 cells under lipotoxic conditions. Analysis of functional parameters, gene expression, and epigenetic alterations were combined to provide a comprehensive insight into the lipotoxic situation in hepatic cells. The analysis revealed mTORC1–G9a–H3K9me2 axis as key regulatory pathway of autophagy in lipotoxic conditions.A–C). The increased lipogenesis and lipid accumulation was associated with downregulation of autophagy as suggested by decreased LC3II/actin ratio in lipotoxic cells as compared with untreated control cells (D and G). The decrease in LC3II levels suggests a defect in autophagosome formation in lipotoxic cells. This was also accompanied by increased accumulation of autophagic cargo p62/SQSTM1 (D and H), which reflects the decrease in autophagic flux or execution. p62/SQSTM1 is a multifunctional adapter protein involved in various cell signaling pathways with significant implications in the development of oxidative stress–mediated cellular damage and tumorigenesis , whose deficiency leads to impaired autophagy, accumulation of p62/SQSTM1, and development of multiple liver tumors (−/−) dies in early embryonic development, which is even more severe than other autophagy-gene–deficient mice , thus providing further evidence that autophagy is impaired in palmitate-treated cells.Lipotoxicity is the part of metabolic syndrome that results from the accumulation of lipids and their intermediates in nonadipose tissue, such as liver, kidney, heart, and skeletal muscle leading to cellular dysfunction and cell death . Lipotoxol cells , D and G2/SQSTM1 , D and Higenesis . Our obsr tumors . Beclin1r tumors . The nucent mice . We testOne of the processes that has been consistently linked with suppressed autophagy is mTORC1 activation , 42. mTOvia unknown mechanism (E and F). Furthermore, inhibition of the mTORC1 in palmitate-treated cells increased the RNA polymerase II levels at the promoter regions of Atg7 and Beclin1 (G and H). We also checked the relative gene expression of Atg7 and Beclin1 in different experimental groups, and our results indicate increased mRNA and protein levels of Atg7 and Beclin1 in palmitate + rapamycin-treated cells as compared with palmitate-treated cells , 15 mM Hepes, 200 U/ml penicillin, 270 μg/ml streptomycin, 1% (v/v) nonessential amino acids, 10% (v/v) obtained from Sigma–Aldrich, and 10% (v/v) fetal bovine serum, obtained from Gibco at 37 °C in a humidified atmosphere of 5% CO2. AML 12 cells were grown in Dulbecco's modified Eagle's medium F-12 containing 200U/ml penicillin, 270 μg/ml streptomycin, 1% (v/v), 10% (v/v) fetal bovine serum, obtained from Gibco and supplemented with Insulin Transferrin Selenium (ITS) obtained from Sigma Aldrich, St Louis, MO. For all experiments cells were used at density of 7 × 105 cells/6 well plate or 1.2 × 106 cells/60 mm dish, unless otherwise mentioned. All the studies were conducted using 70 to 80% confluent cells, which were treated with indicated concentrations of palmitate  for various time points as indicated. For mTORC1 inhibition with rapamycin (100 nM), AMPK activation with Metformin (2 mM) , cells were pre-treated 2 h prior to palmitate exposure. For G9a inhibition experiments, HepG2 and AML 12 cells were pretreated with indicated concentration of G9a inhibitor UNC 0642  for 2 h and 12 h respectively prior to palmitate exposure.Human hepatoma HepG2 cell line was obtained from National Centre for Cell Science, Department of Biotechnology, India. Immortalized mouse hepatocyte AML 12 cells were obtained from American Type Culture Collection. HepG2 Cells were grown and maintained in minimum essential medium containing Palmitate was complexed to fatty acid free BSA  as mentioned previously . Briefly4 cells per well. The cells were incubated for 24 h with different concentrations of palmitate (0.1–0.5 mM). After 24 h, medium was removed, and the cells were incubated in fresh medium containing 250 μg/ml 3--2,5-diphenyltetrazolium bromide for 3 h at 37 °C. Cytotoxicity/cell viability was evaluated by assaying the ability of mitochondria to catalyze the reduction of thiazolyl blue tetrazolium bromide -2,5-diphenyltetrazolium bromide) to a formazan salt by mitochondrial dehydrogenases.Cytotoxicity test was done to identify the cytotoxic potential of palmitate as mentioned previously . BrieflyThe mRNA level of different target genes was studied using RT–qPCR. Total RNA from control and treated cells was isolated using TRIzol reagent. For complementary DNA synthesis, 2 μg of total RNA was processed using RevertAid first strand complementary DNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. qRT–PCR was performed in 7500 Real Time PCR System (Applied Biosystem) using SYBR Green PCR master mix (Thermo Fisher Scientific). The PCR amplification was carried out using the primers listed in Human hepatoma HepG2 cells and AML 12 cells treated with palmitate, with or without rapamycin and UNC-0642 for indicated time point(s), were scrapped and suspended in radioimmunoprecipitation assay buffer containing protease/phosphatase inhibitor cocktail and phenylmethylsulfonyl fluoride. Cells were sonicated for 30 s using ultrasonic processor (Vibracell) with 40% amplitude and 5 s ON and 10 s OFF cycle. The lysate was centrifuged at 14,000 rpm for 30 min at 4 °C to remove cellular debris. Protein concentrations were determined by Bradford assay. For Western blotting, 30 μg of protein were denatured at 100 °C for 5 min in Laemmli buffer. Protein samples were resolved on 7 to 18% SDS gels at 100 V. Proteins were electrotransferred to polyvinylidene difluoride membrane  using Bio-Rad Mini Transblot Electrophoretic transfer Cell. Membranes were blocked in 5% fat-free milk in 50 mM Tris, pH 8.0 with 150 mM sodium chloride and 0.1% Tween-20 for 2 h. Rabbit: anti-LC3II, anti-Beclin1, anti-Atg7, anti-pP70S6 kinase, anti-P70S6 kinase, anti-G9a, anti-pAMPK-Thr172, anti-p4-EBP1, anti-4-EBP1 , anti-SQSTM1/p62 (Santa Cruz Biotechnologies), mouse: anti-β-actin (Sigma–Aldrich), antibodies were used as primary antibodies in 3% BSA overnight at 4 °C. Anti-H3K9me2, anti-H3, and anti-Pol II antibodies obtained from abcam were used for ChIP experiments. Goat anti-rabbit and goat antimouse immunoglobulin G antibodies conjugated with horseradish peroxidase  were used as secondary antibodies. Chemiluminescence was detected by ECL Start Western blotting detection reagent  and visualized by Odyssey CLx Imaging System. Densitometric measurement of the bands was performed using ImageJ software , and the band detection was within the linear range.ChIP assays were performed in cells treated with palmitate, with or without rapamycin and UNC-0642 for indicated time point(s). Cells were crosslinked using 1% formaldehyde for 15 min followed by quenching using 125 mM glycine for 5 min. Cells were lysed, and cross-linked chromatin was sonicated for 5 min using ultrasonic processor (Vibracell) with 40% amplitude and 15 s ON and 30 s OFF cycle to obtain DNA fragments of 400 to 700 bp in size. Immunoprecipitations were carried out using anti-H3K9me2 antibody, anti-H3 antibody, and anti-Pol II antibody. All the ChIP experiments were performed thrice with similar results. qPCR was performed using SYBR Green reagent (Thermo Scientific) according to the manufacturer’s protocol using Real Time Cycler (Applied Biosystems). The results shown are based on three independent experiments with standard errors. Primers used in the ChIP experiment are listed in p < 0.05 was considered as statistically significant.Data are presented as mean ± SD from three independent experiments. Statistical comparisons between the groups were determined by using one-way ANOVA followed by Bonferroni multiple comparison test using GraphPad Prism 8.0.1 statistical software. All the data described in this study are contained within the article.This article contains The authors declare that they have no conflict of interest with the contents of this article.