One stone, two birds: CDK9-directed activation of UBE2A regulates monoubiquitination of both H2B and PCNA.

The modification of proteins by ubiquitin is a highly conserved process, requiring E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligating enzymes. Protein ubiquitination plays an essential role in regulating key biological processes, including the cellular response to DNA damage.

UBE2A, an E2, is the human ortholog of yeast Rad6 and is essential for the ubiquitination of histone H2B and proliferating cell nuclear antigen (PCNA). Histone H2B monoubiquitination (H2Bub1) is associated with transcriptional elongation on active genes but has also been shown to function in DNA double-strand break (DSB) repair., PCNA is ubiquitinated at lysine 164 in response to genotoxic stess, which promotes the recruitment of DNA polymerase η to activate the translesion synthesis DNA repair pathway., Both of these processes imply a role for UBE2A in the maintenance of genome integrity.

Cyclin-dependent kinase 9 (CDK9) is a well-characterized component of the positive elongation factor b (P-TEFb) complex, which is involved in transcriptional elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II, as well as suppressor of Ty homolog 5 (SUPT5H) and negative elongation factor E (NELF-E). CDK9 also functions in co-transcriptional histone modification,, mRNA (mRNA) processing, mRNA export and the cellular response to replication stress, in a manner that is evolutionarily conserved. Loss of CDK9 activity causes an increase in spontaneous levels of DNA damage signaling in replicating cells and a decreased ability to recover from transient replication arrest. CDK9 also localizes to chromatin in response to replication stress, limits the amount of single-stranded DNA in cells under stress and interacts with ATR and other cell cycle checkpoint proteins. However, the mechanism and functional target through which CDK9 functions to maintain genome integrity in response to replication stress has remained unclear. A recent report has identified UBE2A as a novel phosphorylation target for CDK9 and shown that CDK9-dependent activation of UBE2A at conserved serine residue 120 regulates monoubiquitination of both H2B and PCNA, providing mechanistic insight into how CDK9 controls genome integrity.

Pirngruber and Shchebet et al. previously showed that CDK9 activity is essential for maintaining global and gene-associated levels of H2Bub1, at least in part by phosphorylation of serine 2 in the CTD of RNA polymerase II. However, since mutation of serine 2 within the CTD of RNA polymerase II resulted in a milder effect on H2Bub1 than CDK9 knockdown, Shchebet et al. explored whether another CDK9 target might contribute to H2Bub1. Using insight gained from yeast, in which Bur1, the yeast homolog of CDK9, interacts with and phosphorylates Rad6 at serine 120 to promote H2Bub1, they hypothesized that CDK9 may regulate H2Bub1 through UBE2A. Indeed, they found that UBE2A complexes with CDK9 by reciprocal co-immunoprecipitation. In particular, this interaction is specific for CDK9, as another cyclin-dependent kinase, CDK2, does not bind to UBE2A, nor does it regulate H2Bub1. The authors next demonstrated that CDK9 phosphorylates UBE2A at serine 120 both in vitro and in cells. Furthermore, they found that this site-specific phosphorylation by CDK9 regulates UBE2A activity, and CDK9 is required for both UBE2A-dependent H2Bub1 and PCNA monoubiquitination in response to UVC.

The findings in this study provide clarification for how CDK9 regulates H2Bub1. CDK9 phosphorylates RNA polymerase II CTD at serine 2 to recruit the RNF20/40 E3 ubiquitin ligase, which is required for H2Bub1, and phosphorylates UBE2A at serine 120 to increase its activity in regulating H2Bub1. These two pathways are likely not redundant, as abolishing CTD serine 2 phosphorylation leads to an incomplete loss of H2Bub1. However, it will be of interest to further clarify the individual contribution of these two pathways and their potential crosstalk in the maintenance of H2Bub1. This study also provides the first link between CDK9 and PCNA monoubiquitination. The dual function of CDK9-UBE2A signaling in modulating both H2Bub1 and PCNA monoubiquitination provides further evidence for a role for CDK9 in genome maintenance. From a clinical perspective, this study offers a mechanistic rationale for the application of CDK9 inhibitors, such as flavopiridol, in cancer therapy. Moreover, by understanding how CDK9 functions in controlling H2B and/or PCNA monoubiquitination to maintain genome integrity, insights may be gained into targeting these proteins or their interactions to enhance the efficacy of more traditional cancer therapies.