The PRDM9 KRAB domain is required for meiosis and involved in protein interactions.

PR domain-containing protein 9 (PRDM9) is a major regulator of the localization of meiotic recombination hotspots in the human and mouse genomes. This role involves its DNA-binding domain, which is composed of a tandem array of zinc fingers, and PRDM9-dependent trimethylation of histone H3 at lysine 4. PRDM9 is a member of the PRDM family of transcription regulators, but unlike other family members, it contains a Krüppel-associated box (KRAB)-related domain that is predicted to be a potential protein interaction domain. Here, we show that truncation of the KRAB domain of mouse PRDM9 leads to loss of PRDM9 function and altered meiotic prophase and gametogenesis. In addition, we identified proteins that interact with the KRAB domain of PRDM9 in yeast two-hybrid assay screens, particularly CXXC1, a member of the COMPASS complex. We also show that CXXC1 interacts with IHO1, an essential component of the meiotic double-strand break (DSB) machinery. As CXXC1 is orthologous to Saccharomyces cerevisiae Spp1 that links DSB sites to the DSB machinery on the chromosome axis, we propose that these molecular interactions involved in the regulation of meiotic DSB formation are conserved in mouse meiosis.

KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions.

Zinc finger proteins containing the Kruppel associated box (KRAB-ZFPs) constitute the largest individual family of transcriptional repressors encoded by the genomes of higher organisms. KRAB domain, positioned at the NH2 terminus of the KRAB-ZFPs, interacts with a scaffold protein, KAP-1, which is able to recruit various transcriptional factors causing repression of genes to which KRAB ZFPs bind. The relevance of such repression is reflected in the large number of the KRAB zinc finger protein genes in the human genome. However, in spite of their numerical abundance little is currently known about the gene targets and the physiological functions of KRAB- ZFPs. However, emerging evidence links the transcriptional repression mediated by the KRAB-ZFPs to cell proliferation, differentiation, apoptosis and cancer. Moreover, the fact that KRAB containing proteins are vertebrate-specific suggests that they have evolved recently, and that their key roles lie in some aspects of vertebrate development. In this review, we will briefly discuss some regulatory functions of the KRAB-ZFPs in different physiological and pathological states, thus contributing to better understand their biological roles.

High Expression of POGK Predicts Poor Prognosis in Patients with Hepatocellular Carcinoma.

OBJECTIVE: Kruppel-associated box (KRAB) proteins reportedly play a dual role in neoplastic transformation. At present, little is known about the function of the proteins encoded by the human pogo transposable element derived with KRAB domain (POGK) gene. Herein, we evaluated the prognostic significance of POGK expression in patients with hepatocellular carcinoma (HCC). METHODS: The data of HCC patients was downloaded from The Cancer Genome Atlas (TCGA) database. To determine the relationship between POGK and clinical features, logistic regression was applied. Cox regression and Kaplan-Meier analyses were used to evaluate the correlation between POGK and survival rates. Gene ontology (GO) analysis and Gene set enrichment analysis (GSEA) were conducted to identify the enriched pathways and functions associated with POGK. RESULTS: A total of 374 HCC patients were identified in TCGA. POGK was significantly upregulated in HCC and correlated with tumor status (p = 0.036), race (p = 0.025), weight (p = 0.002), body mass index (p = 0.033), histologic grade (p < 0.001), and alpha-fetoprotein (p < 0.001). High POGK expression in HCC patients correlated with a poor outcome in terms of overall survival (p = 0.0018), progression-free survival (p = 0.0087), relapse-free survival (p = 0.045), and disease-specific survival (p = 0.014), according to Kaplan-Meier analysis. Receiver operating characteristic curve analysis showed that the area under the curve of POGK expression for HCC diagnosis was 0.891. GSEA showed that high POGK expression might activate mitotic prometaphase, kinesins, homologous DNA pairing and strand exchange, MET activates PTK2 signaling pathway, G1 to S cell cycle control, Aurora B pathway, ncRNAs involved in WNT signaling pathway, hepatitis C, and ncRNAs involved in the STAT3 signaling pathway. POGK expression correlated with the abundance of adaptive and innate immunocytes in HCC. CONCLUSION: High expression of POGK has high diagnostic and prognostic values in patients with HCC. Moreover, POGK expression is correlated with immune infiltration in HCC.

Knockdown of KRAB domain-associated protein 1 suppresses the proliferation, migration and invasion of thyroid cancer cells by regulating P68/DEAD box protein 5.

