Identification and expression analysis of StGRAS gene family in potato (Solanum tuberosum L.).

The GRAS gene family is a class of plant-specific transcription factors which play pivotal roles in the regulation of plant growth and development. At present, the GRAS gene family has been completely identified in Arabidopsis thaliana, however, there are no systematic research reports in potato. In the present study, we obtained an overview of the GRAS gene family including gene structure, gene expression, chromosome mapping and phylogenetic analysis, and 52 StGRASs were identified in the potato by bioinformatics analysis, which could be divided into eight subfamilies based on phylogeny. More than 90% of genes do not contain introns and the StGRAS family major function is protein binding according to gene ontology analysis (GO).The tissue specific expression analysis showed that StGRAS3, StGRAS35 and StGRAS50 gene had the higher expression in roots, stems and leaves compared with other StGRAS, StGRAS9 and StGRAS28 genes were responded to plant hormones IAA, ABA and GA3 treatment. The result could provide a basis for further studying the function of GRAS genes and GRAS-mediated signal transduction pathways in potato.

Discovery of alkoxy benzamide derivatives as novel BPTF bromodomain inhibitors via structure-based virtual screening.

Bromodomain PHD finger transcription factor (BPTF), a bromodomain-containing protein, plays a crucial role in the regulation of downstream gene expression through the specific recognition of lysine acetylation on bulk histones. The dysfunction of BPTF is closely involved with the development and progression of many human diseases, especially cancer. Therefore, BPTF bromodomain has become a promising drug target for epigenetic cancer therapy. However, unlike BET family inhibitors, few BPTF bromodomain inhibitors have been reported. In this study, by integrating docking-based virtual screening with biochemical analysis, we identified a novel selective BPTF bromodomain inhibitor DCB29 with the IC50 value of 13.2 ±â€¯1.6 µM by homogenous time-resolved fluorescence resonance energy transfer (HTRF) assays. The binding between DCB29 and BPTF was confirmed by NMR and SPR. Molecular docking disclosed that DCB29 occupied the pocket of acetylated H4 peptide substrate and provided detailed SAR explanations for its derivatives. Collectively, DCB29 presented great potential as a powerful tool for BPTF-related biological research and further medicinal chemistry optimization.

The Extract of Leonurus sibiricus Transgenic Roots with AtPAP1 Transcriptional Factor Induces Apoptosis via DNA Damage and Down Regulation of Selected Epigenetic Factors in Human Cancer Cells.

The aim of this study was to determine the anticancer potential of Leonurus sibiricus extract derived from in vitro transgenic roots transformed by Agrobacetrium rhizogenes with AtPAP1 transcriptional factor, and that of transformed roots without construct, on grade IV human glioma cells and the U87MG cell line, and attempt to characterize the mechanism involved in this process. The anticancer effect induced by the tested extracts was associated with DNA damage, PARP cleavage/increased H2A.X histone levels and UHRF-1/DNMT1 down-regulation of mRNA levels. Additionally, we demonstrated differences in the content of compounds in the tested extracts by HPLC analysis with ATPAP1 construct and without. Both the tested extracts showed anticancer properties and the better results were observed for AtPAP1 with transcriptional factor root extract; this effect could be ascribed to the presence of higher condensed phenolic acids such as neochlorogenic acid, chlorogenic acids, ferulic acid, caffeic acid and p-coumaric acid. Further studies with AtPAP1 (with the transcriptional factor from Arabidopisi thaliana) root extract which showed better activities in combination with anticancer drugs are needed.

Identification and functional characterization of MRE-binding transcription factor (MTF) in Crassostrea gigas and its conserved role in metal-induced response.

