Co-regulation of transcription by DIDO3 and PHF3 paralogues

Gene expression is an essential cellular process that copies the genetic information from DNA into RNA. Precise regulation of gene expression is established by dedicated transcription regulators. In this project we will focus on two transcription regulators, DIDO3 (Death inducer obliterator 3) and PHF3 (PHD finger protein 3). These proteins belong to the SPOC (Spen paralogue and orthologue C-terminal) domain family. The SPOC domain binds to phosphorylated C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Pol II CTD is dynamically phosphorylated during the transcription cycle and serves as a code that is recognized by CTD-binding domains. The early stages of transcription are marked by Pol II phosphorylated on Serine-5 (pSer5), whereas productive elongation is characterized by the removal of pSer5 and an increase in phosphorylated Serine-2 (pSer2). For DIDO3 and PHF3, SPOC-dependent recognition of pSer2 CTD is essential for their recruitment onto Pol II during transcription elongation. How exactly these two proteins interact in transcription regulation remains unclear. To address this, we will first generate cell lines in which we can rapidly deplete one or both proteins and examine changes in gene expression. To identify their direct gene targets, we will examine whether they bind to the genes that they regulate. Next, we will study their mechanism of gene expression regulation by analysing the distribution of phosphorylated Pol II CTD and transcription elongation factors. We will further examine whether DIDO3 and PHF3 regulate the activity and distribution of enzymes that deposit or remove phosphorylation marks on Pol II CTD and elongation factors. To establish a direct link between DIDO3/PHF3 and phosphorylation, we will compare transcriptional changes between cell lines in which these proteins are depleted, and cell lines in which the phosphorylation of elongation factors is perturbed. Overall, this project will uncover how DIDO3 and PHF3 co-regulate gene expression by modulating phosphorylation dynamics during the transcription cycle. DIDO3 is essential for mouse development, while PHF3 mutations have been linked with neurological defects including microcephaly and autism, and PHF3 expression levels are frequently reduced in glioblastoma. Determining how DIDO3 and PHF3 regulate gene expression will thus help in understanding how their deregulation leads to impaired development and disease.