C2H2-type Zinc Finger Transcription Factor Research Articles

Aberrant expression of BCL11B in mycosis fungoides and its potential role in interferon‐induced apoptosis

BCL11B is a Kruppel-like C2H2-type zinc finger transcription factor, which has been associated with several human malignancies. Recent evidence showed that overexpressed BCL11B conferred chemoresistance to malignant T cells and that inhibiting BCL11B led to increased apoptosis, suggesting its potential pathogenic relevance in cutaneous T-cell lymphomas (CTCL), which were characterized by the resistance to chemotherapy-induced apoptosis. To investigate the expression pattern of BCL11B in different stages of mycosis fungoides (MF), quantitative reverse transcription polymerase chain reaction and immunohistochemistry were performed to compare the mRNA and protein expression among different stages of MF and benign inflammatory dermatoses (BID), respectively. BCL11B demonstrated significant upregulation in all stages of MF, compared with BID, in both mRNA expression level and protein level. In addition, BCL11B expression increased with advancing lesion tumor stage and overall disease stage. Further, to evaluate the dynamic expression of BCL11B under CTCL-directed treatment, BCL11B expression and cell apoptosis were evaluated after interferon (IFN)-α-2b and methotrexate treatment on CTCL cell line Hut78 cells. IFN-α-2b, but not methotrexate, induced BCL11B inhibition and cell apoptosis, suggesting that BCL11B may play important roles in the anti-CTCL effect of IFN-α-2b. In conclusion, our study demonstrated the overexpression of BCL11B in MF lesions and its potential relevance to disease progression. In addition, we provided evidence for BCL11B inhibitory approaches as a potential treatment to target chemoresistant tumor cells in advanced MF.

The Fermentation Stress Response Protein Aaf1p/Yml081Wp Regulates Acetate Production in Saccharomyces cerevisiae

The production of acetic acid during wine fermentation is a critical issue for wineries since the sensory quality of a wine can be affected by the amount of acetic acid it contains. We found that the C2H2-type zinc-finger transcription factor YML081Wp regulated the mRNA levels of ALD4 and ALD6, which encode a cytosolic acetaldehyde dehydrogenase (ACDH) and a mitochondrial ACDH, respectively. These enzymes produce acetate from acetaldehyde as part of the pyruvate dehydrogenase bypass. This regulation was also reflected in the protein levels of Ald4p and Ald6p, as well as total ACDH activity. In the absence of ALD6, YML081W had no effect on acetic acid levels, suggesting that this transcription factor’s effects are mediated primarily through this gene. lacZ reporter assays revealed that Yml081wp stimulates ALD6 transcription, in large part from a GAGGGG element 590 base pairs upstream of the translation start site. The non-annotated ORF YML081W therefore encodes a transcription factor that regulates acetate production in Saccharomyces cerevisiae. We propose AAF1 as a gene name for the YML081W ORF.

Abstract 286: Development and evaluation of a molecular decoy against SLUG function in the breast cancer cells

Abstract SLUG, also known as SNAI2, is a member of the SNAI superfamily of zinc-finger transcriptional repressors. It is a C2H2-type zinc finger transcription factor that binds to E2-box motif (5′-CAGGTG-3′/5′-CACCTG-3′), and silences gene expression by chromatin remodeling. More than ninety genes are shown to bind tightly to SLUG as was revealed by ChIP-on-chip analysis, showing the large regulatory network involving SLUG. It is recently implicated as the major mediator of the master transcriptional regulator protein TWIST. SLUG represses several genes to directly contribute towards the metastatic transformation of several epithelial cancer cells. Specific inhibitor against SLUG is not known. We describe here the development and evaluation of a nuclease resistant double-stranded DNA decoy that specifically inhibit the repressor activity of SLUG in the human breast cancer cells. The decoy is designed from the VDR gene promoter which is repressed by SLUG. This decoy has two SLUG-binding E2-box sequences. EMSA analysis showed in vitro binding of SLUG to the wild-type decoy but not to the E2-box mutated decoy. In vitro pull down analysis also showed high affinity binding of SLUG to the decoy. Biotin-tagged wild-type decoy DNA could immunopull down SLUG but little SNAIL from the extracts from decoy-transfected cells. The decoy increased the mRNA and protein levels of several SLUG target genes in MDA-MB-231 cells. The decoy also alleviated the repressive activity of SLUG on the SLUG target gene promoters in MDA-MB-231 cells. Since SLUG overexpression is implicated in the drug-resistance and antiapoptosis development in breast cancer cells, the SLUG decoy we developed should be useful to combat breast cancer progression when used with classical anticancer drugs. Supported in parts by the DOD-CDMRP grants BC050641, BC086542 and BC103645 to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 286. doi:1538-7445.AM2012-286

