KRAB-containing Zinc Finger Protein Research Articles

Landscape of evolutionary arms races between transposable elements and KRAB-ZFP family

Transposable elements (TEs) are mobile parasitic sequences that have expanded within the host genome. It has been hypothesized that host organisms have expanded the Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs), which epigenetically suppress TEs, to counteract disorderly TE transpositions. This process is referred to as the evolutionary arms race. However, the extent to which this evolutionary arms race occurred across various TE families remains unclear. In the present study, we systematically explored the evolutionary arms race between TE families and human KRAB-ZFPs using public ChIP-seq data. We discovered and characterized new instances of evolutionary arms races with KRAB-ZFPs in endogenous retroviruses. Furthermore, we found that the regulatory landscape shaped by this arms race contributed to the gene regulatory networks. In summary, our results provide insight into the impact of the evolutionary arms race on TE families, the KRAB-ZFP family, and host gene regulatory networks.

KRAB zinc finger protein ZNF676 controls the transcriptional influence of LTR12-related endogenous retrovirus sequences

BackgroundTransposable element-embedded regulatory sequences (TEeRS) and their KRAB-containing zinc finger protein (KZFP) controllers are increasingly recognized as modulators of gene expression. We aim to characterize the contribution of this system to gene regulation in early human development and germ cells.ResultsHere, after studying genes driven by the long terminal repeat (LTR) of endogenous retroviruses, we identify the ape-restricted ZNF676 as the sequence-specific repressor of a subset of contemporary LTR12 integrants responsible for a large fraction of transpochimeric gene transcripts (TcGTs) generated during human early embryogenesis. We go on to reveal that the binding of this KZFP correlates with the epigenetic marking of these TEeRS in the germline, and is crucial to the control of genes involved in ciliogenesis/flagellogenesis, a biological process that dates back to the last common ancestor of eukaryotes.ConclusionThese results illustrate how KZFPs and their TE targets contribute to the evolutionary turnover of transcription networks and participate in the transgenerational inheritance of epigenetic traits.

Genome-wide CRISPR knockout screen identifies ZNF304 as a silencer of HIV transcription that promotes viral latency.

Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.

The interactome of KRAB zinc finger proteins reveals the evolutionary history of their functional diversification

Krüppel‐associated box (KRAB)‐containing zinc finger proteins (KZFPs) are encoded in the hundreds by the genomes of higher vertebrates, and many act with the heterochromatin‐inducing KAP1 as repressors of transposable elements (TEs) during early embryogenesis. Yet, their widespread expression in adult tissues and enrichment at other genetic loci indicate additional roles. Here, we characterized the protein interactome of 101 of the ~350 human KZFPs. Consistent with their targeting of TEs, most KZFPs conserved up to placental mammals essentially recruit KAP1 and associated effectors. In contrast, a subset of more ancient KZFPs rather interacts with factors related to functions such as genome architecture or RNA processing. Nevertheless, KZFPs from coelacanth, our most distant KZFP‐encoding relative, bind the cognate KAP1. These results support a hypothetical model whereby KZFPs first emerged as TE‐controlling repressors, were continuously renewed by turnover of their hosts’ TE loads, and occasionally produced derivatives that escaped this evolutionary flushing by development and exaptation of novel functions.

Spalt-Like Transcription Factor 1 (SALL1) Gene Expression Inhibits Cell Proliferation and Cell Migration of Human Glioma Cells Through the Wnt/β-Catenin Signaling Pathway.

