A postprandial FGF 19‐ SHP ‐ LSD 1 regulatory axis mediates epigenetic repression of hepatic autophagy
2017; Springer Nature; Volume: 36; Issue: 12 Linguagem: Inglês
10.15252/embj.201695500
ISSN1460-2075
AutoresSangwon Byun, Young‐Chae Kim, Yang Zhang, Bo Kong, Grace L. Guo, Junichi Sadoshima, Jian Ma, Byron Kemper, Jongsook Kim Kemper,
Tópico(s)Cannabis and Cannabinoid Research
ResumoArticle26 April 2017free access Transparent process A postprandial FGF19-SHP-LSD1 regulatory axis mediates epigenetic repression of hepatic autophagy Sangwon Byun orcid.org/0000-0001-5101-8372 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Young-Chae Kim Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Yang Zhang Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Bo Kong Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Grace Guo Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Junichi Sadoshima Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Jian Ma Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA Search for more papers by this author Byron Kemper Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Jongsook Kim Kemper Corresponding Author [email protected] orcid.org/0000-0002-5534-0286 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Sangwon Byun orcid.org/0000-0001-5101-8372 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Young-Chae Kim Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Yang Zhang Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Bo Kong Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Grace Guo Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Junichi Sadoshima Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA Search for more papers by this author Jian Ma Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA Search for more papers by this author Byron Kemper Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Jongsook Kim Kemper Corresponding Author [email protected] orcid.org/0000-0002-5534-0286 Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Author Information Sangwon Byun1, Young-Chae Kim1, Yang Zhang2, Bo Kong3, Grace Guo3, Junichi Sadoshima4, Jian Ma2,5, Byron Kemper1 and Jongsook Kim Kemper *,1 1Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA 2Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA 3Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ, USA 4Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA 5Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA *Corresponding author. Tel: +1 217 333 6317; E-mail: [email protected] EMBO J (2017)36:1755-1769https://doi.org/10.15252/embj.201695500 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Lysosome-mediated autophagy is essential for cellular survival and homeostasis upon nutrient deprivation, but is repressed after feeding. Despite the emerging importance of transcriptional regulation of autophagy by nutrient-sensing factors, the role for epigenetic control is largely unexplored. Here, we show that Small Heterodimer Partner (SHP) mediates postprandial epigenetic repression of hepatic autophagy by recruiting histone demethylase LSD1 in response to a late fed-state hormone, FGF19 (hFGF19, mFGF15). FGF19 treatment or feeding inhibits macroautophagy, including lipophagy, but these effects are blunted in SHP-null mice or LSD1-depleted mice. In addition, feeding-mediated autophagy inhibition is attenuated in FGF15-null mice. Upon FGF19 treatment or feeding, SHP recruits LSD1 to CREB-bound autophagy genes, including Tfeb, resulting in dissociation of CRTC2, LSD1-mediated demethylation of gene-activation histone marks H3K4-me2/3, and subsequent accumulation of repressive histone modifications. Both FXR and SHP inhibit hepatic autophagy interdependently, but while FXR acts early, SHP acts relatively late after feeding, which effectively sustains postprandial inhibition of autophagy. This study demonstrates that the FGF19-SHP-LSD1 axis maintains homeostasis