Region-leucine Zipper Research Articles

A point mutation in VIG1 boosts development and chilling tolerance in rice

The rice paddy-direct seeding system has been widely adopted due to its low cost and convenience, whereas its application is mainly constrained by low seedling vigor, cold sensitivity, eventually resulting in reduced grain yield. Here, we show vig1a and vig1b, two allelic mutants of OsbZIP01, that both demonstrate greatly enhanced seedling vigor and chilling tolerance but differ in final grain production. The vig1a phenotype can be obtained via simultaneous mutation of the genes OsbZIP01 and OsbZIP18, or by selectively manipulating the basic region of OsbZIP01. Destroying the leucine zipper region of OsbZIP01 in vig1a turns vig1a to be vig1b. Further analysis reveals that OsbZIP01 and OsbZIP18 function cooperatively in diverse crucial biological programs that determine seedling establishment, chilling tolerance, and grain yield through their interactions. These findings provide a strategy toward simultaneously improving seedling vigor, chilling tolerance, and grain yield for rice production.

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.

Unraveling the interplay between the leucine zipper and forkhead domains of FOXP2: Implications for DNA binding, stability and dynamics.

FOXP2 is a transcription factor associated with speech and language. Like other FOX transcription factors, it has a DNA binding region called the forkhead domain (FHD). This domain can exist as a monomer or a domain swapped dimer. In addition to the FHD, the leucine zipper region (LZ) of FOXP2 is also believed to be associated with both DNA binding and oligomerization. To better understand the relationship between DNA binding and oligomerization of FOXP2, we investigated its structure, stability and dynamics, focusing specifically on the FHD and the LZ. We did this by using two constructs: one containing the isolated FHD and one containing both the LZ and the FHD (LZ-END). We demonstrate in this work, that while the FHD maintains a monomeric form that is capable of binding DNA, the LZ-END undergoes a dynamic transition between oligomeric states in the presence of DNA. Our findings suggest that FOXP2's LZ domain influences DNA binding affinity through a change in oligomeric state. We show through hydrogen exchange mass spectroscopy that certain parts of the FHD and interlinking region become less dynamic when in the presence of DNA, confirming DNA binding and oligomerization in these regions. Moreover, the detection of a stable equilibrium intermediate state during LZ-END unfolding supports the idea of cooperation between these two domains. Overall, our study sheds light on the interplay between two FOXP2 domains, providing insight into the protein's ability to respond dynamically to DNA, and enriching our understanding of FOXP2's role in gene regulation.

Structural basis for specific DNA sequence recognition by the transcription factor NFIL3

The CCAAT/enhancer-binding proteins (C/EBPs) constitute a family of pivotal transcription factors involved in tissue development, cellular function, proliferation, and differentiation. NFIL3, as one of them, plays an important role in regulating immune cell differentiation, circadian clock system and neural regeneration, yet its specific DNA recognition mechanism remains enigmatic. In this study, we showed by the ITC binding experiments that NFIL3 prefers to bind to the TTACGTAA DNA motif. Our structural studies revealed that the α-helical NFIL3 bZIP domain dimerizes through its leucine zipper region, and binds to DNA via its basic region. The two basic regions of the NFIL3 bZIP dimer were pushed apart upon binding to DNA, facilitating the snug accommodation of the two basic regions within the major grooves of the DNA. Remarkably, our binding and structural data also revealed that both NFIL3 and C/EBPα/β demonstrated a shared preference for the TTACGTAA sequence. Furthermore, our study revealed that disease-associated mutations within the NFIL3 bZIP domain resulted in either reduction or complete disruption of its DNA binding ability. These discoveries not only provide valuable insights into the DNA binding mechanisms of NFIL3 but also elucidate the causal role of NFIL3 mutations in disease pathogenesis.

