C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2-ZF arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. In addition, little is known about whether or how these proteins regulate transcription. Most of the ∼700 human C2H2-ZF proteins also contain at least one KRAB, SCAN, BTB, or SET domain, suggesting that they may have common interacting partners and/or effector functions. Here, we report a multifaceted functional analysis of 131 human C2H2-ZF proteins, encompassing DNA binding sites, interacting proteins, and transcriptional response to genetic perturbation. We confirm the expected diversity in DNA binding motifs and genomic binding sites, and provide motif models for 78 previously uncharacterized C2H2-ZF proteins, most of which are unique. Surprisingly, the diversity in protein–protein interactions is nearly as high as diversity in DNA binding motifs: Most C2H2-ZF proteins interact with a unique spectrum of co-activators and co-repressors. Thus, multiparameter diversification likely underlies the evolutionary success of this large class of human proteins.

Calcium deficiency causes abnormal colonic growth and increases colon cancer risk with poorly understood mechanisms. Here we elucidate a novel signaling mechanism underlying the Ca 2 þ deficiency-induced epithelial proliferation using a unique animal model. The zebrafish larval yolk sac skin contains a group of Ca 2 þ -transporting epithelial cells known as ionocytes. Their number and density increases dramatically when acclimated to low [Ca 2 þ ] environments. BrdU pulse-labeling experiments suggest that low [Ca 2 þ ] stimulates pre-existing ionocytes to re-enter the cell cycle. Low [Ca 2 þ ] treatment results in a robust and sustained activation of IGF1R-PI3K-Akt signaling in these cells exclusively. These ionocytes specifically express Igfbp5a, a high-affinity and specific binding protein for insulin-like growth factors (IGFs) and the Ca 2 þ -selective channel Trpv5/6. Inhibition or knockdown of Igfbp5a, IGF1 receptor, PI3K, and Akt attenuates low [Ca 2 þ ]-induced ionocyte proliferation. The role of Trpv5/6 was investigated using a genetic mutant, targeted knockdown, and pharmacological inhibition. Loss-of-Trpv5/6 function or expression results in elevated pAkt levels and increased ionocyte proliferation under normal [Ca 2 þ ]. These increases are eliminated in the presence of an IGF1R inhibitor, suggesting that Trpv5/6 represses IGF1R-PI3K-Akt signaling under normal [Ca 2 þ ]. Intriguingly, blockade of Trpv5/6 activity inhibits the low [Ca 2 þ ]-induced activation of Akt. Mechanistic analyses reveal that the low [Ca 2 þ ]-induced IGF signaling is mediated through Trpv5/6-associated membrane depolarization. Low extracellular [Ca 2 þ ] results in a similar amplification of IGF-induced PI3K-PDK1-Akt signaling in human colon cancer cells in a TRPV6-dependent manner. These results uncover a novel and evolutionarily conserved signaling mechanism that contributes to the abnormal epithelial proliferation associated with Ca 2 þ deficiency.

Aberrant regulation of the insulin-like growth factor (IGF)/insulin (IIS)-PI3K-AKT-TOR signaling pathway is linked to major human diseases, and key components of this pathway are targets for therapeutic intervention. Current assays are molecular target- or cell culture-based platforms. Due to the great in vivo complexities inherited in this pathway, there is an unmet need for whole organism based assays. Here we report the development of a zebrafish transgenic line, Tg(igfbp5a:GFP), which faithfully reports the mitotic action of IGF1R-PI3K-Akt-Tor signaling in epithelial cells in real-time. This platform is well suited for high-throughput assays and real-time cell cycle analysis. Using this platform, the dynamics of epithelial cell proliferation in response to low [Ca2+] stress and the distinct roles of Torc1 and Torc2 were elucidated. The availability of Tg(igfbp5a:GFP) line provides a whole organism platform for phenotype-based discovery of novel players and inhibitors in the IIS-PI3K-Akt-Tor signaling pathway.

A long-standing question in collective cell migration has been what might be the relative advantage of forming a cluster over migrating individually. Does an increase in the size of a collectively migrating group of cells enable them to sample the chemical gradient over a greater distance because the difference between front and rear of a cluster would be greater than for single cells? We combined theoretical modeling with experiments to study collective migration of the border cells in-between nurse cells in the Drosophila egg chamber. We discovered that cluster size is positively correlated with migration speed, up to a particular point above which speed plummets. This may be due to the effect of viscous drag from surrounding nurse cells together with confinement of all of the cells within a stiff extracellular matrix. The model predicts no relationship between cluster size and velocity for cells moving on a flat surface, in contrast to movement within a 3D environment. Our analyses also suggest that the overall chemoattractant profile in the egg chamber is likely to be exponential, with the highest concentration in the oocyte. These findings provide insights into collective chemotaxis by combining theoretical modeling with experimentation.cell migration | theoretical modeling | three-dimensional | chemotaxis

