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Molecular and Cellular Biology, September 2008, p. 5139-5146, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00287-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

p53-Targeted LSD1 Functions in Repression of Chromatin Structure and Transcription In Vivo{triangledown} ,{dagger}

Wen-Wei Tsai,1 Thi T. Nguyen,1 Yang Shi,2 and Michelle Craig Barton1*

Department of Biochemistry and Molecular Biology, Program in Genes and Development, Graduate School of Biomedical Sciences, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030,1 Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 021152

Received 20 February 2008/ Returned for modification 10 April 2008/ Accepted 10 June 2008


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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.


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INTRODUCTION
 
Numerous analyses of p53 functions, in response to cellular stress, underscore its important, pleiotropic roles in arrest of the cell cycle and promotion of apoptosis (5, 24, 33). Less understood are mechanisms of p53-mediated regulation during differentiation and in maintenance of cellular homeostasis. Previous work from our laboratory established p53 as a major repressor of AFP transcription during hepatic development (60). Developmental repression of AFP, an onco-fetal tumor marker gene, is delayed more than 2 months after birth in p53-null mice, compared to cessation within 2 to 3 weeks in wild-type (WT) mice (45). Transactivating p73 (TA-p73), but not TA-p63, partially compensates for an absence of p53 in p53-null mice, characterized by elevated methylation of histone H3 lysine 4 (H3K4) at the p53/p73 response element of alpha-fetoprotein (AFP) chromatin (12). Therefore, we hypothesized that an H3K4 demethylase is recruited by p53 during hepatic differentiation and that combinatorial alterations in chromatin structure during development dictate the developmental timing of transcription regulation.

Enzymatic removal of methyl groups from lysine residues of histone N-terminal tails correlates with either repression or activation of transcription, as determined by the specific histone substrate, amino acid position, and level of methylation. Loss of methylated H3K4 (H3K4me) is generally associated with repression or cessation of active transcription (7, 14, 55). The first enzyme with the capacity to remove methyl groups from histone lysine residues was identified as BHC110 or histone lysine-specific demethylase 1 (LSD1; recently renamed KDM1) (42), a corepressor of nerve-specific genes in nonneural tissues, where LSD1 is targeted by the repressor proteins REST and Co-REST (50, 51). LSD1 is restricted to demethylation of di- and monomethylated substrates due to chemical limitation of the flavin-dependent oxidation reaction (50, 51). Target specificity of LSD1 is determined by interacting proteins, e.g., interactions with nuclear receptors to demethylate H3K9me2 and effect activation (21, 39, 59, 61) or interactions with REST/Co-REST to demethylate H3K4me2 and repress transcription (50, 51).

Similar to the case for modifications of histone proteins, the specific types, extent, and sites of posttranslational modifications of p53 offer a "combinatorial code" and determine p53 interactions with protein partners as effectors of downstream regulation and p53 protein stability (2, 24). Additionally, the consequences of methylation are degree and residue specific for p53, as with histones. Methylation of p53 alters its ability to regulate transcription and is either repressive, e.g., monomethylation at K370 by Smyd2 (28) or at K382 by SET8 (49), or activating, e.g., dimethylation at K372 by SET9 (9) or at K370 by an unidentified methyltransferase (29). Recently, Berger and colleagues uncovered LSD1 as a demethylase of a nonhistone substrate, i.e., dimethylated p53 (29). They found that LSD1-mediated demethylation of p53K370me2 prevents p53 interaction with 53BP1 as a coactivator, blocking p53 binding to DNA and activation of p21/CIP1 and MDM2 in cultured cells. In this manner, LSD1 functions as a repressor of p53-activated gene expression in the absence of stress stimuli and does so independently of its ability to modify chromatin.

In the current study, we found that LSD1 is recruited by p53 to demethylate H3K4me2 of active chromatin and to repress transcription of AFP during liver development. Interaction with p53 is direct and independent of the enzymatic activity of LSD1. The binding of p53 and recruitment of LSD1 to chromatin are gene specific and reversible, concomitant with degradation of p53 and reactivation of AFP expression during liver regeneration. These results reveal a role for LSD1 complexes in developmental and tissue-specific regulation of nonneural genes, a role that is mediated by p53 in normal cells in vivo.


