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1.  A Novel Zinc Finger Protein 219-like (ZNF219L) is Involved in the Regulation of Collagen Type 2 Alpha 1a (col2a1a) Gene Expression in Zebrafish Notochord 
The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.
PMCID: PMC3805895  PMID: 24155663
zinc finger protein 219; notochord; zebrafish; collagen type 2 alpha 1a; transcriptional regulation.
2.  Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration 
Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulates diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis, which is the main risk factor for hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in human HCC cells and tissue specimens and found a significant up-regulation compared to primary human hepatocytes and corresponding non-tumorous liver tissue. Over-expression of the transcription factor Ets-1 further enhanced ZNF267 expression, and reporter gene assays revealed that mutation of the Ets-1 binding site to the ZNF267 promotor markedly inhibited ZNF267 promotor activity. Hypoxic conditions induced Ets-1 in HCC cells via HIF1alpha activation, and hypoxia induced ZNF267 expression while HIF1alpha inhibition significantly reduced both hypoxia-induced as well as basal ZNF267 expression in HCC cells. It is known that hypoxic conditions in tumorous tissues induce the formation of reactive oxygen species (ROS), and ROS have been identified as important factor in the regulation of Ets-1 expression in tumor cells. Here, we found that ROS induction induced and ROS scavenging reduced ZNF267 expression in HCC cells, respectively. Loss and gain of function analysis applying siRNA directed against ZNF267 or transient transfection revealed that ZNF267 promotes proliferation and migration of HCC cells in vitro. These findings indicate Ets-1 and HIF1alpha as critical regulators of basal and hypoxia- or ROS-induced ZNF267 expression in HCC, and further suggest that the pro-tumorigenic effect of these factors is at least in part mediated via increased ZNF267 expression in HCC. Since ZNF267 is already elevated in cirrhosis, ZNF267 appears as promising target for both prevention as well as treatment of HCC in patients with chronic liver disease.
PMCID: PMC3342774  PMID: 21840307
Hepatocellular carcinoma; ZNF267; Kruppel-like factor
3.  A KRAS-directed transcriptional silencing pathway that mediates the CpG island methylator phenotype 
eLife  2014;3:e02313.
Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in, and the mechanistic basis of, CIMP is not understood. Among the CIMP genes are the tumor suppressors p14ARF, p15INK4B, and p16INK4A, encoded by the INK4-ARF locus. In this study, we perform an RNA interference screen and identify ZNF304, a zinc-finger DNA-binding protein, as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors, ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1, resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304, which drives DNA binding. Finally, we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells.
eLife digest
Colorectal cancer, which affects the large intestine, is a leading cause of cancer deaths worldwide, ranking fourth after cancers of the lung, stomach, and liver. Like these other cancers, this disease is caused by mutations to genes that allow cells to multiply in an out of control manner. Mutations that change the gene encoding a protein called KRAS are found in many different types of cancer. Moreover, about 70% of colorectal cancers with a KRAS mutation also have an excess of small chemical marks on other genes, some of which are known to suppress the growth of tumors. These marks ‘switch off’ these genes, and although the identities of the enzymes that typically leave these marks on DNA are known, the link between these enzymes and the KRAS protein is unknown.
Now Serra, Fang et al. have identified a protein, called ZNF304, that is required by KRAS to switch off a large number of genes, including multiple tumor suppressors. In the absence of ZNF304, these tumor suppressor genes remained switched on in cancer cells with the KRAS mutation, so the growth of the tumor was slowed down. ZNF304 is a protein that binds to stretches of DNA, including regions of DNA at the start of several tumor suppressor genes, and it recruits the enzymes that add the chemical marks that switch off these genes.
Serra, Fang et al. found that the levels of ZNF304 protein were elevated in colorectal cancer cells with the mutated KRAS, and showed that this was due to the combined activities of two other proteins that prevented ZNF304 from being broken down in the cell. Mutant KRAS caused an increase in the levels of these two proteins, which in turn caused the elevated ZNF304 levels and the excessive marking of the DNA in the tumor suppressor genes.
Furthermore, some of these same tumor suppressor genes are switched off in the earliest cells in a human embryo—which have the potential to become any of 200 or so cell types in the human body. In these embryonic stem cells, Serra, Fang et al. showed that ZNF304, but not KRAS, was also involved in keeping these genes switched off until the stem cells started changing into specific types of cells.
Since they are a crucial part of the pathway linking a cancer-causing mutation to increased tumor growth, the proteins identified by Serra, Fang et al. could represent promising targets for the development of new anti-cancer drugs.
PMCID: PMC3949416  PMID: 24623306
CpG island methylator phenotype; INK4-ARF; colorectal cancer; ZNF304; KRAS; DNMT1; human; mouse
4.  A transcript profiling approach reveals the zinc finger transcription factor ZNF191 is a pleiotropic factor 
BMC Genomics  2009;10:241.
