CIZ1 in Xist seeded assemblies at the inactive X chromosome.

There is growing evidence that X-chromosome inactivation is driven by phase-separated supramolecular assemblies. However, among the many proteins recruited to the inactive X chromosome by Xist long non-coding RNA, so far only a minority (CIZ1, CELF1, SPEN, TDP-43, MATR3, PTBP1, PCGF5) have been shown to form Xist-seeded protein assemblies, and of these most have not been analyzed in detail. With focus on CIZ1, here we describe 1) the contribution of intrinsically disordered regions in RNA-dependent protein assembly formation at the inactive X chromosome, and 2) enrichment, distribution, and function of proteins within Xist-seeded assemblies.

CIZ1 aggravates gastric cancer progression via mediating FBXL19-AS1 and miR-339-3p.

Gastric cancer (GC) remains a prevalent malignancy with high morbidity and mortality. CDKN1A interacting zinc finger protein 1 (CIZ1) has been demonstrated to have oncogenic functions in the development of cancers. We detected CIZ1 expression via quantitative real-time PCR (RT-qPCR). The protein level of CIZ1 was measured through Western blot. We noticed that CIZ1 expression was markedly enhanced in GC cells. Furthermore, functional experiments including colony formation assay, EdU staining assay, transwell assay, TUNEL staining assay and flow cytometry analysis uncovered that CIZ1 silencing attenuated cell malignant phenotypes in GC. Through bioinformatics tools and mechanism assays, we explored the up-stream mechanism of CIZ1 and determined that CIZ1 was modulated by FBXL19 antisense RNA 1 (FBXL19-AS1) and microRNA-339-3p (miR-339-3p). Additionally, miR-339-3p exerted a negative role on GC development in vitro, and FBXL19-AS1 depletion also had the inhibitory impacts on the progression of GC in vitro. Eventually, the finding that CIZ1 overexpression reversed the effects of FBXL19-AS1 silencing on GC development was validated by rescue assays. In a word, CIZ1 functioned as a tumor promoter in GC, indicating that CIZ1 might be a promising target for GC treatment.

Epigenetic instability caused by absence of CIZ1 drives transformation during quiescence cycles.

BACKGROUND: Cip1-interacting zinc finger protein 1 (CIZ1) forms RNA-dependent protein assemblies that stabilise epigenetic state, notable at the inactive X chromosome in females. CIZ1 has been linked with a range of human cancers and in mice genetic deletion of CIZ1 manifests as hyperproliferative lymphoid lineages in females. This suggests that its role in maintenance of epigenetic stability is linked with disease. RESULTS: Here, we show that male and female CIZ1-null primary murine fibroblasts have reduced H4K20me1 and that this compromises nuclear condensation on entry to quiescence. Global transcriptional repression remains intact in condensation-deficient CIZ1-null cells; however, a subset of genes linked with chromatin condensation and homology-directed DNA repair are perturbed. Failure to condense is phenotypically mimicked by manipulation of the H4K20me1 methyltransferase, SET8, in WT cells and partially reverted in CIZ1-null cells upon re-expression of CIZ1. Crucially, during exit from quiescence, nuclear decondensation remains active, so that repeated entry and exit cycles give rise to expanded nuclei susceptible to mechanical stress, DNA damage checkpoint activation, and downstream emergence of transformed proliferative colonies. CONCLUSIONS: Our results demonstrate a role for CIZ1 in chromatin condensation on entry to quiescence and explore the consequences of this defect in CIZ1-null cells. Together, the data show that CIZ1's protection of the epigenome guards against genome instability during quiescence cycles. This identifies loss of CIZ1 as a potentially devastating vulnerability in cells that undergo cycles of quiescence entry and exit.

Early chromosome condensation by XIST builds A-repeat RNA density that facilitates gene silencing.

XIST RNA triggers chromosome-wide gene silencing and condenses an active chromosome into a Barr body. Here, we use inducible human XIST to examine early steps in the process, showing that XIST modifies cytoarchitecture before widespread gene silencing. In just 2-4 h, barely visible transcripts populate the large "sparse zone" surrounding the smaller "dense zone"; importantly, density zones exhibit different chromatin impacts. Sparse transcripts immediately trigger immunofluorescence for H2AK119ub and CIZ1, a matrix protein. H3K27me3 appears hours later in the dense zone, which enlarges with chromosome condensation. Genes examined are silenced after compaction of the RNA/DNA territory. Insights into this come from the findings that the A-repeat alone can silence genes and rapidly, but only where dense RNA supports sustained histone deacetylation. We propose that sparse XIST RNA quickly impacts architectural elements to condense the largely non-coding chromosome, coalescing RNA density that facilitates an unstable, A-repeat-dependent step required for gene silencing.

