Ciz1 promotes mammalian DNA replication

Abstract

Using a cell-free system that reconstitutes initiation of mammalian DNA replication, we identified a cyclin A-responsive protein, p21Cip1-interacting zinc finger protein 1 (Ciz1). In cell-free experiments, Ciz1 protein increases the number of nuclei that initiate DNA replication, and in intact cells GFP-tagged Ciz1 stimulates DNA synthesis, in both a wild-type and a p21Cip1 null background. Furthermore, mutation of a putative cyclin-dependent kinase phosphorylation site at threonines 191/2 alters Ciz1 activity in vitro, indicating that this site plays a role in regulating Ciz1. Consistent with a role in DNA replication, endogenous Ciz1 is present in nuclear foci that co-localize with PCNA during S phase, and targeted depletion of Ciz1 transcripts restrains cell proliferation by inhibiting entry to S phase. Ciz1-depleted cells accumulate with chromatin bound Mcm3 and PCNA but fail to synthesize DNA efficiently. These cell-based and cell-free experiments suggest that Ciz1 functions to promote DNA replication after replication complex formation. Finally, alternatively spliced forms of Ciz1 occur in embryonic cells from mouse and man, raising the possibility that Ciz1 splicing contributes to the regulation of DNA replication during development.

CIZ1-F, an alternatively spliced variant of the DNA replication protein CIZ1 with distinct expression and localisation, is overrepresented in early stage common solid tumours

“…The exon 8–12 junction causes a frame-shift in the CIZ1 reading frame, leading to a premature translational stop codon in exon 13, despite the presence of downstream exons in CIZ1 -F mRNA (Figure 1(a)). CIZ1-F protein therefore lacks previously characterized functional domains, including the MH3-domain which anchors CIZ1 to the NM [8], and cyclin-binding motifs through which CIZ1 interacts with cyclin A, and which are essential for its DNA replication activity (Figure 1(b)) [6,7]. Other characterized sites of interaction with CDK2, p21 cip1 and YAP are also excluded [15–17], while interaction with CDC6 [18], ERα [12], and dynein light chain [15] might be retained.…”

“…We and others have shown that CIZ1 is a NM protein, defined by its resistance to extraction of chromatin [3,8], which suggests that its function is connected to nuclear architecture, possibly contributing to the spatial regulation of nuclear processes [6–8,18,24]. Although the NM appears to be corrupted in certain types of cancer [25] previous work [26–28] and our own experiments indicate that MCF-7 cells retain a NM structure to some extent.…”

“…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], (Figure 1(a,b)). Separate sequences in the C-terminus of CIZ1 support interaction with nuclear structures, identifying a salt and DNase-resistant nuclear matrix (NM) interaction domain [8], which may restrict its function to specific sub-nuclear locations.…”

“…(B) Full-length and CIZ1-F protein showing annotated protein domains including glutamine-rich regions (Q), nuclear localization signals (NLS), zinc finger domains (ZF), matrin-3 domain (MH3), and acidic domain (AcD). Characterized functional domains and interaction sites for full-length CIZ1 are also shown, including replication domain (RD) which contains all sequences required for replication activity [6], and anchor domain (AD) which contains sequences that are sufficient to mediate attachment to the nuclear matrix [8]. The CIZ1-F specific sequence, encoded by an alternative reading frame (ARF) of exon 12–13, is highlighted in light blue.…”

“…Despite functional studies that link full-length murine CIZ1 and embryonic variant murine CIZ1 (ECIZ1) with DNA replication [6,7], little is known about the function of other variants. Alternative splicing of CIZ1  may have important consequences for the cell and is known to affect the sub-nuclear localization of the protein (Supplementary Table 1).…”