KRAB domain-associated protein 1 (KAP-1) has been reported to be an oncogene in diverse tumors. KAP-1 was found to have abundant existence in malignant thyroid tissues, but its role in thyroid cancer hasn't been elucidated clearly. This study was carried out to explore the role of KAP-1 in thyroid cancer, and to clarify its molecular mechanism. The expressions of KAP-1 and P68/DEAD box protein 5 (DDX5) were assessed under the help of qRT-PCR and western blot. Then, we downregulated KAP-1 or upregulated DDX5 by cell transfection in TPC-1 cells. A series of cellular experiments on proliferation, apoptosis, migration and invasion were conducted with CCK-8, EdU, TUNEL, wound-healing and Transwell assays. Besides, the relationship between KAP-1 and DDX5 was verified by co-immunoprecipitation (Co-IP). The results showed that both of KAP-1 and DDX5 were upregulated in thyroid cancer cells. Loss-of-function experiments revealed that KAP-1 knockdown imparted suppressive effects on cell proliferation, migration and invasion, but promoted cell apoptosis. Additionally, KAP-1 was demonstrated to interact with DDX5 and positively regulate DDX5 expression. The following rescued experiments exhibited that the inhibitory effects of KAP-1 knockdown on cellular activities of thyroid cancer and Wnt/ß-catenin signaling were also partly reversed by DDX5 overexpression. Moreover, activation of Wnt/ß-catenin signaling retarded the anti-tumor activity of KAP-1 knockdown. In conclusion, the data in this study disclosed that KAP-1 silence helped to repress the cell proliferation, migration and invasion by degrading DDK5, so as to hinder the development of thyroid cancer.

Mapping the interaction between Trim28 and the KRAB domain at the center of Trim28 silencing of endogenous retroviruses.

Transcription of endogenous retroviral elements are tightly regulated during development by members of the KRAB-containing zinc finger proteins (KRAB-ZFPs) and the co-repressor Trim28 (also known as Kap-1 or Tif1ß). KRAB-ZFPs form the largest family of transcription regulators in mammals and initiate transcriptional silencing by tethering Trim28 to a target locus. Subsequently, Trim28 recruits chromatin modifying effectors resulting in the formation of heterochromatin. In the present study, we identify surface exposed residues on the central six turns of the Trim28 coiled-coil region forming the binding interface for the KRAB domain. Using AlphaFold2 (AF2) we provide high confidence models of the interface between Trim28 and the KRAB domain and identified leucine 301 on each chain of the Trim28 monomer to act as a pin extending into a hydrophobic pocket on the KRAB domain surface. Site directed mutations in the Trim28-KRAB binding interface abolished binding to the KRAB domain. Our work provides a detailed understanding of the specific interactions between the KRAB domain and the Trim28 coiled-coil and how this interaction may be regulated during silencing events.

The KRAB Domain-Containing Protein ZFP961 Represses Adipose Thermogenesis and Energy Expenditure through Interaction with PPARα.

Adipose thermogenesis plays a pivotal role in whole-body metabolic homeostasis. Although transcriptional mechanisms that promote thermogenesis are extensively studied, the negative regulatory network is still poorly understood. Here, a Krüppel-associated box (KRAB) domain-containing zinc finger protein, ZFP961, as a potent repressor of the thermogenic program is identified. ZFP961 expression is induced by cold and ß3-adrenergic agonist in adipose tissue. ZFP961 represses brown fat-selective gene expression and mitochondrial respiration without any effect on general adipogenesis in cultured adipocytes. Adipose-specific knockdown and overexpression of ZFP961 produce remarkable and opposite phenotypes of white fat remodeling. ZFP961 knockout mice display robust inguinal white adipose tissue browning, which is abolished by reexpression of full-length ZFP961, but not by KRAB domain-deleted ZFP961 mutant. ZFP961-deficient mice are cold tolerant and resistant to high-fat diet-induced obesity, hyperglycemia, and hepatic steatosis. ZFP961 suppresses thermogenic gene expression by directly interacting with PPARα and blocking its transcriptional activity, which can be completely negated by the PPARα agonist. The findings uncover ZFP961 as a critical physiological brake that limits adipose thermogenesis and provides insights into the regulatory mechanisms that maintain energy balance and tissue homeostasis.

KAP1 phosphorylation promotes the survival of neural stem cells after ischemia/reperfusion by maintaining the stability of PCNA.