BackgroundThe spalt-like transcription factor 1 (SALL1) gene is a member of the Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) and has been shown to modulate the onset and progression of human tumors. This study aimed to investigate the regulatory effects and mechanisms of SALL1 gene expression in human glioblastoma and glioma cells and tissue samples from patients with cerebral glioma.Material/MethodsThe human glioblastoma cell lines, LN229, U87-MG, U-251, U343, and the Hs683 glioma cell line were studied. The cell counting kit-8 (CCK-8) assay, cell cycle assay, wound-healing assay, transwell assay, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate cell proliferation, cell migration, and the cell cycle and expression of SALL1. Expression of SALL1 mRNA in 120 samples of cerebral glioma and 20 samples of normal brain were studied. Overall survival data from patients with cerebral glioma were analyzed.ResultsSALL1 expression was down-regulated in human glioblastoma and glioma cells and in cerebral glioma tissues. Down-regulation of SALL1 expression was associated with reduced overall survival. Overexpression of SALL1 was associated with inhibition of cell proliferation associated with cell cycle arrest at the G0/G1 phase. SALL1 inhibited cell migration by preventing epithelial-mesenchymal transition (EMT) and down-regulating the expression of stem cell markers. Reduced levels of β-catenin and downregulation of c-Myc and cyclin D1 and upregulation of p21and p27 expression were associated with SALL1 expression.ConclusionsIn human glioblastoma cells and cerebral glioma tissues, SALL1 acted as a tumor suppressor gene by inhibiting Wnt/β-catenin signaling.

Hominoid-Specific Transposable Elements and KZFPs Facilitate Human Embryonic Genome Activation and Control Transcription in Naive Human ESCs.

SummaryExpansion of transposable elements (TEs) coincides with evolutionary shifts in gene expression. TEs frequently harbor binding sites for transcriptional regulators, thus enabling coordinated genome-wide activation of species- and context-specific gene expression programs, but such regulation must be balanced against their genotoxic potential. Here, we show that Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFPs) control the timely and pleiotropic activation of TE-derived transcriptional cis regulators during early embryogenesis. Evolutionarily recent SVA, HERVK, and HERVH TE subgroups contribute significantly to chromatin opening during human embryonic genome activation and are KLF-stimulated enhancers in naive human embryonic stem cells (hESCs). KZFPs of corresponding evolutionary ages are simultaneously induced and repress the transcriptional activity of these TEs. Finally, the same KZFP-controlled TE-based enhancers later serve as developmental and tissue-specific enhancers. Thus, by controlling the transcriptional impact of TEs during embryogenesis, KZFPs facilitate their genome-wide incorporation into transcriptional networks, thereby contributing to human genome regulation.

KRAB-containing zinc finger protein ZNF496 inhibits breast cancer cell proliferation by selectively repressing ERα activity

KRAB-containing zinc finger proteins (KZNF) constitute the largest family of transcriptional regulators in humans and play critical roles in normal development and tumorigenesis. However, the function and mechanism of most KZNFs remain unclear. Here, we report that ZNF496, a KZNF family member, interacts with the DNA binding domain (DBD) of estrogen receptor alpha (ERα) via its C2H2 domain. This interaction decreases ERα binding to chromatin DNA and results in the repression of ERα transactivation, the selective suppression of ERα target genes, and ultimately in a reduction of ERα-positive cell growth in the presence of E2. An analysis of clinical data revealed that the downregulation of ZNF496 expression is observed only in ERα-positive and not in ERα-negative breast cancer tissues when compared with that in matched adjacent tissues. Lastly, we also observed that the downregulation of ZNF496 is associated with poor recurrence-free survival among patients with breast cancer. Collectively, our findings demonstrate that ZNF496 is a novel ERα-binding protein that acts as a target gene-specific ERα corepressor and inhibits the growth of ERα-positive breast cancer cells.

ZNF23 Suppresses Cutaneous Melanoma Cell Malignancy via Mitochondria-Dependent Pathway

