p53-targeted LSD1 functions in repression of chromatin structure and transcription in vivo

Abstract

Despite years of study focused on the tumor suppressor p53, little is understood about its functions in normal, differentiated cells. We found that p53 directly interacts with lysine-specific demethylase 1 (LSD1) to alter chromatin structure and confer developmental repression of the tumor marker alpha-fetoprotein (AFP). Chromatin immunoprecipitation (ChIP) and sequential ChIP of developmentally staged liver showed that p53 and LSD1 cooccupy a p53 response element, concomitant with dimethylated histone H3 lysine 4 (H3K4me2) demethylation and postnatal repression of AFP transcription. In p53-null mice, LSD1 binding is depleted, H3K4me2 is increased, and H3K9me2 remains unchanged compared to those of the wild type, underscoring the specificity of p53-LSD1 complexes in demethylation of H3K4me2. We performed partial hepatectomy of wild-type mouse liver and induced a regenerative response, which led to a loss of p53, increased H3K4me2, and decreased LSD1 interaction at AFP chromatin, in parallel with reactivation of AFP expression. In contrast, nuclear translocation of p53 in mouse embryonic fibroblasts led to p53 interaction with p21/CIP1 chromatin, without recruitment of LSD1, and to activation of p21/CIP1. These findings reveal that LSD1 is targeted to chromatin by p53, likely in a gene-specific manner, and define a molecular mechanism by which p53 mediates transcription repression in vivo during differentiation.

FIG. 1.

p53 and LSD1 act together to repress AFP during liver development. (A) RT-PCR analysis of AFP and GAPDH RNA expression in livers excised from 8-day (8d)- and 2-month (2mos)-old WT mice and in Hepa1-6 cells. (B) ChIP analysis of WT livers taken from mice at the ages of 8 days and 2 months. Quantitative real-time PCR (upper panel) and conventional PCR analysis (bottom panel) (28 cycles) were performed to measure relative antibody-bound DNA fragments of the SBE/p53RE region in 8-day- and 2-month-old livers. H3K4me2 levels were normalized to H3 recovery. Each bar represents the average result for three independent ChIP experiments. Error bars show standard deviations. *, P < 0.05; **, P < 0.01. (C) Re-ChIP assay of 2-month-old WT livers. Conventional PCR (32 cycles) analysis was performed with reciprocal re-ChIP assays (primary antibody left of arrow and secondary antibody right of arrow) of p53 and LSD1 interaction at the SBE/p53RE.

FIG. 2.

p53 interacts with LSD1 in vivo and in vitro. (A) Co-IP of endogenous p53 and endogenous LSD1 in HEK293 cells. Normal rabbit IgG was used as a negative control. IP lysate (5%) was used as input. (B) Co-IP of expressed Flag-LSD1 protein or Flag-LSD1 with a carboxy-terminal deletion (LSD1ΔC) and endogenous p53 protein in HEK293 cells. Expression of enhanced green fluorescent protein (EGFP) served as a negative control. IP lysate (5%) was used as input. (C) Purified recombinant p53 and LSD1ΔC proteins interact directly, as shown by co-IP experiments.

FIG. 3.

