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Nucleic acid-binding proteins are traditionally divided into two categories: With the ability to bind DNA or RNA. In the light of new knowledge, such categorizing should be overcome because a large proportion of proteins can bind both DNA and RNA. Another even more important features of nucleic acid-binding proteins are so-called sequence or structure specificities. Proteins able to bind nucleic acids in a sequence-specific manner usually contain one or more of the well-defined structural motifs (zinc-fingers, leucine zipper, helix-turn-helix, or helix-loop-helix). In contrast, many proteins do not recognize nucleic acid sequence but rather local DNA or RNA structures (G-quadruplexes, i-motifs, triplexes, cruciforms, left-handed DNA/RNA form, and others). Finally, there are also proteins recognizing both sequence and local structural properties of nucleic acids (e.g., famous tumor suppressor p53). In this mini-review, we aim to summarize current knowledge about the amino acid composition of various types of nucleic acid-binding proteins with a special focus on significant enrichment and/or depletion in each category.

The highly efficient C 4 photosynthetic pathway is facilitated by 'Kranz' leaf anatomy. In Kranz leaves, closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and mesophyll (outer) cells. Here, we demonstrate that, in the C 4 monocot maize, Kranz patterning is regulated by redundant function of SCARECROW 1 (ZmSCR1) and a previously uncharacterized homeologue: ZmSCR1h. ZmSCR1 and ZmSCR1h transcripts accumulate in ground meristem cells of developing leaf primordia and in Zmscr1;Zmscr1h mutant leaves, most veins are separated by one rather than two mesophyll cells; many veins have sclerenchyma above and/or below instead of mesophyll cells; and supernumerary bundle sheath cells develop. The mutant defects are unified by compromised mesophyll cell development. In addition to Kranz defects, Zmscr1;Zmscr1h mutants fail to form an organized endodermal layer in the root. Collectively, these data indicate that ZmSCR1 and ZmSCR1h redundantly regulate cell-type patterning in both the leaves and roots of maize. Leaf and root pathways are distinguished, however, by the cell layer in which they operate - mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Bladder cancer is among the most common cancers all over the world. The function of basic leucine zipper and W2 domains 2 (BZW2) in tumour progression has been reported. However, the biological function of BZW2 in muscle-invasive bladder cancers (MIBCs) remains to be determined. The aim of the present study was to reveal the expression and roles of BZW2 in human MIBCs and to explore the molecular mechanisms underlying these functions. Clinically, BZW2 expression was higher in MIBC tissues than the adjacent non-tumour tissues. Knocking down BZW2 using shRNA inhibited cell proliferation and G1/S cell cycle progression in vitro, and induced apoptosis in both 5637 and T24 cells. Moreover, in vivo studies with mice xenograft models confirmed the anti-proliferative effects of BZW2-knockdown, providing a future therapeutic target. We also performed biochemical microarray analysis to identify the potential signalling pathways, disease states and functions which could be affected by suppressing BZW2 in MIBC cells. Collectively, our findings suggest BZW2 has an oncogenic role in MIBCs and serves as a promising target for molecular diagnosis and gene therapy., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Breast invasive micropapillary carcinoma (IMPC) is a rare subtype of breast cancer with a high potential of lymph node metastasis, aggressive clinical behavior, and poor disease-free or overall survival. Expression of leucine zipper putative tumor suppressor 1 (LZTS1) was frequently lost or reduced in breast cancer tissues. This study investigated the expression of LZTS1 protein in breast IMPC tissues using immunohistochemistry. In addition, somatic LZTS1 mutations and promoter methylation were assessed to determine an association with clinicopathological data from IMPC patients. LZTS1 protein was downregulated in 62 (62 %) of 100 IMPC tissue samples and was significantly associated with lymph node metastasis (P < 0.05). A LZTS1 exon mutation occurred in one of the 53 IMPC cases analyzed, whereas a LZTS1 intron mutation occurred in 26 of 53 cases. Moreover, LZTS1 promoter was frequently methylated in IMPC samples and was associated with reduced LZTS1 expression levels in IMPC tissues. These data demonstrated that the loss of LZTS1 expression was associated with lymph node metastasis in patients with IMPC, and LZTS1 promoter methylation could be responsible for the loss of LZTS1 expression.

As sessile organisms, plants have to continuously adjust growth and development to ever-changing environmental conditions. At the end of the growing season, annual plants induce leaf senescence to reallocate nutrients and energy-rich substances from the leaves to the maturing seeds. Thus, leaf senescence is a means with which to increase reproductive success and is therefore tightly coupled to the developmental age of the plant. However, senescence can also be induced in response to sub-optimal growth conditions as an exit strategy, which is accompanied by severely reduced yield. Here, we show that class III homeodomain leucine zipper (HD-ZIPIII) transcription factors, which are known to be involved in basic pattern formation, have an additional role in controlling the onset of leaf senescence in Arabidopsis. Several potential direct downstream genes of the HD-ZIPIII protein REVOLUTA (REV) have known roles in environment-controlled physiological processes. We report that REV acts as a redox-sensitive transcription factor, and directly and positively regulates the expression of WRKY53, a master regulator of age-induced leaf senescence. HD-ZIPIII proteins are required for the full induction of WRKY53 in response to oxidative stress, and mutations in HD-ZIPIII genes strongly delay the onset of senescence. Thus, a crosstalk between early and late stages of leaf development appears to contribute to reproductive success., (© 2014. Published by The Company of Biologists Ltd.)

Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in the stramenopile alga, Vaucheria frigida. AUREO1 harbors a basic leucine zipper (bZIP) domain at the N-terminus and a light-oxygen-voltage-sensing (LOV) domain within the C-terminal region, and has been suggested to function as a light-regulated transcription factor. To understand the molecular mechanism of AUREO1, we have prepared three recombinant proteins: a full-length AUREO1 (FL), an N-terminal truncated construct containing bZIP and LOV (ZL) and a LOV-only (LOV) construct. The constructs showed the same absorption and fluorescent spectra in the dark state and underwent the characteristic cyclic reaction as previously observed in LOV domains upon BL excitation. FL and ZL bound to DNA in a sequence-specific manner. BL appeared to induce a shift of the α-helical structure of the LOV domain to a β-sheet structure, but did not alter the hydrodynamic radius (R(H)) of this domain. ZL formed a dimer possibly through disulfide linkages in the bZIP and the linker region between bZIP and LOV. BL induced an approximately 5% increase in the R(H) of ZL, although its secondary structure was unchanged. These results support a schema where BL-induced changes in the LOV domain may cause conformational changes in the bZIP and/or the linker of a dimeric ZL molecule. Since a 5% increase of the R(H) was also observed with the FL construct, BL may induce global conformational changes similar to those observed for ZL, and formation of the FL dimer may facilitate DNA binding.

Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between alpha helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of alpha-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete alpha-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 A resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.

We describe a unique family of human proteins that are capable of binding to the cAMP regulatory element (CRE) and that are homologous to RNA splicing proteins. A human cDNA was isolated that encodes a protein with a distinctive combination of modular domain structures: 2 leucine-zipper-like domains, a DNA-binding zinc-finger-like domain, an RNA-binding zinc-finger-like domain, and 2 coiled-coil protein-protein interaction domains. It also has a serine-arginine-rich domain, commonly found in proteins involved in RNA splicing. The protein was discovered using the CRE as bait in a yeast 1-hybrid assay. It was then shown to bind specifically to the CRE in vitro using gel shift assays. We have named the protein CRE-associated protein (CREAP). We show that it is widely expressed in human tissues but is highly expressed in several fetal tissues and in several regions of the adult brain. CREAP is closely related to 2 human proteins of unknown function. CREAP shows significant homology with a small nuclear ribonucleoprotein of yeast, Luc7p, involved in 5' splice site recognition. The 3 human CREAP proteins form a unique family with the potential to act as transcription factors that link to RNA processing.

