Activation of the p21 promoter by JMJD2D in HEK293T cells.

<p>(<b>A</b>) Activity of a p21 luciferase reporter construct upon cotransfection of control vector pEV3S or wild-type or H192A JMJD2D is depicted. As indicated, empty vector pcDNA3 or pcDNA3-p53 was also transfected. (<b>B</b>) Analogous, response of a CMV or MMP-1 luciferase reporter construct to cotransfection of p53 and/or JMJD2D. (<b>C</b>) Chromatin immunoprecipitation assay in HCT116 cells treated without and with adriamycin.</p

Mapping of interaction domains.

<p>(<b>A</b>) Indicated Flag-tagged amino acids of JMJD2D were coexpressed with p53 in HEK293T cells and complex formation assessed in coimmunoprecipitation assays as in Fig. 1B. A sketch of human JMJD2D highlighting its JmjC domain is presented at the bottom. (<b>B</b>) Comparable amounts of GST or indicated GST-p53 fusion proteins were bound to glutathione agarose. After incubation with Flag-tagged JMJD2D, bound JMJD2D was revealed by anti-Flag Western blotting. The location of the DNA binding domain within p53 is indicated in the sketch at the bottom.</p

JMJD2D protects from apoptosis.

<p>(<b>A</b>) The level of sub-G1 HCT116 cells was determined in cells that expressed control shRNA or JMJD2D shRNA#3. Cells were treated for 72 h with 1 µM adriamycin or DMSO as indicated. (<b>B</b>) The same in case of p53^−/− HCT116 cells. (<b>C</b>) Model of JMJD2D action.</p

Stimulation of p21 expression by JMJD2D in U2OS cells.

<p>(<b>A</b>) Wild-type or H192A JMJD2D or empty vector pEV3S were stably transfected into U2OS cells. Expression of indicated proteins was assessed by Western blotting in cells treated for 24 h with 1 µM adriamycin or DMSO as a control. (<b>B</b>) RNA was isolated from stably transfected U2OS cells and RT-PCR analyses were performed. Shown is the amplification of p21 cDNA and, as a control, GAPDH cDNA.</p

Binding of JMJD2D to p53.

<p>(<b>A</b>) Flag-tagged JMJD2D or HSPBAP1 were coexpressed with p53 in HEK293T cells. After anti-p53 immunoprecipitation (IP), coprecipitated proteins were revealed by anti-Flag immunoblotting (top panel). The bottom two panels show input levels of Flag-tagged proteins or p53. IgH, immunoglobulin heavy chain. (<b>B</b>) Indicated Flag-tagged JMJD proteins were coexpressed with p53 in HEK293T cells. After anti-Flag immunoprecipitation, coprecipitated p53 was detected by anti-p53 Western blotting (top panel). The middle and bottom panels show p53 and Flag-JMJD protein input levels, respectively. (<b>C</b>) HCT116 cell extracts were challenged with no, control or anti-JMJD2D antibodies and coprecipitated p53 detected by immunoblotting (top panel). The bottom panel shows that respective JMJD2D input levels were equal.</p

JMJD2D depletion results in reduced cell proliferation.

<p>(<b>A</b>) HCT116 cells expressing control or JMJD2D shRNA were treated with 1 µM adriamycin or with DMSO as a control for 24 h. Downregulation of JMJD2D was assessed by Western blotting; immunoblotting for actin served as a loading control. (<b>B</b>) Wild-type or p53^−/− HCT116 cells were challenged with control or JMJD2D shRNA (#1 or #3) and then treated without or with 1 µM adriamycin for 72 h. The number of cells were counted and presented as percent of the control shRNA for each wild-type and p53^−/− HCT116 cells. Statistical significance of differences between various experimental conditions is indicated in the graph.</p

JMJD5 is a regulator of circadian gene expression.

Genetic ablation of JMJD5 in (A) MEFs (mean ± SEM, n = 3) or (B) mouse liver disrupts the expression pattern and levels of clock genes (*p p n = 4, one-tailed permutation test). Levels were normalized to mrpl46, and the maximum level of the indicated transcript in the wild-type genotype was set as 1. CT, circadian time; JMJD5, JmjC domain–containing protein 5; MEF, mouse embryo fibroblast.</p

Relationship between CRY1 stability and function.

