Dea Slade, BSc, MSc, Ph.D., Junior group leader, Max F. Perutz Laboratories, University of Vienna speaks about PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC.
Link to Abstract:
https://www.nature.com/articles/s41467-021-26360-2
Abstract:
The C-terminal domain (CTD) of RNA polymerase II (Pol IIbiggest )’s subunit is a transcription and RNA processing control center. PHD-finger protein 3 (PHF3) is a transcription and mRNA stability regulator that binds onto the Pol II CTD via its SPOC domain. SPOC is a CTD reader domain that binds two phosphorylated Serine-2 tags in neighboring CTD repeats preferentially. PHF3 promotes the separation of phosphorylated Pol II into liquid and liquid phases, colocalizes with Pol II clusters, and tracks Pol II along the length of genes. In human cells, PHF3 knockout or SPOC deletion causes greater Pol II pausing, decreased elongation rate, and higher mRNA stability, as well as substantial derepression of neural genes. In Phf3 knock-out mouse embryonic stem cells, key neuronal genes are abnormally expressed, resulting in poor neuronal differentiation. Our findings imply that PHF3 works as a key regulator of neuronal gene expression by bridging the gap between transcription and mRNA degradation.
Introduction:
Recruitment, initiation, pausing, elongation, and termination of RNA Polymerase II (Pol II) are all highly regulated processes in transcription1,2,3. Transcription control is essential for establishing and maintaining cell identity, and transcriptional abnormalities are at the root of cancer and other diseases4. 5,6,7,8,9,10,11,12,13,14,15,16,17 Transcription elongation factors affect Pol II pause release, backtracking, elongation rate or processivity, and associate transcription elongation with co-transcriptional RNA processing.
Transcription regulators interact with the Pol II complex’s physically defined surfaces as well as the unstructured C-terminal domain (CTD) of RPB118,19,20, the biggest Pol II component. During transcription, heptarepeats (Y1S2P3T4S5P6S7) within the Pol II CTD are dynamically phosphorylated, directing the timely recruitment of regulatory components. Pol II phosphorylated on Serine-5 (pS5) marks the early phases of transcription, whereas productive elongation is represented by the elimination of pS5 and a concurrent increase in phosphorylated Serine-2 (pS2) 18. Pol II CTD acts as a docking site for 5′ mRNA capping, splicing, 3’end processing, termination, and mRNA export factors that recognize certain CTD phosphorylation patterns10,21,22,23, and transcription is strongly connected with co-transcriptional RNA processing. The nucleotidyltransferase (NT) domain of yeast and mammalian 5′ mRNA capping enzymes such as Cgt1, Pce1, and Mce1 has been shown to directly bind pS5 within the Pol II CTD, whereas the CTD-interaction domain (CID) of yeast Pcf11 and mammalian SCAF8 has been shown to directly bind the pS2 mark on the Pol II CTD Unphosphorylated Pol II CTD clusters undergo liquid-liquid phase separation (LLPS), which compartmentalizes transcription initiation machinery, whereas phosphorylated Pol II CTD clusters partition with RNA processing factors29,30.
The human Death-Inducer Obliterator (DIDO) and yeast Bypass of Ess-1 (Bye1)31,32 are both members of the PHD-finger protein 3 (PHF3) family of potential transcriptional regulators. A domain that is distantly related to the Pol II–associated domain of the elongation factor TFIIS, dubbed the TFIIS-Like Domain (TLD), and a Plant Homeo Domain (PHD)31 are discovered in this family of proteins. A Spen paralogue and orthologue C-terminal (SPOC) domain has been linked to cancer, apoptosis, and transcription33. Bye1 binds the RPB1 jaw domain via its TLD in vitro and in vivo, similarly to TFIIS31,34. Due to the absence of the TFIIS domain III31,35, Bye1 TLD does not stimulate mRNA breakage during transcriptional proofreading, unlike TFIIS. In vivo, ablation of the PHD and SPOC domains together prevented Bye1 from binding to Pol II34, implying that the Bye1 TLD is required but not sufficient for interaction with Pol II34. PHF3 was discovered in a mass spectrometry search for proteins binding to phosphorylated GST-CTD36, despite the fact that it lacks any traditional CTD-binding domains. The physiological significance of this relationship, as well as whether or not PHF3 controls transcription, are unknown.
The SPOC domain is used to find an unanticipated interaction between PHF3 and the Pol II CTD. We show that the PHF3 SPOC domain is selective for CTD repeats that are phosphorylated on S2, establishing the SPOC domain as a Pol II CTD reader. Furthermore, we discovered that PHF3 associates with Pol II clusters inside cells and generates condensates in vitro that contain phosphorylated CTD and Pol II. PHF3 has a global and gene-specific dual regulatory effect on Pol II transcription and mRNA stability. PHF3 KO HEK293T cells have abnormally low levels of neuronal genes, and Phf3 KO mESCs have poor neuronal development. Overall, our findings imply that PHF3 regulates neuronal gene expression via the CTD as a regulator of Pol II transcription and mRNA stability.
Results:
The SPOC domain of PHF3 interacts with RNA polymerase II.
To investigate the role of PHF3 in transcription, we produced FLAG-PHF3 in HEK293T cells and used co-immunoprecipitation (co-IP) followed by mass spectrometry to identify interacting proteins (Fig. 1a, b and Supplementary Data 1). RPB1 scored first among 40 PHF3 interactors with high confidence (Supplementary Data 1), which included other Pol II transcription elongation regulators (SPT5, SPT6, PAF1C, FACT), as well as RNA processing factors (Fig. 1a, b and Supplementary Data 1). Due to a shortage of adequate commercially available PHF3 antibodies, we verified these findings by co-IP studies of endogenous PHF3 tagged with GFP in HEK293T cells (Fig. 1c, d, and Supplementary Fig. 1a). Within the heptarepeats, endogenously produced PHF3-GFP interacted with Pol II phosphorylated on Serines 2, 5, and/or 7.