AIMS: To explore the function of phosphorylation of KAP1 (p-KAP1) at the serine-824 site (S824) in the proliferation and apoptosis of endogenous neural stem cells (NSCs) after cerebral ischemic/reperfusion (I/R). METHODS: The apoptosis and proliferation of C17.2 cells transfected with the p-KAP1-expression plasmids and the expression of proliferation cell nuclear antigen (PCNA) and p-KAP1 were detected by immunofluorescence and Western blotting after the Oxygen Glucose deprivation/reperfusion model (OGD/R). The interaction of p-KAP1 and CUL4A with PCNA was analyzed by immunoprecipitation. In the rats MCAO model, we performed the adeno-associated virus (AAV) 2/9 gene delivery of p-KAP1 mutants to verify the proliferation of endogenous NSCs and the colocalization of PCNA and CUL4A by immunofluorescence. RESULTS: The level of p-KAP1 was significantly down-regulated in the stroke model in vivo and in vitro. Simulated p-KAP1(S824) significantly increased the proliferation of C17.2 cells and the expression of PCNA after OGD/R. Simulated p-KAP1(S824) enhanced the binding of p-KAP1 and PCNA and decreased the interaction between PCNA and CUL4A in C17.2 cells subjected to OGD/R. The AAV2/9-mediated p-KAP1(S824) increased endogenous NSCs proliferation, PCNA expression, p-KAP1 binding to PCNA, and improved neurological function in the rat MCAO model. CONCLUSIONS: Our findings confirmed that simulated p-KAP1(S824) improved the survival and proliferation of endogenous NSCs. The underlying mechanism is that highly expressed p-KAP1(S824) promotes binding to PCNA, and inhibits the binding of CUL4A to PCNA. This reduced CUL4A-mediated ubiquitination degradation to increase the stability of PCNA and promote the survival and proliferation of NSCs.

C2H2-Type Zinc Finger Proteins in Brain Development, Neurodevelopmental, and Other Neuropsychiatric Disorders: Systematic Literature-Based Analysis.

Neurodevelopmental disorders (NDDs) are multifaceted pathologic conditions manifested with intellectual disability, autistic features, psychiatric problems, motor dysfunction, and/or genetic/chromosomal abnormalities. They are associated with skewed neurogenesis and brain development, in part through dysfunction of the neural stem cells (NSCs) where abnormal transcriptional regulation on key genes play significant roles. Recent accumulated evidence highlights C2H2-type zinc finger proteins (C2H2-ZNFs), the largest transcription factor family in humans, as important targets for the pathologic processes associated with NDDs. In this review, we identified their significant accumulation (74 C2H2-ZNFs: ~10% of all human member proteins) in brain physiology and pathology. Specifically, we discuss their physiologic contribution to brain development, particularly focusing on their actions in NSCs. We then explain their pathologic implications in various forms of NDDs, such as morphological brain abnormalities, intellectual disabilities, and psychiatric disorders. We found an important tendency that poly-ZNFs and KRAB-ZNFs tend to be involved in the diseases that compromise gross brain structure and human-specific higher-order functions, respectively. This may be consistent with their characteristic appearance in the course of species evolution and corresponding contribution to these brain activities.

MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway.

Melanoma-associated antigen A3 (MAGEA3), a member of the cancer-testis antigen (CTA) family, is aberrantly expressed in various cancer types. Accumulating evidence indicates that MAGEA3 plays a vital role in the pathogenesis and development of various cancers. However, the underlying mechanisms behind the tumor-promoting effect of MAGEA3 remain unclear, particularly in cervical cancer (CC). The present study investigated the effects of MAGEA3 on CC cell proliferation and apoptosis as well as the underlying molecular mechanism. Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays were used to evaluate the effects of MAGE-A3 on proliferation, cell cycle, and apoptosis. Co-immunoprecipitation (Co-IP), dual-luciferase reporter, western blotting, and quantitative RT-PCR assays were performed to investigate the regulatory mechanisms of MAGEA3 in CC cells. Compared to the control, MAGE-A3 overexpression markedly promoted the proliferation of SiHa cells in vitro and in vivo, increased the proportion of cells in S phase, and suppressed apoptosis. However, MAGEA3 knockdown inhibited proliferation, blocked the cell cycle in G1 phase, and induced apoptosis in HeLa cells. Further mechanistic study revealed that MAGEA3 interacts with KAP1, thereby suppressing p53 transcriptional activity, thus suppressing p53-mediated regulation of the expression of genes involved in the cell cycle (p21, cyclin D1) and apoptosis (Bax, Bcl-2, and PUMA). Collectively, our results, both in vivo and in vitro, indicate that the expression of MAGEA3 contributes to CC cell proliferation and tumor growth and exerts tumor-promoting effects by regulating the KAP1/p53 signaling pathway.