Identification of a C2H2-type zinc finger transcription factor (ZAT10) from Arabidopsis as a substrate of MAP kinase

Mitogen-activated protein kinases (MAPKs or MPKs) are one of the most important and conserved signaling molecules in plants. MPKs can directly modulate gene expression by the phosphorylation of transcription factors. However, only a few target substrates of MPKs have been isolated. Here, we identified a C(2)H(2)-type zinc finger transcription factor from Arabidopsis, ZAT10, as a substrate of MPKs. Using in vitro and in vivo protein-protein interaction analyses, we demonstrated that ZAT10 directly interacted with MPK3 and MPK6. ZAT10 was phosphorylated by recombinant Arabidopsis MPK3 and MPK6 in a kinase assay. Furthermore, ZAT10 was also phosphorylated by native MPK3 and MPK6 prepared from Arabidopsis plants in an in-gel kinase assay. Mass spectrometry analysis of phosphopeptides was used to determine two MPK phosphorylation sites in ZAT10. These sites were verified by site-directed mutagenesis and in vitro kinase assays.

A tonoplast‐localized half‐size ABC transporter is required for internal detoxification of aluminum in rice

Toxic aluminum enters the root cells rapidly, therefore internal detoxification is required. However, the molecular mechanisms underlying this process are poorly understood. Here we functionally characterized a rice gene, Os03g0755100 (OsALS1), that is regulated by ART1, a C2H2-type zinc finger transcription factor. OsALS1 encodes a half-size ABC transporter that is a member of the TAP (transporter associated with antigen processing) sub-group. Expression of OsALS1 was rapidly and specifically induced by Al in the roots, but not by other metals or low pH. OsALS1 was localized at all cells of the roots. Furthermore, OsALS1 is localized to the tonoplast. These expression patterns and cell specificity of localization are different from those of the homologous gene AtALS1 in Arabidopsis. Knockout of OsALS1 in three independent lines resulted in significant increased sensitivity to Al, but did not affect the sensitivity to other metals and low pH. Comparison of Al accumulation patterns between wild-type and osals1 mutants showed that there was no difference in Al levels in the cell sap of root tips between wild-type and the mutants, but the mutants accumulated more Al in the cytosol and nucleus than the wild-type. Expression of OsALS1 in yeast resulted in increased Al sensitivity due to mis-localization. These results indicate that OsALS1 localized at the tonoplast is responsible for sequestration of Al into the vacuoles, which is required for internal detoxification of Al in rice.

English

The rice line carrying the nonhost gene Rxo1, which was cloned from maize, exhibits a rapid hypersensitive response (HR) to Xanthomonas oryzae pv. oryzicola(Xoc). In this study, a microarray experiment was carried out to analyze the genome-wide gene expression responses to Xoc at the early stage in the rice transgenic line and its wild type. 175 and 379 differentially regulated genes (DRGs) were detected in 9804-Rxo1 and 9804 at 36 h post-inoculation (hpi), respectively. The patterns of DRGs induced by Xoc in the transgenic rice line and its wild type were distinctly different: 92.00% of the DRGs were up-regulated in inoculated 9804-Rxo1 and 48.22% of the DRGs were sorted as defense-related genes. In contrast, 40.69% of the DRGs were up-regulated in inoculated 9804 and 40.00% were sorted as injury-induced genes. Some genes specifically up-regulated in infected 9804-Rxo1 were defense-related, including the genes encoding pathogenesis-related protein, terpene synthase family, transcription factors (TFs) AP2 domain containing protein, myb-like deoxyribonucleic acid (DNA)-binding domain containing protein, and C2H2-type zinc-finger transcription factors. Six peroxidase (POD) genes were significantly up-regulated and POD activity consistently rose in the inoculated 9804-Rxo1, whereas seven POD genes were down-regulated in its wild type, indicating that POD may also play a role in the complex process of resistant response to Xocin the transgenic line. Instead of focusing only on the R-gene-mediated defense, this study also discusses the susceptible response in the wild types to virulent pathogen infection in transcription level.   Key words: Rice bacterial leaf streak, nonhost resistance, expression profile, Rxo1