Adult stem cells commonly give rise to transit-amplifying progenitors, whose progeny differentiate into distinct cell types. It is unclear if stem cell niche signals coordinate fate decisions within the progenitor pool. Here we use quantitative analysis of Wnt, Hh, and Notch signalling reporters and the cell fate markers Eyes Absent (Eya) and Castor (Cas) to study the effects of hyper-activation and loss of niche signals on progenitor development in the Drosophila ovary. Follicle stem cell (FSC) progeny adopt distinct polar, stalk, and main body cell fates. We show that Wnt signalling transiently inhibits expression of the main body cell fate determinant Eya, and Wnt hyperactivity strongly biases cells towards polar and stalk fates. Hh signalling independently controls the proliferation to differentiation transition. Notch is permissive but not instructive for differentiation of multiple cell types. These findings reveal that multiple niche signals coordinate cell fates and differentiation of progenitor cells.

We cloned and characterized the ZmWRKY17 gene from maize. Overexpression of ZmWRKY17 in Arabidopsis led to increased sensitivity to salt stress and decreased ABA sensitivity through regulating the expression of some ABA- and stress-responsive genes. The WRKY transcription factors have been reported to function as positive or negative regulators in many different biological processes including plant development, defense regulation and stress response. This study isolated a maize WRKY gene, ZmWRKY17, and characterized its role in tolerance to salt stress by generating transgenic Arabidopsis plants. Expression of the ZmWRKY17 was up-regulated by drought, salt and abscisic acid (ABA) treatments. ZmWRKY17 was localized in the nucleus with no transcriptional activation in yeast. Yeast one-hybrid assay showed that ZmWRKY17 can specifically bind to W-box, and it can activate W-box-dependent transcription in planta. Heterologous overexpression of ZmWRKY17 in Arabidopsis remarkably reduced plant tolerance to salt stress, as determined through physiological analyses of the cotyledons greening rate, root growth, relative electrical leakage and malondialdehyde content. Additionally, ZmWRKY17 transgenic plants showed decreased sensitivity to ABA during seed germination and early seedling growth. Transgenic plants accumulated higher content of ABA than wild-type (WT) plants under NaCl condition. Transcriptome and quantitative real-time PCR analyses revealed that some stress-related genes in transgenic seedlings showed lower expression level than that in the WT when treated with NaCl. Taken together, these results suggest that ZmWRKY17 may act as a negative regulator involved in the salt stress responses through ABA signalling.

Two species of the DNA virus Torque teno sus virus (TTSuV), TTSuV1 and TTSuV2, have become widely distributed in pig-farming countries in recent years. In this study, we performed a comprehensive analysis of synonymous codon usage bias in 41 available TTSuV2 coding sequences (CDS), and compared the codon usage patterns of TTSuV2 and TTSuV1. TTSuV codon usage patterns were found to be phylogenetically conserved. Values for the effective number of codons (ENC) indicated that the overall extent of codon usage bias in both TTSuV2 and TTSuV1 was not significant, the most frequently occurring codons had an A or C at the third codon position. Correspondence analysis (COA) was performed and TTSuV2 and TTSuV1 sequences were located in different quadrants of the first two major axes. A plot of the ENC revealed that compositional constraint was the major factor determining the codon usage bias for TTSuV2. In addition, hierarchical cluster analysis of 41 TTSuV2 isolates based on relative synonymous codon usage (RSCU) values suggested that there was no association between geographic distribution and codon bias of TTSuV2 sequences. Finally, the comparison of RSCU for TTSuV2, TTSuV1 and the corresponding host sequence indicated that the codon usage pattern of TTSuV2 was similar to that of TTSuV1. However the similarity was low for each virus and its host. These conclusions provide important insight into the synonymous codon usage pattern of TTSuV2, as well as better understangding of the molecular evolution of TTSuV2 genomes.

Insulin-like growth factor binding protein (IGFBP)-5 is a secreted protein that binds to IGF and modulates IGF actions. IGFBP-5 is also found in the nucleus of mammalian cells and has transactivation activity. The structural basis of this transactivation activity and its role in mediating IGF-independent actions are not clear. Here we report that there are 2 igfbp-5 genes in zebrafish and other teleost fish. In zebrafish, igfbp-5a and -5b are expressed in spatially restricted, mostly nonoverlapping domains during early development. The IGF binding site is conserved in both zebrafish IGFBP-5s, and they are both secreted and capable of IGF binding. Both proteins contain a consensus bipartite nuclear localization signal and were found in the nucleus when introduced into cultured cells. Although zebrafish IGFBP-5b possesses transactivation activity, zebrafish IGFBP-5a lacks this activity. Mutational analysis demonstrated that 2 unique amino acids in positions 22 and 56 of IGFBP-5a are responsible for its lack of transactivation activity. These findings suggest that the duplicated zebrafish IGFBP-5s have evolved divergent regulatory mechanisms and distinct biological properties by partitioning of ancestral structural domains and provide new evidence for a conserved role of the IGF binding, nuclear localization, and transactivation domain of this multifunctional IGFBP.