Signal transducer and activator of transcription 2 (STAT2), the critical component of type I interferons signaling, is a prototype latent cytoplasmic signal-dependent transcription factor. Activated tyrosine-phosphorylated STAT2 associates with STAT1 and IRF9 to bind the ISRE elements in the promoters of a subset of IFN-inducible genes (ISGs). In addition to activate hundreds of ISGs, IFN␣ also represses numerous target genes but the mechanistic basis for this dual effect and transcriptional repression is largely unknown. We investigated by ChIP-chip the binding dynamics of STAT2 and "active" phospho(P)-STAT2 on 113 putative IFN␣ direct target promoters before and after IFN␣ induction in Huh7 cells and primary human hepatocytes (PHH). STAT2 is already bound to 62% of our target promoters, including most "classical" ISGs, before IFN␣ treatment. 31% of STAT2 basally bound promoters also show P-STAT2 positivity. By correlating in vivo promoter occupancy with gene expression and changes in histone methylation marks we found that: 1) STAT2 plays a role in regulating ISGs expression, independently from its phosphorylation; 2) P-STAT2 is involved in ISGs repression; 3) "activated" ISGs are marked by H3K4me1 and H3K4me3 before IFN␣; 4) "repressed" genes are marked by H3K27me3 and histone methylation plays a dominant role in driving IFN␣-mediated ISGs repression.Interferons are pleiotropic cytokines induced upon virus infection and other stimuli to modulate host immune response and are classified as type I interferon ␣ and ␤ (IFN␣ and IFN␤), 3 type II (IFN␥), and the recently discovered type III (IFN) (1-3)). Interferons exert their function by phosphorylating latent transcription factors belonging to the signal transducer and activator of transcription (STAT) family after a signaling cascade, which begins with the binding of interferon to their membrane receptors and involves Janus kinases (JAKs). Receptor dimerization or oligomerization leads to Jak apposition and the trans-phosphorylation on tyrosine residues that releases their intrinsic catalytic activity. Tyrosine-phosphorylated cytokine-receptor cytoplasmic domains then provides binding sites for the Src homology-2 (SH2) domain of the STAT proteins, which are recruited to the JAKs and phosphorylated on a single tyrosine residue (Tyr-689 in the case of STAT2). The interaction between phosphorylated-SH2 domains on STAT proteins leads to homo-or hetero-dimerization and nuclear translocation (4 -6). STAT2 Tyr-689 and STAT1 Tyr-701 can also be phosphorylated by non-receptor TKs, including SRC and ABL in the absence of ligand-induced receptor signaling (7). STAT dimers directly activate genes containing the IFN␥ activation site (GAS) element, while the association of STATs with the DNA-binding protein interferon regulatory factor (IRF) 9 expands the range of DNA response elements that can be targeted by the JAK-STAT pathway to interferon-stimulate response element (ISRE) and IRF response element (IRE) (8). Un-phosphorylated STAT2 binds IRF9 and constitutively shuttles i...

The NAD؉ -dependent histone deacetylase hSirT1 regulates cell survival and stress responses by inhibiting p53-, NF-B-, and E2F1-dependent transcription. Here we show that the hSirT1/PCAF interaction controls the E2F1/p73 apoptotic pathway. hSirT1 represses E2F1-dependent P1p73 promoter activity in untreated cells and inhibits its activation in response to DNA damage. hSirT1, PCAF, and E2F1 are corecruited in vivo on theP1p73 promoter. hSirT1 deacetylates PCAF in vitro and modulates PCAF acetylation in vivo. In cells exposed to apoptotic DNA damage, nuclear NAD ؉ levels decrease and inactivate hSirT1 without altering the hSirT1 interaction with PCAF and hSirT1 binding to the P1p73 promoter. hSirT1, the mammalian homologue of Sir2 (silent information regulator 2), is a NAD-dependent class III deacetylase (15, 33) that regulates cell survival, stress responses, and metabolism by inhibiting p53 (3, 18, 19, 28)-, E2F1 (1, 30)-, NF-B (31)-, and Forkhead (2)-dependent transcription. The role of hSirT1 in the regulation of mammalian cell survival in response to DNA damage is supported by several observations. hSirT1-deficient mice display increased levels of radiation-induced apoptosis and p53 hyperacetylation (4). hSirT1-dependent deacetylation attenuates the ability of p53 to trans-activate the cell cycle (p21) and apoptotic (bax) target genes (19,28). The constitutive expression of the tumor suppressor hypermethylated in cancer 1 (Hic1) represses hSirT1 transcription, thereby allowing the accumulation of acetylated p53 species and the enhancement of p53-mediated growth arrest and apoptosis in response to DNA damage (3). hSirT1 deacetylase function also modulates p53-independent pathways involved in the DNA damage response. The targeted disruption of the SirT1 gene in p53-deficient cells strongly sensitizes cells to radiation-, cisplatin-, and etoposide-induced cell death (20). hSirT1 deacetylates the DNA damage repair protein Ku70, and deacetylated Ku70 prevents Bax translocation to mitochondria to initiate apoptosis (5). hSirT1 maintains the Nijmegen breakage syndrome protein (NBS1) hypoacetylated and susceptible to be phosphorylated by ATM in response to DNA damage (32). Finally, etoposide treatment results in the E2F1-dependent induction of hSirT1 expression, and the abrogation of hSirT1 expression sensitizes cells to E2F1-dependent apoptosis (30).The E2F family of transcription factors has critical roles in the control of cell proliferation and apoptosis (7). E2F transcriptional activity is tightly regulated during the cell cycle through the association with pRb or the related pocket proteins p107 and p130, leading, in quiescent cells, to the recruitment of transcriptional corepressors, including histone deacetylases (HDACs), methyltransferases, and polycomb group proteins, onto the promoters of proliferation-associated E2F target genes (7). As cells progress into the cell cycle, cyclin-dependent kinases phosphorylate pRb, releasing free E2F and allowing it to interact with transcriptional coactivators, and direc...