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MATERIALS AND METHODS
 
ChIP and RNA analyses of solid tissue and cultured cells. Liver tissue was isolated and flash-frozen from WT and p53–/– mice of a mixed C57BL/6J:129sv background. Chromatin immunoprecipitation (ChIP) and re-ChIP assays of liver tissue or cultured cells were performed as described previously (12, 45), with minimal modifications. The fragmented, precleared chromatin lysate was incubated overnight with the following specific antibodies for ChIP: antibodies to histone H3 (Abcam), H3K4me2 (Upstate/Millipore), H3K4me3 (Abcam), H3K4me1 (Abcam), H3K9me2 (Upstate/Millipore and Abcam), acetylated H3K9 (H3K9ac) (Upstate/Millipore), H4ac (Upstate/Millipore), p53 (Oncogene and Novocastra), p73 (Santa Cruz), LSD1 (Abcam), Foxa1 (Abcam), and normal sheep immunoglobulin G (IgG) (Upstate/Millipore). The specificity of the LSD1 antibody was confirmed by immunoblotting of mouse liver nuclear extract (see Fig. S1 in the supplemental material).

To analyze specific and antibody- and protein-bound DNAs, we performed conventional PCR, real-time quantitative PCR (qPCR), and quantitative TaqMan (Applied Biosystems, ABI, Foster City, CA) real-time PCR. Conventional PCR primers were generated to detect the AFP Smad binding element/p53 response element (SBE/p53RE) and calbindin RE-1 regions as described previously (4, 45). The following TaqMan real-time PCR primers and probe were used for the AFP SBE/p53RE region: forward primer, 5'-CTACATATGAAGCCTTAGCAAACATGT-3'; reverse primer, 5'-ACTCAGACGTTGGCGTGTCA-3'; and probe, 6-carboxyfluorescein (FAM)-CCTCTAGACACACAGACT-MGB. The following real-time PCR primers were used to detect the p21 gene 5' p53 binding element: forward primer, 5'-CCTTTCTATCAGCCCCAGAGGATACC-3'; and reverse primer, 5'-GACCCCAAAATGACAAAGTGACAA-3'. Primers and reverse transcription-PCR (RT-PCR) determinations of RNA expression were performed as previously described (45). The following primers were used to detect expression of the LSD1 gene: forward primer, 5'-GGAATCCCATGGCTGTCGTCA-3'; and reverse primer, 5'-GATATCTCTGGGCGGCTTCACTT-3'. qPCRs were conducted in a model 7500 FAST ABI instrument.

Liver regeneration. Partial hepatectomy (PH) to remove 60% to 70% of total liver tissue or control, sham surgery of three to five mice for each experimental condition was performed by IACUC-approved procedures, as previously described (20). Mice were sacrificed 24 h or 7 days following PH; remnant liver tissue was harvested, flash-frozen, and processed for RNA and ChIP analyses.

Cell culture and coimmunoprecipitation (co-IP) assays. Hepa1-6 murine hepatoma cells, HEK293 human embryonic kidney cells, and U2OS human osteosarcoma cells were obtained from ATCC and cultured under suggested conditions. AML12 cells were obtained from J. Clark and N. Fausto (University of Washington, Seattle). Val5 mouse embryonic fibroblasts (MEFs) were obtained from M. Murphy (Fox Chase Cancer Center, Philadelphia, PA) (44). Plasmids encoding human LSD1, LSD1{Delta}C, and TA-p73{alpha} have been described (15, 50, 51). HEK293 cells were transfected with 2 µg of total DNA in six-well plates by standard Ca(PO4) methodology (22). U2OS cells were transfected with 1 µg of total DNA in six-well plates, using Effectene (Qiagen) as recommended by the manufacturer.