by suppressing unnecessary autophagic breakdown of cellular components, including lipids, under nutrient-rich postprandial conditions. Synopsis Nutrient state affects autophagy via distinct pathways. The fed-state hormone FGF19 triggers SHP-dependent recruitment of LSD1 to CREB-bound autophagy genes, leading to repressive histone modifications, thus revealing an epigenetic inhibition of hepatic autophagy under nutrient-rich conditions. SHP represses hepatic autophagy, including autophagy-mediated lipid catabolism, "lipophagy", in response to a late fed-state hormone, FGF19 (human FGF19, mouse FGF15). Feeding-mediated inhibition of hepatic autophagy is blunted in mice lacking SHP or FGF15. SHP mediates postprandial epigenetic repression of CREB-bound autophagy genes by recruiting LSD1 histone demethylase. Feeding-sensing nuclear receptors FXR and SHP inhibit hepatic autophagy in a mutually dependent manner. Introduction Lysosome-mediated autophagy is a highly conserved homeostatic process that recycles cellular components and mobilizes energy stores, including lipid droplets, in response to nutrient deprivation (Klionsky, 2007; Mizushima, 2009; Singh et al, 2009; Rabinowitz & White, 2010; Singh & Cuervo, 2012). Autophagy is known to occur under extremely stressful starvation conditions, but increasing evidence indicates that it also occurs during feeding/fasting cycles under normal physiological conditions (Settembre et al, 2013; Lee et al, 2014; Seok et al, 2014). Autophagy is regulated acutely by nutrient-sensing cytoplasmic kinases, such as mTOR and AMPK (Egan et al, 2011; Kim et al, 2011), but recent studies demonstrate that nuclear events are also important for sustained autophagy regulation. Nutrient-sensing transcriptional factors, including fasting-activated CREB, PPARα, and TFEB, and feeding-activated FXR, dynamically activate or repress macroautophagy, including autophagy-mediated lipid breakdown, "lipophagy" (Settembre et al, 2013; Lee et al, 2014; Seok et al, 2014). Epigenetic modifications, including histone modifications, play a crucial role in linking environmental cues, such as changes in metabolite levels, to regulation of gene expression to maintain homeostasis (Teperino et al, 2010; Lu & Thompson, 2012; Smith et al, 2012). Despite the emerging importance of transcriptional regulation in autophagy, only a few studies have examined epigenetic control of autophagy (Artal-Martinez de Narvajas et al, 2013; Fullgrabe et al, 2013; Shin et al, 2016), and its role in directly linking nutritional status to autophagy-mediated lipid degradation in animals in vivo has not been described. An orphan nuclear receptor, Small Heterodimer Partner (SHP, NR0B2), is a key transcriptional regulator that links changes in hepatic bile acid levels to epigenetic repression of bile acid synthetic genes to maintaining bile acid homeostasis (Kerr et al, 2002; Wang et al, 2002; Smith et al, 2012; Kim et al, 2016). In response to elevated hepatic bile acid levels and bile acid-induced fibroblast growth factor-19 (hFGF19, mFGF15) signaling after feeding (Inagaki et al, 2005; Kir et al, 2011), SHP epigenetically represses transcription of bile acid synthetic genes, Cyp7a1 and Cyp8b1, by recruiting repressive histone-modifying enzymes, including LSD1 histone demethylase (Kemper et al, 2004; Fang et al, 2007; Kim et al, 2015b). Based on published ChIP-seq analysis of mice treated with a late fed-state hormone FGF19 (Kim et al, 2015a), SHP binding peaks were detected at over 80 autophagy-related genes in liver, suggesting a previously unrecognized role for SHP in epigenetic control of autophagy. Here, we identify a new function of an FGF19-SHP-LSD1 axis in transcriptional and epigenetic repression of hepatic autophagy in the late fed-state. After treatment with FGF19 or feeding, SHP recruits LSD1 to a group of CREB-target autophagy-related genes, including Tfeb, resulting in disruption of the