a b s t r a c t TP53 belongs to a small gene family that includes, in mammals, two additional paralogs, TP63 and TP73. The p63 and p73 proteins are structurally and functionally similar to p53 and their activity as transcription factors is regulated by a wide repertoire of shared and unique post-translational modifications and interactions with regulatory cofactors. p63 and p73 have important functions in embryonic development and differentiation but are also involved in tumor suppression. The biology of p63 and p73 is complex since both TP63 and TP73 genes are transcribed into a variety of different isoforms that give rise to proteins with antagonistic properties, the TA-isoforms that act as tumorsuppressors and DN-isoforms that behave as proto-oncogenes. The p53 family as a whole behaves as a signaling ''network'' that integrates developmental, metabolic and stress signals to control cell metabolism, differentiation, longevity, proliferation and death. Despite the progress of our knowledge, the unresolved puzzle of complexity, redundancy and hierarchy in the p53 family continues to represent a formidable challenge.

The DNp73 proteins act as trans-repressors of p53 and p73-dependent transcription and exert both anti-apoptotic activity and pro-proliferative activity. DNp73s are frequently up-regulated in a variety of human cancers, including human hepatocellular carcinomas (HCCs). Increased levels of DNp73 proteins confer to HCC cells resistance to apoptosis and, irrespective to p53 status, a chemoresistant phenotype. Here, we show that interferon (IFN)α down-regulates DNp73 expression in primary human hepatocytes (PHHs) and HCC cell lines. IFNα has been used as pro-apoptotic agent in the treatment of malignancies and there is increasing evidence of IFNα effectiveness in HCC treatment and prevention of recurrence. The precise mechanisms by which class I IFNs exert their anti-proliferative and anti-tumor activity remain unclear. IFNα binding to its receptor activates multiple intracellular signaling cascades regulating the transcription of numerous direct target genes through the recruitment of a complex comprising of STAT1, STAT2 and IFN regulatory factor (IRF)9 to their promoters. We found that, in response to IFNα, the P2p73 promoter undergoes substantial chromatin remodeling. Histone deacetylases (HDACs) replace histone acetyl transferases. STAT2 is recruited onto the endogenous P2p73 promoter together with the polycomb group protein Ezh2, leading to increased H3K27 methylation and transcriptional repression. The reduction of DNp73 levels by IFNα is paralleled by an increased susceptibility to IFNα-triggered apoptosis of Huh7 hepatoma cells. Our results show, for the first time, that IFN-stimulated gene factor 3 recruitment may serve both in activating and repressing gene expression and identify the down-regulation of DNp73 as an additional mechanism to counteract the chemoresistance of liver cancer cells.

Reactivation of the HMGA1 protoncogene is very frequent in human cancer, but still very little is known on the molecular mechanisms leading to this event. Prompted by the finding of putative E2F binding sites in the human HMGA1 promoter and by the frequent deregulation of the RB/E2F1 pathway in human carcinogenesis, we investigated whether E2F1 might contribute to the regulation of HMGA1 gene expression. Here we report that E2F1 induces HMGA1 by interacting with a 193 bp region of the HMGA1 promoter containing an E2F binding site surrounded by three putative Sp1 binding sites. Both gain and loss of function experiments indicate that Sp1 functionally interacts with E2F1 to promote HMGA1 expression. However, while Sp1 constitutively binds HMGA1 promoter, it is the balance between different E2F family members that tunes the levels of HMGA1 expression between quiescence and proliferation. Finally, we found increased HMGA1 expression in pituitary and thyroid tumors developed in Rb(+/-) mice, supporting the hypothesis that E2F1 is a novel important regulator of HMGA1 expression and that deregulation of the RB/E2F1 path might significantly contribute to HMGA1 deregulation in cancer.

Thyroid iodide accumulation via the sodium/iodide symporter (NIS; SLC5A5) has been the basis for the longtime use of radio-iodide in the diagnosis and treatment of thyroid cancers. NIS is also expressed, but poorly functional, in some non-thyroid human cancers. In particular, it is much more strongly expressed in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines than in primary human hepatocytes (PHH). The transcription factors and signaling pathways that control NIS overexpression in these cancers is largely unknown. We identified two putative regulatory clusters of p53-responsive elements (p53REs) in the NIS core promoter, and investigated the regulation of NIS transcription by p53-family members in liver cancer cells. NIS promoter activity and endogenous NIS mRNA expression are stimulated by exogenously expressed p53-family members and significantly reduced by member-specific siRNAs. Chromatin immunoprecipitation analysis shows that the p53–REs clusters in the NIS promoter are differentially occupied by the p53-family members to regulate basal and DNA damage-induced NIS transcription. Doxorubicin strongly induces p53 and p73 binding to the NIS promoter, leading to an increased expression of endogenous NIS mRNA and protein in HCC and CCA cells, but not in PHH. Silencing NIS expression reduced doxorubicin-induced apoptosis in HCC cells, pointing to a possible role of a p53-family-dependent expression of NIS in apoptotic cell death. Altogether, these results indicate that the NIS gene is a direct target of the p53 family and suggests that the modulation of NIS by DNA-damaging agents is potentially exploitable to boost NIS upregulation in vivo.

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