Transfected or control HEK293 cells, U2OS cells, and AML12 cells were lysed in NTEP buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.5% NP-40 plus freshly added 1x protease inhibitor cocktail set I [Calbiochem] with 1 mM phenylmethylsulfonyl fluoride [Sigma]) and sonicated for a few seconds. The cell lysate was incubated with 100 µg/ml ethidium bromide for 1 h and precleared by incubation with 20 µl protein G beads (50% slurry; Sigma) for 1 h. The precleared lysate was incubated overnight with anti-Flag M2 beads (Sigma) or the following specific antibodies: anti-p53 (Santa Cruz, Oncogene, and Novocastra), anti-LSD1 (Abcam), anti-rabbit IgG (Upstate/Millipore), anti-RBP2 (Bethyl Laboratories), and anti-Flag M2 (Sigma). Next, 25 µl protein G beads (50% slurry) was added and incubated for 2 h at 4°C. Protein-bound beads were recovered by centrifugation and washed three times with NTEP, first with 500 mM NaCl, then with 0.5% sodium dodecyl sulfate, and then with NTEP buffer alone. Input lysate, equivalent to 1/20 of the immunoprecipitation lysate, was analyzed alongside bead-bound proteins by sodium dodecyl sulfate-polyacrylamide electrophoresis and immunoblot analyses, as previously described (60). The primary antibodies for immunoblotting were as follows: anti-Co-REST (Upstate), anti-RB (Santa Cruz), antihemagglutinin (Roche), and anti-Flag M2 (Sigma).

RNA interference assays. Small interfering RNA (siRNA) oligonucleotide pools targeting murine LSD1 and nonspecific and nontarget siRNAs were purchased from Dharmacon/Thermo Fisher Scientific (Chicago, IL). Transient transfection of siRNAs into AML12 cells was carried out using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. RNAs were isolated 48 h after transfection, and protein lysates were prepared 72 h after transfection. Primers and RT-PCR determinations of RNA expression were as previously described (45). qPCRs were conducted in a model 7500 FAST ABI instrument.

Statistical analyses. GraphPad Prism5 software (GraphPad Software, Inc.) was used for analysis of P values based on at least two independent experiments with three independent PCRs. The two-tailed paired t test was used to compare the differences in relative changes between two groups (see Fig. 1B, 3B, 4A, 5D, and 6A). The two-tailed unpaired t test was used to compare the differences in actual percentages between two groups (see Fig. 3A and 6B). P values of <0.05 were considered statistically significant.


Figure 1
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  		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.


Figure 3
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  		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).


Figure 4
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  		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.


Figure 5
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  		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).


Figure 6
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  		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.


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RESULTS
 
Developmental repression of AFP is marked by p53 and LSD1 interaction with chromatin. We performed ChIP analyses of developmentally staged mouse liver tissue to determine relative levels of endogenous p53, LSD1, and H3K4me2 present at the developmental repressor region (SBE/p53RE) (60) of AFP chromatin. Our previous work showed that this intercalated Smad/p53 response element is essential for p53-mediated repression of AFP in cultured cells and by in vitro transcription (11, 34). Shortly after birth, when hepatic AFP expression is robust (Fig. 1A, lane 8d), H3K4me2 levels are high at the SBE/p53RE repressor region, but p53 and LSD1 are barely detectable (Fig. 1B). Quantitative real-time PCR analyses of multiple ChIP assays showed that a 10-fold decrease in H3K4me2 level occurs alongside a 4- to 6-fold increase in p53 and LSD1 binding at 2 months of age (Fig. 1B), when AFP expression is completely repressed (Fig. 1A). Absolute levels of bound DNA amplified by nonquantitative PCR and the lack of nonspecific DNA associated with the IgG control are shown compared to undiluted input DNA (Fig. 1B, bottom panel). Overexpression of Flag-tagged p53 or LSD1 in U2OS cells, which express endogenous p53 (1), does not cause a generalized decrease in cellular H3K4me2 levels (see Fig. S2A and B in the supplemental material).

p53 interacts with LSD1 to mediate AFP repression. To determine if p53 and LSD1 simultaneously bind to the same DNA element in vivo, we performed sequential ChIP or re-ChIP experiments to analyze 2-month-old mouse liver tissue. The results show that p53 and LSD1 cooccupy chromatin at this stage of development, as revealed by sequential ChIP with an antibody recognizing mouse p53 protein, followed by precipitation of p53-bound chromatin with an LSD1-specific antibody. These antibodies differ in their relative affinities for protein-bound chromatin, so elution is not likely quantitative; however, reversal of the order of antibodies in re-ChIP yielded equivalent results, with no nonspecific background binding to IgG (Fig. 1C). These results suggest that p53 and LSD1 act together to mediate repression of AFP during development.