CREB-CRTC2 transactivation complex and an epigenetic cascade leading to gene repression. We further show that both feeding-sensing FXR and SHP inhibit autophagy in a mutually dependent manner, but FXR acts predominantly early after feeding, while SHP acts relatively late, which effectively sustains postprandial inhibition of autophagy. Results SHP inhibits hepatic autophagy in vivo From liver ChIP-seq studies of FGF19-treated mice (Kim et al, 2015a), SHP binding peaks were detected within 10 kb of the transcription start sites (TSS) in 85 of the 230 autophagy-related genes in the Human Autophagy Database (http://autophagy.lu/) (Appendix Fig S1 and Appendix Table S1). In ChIP analyses, treatment with FGF19 increased SHP occupancy at all tested autophagy-related genes and a known SHP-target gene, Cyp7a1 (Fig 1A), and decreased mRNA levels of most of these autophagy genes in WT mice (Fig 1B). In contrast, in SHP−/− mice, the basal expression of these genes was elevated and the inhibition by FGF19 was blunted (Fig 1B), suggesting that SHP is required for FGF19 inhibition of autophagy genes. Figure 1. SHP inhibits hepatic autophagy by FGF19 treatment or feeding A–D. Mice were fasted for 12 h and injected via the tail vein with FGF19 (1 mg/kg) or vehicle for 2 h (A) or 6 h (B–D). (A) SHP occupancy in liver was detected by ChIP (n = 3), and (B) mRNA levels were detected by qRT–PCR (n = 6). (C) LC3 and p62 detected by IHC analysis of liver sections from WT or SHP−/− mice. Representative images of LC3-II puncta and quantitation are shown (n = 30 hepatocytes). (D) WT or SHP−/− mice were injected via the tail vein with Ad-GFP-LC3, and then, 1 week later the mice were treated with FGF19 or vehicle. Representative images of GFP-LC3-II puncta are shown. E. LC3 and p62 detected by IHC analysis of liver sections from fasted or fed WT or SHP−/− mice. Representative images of LC3-II puncta and quantitation are shown (n = 30 hepatocytes). F. Mice adenovirally expressing GFP-LC3 were fasted or fasted and refed, and representative images of GFP-LC3-II puncta in liver sections are shown. G, H. LC3 and p62 levels in liver extracts from fasted or fed WT or SHP−/− (G) or from fasted or fed WT or FGF15−/− (H) mice were measured by IB, and LC3-I and LC3-II intensities were quantified and the relative ratio of LC3-II to LC3-I was set to 1 for fasted WT mice (right, n = 3). Data information: Means ± SD are shown, and statistical significance was measured using two-way ANOVA with the Bonferroni post-test. *P < 0.05, **P < 0.01, and NS, statistically not significant. Download figure Download PowerPoint To determine whether FGF19 treatment inhibits hepatic autophagy, we examined the expression of LC3 and p62, markers of autophagy (Mizushima et al, 2010). Phosphatidylethanolamine-conjugated LC3-II is present on autophagosome membranes, and the ratio of LC3-II to LC3-I is an indicator of autophagic flux. Conversely, the autophagosome adaptor protein, p62/SQSTM1, is degraded by autophagy, and thus, p62 accumulates when autophagy is inhibited. We examined hepatic autophagy by immunohistochemistry (IHC) and immunofluorescence (IF). LC3-I appears diffuse in the cytoplasm and LC3-II appears as distinct puncta. FGF19 treatment decreased the number of endogenous LC3-II puncta (Fig 1C) or exogenous GFP-LC3-II puncta (Fig 1D) and increased p62 levels (Fig 1C) in WT mouse liver, whereas these changes were blunted in livers of SHP−/− mice. FGF19 signaling assessed by p-ERK levels (Kir et al, 2011) was normal in SHP-KO mice (Appendix Fig S2A). These results demonstrate that FGF19 inhibition of autophagy is SHP-dependent. Nutrient-sensing mTOR complex 1 (mTORC1) was shown to negatively regulate autophagy by phosphorylation of ULK1 at S757 (Kim et al, 2011), so it is possible that FGF19 inhibition of autophagy is affected by mTOR signaling. To test this idea, we examined the effect of rapamycin, an allosteric inhibitor