Mice with a keratinocyte-restricted deletion of the actin polymerization-promoting molecule, N-WASP, display cyclic hair loss and skin inflammation. Here, we showed that these mice were also resistant to 7,12-dimethylbenz(a) anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumor formation. This resistance correlated with decreased expression of the senescence regulator, DNMT1, and increased expression of the senescence marker, p16Ink4a, in N-WASP-deficient epidermis. Moreover, primary N-WASP-null keratinocytes displayed a premature senescence phenotype in vitro. Expression and activation of p53, a major inducer of senescence, was not significantly altered in N-WASP-null keratinocytes. However, impairment of p53 function effectively rescued the senescence phenotype, indicating that N-WASP was an inhibitor of p53-induced senescence. Mechanistically, N-WASP regulated senescence by preventing p53-dependent degradation of the H3K9 methyltransferases, G9a/GLP, and the DNA methyltransferase, DNMT1, which both control keratinocyte senescence. This pathway collaborated with other N-WASP-independent, senescence-promoting signaling downstream of p53 and allowed the fine tuning of p53induced senescence by N-WASP. Collectively, these data reveal N-WASP as an inhibitor of p53-induced senescence, which might be of importance for skin tumor formation and cellular aging of keratinocytes.Significance: These findings demonstrate that N-WASP regulates p53-dependent senescence in keratinocytes in vitro and in vivo. Gene expression analysisRNA from skin and keratinocytes culture was prepared using the GeneElute Mammalian Total RNA Miniprep Kit (RNT350, Sigma) according to the instructions of the manufacturer. Skin Li et al.

<p>Legends to Supplementary Data</p>

<p>No SA-β-gal activity in N-WASP ko primary keratinocytes</p>

<p>No SA-β-gal activity in N-WASP ko primary keratinocytes</p>

<p>Senescence of N-WASP ko keratinocytes is dependent on p16Ink4a and p21</p>