The human zinc finger protein 191 (ZNF191) is a member of the SCAN domain family of Krüppel-like zinc finger transcription factors. ZNF191 shows 94% identity to its mouse homologue zinc finger protein 191(Zfp191), which is the most highly conserved among the human-mouse SCAN family member orthologues pairs. Zfp191 is widely expressed during early embryogenesis and in adult organs. Moreover, Zfp191-/- embryos have been shown to be severely retarded in development and die approximately at embryonic day E7.5. ZNF191 can specifically interact with the widespread TCAT motif which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. In addition, ZNF191 displays a suppressive effect on the transcription; however, little downstream targets have identified.
We searched for ZNF191 target genes by using a transient overexpression and knockdown strategy in the human embryo kidney (HEK293) cells. Microarray analyses identified 6094 genes modulated by overexpression of ZNF191 and 3332 genes regulated by knockdown of ZNF191, using a threshold of 1.2-fold. Several interested candidate genes, validated by real time RT-PCR, were correlated well with the array data. Interestingly, 1456 genes were identified in both transient overexpression and transient knockdown strategies. The GenMAPP and MappFinder software packages were further used for pathway analysis of these significantly altered genes. Several gene pathways were found to be involved in processes of the regulation of kinase activity, transcription, angiogenesis, brain development and response to DNA damage.
Our analysis reveals for the first time that ZNF191 is a pleiotropic factor that has a role in hematopoiesis, brain development and cancers.
PMCID: PMC2694838  PMID: 19463170
5.  Genome-wide evidence for an essential role of the human Staf/ZNF143 transcription factor in bidirectional transcription 
Nucleic Acids Research  2010;39(8):3116-3127.
In the human genome, ∼10% of the genes are arranged head to head so that their transcription start sites reside within <1 kbp on opposite strands. In this configuration, a bidirectional promoter generally drives expression of the two genes. How bidirectional expression is performed from these particular promoters constitutes a puzzling question. Here, by a combination of in silico and biochemical approaches, we demonstrate that hStaf/ZNF143 is involved in controlling expression from a subset of divergent gene pairs. The binding sites for hStaf/ZNF143 (SBS) are overrepresented in bidirectional versus unidirectional promoters. Chromatin immunoprecipitation assays with a significant set of bidirectional promoters containing putative SBS revealed that 93% of them are associated with hStaf/ZNF143. Expression of dual reporter genes directed by bidirectional promoters are dependent on the SBS integrity and requires hStaf/ZNF143. Furthermore, in some cases, functional SBS are located in bidirectional promoters of gene pairs encoding a noncoding RNA and a protein gene. Remarkably, hStaf/ZNF143 per se exhibits an inherently bidirectional transcription activity, and together our data provide the demonstration that hStaf/ZNF143 is indeed a transcription factor controlling the expression of divergent protein–protein and protein–non-coding RNA gene pairs.
PMCID: PMC3082894  PMID: 21177654
6.  The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues 
BMC Genomics  2010;11:206.
Expansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation.
Here, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities.
The ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny.
PMCID: PMC2865497  PMID: 20346131
7.  ZNF143 transcription factor mediates cell survival through upregulation of the GPX1 activity in the mitochondrial respiratory dysfunction 
Cell Death & Disease  2012;3(11):e422-.
Mitochondrial respiratory dysfunction has intimate relationship with redox regulation. The key mechanism about how the mitochondrial respiration-defective cells survive oxidative stress is still elusive. Here, we report that transcription factor zinc-finger protein 143 (ZNF143) expression and glutathione peroxidase (GPX) activity are markedly increased in the mitochondrial respiratory-defective cells induced by dominant-negative DNA polymerase γ (POLGdn). In this work, investigation of the cellular antioxidant glutathione (GSH) and enzyme GPX activity in the mitochondrial dysfunction revealed the presence of an increased synthesis of GSH through the activation of GCLC (glutamate–cysteine ligase catalytic subunit) and GCLM (glutamate–cysteine ligase regulatory subunit) gene expression, and also a positive upregulation of glutathione peroxidase 1 (GPX1) activity by the transcription factor ZNF143. Significant increase in gene expression of SepSecS, the key enzyme responsible for selenocysteine transfer RNA (tRNA) synthesis, further confirmed the activation of the selenocysteine synthesis pathway. By using both GPX1 and ZNF143 knockdown, we provided insight into the involvement of ZNF143 in promoting GPX1 activity and protecting cells from oxidative damage and cisplatin treatment in the mitochondrial dysfunction. Furthermore, we reported the possible regulation of mitochondrial transcription factor A (TFAM) in the mitochondrial dysfunction. Our findings delineate an important antioxidant survival pathway that allows the mitochondrial-defective cells to survive oxidative stress and cisplatin treatment.
PMCID: PMC3542592  PMID: 23152058
ZNF143; mitochondrial dysfunction; GPX1; cisplatin; ROS
8.  Knockdown of ZNF268, which Is Transcriptionally Downregulated by GATA-1, Promotes Proliferation of K562 Cells 
PLoS ONE  2012;7(1):e29518.