p53- and LSD1-mediated AFP repression and chromatin modification are reversed during liver regeneration. (A) (Right) RT-PCR was conducted to detect the RNA levels of AFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at different time points after PH and in sham control tissue. (Left) qPCR was performed to quantify RNA levels. AFP levels were normalized to GAPDH levels. Each bar represents the average result for three independent RT-PCR experiments. Error bars show standard deviations. *, P < 0.05; ns, no statistical significance. (B) ChIP analysis of mouse livers undergoing regeneration. qPCR was performed to quantify antibody-bound protein-DNA of SBE/p53RE from livers at 24 h post-PH and 7 days post-PH compared to sham levels. Each bar represents the average result for at least six independent PCRs from two independent ChIP experiments. Error bars show standard deviations. *, P < 0.05; **, P < 0.01. (C) ChIP analysis of mouse livers undergoing regeneration. Conventional PCR (29 cycles) of the SBE/p53RE region was performed after ChIP at 24 h post-PH (24h PH) and with sham-treated liver (24h Sham). (D) Protein levels of p53 were analyzed by immunoblotting of liver nuclear extracts isolated at different stages of development and at 24 h post-PH. Recombinant His-p53 was used as a positive control for antibody detection, and the blot was reprobed for albumin (ALB) expression for loading of each extract. The nuclear extract from 24 h post-PH was overloaded to attempt detection of p53; albumin levels remain unchanged in liver at 24 h post-PH (57a). The slower migrating band positive for p53 (*) in the 24-h PH lysate is likely nonspecific, as it did not appear with a different p53 antibody (CM5) (data not shown).

FIG. 4.

p53 is required for LSD1 recruitment to the developmental repressor region of AFP. (A) ChIP analyses of 2-month-old WT and p53^−/− mouse liver tissues. qPCR was performed to assay relative antibody-bound SBE/p53RE from WT and p53^−/− mouse liver tissues. Each bar is an average result for three independent ChIP experiments. Error bars show standard deviations. **, P < 0.01. (B) ChIP analyses of 2-month-old WT and p53^−/− mouse liver tissues. Conventional PCRs (30 cycles) of the SBE/p53RE region and transcription start site of AFP were conducted to determine the binding of p53 and LSD1. (C) RT-PCR (left) and immunoblot (right) analyses were performed with RNAs and nuclear extracts isolated from liver tissues excised from 2-month-old WT and p53^−/− mice to determine the relative levels of LSD1 and GAPDH RNA and protein expression. (D) Co-IP of expressed Flag-LSD1 protein and hemagglutinin-p73 (HA-p73) protein in U2OS cells. Expression of enhanced green fluorescent protein (EGFP) served as a negative control. IP lysate (5%) was used as input.

FIG. 5.

LSD1 is required for repression of AFP in hepatocytes. (A) RT-PCR analyses were performed with RNAs isolated from Hepa1-6 and AML12 cells to determine the relative levels of AFP, albumin, and GAPDH expression. (B) Co-IP of endogenous p53 and endogenous LSD1 in AML12 cells. Normal rabbit IgG was used as a negative control. IP lysate (5%) was used as input. (C) ChIP analyses of AML12 cells. Conventional PCR (29 cycles) of the SBE/p53RE region of AFP was performed to determine the binding of p53 and LSD1. (D) (Top) Quantitative RT-PCR of AFP, albumin (ALB), LSD1, and actin RNA levels after nonspecific and LSD1 siRNA treatment of AML12 cells. Each bar represents the average result for three independent RNA knockdown experiments. Error bars show standard deviations. **, P < 0.01. (Bottom) Protein levels of LSD1 and actin were analyzed by immunoblotting of whole-cell lysates after nonspecific, nontarget, and LSD1 siRNA treatment of AML12 cells. (E) ChIP analyses of Hepa1-6 cells. Conventional PCRs were performed to analyze the SBE/p53RE region of AFP (28 cycles) and the calbindin REST element (RE1) region (29 cycles).

FIG. 6.

p53, but not LSD1, binds the p21 promoter after activation of p53. (A) Quantitative RT-PCRs were conducted to detect the relative RNA levels of p21 and GAPDH in Val5 cells 3 h after a shift to 32°C. Each bar represents the average result for three independent RT-PCR experiments. Error bars show standard deviations. **, P < 0.01. (B) ChIP analyses were performed to compare Val5 cells prior to and 3 h after a shift to 32°C. The percentage of input bound by antibodies was determined by qPCR for the p21 gene 5′ p53 binding element. Each bar represents the average result for at least six independent PCRs from two independent ChIP experiments. Error bars show standard deviations. *, P < 0.05; **, P < 0.01; ns, no statistical significance.