Background/Aims: Cutaneous melanoma is one of the leading causes of cancer deaths with an increasing incidence worldwide. A KRAB-containing zinc finger protein member, zinc finger 23 (ZNF23), was reduced in some types of tumors and inhibited cell growth by inducing cell cycle arrest. However, the role of ZNF23 expression is still poorly understood in melanoma. Methods: The level of ZNF23 expression was detected in cutaneous melanoma, adjacent normal skin tissues and cutaneous melanoma cell lines using immunohistochemistry and western blotting. The correlations between ZNF23 expression and other clinicopathologic parameters were analyzed in melanoma patients. Ectopic expression of ZNF23 plasmid was transfected into melanoma cells, SK-MEL-1 and SK-MEL-28. MTT, flow cytometry and transwell assay were used to measure cell proliferation, apoptosis, invasion and migration abilities, respectively. Mitochondrial functions and structures were detected by mitochondrial membrane potential assay and Transmission electron microscopy (TEM) method in melanoma cells transfected with overexpressing ZNF23 plasmid or empty vector. Western blotting was performed to detect the levels of ZNF23, p53, p27, Bcl-2 and cleaved caspase-3 after overexpressing of ZNF23 in melanoma cells. Results: ZNF23 was elevated in adjacent normal skin tissues compared with melanoma tissues. Patients with low level of ZNF23 expression exhibited higher incidence of lymphoid metastasis, thicker size of tumors and worse outcome. By using Cox’s regression analysis, ZNF23 expression, tumor thickness and lymph node metastasis were the independent prognostic factors for overall survival (p < 0.05). Results from cellular experiments indicated that ectopic expression of ZNF23 induced cell apoptosis by activation of caspase-3, p27, p53 expression and down-regulation of Bcl-2 through mitochondria-dependent pathway. Conclusions: Decreased ZNF23 was contributed to melanoma progression and poor survival with mitochondria-dependent pathway. It indicated that ZNF23 could be a promising therapeutic biomarker candidate for cutaneous melanoma.

PEG3 Interacts with KAP1 through KRAB-A.

Peg3 (Paternally Expressed Gene 3) is an imprinted gene that encodes a zinc finger DNA-binding protein. Peg3 itself is localized in the middle of a KRAB-A (Kruppel-Associated Box) zinc finger gene cluster. The amino acid sequence encoded by its exon 7 also shows sequence similarity to that of KRAB-A, suggesting Peg3 as a KRAB-containing zinc finger gene. As predicted, the PEG3 protein was co-immunoprecipitated with KAP1, a co-repressor that interacts with KRAB-A. A series of follow-up experiments further demonstrated that the exon 7 of PEG3 is indeed responsible for its physical interaction with KAP1. ChIP and promoter assays also indicated that PEG3 likely controls its downstream genes through the KAP1-mediated repression mechanism. Overall, the current study identifies PEG3 as a KRAB-containing zinc finger protein that interacts with the co-repressor protein KAP1.

Overexpression of zinc-finger protein 777 (ZNF777) inhibits proliferation at low cell density through down-regulation of FAM129A.

Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) regulate a wide range of cellular processes. KRAB-ZFPs have a KRAB domain, which binds to transcriptional corepressors, and a zinc finger domain, which binds to DNA to activate or repress gene transcription. Here, we characterize ZNF777, a member of KRAB-ZFPs. We show that ZNF777 localizes to the nucleus and inducible overexpression of ZNF777 inhibits cell proliferation in a manner dependent on its zinc finger domain but independent of its KRAB domain. Intriguingly, ZNF777 overexpression drastically inhibits cell proliferation at low cell density but slightly inhibits cell proliferation at high cell density. Furthermore, ZNF777 overexpression decreases the mRNA level of FAM129A irrespective of cell density. Importantly, the protein level of FAM129A strongly decreases at low cell density, but at high cell density the protein level of FAM129A does not decrease to that observed at low cell density. ZNF777-mediated inhibition of cell proliferation is attenuated by overexpression of FAM129A at low cell density. Furthermore, ZNF777-mediated down-regulation of FAM129A induces moderate levels of the cyclin-dependent kinase inhibitor p21. These results suggest that ZNF777 overexpression inhibits cell proliferation at low cell density and that p21 induction by ZNF777-mediated down-regulation of FAM129A plays a role in inhibition of cell proliferation.

Transposable Elements, Polydactyl Proteins, and the Genesis of Human-Specific Transcription Networks.

