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.

HO-1 and Heme: G-Quadruplex Interaction Choreograph DNA Damage Responses and Cancer Growth

“…The above data indicate that nuclear HO-1 is an important effector protein in response to heme and may modulate G4 function by supporting temporary or permanent G4 unwinding. Our results are also in agreement with recent data showing that nuclear HO-1 co-localizes with G-quadruplexes and, similar to G4-unwinding helicases, it might be associated with lower G4 staining [78].…”

“…These questions will be addressed in future studies. Prior work demonstrated lower levels of G4 in normal hematopoietic cells deficient in HO-1 compared to wild type cells, which correlated with higher overall helicase levels (i.e., Brip1, Pif1) [ 78 ]. By using PLA technique in HEK293 cell line, it was demonstrated that HO-1 was localized in a proximity to G4 in the cytosol and to the much lesser extend in the nucleus and did not interact directly with G4 structures [ 78 ].…”

“…Prior work demonstrated lower levels of G4 in normal hematopoietic cells deficient in HO-1 compared to wild type cells, which correlated with higher overall helicase levels (i.e., Brip1, Pif1) [ 78 ]. By using PLA technique in HEK293 cell line, it was demonstrated that HO-1 was localized in a proximity to G4 in the cytosol and to the much lesser extend in the nucleus and did not interact directly with G4 structures [ 78 ]. Here, we provided an explanation how HO-1 by interacting with γH2AX foci in cancer cells may impact G4 structures as G4 are marked by γH2AX positive foci [ 80 ].…”

HO-1 Modulates Aerobic Glycolysis through LDH in Prostate Cancer Cells

“…Considering that HO-1 partially localizes in the nucleus in PCa human samples, and partially translocates to the nucleus under induction in different cell types [ 52 , 53 ], it might need to partner up with other transcription factors or co-regulators of transcription to exert its regulatory function, given that it does not contain DNA binding motifs. Moreover, it has been recently reported that nuclear HO-1 might influence gene transcription by regulating the stability of DNA G-quadruplexes structures [ 54 ].…”

The non-canonical effects of heme oxygenase-1, a classical fighter against oxidative stress

“…Heme binding to G4 can stabilize G4 maintain normal levels of free heme in the nucleus [ 53 ]. But numerous G4-heme binding can lead to increased oxidative stress on the DNA and G4 toxicity [ 55 ]. When the high-affinity ligand of G4 was used to compete with heme for G4 binding, the isolated heme in the nucleus was released and the expression of downstream target genes was regulated [ 53 ].…”