NEW TARGET GENES FOR TUMOR SUPPRESSORS p53 AND p73 IN REGENERATING LIVER

The p53-family of proteins regulates expression of target genes during tissue development and differentiation. Within the p53-family, p53 and p73 have hepatic-specific functions in development and tumor suppression. Despite a growing list of p53/p73 target genes, very few of these have been studied in vivo, and the knowledge regarding functions of p53 and p73 in normal tissues remains limited. p53+/-p73+/- mice develop hepatocellular carcinoma (HCC), whereas overexpression of p53 in human HCC leads to tumor regression. However, the mechanism of p53/p73 function in liver remains poorly characterized. Here, the model of mouse liver regeneration is used to identify new target genes for p53/p73 in normal quiescent vs. proliferating cells. In response to surgical removal of ~2/3 of liver mass (partial hepatectomy, PH), the remaining hepatocytes exit G0 of cell cycle and undergo proliferation to reestablish liver mass. The hypothesis tested in this work is that p53/p73 functions in cell cycle arrest, apoptosis and senescence are repressed during liver regeneration, and reactivated at the end of the regenerative response. Chromatin immunoprecipitation (ChIP), with a p73-antibody, was used to probe arrayed genomic sequences (ChIP-chip) and uncover 158 potential targets of p73-regulation in normal liver. Global microarray analysis of mRNA levels, at T=0-48h following PH, revealed sets of genes that change expression during regeneration. Eighteen p73-bound genes changed expression after PH. Four of these genes, Foxo3, Jak1, Pea15, and Tuba1 have p53 response elements (p53REs), identified in silico within the upstream regulatory region. Forkhead transcription factor Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative, cellular proliferation. p53 and p73 bind a Foxo3 p53RE and maintain active expression in quiescent liver. During liver regeneration, binding of p53 and p73, recruitment of acetyltransferase p300, and an active chromatin structure of Foxo3 are disrupted, alongside loss of Foxo3 expression. These parameters of Foxo3 regulation are reestablished at completion of liver growth and regeneration, supporting a temporary suspension of p53 and p73 regulatory functions in normal cells during tissue regeneration

Genome-Wide Location Analysis Reveals Distinct Transcriptional Circuitry by Paralogous Regulators Foxa1 and Foxa2

Gene duplication is a powerful driver of evolution. Newly duplicated genes acquire new roles that are relevant to fitness, or they will be lost over time. A potential path to functional relevance is mutation of the coding sequence leading to the acquisition of novel biochemical properties, as analyzed here for the highly homologous paralogs Foxa1 and Foxa2 transcriptional regulators. We determine by genome-wide location analysis (ChIP-Seq) that, although Foxa1 and Foxa2 share a large fraction of binding sites in the liver, each protein also occupies distinct regulatory elements in vivo. Foxa1-only sites are enriched for p53 binding sites and are frequently found near genes important to cell cycle regulation, while Foxa2-restricted sites show only a limited match to the forkhead consensus and are found in genes involved in steroid and lipid metabolism. Thus, Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the adult liver, ensuring the maintenance of both genes during evolution.Institute for Diabetes, Obesity and Metabolism. Diabetes Research Center (Functional Genomics Core P30-DK19525

p53 regulates a mitotic transcription program and determines ploidy in normal mouse liver

Functions of p53 during mitosis reportedly include prevention of polyploidy and transmission of aberrant chromosomes. However, whether p53 plays these roles during genomic surveillance in vivo and, if so, by direct or indirect means remain unknown. The ability of normal, mature hepatocytes to respond to stimuli, reenter cell cycle and regenerate liver mass offers an ideal setting to assess mitosis in vivo. In quiescent liver, normally high ploidy levels in adult mice increased with loss of p53. Following partial hepatectomy, p53(−/−) hepatocytes exhibited early entry into cell cycle and prolonged proliferation with an increased number of polyploid mitoses. Ploidy levels increased during regeneration of both WT and p53(−/−) hepatocytes, but only WT hepatocytes were able to dynamically resolve ploidy levels and return to normal by the end of regeneration. We identified multiple cell cycle and mitotic regulators, including Foxm1, Aurka, Lats2, Plk2 and Plk4, as directly regulated by chromatin interactions of p53 in vivo. Over a time course of regeneration, direct and indirect regulation of expression by p53 is mediated in a gene-specific manner. CONCLUSION: Our results show that p53 plays a role in mitotic fidelity and ploidy resolution in hepatocytes of normal and regenerative liver