(A–F) Real-time luciferase measurements of Per1 and Per2 promoter activity in non-oscillating HEK293T cells show repression of CB by WT CRY1 or AA (mean ± SD, counts ×103). (G) CRY1 levels observed in transfections with the indicated plasmid amounts (mean ± SD, n = 3). (H) JMJD5 (denoted by a “J”) cooperates with WT CRY1 to repress CB and (I) compensates for the decreased repressive activity of AA. (J) JMJD5 destabilizes FLAG-AA in a CHX chase assay in HEK293T cells. (K) Quantification of comparing effect of JMD5 on CRY1 AA (J) versus WT CRY1 (data from Fig 3A). §, AA versus AA+JMJD5 p p p p p = 0.05 (WT n = 8, others n = 5, mean ± SEM, one-tailed Mann-Whitney U-test). AA, CRY171A/280A mutant; CB, circadian locomotor output cycles protein kaput–brain and muscle ARNT-like protein 1; CHX, cycloheximide; CRY, CRYPTOCHROME; HEK293T, human embryonic kidney 293T; JMJD5, JmjC domain–containing protein 5; V, vector; WT, wild-type.</p

JMJD5 forms a complex with CRY1–FBXL3 and leads to CRY1 degradation.

(A) JMJD5 interacts with CRY1. Co-IP experiments show the binding profile of (B) FBXL3 and (C) JMJD5 with CRY1 WT and the indicated CRY1 mutants. Inputs for (B) and (C) are shown in S7 Fig. (D) Knockdown of FBXL3 decreases JMJD5 co-IP with CRY1. Densitometric analysis showing ratio of JMJD5 to CRY1 signals from these experiments (mean ± SEM, n = 4) are shown in (E). (F) CRY1 knockdown has no effect on JMJD5–FBXL3 interactions. (G) JMJD5 knockdown does not alter CRY1–FBXL3. (H) CRY1 accumulation in response to MG132 treatment is decreased in JMJD5-null cells compared to controls, whereas ubiquitylation is unaffected (S10 Fig). (I) Quantification of four independent MG132 block experiments shown as baseline-normalized of total CRY1 signal (mean ± SEM, n = 4). Regions used for quantification are described in S10 Fig. Twelve-hour time points were omitted as significant cell death was observed. (J) Co-IP of HA-RPN1 and endogenous CRY1 shows diminished binding Jmjd5−/− MEFs, quantified in (K), presented as CRY1:RPN1 signal ratios (mean ± SEM, n = 6). CRY, CRYPTOCHROME; FBXL3, F-box/leucine-rich repeat protein 3; IP, immunoprecipitation; KO, knockout; JMJD5, JmjC domain–containing protein 5; MEF, mouse embryo fibroblast; RPN1, proteasome regulatory particle non-ATPase 1; shRNA, short hairpin RNA; siRNA, small interfering RNA; WT, wild type.</p

JMJD5 is a repressor of the circadian clock.

(A, B) Dose-response repression of CLOCK and BMAL1 (“CB”) by JMJD5 (“J”). Real-time luciferase measurements of Per1 and Per2 promoter activity in non-oscillating HEK293T cells show repression of CB by JMJD5. Shown: counts normalized to maximal CB activation of the indicated reporter (mean ± SD) (C) JMJD5 repression is E-box mediated. JMJD5 represses Per1-Luc with a wt but not one with a mutant (“mut”) E-box (mean ± SD). (D) JMJD5 effect on wt and E-box mutant Per1 reporters in the absence of CB. Shown: counts normalized to maximum signal obtained with wt reporter in the absence of JMJD5. (E) Per1 and (F) Per2 promoter is independent of the catalytic activity of JMJD5, normalizations performed as in A (mean ± SD). Please note that data plotted in A and E and in B and F were collected simultaneously; V, CB, and CB+JMJD5 in E and F are the same data as in A and B, respectively. BMAL1, brain and muscle ARNT-like protein 1; CLOCK, circadian locomotor output cycles protein kaput; HEK293T, human embryonic kidney 293T; JMDJ5, JmjC domain–containing protein 5; V, vector; wt, wild type.</p