An Al‐inducible MATE gene is involved in external detoxification of Al in rice

A number of plant species, including rice, secretes citrate from roots in response to Al stress. Here we characterized the functions of a gene, OsFRDL4 (Os01g0919100) that belongs to the multidrug and toxic compound extrusion (MATE) family in rice (Oryza sativa). Heterologous expression in Xenopus oocyte showed that the OsFRDL4 protein was able to transport citrate and was activated by Al. The expression level of the OsFRDL4 gene in roots was very low in the absence of Al, but was greatly enhanced by Al after short exposure. Furthermore, the OsFRDL4 expression was regulated by ART1, a C2H2-type zinc finger transcription factor for Al tolerance. Transient expression of OsFRDL4 in onion epidermal cells showed that it localized to the plasma membrane. Immunostaining showed that OsFRDL4 was localized in all cells in the root tip. These expression patterns and cell specificity of localization of OsFRDL4 are different from other MATE members identified previously. Knockout of OsFRDL4 resulted in decreased Al tolerance and decreased citrate secretion compared with the wild-type rice, but did not affect citrate concentration in the xylem sap. Furthermore, there is a positive correlation between OsFRDL4 expression level and the amount of citrate secretion in rice cultivars that are differing in Al tolerance. Taken together, our results show that OsFRDL4 is an Al-induced citrate transporter localized at the plasma membrane of rice root cells and is one of the components of high Al tolerance in rice.

Identification of a Cis-Acting Element of ART1, a C2H2-Type Zinc-Finger Transcription Factor for Aluminum Tolerance in Rice

Rice (Oryza sativa) is one of the most aluminum (Al)-tolerant species among small-grain cereals. Recent identification of a transcription factor AL RESISTANCE TRANSCRIPTION FACTOR1 (ART1) revealed that this high Al tolerance in rice is achieved by multiple genes involved in detoxification of Al at different cellular levels. ART1 is a C2H2-type zinc-finger transcription factor and regulates the expression of 31 genes in the downstream. In this study, we attempted to identify a cis-acting element of ART1. We used the promoter region of SENSITIVE TO AL RHIZOTOXICITY1, an Al tolerance gene in the downstream of ART1. With the help of gel-shift assay, we were able to identify the cis-acting element as GGN(T/g/a/C)V(C/A/g)S(C/G). This element was found in the promoter region of 29 genes among 31 ART1-regulated genes. To confirm this cis-acting element in vivo, we transiently introduced this element one or five times tandemly repeated sequence with 35S minimal promoter and green fluorescent protein reporter together with or without ART1 gene in the tobacco (Nicotiana tabacum) mesophyll protoplasts. The results showed that the expression of green fluorescent protein reporter responded to ART1 expression. Furthermore, the expression increased with repetition of the cis-acting element. Our results indicate that the five nucleotides identified are the target DNA-binding sequence of ART1.

Transcription factor families inferred from genome sequences of photosynthetic stramenopiles

• By comparative analyses we identify lineage-specific diversity in transcription factors (TFs) from stramenopile (or heterokont) genome sequences. We compared a pennate (Phaeodactylum tricornutum) and a centric diatom (Thalassiosira pseudonana) with those of other stramenopiles (oomycetes, Pelagophyceae, and Phaeophyceae (Ectocarpus siliculosus)) as well as to that of Emiliania huxleyi, a haptophyte that is evolutionarily related to the stramenopiles. • We provide a detailed description of diatom TF complements and report numerous peculiarities: in both diatoms, the heat shock factor (HSF) family is overamplified and constitutes the most abundant class of TFs; Myb and C2H2-type zinc finger TFs are the two most abundant TF families encoded in all the other stramenopile genomes investigated; the presence of diatom and lineage-specific gene fusions, in particular a class of putative photoreceptors with light-sensitive Per-Arnt-Sim (PAS) and DNA-binding (basic-leucine zipper, bZIP) domains and an HSF-AP2 domain fusion. • Expression data analysis shows that many of the TFs studied are transcribed and may be involved in specific responses to environmental stimuli. • Evolutionary and functional relevance of these observations are discussed.