Transposable elements (TEs) may account for up to two-thirds of the human genome, and as genomic threats they are subjected to epigenetic control mechanisms engaged from the earliest stages of embryonic development. We previously determined that an important component of this process is the sequence-specific recognition of TEs by KRAB (Krüppel-associated box)-containing zinc-finger proteins (KRAB-ZFPs), a large family of tetrapod-restricted transcription factors that act by recruiting inducers of heterochromatin formation and DNA methylation. We further showed that KRAB-ZFPs and their cofactor KAP1 exert a marked influence on the transcription dynamics of embryonic stem cells via their docking of repressor complexes at TE-contained regulatory sequences. It is generally held that, beyond this early embryonic period, TEs become permanently silenced, and that the evolutionary selection of KRAB-ZFPs and other TE controllers is the result of a simple evolutionary arms race between the host and these genetics invaders. Here, I discuss recent evidence that invalidates this dual assumption and instead suggests that KRAB-ZFPs are the instruments of a massive enterprise of TE domestication, whereby transposon-based regulatory sequences and their cellular ligands establish species-specific transcription regulation networks that influence multiple aspects of human development and physiology.

Zinc Fingers Function Cooperatively with KRAB Domain for Nuclear Localization of KRAB-Containing Zinc Finger Proteins

Multiple nuclear localization domains have been identified in nuclear proteins, and they finely control nuclear import and functions of those proteins. ZNF268 is a typical KRAB-containing zinc finger protein (KRAB-ZFP), and previous studies have shown that the KRAB domain reinforces nuclear localization of KRAB-ZFPs by interacting with KAP1. In this study, we find that some of 24 zinc fingers of ZNF268 also possess nuclear localization activity. Results of mutagenesis studies suggest that KRAB and zinc fingers are both necessary, and they function both independently and cooperatively for the nuclear localization of ZNF268. However, the subnuclear targeting activities of KRAB and zinc fingers are different. KRAB targets proteins in nucleoplasm, but not in the nucleolus, which is mediated by interaction with KAP1, while zinc fingers target proteins in the whole nucleus uniformly. The cooperative activities of KAP1-KRAB-zinc fingers result in the precise nucleoplasmic, but not nucleolar localization of KRAB-ZFPs. Our studies reveal a novel mechanism for the subcellular localization of KRAB-ZFPs and may help us to further explore their biological functions.

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

KAP1/TRIM28/TIF1β 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.

Abstract 197: The KRAB-containing zinc finger protein ZNF545 is a functional tumor suppressor exerting proapoptotic and antiproliferation abilities with frequent epigenetic inactivation in multiple carcinomas

Abstract Epigenetic silencing of tumor suppressor gene is frequently involved in tumor development. Some zinc finger proteins (ZFPs) involved in tumorigenesis are down-regulated through CpG methylation in multiple carcinomas. Here, through genome wide-screening, we identified a novel KRAB domain-containing zinc finger protein (KRAB-ZFP), ZNF545, downregulated in multiple tumor cell lines including nasopharyngeal, esophageal, colon, stomach, liver, kidney, breast, cervix, and prostate carcinomas, in contrast to its broad expression in normal tissues. The promoter of ZNF545 contains a typical CpG island and is frequently methylated in multiple carcinoma cell lines. Transcriptional silencing of ZNF545 could be reversed by pharmacologic demethylation or genetic demethylation, indicating a direct epigenetic silencing mechanism. ZNF545 was also frequently methylated in primary tumors of NPC and ESCC, but less frequently in normal epithelial tissues and paired surgical marginal normal tissues. ZNF545 was found to be a transcriptional repressor when fused to Gal4-binding domain, as determined by the luciferase assay. Ectopic expression of ZNF545 in silenced cancer cell lines significantly inhibited their colony formation, proliferation and induced apoptosis, indicating that ZNF545 can function as a tumor suppressor. ZNF545 also can inhibit the activity of NF-KappaB and AP-1, which play an important role in tumorigenesis. We further found that ZNF545 was located in the nucleus and sequestered in the nucleolus in some cells. ZNF545 could co-localize with KAP1 and heterochromatin protein, so we proposed that ZNF545 could inhibit tumorigenesis, at least partially through downregulating the transcription of target genes, or regulating nucleolus function such as ribosome biogenesis. The tumor specific-silencing of ZNF545 could serve as an epigenetic biomarker for common cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 197.

