The Role Of Drosophila Insulator Protein Dwg In Autophagy

Autophagy plays a critical role in maintaining cell homeostasis in physiological conditions or stress.Autophagy is highly conserved from yeast to humans.Malfunctions of autophagy were related to multiple diseases including metabolic diseases,neurodegenerative diseases,cancer and so on.Therefore,exploring the regulation mechanism of autophagy is of great significance for solving major human health problems.In a mosaic screen aiming to isolate mutants with autophagy defects,we identified a mutation in dwg that led to reduction of GFP-Atg8 a puncta upon starvation.Further studies found that the starvation induced puncta of multiple autophagy markers such as GFP-Atg9,GFP-Atg18 a,GFP-Syx17,and GFP-Rab7 were increased in dwg mutant clones.In addition,the red puncta of GFP-RFP-Atg8 a were increased in the dwg mutant clones upon starvation,indicating autophagy flux were increased.Consistent with this,the autophagy substrate P62 was reduced in dwg mutant cells.When the fusion between autophagosomes and lysosomes was blocked,the dwg RNAi tissues accumulated more autophagosomes than the control wild type tissues.These data suggested that the loss of dwg leads to enhanced autophagy upon starvation.Further analysis showed that the mRNA levels of Atg8,Atg9,Atg18 a,syx17,and Lamp were increased significantly when dwg was knockdown by RNAi.Indicating dwg might regulate autophagy at transcriptional level.Moreover,Dwg proteins were clustered inside nuclei when they were overexpressed.The overexpression of Dwg inhibited cell growth.Dwg co-localized with Atg8 a in the nuclei.Immunoprecipitation assay showed that Atg8 a and Dwg interacted with each other in one direction.Both Dwg and BEAF-32 are insulator binding proteins that interact with each other to mediate boundary elements on DNA forming loops.However,BEAF-32 RNAi did not lead to similar autophagy defects caused by dwg RNAi.In conclusion,we identified a novel autophagy regulator Dwg.Upon starvation,Dwg suppresses autophagy through inhibiting the transcription of the key autophagy related genes to prevent autophagy out of control.