PKR Regulates B56 alpha-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic, scaffold, and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56 is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56 at serine 28 in vitro, but it has been unclear how PKRmight regulate the BCL2 phosphatase. In the present study, PKR regulation of B56 in REH cells was examined, because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56 -mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56 phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells, as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56 proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56 protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2 activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56 , because wild-type but not mutant S28A B56 promoted mitochondrial PP2A activity. Cells expressing wildtype B56 but not S28A B56 were sensitized to etoposide. These results suggest that PKR regulates B56 -mediated PP2A signaling in REH cells. Originally published Journal of Biological Chemistry, Vol. 283, No. 51, Dec 200

PKR Regulates B56 alpha-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic scaffold and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56 is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56 at serine 28 in vitro but it has been unclear how PKRmight regulate the BCL2 phosphatase. In the present study PKR regulation of B56 in REH cells was examined because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56 -mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56 phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56 proteolysis which could be blocked by a proteasome inhibitor. However the mechanism by which PKR supports B56 protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2 activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56 because wild-type but not mutant S28A B56 promoted mitochondrial PP2A activity. Cells expressing wildtype B56 but not S28A B56 were sensitized to etoposide. These results suggest that PKR regulates B56 -mediated PP2A signaling in REH cells. Originally published Journal of Biological Chemistry Vol. 283 No. 51 Dec 200

Identification of Genomic Targets of Foxa1 and Foxa2 in Adult Liver.

<p>(A) Venn diagram showing the results of genome-wide location analysis for Foxa1 and Foxa2 in the adult liver, identifying 5,562 binding sites for Foxa1 and 11,097 for Foxa2, of which 3,120 were called bound by both factors by the GLITR algorithm (certain common targets). Many of the apparently Foxa1- or Foxa2-specific regions contained overlapping reads from the opposite factor, indicating that the sites may be common, but the reads did not reach significant depth to be called by GLITR. We defined more stringent criteria for Foxa1-only and Foxa2-only binding sites. The sets of unique targets (yellow circles) contain peaks for the first factor that include at most one tag per million per KB for the other factor (1,816 Foxa1 sites with one or no Foxa2 tag and 5,682 Foxa2 sites with one or no Foxa1 tag). (B) Foxa1 and Foxa2 can occupy common binding sites (top panel) in the adult liver, or sites specific to either factor (Foxa1-only binding site, middle panel; Foxa2-only binding site, bottom panel). (C) Both common and unique binding sites for Foxa1 and Foxa2 can co-occur at a single genomic locus. (D) Comparison of Foxa1 and Foxa2 binding sites to a profile of H3K3me1 regions (purple circle) in the adult mouse liver.</p

Foxa1-Specific Targets Are Enriched for p53 Binding Sites.

<p>(A) A set of Foxa1-only targets was defined as those sequences that had one or fewer sequence tags Foxa2 ChIP-Seq data set (yellow circle). Motif analysis of these sequences found in a weaker <i>forkhead</i> consensus. In addition to the <i>forkhead</i> consensus, several other motifs appeared in this set of sequences. The first (ACATG and ATG repeats with a spacer in the middle) comprises parts of the positional weight matrix (PWM) for p53. The second closely resembles the PWM of Klf12, also known as repressor of AP-2alpha (Tfap2a). Orthogonal analysis of sites bound only bound by Foxa1 (yellow circle) and gene expression changes in livers of Foxa1-deficient mice (light purple circle) shows that thirty-seven are direct targets of Foxa1. (B) Verification of Foxa1-only targets by qPCR. Filled bars, ChIP of liver chromatin with an anti-Foxa1 antibody, open bars, ChIP with an anti-Foxa2 antibody. Three negative control regions (Nc1, Nc2, and Nc3) with a low amount of reads are included Binding is expressed as enrichment of the PCR amplicon relative to input DNA in liver chromatin. (C) Binding of p53 to <i>cis</i>-regulatory elements of its previously identified targets (positive controls), the alpha-fetoprotein (AFP) and TNF receptor superfamily <i>Fas</i> genes and twelve additional Foxa1-only targets, including cell-cycle associated <i>Aatf</i> and <i>Zwint</i> genes in both wildtype (black bars) and Foxa1 mutant livers (white bars) (left panel) by ChIP of liver chromatin followed by qPCR. None of Foxa2-only sites tested were bound by p53 in wiltype livers (right panel) (D) Quantitative RT- PCR analysis for mRNA of Zwint, Zwint mRNA levels are significantly downregulated in livers of Foxa1 mutant mice by 30%, Values are represented as means plus standard error. P values were determined by Student's <i>t</i> test. * p-value<0.05 (E) Foxa1 targets are enriched in genes involved in cell differentiation, morphogenesis, movement, and cellular cycle. (F) A regulatory feed-forward loop involving Foxa1. In the set of Foxa1-specific targets, Foxa1 binds to regulatory elements of the nuclear receptor Esr2 (estrogen receptor), its co-activator Smyd3, and target gene Pgr (progesterone receptor). (G) Foxa1 only sites are more distributed more broadly surrounding TSS (+/−100 Kb) than dual Foxa1/Foxa2 targets (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002770#pgen-1002770-g004" target="_blank">Figure 4E</a>).</p