Dihydroartemisinin inhibits the tumorigenesis and metastasis of breast cancer via downregulating CIZ1 expression associated with TGF-ß1 signaling.

AIMS: Dihydroartemisinin (DHA) is currently considered as the promising cancer therapeutic drug. In this study, we aimed to investigate the anti-proliferative and anti-metastasis effects of DHA. MAIN METHODS: Utilizing breast cancer cells MCF-7, MDA-MB-231 and BT549, cell proliferation, migration and invasion were detected. RT-qPCR was performed to detect CIZ1, TGF-ß1 and Snail expression, and the interactions of these related molecules were analyzed by GeneMANIA database. Western blot detected CIZ1, TGF-ß1/Smads signaling and Snail expression in DHA-treated cells, in TGFß1-induced cells with enhanced metastatic capacity, and in cells treated with DHA plus TGFß1/TGFß1 inhibitor SD-208. KEY FINDINGS: Results indicated DHA inhibited breast cancer cell proliferation and migration, with more potent effects compared with that of artemisinin. RT-qPCR and Western blot showed DHA inhibited CIZ1, TGF-ß1 and Snail expression, and these molecules were shown to have protein-protein interactions by bioinformatics. Furthermore, TGFß1-treatment enhanced MCF-7 migration and invasion, and CIZ1, TGF-ß1/Smads signaling and snail activities; DHA, SD-208, combination of DHA and SD-208 reversed these conditions, preliminarily proving the cascade regulation between TGF-ß1 signaling and CIZ1. MCF-7 xenografts model demonstrated the inhibition of DHA on tumor burden, and its mechanisms and well-tolerance in vivo; combination of DHA and SD-208 tried by us for the first time showed better treatment effects, but possible liver impairment made its use still keep cautious. SIGNIFICANCE: DHA treatment inhibits the proliferation and metastasis of breast cancer, through suppressing TGF-ß1/Smad signaling and CIZ1, suggesting the promising potential of DHA as a well-tolerated antitumor TGF-ß1 pathway inhibitor.

CIZ1 knockdown suppresses the proliferation of bladder cancer cells by inducing apoptosis.

Bladder cancer (BC) is the ninth most frequent malignancy and the thirteenth leading cause of cancer-related death worldwide. CDKN1A-interacting zinc finger protein 1 (CIZ1) is involved in the development of various cancers, while its role in BC remains unclear. In this study, we found that CIZ1 was up-regulated in BC tissues and cells. Knockdown of CIZ1 suppressed the proliferation and growth of T24 and 5637 cells. Apoptosis and caspase 3/7 activity were enhanced after CIZ1 silencing in both cells. At the molecular level, PCNA, Ki67, HIF-1α, survivin, CCND1 and CCNB1 were down-regulated, and caspase-3, p53, p21, caspase-9 were up-regulated by CIZ1 knockdown. This suggested that silencing of CIZ1 suppressed BC cell proliferation through inducing apoptosis and reducing cell cycle progression. Our study suggests that targeting CIZ1 maybe a potential therapeutic strategy for BC.

Screening Gene Mutations in Chinese Patients With Benign Essential Blepharospasm.

Objective: This study aimed to screen gene mutations in Chinese patients with benign essential blepharospasm (BEB) to understand its etiology. Methods: Twenty BEB patients diagnosed by clinical manifestations between April 2015 and October 2015 were enrolled. All the cases were investigated by questionnaires about general conditions, social behavioral factors, environmental factors, psychological factors, genetic factors, and previous diseases. In each patient, a total of 151 genes related to movement disorders were analyzed by second-generation sequencing. Results: Two patients had a family history of BEB, and they had SYNE1 and Cdkn1A-interacting zinc finger protein 1 (CIZ1) mutation, respectively. We found the SYNE1 mutation in seven patients, the CIZ1 mutation in two patients, the CACNA1A mutation in two patients, the LRRK2 mutation in two patients, and the FUS mutation in two patients. The C10orf2, TPP1, SLC1A3, PNKD, EIF4G1, SETX, PRRT2, SPTBN2, and TTBK2 mutations were found in only one patient, respectively, while not any mutation in the 151 genes were found in two patients. Some patients had mutations in two genes. Conclusion: Genetic factors, especially SYNE1 and CIZ1 mutations, contribute to the etiology of BEB.

