Characterization of Critical Domains within the Tumor Suppressor CASZ1 Required for Transcriptional Regulation and Growth Suppression

Abstract

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.

Essential Role of the Zinc Finger Transcription Factor Casz1 for Mammalian Cardiac Morphogenesis and Development

“…Our studies confirmed the relatively high levels of Casz1 in murine heart and showed ele-vated CASZ1 levels in human heart (11,12). Our studies also mapped CASZ1 to the region of chromosome 1p36 loss of heterozygosity in neuroblastoma tumors and elucidated its function as a mammalian regulator of differentiation with tumor suppressor functions (11)(12)(13)(14)(15). Furthermore, a 1p36 deletion syndrome is associated with CHD, including noncompaction cardiomyopathy and ventricular septal defect (2,16).…”

“…1A), resulting in a truncated Casz1 allele in which 10 of the 11 zinc fingers of full-length Casz1 are lost. The zinc fingers are critical domains for Casz1 function (14). Using primers specific for the WT (Casz1 ϩ/ϩ ) allele and the trapped allele, we found that Casz1 ϩ/ϩ mice only have the unaltered Casz1 allele, whereas Casz1 heterozygous (Casz1 ) and 120 (64.6%) mice are heterozygous (Casz1 ϩ/␤geo ), but none are homozygous (Casz1 ␤geo/␤geo ).…”

“…For overexpression expression experiments we used the human CASZ1b cDNA construct because it is the most evolutionarily conserved isoform (11,14). First, we used human cardiac fibroblasts whose level of Casz1 expression is not detectable by routine qRT-PCR.…”

CASZ1 (castor zinc finger 1)

“…For CASZ1a, six other zinc fingers (ZF) are present along with a third nuclear localization signal between ZF8 and ZF9. CASZ1b totals 1166 amino acids and CASZ1a totals 1794 amino acids (Liu et al, 2006;Virden et al, 2012).…”

“…Restoration of CASZ1 in NB cells induces cell differentiation, inhibits cell migration and suppresses NB growth (Liu et al, 2011a;Liu et al, 2011b). Structural and functional studies show that either deletion of the N-terminus or mutation in any of the first four zinc fingers of CASZ1b results in a drastic loss in transcriptional activity, which also leads to a decreased tumor suppression activity (Virden et al, 2012). Additionally, single nucleotide polymorphisms in this gene are associated with blood pressure variations in East Asian populations (Takeuchi et al, 2010).…”