PMID- 32726170 OWN - NLM STAT- MEDLINE DCOM- 20210622 LR - 20231213 IS - 1939-4586 (Electronic) IS - 1059-1524 (Print) IS - 1059-1524 (Linking) VI - 31 IP - 20 DP - 2020 Sep 15 TI - The HIF target ATG9A is essential for epithelial barrier function and tight junction biogenesis. PG - 2249-2258 LID - 10.1091/mbc.E20-05-0291 [doi] AB - Intestinal epithelial cells (IECs) exist in a metabolic state of low oxygen tension termed "physiologic hypoxia." An important factor in maintaining intestinal homeostasis is the transcription factor hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and mediates IEC homeostatic responses to low oxygen tension. To identify HIF transcriptional targets in IEC, chromatin immunoprecipitation (ChIP) was performed in Caco-2 IECs using HIF-1alpha- or HIF-2alpha-specific antibodies. ChIP-enriched DNA was hybridized to a custom promoter microarray (termed ChIP-chip). This unbiased approach identified autophagy as a major HIF-1-targeted pathway in IEC. Binding of HIF-1 to the ATG9A promoter, the only transmembrane component within the autophagy pathway, was particularly enriched by exposure of IEC to hypoxia. Validation of this ChIP-chip revealed prominent induction of ATG9A, and luciferase promoter assays identified a functional hypoxia response element upstream of the TSS. Hypoxia-mediated induction of ATG9A was lost in cells lacking HIF-1. Strikingly, we found that lentiviral-mediated knockdown (KD) of ATG9A in IECs prevents epithelial barrier formation by >95% and results in significant mislocalization of multiple tight junction (TJ) proteins. Extensions of these findings showed that ATG9A KD cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein. These results implicate ATG9A as essential for multiple steps of epithelial TJ biogenesis and actin cytoskeletal regulation. Our findings have novel applicability for disorders that involve a compromised epithelial barrier and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention. FAU - Dowdell, Alexander S AU - Dowdell AS AD - Mucosal Inflammation Program and Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO 80045. FAU - Cartwright, Ian M AU - Cartwright IM AD - Mucosal Inflammation Program and Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO 80045. FAU - Goldberg, Matthew S AU - Goldberg MS AD - Mucosal Inflammation Program and Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO 80045. FAU - Kostelecky, Rachael AU - Kostelecky R AD - Mucosal Inflammation Program and Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO 80045. FAU - Ross, Tyler AU - Ross T AD - Mucosal Inflammation Program and Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO 80045. FAU - Welch, Nichole AU - Welch N AD - Mucosal Inflammation Program and Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO 80045. FAU - Glover, Louis E AU - Glover LE AD - Mucosal Inflammation Program and Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO 80045. AD - School of Biochemistry and Immunology, Trinity College Dublin, Ireland. FAU - Colgan, Sean P AU - Colgan SP AD - Mucosal Inflammation Program and Division of Gastroenterology and Hepatology, University of Colorado, Aurora, CO 80045. LA - eng GR - P30 DK048520/DK/NIDDK NIH HHS/United States GR - R01 DK050189/DK/NIDDK NIH HHS/United States GR - I01 BX002182/BX/BLRD VA/United States GR - R01 DK104713/DK/NIDDK NIH HHS/United States GR - R01 DK103712/DK/NIDDK NIH HHS/United States GR - R37 DK050189/DK/NIDDK NIH HHS/United States GR - R01 DK095491/DK/NIDDK NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20200729 PL - United States TA - Mol Biol Cell JT - Molecular biology of the cell JID - 9201390 RN - 0 (ATG9A protein, human) RN - 0 (Autophagy-Related Proteins) RN - 0 (Hypoxia-Inducible Factor 1, alpha Subunit) RN - 0 (Membrane Proteins) RN - 0 (Tight Junction Proteins) RN - 0 (Transcription Factors) RN - 0 (Vesicular Transport Proteins) SB - IM MH - Autophagy-Related Proteins/genetics/*metabolism MH - Caco-2 Cells MH - Cell Hypoxia/physiology MH - Cell Line MH - Epithelial Cells/metabolism MH - Gene Expression Regulation/genetics MH - HeLa Cells MH - Humans MH - Hypoxia/metabolism MH - Hypoxia-Inducible Factor 1, alpha Subunit/metabolism MH - Intestinal Mucosa/*metabolism MH - Membrane Proteins/genetics/*metabolism MH - Promoter Regions, Genetic/genetics MH - Response Elements/genetics MH - Tight Junction Proteins/metabolism MH - Tight Junctions/*metabolism MH - Transcription Factors/metabolism MH - Vesicular Transport Proteins/genetics/*metabolism PMC - PMC7550696 EDAT- 2020/07/30 06:00 MHDA- 2021/06/23 06:00 PMCR- 2020/11/30 CRDT- 2020/07/30 06:00 PHST- 2020/07/30 06:00 [pubmed] PHST- 2021/06/23 06:00 [medline] PHST- 2020/07/30 06:00 [entrez] PHST- 2020/11/30 00:00 [pmc-release] AID - E20-05-0291 [pii] AID - 10.1091/mbc.E20-05-0291 [doi] PST - ppublish SO - Mol Biol Cell. 2020 Sep 15;31(20):2249-2258. doi: 10.1091/mbc.E20-05-0291. Epub 2020 Jul 29.