Fig 2: H2Bub1 modulates the transcription of autophagy-related genes. (A) Total RNA from 293T cells transfected with a control siRNA, an RNF20-specific siRNA or an H2BK120R mutant plasmid, or cells treated with starvation for 10 h was prepared and then subjected to Affymetrix microarray analysis. (B) Differentially expressed genes with similar change patterns were selected and counted. (C) Genes with similar change patterns were selected and subjected to GO and Pathway annotation analyses. (D) Thirty two autophagy-related genes were selected from the genes with similar change patterns. (E) Total RNA was isolated from control, RNF20 RNAi-transfected and H2BK120R mutant plasmid-transfected 293T cells, and HBSS-starved 293T cells. RT-PCR analyses were performed using specific primers, as indicated. At least three biological replicates were analyzed (n > 3). (F) Chromatin immunoprecipitation (ChIP) assays were performed in control or HBSS-starved HEK293T cells using anti-H2Bub1 antibody, followed by a PCR analysis using the indicated primers. At least three biological replicates were analyzed (n > 3). (G) Total RNAs from control, USP44 RNAi, and HBSS-starved control 293T cells, and HBSS-starved USP44 RNAi 293T cells were isolated. RT-PCR analyses were then performed using specific primers, as indicated. At least three biological replicates were analyzed (n > 3).

Fig 3: Starvation-induced USP44 up-regulation is mediated by DNA methylation. (A) 293T cells were starved by treating with HBSS for the indicated periods. Cell extracts were then prepared and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (B) 293T cells were treated with or without starvation for 10 h by incubating with HBSS. Protein extracts and total RNA were then prepared and subjected to western blot and RT-PCR analyses usingspecific antibodies and primers,as indicated. The experiments were repeated more than three times (n > 3). (C) Methylation of the USP44 gene is decreased in response to starvation. DNA methylation profiling analysis was employed in control and HBSS-starved 293T cells. The detailed procedure was performed according to the manufacturer's instructions (Millipore, Lot#: 17-10451). (D) 293T cells were transfected with a control or a DNMT3a- or DNMT3b-specific siRNA for 48 h. The total RNA was then isolated and subjected to RT-PCR analysis using the indicated primers. At least three biological replicates were analyzed (n > 3). (E) 293T cells were treated with or without starvation for 10 h. The cells were then lysed and subjected to chromatin immunoprecipitation (ChIP) assay using antibodies against DNMT3a and DNMT3b or normal control IgG (as a negative control), followed by real-time PCR with specific primers for the USP44 gene CpG region, as indicated. At least three biological replicates were analyzed (n > 3). (F) 293T cells were treated with starvation or starvation together with MG132 (25 µM), 3-MA (10 mM) or chloroquine (100 µM) and leupeptin (50 µM) for 10 h. The cells were then lysed and subjected to western blot analysis using the indicated antibodies. The experiments were repeated three times (n = 3). (G) In vivo ubiquitination assays were performed in control and HBSS-starved 293T cells together with MG132 incubation. The immunoprecipitation was performed under denaturing conditions using antibodies against DNMT3a and DNMT3b, followed by western blot analysis using the indicated antibodies. The experiments were repeated three times (n = 3).

Fig 4: The inhibition of H2Bub1 induces autophagy activation. (A) 293T cells were transfected with a control or H2BK120R mutant plasmid for 48 h. The cells were then lysed and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (B) Control plasmid- or H2BK120R mutant plasmid-transfected 293T cells were cotransfected with GFP-LC3 or stained with anti-LC3B antibody. The cells were then subjected to microscopy analysis. The experiments were repeated more than three times (n > 3), and 500 cells were analyzed each time. (C) 293T cells were transfected with a control siRNA or an RNF20-specific siRNA for 48 h. The cells were then lysed and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (D) Control siRNA- or RNF20-specific siRNA-transfected 293T cells were cotransfected with GFP-LC3 or stained with anti-LC3B antibody. The cells were then subjected to microscopy analysis. The experiments were repeated more than three times (n > 3), and 500 cells were analyzed each time. (E) 293T cells were transfected with a control or USP44 plasmid for 48 h. The cells were then lysed and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (F) Control plasmid- or USP44 plasmid-transfected 293T cells were cotransfected with GFP-LC3 or stained with anti-LC3B antibody. The cells were then subjected to microscopy analysis. The experiments were repeated more than three times (n > 3), and 500 cells were analyzed each time. (G) 293T cells were transfected with a control or H2BK120R mutant plasmid for 48 h. The cells were then lysed and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (H) 293T cells were transfected with a control siRNA or an RNF20-specific siRNA for 48 h. The cells were then lysed and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (I) 293T cells were transfected with a control or USP44 plasmid for 48 h. The cells were then lysed and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (J) Inhibition of H2Bub1 does not affect the mRNA levels of p62. H2BK120R mutant plasmid-, RNF20-specific siRNA- and USP44 plasmid-transfected 293T cells and their related control cells were lysed, and total RNA was prepared and subjected to RT-PCR assays using the indicated primers (n = 3).

Fig 5: H2Bub1 levels are decreased during starvation-induced autophagy activation mediated by its deubiquitinase USP44. (A) H2Bub1 is decreased in response to starvation as analyzed by western blot assay. 293T cells were starved by HBSS treatment for the indicated periods to activate autophagy. Cell extracts were then prepared and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (B) H2Bub1 is decreased in response to starvation as analyzed by immunofluorescence assay. Immunofluorescence assays were performed using control or starved 293T cells with anti-H2Bub1 antibody. DAPI staining was used to indicate the cell nucleus. (C) USP44 is increased in response to starvation. Control or HBSS-treated (starvation) 293T cells were lysed and subjected to western blot and RT-PCR analyses using specific antibodies or primers, as indicated. The experiments were repeated more than three times (n > 3). (D) USP44 regulates the starvation-induced down-regulation of H2Bub1. 293T cells were transfected with control or USP44-specific shRNAs for 48 h. The cells were then treated with or without HBSS (starvation) for the indicated periods. Cell extracts were prepared and subjected to western blot analysis using the indicated antibodies. The experiments were repeated more than three times (n > 3). (E) Loss of H2Bub1 does not depend on autophagy activity. Control and ATG5 RNAi 293T cells were treated with or without starvation for 10 h. The cells were then lysed and subjected to western blot assays using the indicated antibodies. The experiments were repeated more than three times (n > 3). (F) H2Bub1 does not respond to rapamycin treatment. 293T cells were treated with starvation or 2 μM rapamycin for 10 h. The cells were then lysed and subjected to western blot assays using the indicated antibodies. The experiments were repeated more than three times (n > 3). (G) H2Bub1 likely functions upstream of H4K16ac in the regulation of autophagy. Cell extracts from 293T cells transfected with RNF20 siRNA, H2BK120R mutant plasmid, ATG5 siRNA, or hMof siRNA were prepared. Western blot analysis was then performed using the indicated antibodies. The experiments were repeated more than three times (n > 3).