The human ZNF268 gene encodes a typical KRAB-C2H2 zinc finger protein that may participate in hematopoiesis and leukemogenesis. A recent microarray study revealed that ZNF268 expression continuously decreases during erythropoiesis. However, the molecular mechanisms underlying regulation of ZNF268 during hematopoiesis are not well understood. Here we found that GATA-1, a master regulator of erythropoiesis, repressed the promoter activity and transcription of ZNF268. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that GATA-1 directly bound to a GATA binding site in the ZNF268 promoter in vitro and in vivo. Knockdown of ZNF268 in K562 erythroleukemia cells with specific siRNA accelerated cellular proliferation, suppressed apoptosis, and reduced expression of erythroid-specific developmental markers. It also promoted growth of subcutaneous K562-derived tumors in nude mice. These results suggest that ZNF268 is a crucial downstream target and effector of GATA-1. They also suggest the downregulation of ZNF268 by GATA-1 is important in promoting the growth and suppressing the differentiation of K562 erythroleukemia cells.
PMCID: PMC3250450  PMID: 22235304
9.  Proteins ZNF198 and SUZ12 are down-regulated in Hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication 
Hepatology (Baltimore, Md.)  2011;53(4):1137-1147.
Chronic Hepatitis B Virus (HBV) infection is a major etiologic factor in hepatocellular carcinoma (HCC) pathogenesis, involving effects of chronic liver inflammation and of the weakly oncogenic HBV X protein (pX). pX-mediated hepatocyte transformation requires Polo-like kinase1 (Plk1) activity, but the mechanism is not fully understood. We identified by a genome-wide shRNA library screen the genes ZNF198 and SUZ12 whose protein depletion rescues pX-expressing cells from DNA damage-induced apoptosis. ZNF198 and SUZ12 are components of chromatin remodeling complexes and associate with promyelocytic leukemia (PML) nuclear bodies. Knockdown of ZNF198 and SUZ12 by siRNA reduced p53 stability and DNA repair, rescued pX-expressing hepatocytes from DNA damage-induced apoptosis, and increased pX-induced polyploidy and oncogenic transformation, suggesting ZNF198 and SUZ12 have a role in pX-mediated transformation. Interestingly, during pX-mediated transformation the protein but not mRNA levels of ZNF198 and SUZ12 progressively decreased, while Plk1 levels increased. Inhibition of Plk1 activity restored protein levels of ZNF198 and SUZ12. In addition, transfected Polo-box-domain (PBD) of Plk1 co-immunoprecipitated with ZNF198 and SUZ12, suggesting these proteins are Plk1 substrates. Elevated Plk1 and reduced protein levels of ZNF198 and SUZ12 were also observed in human liver cancer cell lines derived from HBV-related tumors and in the presence of HBV replication. Importantly, knockdown by siRNA of ZNF198 and SUZ12 enhanced HBV replication.
Reduced protein levels of ZNF198 and SUZ12 and elevated Plk1 occur during pX-mediated hepatocyte transformation, in human liver cancer cell lines, as well as during HBV replication, underscoring the significance of these genes both in HBV-mediated HCC pathogenesis and HBV replication. We propose Plk1 activity down-regulates ZNF198 and SUZ12 thereby enhancing both HBV replication and pX-mediated oncogenic transformation.
PMCID: PMC3079326  PMID: 21480320
Hepatitis B Virus X protein; hepatocellular carcinoma; HBV replication; SUZ12; ZNF198; p53; Polo-like kinase 1
10.  Identification of a recurrent transforming UBR5–ZNF423 fusion gene in EBV-associated nasopharyngeal carcinoma 
The Journal of Pathology  2013;231(2):158-167.
Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is prevalent in southern China, south-east Asia and northern Africa. The development and stepwise progression of NPC involves accumulation of multiple gross genetic changes during the clonal expansion of Epstein–Barr virus (EBV)-infected nasopharyngeal epithelial cell population. Here, using paired-end whole-transcriptome sequencing, we discovered a number of chimeric fusion transcripts in a panel of EBV-positive tumour lines. Among these transcripts, a novel fusion of ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and zinc finger protein 423 (ZNF423) on 16q12.1, identified from the NPC cell line C666-1, was recurrently detected in 12/144 (8.3%) of primary tumours. The fusion gene contains exon 1 of UBR5 and exons 7–9 of ZNF423 and produces a 94 amino acid chimeric protein including the original C-terminal EBF binding domain (ZF29-30) of ZNF423. Notably, the growth of NPC cells with UBR5–ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5–ZNF423 by fusion-specific siRNA significantly inhibited the cell proliferation and colony-forming ability of C666-1 cells. The transforming ability of UBR5–ZNF423 fusion was also confirmed in NIH3T3 fibroblasts. Constitutive expression of UBR5–ZNF423 in NIH3T3 fibroblasts significantly enhanced its anchorage-independent growth in soft agar and induced tumour formation in a nude mouse model. These findings suggest that expression of UBR5–ZNF423 protein might contribute to the transformation of a subset of NPCs, possibly by altering the activity of EBFs (early B cell factors). Identification of the oncogenic UBR5–ZNF423 provides new potential opportunities for therapeutic intervention in NPC. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
PMCID: PMC4166696  PMID: 23878065
UBR5–ZNF423 fusion; transcriptome sequencing; nasopharyngeal carcinoma; oncogene; gene rearrangement; Epstein–Barr virus
11.  Modulation of gene expression via overlapping binding sites exerted by ZNF143, Notch1 and THAP11 
Nucleic Acids Research  2013;41(7):4000-4014.