We used co-IP analysis to determine if LSD1 and p53 interact and associate as a soluble protein complex. We found a specific, DNA-independent interaction between endogenous p53 and LSD1 proteins in p53-positive HEK293 cells (Fig. 2A; see Fig. S1B in the supplemental material). We detected no interaction between p53 and RBP2 (Fig. 2A), a member of the JmjC family that can demethylate H3K4me2 (8, 32). Protein-protein interactions between LSD1 and p53 do not require enzymatic activity of the demethylase, as endogenous p53 associates with either a full-length or C-terminally truncated (LSD1{Delta}C) version of LSD1 (Fig. 2B). We next determined if p53 interacts directly with LSD1 by using recombinant proteins. We performed in vitro co-IP experiments with recombinant His-tagged p53 and recombinant Flag-tagged LSD1{Delta}C proteins (Fig. 2C). The results show that the p53-LSD1 interaction is direct and does not require the catalytic domain of the LSD1 protein.


Figure 2
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  		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 (LSD1{Delta}C) 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 LSD1{Delta}C proteins interact directly, as shown by co-IP experiments.

LSD1- and p53-mediated chromatin modification is reversed during liver regeneration. Two-thirds PH of adult mouse liver induces a complex network of signal transduction pathways, which trigger hepatocytes to reenter the cell cycle in a synchronized wave of proliferation and compensatory growth until the liver mass is fully regenerated (20, 40). During liver regeneration, differentiation-associated repression of AFP gene expression is reversed, and transcription is reactivated (Fig. 3A) (23, 37, 53). Within 24 h of PH, p53 and LSD1 interactions with AFP chromatin are significantly reduced, coincident with derepression of AFP expression (Fig. 3A and B). Along with a loss of repressive p53, H3K4me2 levels increase and Foxa1, a transactivator that opposes p53-mediated repression of AFP expression (34, 45), binds its response element, intercalated within the SBE/p53RE (Fig. 3C).

The inverse relationship between binding of p53 and LSD1 and H3K4me2 modification of chromatin at the repressor region of AFP is further supported by ChIP analyses of fully regenerated liver 7 days after PH, when AFP expression is repressed once more (Fig. 3A and B). The SBE/p53RE repressor is a complex regulatory element where Foxa factors, transforming growth factor beta effectors Smad and SnoN, and p53 family members interact to regulate AFP expression. When p53 and LSD1 are significantly depleted at the SBE/p53RE repressor, during the regenerative response, multiple changes in histone modifications occur in this region of chromatin, including demethylation of H3K9me2, a <2-fold increase in H3K4me1 and H4ac, and relatively no change in H3K4me3 at this distal regulatory site (data not shown; see Fig. S3 in the supplemental material).

The physiological roles of p53 during liver regeneration are poorly understood, as the driving forces of regeneration restore full liver mass even in p53-null mice and p53-compromised livers (18; our unpublished data). We found, by immunoblotting of liver nuclear extract with an antibody that detects normal p53, that p53 is undetectable within 24 h of PH (Fig. 3D, 24-h PH extract overloaded for detection of p53). We are currently investigating the signaling pathways that inactivate and/or degrade p53 at peak times of proliferation during liver regeneration (24 to 36 h after PH) (20; data not shown) and those that restore p53 function once the liver mass is restored.

p53 is required for LSD1-mediated repression of AFP. We determined if p53 is essential for recruitment of LSD1 in vivo by ChIP analysis of liver tissue from p53-null mice. These mice, disrupted in Trp53 by homologous recombination and lacking expression of p53 (17, 38), maintain expression of AFP at 2 months of age, well beyond the developmental window of repression observed in WT mice (45). Comparison of WT and p53-null livers showed that in the absence of p53, LSD1 is significantly reduced (Fig. 4A). Nonquantitative PCR analysis of SBE/p53RE and the transcription start site supports the sequence specificity of the p53 and LSD1 interaction with chromatin and the lack of nonspecific binding compared to undiluted input DNA (Fig. 4B). The decrease in LSD1 interaction with AFP chromatin is not due to decreased expression of LSD1, which is not altered in p53-null mice (Fig. 4C).

