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Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor FOXA1, activating its transcriptional network while promoting WNT/β-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and β-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis. Significance: Oncogenic KRAS and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic KRAS effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis. This article is highlighted in the In This Issue feature, p. 1861

Background The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. Methods We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. Results We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8+ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8+ T-cell function. TCGA pan-cancer data confirmed that CD8lowPlatelethigh patients have a significantly lower survival probability compared to CD8highPlateletlow. Conclusions CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function.

Acute myeloid leukemia (AML) with a mutation in the fms-like tyrosine kinase receptor 3 gene (FLT3) confers poor survival for pediatric patients, indicating a significant need to improve treatment efficacy. A significant percentage of pediatric patients consume diets high in fat and sugar content. These macronutrients modulate oxidative stress yet their impact on AML treatment is unexplored. Studies have implicated a role for BMAL1 rhythmicity, a circadian clock protein, in leukemogenesis. However, BMAL1 has not been studied in FLT3 mutant AML. Given that both nutrition and circadian rhythmicity can be modulated by behavior, we hypothesized that modulating redox and BMAL1 status through diet composition and timing would enhance treatment efficacy in an orthotopic mouse model. In all in vivo experiments, mice were injected via tail vein with 100,000 MOLM13 luciferase labelled cells, a FLT3 AML human cell line. After engraftment was confirmed 3 days later, mice were put on diet modification regimens. In three subsequent experiments, distinct diet interventions were delivered: low fat (30%)/low sucrose (10%), low sucrose alone (10%), or an isocaloric time feeding regimen (TRF) restricted to mice’s active phase (7 PM to 7 AM). Standard chow, high sucrose diet (45%) and ad libitum feeding were controls, respectively. In each experiment, doxorubicin (2 mg/kg) or PBS was injected twice weekly via tail vein. Tumor burden was monitored via bioluminescent imaging. Reactive oxygen species (ROS) levels were assessed with flow cytometry, and changes in circadian, redox, and apoptotic protein expression were measured by western blot and mass cytometry. On day 17, in the presence of doxorubicin, mice on a low fat/low sucrose diet showed significantly lower tumor burden compared to control. Doxorubicin induced oxidative stress was reduced in the mice fed the modified diet as demonstrated by a decrease in ROS levels in peripheral blood mononuclear cells. Even in the absence of doxorubicin treatment, the modified diet group showed significantly lower tumor burden on day 17. For mice on a low sucrose diet, changes in redox and circadian protein expression were observed on day 17. Of note, total BMAL1 protein showed a two-fold increase in spleen from the low sucrose group. In the timed feeding experiments, we observed TRF mice had significantly reduced leukemic burden on day 14. Importantly, doxorubicin efficacy was not compromised by TRF. In addition, a significant increase in total BMAL1 protein expression was observed in liver from these TRF mice on day 14. These results point to a novel finding: diet composition and timing slows progression of FLT3 AML growth in vivo and are accompanied by BMAL1 modulation. In future studies, we aim to further define the role of circadian rhythmicity and diet composition in AML progression and identify methods to complement treatment to positively impact survival outcomes for patients diagnosed with AML. Citation Format: Megan Rodriguez, Mary Figueroa, Brianna Murphy, Tiewei Cheng, Baharan Fekry, Kendra Allton, Michelle Barton, Marina Konopleva, Kristin Mahan, Joya Chandra. Effects of diet composition and timing on treatment for FLT3 positive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 726.

SUMMARY β-hydroxybutyrate (β-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine β-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5+ stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of β-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting., Graphical Abstract, In brief Terranova et al. demonstrate that fasting induces production of HMGCS2 and β-hydroxybutyrate in small intestine (SI) crypt cells. This causes enrichment of H3K9bhb within regulatory regions of critical metabolic genes in crypt epithelial cells. Loss of intestinal Hmgcs2 impairs H3K9bhb enrichment and affects expression of H3K9bhb-associated metabolic gene programs.

DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53−/−) mESCs is the result of augmented overall DNA methylation as well as increased methylation landscape heterogeneity. In differentiating p53−/− mESCs, elevated methylation persists, albeit more mildly. Importantly, concomitant with DNA methylation heterogeneity, p53−/− mESCs display increased cellular heterogeneity both in the “naïve” state and upon induced differentiation. This impact of p53 loss on 5-methylcytosine (5mC) heterogeneity was also evident in human ESCs and mouse embryos in vivo. Hence, p53 helps maintain DNA methylation homeostasis and clonal homogeneity, a function that may contribute to its tumor suppressor activity.