The full-length cDNA that encodes the MRE-binding transcription factor (MTF) was cloned from the Pacific oyster (Crassostrea gigas) using reverse transcription polymerase chain reaction and the rapid amplification of cDNA ends. The cgMTF cDNA sequence is 2892 bp long, with a 2508 bp open reading frame that encodes an 835-amino acid polypeptide. Multiple alignment revealed that cgMTF has four putative zinc finger-like regions in cgMTF with three C2C2-type zinc fingers and one C2H2-type zinc finger. After 12 h of exposure to Cd(2+), the cgMTF mRNA level was increased in a dose-dependent manner, which then subsided with time. cgMTF stimulates the cgMT promoter reporter in the HEK293 cell line in a dose-dependent manner. When either of the metal-responsive elements (MRE1 or MRE2) of the cgMT promoter was mutated, the cgMT promoter reporter activity was significantly reduced. After the two MREs were mutated simultaneously, the promoter activity was completely abolished. In conclusion, we identified an MTF in C. gigas and revealed the presence of an evolutionarily conserved molecular mechanism for coping with environmental metal stress.

Isolation and molecular characterization of thirteen R2R3-MYB transcription factors from Epimedium sagittatum.

Epimedium sagittatum (Sieb. et Zucc.) Maxim, a popular traditional Chinese medicinal plant, has been widely used for treating sexual dysfunction and osteoporosis in China. The main bioactive components in herba epimedii are prenylated flavonol glycosides, which are end products of a branch of the flavonoid biosynthetic pathway. The MYB transcription factors (TF) act as activators or repressors to regulate the flavonoid pathway. In this study, 13 full-length cDNA clones of R2R3-MYB TFs from E. sagittatum (designated as EsMYB1 to EsMYB13) were isolated and characterized. Sequence similarity and phylogenetic analysis placed nine R2R3-MYB members of epimedii into five subgroups of the Arabidopsis R2R3-MYB family, while four members were not clustered into a defined subgroup. The number and length of introns from epimedii R2R3-MYB genes varied significantly, but intron positions and phases were well conserved. Expression patterns of epimedii R2R3-MYB genes in various tissues showed diverse. Finally, it is suggested that five epimedii R2R3-MYB genes may be involved in regulating the flavonoid pathway and could be used as valuable candidate genes for metabolic engineering studies in future. Sequence information of 13 R2R3-MYB genes discovered here will also provide an entry point into the overview of whole R2R3-MYB family in epimedii.

Ectopic expression of dehydration responsive element binding proteins (StDREB2) confers higher tolerance to salt stress in potato.

Dehydration responsive element binding proteins (DREB) are members of a larger family of transcription factors, many of which have been reported to contribute to plant responses to abiotic stresses in several species. While, little is known about their role in potato (Solanum tuberosum). This report describes the cloning and characterization of a DREB transcription factor cDNA, StDREB2, isolated from potato (cv Nicola) plants submitted to salt treatment. Based on a multiple sequence alignment, this protein was classified into the A-5 group of DREB subfamily. Expression studies revealed that StDREB2 was induced in leaves, roots and stems upon various abiotic stresses and in response to exogenous treatment with abscisic acid (ABA). In agreement with this expression pattern, over-expression of StDREB2 in transgenic potato plants resulted in enhanced tolerance to salt stress. These data suggest that the isolated StDREB2 encodes a functional protein involved in plant response to different abiotic stresses. An electrophoretic mobility shift assay (EMSA) indicated that the StDREB2 protein bound specifically to the DRE core element (ACCGAGA) in vitro. Moreover, Semi quantitative RT-PCR analysis revealed that the transcript level of a putative target gene i.e. δ(1)-pyrroline-5-carboxylate synthase (P5CS) was up-regulated in transgenic plants submitted to salt stress conditions. A concomitant increase in proline accumulation was also observed under these conditions. Taking together, all these data suggest that StDREB2 takes part in the processes underlying plant responses to abiotic stresses probably via the regulation of ABA hormone signaling and through a mechanism allowing proline synthesis.

Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction.

BACKGROUND: Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details. RESULTS: In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg2+/Mn2+, and that neither Zn2+ or nor Ca2+ can activate hTDP2. Mg2+ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg2+ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity. CONCLUSION: These results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.

Expression of StMYB1R-1, a novel potato single MYB-like domain transcription factor, increases drought tolerance.

