Class Of DNA-binding Proteins Research Articles

Tomato SlSTK is involved in glucose response and regulated by the ubiquitin ligase SlSINA4

Glucose signaling plays an essential role in plant growth, development and stress response. Previous studies have shown that STOREKEEPER (STK) is a new class of DNA binding protein that regulates patatin expression in potato tubers and confers elevated sensitivity to glucose response in Arabidopsis thaliana. However, the biological functions of STK gene in tomato (Solanum lycopersicum) have not been studied. Here, we characterized the tomato SlSTK and determined its role in glucose signaling. The SlSTK protein was localized in the nucleus and the expression of the SlSTK gene was induced by the glucose treatment. Overexpression of SlSTK in tomato enhanced glucose sensitivity, as manifested by reduced seed germination rate and arrested growth at the early seedling stage. In contrast, the SlSTK-knockout plants generated via the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (CRISPR-Cas9) technique attenuated the sensitivity to glucose. In addition, SlSTK was ubiquitinated in plant cells and interacted with the tomato ubiquitin ligase SEVEN IN ABSENTIA4 (SlSINA4) that degrades SlSTK in a ligase-dependent manner. Taken together, these results suggest that SlSTK is involved in glucose signaling and its stability is regulated by the ubiquitin ligase SlSINA4.

The Effect of New Therapy Zinc on Perinatal Mortality, Prematurity and Placental Ablation

Zinc is essential for life and is a part of zinc finger proteins, which perhaps represent the largest class of DNA-binding proteins. Zinc finger protein controlled gene expression may be a fundamental aspect of development as well as other processes. Nearly 30 elements are accepted as being essential for some or all organisms. Knowledge of the amounts of zinc in food is necessary for the understanding of the very complex system of zinc metabolism. Zinc content in leucocytes, mononuclear cells, red cells and platelets could be used as a measure of possible zinc deficiency. Almost 300 different enzymes, which include the synthesis and/or degradation of all major metabolites, have zinc as an integral component. AE has been interpreted as being a zinc-deficiency disease. The signs and symptoms of AE are reserved by zinc therapy. The recommended daily intake of zinc in pregnancy and lactation is 15 mg in Sweden and the Scandinavian countries. A woman on continuous zinc therapy for AE had two normal pregnancies. Other contributors to this volume will review these data.

Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family

SummarySpecific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.

Abstract P3-07-16: ZBTB2 is a novel therapeutic target for cisplatin-resistance in metastatic breast cancer

Abstract Background: Zinc finger and BTB/POZ domain containing proteins (ZBTB) belong to a class of DNA-binding proteins that involved in development, differentiation and carcinogenesis. Our earlier findings showed that ZBTB2 expression was upregulated in cisplatin-resistant cells when compared with naïve cells. Increase in ZBTB2 expression was associated with ABCG2 drug transporter in drug resistance. However, the molecular mechanism of ZBTB2 in drug resistance and breast cancer metastasis remain largely unclear. Methods: A cisplatin-resistant triple-negative breast cancer (TNBC) cell line, MDA-MB-231/cis, was used to examine the involvement of drug transporter genes and the drug resistance pathway. Stem cell related genes were studied by RNA-sequencing. Characterization of ZBTB2 on cell proliferation, invasion and epithelial-mesenchymal transition (EMT) process were performed in ZBTB2 siRNA transfected cells. Stem-like cells properties were validated using aldehyde dehydrogenase (ALDH) activity, tumorsphere formation ability and stem cell markers. Metastatic animal model was used to study the genetic changes during metastasis and associated with the findings in clinical samples. Results: ZBTB2 has a higher expression in TNBC than other breast cancer subtypes. Also, the ZBTB2 expression is more prominent in primary breast tumor tissues when compared with non-tumor counterparts from TNBC patients. Silencing of ZBTB2 significantly reduced cell proliferation, invasion and sensitized cells to cisplatin through induction of apoptosis. Induction of ZBTB2 has been associated with breast cancer metastasis in mice model, where higher expression was seen in metastatic tumors when compared with primary tumors from tumor xenografts. ABCG2 and some epithelial-mesenchymal transition (EMT) markers (Twist 1, Snai2 and MMP-9) have been increased in metastatic tumors. Further, RNA sequencing analysis showed that ABCG2, IL6ST and FGFR1 were upregulated in MDA-MB-231/cis cells and downregulated in ZBTB2 siRNA transfected cells. Silencing of ZBTB2 reduced ALDH activity and tumorsphere formation ability. Treatment with inhibitor of NFkB (Bay11-7085) further decreased cell proliferation in cisplatin-resistant cells when compared with siZBTB2 alone. Expression of Stat3 and b-catenin were reduced upon knockdown of ZBTB2. Conclusion: Taken together, silencing of ZBTB2 sensitize cancer cells to cisplatin through regulation of EMT markers and stem-like cell properties in TNBC. These findings suggest a novel molecular pathway targeting ZBTB2/stat3/NFkB signaling to combat drug resistance in metastatic TNBC. Citation Format: Shin V, Siu MT, Cheuk I, Ho J, Chen J, Kwong A. ZBTB2 is a novel therapeutic target for cisplatin-resistance in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-16.

