C2H2 Transcription Factor Research Articles

Specific Inhibition of the Transcription Factor Ci by a Cobalt(III) Schiff Base–DNA Conjugate

We describe the use of Co(III) Schiff base-DNA conjugates, a versatile class of research tools that target C2H2 transcription factors, to inhibit the Hedgehog (Hh) pathway. In developing mammalian embryos, Hh signaling is critical for the formation and development of many tissues and organs. Inappropriate activation of the Hedgehog (Hh) pathway has been implicated in a variety of cancers including medulloblastomas and basal cell carcinomas. It is well-known that Hh regulates the activity of the Gli family of C2H2 zinc finger transcription factors in mammals. In Drosophila the function of the Gli proteins is performed by a single transcription factor with an identical DNA binding consensus sequence, Cubitus Interruptus (Ci). We have demonstrated previously that conjugation of a specific 17 base-pair oligonucleotide to a Co(III) Schiff base complex results in a targeted inhibitor of the Snail family C2H2 zinc finger transcription factors. Modification of the oligonucleotide sequence in the Co(III) Schiff base-DNA conjugate to that of Ci's consensus sequence (Co(III)-Ci) generates an equally selective inhibitor of Ci. Co(III)-Ci irreversibly binds the Ci zinc finger domain and prevents it from binding DNA in vitro. In a Ci responsive tissue culture reporter gene assay, Co(III)-Ci reduces the transcriptional activity of Ci in a concentration dependent manner. In addition, injection of wild-type Drosophila embryos with Co(III)-Ci phenocopies a Ci loss of function phenotype, demonstrating effectiveness in vivo. This study provides evidence that Co(III) Schiff base-DNA conjugates are a versatile class of specific and potent tools for studying zinc finger domain proteins and have potential applications as customizable anticancer therapeutics.

FlbC is a putative nuclear C2H2 transcription factor regulating development in Aspergillus nidulans

Asexual development (conidiation) in Aspergillus is governed by multiple regulators. Here, we characterize the upstream developmental activator FlbC in Aspergillus nidulans. flbC mRNA is detectable throughout the life cycle, at relatively high levels during vegetative growth, early asexual and late sexual developmental phases. The deletion of flbC causes a delay/reduction in conidiation, brlA and vosA expression, and conidial germination. While overexpression of flbC (OEflbC) does not elaborate conidiophores, it inhibits hyphal growth and activates expression of brlA, abaA and vosA, but not wetA. FlbC is conserved in filamentous Ascomycetes containing two C(2) H(2) zinc fingers at the C-terminus and a putative activation domain at the N-terminus. FlbC localizes in the nuclei of both hyphae and developmental cells. Localization and expression of FlbC are not affected by the absence of FlbB or FlbE, and vice versa. Importantly, overexpression of flbC causes growth inhibition and activation of abaA and vosA in the absence of brlA and abaA respectively. In vitro DNA-binding assay reveals that FlbC binds to the brlA, abaA and vosA, but not the wetA, promoters. In summary, FlbC is a putative nuclear transcription factor necessary for proper activation of conidiation, and its balanced activity is crucial for governing growth and development in A. nidulans.

Role of Aspergillus fumigatus DvrA in Host Cell Interactions and Virulence

The transcription factors that regulate Aspergillus fumigatus interactions with host cells and virulence are incompletely defined. We investigated the role of the putative C2H2 transcription factor DvrA in governing these processes. Although DvrA was identified by its limited homology to Candida albicans Bcr1, a ΔdvrA mutant strain of A. fumigatus had wild-type adherence to host constituents in vitro. However, it had increased capacity to damage both endothelial cells and a pulmonary epithelial cell line compared to the ability of the wild-type strain and a ΔdvrA::dvrA-complemented strain. This increase in damage required direct contact between the mutant and host cells. The ΔdvrA mutant also stimulated greater CCL20, interleukin-8, and tumor necrosis factor mRNA expression in a pulmonary epithelial cell line compared to levels induced by the control strains. Also, it was resistant to nikkomycin Z, suggesting an altered cell wall composition. As predicted by these in vitro results, the ΔdvrA mutant had increased virulence and stimulated a greater pulmonary inflammatory response than the wild-type strain and ΔdvrA::dvrA-complemented strains in the nonneutropenic mouse model of invasive pulmonary aspergillosis. These results indicate that DvrA influences A. fumigatus virulence as well as its capacity to damage host cells and stimulate a proinflammatory response.