Homozygous CEBPA Mutations Predict Favorable Outcomes in AML Patients

Background: Loss of heterozygosity (LOH) of CEBPA has been sporadically reported in acute myeloid leukemia (AML), resulting in a complete or nearly complete homozygosity of CEBPA mutations ( CEBPA homo). However, its clinical significance has been underappreciated. Methods: We retrospectively analyzed the prognostic impact of CEBPA homo in 1223 adult AML patients, where CEBPA mutations were detected using whole exome sequencing (n=296) or targeted sequencing (n=927) technology. CEBPA homo is defined as the variant allele frequency (VAF) of a CEBPA mutation over 50%, and CEBPA heter is the opposite. Propensity score matching was used to reduce selection bias when comparing the survival difference between two groups. Results: CEBPA mutations were identified in 106 patients (8.7%), 47(44.3%) of which carried the C-terminal DNA-binding or basic leucine zipper region ( CEBPA bZIP). Compared to patients with other CEBPA mutations ( CEBPA others), patients carrying CEBPA bZIP were significantly younger (40.38±14.80 vs. 52.05±17.44 years, P<0.001) and had higher hemoglobin [89(65.5, 98) vs 70(58, 81) g/dL, P=0.001], lower platelet counts [35(17.5, 43) vs. 44(24, 78.5) 10 9/L, P=0.019) and higher proportion of peripheral blast cells [80(52.5, 90.5) vs. 51(14, 86.5) %, P=0.001] at diagnosis. CEBPA bZIP was associated with a higher complete remission rate (CR; 85% vs. 61%, P=0.012) in AML patients but did not reduce the relapse rate (32% vs. 39%, P=0.582). CEBPA bZIP significantly improved 4-year overall survival (OS; 0.70±0.09 vs. 0.30±0.13, P=0.013), 2-year event-free survival (EFS; 0.47±0.09 vs. 0.2±0.09, P=0.01), and 3-year pre-transplantation survival (PTS; 0.54±0.09 vs. 0.38±0.09, P=0.003) in AML patients compared to those with CEBPA others. These data are consistent with previous reports, suggesting the representative of this cohort. We further evaluated the clinical significance of CEBPA homo in this cohort and found that patients with CEBPA homo had higher white blood cell counts [29.37 (9.16, 76.11) vs. 10.37 (3.38, 43.43) 10 9/L, P=0.048] than those with CEBPA heter. Although CEBPA homo was not associated with CR rate (65% vs. 80%, P=0.145), it did reduce relapse rate (26% vs. 47%, P=0.045) in AML patients compared to those with CEBPA heter. CEBPA homo was significantly associated with improved 5-year OS (0.68±0.10 vs. 0.29±0.10, P=0.006), 5-year EFS (0.30±0.09 vs. 0.10±0.08, P=0.018), and 5-year PTS (0.40±0.10 vs. 0.14±0.07, P=0.046) compared to CEBPA heter. There is no difference in CEBPA VAF between patients carrying CEBPA bZIP and CEBPA other, suggesting similar distribution of CEBPA homo between these two groups. Conclusions: The clinical significance of CEBPA homo is different from CEBPA bZIP, which suggests a distinct biological meaning. Our study would add one new layer to the current stratification of CEBPA-mutated AML based on the prognosis. More cases are needed to further examine the clinical significance of the concurrency of CEBPA homo and CEBPA bZIP.

Exploring Large MAF Transcription Factors: Functions, Pathology, and Mouse Models with Point Mutations.

Large musculoaponeurotic fibrosarcoma (MAF) transcription factors contain acidic, basic, and leucine zipper regions. Four types of MAF have been elucidated in mice and humans, namely c-MAF, MAFA, MAFB, and NRL. This review aimed to elaborate on the functions of MAF transcription factors that have been studied in vivo so far, as well as describe the pathology of human patients and corresponding mouse models with c-MAF, MAFA, and MAFB point mutations. To identify the functions of MAF transcription factors in vivo, we generated genetically modified mice lacking c-MAF, MAFA, and MAFB and analyzed their phenotypes. Further, in recent years, c-MAF, MAFA, and MAFB have been identified as causative genes underpinning many rare diseases. Careful observation of human patients and animal models is important to examine the pathophysiological mechanisms underlying these conditions for targeted therapies. Murine models exhibit phenotypes similar to those of human patients with c-MAF, MAFA, and MAFB mutations. Therefore, generating these animal models emphasizes their usefulness for research uncovering the pathophysiology of point mutations in MAF transcription factors and the development of etiology-based therapies.