of mTORC1, FGF19, or both, on hepatic autophagy in mice. Treatment with rapamycin decreased p-S757-ULK1 levels and increased autophagic flux as assessed by the increased ratio of LC3-II to LC-I as expected (Appendix Fig S2B). FGF19 treatment inhibited autophagic flux without changing the levels of mTORC1 targets, p-S6K or p-ULK1, and co-treatment with rapamycin and FGF19 largely blunted the rapamycin-induced autophagic flux without changing the p-S757-ULK1 levels. FGF19 treatment also increased p-ERK levels as expected, whereas p-AMPK levels were not changed (Appendix Fig S2C). Further, in primary mouse hepatocytes, treatment with a lysosomal inhibitor, bafilomycin-A1, did not block the decreased ratio of LC3-II to LC-I after FGF19 treatment (Appendix Fig S2D). These results suggest that FGF19 inhibition of autophagy is likely independent of mTORC1 or lysosomal function. SHP represses hepatic autophagy after feeding in an FGF15-dependent manner FGF15/19 is a late fed-state hormone that is induced by the intestinal bile acid nuclear receptor FXR and acts at the liver to mediate postprandial metabolic responses independent of insulin action (Inagaki et al, 2005; Kir et al, 2011). Serum FGF15/19 levels peak about 3 h after feeding (Lundasen et al, 2006), which is considerably later than the peaks of insulin or bile acids. To determine the physiological relevance of SHP-dependent FGF15/19 inhibition of autophagy, we next examined whether SHP is required for feeding-mediated inhibition of hepatic autophagy. Numbers of LC3-II puncta in liver were elevated in SHP−/− mice, the feeding-mediated decrease in the number of LC3-II puncta in WT mice was substantially attenuated in SHP−/− mice (Fig 1E and F), and the decreased ratio of LC3-II to LC3-I after feeding in WT mice was largely absent in SHP−/− mice (Fig 1G). Levels of p62 were reduced in SHP−/− mice and the increase in p62 after feeding in WT mice was not observed in SHP−/− mice (Fig 1E and G). Consistent with these results, mRNA levels of SHP-target autophagy genes were also decreased by feeding in WT, but not SHP−/− mice (Appendix Fig S3A). Importantly, feeding-mediated decreases in the LC3-II/LC3-I ratio (Fig 1H) and gene expression (Appendix Fig S3B) were largely abolished in FGF15−/− mice (Kong et al, 2014), indicating the dependence of SHP-mediated postprandial repression of hepatic autophagy on FGF15. These results demonstrate that SHP acts as a key physiological repressor of autophagy in response to FGF15/19 in the late fed-state. SHP antagonizes the CREB transactivation of autophagy genes To identify potential transcription factors that might recruit the non-DNA binding SHP to autophagy genes, we compared binding sites for the activators of autophagy, CREB (Everett et al, 2013), PPARα (Boergesen et al, 2012), and SREBP-2 (Seo et al, 2011) with those of SHP (Kim et al, 2015a) from published liver ChIP-seq data. CREB binding peaks overlapped with 63% of SHP binding peaks at autophagy genes, a substantially higher percentage than the overlap with PPARα and SREBP-2 (Fig 2A and Appendix Tables S2–S4), and one or more CREB binding motifs were present within the SHP binding regions (Appendix Table S5). We therefore focused on CREB, a master transcriptional activator that drives the fasting response (Herzig et al, 2001; Koo et al, 2005). Notably, nearly all of the binding peaks for SHP (Kim et al, 2015a) and CREB (Everett et al, 2013; Seok et al, 2014) were found within 1 kb of the TSS of autophagy-related genes (Appendix Table S2), including the key autophagy component genes Atg3 and Atg10, and a key transcriptional activator of autophagy, Tfeb (Fig 2B). These results suggest that CREB may recruit SHP to autophagy-related genes. Figure 2. SHP represses CREB-target autophagy genes Venn diagrams showing autophagy-related genes that contain binding peaks of SHP compared with those of activators of autophagy, CREB, PPARα, and