CIZ1 Expression Is Upregulated in Hemangioma of the Tongue.

Hemangioma is a vascular neoplasm and one of the most common benign tumors. The pathogenesis of hemangioma has not been fully understood. CIZ1 (Cip1-interacting zinc finger protein 1) is a nuclear protein and the binding partner of p21. Dysregulation of CIZ1 expression has been reported in various types of cancerous tissues. In this study, we examined CIZ1 expression in hemangioma of the tongue and explored its function in vascular endothelial cells, the proliferative cell type in hemangioma. Immunohistochemistry showed that CIZ1 was highly expressed in hemangioma of the tongue while its expression is minimal in the normal tongue tissues. In vitro, knockdown of CIZ1 expression by shRNA transfection significantly reduced the proliferation and migration of human umbilical vein endothelium cells (HUVECs), suggesting a positive role of CIZ1 in endothelial cell proliferation and migration. Therefore, CIZ1 might involve in pathogenesis of hemangioma of the tongue by regulation of endothelial cell functions.

Visualization of Hidden Epitopes at the Inactive X Chromosome.

Immunodetection of nuclear antigens is often complicated by epitope masking, so that proteins known to function in the nucleus are sometimes not easily detected at their sites of action. Moreover, protein populations that are detected before unmasking can be very different to those seen after removal of nucleic acids. This is particularly true for components of the nuclear matrix, including those known to function at the inactive X chromosome. Here we describe an unmasking protocol that reveals previously undetected proteins at the inactive X chromosome in mouse fibroblasts.

Consequences of Cre-mediated deletion of Ciz1 exon 5 in mice.

CIZ1 plays a role in DNA synthesis at the G1/S checkpoint. Ciz1 gene-trap null mice manifest motor dysfunction, cell-cycle abnormalities, and DNA damage. In contrast, it has previously been reported that mouse embryonic fibroblasts derived from presumed Ciz1 knock-out mice (Ciz1tm1.1Homy/tm1.1Homy ) generated by crossing Cre-expressing mice with exon 5-floxed mice (Ciz1tm1Homy/tm1Homy ) do not exhibit evidence of enhanced DNA damage following γ-irradiation or cell-cycle defects. Here, we report that Ciz1tm1.1Homy/tm1.1Homy mice show loss of Ciz1 exon 5 but are neurologically normal and express abnormal transcripts (Ciz1ΔE5/ΔE5 mice) that are translated into one or more proteins of approximate wild-type size. Therefore, Ciz1tm1.1Homy/tm1.1Homy mice (Ciz1ΔE5/ΔE5 ) lose residues encoded by exon 5 but may gain function from novel amino acid sequences.

DNA damage and neurodegenerative phenotypes in aged Ciz1 null mice.

Cell-cycle dysfunction and faulty DNA repair are closely intertwined pathobiological processes that may contribute to several neurodegenerative disorders. CDKN1A interacting zinc finger protein 1 (CIZ1) plays a critical role in DNA replication and cell-cycle progression at the G1/S checkpoint. Germline or somatic variants in CIZ1 have been linked to several neural and extra-neural diseases. Recently, we showed that germline knockout of Ciz1 is associated with motor and hematological abnormalities in young adult mice. However, the effects of CIZ1 deficiency in much older mice may be more relevant to understanding age-related declines in cognitive and motor functioning and age-related neurologic disorders such as isolated dystonia and Alzheimer disease. Mouse embryonic fibroblasts from Ciz1-/- mice showed abnormal sensitivity to the effects of γ-irradiation with persistent DNA breaks, aberrant cell-cycle progression, and apoptosis. Aged (18-month-old) Ciz1-/- mice exhibited marked deficits in motor and cognitive functioning, and, in brain tissues, overt DNA damage, NF-κB upregulation, oxidative stress, vascular dysfunction, inflammation, and cell death. These findings indicate that the deleterious effects of CIZ1 deficiency become more pronounced with aging and suggest that defects of cell-cycle control and associated DNA repair pathways in postmitotic neurons could contribute to global neurologic decline in elderly human populations. Accordingly, the G1/S cell-cycle checkpoint and associated DNA repair pathways may be targets for the prevention and treatment of age-related neurodegenerative processes.