Prognostic Predictability of 2022 European Leukemianet (ELN) Risk Stratification in the Real World

Introduction The European LeukemiaNet (ELN) reported 2022 guideline of the recommendation for diagnosis and management of acute myeloid leukemia (AML). Compared to 2017, ELN 2022 recommendation has some significant changes in risk classification. First, an exclusion of the FLT3-ITD allelic ratio in the risk classification, and FLT-ITD without NPM1 mutation is no longer classified as adverse risk because of the incorporation of FLT3 inhibitor. Next, in-frame mutations affecting the basic leucine zipper region (bZIP) of CEBPA regardless of monoallelic or biallelic is classified as favorable risk group. Another important change is the inclusion of myelodysplasia-related gene mutations, and patients with these gene mutations are now categorized as adverse risk group. The purpose of this study was to verify and compare the prognostic predictability between previous and new recommendation in the real world. Patients and methods A total of 624 patients who were newly diagnosed with AML from October 1998 to October 2014 in seven institutions were included in this study. Patients who achieved complete remission (CR) received consolidation chemotherapy with or without allogeneic hematopoietic stem cell transplantation (HCT), depending on the availability of a matched donor. Genetic factors were not considered while considering allogeneic HCT. Cryopreserved bone marrow or peripheral blood samples obtained at diagnosis were archived. Genomic DNA was extracted using QIAamp DNA blood mini-kits (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Genetic profiling included the targeted deep sequencing of 45 genes, which had been selected based on recurrent driver mutations from previous studies. Agilent custom probes were designed to cover the entire exon regions of targeted genes and sequenced using an Illumina HiSeq 2000 sequencer. The threshold of mutation positivity was defined as a variant allele frequency≥2%. Results All of the 624 patients were older than 18 years and age with median age was 51 years (range 18-84). All patients were treated with induction chemotherapy using a standard 3+7 protocol, and 523 patients (83.8%) achieved CR. Among them, 235 patients (44.9%) proceeded to allogeneic HCT. After median follow-up of 84.4 months, median overall survival (OS) was 22.0 months and median event-free survival (EFS) was 16.2 months in all patients. By applying the 2022 ELN risk classification, each risk groups showed significant prognostic difference with Kaplan-Meier analysis of OS (P<0.001) and EFS (P<0.001) in all patients. According to ELN 2017 risk stratification, 218 patients (34.9%) were in favorable risk group, 298 patients (47.8%) were in intermediate risk group, and 108 patients (17.3%) were in adverse risk group. Among the previously favorable group, 31 patients were reclassified as intermediate risk group and 3 patients as adverse risk group, and there was no significant prognostic difference in OS (P=0.289) and EFS (P=0.279) between patients newly classified as favorable risk group and those reclassified as intermediate or adverse risk groups. But in patients without allogeneic HCT, favorable group by 2022 edition showed significantly longer OS (P=0.003) and EFS (P<0.000) than intermediate or adverse groups. Among the previously intermediate risk group patients, 21 patients were reclassified as favorable group and 46 patients as adverse risk group, and each group showed significant difference of OS (P<0.001) and EFS (P<0.001). Also, in adverse risk group patients by the 2017 edition, 33 patients were reclassified as intermediate risk group. Because the anti-FLT3 inhibitor therapy was not available at that times, patients who reclassified as intermediate group showed no survival difference compared to the patients remaining in the adverse group. The survival analyses comparing ELN 2017 and ELN 2022 are described in figure 1. Conclusion ELN 2022 guideline is superior to the ELN 2017 for risk stratification and better predicts the prognosis of patients with AML in the real world. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The binding specificity of bZIP/DNA from a knob-socket analysis

The basic leucine zipper motif (bZIP) is a simple DNA-binding domain recognizing consensus sequences through interactions between α-helices and DNA. This α-helical domain consists of an N-terminal DNA-binding basic and C-terminal leucine zipper region forming a coiled-coil. As a transcription factor, bZIPs exhibit a high degree of variance in the DNA binding region. This study applies a knob-socket analysis to characterize the multibody determinants of the variation in specificity between individual bZIP monomers and their respective four-base half-site.

