CHARACTERIZATION OF THE DYRK1A PROTEIN-PROTEIN INTERACTION NETWORK

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is a protein kinase encoded by a dosage-dependent gene. An extra copy of DYRK1A contributes to Down syndrome (DS) pathogenesis while loss of one allele causes severe mental retardation and autism. DYRK1A is involved in phosphorylation of several proteins that regulate cell cycle control and tumor suppression. However, the function and regulation of this kinase is not well understood and current knowledge does not fully explain dosage-dependent function of this important kinase. Our previous proteomic studies identified several novel DYRK1A interacting proteins including RNF169, FAM117B, TROAP, LZTS1, LZTS2 and DCAF7. In this dissertation, we report the proteomic, biochemical and functional characterization of this DYRK1A protein-protein interaction network. Firstly, we show that DYRK1A regulates that recruitment of 53BP1 to DNA double strand breaks (DSBs) in part through its interaction with RNF169. This revealed a novel role of DYRK1A in DSB repair pathway choice. Secondly, we identify LZTS1 and LZTS2 as novel regulators of DYRK1A activity towards LIN52. Thirdly, we observed that DCAF7 interacts with several other DYRK1A-binding proteins including RNF169, TROAP, FAM117B, LZTS1 and LZTS2 giving rise to various multi-subunit protein complexes, but it does not act as a scaffold for these interactions. We also observed an unexpected role of DYRK1A in mediating the interaction between these DYRK1A-binding proteins and DCAF7, which could explain some aspects of the dosage-dependent function of DYRK1A. As DCAF7 was the most highly enriched DYRK1A-interacting protein, we generated and analyzed the DCAF7 interactome in order to understand the functional significance of the DYRK1A-DCAF7 interaction. We show that DCAF7 interacts with the components of a multi-subunit Polycomb Repressive Complex 1.3/5 (PRC1.3/5) independent of DYRK1A, but DYRK1A could influence the molecular size of the PRC1.3/5 complex. Furthermore, our data suggest that DYRK1A and DCAF7 regulate the monoubiquitination of H2A at K119 by PRC1.3/5. Using RNA-seq analysis, we identified a common set of genes regulated by DYRK1A and DCAF7. Our data shows that DCAF7 requires DYRK1A for its transcriptional effect. Future studies will be focused to determine the molecular mechanism by which DYRK1A and DCAF7 regulate the transcription of the PRC1.5 target genes. In conclusion, DYRK1A interacting proteins could regulate the activity and function of DYRK1A and play a role in its biological functions including tumor suppression, the DNA damage response and transcription

Molecular Mechanisms of the DYRK1A-regulated DNA Repair

Molecular Mechanisms of the DYRK1A-regulated DNA Repair Polina Bukina, Dept. of Biology, with Dr. Sarah Golding, Dept. of Biology The functions of human Dual-specificity tyrosine (Y)-Regulated Kinase 1A, or DYRK1A, include cell cycle control and differentiation. DYRK1A is required for assembly of the DREAM complex and repression of the cell cycle-dependent genes, such as BRCA1 and RAD51, in quiescence. Our lab previously reported that overexpression of DYRK1A inhibits the accumulation of a DNA repair protein 53BP1, at the DNA double-stranded breaks (DSB). Accumulation of 53BP1 is attributed to repair by non-homologous end joining (NHEJ) over homologous recombination (HRR). The function of 53BP1 is opposed by RNF169, a ubiquitin-binding protein that also accumulates at the DSB sites and promotes HRR. It was found that DYRK1A interacts with RNF169 to regulate the displacement of 53BP1 from the DSB sites. This study focuses on RNF169 in order to understand the role of DYRK1A in DNA damage response. We used the Multi-Dimensional Protein Identification Technology (MudPIT) proteomic analysis to identify RNF169-interacting proteins. Human cancer U-2 OS cells stably expressing HA-tagged RNF169, as well as control cells were used for immunoprecipitation. The samples were sent to Stowers Institute for Medical Research for MudPIT proteomic analysis. In order to understand the regulation of DNA repair by DYRK1A, the RNA sequencing dataset was analyzed as part of other studies in the lab. The expression of the mRNA for repair factors RAD51 and BRCA1 was found to be regulated by DYRK1A. To determine the significance of this finding, an experiment was designed to assess BRCA1 and RAD51 protein levels in the normal U-2 OS cells and in the cells lacking DYRK1A (U-2 OS DYRK1A knockout cells) after inducing DNA damage by gamma irradiation. It was found that the levels of RAD51, BRCA1 and 53BP1 levels were increased with DYRK1A KO. These results were consistent with the finding that DNA repair efficiency is increased with DYRK1A KO. Further studies can help to understand if these effects are mediated by DYRK1A-regulated DREAM complex.https://scholarscompass.vcu.edu/uresposters/1357/thumbnail.jp

UNDERSTANDING THE FUNCTION OF DYRK1A THROUGH CHARACTERIZATION OF ITS INTERACTING PROTEINS

DYRK1A is a protein kinase encoded by a gene implicated in Down syndrome pathogenesis. Loss of DYRK1A could promote oncogenic transformation. However, the regulation and substrates of DYRK1A are not fully understood. MudPIT proteomic analysis revealed novel DYRK1A interacting proteins with poorly characterized or even unknown functions. Therefore, the aim of this thesis was to understand the function of DYRK1A through the characterization of its interacting proteins. To achieve this aim, we established stable cell lines expressing these proteins and confirmed the interactions between DYRK1A and seven candidate binding partners. Furthermore, we found that all novel DYRK1A-interacting proteins also bind DCAF7, a previously reported DYRK1A-binding scaffold protein that binds to the N-terminus of DYRK1A. Using cyto-nuclear fractionation and immunostaining we found that DYRK1A-interacting proteins were present in different cellular compartments, suggesting that DYRK1A could play distinct roles in the cell depending on its localization. DYRK1A has been shown to regulate cell proliferation and actin cytoskeleton therefore we used cell proliferation assays and actin staining to determine the role of DYRK1A-interacting proteins in these processes. Here we report functional characterization of the interacting partners of DYRK1A and present cell-based models that will help to understand the function and regulation of this important protein kinase

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I–low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC