Abhinav K. Jain

Trim24 targets endogenous p53 for degradation.

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 regulates cell cycle and micrornas to promote differentiation of human embryonic stem cells

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.

Genome-wide profiling reveals stimulus-specific functions of p53 during differentiation and DNA damage of human 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.

Regulation of p53: TRIM24 enters the RING

Negative regulation of p53 in normal, unstressed cells maintains levels of this tumor suppressor below a threshold for cell cycle arrest or apoptosis, and is rapidly reversed in the face of cellular stresses to permit p53 response. Recently, we created a new mouse and stem cell model by knock-in addition of an epitope tag at Trp53. Biochemical purification of endogenous, tagged p53-protein complexes from mouse embryonic stem cells, and peptide analysis by mass spectrometry, revealed a new RING-domain E3-ubiquitin ligase TRIM24 that targets p53 for degradation. Depletion of TRIM24, formerly named TIF1α, in tumor-derived cells induces p53-dependent apoptosis. In Drosophila, bonus is a single copy gene homologous to the mammalian Tif1 family. Mosaic deletion of bonus induces cell death in vivo, which is rescued by depletion of D-p53. Bonus is the first identified regulator of p53 protein levels in Drosophila, which lacks an ortholog of Mdm2. TRIM24/bonus may be the ancestral precursor of the large group of mammalian E3-ligases that target p53 for ubiquitin modification. Understanding the specific roles that these numerous E3-ligases have in the hierarchy of p53-regulation remains a challenge for the field. We discuss various scenarios for selectivity in choice of E3-ligase targeting p53 for degradation.

Analysis of epigenetic alterations to chromatin during development

Each cell within a multicellular organism has distinguishable characteristics established by its unique patterns of gene expression. This individual identity is determined by the expression of genes in a time and place‐dependent manner, and it is becoming increasingly clear that chromatin plays a fundamental role in the control of gene transcription in multicellular organisms. Therefore, understanding the regulation of chromatin and how the distinct identity of a cell is passed to daughter cells during development is paramount. Techniques with which to study chromatin have advanced rapidly over the past decade. Development of high throughput techniques and their proper applications has provided us essential tools to understand the regulation of epigenetic phenomena and its effect on gene expression. Understanding the changes that occur in chromatin during the course of development will not only contribute to our knowledge of normal gene expression, but will also add to our knowledge of how gene expression goes awry during disease. This review opens with an introduction to some of the key premises of epigenetic regulation of gene expression. A discussion of experimental techniques with which one can study epigenetic alterations to chromatin during development follows, emphasizing recent breakthroughs in this area. We then present examples of epigenetic mechanisms exploited in the control of developmental cell fate and regulation of tissue‐specific gene expression. Finally, we discuss some of the frontiers and challenges in this area of research. genesis 47:559–572, 2009.

Making sense of ubiquitin ligases that regulate p53

The functions of p53 most highly associated with the well-studied tumor suppressor are its abilities to induce cell cycle arrest and apoptosis in response to cellular stresses. Recent progress underscores that p53 is a multi-functional protein with activities that range beyond tumor suppression to normal homeostasis, metabolism, fertility and differentiation. A unifying theme of these studies is that p53 is first and foremost a transcription factor; and control of p53 protein stability determines its ability to carry out this task. There are an expanding number of E3-ubiquitin ligase proteins that target p53 for ubiquitin tagging and protein degradation. This review discusses these many effectors of p53 protein degradation, and our task is to provide some level of understanding as to their differences and their similarities. Further, we propose how some degree of specialization may be assigned to the E3-ligases, in their navigation toward a common goal of regulating p53 protein levels, and emphasize that better understanding of the mechanisms involved in E3-ligase functions is needed to further their potential as therapeutic targets.

TRIM24 Is a p53-Induced E3-Ubiquitin Ligase That Undergoes ATM-Mediated Phosphorylation and Autodegradation during DNA Damage

ABSTRACT 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.