Genome wide analysis of stress responsive WRKY transcription factors in Arabidopsis thaliana

WRKY transcription factors are a class of DNA-binding proteins that bind with a specific sequence C/TTGACT/C known as W-Box found in promoters of genes which are regulated by these WRKYs. From previous studies, 43 different stress responsive WRKY transcription factors in Arabidopsis thaliana, identified and then categorized in three groups viz., abiotic, biotic and both of these stresses. A comprehensive genome wide analysis including chromosomal localization, gene structure analysis, multiple sequence alignment, phylogenetic analysis and promoter analysis of these WRKY genes was carried out in this study to determine the functional homology in Arabidopsis. This analysis led to the classification of these WRKY family members into 3 major groups and subgroups and showed evolutionary relationship among these groups on the base of their functional WRKY domain, chromosomal localization and intron/exon structure. The proposed groups of these stress responsive WRKY genes and annotation based on their position on chromosomes can also be explored to determine their functional homology in other plant species in relation to different stresses. The result of the present study provides indispensable genomic information for the stress responsive WRKY transcription factors in Arabidopsis and will pave the way to explain the precise role of various AtWRKYs in plant growth and development under stressed conditions.

Visualizing Specific Genomic Loci using Fluorescently Labeled Transcription Activator-Like Effectors

Understanding how gene transcription is spatially regulated by cis-regulatory elements (CREs) is both genetically and biomedically important. Cis-regulatory elements can be located thousands to millions of base pairs away from the promoter site, yet they somehow control and regulate transcription. Here we outline a new live cell, high resolution imaging methodology that allows for the visualization and quantification of these critical regulatory interactions. The method is based on the insertion of DNA sequences highly specific for a class of DNA binding proteins, known as Transcription Activator-Like Effectors (TALEs) into the human genome. TALEs are known to have high binding affinity and specificity to their target sites, and through conditional expression of fluorescently labeled TALEs, we are able to image specific chromosomal loci with improved signal-to-background and excellent localization precision. By using multicolor enhancer and promoter targeted TALEs, the technique can be coupled with super-resolution microscopy to detect and quantify subtle spatial changes occurring in the nucleus prior to or during transcriptional activation. This new methodology will help answer important questions on when, where and how genes are regulated during the process of cell differentiation or from external stresses and stimuli.

BinDNase: a discriminatory approach for transcription factor binding prediction using DNase I hypersensitivity data.