CEBPA mutations in 4708 patients with acute myeloid leukemia: differential impact of bZIP and TAD mutations on outcome

AbstractBiallelic mutations of the CEBPA gene (CEBPAbi) define a distinct entity associated with favorable prognosis; however, the role of monoallelic mutations (CEBPAsm) is poorly understood. We retrospectively analyzed 4708 adults with acute myeloid leukemia (AML) who had been recruited into the Study Alliance Leukemia trials, to investigate the prognostic impact of CEBPAsm. CEBPA mutations were identified in 240 patients (5.1%): 131 CEBPAbi and 109 CEBPAsm (60 affecting the N-terminal transactivation domains [CEBPAsmTAD] and 49 the C-terminal DNA-binding or basic leucine zipper region [CEBPAsmbZIP]). Interestingly, patients carrying CEBPAbi or CEBPAsmbZIP shared several clinical factors: they were significantly younger (median, 46 and 50 years, respectively) and had higher white blood cell (WBC) counts at diagnosis (median, 23.7 × 109/L and 35.7 × 109/L) than patients with CEBPAsmTAD (median age, 63 years, median WBC 13.1 × 109/L; P < .001). Co-mutations were similar in both groups: GATA2 mutations (35.1% CEBPAbi; 36.7% CEBPAsmbZIP vs 6.7% CEBPAsmTAD; P < .001) or NPM1 mutations (3.1% CEBPAbi; 8.2% CEBPAsmbZIP vs 38.3% CEBPAsmTAD; P < .001). CEBPAbi and CEBPAsmbZIP, but not CEBPAsmTAD were associated with significantly improved overall (OS; median 103 and 63 vs 13 months) and event-free survival (EFS; median, 20.7 and 17.1 months vs 5.7 months), in univariate and multivariable analyses. Additional analyses revealed that the clinical and molecular features as well as the favorable survival were confined to patients with in-frame mutations in bZIP (CEBPAbZIP-inf). When patients were classified according to CEBPAbZIP-inf and CEBPAother (including CEBPAsmTAD and non-CEBPAbZIP-inf), only patients bearing CEBPAbZIP-inf showed superior complete remission rates and the longest median OS and EFS, arguing for a previously undefined prognostic role of this type of mutation.

Structural basis for the dimerization mechanism of human transcription factor E3

The transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) is a member of the microphthalmia (MiT/TFE) transcription factor family. Dysregulation of TFE3 due to chromosomal abnormalities is associated with a subset of human renal cell carcinoma. Little structural information of this key transcription factor has been reported. In this study, we determined the crystal structure of the helix-loop-helix leucine zipper (HLH-Lz) domain of human TFE3 to a resolution of 2.6 Å. The HLH-Lz domain is critical for the dimerization and function of TFE3. Our structure showed that the conserved HLH region formed a four-helix bundle structure with a predominantly hydrophobic core, and the leucine zipper region contributed to the function of TFE3 by promoting dimer interaction and providing partner selectivity. Together, our results elucidated the dimerization mechanism of this important transcription factor, providing the structural basis for the development of inhibiting strategies for treating TFE3 dysregulated diseases.

Structural characteristics of coiled-coil regions in AF10-DOT1L and AF10-inhibitory peptide complex.

The interaction of the solo H3K79 methyltransferase DOT1-like (DOT1L) and its regulatory factor ALL1-fused gene from chromosome 10 protein (AF10) is crucial for the transcription of developmental genes such as HOXA in acute leukemia. The octapeptide motif and leucine zipper region of AF10 is responsible for binding DOT1L and catalyzing H3K79 monomethylation to demethylation. However, the characteristics of the mechanism between DOT1L and AF10 are not clear. Here, we present the crystal structures of coiled-coil regions of DOT1L-AF10 and AF10-inhibitory peptide, demonstrating the inhibitory peptide could form a compact complex with AF10 via a different recognition pattern. Furthermore, an inhibitory peptide with structure-based optimization is identified and decreases the HOXA gene expression in a human cell line. Our studies provide an innovative pharmacologic basis for therapeutic intervention in leukemia.

Functional analysis of large MAF transcription factors and elucidation of their relationships with human diseases.

The large MAF transcription factor group is a group of transcription factors with an acidic region, a basic region, and a leucine zipper region. Four types of MAF, MAFA, MAFB, c-MAF, and NRL, have been identified in humans and mice. In order to elucidate the functions of the large MAF transcription factor group in vivo, our research group created genetically modified MAFA-, MAFB-, and c-MAF-deficient mice and analyzed their phenotypes. MAFA is expressed in pancreatic β cells and is essential for insulin transcription and secretion. MAFB is essential for the development of pancreatic endocrine cells, formation of inner ears, podocyte function in the kidneys, and functional differentiation of macrophages. c-MAF is essential for lens formation and osteoblast differentiation. Furthermore, a single-base mutation in genes encoding the large MAF transcription factor group causes congenital renal disease, eye disease, bone disease, diabetes, and tumors in humans. This review describes the functions of large MAF transcription factors in vivo and their relationships with human diseases.

