Ananthapadmanabhan, V. | Search bioRxiv

bioRxiv2018

DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169

Human DYRK1A gene encoding Dual-specificity tyrosine (Y)- Regulated Kinase 1A (DYRK1A) is a dosage-dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in the DNA double-strand break (DSB) repair signaling. This novel function of DYRK1A is mediated in part by its interaction with ubiquitin-binding protein RNF169 that regulates the choice between homologous recombination (HR) and non-homologous end joining (NHEJ) DSB repair. Accumulation of RNF169 at the DSB sites promotes homologous recombination (HR) by limiting the recruitment of the scaffold protein 53BP1 that promotes NHEJ by protecting the DNA ends from resection. Inducible overexpression of active, but not the kinase inactive, DYRK1A in U-2 OS cells inhibited accumulation of 53BP1 at the DSB sites in RNF169-dependent manner. Mutation of DYRK1A phosphorylation sites in RNF169 or pharmacological inhibition of DYRK1A using harmine decreased the ability of RNF169 to displace 53BP1 from radiation-induced DSB sites. In order to further investigate the role of DYRK1A in regulation of DNA repair, we used CRISPR-Cas9 mediated knockout of DYRK1A in human and mouse cells. Interestingly, knockout of DYRK1A also caused a defect in 53BP1 DSB recruitment that was independent of RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through several mechanisms. U-2 OS cells devoid of DYRK1A displayed an increased DNA repair and HR efficiency, and showed a decreased sensitivity to the PARP inhibitor olaparib when compared to control cells. Given evidence of its altered expression in human cancers, DYRK1A levels could represent a significant determinant of the DNA damaging therapy response.

molecular biology↗

bioRxiv2017

The cell cycle regulatory DREAM complex is disrupted by high expression of oncogenic B-Myb

The oncogene MYBL2 (encoding B-Myb) is a poor prognostic biomarker in many cancers. B-Myb interacts with the MuvB core of five proteins (LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54) to form the MMB (Myb-MuvB) complex and promotes expression of late cell cycle genes necessary for progression through mitosis. Both MYBL2 amplification and over-expression are associated with deregulation of the cell cycle and increased cell proliferation. Alternatively, by interacting with E2F4-DP1 and p130 or p107, the MuvB core becomes part of the DREAM complex (DP, RB-like, E2F, and MuvB). The DREAM complex opposes MMB by globally repressing cell cycle genes in G0/G1, maintaining the cell in a quiescent state. However, the specific mechanism by which B-Myb alters the cell cycle is not well understood. Our analysis of The Cancer Genome Atlas data revealed significant upregulation of DREAM and MMB target genes in breast and ovarian cancer with MYBL2 gain. Given that most of the DREAM target genes are not directly regulated by B-Myb, we investigated the effects of B-Myb on DREAM formation. We found that depletion of B-Myb results in increased DREAM formation in human cancer cells, while its overexpression inhibits DREAM formation in the non-transformed cells. Since the MuvB core subunit LIN52 is essential for assembly of both the DREAM and MMB complexes, we tested whether B-Myb disrupts DREAM by sequestering LIN52. Overexpression of LIN52 did not increase either DREAM or MMB formation, but instead increased the turnover rate of the endogenous LIN52 protein. Interestingly, co-expression of B-Myb increased the expression of both endogenous and overexpressed LIN52 while knockdown of B-Myb had an opposite effect. We found that regulation of LIN52 occurs at the protein level, and that activity of DYRK1A kinase, the enzyme that triggers DREAM complex formation by phosphorylating LIN52, is required for this regulation. These findings are the first to implicate B-Myb in the disassembly of the DREAM complex and offer insight into the underlying mechanisms of poor prognostic value of MYBL2 amplification in cancer. We conclude that B-Myb mediates its oncogenic effects not only by increasing mitotic gene expression by the MMB complex, but also by broad disruption of cell cycle gene regulatory programs through compromised DREAM formation.

cancer biology↗