Transcription factors (TFs) are a class of DNA-binding proteins that have a central role in regulating gene expression. To reveal mechanisms of transcriptional regulation, a number of computational tools have been proposed for predicting TF-DNA interaction sites. Recent studies have shown that genome-wide sequencing data on open chromatin sites from a DNase I hypersensitivity experiments (DNase-seq) has a great potential to map putative binding sites of all transcription factors in a single experiment. Thus, computational methods for analysing DNase-seq to accurately map TF-DNA interaction sites are highly needed. Here, we introduce a novel discriminative algorithm, BinDNase, for predicting TF-DNA interaction sites using DNase-seq data. BinDNase implements an efficient method for selecting and extracting informative features from DNase I signal for each TF, either at single nucleotide resolution or for larger regions. The method is applied to 57 transcription factors in cell line K562 and 31 transcription factors in cell line HepG2 using data from the ENCODE project. First, we show that BinDNase compares favourably to other supervised and unsupervised methods developed for TF-DNA interaction prediction using DNase-seq data. We demonstrate the importance to model each TF with a separate prediction model, reflecting TF-specific DNA accessibility around the TF-DNA interaction site. We also show that a highly standardised DNase-seq data (pre)processing is a requisite for accurate TF binding predictions and that sequencing depth has on average only a moderate effect on prediction accuracy. Finally, BinDNase's binding predictions generalise to other cell types, thus making BinDNase a versatile tool for accurate TF binding prediction. R implementation of the algorithm is available in: http://research.ics.aalto.fi/csb/software/bindnase/. juhani.kahara@aalto.fi Supplemental data are available at Bioinformatics online.

Integrative Modeling of eQTLs and Cis-Regulatory Elements Suggests Mechanisms Underlying Cell Type Specificity of eQTLs

Genetic variants in cis-regulatory elements or trans-acting regulators frequently influence the quantity and spatiotemporal distribution of gene transcription. Recent interest in expression quantitative trait locus (eQTL) mapping has paralleled the adoption of genome-wide association studies (GWAS) for the analysis of complex traits and disease in humans. Under the hypothesis that many GWAS associations tag non-coding SNPs with small effects, and that these SNPs exert phenotypic control by modifying gene expression, it has become common to interpret GWAS associations using eQTL data. To fully exploit the mechanistic interpretability of eQTL-GWAS comparisons, an improved understanding of the genetic architecture and causal mechanisms of cell type specificity of eQTLs is required. We address this need by performing an eQTL analysis in three parts: first we identified eQTLs from eleven studies on seven cell types; then we integrated eQTL data with cis-regulatory element (CRE) data from the ENCODE project; finally we built a set of classifiers to predict the cell type specificity of eQTLs. The cell type specificity of eQTLs is associated with eQTL SNP overlap with hundreds of cell type specific CRE classes, including enhancer, promoter, and repressive chromatin marks, regions of open chromatin, and many classes of DNA binding proteins. These associations provide insight into the molecular mechanisms generating the cell type specificity of eQTLs and the mode of regulation of corresponding eQTLs. Using a random forest classifier with cell specific CRE-SNP overlap as features, we demonstrate the feasibility of predicting the cell type specificity of eQTLs. We then demonstrate that CREs from a trait-associated cell type can be used to annotate GWAS associations in the absence of eQTL data for that cell type. We anticipate that such integrative, predictive modeling of cell specificity will improve our ability to understand the mechanistic basis of human complex phenotypic variation.

DNA Sequence Preferences of Transcriptional Activators Correlate More Strongly than Repressors with Nucleosomes

SummaryTranscription factors (TFs) and histone octamers are two abundant classes of DNA binding proteins that coordinate the transcriptional program in cells. Detailed studies of individual TFs have shown that TFs bind to nucleosome-occluded DNA sequences and induce nucleosome disruption/repositioning, while recent global studies suggest this is not the only mechanism used by all TFs. We have analyzed to what extent the intrinsic DNA binding preferences of TFs and histones play a role in determining nucleosome occupancy, in addition to nonintrinsic factors such as the enzymatic activity of chromatin remodelers. The majority of TFs in budding yeast have an intrinsic sequence preference overlapping with nucleosomal histones. TFs with intrinsic DNA binding properties highly correlated with those of histones tend to be associated with gene activation and might compete with histones to bind to genomic DNA. Consistent with this, we show that activators induce more nucleosome disruption upon transcriptional activation than repressors.

Computational Modeling of the Binding Mode of Transcription Activator-Like Effectors to DNA

Transcription activator-like effectors (TALEs) form a novel class of DNA-binding proteins with predictable specificity. The TALE protein for avrBs3 is composed of 17.5 repeat domains of 34 amino acid residues, and each repeat domain recognizes one specific base-pair. The specificity is encoded by the repeat-variable diresidue (RVD) of each repeat domain, and this modular nature of DNA base recognition enables useful applications in biotechnology. However, there is very little available structural information about the binding mode and the specificity. In this study, we attempt to generate possible binding modes of the TALE with avrBs3 using protein structure modeling, protein-DNA docking, and molecular dynamics simulation. We discuss the observed binding specificities in terms of the molecular interactions found in the models.

