Fig 2: HNF4A-AS1 facilitates growth and invasion of NB cells via hnRNPU-mediated transactivation of CTCF. a Volcano plots of RNA-seq revealing alteration of gene expression (fold change > 2.0, P < 0.05) in SH-SY5Y cells stably transfected with empty vector (mock) or HNF4A-AS1. b By analyzing with ChIP-X program and BioGRID database, venn diagram (left panel) showing identification of hnRNPU-interacting transcription factors (TFs) regulating genes altered in RNA-seq and correlated with HNF4A-AS1 in a public dataset (GSE45547). Gene network (right panel) revealing identified TFs and target genes. c Co-IP and Western blot assays indicating the interaction between hnRNPU and CTCF in SH-SY5Y and BE(2)-C cells stably transfected with mock, HNF4A-AS1, scramble shRNA (sh-Scb), or sh-HNF4A-AS1 #1. d Confocal images of BiFC assay showing direct interaction between hnRNPU and CTCF (arrowheads) within SH-SY5Y and BE(2)-C cells co-transfected with pBiFC-VN173-hnRNPU and pBiFC-VC155-CTCF, and those stably transfected with mock, HNF4A-AS1, sh-Scb, or sh-HNF4A-AS1 #1. Scale bars, 10 µm. e ChIP and real-time qPCR (normalized to input) assays indicating the CTCF enrichment on target gene promoters in SH-SY5Y cells stably transfected with mock or HNF4A-AS1, and those co-transfected with sh-hnRNPU (n = 5). f and g Real-time qRT-PCR (f, normalized to ß-actin, n = 4) and Western blot (g) assays showing the levels of HNF4A-AS1, hnRNPU, CTCF, and target genes in SH-SY5Y cells stably transfected with mock or HNF4A-AS1, and those co-transfected with sh-Scb, sh-hnRNPU #1, or sh-CTCF #3. h and i Representative images (left panel) and quantification (right panel) of soft agar (h) and matrigel invasion (i) assays indicating anchorage-independent growth and invasion of SH-SY5Y cells stably transfected with mock or HNF4A-AS1, and those co-transfected with sh-hnRNPU #1, sh-CTCF #3, or sh-HNF4A #2 (n = 5). ANOVA compared the difference in e, f, h, and i. *P < 0.05 vs. mock+sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c–i

Fig 3: C280 SAF-A mutant displaces specific nuclear proteins and not others. For all images: color channels are separated in black and white. Scale bars 5 µm. Cell types: normal interphase human fibroblasts (Tig-1). A–B Expression of the C280-GFP SAF-A mutant had no effect on the overall nuclear distribution of LaminB1 (B) or NuMA (A) (top cell was transfected with the C280 vector while bottom cell was not). However, the deletion mutant frequently led to reduced levels of NuMA. Notably, in many cells the deletion mutant disrupts the normal distribution of FUS (C), hnRNPc (D), and SAFB1 (E). All nuclei shown are in interphase (not prophase or mitosis) and changes in DNA corresponded to presence of SAF-A mutant. Control cells lack green C280-GFP signal. Additional images in Supplemental Fig. 2 and 3

Fig 4: Nup98 depletion alters the intranuclear distribution of DHX9, but not hnRNP U.HEK293T cells were transfected with shRNA targeting Nup98 or with a control shRNA. Four days later the cellular distributions of Nup98 and either DHX9 or hnRNP U were examined by indirect immunofluorescence. Cells depleted of Nup98 show partial relocation of DHX9 into intranuclear foci (white arrows). Merged images show DHX9 or hnRNP U (red), Nup98 (green), and DAPI-stained DNA (blue). Scale bars, 5 µm. The cellular localization of Nup98 is not affected by depletion of DHX9 (see Figure 4—figure supplement 1). Protein depletion in these experiments was confirmed by immunoblot analysis (Figure 4—figure supplement 2).DOI: http://dx.doi.org/10.7554/eLife.18825.006

Fig 5: HNF4A-AS1 interacts with hnRNPU protein in NB cells. a Coomassie blue staining (left panel) and mass spectrometry (MS) assay (right panel) of indicated electrophoretic bands revealing the identification of protein pulled down by biotin-labeled HNF4A-AS1 in BE(2)-C cells. b Biotin-labeled RNA pull-down and Western blot assays showing the hnRNPU protein pulled down by sense or antisense (AS) HNF4A-AS1 from lysates of BE(2)-C cells. The HNF4A-AS1 AS- and bead-bound protein served as negative controls. c Dual RNA-FISH and immunofluorescence staining assay indicating the co-localization of hnRNPU and HNF4A-AS1 in the nuclei of SH-SY5Y cells stably transfected with empty vector (mock) or HNF4A-AS1. d RIP (upper right panel) and Western blot (lower right panel) assays using hnRNPU antibody showing the interaction between HNF4A-AS1 and hnRNPU protein in SH-SY5Y cells transfected with a series truncations of HNF4A-AS1 (left panel). The IgG-bound RNA was taken as a negative control. e Biotin-labeled RNA pull-down assay revealing the interaction between HNF4A-AS1 truncations and hnRNPU protein in BE(2)-C cells. Bead-bound protein served as a negative control. f RNA EMSA assay using biotin-labeled probes indicating the interaction of HNF4A-AS1 truncations with recombinant GST-tagged hnRNPU protein, with or without competition using an excess of unlabeled homologous probe. g Biotin-labeled RNA pull-down and Western blot assays showing the recovered hnRNPU truncations (upper panel) after incubation of biotin-labeled HNF4A-AS1 with full-length or truncated forms of GST-tagged recombinant hnRNPU protein (lower panel). h RIP assay using FLAG antibody indicating the interaction between HNF4A-AS1 truncations and FLAG-tagged hnRNPU protein in BE(2)-C cells. The IgG was applied as a negative control. Data are representative of three independent experiments in b–h