Aymone Gurtner

Direct p53 transcriptional repression: in vivo analysis of CCAAT-containing G2/M promoters.

ABSTRACT In response to DNA damage, p53 activates G1/S blocking and apoptotic genes through sequence-specific binding. p53 also represses genes with no target site, such as those for Cdc2 and cyclin B, key regulators of the G2/M transition. Like most G2/M promoters, they rely on multiple CCAAT boxes activated by NF-Y, whose binding to DNA is temporally regulated during the cell cycle. NF-Y associates with p53 in vitro and in vivo through the αC helix of NF-YC (a subunit of NF-Y) and a region close to the tetramerization domain of p53. Chromatin immunoprecipitation experiments indicated that p53 is associated with cyclin B2, CDC25C, and Cdc2 promoters in vivo before and after DNA damage, requiring DNA-bound NF-Y. Following DNA damage, p53 is rapidly acetylated at K320 and K373 to K382, histones are deacetylated, and the release of PCAF and p300 correlates with the recruitment of histone deacetylases (HDACs)—HDAC1 before HDAC4 and HDAC5—and promoter repression. HDAC recruitment requires intact NF-Y binding sites. In transfection assays, PCAF represses cyclin B2, and a nonacetylated p53 mutant shows a complete loss of repression potential, despite its abilities to bind NF-Y and to be recruited on G2/M promoters. These data (i) detail a strategy of direct p53 repression through associations with multiple NF-Y trimers that is independent of sequence-specific binding of p53 and that requires C-terminal acetylation, (ii) suggest that p53 is a DNA damage sentinel of the G2/M transition, and (iii) delineate a new role for PCAF in cell cycle control.

HDAC2 blockade by nitric oxide and histone deacetylase inhibitors reveals a common target in Duchenne muscular dystrophy treatment

The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy.

The cyclin B2 promoter depends on NF-Y, a trimer whose CCAAT-binding activity is cell-cycle regulated

Cyclin B2 is a regulator of p34cdc2 kinase, involved in G2/M progression of the cell cycle, whose gene is strictly regulated at the transcriptional level in cycling cells. The mouse promoter was cloned and three conserved CCAAT boxes were found. In this study, we analysed the mechanisms leading to activation of the cyclin B2 CCAAT boxes: a combination of (i) genomic footprinting, (ii) transfections with single, double and triple mutants, (iii) EMSAs with nuclear extracts, antibodies and NF-Y recombinant proteins and (iv) transfections with an NF-YA dominant negative mutant established the positive role of the three CCAAT sequences and proved that NF-Y plays a crucial role in their activation. NF-Y, an ubiquitous trimer containing histone fold subunits, activates several other promoters regulated during the cell cycle. To analyse the levels of NF-Y subunits in the different phases of the cycle, we separated MEL cells by elutriation, obtaining fractions >80% pure. The mRNA and protein levels of the histone-fold containing NF-YB and NF-YC were invariant, whereas the NF-YA protein, but not its mRNA, was maximal in mid-S and decreased in G2/M. EMSA confirmed that the CCAAT-binding activity followed the amount of NF-YA, indicating that this subunit is limiting within the NF-Y complex, and suggesting that post-transcriptional mechanisms regulate NF-YA levels. Our results support a model whereby fine tuning of this activator is important for phase-specific transcription of CCAAT-containing promoters.

The Transcriptional Repressor ZEB Regulates p73 Expression at the Crossroad between Proliferation and Differentiation

ABSTRACT The newly discovered p73 gene encodes a nuclear protein that has high homology with p53. Furthermore, ectopic expression of p73 in p53+/+ and p53−/− cancer cells recapitulates some of the biological activities of p53 such as growth arrest, apoptosis, and differentiation. p73−/−-deficient mice exhibit severe defects in proper development of the central nervous system and pheromone sensory pathway. They also suffer from inflammation and infections. Here we studied the transcriptional regulation of p73 at the crossroad between proliferation and differentiation. p73 mRNA is undetectable in proliferating C2C12 cells and is expressed at very low levels in undifferentiated P19 and HL60 cells. Conversely, it is upregulated during muscle and neuronal differentiation as well as in response to tetradecanoyl phorbol acetate-induced monocytic differentiation of HL60 cells. We identified a 1-kb regulatory fragment located within the first intron of p73, which is positioned immediately upstream to the ATG codon of the second exon. This fragment exerts silencer activity on p73 as well as on heterologous promoters. The p73 intronic fragment contains six consensus binding sites for transcriptional repressor ZEB, which binds these sites in vitro and in vivo. Ectopic expression of dominant-negative ZEB (ZEB-DB) restores p73 expression in proliferating C2C12 and P19 cells. Thus, transcriptional repression of p73 expression by ZEB binding may contribute to the modulation of p73 expression during differentiation.

Repression of the Antiapoptotic Molecule Galectin-3 by Homeodomain-Interacting Protein Kinase 2-Activated p53 Is Required for p53-Induced Apoptosis

ABSTRACT Galectin 3 (Gal-3), a member of the β-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3.

The cyclin B1 gene is actively transcribed during mitosis in HeLa cells

In mammalian cells, the expression level of the cyclin B1 gene plays a critical role in the progression through mitosis. Here we demonstrate that the transcriptional activity of the human cyclin B1 promoter, as well as the rate of gene transcription, is high during mitosis. Indeed, the cyclin B1 promoter maintains an open chromatin configuration at the mitotic stage. Consistent with this, we show that the cyclin B1 promoter is occupied and bound to NF‐Y during mitosis in vivo. Our results provide the first example of RNA polymerase II‐dependent transcription during mitosis in mammalian cells.