Characteristics and Expression Pattern of MYC Genes in Triticum aestivum, Oryza sativa, and Brachypodium distachyon.

Myelocytomatosis oncogenes (MYC) transcription factors (TFs) belong to basic helix-loop-helix (bHLH) TF family and have a special bHLH_MYC_N domain in the N-terminal region. Presently, there is no detailed and systematic analysis of MYC TFs in wheat, rice, and Brachypodium distachyon. In this study, 26 TaMYC, 7 OsMYC, and 7 BdMYC TFs were identified and their features were characterized. Firstly, they contain a JAZ interaction domain (JID) and a putative transcriptional activation domain (TAD) in the bHLH_MYC_N region and a BhlH region in the C-terminal region. In some cases, the bHLH region is followed by a leucine zipper region; secondly, they display tissue-specific expression patterns: wheat MYC genes are mainly expressed in leaves, rice MYC genes are highly expressed in stems, and B. distachyon MYC genes are mainly expressed in inflorescences. In addition, three types of cis-elements, including plant development/growth-related, hormone-related, and abiotic stresses-related were identified in different MYC gene promoters. In combination with the previous studies, these results indicate that MYC TFs mainly function in growth and development, as well as in response to stresses. This study laid a foundation for the further functional elucidation of MYC genes.

The Ellis van Creveld protein 1 binds and hydrolyzes GTP to a mixture of GMP and GDP and possesses the characteristics of large GTPases

Mutations in the genes that code for EVC1 and EVC2 proteins cause Ellis van Creveld (EvC) syndrome and its milder form, Weyers acrofacial dysostosis (WAD). EVC1 physically binds to EVC2 and together, they are tethered to the base of the primary cilium by the EFCAB7/IQCE protein complex. This multi‐protein assembly positively regulates the Sonic and Indian hedgehog (Hh) signaling pathways by promoting downstream processes after activation of the receptor, smoothened (smo), by the hedgehog proteins. Bioinformatic analysis revealed the presence of several structural motifs in EVC1 sequences including P‐loop, leucine zipper, transmembrane and nuclear localization signal. Using a fragment of EVC1 from mice and humans that contains P‐loop and leucine zipper regions, we present experimental evidence to show that EVC1 protein possesses a high intrinsic GTPase activity with a kcat of ~10 min−1. In addition, dimerization of EVC1 via its leucine zipper causes the protein to hydrolyze GTP to a mixture of GDP and GMP at unequal ratios. The protein binds GTP at milli to micro molar concentrations, indicating low substrate affinity. These kinetic parameters are similar to those displayed by large GTPases such as Dynamin. We propose that the GTP‐hydrolyzing activity of EVC1 protein is an important mechanistic step in the regulation of the hedgehog signaling pathways.Support or Funding InformationWelch Foundation Grant # AN‐0008This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Insights from the crystal structure of the chicken CREB3 bZIP suggest that members of the CREB3 subfamily transcription factors may be activated in response to oxidative stress

cAMP response element binding Protein 3 (CREB3) is an endoplasmic reticulum (ER) membrane‐bound transcription factor, which belongs to the basic leucine zipper (bZIP) superfamily of eukaryotic transcription factors. CREB3 plays a role in the ER‐stress induced unfolded protein response (UPR) and is a multifunctional cellular factor implicated in a number of biological processes including cell proliferation and migration, tumor suppression, and immune‐related gene expression. To gain structural insights into the transcription factor, we determined the crystal structure of the conserved bZIP domain of chicken CREB3 (chCREB3) to a resolution of 3.95 Å. The X‐ray structure provides evidence that chCREB3 can form a stable homodimer. The chCREB3 bZIP has a structured, pre‐formed DNA binding region, even in the absence of DNA, a feature that could potentially enhance both the DNA binding specificity and affinity of chCREB3. Significantly, the homodimeric bZIP possesses an intermolecular disulfide bond that connects equivalent cysteine residues of the parallel helices in the leucine zipper region. This disulfide bond in the hydrophobic core of the bZIP may increase the stability of the homodimer under oxidizing conditions. Moreover, sequence alignment of bZIP sequences from chicken, human, and mouse reveals that only members of the CREB3 subfamily contain this cysteine residue, indicating that it could act as a redox‐sensor. Taken together, these results suggest that the activity of these transcription factors may be redox‐regulated and they may be activated in response to oxidative stress.