Potato (Solanum tuberosum) is relatively vulnerable to abiotic stress conditions such as drought, but the tolerance mechanisms for such stresses in potato are largely unknown. To identify stress-related factors in potato, we previously carried out a genetic screen of potato plants exposed to abiotic environmental stress conditions using reverse northern-blot analysis. A cDNA encoding a putative R1-type MYB-like transcription factor (StMYB1R-1) was identified as a putative stress-response gene. Here, the transcript levels of StMYB1R-1 were enhanced in response to several environmental stresses in addition to drought but were unaffected by biotic stresses. The results of intracellular targeting and quadruple 9-mer protein-binding microarray analysis indicated that StMYB1R-1 localizes to the nucleus and binds to the DNA sequence (G)/(A)GATAA. Overexpression of a StMYB1R-1 transgene in potato plants improved plant tolerance to drought stress while having no significant effects on other agricultural traits. Transgenic plants exhibited reduced rates of water loss and more rapid stomatal closing than wild-type plants under drought stress conditions. In addition, overexpression of StMYB1R-1 enhanced the expression of drought-regulated genes such as AtHB-7, RD28, ALDH22a1, and ERD1-like. Thus, the expression of StMYB1R-1 in potato enhanced drought tolerance via regulation of water loss. These results indicated that StMYB1R-1 functions as a transcription factor involved in the activation of drought-related genes.

Characterization of a chickpea (Cicer arietinum L.) NAC family gene, CarNAC5, which is both developmentally- and stress-regulated.

It has been documented that the plant-specific NAC (for NAM, ATAF1,2 and CUC2) transcription factors play an important role in plant development and stress responses. In this study, a chickpea NAC gene CarNAC5 (for Cicer arietinum L. NAC gene 5) was isolated from a cDNA library from chickpea leaves treated by polyethylene glycol (PEG). CarNAC5, as a single/low copy gene, contained three exons and two introns within genomic DNA sequence and encoded a polypeptide with 291 amino acids. CarNAC5 protein had a conserved NAC domain in the N-terminus and showed high similarity to other NACs, especially ATAF subgroup members. The CarNAC5:GFP fusion protein was localized in the nucleus of onion epidermal cells. Furthermore, CarNAC5 protein activated the reporter genes LacZ and HIS3 in yeast. The transactivation activity was mapped to the C-terminal region. The transcripts of CarNAC5 appeared in many chickpea tissues including seedling leaves, stems, roots, flowers, seeds and pods, but mostly accumulated in flowers. Meanwhile, CarNAC5 was strongly expressed during seed maturation and in embryos of the early germinating seeds. It was also significantly induced by drought, heat, wounding, salicylic acid (SA), and indole-3-acetic acid (IAA) treatments. Our results suggest that CarNAC5 encodes a novel NAC-domain protein and acts as a transcriptional activator involved in plant developmental regulation and various stress responses.

Isolation and characterization of DMRT1 and its putative regulatory region in the protogynous wrasse, Halichoeres tenuispinis.

A full-length cDNA of doublesex and mab-3-related transcription factor 1 gene (DMRT1) from wrasse testis was isolated by cDNA library screening. Wrasse DMRT1 was 3116 bp in size and contained the DM domain, with a zinc finger DNA-binding motif, and the male-specific motif. Northern blot analysis identified a 3.2-kb transcript approximately equal in size to the DMRT1 nucleotide sequence detected in the testis, but not in the ovary, confirming that this sequence is male-specific in protogynous wrasse. Southern blot analysis suggested that the wrasse genome contains two copies of the DMRT1 gene. The ORF consisted of five exons and four introns with conserved donor-acceptor splice sites at all exon-intron junctions. The 5'-flanking region of the wrasse DMRT1 gene was isolated by DNA walking, and putative regulatory sites were identified by searching data bases. The 5'-flanking region was divided into 9 elements, then 17 DMRT-luciferase chimeric plasmids were constructed. By transient transfection into Cos-1 and TM4 cells, distal element I which contains GATA-binding sites and proximal element B containing the sex-determining region on Y chromosome gene (SRY) binding site were revealed to have an important role in transcriptional regulation of the wrasse DMRT1 when an enhancer sequence was provided.