Recent studies indicate that NF-E2 related factor 2 (Nrf2) is a substrate for the ubiquitin-proteasome pathway. The present study is aimed to determine whether increased protein stability is a mechanism by which quinone compounds, like tert-butylhydroquinone (tBHQ), may enhance Nrf2-mediated transcriptional activation and subsequent antioxidant protection. H2O2-induced necrotic cell death, evidenced by transmission electronic microscope (TEM) imaging with no caspase 3 activation and PARP cleavage, was significantly attenuated by pretreatment with tBHQ or overexpression of Nrf2 through advenovirus-mediated infection in human neural stem cells (hNSCs). Microarray analysis showed that those identified antioxidant genes, responsible for antiapoptotic action in IMR-32 cells (J. Li et al., 2002, J. Biol. Chem. 277, 388-394), were also coordinately upregulated through Nrf2-dependent antioxidant responsive element (ARE) activation in hNSC. The stabilization of Nrf2 by tBHQ in IMR-32 cells was evidenced by a pulse-chase assay showing no significant increase in Nrf2 protein synthesis after tBHQ treatment, and by ubiquitin immunoprecipitation showing that tBHQ stabilized ubiquitinated Nrf2. An in vitro proteasomal activity assay showed that tBHQ did not act as a 20S/26S proteasome inhibitor. Nrf2 stabilization by tBHQ also was observed in hNSCs. Taken together, this study suggests that identified antioxidant genes, which were upregulated through tBHQ induced Nrf2 stabilization, confer protection on target cells against H2O2-induced apoptotic cell death in neuroblastoma cells as well as the necrotic cell death in the hNSC. Nrf2 stabilization by pharmacological modulation or adenovirus-mediated Nrf2 overexpression, therefore, might be viable strategies to prevent a wide-spectrum of oxidative stress-related neuronal cell injuries.

Chalcone synthase (CHS), chalcone flavanone isomerase (CFI), flavanone 3-hydroxylase (F3H) and flavonol synthase (FLS) catalyze successive steps in the biosynthetic pathway leading to the production of flavonols. We show that in Arabidopsis thaliana all four corresponding genes are coordinately expressed in response to light, and are spatially coexpressed in siliques, flowers and leaves. Light regulatory units (LRUs) sufficient for light responsiveness were identified in all four promoters. Each unit consists of two necessary elements, namely a MYB-recognition element (MRE) and an ACGT-containing element (ACE). C1 and Sn, a R2R3-MYB and a BHLH factor, respectively, known to control tissue specific anthocyanin biosynthesis in Z. mays, were together able to activate the AtCHS promoter. This activation of the CHS promoter required an intact MRE and a newly identified sequence designated R response element (RREAtCHS) containing the BHLH factor consensus binding site CANNTG. The RRE was dispensable for light responsiveness, and the ACE was not necessary for activation by C1/Sn. These data suggest that a BHLH and a R2R3-MYB factor cooperate in directing tissue-specific production of flavonoids, while an ACE-binding factor, potentially a BZIP, and a R2R3-MYB factor work together in conferring light responsiveness.

In a previous study, a gst gene was isolated from the fission yeast Schizosaccharomyces pombe. This gene was dubbed gst I, and was characterized using the gstI-lacZ fusion plasmid pYSH2000. In this work, four additional fusion plasmids, pYSHSD1, pYSHSD2, pYSHSD3 and pYSHSD4, were constructed, in order to carry (respectively) 770, 551, 358 and 151 bp upstream regions from the translational initiation point. The sequence responsible for induction by aluminum, mercury and hydrogen peroxide was located in the range between -1,088 and -770 bp upstream of the S. pombe gst I gene. The same region was identified to contain the nucleotide sequence responsible for regulation by Pap1, and has one putative Pap1 binding site, TTACGTAT, located in the range between -954 approximately -947 bp upstream of the gst I gene. Negatively acting sequences are located between -1,088 and -151 bp. These findings imply that the Pap1 protein is involved in basal and inducible transcription of the gst I gene in the fission yeast S. pombe.

Background & Objective: Papillary thyroid carcinoma is characterized by RET (rearranged during transfection)/PTC (papillary thyroid carcinoma) rearrangements. However, the function of RET/PTC in carcino- genesis is not well understood. This study was designed to investigate the interaction between DNA double-strand break sensor ATM (mutated in ataxia telangiectasia) kinase and PTC1, a rearranged form of proto-oncogene ret, to explore the role of ret rearrangements in carcinogenesis., Methods: RET TK phosphorylation was determined by in vitro kinase assay using the immunoprecipitation with anti-ATM antibody as kinase and the immunoprecipitation of HA-tagged TK as substrate. The location of ATM-LZPR in COS7 cells coexpressed with PTC1 was investigated by protein extracts of cytoplasm and nucleus. The phosphorylation level of p53, was determined by Western blot analysis with the antibody against phosphorylated p53, and the cell cycle was determined by flow cytometry when PTC1 overexpressed., Results: ATM directly phosphorylated TK domain of PTC1 in vitro kinase assay. ATM-LZPR was located in both cell cytoplasm and nucleus when PTC1 was not expressed, however, co-overexpression of PTC1 and ATM-LZPR made the latter locate only in the cytoplasm. In addition, overexpression of PTC1 inhibited the phosphorylation level of p53 by ATM and caused G(1)/S phase arrest of cell cycle., Conclusions: PTC1 may remain ATM kinase in cytoplasm and inhibit the phosphorylation of p53 by ATM. PTC1, a rearrangement form of ret, may result in the disorder of cell damage repair and cell cycle checkpoint and destroy cell homeostasis.

Lipid synthesis is under tight transcriptional control involving sterol regulatory element binding proteins (SREBP1, SREBP2). Rising cellular cholesterol levels prevent SREBP2 from entering the nucleus to directly activate the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr), the key enzyme of cholesterol synthesis. The failure to down-regulate cholesterol synthesis in gallstone-susceptible C57L/J but not AKR/J mice prompted us to study the processing of SREBP2 in these mice. Male mice of each strain received a control or lithogenic diet for 28 days. Membrane and nuclear extracts of pooled livers were prepared for immunoblot analysis of SREBP. Steady-state mRNA levels of Hmgcr, Srebp1, Srebp2 and the SREBP cleavage activating protein (Scap) were measured from individual livers. There was no marked difference between the two strains with regard to processing of SREBP1 as well as steady-state mRNA levels of Srebp1, Srebp2 and Scap. However, a near-complete suppression of nuclear SREBP2 related to low Hmgcr mRNA levels was noticed only for gallstone-resistant AKR mice. Abnormal regulation of SREBP2 appears to be responsible for the failure to suppress cholesterol synthesis in genetically cholesterol gallstone-susceptible mice. This defect may contribute to cholesterol hypersecretion and gallstone formation.

