Abstract
ABSTRACTCIZ1 promotes cyclin-dependent DNA replication and resides in sub-nuclear foci that are part of the protein nuclear matrix (NM), and in RNA assemblies that are enriched at the inactive X chromosome (Xi) in female cells. It is subjected to alternative splicing, with specific variants implicated in adult and pediatric cancers. CIZ1-F is characterized by a frame shift that results from splicing exons 8–12 leading to inclusion of a short alternative reading frame (ARF), excluding the previously characterized C-terminal NM anchor domain. Here, we apply a set of novel variant-selective molecular tools targeted to the ARF to profile the expression of CIZ1-F at both transcript and protein levels, with focus on its relationship with the RNA-dependent and -independent fractions of the NM. Unlike full-length CIZ1, CIZ1-F does not accumulate at Xi, though like full-length CIZ1 it does resist extraction with DNase. Notably, CIZ1-F is sensitive to RNase identifying it as part of the RNA-fraction of the NM. In quiescent cells CIZ1-F transcript expression is suppressed and CIZ1-F protein is excluded from the nucleus, with re-expression not observed until the second cell cycle after exit from quiescence. Importantly, CIZ1-F is over-expressed in common solid tumors including colon and breast, pronounced in early stage but not highly-proliferative late stage tumors. Moreover, expression was significantly higher in hormone receptor negative breast tumors than receptor positive tumors. Together these data show that CIZ1-F is expressed in proliferating cells in an unusual cell cycle-dependent manner, and suggest that it may have potential as a tumor biomarker.
Highlights
CDKN1A interacting zinc finger protein 1 (CIZ1) is subject to extensive alternative splicing to yield multiple transcript variants.[1,2,3,4,5] Functional analysis of discrete protein fragments has identified domains and regulatory sites in the N-terminal half of the protein that are involved in DNA replication[6, 7] and interaction with cell cycle regulators[7] (Fig. 1A, B)
CIZ1-F protein lacks previously characterised functional domains, including the MH3-domain which anchors CIZ1 to the nuclear matrix (NM),[8] and cyclin-binding motifs through which CIZ1 interacts with cyclin A, and which are essential for its DNA replication activity (Fig. 1B).[6, 7]
Evaluation of cultured tumour-derived cell lines showed that CIZ1-F transcript is elevated in MCF7 breast cancer cells compared to normal MCF-10A breast cells (Supplementary Fig. 1D), and is one of the main CIZ1 PCRproducts (Supplementary Fig. 1A), identifying MCF7 cells as a model cell line to study the function of CIZ1-F in cell proliferation
Summary
CDKN1A interacting zinc finger protein 1 (CIZ1) is subject to extensive alternative splicing to yield multiple transcript variants.[1,2,3,4,5] Functional analysis of discrete protein fragments has identified domains and regulatory sites in the N-terminal half of the protein that are involved in DNA replication[6, 7] and interaction with cell cycle regulators[7] (Fig. 1A, B). We and others showed that CIZ1 localises to the inactive X chromosome (Xi) and is required to ensure retention of Xist long non-coding RNA (lncRNA) at Xi in fibroblasts.[9, 10] Alternative splicing affects both the replication domain (RD) and NM anchor domain (AD) of CIZ1, and several splicing events have been implicated in cancer, including mutation driven exclusion of exon 4 (CIZ1-Δ4) in Ewing’s Tumour cell lines[2] and exclusion of 24 nucleotides of exon 14 in lung cancer (CIZ1B).[5] A unique splice-junction epitope from CIZ1-B is a circulating biomarker for patients with small cell and non-small cell lung cancer.[5, 11] CIZ1 has been reported to be involved in the development of breast cancer,[12] the most common type of cancer in women.[13]. Alternative splicing of CIZ1 is implicated in other chronic diseases including upregulation of a form in which part of exon 8 is excluded (CIZ1-S) in Alzheimer’s disease,[3] and in cervical dystonia where mutations in an exonic splicing enhancer may affect splicing patterns.[14]
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