Previously, we saw that p53 and its family member TA-p73 are bound at the developmental repressor of AFP, concomitant with developmental repression of AFP chromatin structure and expression. In p53-null liver, repression of AFP is greatly delayed, from 2 to 3 weeks to 4 months, when chromatin-bound TA-p73 levels increase compared to those in the WT. When AFP is fully repressed at 4 months in the p53-null mouse, H3K4me2 levels at the SBE-p53RE region remain higher than those in 4-month-old WT mice, but H3K9me2 levels are equivalent to those of WT mice (12). Partial compensation by p73 for loss of p53, which leads to a significant temporal delay but not a reversal of repression, may be due in part to significantly decreased but measurable recruitment of LSD1 in p53-null liver (Fig. 4A). We saw that p73 and LSD1 interact in a protein complex when they are expressed ectopically in p53-positive U2OS cells (Fig. 4D), though an endogenous association was undetectable (data not shown).

LSD1 is required for AFP repression in hepatocytes. We used a nontransformed hepatocyte cell line to manipulate levels of LSD1 expression and determine whether LSD1 is required for AFP repression in isolated hepatocyte-derived cells. AML12 cells were established by continuous growth of primary hepatocytes derived from liver tissue of a mouse transgenic for human transforming growth factor alpha expression (62). These immortalized hepatic cells recapitulate repression of AFP and expression of albumin (ALB), as occurs in fully differentiated liver tissue (Fig. 5A). The results of co-IP experiments showed that endogenous p53 and LSD1 interact in these cells (Fig. 5B), and ChIP analysis showed binding of p53 and LSD1 at the SBE/p53RE region of endogenous AFP chromatin (Fig. 5C). We transiently transfected siRNA oligonucleotides to target LSD1 expression and found that depletion of LSD1 by siRNA led to derepression of AFP and no change in expression of ALB (Fig. 5D), reflecting the target specificity of LSD1 depletion and its requirement in repression of AFP.

Dysfunction of p53 occurs in more than 50% of all human cancers (6, 52, 58). We therefore determined if expression of AFP as a tumor marker in hepatoma cells (Fig. 1A) was consistent with a lack of p53 and LSD1 interaction with chromatin at the repressor region of AFP. We performed ChIP analyses to detect binding of p53 and LSD1 on AFP chromatin in Hepa1-6 cells, which were originally cultured from a clonally derived mouse hepatoma tumor (13) and express signal-responsive p53 (see Fig. S4 in the supplemental material). We found that p53 and LSD1 do not interact with AFP chromatin and that H3K4me2 levels are readily detectable at the SBE/p53RE (Fig. 5E). As a positive control, LSD1 did associate with a REST response element of calbindin chromatin (Fig. 5E, lower panel). Previously, REST and Co-REST were shown to interact at this site within the calbindin gene, which is expressed only in neural tissues (3, 4).

LSD1 is recruited by p53 in a gene-specific manner. To determine whether p53-dependent recruitment of LSD1 is gene specific or occurs at other p53 target genes, we performed ChIP analyses of the p21/CIP1 gene in immortalized MEFs that express a temperature-regulated form of p53 (Val5 MEFs [44]) (Fig. 6). A temperature shift from 37°C to 32°C promotes translocation of p53R135V from the cytoplasm to the nuclei of Val5 MEFs, where p21/CIP1 is robustly activated within 3 h (Fig. 6A). Concomitant with p53 binding to chromatin and activation of p21/CIP1 expression, nucleosome occupancy at the distal p53 response element is significantly reduced, H3K4me2 levels rise, and LSD1 is not recruited (Fig. 6B). This lack of p53-mediated recruitment of LSD1 and our inability to detect either p53R135V or LSD1 at inactive p21/CIP1 chromatin at 37°C support a model of gene-specific outcomes for p53 and LSD1 interaction that differ between targets of p53-mediated activation (29; this study) and when p53 is an active repressor of transcription.


