Mammary-specific expression of Trim24 establishes a mouse model of human metaplastic breast cancer.

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.

p53: emerging roles in stem cells, development and beyond.

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.

Regulation of gene expression in human cancers by TRIM24.

Tripartite Motif-containing protein 24 (TRIM24) functions as an E3 ligase targeting p53 for ubiquitination, a histone 'reader' that interacts with a specific signature of histone post-translational modifications and a co-regulator of nuclear receptor-regulated transcription. Although mouse models of Trim24 depletion suggest that TRIM24 may be a liver-specific tumor suppressor, several studies show that human TRIM24 is an oncogene when aberrantly over expressed. This review focuses on the mechanisms of TRIM24 functions in oncogenesis and metabolic reprogramming, which underlie recent interest in therapeutic targeting of aberrant TRIM24 in human cancers.

An essential role for UTX in resolution and activation of bivalent promoters.

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.

Minimal role of base excision repair in TET-induced global DNA demethylation in HEK293T cells.

Oxidation of 5-methylcytosine by TET family proteins can induce DNA replication-dependent (passive) DNA demethylation and base excision repair (BER)-based (active) DNA demethylation. The balance of active vs. passive TET-induced demethylation remains incompletely determined. In the context of large scale DNA demethylation, active demethylation may require massive induction of the DNA repair machinery and thus compromise genome stability. To study this issue, we constructed a tetracycline-controlled TET-induced global DNA demethylation system in HEK293T cells. Upon TET overexpression, we observed induction of DNA damage and activation of a DNA damage response; however, BER genes are not upregulated to promote DNA repair. Depletion of TDG (thymine DNA glycosylase) or APEX1 (apurinic/apyrimidinic endonuclease 1), two key BER enzymes, enhances rather than impairs global DNA demethylation, which can be explained by stimulated proliferation. By contrast, growth arrest dramatically blocks TET-induced global DNA demethylation. Thus, in the context of TET-induction in HEK293T cells, the DNA replication-dependent passive mechanism functions as the predominant pathway for global DNA demethylation. In the same context, BER-based active demethylation is markedly restricted by limited BER upregulation, thus potentially preventing a disastrous DNA damage response to extensive active DNA demethylation.

TRIM24 suppresses development of spontaneous hepatic lipid accumulation and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Aberrantly high expression of TRIM24 occurs in human cancers, including hepatocellular carcinoma. In contrast, TRIM24 in the mouse is reportedly a liver-specific tumour suppressor. To address this dichotomy and to uncover direct regulatory functions of TRIM24 in vivo, we developed a new mouse model that lacks expression of all Trim24 isoforms, as the previous model expressed normal levels of Trim24 lacking only exon 4. METHODS: To produce germline-deleted Trim24(dlE1) mice, deletion of the promoter and exon 1 of Trim24 was induced in Trim24(LoxP) mice by crossing with a zona pellucida 3-Cre line for global deletion. Liver-specific deletion (Trim24(hep)) was achieved by crossing with an albumin-Cre line. Phenotypic analyses were complemented by protein, gene-specific and global RNA expression analyses and quantitative chromatin immunoprecipitation. RESULTS: Global loss of Trim24 disrupted hepatic homeostasis in 100% of mice with highly significant, decreased expression of oxidation/reduction, steroid, fatty acid, and lipid metabolism genes, as well as increased expression of genes involved in unfolded protein response, endoplasmic reticulum stress and cell cycle pathways. Trim24(dlE1/dlE1) mice have markedly depleted visceral fat and, like Trim24(hep/hep) mice, spontaneously develop hepatic lipid-filled lesions, steatosis, hepatic injury, fibrosis and hepatocellular carcinoma. CONCLUSIONS: TRIM24, an epigenetic co-regulator of transcription, directly and indirectly represses hepatic lipid accumulation, inflammation, fibrosis and damage in the murine liver. Complete loss of Trim24 offers a model of human non-alcoholic fatty liver disease, steatosis, fibrosis and development of hepatocellular carcinoma in the absence of high-fat diet or obesity.

TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells.

TET1 is a 5-methylcytosine dioxygenase and its DNA demethylating activity has been implicated in pluripotency and reprogramming. However, the precise role of TET1 in DNA methylation regulation outside of developmental reprogramming is still unclear. Here, we show that overexpression of the TET1 catalytic domain but not full length TET1 (TET1-FL) induces massive global DNA demethylation in differentiated cells. Genome-wide mapping reveals that 5-hydroxymethylcytosine production by TET1-FL is inhibited as DNA methylation increases, which can be explained by the preferential binding of TET1-FL to unmethylated CpG islands (CGIs) through its CXXC domain. TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs. We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading into CGIs in differentiated cells.

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.

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

UNLABELLED: 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, whether this is done via direct or indirect means remain unknown. The ability of normal, mature hepatocytes to respond to stimuli, reenter the 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 the cell cycle and prolonged proliferation with an increased number of polyploid mitoses. Ploidy levels increased during regeneration of both wild-type (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.

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 G(1) 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.

Integrative genomics: liver regeneration and hepatocellular carcinoma.

