Order and disorder – the two faces of a protein

Kinkley lab gains new insight into how an epigenetic reader regulates its ability to bind chromatin

PHF13 is an epigenetic reader implicated in various cellular processes, including transcription, the response to DNA damage and cell division, however details about its inner workings and how exactly it exerts its different functions remains elusive. The group of Sarah Kinkley, in collaboration with the Vingron and Hnisz labs at the MPIMG, has now discovered that PHF13 self-assembles into more complex structures in two different ways. Depending on the method, the protein changes its affinity to bind to chromatin.

The simplest proteins are built from single chains of amino acids that fold into a three-dimensional structures. Larger proteins often form more elaborate structures where multiple protein subunits assemble into complexes. This step often renders a protein functional or affects its properties and interactions within the cell. The lab of Sarah Kinkley has previously identified the domains of PHF13 that enable the protein to interact with chromatin. In their current study, the team found two different mechanisms by which the protein self-assembles and that help PHF13 to modulate its chromatin interactions. “In essence we can now demonstrate how different domains dictate the function of this protein and the mechanism that enables it to perform various functions within the cell," explains Francesca Rossi, the study's first author, who conducted the research during her doctoral studies at the MPIMG.

Apart from the important N- and C-terminal domains of PHF13, the scientists have now turned their attention to the intrinsically disordered regions of the protein. These sequences are found in about 40-50 % of the eukaryotic proteome. They are unique in that they do not form a fixed 3D structure, but rather can take different conformations. “We demonstrated that the protein forms long oligomers with alternating DNA and histone binding domains, greatly increasing its chromatin affinity and resulting in phenotypic consequences,” explains group leader Sarah Kinkley. “However, independently of this process, we found that the intrinsically disordered domains in the protein can also self-associate. When these regions are engaged, the protein forms biomolecular condensates and has a weaker chromatin affinity”.

The scientists compared the impact of the two mechanisms on chromatin and transcription states, showing that different transcriptional responses can be observed depending on how the protein self-associates. They also discovered previously unknown roles for PHF13. When the protein was overexpressed in its oligomeric form, it compacted chromatin. This phenonemon is usually only observed during the formation of chromosomes in cell division, suggesting an unrecognized role for PHF13 in these processes. Another novel role for PHF13 relates to one of the most important cellular processes: protein synthesis. Ribosomal RNA, which is transcribed by RNA polymerase I (RNAPol I), is an important building block of ribosomes. “The most exciting thing that popped out was that PHF13 seems to be activating RNAPol I while repressing RNAPol II”, says Sarah Kinkley. “This is an unexpected finding that opens up a lot of questions to be explored, as there are not a lot of factors to the best of my knowledge, known to activate RNAPol I and simultaneously repress RNAPol II.