Li-jun Xiong

The histone H3 Lys 27 (H3K27) demethylase JMJD3 has been shown to play important roles in transcriptional regulation and cell differentiation. However, the mechanism underlying JMJD3-mediated transcriptional regulation remains incompletely understood. Here we show that JMJD3 is associated with KIAA1718, whose substrates include dimethylated H3K27 (H3K27me2), and proteins involved in transcriptional elongation. JMJD3 and KIAA1718 directly bind to and regulate the expression of a plethora of common target genes in both a demethylase activity-dependent and -independent manner in the human promyelocytic leukemia cell line HL-60. We found that JMJD3 and KIAA1718 collaborate to demethylate trimethylated H3K27 (H3K27me3) on a subset of their target genes, some of which are bivalently marked by H3K4me3 and H3K27me3 and associated with promoter-proximal, paused RNA polymerase II (Pol II) before activation. Reduction of either JMJD3 or KIAA1718 diminishes Pol II traveling along the gene bodies of the affected genes while having no effect on the promoter-proximal Pol II. Furthermore, JMJD3 and KIAA1718 also play a role in localizing elongation factors SPT6 and SPT16 to the target genes. Our results support the model whereby JMJD3 activates bivalent gene transcription by demethylating H3K27me3 and promoting transcriptional elongation. Taken together, these findings provide new insight into the mechanisms by which JMJD3 regulates gene expression.

<p>(A) CDC25A and FOXM1 proteins physically interact. The protein-protein interaction of FOXM1 and CDC25A was confirmed by co-immunoprecipitation. HEK293T cells were co-transfected with p3×FLAG-CMV-14-FOXM1 (FOXM1-3×FLAG) or the empty vector control p3×FLAG-CMV-14, along with pCMV3Tag9-CDC25A (CDC25A-3×MYC) or the empty vector control (pCMV3Tag9). Lysates were immunoprecipitated with anti-FLAG agarose resin, separated by PAGE, and electroblotted to PVDF. Western blot analysis showed that CDC25A-3×MYC co-immunoprecipitates with FOXM1-3×FLAG, supporting the hypothesis that these two expressed proteins interact. (B) The native protein interaction between FOXM1 and CDC25A was detected by co-immunoprecipitation. 6×10<sup>6</sup> U2OS cells were lysed in MPER buffer containing 1× protease/phosphatase inhibitors. Whole cell lysates were pre-cleared with protein G sepharose and normal rabbit IgG overnight at 4°C with end-over-end mixing. The cell lysates were incubated with anti-CDC25A antibody overnight at 4°C and then with protein G sepharose for 1 h at 4°C. The resin was washed three times, and the eluted protein complex was separated by PAGE and analyzed using the anti-FOXM1 antibody by Western blotting. (C) Schematic diagram showing FOXM1 deletion constructs used to identify FOXM1 domains critical to the interaction with CDC25A. Sequences encoding the specific FOXM1 deletion constructs were subcloned in pBIND. (D) A mammalian two-hybrid assay reveals a critical role for the FOXM1 C-terminus in interactions with CDC25A. pBIND construct (FOXM1, deletion, or empty) was co-transfected with pACT construct (CDC25A or empty) and pG5luc reporter. Data represent the mean ± SD, normalized to <i>Renilla</i> luciferase activities (N = 3). Co-expression of the C-terminal FOXM1 and CDC25A revealed a robust interaction. In a construct lacking the C-terminus, the interaction between FOXM1 and CDC25A was significantly diminished. *** p<0.001. (E) Schematic diagram showing the mutations of the CDK/cyclin phosphorylation sites and LXL motif at the C-terminal of FOXM1 protein. pBIND construct (FOXM1 wild-type and FOXM1 with mutations of phosphorylation sites/LXL motifs) was co-transfected with pACT construct (CDC25A or empty) and pGL5-luc reporter. Data represent the mean ± SD, normalized to <i>Renilla</i> luciferase activities (N = 3). Data were subjected to one-way ANOVA (significance level α = 0.05) and Dunnett's multi-comparison post-hoc tests (mutations vs. wild-type). *** p<0.001. (F) Mutation of T600 and T611 diminishes FOXM1 protein association with CDC25A by co-immunoprecipitation. U2OS cells were transiently transfected with either FOXM1-3×FLAG construct encoding wild-type FOXM1 (lane 1) or FOXM1 with the following mutations: T600A (lane 2), T611A (lane 3), T620A (lane 4) and L656A (lane 5). U2OS cell lysates were prepared 48 hours after transfection and FOXM1 expression in these cell lysates was detected by Western blot. The same amount of protein was co-immunoprecipitated with anti-CDC25A antibody, and the co-immunoprecipitated proteins were subjected to Western blot with an anti-FLAG antibody. The CDC25A expression in the co-immunoprecipitated proteins was used to test the efficiency of co-immunoprecipitation. The phosphorylation of T600 and T611 enhanced the protein interaction of FOXM1 and CDC25A.</p>