The antioxidant responsive element (ARE) is a cis-acting regulatory element of genes encoding phase II detoxification enzymes and antioxidant proteins, such as NAD(P)H: quinone oxidoreductase 1, glutathione S-transferases, and glutamate-cysteine ligase. Interestingly, it has been reported that Nrf2 (NF-E2-related factor 2) regulates a wide array of ARE-driven genes in various cell types. Nrf2 is a basic leucine zipper transcription factor, which was originally identified as a binding protein of locus control region of beta-globin gene. The DNA binding sequence of Nrf2 and ARE sequence are very similar, and many studies demonstrated that Nrf2 binds to the ARE sites leading to up-regulation of downstream genes. The function of Nrf2 and its downstream target genes suggests that the Nrf2-ARE pathway is important in the cellular antioxidant defense system. In support of this, many studies showed a critical role of Nrf2 in cellular protection and anti-carcinogenicity, implying that the Nrf2-ARE pathway may serve as a therapeutic target for neurodegenerative diseases and cancers, in which oxidative stress is closely implicated.

A 6-bp sequence, ACTCAT, acts as a cis-acting element involved in hypoosmolarity- and proline-responsive expression of an Arabidopsis proline dehydrogenase (ProDH) gene. Search of the database for plant cis-acting elements revealed that the ACTCAT sequence is similar to the GCN4 motif [ATGA(C/G)TCAT] that is recognized by bZIP transcription factors. To identify transcription factor(s) for regulation of ProDH, we examined whether Arabidopsis bZIPs function as transcription factors for the ACTCAT sequence. Transient expression analysis revealed that the four proteins in Group S bZIPs, AtbZIP11/ATB2, AtbZIP44, AtbZIP2/GBF5 and AtbZIP53, formed an ATB2 subgroup that activated expression of the GUS reporter gene driven by the ACTCAT sequence while other bZIPs and different families of plant transcription factors did not. The transactivation activity of the ATB2 subgroup was enhanced in a hypoosmotic condition. In a gel mobility shift assay, the recombinant proteins of the ATB2 subgroup specifically bound to the ACTCAT sequence. RNA gel blot analysis indicated that the expression of AtbZIP2/GBF5 and AtbZIP53, as well as that of ProDH, is induced by hypoosmolarity. Moreover, we showed that the sGFP::AtbZIP11/ATB2 fusion protein is localized in the nucleus. These results suggest that the ATB2 subgroup functions as a transcriptional activator for hypoosmolarity-inducible ProDH in Arabidopsis:

The truncated myc and max proteins, only containing basic regions and helix-loop-helix/zipper (b/HLH/Zip) regions were over-expressed in E. coli and used for the determination of the binding constant and of the inhibitory mechanism on myc-max (or max-max)-DNA complex formation. The association kinetic constants (k(1) and k(-1)) of truncated max-max or myc-max dimer and DNA were determined as k(1)=(1.7+/-0.6)x10(5) M(-1) s(-1), k(-1)=(3.4+/-1.2)x10(-2) s(-1) for max-max and DNA or k(1)=(2.1+/-0.7)x10(5) M(-1) s(-1), k(-1)=(3.2+/-1.4)x10(-2) s(-1) for myc-max and DNA. The equilibrium binding constant (K(1)) was determined using these kinetic parameters [K(XXD)=(7.8+/-2.6)x10(6) M(-1) for max-max and DNA or K(XYD)=(6.9+/-2.2)x10(6) M(-1) for myc-max and DNA]. The binding constants of myc-max or max-max dimer formation were K(XX)=(2.6+/-0.9)x10(5) M(-1) or K(XY)=(1.3+/-0.4)x10(4) M(-1), respectively. When truncated proteins were used, the max-max dimer formation was easier than the myc-max dimer formation, contrary to the physiologically determined case. This leads us to deduce that domains other than b/HLH/Zip are very important for the transcriptional regulatory activity in physiological conditions. The truncated myc and max proteins, which were expressed in E. coli and contained only b/HLH/Zip regions were also used for the screening of inhibitors of myc-max-DNA complex formation. A synthesized curcuminoid, 1,7-bis(4-methyl-3-nitrophenyl)-1,6-heptadiene-3,5-dione (curcuminoid 004), showed the most potent inhibition out of the synthesized curcuminoids, in competition with DNA. The dissociation constant of max-max dimer and the inhibitor was 9 microM, when investigated using in vitro expressed b/HLH/Zip dimer proteins. The curcuminoid 004 showed an inhibitory effect on the binding of myc-max protein to the E-box element in SNU16 cells, and suppressed the expression of myc target genes including ornithine decarboxylase (ODC), cdc25a and c-myc in myc over-expressed human stomach cancer cell line SNU16.

Foxp1, Foxp2, and Foxp4 are large multidomain transcriptional regulators belonging to the family of winged-helix DNA binding proteins known as the Fox family. Foxp1 and Foxp2 have been shown to act as transcriptional repressors, while regulatory activity of the recently identified Foxp4 has not been determined. Given the importance of this Fox gene subfamily in neural and lung development, we sought to elucidate the mechanisms by which Foxp1, Foxp2, and Foxp4 repress gene transcription. We show that like Foxp1 and Foxp2, Foxp4 represses transcription. Analysis of the N-terminal repression domain in Foxp1, Foxp2, and Foxp4 shows that this region contains two separate and distinct repression subdomains that are highly homologous termed subdomain 1 and subdomain 2. However, subdomain 2 is not functional in Foxp4. Screening for proteins that interact with subdomains 1 and 2 of Foxp2 using yeast two-hybrid analysis revealed that subdomain 2 binds to C-terminal binding protein 1, which can synergistically repress transcription with Foxp1 and Foxp2, but not Foxp4. Subdomain 1 contains a highly conserved leucine zipper similar to that found in N-myc and confers homo- and heterodimerization to the Foxp1/2/4 family members. These interactions are dependent on the conserved leucine zipper motif. Finally, we show that the integrity of this subdomain is essential for DNA binding, making Foxp1, Foxp2, and Foxp4 the first Fox proteins that require dimerization for DNA binding. These data reveal a complex regulatory mechanism underlying Foxp1, Foxp2, and Foxp4 activity, demonstrating that Foxp1, Foxp2, and Foxp4 are the first Fox proteins reported whose activity is regulated by homo- and heterodimerization.

The Arabidopsis NPR1 protein is essential for regulating salicylic acid-dependent gene expression during systemic acquired resistance. NPR1 interacts differentially with members of the TGA class of basic domain/Leu zipper transcription factors and regulates their DNA binding activity. Here, we report that although TGA1 does not interact with NPR1 in yeast two-hybrid assays, treatment with salicylic acid induces the interaction between these proteins in Arabidopsis leaves. This phenomenon is correlated with a reduction of TGA1 Cys residues. Furthermore, site-directed mutagenesis of TGA1 Cys-260 and Cys-266 enables the interaction with NPR1 in yeast and Arabidopsis. Together, these results indicate that TGA1 relies on the oxidation state of Cys residues to mediate the interaction with NPR1. An intramolecular disulfide bridge in TGA1 precludes interaction with NPR1, and NPR1 can only stimulate the DNA binding activity of the reduced form of TGA1. Unlike its animal and yeast counterparts, the DNA binding activity of TGA1 is not redox regulated; however, this property is conferred by interaction with the NPR1 cofactor.

