TIS7, methylation and muscle differentiation

TPA Induced Sequence 7 (TIS7) was characterized by our laboratory as a transcriptional co-repressor, acting via its interaction with a histone deacetylase complex. Our studies have shown that the exogenous expression of TIS7 causes mainly down-regulation of gene expression in several cellular systems requiring cell polarity and differentiation. Among the characterized cell systems where mammary epithelial cells, muscles and peripheral neurons. Following the exogenous TIS7 expression we have shown besides morphological changes and changes in gene expression also the effects of TIS7 on Wnt signaling pathway, a conserved regulatory pathway important for both development and differentiation. The aim of our further studies was to identify the biological role of TIS7 and mainly the mechanism of its action. In order to do so we have generated TIS7 knockout mice and shown that TIS7 is involved in processes of cell differentiation and regeneration in the adult organism. Furthermore, TIS7 knockout mice became a valuable model for a human disease, namely cystic fibrosis. The proposed project is based on an interesting observation that we made in a pull-down experiment followed by mass spectrometry of TIS7 interacting-proteins. We identified all known major components of the methylosome complex to be co-immunoprecipitated with TIS7. Since methylation represents an important regulatory mechanism of the gene expression in general, and plays a crucial role in myogenesis in particular, our recent findings motivated us to study the interaction between TIS7, the methylosome complex, and methylation mechanisms in the process of muscle differentiation more in detail. Our working hypothesis is that TIS7 interacts with the methylosome complex and thereby regulates muscle differentiation. The principal goal of this proposal is to prove whether TIS7 regulates muscle differentiation via methylation events. In order to answer this question we will specify: the TIS7-interacting proteins; distinguish between direct and indirect TIS7-binding protein partners; test whether in vitro TIS7-binding partners co-localize and bind also in vivo and search for biological targets of TIS7-regulated methylation.

In our previous studies we have identified that the protein TPA Induced Sequence 7 (TIS7) acts as a transcriptional co-regulator controlling the expression of genes involved in differentiation of various cell types, including skeletal myoblasts. In this project we have unveiled the molecular mechanisms used by TIS7 to regulate adult myogenesis, in particular its role in regulation of MyoD, one of the essential myogenic regulatory factors. Although in the past multiple steps in which TIS7 was involved in the regulation of the MyoD activity were shown, our current project presents a completely new, detailed molecular insight into TIS7- specific control of MyoD gene expression and adult skeletal muscle differentiation. Secondly, in this project we have identified the protein ICln as the specific, novel protein downstream of TIS7, controlling myogenesis. ICln was so far known for having multiple physiological functions; however, our results show for the first time that ICln plays a regulatory role in the process of adult skeletal myogenesis. Using several independent technical approaches we documented in this project for the first time that TIS7 together with ICln epigenetically regulates myoD expression by controlling protein methyl transferase activity. In particular, ICln regulates MyoD expression via its interaction with the enzyme protein arginine methyl transferase 5 (PRMT5) resulting in epigenetic modification that utilizes symmetrical di- methylation of histone H3 on arginine 8. Finally, we provide multiple evidences for a novel functional characteristic of TIS7, namely that TIS7 possesses the ability to directly bind DNA, which is a functional feature necessary for its role in transcriptional regulation. Taken together, as a result of the focused effort in this project we are able now to present a novel regulatory pathway in which TIS7 controls MyoD expression and thereby skeletal myoblast differentiation via ICln, a protein playing a positive regulatory role in this physiological process. Furthermore, we provide an exact mechanism of the TIS7/ICln axis mediated epigenetic regulation of myoD gene expression.