Effect of <i>MIG1</i> Gene Deletion on Glucose Repression in Baker’s Yeast

Mig1p, a zinc finger class of DNA-binding protein, plays a critical role in glucose repression for maltose utilization in Baker’s yeast. Maltose is the hydrolyzate of starch, which is the main component of dough. In this study, the recombinant yeast strains with MIG1 gene deletion were constructed, and the maltose metabolism of the parental and mutant strains in batch cultivations were investigated. Our results show that the degree of glucose repression of mutants △MIG1α and △MIG1a are reduced by 49.88% and 41.59% respectively compared to their parental strains, suggesting that MIG1 deletion can partially relieve glucose repression of maltose metabolism.

E box motifs as mediators of proviral latency of human retroviruses

The palindromic sequence motifs (CANNTG) known as E boxes are considered as binding sites for the basic helix-loop-helix (bHLH) class of DNA-binding proteins. Their presence has been reported in the long terminal repeats (LTR) of the HIV-1 and HTLV-1 proviruses. Their close proximity with the TATA region of both LTRs indicates that the bHLH proteins may act as important regulators of the function of proviral transcription. Indeed, observations on HIV-1 and recent results on HTLV-1 underline that these E boxes may be critically involved in the regulation of the proviral transcription of these human retroviruses. Indeed, of the two E boxes flanking the TATA sequences of the HIV-1 provirus, the 3' E box has been implicated in the transcriptional inhibition of viral gene expression. Such a role might also be played by the unique 5' E box present in the HTLV-1 LTR. In both cases, the expression of tissue-specfic bHLH proteins, like TAL1 might counteract the inhibitory effect exerted by E box proteins, thereby increasing proviral transcription. Finally, a phylogenetic study encompassing several subtypes of these two human retroviruses underlines that these E box motifs have recently appeared in the proviral LTRs and may be considered as potential mediators in the establishment of proviral latency.

Silent chromatin at the middle and ends: lessons from yeasts

Eukaryotic centromeres and telomeres are specialized chromosomal regions that share one common characteristic: their underlying DNA sequences are assembled into heritably repressed chromatin. Silent chromatin in budding and fission yeast is composed of fundamentally divergent proteins tat assemble very different chromatin structures. However, the ultimate behaviour of silent chromatin and the pathways that assemble it seem strikingly similar among Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe) and other eukaryotes. Thus, studies in both yeasts have been instrumental in dissecting the mechanisms that establish and maintain silent chromatin in eukaryotes, contributing substantially to our understanding of epigenetic processes. In this review, we discuss current models for the generation of heterochromatic domains at centromeres and telomeres in the two yeast species.

CEBPA polymorphisms and mutations in patients with acute myeloid leukemia, myelodysplastic syndrome, multiple myeloma and non-Hodgkin's lymphoma

The transcription factor CCAAT/enhancer binding protein (C/EBP)α is a myeloid-specific transcription factor which is required for normal myeloid differentiation. C/EBPα is encoded by an intronless gene that is 2783 bp long and maps to human chromosome 19q13.1. C/EBPα is a member of the basic region leucine zipper (bZIP) class of DNA-binding proteins. The loss of function of C/EBPα has leukemogenic potential. Four types of polymorphisms and 25 mutations (3 already known mutations and 22 novel mutations) were detected in CEBPA (gene for the transcription factor CCAAT/enhancer binding protein (C/EBP) α) in analysed samples from 390 patients with myelodysplastic syndrome (MDS) and hematologic malignancies. CEBPA mutations were found in 14/152 (9.2%) of acute myeloid leukemia (AML) patients' samples, 6/143 (4.2%) of MDS patients' samples, 2/56 (3.6%) of non-Hodgkin's lymphoma (NHL) patients' samples and 2/39 (5.1%) of multiple myeloma (MM) patients' samples. No C/EBPα mutations were detected in healthy donors (41 individuals). We discuss how these mutations can affect the cellular function of C/EBPα and block the myeloid differentiation.

