KRas-ERK signalling promotes the onset and maintenance of uveal melanoma through regulating JMJD6-mediated H2A.X phosphorylation at tyrosine 39

Figures & data

Figure 1. H2A.X^Y39ph was regulated via Ras-ERK1/2 signalling. MP65 cells were transfected with pEGFP-N1 vector (an empty plasmid), Ras^G12D/T35S plasmid, or Ras WT plasmid. H2A.X^Y39ph expression was examined through (A) Western blot followed by (B) densitometry analysis. **p < .01 (n = 3), pEGFP-N1 versus Ras^G12D/T35S. (C) ERK1/2 expression was measured by Western blot.

Figure 2. Silence of H2A.X^Y39ph contributed to the regulation of uveal melanoma cell phenotypes. pEGFP-H2A.X, Ras^G12D/T35S plasmid, or increasing amounts of H2A.X^Y39A plasmid (0.5, 1, and 2 g) were transfected into MP65 cells. H2A.XY39A plasmids were constructed to mimic H2A.X^Y39ph. (A) OD values in MTT assay reflected cell viability. (B) Colonies numbers were measured through soft-agar colony formation experiment. (C) OD values in Transwell assay reflected cell migration. **p < .01, Ras + H2A.X versus Ras + H2A.X^Y39A, ***p < .001 (n = 3).

Figure 3. H2A.X^Y39ph modulated ERK1/2 downstream genes’ transcription. (A) RNA levels of several genes were tested through qRT-PCR after transfection with pEGFP-H2A.X, Ras^G12D/T35S plasmid + pEGFP-H2A.X, or Ras^G12D/T35S plasmid + pEGFP-H2A.X ^Y39A, respectively. (B) These genes’ transcription was examined through ChIP assay after transfection with pEGFP-H2A.X, or Ras^G12D/T35S plasmid + pEGFP-H2A.X. *p < .05, **p < .01, GFP + H2A.X versus Ras + H2A.X, Ras + H2A.X versus Ras + H2A.X^Y39A, ***p < .001 (n = 3).

Figure 4. Overexpression of JMJD6 recovered H2A.X^Y39ph expression and uveal melanoma cell phenotypes. (A) JMJD6-HA (a HA-tagged plasmid for expression of JMJD6) or an empty vector was transfected into MP65 cells. Gel electrophoresis showed the mRNA level of JMJD6. (B) Ras^G12D/T35S plasmid, JMJD6-HA, or empty vector was transfected into MP65 cells. The expression of H2A.X^Y39ph was examined through Western blot. (C) Cell activity was examined through MTT experiment. (D) Colonies numbers were measured through soft-agar colony formation experiment, and (E) cell cycle progression tested through flow cytometry were done after transfection with Ras^G12D/T35S plasmid, JMJD6-HA (with raising concentrations, i.e. 0.5 and 1 g), or empty vector. *p < .05, ***p < .001 (n = 3).

Figure 5. Activation of ERK1/2 induced the degradation of JMJD6 and led to the down-regulation of H2A.X^Y39ph. (A) Ras^G12D/T35S plasmid or an empty vector was transfected into MP65 cells. Gel electrophoresis showed the mRNA level of JMJD6. (B) MP65 cells were transfected with JMJD6-HA, Ras^G12D/T35S plasmid or empty vector. JMJD6 expression was examined through Western blot. (C) Ras^G12D/T35S plasmid or an empty vector was transfected into MP65 cells. Protein levels of JMJD6 and H2A.X^Y39ph were examined through Western blot analysis. (D) If JMJD6 bind with the promoter of ERK1/2 downstream genes was examined through ChIP experiment. (E) JMJD6 expression was measured through Western blot after treating with MG132. (F) H2A.X^Y39ph expression was measured through Western blot after 0–60 h transfection. (G) H2A.X^Y39ph expression was examined through Western blot after treating with MG132 for 0–12 h. *p<.05, **p<.01, GFP versus Ras, ***p<.001 (n = 3).

Figure 6. Activation of ERK1/2 degraded JMJD6 via modulating MDM2. Ras^G12D/T35S, JMJD6-HA, MDM2-His (a His-tagged plasmid for expression of MDM2), or empty vector was transfected into MP65 cells. (A) Exogenous and (B) endogenous expression of JMJD6 was examined through Western blot. MDM2-His was treated with increasing concentrations (0.5, 1.0 and 2.0 g). Subsequently, MDM2-His or MDM2-MU (the mutated MDM2) were transfected into cell and the (C) exogenous and (D) endogenous expression of JMJD6 was measured through Western blot. (E) The expression of MDM2 and H2A.X^Y39ph were examined after transfection. (F) siRNA transfection was done to repress MDM2 expression. (G) The expression changes of H2A.X^Y39ph were tested when MDM2 was silenced. All protein levels were tested by Western blot analysis.

Figure 7. Diagram was made to explain how Ras-ERK1/2 and JMJD6-MDM2 regulate H2A.X^Y39ph.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.