ZNF143 is a zinc-finger protein involved in the transcriptional regulation of both coding and non-coding genes from polymerase II and III promoters. Our study deciphers the genome-wide regulatory role of ZNF143 in relation with the two previously unrelated transcription factors Notch1/ICN1 and thanatos-associated protein 11 (THAP11) in several human and murine cells. We show that two distinct motifs, SBS1 and SBS2, are associated to ZNF143-binding events in promoters of >3000 genes. Without co-occupation, these sites are also bound by Notch1/ICN1 in T-lymphoblastic leukaemia cells as well as by THAP11, a factor involved in self-renewal of embryonic stem cells. We present evidence that ICN1 binding overlaps with ZNF143 binding events at the SBS1 and SBS2 motifs, whereas the overlap occurs only at SBS2 for THAP11. We demonstrate that the three factors modulate expression of common target genes through the mutually exclusive occupation of overlapping binding sites. The model we propose predicts that the binding competition between the three factors controls biological processes such as rapid cell growth of both neoplastic and stem cells. Overall, our study establishes a novel relationship between ZNF143, THAP11 and ICN1 and reveals important insights into ZNF143-mediated gene regulation.
PMCID: PMC3627581  PMID: 23408857
12.  The paracrine effect of exogenous growth hormone alleviates dysmorphogenesis caused by tbx5 deficiency in zebrafish (Danio rerio) embryos 
Dysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome.
Using an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5 morphants, we demonstrated that GH and some GH-related genes were markedly downregulated. Zebrafish embryos microinjected with tbx5-morpholino (MO) antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group.
The attenuating effects of GH in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors (erk1, akt2), and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes (amhc, vmhc, and cmlc2).
Based on our results, tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner.
PMCID: PMC3407474  PMID: 22776023
tbx5; Growth hormone; Apoptosis; Embryogenesis; Zebrafish
13.  Sequencing of Pax6 Loci from the Elephant Shark Reveals a Family of Pax6 Genes in Vertebrate Genomes, Forged by Ancient Duplications and Divergences 
PLoS Genetics  2013;9(1):e1003177.
Pax6 is a developmental control gene essential for eye development throughout the animal kingdom. In addition, Pax6 plays key roles in other parts of the CNS, olfactory system, and pancreas. In mammals a single Pax6 gene encoding multiple isoforms delivers these pleiotropic functions. Here we provide evidence that the genomes of many other vertebrate species contain multiple Pax6 loci. We sequenced Pax6-containing BACs from the cartilaginous elephant shark (Callorhinchus milii) and found two distinct Pax6 loci. Pax6.1 is highly similar to mammalian Pax6, while Pax6.2 encodes a paired-less Pax6. Using synteny relationships, we identify homologs of this novel paired-less Pax6.2 gene in lizard and in frog, as well as in zebrafish and in other teleosts. In zebrafish two full-length Pax6 duplicates were known previously, originating from the fish-specific genome duplication (FSGD) and expressed in divergent patterns due to paralog-specific loss of cis-elements. We show that teleosts other than zebrafish also maintain duplicate full-length Pax6 loci, but differences in gene and regulatory domain structure suggest that these Pax6 paralogs originate from a more ancient duplication event and are hence renamed as Pax6.3. Sequence comparisons between mammalian and elephant shark Pax6.1 loci highlight the presence of short- and long-range conserved noncoding elements (CNEs). Functional analysis demonstrates the ancient role of long-range enhancers for Pax6 transcription. We show that the paired-less Pax6.2 ortholog in zebrafish is expressed specifically in the developing retina. Transgenic analysis of elephant shark and zebrafish Pax6.2 CNEs with homology to the mouse NRE/Pα internal promoter revealed highly specific retinal expression. Finally, morpholino depletion of zebrafish Pax6.2 resulted in a “small eye” phenotype, supporting a role in retinal development. In summary, our study reveals that the pleiotropic functions of Pax6 in vertebrates are served by a divergent family of Pax6 genes, forged by ancient duplication events and by independent, lineage-specific gene losses.