β-hydroxybutyrate (β-OHB) provides an essential metabolic energy source during fasting and functions as an epigenetic regulator by lysine β-hydroxybutyrylation (Kbhb) modification of core histones H3 and H4. We report that H3K9bhb, but not H4K12bhb, becomes highly enriched at proximal promoters of genes regulating lipolytic and ketogenic metabolic programs in small intestinal (SI) crypts during fasting and these pathways are transcriptionally activated in Lgr5 + SI stem cells. Similar Kbhb enrichment is observed in Lgr5 + stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb enrichment is associated with active chromatin states and that fasting enriches for an H3K9bhb–H3K27ac signature at active metabolic gene promoters and distal DNA enhancer elements. Intestinal knockout of Hmgcs2 results in a marked loss of H3K9bhb associated fasting-induced gene programs suggesting that local production of β-OHB is responsible for epigenetic reprogramming. We conclude that specific modulation of H3K9bhb in SI crypts is a key epigenetic event in response to fasting.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most p53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis. However, the extent of direct cooperation or synergism between KRAS effectors and mutant p53 remain undefined. To study how KRAS effectors and mutant p53 cooperate to drive PDAC development, we developed a novel somatic model that expresses mutant KRAS and p53 only in pancreas tumor cells, leaving the tumor microenvironment and immune system intact. PDAC genetically engineered mouse models (GEMMs) were generated by crossing a conditional oncogenic KRasG12D (K) allele with a conditional null p53 allele (Pfl) or a novel, conditional knock-in mutant p53R172H allele (Pwm) that selectively expresses mutant p53 in tumor cells while preserving wildtype p53 expression in all other animal cells. The incidence of metastatic lesions in the liver and lungs of KPwmC mice was over 2-fold greater (p=.03) relative to KPflC mice. Through orthogonal analyses of this model and human PDAC samples, we show that oncogenic KRAS effectors phosphorylate and activate cyclic AMP responsive element binding protein 1 (CREB1), allowing physical interactions with mutant p53 to transcriptionally upregulate the pro-metastatic transcription factor, FOXA1, which enhances β-catenin stabilization and activity. Targeting KRAS and mutant p53 cooperativity through pharmacologic inhibition of CREB1 dramatically dampens FOXA1 expression and PDAC metastasis. Our findings demonstrate a direct, mechanistic link between key oncogenic KRAS signaling elements and mutant p53 that result in a broad, multiplexed activation of cancer-associated transcriptional networks. Moreover, we identify CREB1 as a viable therapeutic strategy to undermine oncogenic KRAS and mutant p53 cooperation to mitigate PDAC metastasis. Citation Format: Michael Paul Kim, Xinqun Li, Jenying Deng, Yun Zhang, Bingbing Dai, Kendra Allton, Tara Hughes, Christian Siangco, Jithesh Augustine, Yaan Kang, Joy M. McDaniel, Shunbin Xiong, Eugene Koay, Florencia McAllister, Christopher Bristow, Timothy Heffernan, Anirban Maitra, Bin Liu, Michelle Barton, Amanda Wasylischen, Jason Fleming, Guillermina lozano. Mutant p53 and oncogenic KRAS converge on CREB1 to drive pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2417.

Recent evidence suggests that lncRNAs play an integral regulatory role in numerous functions, including determination of cellular identity. We determined global expression (RNA-seq) and genome wide profiles (ChIP-seq) of histone post-translational modifications and p53 binding in human embryonic stem cells (hESCs) undergoing differentiation to define a high-confidence set of 40 lncRNAs, which are p53 transcriptional targets. We focused on lncRNAs highly expressed in pluripotent hESCs and repressed by p53 during differentiation to identify lncPRESS1 as a p53-regulated transcript that maintains hESC pluripotency in concert with core pluripotency factors. RNA-seq of hESCs depleted of lncPRESS1 revealed that lncPRESS1 controls a gene network that promotes pluripotency. Further, we found that lncPRESS1 physically interacts with SIRT6 and prevents SIRT6 chromatin localization, which maintains high levels of histone H3K56 and H3K9 acetylation at promoters of pluripotency genes. In summary, we describe a p53-regulated, pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity.

Background Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy. Results To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells. Discussion Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway. Conclusions Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment. Electronic supplementary material The online version of this article (10.1186/s12864-018-4533-0) contains supplementary material, which is available to authorized users.