Two potential breast cancer susceptibility genes, encoding the BRCA1-interacting proteins ZNF350 (or ZBRK1) and BRIP1 (or BACH1), have been identified in yeast two-hybrid screens. We sequenced these genes in probands from 21 families with potentially inherited breast/ovarian cancer, all of which were negative for BRCA1/BRCA2 mutations. Families had at least one case of male breast cancer, two cases of ovarian cancer, or three or more cases of breast and ovarian cancer. In addition, 58 early-onset (before age 35) breast cancer cases and 30 reference individuals were analyzed. Of 17 variants detected in ZBRK1, a missense mutation Val524Ile was identified in the proband of one high-risk family, but no other family members were available for testing. Of 25 variants identified in BRIP1, in addition to four common silent or missense mutations, we identified Gln540Leu, a non-conservative amino acid change, in a single familial proband with inflammatory breast cancer, but this mutation was not present in her three relatives with breast cancer. Haplotype analysis suggests that all ZBRK1 SNPs fall within a single block with two SNPs capturing 92% of the haplotype diversity, while the BRIP1 SNPs fall in two blocks, with five SNPs capturing 89% of the haplotype diversity. Based on sequencing of ZBRK1 and BRIP1 in 21 BRCA1/2-negative probands from inherited breast/ovarian cancer families, it appears unlikely that mutations in these genes account for a significant fraction of inherited breast cancer. Further analysis in unselected cases will be required to know whether the identified variants play a role in genetic predisposition to breast cancer in the general population. Hum Mutat 22:121-128, 2003. Published 2003 Wiley-Liss, Inc.

Parathyroid hormone (PTH), a major regulator of bone metabolism, activates the PTHR1 receptor on the osteoblast plasma membrane to initiate signaling and induce transcription of primary response genes. Subsequently, primary genes with transcriptional activity regulate expression of downstream PTH targets. We have identified the adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4bp4) as a PTH-induced primary gene in osteoblasts. E4BP4 is a basic leucine zipper (bZIP) transcription factor that represses or activates transcription in non-osteoblastic cells. We report here that PTH rapidly and transiently induced E4bp4 mRNA in osteoblastic cells and that this induction did not require protein synthesis. PTH also induced E4BP4 protein synthesis and E4BP4 binding to a consensus but not to a mutant E4BP4 response element (EBPRE). E4BP4 overexpression inhibited an EBPRE-containing promoter-reporter construct, whereas PTH treatment attenuated activity of the same construct in primary mouse osteoblasts. Finally, E4BP4 overexpression inhibited PTH-induced activity of a cyclooxygenase-2 promoter-reporter construct. Our data suggest a role for E4BP4 in attenuation of PTH target gene transcription in osteoblasts.

Background: We previously reported an Ser50Thr mutation in the NRL gene as a cause of autosomal dominant retinitis pigmentosa., Objective: To determine the characteristic features of the autosomal dominant retinitis pigmentosa phenotype associated with the NRL Ser50Thr mutation in affected individuals from 4 related families., Methods: Clinical records were available for 21 affected individuals; 7 underwent more extensive electrophysiologic and psychophysical testing., Results: Night blindness was the first symptom to manifest, with onset between birth and age 16 years. Difficulty with peripheral vision was experienced between 20 and 37 years of age. Visual acuity was well preserved in younger individuals, but those older than 30 years frequently had substantial visual loss (6/36 or worse) associated with macular atrophy. Electrophysiologic testing revealed a nondetectable scotopic electroretinogram with relative preservation of the photopic electroretinogram and pattern electroretinography in the 3 youngest patients tested (aged 15-18 years). In older individuals, all components of the electroretinogram were nondetectable. Dark-adapted visual fields in younger individuals were greatly impaired, but their photopic fields remained relatively well preserved. Older patients had photopic fields limited to just a few degrees. Distinctive peripapillary chorioretinal atrophy seems to develop as the disorder progresses., Conclusions: The NRL Ser50Thr mutation is associated with selective loss of scotopic function before age 20 years. With time, however, the photopic system becomes affected, leading to loss of the photopic visual field and of visual acuity.

A novel leucine-zipper gene, leucine zipper protein 2 (Luzp2), has been cloned as part of an aberrant deletion-fusion transcript in the chromosomal interval between Gas2 and Herc2 on mouse Chromosome 7 (Chr 7). Luzp2 is normally expressed only in brain and spinal cord. The human homolog of Luzp2 maps to Chr 11p13-11p14 by radiation-hybrid mapping and is deleted in some patients with Wilms tumor-Aniridia-Genitourinary anomalies-mental Retardation (WAGR) syndrome. Disruption of Luzp2 by gene targeting in mice did not result in any obvious abnormal phenotypes.

Helix-loop-helix (HLH) and helix-loop-helix-leucine zipper (HLHZip) are dimerization domains that mediate selective pairing among members of a large transcription factor family involved in cell fate determination. To investigate the molecular rules underlying recognition specificity and to isolate molecules interfering with cell proliferation and differentiation control, we assembled two molecular repertoires obtained by directed randomization of the binding surface in these two domains. For this strategy we selected the Heb HLH and Max Zip regions as molecular scaffolds for the randomization process and displayed the two resulting molecular repertoires on lambda phage capsids. By affinity selection, many domains were isolated that bound to the proteins Mad, Rox, MyoD, and Id2 with different levels of affinity. Although several residues along an extended surface within each domain appeared to contribute to dimerization, some key residues critically involved in molecular recognition could be identified. Furthermore, a number of charged residues appeared to act as switch points facilitating partner exchange. By successfully selecting ligands for four of four HLH or HLHZip proteins, we have shown that the repertoires assembled are rather general and possibly contain elements that bind with sufficient affinity to any natural HLH or HLHZip molecule. Thus they represent a valuable source of ligands that could be used as reagents for molecular dissection of functional regulatory pathways.

Retroviral late-budding (L) domains are required for the efficient release of nascent virions. The three known types of L domain, designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL), each bind distinct cellular cofactors. We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses, such as human immunodeficiency virus type 1 (HIV-1) and Ebola virus, that encode a PTAP-type L domain, but subsequent events remain undefined. In this study, we demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C terminus and is therefore recruited to the plasma membrane by HIV-1 Gag. In addition, we show that Tsg101 exhibits a multimerization activity. Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28 binding site within Tsg101 is required for particle budding. In addition, mutation of a putative leucine zipper or residues important for Tsg101 multimerization also impairs the ability of Tsg101 to support HIV-1 budding. A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding but does not inhibit YPDL-type or PPXY-type L-domain function. Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase VPS4. These results indicate that all three classes of retroviral L domains require a functioning class E VPS pathway in order to effect budding. However, the PTAP-type L domain appears to be unique in its requirement for an intact, or nearly intact, ESCRT-I complex.

Purpose: To determine if mutations in the retinal transcription factor gene NRL are associated with retinopathies other than autosomal dominant retinitis pigmentosa (adRP)., Methods: Genomic DNA was isolated from blood samples obtained from 50 patients with Leber Congenital Amaurosis (LCA), 17 patients with the Enhanced S-Cone Syndrome (ESCS), and a patient with an atypical retinal degeneration that causes photoreceptor rosettes with blue cone opsin. The 5' upstream region (putative promoter), untranslated exon 1, coding exons 2 and 3, and exon-intron boundaries of the NRL gene were analyzed by direct sequencing of the PCR-amplified products., Results: Complete sequencing of the NRL gene in DNA samples from this cohort of patients revealed only one nucleotide change. The C->G transversion at nucleotide 711 of NRL exon 3 was detected in one LCA patient; however, this change did not alter the amino acid (L237L)., Conclusions: No potential disease causing mutation was identified in the NRL gene in patients with LCA, ESCS, or the atypical retinal degeneration. Together with previous studies, our results demonstrate that mutations in the NRL gene are not a major cause of retinopathy. To date, only missense changes have been reported in adRP patients, and sequence variations are rare. It is possible that the loss of NRL function in humans is associated with a more complex clinical phenotype due to its expression in pineal gland in addition to rod photoreceptors.