KAP1 silencing relieves OxLDL-induced vascular endothelial dysfunction by down-regulating LOX-1.

KRAB domain-associated protein 1 (KAP1) is highly expressed in atherosclerotic plaques. Here, we studied the role of KAP1 in atherosclerosis development using a cell model of endothelial dysfunction induced by oxidative low-density lipoprotein (OxLDL). The phosphorylation and protein levels of KAP1 were similar between OxLDL-treated and non-treated endothelial cells (ECs). KAP1 depletion significantly inhibited the production of OxLDL-enhanced reactive oxygen species and the expression of adhesion molecules in ECs. Treatment with OxLDL promoted the proliferation and migration of ECs, which was also confirmed by the elevated levels of the proliferative markers c-Myc and PCNA, as well as the migratory marker MMP-9. However, these effects were also abrogated by KAP1 depletion. Moreover, the depletion of KAP1 in OxLDL-treated ECs resulted in decreases in the LOX-1 level and increases in eNOS expression. Generally, the data suggest that strategies targeting KAP1 depletion might be particularly useful for the prevention or treatment of atherosclerosis.

Interrogating the Functions of PRDM9 Domains in Meiosis.

Homologous recombination is required for proper segregation of homologous chromosomes during meiosis. It occurs predominantly at recombination hotspots that are defined by the DNA binding specificity of the PRDM9 protein. PRDM9 contains three conserved domains typically involved in regulation of transcription; yet, the role of PRDM9 in gene expression control is not clear. Here, we analyze the germline transcriptome of Prdm9-/- male mice in comparison to Prdm9+/+ males and find no apparent differences in the mRNA and miRNA profiles. We further explore the role of PRDM9 in meiosis by analyzing the effect of the KRAB, SSXRD, and post-SET zinc finger deletions in a cell culture expression system and the KRAB domain deletion in mice. We found that although the post-SET zinc finger and the KRAB domains are not essential for the methyltransferase activity of PRDM9 in cell culture, the KRAB domain mutant mice show only residual PRDM9 methyltransferase activity and undergo meiotic arrest. In aggregate, our data indicate that domains typically involved in regulation of gene expression do not serve that role in PRDM9, but are likely involved in setting the proper chromatin environment for initiation and completion of homologous recombination.

Discovery of a novel oocyte-specific Krüppel-associated box domain-containing zinc finger protein required for early embryogenesis in cattle.

Zinc finger (ZNF) transcription factors interact with DNA through zinc finger motifs and play important roles in a variety of cellular functions including cell growth, proliferation, development, apoptosis, and intracellular signal transduction. One-third of ZNF proteins in metazoans contain a highly conserved N-terminal motif known as the Krüppel-associated box (KRAB) domain, which acts as a potent, DNA-binding dependent transcriptional repression module. Analysis of RNA-Seq data generated from a bovine oocyte cDNA library identified a novel transcript, which encodes a KRAB-containing ZNF transcription factor (named ZNFO). Characterization of ZNFO mRNA expression revealed that it is exclusively expressed in bovine oocytes and early embryos. A GFP reporter assay demonstrated that ZNFO protein localizes specifically to the nucleus, supporting its role in transcriptional regulation. To test the role of ZNFO in early embryonic development, zygotes were generated by in vitro maturation and fertilization of oocytes, and injected with small interfering RNA (siRNA) designed to knockdown ZNFO. Cleavage rates were not affected by ZNFO siRNA injection. However, embryonic development to 8- to 16-cell stage and blastocyst stage was significantly reduced relative to the uninjected and negative control siRNA-injected embryos. Further, interaction of ZNFO with the highly conserved co-factor, KRAB-associated protein-1 (KAP1), was demonstrated, and evidence supporting transcriptional repression by ZNFO was demonstrated using a GAL4-luciferase reporter system. Results of described studies demonstrate that ZNFO is a maternally-derived oocyte-specific nuclear factor required for early embryonic development in cattle, presumably functioning by repressing transcription.