Repeat E anchors Xist RNA to the inactive X chromosomal compartment through CDKN1A-interacting protein (CIZ1).

X chromosome inactivation is an epigenetic dosage compensation mechanism in female mammals driven by the long noncoding RNA, Xist. Although recent genomic and proteomic approaches have provided a more global view of Xist's function, how Xist RNA localizes to the inactive X chromosome (Xi) and spreads in cis remains unclear. Here, we report that the CDKN1-interacting zinc finger protein CIZ1 is critical for localization of Xist RNA to the Xi chromosome territory. Stochastic optical reconstruction microscopy (STORM) shows a tight association of CIZ1 with Xist RNA at the single-molecule level. CIZ1 interacts with a specific region within Xist exon 7-namely, the highly repetitive Repeat E motif. Using genetic analysis, we show that loss of CIZ1 or deletion of Repeat E in female cells phenocopies one another in causing Xist RNA to delocalize from the Xi and disperse into the nucleoplasm. Interestingly, this interaction is exquisitely sensitive to CIZ1 levels, as overexpression of CIZ1 likewise results in Xist delocalization. As a consequence, this delocalization is accompanied by a decrease in H3K27me3 on the Xi. Our data reveal that CIZ1 plays a major role in ensuring stable association of Xist RNA within the Xi territory.

The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory.

The nuclear matrix protein Cip1-interacting zinc finger protein 1 (CIZ1) promotes DNA replication in association with cyclins and has been linked to adult and pediatric cancers. Here we show that CIZ1 is highly enriched on the inactive X chromosome (Xi) in mouse and human female cells and is retained by interaction with the RNA-dependent nuclear matrix. CIZ1 is recruited to Xi in response to expression of X inactive-specific transcript (Xist) RNA during the earliest stages of X inactivation in embryonic stem cells and is dependent on the C-terminal nuclear matrix anchor domain of CIZ1 and the E repeats of Xist CIZ1-null mice, although viable, display fully penetrant female-specific lymphoproliferative disorder. Interestingly, in mouse embryonic fibroblast cells derived from CIZ1-null embryos, Xist RNA localization is disrupted, being highly dispersed through the nucleoplasm rather than focal. Focal localization is reinstated following re-expression of CIZ1. Focal localization of Xist RNA is also disrupted in activated B and T cells isolated from CIZ1-null animals, suggesting a possible explanation for female-specific lymphoproliferative disorder. Together, these findings suggest that CIZ1 has an essential role in anchoring Xist to the nuclear matrix in specific somatic lineages.

Cardiomyocyte--specific expression of the nuclear matrix protein, CIZ1, stimulates production of mono-nucleated cells with an extended window of proliferation in the postnatal mouse heart.

Myocardial injury in mammals leads to heart failure through pathological cardiac remodelling that includes hypertrophy, fibrosis and ventricular dilatation. Central to this is inability of the mammalian cardiomyocyte to self-renew due to entering a quiescent state after birth. Modulation of the cardiomyocyte cell-cycle after injury is therefore a target mechanism to limit damage and potentiate repair and regeneration. Here, we show that cardiomyocyte-specific over-expression of the nuclear-matrix--associated DNA replication protein, CIZ1, extends their window of proliferation during cardiac development, delaying onset of terminal differentiation without compromising function. CIZ1-expressing hearts are enlarged, but the cardiomyocytes are smaller with an overall increase in number, correlating with increased DNA replication after birth and retention of an increased proportion of mono-nucleated cardiomyocytes into adulthood. Furthermore, these CIZ1 induced changes in the heart reduce the impact of myocardial injury, identifying CIZ1 as a putative therapeutic target for cardiac repair.

A quantitative immunoassay for lung cancer biomarker CIZ1b in patient plasma.

