CASZ1 is a zinc finger (ZF) transcription factor that is critical for controlling the normal differentiation of subtypes of neural and cardiac muscle cells. In neuroblastoma tumors, loss of CASZ1 is associated with poor prognosis and restoration of CASZ1 function suppresses neuroblastoma tumorigenicity. However, the key domains by which CASZ1 transcription controls developmental processes and neuroblastoma tumorigenicity have yet to be elucidated. In this study, we show that loss of any one of ZF1 to ZF4 resulted in a 58 to 79% loss in transcriptional activity, as measured by induction of tyrosine hydroxylase promoter-luciferase activity, compared to that of wild-type (WT) CASZ1b. Mutation of ZF5 or deletion of the C-terminal sequence of amino acids (aa) 728 to 1166 (a truncation of 38% of the protein) does not significantly alter transcriptional function. A series of N-terminal truncations reveals a critical transcriptional activation domain at aa 31 to 185 and a nuclear localization signal at aa 23 to 29. Soft agar colony formation assays and xenograft studies show that WT CASZ1b is more active in suppressing neuroblastoma growth than CASZ1b with a ZF4 mutation or a deletion of aa 31 to 185. This study identifies key domains needed for CASZ1b to regulate gene transcription. Furthermore, we establish a link between loss of CASZ1b transcriptional activity and attenuation of CASZ1b-mediated inhibition of neuroblastoma growth and tumorigenicity.
As a transcription factor, localization to the nucleus and the recruitment of co-factors to regulate gene transcription is essential. Nuclear localization and nucleosome remodeling and histone deacetylase (NuRD) complex binding are required for the zinc finger transcription factor CASZ1 to function as neuroblastoma (NB) tumor suppressor. However, the critical amino acids (AAs) that are required for CASZ1 interaction with NuRD complex and the regulation of CASZ1 subcellular localization have not been characterized. Through alanine scanning, immunofluorescence cell staining and co-immunoprecipitation we define a critical region at the CASZ1 N-terminus (AA23-40) that mediates the CASZ1b nuclear localization and NuRD interaction. Furthermore, we identify a nuclear export signal (NES) at the N-terminus (AA176-192) that contributes to CASZ1 nuclear-cytoplasmic shuttling in a chromosomal maintenance 1 (CRM1)-dependent manner. An analysis of CASZ1 protein expression in a primary neuroblastoma tissue microarray shows that high nuclear CASZ1 staining is detected in tumors from NB patients with good prognoses. In contrast, cytoplasmic-restricted CASZ1 staining or low nuclear CASZ1 staining is found in tumors from patients with poor prognoses. These findings provide insight into mechanisms by which CASZ1 regulates transcription, and suggests that regulation of CASZ1 subcellular localization may impact its function in normal development and pathologic conditions such as neuroblastoma tumorigenesis.
This study presents data describing the current practice of medical student documentation in academic pediatric EDs in the United States. There is a strong consensus among educators and students on the usefulness of medical student documenting patient encounters in the ED.
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