Fig. 6: Endogenous Golgin45 enters the nucleus and regulates gene transcription in response to heat stress.

a Schematic illustration of CRISPR-Cas9-mediated mutation of R375A in endogenous Golgin45. b HeLa wildtype and R375 knockin cells expressing myc-SUMO3 and Flag-UBC9 were subjected to immunoprecipitation using anti-Golgin45 antibody. c HeLa wildtype and R375A knockin cells were subjected to subcellular fractionation. Cells were treated with 5 μM XAV939 for 12 h, prior to heat shock (HS) or no heat shock (NHS) treatment for 2 h. The cytoplasmic and nuclear fractions of cell lysis were determined by western blotting using specific antibodies. W, whole cell lysate; C, cytoplasm; N, nucleus. d Relative protein level of Golgin45-WT or R375A in nucleus and cytoplasm compared to their own WCL was analyzed by two-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. n = 3 independent experiments. **P < 0.01. *P < 0.05. e, f Gene expression profiling of HeLa wildtype and Golgin45 KO cells before and after heat shock. Significantly upregulated genes in HeLa + HS vs. HeLa + NHS cells and HeLa + HS vs. HeLa Golgin45 KO + HS cells were subjected to GO enrichment analysis. g The transcription of ABHD16A, ACAD11, and SREBF1 were reduced in R375KI cells. Indicated genes were tested for mRNA expression levels in HeLa wildtype, R375A KI cells with or without heat stress treatment. Relative enrichment of genes was quantified and analyzed by one-way ANOVA with Tukey’s multiple comparisons. n = 3 independent experiments. ***P < 0.001. ****P < 0.0001. h Model for Golgin45 SUMOylation and its role in cellular stress response. Golgin45 gets imported into the nucleus via its interaction with Importin-β2. Golgin45 is conjugated to SUMO1 under steady state condition. Cellular stress (such as heat shock) promotes SUMO2/3 conjugation to Golgin45, leading to its inclusion into PML-NBs.