Journal: Nature Communications

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

doi: 10.1038/s41467-020-19579-y

Figure Lengend Snippet: a The chromatin binding sites and enrichment of 33 responsive OCTFs generated through a motif enrichment analysis of MCF-7- and ADR-biased COGC-seq peaks are shown in a heat map. The color of the dots represents the TF motif enrichment. More TF-binding sites are indicated by a large dot size. b Average enrichment profiles of NRF1 ChIP-seq reads (MCF-7 and ADR cells) and published HCF-1 ChIP-seq reads (MCF-7, GSE91992 ) at differential quantitative COGC-seq peaks. c COGC-seq peaks in MCF-7 and ADR cells overlap with NRF1 and a published HCF-1 ( GSE91992 ) ChIP-seq dataset. d O-GlcNAc NRF1 was upregulated in MCF-7 cells after transient stimulation with 100 nM Adm. IP of NRF1 was performed, and the immunoprecipitated fractions were analyzed by immunoblotting for O-GlcNAc (CTD110.6). e The NRF1-HCF-1 interaction is increased in ADR cells compared with MCF-7 cells. NRF1 co-IP was performed, and the immunoprecipitated fractions were analyzed by immunoblotting for the indicated proteins. f NRF-1 is O-GlcNAcylated at Ser448/Ser451. After treatment with PugNAc (Pug, 100 μM) and glucose (Glu, 25 mM) for 24 h, MCF-7 cells stably expressing Flag-WT-NRF1 or Flag-AA-NRF1 were immunoprecipitated with anti-Flag magnetic beads. O-GlcNAcylation (CTD110.6) was analyzed by immunoblotting. WT, wild-type NRF1; AA, Ser447/Ser450 → Ala mutational NRF1. Mock, cells transfected with empty pCMVPuro64 vector. g O-GlcNAc promotes the interaction of HCF-1 and OGT with NRF1. Immunoblotting showing the PPIs of endogenous HCF-1 and OGT with NRF1 in ADR cells. ADR cells stably expressing Flag-WT-NRF1 or Flag-AA-NRF1 were immunoprecipitated with anti-Flag magnetic beads. h O-GlcNAc inhibition expedites the degradation of NRF1. ADR cells expressing Flag-WT-NRF1 or Flag-AA-NRF1 were incubated with 50 μM cycloheximide (CHX) for up to 12 h. The expression levels of Flag-NRF1 were monitored by immunoblotting. i O-GlcNAc enhances the chromatin binding of NRF1. The crosslinked chromatin proteins were extracted, and the levels of Flag-NRF1 were detected by immunoblotting. For ( d – i ), all blots are representative of at least two biologically independent experiments. a , d – i Source Data are provided as a Source Data file.

Article Snippet: SP1 siRNA (#sc-29487), KLF5 siRNA (#sc-37718), NRF1 shRNA (#sc-38105-SH) and NSMCE2 siRNA (#sc-77813) were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Generated, ChIP-sequencing, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Magnetic Beads, Transfection, Plasmid Preparation, Inhibition, Incubation

Journal: Nature Communications

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

doi: 10.1038/s41467-020-19579-y

Figure Lengend Snippet: a NRF-1 ChIP-seq signal in NRF-1 uniquely bound sites identified by overlapping MCF-7 and ADR NRF-1 peaks. Upper panel: Venn diagram showing the overlap of NRF-1 peaks in MCF-7 and ADR cells. The percentage of peaks annotated to promoter regions is indicated. Lower panel: Heat map representation of NRF-1 signal enrichment (red, low; blue, high) at NRF-1 uniquely bound sites. The enrichment levels were profiled ±3 kb from the peak center. b Heat map representation of COGC-seq signal enrichment (red, low; blue, high) at NRF-1 uniquely bound sites. The enrichment levels were profiled ±3 kb from the peak center. c Average enrichment profiles of published H3K27ac, H3K4me3 ( GSE97481 ), H3K27me3 ( GSE96363 ) and H3K4me1 ( GSE86714 ) ChIP-seq reads at NRF-1 uniquely bound sites. d The box plots showing the mRNA expression changes (RNA-seq FPKM) of NRF1-binding genes associated with MCF-7- and ADR-biased peaks. The box plots show the medians (black lines), 25th and 75th percentiles (boundaries), and minimum/maximum values (whiskers). The p value (0.0000006, two-sided unpaired Student’s t -test, calculated between multiple genes in each group) is indicated. n = 2 biologically independent RNA-seq replicates. Source Data are provided as a Source Data file. e Heat map representation of WT-NRF-1 and AA-NRF-1 signal enrichment (red, low; blue, high) at NRF-1 binding sites in MCF-7 and ADR cells. The enrichment levels were profiled ±3 kb from the peak center.

