IGF1 activates cell cycle arrest following irradiation by reducing binding of ΔNp63 to the p21 promoter

Radiotherapy for head and neck tumors often results in persistent loss of function in salivary glands. Patients suffering from impaired salivary function frequently terminate treatment prematurely because of reduced quality of life caused by malnutrition and other debilitating side-effects. It has been previously shown in mice expressing a constitutively active form of Akt (myr-Akt1), or in mice pretreated with IGF1, apoptosis is suppressed, which correlates with maintained salivary gland function measured by stimulated salivary flow. Induction of cell cycle arrest may be important for this protection by allowing cells time for DNA repair. We have observed increased accumulation of cells in G2/M at acute time-points after irradiation in parotid glands of mice receiving pretreatment with IGF1. As p21, a transcriptional target of the p53 family, is necessary for maintaining G2/M arrest, we analyzed the roles of p53 and p63 in modulating IGF1-stimulated p21 expression. Pretreatment with IGF1 reduces binding of ΔNp63 to the p21 promoter after irradiation, which coincides with increased p53 binding and sustained p21 transcription. Our data indicate a role for ΔNp63 in modulating p53-dependent gene expression and influencing whether a cell death or cell cycle arrest program is initiated

Chromatin Immunoprecipitation-Based Screen To Identify Functional Genomic Binding Sites for Sequence-Specific Transactivators

In various human diseases, altered gene expression patterns are often the result of deregulated gene-specific transcription factor activity. To further understand disease on a molecular basis, the comprehensive analysis of transcription factor signaling networks is required. We developed an experimental approach, combining chromatin immunoprecipitation (ChIP) with a yeast-based assay, to screen the genome for transcription factor binding sites that link to transcriptionally regulated target genes. We used the tumor suppressor p53 to demonstrate the effectiveness of the method. Using primary and immortalized, nontransformed cultures of human mammary epithelial cells, we isolated over 100 genomic DNA fragments that contain novel p53 binding sites. This approach led to the identification and validation of novel p53 target genes involved in diverse signaling pathways, including growth factor signaling, protein kinase/phosphatase signaling, and RNA binding. Our results yield a more complete understanding of p53-regulated signaling pathways, and this approach could be applied to any number of transcription factors to further elucidate complex transcriptional networks

p63 maintains keratinocyte proliferative capacity through regulation of Skp2–p130 levels

p63 is a master regulator of proliferation and differentiation in stratifying epithelia, and its expression is frequently altered in carcinogenesis. However, its role in maintaining proliferative capacity remains unclear. Here, we demonstrate that hypoproliferation and loss of differentiation in organotypic raft cultures of primary neonatal human foreskin keratinocytes (HFKs) depleted of the α and β isoforms of p63 result from p53–p21-mediated accumulation of retinoblastoma (Rb) family member p130. Hypoproliferation in p63-depleted HFKs can be rescued by depletion of p53, p21CIP1 or p130. Furthermore, we identified the gene encoding S-phase kinase-associated protein 2 (Skp2), the recognition component of the SCFSkp2 E3 ubiquitin ligase, as a novel target of p63, potentially influencing p130 levels. Expression of Skp2 is maintained by p63 binding to a site in intron 2 and mRNA levels are downregulated in p63-depleted cells. Hypoproliferation in p63-depleted cells can be restored by re-expression of Skp2. Taken together, these results indicate that p63 plays a multifaceted role in maintaining proliferation in the mature regenerating epidermis, in addition to being required for differentiation

STAT5 requires the N-domain for suppression of miR15/16, induction of bcl-2, and survival signaling in myeloproliferative disease

Phosphorylated signal transducer and activator of transcription 5 (STAT5) is a biomarker and potential molecular target for hematologic malignancies. We have shown previously that lethal myeloproliferative disease (MPD) in mice mediated by persistently activated STAT5 (STAT5aS711F) requires the N-domain, but the mechanism was not defined. We now demonstrate by retrovirally complementing STAT5abnull/null primary mast cells that relative to wild-type STAT5a, STAT5a lacking the N-domain (STAT5aΔN) ineffectively protected against cytokine withdrawal-induced cell death. Both STAT5a and STAT5aΔN bound to a site in the bcl-2 gene and both bound near the microRNA 15b/16 cluster. However, only STAT5a could effectively induce bcl-2 mRNA and reciprocally suppress miR15b/16 leading to maintained bcl-2 protein levels. After retroviral complementation of STAT5abnull/null fetal liver cells and transplantation, persistently active STAT5aS711F lacking the N-domain (STAT5aΔNS711F) was insufficient to protect c-Kit+Lin−Sca-1+ (KLS) cells from apoptosis and unable to induce bcl-2 expression, whereas STAT5aS711F caused robust KLS cell expansion, induction of bcl-2, and lethal MPD. Severe attenuation of MPD by STAT5aΔNS711F was reversed by H2k/bcl-2 transgenic expression. Overall, these studies define N-domain–dependent survival signaling as an Achilles heel of persistent STAT5 activation and highlight the potential therapeutic importance of targeting STAT5 N-domain–mediated regulation of bcl-2 family members