Trimethylated histone H3 lysine 27 (H3K27me3) is linked to gene silencing, whereas H3K4me3 is associated with gene activation. These two marks frequently co-occupy gene promoters, forming bivalent domains. Bivalency signifies repressed but activatable states of gene expression and can be resolved to active, H3K4me3-prevalent states during multiple cellular processes, including differentiation, development and epithelial mesenchymal transition. However, the molecular mechanism underlying bivalency resolution remains largely unknown. Here, we show that the H3K27 demethylase UTX (also called KDM6A) is required for the resolution and activation of numerous retinoic acid (RA)-inducible bivalent genes during the RA-driven differentiation of mouse embryonic stem cells (ESCs). Notably, UTX loss in mouse ESCs inhibited the RA-driven bivalency resolution and activation of most developmentally critical homeobox (Hox) a-d genes. The UTX-mediated resolution and activation of many bivalent Hox genes during mouse ESC differentiation were recapitulated during RA-driven differentiation of human NT2/D1 embryonal carcinoma cells. In support of the importance of UTX in bivalency resolution, Utx-null mouse ESCs and UTX-depleted NT2/D1 cells displayed defects in RA-driven cellular differentiation. Our results define UTX as a bivalency-resolving histone modifier necessary for stem cell differentiation.

Noncoding transcription is a defining feature of active enhancers, linking transcription factor (TF) binding to the molecular mechanisms controlling gene expression. To determine the relationship between enhancer activity and biological outcomes in breast cancers, we profiled the transcriptomes (using GRO-seq and RNA-seq) and epigenomes (using ChIP-seq) of 11 different human breast cancer cell lines representing five major molecular subtypes of breast cancer, as well as two immortalized (“normal”) human breast cell lines. In addition, we developed a robust and unbiased computational pipeline that simultaneously identifies putative subtype-specific enhancers and their cognate TFs by integrating the magnitude of enhancer transcription, TF mRNA expression levels, TF motif P-values, and enrichment of H3K4me1 and H3K27ac. When applied across the 13 different cell lines noted above, the Total Functional Score of Enhancer Elements (TFSEE) identified key breast cancer subtype-specific TFs that act at transcribed enhancers to dictate gene expression patterns determining growth outcomes, including Forkhead TFs, FOSL1, and PLAG1. FOSL1, a Fos family TF, (1) is highly enriched at the enhancers of triple negative breast cancer (TNBC) cells, (2) acts as a key regulator of the proliferation and viability of TNBC cells, but not Luminal A cells, and (3) is associated with a poor prognosis in TNBC breast cancer patients. Taken together, our results validate our enhancer identification pipeline and reveal that enhancers transcribed in breast cancer cells direct critical gene regulatory networks that promote pathogenesis.

Tumor suppressor p53 protects cells from genomic insults and is a target of mutation in more than 50% of human cancers. Stress-mediated modification and increased stability of p53 promote p53 interaction with chromatin, which results in transcription of target genes that are critical for the maintenance of genomic integrity. We recently discovered that TRIM24, an E3-ubiquitin ligase, ubiquitinates and promotes proteasome-mediated degradation of p53. Here, we show that TRIM24 is destabilized by ATM-mediated phosphorylation of TRIM24S768 in response to DNA damage, which disrupts TRIM24-p53 interactions and promotes the degradation of TRIM24. Transcription of TRIM24 is directly induced by damage-activated p53, which binds p53 response elements and activates expression of TRIM24. Newly synthesized TRIM24 interacts with phosphorylated p53 to target it for degradation and termination of the DNA damage response. These studies indicate that TRIM24, like MDM2, controls p53 levels in an autoregulatory feedback loop. However, unlike MDM2, TRIM24 also targets activated p53 to terminate p53-regulated response to DNA damage.

DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53

Epigenetic control of genes that are silent in embryonic stem cells, but destined for expression during differentiation, includes distinctive hallmarks, such as simultaneous activating/repressing (bivalent) modifications of chromatin and DNA hypomethylation at enhancers of gene expression. Although alpha-fetoprotein (Afp) falls into this class of genes, as it is silent in pluripotent stem cells and activated during differentiation of endoderm, we find that Afp chromatin lacks bivalent histone modifications. However, critical regulatory sites for Afp activation, overlapping Foxa1/p53/Smad-binding elements, are located within a 300-bp region lacking DNA methylation, due to transposed elements underrepresented in CpG sequences: a short interspersed transposable element and a medium reiterated sequence 1 element. Forkhead family member Foxa1 is activated by retinoic acid treatment of embryonic stem cells, binds its DNA consensus site within the short interspersed transposable/medium reiterated sequence 1 elements, and displaces linker histone H1 from silent Afp chromatin. Small interfering RNA depletion of Foxa1 showed that Foxa1 is essential in providing chromatin access to transforming growth factor beta-activated Smad2 and Smad4 and their subsequent DNA binding. Together these transcription factors establish highly acetylated chromatin and promote expression of Afp. Foxa1 acts as a pioneer transcription factor in de novo activation of Afp, by exploiting a lack of methylation at juxtaposed transposed elements, to bind and poise chromatin for intersection with transforming growth factor beta signaling during differentiation of embryonic stem cells.

How tumor suppressor p53 selectively responds to specific signals, especially in normal cells, is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation, induced by retinoic acid, versus DNA damage, caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and, in pluripotent hESCs, are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases, UTX and JMJD3, to chromatin. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation, a process highly distinct from stress-induced p53 response in hESCs.

Recent work shows that long non-coding RNAs (lncRNAs), transcribed from 80% of our genomes, play important roles in gene regulation and are associated with disease pathogenesis. However, there is a broad gap in our knowledge regarding any contributions of lncRNAs in mediating p53 response or regulating stem cell state. Here, we report our discovery of lncRNAs that fine-tune p53's transcriptional outcomes in differentiating human embryonic stem cells (hESCs). By integrating RNA-Seq with p53 ChIP-Seq analyses along with active or repressive histone marks of hESCs undergoing differentiation, we defined a high-confidence set of 40 lncRNAs that are p53 transcriptional targets. We found that p53 directly regulates nuclear lncRNAs, which are either activated (HOTAIRM1) or repressed (lncPRESS-1) during hESC differentiation. Single-cell gene expression analysis during lineage-specific hESC differentiation revealed that lncPRESS-1 is a novel pluripotency-specific, p53-regulated transcript with expression tightly correlated with OCT4, NANOG and PRDM14. Loss-of-function of lncPRESS-1 results in diminished pluripotency and spontaneous differentiation of hESCs. In addition, mass cytometry-based multi-parametric analysis at a single cell level revealed that depletion of lncPRESS-1 results in overall loss of hESC pluripotency with marked induction of an ectoderm specific gene-signature. Further, we show that lncPRESS-1 physically interacts with SIRT6 and restrains its chromatin localization to maintain high levels of histone H3 Lys-56 (H3K56) and Lys-9 (H3K9) acetylation at promoters of pluripotency genes to sustain a chromatin architecture that favors pluripotency. In summary, we describe a novel pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity. Since lncPRESS-1 is repressed by p53, we believe that cancers that harbor mutant TP53 will have robust expression of this lncRNA. Further investigations are underway to determine the disease relevance of lncPRESS-1 and correlation with p53-mutations. Citation Format: Abhinav K. Jain, Kadir C. Akdemir, Yuanxin Xi, Ryan L. McCarthy, Kendra Allton, Bruce Aronow, Chunru Lin, Wei Li, Liuqing Yang, Michelle C. Barton. Characterization of a novel p53-regulated embryonic stem cell-specific lncRNA that safeguards pluripotency. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A06.

Numerous studies focus on the tumor suppressor p53 as a protector of genomic stability, mediator of cell cycle arrest and apoptosis, and target of mutation in 50% of all human cancers. The vast majority of information on p53, its protein-interaction partners and regulation, comes from studies of tumor-derived, cultured cells where p53 and its regulatory controls may be mutated or dysfunctional. To address regulation of endogenous p53 in normal cells, we created a mouse and stem cell model by knock-in (KI) of a tandem-affinity-purification (TAP) epitope at the endogenous Trp-53 locus. Mass spectrometry of TAP-purified p53-complexes from embryonic stem cells revealed Tripartite-motif protein 24 (Trim24), a previously unknown partner of p53. Mutation of TRIM24 homolog, bonus , in Drosophila led to apoptosis, which could be rescued by p53-depletion. These in vivo analyses establish TRIM24/bonus as a pathway that negatively regulates p53 in Drosophila . The Trim24-p53 link is evolutionarily conserved, as TRIM24 depletion in human breast cancer cells caused p53-dependent, spontaneous apoptosis. We found that Trim24 ubiquitylates and negatively regulates p53 levels, suggesting Trim24 as a therapeutic target to restore tumor suppression by p53.

