ZNF131 facilitates the growth of hepatocellular carcinoma by acting as a transcriptional activator of SMC4 expression.

ZNF131 is a Zinc finger protein that acts as a transcription factor with oncogenic effects in multiple cancers. In this study, we aimed to explore the alternative splicing profile of ZNF131 in hepatocellular carcinoma (HCC), its regulatory effects on cell-cycle progression, and the downstream effectors. ZNF131 transcriptional profile and HCC survival analysis were conducted using data from the Cancer Genome Atlas (TCGA)-Liver Hepatocellular Cancer (LIHC) dataset. Chromatin immunoprecipitation (ChIP)-qPCR and dual-luciferase reporter assays were utilized to explore transcriptional regulation. CCK-8, colony formation and xenograft tumor models were used to study HCC tumor growth. Results showed that ZNF131 isoform 2 is upregulated in HCC tissues and its upregulation was associated with unfavorable overall survival (OS) and progression-free interval (PFI). Knockdown of endogenous ZNF131 inhibits HCC cell growth and induces G2/M cell-cycle arrest. ZNF131 binds to the SMC4 promoter by interacting with ZBTB33 and the ZBTB33 recognizing motif. ZNF131 transcriptionally activates SMC4 expression in HCC cells. The tumor-suppressive effects of ZNF131 shRNA could be partially reversed by enforced SMC4 overexpression. In summary, this study highlights the ZNF131/ZBTB33/SMC4 axis as a driver of pathological cell cycling and proliferation in HCC.

Temporal and differential regulation of KAISO-controlled transcription by phosphorylated and acetylated p53 highlights a crucial regulatory role of apoptosis.

Transcriptional regulator KAISO plays a critical role in cell cycle arrest and apoptosis through modulation of p53 acetylation by histone acetyltransferase p300. KAISO potently stimulates apoptosis in cells expressing WT p53, but not in p53-mutant or p53-null cells. Here, we investigated how KAISO transcription is regulated by p53, finding four potential p53-binding sites (p53-responsive DNA elements; p53REs) located in a distal 5'-upstream regulatory element, intron 1, exon 2 coding sequence, and a 3'-UTR region. Transient transcription assays of pG5-p53RE-Luc constructs with various p53REs revealed that p53 activates KAISO (ZBTB33) transcription by acting on p53RE1 (-4326 to -4227) of the 5'-upstream region and on p53RE3 (+2929 to +2959) of the exon 2 coding region during early DNA damage responses (DDRs). ChIP and oligonucleotide pulldown assays further disclosed that p53 binds to the p53RE1 and p53RE3 sites. Moreover, ataxia telangiectasia mutated (ATM) or ATM-Rad3-related (ATR) kinase-mediated p53 phosphorylation at Ser-15 or Ser-37 residues activated KAISO transcription by binding its p53RE1 or p53RE3 sites during early DDR. p53RE1 uniquely contained three p53-binding half-sites, a structural feature important for transcriptional activation by phosphorylated p53 Ser-15·Ser-37. During the later DDR phase, a KAISO-mediated acetylated p53 form (represented by a p53QRQ acetyl-mimic) robustly activated transcription by acting on p53RE1 in which this structural feature is not significant, but it provided sufficient KAISO levels to confer a p53 "apoptotic code." These results suggest that the critical apoptosis regulator KAISO is a p53 target gene that is differently regulated by phosphorylated p53 or acetylated p53, depending on DDR stage.

Cell-specific Kaiso (ZBTB33) Regulation of Cell Cycle through Cyclin D1 and Cyclin E1.

The correlation between aberrant DNA methylation with cancer promotion and progression has prompted an interest in discerning the associated regulatory mechanisms. Kaiso (ZBTB33) is a specialized transcription factor that selectively recognizes methylated CpG-containing sites as well as a sequence-specific DNA target. Increasing reports link ZBTB33 overexpression and transcriptional activities with metastatic potential and poor prognosis in cancer, although there is little mechanistic insight into how cells harness ZBTB33 transcriptional capabilities to promote and progress disease. Here we report mechanistic details for how ZBTB33 mediates cell-specific cell cycle regulation. By utilizing ZBTB33 depletion and overexpression studies, it was determined that in HeLa cells ZBTB33 directly occupies the promoters of cyclin D1 and cyclin E1, inducing proliferation by promoting retinoblastoma phosphorylation and allowing for E2F transcriptional activity that accelerates G1- to S-phase transition. Conversely, in HEK293 cells ZBTB33 indirectly regulates cyclin E abundance resulting in reduced retinoblastoma phosphorylation, decreased E2F activity, and decelerated G1 transition. Thus, we identified a novel mechanism by which ZBTB33 mediates the cyclin D1/cyclin E1/RB1/E2F pathway, controlling passage through the G1 restriction point and accelerating cancer cell proliferation.

Comprehensive analysis of the REST transcription factor regulatory networks in IDH mutant and IDH wild-type glioma cell lines and tumors.

