Pascarella, Angela (2016) Functional analysis of Dnajc17 gene function: in vivo and in vitro studies. [Tesi di dottorato]

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Congenital hypothyroidism (CH) is the most frequent endocrinological congenital disorder often due to thyroid dysgenesis (TD). Mutations in genes involved in thyroid development such as Nxk2.1, Pax8, Foxe1, and Tshr cause TD in animal models, although in humans loss of function mutations in these genes have been identified only in few cases of CH with TD, suggesting the involvement of additional loci. A polygenic mouse model of CH with TD allowed our laboratory to identify Dnajc17, a member of the type III heat-shock protein-40 (Hsp40) family, as a candidate TD modifier gene. Our laboratory demonstrated that homozygous null mutation in Dnajc17 gene are early embryo lethal. Moreover, as DNAJC17 is still poorly characterized, we started a detailed study in vitro. First, we studied its subcellular localization, showing that it localizes predominantly in the nucleus. Then, to uncover DNAJC17 function we used two different in vitro approaches: 1) analysis of DNAJC17 interactome by CO-IP and mass spectrometry, 2) analysis of gene expression profile induced by Dnajc17 knock-down, both performed in HeLa cells. Interactome studies show that DNAJC17 interacts with several proteins involved in splicing. Some of these proteins are essential for embryonic development, similarly to Dnajc17 (PPR19, PLRG-1, snRNP200, XAB2). SNRNP200 (U5) is a component of the U5 snRNP and U4/U6-U5 tri-snRNP complexes and XAB2 that is involved in transcription-coupled repair and transcription. PRP19 and PLRG-1 belong to Prp19/CDC5L complex. Depletion of this complex leads to a block in splicing before the first transesterfication step in splicing and lariat formation of the pre-mRNA. Furthermore, the confirmation of DNAJC17 interaction with splicing machinery components comes from immunofluorescence demonstrating that DNAJC17 colocalize whith SC35, a spliceosome assembly factor localized in nuclear speckles. Consistently, gene expression profile of cells knocked-down for Dnajc17 displays alterations in the balance between alternatively spliced isoforms of many genes. We are currently characterizing differentially spliced genes. To elucidate the functional role of Dnajc17 in thyroid we generated a conditional knock-out mouse in which Dnajc17 has been disrupted specifically in the thyroid.

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