Author Summary
Pax6 is a highly conserved transcription factor with key roles in eye, brain, pancreas, and olfactory system development. In mammals multiple Pax6 isoforms are encoded by a single Pax6 gene, embedded within a complex regulatory landscape. Here we provide evidence for the presence of multiple Pax6 loci in other vertebrate species. We show that two Pax6 genes (Pax6.1 and Pax6.2) are present in the genome of elephant shark (a cartilaginous fish). Pax6.1 is highly similar to mammalian Pax6 in terms of structure of the gene locus, protein sequence, and expression pattern; whereas the second gene, Pax6.2, codes for a protein lacking the paired domain. We identify orthologs of Pax6.2 in other vertebrate genomes, such as lizard, Xenopus, and teleost fishes, and show it is important for eye development in zebrafish. Additionally, we have characterised a third Pax6 (Pax6.3) present only in some teleost fishes. Phylogenetic analyses indicate that the evolutionary history of the Pax6 gene family in vertebrates is a result of ancient duplications followed by independent gene losses in different lineages. Sequence comparison of the cis-regulatory landscapes around the genes has led to the identification of novel Pax6 enhancers that provide a link between the diverged Pax6 family members.
PMCID: PMC3554528  PMID: 23359656
14.  The Hypoxia-Inducible Transcription Factor ZNF395 Is Controlled by IĸB Kinase-Signaling and Activates Genes Involved in the Innate Immune Response and Cancer 
PLoS ONE  2013;8(9):e74911.
Activation of the hypoxia inducible transcription factor HIF and the NF-ĸB pathway promotes inflammation-mediated tumor progression. The cellular transcription factor ZNF395 has repeatedly been found overexpressed in various human cancers, particularly in response to hypoxia, implying a functional relevance. To understand the biological activity of ZNF395, we identified target genes of ZNF395 through a genome-wide expression screen. Induced ZNF395 expression led to the upregulation of genes known to play a role in cancer as well as a subset of interferon (IFN)-stimulated genes (ISG) involved in antiviral responses such as IFIT1/ISG56, IFI44 and IFI16. In cells that lack ZNF395, the IFN-α-mediated stimulation of these factors was impaired, demonstrating that ZNF395 is required for the full induction of these antiviral genes. Transient transfections revealed that ZNF395-mediated activation of the IFIT1/ISG56 promoter depends on the two IFN-stimulated response elements within the promoter and on the DNA-binding domain of ZNF395, a so-called C-clamp. We also show that IĸBα kinase (IKK)-signaling is necessary to allow ZNF395 to activate transcription and simultaneously enhances its proteolytic degradation. Thus, ZNF395 becomes activated at the level of protein modification by IKK. Moreover, we confirm that the expression of ZNF395 is induced by hypoxia. Our results characterize ZNF395 as a novel factor that contributes to the maximal stimulation of a subset of ISGs. This transcriptional activity depends on IKK signaling further supporting a role of ZNF395 in the innate immune response. Given these results it is possible that under hypoxic conditions, elevated levels of ZNF395 may support inflammation and cancer progression by activating the target genes involved in the innate immune response and cancer.
PMCID: PMC3781154  PMID: 24086395
15.  ZNF667/Mipu1 Is a Novel Anti-Apoptotic Factor That Directly Regulates the Expression of the Rat Bax Gene in H9c2 Cells 
PLoS ONE  2014;9(11):e111653.
ZNF667/Mipu1, a C2H2-type zinc finger transcription factor, was suggested to play an important role in oxidative stress. However, none of the target genes or potential roles of ZNF667 in cardiomyocytes have been elucidated. Here, we investigated the functional role of ZNF667 in H9c2 cell lines focusing on its molecular mechanism by which it protects the cells from apoptosis. We found that ZNF667 inhibited the expression and the promoter activity of the rat proapoptotic gene Bax gene, and at the same time prevented apoptosis of H9c2 cells, induced by H2O2 and Dox. Western immunoblotting analysis revealed that ZNF667 also inhibited Bax protein expression, accompanied by attenuation of the mitochondrial translocation of Bax protein, induced by H2O2. EMSA and target detection assay showed that the purified ZNF667 fusion proteins could interact with the Bax promoter sequence in vitro, and this interaction was dependent upon the ZNF667 DNA binding sequences or its core sequence in the promoter. Furthermore, ChIP assay demonstrated that a stimulus H2O2 could enhance the ability of ZNF667 protein binding to the promoter. Finally, a reporter gene assay showed that ZNF667 could repress the activity of the Bax gene promoter, and the repression was dependent upon its binding to the specific DNA sequence in the promoter. Our work demonstrates that ZNF667 that confers cytoprotection is a novel regulator of the rat Bax gene, mediating the inhibition of the Bax mRNA and protein expression in H9c2 cardiomyocytes in response to H2O2 treatment.
PMCID: PMC4232351  PMID: 25397408
16.  Global analysis of ZNF217 chromatin occupancy in the breast cancer cell genome reveals an association with ERalpha 
BMC Genomics  2014;15(1):520.