Evolution of Vertebrate Foxa Paralogs and Functional Redundancy in the Fetal Liver.

<p>(A) Phylogenetic tree of the Foxa subfamily of transcriptional regulators. The putative Foxa4 gene was lost in vertebrates during evolution. (B) Sequence alignment of mouse Foxa1 and Foxa2 proteins by ClustalW2 algorithm. The winged-helix DNA binding domain is highlighted in blue. ‘*’, identical residues in all sequences, ‘:’ highly conserved amino acids; ‘.’ weakly conserved amino acids. (C) Venn diagram of the number of genes that are differentially expressed in fetal livers of Foxa1 and Foxa2 single mutants, as well as the double mutant (shaded in blue) on embryonic day 18.5. The number of genes dependent on each single factor is small compared to the number of genes that are differentially expressed in the double mutant.</p

Foxa1 and Foxa2 Regulate Different Sets of Target Genes in the Adult Liver.

<p>(A,B) Expression of Foxa factors in reciprocal mutant mice. Black bar, wild-type liver, white bar, Foxa1-deficient liver, and grey bar, Foxa2-deficient liver. As expected, Foxa1 mRNA is undetectable in the Foxa1 Foxa1-deficient liver, while expression of Foxa2 is not changed in absence of Foxa1 compared to control mice on both the mRNA and protein level. Similarly, expression of Foxa2 is near background levels in Foxa2-deficient livers, while expression of Foxa1 is comparable to that in wild-type littermates. (C) Venn diagrams of the number of genes that are differentially expressed (|FC|> = 1.5 and FDR = 15%) in adult livers (total, downregulated, and upregulated) of Foxa1 (pink circles) and Foxa2 mutant (yellow circles) mice. (D) Confirmation of seven microarray targets changed in expression in the Foxa1-deficient liver by quantitative real-time PCR (qRT-PCR) (top panel). When the mRNA levels of the same genes were determined in the Foxa2-deficient liver (bottom panel), these targets were either not Foxa2-dependent at all or regulated in the opposite direction. Values are represented as means plus standard error. P values were determined by Student's <i>t</i> test. * p-value<0.05. (E) mRNA levels of previously identified Foxa2 targets in Foxa1 mutant mice. Values are represented as means plus standard error.</p

Foxa2-Specific Targets Contain a Medium-Strength <i>Forkhead</i> Consensus and Control Genes in Steroid and Lipid Metabolism.

<p>(A) The motifs found by <i>de novo</i> analysis for both Foxa2-only bound set (yellow circle) resemble a <i>forkhead</i> binding site. Intersection of Foxa2-bound regions and genes differentially expressed in livers of Foxa2-deficient mice (light purple circle) identified three-hundred thirty-three direct targets. (B) Validation of Foxa2-only targets by qPCR. Filled bars, ChIP of liver chromatin with an anti-Foxa1 antibody, open bars, ChIP with an anti-Foxa2 antibody. Binding is expressed as enrichment of the PCR amplicon relative to input DNA in liver chromatin. Negative controls (Nc1, Nc2, Nc3) are regions with a low amount of reads. (C) Foxa2 targets are enriched in genes involved in lipid and steroid metabolism, protein modification, and carbohydrate metabolism. (D) Histogram of the distribution of Foxa2-only sites relative to TSS. Foxa2-only sites, similar to the Foxa1/Foxa2 dual targets (compare <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002770#pgen-1002770-g004" target="_blank">Figure 4E</a>), are distributed normally near transcription start sites (TSS), with most sites within ten kilobases (Kb) from TSS.</p