The role of DNase1L2 in epidermal DNA degradation

Keratinocytes of the epidermis form epidermal appendages such as hair and nail as well as the stratum corneum, which functions as the barrier to the environment. During these cell differentiation processes the nuclear DNA of the keratinocytes is degraded. We have recently identified deoxyribonuclease 1-like 2 (DNase1L2) as the first skin- specific endonuclease and have shown by siRNA-mediated knockdown of DNase1L2 in human skin equivalents that DNase1L2 is required for stratum corneum formation in vitro. To provide the basis for in vivo studies of DNase1L2 functions, we have generated a mouse model in which the DNase1L2 gene is disrupted. In the present project we will test the hypotheses that DNase1L2 is required for the degradation of DNA in one or several keratinocyte differentiation products in vivo and that aberrantly retained DNA in terminally differentiated keratinocytes impairs the function of stratum corneum, hair and / or nail. The epidermis and the epidermial appendages of DNase1L2-deficient mice and normal DNase1L2-expressing mice will be compared with regard to morphology and function under homeostatic and stressed conditions. This study will, for the first time, provide insights into the mechanism and physiological role of DNA breakdown in differentiated keratinocytes in vivo. The results may explain structural features of hair and nail and may improve our understanding of the molecular mechanisms underlying aberrant keratinocyte DNA degradation in skin diseases such as psoriasis.

The outermost layer of the epidermis, i.e. the stratum corneum, as well as hair and nails consist of cornified keratinocytes. In the course of the formation of these epidermal structures the nuclear DNA of keratinocytes is almost completely degraded. The molecular mechanism and the physiological function of DNA breakdown during cornification are not known at present. In a previous study we had shown that keratinocytes express the cell type-specific DNA-degrading enzyme, DNase1L2. To elucidate the function of DNase1L2 in vivo, we inactivated DNase1L2 by targeted gene deletion in the mouse and compared the skin phenotype of mutant mice with that of control animals. DNase1L2-deficient mice failed to degrade DNA in hair and nails as well as in the epitelia of the tongue and the esophagus whereas cornification was not comprised in the interfollicular epidermis. Die aberrant retention of DNA in hair disturbed the regular arrangement of structural hair proteins and caused a significant decrease in the resistance of hair to mechanical stress. In the course of the characterization of DNase1L2-deficient mice we established a novel method for in situ- fluorescence labeling of DNA in hair. This method was also applied in forensic investigations of human hair. We observed high variability in the degree of completeness of DNA breakdown among donors of hair. The individual efficiency of DNA degradation was shown to be highly predictive for the success rate of forensic analyses of shed hairs. Studies on human in vitro skin models and on various knockout mice demonstrated that, besides DNase1L2, other DNases are active in the skin. DNase 2, which so far has been detected in the lysosomes of multiple cell types, was identified as the essential DNA-degrading enzyme on the acidic surface of the skin. The results of this study reveal a surprising link between DNA degradation in cornifying keratinocytes and the mechanical resilience of hair. Moreover, our data suggest that a lysosomal enzyme suppresses the transfer of DNA through the skin. The newly established method of in situ labeling of DNA in hair will be further developed for routine applications in forensic investigations.