Temporal Control of Trichome Distribution by MicroRNA156-TargetedSPLGenes inArabidopsis thaliana

The production and distribution of plant trichomes is temporally and spatially regulated. After entering into the flowering stage, Arabidopsis thaliana plants have progressively reduced numbers of trichomes on the inflorescence stem, and the floral organs are nearly glabrous. We show here that SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes, which define an endogenous flowering pathway and are targeted by microRNA 156 (miR156), temporally control the trichome distribution during flowering. Plants overexpressing miR156 developed ectopic trichomes on the stem and floral organs. By contrast, plants with elevated levels of SPLs produced fewer trichomes. During plant development, the increase in SPL transcript levels is coordinated with the gradual loss of trichome cells on the stem. The MYB transcription factor genes TRICHOMELESS1 (TCL1) and TRIPTYCHON (TRY) are negative regulators of trichome development. We show that SPL9 directly activates TCL1 and TRY expression through binding to their promoters and that this activation is independent of GLABROUS1 (GL1). The phytohormones cytokinin and gibberellin were reported to induce trichome formation on the stem and inflorescence via the C2H2 transcription factors GIS, GIS2, and ZFP8, which promote GL1 expression. We show that the GIS-dependent pathway does not affect the regulation of TCL1 and TRY by miR156-targeted SPLs, represented by SPL9. These results demonstrate that the miR156-regulated SPLs establish a direct link between developmental programming and trichome distribution.

Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen.

Experimentally based contact energies decode interactions responsible for protein–DNA affinity and the role of molecular waters at the binding interface

A major obstacle towards understanding the molecular basis of transcriptional regulation is the lack of a recognition code for protein–DNA interactions. Using high-quality crystal structures and binding data on the promiscuous family of C2H2 zinc fingers (ZF), we decode 10 fundamental specific interactions responsible for protein–DNA recognition. The interactions include five hydrogen bond types, three atomic desolvation penalties, a favorable non-polar energy, and a novel water accessibility factor. We apply this code to three large datasets containing a total of 89 C2H2 transcription factor (TF) mutants on the three ZFs of EGR. Guided by molecular dynamics simulations of individual ZFs, we map the interactions into homology models that embody all feasible intra- and intermolecular bonds, selecting for each sequence the structure with the lowest free energy. These interactions reproduce the change in affinity of 35 mutants of finger I (R2 = 0.998), 23 mutants of finger II (R2 = 0.96) and 31 finger III human domains (R2 = 0.94). Our findings reveal recognition rules that depend on DNA sequence/structure, molecular water at the interface and induced fit of the C2H2 TFs. Collectively, our method provides the first robust framework to decode the molecular basis of TFs binding to DNA.

Characterization of Snail nuclear import pathways as representatives of C2H2 zinc finger transcription factors

Snail proteins are C(2)H(2) class zinc finger transcription factors involved in different processes during embryonic development, as well as in several adult pathologies including cancer and organ fibrosis. The expression of Snail transcription factors is tightly regulated at the transcriptional level and their activity is modulated by their subcellular localization. Given the importance of this gene family in physiology and pathology, it is essential to understand the mechanisms by which Snail proteins are imported into or exported out of the nucleus. Here we show that several importins mediate the nuclear import of the human Snail proteins and we identify a unique nuclear localization signal (NLS), recognized by all the importins, that has been conserved during the evolution of the Snail family. This NLS is characterized by the presence of basic residues at defined positions in at least three consecutive zinc fingers. Interestingly, the consensus residues for importin-binding are also involved in DNA binding, suggesting that importins could prevent non-specific binding of these transcription factors to cytoplasmic polyanions. Importantly, the identified basic residues are also conserved in other families of C(2)H(2) transcription factors whose nuclear localization requires the zinc finger region.

