The paralogous proteins DIDO3 and PHF3 are known to play a key role in fine-tuning gene expression, especially transcription elongation, but precisely what functions they fulfil and how is, as yet, unknown. Both proteins encode a so-called SPOC domain (Spen Paralogue and Orthologue C-terminal domain) which interacts with the C-terminal domain of RNA Polymerase II (Pol II) and plays a role in transcript elongation. Deletion of the SPOC domain of human PHF3 leads to the de-repression of neuronal genes.
DIDO has three isoforms, but the SPOC domain is only present in DIDO3. In embryonic stem cells, DIDO3 actively supports the maintenance of the stem cell state, whereas expression of DIDO1 initiates the process of cell differentiation. Curiously, Dea Slade’s team observed that DIDO3 is transcriptionally upregulated in PHF3 knockout cells, concomitant with a downregulation of DIDO1. The authors hypothesized that DIDO3, but not DIDO1, might be capable of compensating for the loss of PHF3 due to its SPOC domain. “This appears to be the first example of isoform switching serving as a compensatory mechanism”, Dea Slade says.
However, there is more behind the curtain: How PHF3 and DIDO interact during transcription remained unclear until now. Dea Slade and her team’s work shows that, through the common platform of Pol II, both proteins form a macromolecular complex to fine-tune gene expression. DIDO3’s role within the complex is to anchor Pol II to chromatin, whereas PHF3 connects Pol II to RNA processing factors. Their results show that, through their SPOC domains, PHF3 and DIDO3 can connect the transcription machinery with regulators involved in both transcription and co-transcriptional processes.
Gene expression is also regulated by specific, intrinsically disordered regions (IDRs) at the C-terminus of PHF3 and DIDO3. The work of Dea Slade and her lab suggest that IDR deletions in PHF3 and DIDO3 result in transcriptional deregulation and genome reorganization. “When we remove the IDR of PHF3, we start seeing chromocenters in the cells, suggesting that gene downregulation is actually coupled with an increase in heterochromatin”, Dea Slade explains. Chromocenters are condensate-like regions of heterochromatin which can be visualized under a fluorescence microscope as bright foci. While chromocenters have been observed in mouse cells before, their existence had not been documented in human cells. The team’s findings suggest a novel role of PHF3 in regulating genome organization and heterochromatin formation.
A mechanistic understanding of DIDO and PHF3 and their respective roles in gene expression awaits further investigation, a challenge that the Slade lab is embracing. For that purpose, the lab has currently been granted a FWF funding of more than €400,000.