The ZNF217 gene, encoding a C2H2 zinc finger protein, is located at 20q13 and found amplified and overexpressed in greater than 20% of breast tumors. Current studies indicate ZNF217 drives tumorigenesis, yet the regulatory mechanisms of ZNF217 are largely unknown. Because ZNF217 associates with chromatin modifying enzymes, we postulate that ZNF217 functions to regulate specific gene signaling networks. Here, we present a large-scale functional genomic analysis of ZNF217, which provides insights into the regulatory role of ZNF217 in MCF7 breast cancer cells.
ChIP-seq analysis reveals that the majority of ZNF217 binding sites are located at distal regulatory regions associated with the chromatin marks H3K27ac and H3K4me1. Analysis of ChIP-seq transcription factor binding sites shows clustering of ZNF217 with FOXA1, GATA3 and ERalpha binding sites, supported by the enrichment of corresponding motifs for the ERalpha-associated cis-regulatory sequences. ERalpha expression highly correlates with ZNF217 in lysates from breast tumors (n = 15), and ERalpha co-precipitates ZNF217 and its binding partner CtBP2 from nuclear extracts. Transcriptome profiling following ZNF217 depletion identifies differentially expressed genes co-bound by ZNF217 and ERalpha; gene ontology suggests a role for ZNF217-ERalpha in expression programs associated with ER+ breast cancer studies found in the Molecular Signature Database. Data-mining of expression data from breast cancer patients correlates ZNF217 with reduced overall survival.
Our genome-wide ZNF217 data suggests a functional role for ZNF217 at ERalpha target genes. Future studies will investigate whether ZNF217 expression contributes to aberrant ERalpha regulatory events in ER+ breast cancer and hormone resistance.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-520) contains supplementary material, which is available to authorized users.
PMCID: PMC4082627  PMID: 24962896
Breast cancer; ZNF217; ERalpha; GATA3; FOXA1; ChIP-seq; RNA-seq; Endocrine resistance
17.  Identification of genes directly regulated by the oncogene ZNF217 using ChIP-chip assays 
The Journal of biological chemistry  2007;282(13):9703-9712.
It has been proposed that ZNF217, which is amplified at 20q13 in various tumors, plays a key role during neoplastic transformation. ZNF217 has been purified in complexes that contain repressor proteins such as CtBP2, suggesting that it acts as a transcriptional repressor. However, the function of ZNF217 has not been well characterized due to a lack of known target genes. Using a global ChIP-chip approach, we have identified thousands of ZNF217 binding sites in three tumor cell lines (MCF7, SW480, and Ntera2). Further analysis of ZNF217 in Ntera2 cells has shown that many promoters are bound by ZNF217 and CtBP2, and that a subset of these promoters are activated upon removal of ZNF217. Thus, our in vivo studies corroborate the in vitro biochemical analyses of ZNF217-containing complexes and support the hypothesis that ZNF217 functions as a transcriptional repressor. Gene ontology analysis shows that ZNF217 targets in Ntera2 cells are involved in organ development, suggesting that one function of ZNF217 may be to repress differentiation. Accordingly, we show that differentiation of Ntera2 cells with retinoic acid leads to down-regulation of ZNF217. Our identification of thousands of ZNF217 target genes will enable further studies of the consequences of aberrant expression of ZNF217 during neoplastic transformation.
PMCID: PMC2269729  PMID: 17259635
18.  Nlz1/Znf703 acts as a repressor of transcription 
Members of the NET subfamily of zinc-finger proteins are related to the Sp-family of transcription factors and are required during embryogenesis. In particular, Nlz1/Znf703 and Nlz2/Znf503 are required for formation of rhombomere 4 of the vertebrate hindbrain. While NET family proteins have been hypothesized to regulate transcription, it remains unclear if they function as activators or repressors of transcription.
Here we demonstrate that Nlz proteins repress transcription both in cell lines and in developing zebrafish embryos. We first use standard cell culture-based reporter assays to demonstrate that Nlz1/Znf703 represses transcription of a luciferase reporter in four different cell lines. Structure-function analyses and pharmacological inhibition further reveal that Nlz1-mediated repression requires histone deacetylase activity. We next generate a stable transgenic zebrafish reporter line to demonstrate that Nlz1 promotes histone deacetylation at the transgenic promoter and repression of transgene expression during embryogenesis. Lastly, taking a genetic approach we find that endogenous Nlz proteins are required for formation of hindbrain rhombomere 4 during zebrafish embryogenesis by repressing expression of non-rhombomere 4 genes.
We conclude that Nlz1/Znf703 acts as a repressor of transcription and hypothesize that other NET family members function in a similar manner.
PMCID: PMC2588584  PMID: 19014486
19.  Targeted disruption of the mouse testis-enriched gene Znf230 does not affect spermatogenesis or fertility 
Genetics and Molecular Biology  2014;37(4):708-715.