C2H2 Zinc Finger Proteins: The Largest but Poorly Explored Family of Higher Eukaryotic Transcription Factors.

The emergence of whole-genome assays has initiated numerous genome-wide studies of transcription factor localizations at genomic regulatory elements (enhancers, promoters, silencers, and insulators), as well as facilitated the uncovering of some of the key principles of chromosomal organization. However, the proteins involved in the formation and maintenance of the chromosomal architecture and the organization of regulatory domains remain insufficiently studied. This review attempts to collate the available data on the abundant but still poorly understood family of proteins with clusters of the C2H2 zinc finger domains. One of the best known proteins of this family is a well conserved protein known as CTCF, which plays a key role in the establishment of the chromosomal architecture in vertebrates. The distinctive features of C2H2 zinc finger proteins include strong and specific binding to a long and unique DNA recognition target sequence and rapid expansion within various animal taxa during evolution. The reviewed data support a proposed model according to which many of the C2H2 proteins have functions that are similar to those of the CTCF in the organization of the chromatin architecture.

ZNF10 inhibits HIV-1 LTR activity through interaction with NF-κB and Sp1 binding motifs.

Kruppel-associated box-containing zinc finger (KRAB-ZNF) genes constitute the single largest gene family of transcriptional repressors in the genomes of higher organisms. In this study, we isolated 52 cDNA clones of KRAB-ZFPs from U1 cell lines and screened them to identify which were capable of regulating HIV-1 gene expression. We identified 5 KRAB-ZFPs that suppressed ⩾50% of HIV-1 LTR. Of the 5 identified KRAB-ZFPs, the expression of ZNF10 significantly enhanced the transcriptional repression activity of the LTR compared with other ZNFs. In addition, the depletion of endogenous ZNF10 led to the activation of HIV-1 LTR. The repressor activity of ZNF10 was required for TRIM28, SETDB1 and HP1-gamma binding. These results indicate that ZNF10 could be involved in a potent intrinsic antiretroviral defense.

KAPtain in charge of multiple missions: Emerging roles of KAP1.

KAP1/TRIM28/TIF1ß was identified nearly twenty years ago as a universal transcriptional co-repressor because it interacts with a large KRAB-containing zinc finger protein (KRAB-ZFP) transcription factor family. Many studies demonstrate that KAP1 affects gene expression by regulating the transcription of KRAB-ZFP-specific loci, trans-repressing as a transcriptional co-repressor or epigenetically modulating chromatin structure. Emerging evidence suggests that KAP1 also functions independent of gene regulation by serving as a SUMO/ubiquitin E3 ligase or signaling scaffold protein to mediate signal transduction. KAP1 is subjected to multiple post-translational modifications (PTMs), including serine/tyrosine phosphorylation, SUMOylation, and acetylation, which coordinately regulate KAP1 function and its protein abundance. KAP1 is involved in multiple aspects of cellular activities, including DNA damage response, virus replication, cytokine production and stem cell pluripotency. Moreover, knockout of KAP1 results in embryonic lethality, indicating that KAP1 is crucial for embryonic development and possibly impacts a wide-range of (patho)physiological manifestations. Indeed, studies from conditional knockout mouse models reveal that KAP1-deficiency significantly impairs vital physiological processes, such as immune maturation, stress vulnerability, hepatic metabolism, gamete development and erythropoiesis. In this review, we summarize and evaluate current literatures involving the biochemical and physiological functions of KAP1. In addition, increasing studies on the clinical relevance of KAP1 in cancer will also be discussed.

Breaking through an epigenetic wall: re-activation of Oct4 by KRAB-containing designer zinc finger transcription factors.

The gene Oct4 encodes a transcription factor critical for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition, improper re-activation of Oct4 contributes to oncogenic processes. Herein, we describe a novel designer zinc finger protein (ZFP) capable of upregulating the endogenous Oct4 promoter in a panel of breast and ovarian cell lines carrying a silenced gene. In some ovarian tumor lines, the ZFP triggered a strong reactivation of Oct4, with levels of expression comparable with exogenous Oct4 cDNA delivery. Surprisingly, the reactivation of Oct4 required a KRAB domain for effective upregulation of the endogenous gene. While KRAB-containing ZFPs are traditionally described as transcriptional repressors, our results suggest that these proteins could, in certain genomic contexts, function as potent activators and, thus, outline an emerging novel function of KRAB-ZFPs. In addition, we document a novel ZFP that could be used for the epigenetic reprograming of cancer cells.