The zinc finger DNA-binding domain of K-RBP plays an important role in regulating Kaposi's sarcoma-associated herpesvirus RTA-mediated gene expression

K-RBP is a KRAB-containing zinc finger protein with multiple zinc finger motifs and represses Kaposi's sarcoma-associated herpesvirus (KSHV) transactivator RTA-mediated transactivation of several viral lytic gene promoters, including the ORF57 promoter. Whether K-RBP binds DNA through its zinc fingers and the role of zinc finger domain in repressing gene expression are unclear. Here we report that K-RBP binds DNA through its zinc finger domain and the target DNA sequences contain high GC content. Furthermore, K-RBP binds to KSHV ORF57 promoter, which contains a GC-rich motif. K-RBP suppresses the basal ORF57 promoter activity as well as RTA-mediated activation. The zinc finger domain of K-RBP is sufficient for the suppression of ORF57 promoter activation mediated by the viral transactivator RTA. Finally, we show that K-RBP inhibits RTA binding to ORF57 promoter. These findings suggest that the DNA-binding activity of K-RBP plays an important role in repressing viral promoter activity.

ZNF23 induces apoptosis in human ovarian cancer cells

We recently reported that the level of ZNF23, a KRAB-containing zinc finger protein, is reduced in human cancers and it inhibits cell growth by inducing cell cycle arrest. Here we showed that ZNF23 also induces apoptosis in ovarian cancer cells. The protein level of ZNF23 in ovarian cancers was greatly down-regulated compared with that in the normal ovaries. Introduction of ZNF23 into ovarian cancer cells led to apoptosis as demonstrated by activation of caspase-3, nuclear condensation and formation of a sub-G1 peak. This apoptotic process was correlated with loss in mitochondrial membrane potential, cytochrome c release and caspase-9 activation. Furthermore, ZNF23 induced apoptosis partially via down-regulation of Bcl-X L. Thus, our results suggest that ZNF23 may also induce apoptosis to suppress tumor cell growth and points to the possibility that its down-regulation might facilitate ovarian cancer cell survival.

Dynamic and selective interactions of the transcriptional corepressor TIF1β with the heterochromatin protein HP1 isotypes during cell differentiation

Cell differentiation is a multi-step process marked by progressive silencing of gene expression through mechanisms believed to involve heterochromatin. We have previously shown that interaction between the Krüppel associated box-containing zinc finger proteins (KRAB-ZFP) corepressor TIF1β and the heterochromatin proteins HP1 is essential for progression through differentiation of embryonal carcinoma F9 cells. This analysis clearly demonstrated the link between gene specific repressors, components of heterochromatin and cell differentiation. In mammals, there are three HP1 isotypes, HP1α, β, and γ, that appear to be involved in both eu- and heterochromatin, but whose individual functions are still poorly defined. Therefore, the aim of the present study was to determine in vivo (i) which HP1 isotypes interact with TIF1β, (ii) in which sub-nuclear compartments these interactions occur and (iii) whether these interactions are regulated during cell differentiation. To address these questions, we established stable F9 cell lines co-expressing TIF1β fused to the ECFP fluorophore and HP1α, β, or γ fused to the EYFP fluorophore. Using the Föster resonance energy transfer (FRET) technology, we map the physical interaction between TIF1β-CFP and the different HP1-YFP isotypes in living F9 cells. We demonstrate that in non-differentiated cells, TIF1β-CFP/HP1-YFP interaction occurs only within euchromatin and involves selectively HP1β-YFP and HP1γ-YFP, but not HP1α-YFP. Furthermore, in differentiated cells, TIF1β-CFP selectively associates with HP1β-YFP within heterochromatin, while TIF1β-CFP/HP1γ-YFP is exclusively present within euchromatin. No physical TIF1β-CFP/HP1α-YFP interaction is detected in neither non differentiated nor differentiated cells. These results support the notion that, in vivo, HP1 isotypes have specific nonredundant functions and provide evidence for HP1β playing an essential role in the shuttling of TIF1β from eu- to heterochromatin during cell differentiation.