Screening for compounds that affect the interaction between bacterial two-component signal transduction response regulator protein and cognate promoter DNA.

Bacterial signal transduction systems can be used as drug targets. The signal transduction targets fall into two groups--sensor kinases and response regulators. Previously reported studies describe hits that were thought to inactivate sensor kinases but on closer examination were found to act elsewhere instead; a possible reason for this is that full-length sensor kinases are integral membrane proteins whose activity might reflect interaction with the cell membrane or with membrane components. We describe a model system that instead is based on the interaction between a test compound and a response regulator in a homogeneous phase reaction. In this system, response regulator-DNA complex formation and its inhibition by a test compound are measured by fluorescence polarization. The model system should be readily adaptable to drug discovery based on other bacterial two-component s transduction systems.

Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.).

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

The recessive epigenetic swellmap mutation affects the expression of two step II splicing factors required for the transcription of the cell proliferation gene STRUWWELPETER and for the timing of cell cycle arrest in the Arabidopsis leaf.

Generally, cell division can be uncoupled from multicellular development, but more recent evidence suggests that cell cycle progression and arrest is coupled to organogenesis and growth. We describe a recessive mutant, swellmap (smp), with reduced organ size and cell number. This defect is partially compensated for by an increase in final cell size. The mutation causes a precocious arrest of cell proliferation in the organ primordium and possibly reduces the rate of cell division there. The mutation proved to be an epigenetic mutation (renamed smp(epi)) that defined a single locus, SMP1, but affected the expression of both SMP1 and a second very similar gene, SMP2. Both genes encode CCHC zinc finger proteins with similarities to step II splicing factors involved in 3' splice site selection. Genetic knockouts demonstrate that the genes are functionally redundant and essential. SMP1 expression is associated with regions of cell proliferation. Overexpression of SMP1 produced an increase in organ cell number and a partial decrease in cell expansion. The smp(epi) mutation does not affect expression of eukaryotic cell cycle regulator genes CYCD3;1 and CDC2A but affects expression of the cell proliferation gene STRUWWELPETER (SWP) whose protein has similarities to Med150/Rgr1-like subunits of the Mediator complex required for transcriptional activation. Introduction of SWP cDNA into smp(epi) plants fully restored them to wild-type, but the expression of both SMP1 and SMP2 were also restored in these lines, suggesting a physical interaction among the three proteins and/or genes. We propose that step II splicing factors and a transcriptional Mediator-like complex are involved in the timing of cell cycle arrest during leaf development.

The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response.

Regulation of iron uptake is critical for plant survival. Although the activities responsible for reduction and transport of iron at the plant root surface have been described, the genes controlling these activities are largely unknown. We report the identification of the essential gene Fe-deficiency Induced Transcription Factor 1 (FIT1), which encodes a putative transcription factor that regulates iron uptake responses in Arabidopsis thaliana. Like the Fe(III) chelate reductase FRO2 and high affinity Fe(II) transporter IRT1, FIT1 mRNA is detected in the outer cell layers of the root and accumulates in response to iron deficiency. fit1 mutant plants are chlorotic and die as seedlings but can be rescued by the addition of supplemental iron, pointing to a defect in iron uptake. fit1 mutant plants accumulate less iron than wild-type plants in root and shoot tissues. Microarray analysis shows that expression of many (72 of 179) iron-regulated genes is dependent on FIT1. We demonstrate that FIT1 regulates FRO2 at the level of mRNA accumulation and IRT1 at the level of protein accumulation. We propose a new model for iron uptake in Arabidopsis where FRO2 and IRT1 are differentially regulated by FIT1.

Arabidopsis response regulator, ARR22, ectopic expression of which results in phenotypes similar to the wol cytokinin-receptor mutant.