Integration of cytokinin and gibberellin signalling byArabidopsistranscription factors GIS, ZFP8 and GIS2 in the regulation of epidermal cell fate

The effective integration of hormone signals is essential to normal plant growth and development. Gibberellins (GA) and cytokinins act antagonistically in leaf formation and meristem maintenance and GA counteract some of the effects of cytokinins on epidermal differentiation. However, both can stimulate the initiation of defensive epidermal structures called trichomes. To understand how their relative influence on epidermal cell fate is modulated, we investigated the molecular mechanisms through which they regulate trichome initiation in Arabidopsis. The control by cytokinins of trichome production requires two genes expressed in late inflorescence organs, ZFP8 and GIS2, which encode C2H2 transcription factors related to GLABROUS INFLORESCENCE STEMS (GIS). Cytokinin-inducible GIS2 plays a prominent role in the cytokinin response, in which it acts downstream of SPINDLY and upstream of GLABROUS1. In addition, GIS2 and ZFP8 mediate, like GIS, the regulation of trichome initiation by gibberellins. By contrast, GIS does not play a significant role in the cytokinin response. Collectively, GIS, ZFP8 and GIS2, which encode proteins that are largely equivalent in function, play partially redundant and essential roles in inflorescence trichome initiation and in its regulation by GA and cytokinins. These roles are consistent with their pattern of expression and with the regional influence of GA and cytokinins on epidermal differentiation. Our findings show that functional specialization within a transcription factor gene family can facilitate the integration of different developmental cues in the regulation of plant cell differentiation.

Core promoter sequences contribute to ovo-B regulation in the Drosophila melanogaster germline.

Utilization of tightly linked ovo-A vs. ovo-B germline promoters results in the expression of OVO-A and OVO-B, C(2)H(2) transcription factors with different N -termini, and different effects on target gene transcription and on female germline development. We show that two sex-determination signals, the X chromosome number within the germ cells and a female soma, differentially regulate ovo-B and ovo-A. We have previously shown that OVO regulates ovarian tumor transcription by binding the transcription start site. We have explored the regulation of the ovo-B promoter using an extensive series of transgenic reporter gene constructs to delimit cis-regulatory sequences as assayed in wild-type and sex-transformed flies and flies with altered ovo dose. Minimum regulated expression of ovo-B requires a short region flanking the transcription start site, suggesting that the ovo-B core promoter bears regulatory information in addition to a "basal" activity. In support of this idea, the core promoter region binds distinct factors in ovary and testis extracts, but not in soma extracts, suggesting that regulatory complexes form at the start site. This idea is further supported by the evolutionarily conserved organization of OVO binding sites at or near the start sites of ovo loci in other flies.

Molecular events in senescing Arabidopsis leaves.

Senescence is the final stage of leaf development. Although it means the loss of vitality of leaf tissue, leaf senescence is tightly controlled by the development to increase the fitness of the whole plant. The molecular mechanisms regulating the induction and progression of leaf senescence are complex. We used a cDNA microarray, containing 11 500 Arabidopsis DNA elements, and the whole-genome Arabidopsis ATH1 Genome Array to examine global gene expression in dark-induced leaf senescence. By monitoring the gene expression patterns at carefully chosen time points, with three biological replicates each time, we identified thousands of up- or down-regulated genes involved in dark-induced senescence. These genes were clustered and categorized according to their expression patterns and responsiveness to dark treatment. Genes with different expression kinetics were classified according to different biological processes. Genes showing significant alteration of expression patterns in all available biochemical pathways were plotted to envision the molecular events occurring in the processes examined. With the expression data, we postulated an innovative biochemical pathway involving pyruvate orthophosphate dikinase in generating asparagine for nitrogen remobilization in dark-treated leaves. We also surveyed the alteration in expression of Arabidopsis transcription factor genes and established an apparent association of GRAS, bZIP, WRKY, NAC, and C2H2 transcription factor families with leaf senescence.

Fumonisins in Maize: Can We Reduce Their Occurrence?

Fumonisins in Maize: Can We Reduce Their Occurrence?