Article Snippet: SP1 siRNA (#sc-29487), KLF5 siRNA (#sc-37718), NRF1 shRNA (#sc-38105-SH) and NSMCE2 siRNA (#sc-77813) were purchased from Santa Cruz Biotechnology.

Techniques: ChIP-sequencing, Expressing, RNA Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

doi: 10.1038/s41467-020-19579-y

Figure Lengend Snippet: a Effect of NRF1 O-GlcNAc modification on the indicated gene transcription levels in ADR cells. The gene mRNA levels in ADR cells expressing Flag-WT-NRF1 or Flag-AA-NRF1 were analyzed by quantitative PCR (qPCR). b Left panel: IGV tracks showing the signals at the promoter regions of the representative genes. Right panel: Validation of O-GlcNAc NRF1 binding peaks by ChIP-qPCR. qPCR amplification was performed. Each bar represents the fold enrichment of binding relative to the input. IgG and random primers that could not specifically bind the indicated gene promoter regions (off target) were used as negative controls. Mock, cells transfected with empty pCMVPuro64 vector. c O-GlcNAc inhibition reduces the transcriptional activity of NRF1. 293T cells were transfected with a reporter vector consisting of luciferase cDNA fused to the NSMCE2 promoter. The pGL3-basic vector (Mock) was used as a control. d ADR cells were transfected with NSMCE2 siRNA (siNSMCE2) or scrambled siRNA (siScr) and treated with increasing doses of Adm for 48 h. The cell viability was then assessed. Representative images of cell viability determined by crystal violet staining are shown. Results were reproduced in two biologically independent experiments. The protein levels of NSMCE2 were monitored by immunoblotting. All blots are representative of at least two biologically independent experiments. For ( a - c ), the data are presented as the means ± SEM., ( d ) replicates are represented. ( a – d ) n = 3 biologically independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-sided unpaired Student’s t -test). p values: 0.010099, 0.001375, 0.046814, 0.020148 ( a ); 0.010191, 0.000117, 0.000159, 0.001032 ( b ); 0.0065 (WT-NRF1 vs. WT-NRF1 L01), 0.0002 (WT-NRF1 vs. AA-NRF1), 0.0000858 (WT-NRF1 vs. AA-NRF1 L01) ( c ); 0.001575 ( d ). a – d Experiments were repeated independently two times with similar results. Source Data are provided as a Source Data file.

Article Snippet: SP1 siRNA (#sc-29487), KLF5 siRNA (#sc-37718), NRF1 shRNA (#sc-38105-SH) and NSMCE2 siRNA (#sc-77813) were purchased from Santa Cruz Biotechnology.

Techniques: Modification, Expressing, Real-time Polymerase Chain Reaction, Biomarker Discovery, Binding Assay, ChIP-qPCR, Amplification, Transfection, Plasmid Preparation, Inhibition, Activity Assay, Luciferase, Control, Staining, Western Blot

Journal: Nature Communications

Article Title: Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

doi: 10.1038/s41467-020-19579-y

Figure Lengend Snippet: Genotoxicity provokes O-GlcNAc (G) elevation and dynamic changes in multiple OCTF genomic binding sites. The activity of multiple OCTFs, including NRF1, modulates a network of transcriptome upregulation to induce a holistic effect on cell fates in response to genotoxic stress.