IL-12 is a principal activator of both innate and adaptive immunity against infectious agents and malignancies. Regulation of proinflammatory IL-12 gene expression in phagocytes by the anti-inflammatory cytokine IL-10 represents a major homeostatic process underlying host-pathogen and host-self interactions. Delineation of the signaling pathway of IL-10 is crucial to the understanding of immunological regulatory networks. In this study, we report that IL-10 and c-musculoaponeurotic fibrosarcoma (Maf) induce their mutual expression in inflammatory macrophages. We demonstrate that c-Maf is one of the physiological mediators of IL-10's immunosuppressive activities. When overexpressed, c-Maf selectively inhibits transcriptional activation of IL-12 p40 and p35 genes while potently activating IL-10 and IL-4 expression, potentially contributing to the development of a state of anti-inflammation and dichotomy of immunologic polarization. c-Maf induces changes in nuclear DNA-binding activities at multiple sites including the ets, GA-12, NF-kappaB, C/EBP, and AP-1 elements. Nonetheless, the essential c-Maf-responsive element appears to be located elsewhere. Inhibition of IL-12 p40 gene expression by c-Maf requires the N-terminal transactivation domain, suggesting an indirect mechanism of transcriptional inhibition involving the induction of an unidentified repressor. In c-Maf-deficient murine macrophages, IL-10 production is impaired. However, IL-10-mediated inhibition of IL-12 production remains intact, indicating the existence of alternative mediators in the absence of c-Maf, consistent with the observation that a functional AP-1 is required for this pathway.

The Arabidopsis thaliana NIM1/NPR1 gene product is required for induction of systemic acquired resistance (SAR) by pathogens, salicylic acid (SA) or synthetic SA analogs. We identified, in a yeast two-hybrid screen, two NIM1/NPR1 interacting proteins, TGA2 and TGA5, which belong to the basic region, leucine zipper (bZIP) family of transcription factors. Both TGA2 and TGA5 strongly interact with NIM1/NPR1 in yeast and in vitro, and recognize the as-1 cis element found within the promoter of several pathogenesis-related genes, such as PR-1. To determine the role TGA2 and TGA5 may play in NIM1/NPR1-mediated disease resistance, we introduced sense and antisense versions of both genes into transgenic Arabidopsis plants. Characterization of TGA2 transgenic plants revealed that inhibition or overexpression of TGA2 does not significantly affect PR-1 expression or induction of SAR after pathogen infection or INA treatment. Surprisingly, all TGA5-antisense transgenic plants produced showed increased accumulation of TGA5 transcripts compared with untransformed control plants, while the TGA5-sense lines showed no significant increase in TGA5 mRNA levels. Interestingly, the high level of TGA5 mRNA in the antisense lines was accompanied by significant resistance to a highly virulent isolate of the oomycete pathogen Peronospora parasitica. Further, resistance was not coupled to accumulation of products from the SAR-linked PR-1 gene following inoculation with P. parasitica or treatment with INA, indicating that these plants express a robust, PR-1-independent resistance mechanism. Resistance was retained when a TGA5-accumulating line was combined genetically with a nim1-1 mutation or nahG (salicylate hydroxylase) transgene, indicating that resistance in these plants is due to an SA and SAR-independent mechanism.

DivIVA of Bacillus subtilis and FtsZ of Escherichia coli were used to target heterologous protein complexes to cell division sites of E. coli and Agrobacterium tumefaciens. DivIVA and FtsZ that were fused to the dimerizing leucine zipper (LZ) domain of the yeast transcription activator GCN4 directed the green fluorescent protein (GFP) that was fused to an LZ domain to E. coli division sites, resulting in fluorescence patterns identical to those observed with DivIVA::GFP and FtsZ::GFP. These cell division proteins also targeted the VirE1 chaperone and VirE2 secretion substrate complex to division sites of E. coli and A. tumefaciens. Coproduction of the native VirE1 or VirE2 proteins inhibited the dihybrid interaction in both species, as judged by loss of GFP targeting to division sites. The VirE1 chaperone bound independently to N- and C-terminal regions of VirE2, with a requirement for residues 84 to 147 and 331 to 405 for these interactions, as shown by dihybrid studies with VirE1::GFP and DivIVA fused to N- and C-terminal VirE2 fragments. DivIVA also targeted homo- and heterotypic complexes of VirB8 and VirB10, two bitopic inner membrane subunits of the A. tumefaciens T-DNA transfer system, in E. coli and homotypic complexes of VirB10 in A. tumefaciens. VirB10 self-association in bacteria was mediated by the C-terminal periplasmic domain, as shown by dihybrid studies with fusions to VirB10 truncation derivatives. Together, our findings establish a proof-of-concept for the use of cell-location-specific proteins for studies of interactions among cytosolic and membrane proteins in diverse bacterial species.

A small family of novel basic leucine zipper proteins that includes abscisic acid (ABA)-INSENSITIVE 5 (ABI5) binds to the promoter region of the lea class gene Dc3. The factors, referred to as AtDPBFs (Arabidopsis Dc3 promoter-binding factors), were isolated from an immature seed cDNA library. AtDPBFs bind to the embryo specification and ABA-responsive elements in the Dc3 promoter and are unique in that they can interact with cis-elements that do not contain the ACGT core sequence required for the binding of most other plant basic leucine zipper proteins. Analysis of full-length cDNAs showed that at least five different Dc3 promoter-binding factors are present in Arabidopsis seeds; one of these, AtDPBF-1, is identical to ABI5. As expected, AtDPBF-1/ABI5 mRNA is inducible by exogenous ABA in seedlings. Despite the near identity in their basic domains, AtDPBFs are distinct in their DNA-binding, dimerization, and transcriptional activity.

Spermatozoa formation involves drastic morphological and cellular reconstructions. However, the molecular mechanisms driving this process remain elusive. We describe the cloning of a novel murine spermatid-specific gene, designated nurit, identified in a two-hybrid screen for proteins that binds the Nek1 kinase. Nurit protein harbors a leucine-zipper motif, and two additional coiled-coil regions. The C-terminal coiled-coil domain mediates homodimerization of the protein. Nurit homologues are found in primates, pig and rodents. nurit is transcribed through the elongation stage of the spermatids, but is absent from mature spermatozoa. Interestingly, immunogold electron microscopy revealed that the protein is restricted, from its first detectable appearance, to a unique spermatid organelle called the 'flower-like structure'. The function of this structure is unknown, though it may be involved in transporting proteins designated to be discarded via the residual bodies. Nurit is the first marker of the flower-like structure, and its study may provide an excellent opportunity to dissect the function of this organelle., (Copyright 2002 Elsevier Science Ireland Ltd.)

The basic region-leucine zipper (B-ZIP) (bZIP) protein motif dimerizes to bind specific DNA sequences. We have identified 27 B-ZIP proteins in the recently sequenced Drosophila melanogaster genome. The dimerization specificity of these 27 B-ZIP proteins was evaluated using two structural criteria: (1) the presence of attractive or repulsive interhelical g<-->e' electrostatic interactions and (2) the presence of polar or charged amino acids in the 'a' and 'd' positions of the hydrophobic interface. None of the B-ZIP proteins contain only aliphatic amino acids in the'a' and 'd' position. Only six of the Drosophila B-ZIP proteins contain a "canonical" hydrophobic interface like the yeast GCN4, and the mammalian JUN, ATF2, CREB, C/EBP, and PAR leucine zippers, characterized by asparagine in the second 'a' position. Twelve leucine zippers contain polar amino acids in the first, third, and fourth 'a' positions. Circular dichroism spectroscopy, used to monitor thermal denaturations of a heterodimerizing leucine zipper system containing either valine (V) or asparagine (N) in the 'a' position, indicates that the V-N interaction is 2.3 kcal/mole less stable than an N-N interaction and 5.3 kcal/mole less stable than a V-V interaction. Thus, we propose that the presence of polar amino acids in novel positions of the 'a' position of Drosophila B-ZIP proteins has led to leucine zippers that homodimerize rather than heterodimerize.