Mutant p53-induced Up-regulation of Mitogen-activated Protein Kinase Kinase 3 Contributes to Gain of Function

Mitogen-activated protein kinase kinase 3 (MAP2K3) is a member of the dual specificity kinase group. Growing evidence links MAP2K3 to invasion and tumor progression. Here, we identify MAP2K3 as a transcriptional target of endogenous gain-of-function p53 mutants R273H, R175H, and R280K. We show that MAP2K3 modulation occurred at the mRNA and protein levels and that endogenous mutant p53 proteins are capable of binding to and activate the MAP2K3 promoter. In addition, we found that the studied p53 mutants regulate MAP2K3 gene expression through the involvement of the transcriptional cofactors NF-Y and NF-κB. Finally, functional studies showed that endogenous MAP2K3 knockdown inhibits proliferation and survival of human tumor cells, whereas the ectopic expression of MAP2K3 can rescue the proliferative defect induced by mutant p53 knockdown. Taken together, our findings define a novel player through which mutant p53 exerts its gain-of-function activity in cancer cells.

NF-Y Dependent Epigenetic Modifications Discriminate between Proliferating and Postmitotic Tissue

The regulation of gene transcription requires posttranslational modifications of histones that, in concert with chromatin remodeling factors, shape the structure of chromatin. It is currently under intense investigation how this structure is modulated, in particular in the context of proliferation and differentiation. Compelling evidence suggests that the transcription factor NF-Y acts as a master regulator of cell cycle progression, activating the transcription of many cell cycle regulatory genes. However, the underlying molecular mechanisms are not yet completely understood. Here we show that NF-Y exerts its effect on transcription through the modulation of the histone “code”. NF-Y colocalizes with nascent RNA, while RNA polymerase II is I phosphorylated on serine 2 of the YSPTSPS repeats within its carboxyterminal domain and histones are carrying modifications that represent activation signals of gene expression (H3K9ac and PAN-H4ac). Comparing postmitotic muscle tissue from normal mice and proliferating muscles from mdx mice, we demonstrate by chromatin immunoprecipitation (ChIP) that NF-Y DNA binding activity correlates with the accumulation of acetylated histones H3 and H4 on promoters of key cell cycle regulatory genes, and with their active transcription. Accordingly, p300 is recruited onto the chromatin of NF-Y target genes in a NF-Y-dependent manner, as demonstrated by Re-ChIP. Conversely, the loss of NF-Y binding correlates with a decrease of acetylated histones, the recruitment of HDAC1, and a repressed heterochromatic state with enrichment of histones carrying modifications known to mediate silencing of gene expression (H3K9me3, H3K27me2 and H4K20me3). As a consequence, NF-Y target genes are downregulated in this context. In conclusion, our data indicate a role of NF-Y in modulating the structure and transcriptional competence of chromatin in vivo and support a model in which NF-Y-dependent histone “code” changes contribute to the proper discrimination between proliferating and postmitotic cells in vivo and in vitro.

Posttranslational Regulation of NF-YA Modulates NF-Y Transcriptional Activity

NF-Y binds to CCAAT motifs in the promoter region of a variety of genes involved in cell cycle progression. The NF-Y complex comprises three subunits, NF-YA, -YB, and -YC, all required for DNA binding. Expression of NF-YA fluctuates during the cell cycle and is down-regulated in postmitotic cells, indicating its role as the regulatory subunit of the complex. Control of NF-YA accumulation is posttranscriptional, NF-YA mRNA being relatively constant. Here we show that the levels of NF-YA protein are regulated posttranslationally by ubiquitylation and acetylation. A NF-YA protein carrying four mutated lysines in the C-terminal domain is more stable than the wild-type form, indicating that these lysines are ubiquitylated Two of the lysines are acetylated in vitro by p300, suggesting a competition between ubiquitylation and acetylation of overlapping residues. Interestingly, overexpression of a degradation-resistant NF-YA protein leads to sustained expression of mitotic cyclin complexes and increased cell proliferation, indicating that a tight regulation of NF-YA levels contributes to regulate NF-Y activity.

deltaEF1 repressor controls selectively p53 family members during differentiation

The discovery of two new p53 homologs, p73 and p63, has defined a family of transcription factors heavily involved in the control of growth suppression, apoptosis, differentiation and development. While p53-deficient mice undergo spontaneous tumors, p73 and p63 knockout mice exhibit severe developmental defects. We demonstrate here that p73 gene is an in vivo transcriptional target of the muscle regulatory factors MyoD, myogenin, Myf5 and Myf6. Ectopic expression of the transcriptional repressor δEF1/ZEB/zfhx1a counteracts MyoD/Myf5- or MyoD/Myf6-mediated transcriptional activation of p73. A distinct pattern of in vivo recruitment of muscle regulatory factors and δEF1 on p73 regulatory regions was found between proliferating and differentiating muscle cells. We also found that δEF1 plays a role in the transcriptional regulation of p53 family members during keratinocytic differentiation. Mouse embryo fibroblasts derived from δEF1-deficient mice exhibit unbalanced expression of ΔNp63, TAp73 and ΔNp73 but not of TAp63 and p53. The analysis of tissues derived from δEF1+/− mice exhibit a selective enrichment of ΔNp63 in skin.