Numerous genome wide profiles of gene expression changes in human hepatocellular carcinoma (HCC), compared to normal liver tissue, have been reported. Hierarchical clustering of these data reveal distinct patterns, which underscore conservation between human disease and mouse models of HCC, as well as suggest specific classification of subtypes within the heterogeneous disease of HCC. Global profiling of gene expression in mouse liver, challenged by partial hepatectomy to regenerate, reveals alterations in gene expression that occur in response to acute injury, inflammation, and re-entry into cell cycle. When we integrated datasets of gene expression changes in mouse models of HCC and those that are altered at specific times of liver regeneration, we saw shared, conserved alterations in gene expression within specific biological pathways, both up-regulated, for example, cell cycle, cell death, and cellular development, or down-regulated, for example, vitamin and mineral metabolism, lipid metabolism, and molecular transport. Additional molecular mechanisms shared by liver regeneration and HCC, as yet undiscovered, may have important implications in tumor development and recurrence. These comparisons may offer a way to judge how liver resection, in the treatment of HCC, introduces challenges to care of the disease. Further, uncovering the pathways conserved in inflammatory response, hypertrophy, proliferation, and architectural remodeling of the liver, which are shared in liver regeneration and HCC, versus those specific to tumor development and progression in HCC, may reveal new biomarkers or potential therapeutic targets in HCC.

Cascades of transcription regulation during liver regeneration.

An increasing demand for new strategies in cancer prevention and regenerative medicine requires a better understanding of molecular mechanisms that control cell proliferation in tissue-specific manner. Regenerating liver is a unique model allowing use of biochemical, genetic, and engineering tools to uncover molecular mechanisms and improve treatment of hepatic cancers, liver failure, and fibrotic disease. Molecular mechanisms of liver regeneration involve extra- and intracellular factors to activate transcription of genes normally silenced in quiescent liver. While many upstream signaling pathways of the regenerating liver have been extensively studied, our knowledge of the downstream effectors, transcription factors (TFs), remains limited. This review describes consecutive engagement of pre-existing and de novo synthesized TFs, as cascades that regulate expression of growth-related and metabolic genes during liver regeneration after partial hepatectomy in mice. Several previously recognized regulators of regenerating liver are described in the light of recently identified co-activator and co-repressor complexes that interact with primary DNA-binding TFs. Published results of gene expression and chromatin immunoprecipitation analyses, as well as studies of transgenic mouse models, are used to emphasize new potential regulators of transcription during liver regeneration. Finally, a more detailed description of newly identified transcriptional regulators of liver regeneration illustrates the tightly regulated balance of proliferative and metabolic responses to partial hepatectomy.

TRIM24 links a non-canonical histone signature to breast cancer.

Recognition of modified histone species by distinct structural domains within 'reader' proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here we report that chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 (that is, histone H3 unmodified at lysine 4, H3K4me0) and acetylated H3K23 (histone H3 acetylated at lysine 23, H3K23ac) within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumour development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.

Hierarchies of transcriptional regulation during liver regeneration.

The remarkable capacity of the liver to regenerate after severe injury or disease has excited interest for centuries. The goal of harnessing this process in treatment of liver disease, and the appreciation of the parallels between regeneration and tumor development in the liver, remain a major driver for research in this area. Studies of liver regeneration as a model system offer a view of intricate and precisely timed regulatory pathways that drive the process toward completion. Successful regeneration of the liver mass demands a hierarchal and well-controlled balance between proliferative and metabolic functions, which is orchestrated by signaling and regulation of transcription factors. Control and regulation of these cascades of transcriptional activities, necessary for induction, renewal, and cessation of liver growth, are the focus of this chapter.

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.

Direct activation of forkhead box O3 by tumor suppressors p53 and p73 is disrupted during liver regeneration in mice.

UNLABELLED: The p53 family of proteins regulates the expression of target genes that promote cell cycle arrest and apoptosis, which may be linked to cellular growth control as well as tumor suppression. Within the p53 family, p53 and the transactivating p73 isoform (TA-p73) have hepatic-specific functions in development and tumor suppression. Here, we determined TA-p73 interactions with chromatin in the adult mouse liver and found forkhead box O3 (Foxo3) to be one of 158 gene targets. Global profiling of hepatic gene expression in the regenerating liver versus the quiescent liver revealed specific, functional categories of genes regulated over the time of regeneration. Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative cellular proliferation. p53 and TA-p73 bind a Foxo3 p53 response element (p53RE) and maintain active expression in the quiescent liver. During regeneration of the liver, the binding of p53 and TA-p73, the recruitment of acetyltransferase p300, and the active chromatin structure of Foxo3 are disrupted along with a loss of Foxo3 expression. In agreement with the loss of Foxo3 transcriptional activation, a decrease in histone activation marks (dimethylated histone H3 at lysine 4, acetylated histone H3 at lysine 14, and acetylated H4) at the Foxo3 p53RE was detected after partial hepatectomy in mice. These parameters of Foxo3 regulation are reestablished with the completion of liver growth and regeneration and support a temporary suspension of p53 and TA-p73 regulatory functions in normal cells during tissue regeneration. p53-dependent and TA-p73-dependent activation of Foxo3 was also observed in mouse embryonic fibroblasts and in mouse hepatoma cells overexpressing p53, TA-p73alpha, and TA-p73beta isoforms. CONCLUSION: p53 and p73 directly bind and activate the expression of the Foxo3 gene in the adult mouse liver and murine cell lines. p53, TA-p73, and p300 binding and Foxo3 expression decrease during liver regeneration, and this suggests a critical growth control mechanism mediated by these transcription factors in vivo.

Foxa1 functions as a pioneer transcription factor at transposable elements to activate Afp during differentiation of embryonic stem cells.

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.