OBJECTIVES: Non-invasive tests for early detection of lung cancer are an important unmet clinical need. CIZ1b plasma biomarker can discriminate stage 1 lung cancer from within high-risk groups with clinically useful accuracy, with ROC AUCs in excess of 0.9 for two independent retrospective cohorts, and could therefore meet this need. Our aim was to characterise the native state of the biomarker and develop a quantitative immunoassay. DESIGN AND METHODS: Selective denaturation, preparative electrophoresis and mass spectrometry of human plasma were used to characterise the biomarker and interaction partners. A sandwich ELISA was generated, and specificity for CIZ1b biomarker tested on lung cancer patient plasma. RESULTS: CIZ1b biomarker is a denaturation-resistant complex between a C-terminal fragment of CIZ1 bearing the CIZ1b epitope specified by alternative splicing of exon14, and fibrinogen alpha chain. Reconstitution of the biomarker epitope with purified fibrinogen and CIZ1b, but not CIZ1a (non-alternatively spliced exon 14) confirmed the specificity of the results. The endogenous complex is highly stable in lung cancer plasma and can be quantified by pairing of a CIZ1b exon-junction specific antibody with detection of fibrinogen. Application of this sandwich ELISA to a prospectively collected development set of plasmas reveals the same level of accuracy as the western blot used to validate the discriminatory capability of the biomarker. CONCLUSIONS: Unexpected and unusual molecular structure of CIZ1b in native plasma has complicated immunoassay design, and delayed translation of this promising biomarker. However, CIZ1b can now be measured using a high-throughput, hospital-friendly sandwich ELISA format, overcoming an important barrier to further clinical development and application of this blood test for early stage lung cancer.

Motor phenotypes and molecular networks associated with germline deficiency of Ciz1.

A missense mutation in CIZ1 (c.790A>G, p.S264G) was linked to autosomal dominant cervical dystonia in a large multiplex Caucasian pedigree (OMIM614860, DYT23). CIZ1 is a p21((Cip1/Waf1)) -interacting zinc finger protein, widely expressed in neural and extra-neural tissues, and plays a role in DNA synthesis at the G1/S cell-cycle checkpoint. The role of CIZ1 in the nervous system and relative contributions of gain- or loss- of function to the pathogenesis of CIZ1-associated dystonia remain indefinite. Using relative quantitative reverse transcriptase-PCR, cerebellum showed the highest expression levels of Ciz1 in adult mouse brain, over two fold higher than liver, and higher than striatum, midbrain and cerebral cortex. Overall, neural expression of Ciz1 increased with postnatal age. A Ciz1 gene-trap knock-out (KO) mouse model (Ciz1(-/-)) was generated to examine the functional role(s) of CIZ1 in the sensorimotor nervous system and contributions of CIZ1 to cell-cycle control in the mammalian brain. Ciz1 transcripts were absent in Ciz1(-/-) mice and reduced by approximately 50% in Ciz1(+/-) mice. Ciz1(-/-) mice were fertile but smaller than wild-type (WT) littermates. Ciz1(-/-) mice did not manifest dystonia, but exhibited mild motoric abnormalities on balance, open-field activity, and gait. To determine the effects of germline KO of Ciz1 on whole-genome gene expression in adult brain, total RNA from mouse cerebellum was harvested from 6 10-month old Ciz1(-/-) mice and 6 age- and gender- matched WT littermates for whole-genome gene expression analysis. Based on whole-genome gene-expression analyses, genes involved in cellular movement, cell development, cellular growth, cellular morphology and cell-to-cell signaling and interaction were up-regulated in Ciz1(-/-) mice. The top up-regulated pathways were metabolic and cytokine-cytokine receptor interactions. Down-regulated genes were involved in cell cycle, cellular development, cell death and survival, gene expression and cell morphology. Down-regulated networks included those related to metabolism, focal adhesion, neuroactive ligand-receptor interaction, and MAPK signaling. Based on pathway analyses, transcription factor 7-like 2 (TCF7L2), a member of the Wnt/ß-catenin signaling pathway, was a major hub for down-regulated genes, whereas NF-κB was a major hub for up-regulated genes. In aggregate, these data suggest that CIZ1 may be involved in the post-mitotic differentiation of neurons in response to external signals and changes in gene expression may compensate, in part, for CIZ1 deficiency in our Ciz1(-/-) mouse model. Although CIZ1 deficiency was associated with mild motor abnormalities, germline loss of Ciz1 was not associated with dystonia on the C57BL/6J background.