Arabidopsis thaliana has a number of response regulators (ARRs) implicated in the histidine (His)-->aspartate (Asp) phosphorelay signal transduction. According to the current consistent model, both the type-A and type-B ARR family members play crucial roles in the cytokinin signaling circuitry. However, this higher plant has a few extra ARRs, on which no attention has been paid so far. Characterization of these extra ARRs might provide us with new insight into the His-->Asp phosphorelay signal transduction in plants. For this reason, in this study we extensively examined the natures of such a representative (named ARR22). Transcripts of ARR22 were expressed predominantly in reproductive organs, and a GFP::ARR22 fusion protein was localized in the cytoplasmic space in onion epidermal cells. The purified ARR22 protein had the ability to undergo phosphorylation in vitro, when incubated with phospho-AHP5, indicating that ARR22 has the fundamental ability to participate into a His-Asp phosphorelay pathway in its own right. In plants, transgenic lines overexpressing ARR22 were characterized (referred to as ARR22-ox), which showed the characteristic dwarf phenotypes with poorly developed root systems. The results of Northern blot hybridization with selected sets of hormone-responsive genes suggested that cytokinin responses are selectively attenuated in ARR22-ox, while other hormone responses (auxin, ABA and ethylene) occur normally. The results of microarray analyses with cytokinin-treated wild-type and ARR22-ox plants further supported the view that cytokinin responses are globally attenuated in ARR22-ox, at least, at the level of gene regulation. Finally, we demonstrated that the dwarf phenotypes of ARR22-ox are very similar to those of the wooden leg (wol) mutant, which has a severe lesion in the AHK4/CRE1 cytokinin-receptor of histidine protein kinase. These results suggested that ARR22 might also be implicated, directly or indirectly, in the cytokinin-responsive His-->Asp phophorelay signal transduction.

Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes.

Phosphatidylinositol-3-phosphate [PtdIns(3)P] regulates endocytic and autophagic membrane traffic. In order to understand the downstream effects of PtdIns(3)P in these processes, it is important to identify PtdIns(3)P-binding proteins, many of which contain FYVE zinc-finger domains. Here, we describe a novel giant FYVE-domain-containing protein, named autophagy-linked FYVE protein (Alfy). Alfy is ubiquitously expressed, shares sequence similarity with the Chediak-Higashi-syndrome protein and has putative homologues in flies, nematodes and fission yeast. Alfy binds PtdIns(3)P in vitro and partially colocalizes with PtdIns(3)P in vivo. Unlike most other FYVE-domain proteins, Alfy is not found on endosomes but instead localizes mainly to the nuclear envelope. When HeLa cells are starved or treated with a proteasome inhibitor, Alfy relocalizes to characteristic filamentous cytoplasmic structures located close to autophagic membranes and ubiquitin-containing protein aggregates. By electron microscopy, similar structures can be found within autophagosomes. We propose that Alfy might target cytosolic protein aggregates for autophagic degradation.

VOZ; isolation and characterization of novel vascular plant transcription factors with a one-zinc finger from Arabidopsis thaliana.

A 38-bp pollen-specific cis-acting region of the AVP1 gene is involved in the expression of the Arabidopsis thaliana V-PPase during pollen development. Here, we report the isolation and structural characterization of AtVOZ1 and AtVOZ2, novel transcription factors that bind to the 38-bp cis-acting region of A. thaliana V-PPase gene, AVP1. AtVOZ1 and AtVOZ2 show 53% amino acid sequence similarity. Homologs of AtVOZ1 and AtVOZ2 are found in various vascular plants as well as a moss, Physcomitrella patens. Promoter-beta-glucuronidase reporter analysis shows that AtVOZ1 is specifically expressed in the phloem tissue and AtVOZ2 is strongly expressed in the root. In vivo transient effector-reporter analysis in A. thaliana suspension-cultured cells demonstrates that AtVOZ1 and AtVOZ2 function as transcriptional activators in the Arabidopsis cell. Two conserved regions termed Domain-A and Domain-B were identified from an alignment of AtVOZ proteins and their homologs of O. sativa and P. patens. AtVOZ2 binds as a dimer to the specific palindromic sequence, GCGTNx7ACGC, with Domain-B, which is comprised of a functional novel zinc coordinating motif and a conserved basic region. Domain-B is shown to function as both the DNA-binding and the dimerization domains of AtVOZ2. From highly the conservative nature among all identified VOZ proteins, we conclude that Domain-B is responsible for the DNA binding and dimerization of all VOZ-family proteins and designate it as the VOZ-domain.