The mouse testis-enriched Znf230 gene, which encodes a type of RING finger protein, is present primarily in the nuclei of spermatogonia, the acrosome and the tail of spermatozoa. To investigate the role of Znf230 in spermatogenesis, we generated Znf230-deficient mice by disrupting Znf230 exon-5 and exon-6 using homologous recombination. The homozygous Znf230-knockout (KO) mice did not exhibit Znf230 mRNA expression and Znf230 protein production. Znf230 KO mice exhibited no obvious impairment in body growth or fertility. Male Znf230 KO mice had integral reproductive systems and mature sperm that were regular in number and shape. The developmental stages of male germ cells of Znf230 KO mice were also normal. We further examined variations in the transcriptomes of testicular tissue between Znf230 KO and wild-type mice through microarray analysis. The results showed that the mRNA level of one unclassified transcript 4921513I08Rik was increased and that the mRNA levels of three other transcripts, i.e., 4930448A20Rik, 4931431B13Rik and potassium channel tetramerisation domain containing 14(Kctd14), were reduced more than two-fold in Znf230 KO mice compared with wild-type mice. Using our current examination techniques, these findings suggested that Znf230 deficiency in mice may not affect growth, fertility or spermatogenesis.
PMCID: PMC4261971  PMID: 25505846
Znf230; knockout mice; spermatogenesis; Kctd14
20.  Genome-Wide Analysis of KAP1 Binding Suggests Autoregulation of KRAB-ZNFs 
PLoS Genetics  2007;3(6):e89.
We performed a genome-scale chromatin immunoprecipitation (ChIP)-chip comparison of two modifications (trimethylation of lysine 9 [H3me3K9] and trimethylation of lysine 27 [H3me3K27]) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger (ZNF) genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the Polycomb repressive complex 2 is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the Polycomb repressive complex 2, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 corepressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5-kb promoter arrays. We found that a large number of genes of ZNF transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38-array tiling set, identifying ~7,000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 ZNFs, especially those containing Krüppel-associated box (KRAB) domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1.
Author Summary
Methylation of lysines 9 or 27 of histone H3 (H3me3K9 or H3me3K27, respectively) has been associated with silenced chromatin. However, a comprehensive comparison of the regions of the genome bound by these two types of modified histone H3 has not been performed. Therefore, we compared the binding patterns of H3me3K9 and H3me3K27 at ~26,000 human promoters in four different cell populations. Our studies indicated that the two marks segregate differentially with the two most common types of transcriptional regulators; H3me3K27 is highly enriched at homeobox genes and H3me3K9 is highly enriched at zinc-finger genes (ZNFs). We showed that many of the promoters bound by H3me3K9 are also bound by the corepressor KAP1. A genome-wide screen for KAP1 target genes revealed a difference in the location of KAP1 binding sites in the ZNF genes versus other targets. In general, KAP1 binding sites were localized to core promoter regions. However, KAP1 binding sites associated with ZNF genes are near the 3′ end of the coding region. Our results suggest that the KRAB-ZNF family members participate in an autoregulatory loop involving binding of the KAP1 protein to the 3′ end of the ZNF target genes, resulting in trimethylation of H3K9 and transcriptional repression.
PMCID: PMC1885280  PMID: 17542650
21.  Reduction of the rate of Protein Translation in Patients with Myotonic Dystrophy 2 
Myotonic Dystrophy 2 (DM2) is an autosomal dominant, multisystem disease, which primarily affects skeletal muscle. DM2 is caused by CCTGn expansion in the intron 1 of the ZNF9 gene. Expression of the mutant CCUGn RNA changes RNA processing in patients with DM2; however, the role of ZNF9 protein in DM2 pathology has been not elucidated. ZNF9 has been shown to regulate cap-dependent and cap-independent translation. We have examined a possible role of ZNF9 in the regulation of translation in DM2 patients. We have found that ZNF9 interacts with the 5′ UTRs of TOP (terminal oligopyrimidine tract) mRNAs encoding human ribosomal protein, RPS17, poly(A)-binding protein, PABP1, and elongation factors, eEF1A and eEF2. The binding activity of ZNF9 toward these TOP-containing 5′ UTRs is reduced in DM2 muscle. Consistent with the reduction of this activity, the levels of RPS17, PABP, eEF1A and eEF2 proteins are also diminished in DM2 muscle. The reduction of ZNF9 RNA-binding activity in DM2 correlates with a decrease of ZNF9 protein levels in cytoplasm of DM2 muscle cells. We have found that the reduction of ZNF9 is caused by expression of the mutant CCUG repeats. This decrease of proteins of translational apparatus in DM2 correlates with a reduction of a rate of protein synthesis in myoblasts from DM2 patients. We found that the ectopic expression of ZNF9 in DM2 myoblasts corrects rate of protein synthesis suggesting that the alterations in CCUG-ZNF9-TOP mRNAs pathway are responsible for the reduction of the rate of protein translation in DM2 muscle cells.
