Proximity Ligation Assay Detection of Protein-DNA Interactions-Is There a Link between Heme Oxygenase-1 and G-quadruplexes?

Abstract

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1^-/- mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4-protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.

Keywords: G-quadruplex; heme; heme oxygenase-1; proximity ligation assay.

Figure 1

Mouse hematopoietic stem cells from Hmox1^−/− mice had a higher expression of genes associated with G4 unwinding and showed lower G4 staining. The expression of (A) Pif1, (B) Blm, (C) Brip1, (D) Brca1, (E) Wrn, (F) Polq, and (G) Top2a was assessed with RNA-Seq in fluorescence-activated cell-sorting (FACS)-sorted phenotypic bone marrow hematopoietic stem cells (HSCs) (data source: [42]). The box-and-whiskers graphs show the median, 25th and 75th percentile (box) minimum, and maximum values (whiskers) of the FPKM (fragments per kilobase million), n = 4, * p < 0.05, Mann–Whitney test. (H) G4 staining shown as the mean fluorescence intensity (MFI) ratio of cells stained with anti-G4 antibodies and the background fluorescence (secondary antibodies only) in hematopoietic stem and progenitor cells (KLS), HSCs, multipotent progenitors (MPPs), or granulocyte-monocyte progenitors (GMPs) from Hmox1^+/+ and Hmox1^−/− mice, n = 4–5, violin plot, * p < 0.05, two-way ANOVA with a Bonferroni post hoc test, for genotype p = 0.002.

Figure 2

Expression of Slc48A1 and Slc46A1 in mouse Hmox1^+/+or Hmox1^−/− HSCs (A) and mouse Hmox1^+/+ or Hmox1^−/− iPSCs (B). Expression of Pif1 (C) and Brip1 (D) at the mRNA (graphs) and protein (photos) levels in iPSCs. Gene expression was assessed with RNA-seq in FACS-sorted bone marrow HSCs [42] or with real-time RT-PCR in cultured iPSCs. The box-and-whiskers graphs show the median, 25th and 75th percentile (box) minimum, and maximum values (whiskers) of the FPKM (fragments per kilobase million) or relative expression in comparison to house-keeping genes (geometric mean for HPRT, B2M, and β-actin), n = 4, * p < 0.05, ** p < 0.01, Mann–Whitney test (HSCs) or Wilcoxon test (iPSCs). Representative pictures showing Pif1 and Brip1 immunostaining. Nuclei are labeled with DAPI. Scale bars: 20 µm.

Figure 3

G-quadruplexes in iPSCs. (A) G4-specific staining (magenta) of Hmox1^+/+ or Hmox1^−/− iPSCs that were untreated (control) or treated with hemin (2 μmol/L, 4 h) and (B) quantitative analysis of the fluorescence intensity. Analysis of cells from three independent experiments, Kruskall–Wallis test, ** p < 0.01, *** p < 0.001. Scale bars: 20 µm. (C) Staining for Hmox1 (green), G-quadruplexes (red), and DAPI (blue) in Hmox1^−/− and Hmox2^−/− iPSCs with re-introduced cytoplasmic or nuclear Hmox1. Scale bars: 50 µm.

Figure 4

G4 immunolabelling in HEK293T cells. (A) Mouse and (B) goat 1H6 antibodies were used to visualize the G4 structures (magenta). (C) G4-specific staining (mouse antibodies) disappeared after the treatment with DNase but were still visible after the treatment with RNase. Cells were fixed in methanol. Nuclei were counterstained with DAPI (blue) or histone H3 (green), where transmission bright-field (BF) images show the cell morphologies. Scale bars: 10 μm.

Figure 5

In situ proximity ligation assay (PLA) in HEK293T cells. (A) G4-BRIP1 and (B) G4-HMOX1 interactions (red dots) were visualized in the control group and after DNase or RNase digestion. Most of the G4 interactions were localized in the nucleus (arrows), but some were also present in the cytoplasm. Cells that were fixed in methanol and mouse 1H6 antibodies were used. Nuclei were counterstained with DAPI (blue), where the transmission bright-field images (BF) show the cell morphologies. Scale bar: 10 μm.

Figure 6

Detection and quantification of the PLA signal at a single-cell level in HEK293T cells using flow cytometry. (A) G4-BRIP1 and (B) G4-HMOX1 interactions (green dots) were visualized in the control group and after DNase digestion. Most of the G4 interactions were localized in the nucleus (arrows) but some also appeared in the cytoplasm. Nuclei were counterstained with DAPI (blue), where the transmission bright-field images (BF) show the cell morphologies. Cells were fixed in methanol and mouse 1H6 antibodies were used. Scale bars: 10 μm. (C) Mean fluorescence of G4-BRIP1 and G4-HMOX1 PLA signal and (D) DAPI in the control cells and after DNase digestion. Negative controls were cells stained with secondary antibodies only. Data is shown as mean + SEM (standard error of the mean).

Figure 7

Detection of the PLA signal in sorted hematopoietic stem cells. (A) G4–Brip1 and (B) G4–Hmox1 interactions (green dots) were visualized in HSCs and GMP and MPP cells. G4 interactions were visible in the nucleus (arrows) and in the cytoplasm. Nuclei were counterstained with DAPI (blue), where the transmission bright-field images (BF) show the cell morphologies. Cells were fixed in PFA and goat 1H6 antibodies were used. Scale bars: 5 μm.