The mammalian ATF/CREB family of transcription factors comprises a large group of basic-region leucine zipper (bZIP) proteins whose members mediate diverse transcriptional regulatory functions. Here we report that expression of a specific mouse ATF gene, ATFx, is down-regulated in a variety of cells undergoing apoptosis following growth factor deprivation. When stably expressed in an interleukin 3 (IL-3)-dependent cell line, ATFx suppresses apoptosis resulting from cytokine deprivation. Conversely, a dominant-negative ATFx mutant induces apoptosis of cells cultured in the presence of growth factors. We also show that 24p3, a secreted lipocalin that induces apoptosis when added to hematopoietic cells, represses ATFx expression. However, constitutive expression of ATFx renders cells resistant to 24p3-mediated apoptosis. Collectively, our results indicate that ATFx is an anti-apoptotic factor, a novel role for an ATF protein.

The cGvpE protein of Halobacterium salinarum PHH4 has been identified as transcriptional activator for the promoter of the c-gvpA gene encoding the major gas vesicle structural protein cGvpA. Molecular modelling of the carboxy-terminal region of cGvpE suggests that this protein resembles a basic leucine-zipper protein, and mutations in the putative DNA binding domain DNAB completely abolish the activator function in Haloferax volcanii transformants. Mutations in the key residues of the putative leucine-zipper region AH6 of cGvpE confirmed that the three residues V159, L166 and L173 were essential for the activator function of cGvpE at the c-gvpA promoter, whereas the cysteine residue C180 could be altered to a leucine or an aspartate residue without the loss of this function. Mutations in basic residues of helix AH4 demonstrated the importance of the lysine K104 for the activator function of cGvpE. A cGvpE protein containing a his-tag at the C-terminus was still able to activate the expression of c-gvpA in vivo. The cGvpE his-purified from Hf. volcanii formed a dimer in Blue-native polyacrylamide gels that could be resolved into monomers by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Dimers of cGvpE were already seen using SDS-PAGE, but not with cGvpE mutant proteins containing the alterations L166E or L173E/C180L in the leucine zipper. These results imply that the hydrophobic surface of helix AH6 is indeed required for the establishment of cGvpE dimers.

TATA binding protein (TBP) and transcription factor IIB (TFIIB) are key factors for the assembly of eukaryotic transcription initiation complexes. We used a rice whole-cell extract in vitro transcription system to characterize the functional interactions of recombinant plant TBP and TFIIB. Bacterially expressed rice TBP (OsTBP2) bound to the TATA box of the rice pal gene encoding phenylalanine ammonia-lyase, caused DNA bending, and enhanced basal transcription from the pal promoter in a TATA box-dependent manner. Recombinant rice TFIIB (OsTFIIB) stimulated the DNA binding and bending activities of OsTBP2 and synergistically enhanced OsTBP2-mediated transcription from the pal promoter and the promoter of Rice tungro bacilliform virus but not from the barley pr1 promoter. We also demonstrate a physical interaction between OsTBP2 and RF2a, a rice bZIP transcription factor that bound to the box II cis element of the promoter of Rice tungro bacilliform virus, resulting in enhanced transcription from the viral promoter. Enhancement of rice whole-cell extracts with recombinant transcription factors thus provides a powerful tool for the in vitro determination of plant gene regulation mechanisms. We conclude that OsTBP2 undergoes promoter-specific functional interactions with both the basal transcription factor OsTFIIB and the accessory transcription factor RF2a.

Objectives: To search for mutations in the neural retina leucine zipper (NRL) gene in patients with dominant retinitis pigmentosa and to compare the severity of disease in these patients with that observed previously in patients with dominant rhodopsin mutations., Methods: Single-strand conformation analysis was used to survey 189 unrelated patients for mutations. The available relatives of index patients with mutations were also evaluated. In our clinical examination of patients, we measured visual acuity, final dark-adaptation threshold equivalent visual field diameter, and electroretinogram amplitudes among other parameters of visual function. We compared the clinical findings with those obtained earlier from similar evaluations of a group of 39 patients with the dominant rhodopsin mutation Pro23His and a group of 25 patients with the dominant rhodopsin mutation Pro347Leu., Results: We identified 3 novel missense mutations in a total of 4 unrelated patients with dominant retinitis pigmentosa: Ser50Pro, Ser50Leu (2 patients), and Pro51Thr. Each mutation cosegregated with dominant retinitis pigmentosa. None of these mutations were found among 91 unrelated control individuals. The visual acuities among the 4 index patients and 3 relatives with NRL mutations who were clinically evaluated ranged from 20/20 (in a 9-year-old patient) to 20/200 (in a 73-year-old patient). All patients had bone-spicule pigment deposits in their fundi. Average rod-plus-cone and cone-isolated electroretinogram amplitudes were both decreased by 99% or more compared with normal amplitudes. The dark-adaptation thresholds, equivalent visual field diameters, and electroretinogram amplitudes (all corrected for age and refractive error) indicated that the disease caused by the NRL mutations was more severe than that caused by the dominant rhodopsin mutation Pro23His and was similar in severity to that produced by the rhodopsin mutation Pro347Leu., Conclusion: The 3 novel NRL mutations we discovered bring the total number of reported mutations in this gene to 6. Five of the 6 mutations affect residues 50 or 51, suggesting that these residues are important in a structural or functional domain of the encoded protein., Clinical Relevance: Rod and cone function is affected to a similar degree in patients with these mutations. The disease caused by NRL mutations found in this study appears to be more severe than that caused by the rhodopsin mutation Pro23His and is similar in severity to that caused by the rhodopsin mutation Pro347Leu, even after correcting for age.

In Arabidopsis, EDS1 is essential for disease resistance conferred by a structural subset of resistance (R) proteins containing a nucleotide-binding site, leucine-rich-repeats and amino-terminal similarity to animal Toll and Interleukin-1 (so-called TIR-NBS-LRR proteins). EDS1 is not required by NBS-LRR proteins that possess an amino-terminal coiled-coil motif (CC-NBS-LRR proteins). Using virus-induced gene silencing (VIGS) of a Nicotiana benthaminana EDS1 orthologue, we investigated the role of EDS1 in resistance specified by structurally distinct R genes in transgenic N. benthamiana. Resistance against tobacco mosaic virus mediated by tobacco N, a TIR-NBS-LRR protein, was EDS1-dependent. Two other R proteins, Pto (a protein kinase), and Rx (a CC-NBS-LRR protein) recognizing, respectively, a bacterial and viral pathogen did not require EDS1. These data, together with the finding that expression of N. benthamiana and Arabidopsis EDS1 mRNAs are similarly regulated, lead us to conclude that recruitment of EDS1 by TIR-NBS-LRR proteins is evolutionarily conserved between dicotyledenous plant species in resistance against bacterial, oomycete and viral pathogens. We further demonstrate that VIGS is a useful approach to dissect resistance signaling pathways in a genetically intractable plant species.

In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes including pathogen defence, light and stress signalling, seed maturation and flower development. The Arabidopsis genome sequence contains 75 distinct members of the bZIP family, of which approximately 50 are not described in the literature. Using common domains, the AtbZIP family can be subdivided into ten groups. Here, we review the available data on bZIP functions in the context of subgroup membership and discuss the interacting proteins. This integration is essential for a complete functional characterization of bZIP transcription factors in plants, and to identify functional redundancies among AtbZIP factors.

