Gomes NP, Espinosa JMGene-specific repression of the p53 target gene PUMA via intragenic CTCF-Cohesin binding. Genes Dev 24:1022-1034

Howard Hughes Medical Institute, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA.
Genes & development (Impact Factor: 10.8). 05/2010; 24(10):1022-34. DOI: 10.1101/gad.1881010
Source: PubMed


The p53 transcriptional program orchestrates alternative responses to stress, including cell cycle arrest and apoptosis, but the mechanism of cell fate choice upon p53 activation is not fully understood. Here we report that PUMA (p53 up-regulated modulator of apoptosis), a key mediator of p53-dependent cell death, is regulated by a noncanonical, gene-specific mechanism. Using chromatin immunoprecipitation assays, we found that the first half of the PUMA locus (approximately 6 kb) is constitutively occupied by RNA polymerase II and general transcription factors regardless of p53 activity. Using various RNA analyses, we found that this region is constitutively transcribed to generate a long unprocessed RNA with no known coding capacity. This permissive intragenic domain is constrained by sharp chromatin boundaries, as illustrated by histone marks of active transcription (histone H3 Lys9 trimethylation [H3K4me3] and H3K9 acetylation [H3K9Ac]) that precipitously transition into repressive marks (H3K9me3). Interestingly, the insulator protein CTCF (CCCTC-binding factor) and the Cohesin complex occupy these intragenic chromatin boundaries. CTCF knockdown leads to increased basal expression of PUMA concomitant with a reduction in chromatin boundary signatures. Importantly, derepression of PUMA upon CTCF depletion occurs without p53 activation or activation of other p53 target genes. Therefore, CTCF plays a pivotal role in dampening the p53 apoptotic response by acting as a gene-specific repressor.

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Available from: Joaquín M Espinosa, Mar 20, 2015
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    • "Upon p53 activation, resulting in functional PUMA mRNA accumulation confirmed the results of our RT-qPCR analyses (Figure 10(a)). These observations in correlation with the results of other in vitro studies [80] suggested that PUMA a major effector of p53-mediated cell death may play an important role in in vitro regulator of apoptosis when it overexpressed. Moreover the results of the overexpression of p53 mRNA and PUMA mRNA (Figure 10(a)) taken together corroborated the findings expressed by Tsai and Barton [82], suggesting apparently that the branch of apoptosis put in evidence here was the intrinsic mitochondrial pathway. "
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    ABSTRACT: Background: Rhein is a natural anthraquinone molecule enriched in the rhizome of Polygonum multiflorum, a traditional Chinese medicine herb with various bioactivities such as anti-tumor, antibacterial, anti-inflammation and anti-aging. However, liver injuries induced by Polygonum multiflorum have been raising serious concern in recent years, and the mechanisms are still not fully elucidated. The aim of this study was to assess rhein-induced apoptosis and to investigate its molecular mechanisms in primary human hepatic cells. Results: Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25μM, 50μM, 100μM) for 12 h, the detection of apoptotic cells were significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed up-regulation level of the relevant hepatic markers (P<0.01). Caspase activities expressed significant up-regulation of caspase-3, -9 and -8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor and caspase-3 inhibitor respectively. Overproduction of reactive oxygen species, lipid peroxidation and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant up-regulation of p53, PUMA, Apaf-1, Casp-9 and -3 mRNA, with no significant changes of Fas and cytochrome c. Immunoblotting revealed significant cytochrome c release from mitochondria into cytosol, and no change in Fas expression. Conclusion: Taken together, these observations suggested rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.
    Evidence-based Complementary and Alternative Medicine 05/2015; 2015:19 pages. DOI:10.1155/2015/329831 · 1.88 Impact Factor
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    • "Of these, the PUMA RE has moderate transactivation potential based on its sequence features. Interestingly, the discovery of an insulator-type RNA-polymerase II stop site in proximity to exon 3 led to the proposal (82) that there is a post-transcriptional initiation mechanism for the regulation of PUMA expression, suggesting that in this case fine tuning of p53 affinity for the promoter may not be critical. "
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    ABSTRACT: Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein-DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.
    Nucleic Acids Research 07/2013; 41(18). DOI:10.1093/nar/gkt657 · 9.11 Impact Factor
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    • "Cohesin–DNA interactions are known to be influenced by loading factors (Scc2-Scc4) (34,35), transcription factors (e.g. mediator complex or CTCF) (11,36) and the DNA double-strand break repair machinery (9). Here we have shown that the core heterodimer alone (SMC1/3) is able to directly interact with DNA to drive a stepwise compaction process, and to mediate interactions between separate DNA molecules. "
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    ABSTRACT: Cohesin plays a critical role in sister chromatid cohesion, double-stranded DNA break repair and regulation of gene expression. However, the mechanism of how cohesin directly interacts with DNA remains unclear. We report single-molecule experiments analyzing the interaction of the budding yeast cohesin Structural Maintenance of Chromosome (SMC)1-SMC3 heterodimer with naked double-helix DNA. The cohesin heterodimer is able to compact DNA molecules against applied forces of 0.45 pN, via a series of extension steps of a well-defined size ≈130 nm. This reaction does not require ATP, but is dependent on DNA supercoiling: DNA with positive torsional stress is compacted more quickly than negatively supercoiled or nicked DNAs. Un-nicked torsionally relaxed DNA is a poor substrate for the compaction reaction. Experiments with mutant proteins indicate that the dimerization hinge region is crucial to the folding reaction. We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe.
    Nucleic Acids Research 04/2013; 41(12). DOI:10.1093/nar/gkt303 · 9.11 Impact Factor
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