Unmet Expectations: miR-34 Plays No Role in p53-Mediated Tumor Suppression In Vivo

In vivo modeling of tumor suppressor p53 functions and regulation has a history of unexpected and even enigmatic outcomes [1], despite the status of p53 as the most frequently mutated gene or dysfunctional pathway in human cancers [2], [3]. Beginning with the surprising viability of the first mice deleted for Trp53 [4], [5], various hypotheses of compensation, cell type–specificity, stimulus-dependent response, or modifier influences were posed to explain how an exquisitely regulated transcription factor, implicated in a vast array of pathways [6], appeared to have no impact on development. Limited background-specific developmental and fertility problems do occur, especially in female p53-null mice [7], [8], and deletion of potentially compensatory p53 family members, p63 and p73 isoforms, leads to profound developmental and tissue-specific phenotypes [9], [10]. But overall, the most striking result of p53 loss in vivo is early tumor predisposition in p53−/− mice, which lack genomic surveillance provided by p53-mediated regulation of cell cycle arrest, apoptosis, and senescence. As reported by Concepcion et al. in this issue of PLoS Genetics [11], expectations built on cell-based studies of p53 response are again unrealized in mouse models. Previously, multiple in vitro analyses suggested that microRNA (miR)-34 family members are important players in a p53-regulated network of genomic surveillance [12]–[17] (Table 1). Together, these studies strongly supported the view that p53 response to multiple stimuli depended on miR-34, and that ectopic expression of miR-34 was sufficient to elicit p53 response, consistent with miR-34 functioning as a bonafide tumor suppressor. However, Concepcion et al. report that complete inactivation of the entire family of miR-34 genes (miR-34a/b/c) or knockout of each individual miR-34 gene in mice leads to little or no change in p53-mediated functions in tumor suppression [11]. Table 1 A list of different in vitro and in vivo model systems used to study miR-34 functions. Interest in a miR-34 axis as mediator of p53-response begins with the niche that miRNAs fill in regulation of RNA expression. miRNAs are small, regulatory non-coding RNAs that generally mediate post-transcriptional silencing of a number of specific target mRNAs [18]. More than 50% of human miRNA genes are found within cancer-associated or fragile sites of the genome, which suggests that miRNAs play essential roles in tumorigenesis [19]. The identification of miRNAs as regulatory targets of p53 [20] suggested their potential involvement in tumor suppression, and expanded the repertoire of p53 downstream targets to both coding and non-coding genes. Further, the view that p53 both positively and negatively regulates gene expression could now rely on increased expression of miRNAs as a mechanism for p53-mediated, indirect repression of gene expression [13], [20], in addition to the few documented cases of direct repression by p53 binding to chromatin [21]–[25]. The members of the evolutionarily conserved miR-34 family, which arise from three different transcripts at two different gene loci in vertebrates, were the first of several non-coding RNAs identified as directly activated by p53 in response to genotoxic stress [13], [26]. miR-34a is at 1p36, a region commonly deleted in tumors, and miR-34b and miR-34c share a common primary transcript arising from 11q23 [27], [28]. miR-34a, b, and c are expressed at very low levels in several types of cancers [28]. Previous reports show that p53 directly activates miR-34a/b/c expression and, dependent on cellular context, they act downstream of p53 in mediating cell cycle arrest or apoptosis [29]. The current list of validated miR-34 downstream targets includes several genes that are repressed during cell cycle arrest or apoptosis when p53 is activated [28]. Given the rationale provided by these studies in cultured cells (Table 1), multiple laboratories created genetic knockout models of either miR-34a or miR-34b/c, or a compound mutant animal harboring homozygous deletion of all three miR-34 family members (miR-34TKO) [11], [30]. Surprisingly, mice bearing the miR-34 deletion(s) developed normally, are born at the expected Mendelian ratio, and are fertile [11]. The authors subjected the mice and derived mouse embryonic fibroblasts (MEFs) to a battery of tests to assess any impact on p53-dependent tumor suppression. MEFs obtained from mir-34TKO mice have a slightly higher proliferation rate, but reach senescence with kinetics similar to wild-type MEFs. In response to genotoxic threats, miR-34–deficient MEFs are indistinguishable from wild type: they undergo p53-dependent cell cycle arrest and apoptosis. With ectopic expression of oncogenic K-Ras, p53-deficient MEFs are readily transformed, which is not true of K-Ras–expressing miR-34−/− MEFs. In the intact mouse, the story is similar: aging cohorts of mir-34TKO mice remain healthy with no spontaneous tumors, in contrast to p53-null mice [4]. In fact, miR-34–deficient mice remain remarkably healthy and tumor-free for at least 60 weeks after irradiation. Assays of apoptosis in response to irradiation proved positive in tissues of these mice, which additionally exhibited no acceleration of tumor progression in Eμ-models of B-cell lymphomagenesis. All of these assessments of p53 functions in vivo undermine the view that miR-34 functions as a tumor suppressor or is an essential component of the p53-tumor suppression network. Although miR-34 proved nonessential in the most highly studied examples of p53 function (senescence, cell cycle arrest, apoptosis, and tumor suppression), it remains possible that miR-34 is involved in other p53-influenced processes, such as metabolism, autophagy, stem cell quiescence, differentiation, and embryogenesis [6]. For example, specific links between miR-34– and p53-regulated functions have been forged in stem cells [26]. miR-34–deficient MEFs are more efficiently reprogrammed to induced pluripotent stem cells (iPSCs), by expression of pluripotency factors and c-myc [30], compared to wild-type counterparts. While this study of miR-34 as a barrier to reprogramming does not establish a direct tie to p53, it complements multiple reports that depletion of p53 or dysfunctional p53 pathways enhance the efficiency of reprogramming differentiated, somatic cells to iPSCs [31]. Recently, we showed that p53 promotes human embryonic stem cell differentiation by direct activation of p21 and miRNAs, including miR-34a, which repress pluripotency factors and SIRT1 [32]. Taken together, these results indicate that miR-34 has pro-differentiation effects in maintenance of nontransformed, somatic cells, some of which are p53-dependent. In the future, miR-34–deficient mouse models will be valuable in addressing whether miR-34 functions downstream of p53 in a tissue- and/or context-specific manner. miR-34a, miR-34b, and miR-34c share the same seed sequence and target the same RNAs, although differences in target accessibility or binding affinities may dictate their effectiveness. Genome-wide expression analysis may be needed to determine family member–specific effects, such as the reported regulation of c-MYC by miR-34b/c and not miR-34a [33]. Questions of specificity in gene targets for each member of a miRNA family and potential compensation by other miRNAs may be addressed by studies in these and other miRNA mouse models, perhaps still under development. Non-coding RNAs are thought to act in networks that impact diverse cellular pathways, suggesting considerable challenges ahead in asking the right questions and understanding the functional significance of these RNAs.