PMCID: PMC3610333  PMID: 19605641
myotonic dystrophy; CCUG repeats; ZNF9; TOP element; TOP mRNAs; rate of translation
22.  The scaRNA2 is produced by an independent transcription unit and its processing is directed by the encoding region 
Nucleic Acids Research  2009;38(2):370-381.
The C/D box scaRNA2 is predicted to guide specific 2′-O-methylation of U2 snRNA. In contrast to other SCARNA genes, SCARNA2 appears to be independently transcribed. By transient expression of SCARNA2-reporter gene constructs, we have demonstrated that this gene is transcribed by RNA polymerase II and that the promoter elements responsible for its transcription are contained within a 161 bp region upstream of the transcription start site. In mammals, we have identified four cross species conserved promoter elements, a TATA motif, an hStaf/ZNF143 binding site and two novel elements that are required for full promoter activity. Binding of the human hStaf/ZNF143 transcription factor to its target sequence is required for promoter activity, suggesting that hStaf/ZNF143 is a fundamental regulator of the SCARNA2 gene. We also showed that RNA polymerase II continues transcription past the 3′-end of the mature RNA, irrespective of the identity of the Pol II promoter. The 3′-end processing and accumulation are governed by the sole information contained in the scaRNA2 encoding region, the maturation occurring via a particular pathway incompatible with that of mRNA or snRNA production.
PMCID: PMC2811027  PMID: 19906720
23.  MAGE I Transcription Factors Regulate KAP1 and KRAB Domain Zinc Finger Transcription Factor Mediated Gene Repression 
PLoS ONE  2011;6(8):e23747.
Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells but are aberrantly expressed in many cancers. They have been shown to promote tumor survival, aggressive growth, and chemoresistance but the underlying mechanisms and MAGE I functions have not been fully elucidated. KRAB domain zinc finger transcription factors (KZNFs) are the largest group of vertebrate transcription factors and regulate neoplastic transformation, tumor suppression, cellular proliferation, and apoptosis. KZNFs bind the KAP1 protein and direct KAP1 to specific DNA sequences where it suppresses gene expression by inducing localized heterochromatin characterized by histone 3 lysine 9 trimethylation (H3me3K9). Discovery that MAGE I proteins also bind to KAP1 prompted us to investigate whether MAGE I can affect KZNF and KAP1 mediated gene regulation. We found that expression of MAGE I proteins, MAGE-A3 or MAGE-C2, relieved repression of a reporter gene by ZNF382, a KZNF with tumor suppressor activity. ChIP of MAGE I (-) HEK293T cells showed KAP1 and H3me3K9 are normally bound to the ID1 gene, a target of ZNF382, but that binding is greatly reduced in the presence of MAGE I proteins. MAGE I expression relieved KAP1 mediated ID1 repression, causing increased expression of ID1 mRNA and ID1 chromatin relaxation characterized by loss of H3me3K9. MAGE I binding to KAP1 also induced ZNF382 poly-ubiquitination and degradation, consistent with loss of ZNF382 leading to decreased KAP1 binding to ID1. In contrast, MAGE I expression caused increased KAP1 binding to Ki67, another KAP1 target gene, with increased H3me3K9 and decreased Ki67 mRNA expression. Since KZNFs are required to direct KAP1 to specific genes, these results show that MAGE I proteins can differentially regulate members of the KZNF family and KAP1 mediated gene repression.
PMCID: PMC3158099  PMID: 21876767
24.  ZNF265—a novel spliceosomal protein able to induce alternative splicing 
The Journal of Cell Biology  2001;154(1):25-32.
The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-β1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF35. Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265–EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain–containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.
PMCID: PMC2196870  PMID: 11448987
zinc finger protein; RS domain; SR proteins; RNA processing; nuclear localization; renin
25.  Functional Analysis of KAP1 Genomic Recruitment▿† 
Molecular and Cellular Biology  2011;31(9):1833-1847.
TRIM28 (KAP1) is upregulated in many cancers and has been implicated in both transcriptional activation and repression. Using chromatin immunoprecipitation and sequencing, we show that KAP1 binding sites fall into several categories, specifically, the 3′ coding exons of zinc finger (ZNF) genes and promoter regions of ZNFs and other genes. The currently accepted model is that KAP1 is recruited to the genome via interaction of its N-terminal RBCC domain with KRAB ZNFs (KRAB domain containing ZNFs). To determine whether the interaction of KAP1 with KRAB ZNFs is the mechanism by which KAP1 is recruited to genomic binding sites, we analyzed stable cell lines that express tagged wild-type and mutant KAP1. Surprisingly, deletion of the RBCC domain abolished KAP1 binding to the 3′ exons of ZNF genes but KAP1 binding to promoter regions was unaffected. Using KAP1 knockdown cells, we showed that the genes most responsive to KAP1 were not ZNF genes but instead were either indirect targets or had KAP1 bound 10 to 100 kb from the transcription start site. Therefore, our studies suggest that KAP1 plays a role distinct from transcriptional regulation at the majority of its strongest binding sites.
PMCID: PMC3133220  PMID: 21343339

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