The RE1-silencing transcription factor (REST) acts either as a repressor or activator of transcription depending on the genomic and cellular context. REST is a key player in brain cell differentiation by inducing chromatin modifications, including DNA methylation, in a proximity of its binding sites. Its dysfunction may contribute to oncogenesis. Mutations in IDH1/2 significantly change the epigenome contributing to blockade of cell differentiation and glioma development. We aimed at defining how REST modulates gene activation and repression in the context of the IDH mutation-related phenotype in gliomas. We studied the effects of REST knockdown, genome wide occurrence of REST binding sites, and DNA methylation of REST motifs in IDH wild type and IDH mutant gliomas. We found that REST target genes, REST binding patterns, and TF motif occurrence proximal to REST binding sites differed in IDH wild-type and mutant gliomas. Among differentially expressed REST targets were genes involved in glial cell differentiation and extracellular matrix organization, some of which were differentially methylated at promoters or gene bodies. REST knockdown differently impacted invasion of the parental or IDH1 mutant glioma cells. The canonical REST-repressed gene targets showed significant correlation with the GBM NPC-like cellular state. Interestingly, results of REST or KAISO silencing suggested the interplay between these TFs in regulation of REST-activated and repressed targets. The identified gene regulatory networks and putative REST cooperativity with other TFs, such as KAISO, show distinct REST target regulatory networks in IDH-WT and IDH-MUT gliomas, without concomitant DNA methylation changes. We conclude that REST could be an important therapeutic target in gliomas.

Association of Kaiso and partner proteins in oral squamous cell carcinoma.

Objectives: 1. Identification of protein expression and subcellular localization of E-cadherin (E-cad), p120 catenin (P120ctn), and Kaiso in oral cancer (OC). 2. To study the protein expression of cyclin D1 and c-Myc (Kaiso targets) and determine their relationship with the expression and localization of Kaiso. Methods: Histological grading was performed in accordance with Broder's criteria. Expression and localization data for E-cad, p120ctn, Kaiso, cyclin D1, and c-Myc were acquired using immunohistochemistry. Data were analyzed using SPSS version 21. The chi-square test was used to measure the statistical significance of associations, with p < 0.05 as statistically significant. Results: Of 47 OC cases, 36% showed low E-cad expression and 34% showed low p120ctn. Low Kaiso expression was recognized in 78% of tumor specimens. Aberrant cytoplasmic localization of p120ctn was seen in 80.8% cases. Cytoplasmic Kaiso localization was appreciated in 87% of tumor tissues, whereas 29.7% lacked any nuclear Kaiso. Kaiso expression was significantly associated with the expression of cyclin D1 but not with c-Myc. Conclusion: The present study identified a change in the localization of Kaiso in OC. The significance of this in relation to OC and tumor prognosis needs to be investigated with further studies using larger sample sizes and more sensitive molecular tools.

Kaiso Protein in the Regulation of Brain and Behavior.

Kaiso is a bimodal transcriptional repressor. It binds methylated CpG islands or the sequence- specific consensus in the DNA molecule with the Kaiso zinc-finger domain and recruits repressive protein complexes to these DNA fragments by the interaction of the BTB/POZ domain with the complex of NCoR1 corepressor and histone deacetylase, thereby performing transcription repression. Kaiso is involved in epigenetic regulation of transcription. Moreover, the complex Kaiso and catenin p120ctn modulates the transcription of the Wnt-target genes. The review discusses the role of Kaiso in the central nervous system. Kaiso molecules are abundant in the brain. MRI study did not show any alterations in the whole brain, hippocampus and striatum in Kaiso null mice. However, in Kaiso deficient mice the lateral ventricles were three-fold smaller compared with wild-type control. Kaiso deficiency increased the locomotor and exploratory activities as well as the prepuls inhibition of acoustic startle reflex without any adverse effect on anxiety-related behavior, learning and memory. At the same time, Kaiso deficiency produces a marked antidepressant-like effect. Thus, Kaiso involved in the mechanism of locomotion and depressive-like behavior. Kaiso inhibitors are expected to be promising atypical antidepressant drugs.

Knockout Zbtb33 gene results in an increased locomotion, exploration and pre-pulse inhibition in mice.

The Zbtb33 gene encodes the Kaiso protein-a bimodal transcriptional repressor. Here, the effects of Zbtb33 gene disruption on the brain and behaviour of the Kaiso-deficient (KO) and C57BL/6 (WT) male mice were investigated. Behaviour was studied using the open field, novel object, elevated plus maze and acoustic startle reflex tests. Brain morphology was investigated with magnetic resonance imaging. Biogenic amine levels and gene expression in the brain were measured with high-performance liquid chromatography and quantitative real-time RT-PCR, respectively. Zbtb33 gene mRNA was not detected in the brain of KO mice. KO mice exhibited increased locomotion, exploration in the open field, novel object and elevated plus-maze test. At the same time, Zbtb33 gene disruption did not alter anxiety-related behaviour in the elevated plus-maze test. KO mice showed elevated amplitudes and pre-pulse inhibitions of the acoustic startle reflex. These behavioural alterations were accompanied by significant reductions in the volumes of the lateral ventricles without significant alterations in the volumes of the hippocampus, striatum, thalamus and corpus callosum. Norepinephrine concentration was reduced in the hypothalami and hippocampi in KO mice, while the levels of serotonin, dopamine, their metabolites as well as mRNA of the gene coding brain-derived neurotrophic factor were not altered in the brain of KO mice compared to WT mice. This study is the first to reveal the involvement of the Zbtb33 gene in the regulation of behaviour and the central nervous system.