Specific Aim 1. To identify key differentiation genes and characterize their molecular mechanisms of action Part A. Identification, Cloning and Characterization of CASZ1, the human homolog of the drosophila neural fate determination gene castor (dCas). 1. The study of CASZ1 is warranted because it; a) maps to chromosome 1p36.22 a region lost in almost 98% of NB tumors with 1p36 LOH ; b) was a highly evolutionarily conserved neural fate determination gene; c) has not been functionally studied in mammalian model systems; d) is regulated during NB cell differentiation and e) is expressed at high levels in primary tumors of NB patients with good overall survival (p=0.0009). 2. CASZ1 protein expression in Neuroblastoma tumors. As part of our structure/function studies we identified a novel nuclear export signal in the N-terminal domain of CASZ1 that is critical for its transcriptional and tumor suppressive activity. As a transcription factor, localization to the nucleus and the recruitment of co-factors to regulate gene transcription are essential. . We identified a novel nuclear export signal located at the N-terminus (AA 174-209) that contributes to CASZ1 nuclear-cytoplasmic shuttling in a CRM1-dependent manner. CASZ1 protein is expressed in the cytoplasm of some cell lines and tissues. In neuroblastoma (NB) tumors, high nuclear CASZ1 staining is detected in tumors from NB patients with good prognoses. In contrast, cytoplasmic only 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. These studies were published in Liu et al Oncogene, 2016PMID: 27270431 Part B. Mechanism of CASZ1 Function 1. Our previous Structure/Function Study identified a NH2-terminal transactivation domain and a Zn-finger containing region as critical domains regulating CASZ1 transcriptional activity and tumor suppressor activity. More recently we have used a proteomic approach to identify the CASZ1 interactome in neuroblastoma cells. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs. These studies were published in Liu et al Oncotarget 6:27628-40,2015.
Showing the most recent 10 out of 22 publications