Xenopus XsalF: anterior neuroectodermal specification by attenuating cellular responsiveness to Wnt signaling.

Here we show that XsalF, a frog homolog of the Drosophila homeotic selector spalt, plays an essential role for the forebrain/midbrain determination in Xenopus. XsalF overexpression expands the domain of forebrain/midbrain genes and suppresses midbrain/hindbrain boundary (MHB) markers and anterior hindbrain genes. Loss-of-function studies show that XsalF is essential for the expression of the forebrain/midbrain genes and for the repression of the caudal genes. Interestingly, XsalF functions by antagonizing canonical Wnt signaling, which promotes caudalization of neural tissues. XsalF is required for anterior-specific expressions of GSK3beta and Tcf3, genes encoding antagonistic effectors of Wnt signaling. Loss-of-function phenotypes of GSK3beta and Tcf3 mimic those of XsalF while injections of GSK3beta and Tcf3 rescue loss-of-function phenotypes of XsalF. These findings suggest that the forebrain/midbrain-specific gene XsalF negatively controls cellular responsiveness to posteriorizing Wnt signals by regulating region-specific GSK3beta and Tcf3 expression.

NF-kappa B and Oct-2 synergize to activate the human 3' Igh hs4 enhancer in B cells.

In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs) sites with enhancer activity. These include the 3' Igh enhancers, which are located downstream of the Calpha gene(s) in both mouse and human. In vivo experiments have implicated murine 3' enhancers, hs3B and/or hs4, in class switching and somatic hypermutation. We previously reported that murine hs4 was regulated by NF-kappaB, octamer binding proteins, and Pax5 (B cell-specific activator protein). In this study we report that human hs4 is regulated differently. EMSAs and Western analysis of normal B cells before and after stimulation with anti-IgM plus anti-CD40 showed the same complex binding pattern formed by NF-kappaB, Oct-1, and Oct-2 (but not by Pax5). A similar EMSA pattern was detected in mature human B cell lines (BL-2, Ramos, and HS-Sultan) and in diffuse large B cell lymphoma cell lines, although yin yang 1 protein (YY1) binding was also observed. We have confirmed the in vivo association of these transcription factors with hs4 in B cells by chromatin immunoprecipitation assays. The diffuse large B cell lymphoma cell lines had a distinctive slow-migrating complex containing YY1 associated with Rel-B. We have confirmed by endogenous coimmunoprecipitation an association of YY1 with Rel-B, but not with other NF-kappaB family members. Transient transfection assays showed robust hs4 enhancer activity in the mature B cell lines, which was dependent on synergistic interactions between NF-kappaB and octamer binding proteins. In addition, human hs4 enhancer activity required Oct-2 and correlated with expression of Oct coactivator from B cells (OCA-B).

Churchill, a zinc finger transcriptional activator, regulates the transition between gastrulation and neurulation.

Gastrulation generates mesoderm and endoderm from embryonic epiblast; soon after, the neural plate is established within the epiblast-both events require FGF signaling. We describe a zinc finger transcriptional activator, Churchill (ChCh), which acts as a switch between different roles of FGF. FGF induces ChCh slowly; this activates Smad-interacting-protein-1 (Sip1), which blocks further induction of the mesoderm markers brachyury and Tbx6L by FGF. ChCh is first expressed as cells stop migrating through the primitive streak, and we show that it regulates cell ingression. We propose a simple mechanism by which FGF sensitizes cells to BMP signals. These results reveal that neural induction requires cessation of mesoderm formation at the midline in addition to the decision between epidermis and neural plate.