SREBP-2, within 10 kb from the TSS. Display of autophagy-related genes with shared SHP and CREB binding peaks (UCSC genome browser). Mice were treated with FGF19 for 2 h, and three independent ChIP assays were done to detect occupancy of indicated proteins at Tfeb and Atg3. Hepatocytes were transfected with siRNA for CREB, and 48 h later, cells were treated with FGF19 for 2 h and re-ChIP was done with initial immunoprecipitation of CREB, followed by immunoprecipitation of SHP, CREB, and CRTC2 (n = 3). The effects of FGF19 treatment on the interaction of endogenous SHP with CREB in mouse liver nuclear extracts were detected by Co-IP (n = 3). Interactions of GST fusions of CREB with SHP, FXR, or CRTC2 or GST fusions of SHP with CREB were detected. Bound proteins were detected by IB. Domains of CREB and SHP fused to GST are shown above the IB blots. CREB, CRTC2, or SHP was expressed in Hepa1c1c7 cells or the cells were transfected with SHP siRNA as indicated, and luciferase reporter assays were done in triplicate (n = 4). GFP-LC3 and either SHP alone (top panels) or SHP and CREB (bottom panels) were expressed in Hepa1c1c7 cells. C indicates the control pcDNA3 vector. The cells were incubated in HBSS (2 h), and fluorescence was imaged by confocal microscopy. The average number of LC3-II puncta per cell is shown at the right (n = 20 cells). Data information: Means ± SD are shown, and statistical significance was measured using (G) one- or (C, D, H) two-way ANOVA and Bonferroni post-test. *P < 0.05, **P < 0.01, and NS, statistically not significant. Download figure Download PowerPoint After FGF19 treatment, occupancy of SHP at Tfeb and Atg3 increased, that of the CREB coactivator CRTC2 decreased, and CREB occupancy was unaffected while no decrease in CRTC2 occupancy was observed in SHP−/− mice (Fig 2C). Similar results were observed for Atg5, Atg10, and Pten (Appendix Fig S4). These results suggest that the decreased occupancy of CRTC2 is dependent on SHP. Further, in re-CHIP assays, SHP was detected in CREB-bound chromatin indicating the co-occupancy of both SHP and CREB at these genes, and as a control, downregulation of CREB showed that the SHP occupancy in the CREB-bound chromatin was dependent on CREB (Fig 2D and Appendix Fig S6A). Consistent with the findings above that FGF19 inhibition of hepatic autophagy is likely independent of mTORC1 (Appendix Fig S2B–D), FGF19-mediated changes in occupancy of SHP, CREB, and CRTC2 were not affected by co-treatment with rapamycin in mice (Appendix Fig S5). We next examined whether SHP and CREB directly interact. FGF19 treatment increased the interaction of endogenous SHP with CREB in Co-IP assays using liver extracts (Fig 2E). In GST-pull-down assays, SHP interacts through its N-terminal domain with the central transactivation KID domain of CREB (Fig 2F and Appendix Fig S6B). Interestingly, the CREB-interacting proteins, FXR (Seok et al, 2014) and CRTC2 (Koo et al, 2005; Dentin et al, 2007; Altarejos & Montminy, 2011), each interact with CREB through different domains that are distinct from the SHP-interaction domain (Fig 2F, top). Further, FGF19 treatment increased accumulation of SHP in the nucleus (Appendix Fig S6C), where CREB was located, enabling protein interaction. These results indicate that FGF19 treatment increases nuclear localization of SHP and that SHP interacts directly with CREB. To further examine the functional interaction of SHP and CREB, reporter assays were performed using luciferase reporter constructs containing the Tfeb, Atg3, or Atg7 promoter. Exogenous expression of SHP inhibited the reporter activation by CREB-CRTC2, and downregulation of SHP increased it (Fig 2G and Appendix Fig S6D). Consistent with these results, exogenous expression of CREB increased autophagy, and the increase was abolished by exogenous expression of SHP (Fig 2H). These results indicate that SHP inhibits the CREB transactivation of autophagy