Myc phosphorylation in its basic helix–loop–helix region destabilizes transient α-helical structures, disrupting Max and DNA binding

Myelocytomatosis proto-oncogene transcription factor (Myc) is an intrinsically disordered protein with critical roles in cellular homeostasis and neoplastic transformation. It is tightly regulated in the cell, with Myc phosphorylation playing a major role. In addition to the well-described tandem phosphorylation of Thr-52 and Ser-62 in the Myc transactivation domain linked to its degradation, P21 (RAC1)-activated kinase 2 (PAK2)-mediated phosphorylation of serine and threonine residues in the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) region regulates Myc transcriptional activity. Here we report that PAK2 preferentially phosphorylates Myc twice, at Thr-358 and Ser-373, with only a minor fraction being modified at the previously identified Thr-400 site. For transcriptional activity, Myc binds E-box DNA elements, requiring its heterodimerization with Myc-associated factor X (Max) via the bHLH-LZ regions. Using isothermal calorimetry (ITC), we found that Myc phosphorylation destabilizes this ternary protein-DNA complex by decreasing Myc's affinity for Max by 2 orders of magnitude, suggesting a major effect of phosphorylation on this complex. Phosphomimetic substitutions revealed that Ser-373 dominates the effect on Myc-Max heterodimerization. Moreover, a T400D substitution disrupted Myc's affinity for Max. ITC, NMR, and CD analyses of several Myc variants suggested that the effect of phosphorylation on the Myc-Max interaction is caused by secondary structure disruption during heterodimerization rather than by a change in the structurally disordered state of Myc or by phosphorylation-induced electrostatic repulsion in the heterodimer. Our findings provide critical insights into the effects of PAK2-catalyzed phosphorylation of Myc on its interactions with Max and DNA.

Regulatory roles of sterol regulatory element-binding protein (SREBP) on lipid metabolism in marine invertebrate Chlamys nobilis

Sterol regulatory element-binding protein (SREBP) has mainly been studied in vertebrates in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on bivalves, in which lipogenesis occurs essentially in ovary. To investigate the regulatory roles of SREBP on lipid metabolism in mollusks, the cDNA of scallop Chlamys nobilis was sequenced, its expression in different tissues and different developmental ovary stages was studied, and effect of 17β-estradiol (E2) on CnSREBP in ovary was investigated. The predicted scallop SREBP contains a conserved helix-loop-helix domain and a leucine zipper (bHLH-Zip) region, however, the amino acid sequence only shows a 39–45% identity with vertebrate. Phylogenetic tree analysis showed that the putative scallop SREBP (CnSREBP) clustered separately with invertebrate SREBP-1 and SREBP-2. There is a significantly positive correlation between CnSREBP expression level and lipid content in tissues (r = 0.9705, P < 0.001), but a reverse relationship between the gene expression level and ovary mature degree. Moreover, the gene was significantly repressed by 17β-estradiol. Our results indicated that CnSREBP was similar with vertebrate SREBP-1, which could regulate lipid metabolism in the scallops.

Quantitative analyses of the equilibria among DNA complexes of a blue-light-regulated bZIP module, Photozipper.

Aureochrome1 is a blue-light-receptor protein identified in a stramenopile alga, Vaucheria frigida. Photozipper (PZ) is an N-terminally truncated, monomeric, V. frigida aureochrome1 fragment containing a basic leucine zipper (bZIP) domain and a light–oxygen–voltage (LOV)-sensing domain. PZ dimerizes upon photoexcitation and consequently increases its affinity for the target sequence. In the present study, to understand the equilibria among DNA complexes of PZ, DNA binding by PZ and mutational variants was quantitatively investigated by electrophoretic-mobility-shift assay and fluorescence-correlation spectroscopy in the dark and light states. DNA binding by PZ was sequence-specific and light-dependent. The half-maximal effective concentration of PZ for binding to the target DNA sequence was ~40 nM in the light, which was >10-fold less than the value in the dark. By contrast, the dimeric PZ-S2C variant (with intermolecular disulfide bonds) had higher affinity for the target sequence, with dissociation constants of ~4 nM, irrespective of the light conditions. Substitutions of Glu159 and Lys164 in the leucine zipper region decreased the affinity of PZ for the target sequence, especially in the light, suggesting that these residues form inter-helical salt bridges between leucine zipper regions, stabilizing the dimer–DNA complex. Our quantitative analyses of the equilibria in PZ–DNA-complex formation suggest that the blue-light-induced dimerization of LOV domains and coiled-coil formation by leucine zipper regions are the primary determinants of the affinity of PZ for the target sequence.

