PMC

Genomic structure of mir-21. Location of a previously described promoter (miPPR-21) [], our putative regulatory region (GIS) [], a H3K4me3 binding site as determined by previous ChIP-seq [], and a STAT3 binding site according to Loffler et al. []. Both miPPR-21 and GIS regions are highly conserved.

p53-NF-κB mediated mir-21 expression is dependent on STAT3. (a) Cardiac fibroblasts were treated with DFX with or without an inhibitor of STAT3 DNA binding (S3I-201), and cell lysates were incubated with streptavidin-coated beads on which biotinylated GIS duplex (oligo pulldown) or a scrambled sequence duplex (scrambled) was immobilized. Proteins bound to these duplexes were eluted and western blotted (WB) for STAT3, RELA and p53. (b) p53-RELA sequential ChIP was performed on cardiac fibroblasts with or without DFX and S3I-201. Results are presented as mean ± standard error for three independent experiments performed in triplicate. (c) Cardiac fibroblasts were treated with or without DFX and S3I-201, and mir-21 was quantified using the TaqMan miRNA assay. (d) Nuclear extracts (NE) from cardiac fibroblasts with or without DFX and S3I-201 were isolated and western blotted for STAT3, RELA and p53. (e) Using cell lysates from cardiac fibroblasts treated with DFX with or without S3I-201, RELA or control IgG immunoprecipitation (IP) was performed and western blotted for p53, STAT3 and RELA. (f) Stat3^-/- MEF cells and Stat3^-/- MEF cells that were re-constituted with wild-type Stat3 were treated with or without DFX and quantification for mir-21 was performed. All miRNA quantification results are presented as mean ± standard error, from three independent experiments and performed in triplicate. Asterisks represent P < 0.05.

p53 and NF-κB form a complex and occupy the putative GIS regulatory region simultaneously. (a) ChIP was performed on cardiac fibroblasts with or without DFX using antibodies against either p53 or RELA. Results show fold enrichment of real-time qPCR for the putative regulatory sequence (GIS). (b) ChIP using a p53 antibody was performed on cardiac fibroblasts with or without DFX and NFI. ChIP results are presented as mean ± standard error for three independent experiments performed in triplicate. (c) Using cell lysates from cardiac fibroblasts treated with DFX with or without NFI, RELA or control IgG immunoprecipitation (IP) was performed followed by western blotting (WB) for p53 (left), and vice versa (right). Arrows indicate RELA. (d) Cardiac fibroblasts were treated with or without DFX and NFI, and p53 ChIP was performed followed by 'release' of the chromatin, and RELA re-ChIP. Results represent fold enrichment of real-time qPCR for GIS. Re-ChIP results are presented as mean ± standard error for two independent experiments performed in triplicate. (e) Lysates from cardiac fibroblasts treated with or without DFX were incubated with streptavidin-coated beads on which biotinylated GIS duplexes (oligo pulldown) or scrambled sequence duplexes (scrambled) were immobilized. Proteins bound to these duplexes were eluted and western blotted for p53, RELA and NF-κB subunit p50. Asterisks represent P < 0.05 (paired t-test).

The p53-NF-κB complex is present at the GIS regulatory region in human dilated cardiomyopathic hearts. (a-c) Human left ventricular tissue sections immunostained for NF-κB/RELA showed that NF-κB/RELA (in brown) was predominantly cytoplasmic in control left ventricule (a) but nuclear in both myocytes (open arrows) and fibroblasts or non-myocytes (closed arrows) of cardiomyopathic left ventricule (b,c). (d) No primary antibody control. (e,f) Sections were also immunostained for both a marker of oxidative DNA damage (8-oxoG, in brown) and activated p53 (phospho-p53^ser15 (e); phospho-p53^ser20 (f); in black with closed arrows). (g) Myocytes were distinguished from non-myocytes and fibrotic tissue both by their characteristic striations and positive staining for ankyrin (in brown). Bar represents 100 μm. (h) Left ventricular tissues from normal hearts and cardiomyopathic hearts were used for p53-RELA re-ChIP. Results represent fold enrichment of real-time qPCR for GIS and are representative of two replicated experiments using the same eight left ventricular samples. **P < 0.0005. Patient details for these left ventricular samples are in Additional file .

NF-κB forms a complex at the GIS regulatory region with both wild-type p53 and p53 with a DNA-binding domain mutation. (a) p53-deficient Soas2 cells were transfected with wild-type p53 (p53WT), DNA binding domain mutant p53 (p53R175H) or vector control, and cell lysates were incubated with GIS duplexes as in Figure 2e. Proteins bound to the GIS duplex were western blotted (WB) for p53 (left panel). The right panel shows input from transfected Soas2 cell lysates. (b) p53-deficent Soas2 cells were transfected with wild-type p53 (p53WT), mutant p53R175H (p53RH) or vector control. Cell lysates were co-immunoprecipitated with anti-RELA antibody or isotypic IgG control, and western blotting was performed for p53. (c) As in Figure 2c, GIS-luciferase and TK-renilla control were transfected into p53^-/- MEF cells with or without plasmids encoding p53 (WT and RH, respectively) and RELA, and incubated with or without NFI as indicated. Firefly luciferase gene reporter activity was normalized to renilla control. Asterisks represent P < 0.05 for treatment versus control, and with inhibitor versus without inhibitor. Luciferase reporter assays are presented as mean ± standard error for at least three independent replicates.

p53 and NF-κB cooperate to induce mir-21. (a) Primary neonatal rat cardiac fibroblasts were treated with or without DFX and the NF-κB inactivator (NFI; 1 μM quinazoline) and mir-21 was quantified using the TaqMan miRNA assay. (b) Nuclear extracts from cardiac fibroblasts with or without DFX and NFI as in (a) were isolated and western blotted (WB) for p53 and RELA. Splicing factor 2 (SF2), was used to confirm equal protein loading. {(c) GIS-luciferase (GIS-Luc) and TK-renilla control (RL) were transfected into p53^-/- MEF cells with or without plasmids encoding p53 or RELA, and incubated with or without NFI as indicated. Firefly luciferase gene reporter activity was normalized to renilla control. (d) GIS, GIS with an AAA mutation engineered into the putative NF-κB binding site (GISmAAA), and GIS with the NF-κB binding site deleted (GISdel) were cloned upstream of firefly luciferase. Constructs were transiently transfected together with a TK-renilla luciferase plasmid and plasmids encoding p53 and RELA into p53^-/- MEF cells and firefly luciferase reporter gene activity (FL) was quantified and normalized against renilla (RL). Results represent a fold-difference between the three different GIS-constructs. (e) RelA^-/- (-/-) MEF cells and RelA^-/- MEF cells that were reconstituted with RELA using lentiviral overexpression (RA) were treated with or without DFX, and mir-21 quantification was performed. (f) RelA^-/- and reconstituted RelA^-/- MEF cells were treated with or without DFX, and whole cell (WCE) and nuclear (NE) extracts were western blotted for MDM2 (arrow), BAX and RELA. RhoGDI and SF2 were used to demonstrate protein loading. miRNA quantification is shown as mean ± standard error, at least n = 3. Luciferase reporter assays are presented as mean ± standard error for at least four independent replicates. Asterisks represent P < 0.05 (paired t-test).