p53 is a critical guardian of somatic cells in the face of cellular stress and genomic challenges. Recent work shows that functions of transcription factor p53 go beyond its well-studied roles in tumor suppression. In addition to functions in cell cycle arrest and apoptosis, p53 is a functional barrier to reprogramming adult cells into induced pluripotent (iPS) cells that are phenotypically similar to embryonic stem (ES) cells. How p53 is regulated and functions in stem cells, is poorly understood. Previously we have shown that p53 plays a significant role that depends on its transcriptional activity in early differentiation of human ES cells (hESCs), as induced in vitro by retinoic acid (RA). In order to delineate p53 transcriptional functions during differentiation and how p53 selectively responds to specific signals in hESCs we performed genome wide profiling of p53 chromatin interactions and target gene expression in hESCs in response to early differentiation or DNA damage. The majority of p53 binding sites is unique to each state and defines stimulus-specific p53 response in hESCs. p53 binding regions specific to differentiation significantly overlap with those of core-pluripotency factors OCT4 and NANOG. Differentiation-specific p53 targets are highly expressed, and include many developmental transcription factors, poised by OCT4/NANOG and bivalent histone marks (H3K4me3 and H3K27me3) in pluripotent hESCs. Activation of poised genes during differentiation is associated with elimination of H3K27me3 marks, which is largely dependent on p53. During hESC differentiation, p53 interacts with H3K37me3 specific de-methylases (JMJD3 and UTX) that are enriched at the upstream regions of poised genes resulting in alteration of bivalent histone status and subsequent gene activation. These studies suggest that p53 plays essential roles in regulating epigenetic bivalency in ES cells. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Thus, our findings expand the registry of p53-regulated genes to define p53-mediated opposition to pluripotency during early differentiation, a process highly distinct from stress-induced p53 response in hESCs. This abstract is also presented as poster A68. Citation Format: Abhinav K. Jain, Kadir C. Akdemir, Kendra Allton, Bruce Aronow, Xueping Xu, Austin J. Cooney, Wei Li, Michelle C. Barton. Genome wide profiling reveals stimulus-specific functions of p53 in human embryonic stem cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR01.

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells, p53 in hESCs is maintained at low levels in the nucleus, albeit in a deacetylated, inactive state. In response to retinoic acid, CBP/p300 acetylates p53 at lysine 373, which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G1 phase of cell cycle without activation of cell death pathways. In parallel, p53 activates expression of miR-34a and miR-145, which in turn repress stem cell factors OCT4, KLF4, LIN28A, and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation, whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs, independently of retinoic acid. Ectopic expression of p53R175H, a mutated form of p53 that does not bind DNA or regulate transcription, failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state., Author Summary Most cell types in an organism are generated from embryonic stem cells (ESCs), which are able to proliferate an unlimited number of times and have the potential to produce any kind of cell of that organism (this ability is called pluripotency). In order to maintain these properties, ESCs have to remain in a proliferate state, which is achieved by the collaboration of several factors. Expressing combinations of these factors in differentiated cells can result in ESC-like qualities; these induced pluripotent stem cells (iPSCs) can then function like ESCs. Previous studies suggested that p53, generally known for its roles in maintaining genomic integrity by regulating cell cycle and cell death pathways, also acts as a barrier to reprogramming adult cells during the creation of iPSCs; whether this is strictly due to repression of proliferation remains a subject of debate. Here, we show that p53 plays a significant role in actively promoting differentiation of human ESCs (hESCs). We find that, prior to differentiation, p53 is expressed at very low levels in hESCs, held in check by two negative regulators, HDM2 and TRIM24, that trigger p53 degradation. Upon induction of differentiation, lysine 373 of p53 is acetylated, and this disrupts the existing interactions with negative regulators, thus allowing stabilization and activation of p53. Active p53 in turn promotes expression of the cell cycle regulator p21 to slow down the hESC cell cycle; cells in the gap (G1) phase of the cell cycle accumulate, preventing division. In parallel, p53 activates specific microRNAs, miR-34a and miR-145, that inhibit the expression of several stem cell factors and prevent differentiated cells from backsliding to pluripotency. Our results highlight a novel function of p53 in determining the human stem cell state.