Human congenital cataract and ocular anterior segment dysgenesis both demonstrate extensive genetic and phenotypic heterogeneity. We identified a family where ocular developmental abnormalities (cataract, anterior segment dysgenesis and microphthalmia) co-segregated with a translocation, t(5;16)(p15.3;q23.2), in both balanced and unbalanced forms. We hypothesized that this altered the expression of a gene of developmental significance in the human lens and ocular anterior segment. Cloning the 16q23.2 breakpoint demonstrated that it transected the genomic-control domain of MAF, a basic region leucine zipper (bZIP) transcription factor, first identified as an oncogene, which is expressed in vertebrate lens development and regulates the expression of the eye lens crystallins. The homozygous null mutant Maf mouse embryo demonstrates defective lens formation and microphthalmia. Through mutation screening of a panel of patients with hereditary congenital cataract we identified a mutation in MAF in a three-generation family with cataract, microcornea and iris coloboma. The mutation results in the substitution of an evolutionarily highly conserved arginine with a proline at residue 288 (R288P) in the basic region of the DNA-binding domain of MAF. Our findings further implicate MAF/Maf in mammalian lens development and highlight the role of the lens in anterior segment development. The 16q23.2 breakpoint transects the common fragile site, FRA16D, providing a molecular demonstration of a germline break in a common fragile site.

Gene activation mediated by nuclear receptors is regulated in a tissue-specific manner and requires interactions between nuclear receptors and their cofactors. Here, we identified and characterized a tissue-specific coactivator, GT198, that interacts with the DNA-binding domains of nuclear receptors. GT198 was originally described as a genomic transcript that mapped to the human breast cancer susceptibility locus 17q12-q21 with unknown function. We show that GT198 exhibits a tissue-specific expression pattern in which its mRNA is elevated in testis, spleen, thymus, pituitary cells, and several cancer cell lines. GT198 is a 217-amino-acid nuclear protein that contains a leucine zipper required for its dimerization. In vitro binding and yeast two-hybrid assays indicated that GT198 interacted with nuclear receptors through their DNA-binding domains. GT198 potently stimulated transcription mediated by estrogen receptor alpha and beta, thyroid hormone receptor beta1, androgen receptor, glucocorticoid receptor, and progesterone receptor. However, the action of GT198 was distinguishable from that of the ligand-binding domain-interacting nuclear receptor coactivators, such as TRBP, CBP, and SRC-1, with respect to basal activation and hormone sensitivity. Furthermore, protein kinase A, protein kinase C, and mitogen-activated protein kinase can phosphorylate GT198 in vitro, and cotransfection of these kinases regulated the transcriptional activity of GT198. These data suggest that GT198 is a tissue-specific, kinase-regulated nuclear receptor coactivator that interacts with the DNA-binding domains of nuclear receptors.

ICAT inhibits the interaction between beta-catenin and TCF transcription factors. As ICAT might be a tumor suppressor gene with the potential to negatively regulate the WNT - beta-catenin - TCF signaling pathway, ICAT related gene in the human genome draft sequence was searched for. Here, the LZIC gene, a novel gene encoding a 190-amino-acid polypeptide with leucine zipper domain and ICAT homologous domain, was cloned and characterized. Amino-acid identity between LZIC and ICAT in the ICAT homologous domain was 38%. The LZIC gene, consisting of at least 8 exons, was located in the human chromosome 1p36.32-pter region. The major 5.2-kb LZIC mRNA and minor 2.1-, 1.6-, and 1.0-kb LZIC mRNAs were expressed almost ubiquitously in normal human tissues. LZIC was expressed in all cancer cell lines examined in this study, and was significantly up-regulated in a gastric cancer cell line MKN74 and 5 cases of primary gastric cancer. As LZIC contains ICAT homologous domain, LZIC might inhibit the interaction between beta-catenin and TCF transcription factors, just like ICAT, and, up-regulation of LZIC in gastric cancer might be due to a negative feed-back mechanism to inhibit the WNT - beta-catenin - TCF signaling pathway.

Basic-region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a leucine zipper dimerization domain and a basic region that directly contacts DNA. In all naturally occurring bZip proteins, the basic region is positioned N-terminal to the leucine zipper. We have designed a series of model bZip peptides in which the basic region of the yeast transcriptional activator GCN4 is placed C-terminal to its leucine zipper. DNA-binding studies demonstrate that the optimal reverse GCN4 (rGCN4) peptide is able to bind specifically and with wild-type affinity to DNA despite this unnatural arrangement of the two subdomains. These results suggest that a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the dimerization domain is unlikely.

Salicylic acid (SA) is a key regulator for the induction of systemic acquired resistance (SAR), and NPR1 is a critical mediator for the biological effects of SA. Physical interactions between NPR1 and TGA factors, a conserved family of basic-leucine-zipper (bZip) proteins in plants, have suggested a role for these transcription factors in mediating SAR induction via the regulation of defense genes. To elucidate this function, we constructed a trans-dominant mutant that specifically eliminates DNA-binding activities of this class of bZip proteins in transgenic tobacco plants. Our results demonstrate that the loss of TGA DNA-binding activities is correlated with suppression of two xenobiotic-responsive genes, GNT35 and STR246, and enhanced induction of pathogenesis-related (PR) genes by SA. In addition, these TGA-suppressed plants exhibited higher levels of PR gene induction by pathogen challenge and an enhanced SAR. These results suggest that TGA transcription factors serve both negative and positive regulatory roles in mediating plant defense responses.

Induction by low temperature is a common feature of the lip19 subfamily members of the basic region leucine zipper gene family in plants. Here, we characterize two tobacco (Nicotiana tabacum) genes, tbzF and tbz17, belonging to the lip19 subfamily, whose gene products, TBZF and TBZ17, show 73% identity and are located in nuclei. They preferentially bind to DNA fragments spanning A-box/G-box and C-box/G-box hybrid motifs and show transactivation activity in cobombarded tobacco BY-2 cells, indicating they function as transcriptional activators. Transcripts of tbzF were detected at a high level in senescing leaves and flowers. In contrast, tbz17 transcripts could be shown to accumulate in aged leaves but not in flowers. In situ hybridization analysis revealed transcripts of tbzF and tbz17 to be predominantly located in guard cells and vascular tissues of senescing leaves. These results suggest that TBZF and TBZ17 are both involved in controlling gene transcription related to functions of guard cells in senescing leaves and that TBZF bifunctionally acts in floral development.

Transcription of the human nonmuscle myosin II heavy chain-A (NMHC-A) gene is regulated via multiple elements located in intron 1, including element F which contains an E-box. In this study we have identified and characterized the factors that are capable of binding to element F. Yeast one-hybrid screening using element F allowed isolation of cDNAs encoding transcriptional factors TFEC, TFE3, and USF2, each of which contains basic helix-loop-helix and leucine zipper motifs. Furthermore, cDNA cloning by polymerase chain reaction yielded cDNAs for two TFEC isoforms, designated TFEC-l and TFEC-s, which are generated by alternative pre-mRNA splicing. In addition to these four factors, USF1, which is known to share the same DNA binding elements with USF2, was isolated for comparison. Electrophoretic mobility shift assays and cotransfection studies of the expression constructs with reporter gene constructs revealed that the above five factors have different binding activities for element F with different transactivation potencies. USF1 and USF2 demonstrate the highest binding activity to element F, yet show the lowest element F-dependent transactivation. TFE3 has a high transactivation potency but the lowest binding activity. TFEC-l demonstrates a high binding activity with the highest transactivation potency, whereas TFEC-s has the same binding activity as TFEC-l with intermediate transactivation. We also demonstrate that an N-terminal activation domain exists only in TFEC-l, whereas a C-terminal activation domain is common to both the l and s isoforms. This study provides the first evidence of TFEC being an activator of transcription, with two separate activation domains.