p53-independent DUX4 pathology in cell and animal models of facioscapulohumeral muscular dystrophy

ABSTRACT Facioscapulohumeral muscular dystrophy (FSHD) is a genetically dominant myopathy caused by mutations that disrupt repression of the normally silent DUX4 gene, which encodes a transcription factor that has been shown to interfere with myogenesis when misexpressed at very low levels in myoblasts and to cause cell death when overexpressed at high levels. A previous report using adeno-associated virus to deliver high levels of DUX4 to mouse skeletal muscle demonstrated severe pathology that was suppressed on a p53-knockout background, implying that DUX4 acted through the p53 pathway. Here, we investigate the p53 dependence of DUX4 using various in vitro and in vivo models. We find that inhibiting p53 has no effect on the cytoxicity of DUX4 on C2C12 myoblasts, and that expression of DUX4 does not lead to activation of the p53 pathway. DUX4 does lead to expression of the classic p53 target gene Cdkn1a (p21) but in a p53-independent manner. Meta-analysis of 5 publicly available data sets of DUX4 transcriptional profiles in both human and mouse cells shows no evidence of p53 activation, and further reveals that Cdkn1a is a mouse-specific target of DUX4. When the inducible DUX4 mouse model is crossed onto the p53-null background, we find no suppression of the male-specific lethality or skin phenotypes that are characteristic of the DUX4 transgene, and find that primary myoblasts from this mouse are still killed by DUX4 expression. These data challenge the notion that the p53 pathway is central to the pathogenicity of DUX4. Summary: DUX4 is thought to mediate cytopathology through p53. Here, DUX4 is shown to kill primary myoblasts and promote pathological phenotypes in the iDUX4[2.7] mouse model on the p53-null background, calling into question this notion.

p53: emerging roles in stem cells, development and beyond

ABSTRACT Most human cancers harbor mutations in the gene encoding p53. As a result, research on p53 in the past few decades has focused primarily on its role as a tumor suppressor. One consequence of this focus is that the functions of p53 in development have largely been ignored. However, recent advances, such as the genomic profiling of embryonic stem cells, have uncovered the significance and mechanisms of p53 functions in mammalian cell differentiation and development. As we review here, these recent findings reveal roles that complement the well-established roles for p53 in tumor suppression. Summary: This Primer provides an overview of the roles of p53 in development and stem cells, and highlights how these relate to its well-known functions in tumor suppression.