Article Snippet: SP1 siRNA (#sc-29487), KLF5 siRNA (#sc-37718), NRF1 shRNA (#sc-38105-SH) and NSMCE2 siRNA (#sc-77813) were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation

doi: 10.1083/jcb.201708168

Figure Lengend Snippet: Nrf1 and NF-E2 activate p62 transcription in control cells and upon proteasome inhibition. (A) When different p62 promoter segments were fused to luciferase and expressed in M17 cells, the segments (P310 [−310 to 37] and P1068) that contain NFE2-E increased luciferase expression by ∼40% upon BTZ (0.1 µM) treatment for 16 h. However, the segment (P270) lacking NFE2-E showed no effect. n = 4. (B) NF-E2 knockdown by siRNA in HEK293A cells (verified by NF-E2 mRNA measurement in the left panel) reduced p62 mRNA (left) and protein (right) in cells treated or not for 16 h with 0.1 µM BTZ. (C) In M17 cells, knockdown of Nrf1 or NF-E2 , but not Nrf2 , reduced p62 level after BTZ treatment. (D) In HEK293A cells, knockdown of both NF-E2 (by siRNA) and Nrf1 (by stably expressing shRNA) caused additive reduction of p62 mRNA (left) and protein (right) after BTZ treatment. Molecular masses are given in kilodaltons. (E and F) Stable knockdown of Nrf1 with shRNA (E) suppressed p62 mRNA in untreated HAP1 cells or cells treated for 16 h with 100 nM BTZ or CFZ, and in SH-SY5Y cells treated with BTZ at indicated conditions (F). (G–J) Knockdown of Nrf1 in SH-SY5Y (G and H) or HAP1 (I and J) cells suppressed p62 mRNA when treated with 100 nM BTZ for 20 h (G and I) but did not suppress the mRNAs for most other Atg genes and Ub receptors (H and J). * , P < 0.05. Error bars indicate SD.

Article Snippet: Stable knockdown cell lines for Nrf1 (HEK293A and HAP1), Nrf2 (SH-SY5Y), p62 (M17 and SH-SY5Y), Nbr1 (M17 or SH-SY5Y), and GABARAPL1 (M17 or SH-SY5Y) were constructed using lentiviral particles expressing shRNAs for Nrf1 (sc-43575-V; Santa Cruz Biotechnology, Inc.), Nrf2 (sc-156128-V; Santa Cruz Biotechnology, Inc.), p62 (TRCN0000007235; Sigma-Aldrich), Nbr1 (TRCN0000123161; Sigma-Aldrich), or GABARAPL1 (TRCN0000060673; Sigma-Aldrich).

Techniques: Control, Inhibition, Luciferase, Expressing, Knockdown, Stable Transfection, shRNA

Journal: The Journal of Cell Biology

Article Title: Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation

doi: 10.1083/jcb.201708168

Figure Lengend Snippet: Summary of responses to proteasome inhibitors described in this study. When treated with proteasome inhibitors, which rapidly cause the buildup of soluble Ub conjugates, >50% of myeloma cells are killed by 20 h, but most neuroblastoma cells are viable at 40 h. Within 4 h, however, both cell types induce p62 and GABARAPL1 but not most Atg genes. The induction of p62 occurs simultaneously with that of all proteasome genes and also requires Nrf1. p62 promotes survival by sequestering nondegraded proteins in large cytoplasmic ubiquitinated and nuclear sumoylated aggregates, which are evident by 8 h. p62 accumulates only in cytoplasmic aggregates. Many hours later, prolonged treatment with a high concentration of inhibitors causes induction of all Atg genes and some lysosomal genes and activation of autophagy.

Article Snippet: Stable knockdown cell lines for Nrf1 (HEK293A and HAP1), Nrf2 (SH-SY5Y), p62 (M17 and SH-SY5Y), Nbr1 (M17 or SH-SY5Y), and GABARAPL1 (M17 or SH-SY5Y) were constructed using lentiviral particles expressing shRNAs for Nrf1 (sc-43575-V; Santa Cruz Biotechnology, Inc.), Nrf2 (sc-156128-V; Santa Cruz Biotechnology, Inc.), p62 (TRCN0000007235; Sigma-Aldrich), Nbr1 (TRCN0000123161; Sigma-Aldrich), or GABARAPL1 (TRCN0000060673; Sigma-Aldrich).

Techniques: Concentration Assay, Activation Assay