Translin is thought to participate in a variety of cellular activities including chromosomal translocations, translational regulation of mRNA expression, and mRNA transport. It forms an octameric ring structure capable of sequence-specific binding of both DNA and RNA substrates. We have used electron microscopy and single-particle image analysis to generate a three-dimensional reconstruction of the Translin ring. The subunits appear to have two distinct domains that assemble to form an open channel with diameter of approximately 30 A at one end and approximately 50 A at the opposite end. In the presence of either DNA or RNA containing consensus binding sequences, the largest opening into the central cavity is filled with density. Strikingly, although Translin shows significant sequence homology to only one other protein, Translin-associated factor X, the quaternary organization and the dimerization of subunits in the ring are very similar to those observed for hexameric ring helicases. This suggests that many of the structures in DNA and RNA metabolism may have similar quaternary organization., (Copyright 2001 Academic Press.)

Northern blotting has shown that mouse small intestine contains relatively large amounts of the nuclear factor-E2 p45-related factor (Nrf) 2 transcription factor but relatively little Nrf1. Regulation of intestinal antioxidant and detoxication enzymes by Nrf2 has been assessed using a mouse line bearing a targeted disruption of the gene encoding this factor. Both Nrf2-/- and Nrf2+/+ mice were fed a control diet or one supplemented with either synthetic cancer chemopreventive agents [butylated hydroxyanisole (BHA), ethoxyquin (EQ), or oltipraz] or phytochemicals [indole-3-carbinol, cafestol and kahweol palmitate, sulforaphane, coumarin (CMRN), or alpha-angelicalactone]. The constitutive level of NAD(P)H:quinone oxidoreductase (NQO) and glutathione S-transferase (GST) enzyme activities in cytosols from small intestine was typically found to be between 30% and 70% lower in samples prepared from Nrf2 mutant mice fed a control diet than in equivalent samples from Nrf2+/+ mice. Most of the chemopreventive agents included in this study induced NQO and GST enzyme activities in the small intestine of Nrf2+/+ mice. Increases of between 2.7- and 6.2-fold were observed in wild-type animals fed diets supplemented with BHA or EQ; increases of about 2-fold were observed with a mixture of cafestol and kahweol palmitate, CMRN, or alpha-angelicalactone; and increases of 1.5-fold were measured with sulforaphane. Immunoblotting confirmed that in the small intestine, the constitutive level of NQO1 is lower in the Nrf2-/- mouse, and it also showed that induction of the oxidoreductase was substantially diminished in the mutant mouse. Immunoblotting class-alpha and class-mu GST showed that constitutive expression of most transferase subunits is also reduced in the small intestine of Nrf2 mutant mice. Significantly, induction of class-alpha and class-mu GST by EQ, BHA, or CMRN is apparent in the gene knockout animal. No consistent change in the constitutive levels of the catalytic heavy subunit of gamma-glutamylcysteinyl synthetase (GCS(h)) was observed in the small intestine of Nrf2-/- mice. However, although the expression of GCS(h) was found to be increased dramatically in the small intestine of Nrf2+/+ mice by dietary BHA or EQ, this induction was essentially abolished in the knockout mice. It is apparent that Nrf2 influences both constitutive and inducible expression of intestinal antioxidant and detoxication proteins in a gene-specific fashion. Immunohistochemistry revealed that induction of NQO1, class-alpha GST, and GCS(h) occurs primarily in epithelial cells of the small intestine. This suggests that the variation in inducibility of NQO1, Gsta1/2, and GCS(h) in the mutant mouse is not attributable to the expression of the enzymes in distinct cell types but rather to differences in the dependency of these genes on Nrf2 for induction.

The mi transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the development of mast cells. Mast cells of mi/mi genotype express normal amount of abnormal MITF (mi-MITF), whereas mast cells of tg/tg genotype do not express any MITFs. Mast cells of mi/mi mice show more severe abnormalities than those of tg/tg mice, indicating that the mi-MITF possesses the inhibitory function. The MITF encoded by the mi(ce) mutant allele (ce-MITF) lacks the Zip domain. We examined the importance of the Zip domain using mi(ce)/mi(ce) mice. The amounts of c-kit, granzyme B (Gr B), and tryptophan hydroxylase (TPH) messenger RNAs decreased in mast cells of mi(ce)/mi(ce) mice to levels comparable to those of tg/tg mice, and the amounts were intermediate between those of +/+ mice and those of mi/mi mice. Gr B mediates the cytotoxic activity of mast cells, and TPH is a rate-limiting enzyme for the synthesis of serotonin. The cytotoxic activity and serotonin content of mi(ce)/mi(ce) mast cells were comparable to those of tg/tg mast cells and were significantly higher than those of mi/mi mast cells. The phenotype of mi(ce)/mi(ce) mast cells was similar to that of tg/tg mast cells rather than to that of mi/mi mast cells, suggesting that the ce-MITF had no functions. The Zip domain of MITF appeared to be important for the development of mast cells. (Blood. 2001;97:2038-2044)

p45NF-E2 is a member of the cap 'n' collar (CNC)-basic leucine zipper family of transcriptional activators that is expressed at high levels in various types of blood cells. Mice deficient in p45NF-E2 that were generated by gene targeting have high mortality from bleeding resulting from severe thrombocytopenia. Surviving p45nf-e2(-/-) adults have mild anemia characterized by hypochromic red blood cells (RBCs), reticulocytosis, and splenomegaly. Erythroid abnormalities in p45nf-e2(-/-) animals were previously attributed to stress erythropoiesis caused by chronic bleeding and, possibly, ineffective erythropoiesis. Previous studies suggested that CNC factors might play essential roles in regulating expression of genes that protect cells against oxidative stress. In this study, we found that p45NF-E2-deficient RBCs have increased levels of reactive oxygen species and an increased susceptibility to oxidative-stress-induced damage. Deformability of p45NF-E2-deficient RBCs was markedly reduced with oxidative stress, and mutant cells had a reduced life span. One possible reason for increased sensitivity to oxidative stress is that catalase levels were reduced in mutant RBCs. These findings suggest a role for p45NF-E2 in the oxidative-stress response in RBCs and indicate that p45NF-E2 deficiency contributes to the anemia in p45nf-e2(-/-) mice. (Blood. 2001;97:2151-2158)

The flexibility and dynamics of proteins directly influence the processes of protein folding, recognition, and function. NMR spin relaxation methods are used to assess the dynamics and mobility of proteins, for fast ps and ns motions as well as slower microsecond and ms events. The degree of protein flexibility and disorder as well as the changes in protein flexibility can be assessed by NMR spin relaxation methods at individual residues within the protein. In addition to probing protein dynamics, the changes in the NMR-derived order parameters can be used to estimate the entropic contributions of order-disorder transitions. Furthermore, kinetic processes in the ms time regime may be directly investigated to extract the rates of conformational interconversion, ligand binding, and protein folding processes. We show how a variety of dynamical information can be obtained from NMR relaxation measurements. We present examples that illustrate the use of NMR spin relaxation analysis for investigation of folding and disorder in proteins.