CORDIS - EU research results

Transcription and the maintenance of genome integrity

Final Report Summary - TRANSINTEG (Transcription and the maintenance of genome integrity)

DNA damage in genes can give rise to harmful mutations but can also obstruct the progress of transcribing RNA polymerase II (RNAPII), thereby affecting gene expression, also at the level of mRNA splicing. Not surprisingly, repair pathways have evolved that specifically target RNAPII-stalling DNA lesions, so-called transcription-coupled repair pathways. As an alternative ‘last resort’, stalled RNAPII can also be permanently removed by ubiquitylation/degradation, clearing the gene for DNA repair by other means and transcription by other polymerases. Conversely, while transcription is essential and therefore protected by a variety of mechanisms, it also itself comes at a cost for genome integrity. For example, transcription is correlated with breaks at fragile chromosome sites, mutagenesis and elevated levels of DNA recombination. Insight into the regulatory mechanisms at play in the interface between transcription and other DNA-related processes is essential, not least for understanding processes maintaining genome stability.
Over the period of this grant, we investigated connections between transcription and genome stability at several different levels. Much of this activity was focused on the consequences of pauses and stalls during the journey of RNAPII across a gene. Such events, typically unplanned (‘transcription stress’), but sometimes scheduled and evolutionarily important, can be triggered by a number of stochastic events, such as DNA damage, collision with other proteins on DNA, or may even be intrinsic to the DNA sequence being transcribed. Indeed, we discovered a novel protein complex, named DBIRD, which associates with the transcribing polymerase and determines its ability to pass through DNA sequences that are rich in adenine- and thymine-tracts, and thus mRNA splicing in such regions. Much current research is focused on the use of genome-wide exploratory approaches (ChIP-Seq, etc), but a mechanistic interpretation of the ‘maps’ resulting from such endeavours have been lacking. We reported the development of mathematical and computational tools to overcome these restraints. We also investigated poly-ubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a ‘mechanism of last resort’ employed during transcription stress. In yeast, this process is dependent on Def1, through a previously unresolved mechanism. Our new results showed how Def1 becomes activated through a ubiquitylation- and proteasome-dependent processing mechanism. This facilitates poly-ubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Interestingly, cells even use RNAPII ubiquitylation and degradation to deal with detrimental protein collision. We thus reported that RNAPII stops, but does not dissociate upon head-to-head collision on genes, and that removal of collided RNAPII from the DNA template is achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII poly-ubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent genes.
RECQ helicases are important for maintaining genome stability. Among these proteins, RECQL5 is unique by associating with RNA polymerase II (RNAPII), but its function had remained unclear. We showed that RECQL5 is a general elongation factor, which regulates RNAPII speed, and is crucial for preserving genome stability during transcription stress. Importantly, RECQL5 interacts with the protein encoded by the cancer driver MLL2, which encodes a histone methyltransferase. Numerous recent genome-wide cancer studies have shown that MLL2 is also a tumor-suppressor, mutated in a large number of different cancers. Indeed, in a recent survey of frequent cancer mutations, only a few well-studied drivers such as p53, B-raf, K-ras, and PTEN were more frequently mutated. Our work shows that the widespread connection between MLL2 mutation and tumourigenesis can be explained by an important role for MLL2 in suppressing transcription stress and genome instability. We also used a variety of approaches to uncover the molecular basis of the very severe human disorder, Cockayne Syndrome (CS), which had previously been thought of as a ‘repair disease’. We showed that CS is actually caused by transcription defects, which in turn result in a lack of efficient neuronal differentiation, explaining many of the severe CS symptoms. Gratifyingly, our latest results even point to possible pharmacological treatments for this devastating disease. In many of these studies, we took advantage of a novel procedure for isolating and characterizing ubiquitylated proteins (MultiDsk rein), which is now in widespread use in the scientific community.
Together, our results provide new insight into fundamental mechanisms of gene traffic control, transcription stress and the consequences of changing polymerase speed across genes.