genes. SHP epigenetically represses CREB-bound autophagy genes by recruiting LSD1 SHP represses expression of bile acid synthetic genes by recruiting histone-modifying enzymes, including LSD1, a key epigenetic factor that is recruited initially and required for subsequent repressive histone modifications, deacetylation of H3K9/14, and methylation of H3K9 (Kim et al, 2015b). LSD1 (also KDM1A) is a lysine-specific histone demethylase that represses genes by removing gene-activation histone marks, mono- or di-methylated H3K4 (Shi et al, 2004), but also activates genes by removing a gene-repression histone mark, methylated H3K9 (Metzger et al, 2005). To determine whether LSD1 was involved in FGF19/SHP-mediated repression of autophagy, we first examined whether FGF19 treatment affects the interaction of SHP with LSD1. FGF19 treatment increased the interaction of endogenous SHP with LSD1 in liver extracts (Fig 3A). Further, FGF19 treatment increased LSD1 occupancy in SHP-bound chromatin at Tfeb and Atg3 in re-ChIP assays and decreased histone H3K4-me2/3 and RNA polymerase II levels (Fig 3B). The increases in occupancy of LSD1 and decreases in the levels of histone H3K4-me2/3 at Tfeb, Atg3, and Atg10 after FGF19 treatment were absent in SHP−/− mice (Fig 3C and Appendix Fig S7A), although protein levels of CREB, CRTC2, and LSD1 were similar in WT mice and SHP−/− mice (Appendix Fig S7B). These results indicate that FGF19 increases the interaction of SHP with LSD1 and that SHP recruits LSD1 to SHP-target autophagy genes. Figure 3. SHP represses CREB-target autophagy genes by recruiting LSD1 The interaction of SHP with LSD1 in mouse liver nuclear extracts was detected by Co-IP after FGF19 treatment. Mice were treated with FGF19, and re-ChIP assays were done with initial immunoprecipitation of SHP, followed by immunoprecipitation of the indicated proteins at the Tfeb and Atg3 genes (n = 5). WT or SHP−/− mice were treated with FGF19 for 2 h, and liver ChIP assays (n = 3–5) were done to detect occupancy of indicated proteins and histone levels at Tfeb and Atg3. Hepa1c1c7 cells were transfected with expression plasmids and siRNAs as indicated, and reporter assays were done (n = 4). GFP-LC3 was expressed in Hepa1c1c7 cells, and the cells were transfected with control siRNA, siRNA for LSD1 or SHP, and LSD1 expression plasmids as indicated. C indicates the control pcDNA3 vector. The cells were incubated in HBSS (2 h), and fluorescence was imaged by confocal microscopy. The average number of LC3-II puncta per cell is shown at the right (n = 20 cells). Data information: Means ± SD are shown, and statistical significance was measured using (B) Mann–Whitney test, (D) one- or (C, E) two-way ANOVA with the Bonferroni post-test, and (E) Student's t-test. *P < 0.05, **P < 0.01, and NS, statistically not significant. Download figure Download PowerPoint In functional studies, exogenous expression of SHP inhibited CREB transactivation of Tfeb-luc and Atg3-luc reporters, and co-expression of LSD1 enhanced the inhibition, while downregulation of LSD1 reversed the SHP inhibition (Fig 3D, Appendix Fig S7C). Downregulation of SHP eliminated the effects of LSD1 on transcriptional activity of the promoters. Consistent with these results, downregulation of LSD1 increased, while expression of LSD1 decreased autophagy and these effects were absent if SHP was downregulated (Fig 3E). These results demonstrate that LSD1 inhibition of autophagy and autophagy gene expression is dependent on SHP. LSD1 mediates epigenetic repression of SHP-target autophagy genes FGF19 treatment or feeding of mice increased occupancy of LSD1, decreased occupancy of Pol II, decreased levels of gene-activation histone marks, H3K4-me2, H3K4-me3, and H3K9/14-Ac, and increased levels of a gene-repression mark, H3K9-me2, at SHP-target autophagy genes, Tfeb, Atg3, and Atg7 (Fig 4A and B, Appendix Fig S7D and E). Downregulation of LSD1 (Appendix