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DISCUSSION
 
The functions of p53 as a regulatory protein in normal cellular physiology are poorly understood and may be masked or compensated for by activities of its related family members, p63 and p73 (12, 16, 41, 63). One important role of p53 in normal cells was recently reported by Levine and colleagues, who showed that p53 controls basal and transiently upregulated transcription of LIF in uterine tissue, a process essential for embryonic implantation and maternal reproduction (27). In normal liver tissue, p53 is a developmental repressor of AFP transcription, and p73 can partially compensate repression of AFP in p53-null mice (45). Developmentally sustained levels of H3K4me2 in adult p53-null liver, concomitant with delayed, developmental repression of AFP, led us to connect the function of LSD1, a demethylating enzyme of H3K4, and p53-mediated recruitment of LSD1 to AFP chromatin in vivo.

LSD1-p53 interaction and repression of AFP are reversed during liver regeneration in response to two-thirds PH. Regeneration of liver is the result of a complex network of signal transduction pathways that promote differentiated hepatocytes to reenter the cell cycle, grow, and repopulate the liver mass (10, 19, 40, 57). This model of cell cycle reactivation in postmitotic tissue allowed us to establish cause and effect between DNA binding of p53 and LSD1 targeting of demethylation of H3K4 in normal liver tissue. Our finding that p53 protein is undetectable 24 h after PH correlates with the established timing of the first wave of S phase entry during murine liver regeneration (20), reactivation of AFP expression, and loss of LSD1 binding at AFP. These results help to pinpoint p53 as a target of regeneration-induced signaling, whose nature is under active investigation in our laboratory.

Numerous studies of p53 functions focus on transactivation by p53 in response to stress signaling; however, relatively little is known about p53-mediated transcription repression (24, 25). Where repression has been analyzed at the level of chromatin modification, p53-mediated recruitment of mSin3-histone deacetylase (HDAC) complexes and deacetylation of histones occur (26, 36, 43, 45, 46, 54, 56). Interactions of mSin3A-HDAC protein complexes with p53 may have multiple outcomes due to p53-tethered modification of chromatin and/or direct deacetylation of p53 to regulate its activity (30, 31, 47) or protein stability (64). Corepressor activities of LSD1 with p53, like those of mSin3-HDAC complexes, have multiple mechanisms and outcomes. LSD1 may directly interact with p53 protein and directly demethylate p53 to regulate coactivator interactions and block the activation of transcription, independent of chromatin (29). Alternatively, as we show here, p53 may associate with LSD1 and recruit it to chromatin at a gene-specific p53 response element to actively repress transcription.

Our knowledge of chromatin-independent and -dependent functions of LSD1 will increase with further study. Depletion of LSD1 is associated with increased p53-activated transcription and stress responses (29) but also with a delayed p53 response to DNA damage and regulation of prosurvival genes (48). LSD1 is known to display considerable versatility as a member of either activating, chromatin-modifying complexes or repressing enzyme complexes (35, 39, 51). These studies and the results described here suggest that p53 and LSD1 protein complexes, both soluble and chromatin-bound, regulate transcription in a gene-specific manner. Taken together with the ability of p53 to act as either an activator or repressor of transcription (25) and the numerous posttranslational modifications and protein partners of p53, LSD1 interactions with p53 may have multiple, context-specific outcomes, which are further expanded by the number of enzymatically active complexes that count LSD1 as a member.


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ACKNOWLEDGMENTS
 
We acknowledge C. Elferink and R. Behringer for training in PH; G. Lozano for p53-null mice; S. Kurinna, S. Stratton, and J. Piechan for liver extracts; M. Minard for p53 recombinant protein; M. Murphy for Val5 MEF cells; and S. Dent for helpful comments.

This work was supported by grant GM53683 from the National Institutes of Health to M.C.B. and by an NCI Cancer Center support grant to the University of Texas M. D. Anderson Cancer Center.


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FOOTNOTES
 
* Corresponding author. Mailing address: Dept. of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 1000, Houston, TX 77030. Phone: (713) 834-6268. Fax: (713) 834-6273. E-mail: mbarton{at}mdanderson.org Back

{triangledown} Published ahead of print on 23 June 2008. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back


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Molecular and Cellular Biology, September 2008, p. 5139-5146, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00287-08
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