PREDICTION OF DNA-BINDING RESIDUES FROM SEQUENCE FEATURES

Protein-DNA interaction plays a pivotal role in transcriptional regulation, DNA metabolism and chromatin formation. Although structural data are available for a few hundreds of protein-DNA complexes, the molecular recognition pattern is still poorly understood. With the rapid accumulation of sequence data from many genomes, it is important to develop predictive methods for identification of potential DNA-binding residues in proteins. In this study, neural networks have been trained using five sequence-derived features for prediction of DNA-binding residues. These features include the molecular mass, hydrophobicity index, side chain pKa value, solvent accessible surface area and conservation score of an amino acid. Interestingly, the side chain pKa value appears to be the best feature for prediction, suggesting that the ionization state of amino acid side chains is important for DNA-binding. The predictive performance is enhanced by using multiple features for classifier construction. The classifier that has been constructed using all the five features predicts at 72.71% sensitivity and 67.73% specificity. This is by far the most accurate classifier reported for prediction of DNA-binding residues from sequence data. The classifier has also been evaluated by using the Receiver Operating Characteristic curve and by examining the predictions made for different classes of DNA-binding proteins. Supplementary materials including the datasets are available at http://bioinformatics.ksu.edu/pdi/feature.html.

Minor Groove Deformability of DNA: A Molecular Dynamics Free Energy Simulation Study

The conformational deformability of nucleic acids can influence their function and recognition by proteins. A class of DNA binding proteins including the TATA box binding protein binds to the DNA minor groove, resulting in an opening of the minor groove and DNA bending toward the major groove. Explicit solvent molecular dynamics simulations in combination with the umbrella sampling approach have been performed to investigate the molecular mechanism of DNA minor groove deformations and the indirect energetic contribution to protein binding. As a reaction coordinate, the distance between backbone segments on opposite strands was used. The resulting deformed structures showed close agreement with experimental DNA structures in complex with minor groove-binding proteins. The calculated free energy of minor groove deformation was ∼4–6 kcal mol −1 in the case of a central TATATA sequence. A smaller equilibrium minor groove width and more restricted minor groove mobility was found for the central AAATTT and also a significantly (∼2 times) larger free energy change for opening the minor groove. The helical parameter analysis of trajectories indicates that an easier partial unstacking of a central TA versus AT basepair step is a likely reason for the larger groove flexibility of the central TATATA case.

Kinetic analysis of operator binding by the E. coli methionine repressor highlights the role(s) of electrostatic interactions

MetJ is a member of the ribbon-helix-helix class of DNA-binding proteins whose affinity for operators is apparently controlled by an unprecedented long-range electrostatic effect from the tertiary sulphur atom of its co-repressor, S-adenosyl methionine. We report here the results of kinetic assays of DNA binding with MetJ mutant proteins having altered net charges. The results (a) suggest that MetJ locates its operators via a sliding mechanism, (b) support the idea that electrostatic steering is important in the initial DNA binding event and (c) highlight the sensitivity of this system to electrostatic effects.

Transcriptional regulation by calcium, calcineurin, and NFAT.