Genome-Wide Identification of bZIP Family Genes Involved in Drought and Heat Stresses in Strawberry (Fragaria vesca).

Basic leucine zipper (bZIP) genes are known to play a crucial role in response to various processes in plant as well as abiotic or biotic stress challenges. We have performed an identification and characterization of 50 bZIP genes across the woodland strawberry (Fragaria vesca) genome, which were divided into 10 clades according to the phylogenetic relationship of the strawberry bZIP proteins with those in Arabidopsis and rice. Five categories of intron patterns were observed within basic and hinge regions of the bZIP domains. Some additional conserved motifs have been found with the group specificity. Further, we predicted DNA-binding specificity of the basic and hinge regions as well as dimerization properties of leucine zipper regions, which was consistent with our phylogenetic clade and classified into 20 subfamilies. Across the different developmental stages of 15 organs and two types of fruits, the clade A bZIP members showed different tissue-specific expression patterns and the duplicated genes were differentially regulated, indicating a functional diversification coupled with the expansion of this gene family in strawberry. Under normal growth conditions, mrna11837 and mrna30280 of clade A showed very weak expression levels in organs and fruits, respectively; but higher expression was observed with different set of genes following drought and heat treatment, which may be caused by the separate response pathway between drought and heat treatments.

Bioinformatic Analyses of Subgroup-A Members of the Wheat bZIP Transcription Factor Family and Functional Identification of TabZIP174 Involved in Drought Stress Response.

Extensive studies in Arabidopsis and rice have demonstrated that Subgroup-A members of the bZIP transcription factor family play important roles in plant responses to multiple abiotic stresses. Although common wheat (Triticum aestivum) is one of the most widely cultivated and consumed food crops in the world, there are limited investigations into Subgroup A of the bZIP family in wheat. In this study, we performed bioinformatic analyses of the 41 Subgroup-A members of the wheat bZIP family. Phylogenetic and conserved motif analyses showed that most of the Subgroup-A bZIP proteins involved in abiotic stress responses of wheat, Arabidopsis, and rice clustered in Clade A1 of the phylogenetic tree, and shared a majority of conserved motifs, suggesting the potential importance of Clade-A1 members in abiotic stress responses. Gene structure analysis showed that TabZIP genes with close phylogenetic relationships tended to possess similar exon–intron compositions, and the positions of introns in the hinge regions of the bZIP domains were highly conserved, whereas introns in the leucine zipper regions were at variable positions. Additionally, eleven groups of homologs and two groups of tandem paralogs were also identified in Subgroup A of the wheat bZIP family. Expression profiling analysis indicated that most Subgroup-A TabZIP genes were responsive to abscisic acid and various abiotic stress treatments. TabZIP27, TabZIP74, TabZIP138, and TabZIP174 proteins were localized in the nucleus of wheat protoplasts, whereas TabZIP9-GFP fusion protein was simultaneously present in the nucleus, cytoplasm, and cell membrane. Transgenic Arabidopsis overexpressing TabZIP174 displayed increased seed germination rates and primary root lengths under drought treatments. Overexpression of TabZIP174 in transgenic Arabidopsis conferred enhanced drought tolerance, and transgenic plants exhibited lower water loss rates, higher survival rates, higher proline, soluble sugar, and leaf chlorophyll contents, as well as more stable osmotic potential under drought conditions. Additionally, overexpression of TabZIP174 increased the expression of stress-responsive genes (RD29A, RD29B, RAB18, DREB2A, COR15A, and COR47). The improved drought resistance might be attributed to the increased osmotic adjustment capacity. Our results indicate that TabZIP174 may participate in regulating plant response to drought stress and holds great potential for genetic improvement of abiotic stress tolerance in crops.