The Transcriptional Repressor K-RBP Modulates RTA-Mediated Transactivation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus

The replication and transcription activator (RTA) protein of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV)/human herpesvirus 8 functions as the key regulator to induce KSHV lytic replication from latency through activation of the lytic cascade of KSHV. Elucidation of the host factors involved in RTA-mediated transcriptional activation is pivotal for understanding the transition between viral latency and lytic replication. KSHV-RTA binding protein (K-RBP) was previously isolated as a cellular RTA binding protein of unknown function. Sequence analysis showed that K-RBP contains a Kruppel-associated box (KRAB) at the N terminus and 12 adjacent zinc finger motifs. In similarity to other KRAB-containing zinc finger proteins, K-RBP is a transcriptional repressor. Mutational analysis revealed that the KRAB domain is responsible for the transcriptional suppression activity of this protein and that the repression is histone deacetylase independent. K-RBP was found to repress RTA-mediated transactivation and interact with TIF1beta (transcription intermediary factor 1beta), a common corepressor of KRAB-containing protein, to synergize with K-RBP in repression. Overexpression and knockdown experiment results suggest that K-RBP is a suppressor of RTA-mediated KSHV reactivation. Our findings suggest that the KRAB-containing zinc finger protein K-RBP can suppress RTA-mediated transactivation and KSHV lytic replication and that KSHV utilizes this protein as a regulator to maintain a balance between latency and lytic replication.

Orphan Nuclear Receptor Nur77 Induces Zinc Finger Protein GIOT-1 Gene Expression, and GIOT-1 Acts as a Novel Corepressor of Orphan Nuclear Receptor SF-1 via Recruitment of HDAC2

Kruppel-associated box (KRAB) domain-containing proteins consist of potential transcriptional repression modules. Previously, gonadotropin-inducible ovarian transcription factor-1 (GIOT-1) was identified as a novel KRAB-containing zinc finger protein and shown to have transcriptional repression activity. Here, we demonstrate that orphan nuclear receptor Nur77 regulates GIOT-1 gene expression in testicular Leydig cell lines and that GIOT-1 acts as a novel corepressor of the orphan nuclear receptor steroidogenic factor 1 (SF-1). Mutation analysis of the GIOT-1 promoter and overexpression analysis of dominant-negative Nur77 revealed that luteinizing hormone activates GIOT-1 gene expression through Nur77. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that Nur77 directly binds to the GIOT-1 promoter. GIOT-1 represses the SF-1 transactivation, and specific interaction between GIOT-1 and SF-1 was observed. We also demonstrate an interaction between GIOT-1 and histone deacetylase 2 (HDAC2). GIOT-1-mediated transrepression was recovered by down-regulation of HDAC2 expression with small interfering RNA of HDAC2. Knock down of the endogenous GIOT-1 results in significant enhancement of CYP17 expression in Leydig cells. In conclusion, this study of cross-talk between GIOT-1 and orphan nuclear receptors will provide new insights into the role of KRAB-containing zinc finger proteins in nuclear receptor action.

ZNF569, a novel KRAB-containing zinc finger protein, suppresses MAPK signaling pathway

Transcription factors play an essential role in altering gene expression. A great progress about transcription factors has been made towards the understanding of normal physiological processes, embryonic development, and human diseases. Here we report the identification and characterization of a novel KRAB-containing zinc-finger protein, ZNF569, which is isolated from a human embryonic heart cDNA library. ZNF569 encodes a putative protein of 686 amino acids. The protein is conserved across different species during evolution. Expression of ZNF569 was found in most of the examined human adult and embryonic tissues with a higher level in heart and skeletal muscles. The KRAB and ZNF motifs of ZNF569 represent potent repression domains. When ZNF569 is fused to Gal-4 DNA-binding domain and co-transfected with VP-16, ZNF569 protein suppresses transcriptional activity. Overexpression of ZNF569 in COS-7 cells inhibited the transcriptional activities of SRE and AP-1, which may be silenced by siRNA. The results suggest that ZNF569 protein may act as a transcriptional repressor that suppresses MAPK signaling pathway to mediate cellular functions.