Dataset Information

A proteomics-based interaction screen links DYRK1A to RNF169 and to the DNA damage response

ABSTRACT: DYRK1A is a protein kinase whose dysregulation is linked to disease in humans. On the one side, its overexpression in trisomy 21 has been linked to certain Down syndrome pathological traits, while inactivating mutations in just one allele that hinders its function are responsible for a rare clinical syndrome. Moreover, altered expression of this kinase could be at the basis of other human pathologies including cancer and diabetes. Although, a few DYRK1A substrates have been described there is still a poor knowledge on both upstream regulators and downstream targets that could explain DYRK1A cellular activities. Where, we carried out a proteomics screen using antibody-based affinity purification coupled to mass spectrometry to identify cellular proteins that bind directly or indirectly to endogenous DYRK1A. We show that the use of a cell line not expressing DYRK1A, generated by CRISPR/Cas9 technology, was needed to discriminate between true positives and non-specific interactors. Most of the proteins found in the screen are novel DYRK1A interactor candidates, which are linked to a variety of cellular functions. The in-depth characterization of the functional interaction with one of them, the E3 ubiquitin ligase RNF169, has revealed a role forfor the link of DYRK1A inwith the DNA damage response. We found that RNF169 is a DYRK1A substrate and identified several phosphositesof its phosphorylation sites. In particular, one of the sites appears to contribute to the ability of RNF169 to displaced 53BP1 from sites of DNA damage at chromatin. Additionally, depletion of DYRK1A increases cell sensitivity to DNA damage. Therefore, our unbiased proteomic screen has revealed a novel functional activity for DYRK1A expanding the complex role of this kinase in controlling cellular homeostasis.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Eva Borràs

LAB HEAD: Eduard Sabido

PROVIDER: PXD011925 | Pride | 2019-07-15

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	130306_S_JRSL_01_01_Igg1.msf	Msf
	130306_S_JRSL_01_01_Igg1.raw	Raw
	130306_S_JRSL_02_01_Igg2.msf	Msf
	130306_S_JRSL_02_01_Igg2.raw	Raw
	130306_S_JRSL_03_01_Igg3.msf	Msf

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A comprehensive proteomics-based interaction screen that links DYRK1A to RNF169 and to the DNA damage response.

Roewenstrunk Julia J Di Vona Chiara C Chen Jie J Borras Eva E Dong Chao C Arató Krisztina K Sabidó Eduard E Huen Michael S Y MSY de la Luna Susana S

Scientific reports 20190412 1

Dysregulation of the DYRK1A protein kinase has been associated with human disease. On the one hand, its overexpression in trisomy 21 has been linked to certain pathological traits of Down syndrome, while on the other, inactivating mutations in just one allele are responsible for a distinct yet rare clinical syndrome, DYRK1A haploinsufficiency. Moreover, altered expression of this kinase may also provoke other human pathologies, including cancer and diabetes. Although a few DYRK1A substrates have ...[more]

PMID: 30979931

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Project description:The molecular mechanisms that lead to the cognitive defects characteristic of Down syndrome (DS), the most frequent cause of mental retardation, have remained elusive. Here we use a transgenic DS mouse model to show that DYRK1A gene dosage imbalance deregulates chromosomal clusters of genes located near neuron-restrictive silencer factor (REST/NRSF) binding sites. We found that DYRK1A binds the SWI/SNF-complex known to interact with REST/NRSF. Mutation of a REST/NRSF binding site in the promoter of the REST/NRSF target gene L1cam modifies the transcriptional effect of Dyrk1A-dosage imbalance on L1cam. DyrkA dosage imbalance perturbs Rest/Nrsf levels with decreased Rest/Nrsf expression in embryonic neurons and increased expression in adult neurons. We identified a coordinated deregulation of multiple genes that are responsible for the cellular phenotypic traits present in DS such as dendritic growth impairment and microcephaly during prenatal cortex development. Dyrk1a overexpression in primary mouse cortical neurons reduced the neuritic complexity. In the postnatal hippocampus, DYRK1A overexpression suppresses a form of synaptic plasticity that may be sufficient to cause DS cognitive defects. We propose that DYRK1A overexpression-related neuronal gene deregulation generates the brain phenotypic changes that characterize DS, with an accessory role for the gene dosage imbalance of other chromosome 21 genes. Experiment Overall Design: Agilent Whole Mouse Genome oligomicroarrays (GEO accession no. GPL2872, Agilent Technologies, Palo Alto, CA) were used. They contain 60-mer DNA probes synthesized in situ in a 44k format. Of 44,290 spots, 2756 are controls. The remaining 41,534 spots represent 33,661 unique transcripts which correspond to 20,202 unique human genes. Four dependent (152F7, one series of pooled samples) measurements were carried out for each group of biological conditions using exchanged dye-labeled RNA targets (i.e., Cy3 and Cy5 dye-swapping experiments).

Project description:The molecular mechanisms that lead to the cognitive defects characteristic of Down syndrome (DS), the most frequent cause of mental retardation, have remained elusive. Here we use a transgenic DS mouse model to show that DYRK1A gene dosage imbalance deregulates chromosomal clusters of genes located near neuron-restrictive silencer factor (REST/NRSF) binding sites. We found that DYRK1A binds the SWI/SNF-complex known to interact with REST/NRSF. Mutation of a REST/NRSF binding site in the promoter of the REST/NRSF target gene L1cam modifies the transcriptional effect of Dyrk1A-dosage imbalance on L1cam. DyrkA dosage imbalance perturbs Rest/Nrsf levels with decreased Rest/Nrsf expression in embryonic neurons and increased expression in adult neurons. We identified a coordinated deregulation of multiple genes that are responsible for the cellular phenotypic traits present in DS such as dendritic growth impairment and microcephaly during prenatal cortex development. Dyrk1a overexpression in primary mouse cortical neurons reduced the neuritic complexity. In the postnatal hippocampus, DYRK1A overexpression suppresses a form of synaptic plasticity that may be sufficient to cause DS cognitive defects. We propose that DYRK1A overexpression-related neuronal gene deregulation generates the brain phenotypic changes that characterize DS, with an accessory role for the gene dosage imbalance of other chromosome 21 genes. Experiment Overall Design: Agilent Whole Mouse Genome oligomicroarrays (GEO accession no. GPL2872, Agilent Technologies, Palo Alto, CA) were used. They contain 60-mer DNA probes synthesized in situ in a 44k format. Of 44,290 spots, 2756 are controls. The remaining 41,534 spots represent 33,661 unique transcripts which correspond to 20,202 unique human genes. Five independent (Dyrk1A overexpression, five individual samples) measurements were carried out for each group of biological conditions using exchanged dye-labeled RNA targets (i.e., Cy3 and Cy5 dye-swapping experiments).