Figure 1.
a, The number of JMJD3, UTX and p53 peptides identified by mass spectrometry in the tandem anti-Flag-HA purifications of Flag–HA–JMJD3 and Flag-HA-UTX stably expressed in HEK293 cells. b, Phoenix cells were transfected with HA-JMJD3, HA-UTX or an empty vector (Emp), and immunoprecipitated with an antibody against the HA-tag. Western blotting was performed with antibodies against HA or p53. c, Co-immunoprecipitation of endogenous JMJD3 and p53 was performed in Phoenix cells by immunoprecipitating with HA (negative control) or JMJD3 antibody. d, Schematic representation of wild type (wt) p53 and the three p53 deletion mutants lacking the regulatory (REG), the tetramerization (TET) or the transactivating (TA) domains as indicated. e, Pheonix cells were transfected with myc-tagged JMJD3 alone or together with wt p53 or p53 deletion mutants as indicated. Immunoprecipitation was performed with myc- or HA antibody followed by western blotting.
Figure 2.
Genome-wide binding of JMJD3 and p53 before and after IR treatment.
a, Western blot of JMJD3, p53, p-p53 (p53 serine 15 phosphorylation) and vinculin in human immortalized BJ fibroblasts either untreated or 6 hours after exposure to IR. b, The number of identified binding sites and target genes for p53 and JMJD3 (defined as binding of p53 or JMJD3 +/− 5 kb from TSS) in untreated or IR exposed cells. c, The distribution of identified p53 peaks (left panel) or JMJD3 peaks (right panel) in IR treated cells into promoter regions (+/−5 kb from TSS), intragenic regions or intergenic regions (> 5 kb from a gene). d, Gene Ontology analysis of JMJD3 target genes in IR treated BJ cells. e, Heat map of p53 and JMJD3 ChIP-seq data from untreated or radiated cell representing the 3232 identified p53 binding sites. The heat map is ranked according to the FDR-value of the p53 peaks. f, The average distribution of p53, JMJD3 and IgG across the centre of all p53 peaks before and after IR.
Figure 3.
IR responsive recruitment of JMJD3 and p53 to promoter regions.
a, Venn diagram demonstrating significant (p<10−8) overlap between JMJD3 and p53 target genes in the IR treated BJ cells. At least 263 genes were found to be bound by both JMJD3 and p53 in the promoter regions, which included several well-known p53 target genes such as CDKN1A, BBC3, TP53INP1, MDM2, GADD45A, RPS27L, CCNG1, TNFRSF10B, and DDB2. b, Examples of p53 (before and after IR treatment) and JMJD3 (before and after IR treatment) ChIP-seq tracks at BBC3, MDM2, TP53INP1, RPS27L and HOXB9 (negative control). y-axis of binding profiles denotes number of sequence tag reads. c, The corresponding p53 and JMJD3 ChIP-qPCR validations at the TSS of the genes listed above.
Figure 4.
IR responsive recruitment of JMJD3 and p53 to distal enhancer elements.
a, Heat map representing p53 binding sites divided into promoter associated (< 5 kb from a TSS, 487 peaks) or distal binding sites (> 5 kb from a TSS, 2745 peaks). b, Venn diagrams illustrating the overlap between p53 and JMJD3 co-bound distal elements and DNase I hypersensitive sites (left panel) or H3K4me3 positive regions (right panel). c, Examples of p53 (before and after IR treatment) and JMJD3 (before and after IR treatment), H3K4me3 and DNase I-seq [34], [35], [43] tracks at two putative enhancer elements located 11 kb upstream of CDKN1A (upper panel) or 19 kb upstream of GML (lower panel). d, Corresponding ChIP-qPCR validations of the binding of p53, JMJD3 and p300 as well as the levels of histone modifications H3K4me1, H3K4me3, H3K27ac and H3K27me3 at the two distal binding sites listed above.
Figure 5.
JMJD3 binding to p53 binding sites is dependent on p53.
a, Western blot of control (ctrl) or p53 knockdown (shp53) cells. The cells were exposed to IR and harvested for ChIP-qPCR assays after five hours. b and c, ChIP-qPCR data demonstrating the binding of p53 and JMJD3 to the promoters of p53 target genes BBC3, TP53INP1, MDM2, and RPS27L (b) or to the CDKN1A -11 kb and GML -19 kb distal elements (c) in control or p53 knockdown cells. d, ChIP-qPCR data demonstrating the binding of p53 and JMJD3 to HIST1H3E and EIF3B, which are not p53 target genes.
Figure 6.
Model of the potential roles of JMJD3 in regulating p53 target genes.
JMJD3 is recruited to p53 bound promoters and distal enhancer elements via an interaction with the p53 tetramer, which simultaneously recruits the histone acetyltransferase p300. JMJD3 could be involved in adjusting p53 transcriptional regulation by demethylating H3K27me3/me2, demethylating other non-histone proteins, or by having catalytically independent functions. See text for further details.