CIZ1 interacts with YAP and activates its transcriptional activity in hepatocellular carcinoma cells.

Dysregulation of Hippo-Yes-associate protein (YAP) signaling has important roles in the tumorigenesis of hepatocellular carcinoma (HCC). Our previous studies have shown that Cip1 interacting zinc finger protein 1 (CIZ1) activated YAP signaling in the HCC cells and promoted the growth and migration of cancer cells. However, the mechanisms for the activation of YAP signaling by CIZ1 are unknown. In this study, it was found that CIZ1 interacted with the transcriptional factor YAP in HCC cells. The nuclear matrix anchor domain of CIZ1 is responsible for its interaction with YAP. Moreover, CIZ1 enhanced the interaction between YAP and TEAD. Knocking down the expression of CIZ1 impaired the transcriptional activity as well as the biological functions of YAP. Taken together, our study demonstrated that CIZ1 is a positive regulator of YAP signaling, and CIZ1 might be a therapeutic target for HCC.

The Role of Cdkn1A-Interacting Zinc Finger Protein 1 (CIZ1) in DNA Replication and Pathophysiology.

Cdkn1A-interacting zinc finger protein 1 (CIZ1) was first identified in a yeast-2-hybrid system searching for interacting proteins of CDK2 inhibitor p21(Cip1/Waf1). Ciz1 also binds to CDK2, cyclin A, cyclin E, CDC6, PCNA, TCF4 and estrogen receptor-α. Recent studies reveal numerous biological functions of CIZ1 in DNA replication, cell proliferation, and differentiation. In addition, splicing variants of CIZ1 mRNA is associated with a variety of cancers and Alzheimer's disease, and mutations of the CIZ1 gene lead to cervical dystonia. CIZ1 expression is increased in cancers and rheumatoid arthritis. In this review, we will summarize the biological functions and molecular mechanisms of CIZ1 in these physiological and pathological processes.

CIZ1 is upregulated in hepatocellular carcinoma and promotes the growth and migration of the cancer cells.

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and the prognosis for the HCC remains very poor. Although dys-regulation of CIZ1 (Cip1 interacting zinc finger protein 1) has been observed in various cancer types, its expression and functions in HCC remain unknown. In this study, the mRNA level of CIZ1 in the HCC tissues were examined using real-time polymerase chain reaction, and the effects of CIZ1 on the growth, migration, and metastasis of HCC cells were examined by crystal violet assay, Boyden chamber assay, and in vivo image system, respectively. In addition, the molecular mechanisms were investigated by luciferase assay. Upregulation of CIZ1 in the clinical HCC samples was observed. Forced expression of CIZ1 promoted the growth and migration of HCC cells, while knocking down the expression of CIZ1 inhibited the growth, migration, and metastasis of HCC cells. Molecular mechanism studies revealed that CIZ1 activated YAP/TAZ signaling in HCC cells. Taken together, our study demonstrated the oncogenic roles of CIZ1 in HCC cells and CIZ1 might be a promising therapeutic target for HCC.

Cyclin-A-CDK2-mediated phosphorylation of CIZ1 blocks replisome formation and initiation of mammalian DNA replication.

CIZ1 is a nuclear matrix protein that cooperates with cyclin A2 (encoded by CCNA2) and CDK2 to promote mammalian DNA replication. We show here that cyclin-A-CDK2 also negatively regulates CIZ1 activity by phosphorylation at threonines 144, 192 and 293. Phosphomimetic mutants do not promote DNA replication in cell-free and cell-based assays, and also have a dominant-negative effect on replisome formation at the level of PCNA recruitment. Phosphorylation blocks direct interaction with cyclin-A-CDK2 and recruitment of endogenous cyclin A to the nuclear matrix. In contrast, phosphomimetic CIZ1 retains the ability to bind to the nuclear matrix, and its interaction with CDC6 is not affected. Phospho-T192-specific antibodies confirm that CIZ1 is phosphorylated during S phase and G2, and show that phosphorylation at this site occurs at post-initiation concentrations of cyclin-A-CDK2. Taken together, the data suggest that CIZ1 is a kinase sensor that promotes initiation of DNA replication at low kinase levels, when in a hypophosphorylated state that is permissive for cyclin-A-CDK2 interaction and delivery to licensed origins, but blocks delivery at higher kinase levels when it is phosphorylated.