5mC-hydroxylase activity is influenced by the PARylation of TET1 enzyme

TET enzymes are the epigenetic factors involved in the formation of the sixth DNA base 5-hydroxymethylcytosine, whose deregulation has been associated with tumorigenesis. In particular, TET1 acts as tumor suppressor preventing cell proliferation and tumor metastasis and it has frequently been found down-regulated in cancer. Thus, considering the importance of a tight control of TET1 expression, the epigenetic mechanisms involved in the transcriptional regulation of TET1 gene are here investigated. The involvement of poly(ADP-ribosyl)ation in the control of DNA and histone methylation on TET1 gene was examined. PARP activity is able to positively regulate TET1 expressionmaintaining a permissive chromatin state characterized by DNA hypomethylation of TET1 CpG island as well as high levels of H3K4 trimethylation. These epigenetic modifications were affected by PAR depletion causing TET1 down-regulation and in turn reduced recruitment of TET1 protein on HOXA9 target gene. In conclusion, this work shows that PARP activity is a transcriptional regulator of TET1 gene through the control of epigenetic events and it suggests that deregulation of these mechanisms could account for TET1 repression in cancer.

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project ‘MARK-AGE’. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.

PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression

Melanoma, the most aggressive form of skin cancer, is characterized by high rates of metastasis, drug resistance and mortality. Here we investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation. Western blotting, qRT-PCR, Chromatin immunoprecipitation (ChIP) assay, immunohistochemistry of melanoma patient specimens and xenograft tissues, in vitro Transwell assay for cell migration and invasion evaluation, in vitro capillary-like structure formation analysis. A significant correlation of Sema5A mRNA expression and melanoma progression was observed by analyzing GEO profile dataset. Endogenous Sema5A protein was detected in 95% of human melanoma cell lines tested, in 70% of metastatic specimens from patients affected by melanoma, and 16% of in situ melanoma specimens showed a focal positivity. We demonstrated that Sema5A regulates in vitro cell migration and invasion and the formation of vasculogenic structures. We also found an increase of Sema5A at both mRNA and protein level after forced expression of Bcl-2. By use of transcriptional and proteasome inhibitors, we showed that Bcl-2 increases the stability of Sema5A mRNA and protein. Moreover, by ChIP we demonstrated that Sema5A expression is under the control of the transcription factor c-Myb and that c-Myb recruitment on Sema5A promoter is increased after Bcl-2 overexpression. Finally, a concomitant decrease in the expression of Sema5A, Bcl-2 and c-Myb proteins was observed in melanoma cells after miR-204 overexpression. Overall our data provide evidences supporting the role of Sema5A in melanoma progression and the involvement of Bcl-2, miR-204 and c-Myb in regulating its expression.