Phosphoinositides and TATA Binding Protein-Associated Factor 3 (TAF3)-mediated transcription in post-natal muscle differentiation

Despite the large amount of data describing phospholipid-signaling enzymes in the cytoplasm, and their roles in cell homeostasis, the nuclear functions of these molecules are poorly understood. Recently, increasing evidence suggests nuclear phosphoinositides as key molecules in genome regulation including transcription, histone modification, RNA maturation and export. And therefore, their possible involvement in cell-fate decision and differentiation. However, many questions still need to be answered such as how the phospholipid signalling is read by interaction domains within the nucleus or how these networks are coordinated in the regulation of nuclear functions. This dissertation aims to answer part of these questions. In particular, the first research point that I address is if the binding of phosphoinositide to the Plant Homeo Domain of the TBP-associated factor 3 (TAF3) impacts gene transcription regulation during myogenesis. TAF3 is a key component of the basal transcription complex and has been proposed to regulate tissue-specific gene transcription during muscle differentiation. Preliminary results in modified C2C12 myoblast cells line show that the loss of PI-binding impaired TAF3 function as co-activator of transcription during muscle differentiation, leading to a strongly reduced expression of the early markers Pax7, Myf-5 and MyoD prior to the beginning of the process and throughout all subsequent stages. This results in the inability of myocytes to undergo muscle differentiation. Further analyses with RNA sequencing (RNAseq) confirmed a strongly altered transcriptional output when phosphoinositide cannot bind TAF3 possibly due to an impaired assembly of the pre-initiation complex of transcription at specific gene promoters. Therefore, the second aspect I investigated is the role of phosphoinositides in the regulation of TAF3 and Polymerase II recruitment at Pax7, MyoD and Myogenin gene loci. Preliminary Chromatin immune precipitation results show a decrease of the enrichment of both TAF3 and Polymerase II at Pax7 and MyoD gene promoters when the binding of TAF3 with phosphoinositide is lost, associated with a decrease of the active histone mark H3K4me3. This suggests that phosphoinositides, through the binding of TAF3, may act as regulators of the transcription complex assembly and chromatin remodelling during myogenesis raising new insights and fascinating questions on the molecular mechanism of action and impact in gene control of these molecules within the nucleus.