The Eyes Absent (Eya) proteins were first identified as co-activators of the Six homeobox family of transcription factors and are critical in embryonic development. These proteins are also re-expressed in cancers after development is complete, where they drive tumor progression. We have previously shown that the Eya3 N-terminal domain (NTD) contains Ser/Thr phosphatase activity through an interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme, and that this interaction increases the half-life of Myc through pT58 dephosphorylation. Here we showed that Eya3 directly interacted with the NTD of Myc, recruiting PP2A-B55α to Myc. We also showed that Eya3 increased the Ser/Thr phosphatase activity of PP2A-B55α but not PP2A-B56α. Furthermore, we demonstrated that the NTD (∼250 amino acids) of Eya3 was completely disordered, and it used a 38-residue segment to interact with B55α. In addition, knockdown and phosphoproteomic analyses demonstrated that Eya3 and B55α affected highly similar phosphosite motifs with a preference for Ser/Thr followed by Pro, consistent with Eya3’s apparent Ser/Thr phosphatase activity being mediated through its interaction with PP2A-B55α. Intriguingly, mutating this Pro to other amino acids in a Myc peptide dramatically increased dephosphorylation by PP2A. Not surprisingly, MycP59A, a naturally occurring mutation hotspot in several cancers, enhanced Eya3-PP2A-B55α mediated dephosphorylation of pT58 on Myc, leading to increased Myc stability and cell proliferation, underscoring the critical role of this phosphosite in regulating Myc stability.

While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems when used with chemically modified synthetic single guide RNAs (sgRNAs), while exhibiting high target specificity. We show that dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3. Synthetic sgRNAs can also be used with in vitro-transcribed dCas9-SALL1-SDS3 mRNA for short-term delivery into primary cells, including human induced pluripotent stem cells and primary T cells. Finally, we used dCas9-SALL1-SDS3 for functional gene characterization of DNA damage host factors, orthogonally to small interfering RNA, demonstrating the ability of the system to be used in arrayed-format screening.

Protein kinases comprise only ~2.5% of human protein coding genes yet are responsible for the regulation of nearly every cellular process through their catalytic activity. The kinome thus represents one of the most druggable classes of regulatory proteins for human disease. However, despite major advances in protein identification through mass‐spectrometry based techniques, it remains difficult to uncover substrate profiles for individual kinases using pharmacological manipulation. Undesirable off‐target inhibition and diverse secondary events muddle the phosphorylation landscape that can be detected using phosphoproteomic methods. Further, phosphorylation events and target engagement are often cell type specific confounding our ability to discern contributions of off‐target kinases. Using the protein kinase DYRK1A and inhibitor harmine as a model, we utilize orthogonal mass spectrometry‐based approaches to distinguish kinase‐substrates. Thermal proteome profiling revealed that harmine differentially stabilizes proteins in a cell type and dose dependent manner. Drug target stabilization in combination with substrate motif analysis were used to elucidate high‐confidence DYRK1A substrates from a SILAC‐based harmine phosphoproteome. The substrates revealed in this study will be crucial to understanding DYRK1A function and contribution to human health.Support or Funding InformationDARPA cooperative agreement 13‐34‐RTA‐FP‐007 to W.M.OLinda Crnic Institute for Down syndrome‐ Grand Challenge GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Down syndrome, the most common genetic cause of intellectual disability, results from trisomic chromosome 21. A section of chromosome 21 containing 33 genes, known as the Down syndrome critical region, plays a crucial role in producing the cognitive defects characteristic of Down syndrome. The dual‐specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A), located within the Down syndrome critical region, is necessary for normal brain development from flies to humans. Neurological defects in mice arise from an additional DYRK1A gene copy, while heterozygous loss of function causes microcephaly, facial dysmorphia, impaired motor function and behavioral problems in humans, demonstrating the sensitivity of DYRK1A function to gene dosage. However, the molecular mechanisms underlying these phenotypic changes are not fully understood. Defining the protein interacting partners of DYRK1A and subsequent function of these relationships is crucial for understanding the implication of trisomic chromosome 21 on human health.Most known functions and substrates of this kinase are cytoplasmic, leaving the smaller subpopulation of DYRK1A in the nucleus relatively unstudied. To systematically investigate the nuclear interacting partners of DYRK1A, immunoprecipitation of the endogenous protein in conjunction with mass‐spectrometry based proteomics was used to generate a deep interactome. This nuclear interactome reveals interactions between DYRK1A and several E3 ubiquitin ligases that act as negative regulators of DNA double stranded break repair. Efficient repair of these lesions is necessary for maintaining genome stability and is dependent on a ubiquitin signaling cascade. CRISPR knockout of DYRK1A and inhibition of this kinase with the selective inhibitor harmine decreases the efficiency of DNA double strand break repair following ionizing radiation. The persistence of DNA double strand break markers increased in DYRK1A knock out cells and in a dose dependent manner with harmine at late time points following the initial damage.Maintaining genomic stability during expansion of neural progenitors is crucial for brain development. The rapid expansion of progenitors during typical neurogenesis is of particular risk, as early‐born cortical progenitors are hypersensitive to replication‐associated DNA damage. Inaccurate repair of genomic lesions at such a time can result in severe and ill effects on the developing organism. The results from this study strongly implicate DYRK1A gene dosage in the efficient repair of DNA double strand breaks, and in a neural context could contribute to the neurodevelopment defects associated with DYRK1A.Support or Funding InformationBlumenthal Fund Fellowship; Linda Crnic Institute; DARPAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.