A Zinc Finger Transcription Factor ART1 Regulates Multiple Genes Implicated in Aluminum Tolerance in Rice

Aluminum (Al) toxicity is the major limiting factor of crop production on acid soils, but some plant species have evolved ways of detoxifying Al. Here, we report a C2H2-type zinc finger transcription factor ART1 (for Al resistance transcription factor 1), which specifically regulates the expression of genes related to Al tolerance in rice (Oryza sativa). ART1 is constitutively expressed in the root, and the expression level is not affected by Al treatment. ART1 is localized in the nucleus of all root cells. A yeast one-hybrid assay showed that ART1 has a transcriptional activation potential and interacts with the promoter region of STAR1, an important factor in rice Al tolerance. Microarray analysis revealed 31 downstream transcripts regulated by ART1, including STAR1 and 2 and a couple of homologs of Al tolerance genes in other plants. Some of these genes were implicated in both internal and external detoxification of Al at different cellular levels. Our findings shed light on comprehensively understanding how plants detoxify aluminum to survive in an acidic environment.

Cloning and characterization of RZF71 encoding a C2H2-type zinc finger protein from rice

A rice zinc-finger protein gene, RZF71, encoding the C2H2-type zinc-finger transcription factor was isolated from rice (Oryza sativa L. subs. Japonica) by RT-PCR approach. Gene RZF71 encodes a 25 kDa protein with 250 amino acids, which contains two typical C2H2 zinc finger domains. The expression profiling showed that RZF71 was constitutively expressed in roots, culms, leaves, and flowering spikes. The semi-quantitative RT-PCR assay showed RZF71 was strongly induced by high-salinity and 20% PEG6000 treatments, but not regulated by low temperature and ABA (abscisic acid) treatments. Tran-sient expression of the RZF71-GFP protein in onion epidermal cell showed that RZF71 was localized in cell nuclei. These results indicated that the RZF71 may play an important role in rice responses to salt and osmotic stresses as a transcription factor.

Glial and Neuronal Functions of the Drosophila Homolog of the Human SWI/SNF Gene ATR-X (DATR-X) and the jing Zinc-Finger Gene Specify the Lateral Positioning of Longitudinal Glia and Axons

Neuronal-glial communication is essential for constructing the orthogonal axon scaffold in the developing Drosophila central nervous system (CNS). Longitudinal glia (LG) guide extending commissural and longitudinal axons while pioneer and commissural neurons maintain glial survival and positioning. However, the transcriptional regulatory mechanisms controlling these processes are not known. Previous studies showed that the midline function of the jing C2H2-type zinc-finger transcription factor was only partially required for axon scaffold formation in the Drosophila CNS. We therefore screened for gain-of-function enhancers of jing gain of function in the eye and identified the Drosophila homolog of the disease gene of human alpha-thalassemia/mental retardation X-linked (ATR-X) as well as other genes with potential roles in gene expression, translation, synaptic transmission, and cell cycle. jing and DATR-X reporter genes are expressed in both CNS neurons and glia, including the LG. Coexpression of jing and DATR-X in embryonic neurons synergistically affects longitudinal connective formation. During embryogenesis, jing and DATR-X have autonomous and nonautonomous roles in the lateral positioning of LG, neurons, and longitudinal axons as shown by cell-specific knockdown of gene expression. jing and DATR-X are also required autonomously for glial survival. jing and DATR-X mutations show synergistic effects during longitudinal axon formation suggesting that they are functionally related. These observations support a model in which downstream gene expression controlled by a potential DATR-X-Jing complex facilitates cellular positioning and axon guidance, ultimately allowing for proper connectivity in the developing Drosophila CNS.

Methylglyoxal, a Metabolite Derived from Glycolysis, Functions as a Signal Initiator of the High Osmolarity Glycerol-Mitogen-activated Protein Kinase Cascade and Calcineurin/Crz1-mediated Pathway in Saccharomyces cerevisiae

Methylglyoxal (MG) is a typical 2-oxoaldehyde derived from glycolysis, although it inhibits the growth of cells in all types of organism. Hence, it has been questioned why such a toxic metabolite is synthesized via the ubiquitous energy-generating pathway. We have previously reported that expression of GLO1, coding for the major enzyme detoxifying MG, was induced by osmotic stress in a high osmolarity glycerol (HOG)-mitogen-activated protein (MAP) kinase-dependent manner in Saccharomyces cerevisiae. Here we show that MG activates the HOG-MAP kinase cascade. Two osmosensors, Sln1 and Sho1, have been identified to function upstream of the HOG-MAP kinase cascade, and we reveal that MG initiates the signal transduction to this MAP kinase cascade through the Sln1 branch. We also demonstrate that MG activates the Msn2 transcription factor. Moreover, MG activated the uptake of Ca(2+) in yeast cells, thereby stimulating the calcineurin/Crz1-mediated Ca(2+) signaling pathway. We propose that MG functions as a signal initiator in yeast.

Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis.

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148(+/-) mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148(+/-) tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation. Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp148(+/-) embryonic stem cells and in fetal germ cells from chimeric Zfp148(+/-) embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.

The only function of Grauzone required for Drosophila oocyte meiosis is transcriptional activation of the cortex gene.

The grauzone and cortex genes are required for the completion of meiosis in Drosophila oocytes. The grauzone gene encodes a C2H2-type zinc-finger transcription factor that binds to the cortex promoter and is necessary for high-level activation of cortex transcription. Here we define the region of the cortex promoter to which Grauzone binds and show that the binding occurs through the C-terminal, zinc-finger-rich region of the protein. Mutations in two out of the five grauzone alleles result in single amino acid changes within different zinc-finger motifs. Both of these mutations result in the inability of Grauzone to bind DNA effectively. To determine the mechanism by which Grauzone regulates meiosis, transgenic flies were produced with an extra copy of the cortex gene in homozygous grauzone females. This transgene rescued the meiosis arrest of embryos from these mutants and allowed their complete development, indicating that activation of cortex transcription is the primary role of Grauzone during Drosophila oogenesis. These experiments further define a new transcriptional pathway that controls the meiotic cell cycle in Drosophila oocytes.

Completion of meiosis in Drosophila oocytes requires transcriptional control by grauzone, a new zinc finger protein.

Mutations in grauzone or cortex cause abnormal arrest in Drosophila female meiosis. We cloned grauzone and identified it as a C2H2-type zinc finger transcription factor. The grauzone transcript is present in ovaries and at later developmental stages. A Grauzone-GFP fusion protein is functional and localizes to nuclei of both nurse cells and follicle cells during oogenesis. Three lines of evidence indicate that grauzone and cortex interact: reducing cortex function enhanced the grauzone mutant phenotype; cortex transcript abundance is reduced in the absence of grauzone function and Grauzone protein binds to the cortex promoter. These results demonstrate that activation of cortex transcription by grauzone is necessary for the completion of meiosis in Drosophila oocytes, and establish a new pathway that specifically regulates the female meiotic cell cycle.

The Krüppel-type zinc finger family gene, HKR1, is induced in lung cancer by exposure to platinum drugs

To investigate the molecular mechanism associated with the signaling pathway of platinum drug administration, we focused on the C2H2-type zinc finger (ZNF) transcription factor gene family. Here we show cloning of a Krüppel-type ZNF gene, HKR1, which contains Krüppel-associated box (KRAB) domain and ZNF motifs. We found that mRNA expression of the HKR1 gene was induced in lung-cancer cell lines by exposure to cisplatin using Northern blot analysis. Moreover, we also found that HKR1 mRNA expression levels in lung cancers were higher than those in normal lung tissues, and that high expression levels in lung cancers were associated with antemortem platinum drug administration. These results suggest that HKR1 may be associated with the regulation of a signaling pathway involved in the progression of lung cancer or the acquisition of resistance to platinum drugs.

Crystal structure of the BTB domain from PLZF.

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein-protein interaction motif found at the N terminus of 5-10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 A resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.

Studies on Neisseria gonorrhoeae cultured in liquid medium.

Experiments were carried out to determine the minimum amount of inoculum of gonococci that is needed to produce growth in ANM liquid medium. This was found to be less than had previously been thought. Minimum inocula were used to study penicillin sensitivity in liquid medium. This work showed that growth occurred at higher concentrations of penicillin with larger inocula. Evidence was also obtained which suggested that in some circumstances penicillin acts bacteriostatically rather than bacteriocidally. It was also shown that growth of gonococci can be inhibited in liquid medium by the presence of hyperimmune serum, the inhibition being more marked in the presence of fresh complement. This work is continuing in the hope that it may provide a basis for a much-needed serological test for gonorrhoea. This would be especially valuable in chronic assymptomatic infections.