Fig S7F and G) partially or completely reversed these changes in histone modifications. In addition, FGF19- or feeding-mediated decreases in mRNA levels were attenuated in most SHP-target autophagy genes by downregulation of LSD1 in mice, and notably, basal expression of Wipi1, Rb1cc1, and Uvrag was substantially increased (Fig 4C and D). Further, FGF19 or feeding dramatically decreased the number of LC3 puncta in mouse liver, and downregulation of LSD1 substantially increased basal puncta numbers and partially reversed the decrease in the numbers of puncta after FGF19 or feeding from 80 to 50% (Fig 4E and F). FGF19 signaling assessed by p-ERK levels was normal in LSD1-downregulated mice (Appendix Fig S7H). These results indicate that LSD1 mediates the epigenetic repression of autophagy genes by SHP in a gene-selective manner. Figure 4. LSD1 mediates epigenetic repression of SHP-target autophagy genesMice were injected via the tail vein with Ad-GFP or Ad-shRNA for LSD1, and 2 weeks later, mice were fasted for 12 h and then treated with FGF19 for 2 h (A, C, E) or refed for 6 h (B, D, F). A, B. ChIP assays (n = 3–4) for LSD1 and Pol II occupancy and histone modifications were done. C, D. The mRNA levels of indicated genes in liver were measured by qRT–PCR (n = 5 mice). E, F. Endogenous LC3 was detected by IHC in liver sections. Representative images of LC3-II puncta and the average number of puncta per cell are shown (n = 30 hepatocytes). Data information: Means ± SD are shown, and statistical significance was measured using two-way ANOVA with the Bonferroni post-test. *P < 0.05, **P < 0.01, and NS, statistically not significant. Download figure Download PowerPoint SHP and LSD1 inhibit hepatic lipophagy We next examined whether autophagy-mediated lipid catabolism, "lipophagy", is inhibited by SHP and LSD1. The co-localization of lipid droplets and autophagic GFP-LC3 puncta was increased by downregulation of SHP and LSD1 in Hepa1c1c7 cells (Fig 5A). However, these effects were significantly attenuated by downregulation of ATG7, a key component of the autophagosome. In electron microscopic studies in mouse liver, after downregulation of SHP or LSD1 in mice (Fig 5B), autophagic vesicles within lipid droplets were abundant, but were not observed in livers from control mice (Fig 5C and D). Further, lipid droplets detected by Oil Red O staining were reduced and serum and liver triglyceride levels were decreased in SHP−/− mice (Appendix Fig S8A–C). In WT mice, neutral lipid levels were increased in fasted mice but not in SHP−/− mice (Appendix Fig S8D), and fasting-induced accumulation of lipids was also modestly decreased in FGF15-KO mice (Appendix Fig S8E). These results demonstrate a role for SHP and LSD1 in inhibition of autophagy-mediated lipid mobilization. Figure 5. SHP and LSD1 inhibit hepatic lipophagy A. GFP-LC3 (green) was expressed in Hepa1c1c7 cells, and the cells were transfected with control siRNA (siC) or siRNA for SHP, LSD1, or ATG7 as indicated. Cells were stained for lipid droplets with BODIPY (red) and imaged by confocal microscopy. Merged images are shown in the bottom row. Co-localization of LC3 and BODIPY is indicated by white arrows. The average number of fluorescent puncta per cell (right top, n = 20 cells) and the number of fluorescent puncta that co-localized with lipid staining (right bottom, n = 10 cells) are shown. B. Experimental outline. Mice were injected with adenoviral shRNA for SHP and LSD1 or control virus; 2 weeks later, hepatic SHP and LSD1 protein levels were detected by IB. C, D. Mice (n = 6) were injected via the tail vein with Ad-shRNA for SHP or LSD1 or control virus, and 2 weeks later, livers were collected for transmission electron microscopy analysis. Autophagy vesicles inside lipid droplets are indicated by yellow arrows. E. Normalized SHP binding peaks at the promoter region of A
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