The NFAT family of transcription factors encompasses five proteins evolutionarily related to the Rel/NF B family (Chytil and Verdine 1996; Graef et al. 2001b). The primordial family member is NFAT5, the only NFATrelated protein represented in the Drosophila genome. NFAT5 is identical to TonEBP (tonicity element binding protein), a transcription factor crucial for cellular responses to hypertonic stress (Lopez-Rodriguez et al. 1999; Miyakawa et al. 1999). We focus here on the remaining four NFAT proteins (NFAT1–NFAT4, also known as NFATc1–c4; see Table 1), referring to them collectively as NFAT. The distinguishing feature of NFAT is its regulation by Ca and the Ca/calmodulin-dependent serine phosphatase calcineurin. NFAT proteins are phosphorylated and reside in the cytoplasm in resting cells; upon stimulation, they are dephosphorylated by calcineurin, translocate to the nucleus, and become transcriptionally active, thus providing a direct link between intracellular Ca signaling and gene expression. NFAT activity is further modulated by additional inputs from diverse signaling pathways, which affect NFAT kinases and nuclear partner proteins. In the first part of this review, we describe the influence of these multiple inputs on the nuclear–cytoplasmic distribution and transcriptional function of NFAT. Recent structural data emphasize the remarkable versatility of NFAT binding to DNA. At composite NFAT:AP-1 elements found in the regulatory regions of many target genes, NFAT proteins bind cooperatively with an unrelated transcription factor, AP-1 (Fos–Jun; Chen et al. 1998). At DNA elements that resemble NF B sites, NFAT proteins bind DNA as dimers (Giffin et al. 2003; Jin et al. 2003). In the second section of this review, we describe these two modes of DNA binding by NFAT. NFAT also acts synergistically with transcription factors other than Fos and Jun, but the structural basis for synergy remains unknown. Drawing on published structures, we discuss the potential cooperation of NFAT with other classes of DNA-binding proteins. It is clear that NFAT activates transcription of a large number of genes during an effective immune response (Rao et al. 1997; Kiani et al. 2000; Serfling et al. 2000; Macian et al. 2001). In the third part of this review, we present information obtained from these studies, highlighting experimental and bioinformatics approaches to identifying NFAT target genes. We discuss the finding that NFAT and NFAT–Fos–Jun complexes activate distinct subsets of target genes in lymphocytes (Macian et al. 2002). We also describe a novel aspect of gene regulation by NFAT, in which this transcription factor participates in an early phase of chromatin remodeling that occurs at specific genetic loci in differentiating T cells (Avni et al. 2002). There is evidence that NFAT regulates cell differentiation programs in cell types other than immune cells (Crabtree and Olson 2002; Horsley and Pavlath 2002; Graef et al. 2003; Hill-Eubanks et al. 2003). In the last section of this review, we select three differentiation programs—fiber-type specification in differentiated skeletal muscle, cardiac valve development, and osteoclast differentiation—for detailed consideration. We evaluate the evidence for NFAT involvement, point out novel cellular and molecular mechanisms that might regulate this familiar transcription factor, and discuss how NFAT exerts its biological effects. Because the phenotypes of NFAT knockout mice have been reviewed elsewhere (Crabtree and Olson 2002; Horsley and Pavlath 2002), we refer to them only as necessary to illustrate specific points.

Interactions of the Mcm1 MADS box protein with cofactors that regulate mating in yeast.

The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals. Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites. To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast. Our results indicate which residues of Mcm1 are essential for viability and transcriptional regulation with its cofactors in vivo. Most of the mutations in Mcm1 that are lethal affect DNA-binding affinity. Interestingly, the lethality of many of these mutations can be suppressed if the MCM1 gene is expressed from a high-copy-number plasmid. Although many of the alanine substitutions affect the ability of Mcm1 to activate transcription alone or in combination with the alpha 1 and Ste12 cofactors, most mutations have little or no effect on Mcm1-mediated repression in combination with the alpha 2 cofactor. Even nonconservative amino acid substitutions of residues in Mcm1 that directly contact alpha 2 do not significantly affect repression. These results suggest that within the same region of the Mcm1 MADS box domain, there are different requirements for interaction with alpha 2 than for interaction with either alpha1 or Ste12. Our results suggest how a small domain, the MADS box, interacts with multiple cofactors to achieve specificity in transcriptional regulation and how subtle differences in the sequences of different MADS box proteins can influence the interactions with specific cofactors while not affecting the interactions with common cofactors.

Storekeeper defines a new class of plant-specific DNA-binding proteins and is a putative regulator of patatin expression.

The expression of class I patatin genes is restricted to potato tubers but can be induced in other tissues by exogenous sucrose. Here we show that tuber-specific and sucrose-inducible gene expression is reduced in transgenic potato plants by mutations in a conserved 10 base pair motif within the B-box of the patatin promoter. In a southwestern screen, we have isolated a novel DNA-binding protein designated Storekeeper (STK) that specifically recognises the B-box motif in vitro. Gel shift experiments with an STK-specific antibody suggest that STK is the B-box binding protein found in tuber nuclei. We propose that STK, the defining member of a new class of DNA binding proteins, regulates patatin expression in potato tubers via the B-box motif.